key: cord-0004925-hhge62sl authors: Remond, M.; Boireau, P.; Lebreton, F. title: Partial DNA cloning and sequencing of a canine parvovirus vaccine strain: application of nucleic acid hybridization to the diagnosis of canine parvovirus disease date: 1992 journal: Arch Virol DOI: 10.1007/bf01309589 sha: 01c95cf315d321462af88216191620030ea50439 doc_id: 4925 cord_uid: hhge62sl The cloning and sequencing of anEco RI-Pst I fragment derived from the replicative form of a canine parvovirus (CPV) vaccine strain are reported. The variability of the 5′ end of NS 1 protein gene in the genome is confirmed by comparison with previously determined DNA sequences. A 15 nucleotide deletion was also observed in this vaccine strain. In order to improve CPV diagnosis, radioactively labelled RNA or DNA and biotin labelled DNA obtained by random priming of the recombinant plasmid were used as probes mainly on gut or stool samples from naturally infected dogs. Results of filter hybridization correlated well with histopathological diagnosis of parvovirus infection and with hemagglutination tests performed on dog faeces. We propose that nucleic acid hybridization may be an alternative diagnostic method to ascertain the presence of CPV, especially in frozen samples. Canine parvovirus (CPV) like other parvoviruses contains a linear 5 kilobase (kb) single stranded DNA (ss DNA). The viral genome encodes two nonoverlapping transcription units 130]. When replication occurs, DNA is converted into double stranded replicative forms (RF) . Restriction sites have been mapped [22] on the genome and the nucleotide sequence has been determined 1 -30, 32] . Canine parvovirus, in spite of vaccination, remains an important cause of disease and is often implicated in fatal disease in young puppies. CPV diagnosis is best achieved with hemagglutination test on faeces or by the detection of histological changes in gut mucosa 1, 7, 24, 28] . Alternatively, virus can be isolated in cell culture from various organs, but this method is reported to be much less sensitive because of the high lytic properties of the intestinal content. So, even if tests are available CPV diagnosis may be difficult in some cases. A sensitive CPV diagnosis test based on viral nucleic acid hybridization has been developed. The Eco RI-Pst I restriction fragment of the CPV replicative form DNA has been cloned into the multiple cloning site of the pT7T3 18 U plasmid. Viral nucleic acid hybridization was realized using radioactively labelled DNA or RNA probes and also a biotin labelled DNA probe. The biotin labelled probe was found to be 10 fold less sensitive. The cloned DNA was sequenced and compared to the previously described CPV sequences. Short deletion and point mutations were observed, which emphasised the high variability of C terminal region of the non structural (NS 1) protein gene. Cells used for virus propagation were freshly seeded Crandell feline kidney cells maintained with Eagles's minimum essential medium supplemented with 10% foetal bovine serum. The CPV strain used in the present study was derived from the Carmichael strain (CPVb) partially sequenced at passage 88 [8, 32] . It was obtained from a commercial vaccine at passage 108 and six additional passages in cell culture were performed before DNA cloning. Passage 108 and passage 114 were further designated as CPV-b 108 and CPVb 114. Feline panleukopenia virus (FPLV), porcine parvovirus, mink aleutian disease virus (Gorham strain) and Derzsy goose virus (kindly supplied by V. Marius, Laboratoire Central de Recherches Avicole et Porcine, Ploufragan) were used to test the probe specificity. Organs and faeces were collected from diseased puppies with parvovirus-related symptoms. Some of them were kindly supplied by A. MoraiUon, Veterinary School, Maisons-Alfort. Organs from 3 kittens with panleukopenia-like symptoms were also collected and included in this study. Histological analysis or hemagglutination test on faeces were performed on an aliquot of each sample. Samples were then stored at -2 0 °C before being processed. The replicative form of CPV-b 114 DNA was extracted by a modified Hirt procedure as described by McMaster et al. [14, 21] . Viral single stranded DNA was either prepared from purified viral particles [26] or directly from infected cell supernatants. Virus was then treated with 0.2% SDS and proteinase K (50 ~tg/ml) for 2 h at 37 °C, followed by a phenol chloroform extraction and ethanol precipitation. Viral DNA was extracted from organs and faeces of diseased animals as described by Orth [25] . Gut, spleen and faeces were minced and left in lysis buffer (10 mM Tris pH 8, 100 mM NaC1, 50 mM EDTA, SDS 0.5 %) for 1 h at room temperature. Proteinase K was added (1001.tg/ml) and samples were incubated for 2 h at 37 °C. After clarification, the NaC1 concentration of the supernatant was adjusted to 1 M and mixtures were kept on ice overnight. The supernatant was centrifuged for 1 h at 10,000 rpm and treated with an equal volume of phenol for 1 h at room temperature. It was then treated by chloroform and precipitated by ethanol. The DNA pellet was suspended in TE with RNase (20 lag/ml) for 30 min at 37 °C, and 50 gg of this treated DNA were spotted onto a nitrocellulose filter after denaturation by sodium hydroxide. Partial DNA cloning and sequencing of a canine parvovirus vaccine strain 259 DNA cloning DNA was digested with EcoRI and PstI (Boehringer) and ligated into the pT7T3 18U plasmid (Pharmacia). Eseherichia coli NM 522 was transformed with the recombined plasmid as described by Hanahan [13] . Recombinants were identified by in situ hybridization of white colony replicas with a radioactive labelled viral probe obtained from purified virions. The hybridization procedure was performed as described by Maniatis etal. [19] . Recombinant plasmid DNA was also further analyzed by agar gel electrophoresis after digestion with Eco RI and Pst I or with Hind III. DNA sequencing M 13 dideoxynucleotide sequencing was carried out as already described [5] . For direct sequencing of denatured plasmid DNA, we used synthetic primers (Biosearch 8600 apparatus) [17] with the Sequenase Kit (USB). Sequence data were analyzed by using the Microgenie (Beckman, 1988) and PC Gene (Intellegenetics, 1990) computer programs. Hybridization were carried out overnight at 420C in 50% formamide, 5 x SSC (1 x SSC: 150mM sodium chloride, 15 mM sodium citrate), 1 x Denhardt's solution (Denhardt's solution: 1% polyvinyl pyrrolidone, 1% Ficoll, 1% bovine serum albumin), 0.5% SDS and 10 gg/ml of sonicated calf thymus DNA (Sigma). 10% dextran sulfate was also added for biotinylated probe. Filters were washed twice in 0.1% SSC, 0.1% SDS for 1 h at 42 °C. Radioactive filters were exposed to X-ray film (Kodak XAR) at -7 0 °C for 12 h. Biotin labelled probe was detected with streptavidin-alkaline phosphatase conjugate (BRL). After 260 M. Remond etal. incubation with a luminescent substrate, PPD (4 methoxy 4-3-phosphate phenyl spiro 1-2-dioxetane 3.2' adamantan), as recommended by the supplier (Photogene-BRL), light emission was detected by autoradiography for 5 rain on X-ray film. Eight recombinant clones containing CPV sequences were selected by colony hybridization, Six clones contained an insert of the 2 kb; as expected the other two contained 1.5 kb and 1.3 kb inserts. All clones hybridized with purified viral Partial DNA cloning and sequencing of a canine parvovirus vaccine strain It is noteworthy that sequence analysis of Hind III fragment allowed differentiation between the four CPV strains a See [20] b Reversions c Consistent differences between CPV-b 114 and the other CP¥ strains d Unique difference between CPV-N and CPV Parrish del Deletion nc Non coding nd Not done In brackets, amino acids probe after restriction enzyme digestion and Southern transfer. As predicted from previously sequenced CPV [30] , digestion of the six 2kb recombinant plasmids with Hind Ill yielded the predicted 660 bp fragment (Fig. 1) . The sequence of both strands of cloned DNA was determined for three different plasmids after subcloning into the M 13 phage. The resulting 1947 nucleotide sequence represented about 37% of the CPV genome. As expected, it included parts of two major open reading frames (ORFs) which are in the same phase corresponding to the 3' end of NSI gene and the first 785 nucleotides of the VP 1/VP 2 gene (Fig. 1 ) [30] . Comparison of this sequence with that of three other CPV strains [27, 30, 32] and one FPLV strain [20] revealed point mutations between the different isolates (Table 1) . Furthermore, a 15 nucleotides deletion, located in the NS 1 gene, and extending from nucleotide 2040 to nucleotide 2056 of the Norden CPVs strain (CPV-N) sequence [30] was observed in the cloned DNA. To elucidate whether the deletion observed was originally in the vaccine or was generated by cell culture passages in our laboratory, DNA sequence was determined on CPV-b 108 after PCR amplification of a 1000 bp fragment including the deleted region and the two H i n d I I I restriction sites (Fig. 1) . PCR amplification products were then cleaved with Hind III, cloned in pT7T 318U and sequenced. The presence of the deletion in the original vaccine was thus confirmed. Consequently, the modified NS 1 gene did not alter viral replication in cell culture as CPV-b 108 could be multiplied without loss of infectivity. Application of 32p labelled DNA or RNA probe for routine diagnosis DNA from various organs of dogs and cats was extracted, spotted onto nitrocellulose and assayed with probes. Samples came from animals which died of parvovirus infection as could be deduced from histological examinations, expected for three of them which were classified as "suspect"and one gut sample taken from a puppy suffering from distemper which was chosen as a negative control. Out of 15 gut samples from diseased dogs, 13 were positive with DNA or RNA probes. Two spleen extracts were tested: only one hybridized. Four faecal extracts which showed specific hemagglutinating properties hybridized strongly and the fifth one from a suspect dog was negative. Two samples for panleukopenia diagnosis, one from the gut and the other, a mixture of spleen and kidney, did not hybridize. Results presented with the DNA probe in Fig. 3 were identical with the RNA probe (data not shown). Twofold dilutions of purified CPV-DNA were spotted in duplicate and assayed respectively with 32p labelled DNA probe and biotin labelled probe in order to determine the sensitivity of the non-radioactive probe. Results are shown in The vaccine strain used in this study was derived from the Carmichael strain (Fig. 5) . It underwent 26 additional passages in cell culture (CPV-b 114) before being cloned and sequenced. Eco RI and Pst I sites were chosen because they are perfectly conserved among different isolates of CPV and FPLV [20] . Moreover, the Eco RI-Pst I fragment covered partially non-structural protein genes which present a strong homology between some members of the family Parvoviridae, allowing its use for diagnosis of related diseases [-37] . Comparison with the previously published sequence of CPV-b strain [32] showed a high rate of point mutations and one deletion which could have been generated by cell culture passages. This result emphasises the high variability of the CPV genome and sequence comparison was further investigated on three other CPV strains [27, 30, 32] and one FPLV strain [20] . 22 point mutations were observed between the different CPV sequences and 15 between CPV-b 108 and FPLV (Table 1) . 10 reversions appeared in CPV-b 108 strain; the 10 nucleotidic changes were only observed in CPV-b at passage 88, compared to FPLV and no longer existed in the partially sequenced DNA of CPV-b 108. By contrast, no changes were observed in regulatory regions (polyadenylation sites, P38 and P45 promoters, potential splicing sites). Most of the coding mutations appeared in the C terminal part of NS 1 ( Table 1 ) and this is in contrast with the results published for Minute virus of mice [2] . The short deletion observed in CPV-b 108 and CPV-b 114 was also located in this part of the NS 1 gene. Short deletions often appeared in the noncoding part of the genome but were never described in the parvovirus NS 1 gene. This deletion, located 45 bases downstream of the P 38 promoter did not 266 M. Remond et al. R I F A F H G W N Y V K V C H A I C C V L N R Q G G K R N T V L F H 398 ET S K L T N F S L P D T R T C K I F A F H G W N Y V K V C H A I C C V L N R Q G G K R N T V L F H 447 E T S K L A N F S M A S T R T C R I F A E H G W N Y I K V C H A I C C V L N R Q G G K R N T V L F H 398 K P S M L P T F N I S N T R T C K I F S M H N W N Y I K C C H A I T C V L N R Q G G K R N T I L F H 397 D N T K L T N F D L A N S R T C Q I F R M H G W N W I K V C H A I A C V L N R Q G G K R N T V L F H 123 F E Q V M ---C I K D N K I V K L L L C Q N Y D P L L V G Q H V L K W I D K K C G K K N T L W F Y 327 A P H Y D ---C -Q G N L V F K L L N L Q G Y N P W Q V G H W L V M M L S K K T G K R N S T L F Y FPFNI~PNKNILWWEECIMT 359 alter the reading frame and defined a genetic marker for this vaccine strain. The N terminal part of the NS 1 gene appeared more conserved in the analyzed sequences. Furthermore, the alignment of NS 1 protein sequences from different parvovirus allowed identification of a highly conserved sequence already de-scribed as a homologous domain of proteins which used purines nucleotides (Fig. 6 ) [1] . The 2 kb insert has been used as probe on CPV and other parvoviruses available in the laboratory for cross-hybridization in order to measure to what extent the probe could be useful for other parvoviruses. Results were~as expected [23, 29] . CPV and FPLV are closely related but the sensitivity of the probe for FPLV is one hundred fold lower as measured by the difference in titer of the infectious particles detected. The sensitivity of the DNA CPV probe on homologous DNA is comparable to that reported for other viruses: herpesvirus [18] , adenovirus [12] and, rotavirus [10] . Probes for viruses in the family Parvoviridae have only been reported for the human B 19 parvovirus [4, 34] with higher sensitivity in terms of quantity of target DNA detected but the number of infectious particles detected is of the same order. In fact, there is a discrepancy between the number of infectious particles detected (104s CCIDs0) and the quantity of purified DNA spotted and detected: 0.3 ng DNA correspond to 10 s viral particles. This may be explained by the production of defective DNA genomes and/or a greater number of RF copies in the supernatants of infected cell culture which cannot be measured in terms of infectivity. RNA probes have been developped to overcome any background due to hybridization of the plasmid with bacteria present in stools and gut contents but we did not encounter such difficulty. Although RNA probes have been shown to offer up to tenfold more sensitivity than DNA probes, at least on RNA viruses such as enterovirus [15] and on DNA viruses such as B 19 parvovirus [34] , the results obtained in our study did not demonstrate any difference in the sensitivity and specificity of the two types of probes. Data presented on clinical samples demonstrated that CPV nucleic acid probes were effective for diagnosis of parvovirus disease even on specimens which had been stored under unappropriate conditions. We plan to simplify DNA extraction and to use this probe in combination with PCR to improve the sensitivity of the test. 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Boireau, Centre National d'Etudes V6t6rinaires et Alimentaires, Laboratoires Central de Recherches V6t~rinaires, 22 rue Pierre Curie, F-94703 We thank Dr. F. Fontaine and Dr. L. Grosgean for critical review of the manuscript, and V. Del Moral for excellent secretarial help.