# bib

id: cord-344309-6c2wttxg
author: Lin, Huixing
date: 2018-08-20
url: https://doi.org/10.1186/s12917-018-1570-5
doi: 10.1186/s12917-018-1570-5
title:
  Development and application of an indirect ELISA for the detection of antibodies to porcine epidemic diarrhea virus based on a recombinant spike protein

abstract:
  BACKGROUND: As the major causative agent of swine viral diarrhea, porcine epidemic diarrhea virus (PEDV) has caused massive losses to the economies of swine raising countries. Accordingly, the serological detection of corresponding antibodies would be beneficial to diagnose PEDV indirectly to control the disease. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant truncated spike (S) protein of PEDV was developed and validated. RESULTS: The reaction conditions of the developed indirect ELISA were optimized. This indirect ELISA was compared to indirect immunoinfluscent assay (IFA), and the overall coincidence rate was 96.74% based on testing 368 clinical serum samples with different PEDV antibody levels. No cross-reactivity with other common swine pathogens was detected for the developed S1 indirect ELISA. Finally, the S1 indirect ELISA was applied to detect serum antibodies of 3304 field samples collected from different pig farms in eastern China, and it presented an overall substantial agreement on the PEDV infection status. CONCLUSIONS: This established S1 indirect ELISA is capable of detecting serum antibodies against PEDV, and due to its high sensitivity and specificity, it could be applied for serological evaluation and indirect diagnosis of PEDV infection.


id: cord-344421-rmnck42f
author: Theuns, Sebastiaan
date: 2018-06-29
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url: https://doi.org/10.1038/s41598-018-28180-9
doi: 10.1038/s41598-018-28180-9
summary:
  Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding (10(6.42)–10(7.01) copies/swab) and diarrheic signs was not observed during a follow-up study. Third-generation sequencing using MinION (Oxford Nanopore Technologies, ONT) might be a useful and affordable diagnostic tool for swine veterinary medicine as it allows rapid sample preparation and real-time sequence analysis. The shedding of porcine kobuvirus, RVA and rotavirus C (RVC) was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated. The third-generation sequencing device MinION (ONT), holds promise as a diagnostic platform, as it allows real-time sequencing and analyses of all DNA/RNA in a sample, theoretically without needing pre-amplification of viral nucleic acids. In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions.

title:
  Nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus

abstract:
  Enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. The capacities of nanopore sequencing for viral diagnostics were investigated here. First, cell culture-grown porcine epidemic diarrhea virus and rotavirus A were pooled and sequenced on a MinION. Reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths (19.2 to 103.5X) after 3 h. Next, diarrheic feces of a one-week-old piglet was analyzed. Almost all reads (99%) belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages. Moreover, porcine kobuvirus was discovered in the feces for the first time in Belgium. Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding (10(6.42)–10(7.01) copies/swab) and diarrheic signs was not observed during a follow-up study. Retrospective analysis showed the widespread (n = 25, 56.8% positive) of genetically moderately related kobuviruses among Belgian diarrheic piglets. MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted. The true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded.


id: cord-344558-1jgqofbr
title: Completion of the Porcine Epidemic Diarrhoea Coronavirus (PEDV) Genome Sequence
author: Kocherhans, Rolf
date: 2001
txt: ./txt/cord-344558-1jgqofbr.txt
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url: https://www.ncbi.nlm.nih.gov/pubmed/11724265/
doi: 10.1023/a:1011831902219
summary:
  A cloning strategy, which involves primers based on conserved regions in the predicted ORF1 products from other coronaviruses whose genome sequence has been determined, was used to amplify the equivalent, but as yet unknown, sequence of PEDV. Comparative amino acid sequence alignments revealed that PEDV is most closely related to human coronavirus (HCoV)-229E and transmissible gastroenteritis virus (TGEV) and less related to murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). The alignment of the deduced amino acid sequences indicated that PEDV occupies an interesting intermediate position between the two well-characterized members of the group I coronaviruses, transmissible gastroenteritis virus (TGEV) and human coronavirus (HCoV)-229E. In order to obtain the first partial PEDV specific sequences, the predicted amino acid sequences of the HCoV-229E and TGEV polymerase ORFs were aligned and homologous regions identified. The deduced amino acid sequences of ORF1a and ORF1b from PEDV were aligned with the corresponding sequences of HCoV-229E, TGEV, MHV-A59, and IBV using PILEUP.

abstract:
  The sequence of the replicase gene of porcine epidemic diarrhoea virus (PEDV) has been determined. This completes the sequence of the entire genome of strain CV777, which was found to be 28,033 nucleotides (nt) in length (excluding the poly A-tail). A cloning strategy, which involves primers based on conserved regions in the predicted ORF1 products from other coronaviruses whose genome sequence has been determined, was used to amplify the equivalent, but as yet unknown, sequence of PEDV. Primary sequences derived from these products were used to design additional primers resulting in the amplification and sequencing of the entire ORF1 of PEDV. Analysis of the nucleotide sequences revealed a small open reading frame (ORF) located near the 5′ end (no 99–137), and two large, slightly overlapping ORFs, ORF1a (nt 297–12650) and ORF1b (nt 12605–20641). The ORF1a and ORF1b sequences overlapped at a potential ribosomal frame shift site. The amino acid sequence analysis suggested the presence of several functional motifs within the putative ORF1 protein. By analogy to other coronavirus replicase gene products, three protease and one growth factor-like motif were seen in ORF1a, and one polymerase domain, one metal ion-binding domain, and one helicase motif could be assigned within ORF1b. Comparative amino acid sequence alignments revealed that PEDV is most closely related to human coronavirus (HCoV)-229E and transmissible gastroenteritis virus (TGEV) and less related to murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). These results thus confirm and extend the findings from sequence analysis of the structural genes of PEDV.


id: cord-344845-52rehsd5
author: Opriessnig, Tanja
date: 2017-10-26
txt: ./txt/cord-344845-52rehsd5.txt
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url: https://www.ncbi.nlm.nih.gov/pubmed/29073936/
doi: 10.1186/s13567-017-0472-z
summary:
  title: Evaluation of the efficacy of a commercial inactivated genogroup 2b-based porcine epidemic diarrhea virus (PEDV) vaccine and experimental live genogroup 1b exposure against 2b challenge The aim of this study was to compare the ability of G1b-based live virus exposure against use of a commercial G2b–based inactivated vaccine to protect growing pigs against G2b challenge. Under the study conditions a commercial inactivated G2b-based vaccine protected pigs against G2b challenge, as evidenced by reduction of PEDV RNA in feces for 3–4 logs during peak shedding and a shorter viral shedding duration. The oral, but not the intramuscular, experimental G1b-based live virus exposure induced a high anti-PEDV IgA response prior to challenge, which apparently did not impact PEDV shedding compared to POS-CONTROL pigs. Anti-PEDV IgA antibodies in serum samples were first detected in 8/8 EXP-ORAL-1b pigs at dpv 14 ( Figure 3 ).

title:
  Evaluation of the efficacy of a commercial inactivated genogroup 2b-based porcine epidemic diarrhea virus (PEDV) vaccine and experimental live genogroup 1b exposure against 2b challenge

abstract:
  Porcine epidemic diarrhea virus strains from the G1b cluster are considered less pathogenic compared to the G2b cluster. The aim of this study was to compare the ability of G1b-based live virus exposure against use of a commercial G2b–based inactivated vaccine to protect growing pigs against G2b challenge. Thirty-nine PEDV naïve pigs were randomly divided into five groups: EXP-IM-1b (intramuscular G1b exposure; G2b challenge), EXP-ORAL-1b (oral G1b exposure; G2b challenge), VAC-IM-2b (intramuscular commercial inactivated G2b vaccination; G2b challenge), POS-CONTROL (sham-vaccination; G2b challenge) and NEG-CONTROL (sham-vaccination; sham-challenge). Pigs were vaccinated/exposed at 3 weeks of age (day post-vaccination 0, dpv 0), VAC-IM-2b pigs were revaccinated at dpv 14, and the pigs were challenged at dpv 28. Among all groups, VAC-IM-2b pigs had significantly higher anti-PEDV IgG levels on dpv 21 and 28 while EXP-ORAL-1b pigs had significantly higher anti-PEDV IgA levels on dpv 14, 21, 28 and 35. EXP-ORAL-1b also had detectable IgA in feces. Intramuscular PEDV exposure did not result in a detectable antibody response in EXP-IM-1b pigs. The fecal PEDV RNA levels in VAC-IM-2b pigs were significantly lower 5–7 days after challenge compared to the POS-CONTROL group. Under the study conditions a commercial inactivated G2b-based vaccine protected pigs against G2b challenge, as evidenced by reduction of PEDV RNA in feces for 3–4 logs during peak shedding and a shorter viral shedding duration. The oral, but not the intramuscular, experimental G1b-based live virus exposure induced a high anti-PEDV IgA response prior to challenge, which apparently did not impact PEDV shedding compared to POS-CONTROL pigs.


id: cord-345940-adg264vb
author: Wanitchang, Asawin
date: 2018-08-22
url: https://doi.org/10.1007/s00705-018-4001-9
doi: 10.1007/s00705-018-4001-9
title:
  Characterization of influenza A virus pseudotyped with the spike protein of porcine epidemic diarrhea virus

abstract:
  The coronavirus spike protein and the influenza virus hemagglutinin are class I viral membrane fusion proteins. While the two proteins display strong structural conservation and the mechanisms underlying membrane fusion are similar, they share no sequence similarity. Whether they are functionally interchangeable is currently unknown. In this study, we constructed scIAV-S, a single-cycle influenza A virus pseudotyped with the spike protein of porcine epidemic diarrhea virus (PEDV), and demonstrated that this virus could infect cultured cells and trigger massive syncytium formation. Treatment with endocytosis inhibitors did not affect syncytium formation by infected cells. Moreover, the infectivity of scIAV-S was associated with the degree of cell adaptation of PEDV-S. Intriguingly, scIAV-S lacking functional neuraminidase (NA) exhibited substantially higher infectivity, suggesting a pivotal role of the sialic acid in the binding/entry of PEDV. Together, scIAV-S offers a robust platform for the investigation of the entry mechanism of PEDV or, possibly, of other coronaviruses.


id: cord-346457-2mq2aije
author: Wang, Zhilin
date: 2020-06-22
txt: ./txt/cord-346457-2mq2aije.txt
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url: https://doi.org/10.1186/s12917-020-02424-1
doi: 10.1186/s12917-020-02424-1
summary:
  RESULTS: In the study, a differential one-step quantitative real-time fluorescent reverse transcription recombinase polymerase amplification (real-time RT-RPA) method based on the PEDV universal real-time RT-RPA assay was established according to the ORF1 deletion sequences of three classical attenuated vaccine strains (PEDV attenuated vaccine KC189944, attenuated CV777 and DR13) and five Vero cell-adapted isolates (JS2008, SDM, SQ2014, SC1402, HLJBY), which could effectively differentiate PEDV classical attenuated vaccine strains from wild-type strains (PEDV classical wild strains and variant strains). The detection limits of PEDV RNA in the both PEDV real-time RT-RPA assays were 300 copies within 20 min at 39 °C, and the detection limits of classical attenuated vaccine strain CV777, Vero-cell-adapted isolate JS2008, and PEDV wild-type strain DX were 10(0.5) TCID(50)/100 μL, 10(1.1) TCID(50)/100 μL, and 10(1.2) TCID(50)/100 μL, respectively. CONCLUSION: This RPA method we developed is simple, time-effective, and safe and provides a reliable technical tool for the differential diagnosis and clinical epidemic surveillance of PEDV classical attenuated vaccine strains and wild-type strains.

title:
  Rapid differentiation of PEDV wild-type strains and classical attenuated vaccine strains by fluorescent probe-based reverse transcription recombinase polymerase amplification assay

abstract:
  BACKGROUND: Porcine epidemic diarrhea virus (PEDV), an intestinal coronavirus that causes acute diarrhea and high mortality in suckling piglets, can result in high economic losses in the swine industry. In recent years, despite the use of China’s current vaccine immunization strategy, multiple types of PEDV strains were still found in immunized swine herds. Our research aims to explore a new rapid differentiation method to distinguish the different types of PEDV strains and assess the safety evaluation of classical attenuated vaccine strains in swine herds. RESULTS: In the study, a differential one-step quantitative real-time fluorescent reverse transcription recombinase polymerase amplification (real-time RT-RPA) method based on the PEDV universal real-time RT-RPA assay was established according to the ORF1 deletion sequences of three classical attenuated vaccine strains (PEDV attenuated vaccine KC189944, attenuated CV777 and DR13) and five Vero cell-adapted isolates (JS2008, SDM, SQ2014, SC1402, HLJBY), which could effectively differentiate PEDV classical attenuated vaccine strains from wild-type strains (PEDV classical wild strains and variant strains). The detection limits of PEDV RNA in the both PEDV real-time RT-RPA assays were 300 copies within 20 min at 39 °C, and the detection limits of classical attenuated vaccine strain CV777, Vero-cell-adapted isolate JS2008, and PEDV wild-type strain DX were 10(0.5) TCID(50)/100 μL, 10(1.1) TCID(50)/100 μL, and 10(1.2) TCID(50)/100 μL, respectively. Both assays were highly specific for PEDV, showing no cross-reactivity with other enteral viruses. CONCLUSION: This RPA method we developed is simple, time-effective, and safe and provides a reliable technical tool for the differential diagnosis and clinical epidemic surveillance of PEDV classical attenuated vaccine strains and wild-type strains.


id: cord-346872-k5d5793a
author: Yuan, Peng
date: 2018-09-03
url: https://www.ncbi.nlm.nih.gov/pubmed/30176660/
doi: 10.1159/000492424
title:
  Three Main Inducers of Alphacoronavirus Infection of Enterocytes: Sialic Acid, Proteases, and Low pH

abstract:
  Transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are similar coronaviruses, causing diseases characterized by vomiting, diarrhea, and death from severe dehydration in piglets. Thus, they have caused huge losses to the swine-breeding industry worldwide. Nowadays, they are easily transmitted among the continents via vehicles, equipment, and cargo. Both viruses establish an infection in porcine enterocytes in the small intestine, and their spike (S) proteins play a key role in the virus-cell binding process under unfavorable conditions when the intestine with a low pH is filled with a thick layer of mucus and proteases. Sialic acid, proteases, and low pH are three main inducers of coronavirus infection. However, the details of how sialic acid and low pH affect virus binding to the host cell are not determined, and the functions of the proteases are unknown. This review emphasizes the role of three factors in the invasion of TGEV and PEDV into porcine enterocytes and offers more insights into Alphacoronavirus infection in the intestinal environment.


id: cord-347475-ttmactz0
title: Outbreak of Porcine Epidemic Diarrhea Virus in Portugal, 2015
author: Mesquita, J. R.
date: 2015-09-07
url: https://www.ncbi.nlm.nih.gov/pubmed/26344708/
doi: 10.1111/tbed.12409
abstract:
  An outbreak of porcine epidemic diarrhea virus (PEDV) in the South of Portugal in January 2015 and the spread of PEDV northwards in the territory are described. Comparative analysis of the amplified sequences showed a very high (99.0%) identity with the PEDV variant most recently reported in the United States and also show complete (100%) identity to the strains recently reported in Germany, supporting the hypothesis that a unique strain is currently circulating in Europe. The origin of this PEDV variant still needs to be elucidated and further studies in the remaining European countries may contribute to the knowledge.


id: cord-348669-mizygp4j
author: Beall, Anne
date: 2016-01-05
url: https://doi.org/10.1128/mbio.01451-15
doi: 10.1128/mbio.01451-15
title:
  Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A

abstract:
  Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic alphacoronavirus. In the United States, highly virulent PEDV strains cause between 80 and 100% mortality in suckling piglets and are rapidly transmitted between animals and farms. To study the genetic factors that regulate pathogenesis and transmission, we developed a molecular clone of PEDV strain PC22A. The infectious-clone-derived PEDV (icPEDV) replicated as efficiently as the parental virus in cell culture and in pigs, resulting in lethal disease in vivo. Importantly, recombinant PEDV was rapidly transmitted to uninoculated pigs via indirect contact, demonstrating virulence and efficient transmission while replicating phenotypes seen in the wild-type virus. Using reverse genetics, we removed open reading frame 3 (ORF3) and replaced this region with a red fluorescent protein (RFP) gene to generate icPEDV-ΔORF3-RFP. icPEDV-ΔORF3-RFP replicated efficiently in vitro and in vivo, was efficiently transmitted among pigs, and produced lethal disease outcomes. However, the diarrheic scores in icPEDV-ΔORF3-RFP-infected pigs were lower than those in wild-type-virus- or icPEDV-infected pigs, and the virus formed smaller plaques than those of PC22A. Together, these data describe the development of a robust reverse-genetics platform for identifying genetic factors that regulate pathogenic outcomes and transmission efficiency in vivo, providing key infrastructural developments for developing and evaluating the efficacy of live attenuated vaccines and therapeutics in a clinical setting.


id: cord-349249-jwvz1ux2
author: Singh, Gagandeep
date: 2019-10-22
txt: ./txt/cord-349249-jwvz1ux2.txt
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url: https://doi.org/10.3389/fvets.2019.00347
doi: 10.3389/fvets.2019.00347
summary:
  To combine the safety and efficacy advantages of inactivated and attenuated PEDV vaccines, respectively, in this study, we tested the hypothesis that subjecting PEDV virions to heat treatment at 44°C for 10 min to reversibly unfold structural proteins, followed by exposure to RNAse to fragment the genome, would result in a vaccine preparation with intact viral structure/antigenicity but highly diminished replicative abilities. to that of the spike protein-specific Abs. Strong virus neutralizing Ab responses, were detected in animals vaccinated with the heat and RNAse treated virions but not in the pigs which received the irradiated viral vaccine. Although the heat and RNAse treated virus culture was amplified after 3 passages in cell culture (Figure 2) , the absence its detection by RT-qPCR (Figure 4) , or immunohistochemistry (Table 1 and Figure 5 ) and the lack of strong Ab responses to the non-structural NP (Figure 3) , in vaccinated pigs prior to challenge indicates that active vaccine viral replication was absent in the host or was undetectable by the techniques used.

title:
  A Minimally Replicative Vaccine Protects Vaccinated Piglets Against Challenge With the Porcine Epidemic Diarrhea Virus

abstract:
  Porcine epidemic diarrhea virus (PEDV), is an economically important enteric coronavirus, with over a 90% mortality rate in neonatal piglets. The virus emerged in the US in 2013, resulting in severe production losses. Effective vaccine development against PEDV is a challenge. Inactivated vaccines are of questionable efficacy. Attenuated vaccines, while more effective, require a relatively long lead development time, are associated with safety concerns and are also unable to prevent new field outbreaks. To combine the safety and efficacy advantages of inactivated and attenuated PEDV vaccines, respectively, in this study, we tested the hypothesis that subjecting PEDV virions to heat treatment at 44°C for 10 min to reversibly unfold structural proteins, followed by exposure to RNAse to fragment the genome, would result in a vaccine preparation with intact viral structure/antigenicity but highly diminished replicative abilities. We expected the vaccine to be both safe and effective in a piglet challenge model. Following the heat and RNAse treatment, PEDV virions had an intact electron microscopic ultrastructure and were amplified only in the 3rd passage in Vero cells, indicating that diminished replication was achieved in vitro. Strong PEDV spike-protein specific and virus neutralizing antibody responses were elicited in vaccinated piglets. Upon challenge, all vaccinated pigs were protected against fecal viral shedding and intestinal pathology, while the unvaccinated controls were not. The vaccine virus was not detected in the fecal matter of vaccinated pigs prior to challenge; nor did they develop intestinal lesions. Thus, the described approach has significant promise in improving current approaches for PEDV immunization.


id: cord-353245-es7b1rs0
author: Song, Deping
date: 2015-03-19
txt: ./txt/cord-353245-es7b1rs0.txt
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url: https://doi.org/10.1371/journal.pone.0120310
doi: 10.1371/journal.pone.0120310
summary:
  Compared to the PEDV CV777 strain, CH/JX-1/2013 and CH/JX-2/2013 had some unique genetic characteristics in the proximal region of the 5''-UTRs. Phylogenetic analysis of the complete genomes and the structural proteins revealed that CH/JX-1/2013 and CH/JX-2/2013 had a close relationship with post-2010 Chinese PEDV strains and US strains identified in 2013. Genetic characteristics were observed between the two groups: 1) compared to genome sequences of the members in G1, four insertions, 20803G, 20810CAGGGTGTCAA20820, 20830G, 21042AAT21044 and two deletions, 20842A, 21097CGTGAT21102, existed in the N-terminal domain (NTD) of the S protein in G 2 members; 2) the three field PEDV strains of JS2008, JS2008new and SD-M together with two attenuated PEDV strains, DR13 and vaccine_KC189944, were clustered into subgroup 1b. Notably, an amino acid substitution was found in the middle of one neutralizing epitope Phylogenetic trees based on the complete genome, aa sequences of structural proteins and ORF3 of PEDV strains. Sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (PEDV) strains in China

title:
  Molecular Characterization and Phylogenetic Analysis of Porcine Epidemic Diarrhea Viruses Associated with Outbreaks of Severe Diarrhea in Piglets in Jiangxi, China 2013

abstract:
  Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is a highly contagious, acute enteric viral disease of swine characterized by vomiting, watery diarrhea, dehydration and death. To identify and characterize the field PEDVs associated with the outbreaks of severe diarrhea in piglets in Jiangxi, 2013, the complete genome sequences of two representative strains of PEDV, designated CH/JX-1/2013 and CH/JX-2/2013, were determined and analyzed. The genome sequences of both emergent Jiangxi PEDV strains, CH/JX-1/2013 and CH/JX-2/2013, were 28,038 nucleotides in length excluding 3’ poly (A) tail. Compared to the PEDV CV777 strain, CH/JX-1/2013 and CH/JX-2/2013 had some unique genetic characteristics in the proximal region of the 5´-UTRs. Phylogenetic analysis of the complete genomes and the structural proteins revealed that CH/JX-1/2013 and CH/JX-2/2013 had a close relationship with post-2010 Chinese PEDV strains and US strains identified in 2013. The nucleotide identity between the two Jiangxi strains (CH/JX-1/2013 and CH/JX-2/2013) and 30 strains of PEDV identified ante-2010 and post-2010 ranged from 96.3–97.0% and 97.3–99.7%, respectively. Multiple nucleotide and deduced amino acid mutations were observed in the ORF1a/b, S, ORF3, E, M and N genes among the current field PEDV strains when compared to the CV777 strain. Some of the mutations altered the amino acid charge and hydrophilicity, and notably, there was an amino acid substitution in the middle of one neutralizing epitope (L1371I) of the S gene of both CH/JX-1/2013 and CH/JX-2/2013. Taken together, the accumulated genetic variations of the current field PEDV strains might have led to antigenic changes of the viruses, which might confer the less effectiveness or failure of the CV777-based vaccines currently being widely used in Jiangxi, China.


id: cord-354052-x4ckzw64
title: Manipulation of the Porcine Epidemic Diarrhea Virus Genome Using Targeted RNA Recombination
author: Li, Chunhua
date: 2013-08-02
url: https://doi.org/10.1371/journal.pone.0069997
doi: 10.1371/journal.pone.0069997
abstract:
  Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines.


id: cord-354729-dpaz01np
author: Huan, Changchao
date: 2019-08-22
txt: ./txt/cord-354729-dpaz01np.txt
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mime: text/plain
url: https://doi.org/10.1111/tbed.13321
doi: 10.1111/tbed.13321
summary:
  title: Characterization and evolution of the coronavirus porcine epidemic diarrhoea virus HLJBY isolated in China A strain of porcine epidemic diarrhoea virus (PEDV), namely HLJBY, was isolated in Heilongjiang province, China. To investigate the molecular characteristics of PEDV HLJBY strain, UTR (5'' and 3'') and the nucleotide and predicted amino acid sequences of the nonstructural and structural proteins (replicase ORF1a/1b, S, ORF3, E, M, N) of PEDV HLJBY strain were compared with CV777. 399 nt deletions exhibited in ORF3 of PEDV HLJBY (Figure 2d ), and nucleotide sequence identity was 91.7%, and the amino acid sequence identity was 90.1% (Table 3 ). To investigate the evolution of PEDV, phylogenetic analysis based on the entire genomic nucleotide sequences of PEDV HLJBY strain The group Ⅲ was further divided into subgroup Ⅲa and Ⅲb. Characterization and evolution of the coronavirus porcine epidemic diarrhoea virus HLJBY isolated in China

title:
  Characterization and evolution of the coronavirus porcine epidemic diarrhoea virus HLJBY isolated in China

abstract:
  A strain of porcine epidemic diarrhoea virus (PEDV), namely HLJBY, was isolated in Heilongjiang province, China. To provide insight into the understanding of the phylogenetic and the current epidemiological status of PEDV, PEDV HLJBY was compared with CV777 and other PEDV strains deposited in the GenBank. The homology between the entire genomic nucleotide sequences of PEDV HLJBY and CV777 was 97.7%. The homology of M gene was the highest (99.0%). However, the homology of ORF3 gene was 97.7%, and protein of ORF3 was 90.1%. In addition, HLJBY showed the highest nucleotide identity (99.9%) with PEDV‐SX/China/2017 strain and lowest similarity (91.2%) to PEDV/Belgorod/dom/2008 strain. We analysed the changes in S gene and its protein of PEDV HLJBY with 65 historic PEDV strains. The highest nucleotide identity was 99.9% compared with PEDV‐SX/China/2017 strain, and the lowest nucleotide identity was 60.0% compared with PEDV/Belgorod/dom/2008 strain. The length of deduced amino acid sequences of S proteins varied from 1,372 to 1,390 amino acids (aa). Compared with most aa sequences of S proteins, HLJBY exhibited 5 aa deletions (position 55, 59–61, 144). Analysis and comparison of open reading frame 3 (ORF3) proteins between HLJBY strain and other PEDV strains were also focused in this study. We revealed that the length of deduced amino acid sequences of ORF3 proteins was 80–224 aa among tested strains and the identity of HLJBY ORF3 amino acids with other PEDV strains was 71.4%–98.9%. ORF3 protein of both HLJBY strain and PEDV‐SX/China/2017 strain consists of 91 aa, with 133 aa deletions at their C' end in relation to the other tested PEDV strains. The phylogenetic tree based on different proteins or genes resulted in different phylogenetic groups. For pathogenicity evaluation of PEDV HLJBY strain, colostrum deprivation piglets were challenged with PEDV HLJBY, and PEDV reference strain CV777 as a control, the results showed that animals challenged with either of these PEDV strains developed diarrhoea, and histopathological examination of small intestines of challenged animals showed acute viral enteritis with villous atrophy in either PEDV HLJBY‐P10 or PEDV CV777‐P8 inoculated piglets.


id: cord-355119-sdg9zdc1
author: Lin, Huixing
date: 2016-04-22
url: https://doi.org/10.1186/s12985-016-0529-z
doi: 10.1186/s12985-016-0529-z
title:
  Epidemic strain YC2014 of porcine epidemic diarrhea virus could provide piglets against homologous challenge

abstract:
  BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is the main causative agent of porcine epidemic diarrhea (PED). Since December 2010, a large-scale outbreak of diarrhea has been observed in swine farms in China. Accumulated evidence indicates that this large-scale outbreak of diarrhea were caused by highly virulent PEDV variants. METHODS: A PEDV strain, YC2014, was isolated from intestinal samples of suckling piglets with acute diarrhea in 2014. The complete genomic sequence of YC2014 and the nucleotide sequence of S gene were aligned with sequences of published isolates using MEGA 5.1 software. The immune protective efficiency of YC2014 were determined by testing PEDV neutralizing antibodies in sera, the colostrum and the milk on 7th day after farrowing of the immunized sows. The diarrhea symptoms of piglets after challenge were also observed. RESULTS: Phylogenetic analysis of the complete genomic sequence of YC2014 and the nucleotide sequence of S gene demonstrated that the YC2014 PEDV strain was clustered with the PEDV epidemic strains, with >99 % nucleotide identity to these PEDV strains. The S gene sequence of YC2014 shared only 93.9 % ~ 94.4 % identities with classical CV777, DR13 and JS2008 strains, with 15 nucleotide insertion in three sites and three nucleotide deletion in one site. The amino acid (AA) sequence of S gene of YC2014 shared only 92.8 % ~ 93.4 % identities with classical CV777, DR13 and JS2008 strains, with 5 AA insertion in two sites and 1 AA deletion in one site. In the immune protective efficiency tests, the neutralizing antibody titers in sera, the colostrum and the milk on 7th day after farrowing of the inactivated YC2014 PEDV strain immunized group were significantly higher than the inactivated CV777 immunized group and the inactivated DR13 immunized group (P < 0.05). The traditional inactivated PEDV vaccines made from CV777 or DR13 could not protect piglets from YC2014 challenge, while inactivated YC2014 could provide piglets with 100 % protection against YC2014 challenge. CONCLUSIONS: The results showed that, great antigenicity variation had occurred to this YC2014 PEDV strain. The YC2014 PEDV strain could provide piglets against homologous challenge. It is critical for future pathogenic and antigenic studies, as well as for the development of effective preventive and control vaccines against PEDV.


id: cord-355465-qjtifwhd
author: Van Diep, Nguyen
date: 2018-03-14
url: https://doi.org/10.1186/s12917-018-1409-0
doi: 10.1186/s12917-018-1409-0
title:
  Molecular characterization of US-like and Asian non-S INDEL strains of porcine epidemic diarrhea virus (PEDV) that circulated in Japan during 2013–2016 and PEDVs collected from recurrent outbreaks

abstract:
  BACKGROUND: Since late 2013, porcine epidemic diarrhea virus (PEDV) has reemerged in Japan and caused severe economic losses to the swine industry. Although PEDV vaccines have been used widely, the disease has swept rapidly across the county, and is commonly observed in PED-vaccinated farms, and has recurred in domestic herds. To better understand PEDVs responsible for the reemerging outbreaks in Japan, full-length spike (S), membrane (M), and nucleocapsid (N) genes of 45 PEDVs collected in Japan during 2013–2016, were sequenced and analyzed. RESULTS: Phylogenetic analysis based on S gene sequences revealed that all the recent field PEDVs were genetically distinct from the classical Japanese strains, and were classified into three genotypes: North American (NA), S INDEL, and Asian non-S INDEL. Our data suggested a possibility that multiple parental PEDV strains were introduced into Japan from abroad at the same time or similar times. The newly identified Japanese strains showed the closest relationship to the US strains. Two sublineages of Japanese strains circulating in Japan were similar to two sublineages identified in the US, suggesting common ancestors for these strains. In comparison with two vaccine strains used in Japan, the field strains had various changes in epitope regions, glycosylation sites, and phosphorylation sites. These substitutions, particularly observed in epitope regions of the S (521, 553, 568, and 570), M (5), and N (123, 252, and 255) proteins, may have affected antigenicity and vaccine efficacy, resulting in an unsuccessful PEDV control. Sequence comparisons between PEDVs collected from primary and secondary outbreaks in three herds revealed that the disease has developed to an endemic stage in which PEDV could persist for nearly two years in the herds or local regions, causing subsequent epidemics. CONCLUSIONS: These results elucidate the genetic characteristics, origin, and molecular epidemiology of PEDVs circulating in Japan, as well as the PEDV strains causing recurrent outbreaks. This study provides a better insight into the PEDVs responsible for recent outbreaks in Japan, and could potentially help to develop measures for controlling and preventing the disease. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12917-018-1409-0) contains supplementary material, which is available to authorized users.


id: cord-355991-4zu69e0y
author: Piñeyro, Pablo Enrique
date: 2018-09-24
url: https://www.ncbi.nlm.nih.gov/pubmed/30249258/
doi: 10.1186/s12917-018-1615-9
title:
  First retrospective studies with etiological confirmation of porcine transmissible gastroenteritis virus infection in Argentina

abstract:
  BACKGROUND: In 2014, a notification of porcine transmissible gastroenteritis virus (TGEV) was made by the National Services of Animal Health of Argentina (SENASA) to the World Organization of Animal Health (OIE). The notification was based on a serological diagnosis in a small farm with a morbidity rate of 2.3% without enteric clinical signs. In order to determine if TGEV was circulating before the official report, a retrospective study on cases of neonatal diarrhea was performed. The selection criteria was a sudden increase in mortality in 1- to 21-day-old piglets with watery diarrhea that did not respond to antibiotics. Based on these criteria, three clinical cases were identified during 2010–2015. RESULTS: All animals that were evaluated presented histological lesions consistent with enteric viral infection. The feces and ultrathin sections of intestine that were evaluated by electron microscopy confirmed the presence of round particles of approximately 80 nm in size and characterized by finely granular electrodense nucleoids consistent with complete particles of coronavirus. The presence of the TGEV antigen was confirmed by monoclonal specific immunohistochemistry, and final confirmation of a metabolically-active virus was performed by in situ hybridization to detect a TGE mRNA encoding spike protein. All sections evaluated in this case were negative for PEDV and rotavirus A. CONCLUSIONS: This is the first case series describing neonatal mortality with etiological confirmation of TGEV in Argentina. The clinical diagnosis of TGEV infections in endemic regions is challenging due to the epidemiological distribution and coinfection with other enteric pathogens that mask the clinical presentation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12917-018-1615-9) contains supplementary material, which is available to authorized users.


