# ent

id: cord-271915-nvilxnzl
type: ORG
entity:
  Toronto World Health Organization Multicentre Collaborative Network for Severe Acute Respiratory Syndrome SARS Diagnosis


id: cord-277186-sj8ngpk8
type: PROTEIN
entity:
  SARS CoV nucleocapsid protein He Q Chong K H Chang H H Leung B Ling A E Wei T Chan S W Ooi E E Kwang J 2004 Development


id: cord-258057-ti0rpt0q
type: CHEMICAL
entity:
  dNTPs 0 4 μM each of F3 and B3 2 5 U Taq DNA polymerase J o u r n a l P r e p r o o f TaKaRa biotechnology Co Ltd Dalian China and 1 μL template


id: cord-257785-jzdlvo7p
type: TAXON
entity:
  adenovirus coronavirus 229E OC43 NL63 HKU1 parainfluenza 1 4 rhinovirus respiratory syncytial virus human metapneumovirus


id: cord-257785-jzdlvo7p
type: DISEASE
entity:
  influenza B adenovirus coronavirus 229E OC43 NL63 HKU1 parainfluenza 1 4 rhinovirus respiratory syncytial virus human metapneumovirus


id: cord-257785-jzdlvo7p
type: DISEASE
entity:
  influenza A H1N1 2009 H275Y positive clinical sample Each dilution was tested in duplicate This was carried out to ensure that the addition of the H275Y assay to the universal influenza A H1N1 2009 duplex


id: cord-257785-jzdlvo7p
type: DISEASE
entity:
  influenza A H1N1 2009 To assess inter assay variability reproducibility long term precision positive extraction run controls for FLUA N1 and H275Y were monitored over 20 PCR runs To assess intra assay variability repeatability short term precision a positive extraction run control for FLUA N1 and H275Y were tested in 20 wells on the same PCR run Finally the ability of the triplex


id: cord-257785-jzdlvo7p
type: DISEASE
entity:
  influenza A H1N1 2009 and seasonal H1N1 and H3N2 viruses Table 3 When testing the dilution series of the influenza A H1N1 2009 sample the influenza A component of the duplex detected the 10 5 dilution in 1 out of 2 occasions whereas the influenza A component of the triplex


id: cord-257785-jzdlvo7p
type: DISEASE
entity:
  influenza A H1N1 2009 virus The emergence of oseltamivir resistant pandemic influenza A H1N1 2009 virus


id: cord-277057-ww41t4k2
type: EVENT
entity:
  Clinical Infectious Diseases An Official Publication of the Infectious Diseases Society of America Clusters


id: cord-277057-ww41t4k2
type: DISEASE
entity:
  rhinovirus coronavirus adenovirus parainfluenza viruses human metapneumovirus respiratory syncytial virus and influenza A B viruses Wallace 2003


id: cord-265634-7n4cvgs4
type: DISEASE
entity:
  infectious hypodermal and hematopoietic necrosis virus IHHNV resistance in shrimp Penaeus stylirostris Sindrome


id: cord-257850-x7qtxaum
type: PROTEIN
entity:
  structural protein 1 HA hemagglutinin NA neuraminidase M2 1 matrix protein F fusion glycoprotein P phosphoprotein


id: cord-257850-x7qtxaum
type: PROTEIN
entity:
  structural protein 1 HA hemagglutinin NA neuraminidase M2 1 matrix protein F fusion glycoprotein P phosphoprotein c


id: cord-252268-o63ep08b
type: ORG
entity:
  A Language and Environment for Statistical Computing R Foundation for Statistical Computing Alterations


id: cord-261134-zarq507s
type: PROTEIN
entity:
  CP1 4 cowpox virus Brighton 5 cowpox virus EP1 6 cowpox virus Norway 7 ectromelia virus MP1 8 monkeypox virus Z1 9


id: cord-284644-9k2oox64
type: CHEMICAL
entity:
  influenza D virus in cattle Rapid and specific detection of H3 swine influenza virus using reverse transcription


id: cord-270421-ytrkob0h
type: PROTEIN
entity:
  Polio virus goat anti G10 CA16 serum mouse anti FY523 EV71 serum mouse anti Coxsackievirus B5 CVB5 serum mouse anti Hepatitis A virus HAV


id: cord-270421-ytrkob0h
type: DNA
entity:
  mouse anti coxsackievirus B5 CVB5 serum mouse anti hepatitis A virus HAV mouse anti FY523 EV71 serum goat anti


id: cord-275787-5s442sy2
type: PROTEIN
entity:
  monoclonal anti cytokeratin 8 13 clone K8 13 mouse ascites fluid IgG2a isotype Sigma USA Secondary antibodies


id: cord-275787-5s442sy2
type: ORG
entity:
  the Committee on Animal Care and Supply of the University of Saskatchewan Animal Research Ethics Board


id: cord-259738-yuqc6dk0
type: DNA
entity:
  N forward primer 5 CATCTCGAGTCTT TTATCATGGCAAGC 3 reverse primer 5 GGCGAATTCA TTAGAGTTCATTTTCAC 3 IL 2 forward primer 5 CCAGGATCCACCATGATGTGCAAAG 3 reverse primer 5 GAAGCGGCCGCAGATTAGTTAGC 3


id: cord-259738-yuqc6dk0
type: CHEMICAL
entity:
  N gene and IL 2 as listed N forward primer 5 CATCTCGAGTCTT TTATCATGGCAAGC 3 reverse primer 5 GGCGAATTCA TTAGAGTTCATTTTCAC 3 IL 2 forward primer 5 CCAGGATCCACCATGATGTGCAAAG 3 reverse primer 5 GAAGCGGCCGCAGATTAGTTAGC 3 The medium


id: cord-286360-wrrqb387
type: CELL_LINE
entity:
  CCV1 5 TCC AGA TAT GTA ATG TTC GG 3 sense primer 337 356 nucleotides CCV2 5 TCT GTT GAG TAA TCA CCA GCT 3


id: cord-307304-irji8owi
type: DNA
entity:
  dominant selectable marker gene Escherichia coli guanine Xanthine phosphoribosyltransferase Ecogpt Mulligan


id: cord-307304-irji8owi
type: DISEASE
entity:
  infectious positive stranded RNA viruses including picornaviruses caliciviruses alphaviruses flaviviruses arteriviruses


id: cord-276718-3lujp0oy
type: CL
entity:
  stem lengths and cauliflower like structures with multiple loops formed by annealing between alternately inverted repeats of the target sequence


id: cord-297160-tqw9vx2b
type: DNA
entity:
  base pairs IB D274 5 ATACAATTATATCAAACCAGC 3 5 AATACAGATTGCTTACAACCACC 3 211 IB H120 5 AATACTACTTTTACGTTACAC 3 5 AATACAGATTGCTTACAACCACC 3 299


id: cord-255983-3dq99xz9
type: DNA
entity:
  InfV A influenza virus B InfV B human coronaviruses 229E OC43 human metapneumovirus hMPV parainfluenza virus 1 2 and 3 PIV1 2 3 human rhinovirus hRV A


id: cord-279541-rjp2d1u9
type: DNA
entity:
  B human metapneumovirus HMPV coronavirus OC43 coronavirus 229E infectious bursal disease virus IBDV and infectious bronchitis virus IBV


id: cord-279541-rjp2d1u9
type: ORG
entity:
  the Department of Livestock Development Bangkok Thailand 3 Faculty of Veterinary Science Kasetsart University


id: cord-264335-c2hfh3dq
type: DISEASE
entity:
  influenza B influenza C parainfluenza 1 4 human metapneumovirus respiratory syncytial virus Mycoplasma


id: cord-281999-jc4ckqy7
type: DNA
entity:
  nucleotides 1597 1625 GAPDH forward primer 5 CATTGACCTCAACTACATGG 3 GAPDH reverse primer 5 TTGCCCA CAGCCTTGGCAGC 3


id: cord-273074-k8m917i4
type: ORG
entity:
  Trevor Bagust Faculty of Veterinary Science University of Melbourne and Cui Xianlan Landcare Research


id: cord-258008-t78svobg
type: TAXON
entity:
  virus respiratory syncytial virus human metapneumovirus rhinovirus coronaviruses parainfluenza viruses


id: cord-258008-t78svobg
type: DISEASE
entity:
  influenza B viruses respiratory syncytial virus and parainfluenza viruses 1 2 3 and 4 Frequent detection of human coronaviruses


id: cord-302663-gb2vgs97
type: DNA
entity:
  X174 Hinc II digest lanes 2 7 10 1 10 2 10 3 10 4 10 5 and 10 6 respectively B 0 5 M NaOH method lane 1 molecular size marker X174 Hinc II digest lanes 2 5 10 2 10 3 10 4 and 10 5 RT


id: cord-278377-jgq3dz3u
type: DISEASE
entity:
  influenza B parainfluenza 1 4 and respiratory syncytial virus RSV The panel also detects 3 bacteria Mycoplasma


id: cord-278377-jgq3dz3u
type: DISEASE
entity:
  influenza A with antigen detection and cell cultures which depends on the age of patients can be explained by the higher viral shedding


id: cord-294454-uzfsv2df
type: DISEASE
entity:
  influenza B virus 0 88 to 1 for hRSV 0 2 to 1 for PIV 1 0 4 to 1 for PIV 2 0 70 to 1 for PIV 3 and 0 33 to 1 for HCoV OC43


id: cord-275225-fvq8hezk
type: DNA
entity:
  Ákos Bálint Ádám Farsang Attila Balka Gyula Hakhverdyan Mikhayil Rasmussen Thomas Bruun Blomberg Jonas Belák Sándor title


id: cord-270788-w0pewq52
type: PROTEIN
entity:
  N terminal amino acid residues 270 510 Babcock et al 2004 303 537 Xiao et al 2003 and 318 510 Wong et al 2004 In


id: cord-292831-oihcay6w
type: DISEASE
entity:
  influenza B seasonal influenza A H1N1 H3N2 H1N1pdm09 RSV A and B HMPV PIV 1 2 3 and enterovirus Clinical samples positive for PIV 4 rhinovirus human corona virus OC43 HKU1 and 229E NL63


id: cord-310771-tnwfp1je
type: TAXON
entity:
  transmissible gastroenteritis virus TGEV porcine respiratory coronavirus PRCV Chlamydophila Brucella


id: cord-293651-96cmduez
type: DNA
entity:
  5 primer A Beau U30 5 TACACTAGCCTTGCGC TAGA 3 and 3 primer A Beau L5447 5 GCACGCCA AAGTCCCATAG 3 Briefly RNA


id: cord-276541-u9ebql5a
type: DNA
entity:
  shN1 nt7004 7024 Sense 5 caccGGTACTGGTACAGACACAACAttcaagagaTGTTGTGTCTGTACCAGTACCTTTTTTg 3 Antisense 5 gatccAAAAAAGGTACTGGTACAGACACAACAtctcttgaaTGTTGTGTCTGTACCAGTACC 3 shN2 nt7218 7238 Sense 5 caccGGCTATTCCGACTAGGTTTCCttcaagagaGGAAACCTAGTCGGAATAGCCTTTTTTg 3 Antisense 5 gatccAAAAAAGGCTATTCCGACTAGGTTTCCtctcttgaaGGAAACCTAGTCGGAATAGCC 3


id: cord-305640-tgowzrqo
type: ORG
entity:
  the Rapid Diagnosis of Emerging Infections Case Definitions for Surveillance of Severe Acute Respiratory Syndrome SARS


id: cord-308338-lhe51ws7
type: CELL_LINE
entity:
  HKU1 human metapneumovirus hMPV lineages 1 and 2 human rhinovirus 1B coxsackievirus A16 echovirus 2 human adenovirus type 3 human bocavirus Chlamydophila pneumoniae Legionella pneumophila


id: cord-308338-lhe51ws7
type: TAXON
entity:
  coxsackievirus A16 echovirus 2 human adenovirus type 3 human bocavirus Chlamydophila pneumoniae Legionella pneumophila


id: cord-308338-lhe51ws7
type: DISEASE
entity:
  HKU1 human metapneumovirus hMPV lineages 1 and 2 human rhinovirus 1B coxsackievirus A16 echovirus 2 human adenovirus type 3 human bocavirus Chlamydophila


id: cord-298922-k568hlf4
type: PROTEIN
entity:
  polyclonal antibodies anti tubulin anti integrin 3 anti cystatin C anti protein S100 A2 anti apolipoprotein E4


id: cord-286117-m1rlmlun
type: ORG
entity:
  Diagnosis of Feline Infectious Peritonitis A Review of the Current Literature Diagnostic Features of Clinical Neurologic Feline Infectious Peritonitis Feline


id: cord-277804-ujabzic4
type: ORG
entity:
  Chicken Embryos United States Department of Agriculture Animal and Plant Health Inspection Service Protection


id: cord-299585-fkg8d6ym
type: PROTEIN
entity:
  structural proteins hemagglutinin esterase protein HE spike glycoprotein S envelope protein E membrane protein M nucleocapsid protein N


id: cord-302024-zz7mt6be
type: DNA
entity:
  HRSV F gene sequences GenBank cation 94 C for 30 s 50 C for 30 s data collected 72 C for 45 s A 10 fold dilution


id: cord-321886-0b3ocoh9
type: CHEMICAL
entity:
  MgCl 2 1 l each of outer primers 10 M 1 l each of inner primers 40 M 1 l each of loop primers 20 M 2 5 l of dNTP mixture 2 5 mM each 2 5 l of betaine 5 M 1 l of extracted template


id: cord-311639-zij2wbzs
type: PROTEIN
entity:
  KBPV VR 58 type 3 KBPV VR 2 type 4 KBPV VR 60 type 8 KBPV VR 3 type 18 KBPV VR 4 type 23 KBPV VR 5 enterovirus type 71


id: cord-311639-zij2wbzs
type: PROTEIN
entity:
  RIDAQUICK 57 1 83 QuickNavi Noro 88 6 IP NORO 73 7 78 9 IC Kit 75 4 RIDASCREEN 36 92 IDEIA 38 76 9 and Denka ELISA 80 90 4


id: cord-288701-nx9fg4yn
type: DISEASE
entity:
  HoBi like pestivirus The amount of RNA detected in the samples covered a wide range of copies per microlitre of template ranging from 4 02 10 1 to 7 26 10 7 BVDV 1 from 5 78 10 1 to 8 45 10 6 BVDV 2


id: cord-317462-nvrl0vyi
type: CHEMICAL
entity:
  MgCl 2 2 L 10 RT Buffer 1 L RNase Free dH 2 O 4 75 L dNTP Mixture 1 L RNase Inhibitor 0 25 L AMV Reverse Transcriptase 0 5 L Random 9 mer 0 5 L antisense primer 0 5 L


id: cord-259593-shrd1s7r
type: PROTEIN
entity:
  short amino terminal ectodomain residues 1 14 three transmembrane helices residues 15 37 50 72 and 77 99


id: cord-317307-q5mgue5z
type: DNA
entity:
  Primer Express 3 0 software Applied Biosystems and OligoPerfect TM Designer software Invitrogen Table 2 Primer


id: cord-331509-p19dg1jw
type: PROTEIN
entity:
  Beitrag zur kollektiven Behandlung pharmakologisher Reihenversuche MAFFT multiple sequence alignment software version 7


id: cord-322410-k23engcx
type: DNA
entity:
  Massachusetts D3128 D274 D 08880 and 4 91 genotypes Abdel Moneim et al 2006 Jackwood 2012 Sheble et al 1986 In 2001 a unique Egyptian variant type isolate Egypt Beni Suef 01 closely


id: cord-322410-k23engcx
type: ORG
entity:
  the National Laboratory for Veterinary Quality Control on Poultry Production NLQP Ministry of Agriculture Giza


id: cord-322410-k23engcx
type: DISEASE
entity:
  Egyptian variant II sequences Fig 2 Within HVR 1 2 sequences however the existence of two distinct Table 3 RT


id: cord-332522-adul9nzf
type: PROTEIN
entity:
  Taq DNA polymerase Promega 0 1 mM dNTPs 10 mM Tris HCl pH 8 3 50 mM KCl 1 25 mM MgCl 2 and 1 25 l of SYBR Green I or 0 5 l of Taqman probe 5 M


id: cord-322234-1zyy536y
type: TAXON
entity:
  transmissible gastroenteritis virus TGEV PRRSV porcine circovirus type 2 PCV2 porcine adenovirus porcine parvovirus


id: cord-322234-1zyy536y
type: CHEMICAL
entity:
  PRCoV TGEV PRRSV porcine circovirus type 2 swine adenovirus porcine parvovirus blue eye paramyxovirus and pseudorabies virus Cross reaction


id: cord-324213-3uqlimov
type: DISEASE
entity:
  influenza A H5N1 virus Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples Surveillance


id: cord-336639-jaue41mv
type: DISEASE
entity:
  gastroenteritis coronavirus nucleocapsid protein gene Histopathological alterations of lymphatic tissues


id: cord-258468-52gej3co
type: DNA
entity:
  dsDNA adaptor sense oligonucleotide 5 CATGACCTATCCGCGCCGCCGTTACC GCCGCCGCCGCCACCGCCCGCGCAGCCATCTGGG 3


id: cord-313271-2e8vjtop
type: DNA
entity:
  HCoV NL63 sense 5 GCGTGTTCCTACCAGAGA GGA 3 anti sense 5 GCTGTGGAAAACCTTTGGCA 3 probe 5 FAM ATGTTATTCAGTGCTTTGGTCCTCGTGAT TAM RA 3


id: cord-261329-k1p7fo0e
type: PROTEIN
entity:
  BHKPI89165 AY175672 Herts 33 M24702 TEXAS M33855 Mukteswar AY175676 Miyadera 51 M24701 Australia 32 M21881 AR5 Table 1 AR8 Table 1 AR3 Table 1 AR2 Table 1 AR7 Table 1 AR4 Table 1 Italy Table 1 GB1168 84 AF109885 CH1 95


id: cord-007648-tm0hn0hz
type: GGP
entity:
  glycopolypeptides S 1 and 2 which have molecular weights of 90 kDa and 84 kDa respectively Cavanagh 1981 The membrane protein


# bib

id: cord-007427-iqwojhq2
title: Development and Evaluation of a One-Step Quantitative RT-PCR Assay for Detection of Lassa Virus
author: Dedkov, Vladimir G.
date: 2019-06-03
txt: ./txt/cord-007427-iqwojhq2.txt
cache: ./cache/cord-007427-iqwojhq2.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113850/
doi: 10.1016/j.jviromet.2019.113674
summary:
  Based on sequencing data, LASV-specific assay was developed using synthetic MS2-phage-based armored RNA particles, RNA from Lassa virus strain Josiah, and further, evaluated in field conditions using samples from patients and Mastomys natalensis rodents. Viral RNAs were examined for Lassa immediately after extraction by the staff of the Virology Laboratory of Hemorrhagic Fevers Research Project of Gamal Abdel Nasser University of Conakry, Guinea and were then used to assess diagnostic sensitivity and specificity. In addition, LOD was assessed using a series of 10-fold dilutions of ARPs. For this purpose, eight LASV sequences of a maximal number of mismatches in the targeting region of L gene were selected (including a sequence of the strain Josiah, which was also used for the generation of the positive controls) and generated for the production of ARPs as described above (Table 2 ) .

abstract:
  Lassa fever is a severe viral hemorrhagic illness caused by Lassa virus. Based on estimates, the number of LASV infections ranges from 300,000 to 500,000 cases in endemic areas with a fatality rate of 1%. Development of fast and sensitive tools for the control and prevention of Lassa virus infection as well as for clinical diagnostics of Lassa fever are crucial. Here we reported development and evaluation of a one-step quantitative RT-qPCR assay for the Lassa virus detection – LASV-Fl. This assay is suitable for the detection of lineages I-IV of Lassa virus. The limit of detection of the assay ranged from 10(3) copies/ml to 10(5) copies/ml and has 96.4% diagnostic sensitivity, whereas analytical and diagnostic specificities both were 100%. Serum, whole blood and tissue are suitable for use with the assay. The assay contains all the necessary components to perform the analysis, including an armored positive control (ARC+) and an armored internal control (IC). The study was done during the mission of specialized anti-epidemic team of the Russian Federation (SAET) in the Republic of Guinea in 2015-2018. Based on sequencing data, LASV-specific assay was developed using synthetic MS2-phage-based armored RNA particles, RNA from Lassa virus strain Josiah, and further, evaluated in field conditions using samples from patients and Mastomys natalensis rodents.


id: cord-007644-7bsixsgd
author: Chirnside, E.D.
date: 2000-04-04
txt: ./txt/cord-007644-7bsixsgd.txt
cache: ./cache/cord-007644-7bsixsgd.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7119792/
doi: 10.1016/0166-0934(95)00020-u
summary:
  authors: Chirnside, E.D.; Francis, P.M.; De Vries, A.A.F.; Sinclaira, R.; Mumford, J.A. title: Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus A recombinant glutathione-S-transferase fusion protein expressing amino acids 55–98 of equine arteritis virus (EAV) G(L) (rG(L)55–98) was tested in an ELISA for its ability to detect serum antibodies to EAV. This paper describes an indirect ELISA using a recombinant glutathione-Stransferase fusion protein (Smith and Johnson, 1988) as an antigen to screen equine sera for the presence of antibodies to EAV, and its evaluation as a diagnostic test with large numbers of equine serum samples. By testing > 1500 equine sera in ELISA to G,55-98, we have demonstrated that amino acid residues 55-98 of the Bucyrus strain of EAV G,_ encompass a highly immunoreactive antigen which correlates closely with the host virus neutralizing response.

title:
  Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus

abstract:
  A recombinant glutathione-S-transferase fusion protein expressing amino acids 55–98 of equine arteritis virus (EAV) G(L) (rG(L)55–98) was tested in an ELISA for its ability to detect serum antibodies to EAV. Host antibodies induced following EAV infection bound the recombinant antigen by ELISA. The ELISA specificity and sensitivity were determined with a panel of equine sera including postinfection and postvaccination samples. A good correlation existed between EAV neutralizing antibody titers and ELISA absorbance values (r = 0.827). The sensitivity and specificity of the ELISA were 99.6 and 90.1%, respectively, compared with the EAV neutralization test and the recombinant antigen did not crossreact in ELISA with equine sera directed against other common equine respiratory viruses. Three post-EAV infection equine sera raised against different EAV isolates reacted strongly in the ELISA, as did two equine sera raised against EAV vaccines, indicating that the viral epitope was conserved between the viruses tested. Following vaccination with an inactivated whole virus vaccine, antibody detected with the recombinant antigen ELISA preceded the development of a virus-neutralizing response. The study demonstrates the potential application of rG(L)55–98 as a diagnostic antigen.


id: cord-007648-tm0hn0hz
title: Envelope proteins of avian infectious bronchitis virus: Purification and biological properties
author: Mockett, A.P.Adrian
date: 2002-12-20
txt: ./txt/cord-007648-tm0hn0hz.txt
cache: ./cache/cord-007648-tm0hn0hz.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7119818/
doi: 10.1016/0166-0934(85)90138-7
summary:
  Immunoadsorbents, made with monoclonal antibodies, were used to purify the spike and membrane proteins of infectious bronchitis virus (IBV). A second inoculation of virus at 42 days induced an anamnestic antibody response to the spike and membrane proteins and also for the neutralizing antibodies. The objectives of this work were to produce immunoadsorbents using monoclonal antibodies which have been prepared previously (Mockett et al., 1984) and to purify the virus-coded proteins of the viral envelope in a single step in relatively large amounts. In addition the sequential humoral antibody response of chickens after IBV infection has been studied using the purified viral proteins and whole virus in ELISAs and compared to the results using the neutralization test. Thc chicken, about 10 days after an IBV infection, has antibodies to both the spike and membrane proteins in its serum but only very low concentrations of neutralizing antibodies.

abstract:
  Immunoadsorbents, made with monoclonal antibodies, were used to purify the spike and membrane proteins of infectious bronchitis virus (IBV). The purified proteins were inoculated into rabbits to produce antisera. The rabbit anti-spike sera neutralized the infectivity of the virus whereas the anti-membrane sera did not. IBV-infected chickens produced antibodies to both the spike and membrane proteins. Both these antibodies were at their highest concentration about 9–11 days after inoculation, whereas neutralizing antibodies were present only at very low concentrations at that time. Neutralizing antibodies were at their highest concentration 21 days after inoculation. A second inoculation of virus at 42 days induced an anamnestic antibody response to the spike and membrane proteins and also for the neutralizing antibodies. The neutralizing, anti-spike and anti-membrane antibodies all reached highest concentrations 7–11 days after this inoculation. The advantages of purifying viral proteins using affinity chromatography with monoclonal antibodies are discussed.


id: cord-015936-4fwkf8fn
title: SUBJECT INDEX, volumes 123-130
author: nan
date: 2005-11-04
txt: ./txt/cord-015936-4fwkf8fn.txt
cache: ./cache/cord-015936-4fwkf8fn.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7120062/
doi: 10.1016/s0166-0934(05)00346-0
abstract: nan
summary:
  key: cord-015936-4fwkf8fn authors: nan title: SUBJECT INDEX, volumes 123-130 date: 2005-11-04 journal: J Virol Methods DOI: 10.1016/s0166-0934(05)00346-0 sha: doc_id: 15936 cord_uid: 4fwkf8fn nan HIV; 2 LTR circles; New marker (125) 11 HIV antigen/antibody combined assay; HIV; Serology; HIV-1 p24 antigen assay (127) (127) (128) (128) 67 Yeast expression; Pichia pastoris; SARS-CoV; N protein (130) 83 enzyme analysis; Flaviviruses; RT-PCR


id: cord-251974-2zwrqjj9
author: Geller, Chloé
date: 2009-04-05
txt: ./txt/cord-251974-2zwrqjj9.txt
cache: ./cache/cord-251974-2zwrqjj9.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2009.03.023
doi: 10.1016/j.jviromet.2009.03.023
summary:
  The European Standard suggests other methods, based on gel-filtration techniques, using products as Sephadex TM LH-20, Microspin TM columns S400HR or Minicon ® concentrators, but they lead to problems of contact times, separation capacity and cost (supplementary data, Fig. 1 ). These tests were also performed with solutions of CHX and HXM at various concentrations and after their filtration on the "in-house" Sephadex TM columns to evaluate their retention and the elimination of potential cytotoxicity. Three types of controls were performed in each experiment to validate the results: (i) non-retention of viruses after filtration on Sephadex TM columns, (ii) neutralization of the potential antiviral activity of the product tested, and (iii) elimination of its cytotoxicity. To assess the non-retention of viruses, e.g. HCoV 229E, viral suspensions were mixed with sterile distilled water for the contact time defined for the experiment, filtered on the "in-house" Sephadex TM columns and titrated as describe above (Section 2.3).

title:
  A new Sephadex™-based method for removing microbicidal and cytotoxic residues when testing antiseptics against viruses: Experiments with a human coronavirus as a model

abstract:
  The relative lack of efficient methods for evaluating antiseptic antiviral activity, together with weaknesses in the existing European Standard (i.e. NF EN 14476+A1), underlines the need to seek a new method which could allow a more precise evaluation of the antiseptic antiviral activity of chemical agents. This protocol is based on an original gel-based filtration method, using “in-house” G-25 and G-10 Sephadex™ columns. This method allows the neutralization of both the activity and the cytotoxicity of a large range of molecules, according to their molecular size, in only 1 min. The viral model used was the human coronavirus (HCoV) 229E chosen for (i) its increasing medical interest, (ii) its potential resistance and (iii) its representing enveloped viruses mentioned in the European Standard. First, the protocol was validated and it was demonstrated that it was fully operational for evaluating antiviral antiseptic potentiality and useful to screen potentially antiseptic molecules. Second, chlorhexidine (CHX) and hexamidine (HXM) were assessed for their potential anti-HCoV 229E antiseptic activities. It was demonstrated clearly that (i) HXM had no activity on the HCoV 229E and (ii) CHX showed a moderate anti-HCoV 229E activity but insufficient to be antiseptic.


id: cord-251991-ghbpga1s
title: Evaluation of the Calu-3 cell line as a model of in vitro respiratory syncytial virus infection()
author: Harcourt, Jennifer L.
date: 2011-03-31
txt: ./txt/cord-251991-ghbpga1s.txt
cache: ./cache/cord-251991-ghbpga1s.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093411001182
doi: 10.1016/j.jviromet.2011.03.027
summary:
  Respiratory syncytial virus (RSV) replication is primarily limited to the upper respiratory tract epithelium and primary, differentiated normal human bronchial epithelial cells (NHBE) have, therefore, been considered a good system for in vitro analysis of lung tissue response to respiratory virus infection and virus–host interactions. Polarized Calu-3 are susceptible to RSV infection and release infectious virus primarily from the apical surface, consistent with studies in NHBE cells. The mechanisms of cellular responses to RSV infection have been studied extensively in vitro in a variety of immortalized epithelial cell lines grown in monolayer cultures, including but not limited to Vero, Hep-2, A549, and ଝ The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of CDC. Consistent with previous studies in polarized MDCK (Roberts et al., 1995) and in differentiated NHBE, polarized Calu-3 released infectious virus primarily from the apical surface, and infection was persistent, detectable for at least 6 weeks post-infection.

abstract:
  Respiratory syncytial virus (RSV) replication is primarily limited to the upper respiratory tract epithelium and primary, differentiated normal human bronchial epithelial cells (NHBE) have, therefore, been considered a good system for in vitro analysis of lung tissue response to respiratory virus infection and virus–host interactions. However, NHBE cells are expensive, difficult to culture, and vary with the source patient. An alternate approach is to use a continuous cell line that has features of bronchial epithelial cells such as Calu-3, an epithelial cell line derived from human lung adenocarcinoma, as an in vitro model of respiratory virus infection. The results show that Calu-3 fully polarize when grown on permeable supports as liquid-covered cultures. Polarized Calu-3 are susceptible to RSV infection and release infectious virus primarily from the apical surface, consistent with studies in NHBE cells. The data demonstrate that polarized Calu-3 may serve as a useful in vitro model to study host responses to RSV infection.


id: cord-252268-o63ep08b
title: TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses
author: Carolan, Louise A.
date: 2014-09-01
txt: ./txt/cord-252268-o63ep08b.txt
cache: ./cache/cord-252268-o63ep08b.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2014.04.014
doi: 10.1016/j.jviromet.2014.04.014
summary:
  As this equation relies on consistency between samples in the RNA quantity assayed, and the optimum reaction efficiency, as well as minimal fluctuation in the expression of housekeeping genes (endogenous controls) (Peters et al., 2007; Mane et al., 2008; Bruder et al., 2010) , these parameters were optimized using RNA extracted from cultured ferret lymph node cells stimulated with mitogens or with influenza virus. To test the ability of ferret leukocytes to produce mRNA cytokines and chemokines, lymph node cells from naïve ferrets were cultured with various mitogens known to activate T and B lymphocyte and macrophage/monocyte responses (Fig. 5) and with live or heat inactivated influenza virus (Fig. 6 ) and the cytokine and chemokine expression profiles were determined (summarized in (ConA) or Phytohaemagglutinin (PHA), which act by cross linking T cell receptors via sugars on the surface of human T lymphocytes (Chilson and Kelly-Chilson, 1989) , induced similar cytokine profiles, increasing expression of IL2, IL4, IL6, IL10, IL17, Granzyme A, TNF␣ and IFN␥, most with high fold changes, consistent with effective stimulation of T lymphocytes (Fig. 5) .

abstract:
  The ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1 ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states.


id: cord-254210-3mi2aop5
title: Silencing of HTLV-1 gag and env genes by small interfering RNAs in HEK 293 cells
author: Haddad, Rodrigo
date: 2011-01-26
txt: ./txt/cord-254210-3mi2aop5.txt
cache: ./cache/cord-254210-3mi2aop5.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093411000413
doi: 10.1016/j.jviromet.2011.01.012
summary:
  title: Silencing of HTLV-1 gag and env genes by small interfering RNAs in HEK 293 cells Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. In the present study, siRNAs were used for inhibition of the expression of the HTLV-1 structural genes gag and env. Forty-eight hours after transfection, the expression of EGFP-target (gag or env) fusion proteins was observed directly under an inverted fluorescence microscope. HEK 293 cells were observed 48 h posttransfection under a fluorescence microscope to determine the silencing effect of siRNAs on Gag and Env protein expression in cultured cells (Fig. 2B and C) . Based on the fluorescence data, flow cytometry and real time quantitative PCR, two siRNAs targeted to the HTLV-1 gag gene reduced efficiently gene expression by different amounts compared to the negative siRNA, scrambled siRNAs and controls without siRNA.

abstract:
  Since the discovery of RNAi technology, several functional genomic and disease therapy studies have been conducted using this technique in the field of oncology and virology. RNAi-based antiviral therapies are being studied for the treatment of retroviruses such as HIV-1. These studies include the silencing of regulatory, infectivity and structural genes. The HTLV-1 structural genes are responsible for the synthesis of proteins involved in the entry, assembly and release of particles during viral infection. To examine the possibility of silencing HTLV-1 genes gag and env by RNA interference technology, these genes were cloned into reporter plasmids. These vectors expressed the target mRNAs fused to EGFP reporter genes. Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. The plasmids and siRNAs were co-transfected into HEK 293 cells. The results demonstrated that the expression of the HTLV-1 gag and env genes decreased significantly in vitro. Thus, siRNAs can be used to inhibit HTLV-1 structural genes in transformed cells, which could provide a tool for clarifying the roles of HTLV-1 structural genes, as well as a therapy for this infection.


id: cord-255545-nycdhdsd
author: Schoenike, Barry
date: 1999-03-10
txt: ./txt/cord-255545-nycdhdsd.txt
cache: ./cache/cord-255545-nycdhdsd.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/10204695/
doi: 10.1016/s0166-0934(98)00167-0
summary:
  In theory, sense-specific measurement of viral RNAs may be achieved by reverse transcription polymerase chain reaction (RT-PCR) assays which utilize primers of defined polarity during the RT step. Key RT-PCR parameters which were optimized include the design of tagged primers, DNase treatment of in vitro transcribed RNA standards, specification of temperature differences between RT and PCR annealing steps, and use of competitive RNA templates for quantitative assays. In fact, several studies have demonstrated that RT-PCR assays based on specific RNA template recognition by RT primers of defined polarity will not reliably distinguish between viral RNAs of positive or negative sense. Despite the findings above, many published research reports are based on conventional RT-PCR assays, relying on the polarity of primers added to the RT step in putative sense-specific measurements of viral RNAs; rarely are control reactions performed to rigorously show that this method is in fact sense-specific.

title:
  Quantitative sense-specific determination of murine coronavirus RNA by reverse transcription polymerase chain reaction

abstract:
  In many applications, it is useful to know the sense and amount of viral RNAs present in a sample. In theory, sense-specific measurement of viral RNAs may be achieved by reverse transcription polymerase chain reaction (RT-PCR) assays which utilize primers of defined polarity during the RT step. However, in practice, it has been shown that such assays are prone to artifacts, such as non-specific priming, which drastically diminish their reliability. Using murine coronavirus MHV-4 as a model, we describe and validate several modifications of the RT-PCR procedure which eliminate these artifacts. Key RT-PCR parameters which were optimized include the design of tagged primers, DNase treatment of in vitro transcribed RNA standards, specification of temperature differences between RT and PCR annealing steps, and use of competitive RNA templates for quantitative assays. The assays described may be used to determine the sense and abundance of any viral or host RNA of interest in complex biological specimens.


id: cord-255983-3dq99xz9
title: A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus
author: Do, Lien Anh Ha
date: 2012-01-17
txt: ./txt/cord-255983-3dq99xz9.txt
cache: ./cache/cord-255983-3dq99xz9.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2011.11.012
doi: 10.1016/j.jviromet.2011.11.012
summary:
  The quantitative assay was compared to a commercial conventional multiplex PCR method (Seeplex TM RV detection kit, Seegene, Inc., Seoul, Korea) (Kim et al., 2009; Roh et al., 2008) respiratory samples from a study (Do et al., 2011) on acute respiratory infection in children at the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam. All samples were analyzed in parallel by the commercial multiplex Seeplex TM RV detection kit (Seegene, Inc., Seoul, Korea) according to the manufacturer''s instructions, to determine the presence of 12 respiratory viruses: human RSV subgroups A and B (RSV A, RSV B); influenza virus A (InfV A); influenza virus B (InfV B); human coronaviruses (229E, OC43), human metapneumovirus (hMPV), parainfluenza virus 1, 2 and 3 (PIV1, 2, 3), human rhinovirus (hRV A) and adenovirus (AdV) (Kim et al., 2009; Roh et al., 2008) and by the newly developed RSV LNA real-time RT-PCR.

abstract:
  Improved diagnostic tools for rapid detection, quantitation, and subgrouping of human respiratory syncytial virus (RSV) are needed to aid the development and evaluation of novel intervention strategies. A quantitative real-time RT-PCR using specific locked nucleic acid (LNA) probes was developed to identify RSV and to distinguish RSV subgroups A and B (RSV LNA assay). RSV subgroup diversity and the relationship between viral load and disease severity in confirmed RSV infections were also explored. 264 archived respiratory specimens from pediatric patients were tested in parallel using the commercial multiplex Seeplex™ RV detection kit (Seegene) and the novel RSV LNA assay. The LNA assay demonstrated a significantly higher sensitivity than Seeplex, improving overall detection rates from 24% (64/264) to 32% (84/264). Detection limits of 9.0 × 10(1) and 6.0 × 10(2) copies/mL were observed for RSV A and B, respectively. RSV A was detected in 53/84 (63%) cases, and 31/84 (37%) were positive for RSV B. This novel method offers a rapid, quantitative, highly specific and sensitive approach to laboratory diagnosis of RSV.


id: cord-256355-muskjaw3
author: Black, Elizabeth M
date: 2002-05-14
txt: ./txt/cord-256355-muskjaw3.txt
cache: ./cache/cord-256355-muskjaw3.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093402000629
doi: 10.1016/s0166-0934(02)00062-9
summary:
  A rapid and sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay incorporating TaqMan™ probes has been developed that can distinguish among the six established rabies and rabies-related virus genotypes. TaqMan™ probes were designed and validated against 106 rabies and rabies-related virus isolates, one isolate of the Australian bat Lyssaviruses (genotype 7), and 18 other non-rabies viruses important in the veterinary field. By combining RT-PCR with TaqMan™ genotyping probes suspect rabies virus isolates can be identified in a single closed tube system that prevents potential PCR-product carry over contamination. A hemi-nested reverse transcriptase PCR (hnRT-PCR) assay that uses a cocktail of primers capable of detecting the six established genotypes of rabies and rabies-related viruses has been described (Heaton et al., 1997) . Using the sequence data from a number of isolates from each of the rabies and rabies-related virus genotypes, TaqMan™ probes were designed to distinguish the six established genotypes of rabies and rabies-related viruses.

title:
  A rapid RT-PCR method to differentiate six established genotypes of rabies and rabies-related viruses using TaqMan™ technology

abstract:
  A rapid and sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay incorporating TaqMan™ probes has been developed that can distinguish among the six established rabies and rabies-related virus genotypes. TaqMan™ probes were designed and validated against 106 rabies and rabies-related virus isolates, one isolate of the Australian bat Lyssaviruses (genotype 7), and 18 other non-rabies viruses important in the veterinary field. The N gene was used as the target for the probes as it is well conserved and has been intensively used to genotype rabies isolates. Additionally, it was found to contain regions specific to each genotype conducive to probe design. The RT-PCR assay described amplifies a portion of the nucleoprotein gene of all 107 rabies and rabies-related viruses, but none of the other viruses tested. Inclusion of TaqMan™-genotype-specific probes in the RT-PCR assay permits rapid identification of the virus present. By combining RT-PCR with TaqMan™ genotyping probes suspect rabies virus isolates can be identified in a single closed tube system that prevents potential PCR-product carry over contamination.


id: cord-256608-ajzk86rq
title: PCR diagnostics: In silico validation by an automated tool using freely available software programs
author: van Weezep, Erik
date: 2019-05-13
txt: ./txt/cord-256608-ajzk86rq.txt
cache: ./cache/cord-256608-ajzk86rq.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2019.05.002
doi: 10.1016/j.jviromet.2019.05.002
summary:
  An alignment search was performed with the default expectancy threshold value on all fasta files using primers and probes of the PCR test as search queries and the program SSEARCH available in the FASTA sequence analysis package (Brenner et al., 1998; Pearson, 1991; Pearson et al., 2017; . The in silico specificity is expressed as the percentage of specific hits of taxonomy classified sequences with a maximum of one mismatch per primer or probe as these are considered to be detected with the respective PCR test. To demonstrate the suitability of our in-house developed software tool PCRv, we determined the in silico sensitivity and specificity of three PCR tests for West Nile virus (WNV) recommended by the World Organisation for Animal Health (OIE) (Eiden et al., 2010; Johnson et al., 2001) .

abstract:
  PCR diagnostics are often the first line of laboratory diagnostics and are regularly designed to either differentiate between or detect all pathogen variants of a family, genus or species. The ideal PCR test detects all variants of the target pathogen, including newly discovered and emerging variants, while closely related pathogens and their variants should not be detected. This is challenging as pathogens show a high degree of genetic variation due to genetic drift, adaptation and evolution. Therefore, frequent re-evaluation of PCR diagnostics is needed to monitor its usefulness. Validation of PCR diagnostics recognizes three stages, in silico, in vitro and in vivo validation. In vitro and in vivo testing are usually costly, labour intensive and imply a risk of handling dangerous pathogens. In silico validation reduces this burden. In silico validation checks primers and probes by comparing their sequences with available nucleotide sequences. In recent years the amount of available sequences has dramatically increased by high throughput and deep sequencing projects. This makes in silico validation more informative, but also more computing intensive. To facilitate validation of PCR tests, a software tool named PCRv was developed. PCRv consists of a user friendly graphical user interface and coordinates the use of the software programs ClustalW and SSEARCH in order to perform in silico validation of PCR tests of different formats. Use of internal control sequences makes the analysis compliant to laboratory quality control systems. Finally, PCRv generates a validation report that includes an overview as well as a list of detailed results. In-house developed, published and OIE-recommended PCR tests were easily (re-) evaluated by use of PCRv. To demonstrate the power of PCRv, in silico validation of several PCR tests are shown and discussed.


id: cord-256845-5pjam7em
author: Stranieri, Angelica
date: 2017-01-18
txt: ./txt/cord-256845-5pjam7em.txt
cache: ./cache/cord-256845-5pjam7em.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093416306656
doi: 10.1016/j.jviromet.2017.01.009
summary:
  In the case the sensitivity of the test might be ameliorated through further studies, the RT-LAMP could be extremely useful, due to its low costs and rapidity, in those situations where the Table 2 Results obtained on the samples tested with RT-nPCR (PCR) and LAMP and evaluated with agarose gel electrophoresis (LAMP GEL) and hydroxynaphtol blue dye (LAMP HNB detection of FCoV must be repeated over time and on a high number of cats (e.g. breeding catteries). Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis Feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis

title:
  Reverse transcriptase loop-mediated isothermal amplification for the detection of feline coronavirus

abstract:
  The Feline coronavirus (FCoV) is the etiological agent of feline infectious peritonitis (FIP), a lethal disease of felids. The role of molecular methods is controversial for the diagnosis of FIP, while essential for the identification of the shedders. Thus, a fast and inexpensive method for the detection of FCoV could be beneficial, especially in multicat environments. A reverse transcription loop mediated isothermal amplification (RT-LAMP) assay was developed. RNA extraction and RT-nPCR for FCoV were performed on thirty-two samples (11 faeces, 9 blood, 8 effusions, and 4 lymph nodes) collected from 27 cats. Six RT-LAMP primers were designed from the same conserved region of RT-nPCR, and the assay was run at 63 °C for one hour. Results were evaluated through both agarose gel run and hydroxynapthol blue (HNB) dye and then compared with RT-nPCR results for the assessment of sensitivity and specificity. The overall specificity was 100%, but the sensitivity was 50% and 54.5% for agarose gel and HNB respectively. Therefore, RT-LAMP seems optimal to confirm the presence of the virus, but not applicable to exclude it.


id: cord-257284-dash9udv
author: Decaro, Nicola
date: 2010-07-30
url: https://api.elsevier.com/content/article/pii/S0166093410002636
doi: 10.1016/j.jviromet.2010.07.021
title:
  Development and validation of a real-time PCR assay for specific and sensitive detection of canid herpesvirus 1

abstract:
  A TaqMan-based real-time PCR assay targeting the glycoprotein B-encoding gene was developed for diagnosis of canid herpesvirus 1 (CHV-1) infection. The established assay was highly specific, since no cross-reactions were observed with other canine DNA viruses, including canine parvovirus type 2, canine minute virus, or canine adenovirus types 1 and 2. The detection limit was 10(1) and 1.20 × 10(1) DNA copies per 10 μl(−1) of template for standard DNA and a CHV-1-positive kidney sample, respectively: about 1-log higher than a gel-based PCR assay targeting the thymidine kinase gene. The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. CHV-1 isolates of different geographical origins were recognised by the TaqMan assay. Tissues and clinical samples collected from three pups which died of CHV-1 neonatal infection were also tested, displaying a wide distribution of CHV-l DNA in their organs. Unlike other CHV-1-specific diagnostic methods, this quantitative assay permits simultaneous detection and quantitation of CHV-1 DNA in a wide range of canine tissues and body fluids, thus providing a useful tool for confirmation of a clinical diagnosis, for the study of viral pathogenesis and for evaluation of the efficacy of vaccines and antiviral drugs.


id: cord-257785-jzdlvo7p
author: Bennett, Susan
date: 2010-10-14
txt: ./txt/cord-257785-jzdlvo7p.txt
cache: ./cache/cord-257785-jzdlvo7p.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093410003605
doi: 10.1016/j.jviromet.2010.10.005
summary:
  The endpoint detection limit of the universal influenza A and H1N1 (2009) components of the triplex was directly compared to the published duplex using a dilution series of an influenza A H1N1 (2009) clinical sample and clinical samples containing seasonal H1N1 and H3N2 viruses. The endpoint detection limit of the H275Y component of the triplex was compared directly to the H275Y single assay using a dilution series of influenza A H1N1 (2009) H275Y positive clinical sample. Finally the ability of the triplex assay to detect minor populations of the oseltamivir resistant influenza A virus in samples containing a mixture of resistant and sensitive viruses was assessed. The endpoint detection limit of the universal influenza A and the H1N1/2009 component of the triplex assay in comparison to the duplex assay was assessed using serial dilutions of clinical samples containing influenza A H1N1 (2009) and seasonal H1N1 and H3N2 viruses (Table 3) .

title:
  The validation of a real-time RT-PCR assay which detects influenza A and types simultaneously for influenza A H1N1 (2009) and oseltamivir-resistant (H275Y) influenza A H1N1 (2009)

abstract:
  Influenza A H1N1 (2009) was declared by the World Health Organisation (WHO) as the first influenza pandemic of the 21st century. Rapid detection of influenza A and differentiation of influenza A H1N1 (2009) and seasonal influenza A is beneficial. In addition the rapid detection of antiviral resistant strains of influenza A H1N1 (2009) would be useful for clinicians to allow for change to an effective treatment at a much earlier stage if resistance is found. It was the aim of this study to develop a real-time RT-PCR that can detect all influenza A viruses and type simultaneously for influenza A H1N1 (2009) and oseltamivir resistant (H275Y) influenza A H1N1 (2009). This multiplex assay will allow laboratories to screen respiratory samples for all types of influenza A, influenza A H1N1 (2009) virus and oseltamivir resistant (H275Y) influenza A H1N1 (2009) virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. Since most virology laboratories already offer a molecular service for influenza A this assay could easily be implemented into most areas at little cost therefore increasing local access to resistance testing.


id: cord-257850-x7qtxaum
author: Majchrzykiewicz-Koehorst, Joanna A.
date: 2015-03-01
txt: ./txt/cord-257850-x7qtxaum.txt
cache: ./cache/cord-257850-x7qtxaum.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093414004443
doi: 10.1016/j.jviromet.2014.11.014
summary:
  The development of an improved and rapid sample preparation method allowed generic and rapid identification of cultured respiratory viruses by mass spectrometry. In two independent experiments, all ten influenza strains were correctly identified as either H1N1 or H3N2 influenza A virus, with an identification limit of 7 × 10 6 genome copies in the total sample volume subjected to LC-MS/MS analysis (corresponding to a CT value of approximately 30-32; Table 3 ). At this viral titer, the influenza viruses were typed and subtyped based on the identification of peptides derived from the nucleoprotein protein, indicated in boldface in Table 4 . The identification limit at which the RSV strain was identified correctly, was 3.6 × 10 7 genome copy equivalents in the total sample volume subjected to LC-MS/MS analysis (corresponding to a CT value of approximately 28; Tables 5 and S1).

title:
  Rapid and generic identification of influenza A and other respiratory viruses with mass spectrometry

abstract:
  The rapid identification of existing and emerging respiratory viruses is crucial in combating outbreaks and epidemics. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid and reliable identification method in bacterial diagnostics, but has not been used in virological diagnostics. Mass spectrometry systems have been investigated for the identification of respiratory viruses. However, sample preparation methods were laborious and time-consuming. In this study, a reliable and rapid sample preparation method was developed allowing identification of cultured respiratory viruses. Tenfold serial dilutions of ten cultures influenza A strains, mixed samples of influenza A virus with human metapneumovirus or respiratory syncytial virus, and reconstituted clinical samples were treated with the developed sample preparation method. Subsequently, peptides were subjected to MALDI-TOF MS and liquid chromatography tandem mass spectrometry (LC–MS/MS). The influenza A strains were identified to the subtype level within 3 h with MALDI-TOF MS and 6 h with LC–MS/MS, excluding the culturing time. The sensitivity of LC–MS/MS was higher compared to MALDI-TOF MS. In addition, LC–MS/MS was able to discriminate between two viruses in mixed samples and was able to identify virus from reconstituted clinical samples. The development of an improved and rapid sample preparation method allowed generic and rapid identification of cultured respiratory viruses by mass spectrometry.


id: cord-258008-t78svobg
author: Bruijnesteijn van Coppenraet, L.E.S.
date: 2010-08-05
txt: ./txt/cord-258008-t78svobg.txt
cache: ./cache/cord-258008-t78svobg.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/20691735/
doi: 10.1016/j.jviromet.2010.07.032
summary:
  Two molecular assays were compared with real-time RT-PCR and viral culture for simultaneous detection of common viruses from respiratory samples: a multiplex ligation-dependant probe amplification (MLPA) and a dual priming oligonucleotide system (DPO). A panel of 168 culture-positive and negative samples was tested by the molecular assays for the presence of influenza A and B virus, respiratory syncytial virus, human metapneumovirus, rhinovirus, coronaviruses, parainfluenza viruses and adenovirus. Both molecular assays are comparable with real-time RT-PCR, more sensitive than viral culture and can detect dual infections easily. In this study, two commercial molecular assays, both designed for simultaneous detection of the most common viruses from a variety of respiratory samples, were compared with real-time RT-PCR and viral culture: a multiplex ligation-dependant probe amplification (MLPA) and a dual priming oligonucleotide system (DPO). Defined as true positives were samples that yielded positive viral detections by more than one method (culture, DPO, MLPA or real-time RT-PCR).

title:
  Comparison of two commercial molecular assays for simultaneous detection of respiratory viruses in clinical samples using two automatic electrophoresis detection systems

abstract:
  Two molecular assays were compared with real-time RT-PCR and viral culture for simultaneous detection of common viruses from respiratory samples: a multiplex ligation-dependant probe amplification (MLPA) and a dual priming oligonucleotide system (DPO). In addition, the positive detections of MLPA and DPO were identified using two different automatic electrophoresis systems. A panel of 168 culture-positive and negative samples was tested by the molecular assays for the presence of influenza A and B virus, respiratory syncytial virus, human metapneumovirus, rhinovirus, coronaviruses, parainfluenza viruses and adenovirus. One hundred and twenty-nine (77%) samples were positive as detected by at least one method. Sixty-nine (41%) samples were positive by cell culture (excluding human metapneumovirus and coronaviruses), 116 (69%) by RT-PCR, 127 (76%) by MLPA and 100 (60%) by DPO. The MLPA yielded results in one attempt for all samples included while 12 (7.2%) samples had to be repeated by the DPO assay due to inconclusive results. The MLPA assay performed well in combination with either electrophoresis system, while the performance of the DPO assay was influenced by the electrophoresis systems. Both molecular assays are comparable with real-time RT-PCR, more sensitive than viral culture and can detect dual infections easily. Results can be obtained within 1 day.


id: cord-258057-ti0rpt0q
title: Establishment of a Porcine Parvovirus (PPV) LAMP Visual Rapid Detection Method
author: Zhao, Kai
date: 2020-07-01
txt: ./txt/cord-258057-ti0rpt0q.txt
cache: ./cache/cord-258057-ti0rpt0q.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32621958/
doi: 10.1016/j.jviromet.2020.113924
summary:
  A loop-mediated isothermal amplification (LAMP) assay was established to detect PPV infection. The optimized LAMP program was as follows: 50 min at 59 °C followed by 3 min at 80 °C.The amplified products were analyzed both by visual inspection after staining with SYBR Green I dye and by conventional agarose gel electrophoresis. To verify the specificity of PPV detection by LAMP, the DNA (PPV, PCV2, PRV) and cDNA samples (PRRSV, CSFV) were amplified as sample templates by the LAMP reaction at 59.0℃ for 50 min and terminated at 80℃ for 3 min, respectively. The LAMP detection limit for PPV based on visual observation by addition of J o u r n a l P r e -p r o o f SYBR Green I and by gel electrophoresis analysis was 10 1 copies. Rapid detection of porcine parvovirus DNA by sensitive loop-mediated isothermal amplification Rapid and sensitive diagnosis of porcine parvovirus by loop-mediated isothermal amplification (LAMP) method

abstract:
  Porcine parvovirus (PPV) is one of the major causes of reproductive pig disease. Due to its serious nature, wide spread and consequent great damage to the swine industry, an effective, rapid and convenient method for its detection is needed. A loop-mediated isothermal amplification (LAMP) assay was established to detect PPV infection. Two pairs of primers were specifically designed to recognize the six different sequences of open reading frame1 (ORF1) gene. The optimized LAMP program was as follows: 50 min at 59 °C followed by 3 min at 80 °C.The amplified products were analyzed both by visual inspection after staining with SYBR Green I dye and by conventional agarose gel electrophoresis. Both methods showed the same sensitivity. The limit of detection (LOD) for PPV by LAMP was 10 copies, which is 100-fold lower than conventional PCR. Our LAMP assay did not cross-react with other viruses. We used the established LAMP system to test 1100 field samples and detected 660 positives. The LAMP detection method for PPV represents a visual, sensitive and rapid assay which can detect the virus in the field, offering an attractive alternative for the PPV detection methods currently in use.


id: cord-258468-52gej3co
author: Marcekova, Zuzana
date: 2009-08-05
txt: ./txt/cord-258468-52gej3co.txt
cache: ./cache/cord-258468-52gej3co.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093409003541
doi: 10.1016/j.jviromet.2009.07.028
summary:
  title: Heterologous expression of full-length capsid protein of porcine circovirus 2 in Escherichia coli and its potential use for detection of antibodies coli-expressed Cap protein to self-assemble into virus-like particles (VLPs), the immunization of mice with recombinant Cap yielded antibodies with the same specificity as those raised against native PCV 2 virions. In addition, the antigenic properties of the purified Cap protein were employed in a subunit-based indirect ELISA to monitor the levels of PCV 2 specific antibodies in piglets originating from a herd which was experiencing PCV 2 infection. In order to eliminate the cluster of rare codons from the 5 end of the cap gene, the expression vector encoding a truncated variant of Cap protein ( Cap-His), which was lacking the first 16 amino acid residues, was constructed (Fig. 1B) . In summary, a bacterial expression system has been developed for the production of the full-length recombinant capsid protein of porcine circovirus type 2.

title:
  Heterologous expression of full-length capsid protein of porcine circovirus 2 in Escherichia coli and its potential use for detection of antibodies

abstract:
  A capsid protein of porcine circovirus 2 (PCV 2) serves as a diagnostic antigen for the detection of PCV 2-associated disease known as a postweaning multisystemic wasting syndrome (PMWS). In this report, a bacterial expression system was developed for the expression and purification of the full-length PCV 2 capsid (Cap) protein from a codon-optimized cap gene. Replacement of rare arginine codons located at the 5′ end of the cap reading frame with codons optimal for E. coli was found to overcome the poor expression of the viral protein in the prokaryotic system. The Cap protein was purified to greater than 95% homogeneity by using a single cation-exchange chromatography at a yield of 10 mg per litre of bacterial culture. Despite the failure of the E. coli-expressed Cap protein to self-assemble into virus-like particles (VLPs), the immunization of mice with recombinant Cap yielded antibodies with the same specificity as those raised against native PCV 2 virions. In addition, the antigenic properties of the purified Cap protein were employed in a subunit-based indirect ELISA to monitor the levels of PCV 2 specific antibodies in piglets originating from a herd which was experiencing PCV 2 infection. These results pave the way for a straightforward large-scale production of the recombinant PCV 2 capsid protein and its use as a diagnostic antigen or a PCV 2 subunit vaccine.


id: cord-259212-pj8p2x9l
title: Genetic diversity of a canine coronavirus detected in pups with diarrhoea in Italy
author: Pratelli, Annamaria
date: 2003-06-09
url: https://api.elsevier.com/content/article/pii/S0166093403000818
doi: 10.1016/s0166-0934(03)00081-8
abstract:
  The sequence of the S gene of a field canine coronavirus (CCoV), strain Elmo/02, revealed low nucleotide (61%) and amino acid (54%) identity to reference CCoV strains. The highest correlation (77% nt and 81.7% aa) was found with feline coronavirus type I. A PCR assay for the S gene of strain Elmo/02 detected analogous CCoVs of different geographic origin, all which exhibited at least 92–96% nucleotide identity to each other and to strain Elmo/02. The evident genetic divergence between the reference CCoV strains and the newly identified Elmo/02-like CCoVs strongly suggests that a novel genotype of CCoV is widespread in the dog population.


id: cord-259593-shrd1s7r
author: Qin, Zhao-ling
date: 2007-06-27
txt: ./txt/cord-259593-shrd1s7r.txt
cache: ./cache/cord-259593-shrd1s7r.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S016609340700198X
doi: 10.1016/j.jviromet.2007.05.017
summary:
  title: siRNAs targeting terminal sequences of the SARS-associated coronavirus membrane gene inhibit M protein expression through degradation of M mRNA To study M protein function, three candidate small interfering RNAs (siRNAs) corresponding to M gene sequences were designed, transcribed in vitro, and then tested for their ability to silence M protein expression. The results showed that the mean green fluorescence intensity and M RNA transcripts were significantly reduced, and that the expression of M glycoprotein was strongly inhibited in those cells co-transfected with M-specific siRNAs. These findings demonstrated that the three M-specific siRNAs were able to specifically and effectively inhibit M glycoprotein expression in cultured cells by blocking the accumulation of mRNA, which provides an approach for studies on the functions of M protein and for the development of novel prophylactic or therapeutic agents for SCoV infection.

title:
  siRNAs targeting terminal sequences of the SARS-associated coronavirus membrane gene inhibit M protein expression through degradation of M mRNA

abstract:
  SARS-associated coronavirus (SCoV) M protein plays a key role in viral assembly and budding. Recent studies revealed that M protein could interact with N protein in the Golgi complex. In this study, we showed that SCoV M protein co-localized in the Golgi apparatus with a Golgi vector marker. To study M protein function, three candidate small interfering RNAs (siRNAs) corresponding to M gene sequences were designed, transcribed in vitro, and then tested for their ability to silence M protein expression. The plasmid, pEGFP-M, encoding SCoV M protein as a fusion protein with EGFP, was used for silencing and for reporter gene detection in HEK 293T cells transfected with siRNA constructs. The results showed that the mean green fluorescence intensity and M RNA transcripts were significantly reduced, and that the expression of M glycoprotein was strongly inhibited in those cells co-transfected with M-specific siRNAs. These findings demonstrated that the three M-specific siRNAs were able to specifically and effectively inhibit M glycoprotein expression in cultured cells by blocking the accumulation of mRNA, which provides an approach for studies on the functions of M protein and for the development of novel prophylactic or therapeutic agents for SCoV infection.


id: cord-259738-yuqc6dk0
author: Tang, Mengjun
date: 2008-03-07
txt: ./txt/cord-259738-yuqc6dk0.txt
cache: ./cache/cord-259738-yuqc6dk0.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/18329109/
doi: 10.1016/j.jviromet.2008.01.017
summary:
  title: Enhancement of the immunogenicity of an infectious bronchitis virus DNA vaccine by a bicistronic plasmid encoding nucleocapsid protein and interleukin-2 A DNA vaccine against infectious bronchitis virus (IBV) can induce specific humoral and cell-mediated immunity. When chickens were challenged with a virulent strain of IBV, the protective efficacy could be enhanced significantly after immunization with bicistronic DNA vaccine. In the present study, a bicistronic plasmid encoding the nucleocapsid protein and immune-stimulatory interleukin-2 has been constructed and its immunogenicity and protective effect in chickens has been evaluated. These recombinant plasmids were inoculated in chickens and tested in a protection-challenge experiment, demonstrating that vaccination with the co-expression plasmid pIRES-N/IL2 can induce stronger immune response than vaccination with pIRES-N. These results suggested that vaccination with the co-expression plasmid of N protein gene and IL-2 may have increased the protection rate against challenge. Partial protection against infectious bursal disease virus through DNA-mediated vaccination with the VP2 capsid protein and chicken IL-2 genes

title:
  Enhancement of the immunogenicity of an infectious bronchitis virus DNA vaccine by a bicistronic plasmid encoding nucleocapsid protein and interleukin-2

abstract:
  A DNA vaccine against infectious bronchitis virus (IBV) can induce specific humoral and cell-mediated immunity. However, compared to conventional vaccines, DNA vaccines usually induce poor antibody responses. To develop a more potent IBV DNA vaccine formulations, a monocistronic vector encoding the nucleocapsid protein of IBV and a bicistronic vector separately encoding the nucleocapsid protein and immune-stimulatory interleukin-2 were constructed. When the DNA vaccines were administered to the quadriceps muscle of chickens, the induced humoral and cellular responses were evaluated. There was a significant difference in ELISA antibody levels elicited by either monocistronic or bicistronic DNA vaccines. The percentage of CD3(+), CD3(+)CD8(+) and CD3(+)CD4(+) subgroups of peripheral blood T-lymphocytes in chickens immunized with bicistronic DNA vaccine were higher than those in chickens immunized with monocistronic DNA vaccine. When chickens were challenged with a virulent strain of IBV, the protective efficacy could be enhanced significantly after immunization with bicistronic DNA vaccine. These results demonstrated that bicistronic DNA vaccine is an effective approach to increase IBV DNA vaccine immunogenicity.


id: cord-260208-fvdq0yes
author: Wang, Jinfeng
date: 2018-03-14
txt: ./txt/cord-260208-fvdq0yes.txt
cache: ./cache/cord-260208-fvdq0yes.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093417306882
doi: 10.1016/j.jviromet.2018.03.005
summary:
  title: Rapid detection of transmissible gastroenteritis virus in swine small intestine samples using real-time reverse transcription recombinase polymerase amplification A rapid and specific real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed to detect the transmissible gastroenteritis virus (TGEV) in this study. Six primers were needed in the RT-LAMP assays for TGEV, the reaction time was 30 or 60 min, and the results were visualized by agarose gel electrophoresis (Chen et al., 2010; Li and Ren, 2011 T Recombinase polymerase amplification (RPA) is an isothermal gene amplification technique that has been demonstrated to be a rapid, specific, sensitive, and cost-effective molecular diagnostic method (Daher et al., 2016; Piepenburg et al., 2006) . In this study, we developed and evaluated the userfriendly on-site detection platform integrating the real-time RPA technology and a field-deployable device (Genie III tube scanner) for the rapid detection of TGEV.

title:
  Rapid detection of transmissible gastroenteritis virus in swine small intestine samples using real-time reverse transcription recombinase polymerase amplification

abstract:
  A rapid and specific real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed to detect the transmissible gastroenteritis virus (TGEV) in this study. The primers and exo probe were designed to be specific for a portion of spike (S) gene conserved in TGEV, but absent in the closely related porcine respiratory coronavirus (PRCV). The amplification was performed at 40 °C for 20 min. The assay could only detect the TGEV, and there was no cross-reaction with other pathogens tested. Using the in vitro transcribed TGEV RNA as template, the limit of detection of the developed RT-RPA was 100 copies per reaction. The assay performance was evaluated by testing 76 clinical samples by RT-RPA and a real-time RT-PCR. Fourteen samples were TGEV RNA positive in RT-RPA (18.4%, 14/76), which were also positive in the real-time RT-PCR. The diagnostic agreement between the two assays was 100% (76/76). The R(2) value of RT-RPA and real-time RT-PCR was 0.959 by linear regression analysis. The developed RT-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of TGEV in resource-limited diagnostic laboratories and on-site facilities.


id: cord-260250-t48y27wg
title: Quantitation of canine coronavirus RNA in the faeces of dogs by TaqMan RT-PCR
author: Decaro, Nicola
date: 2004-05-07
txt: ./txt/cord-260250-t48y27wg.txt
cache: ./cache/cord-260250-t48y27wg.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093404001028
doi: 10.1016/j.jviromet.2004.03.012
summary:
  A TaqMan(®) fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for the detection and quantitation of canine coronavirus (CCoV) RNA in the faeces of naturally or experimentally infected dogs. The CCoV fluorogenic RT-PCR assay, which targeted the ORF5 (M gene), was more sensitive than a conventional RT-PCR assay targeting the same gene, showing a detection limit of 10 copies of CCoV standard RNA, and was linear from 10 to 10(8) copies, allowing quantitation of samples with a wide range of CCoV RNA loads. As shown in Table 2 , the detec-tion limit of the TaqMan RT-PCR was 1-2 log higher than that of conventional RT-PCR, ranging around 10 1 copies/l and 10 −1.50 TCID 50 /50 l for standard RNA and CCoV strain, respectively, with a detection rate of 100% for each positive dilution. The results of the conventional amplification and real-time analysis carried out on the faecal samples of the CCoV experimentally infected dog are summarized in Fig. 3 .

abstract:
  A TaqMan(®) fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for the detection and quantitation of canine coronavirus (CCoV) RNA in the faeces of naturally or experimentally infected dogs. The CCoV fluorogenic RT-PCR assay, which targeted the ORF5 (M gene), was more sensitive than a conventional RT-PCR assay targeting the same gene, showing a detection limit of 10 copies of CCoV standard RNA, and was linear from 10 to 10(8) copies, allowing quantitation of samples with a wide range of CCoV RNA loads. A total of 78 faecal samples of diarrhoeic dogs were tested simultaneously by conventional and fluorogenic RT-PCR: 29 were negative by both techniques, whereas 27 tested positive by conventional RT-PCR and 48 by the established CCoV fluorogenic assay. One sample, which was positive by conventional RT-PCR, gave no signal in the fluorogenic assay. In addition, by the fluorogenic assay CCoV shedding in the faecal samples of an experimentally infected dog was monitored for 28 days. The high sensitivity, simplicity and reproducibility of the CCoV fluorogenic RT-PCR assay, combined with its wide dynamic range and high throughput, make this method especially suitable for efficacy trials on CCoV vaccines.


id: cord-261089-aul4ifso
author: Yuan, Wen
date: 2016-07-07
txt: ./txt/cord-261089-aul4ifso.txt
cache: ./cache/cord-261089-aul4ifso.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093416300878
doi: 10.1016/j.jviromet.2016.07.004
summary:
  The aim of this study is to develop a rapid, sensitive and specific duplex real-time RT-PCR assay for the simultaneous detection of TMEV and RTV, and provide a useful tool for the routine health monitoring of these two viruses in laboratory rodents and for the screening of contaminated biological materials. In addition, twenty cecum content and spleen samples collected from eight specific pathogen free (SPF) mouse strains (BALB/c, C57BL/6, DBA, FVB, 129, ICR, KM and NIH) and two rat strains (SD and Wistar) were used to evaluate specificity of the duplex real-time RT-PCR assay, these animals were reared under barrier colonies and were confirmed as serology negative for TMEV and RTV by a commercial ELISA kit (XpressBio, Maryland, USA). The specificity of the duplex real-time RT-PCR assay was determined by evaluation of RNA extracted from positive cultures of the following rodent viral pathogens: TMEV, RTV, MHV, Reo-3, RCV, Sendai, PVM, MNV and LCMV, and from twenty cecum content and spleen samples of ten SPF rodent strains.

title:
  Development of a duplex real-time RT-PCR for the simultaneous detection and differentiation of Theiler’s murine encephalomyelitis virus and rat theilovirus

abstract:
  Theiler’s murine encephalomyelitis virus (TMEV) and rat theilovirus (RTV), the member of the genus Cardiovirus, are widespread in laboratory mice and rats, and are potential contaminants of biological materials. Cardioviruses infection may cause serious complications in biomedical research. To improve the efficiency of routine screening for Cardioviruses infection, a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection and differentiation of TMEV and RTV. The duplex assay was specific for reference strains of TMEV and RTV, and no cross-reaction was found with seven other rodent viruses. The limits of detection of both TMEV and RTV were 4 × 10(1) copies RNA/reaction. Reproducibility was estimated using standard dilutions, with coefficients of variation <3.1%. 439 clinical samples were evaluated by both duplex real-time RT-PCR and conventional RT-PCR. For 439 clinical samples，95 samples were positive for TMEV and 72 samples were positive for RTV using duplex real-time RT-PCR approach, whereas only 77 samples were positive for TMEV and 66 samples were positive for RTV when conventional RT-PCR was applied. Mixed infections were found in 20 samples when analyzed by conventional RT-PCR whereas 30 samples were found to be mixed infection when duplex real-time RT-PCR was applied. This duplex assay provides a useful tool for routine health monitoring and screening of contaminated biological materials of these two viruses.


id: cord-261134-zarq507s
title: Amplification refractory mutation system PCR assays for the detection of variola and Orthopoxvirus
author: Pulford, David
date: 2004-02-13
txt: ./txt/cord-261134-zarq507s.txt
cache: ./cache/cord-261134-zarq507s.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/15019263/
doi: 10.1016/j.jviromet.2004.01.001
summary:
  PCR assays that can identify the presence of variola virus (VARV) sequences in an unknown DNA sample were developed using principles established for the amplification refractory mutation system (ARMS). When a variola virus specific primer was used with a consensus primer in an ARMS assay with different Orthopoxvirus genomes, a PCR product was only amplified from variola virus DNA. Incorporating a second consensus primer into the assay produced a multiplex PCR that provided Orthopoxvirus generic and variola-specific products with variola virus DNA. The variola virus specific primers did not produce amplicons with either assay format when tested with 50 other Orthopoxvirus DNA samples. These multiplex assays employ three primers; two consensus primers generate an amplicon diagnostic of an Old World Orthopoxvirus and the third primer simultaneously binds to a variola-specific polymorphism and initiates extension of a shorter PCR product to detect the presence of variola virus.

abstract:
  PCR assays that can identify the presence of variola virus (VARV) sequences in an unknown DNA sample were developed using principles established for the amplification refractory mutation system (ARMS). The assay’s specificity utilised unique single nucleotide polymorphisms (SNP) identified among Orthopoxvirus (OPV) orthologs of the vaccinia virus Copenhagen strain A13L and A36R genes. When a variola virus specific primer was used with a consensus primer in an ARMS assay with different Orthopoxvirus genomes, a PCR product was only amplified from variola virus DNA. Incorporating a second consensus primer into the assay produced a multiplex PCR that provided Orthopoxvirus generic and variola-specific products with variola virus DNA. We tested two single nucleotide polymorphisms with a panel of 43 variola virus strains, collected over 40 years from countries across the world, and have shown that they provide reliable markers for variola virus identification. The variola virus specific primers did not produce amplicons with either assay format when tested with 50 other Orthopoxvirus DNA samples. Our analysis shows that these two polymorphisms were conserved in variola virus genomes and provide a reliable signature of Orthopoxvirus species identification.


id: cord-261329-k1p7fo0e
author: Nidzworski, Dawid
date: 2010-12-28
txt: ./txt/cord-261329-k1p7fo0e.txt
cache: ./cache/cord-261329-k1p7fo0e.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2010.12.015
doi: 10.1016/j.jviromet.2010.12.015
summary:
  A rapid diagnostic method based on the melting curve SYBR Green I real-time PCR analysis was developed to detect and differentiate Newcastle disease virus (NDV) strains. The results obtained in this study demonstrate the possible applications for melting curve real-time PCR analysis in laboratory practice for the diagnosis and differentiation of avirulent and virulent strains of Newcastle disease virus. (2005) described a SYBR Green I real-time PCR melting curve analysis assay for differentiation, although the differences in the Tm values between the three genotypes were not very significant and could cause false characterization of the virus. Using the SYBR Green I real-time PCR melting peak analysis, it was possible to detect and differentiate virulent and avirulent strains of Newcastle disease virus. In this study, a method for the rapid detection and differentiation of Newcastle disease virus by SYBR Green I melting-curve analysis was described.

title:
  Detection and differentiation of virulent and avirulent strains of Newcastle disease virus by real-time PCR

abstract:
  A rapid diagnostic method based on the melting curve SYBR Green I real-time PCR analysis was developed to detect and differentiate Newcastle disease virus (NDV) strains. Degenerated primers based on the cleavage site sequence of the F0 gene were designed to detect specific sequences characteristic of virulent and avirulent strains of NDV. Eighteen strains of NDV from four lineages were identified and grouped into virulent and avirulent strains. Peaks on the melting temperature graph with melting temperature values between 80.00 and 83.80 °C were observed for lentogenic (avirulent) strains. T(m) values higher than 83.80 were observed for virulent (mesogenic and velogenic) strains. The detection limit of real-time PCR was 2 × 10(2) plasmid copies per reaction or 10(2) EID(50) for velogenic strains and 10(3) EID(50) for lentogenic strains. The results obtained in this study demonstrate the possible applications for melting curve real-time PCR analysis in laboratory practice for the diagnosis and differentiation of avirulent and virulent strains of Newcastle disease virus.


id: cord-261735-03hvi4el
title: Development of a one step real time RT-PCR assay to detect and quantify Dugbe virus
author: Rodrigues, R.
date: 2011-06-14
txt: ./txt/cord-261735-03hvi4el.txt
cache: ./cache/cord-261735-03hvi4el.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/21703306/
doi: 10.1016/j.jviromet.2011.06.003
summary:
  A one-step real time quantitative RT-PCR (qRT-PCR) assay was developed to detect all published Dugbe virus (DUGV) genomes of the Nairovirus genus. Frequently detected in tick-borne virus surveys in Africa (Guilherme et al., 1996) , DUGV is a tri-segmented single-stranded negative RNA enveloped virus and is considered endemic in arid regions (Burt et al., 1996) . The aim of this study was to develop a sensitive, specific and rapid one-step quantitative real time RT-PCR assay (qRT-PCR) to detect DUGV in infected cell supernatants, ticks or serum samples. The specificity was evaluated by using RNA extracted from supernatants from CCHFV, Hazara virus, Coronavirus and Influenza A virus infected cells. Among the 498 captured ticks, one Dugbe virus RNA was detected in one tick using the qRT-PCR assay (0.2%). In conclusion, a sensitive and specific qRT-PCR assay was developed to detect and quantify DUGV RNAs in infected cell supernatants, extracts from ticks and potentially sera.

abstract:
  A one-step real time quantitative RT-PCR (qRT-PCR) assay was developed to detect all published Dugbe virus (DUGV) genomes of the Nairovirus genus. Primers and probes were designed to detect specific sequences on the most conserved regions of the S segment. The limit of detection of the assay was 10 copies per reaction which is an improvement of 3 log(10) FFU/mL over the sensitivity of conventional RT-PCR. The specificity of the primers and probe was confirmed with the closely related Nairoviruses CCHFV and Hazara virus, and on the non-related viruses Coronavirus and Influenza A virus. This qRT-PCR assay was used to screen nucleic acids extracted from 498 ticks collected in the Republic of Chad. One sample was found positive suggesting that DUGV is present in this part of the world. The molecular assay developed in this study is sensitive, specific and rapid and can be used for research and epidemiological studies.


id: cord-262991-j36vajdi
author: Pratelli, Annamaria
date: 2002-01-22
url: https://www.sciencedirect.com/science/article/pii/S0166093401004505
doi: 10.1016/s0166-0934(01)00450-5
title:
  Prevalence of canine coronavirus antibodies by an enzyme-linked immunosorbent assay in dogs in the south of Italy

abstract:
  An enzyme-linked immunosorbent assay (Elisa), using as antigen canine coronavirus-infected CrFK cell supernatant, was developed to detect antibodies against canine coronavirus (CCoV). Out of a total of 109 dog serum samples, 80 which were positive by routine virus neutralisation test were also Elisa positive. Seventeen samples which were negative by the virus neutralisation test, were positive by Elisa and by the confirmatory Western blotting test. The Elisa was substantially more sensitive than the virus neutralisation test in detecting antibodies to CCoV and may be used as an alternative technique to virus neutralisation.


id: cord-263570-6notzm6s
title: Detection of infectious canine parvovirus type 2 by mRNA real-time RT-PCR
author: Elia, Gabriella
date: 2007-08-10
txt: ./txt/cord-263570-6notzm6s.txt
cache: ./cache/cord-263570-6notzm6s.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/17692932/
doi: 10.1016/j.jviromet.2007.06.017
summary:
  The load and distribution of CPV-2 mRNA in samples from infected dogs were estimated in comparison with the load of virus DNA, as evaluated by real-time PCR. The analytic specificity of spliced CPV-2 mRNA detection by real-time RT-PCR was assessed by testing RNA and DNA preparations of other canine pathogens, including canine coronavirus types I and II (CCoVI, CCoVII) (Decaro et al., 2005d) , canine distemper virus (CDV) (Elia et al., 2006) , canine adenovirus (CAdV) (Decaro et al., 2006a) , reoviruses (Decaro et al., 2005b) and rotaviruses . To compare the amount of viral DNA in the tissue samples and in the infected cell cultures, a real-time PCR assay was used as described previously (Decaro et al., 2005c) . The newly developed real-time RT-PCR assay was applied to determine the viral mRNA loads in different tissues of CPV-2 naturally infected dogs. A real-time PCR assay for rapid detection and quantitation of canine parvovirus type 2 DNA in the feces of dogs

abstract:
  A TaqMan real-time RT-PCR assay was developed for detection of RNA transcripts produced by replicating CPV-2. A pair of primers and a TaqMan probe targeting the spliced NS2 mRNA were designed. A synthetic DNA fragment was constructed to mimic the spliced NS2 mRNA by PCR-based gene assembly and was used for generation of standard RNAs. The detection limit of the assay was 1 × 10(2) RNA copies and standard curve displayed a linear range from 1 × 10(2) to 1 × 10(9) copies and a good reproducibility. The assay was then applied to determine the mRNA loads in the tissues of dogs naturally infected by CPV-2. mRNA was detected in a variety of tissues, including the central nervous system.


id: cord-264335-c2hfh3dq
author: Gunson, Rory
date: 2009-10-23
txt: ./txt/cord-264335-c2hfh3dq.txt
cache: ./cache/cord-264335-c2hfh3dq.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093409004455
doi: 10.1016/j.jviromet.2009.10.006
summary:
  In order to detect and then type influenza A viruses most laboratories use a two tier testing system comprising of a universal influenza A screening assay complemented with a suite of subtyping assays that determine whether the sample is seasonal influenza A (human H1N1 and H3N2), avian H5N1 or the influenza A/H1N1/2009 virus. This article describes the development of a multiplex real-time reverse transcription polymerase chain reaction (rtPCR) that allows universal detection of all influenza A viruses and simultaneously subtypes all that are influenza A/H1N1/2009. Use of this assay will allow laboratories to screen respiratory samples for influenza A/H1N1/2009 virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. This article describes the development of a rapid, specific and sensitive multiplex rtPCR assay that detects all influenza A types and simultaneously identifies samples that contain the pandemic influenza A/H1N1/2009 virus.

title:
  Development of a multiplex real-time RT-PCR that allows universal detection of influenza A viruses and simultaneous typing of influenza A/H1N1/2009 virus

abstract:
  On June 11, 2009, the World Health Organization declared that the influenza A/H1N1/2009 virus had become the first influenza pandemic of the 21st century. Rapid detection and differentiation from seasonal and avian influenza would be beneficial for patient management and infection control. It was the aim of this study to develop a real-time RT-PCR that can detect all influenza A viruses and offer simultaneous typing for influenza A/H1N1/2009. This would be a useful addition to existing diagnostic protocols for influenza A. Its routine use would allow laboratories to screen out influenza A/H1N1/2009 positive samples rapidly and would reduce overall testing costs.


id: cord-265634-7n4cvgs4
author: Dhar, Arun K.
date: 2002-03-26
txt: ./txt/cord-265634-7n4cvgs4.txt
cache: ./cache/cord-265634-7n4cvgs4.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/12020794/
doi: 10.1016/s0166-0934(02)00042-3
summary:
  title: Quantitative assay for measuring the Taura syndrome virus and yellow head virus load in shrimp by real-time RT-PCR using SYBR Green chemistry The current diagnostic methods for TSV and YHV include bioassay using indicator hosts, monitoring clinical signs, histopathology, dot blot, in situ hybridization using virus specific gene probe, immunohistochemistry and by the polymerase chain reaction (PCR) (Lightner and Redman, 1998) . SYBR Green RT-PCR was performed in a 96 well plate using 1 ml of each of the cDNA dilutions for TSV and YHV detection along with EF-1a and b-actin controls following the reaction parameters as described above. The analytical sensitivity of SYBR Green PCR was determined by using a serial dilution of TSV and YHV plasmid DNA as template for amplification. The SYBR Green RT-PCR was not only highly sensitive but also very specific for detecting TSV, YHV and the internal control genes, EF-1a and b-actin.

title:
  Quantitative assay for measuring the Taura syndrome virus and yellow head virus load in shrimp by real-time RT-PCR using SYBR Green chemistry

abstract:
  Taura syndrome virus (TSV) and yellow head virus (YHV) are the two RNA viruses infecting penaeid shrimp (Penaeus sp.) that have caused major economic losses to shrimp aquaculture. A rapid and highly sensitive detection and quantification method for TSV and YHV was developed using the GeneAmp(®) 5700 Sequence Detection System and SYBR Green chemistry. The reverse transcriptase polymerase chain reaction (RT-PCR) mixture contained a fluorescent dye, SYBR Green, which exhibits fluorescence enhancement upon binding to double strand cDNA. The enhancement of fluorescence was found to be proportional to the initial concentration of the template cDNA. A linear relationship was observed between input plasmid DNA and cycle threshold (C(T)) values for 10(6) down to a single copy of both viruses. To control for the variation in sample processing and in reverse transcription reaction among samples, shrimp β-actin and elongation factor-1α (EF-1α) genes were amplified in parallel with the viral cDNA. The sensitivity and the efficiency of amplification of EF-1α was greater than β-actin when compared to TSV and YHV amplification efficiency suggesting that EF-1α is a better internal control for the RT-PCR detection of TSV and YHV. In addition, sample to sample variation in EF-1α C(T) value was lower than the variation in β-actin C(T) value of the corresponding samples. The specificity of TSV, YHV, EF-1α and β-actin amplifications was confirmed by analyzing the dissociation curves of the target amplicon. The C(T) values of TSV and YHV samples were normalized against EF-1α C(T) values for determining the absolute copy number from the standard curve of the corresponding virus. The method described here is highly robust and is amenable to high throughput assays making it a useful tool for diagnostic, epidemiological and genetic studies in shrimp aquaculture.


id: cord-266571-qbskh1uu
author: de Arriba, M.L
date: 2002-06-04
txt: ./txt/cord-266571-qbskh1uu.txt
cache: ./cache/cord-266571-qbskh1uu.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093402000630
doi: 10.1016/s0166-0934(02)00063-0
summary:
  Lymphocyte proliferative responses were evaluated in mucosal (mesenteric lymph nodes) and systemic (spleen and blood) lymphoid tissues of conventional piglets inoculated with the virulent or attenuated isolates of porcine epidemic diarrhoea virus (PEDV) strain CV-777 and challenged 21 days later with the virulent isolate of the same virus. Virus-specific lymphoproliferative responses of systemic tissues (spleen and blood) and mesenteric lymph nodes were studied in conventional piglets after primary inoculation with the virulent, wild type, strain CV-777 of PEDV or its cell culture attenuated form and after challenge, 3 weeks later, with a high dose of the virulent virus. Correlations between lymphocyte proliferative responses in mononuclear cells collected from mesenteric lymph nodes, blood and spleen from pigs inoculated with virulent or attenuated PEDV or mock-inoculated and protection against challenge 21 days later with virulent PEDV.

title:
  Lymphoproliferative responses and protection in conventional piglets inoculated orally with virulent or attenuated porcine epidemic diarrhoea virus

abstract:
  Lymphocyte proliferative responses were evaluated in mucosal (mesenteric lymph nodes) and systemic (spleen and blood) lymphoid tissues of conventional piglets inoculated with the virulent or attenuated isolates of porcine epidemic diarrhoea virus (PEDV) strain CV-777 and challenged 21 days later with the virulent isolate of the same virus. A lymphoproliferative assay was developed in which mononuclear cells isolated from lymphoid tissues at different postinoculation and postchallenge days underwent a secondary in vitro stimulation with semipurified antigen obtained from PEDV-infected cell cultures. Vigorous lymphocyte proliferative responses were detected in the pigs inoculated with the virulent PEDV at postinoculation days 4–21, especially in the mesenteric lymph nodes and the blood; however, in the spleen this response was lower and less regular. The pigs inoculated with the attenuated virus showed a less intense response, the higher lymphocyte proliferation also corresponded to the mononuclear cells from mesenteric lymph nodes. Lymphocyte proliferation responses showed high correlations with protection against homologous challenge with virulent PEDV, and this correlation was higher in the gut associated lymphoid tissues (mesenteric lymph nodes). The cell proliferation response detected in blood mirrored that detected in the mesenteric lymph nodes, and showed also good correlation with protection. The results confirm that T-cell-helper function, assessed by lymphocyte proliferation responses, contributes to establishing a protective immune response against PEDV infections.


id: cord-267588-ruuzr6l1
author: Garnett, Lauren
date: 2020-08-08
txt: ./txt/cord-267588-ruuzr6l1.txt
cache: ./cache/cord-267588-ruuzr6l1.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093420301993
doi: 10.1016/j.jviromet.2020.113947
summary:
  This study aimed to examine the efficacy of six different swabs that are commonly found in hospital settings (PurFlock Ultra, FLOQSwab, Puritan Pur-Wraps cotton tipped applicators, Puritan polyester tipped applicators, MedPro 6" cotton tipped applicators, and HOLOGIC Aptima), along with more readily available alternative transport mediums (DMEM, PBS, 100% ethanol, 0.9% normal saline and VTM) for their use in molecular detection of SARS-CoV-2. Therefore, our results suggest that the cotton and wood For the portion of the study focusing on alternative transport media, we assessed the ability of DMEM, PBS, 0.9% Normal Saline, and 100% ethanol compared to VTM to be used as medium for the preservation and recovery of viral RNA to be quantified by molecular detection. Despite finding similar levels of viral RNA collected using different swabs and transport media, there is variation when evaluating different respiratory clinical samples while testing for SARS-CoV-2.

title:
  Comparison analysis of different swabs and transport mediums suitable for SARS-CoV-2 testing following shortages

abstract:
  On March 11, 2020, the World Health Organization (WHO) assessed COVID-19, caused by SARS-CoV-2, as a pandemic. As of June 1, 2020, SARS-CoV-2 has had a documented effect of over 6 million cases world-wide, amounting to over 370,000 deaths (World Health Organization, 2020. Novel Coronavirus (COVID-19) Situation. http://https://covid19.who.int/). Consequently, the high demand for testing has resulted in a depletion of commercially available consumables, including the recommended swabs and viral transport media (VTM) required for nasopharyngeal sampling. Therefore, the potential use of unvalidated alternatives must be explored to address the global shortage of testing supplies. To tackle this issue, we evaluated the utility of different swabs and transport mediums for the molecular detection of SARS-CoV-2. This study compared the performance of six swabs commonly found in primary and tertiary health care settings (PurFlock Ultra, FLOQSwab, Puritan Pur-Wraps cotton tipped applicators, Puritan polyester tipped applicators, MedPro 6” cotton tipped applicators, and HOLOGIC Aptima) for their efficacy in testing for SARS-CoV-2. Separately, the molecular detection of SARS-CoV-2 was completed from different transport mediums (DMEM, PBS, 100 % ethanol, 0.9 % normal saline and VTM), which were kept up to three days at room temperature (RT). The results indicate that there is no meaningful difference in viral yield from different swabs and most transport mediums for the collection and detection of SARS-CoV-2, indicating swab and medium alternatives could be used if supplies run out.


id: cord-267744-asjvf123
title: Chicken single-chain variable fragments against the SARS-CoV spike protein
author: Lee, Yu-Ching
date: 2007-07-23
txt: ./txt/cord-267744-asjvf123.txt
cache: ./cache/cord-267744-asjvf123.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093407002236
doi: 10.1016/j.jviromet.2007.06.010
summary:
  Following the immunization of chickens with these recombinant spike proteins, two single-chain variable fragment (scFv) antibody libraries were established with short or long linkers to contain 5 × 10(7) and 9 × 10(6) transformants, respectively. In a comparison of nucleotide sequences with the chicken germline gene, we found that all clones varied in the complementarity-determining regions, that two scFv antibodies reacted significantly with SARS-CoV-infected Vero cells, and that those two specific scFv antibodies recognized the same region of the spike protein spanning amino acid residues 750–1000. The current study aimed to show that monoclonal IgY scFv antibodies which bind specifically to the S protein and SARS-CoV-infected Vero cells can be isolated from chickens immunized with Escherichia coli-derived S proteins. Cellular lysates containing single-chain variable fragment (scFv) antibodies from various Ssc (A) and Lsc (B) library clones were examined for their binding to SARS-CoV-infected cell lysates using a commercially available kit.

abstract:
  The major concern for severe acute respiratory syndrome (SARS), caused by the SARS-associated coronavirus (SARS-CoV), is the lack of diagnostic and therapeutic agents. Using a phage display technology in a chicken system, high-affinity monoclonal antibody fragments against the SARS-CoV spike protein were characterized. Ten truncated spike protein gene fragments were expressed in Escherichia coli cells. Following the immunization of chickens with these recombinant spike proteins, two single-chain variable fragment (scFv) antibody libraries were established with short or long linkers to contain 5 × 10(7) and 9 × 10(6) transformants, respectively. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were demonstrated by Coomassie blue staining, and verified by western blot analysis. In a comparison of nucleotide sequences with the chicken germline gene, we found that all clones varied in the complementarity-determining regions, that two scFv antibodies reacted significantly with SARS-CoV-infected Vero cells, and that those two specific scFv antibodies recognized the same region of the spike protein spanning amino acid residues 750–1000. In conclusion, the results suggest that the chicken scFv phage display system can be a potential model for mass production of high-affinity antibodies against the SARS-CoV spike protein.


id: cord-267941-nrluar4e
title: Production of Ebola virus-like particles in Drosophila melanogaster Schneider 2 cells
author: Park, Eun-Mee
date: 2018-08-23
url: https://api.elsevier.com/content/article/pii/S0166093418304208
doi: 10.1016/j.jviromet.2018.08.016
abstract:
  In this study, we generated recombinant virus-like particles (VLPs) against family Filoviridae, genus Ebolavirus, species Zaire ebolavirus, strain Makona (EBOV) in Drosophila melanogaster Schneider 2 (S2) cells using the EBOV Makona. S2 cells were cotransfected with four viral plasmids encoding EBOV Makona proteins and protein expression was analyzed by immunoblotting. We confirmed that EBOV Makona proteins were successfully expressed in S2 cells. Additionally, we further examined the formation of intracellular and extracellular VLPs by electron microscopy. eVLPs were produced by sucrose gradient ultracentrifugation of S2 cells transfected with EBOV Makona genes, and production of VLPs was confirmed by immunoblot analysis. Collectively, our findings showed that the S2 cell system could be a promising tool for efficient production of eVLPs.


id: cord-270421-ytrkob0h
title: A rapid and quantitative assay for measuring neutralizing antibodies of Coxsackievirus B3
author: Chen, Pan
date: 2016-03-04
txt: ./txt/cord-270421-ytrkob0h.txt
cache: ./cache/cord-270421-ytrkob0h.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093415300410
doi: 10.1016/j.jviromet.2016.02.010
summary:
  The CVB3 pseudovirus system was used for quantifying neutralizing antibody (NtAb) levels of 720 human serum samples and showed superior specificity and sensitivity comparing traditional cytopathic effect (CPE) assay. For the lack of CVB3 national antibody standard, we used a mouse serum collected from mice immunized with formalin inactivated CVB3 virus 112 strain and quantified its neutralizing activity against CVB3 (Nancy)-luc pseudovirus in duplicate on the same plate in six independent tests. We next used this pseudovirus assay to measure CVB3 neutralizing antibodies titers in serum samples collected from health adults (18-65 years old). In summary, we established a single round infection system of CVB3 and developed an in vitro assay for detecting neutralizing antibodies in clinical serum samples, and it was a superior surrogate of the assays using wild type viruses including traditional CPE assay and enzyme-linked immunosorbent spot assay.

abstract:
  Coxsackievirus B3 (CVB3) infection has been found to account for an increasing proportion cases of hand, foot and mouth disease (HFMD) in recent epidemiology studies. CVB3 is a single stranded, non-enveloped RNA virus and the infection can cause prominent health threat to pre-school children. Here, by taking approaches of reverse genetics, we established a single-round infection system for CVB3. The pseudovirus was produced by sequential transfection of CVB3 capsid expresser plasmid and CVB3 replicon RNA bearing firefly luciferase as a reporter. The CVB3 pseudovirus system was used for quantifying neutralizing antibody (NtAb) levels of 720 human serum samples and showed superior specificity and sensitivity comparing traditional cytopathic effect (CPE) assay. Furthermore, we compared the seroprevalence of CVB3 NtAbs in pre-school children and healthy adults, and found that only 11.94% of pre-school children were NtAbs positive which suggested that most children were naive to CVB3 infection; while there is much higher positive rate in adults (60%) indicating that most adults have experienced CVB3 infection during childhood. This rapid and quantitative assay greatly facilitates evaluating the level of NtAbs against CVB3 in populations and will help to advance CVB3 vaccine development.


id: cord-270526-o4hsr4pm
author: An, Dong-Jun
date: 2007-10-24
txt: ./txt/cord-270526-o4hsr4pm.txt
cache: ./cache/cord-270526-o4hsr4pm.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093407003618
doi: 10.1016/j.jviromet.2007.09.006
summary:
  The sensitivity and specificity of this assay were compared with a nested PCR assay by testing conjunctival swabs, nasal irrigation fluid, and blood lymphocyte samples from dogs suspected to have CD. The antemortem diagnosis of canine distemper is based on the demonstration of viral antigens in scrapings and body fluids such as conjunctival and vaginal smears, tracheal washings and Table 2 Clinical signs, age and breed of CD-positive specimens Table 3 Analysis of the sensitivity and specificity of the IC assay relative to nested PCR results in detecting CDV in specimens from dogs suspected to have CD a IC assay result One study using nested PCR analysis revealed that of 22 blood, 20 urine, 25 saliva, and 27 nasal swab samples from dogs suspected to have CD, 81.8%, 75%, 56%, and 70.3% tested positive, respectively (Shin et al., 2004) .

title:
  An immunochromatography assay for rapid antemortem diagnosis of dogs suspected to have canine distemper

abstract:
  A new assay was developed for rapid and antemortem diagnosis of canine distemper (CD). This immunochromatography (IC)-based assay, which employs two monoclonal anti-CDV antibodies, was compared with nested PCR. When serial dilutions of purified CDV were tested, the CDV detection limits of the nested PCR and IC assays were 2 × 10(2) TCID(50)/ml and 5 × 10(2) TCID(50)/ml, respectively. Nasal irrigation fluid, conjunctival swabs, and blood lymphocytes from 66 dogs suspected to have CD were tested. Preliminary IC experiments revealed that the optimal sample volume and reaction time were 100 μl and 5 min, respectively. Relative to nested PCR, the sensitivity and specificity of the IC assay was maximal (100% and 100%, respectively) when conjunctival swabs were tested. This is significant because conjunctival swab specimens are easy to obtain in the early phase of CD infection. However, with blood lymphocytes and nasal samples, the IC assay was slightly less sensitive (89.7% and 85.7%, respectively) and specific (94.6% and 100%, respectively) than nested PCR. Since this novel IC assay does not require special instruments, it is a simple enough for dog owners to use. Since early detection of CD would allow appropriate treatment and quarantine to be instituted quickly, such a test would help reduce the morbidity and mortality associated with CD help to prevent its spread to other animals.


id: cord-270788-w0pewq52
author: Chou, Chih-Fong
date: 2004-11-05
url: https://api.elsevier.com/content/article/pii/S0166093404002654
doi: 10.1016/j.jviromet.2004.09.008
title:
  A novel cell-based binding assay system reconstituting interaction between SARS-CoV S protein and its cellular receptor

abstract:
  Severe acute respiratory syndrome (SARS), a life-threatening disease, is caused by the newly identified virus SARS coronavirus (SARS-CoV). In order to study the spike (S) protein of this highly contagious virus, we established a clonal cell-line, CHO-SG, from the Chinese hamster ovary cells that stably expresses C-terminally EGFP-tagged SARS-CoV S protein (S-EGFP). The ectodomain of the S glycoprotein is localized on the surface of CHO-SG cells with N-acetyl-glucosamine-terminated carbohydrate structure. CHO-SG cells associated tightly with Vero E6 cells, a SARS-CoV receptor (ACE2) expressing cell-line, and the interaction remained stable under highly stringent condition (1 M NaCl). This interaction could be blocked by either the serum from a SARS convalescent patient or a goat anti-ACE2 antibody, indicating that the interaction is specific. A binding epitope with lesser degree of glycosylation and native conformation was localized by using rabbit anti-sera raised against five denatured recombinant S protein fragments expressed in Escherichia coli. One of the sera obtained from the fragment encompassing amino acids 48-358 significantly blocked the interaction between CHO-SG and Vero E6 cells. The region is useful for studying neutralizing antibodies in future vaccine development. This paper describes an easy and safe cell-based assay suitable for studying the binding between SARS-CoV S protein and its receptor.


id: cord-271915-nvilxnzl
author: Adachi, D.
date: 2004-12-01
txt: ./txt/cord-271915-nvilxnzl.txt
cache: ./cache/cord-271915-nvilxnzl.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093404002162
doi: 10.1016/j.jviromet.2004.07.008
summary:
  The SARS-associated human coronavirus (SARS-HCoV) is a newly described, emerging virus conclusively established as the etiologic agent of the severe acute respiratory syndrome (SARS). This study presents a single-tube RT-PCR assay that can detect with high analytical sensitivity the SARS-HCoV, as well as several other coronaviruses including other known human respiratory coronaviruses (HCoV-OC43 and HCoV-229E). Species identification is provided by sequencing the amplicon, although a rapid screening test by restriction enzyme analysis has proved to be very useful for the analysis of samples obtained during the SARS outbreak in Toronto, Canada. This single-tube RT-PCR is based on consensus primers targeting conserved regions of coronavirus genome sequences and allows for the detection and species identification of several coronaviruses including SARS-HCoV, with high analytical sensitivity. Aliquots of a 10-fold serial RNA dilution prepared from a lung biopsy sample of a patient with SARS (see Section 2) were used to compare our assay with the RealArt HPA coronavirus RT-PCR (Artus GmbH).

title:
  Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay

abstract:
  The SARS-associated human coronavirus (SARS-HCoV) is a newly described, emerging virus conclusively established as the etiologic agent of the severe acute respiratory syndrome (SARS). This study presents a single-tube RT-PCR assay that can detect with high analytical sensitivity the SARS-HCoV, as well as several other coronaviruses including other known human respiratory coronaviruses (HCoV-OC43 and HCoV-229E). Species identification is provided by sequencing the amplicon, although a rapid screening test by restriction enzyme analysis has proved to be very useful for the analysis of samples obtained during the SARS outbreak in Toronto, Canada.


id: cord-273074-k8m917i4
title: Preparation and evaluation of anti-SARS coronavirus IgY from yolks of immunized SPF chickens
author: Fu, Chao-Yang
date: 2005-12-01
txt: ./txt/cord-273074-k8m917i4.txt
cache: ./cache/cord-273074-k8m917i4.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/16325277/
doi: 10.1016/j.jviromet.2005.10.027
summary:
  title: Preparation and evaluation of anti-SARS coronavirus IgY from yolks of immunized SPF chickens SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and neutralization test results showed that the IgY obtained was of a high purity and had a strong reactive activity with a neutralization titer of 1:640. In this study, we have successfully immunized specific pathogen-free (SPF) chickens, and then purified a high-titer anti-SARS coronavirus yolk immunoglobulin (IgY) with neutralizing activity against SARS coronavirus. The activity of IgY in sera and yolks diluted at 1:200 in phosphate buffer saline (PBS) from immunized animals was assessed using an indirect ELISA assay as described previously (Huang et al., 2005) (Fig. 1) . The development of high-titer anti-SARS coronavirus IgY described in this study would appear to have potential as a new anti-SARS biological product for passive immunization, as it effectively neutralized the SARS coronavirus.

abstract:
  Severe acute respiratory syndrome (SARS) is a recently discovered viral disease, characterized by fever, cough, acute fibrinous pneumonia and high infectivity. Specific pathogen-free (SPF) chickens were immunized with inactivated SARS coronavirus and their eggs were harvested at regular intervals. Yolk immunoglobulin (IgY) was extracted using the water dilution method, followed by further purification on a Sephadex G-75 column. SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and neutralization test results showed that the IgY obtained was of a high purity and had a strong reactive activity with a neutralization titer of 1:640. Lyophilization and stability tests showed that lyophilized anti-SARS coronavirus IgY had promising physical properties, with no significant reduction in reactive activity and good thermal stability. All these data suggest that the anti-SARS coronavirus IgY could be a new useful biological product for specific antiviral therapy against SARS.


id: cord-273846-l0elcfe8
author: Ganapathy, Kannan
date: 2005-02-24
txt: ./txt/cord-273846-l0elcfe8.txt
cache: ./cache/cord-273846-l0elcfe8.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/15847923/
doi: 10.1016/j.jviromet.2005.01.024
summary:
  title: Effects of cold storage on detection of avian infectious bronchitis virus in chicken carcasses and local antibodies in tracheal washes In order to test the survivability of infectious bronchitis virus (IBV) in dead chicken carcasses during 24 h of cold storage, 7 week-old specific-pathogen-free chickens were infected with virulent IBV Massachusetts strain M41, and were killed humanely 10 days later. Trachea, lung, kidney and rectum were collected for virus isolation by tracheal organ culture (TOC) or embryonated chicken eggs (ECE), and detection by nested reverse-transcriptase polymerase chain reaction (RT-PCR). Diagnosis of infectious bronchitis virus (IBV) is confirmed by isolation of the virus using either chicken embryonated eggs (ECE) or tracheal organ culture (TOC) and detection by reverse-transcriptase polymerase chain reaction (RT-PCR) (Cavanagh and Naqi, 2003; Gelb and Jackwood, 1998) . The use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis virus

title:
  Effects of cold storage on detection of avian infectious bronchitis virus in chicken carcasses and local antibodies in tracheal washes

abstract:
  In order to test the survivability of infectious bronchitis virus (IBV) in dead chicken carcasses during 24 h of cold storage, 7 week-old specific-pathogen-free chickens were infected with virulent IBV Massachusetts strain M41, and were killed humanely 10 days later. Carcasses were stored in a cold room at 4 °C. After 1, 3, 6, 9, 12 or 24 h of storage, necropsies were carried out. Trachea, lung, kidney and rectum were collected for virus isolation by tracheal organ culture (TOC) or embryonated chicken eggs (ECE), and detection by nested reverse-transcriptase polymerase chain reaction (RT-PCR). IBV was detected by RT-PCR at all sampling times, except for 1 and 6 h of storage in kidney and 9 h of storage in kidney and rectum. For ECE, isolation was obtained at all sampling points, except at 1 and 24 h of storage in lungs. Isolation by tracheal organ cultures was less successful, except from rectum. In addition to sampling for virus, tracheal washes were collected from each carcass to measure the ability to detect local antibodies after storage. Levels of IgA in tracheal washes remained high for up to 9 h of storage, suggesting that accurate sampling for research purposes when required must be carried out within this time.


id: cord-274289-8g9tuyrc
author: Liang, Xiao
date: 2014-06-15
txt: ./txt/cord-274289-8g9tuyrc.txt
cache: ./cache/cord-274289-8g9tuyrc.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093414000755
doi: 10.1016/j.jviromet.2014.02.022
summary:
  The study assessed the stability of nucleic acids stored on FTA cards at a temperature range representing the extremes of environmental heat (13 • to 46 • C) and cold specimen handling conditions (−7 • to −27 • C), and a timeframe from specimen collection to laboratory processing consistent with the expected extremes of diagnostic sample shipping (7-14 days). Bovine Coronavirus was the most prevalent virus detected by realtime PCR during this phase of the study, with 60% animals testing Table 2 Kappa values (95% CI) and percentage agreement for specimens collected into viral transport media compared to specimens collected onto FTA Cards. Bovine Respiratory Syncytial virus was detected using both the specimens in viral transport media and those on FTA Cards for 100% agreement; however the realtime PCR positive test result was for only one animal (Tables 1 and 2) .

title:
  Evaluation of Fast Technology Analysis (FTA) Cards as an improved method for specimen collection and shipment targeting viruses associated with Bovine Respiratory Disease Complex

abstract:
  In order to improve the analytic quality of respiratory specimens collected from cattle for nucleic acid-based diagnosis, a study was undertaken to verify realtime PCR efficiency of specimens collected and stabilized on FTA Cards™, filter paper which is treated chemically. Nucleic acids collected using FTA Cards without the need for a cold-chain or special liquid media handling provided realtime PCR results consistent (96.8% agreement, kappa 0.923 [95% CI = 0.89–0.96]) with the same specimens collected using traditional viral transport media and shipped on ice using the U.S. Department of Transportation mandated liquid handling requirements. Nucleic acid stabilization on FTA Cards was evaluated over a temperature range (−27 °C to +46 °C) for up to 14 days to mimic environmental conditions for diagnostic sample handling between collection and processing in a routine veterinary laboratory. No significant difference (P ≥ 0.05) was observed in realtime PCR cycle threshold values over the temperature range and time storage conditions for Bovine Viral Diarrhea virus, Bovine Respiratory Syncytial virus, Bovine Coronavirus, and Bovine Herpesvirus I. The four viruses evaluated in the study are associated with Bovine Respiratory Disease Complex where improvements in ease and reliability of specimen collection and shipping would enhance the diagnostic quality of specimens collected in the field, and ultimately improve diagnostic efficiency.


id: cord-274954-06c3ymc3
author: Huang, Yu-Liang
date: 2009-05-04
txt: ./txt/cord-274954-06c3ymc3.txt
cache: ./cache/cord-274954-06c3ymc3.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/19414034/
doi: 10.1016/j.jviromet.2009.04.029
summary:
  title: Development of a reverse transcription multiplex real-time PCR for the detection and genotyping of classical swine fever virus A reverse transcription multiplex real-time PCR (RT-MRT-PCR) was developed for rapid detection and genotyping of classical swine fever virus (CSFV). For the analytical sensitivity experiment, 100 samples of 14 CSFV genotype 1 strains and 86 samples from CSFV outbreak farms were all detected as CSFV-positive by RT-MRT-PCR, and the genotype results were consistent with the results of sequencing from a previous study. 10-fold serially diluted samples of C-strain, Q90-278, and 83-19 strains were determined to be CSFV-positive by viral isolation, RT-PCR, RT-nPCR, and RT-MRT-PCR. a A total of 169 clinical samples was detected for the presence of CSFV and the results included and used to evaluate agreement among viral isolation, RT-PCR, RT-nPCR, and RT-MRT-PCR. It was shown that the sensitivity of RT-MRT-PCR is comparable to those of RT-nPCR and viral isolation, and higher than RT-PCR for the samples of CSFV diluted serially (Table 4 ).

title:
  Development of a reverse transcription multiplex real-time PCR for the detection and genotyping of classical swine fever virus

abstract:
  A reverse transcription multiplex real-time PCR (RT-MRT-PCR) was developed for rapid detection and genotyping of classical swine fever virus (CSFV). The universal primers and specific TaqMan probes for each of the three genotypes, genotypes 1, 2, and 3, were designed within the 3′-UTR of the CSFV. Non-CSFV swine virus and clinical samples from specific pathogen-free (SPF) pigs were both demonstrated to be CSFV-negative by RT-MRT-PCR. The diagnostic sensitivity of RT-MRT-PCR was determined to be 1 viral copy/μl for each genotype of standard plasmid. For the analytical sensitivity experiment, 100 samples of 14 CSFV genotype 1 strains and 86 samples from CSFV outbreak farms were all detected as CSFV-positive by RT-MRT-PCR, and the genotype results were consistent with the results of sequencing from a previous study. The intra-assay and inter-assay variations of RT-MRT-PCR were below 3% in all experiments. The sensitivity of RT-MRT-PCR was the same as the reverse transcription nested PCR (RT-nPCR) and higher than reverse transcription PCR (RT-PCR) and viral isolation from clinical samples. This assay was used further to evaluate the duration of viremia of wild-type CSFV in vaccinated exposed pigs. The results indicated that pigs vaccinated with the E2 subunit vaccine had longer viremia than pigs given the C-strain vaccine, which is compatible with the findings of previous studies. Thus, the new RT-MRT-PCR is a rapid, reproducible, sensitive, and specific genotyping tool for CSFV detection.


id: cord-275225-fvq8hezk
author: Hornyák, Ákos
date: 2012-02-18
txt: ./txt/cord-275225-fvq8hezk.txt
cache: ./cache/cord-275225-fvq8hezk.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093412000365
doi: 10.1016/j.jviromet.2012.01.022
summary:
  The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale. The detailed analysis of the feline infectious peritonitis positive cat samples by SYBR-Green QPCR method revealed that the following organs harbour FCoV most frequently: lungs 6/6 (100%), liver 6/6 (100%), kidney 10/11 (90.9%), mesenteric lymph node 18/20 (90%), spleen 16/20 (80%) and gut 15/10 (66.7%).

title:
  Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR)

abstract:
  Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10–50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale.


id: cord-275787-5s442sy2
author: Banerjee, Arinjay
date: 2016-09-14
txt: ./txt/cord-275787-5s442sy2.txt
cache: ./cache/cord-275787-5s442sy2.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093416302440
doi: 10.1016/j.jviromet.2016.09.008
summary:
  title: Generation and Characterization of Eptesicus fuscus (Big brown bat) kidney cell lines immortalized using the Myotis polyomavirus large T-antigen Here we describe a method to establish and immortalize big brown bat (Eptesicus fuscus) kidney (Efk3) cells using the Myotis polyomavirus T-antigen. Cell clones expressed interferon beta in response to poly(I:C) stimulation and supported the replication of four different viruses, namely, vesicular stomatitis virus (VSV), porcine epidemic diarrhea coronavirus (PED-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and herpes simplex virus (HSV). The parental cell line and clones were capable of expressing IFN beta and supported the replication of viruses such as vesicular stomatitis virus (VSV; family Rhabdoviridae, genus Vesiculovirus), herpes simplex virus (HSV; family Herpesviridae, subfamily Alphaherpesvirinae, genus Herpesvirus), PED-CoV and MERS-CoV. Bat kidney cells were immortalized by using ViaFect (Promega, USA) to transfect cells with either 2.5 g of pcDNA3 (Invitrogen, USA) empty vector or plasmids expressing either SV40 large T-antigen (SV40Tag) or Myotis polyomavirus large T-antigen (MyPVTag).

title:
  Generation and Characterization of Eptesicus fuscus (Big brown bat) kidney cell lines immortalized using the Myotis polyomavirus large T-antigen

abstract:
  It is speculated that bats are important reservoir hosts for numerous viruses, with 27 viral families reportedly detected in bats. Majority of these viruses have not been isolated and there is little information regarding their biology in bats. Establishing a well-characterized bat cell line supporting the replication of bat-borne viruses would facilitate the analysis of virus-host interactions in an in vitro model. Currently, few bat cell lines have been developed and only Tb1-Lu, derived from Tadarida brasiliensis is commercially available. Here we describe a method to establish and immortalize big brown bat (Eptesicus fuscus) kidney (Efk3) cells using the Myotis polyomavirus T-antigen. Subclones of this cell line expressed both epithelial and fibroblast markers to varying extents. Cell clones expressed interferon beta in response to poly(I:C) stimulation and supported the replication of four different viruses, namely, vesicular stomatitis virus (VSV), porcine epidemic diarrhea coronavirus (PED-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and herpes simplex virus (HSV). To our knowledge, this is the first bat cell line from a northern latitude insectivorous bat developed using a novel technology. The cell line has the potential to be used for isolation of bat viruses and for studying virus-bat interactions in culture.


id: cord-275793-k0uvqcmp
author: Xia, Hongyan
date: 2010-04-18
txt: ./txt/cord-275793-k0uvqcmp.txt
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extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2010.04.009
doi: 10.1016/j.jviromet.2010.04.009
summary:
  title: A microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies This study describes a novel blocking microsphere-based immunoassay for highly sensitive and specific detection of antibodies against bovine viral diarrhoea virus (BVDV). The objectives of this study were to develop a blocking microsphere-based immunoassay (bMIA) for detection of antibodies against BVDV, and to compare the performance of the assay with a commercial ELISA kit. This study describes the development and evaluation of a microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies. The diagnostic performance of the new bMIA was compared to that of a commercial blocking ELISA system, by testing a large panel of 509 bovine sera. A simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (BVDV) specific antibodies in cattle serum, plasma and bulk milk

title:
  A microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies

abstract:
  This study describes a novel blocking microsphere-based immunoassay for highly sensitive and specific detection of antibodies against bovine viral diarrhoea virus (BVDV). The intra- and inter-assay variability are 4.9% and less than 7%, respectively, and variability of bead conjugations is less than 6.6%. The diagnostic performance of the assay was evaluated by testing a total of 509 serum samples. Based on a negative/positive cut-off value of 30.3%, the assay has a sensitivity of 99.4% and a specificity of 98.3% relative to ELISA. The new microsphere immunoassay provides an alternative to conventional ELISA systems and can be used for high-throughput screening in the BVD control programmes.


id: cord-276368-c9e93h0u
author: Hosmillo, Myra D.T.
date: 2010-06-15
txt: ./txt/cord-276368-c9e93h0u.txt
cache: ./cache/cord-276368-c9e93h0u.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093410002132
doi: 10.1016/j.jviromet.2010.06.001
summary:
  Currently, methods such as real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) have not yet been developed for the rapid detection and quantitation of bovine (BToV) and porcine (PToV) toroviruses. Table 1 List of primers used for conventional RT-PCR, nested PCR and SYBR Green real-time RT-PCR assay for the detection and quantitation of bovine and porcine toroviruses in the fecal specimens from diarrheic calves and piglets. The present study developed, optimized and validated a SYBR Green real-time RT-PCR assay using a universal primer pair for the detection and quantitation of both BToVs and PToVs in archived stool samples. The efficacy and reliability of the SYBR Green real-time RT-PCR assay for the detection and quantitation of BToV and PToV in the 121 bovine and 86 porcine stool samples were evaluated. In conclusion, a rapid, sensitive, specific and reproducible onestep SYBR Green real-time RT-PCR was developed for the detection and quantitation of BToV and PToV in bovine and porcine stool samples.

title:
  Development of universal SYBR Green real-time RT-PCR for the rapid detection and quantitation of bovine and porcine toroviruses

abstract:
  Toroviruses (ToVs) are a group of emerging viruses that cause gastroenteritis in domestic animals and humans. Currently, methods such as real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) have not yet been developed for the rapid detection and quantitation of bovine (BToV) and porcine (PToV) toroviruses. Using BToV and PToV RNA standards generated by in vitro transcription, the detection limit of the SYBR Green real-time RT-PCR assay was 2.54 × 10(2) BToV and 2.17 × 10(3) PToV copies/reaction (correlation coefficiency = 0.99 and 0.97, respectively), whereas those of RT-PCR and nested PCR were 2.54 × 10(5) and 2.54 × 10(4) (BToV) and 2.17 × 10(7) and 2.17 × 10(5) (PToV) cRNA viral copies/reaction, respectively. Archived diarrhea specimens of calves (n = 121) and piglets (n = 86) were subjected to RT-PCR, nested PCR and SYBR Green real-time RT-PCR. By conventional RT-PCR, 1 (0.8%) bovine and 7 (8.1%) porcine samples tested positive to BToV and PToV, respectively. With nested PCR, 13 (10.7%) bovine and 17 (19.8%) porcine samples tested positive. SYBR Green real-time RT-PCR assay detected BToV and PToV in 22 of 121 (18.2%) bovine and 31 of 86 (36.0%) porcine samples. These results indicate that SYBR Green real-time RT-PCR (P < 0.05) is a more sensitive assay, which can be reproduced as a reliable, sensitive, and rapid tool for the detection and quantitation of toroviruses.


id: cord-276503-bh7uugwy
title: Flow cytometric detection and serotyping of enterovirus for the clinical laboratory
author: She, Rosemary C.
date: 2009-09-04
txt: ./txt/cord-276503-bh7uugwy.txt
cache: ./cache/cord-276503-bh7uugwy.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2009.08.018
doi: 10.1016/j.jviromet.2009.08.018
summary:
  Primary rhesus monkey kidney (PMK) cells were inoculated with several enterovirus serotypes and stained with enterovirus-specific antibodies for flow cytometry and indirect fluorescence antibody testing (IFA). Previous studies have focused on using flow cytometry to quantitate poliovirus infection in neuronal cells (Daley et al., 2005) , characterize enterovirus binding to host cell surfaces (Freistadt and Eberle, 2006; Mbida et al., 1991; Triantafilou et al., 2001) , confirm cytomegalovirus infection of tissue culture cells with a genetically engineered fluorescence reporter system (Kung et al., 2000) , and serotype human immunodeficiency virus type 1 (Zolla-Pazner et al., 1995) . In this study, flow cytometry proved to be a sensitive method for detecting fluorescently stained enterovirus-infected cells. False positives did not occur by flow cytometric analysis in that infected cells were negative after staining with isotype control antibodies and antibodies directed against other enterovirus serotypes.

abstract:
  Culture and serotyping of human enteroviruses by fluorescence microscopy are time-consuming and labor-intensive. Flow cytometry has the potential of being more rapid, sensitive, and objective but has not been used for these purposes in a clinical laboratory. Primary rhesus monkey kidney (PMK) cells were inoculated with several enterovirus serotypes and stained with enterovirus-specific antibodies for flow cytometry and indirect fluorescence antibody testing (IFA). Kinetic studies of coxsackievirus B1 and echovirus 30 infection of PMK cells were performed on days 1–4 after inoculation. Flow cytometry results for echovirus 6, 9, 11, and 30 and coxsackievirus B1 correlated with IFA in all cases. Coxsackievirus B1 and echovirus 30 infections were detected 1 day earlier by flow cytometry than IFA. Flow cytometry can be effectively used for detecting enterovirus-infected cells in a clinical laboratory with the advantages of better quantitation of low levels of infection and earlier detection of virally infected cells in culture systems.


id: cord-276541-u9ebql5a
author: Lan, Yungang
date: 2011-11-25
txt: ./txt/cord-276541-u9ebql5a.txt
cache: ./cache/cord-276541-u9ebql5a.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093411004447
doi: 10.1016/j.jviromet.2011.11.007
summary:
  title: Inhibition of porcine hemagglutinating encephalomyelitis virus replication by short hairpin RNAs targeting of the nucleocapsid gene in a porcine kidney cell line Here, we constructed a single short hairpin RNA (shRNA) plasmid expression system that targeted the N gene and investigated whether shRNAmediated RNA interference could inhibit PHEV replication in PK-15 cells. To study the inhibitory effects of RNA interference on PHEV replication, the level of viral antigen produced in the PK-15 cells after shRNA transfection and viral infection was examined 48 h post-infection by an indirect immunofluorescence assay (IFA) using anti-PHEV serum. To quantify the effect of shRNA on viral replication at 48 h postviral infection, the viral genome copy number was determined by real-time PCR, using the serially diluted plasmid pT-N as a standard. It is clear from this study that the DNA vector-based shRNA approach, that is, the use of RNAi expression plasmids directed against the PHEV N gene, could effectively block expression of the viral target gene and inhibit viral replication.

title:
  Inhibition of porcine hemagglutinating encephalomyelitis virus replication by short hairpin RNAs targeting of the nucleocapsid gene in a porcine kidney cell line

abstract:
  Porcine hemagglutinating encephalomyelitis virus (PHEV), which causes porcine encephalomyelitis and is widespread among swine worldwide. RNA interference (RNAi) pathways have emerged as important regulators of virus–host cell interactions. In this study, two siRNA expression plasmids (shN1 and shN2) were generated to target two different coding regions of the nucleocapsid protein (N) of PHEV. The shRNAs were transiently transfected into a porcine kidney cell line, PK-15, to determine whether these constructs inhibited PHEV production. Our results revealed that both shRNAs were highly capable of inhibiting viral RNA genome replication, especially shN2. Next, stable transfection of shN2 was used to produce two siRNA stably expressing PK-15 cell clones (shN2-1 and shN2-2), and these two lines were infected with PHEV. The analysis of cytopathic effects (CPE) demonstrated that shN2-1 and shN2-2 were capable of protecting cells against PHEV infection with high specificity and efficiency. Furthermore, effective inhibition of viral replication persisted for up to 120 h by a TCID(50) assay. These results indicated that RNAi targeting of the N gene could facilitate studies of the specific function of viral genes associated with PHEV replication and may have potential therapeutic applications.


id: cord-276718-3lujp0oy
author: Neeraja, M.
date: 2014-10-24
txt: ./txt/cord-276718-3lujp0oy.txt
cache: ./cache/cord-276718-3lujp0oy.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/25455901/
doi: 10.1016/j.jviromet.2014.10.005
summary:
  title: Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India In the present study, the standardization and validation of a one step, four tube reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of the DENV targeting NS1 gene using the Genie® II flourometer was carried out. In the present study, the RT-LAMP assay was developed for the detection and serotyping of DENV infection targeting the serotype specific regions of the NS1 gene using a real-time flourometer (Genie ® II from Optigene, U.K.). The performance of the RT-LAMP assay was validated by testing the samples simultaneously by the CDC real time PCR that is most sensitive and specific method for detection and differentiation of the DENV (CDC Dengue). Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay

title:
  Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India

abstract:
  Early and rapid detection of dengue virus (DENV) infection during the acute phase of illness is crucial for proper patient management and prevention of the spread of the infection. In the present study, the standardization and validation of a one step, four tube reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of the DENV targeting NS1 gene using the Genie® II flourometer was carried out. The performance of the RT-LAMP was compared to RT-PCR, CDC 1-4 Real time PCR and the NS1 antigen ELISA, IgM and IgG anti DENV antibodies. Acute DENV infection was confirmed in 250/300 patients suspected clinically of DENV infection. RT- LAMP and CDC 1-4 Real time PCR assay was positive in 148/250 patients, while 92/250 patients were positive for anti- Dengue IgM and IgG antibodies. The RT-LAMP assay and the CDC real-time RT-PCR assay showed high concordance (k = 1.0). The detection rate of acute DENV infection improved to 96% (240/250) when the results of RT-LAMP were combined with NS1 Ag, IgM and IgG ELISA. The RT-LAMP had a detection limit of 100 copies for DEN-1 and DEN-2, 10 copies for DEN-3 and DEN-4 compared to 1000 copies for DEN-1 and DEN-2, 100 copies for DEN-3 and DEN-4 by the conventional RT-PCR. The assay showed 100% specificity. The RT-LAMP assay developed in this study has potential use for early clinical diagnosis, serotyping and surveillance of DENV infection in endemic countries such as India.


id: cord-276739-84vf5bts
title: Rapid typing of influenza viruses using super high-speed quantitative real-time PCR
author: Sakurai, Akira
date: 2011-08-22
txt: ./txt/cord-276739-84vf5bts.txt
cache: ./cache/cord-276739-84vf5bts.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093411003491
doi: 10.1016/j.jviromet.2011.08.015
summary:
  Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. This method offers high sensitivity and selectivity, but generally requires approximately 2 h per run; therefore, qRT-PCR is not appropriate for rapid virus detection or subtyping in outbreaks of fast-spreading and/or highly pathogenic viruses at public health centers, hospitals, airports, and other public transportation hubs. The commercial reaction mixture was from the qRT-PCR kit, RNA-Direct TM SYBR ® Green Real-time PCR Master Mix (Toyobo, Osaka, Japan; http://www.toyobo.co.jp/e/). The SHRT-PCR system detects the highly conserved sequence of the corresponding viral genome, and the newly designed primer sets targeted for typing MP segments do not exhibit any cross reactions among other influenza viruses (Table 5 ).

abstract:
  The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2 h per run. Detection assays were performed with super high-speed RT-PCR (SHRT-PCR) developed according to a newly designed heating system. The new method uses a high-speed reaction (18 s/cycle; 40 cycles in less than 20 min) for typing influenza viruses. The detection limit of SHRT-PCR was 1 copy/reaction and 10(−1) plaque-forming unit/reaction for viruses in culture supernatants during 20 min. Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. Twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between SHRT-PCR and immunochromatography exhibited 100% agreement for both positive and negative results. The rapid reaction time and high sensitivity of SHRT-PCR makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks.


id: cord-276989-441aclcc
author: Liu, Jianbo
date: 2020-03-12
txt: ./txt/cord-276989-441aclcc.txt
cache: ./cache/cord-276989-441aclcc.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093420301075
doi: 10.1016/j.jviromet.2020.113855
summary:
  title: Development of a rapid immunochromatographic strip test for the detection of porcine epidemic diarrhea virus specific SIgA in colostrum On the strip, a gold-labeled anti-SIgA SC mAb was applied to a conjugate pad; purified PEDV particles and goat anti-mouse antibodies were blotted onto a nitrocellulose membrane to form the test and control lines, respectively. Therefore, the aim of this study was to develop a simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled anti-SIgA SC mAb probe for the detection of anti-PEDV-specific SIgA in swine, and to compare its performance with an indirect SIgA ELISA based on the whole PEDV virus (Cong et al., 2019) . To test for colostrum, we diluted the samples 1: 20 in sample buffer and mixed thoroughly then, 100 μL of the solution was dispensed onto the sample pad well of the strip apparatus to determine the presence of PEDV specific SIgA, as described above.

title:
  Development of a rapid immunochromatographic strip test for the detection of porcine epidemic diarrhea virus specific SIgA in colostrum

abstract:
  Porcine epidemic diarrhea virus (PEDV) causes very high mortality in newborn piglets. The mucosal immune system in the gut must eliminate potential pathogens while maintaining a mutually beneficial relationship with the commensal microbiota. Antibodies derived from the secretory immunoglobulin A (SIgA) class, act as the first line of antigen-specific immunity in the gut by recognizing both pathogens and commensals. Therefore, the measurement of SIgA levels is an important index in evaluating PEDV infections and immune status. A simple and rapid method for the detection of PEDV-specific SIgA using an immunochromatographic test strip has been developed; incorporating a colloidal gold-labeled anti-SIgA secretory component (SC) mAb probe for the detection of anti-PEDV-specific SIgA in swine. On the strip, a gold-labeled anti-SIgA SC mAb was applied to a conjugate pad; purified PEDV particles and goat anti-mouse antibodies were blotted onto a nitrocellulose membrane to form the test and control lines, respectively. Results showed that the immunochromatographic test strip had high sensitivity and specificity. When compared with enzyme-linked immunosorbent assay, kappa value suggesting that the strip could be used to detect PEDV specific SIgA in colostrum samples. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. We found that the immunochromatographic test strip was a rapid, sensitive, and reliable method for the identification of PEDV specific SIgA, indicating its suitability for epidemiological surveillance as well as vaccine immunity when studying PEDV.


id: cord-277057-ww41t4k2
author: Sakthivel, Senthilkumar K.
date: 2012-07-11
txt: ./txt/cord-277057-ww41t4k2.txt
cache: ./cache/cord-277057-ww41t4k2.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/22796035/
doi: 10.1016/j.jviromet.2012.07.010
summary:
  title: Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assays for comprehensive detection of human respiratory viruses() Individual FTDRP assays for adenovirus, respiratory syncytial virus and rhinovirus showed the lowest comparative sensitivities with in-house assays, with most discrepancies occurring with specimens containing low virus loads and failed to detect some rhinovirus strains, even when abundant. This study reports the results of a comparison of the FTDRP multiplex assay with a panel of validated in-house singleplex real-time RT-PCR assays developed at the Centers for Disease Control and Prevention (CDC). These included 26 laboratory reference virus strains and field isolates and 265 geographically (U.S., Central and South America and Africa) and compositionally diverse specimens [nasopharyngeal and oropharyngeal swabs (223), nasal washes and aspirates (21), sputum (1), lung autopsy tissue (1) and unidentified (19)] collected from children and adults with acute respiratory illnesses (ARIs) acquired between 2008 and 2011 and previously testing positive for respiratory viruses by the in-house singleplex assays.

title:
  Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assays for comprehensive detection of human respiratory viruses()

abstract:
  Fast-track Diagnostics respiratory pathogens (FTDRP) multiplex real-time RT-PCR assay was compared with in-house singleplex real-time RT-PCR assays for detection of 16 common respiratory viruses. The FTDRP assay correctly identified 26 diverse respiratory virus strains, 35 of 41 (85%) external quality assessment samples spiked with cultured virus and 232 of 263 (88%) archived respiratory specimens that tested positive for respiratory viruses by in-house assays. Of 308 prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, 270 (87.7%) and 265 (86%) were positive by FTDRP and in-house assays for one or more viruses, respectively, with combined test results showing good concordance (K = 0.812, 95% CI = 0.786–0.838). Individual FTDRP assays for adenovirus, respiratory syncytial virus and rhinovirus showed the lowest comparative sensitivities with in-house assays, with most discrepancies occurring with specimens containing low virus loads and failed to detect some rhinovirus strains, even when abundant. The FTDRP enterovirus and human bocavirus assays appeared to be more sensitive than the in-house assays with some specimens. With the exceptions noted above, most FTDRP assays performed comparably with in-house assays for most viruses while offering enhanced throughput and easy integration by laboratories using conventional real-time PCR instrumentation.


id: cord-277186-sj8ngpk8
author: He, Qigai
date: 2005-04-19
txt: ./txt/cord-277186-sj8ngpk8.txt
cache: ./cache/cord-277186-sj8ngpk8.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093405000856
doi: 10.1016/j.jviromet.2005.03.004
summary:
  Specific binding of the MAb S-A5D5 to both purified N195 and SARS CoV nucleocapsid antigen was effectively inhibited by human SARS positive serum and guinea pig anti-N195 serum. The monoclonal antibodies were characterized by SARS CoV-infected Vero cells and nucleocapsid-spike fusion protein-based IFA, Western blot, and N195 proteinbased ELISA. The isotype of the promising monoclonal antibody, designated as S-A5D5, was determined and was further applied to develop a specific and sensitive antigen capture ELISA for the detection of SARS CoV. The specific reactivity of the MAb S-A5D5 with purified N195 protein (Fig. 3A ) was identical to that of the human SARS positive serum (Fig. 3B) , while no reaction was observed when non-antibody secreting hybridoma was tested (Fig. 3C) . Therefore, this antigen capture ELISA, based on MAb to N protein, might provide a more sensitive method for early detection of SARS CoV infection.

title:
  Characterization of monoclonal antibody against SARS coronavirus nucleocapsid antigen and development of an antigen capture ELISA

abstract:
  This report describes the production of several MAbs against N195 protein, a major immunodomain of SARS CoV nucleocapsid protein [He, Q., Chong, K.H., Chang, H.H., Leung, B., Ling, A.E., Wei, T., Chan, S.W., Ooi, E.E., Kwang, J., 2004. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin. Diagn. Lab. Immunol. 11 (2) 417–422.]. One representative IgG1 monoclonal antibody (MAb), S-A5D5, was selected and characterized. S-A5D5 reacted specifically react with both recombinant and native nucleocapsid protein of SARS CoV. The reactivity of S-A5D5 with purified N195 protein and utilization of the MAb as a detector antibody to develop an antigen capture ELISA was assessed. As little as 37.5 pg of purified N protein and 50 TCID(50) of SARS CoV could be detected by the antigen capture ELISA. Specific binding of the MAb S-A5D5 to both purified N195 and SARS CoV nucleocapsid antigen was effectively inhibited by human SARS positive serum and guinea pig anti-N195 serum. The N protein in N195-spike recombinant baculovirus-infected Sf-9 cells could also be identified. N protein was detected in 18 IFA IgM-positive serum samples collected from SARS confirmed patients, but not in nine samples collected from SARS recovery patient. No false positive results were given when 60 samples from healthy individuals were tested, and no cross-reaction occurred when infectious bronchitis virus (IBV), chicken coronavirus, was tested. This monoclonal antibody-based antigen capture ELISA is thus a powerful tool for early diagnosis of SARS CoV infection.


id: cord-277804-ujabzic4
author: Yuk, Seong-su
date: 2016-01-21
url: https://doi.org/10.1016/j.jviromet.2016.01.008
doi: 10.1016/j.jviromet.2016.01.008
title:
  Comparison between dot-immunoblotting assay and clinical sign determination method for quantifying avian infectious bronchitis virus vaccine by titration in embryonated eggs

abstract:
  A sensitive and specific method for measuring the vaccine titer of infectious bronchitis virus (IBV) is important to commercial manufacturers for improving vaccine quality. Typically, IBV is titrated in embryonated chicken eggs, and the infectivity of the virus dilutions is determined by assessing clinical signs in the embryos as evidence of viral propagation. In this study, we used a dot-immunoblotting assay (DIA) to measure the titers of IBV vaccines that originated from different pathogenic strains or attenuation methods in embryonated eggs, and we compared this assay to the currently used method, clinical sign evaluation. To compare the two methods, we used real-time reverse transcription-PCR, which had the lowest limit of detection for propagated IBV. As a clinical sign of infection, dwarfism of the embryo was quantified using the embryo: egg (EE) index. The DIA showed 9.41% higher sensitivity and 15.5% higher specificity than the clinical sign determination method. The DIA was particularly useful for measuring the titer of IBV vaccine that did not cause apparent stunting but propagated in embryonated chicken eggs such as a heat-adapted vaccine strain. The results of this study indicate that the DIA is a rapid, sensitive, reliable method for determining IBV vaccine titer in embryonated eggs at a relatively low cost.


id: cord-278176-o9glkhyv
author: Houng, Huo-Shu H
date: 2004-09-01
txt: ./txt/cord-278176-o9glkhyv.txt
cache: ./cache/cord-278176-o9glkhyv.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/15234807/
doi: 10.1016/j.jviromet.2004.04.008
summary:
  The SARS-CoV cDNA preparations derived from viral RNA extract and the cloned recombinant plasmid both exhibit the identical amplification characteristics, i.e. amplification efficacy using the same PCR formulation developed in this study. The 3′-NCR based SARS-CoV assay demonstrated 100% diagnostic specificity testing samples of patients with acute respiratory disease from a non-SARS epidemic region. It was demonstrated that the RT-PCR assay with 91% amplification efficiency could be used for consistent detect ion of the SARS-CoV viral RNA extracted from samples containing as little as 0.005 pfu per reaction with an anticipated C T value of 40 cycles (data not shown). It was demonstrated in this study that the cloned pHCV1 plasmid could be used to replace viral cDNA as a stable and rational SARS-CoV copy number standard for the SARS-CoV RT-PCR assay. Detection of SARS coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-PCR assays

title:
  Development and evaluation of an efficient 3′-noncoding region based SARS coronavirus (SARS-CoV) RT-PCR assay for detection of SARS-CoV infections

abstract:
  The severe acute respiratory syndrome (SARS) epidemic originating from China in 2002 was caused by a previously uncharacterized coronavirus that could be identified by specific RT-PCR amplification. Efforts to control future SARS outbreaks depend on the accurate and early identification of SARS-CoV infected patients. A real-time fluorogenic RT-PCR assay based on the 3′-noncoding region (3′-NCR) of SARS-CoV genome was developed as a quantitative SARS diagnostic tool. The ideal amplification efficiency of a sensitive SARS-CoV RT-PCR assay should yield an E value (PCR product concentration increase per amplification cycle) equal to 2.0. It was demonstrated that the 3′-NCR SARS-CoV based RT-PCR reactions could be formulated to reach excellent E values of 1.81, or 91% amplification efficacy. The SARS-CoV cDNA preparations derived from viral RNA extract and the cloned recombinant plasmid both exhibit the identical amplification characteristics, i.e. amplification efficacy using the same PCR formulation developed in this study. The viral genomic copy (or genomic equivalences, GE) per infectious unit (GE/pfu) of SARS-CoV used in this study was also established to be approximate 1200–1600:1. The assay’s detection sensitivity could reach 0.005 pfu or 6–8 GE per assay. It was preliminarily demonstrated that the assay could efficiently detect SARS-CoV from clinical specimens of SARS probable and suspected patients identified in Taiwan. The 3′-NCR based SARS-CoV assay demonstrated 100% diagnostic specificity testing samples of patients with acute respiratory disease from a non-SARS epidemic region.


id: cord-278377-jgq3dz3u
title: Prospective evaluation of diagnostic tools for respiratory viruses in children and adults
author: Busson, L.
date: 2019-01-15
txt: ./txt/cord-278377-jgq3dz3u.txt
cache: ./cache/cord-278377-jgq3dz3u.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093418303203
doi: 10.1016/j.jviromet.2019.01.006
summary:
  METHODS: Two hundred ninety-nine respiratory samples were prospectively explored using multiplex molecular techniques (FilmArray Respiratory Panel, Clart Pneumovir), immunological techniques (direct fluorescent assay, lateral flow chromatography) and cell cultures. Meanwhile, important questions concerning these ''new'' expensive rapid molecular techniques remain unanswered, such as their cost-effectiveness in terms of patient''s management, or the clinical significance of detecting nucleic acids of micro-organisms that could be non-infectious at the time the sample is collected. The objective of this work was to compare the performances of antigen detection and cell cultures techniques routinely used since years for the diagnosis of respiratory viral infections in the setting of a tertiary care hospital to those of newer molecular techniques (Clart Pneumovir, Genomica, Coslada, Spain and FilmArray Respiratory Panel, Biofire, Biomérieux, Marcy L''Etoile, France). False negative results with molecular techniques were significantly more frequent in samples with codetections compared to those with only one pathogen: 12% vs 3% for the FilmArray test (p = 0.034) and 76% vs 11% for the Clart Pneumovir test (p < 0.001).

abstract:
  AIM: To compare the performances of molecular and non-molecular tests to diagnose respiratory viral infections and to evaluate the pros and contras of each technique. METHODS: Two hundred ninety-nine respiratory samples were prospectively explored using multiplex molecular techniques (FilmArray Respiratory Panel, Clart Pneumovir), immunological techniques (direct fluorescent assay, lateral flow chromatography) and cell cultures. FINDINGS: Molecular techniques permitted the recovery of up to 50% more respiratory pathogens in comparison to non-molecular methods. FilmArray detected at least 30% more pathogens than Clart Pneumovir which could be explained by the differences in their technical designs. The turnaround time under 2 hours for the FilmArray permitted delivery of results when patients were still in the emergency room.


id: cord-279541-rjp2d1u9
author: Chutinimitkul, Salin
date: 2006-10-19
txt: ./txt/cord-279541-rjp2d1u9.txt
cache: ./cache/cord-279541-rjp2d1u9.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093406003302
doi: 10.1016/j.jviromet.2006.09.007
summary:
  Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. The nucleotide sequences (N = 246) of the neuraminidase gene of influenza A virus (H5N1) were taken from the Genbank database going back as far as 2003-2006 and hence, comprising entries isolated from various species, such as avian, cats, dog, tigers, swine and humans, including DQ250165, the sequence of one Vietnamese Oseltamivir-resistant patient (A/Vietnam/CL2009/2005(H5N1)). In conclusion, real-time PCR using two labeled TaqMan probes provides a highly specific and sensitive method to detect the amino acid alteration at position 274 of the influenza A subtype H5N1 neuraminidase gene causing oseltamivir resistance.

title:
  H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes

abstract:
  A single amino acid substitution, from histidine to tyrosine at position 274 of the neuraminidase gene has converted Oseltamivir sensitive H5N1 influenza A virus into a resistant strain. Currently, Oseltamivir is being stockpiled in many countries potentially affected by the influenza A virus subtype H5N1 epidemic. To identify this change in Oseltamivir-treated patients, a method based on real-time PCR using two labeled TaqMan probes was developed for its rapid detection. In order to validate the method, Oseltamivir specimen from treated (Oseltamivir-resistant strain from a Vietnamese patient, two Oseltamivir-treated tigers) and untreated subjects have been used for this study. The results thus obtained as well as those derived from clone selection and sequencing showed that TaqMan probes could clearly discriminate wild type H274 from the mutant 274Y variant. The sensitivity of this assay was as low as 10 copies/μl and allowed the detection of the mutation in a mixture of wild type and mutant. Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. Such studies can address potential differences in the diagnostic outcomes between patients who develop detectable Oseltamivir resistance and those who retain only the wild type strain of H5N1.


id: cord-279903-z0wf1wli
author: Wang, Leyi
date: 2014-07-12
url: https://doi.org/10.1016/j.jviromet.2014.07.005
doi: 10.1016/j.jviromet.2014.07.005
title:
  Development and evaluation of a duplex real-time RT-PCR for detection and differentiation of virulent and variant strains of porcine epidemic diarrhea viruses from the United States

abstract:
  Porcine epidemic diarrhea virus (PEDV) has caused significant economic losses in the US swine industry since May 2013. A new variant strain of PEDV emerged in the US in the late December, 2013. This variant strain of PEDV differs from the virulent strain of PEDV currently circulating in the US in 1170 nt of the 5’end of the S1 domain in the spike gene. Importantly, the variant PEDV caused significantly less mortality in piglets than the virulent PEDV, based on clinical observations. This suggests it may be a potential vaccine candidate for PED. Variant PEDV has been detected in samples from multiple states by our laboratory as well as other laboratories in the US. It is critical to detect and differentiate variant PEDV from the virulent PEDV during outbreaks to enhance control and to prevent PED associated disease. In this study, the development and validation of a duplex real-time RT-PCR assay for detection and differentiation of the variant and the virulent strains of PEDV currently circulating in the US was reported.


id: cord-281162-2pu7x5rj
author: Etemadi, Mohammad Reza
date: 2019-03-22
txt: ./txt/cord-281162-2pu7x5rj.txt
cache: ./cache/cord-281162-2pu7x5rj.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093418305937
doi: 10.1016/j.jviromet.2019.03.013
summary:
  The aim of this study was to use conventional and molecular detection methods to assess the epidemiology of respiratory viral infections in children less than five years of age that were hospitalized with ALRTIs. Methods: The cross-sectional study was designed to investigate the occurrence of respiratory viruses including respiratory syncytisl virus (RSV), human metapneumovirus (HMPV), influenza virus A and B (IFV-A and B), parainfluenzavirus 1, 2, 3 and 4 (PIV 1, 2, 3 and 4), human rhinoviruses (HRV), human enterovirus (HEV), human coronaviruses (HCoV) 229E and OC43, human bocavirus (HBoV) and human adenovirus (HAdV) in hospitalized children with ALRTIs, at Hospital Serdang, Malaysia, from June 16 to December 21, 2009.

title:
  Diversity of respiratory viruses detected among hospitalized children with acute lower respiratory tract infections at Hospital Serdang, Malaysia

abstract:
  BACKGROUND: The role of respiratory viruses as the major cause of acute lower respiratory tract infections (ALRTIs) in children is becoming increasingly evident due to the use of sensitive molecular detection methods. The aim of this study was to use conventional and molecular detection methods to assess the epidemiology of respiratory viral infections in children less than five years of age that were hospitalized with ALRTIs. METHODS: The cross-sectional study was designed to investigate the occurrence of respiratory viruses including respiratory syncytisl virus (RSV), human metapneumovirus (HMPV), influenza virus A and B (IFV-A and B), parainfluenzavirus 1, 2, 3 and 4 (PIV 1, 2, 3 and 4), human rhinoviruses (HRV), human enterovirus (HEV), human coronaviruses (HCoV) 229E and OC43, human bocavirus (HBoV) and human adenovirus (HAdV) in hospitalized children with ALRTIs, at Hospital Serdang, Malaysia, from June 16 to December 21, 2009. The study was also designed in part to assess the performance of the conventional methods against molecular methods. RESULTS: Viral pathogens were detected in 158 (95.8%) of the patients. Single virus infections were detected in 114 (67.9%) patients; 46 (27.9%) were co-infected with different viruses including double-virus infections in 37 (22.4%) and triple-virus infections in 9 (5.5%) cases. Approximately 70% of samples were found to be positive using conventional methods compared with 96% using molecular methods. A wide range of respiratory viruses were detected in the study. There was a high prevalence of RSV (50.3%) infections, particularly group B viruses. Other etiological agents including HAdV, HMPV, IFV-A, PIV 1–3, HBoV, HCoV-OC43 and HEV were detected in 14.5, 9.6, 9.1, 4.8, 3.6, 2.4 and 1.8 percent of the samples, respectively. CONCLUSION: Our results demonstrated the increased sensitivity of molecular detection methods compared with conventional methods for the diagnosis of ARTIs in hospitalized children. This is the first report of HMPV infections in Malaysia.


id: cord-281174-3c1vue0y
title: Evaluation of the NucliSens® Basic Kit assay for detection of Norwalk virus RNA in stool specimens
author: Greene, Shermalyn R
date: 2003-01-16
url: https://www.sciencedirect.com/science/article/pii/S0166093402002860
doi: 10.1016/s0166-0934(02)00286-0
abstract:
  Norwalk-like viruses (NLVs) are a genetically diverse group of human caliciviruses that are the most common cause of epidemic gastroenteritis and are detected typically in stool by reverse transcription (RT)-PCR or electron microscopy (EM). The application of a rapid nucleic acid sequence-based amplification (NASBA) assay for the detection of NLV RNA in stool is described using the NucliSens® Basic Kit. Primers and probes for the NLV Basic Kit assay were based on the RNA polymerase region of the prototype NLV, Norwalk virus (NV) genome and could consistently detect 10(4) RT-PCR detectable units of NV RNA in a stool filtrate. When compared directly with RT-PCR on a dilution series of NV stool filtrate, the NucliSens® Basic Kit assay was equally sensitive. Cross-reactivity studies with a representative panel of other enteric pathogens were negative. When applied to 15 stool specimens from NV-challenged volunteers, the NASBA Basic Kit application for NV detection yielded 100% sensitivity, 50% specificity, and 67% concordance, using RT-PCR as the ‘gold standard’. Despite the specificity of the NASBA primer/probe sequences for NV, other representatives from both NLV genogroups I and II could be detected by the Basic Kit assay in outbreak stool specimens, although the results were inconsistent. Our results suggest that the NucliSens® Basic Kit assay provides a rapid and sensitive alternative to RT-PCR for detecting NV RNA in stool specimens. However, improvements in test specificity and primer design will be needed before the assay can be used routinely in the clinical setting.


id: cord-281999-jc4ckqy7
title: Proteasome inhibition reduces avian reovirus replication and apoptosis induction in cultured cells
author: Chen, Yu T.
date: 2008-05-02
txt: ./txt/cord-281999-jc4ckqy7.txt
cache: ./cache/cord-281999-jc4ckqy7.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093408001031
doi: 10.1016/j.jviromet.2008.03.016
summary:
  The interplay between avian reovirus (ARV) replication and apoptosis and proteasome pathway was studied in cultured cells. In the present study, attempts were made to explore the role of proteasome inhibition in ARV infectivity and the mechanisms involved in the proteasome inhibitor suppression of ARV replication and apoptosis induction in cultured cells. Using the proteasome inhibitor MG132 to inhibit the cellular proteasome pathway, it was found that MG132 could reduce ARV-induced apoptosis, cytopathic effect (CPE), virus titer, and protein expression. The results indicated that the expression of A, C, and NS was reduced significantly in BHK-21cells either pre-incubated 30 min before infection or 2 h.p.i. with MG132 (Fig. 2D) , suggesting that the ubiquitin-proteasome system is not involved in virus internalization. In conclusion, it was shown that proteasome inhibitor reduces ARV replication through inhibition of viral RNA transcription and protein synthesis, thus preventing ARV-induced apoptosis.

abstract:
  The interplay between avian reovirus (ARV) replication and apoptosis and proteasome pathway was studied in cultured cells. It is shown that inhibition of the proteasome did not affect viral entry and host cell translation but had influence on ARV replication and ARV-induced apoptosis. Evidence is provided to demonstrate that ubiquitin-proteasome blocked ARV replication at an early step in viral life cycle. However, viral transcription and protein translation were also reduced markedly after addition of proteasome inhibitor MG132. Treatment of BHK-21 cells with the MG132 markedly decreased virus titer as well as prevented virus-induced apoptosis. The expression of ARV proteins σC, σA, and σNS was also reduced markedly, suggesting that suppression of virus replication is due to down-regulation of these ARV proteins by ubiquitin-proteasome system. MG132 was also shown to suppress ARV σC-induced phosphrylation of p53 on serine 46, caspase 3 activities, and DNA fragmentation leading to complete inhibition of ARV-induced apoptosis.


id: cord-284644-9k2oox64
author: Sharma, Vikrant
date: 2017-12-15
txt: ./txt/cord-284644-9k2oox64.txt
cache: ./cache/cord-284644-9k2oox64.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/29253497/
doi: 10.1016/j.jviromet.2017.12.005
summary:
  Optimized RT-LAMP assays were applied on clinical samples from patients having influenza like illness and results were compared with conventional one-step RT-PCR and real-time RT-PCR. CONCLUSIONS: RT-LAMP assay is rapid, sensitive, specific and cost effective method for detection of influenza A viruses than conventional one-step RT-PCR and it can serve as a good alternate for diagnosis and surveillance studies during influenza outbreaks in resource-limited setups of developing countries. The objectives of the current study were to (1) optimize RT-LAMP assay for detection of influenza A viruses and their subtypes (H1N1, H3N2 and pdm09/H1N1); (2) determine sensitivity and specificity of RT-LAMP assay; (3) clinical evaluation of RT-LAMP assay and conventional one-step RT-PCR in comparison to WHO recommended rRT-PCR taken as standard. Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of the swine-origin influenza A H1N1 virus

title:
  Evaluation of clinical applicability of reverse transcription-loop-mediated isothermal amplification assay for detection and subtyping of Influenza A viruses

abstract:
  BACKGROUND: Influenza A viruses (IAVs) have always remain a serious concern for the global economy and public health. A rapid, specific and sensitive detection method is always needed to control the influenza in its early stages by timely intervention of therapy and early clinical management. OBJECTIVES: To develop RT-LAMP assays for detection of influenza A viruses, their further subtyping into seasonal (H1N1, H3N2) and novel pandemic H1N1 viruses and to evaluate clinical applicability of optimized RT-LAMP assays on patients’ samples. STUDY DESIGN: In this study, we optimized RT-LAMP assay to detect IAVs by using primers against matrix gene and subtyping of IAVs was done by using primers against hemagglutinin gene. Optimized RT-LAMP assays were applied on clinical samples from patients having influenza like illness and results were compared with conventional one-step RT-PCR and real-time RT-PCR. RESULTS: RT-LAMP assays successfully detected and differentiated IAVs into H1N1, H3N2 and pdm09/H1N1 subtypes. One hundred and sixty seven clinical swab samples from influenza suspected patients were taken and tested with RT-LAMP assay, detecting 30 (17.9%) samples positive for Influenza A virus. Out of 30 samples, 21, 7 and 2 were found positive for pdm09/H1N1, H3N2 and seasonal H1 respectively. Conventional one-step RT-PCR detected a total of 27 (16.2%) samples for influenza A and further subtyping showed 20 and 7 samples positive for pdm09/H1N1 and H3N2 virus respectively whereas none was found positive for seasonal H1N1. RT-LAMP assay demonstrated higher sensitivity (93.8%) than conventional RT-PCR (84.4%) for influenza A viruses detection in clinical samples. CONCLUSIONS: RT-LAMP assay is rapid, sensitive, specific and cost effective method for detection of influenza A viruses than conventional one-step RT-PCR and it can serve as a good alternate for diagnosis and surveillance studies during influenza outbreaks in resource-limited setups of developing countries.


id: cord-286117-m1rlmlun
title: High-throughput viral microneutralization method for feline coronavirus using image cytometry
author: Pearson, Morgan
date: 2020-09-23
txt: ./txt/cord-286117-m1rlmlun.txt
cache: ./cache/cord-286117-m1rlmlun.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093420302317
doi: 10.1016/j.jviromet.2020.113979
summary:
  To address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. The Celigo Image Cytometer has demonstrated automated enumeration of viral plaques, foci, and individual virus-infected cells in a 96-well microplates using bright field or fluorescence imaging in less than 5 or 10 min, respectively, which can significantly reduce the time required for counting and analysis as well as eliminate operator-to-operator variation (Ramos et al., 2019; Rosen et al., 2019; Viedma and Pickett, 2018) . Development of the image cytometry-based high-throughput FCoV infection detection method involved the optimization of key variables: host cell seeding density, fluorescent labeling, assay buffer, microplate surface coating, virus concentration, and incubation time. After the optimization of each parameter for the high-throughput virus-infected cell counting method, the image cytometer performed the neutralization assay at approximately 15 min per 96-well plate for simultaneously acquiring and analyzing whole well images in bright field and fluorescence, equivalent to ~9 sec per sample.

abstract:
  Feline coronaviruses (FCoV) are members of the alphacoronavirus genus that are further characterized by serotype (types I and II) based on the antigenicity of the spike (S) protein and by pathotype based on the associated clinical conditions. Feline enteric coronaviruses (FECV) are associated with the vast majority of infections and are typically asymptomatic. Within individual animals, FECV can mutate and cause a severe and usually fatal disease called feline infectious peritonitis (FIP), the leading infectious cause of death in domestic cat populations. There are no approved antiviral drugs or recommended vaccines to treat or prevent FCoV infection. The plaque reduction neutralization test (PRNT) traditionally employed to assess immune responses and to screen therapeutic and vaccine candidates is time-consuming, low-throughput and typically requires 2 – 3 days for the formation and manual counting of cytolytic plaques. Host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, thereby reducing the sensitivity of the assay. In addition, operator-to-operator variation can generate uncertainty in the results and digital records are not automatically created. To address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. Host cell seeding density, microplate surface coating, virus concentration and incubation time, wash buffer and fluorescent labeling were optimized. Subsequently, this FCoV viral neutralization assay was used to explore immune correlates of protection using plasma from naturally FECV-infected cats. We demonstrate that the high-throughput viral neutralization assay using the Celigo Image Cytometer provides a robust and efficient method for the rapid screening of therapeutic antibodies, antiviral compounds and vaccines. This method can be applied to various viral infectious diseases to accelerate vaccine and antiviral drug discovery and development.


id: cord-286360-wrrqb387
title: Development of a nested PCR assay for the detection of canine coronavirus
author: Pratelli, A
date: 1999-06-02
txt: ./txt/cord-286360-wrrqb387.txt
cache: ./cache/cord-286360-wrrqb387.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/10403671/
doi: 10.1016/s0166-0934(99)00017-8
summary:
  A diagnostic test for canine coronavirus (CCV) infection based on a nested polymerase chain reaction (n-PCR) assay was developed and tested using the following coronavirus strains: CCV (USDA strain), CCV (45/93, field strain), feline infectious peritonitis virus (FIPV, field strain), trasmissible gastroenteritis virus (TGEV, Purdue strain), bovine coronavirus (BCV, 9WBL-77 strain), infectious bronchitis virus (IBV, M-41 strain) and fecal samples of dogs with CCV enteritis. The test described in the present study was able to amplify both CCV and TGEV strains and also gave positive results on fecal samples from CCV infected dogs. Recently a nested polymerase chain reaction (n-PCR) assay was developed for detection of feline infectious peritonitis virus (FIPV), a coronavirus closely related to CCV, in clinical specimens (Gamble et al., 1997) . PCR carried out with CCV1 and CCV2 primers specific for the target sequence 337 -746 of M gene revealed high sensitivity; tests performed on corresponding viral dilutions, which also were inoculated into cell cultures, gave positive results to the 10 − 4 dilution (approximately 10 -50 TCID 50 of virus).

abstract:
  A diagnostic test for canine coronavirus (CCV) infection based on a nested polymerase chain reaction (n-PCR) assay was developed and tested using the following coronavirus strains: CCV (USDA strain), CCV (45/93, field strain), feline infectious peritonitis virus (FIPV, field strain), trasmissible gastroenteritis virus (TGEV, Purdue strain), bovine coronavirus (BCV, 9WBL-77 strain), infectious bronchitis virus (IBV, M-41 strain) and fecal samples of dogs with CCV enteritis. A 230-bp segment of the gene encoding for transmembrane protein M of CCV is the target sequence of the primer. The test described in the present study was able to amplify both CCV and TGEV strains and also gave positive results on fecal samples from CCV infected dogs. n-PCR has a sensitivity as high as isolation on cell cultures, and can therefore be used for the diagnosis of CCV infection in dogs.


id: cord-286451-ujo72w06
author: Bennett, Susan
date: 2012-09-18
txt: ./txt/cord-286451-ujo72w06.txt
cache: ./cache/cord-286451-ujo72w06.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093412002996
doi: 10.1016/j.jviromet.2012.08.020
summary:
  title: The development of a multiplex real-time PCR for the detection of herpes simplex virus 1 and 2, varizella zoster virus, adenovirus and Chlamydia trachomatis from eye swabs A multiplex real-time PCR assay for rapid and simultaneous detection of HSV 1 and 2, VZV, adenovirus and Chlamydia trachomatis (C. Screening clinical samples by sample type/syndrome based multiplex real time PCR would allow for rapid detection of a variety of pathogens simultaneously, which will in turn aid in the treatment and clinical management of the patient. Screening clinical samples by sample type/syndrome based multiplex real time PCR would allow for rapid detection of a variety of pathogens simultaneously, which will in turn aid in the treatment and clinical management of the patient. A multiplex real-time PCR assay for the rapid and simultaneous detection of HSV 1 and 2, VZV, adenovirus and C. This paper describes the development and validation of a multiplex real-time PCR assay, which will allow rapid and simultaneous detection of HSV1/2, VZV, adenovirus and C.

title:
  The development of a multiplex real-time PCR for the detection of herpes simplex virus 1 and 2, varizella zoster virus, adenovirus and Chlamydia trachomatis from eye swabs

abstract:
  Infectious conjunctivitis can be difficult to distinguish clinically due to the considerable overlap in clinical presentation so clinical diagnosis of conjunctivitis is often insufficient. It is therefore necessary to have a rapid diagnostic test that differentiates between the different causes of infectious conjunctivitis. Screening clinical samples by sample type/syndrome based multiplex real time PCR would allow for rapid detection of a variety of pathogens simultaneously, which will in turn aid in the treatment and clinical management of the patient. A multiplex real-time PCR assay for rapid and simultaneous detection of HSV 1 and 2, VZV, adenovirus and Chlamydia trachomatis (C. trachomatis) from eye swabs was developed and evaluated. The multiplex assay was shown to be sensitive, specific and robust. Reductions in sample turn around times have been achieved by reducing the amount of separate tests needed to be carried out.


id: cord-288701-nx9fg4yn
author: Mari, Viviana
date: 2015-12-17
txt: ./txt/cord-288701-nx9fg4yn.txt
cache: ./cache/cord-288701-nx9fg4yn.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093415003870
doi: 10.1016/j.jviromet.2015.12.003
summary:
  The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. To overcome these limitations, we have developed a multiplex real-time RT-PCR assay for simultaneous detection of the different species of bovine pestiviruses, including the emerging HoBi-like group, allowing a rapid, sensitive and specific diagnosis of pestivirus infection and characterisation of the viral species.

title:
  Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus

abstract:
  HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)–10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples.


id: cord-289676-tjy7f9rk
author: Park, Sang-Ik
date: 2009-03-14
url: https://api.elsevier.com/content/article/pii/S0166093409001050
doi: 10.1016/j.jviromet.2009.03.001
title:
  Development of SYBR Green real-time RT-PCR for rapid detection, quantitation and diagnosis of unclassified bovine enteric calicivirus

abstract:
  Unclassified bovine enteric calicivirus (BECV) is a newly recognized bovine enteric calicivirus that differs from bovine norovirus, and which causes diarrhea in the small intestines of calves. To date, methods such as real-time reverse transcription-polymerase chain reaction (RT-PCR) have not been developed for the rapid detection, quantitation and diagnosis of BECV. Presently, a BECV-specific SYBR Green real-time RT-PCR assay was evaluated and optimized. Diarrheic specimens (n = 118) collected from 2004 to 2005 were subjected to RT-PCR, nested PCR and SYBR Green real-time RT-PCR. By conventional RT-PCR and nested PCR, 9 (7.6%) and 59 (50%) samples tested positive, respectively, whereas the SYBR Green assay detected BECV in 91 (77.1%) samples. Using BECV RNA standards generated by in vitro transcription, the SYBR Green real-time RT-PCR assay sensitively detected BECV RNA to 1.1 × 10(0) copies/μl (correlation coefficiency = 0.98). The detection limits of the RT-PCR and nested PCR were 1.1 × 10(5) and 1.1 × 10(2) copies/μl, respectively. These results indicate that the SYBR Green real-time RT-PCR assay is more sensitive than conventional RT-PCR and nested PCR assays, and has potential as a reliable, reproducible, specific, sensitive and rapid tool for the detection, quantitation and diagnosis of unclassified BECV.


id: cord-290831-45cu8alm
author: Zheng, Yuan Zhi
date: 1999-06-02
txt: ./txt/cord-290831-45cu8alm.txt
cache: ./cache/cord-290831-45cu8alm.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093498001700
doi: 10.1016/s0166-0934(98)00170-0
summary:
  Immunosorbent electron microscopy was used to quantify recombinant baculovirus-generated bluetongue virus (BTV) core-like particles (CLP) in either purified preparations or lysates of recombinant baculovirus-infected cells. French and Roy (1990) constructed a dual-recombinant baculovirus containing genes that encode VP3 and VP7 and their expression in insect cell culture resulted in the synthesis of large quantities of VP3 and VP7 and the concomitant formation of core-like particles (CLP) that lack RNA. Ovine polyclonal antiserum was raised to core particles of BTV-1 isolated from virus-infected cells and purified by equilibrium density centrifugation in cesium chloride (personal communication, B.T. Eaton). The number of crude CLP per negative film was determined by the ISEM method and the concentration of particles calculated by multiplying the number of CLP by the capture efficiency. Serial dilutions of purified CLP were made in uninfected Sf9 cell lysates and 5 ml aliquots of each dilution added to grids coated with a 1:100 dilution of antibody 20E9.

title:
  Quantification of recombinant core-like particles of bluetongue virus using immunosorbent electron microscopy

abstract:
  Immunosorbent electron microscopy was used to quantify recombinant baculovirus-generated bluetongue virus (BTV) core-like particles (CLP) in either purified preparations or lysates of recombinant baculovirus-infected cells. The capture antibody was an anti-BTV VP7 monoclonal antibody. The CLP concentration in purified preparations was determined to be 6.6×10(15) particles/l. CLP concentration in lysates of recombinant baculovirus-infected cells was determined at various times post-infection and shown to reach a value of 3×10(15) particles/l of culture medium at 96 h post-infection. The results indicated that immunosorbent electron microscopy, aided by an improved particle counting method, is a simple, rapid and accurate technique for the quantification of virus and virus-like particles produced in large scale in vitro systems.


id: cord-292643-n6xp5mlz
author: Hall, Richard J.
date: 2013-09-13
url: https://doi.org/10.1016/j.jviromet.2013.08.035
doi: 10.1016/j.jviromet.2013.08.035
title:
  Evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery

abstract:
  The discovery of new or divergent viruses using metagenomics and high-throughput sequencing has become more commonplace. The preparation of a sample is known to have an effect on the representation of virus sequences within the metagenomic dataset yet comparatively little attention has been given to this. Physical enrichment techniques are often applied to samples to increase the number of viral sequences and therefore enhance the probability of detection. With the exception of virus ecology studies, there is a paucity of information available to researchers on the type of sample preparation required for a viral metagenomic study that seeks to identify an aetiological virus in an animal or human diagnostic sample. A review of published virus discovery studies revealed the most commonly used enrichment methods, that were usually quick and simple to implement, namely low-speed centrifugation, filtration, nuclease-treatment (or combinations of these) which have been routinely used but often without justification. These were applied to a simple and well-characterised artificial sample composed of bacterial and human cells, as well as DNA (adenovirus) and RNA viruses (influenza A and human enterovirus), being either non-enveloped capsid or enveloped viruses. The effect of the enrichment method was assessed by both quantitative real-time PCR and metagenomic analysis that incorporated an amplification step. Reductions in the absolute quantities of bacteria and human cells were observed for each method as determined by qPCR, but the relative abundance of viral sequences in the metagenomic dataset remained largely unchanged. A 3-step method of centrifugation, filtration and nuclease-treatment showed the greatest increase in the proportion of viral sequences. This study provides a starting point for the selection of a purification method in future virus discovery studies, and highlights the need for more data to validate the effect of enrichment methods on different sample types, amplification, bioinformatics approaches and sequencing platforms. This study also highlights the potential risks that may attend selection of a virus enrichment method without any consideration for the sample type being investigated.


id: cord-292831-oihcay6w
author: Choudhary, Manohar L.
date: 2013-01-08
txt: ./txt/cord-292831-oihcay6w.txt
cache: ./cache/cord-292831-oihcay6w.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093413000049
doi: 10.1016/j.jviromet.2012.12.017
summary:
  The purpose of this study was to develop a one step mRT-PCR that could detect respiratory viruses including influenza A viruses, H1N1pdm09, seasonal H1N1, H3N2, influenza B viruses, respiratory syncytial virus (RSV) A and B, human metapneumovirus (HMPV), parainfluenza viruses (PIV) 1, 2, 3, 4, rhinovirus, enterovirus, corona viruses OC43, 229E, NL63 and HKU1 in three sets in human clinical samples and to compare it with rRT-PCR. The specificity of the multiplex PCR assay was evaluated by cross reaction tests with known viral isolates and different panels of sequence confirmed known clinical respiratory samples as reference material. Specificity of the mRT-PCR assay was evaluated by cross reaction tests against known respiratory virus isolates/positive samples and a WHO QA/QC panel for influenza viruses showed no cross reactivity amongst different viruses. For validation of set 1 of the multiplex assay, 114 respiratory clinical specimens previously positive for influenza viruses by rRT-PCR were used and results were 100% concordant.

title:
  Development of a multiplex one step RT-PCR that detects eighteen respiratory viruses in clinical specimens and comparison with real time RT-PCR

abstract:
  Rapid and accurate diagnosis of viral respiratory infections is crucial for patient management. Multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) is used increasingly to diagnose respiratory infections and has shown to be more sensitive than viral culture and antigen detection. Objective of the present study was to develop a one-step mRT-PCR that could detect 18 respiratory viruses in three sets. The method was compared with real time RT-PCR (rRT-PCR) for its sensitivity and specificity. Clinical specimens from 843 pediatric patients with respiratory symptoms were used in the study. 503 (59.7%) samples were detected positive by mRT-PCR. Of these 462 (54.8%) exhibited presence of a single pathogen and 41 (4.9%) had multiple pathogens. rRT-PCR detected 439 (52.1%) positive samples, where 419 (49.7%) exhibited one virus and 20 (2.4%) showed co-infections. Concordance between mRT-PCR and rRT-PCR was 91.9% and kappa correlation 0.837. Sensitivity and specificity of mRT-PCR were 99.5% and 83.7% while that of rRT-PCR was 86.9% and 99.4% respectively. Rhinovirus (17.2%) was the most frequently detected virus followed by respiratory syncytial virus B (15.4%), H1N1pdm09 (8.54%), parainfluenza virus-3 (5.8%) and metapneumovirus (5.2%). In conclusion, mRT-PCR is a rapid, cost effective, specific and highly sensitive method for detection of respiratory viruses.


id: cord-293651-96cmduez
author: Callison, Scott A.
date: 2006-08-28
url: https://api.elsevier.com/content/article/pii/S0166093406002734
doi: 10.1016/j.jviromet.2006.07.018
title:
  Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens

abstract:
  It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. In this study, we report the development and testing of a real-time RT-PCR assay using a Taqman(®)-labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5′-UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as two Turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Evaluation of the assay was completed with 1329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. The real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for control of this highly infectious virus. The test was extremely sensitive and specific, and can be used to quantitate viral genomic RNA in clinical samples.


id: cord-294454-uzfsv2df
title: Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses
author: Bellau-Pujol, S.
date: 2005-02-24
txt: ./txt/cord-294454-uzfsv2df.txt
cache: ./cache/cord-294454-uzfsv2df.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093405000352
doi: 10.1016/j.jviromet.2005.01.020
summary:
  Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1–4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses. The aim of this study was to develop rapid, sensitive and specific molecular methods for the detection of a large panel of respiratory RNA viruses that are more powerful than Co-infections hRV + PIV-3 1 hRV + PIV-1 1 hRV + PIV-1 + hMPV 1 hRV + hRSV + hMPV 1 hRV + influenza A virus 1 hRV + hRSV 2 Total no. Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-PCR assay

abstract:
  Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1–4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.


id: cord-295316-ccdj7137
title: Direct diagnosis of human respiratory coronaviruses 229E and OC43 by the polymerase chain reaction
author: Vabret, Astrid
date: 2001-07-30
url: https://api.elsevier.com/content/article/pii/S0166093401003433
doi: 10.1016/s0166-0934(01)00343-3
abstract:
  An RT-PCR-hybridization was developed that amplified genetic material from the M protein gene of HCoV-229E and HCoV-OC43. The analytic sensitivity of these original primers were compared with primers defined in the N gene and described previously. The results show that 0.05 TCID(50) of HCoV-229E and 0.01 TCID(50) of HCoV-OC43 can be detected by this molecular method using the original method. Detection of HCoV-229E and HCoV-OC43 in clinical specimens is possible using this method: 348 respiratory specimens (202 sputum and 146 nasal aspirates) were tested with this RT-PCR-hybridization and 12 human coronavirus are detected (3%). The method could provide a useful tool for demonstrating the role of human coronavirus in infections of the respiratory tract.


id: cord-295401-3p6q92x4
author: Gueudin, M
date: 2003-02-18
url: https://www.ncbi.nlm.nih.gov/pubmed/12668266/
doi: 10.1016/s0166-0934(03)00042-9
title:
  Quantitation of respiratory syncytial virus RNA in nasal aspirates of children by real-time RT-PCR assay

abstract:
  A method was developed for the quantitation of respiratory syncytial virus (RSV) based on real-time RT-PCR using a LightCycler instrument. A control real-time RT-PCR was undertaken on GAPDH mRNA (a human housekeeping gene) was carried out to standardise the non-homogeneous respiratory samples. The real-time RT-PCR method was one log more sensitive for the detection of RSV according to the endpoint dilution technique than the culture method or a conventional qualitative RT-PCR-hybridization-EIA. No cross-reactivity was observed with any of the viruses that could be found in the respiratory tract. RSV and GAPDH were quantified in nasal aspirates from 75 children hospitalised for acute respiratory tract disease: 31 (41.3%) were positive according to the immunofluorescence assay (IFA), 34 (45.3%) were culture-positive and 42 (56%) were positive according to our real-time RT-PCR method. The sensitivity, specificity, positive and negative predictive values of the real-time RT-PCR were 100, 90, 92, 100%, respectively. The samples found to be positive for RSV were classified according to the severity of the disease. The mean number of RSV RNA copies was higher in the severe disease group than in the non-severe group 4.05×10(7) vs 9.1×10(6) (P=0.055). However, the mean ratio of RSV RNA copies to GAPDH mRNA copies was 42.8 in the severe group, and 22.2 in non-severe group (P=NS).


id: cord-295491-zlah6u5s
author: Günther, Sonja
date: 2018-03-11
txt: ./txt/cord-295491-zlah6u5s.txt
cache: ./cache/cord-295491-zlah6u5s.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/29540320/
doi: 10.1016/j.jviromet.2018.03.003
summary:
  The aim of this study was to test two commercially available reaction mixtures in a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect feline Coronavirus (FCoV) in body cavity effusions of cats with and without FIP, in order to minimize the time from sampling to obtaining results. The aim of this study was to test specificity and sensitivity of two commercially available reaction mixtures in a reverse transcription LAMP (RT-LAMP) to detect FCoV in body cavity effusions of cats with and without FIP, and to minimize the time from sampling to obtaining results. The FIP group (n = 34) included cats with a definitive diagnosis of FIP by one or more methods: All effusions of cats with FIP tested positive for FCoV by RT-PCR by a commercial laboratory, and in 26/34 samples putative disease-causing mutations could be detected.

title:
  Detection of feline Coronavirus in effusions of cats with and without feline infectious peritonitis using loop-mediated isothermal amplification

abstract:
  Feline infectious peritonitis (FIP) is a fatal disease in cats worldwide. The aim of this study was to test two commercially available reaction mixtures in a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect feline Coronavirus (FCoV) in body cavity effusions of cats with and without FIP, in order to minimize the time from sampling to obtaining results. RNA was extracted from body cavity effusion samples of 71 cats, including 34 samples from cats with a definitive diagnosis of FIP, and 37 samples of control cats with similar clinical signs but other confirmed diseases. Two reaction mixtures (Isothermal Mastermix, OptiGene Ltd.and PCRun™ Molecular Detection Mix, Biogal) were tested using the same primers, which were designed to bind to a conserved region of the FCoV membrane protein gene. Both assays were conducted under isothermal conditions (61 °C–62 °C). Using the Isothermal Mastermix of OptiGene Ltd., amplification times ranged from 4 and 39 min with a sensitivity of 35.3% and a specificity of 94.6% for the reported sample group. Using the PCRun™ Molecular Detection Mix of Biogal, amplification times ranged from 18 to 77 min with a sensitivity of 58.8% and a specificity of 97.3%. Although the RT-LAMP assay is less sensitive than real time reverse transcription PCR (RT-PCR), it can be performed without the need of expensive equipment and with less hands-on time. Further modifications of primers might lead to a suitable in-house test and accelerate the diagnosis of FIP.


id: cord-297160-tqw9vx2b
author: Geerligs, H.J.
date: 2013-07-01
txt: ./txt/cord-297160-tqw9vx2b.txt
cache: ./cache/cord-297160-tqw9vx2b.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2013.06.029
doi: 10.1016/j.jviromet.2013.06.029
summary:
  After the incubation period of seven days standard practice is for the embryos to be taken from each egg and examined visually for IB specific lesions; these readings are used to determine an end-point in viral titrations. In order to determine end-points in the titration for each of the two strains, we collected the allantoic fluids from each egg after the incubation period and tested these for the presence of IB H120 and IB D274 by a strain specific reverse phase PCR. We used PCR for detection of two different IB virus strains in the allantoic fluids from all the individual eggs in a titration of a live IB combination vaccine. Based on the results of the PCR we determined whether an allantoic fluid was positive for one or both of the viruses or not, and as such we were able to define a separate endpoint in the live virus titration for each of the two strains.

title:
  The use of RT-PCR for determination of separate end-points for the strains IB H120 and IB D274 in titration of the combination vaccine Poulvac IB(®) primer

abstract:
  Poulvac IB(®) Primer is a lyophilized vaccine containing two attenuated infectious bronchitis strains in one vial, IB H120 and IB D274. For quantification of the viral content of the vaccine, dilution series of the final product are inoculated in embryonated chicken eggs. After the incubation period of seven days standard practice is for the embryos to be taken from each egg and examined visually for IB specific lesions; these readings are used to determine an end-point in viral titrations. The result is a titre value to which both strains contribute. However, it is not clear what the live virus titre is for strain IB H120 and for strain IB D274. In order to determine end-points in the titration for each of the two strains, we collected the allantoic fluids from each egg after the incubation period and tested these for the presence of IB H120 and IB D274 by a strain specific reverse phase PCR. Based on the data obtained by PCR we were able to determine an end-point for each of the two strains. For a given commercial batch of Poulvac IB primer we determined titres of 10(6.31) EID(50) per vial for IB H120 and 10(6.59) EID(50) for IB D274 using PCR for end-point determination. These end-points matched well with the end-point determined for both strains cumulatively after visual examination, i.e. 10(6.67) EID(50) per vial. It is concluded that PCR is a suitable means to determine end-points in titrations of live viruses.


id: cord-298922-k568hlf4
author: Sun, Dongbo
date: 2015-06-15
url: https://www.sciencedirect.com/science/article/pii/S0166093415000695
doi: 10.1016/j.jviromet.2015.03.002
title:
  Analysis of protein expression changes of the Vero E6 cells infected with classic PEDV strain CV777 by using quantitative proteomic technique

abstract:
  Recent outbreaks of porcine epidemic diarrhea virus (PEDV) have caused widespread concern. The identification of proteins associated with PEDV infection might provide insight into PEDV pathogenesis and facilitate the development of novel antiviral strategies. We analyzed the differential protein profile of PEDV-infected Vero E6 cells using mass spectrometry and an isobaric tag for relative and absolute quantification. A total of 126 proteins were identified that were differentially expressed between the PEDV-infected and mock-infected groups (P < 0.05, quantitative ratio ≥1.2), among which the expression of 58 proteins was up-regulated and that of 68 proteins was down-regulated in the PEDV-infected Vero E6 cells, involving in integrin β2/β3, cystatin-C. The Gene Ontology analysis indicated that the molecular function of the differentially expressed proteins (DEPs) was primarily related to binding and catalytic activity, and that the biological functions in which the DEPs are involved included metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases. Among the disease-related functions, certain anti-viral pathways and proteins, such as the RIG-I-like receptor, Rap1, autophagy, mitogen-activated protein kinase, PI3K-Akt and Jak-STAT signaling pathways, and integrin β2/β3 and cystatin-C proteins, represented potential factors in PEDV infection. Our findings provide valuable insight into PEDV-Vero E6 cell interactions.


id: cord-299585-fkg8d6ym
author: Wang, Leyi
date: 2019-04-05
url: https://doi.org/10.1016/j.jviromet.2019.04.008
doi: 10.1016/j.jviromet.2019.04.008
title:
  Development of a triplex real-time RT-PCR assay for detection and differentiation of three US genotypes of porcine hemagglutinating encephalomyelitis virus

abstract:
  Porcine hemagglutinating encephalomyelitis virus (PHEV) is a single-stranded, positive-sense RNA virus. PHEV mainly causes two types of clinical manifestations representing vomiting and wasting and encephalomyelitis in piglets. However, our recent findings provide strong evidence that PHEV can also cause respiratory disease in older pigs. Genomic analysis of new PHEV strains identified in our former study further classifies PHEV into three genotypes. Detection and differentiation of these new mutants are critical in monitoring PHEV evolution in the field. In the present study, we report the development of a triplex real-time RT-PCR assay for detection and differentiation of three PHEV genotypes, 1, 2, and 3. Three sets of primers and probes were designed; one set of primers and probe targeting the conserved regions of the 3′ end nucleocapsid for detection of all three genotypes and another two sets of primers and probes targeting the regions of NS2 with different patterns of deletions for detection of both genotypes 1 and 3, or genotype 3 only. Genotype 1 was positive when two probe dyes showed signals, genotype 2 was positive when only one probe dye showed a signal, and genotype 3 was positive when all three probes showed signals. The detection limit of the developed triplex real-time RT-PCR was as low as 8 or 9 DNA copies for three sets of primers and probes. The specificity test showed no cross reaction with other porcine viruses. Positive field-samples were correctly typed by this new assay, which was further confirmed by DNA sequencing. The triplex real-time RT-PCR provides a rapid and sensitive method to detect and differentiate all three US genotypes of PHEV from clinical samples.


id: cord-301355-9lswjro2
author: Fan, Jing-Hui
date: 2015-12-01
url: https://api.elsevier.com/content/article/pii/S0166093415002645
doi: 10.1016/j.jviromet.2015.07.021
title:
  Development of an enzyme-linked immunosorbent assay for the monitoring and surveillance of antibodies to porcine epidemic diarrhea virus based on a recombinant membrane protein

abstract:
  The recent dramatic increase in reported cases of porcine epidemic diarrhea (PED) in pig farms is a potential threat to the global swine industry. Therefore, the accurate diagnosis, serological monitoring, and surveillance of specific antibodies in pigs resulting from porcine epidemic diarrhea virus (PEDV) infection or vaccination would be essential in helping to control the spread of PED. We developed and validated an indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant membrane (M) protein of PEDV. To detect PEDV antibodies in eight herds, 382 serum samples were collected from sows that had been immunized with a PED vaccine, and screened using the developed ELISA in parallel with a serum neutralization (SN) assay. Of the tested samples, 276 were positive for the presence of PEDV antibodies according to both assays, while 98 were negative. An excellent agreement between the ELISA and the SN assay was observed (kappa = 0.947; 95% confidence interval = 0.910–0.984; McNemar's test, P = 0.727). No cross-reaction was detected for the developed ELISA with other coronaviruses or other common pig pathogens. The developed ELISA could be used for serological evaluation and indirect diagnosis of PED infection.


id: cord-301430-gzou8b9k
title: Establishment of a new bovine leukosis virus producing cell line
author: Beier, D.
date: 2004-08-17
url: https://www.sciencedirect.com/science/article/pii/S016609340400196X
doi: 10.1016/j.jviromet.2004.06.017
abstract:
  Due to the prevalence of different bovine leukosis virus (BLV) species in the cattle population in Europe, problems may arise in the serological diagnosis of BLV infections. In addition, earlier investigations demonstrated that contamination of the BLV antigen-producing cell culture systems by bovine viral diarrhea virus (BVDV) may give rise to misinterpretation of serological test results after BVDV vaccination of cattle. By co-cultivation of peripheral leukocytes of a BLV-infected cow with a permanent sheep kidney cell line, a new BLV-producing cell line named PO714 was established. This line carries a BLV provirus of the Belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. Investigations of a panel of well-characterised sera by agar gel immunodiffusion (AGID) and capture ELISA (cELISA) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line FLK/BLV yielded comparable results. False positive results caused by BVDV cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line.


id: cord-301974-4wn40ivq
title: Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus
author: Berry, Jody D
date: 2004-09-01
url: https://www.ncbi.nlm.nih.gov/pubmed/15234813/
doi: 10.1016/j.jviromet.2004.04.009
abstract:
  There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.


id: cord-302024-zz7mt6be
author: Hakhverdyan, Mikhayil
date: 2004-11-17
url: https://www.ncbi.nlm.nih.gov/pubmed/15620402/
doi: 10.1016/j.jviromet.2004.09.016
title:
  Evaluation of a single-tube fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in clinical samples

abstract:
  Bovine respiratory syncytial virus (BRSV) causes severe disease in naïve cattle of all ages and is a common pathogen in the respiratory disease complex of calves. Simplified methods for rapid BRSV diagnosis would encourage sampling during outbreaks and would consequently lead to an extended understanding of the virus. In this study, a BRSV fluorogenic reverse transcription PCR (fRT-PCR) assay, based on TaqMan principle, was developed and evaluated on a large number of clinical samples, representing various cases of natural and experimental BRSV infections. By using a single-step closed-tube format, the turn-around time was shortened drastically and results were obtained with minimal risk for cross-contamination. According to comparative analyses, the detection limit of the fRT-PCR was on the same level as that of a nested PCR and the sensitivity relatively higher than that of a conventional PCR, antigen ELISA (Ag-ELISA) and virus isolation (VI). Interspersed negative control samples, samples from healthy animals and eight symptomatically or genetically related viruses were all negative, confirming a high specificity of the assay. Taken together, the data indicated that the fRT-PCR assay can be applied to routine virus detection in clinical specimens and provides a rapid and valuable tool in BRSV research.


id: cord-302663-gb2vgs97
title: Detection of yellow head virus in shrimp by loop-mediated isothermal amplification (LAMP)
author: Mekata, Tohru
date: 2006-04-04
txt: ./txt/cord-302663-gb2vgs97.txt
cache: ./cache/cord-302663-gb2vgs97.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093406000656
doi: 10.1016/j.jviromet.2006.02.012
summary:
  Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the structural glycoprotein gene of yellow head virus (YHV). The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. The development of a loop-mediated isothermal amplification (LAMP) assay for detection of white spot disease virus (WSDV) DNA was described by Kono et al. Ten-fold serial dilutions (10 −1 to 10 −8 diluted) of RNA extracted from YHV-infected shrimp was used as a template for RT-LAMP according to determined conditions. In order to determine the sensitivity of detection limit, RT-LAMP and nested RT-PCR were carried out using various concentrations (10 −1 to 10 −8 dilution) of RNA extracted from YHV-infected shrimp as template.

abstract:
  Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the structural glycoprotein gene of yellow head virus (YHV). The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. The whole procedure is very simple and rapid, and reaction time and temperatures were optimized for 60 min at 65 °C, respectively. Detection of gene amplification could be accomplished by agarose gel electrophoresis. The standardized RT-LAMP procedure was used to detect YHV in the heart and gill from infected shrimp. Thus, the RT-LAMP assay is extremely rapid, cost-effective, sensitive and specific and has potential usefulness for rapid diagnosis for YHV detection in shrimp.


id: cord-302829-1o1jo8uk
title: Rapid detection of porcine circovirus type 2 by loop-mediated isothermal amplification
author: Chen, Hao-tai
date: 2008-03-19
txt: ./txt/cord-302829-1o1jo8uk.txt
cache: ./cache/cord-302829-1o1jo8uk.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/18355932/
doi: 10.1016/j.jviromet.2008.01.023
summary:
  A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). DNA was extracted from blood, lymph nodes, lung, liver, kidney, heart and spleen samples taken from PCV2-infected and healthy pigs, using a DNeasy Tissue Kit (Qiagen) according to the manufacturer''s instructions. In order to evaluate the optimal tissues for viral detection and to compare the sensitivity of PCV2 detection by LAMP and PCR, DNAs from tissue samples of blood, heart, lung, liver, kidney, lymph nodes and spleen from PCV2-infected pigs were extracted and subjected to LAMP and PCR. Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction

abstract:
  A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). The amplification could be finished in 60 min under isothermal condition at 64 °C by employing a set of four primers targeting the cap gene of PCV2. The LAMP assay showed higher sensitivity than the conventional PCR, with a detection limit of five copies per tube of purified PCV2 genomic DNA. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1 (PCV1), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV). The detection rate of PCV2 LAMP for 86 clinical samples was 96.5% and appeared greater than that of the PCR method. The LAMP assay reported can provide a rapid yet simple test of PCV2 suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.


id: cord-305399-98sqovwb
author: Li, Hao
date: 2019-04-22
url: https://doi.org/10.1016/j.jviromet.2019.04.019
doi: 10.1016/j.jviromet.2019.04.019
title:
  Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus

abstract:
  A simple and accurate reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated for the detection of porcine pegivirus (PPgV). The specific RT-LAMP primers targeting the conserved regions of NS5A genes were designed and used to detect PPgV. The optimal reaction parameter for RT-LAMP assay was 63℃ for 60 min. The detection limit of the RT-LAMP assay was 10 copies of PPgV genome, which was 100 times more sensitive than that of the conventional RT-PCR and comparable to nested RT-PCR and quantitative RT-PCR (qRT-PCR). There was no cross amplification with other related RNA viruses. In the clinical evaluation, the RT-LAMP assay exhibited a similar sensitivity with nested RT-PCR and qRT-PCR. The results indicated that RT-LAMP assay developed in this study could be a highly specific, sensitive, and cost-effective alternative for a rapid detection of PPgV in field settings.


id: cord-305640-tgowzrqo
author: Li, Yong-Hua
date: 2005-07-15
txt: ./txt/cord-305640-tgowzrqo.txt
cache: ./cache/cord-305640-tgowzrqo.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/16024098/
doi: 10.1016/j.jviromet.2005.06.001
summary:
  A capture enzyme-enhanced chemiluminescence immunoassay (ECLIA) based on three specific monoclonal antibodies to detect the nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in the serial serum samples from SARS patients was developed. The N protein is an extensively phosphorylated, highly basic protein, which interacts with viral RNA and makes up the viral core and nucleocapsid (Lai, 2003 the diagnosis of SARS depends basically upon detecting SARS-CoV RNA by RT-PCR and/or testing specific antibodies directed against SARS-CoV by assays based on cultured virus or recombinant viral antigens. In the present study, a capture ECLIA was developed based on three monoclonal antibodies directed against the N protein of SARS-CoV, and the N protein in the longitudinal serum samples from the SARS patients were detected with this method. The detection of the N protein of SARS-CoV in serum samples by ECLIA appears to be superior to the detection of the viral RNA by RT-PCR in rapid diagnosis of SARS patients.

title:
  Detection of the nucleocapsid protein of severe acute respiratory syndrome coronavirus in serum: Comparison with results of other viral markers

abstract:
  A capture enzyme-enhanced chemiluminescence immunoassay (ECLIA) based on three specific monoclonal antibodies to detect the nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in the serial serum samples from SARS patients was developed. The anti-SARS-CoV IgG and the viral RNA were also detected in the sera by ELISA and RT-PCR, respectively. During the first 10 days after onset, anti-SARS-CoV IgG, SARS-CoV RNA and the N protein were detected in 21.4, 42.9, and 90% of the patients’ sera, respectively. The detection rate of the N protein during days 11–15 of the disease was still significantly higher than those of anti-SARS-CoV IgG and SARS-CoV RNA. The data demonstrated that detection of the N protein with the capture ECLIA appears to be more useful than detection of other viral makers for rapid diagnosis of SARS in patients.


id: cord-307304-irji8owi
author: Britton, Paul
date: 2004-11-05
txt: ./txt/cord-307304-irji8owi.txt
cache: ./cache/cord-307304-irji8owi.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/15620403/
doi: 10.1016/j.jviromet.2004.09.017
summary:
  To demonstrate the feasibility of the method we exchanged the ectodomain of the Beaudette spike gene for the corresponding region from IBV M41 and generated two recombinant infectious bronchitis viruses (rIBVs) expressing the chimaeric S protein, validating the method as an alternative way for generating rIBVs. Avian infectious bronchitis virus (IBV), a group three member of the genus Coronavirus (order Nidovirales, family Coronaviridae), is a highly infectious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of the gut, kidney and oviduct (Cavanagh, 2001; Cavanagh and Naqi, 2003; Cook et al., 2001) . In an alternative strategy infectious IBV was recovered following transfection of restricted vaccinia virus DNA, containing the IBV full-length cDNA, into cells infected with recombinant fowlpox virus expressing T7 RNA polymerase (Casais et al., 2001) .

title:
  Generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection

abstract:
  A reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) has been described in which a full-length cDNA, corresponding to the IBV (Beaudette-CK) genome, was inserted into the vaccinia virus genome following in vitro assembly of three contiguous cDNAs [Casais, R., Thiel, V., Siddell, S.G., Cavanagh, D., Britton, P., 2001. Reverse genetics system for the avian coronavirus infectious bronchitis virus. J. Virol. 75, 12359–12369]. The method has subsequently been used to generate a recombinant IBV expressing a chimaeric S gene [Casais, R., Dove, B., Cavanagh, D., Britton, P., 2003. Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism. J. Virol. 77, 9084–9089]. Use of vaccinia virus as a vector for the full-length cDNA of the IBV genome has the advantage that modifications can be made to the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. We describe the use of homologous recombination as a method for modifying the Beaudette full-length cDNA, within the vaccinia virus genome, without the requirement for in vitro assembly of the IBV cDNA. To demonstrate the feasibility of the method we exchanged the ectodomain of the Beaudette spike gene for the corresponding region from IBV M41 and generated two recombinant infectious bronchitis viruses (rIBVs) expressing the chimaeric S protein, validating the method as an alternative way for generating rIBVs.


id: cord-308338-lhe51ws7
author: Wong, Sallene
date: 2011-02-22
url: https://api.elsevier.com/content/article/pii/S0166093411000802
doi: 10.1016/j.jviromet.2011.02.014
title:
  Development of a real-time RT-PCR assay for detection of resistance to oseltamivir in influenza A pandemic (H1N1) 2009 virus using single nucleotide polymorphism probes

abstract:
  Resistance to oseltamivir in pandemic (H1N1) 2009 influenza A virus is linked to an amino acid change from histidine (H) to tyrosine (Y) at position 275 in the neuraminidase protein (NA). A real-time one step RT-PCR assay using single nucleotide polymorphism (SNP) probes was developed to detect this mutation in respiratory specimens. The limit of detection was 47.6 copies/reaction for wild-type H275 RNA and 52.9 copies/reaction for the mutant H275Y RNA. The assay did not cross-react with other respiratory pathogens. The clinical sensitivity and specificity of the assay was compared to the gold standard Sanger sequencing method using 25 sensitive, 15 resistant and 20 negative samples. The sensitivity and specificity was 88.0% and 100% respectively with the SOIV_Osel_SEN probe designed to detect the H275 allele and 100% for the SOIV_Osel_RES probe detecting the 275Y allele. The sensitivity of the assay using nine admixtures of sensitive and resistant alleles was 88.9% and 77.8% with the SOIV_Osel_SEN probe and SOIV_Osel_RES probe respectively. The presence of mixed sensitive and resistant alleles in patient samples and mixtures of in vitro RNA were detected reproducibly. This assay can be used for screening of original samples for oseltamivir resistance without the need for culture and phenotypic testing.


id: cord-310771-tnwfp1je
author: Revilla-Fernández, Sandra
date: 2005-02-23
txt: ./txt/cord-310771-tnwfp1je.txt
cache: ./cache/cord-310771-tnwfp1je.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/15847915/
doi: 10.1016/j.jviromet.2005.01.018
summary:
  A method was developed for qualitative and quantitative detection of the seminal cell-associated PRRSV RNA in relation to endogenous and exogenous reference RNAs. As endogenous control for one-step real-time reverse transcription (RT)-PCR UBE2D2 mRNA was selected. Particularly for the analysis of persistent infections associated with low copy numbers of PRRSV RNA, UBE2D2 mRNA is an ideal control due to its low expression in seminal cells and its detection in all samples analysed (n = 36). For the development of one-step real-time RT-PCR for four endogenous reference RNAs (HPRT, UBE2D2, PPIA, and HMBS) appropriate target regions were selected and the assay conditions were optimised for amplification efficiency. One-step real-time RT-PCR assays for PRRSV-1 and -2 RNA allowed quantitation with optimal efficiency (Fig. 1a ; standard curves for two additional viremic pigs infected with PRRSV-1 (data not shown)) as achieved for endogenous and Fig. 1 .

title:
  The use of endogenous and exogenous reference RNAs for qualitative and quantitative detection of PRRSV in porcine semen

abstract:
  Semen is known to be a route of porcine reproductive and respiratory syndrome virus (PRRSV) transmission. A method was developed for qualitative and quantitative detection of the seminal cell-associated PRRSV RNA in relation to endogenous and exogenous reference RNAs. As endogenous control for one-step real-time reverse transcription (RT)-PCR UBE2D2 mRNA was selected. Particularly for the analysis of persistent infections associated with low copy numbers of PRRSV RNA, UBE2D2 mRNA is an ideal control due to its low expression in seminal cells and its detection in all samples analysed (n = 36). However, the amount of UBE2D2 mRNA in porcine semen varied (up to 106-fold), thus its use is limited to qualitative detection of PRRSV RNA. For quantitation, a synthetic, non-metazoan RNA was added to the RNA isolation reaction at an exact copy number. The photosynthesis gene ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from Arabidopsis thaliana was used as an exogenous spike. Unexpectedly, PRRSV RNA was detected in a herd of specific pathogen-free (SPF) boars which were tested ELISA-negative for anti-PRRSV antibodies. Therefore, RT-PCR for seminal cell-associated PRRSV is a powerful tool for managing the SPF status during quarantine programs and for routine outbreak investigations.


id: cord-311410-lgqup9ug
title: A single tube RT-PCR assay for the detection of mosquito-borne flaviviruses
author: Ayers, M.
date: 2006-05-02
url: https://www.ncbi.nlm.nih.gov/pubmed/16650488/
doi: 10.1016/j.jviromet.2006.03.009
abstract:
  Mosquito-borne flaviviruses include several important agents of human disease and have provided striking examples of emerging infections. In this study we present the design and validation of a single tube RT-PCR assay using a pair of consensus primers for the detection of mosquito-borne flaviviruses. Sequencing of the amplicons permits the species identification. The assay was validated using RNA from the yellow fever virus vaccine strain and from representative strains of dengue viruses 1, 2, 3 and 4, West Nile virus, Kunjin virus (a clade of West Nile virus), and St. Louis encephalitis virus.


id: cord-311639-zij2wbzs
author: Kim, Hyun Soo
date: 2012-08-30
txt: ./txt/cord-311639-zij2wbzs.txt
cache: ./cache/cord-311639-zij2wbzs.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093412002935
doi: 10.1016/j.jviromet.2012.08.014
summary:
  This study was performed to evaluate the analytical and clinical performance of a newly developed rapid ICG test (SD Bioline Norovirus test) for detecting human norovirus genogroups GI and GII in stool specimens. In samples with negative ICG and positive real-time PCR results, 200 L of fecal suspension was mixed with 200 L diluent (1:1 dilution) instead of 1 mL diluent, and the test was repeated. In this study, therefore, in the case of samples with negative ICG and positive real-time PCR results, 200 L of fecal suspension was mixed with 200 L diluent instead of 1 mL diluent (total dilution titer was 1:10-1:20 dilution, which is similar to that of the original procedure of this assay using stool), and the test was repeated. Evaluation of rapid immunochromatography test for the detection of norovirus infection: comparison with ELISA and real time quantitative reverse transcription PCR assays

title:
  Evaluation of the SD Bioline Norovirus rapid immunochromatography test using fecal specimens from Korean gastroenteritis patients

abstract:
  The analytical and clinical performance of a new rapid immunochromatography test, the SD Bioline Norovirus test, was evaluated for the detection of human norovirus in fecal specimens. The analytical performance studies were performed for detection limit, reproducibility, cross-reactivity, and interference. For comparison, 92 norovirus-positive stool samples and 126 norovirus-negative samples for which the results were confirmed by 2 different real-time PCR kits were used. The rapid immunochromatography test detected the equivalent of 4.48 × 10(6) copies/mL of the norovirus genome in stool samples. On performing the repeatability/reproducibility test, samples above this concentration all provided positive results (100%) and 97.8% of the samples slightly below this concentration (2.45 × 10(6) copies/mL) provided negative results. No cross-reactivity or interference was detected. Positive percent agreement (sensitivity), negative percent agreement (specificity), and overall percent agreement of the rapid immunochromatography test compared with testing by real-time PCR were 90.2%, 100%, and 95.9%, respectively. In addition, the rapid immunochromatography test was completed within 20 min. The SD Bioline Norovirus test was, therefore, easier and more rapid to perform and showed excellent reproducibility, no cross-reactivity, no interference, and high agreement compared with real-time PCR. Thus, this test is useful for rapid screening to identity norovirus infection.


id: cord-311801-m2otfdjw
title: Combination of Serological Total Antibody and RT-PCR Test for Detection of SARS-CoV-2 Infections
author: Wang, Pei
date: 2020-06-15
url: https://www.ncbi.nlm.nih.gov/pubmed/32554043/
doi: 10.1016/j.jviromet.2020.113919
abstract:
  The purpose of this study was to investigate the feasibility of serological total antibody tests combined with RT-PCR for detection of SARS-CoV-2. We conducted a retrospective study in which 375 patients were enrolled during the outbreak of SARS-CoV-2 from 25th January to 16th March 2020. Patients were divided into a COVID-19 group (n = 141) and a control group (n = 234). Serum samples and throat swabs were collected from 375 patients for total antibody testing against SARS-CoV-2 and RT-PCR analysis, respectively. The results indicated that diagnostic sensitivity and specificity were 95.7% and 98.7%, 92.2% and 100% by total antibody tests and RT-PCR, respectively. The sensitivity and specificity of total antibody tests combined with RT-PCR were 98.6% and 98.7%. The sensitivity of the combined method was significantly higher than RT-PCR (X(2) = 5.16, P < 0.05), and similar to that of total antibody tests (X(2) = 1.15, P > 0.05). This study supported the advantage of the combined method for detection of SARS-CoV-2 with a high degree of sensitivity and specificity, as a useful tool for accurate diagnosis and timely treatment of suspected patients, epidemiological investigation, as well as monitoring ongoing outbreaks of infections with SARS-CoV-2.


id: cord-312456-6lxc2rj2
author: Soltan, Mohamed A.
date: 2016-05-11
url: https://www.ncbi.nlm.nih.gov/pubmed/27180038/
doi: 10.1016/j.jviromet.2016.05.006
title:
  Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species

abstract:
  There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3–100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3–4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA.


id: cord-313271-2e8vjtop
title: Identification of cell lines permissive for human coronavirus NL63
author: Schildgen, Oliver
date: 2006-09-07
url: https://api.elsevier.com/content/article/pii/S0166093406002795
doi: 10.1016/j.jviromet.2006.07.023
abstract:
  Six cell lines routinely used in laboratories were tested for permissiveness to the infection with the newly identified human coronavirus NL63. Two monkey epithelial cell lines, LLC-MK2 and Vero-B4, showed a cytopathic effect (CPE) and clear viral replication, whereas no CPE or replication was observed in human lung fibroblasts MRC-5s. In Rhabdomyosarcoma cells, Madin–Darby–Canine-kidney cells and in an undefined monkey kidney cell line some replication was observed but massive exponential rise in virus yield lacked The results will lead to an improved routine diagnostic algorithm for the detection of the human coronavirus NL63.


id: cord-313541-fpqwzf9k
author: Ulloa, S.
date: 2020-08-22
url: https://doi.org/10.1016/j.jviromet.2020.113960
doi: 10.1016/j.jviromet.2020.113960
title:
  A simple method for SARS-CoV-2 detection by rRT-PCR without the use of a commercial RNA extraction kit

abstract:
  The World Health Organization (WHO) has declared a pandemic caused by a new coronavirus named SARS-CoV-2. The growing demand for commercial kits used for automated extraction of SARS-CoV-2 RNA, a key step before rRT-PCR diagnosis, could cause a shortage of stocks that hinders the rapid processing of samples. Although the recommendation is to use automated methods for nucleic acid extraction, alternatives are necessary to replace commercial kits. However, these alternatives should be as reliable as automated methods. This work describes a simple method to detect SARS-CoV-2 from specimens collected in different preservation media. Samples were previously inactivated by heating and precipitating with a PEG/NaCl solution before rRT-PCR assays for Orf1ab, N and S genes. The new method was compared with an automated protocol of nucleic acid extraction. Both procedures showed similar analytical results. Consequently, this simple and inexpensive method is a suitable procedure for laboratory diagnosis of SARS-CoV-2 infection.


id: cord-317307-q5mgue5z
author: Terlizzi, Maria Elena
date: 2009-05-13
txt: ./txt/cord-317307-q5mgue5z.txt
cache: ./cache/cord-317307-q5mgue5z.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2009.04.039
doi: 10.1016/j.jviromet.2009.04.039
summary:
  For evaluating IFA sensitivity, 10-fold dilutions (ranging from 10 2 to 10 −2 TCID 50 /200 l) of titrated virus were obtained and different variables, such as days of incubation (2-4 days) and primary monoclonal antibody dilutions (MAb; 1:40-1:80-1:160 in albumin supplemented with PBS 1%), were examined. Different concentrations of TCID 50 (ranged from 10 2 to 10 −5 /reaction) were amplified by the RT Real Time Qt-PCRs in order to compare sensitivity and specificity of the two diagnostic approaches. For the RT Real Time Qt-PCRs, the optimal parameters in obtaining the lowest detection limit with a high specificity resulted in the following concentrations: both primers 0.9 mM and probe 0.25 mM for HPIV-1 and HPIV-2; forward primer 1 mM and reverse 0.9 mM, and probe 0.25 mM for HPIV-3 amplification. Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4

title:
  Quantitative RT Real Time PCR and indirect immunofluorescence for the detection of human parainfluenza virus 1, 2, 3

abstract:
  Human parainfluenza viruses (HPIVs) are distributed worldwide and are involved mainly in the pathogenesis of respiratory tract infections. The development and optimization of three quantitative reverse transcription real time polymerase chain reactions (RT Real Time Qt-PCRs) and an indirect immunofluorescence (IFA) for the detection and quantitation of HPIV-1, -2 and -3 in clinical samples are described. Efficiency, sensitivity, specificity, inter- and intra-assay variability and turnaround time of the two methods were compared. These assays have been validated on 131 bronchoalveolar lavage specimens. Based on the results obtained, the molecular methods represent a valid and rapid tool for clinical management and should be included in diagnostic panels aimed to evaluate suspected respiratory tract infections.


id: cord-317462-nvrl0vyi
author: Song, Zhenhui
date: 2016-09-28
txt: ./txt/cord-317462-nvrl0vyi.txt
cache: ./cache/cord-317462-nvrl0vyi.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2016.09.018
doi: 10.1016/j.jviromet.2016.09.018
summary:
  To gain a better understanding of the replication, proliferation and infection characteristics of porcine transmissible gastroenteritis virus (TGEV) in porcine intestinal epithelial cells (IECs), this study established a cell model of IECs infected with the Chongqing (CQ) strain of TGEV. The results also showed that from 0 to 12 h after TGEV infection of porcine IECs, the intracellular viral RNA content did not change significantly. Thus, we developed an in vitro model based on porcine intestinal epithelial cells (IEC) infected with TGEV, and used transmission electron microscopy (TEM), indirect immunofluorescence assay (IFA) and real-time fluorescence quantitative PCR (FQ-PCR) to investigate the infection mechanism of TGEV. TGEV CQ strain was used to infect porcine IECs, and the supernatant and cells were collected at different time points for FQ-PCR. The one-step growth curve of TGEV in porcine IECs showed that the amount of viral RNA did not change significantly from 0 to 12 h post-infection, whereas Dai et al.

title:
  Morphogenesis and proliferative rule of porcine transmissible gastroenteritis virus in porcine intestinal epithelial cells

abstract:
  To gain a better understanding of the replication, proliferation and infection characteristics of porcine transmissible gastroenteritis virus (TGEV) in porcine intestinal epithelial cells (IECs), this study established a cell model of IECs infected with the Chongqing (CQ) strain of TGEV. The morphogenesis and proliferative rule of TGEV in porcine IECs were investigated using transmission electron microscopy, indirect immunofluorescence assays and real-time fluorescence quantitative PCR. Observations under the TEM indicated that the enveloped viral particles were roughly spherical, with diameters of between 80 and 120 nm. The virions entered porcine IECs by membrane fusion and the mature viruses in the vacuoles were transported to the cell membrane before release. The results also showed that from 0 to 12 h after TGEV infection of porcine IECs, the intracellular viral RNA content did not change significantly. Logarithmic growth occurred from 12 to 36 h, after which it gradually decreased. Moreover, the extracellular RNA content began to rise at 24 h after inoculation and then reduced gradually at approximately 48 h. This study provided a theoretical foundation for further study on the infection characteristics of TGEV in target cells.


id: cord-319392-zg7gkf0j
author: Yi, Li
date: 2011-11-18
url: https://www.ncbi.nlm.nih.gov/pubmed/22108430/
doi: 10.1016/j.jviromet.2011.11.011
title:
  Development of a combined canine distemper virus specific RT-PCR protocol for the differentiation of infected and vaccinated animals (DIVA) and genetic characterization of the hemagglutinin gene of seven Chinese strains demonstrated in dogs

abstract:
  A combined reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of the canine distemper virus (CDV). A pair of primers (P1/P2) was used to detect both CDV wild-type strains and vaccines. Another pair (P3/P4) was used to detect only CDV wild-type strains. A 335 bp fragment was amplified from the genomic RNA of the vaccine and wild-type strains. A 555 bp fragment was amplified specifically from the genomic RNA of the wild-type strains. No amplification was achieved for the uninfected cells, cells infected with canine parvovirus, canine coronavirus, or canine adenovirus. The combined RT-PCR method detected effectively and differentiated the CDV wild-type and vaccine strains by two separate RT-PCRs. The method can be used for clinical detection and epidemiological surveillance. The phylogenetic analysis of the hemagglutinin gene of the local wild-type CDV strains revealed that the seven local isolates all belonged to the Asia-1 lineage, and were clustered closely with one another at the same location. These results suggested that the CDV genotype Asia-1 is circulating currently in domestic dogs in China.


id: cord-320492-1xyjrjpf
title: A novel assay for detecting canine parvovirus using a quartz crystal microbalance biosensor
author: Kim, Yong Kwan
date: 2015-03-23
url: https://www.sciencedirect.com/science/article/pii/S0166093415001056
doi: 10.1016/j.jviromet.2015.03.015
abstract:
  Rapid and accurate diagnosis is crucial to reduce both the shedding and clinical signs of canine parvovirus (CPV). The quartz crystal microbalance (QCM) is a new tool for measuring frequency changes associated with antigen–antibody interactions. In this study, the QCM biosensor and ProLinker™ B were used to rapidly diagnosis CPV infection. ProLinker™ B enables antibodies to be attached to a gold-coated quartz surface in a regular pattern and in the correct orientation for antigen binding. Receiver operating characteristics (ROC) curves were used to set a cut-off value using reference CPVs (two groups: one CPV-positive and one CPV-negative). The ROC curves overlapped and the point of intersection was used as the cut-off value. A QCM biosensor with a cut-off value of −205 Hz showed 95.4% (104/109) sensitivity and 98.0% (149/152) specificity when used to test 261 field fecal samples compared to PCR. In conclusion, the QCM biosensor described herein is eminently suitable for the rapid diagnosis of CPV infection with high sensitivity and specificity. Therefore, it is a promising analytical tool that will be useful for clinical diagnosis, which requires rapid and reliable analyses.


id: cord-321886-0b3ocoh9
author: Zhang, Chao-fan
date: 2010-08-04
txt: ./txt/cord-321886-0b3ocoh9.txt
cache: ./cache/cord-321886-0b3ocoh9.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2010.07.034
doi: 10.1016/j.jviromet.2010.07.034
summary:
  title: Loop-mediated isothermal amplification for rapid detection and differentiation of wild-type pseudorabies and gene-deleted virus vaccines A loop-mediated isothermal amplification (LAMP) assay was developed specifically for detection and differentiation of pseudorabies virus (PRV). Because of its sensitivity, specificity, and simplicity, the LAMP assay could be a useful method for early and rapid differentiation of swine vaccinated with PRV gE-deleted vaccine from swine infected with wild virus. Based on PRV-gG-LAMP, 26 of the 70 piglets were infected with wild-type PRV and may or may not have been vaccinated. Many different PCR assays can differentiate between the wild-type PRV and gene-deleted virus vaccines (Liu et al., 2007) , but LAMP is easier to perform and provides more rapid results. (2008) described a LAMP system for PRV detection but that LAMP system could not differentiate between swine infected with wild-type PRV and swine vaccinated with PRV gE-deleted.

title:
  Loop-mediated isothermal amplification for rapid detection and differentiation of wild-type pseudorabies and gene-deleted virus vaccines

abstract:
  A loop-mediated isothermal amplification (LAMP) assay was developed specifically for detection and differentiation of pseudorabies virus (PRV). One group of primers was designed to detect wild-type strains (i.e., strains with the gE gene) and the other group of primers was designed to detect both PRV gE-vaccine and wild-type strains (i.e., strains with the gG gene and with or without the gE gene). After amplification by Bst enzyme at a constant temperature of 65 °C, a laddering of bright products was visible following electrophoresis on a 2% agarose gel. LAMP was 100–1000-fold more sensitive than the standard PCR. The assay was specific in that it did not amplify other porcine viruses including porcine parvovirus, porcine circovirus type 1, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, classical swine fever virus, swine transmissible gastroenteritis coronavirus, and porcine epidemic diarrhea virus. Because of its sensitivity, specificity, and simplicity, the LAMP assay could be a useful method for early and rapid differentiation of swine vaccinated with PRV gE-deleted vaccine from swine infected with wild virus.


id: cord-322143-hkh1grys
title: Sampling methods for recovery of human enteric viruses from environmental surfaces
author: Turnage, Nicole L.
date: 2017-06-17
txt: ./txt/cord-322143-hkh1grys.txt
cache: ./cache/cord-322143-hkh1grys.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/28633964/
doi: 10.1016/j.jviromet.2017.06.008
summary:
  For instance, understanding the persistence of human enteric viruses on inanimate fomite surfaces in relation to various environmental conditions could provide insight on ways to limit and prevent virus transmission and subsequent outbreaks. Overall, the higher the inoculum level for all enteric viruses, the higher the mean recovery rate regardless of the variability among methods, PA = plaque assay; PBS = phosphate buffered saline; PBST = PBS + 0.02% Tween 80; PCRU = polymerase chain reaction units; PE = polyethylene; PF = porous formic; PFU = plaque forming units; RH = relative humidity; RB = rubberized surface; RT-qPCR = reverse transcription quantitative PCR; RT = room temperature; SS = stainless steel. Additionally, some studies found other tools and methods such as biowipes and cell scraper-aspiration methods to be potentially more efficient for enteric virus recovery from surfaces in comparison to cotton and/or polyester swabs.

abstract:
  Acute gastroenteritis causes the second highest infectious disease burden worldwide. Human enteric viruses have been identified as leading causative agents of acute gastroenteritis as well as foodborne illnesses in the U.S. and are generally transmitted by fecal-oral contamination. There is growing evidence of transmission occurring via contaminated fomite including food contact surfaces. Additionally, human enteric viruses have been shown to remain infectious on fomites over prolonged periods of time. To better understand viral persistence, there is a need for more studies to investigate this phenomenon. Therefore, optimization of surface sampling methods is essential to aid in understanding environmental contamination to ensure proper preventative measures are being applied. In general, surface sampling studies are limited and highly variable among recovery efficiencies and research parameters used (e.g., virus type/density, surface type, elution buffers, tools). This review aims to discuss the various factors impacting surface sampling of viruses from fomites and to explore how researchers could move towards a more sensitive and standard sampling method.


id: cord-322234-1zyy536y
author: Lorusso, Alessio
date: 2009-12-17
url: https://www.sciencedirect.com/science/article/pii/S0166093409005205
doi: 10.1016/j.jviromet.2009.12.002
title:
  One-step real-time RT-PCR for pandemic influenza A virus (H1N1) 2009 matrix gene detection in swine samples

abstract:
  In the spring of 2009, a novel (H1N1) influenza A virus began to spread among humans worldwide. Although the 2009 H1N1 is related genetically to swine influenza viruses, human infection has not been connected to pig exposure. Because the virus is now circulating widely in the human population, swine herds are at increased risk of becoming infected. In order to investigate potential outbreaks of the 2009 pandemic virus in pigs, a quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) for the detection of the (H1N1) 2009 RNA in clinical specimens was developed. To evaluate the applicability of the test as a diagnostic tool in the screening of field specimens from swine, 64 field isolates of North American swine, 5 equine and 48 avian influenza viruses collected during diagnostic investigations were analyzed retrospectively as well as samples collected during an experimental in vivo infection with two novel H1N1 isolates, A/California/04/2009 (H1N1)v virus and A/Mexico/4108/2009 (H1N1)v. The sensitivity of the qRT-PCR was shown to be higher with respect to standard techniques such as virus isolation and the reproducibility was satisfactory. The present unique and highly sensitive assay is able to detect as little as 1 × 10(1) copies of RNA per μl of template and it represents a rapid and useful approach for the screening and quantitation of (H1N1) 2009 RNA in porcine specimens.


id: cord-322410-k23engcx
author: Naguib, Mahmoud M.
date: 2017-03-21
txt: ./txt/cord-322410-k23engcx.txt
cache: ./cache/cord-322410-k23engcx.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/28336367/
doi: 10.1016/j.jviromet.2017.02.018
summary:
  title: New real time and conventional RT-PCRs for updated molecular diagnosis of infectious bronchitis virus infection (IBV) in chickens in Egypt associated with frequent co-infections with avian influenza and Newcastle Disease viruses Conventional RT-PCRs amplifying hypervariable regions (HVRs 1–2 and 3) of the IBV S1 gene were developed and amplificates used for nucleotide sequence-based typing of IBV field strains in Egyptian chickens directly from clinical samples. The specificity of RT-qPCR primer set was evaluated by examining different avian respiratory viruses circulating in poultry in Egypt including AIV H5N1, H9N2 and NDV as well as different coronaviruses including a panel of IBV reference strains as listed in the materials section. Within HVR 1, 2 sequences, however, the existence of two distinct Table 3 RT-qPCRs reveal frequent co-infections in Egyptian poultry samples with avian influenza (AIV), Newcastle Disease (NDV) and infectious bronchitis viruses (IBV).

title:
  New real time and conventional RT-PCRs for updated molecular diagnosis of infectious bronchitis virus infection (IBV) in chickens in Egypt associated with frequent co-infections with avian influenza and Newcastle Disease viruses

abstract:
  In Egypt, currently two geographically restricted genotypes of the infectious bronchitis coronavirus (IBV) are circulating with detrimental effects for poultry industry. A sensitive real-time RT-PCR assay targeting the IBV nucleocapsid gene (N) was developed to screen clinical samples for presence of IBV. Conventional RT-PCRs amplifying hypervariable regions (HVRs 1–2 and 3) of the IBV S1 gene were developed and amplificates used for nucleotide sequence-based typing of IBV field strains in Egyptian chickens directly from clinical samples.


id: cord-323072-4rsgeag7
author: Han, Xueqing
date: 2004-12-01
txt: ./txt/cord-323072-4rsgeag7.txt
cache: ./cache/cord-323072-4rsgeag7.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093404002472
doi: 10.1016/j.jviromet.2004.08.015
summary:
  Since the outbreak of SARS in 2003, several laboratory diagnostic methods have been established, including real-time RT-PCR assay, whole-virus-based immunofluorescence assay (IFA), recombinant protein-based enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests, antigencapturing enzyme-linked immunosorbent assay, and Western blot (WB) assay. To test whether the recombinant M protein is effective as an ELISA antigen for detecting SARS-CoV patient serum, the sera from four healthy people and four SARS patients were used. Detection results of eight human sera by ELISA using purified recombinant M protein as antigen.# 1-4: sera from four healthy people, respectively, # 5-8: sera from four SARS patients, respectively. The results were in complete accordance with those of other assays, thus indicating that the recombinant M protein may be useful as an ELISA antigen for detecting specific antibodies to SARS-CoV in human sera. Recombinant protein-based enzyme-linked imunosorbent assay and immunochromatographic tests for detection of immunoglobulin G antibodies to severe acute respiratory syndrome (SARS) coronavirus in SARS patients

title:
  The expression of SARS–CoV M gene in P. Pastoris and the diagnostic utility of the expression product

abstract:
  High-level protein expression is an important means of obtaining large amounts of viral proteins to investigate further their biological properties. To express the membrane (M) protein of SARS–CoV at high-level in vitro, the M gene fragment was amplified and cloned it into the Pichia Pastoris expression vector pPICZαA. SDS–PAGE and Western blotting analysis of the induced products of recombinant yeast transformant indicated that successful high-level expression of M protein was achieved, and that the expression product was similar antigenically to the natural protein. Purified recombinant M protein was used subsequently as an ELISA antigen for detection of eight serum samples screened previously by whole virus ELISA and immunofluorescence assay, and consistent results were obtained. These findings suggest that the recombinant M protein may be useful as a diagnostic reagent.


id: cord-324213-3uqlimov
author: Bolotin, S.
date: 2009-01-30
txt: ./txt/cord-324213-3uqlimov.txt
cache: ./cache/cord-324213-3uqlimov.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093409000251
doi: 10.1016/j.jviromet.2009.01.016
summary:
  To evaluate this method, 40 oseltamivir resistant and 61 oseltamivir sensitive H1N1 influenza isolates were tested using Sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel H275Y RT-PCR assay. To evaluate this method, 40 oseltamivir resistant and 61 oseltamivir sensitive H1N1 influenza isolates were tested using Sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel H275Y RT-PCR assay. To determine the limit of detection (LOD) of the H275Y and N1 control RT-PCR assay, serial ten-fold dilutions of nucleic acid from an oseltamivir-sensitive clinical influenza A/Brisbane/57/2007 H1N1 isolate were tested. This study evaluated the use of a novel H275Y RT-PCR assay for detection of H275Y-positive isolates in comparison to Sanger sequencing and pyrosequencing, and found that while all three methods may have a role in influenza diagnostic testing, the H275Y RT-PCR assay is a rapid and effective test for the detection of oseltamivir resistance through mutation at residue 275.

title:
  Development of a novel real-time reverse-transcriptase PCR method for the detection of H275Y positive influenza A H1N1 isolates

abstract:
  During the 2007–2008 influenza season global strain surveillance for antiviral resistance revealed the sudden emergence of oseltamivir resistance in influenza A H1N1 isolates. Although oseltamivir resistance rates vary from region to region, 16% of isolates tested globally were found to be oseltamivir resistant by a histidine to tyrosine mutation of residue 275 of the neuraminidase gene of influenza A. In order to implement effective resistance testing locally a novel real-time reverse-transcriptase PCR (RT-PCR) assay was developed for the detection of the H275Y mutation. To evaluate this method, 40 oseltamivir resistant and 61 oseltamivir sensitive H1N1 influenza isolates were tested using Sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel H275Y RT-PCR assay. In comparison to Sanger sequencing, the sensitivity and specificity of the H275Y RT-PCR assay were 100% (40/40) and 100% (61/61) respectively, while the sensitivity and specificity of pyrosequencing were 100% (40/40) and 97.5% (60/61) respectively. Although all three methods were effective in detecting the H275Y mutation associated with oseltamivir resistance, the H275Y RT-PCR assay was the most rapid and could easily be incorporated into an influenza subtyping protocol.


id: cord-326225-crtpzad7
title: Simultaneous rapid sequencing of multiple RNA virus genomes
author: Neill, John D.
date: 2014-06-01
txt: ./txt/cord-326225-crtpzad7.txt
cache: ./cache/cord-326225-crtpzad7.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2014.02.016
doi: 10.1016/j.jviromet.2014.02.016
summary:
  This procedure utilized primers composed of 20 bases of known sequence with 8 random bases at the 3′-end that also served as an identifying barcode that allowed the differentiation each viral library following pooling and sequencing. There is a wealth of information in these isolates, but up till now, it has been time consuming and expensive to sequence these viral genomes, often requiring sets of strain-specific primers for PCR amplification and sequencing. These primers were developed so that the 20 base known sequence was used for PCR amplification of the library as well as served as a barcode for identifying each viral library following pooling and sequencing. This virus, a BVDV 1b strain isolated from alpaca (GenBank accession JX297520.1; Table 2 , library 3, barcode 10), was assembled from Ion Torrent data and was found to have only 1 base difference from the sequence determined earlier (data not shown). One virus, library 1, barcode 9, had only 658 viral sequence reads but 94.4% of the genome was assembled.

abstract:
  Comparing sequences of archived viruses collected over many years to the present allows the study of viral evolution and contributes to the design of new vaccines. However, the difficulty, time and expense of generating full-length sequences individually from each archived sample have hampered these studies. Next generation sequencing technologies have been utilized for analysis of clinical and environmental samples to identify viral pathogens that may be present. This has led to the discovery of many new, uncharacterized viruses from a number of viral families. Use of these sequencing technologies would be advantageous in examining viral evolution. In this study, a sequencing procedure was used to sequence simultaneously and rapidly multiple archived samples using a single standard protocol. This procedure utilized primers composed of 20 bases of known sequence with 8 random bases at the 3′-end that also served as an identifying barcode that allowed the differentiation each viral library following pooling and sequencing. This conferred sequence independence by random priming both first and second strand cDNA synthesis. Viral stocks were treated with a nuclease cocktail to reduce the presence of host nucleic acids. Viral RNA was extracted, followed by single tube random-primed double-stranded cDNA synthesis. The resultant cDNAs were amplified by primer-specific PCR, pooled, size fractionated and sequenced on the Ion Torrent PGM platform. The individual virus genomes were readily assembled by both de novo and template-assisted assembly methods. This procedure consistently resulted in near full length, if not full-length, genomic sequences and was used to sequence multiple bovine pestivirus and coronavirus isolates simultaneously.


id: cord-328961-waxtb759
title: PCR assay for the detection and the identification of atypical canine coronavirus in dogs
author: Pratelli, Annamaria
date: 2002-10-01
txt: ./txt/cord-328961-waxtb759.txt
cache: ./cache/cord-328961-waxtb759.txt
extension: .txt
mime: text/plain
url: https://api.elsevier.com/content/article/pii/S0166093402001659
doi: 10.1016/s0166-0934(02)00165-9
summary:
  Comparative sequence analysis of the PCR products of the M gene and fragments of the pol1a and pol1b genes of canine coronavirus (CCoV) have demonstrated that two separate clusters of CCoV are present in dogs. The sequence analysis of the PCR products of the M and S genes carried out on the faecal samples from the two pups confirmed that the FCoV-like CCoV had caused the disease (personal observations). On the basis of these preliminary results, a PCR assay was developed to detect and identify the FCoV-like CCoV strains from faecal samples of infected dogs. In a previous study, similar nucleotide substitutions in the binding site of the internal primer CCoV3 used for the n-PCR were demonstrated in the sequence analysis of the PCR products from five faecal samples of pups with diarrhoea.

abstract:
  Comparative sequence analysis of the PCR products of the M gene and fragments of the pol1a and pol1b genes of canine coronavirus (CCoV) have demonstrated that two separate clusters of CCoV are present in dogs. This note describes a PCR assay to identify atypical CCoV strains with nucleotide substitutions in the M gene. A total of 177 faecal samples from dogs CCoV positive previously with the PCR assay were analysed. Sixty-two of the 177 samples were amplified with the PCR described in the present study and were thus considered atypical CCoVs. The specificity of the PCR typing assay was confirmed by sequence analysis of the PCR products.


id: cord-331509-p19dg1jw
author: Bigault, Lionel
date: 2020-05-31
txt: ./txt/cord-331509-p19dg1jw.txt
cache: ./cache/cord-331509-p19dg1jw.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093420301580?v=s5
doi: 10.1016/j.jviromet.2020.113906
summary:
  title: Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR In this study we described the development and validation of a SYBR™ Green one-step RT-qPCR according to the French norm NF U47-600, for the detection and quantification of PEDV viral RNA. Specificity and sensitivity, limit of detection (LoD), limit of quantification (LQ), linearity, intra and inter assay variability were evaluated using transcribed RNA and fecal and jejunum matrices spiked with virus. For a rapid, accurate and reliable diagnosis of PED in the veterinary laboratory, a method for the detection of PEDV viral RNA has been developed and more importantly validated according to the "Association Francaise de NORmalisation" (AFNOR) French NF U47-600 norm entitled "requirement and recommendation for the implementation, development and validation of PCR in animal health" (AFNOR, 2015a; AFNOR, 2015b) . This method should help harmonize detection and quantification of viral RNA from PEDV belonging to both S-non-INDEL and S-INDEL strains in both field and experimental settings.

title:
  Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR

abstract:
  Since 2014, porcine epidemic diarrhea virus (PEDV) has reemerged in Europe. RT-PCR methods have been described for the detection of PEDV, but none have been validated according to a norm. In this study we described the development and validation of a SYBR™ Green one-step RT-qPCR according to the French norm NF U47-600, for the detection and quantification of PEDV viral RNA. The method was validated from sample preparation (feces or jejunum) through to nucleic acid extraction and RT-qPCR detection. Specificity and sensitivity, limit of detection (LoD), limit of quantification (LQ), linearity, intra and inter assay variability were evaluated using transcribed RNA and fecal and jejunum matrices spiked with virus. The analytical and diagnostic specificities and sensitivities of this RT-qPCR were 100% in this study. A LoD of 50 genome copies/5 µl of extract from fecal matrices spiked with virus or RNA transcript and 100 genome copies/5 µl of extract from jejunum matrices spiked with virus were obtained. The Lower LQ (LLQ) was 100 genome copies/5 µl and the Upper LQ (ULQ) 10(8) copies/5 µl. This method is the first, validated according a norm for PEDV and may serve as a global reference method to harmonize detection and quantification of PEDV viral RNA in both field and experimental settings.


id: cord-332522-adul9nzf
title: Development of Taqman RT-nested PCR system for clinical SARS-CoV detection
author: Wu, Qingfa
date: 2004-04-02
txt: ./txt/cord-332522-adul9nzf.txt
cache: ./cache/cord-332522-adul9nzf.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/15109816/
doi: 10.1016/j.jviromet.2004.02.011
summary:
  In this study, 12 sets of nested primers covering the SARS-CoV genome have been screened and showed sufficient sensitivity to detect SARS-CoV in RNA isolated from virus cultured in Vero 6 cells. To optimize further the reaction condition of those nested primers sets, seven sets of nested primers have been chosen to compare their reverse transcribed efficiency with specific and random primers, which is useful to combine RT with the first round of PCR into a one-step RT-PCR. Through investigations on a test panel of whole blood obtained from 30 SARS patients and 9 control persons, the specificity and sensitivity of the Taqman RT-nested PCR system was found to be 100 and 83%, respectively, which suggests that the method is a promising one to diagnose SARS in early stages. To compare the sensitivities of these 12 sets of nested primers, serial 10-fold di-lution genome cDNA of BJ01 that reverse transcribed with random primer was used as the template to carry out the nested PCR.

abstract:
  Severe acute respiratory syndrome (SARS) is an acute newly emerged infectious respiratory illness. The etiologic agent of SARS was named ‘SARS-associated coronavirus’ (SARS-CoV) that can be detected with reverse transcription-polymerase chain reaction (RT-PCR) assays. In this study, 12 sets of nested primers covering the SARS-CoV genome have been screened and showed sufficient sensitivity to detect SARS-CoV in RNA isolated from virus cultured in Vero 6 cells. To optimize further the reaction condition of those nested primers sets, seven sets of nested primers have been chosen to compare their reverse transcribed efficiency with specific and random primers, which is useful to combine RT with the first round of PCR into a one-step RT-PCR. Based on the sensitivity and simplicity of results, the no. 73 primer set was chosen as the candidate primer set for clinical diagnoses. To specify the amplicon to minimize false positive results, a Taqman RT-nested PCR system of no. 73 nested primer set was developed. Through investigations on a test panel of whole blood obtained from 30 SARS patients and 9 control persons, the specificity and sensitivity of the Taqman RT-nested PCR system was found to be 100 and 83%, respectively, which suggests that the method is a promising one to diagnose SARS in early stages.


id: cord-336639-jaue41mv
title: A mRNA PCR for the diagnosis of feline infectious peritonitis
author: Simons, Fermin A.
date: 2004-12-21
txt: ./txt/cord-336639-jaue41mv.txt
cache: ./cache/cord-336639-jaue41mv.txt
extension: .txt
mime: text/plain
url: https://www.sciencedirect.com/science/article/pii/S0166093404003477
doi: 10.1016/j.jviromet.2004.11.012
summary:
  A reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of feline coronavirus (FCoV) messenger RNA in peripheral blood mononuclear cells (PBMCs) is described. The reason for this discrepancy became clear when the biological and genetic properties of FECV and FIPV isolates had been studied (Addie and Jarrett, 1992; Hohdatsu et al., 1992; Horzinek and Osterhaus, 1979) : the avirulent FCoV strains causing inconspicuous infections are responsible for the high seroprevalence; in cats experiencing some immunosuppressive event, expansion of the quasispecies cloud and mutations in the FECV genome lead to virulent variants that induce FIP (Vennema et al., 1998) . Detection of feline coronaviruses by culture and reverse transcriptase-polymerase chain reaction of blood samples from healthy cats and cats with clinical feline infectious peritonitis

abstract:
  A reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of feline coronavirus (FCoV) messenger RNA in peripheral blood mononuclear cells (PBMCs) is described. The assay is evaluated as a diagnostic test for feline infectious peritonitis (FIP). It is based on a well-documented key event in the development of FIP: the replication of virulent FCoV mutants in monocytes/macrophages. To detect most feline coronavirus field strains, the test was designed to amplify subgenomic mRNA of the highly conserved M gene. The test was applied to 1075 feline blood samples (424 from healthy, 651 from sick cats suspected of FIP) and returned 46% of the diseased cats as positive for feline coronavirus mRNA in their peripheral blood cells; of the healthy cats, 5% tested positive. Of a group of 81 animals in which FIP had been confirmed by post-mortem examination, 75 (93%) tested positive, whereas 17 cats with different pathologies (non-FIP cases) all tested negative. In view of the low rate of false-positive results (high specificity) the mRNA RT-PCR may be a valuable addition to the diagnostic arsenal for FIP.


id: cord-343136-kftffes0
title: Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2
author: Mohon, Abu Naser
date: 2020-09-15
txt: ./txt/cord-343136-kftffes0.txt
cache: ./cache/cord-343136-kftffes0.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32941977/
doi: 10.1016/j.jviromet.2020.113972
summary:
  A novel reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2 has been developed. Limit of detection of the LAMP assay was evaluated by using a nasopharyngeal (NP) swab sample infected with SARS-CoV-2 for which the viral load was quantified using digital droplet PCR (see Supplementary Methods). Twenty four replicates from a serial dilution containing 25-50 copies of SARS-CoV-2 which equates to 1X LOD (patient sample NP swab in VTM viral load confirmed by digital droplet PCR) per reaction were tested using dual-target RT-LAMP (Table 3) . The dual-target RT-LAMP test for SARS-CoV-2 developed in this study has comparable analytical sensitivity and specificity, limit of detection, precision, and achieved excellent agreement compared to the reference RT-PCR methods used internationally.

abstract:
  A novel reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2 has been developed. The LAMP assay achieves a comparable limit of detection (25 copies per reaction) as commonly used RT-PCR protocols using clinical samples quantified by digital droplet PCR. Precision, cross-reactivity, inclusivity, and limit of detection studies were performed according to regulatory standards. Clinical validation of dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 98.48% (95% CI 91.84% to 99.96%) and NPA 100.00% (95% CI 93.84% to 100.00%) based on the E gene and N2 gene reference RT-PCR methods. The method has implications for development of point of care technology using isothermal amplification.


id: cord-343441-z849jvq5
author: Li, Yan
date: 2013-09-01
txt: ./txt/cord-343441-z849jvq5.txt
cache: ./cache/cord-343441-z849jvq5.txt
extension: .txt
mime: text/plain
url: https://doi.org/10.1016/j.jviromet.2013.08.021
doi: 10.1016/j.jviromet.2013.08.021
summary:
  In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. The analytic sensitivity of the duplex TaqMan rRT-PCR assay was compared with the WHO TaqMan assay and a commercial single H7N9 rRT-PCR kit (bioPerfectus technologies, Taizhou, China) with a 10-fold dilution series of a nasopharyngeal aspirate (NPA) from a patient infected with the H7N9 virus (approximately 4.8 × 10 6 copies of the viral genome/mL). To determine the actual detection limit (number of copies per reaction) of the duplex TaqMan rRT-PCR assay, in vitro RNA transcripts of HA and NA genes from the H7N9 virus were prepared with T7 RNA polymerase (TaKaRa Biotechnology Co. Ltd., Dalian, China) according to the manufacturer''s instructions using influenza A/Nanjing/1/2013 (H7N9) RNA as a template.

title:
  Simultaneous detection of hemagglutinin and neuraminidase genes of novel influenza A (H7N9) by duplex real-time reverse transcription polymerase chain reaction

abstract:
  A novel reassortant influenza A (H7N9) virus emerged recently in China. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. The sensitivity of the assay was determined to be 10 RNA copies per reaction for both HA and NA genes. No cross-reactivity was observed with other influenza virus subtypes or respiratory tract viruses. One hundred and forty-six clinical and environmental specimens were tested and compared with reference methods and were found to be consistent. The assay is suitable for large-scale screening due to short turnaround times and high specificity, sensitivity, and reproducibility.


id: cord-350753-qbm145tr
title: Determination of SARS-CoV-2 antibodies with assays from Diasorin, Roche and IDvet
author: Krüttgen, Alexander
date: 2020-09-23
txt: ./txt/cord-350753-qbm145tr.txt
cache: ./cache/cord-350753-qbm145tr.txt
extension: .txt
mime: text/plain
url: https://www.ncbi.nlm.nih.gov/pubmed/32979407/
doi: 10.1016/j.jviromet.2020.113978
summary:
  Using 75 sera from patients tested positive or negative by SARS-CoV-2 PCR, we investigated the sensitivity and specificity of the Liaison SARS-CoV-2 S1/S2 IgG assay (DiaSorin), the Elecsys Anti-SARS-CoV-2 assay (Roche), and the ID Screen SARS-CoV-2-N IgG indirect kit (IDVet). We and others have published results of the assessment of the first commercially available serological assays, such as the Anti SARS-CoV-2 ELISA (IgG) from Euroimmun (Krüttgen et al., 2020; Okba et al., 2020) . We therefore compared these three new assays with respect to their sensitivity and specificity to detect SARS-CoV-2 specific antibodies using a collection of serum samples employed previously for the analysis of four other assays. Our comparative approach to test in total seven different SARS-CoV-2 antibody assays with an identical collection of serum samples allowed for the first time the direct comparison of performance indicators of such a large number of automated assays.

abstract:
  There is an ongoing need for highly reliable serological assays to detect individuals with past SARS-CoV-2 infection. Using 75 sera from patients tested positive or negative by SARS-CoV-2 PCR, we investigated the sensitivity and specificity of the Liaison SARS-CoV-2 S1/S2 IgG assay (DiaSorin), the Elecsys Anti-SARS-CoV-2 assay (Roche), and the ID Screen SARS-CoV-2-N IgG indirect kit (IDVet). We determined a sensitivity of 95.5%, 95.5%, and 100% and a specificity of 90.5%, 96.2%, and 92,5% for the DiaSorin assay, the Roche assay, and the IDVet assay, respectively. We conclude that serologic assays combining very high sensitivity and specificity are still not commercially available for SARS-CoV-2. For maximizing sensitivity and specificity of SARS-CoV-2 serological diagnostics, the combination of two assays may be helpful.


