Characterization of a parvovirus isolated from the diarrheic feces of a pig

A small DNA virus was isolated from the feces of a sow with diarrhea and identified as a parvovirus on the basis of its properties. The virus replicated preferentially in cell cultures of swine origin, including primary porcine thyroid gland and kidney cell cultures in which the cytopathic effect developed. The virus agglutinated erythrocytes of guinea pig, mouse and human group O but not these of chicken. The growth of the virus was inhibited by 5-iodo-2'-deoxyuridine. The virus was resistant to ether and heating at 56 degrees C for 30 min and stable at pH 3.0. The buoyant density of the infectious particles was 1.40 g/ml in CsCl density gradient, and the virions were 27 nm in diameter by electron microscopy. The viral protein seemed to be separated into four polypeptides with molecular weights of 81k, 70k, 66k and 62k daltons respectively. Cross serum neutralization test demonstrated that the virus was antigenically different from porcine parvovirus as well as bovine and canine parvoviruses. These findings and the survey on neutralizing antibody distribution indicated indirectly that another parvovirus which could be antigenically distinguished from well-known porcine parvovirus had been widespread among swine in Japan.     

Comparison of different in-house test systems to detect parvovirus in faeces of cats

In-house tests for the identification of faecal parvovirus antigen are now available. The majority of these are licensed for canine parvovirus only; but anecdotal information suggests that they will detect feline panleukopenia virus (FPV) as well. This prospective study was designed to compare five commercially available test systems. In total, 200 faecal samples from randomly selected healthy cats (148) and cats with diarrhoea (52) were tested and compared with the results of examination by electron microscopy. Ten cats were positive for FPV and all of these had diarrhoea. In-house canine parvovirus tests can be used to detect FPV. All tests were suitable to screen cats for faecal parvovirus excretion (positive predictive values for the Witness Parvo, the Snap Parvo, the SAS Parvo, the Fastest Parvo Strip, and the Speed Parvo were 100.0, 100.0, 57.1, 38.9, and 100%, respectively, negative predictive values for the Witness Parvo, the Snap Parvo, the SAS Parvo, the Fastest Parvo Strip, and the Speed Parvo were 97.4, 97.9, 98.9, 98.4, and 97.4%, respectively). In-house parvovirus tests may be positive up to 2 weeks after vaccination, and therefore, in recently vaccinated cats positive results do not necessarily mean infection.     

A solid-phase fluorescent immunoassay for detecting canine or mink enteritis parvoviruses in faecal samples

Mink enteritis virus (MEV) and canine parvovirus (CPV) were detected in faecal samples from experimentally or naturally infected minks and dogs, respectively, using antibody-coated polyacrylamide beads (immunobeads, IB) as the solid phase for immunofluorescence (IF) tests. The specificity and sensitivity of the immunobead assay (IBA) were studied by comparing it with an enzyme-linked immunoassay (ELISA), a haemagglutination (HA) test and an IF test using tissue cultures. The IBA was as sensitive as ELISA, but more sensitive than the HA test and the IF test. Furthermore, the use of IB as the matrix for the immunological reactions allows FITC- or enzyme-conjugated antibodies to be used as indicators of the reactions and a simultaneous investigation of several pathogenic agents.     

Unusual characteristics of a parvovirus isolated from a clinically ill steer

A parvovirus was isolated from the feces of an 8- and 9-month-old steer that died acutely with hemorrhagic diarrhea and microscopic evidence of a coccidial infection. The concurrent intestinal parasitism in this steer appeared to play a role in the development of clinical disease. The viral isolate was identified as a bovine parvovirus (BPV) on the basis of its size (22 nm) and icosahedral morphology, the neutralization of viral cytopathology by antiserum to BPV, a strong immunofluorescent reaction with fluorescein-labeled antiserum to BPV, and the inhibition of viral hemagglutination of guinea-pig erythrocytes by antiserum to BPV. Cell cultures infected with this isolate showed a slight nuclear fluorescent reaction with fluorescein-labeled antiserum to canine parvovirus, suggesting an antigenic relationship to canine parvovirus. Patterns of hemagglutination for this isolate with human erythrocytes from 20 donors of various blood types differed from those obtained with the reference Abinanti strain of BPV. These results indicate that blood from multiple donors of a species may be necessary to confirm the presence or absence of viral hemagglutinating activity with clinical isolates of BPV.     

Canine parvovirus serology: a collaborative assay

Fifteen laboratories were supplied with coded samples of canine sera for testing for the presence of antibodies against canine parvoviruses. One of these sera had been designated as a potential British standard canine parvovirus antiserum. Most of these laboratories were either providing a canine parvovirus serology service, or represented pharmaceutical companies which manufacture canine parvovirus vaccines for the United Kingdom market. No attempt was made to influence the test methods used. Thirteen of the laboratories used a haemagglutination inhibition test, three an enzyme-linked immunosorbent assay (ELISA), and two performed serum neutralisation tests. Three laboratories used two different techniques. Adequate analysis was possible only with the results of the haemagglutination inhibition tests. The variability of the results between laboratories could be partly controlled by the use of the standard serum. Much of the residual variability was associated with particular laboratories. The results from the vaccine manufacturers tended to be less variable than those from the diagnostic laboratories.     

Polymerase chain reaction analysis for viruses in paraffin-embedded myocardium from dogs with dilated cardiomyopathy or myocarditis

OBJECTIVE: To perform polymerase chain reaction (PCR) analysis on paraffin-embedded myocardium from dogs with dilated cardiomyopathy (DCM) and dogs with myocarditis to screen for canine parvovirus, adenovirus types 1 and 2, and herpesvirus. SAMPLE POPULATION: Myocardial specimens from 18 dogs with an antemortem diagnosis of DCM and 9 dogs with a histopathologic diagnosis of myocarditis were evaluated. PROCEDURE: Paraffin-embedded myocardial specimens were screened for viral genome by PCR analysis. Positive-control specimens were developed from cell cultures as well as paraffin-embedded tissue specimens from dogs with clinical and histopathologic diagnoses of viral infection with canine parvovirus, adenovirus types 1 and 2, and herpesvirus. The histologic characteristics of all myocardial specimens were classified regarding extent, location, and type of inflammation and fibrosis. RESULTS: Canine adenovirus type 1 was amplified from 1 specimen from a dog with DCM. Canine parvovirus, adenovirus type 2, and herpesvirus were not amplified from any myocardial specimens. Histologic analysis of specimens from dogs with DCM revealed variable amounts of fibrosis; myocardial inflammation was observed in 1 affected dog. Histopathologic analysis of specimens from dogs with myocarditis disclosed variable degrees of inflammation and fibrosis. CONCLUSIONS AND CLINICAL RELEVANCE: Viral agents canine parvovirus, adenovirus types 1 and 2, and herpesvirus are not commonly associated with DCM or active myocarditis in dogs. Additional studies evaluating for nucleic acid from viruses that less commonly affect dogs or different types of infectious agents may be warranted to gain insight into the cause of DCM and myocarditis in dogs.     

Effect of vaccination on parvovirus antigen testing in kittens

OBJECTIVE: To determine the frequency and duration of feline panleukopenia virus (FPV) vaccine-induced interference with fecal parvovirus diagnostic testing in cats. DESIGN: Prospective controlled study. ANIMALS: Sixty-four 8- to 10-week-old specific-pathogen-free kittens. PROCEDURES: Kittens were inoculated once with 1 of 8 commercial multivalent vaccines containing modified-live virus (MLV) or inactivated FPV by the SC or intranasal routes. Feces were tested for parvovirus antigen immediately prior to vaccination, then daily for 14 days with 3 tests designed for detection of canine parvovirus. Serum anti-FPV antibody titers were determined by use of hemagglutination inhibition prior to vaccination and 14 days later. RESULTS: All fecal parvovirus test results were negative prior to vaccination. After vaccination, 1 kitten had positive test results with test 1, 4 kittens had positive results with test 2, and 13 kittens had positive results with test 3. Only 1 kitten had positive results with all 3 tests, and only 2 of those tests were subjectively considered to have strongly positive results. At 14 days after vaccination, 31% of kittens receiving inactivated vaccines had protective FPV titers, whereas 85% of kittens receiving MLV vaccines had protective titers. CONCLUSIONS AND CLINICAL RELEVANCE: Animal shelter veterinarians should select fecal tests for parvovirus detection that have high sensitivity for FPV and low frequency of vaccine-related test interference. Positive parvovirus test results should be interpreted in light of clinical signs, vaccination history, and results of confirmatory testing. Despite the possibility of test interference, the benefit provided by universal MLV FPV vaccination of cats in high-risk environments such as shelters outweighs the impact on diagnostic test accuracy.     

Mapping of porcine parvovirus DNA and development of a diagnostic DNA probe

Dimeric and monomeric replicative forms of DNA of porcine parvovirus (PPV) strain NADL-2 were isolated and examined by restriction enzyme analysis and reciprocal Southern blot hybridization during development of a DNA probe for PPV. Genomic single stranded PPV DNA was 5.0 kb long, and results substantiated the rolling-hairpin model of parvovirus DNA replication with the primer sequence located in the 3' terminal hairpin loop. An additional finding was the generation of a 4.7 kb species of viral DNA which was considered to be a 0.3 kb deletion variant of genomic PPV DNA. A 3.0 kb DNA fragment obtained by Pst I/Hind III digestion of monomer replicative form DNA was cloned into a plasmid vector, pUC 19. The cloned fragment, recovered from transformed Escherichia coli strain TB1 and labelled with [32P] dCTP, was evaluated by dot hybridization as a probe for PPV in infected cell cultures. The probe was specific for PPV infected cells, and was 100 times more sensitive than the standard hemagglutination test.     

Efficacy of spray-drying to reduce infectivity of pseudorabies and porcine reproductive and respiratory syndrome (PRRS) viruses and seroconversion in pigs fed diets containing spray-dried animal plasma

Three experiments were conducted to evaluate viral inactivation by the spray-drying process used in the production of spray-dried animal plasma (SDAP). In Exp. 1, bovine plasma was inoculated with pseudorabies virus (PRV) grown in PK 15 cells. Three 4-L batches were spray-dried in the same manner and conditions of industrial SDAP production but with laboratory spray-drying equipment. Presence of infectivity was determined before and after spray-drying by microtiter assay in PK 15 cell cultures. Before spray-drying, all three samples contained 10(5.3) tissue culture infectious dose50 (TCID50)/mL of PRV. After four consecutive passages, no viable virus was detected in samples of spray-dried bovine plasma. In Exp. 2, bovine plasma was inoculated with porcine respiratory and reproductive syndrome (PRRS) virus propagated previously in MARC cell culture to provide approximately 10(6.3) TCID50/mL. Three 4-L batches were spray-dried in the same manner as Exp. 1. Before spray-drying, samples contained TCID50 of 10(4.0), 10(3.5), and 10(3.5)/mL, respectively. After four consecutive passages in MARC cell cultures, no viable virus was detected in spray-dried bovine plasma. In Exp. 3, 36 weaned piglets (28 d of age) were fed a common diet for 14 d and were determined to be negative for PRV, PRRS, and porcine parvovirus titer. Afterwards, pigs were allotted to six pens with six pigs per pen and fed diets containing either 0 or 8% SDAP (as-fed basis) for 63 d. The SDAP used in the feed contained antibody (titer 1:400) against porcine parvovirus. Blood samples were collected from pigs on d 0 and 63 to determine whether feeding SDAP caused seroconversion and development of antibodies against parvovirus, PRRS, or PRV. Inclusion of SDAP in the diet improved growth of pigs without seroconversion. Spray-drying conditions used in this study were effective in eliminating viable pseudorabies and PRRS viruses from bovine plasma. In this study, feeding SDAP that contained functional antibodies did not promote seroconversion in na?ve animals.     

The growth of attenuated strains of canine parvovirus, mink enteritis virus, feline panleukopenia virus, and rabies virus on various types of cell cultures

The growth characteristics were studied in the attenuated strains of canine parvovirus CPVA-BN 80/82, mink enteritis virus MEVA-BN 63/82 and feline panleucopenia virus FPVA-BN 110/83 on the stable feline kidney cell line FE, and in the attenuated canine distemper virus CDV-F-BN 10/83 on chicken embryo cell cultures (KEB) and cultures of the stable cell line VERO. When the FE cultures were infected with different parvoviruses in cell suspension at MOI 2-4 TKID50 per cell, the first multiplication of the intracellular virus was recorded 20 hours p. i. In the canine parvovirus, the content of intracellular and extracellular virus continued increasing parallelly until the fourth day; then, from the fourth to the sixth day, the content of extracellular virus still increased whereas that of intracellular virus fell rapidly. In the case of the mink enteritis virus the release of the virus into the culture medium continued parallelly with the production of the cellular virus until the sixth day. In the case of the feline panleucopenia virus the values concerning free virus and virus bound to cells were lower, starting from the second day p. i. When KEB or VERO cultures were infected in cell suspension with the canine distemper virus at MOI about 0.004 per 1 cell, the replicated intracellular virus was first recorded in the KEB cultures five hours after infection but in the VERO cultures only 20 hours after infection, with a timely release of the virus into the culture medium in both kinds of tissue. In the KEB and VERO cultures the highest values of infection titres were recorded on the fourth day p. i., the course of virus multiplication on the cells being parallel with its release into the culture medium.     

Equine parvovirus: initial isolation and partial characterization.

A viral agent was isolated from the fetal liver of an aborted equine fetus. The isolate hemagglutinated red blood cells from guinea pig, rhesus monkey and rooster. By hemagglutination inhibition tests, the isolate was shown to be antigenically distinct from parvoviruses of bovine and canine origin. Specific hemagglutination inhibiting antibody against the viral isolate was exhibited by 26 of 136 horse sera tested. The isolated virus showed properties compatible with those of an autonomous parvovirus including size, morphology, stability to ether treatment and heating to 56 degrees C, the presence of a 5300 base DNA genome, characteristic protein composition and density (1.405 g/mL). The virus was classified as an equine parvovirus.     

犬细小病毒DD株的分离鉴定和VP2基因序列分析

本实验室对从疑似犬细小病毒感染的发病犬分离的病毒采用同步培养法接种猫肾细胞（CRFK）增殖,通过PCR试验、IFA试验和VP2基因测序分析等方法进行鉴定并分型,获得一株犬细小病毒强毒株,命名为CPV-DD株。对分离病毒进行PCR扩增,可扩增出特异性DNA片段（1 163 bp）;盲传至第6代时,病毒液的HA效价为1∶1...     

Detection of PI-3 viruses, BVD-MD viruses and parvoviruses in bovine fetuses after experimental intrauterine infections

Growing organ cultures prepared from foetuses experimentally infected in utero by the viruses PI-3 and BVD-MD or bovine parvovirus proved to be suitable in tests with the re-isolation of these viruses 7, 14, 21, 35, 42 and 70 days after infection. The attempts at demonstrating them by the traditional method of the inoculation of the primary cell culture of foetal kidneys were successful only at the re-isolation of the PI-3 virus seven days after infection. The PI-3 virus without cytopathic effect was demonstrated in the primary cell culture prepared from the spleen, lungs, kidneys and testes of foetuses delivered seven days from infection. The cytopathic effect manifested itself after cell transplantation in the first passage when further multiplication of the virus occurred.     

Canine parvovirus in asymptomatic feline carriers

Canine parvovirus (CPV) and feline panleukopaenia virus (FPLV) are two closely related viruses, which are known to cause severe disease in younger unvaccinated animals. As well as causing disease in their respective hosts, CPV has recently acquired the feline host range, allowing it to infect both cats and dogs. As well as causing disease in dogs, there is evidence that under some circumstances CPV may also cause disease in cats. This study has investigated the prevalence of parvoviruses in the faeces of clinically healthy cats and dogs in two rescue shelters. Canine parvovirus was demonstrated in 32.5% (13/50) of faecal samples in a cross sectional study of 50 cats from a feline only shelter, and 33.9% (61/180) of faecal samples in a longitudinal study of 74 cats at a mixed canine and feline shelter. Virus was isolated in cell cultures of both canine and feline origin from all PCR-positive samples suggesting they contained viable, infectious virus. In contrast to the high CPV prevalence in cats, no FPLV was found, and none of 122 faecal samples from dogs, or 160 samples collected from the kennel environment, tested positive for parvovirus by PCR. Sequence analysis of major capsid VP2 gene from all positive samples, as well as the non-structural gene from 18 randomly selected positive samples, showed that all positive cats were shedding CPV2a or 2b, rather than FPLV. Longitudinally sampling in one shelter showed that all cats appeared to shed the same virus sequence type at each date they were positive (up to six weeks), despite a lack of clinical signs. Fifty percent of the sequences obtained here were shown to be similar to those recently obtained in a study of sick dogs in the UK (Clegg et al., 2011). These results suggest that in some circumstances, clinically normal cats may be able to shed CPV for prolonged periods of time, and raises the possibility that such cats may be important reservoirs for the maintenance of infection in both the cat and the dog population.     

Characterization of biological and antigenic properties of raccoon dog and blue fox parvoviruses: a monoclonal antibody study

Parvovirus isolates from blue foxes and raccoon dogs were characterized by studying their haemagglutination properties, host range in vitro and antigenic structure. In all 3 characters, raccoon dog parvovirus resembled canine parvovirus (CPV), while blue fox parvovirus was similar to mink enteritis virus (MEV). Monoclonal antibodies (MAbs) were prepared against both viruses. Raccoon dog parvovirus, while resembling CPV, had a unique antigenic site which could be specified by MAbs. The pattern of MAbs prepared against blue fox parvovirus indicated that it is a member of Type 2 MEV.     

二乙烯亚胺对猪细小病毒的灭活作用

使用新型灭活剂二乙烯亚胺(binary ethylenimine,BEI)对猪细小病毒(Porcine parvovirus,PPV)进行了灭活试验,通过ST传代细胞接种法观察病毒灭活后是否出现细胞病变,并结合血凝试验检测灭活效果,确定最佳灭活方法。用3～5日龄乳鼠检测BEI灭活后的病毒培养物和相应制备疫苗的安全性,并用豚鼠检测该灭活工艺制备疫苗的效果,与传统甲醛灭活进行了比较。结果显示,终浓度为1‰的BEI在32℃情况下经20 h即可彻底灭活PPV病毒;BEI灭活的病毒制备的疫苗免疫豚鼠较甲醛灭活病毒产生较高的血凝抑制抗体。     

一株内源性猪细小病毒的分离与鉴定

本研究从不明病毒污染的ＰＫ１５细胞培养物中分离到了一株猪细小病毒,从形态学、血清学、分子生物学几个方面对其进行了鉴定,同时克隆了该毒株ＶＰ２基因,并测定了其核苷酸序列。将该毒株命名为ＳＹ－９９株。     

Comparison of feline parvovirus subspecific strains using monoclonal antibodies against a feline panleukopenia virus

Four monoclonal antibodies (mAb) against a feline panleukopenia virus (FPLV) TU 1 strain, one of the host range variants of feline parvovirus (FPV), were produced and applied for antigenic analysis of FPLV, canine parvovirus (CPV) and mink enteritis virus (MEV). All mAbs were considered to be directed at epitopes on the virus capsid surface because they neutralized the infectivity and inhibited the hemagglutination (HA) of the homologous virus as well as other FPV strains. They were of the mouse IgG1 type. High antigenic homogeneity among FPLV strains was confirmed by HA-inhibition (HI) test with the mAbs and polyclonal immune sera against FPLV or CPV. But the TU 11 strain of FPLV was antigenically distinguished from the remaining 14 FPLV strains by both the HI test and the micro-neutralization test with one of the mAbs produced. MEV Abashiri strain was found to be antigenically indistinguishable from FPLV. Most of the CPV strains isolated after 1981 were considered to be antigenically different from earlier CPV isolates when some mAbs were applied in the serological tests, confirming the replacement of CPV by an antigenic variant in Japan. However, antigenically different CPVs were detected at the end of 1984 from unrelated epizootics occurred a month apart in the same area.     

Assay for Antibody in Pig Fetuses Infected with Porcine Parvovirus

Fetal fluids from field cases of fetal death were assayed for antibody to porcine parvovirus (PPV) using 3 different techniques. An indirect immunofluorescent antibody test, a counter immunoelectrophoresis test and a hemagglutination inhibition test were compared. The indirect immunofluorescent antibody test was found to be the most sensitive of the tests employed. The hemagglutination inhibition test apparently suffered from the occurrence of false positive results.     

Genomic Typing of Canine Parvovirus Circulating in the State of Rio de Janeiro,Brazil from 1995 to 2001 Using Polymerase Chain Reaction Assay

In this study, the genomic types of canine parvovirus (CPV) circulating in the State of Rio de Janeiro, Brazil, from 1995 to 2001, were investigated using the polymerase chain reaction assay (PCR). A total of 78 faecal samples from gastroenteritic puppies, confirmed as positive for canine parvovirus by haemagglutination/haemagglutination inhibition tests or virus isolation in cell culture (MDCK), were examined. The viral DNA was extracted from faecal samples using a combination of phenol– chloroform and silica–guanidine thiocyanate methods. PCR was carried out with differential pairs of primers to distinguish the old (CPV-2) and new types of virus (CPv-2a or CPV-2b). Specific amplicons were observed for all samples using the primer pair P2ab, which detects CPV-2a and CPV-2b. Seventy-six from a total of 78 samples (97%) were considered as CPV-2b because of their reaction with the primer pair P2b. Thirty samples (30/78) were from previously vaccinated puppies and in 15 of them the enteritis symptoms began from 1 to 12 days after vaccination. PCR confirmed the infection by wild virus (CPV-2b) in 5 of these 15 puppies who had received old-type vaccines. Our results show that CPV-2b was the prevalent type circulating in the State of Rio de Janeiro from 1995 to 2001.