Detection and molecular characterization of two canine circovirus genotypes co-circulating in Vietnam

BackgroundCanine circovirus is reported in dogs in many countries, including the USA, China and Thailand. It has been detected in healthy dogs and dogs with diarrhea, hemorrhagic gastroenteritis, and vasculitis. It comprises five genotypes and is frequently found as a coinfection with canine parvovirus-2 (CPV-2).AimTo characterize canine circovirus genotypes co-circulating with CPV-2 in Vietnam.MethodPCR assessment of 81 CPV-2-positive fecal samples from Vietnamese diarrheic dogs up to seven months of age for other viral enteric pathogens, including canine bocavirus, canine adenovirus, paramyxovirus, canine coronavirus, porcine circovirus-3 and canine circovirus. In addition, eight selected full genome sequences of Vietnamese canine circovirus were analyzed and used for phylogeny.ResultsIn total 19.8% of samples were found to be positive for canine circovirus. Phylogeny revealed that the Vietnamese canine circovirus strains were clustered in two different genotypes (genotype-1 and -3). The genetic diversity among Vietnamese canine circovirus was 86.0–87.2%. The nucleotide discrepancy among both genotypes altered the deduced amino acid sequence in 14 and ten residues of the replicase and capsid proteins, respectively. Genetic recombination analysis revealed that the Vietnamese canine circovirus-6 strain has the American and Chinese canine circovirus as its major and minor parents, respectively. Only a single dog revealed triple detections of CPV-2c, Canine circovirus and canine adenovirus (1.2%).ConclusionThe co-circulation of two different genotypes of canine circovirus and CPV-2c in dogs in Vietnam has been illustrated.Clinical relevanceThe mortality rate with CPV-2 only (22%) doubled in dogs with canine circovirus and CPV-2 co-infection.     

兔抗犬细小病毒2a型多克隆抗体交叉保护研究

为探究犬细小病毒（canine parvovirus,CPV）血清型只有一种的条件下,常用疫苗CPV-2型和CPV-2a病毒免疫血清抗体对国内流行CPV-2a病毒的中和率,试验将藏獒源CPV-2a毒株（10⁵ TCID₅₀/100 μL）和CPV-2（10^4.5 TCID₅₀/100 μL）疫苗接于F81细胞增殖,PEG6000法浓缩制成免疫原,各免疫新西兰长白兔3只,间隔2周1次,共免疫4次。收集血清纯化制备多克隆抗体,通过血凝抑制试验（HI）和血清中和试验（SN）分别用2种抗体中和CPV-2、CPV-2a病毒,对两者保护率进行初步评价,并对本地感染CPV-2a的4只犬,每组2只进行治疗,每日跟踪白细胞消长规律。结果显示,CPV-2a多抗中和国内流行病毒CPV-2a的HI抗体、中和抗体水平都极显著高于CPV-2多抗（P<0.01）,而中和CPV-2病毒的HI抗体、中和抗体水平两者差异不显著（P>0.05）,CPV-2a多抗组治疗能较快恢复正常值,2组白细胞数有显著差异（P＜0.05）。结果表明,CPV-2a多抗与常用CPV-2型疫苗免疫抗体相比能高滴度的中和CPV-2a,更适用于中国犬细小病毒的防制,对研发新型生物制品有一定意义。     

Chronological analysis of canine parvovirus type 2 isolates in Japan

Fifty-five canine parvovirus type 2 (CPV) samples, 12 fecal specimens and 43 cell culture isolates, were examined for their genetic characteristics of VP2 gene. They were collected from the diseased dogs at various districts of Japan during 27 years from 1980 to 2006. A fragment of VP2 gene was analyzed by restriction fragment length polymorphism assay and DNA sequencing. The original antigenic type 2 of CPV (CPV-2) was no longer found in the samples since 1984, and two antigenic variants CPV-2a and CPV-2b replaced CPV-2 as predominant types for about 5 years from 1982. A new genetic variant of prototype CPV-2a with non-synonymous substitution at the VP2 amino acid residue 297 from Ser to Ala was first detected in 1987. New CPV-2b with the same amino acid substitution at position 297 as new CPV-2a was also detected from the samples collected in 1997. Since then new CPV-2b has been the predominant CPV over the field of Japan. Several additional amino acid substitutions were detected in the VP2 gene of some recent CPV strains. Neither CPV-2c(a), CPV-2c(b), nor "Glu-426" of the antigenic variants previously found outside the country was detected in any samples tested. Reactivity of new CPV-2a and 2b variants against antibodies produced by the current vaccine products was determined by a cross hemagglutination-inhibition test. The recent field CPV isolates reacted more efficiently to the antibodies produced in dogs vaccinated with the new CPV-2b vaccine strain than the conventional CPV-2 vaccine strain.     

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.     

Characterisation of canine parvovirus strains isolated from cats with feline panleukopenia

Unlike the original canine parvovirus type 2 (CPV-2), CPV-2 variants have gained the ability to replicate in vivo in cats but there is limited information on the disease patterns induced by these variants in the feline host. During 2008, two distinct cases of parvoviral infection were diagnosed in our laboratories. A CPV-2a variant was identified in a 3-month-old Persian kitten displaying clinical sign of feline panleukopenia (FPL) (acute gastroenteritis and marked leukopenia) and oral ulcerations, that died eight days after the onset of the disease. Two pups living in the same pet shop as the cat were found to shed a CPV-2a strain genetically identical to the feline virus and were likely the source of infection. Also, non-fatal infection by a CPV-2c strain occurred in a 2.5-month-old European shorthair kitten displaying non-haemorrhagic diarrhoea and normal white blood cell counts. By sequence analysis of the major capsid protein (VP2) gene, the feline CPV-2c strain showed 100% identity to a recent canine type-2c isolate. Both kittens had been administered multivalent vaccines against common feline pathogens including FPL virus. Whether and to which extent the FPL vaccines can protect cats adequately from the antigenic variants of CPV-2 should be assessed.     

Detection and genotyping of canine coronavirus RNA in diarrheic dogs in Japan

To clarify the prevalence of canine coronavirus (CCoV) infection in Japan, faecal samples from 109 dogs with diarrhoea were examined for CCoV RNA together with canine parvovirus type 2 (CPV-2) DNA. The detection rates of CCoV and CPV-2 for dogs aged less than 1 year were 66.3% and 43.8%, while those for dogs aged 1 year or older were 6.9% and 10.3%, respectively, which were significantly different (p < 0.0001 and p = 0.0003, respectively), indicating not CPV-2 but CCoV is an important diarrhoea-causing organism in juvenile dogs. Among the CCoV-positive dogs, 65.5% and 72.7% showed to be positive for CCoV types I and II, respectively, and simultaneous detection rate of both types was high at 40.0%. Furthermore, transmissible gastroenteritis virus (TGEV)-like CCoV RNA was detected from 8 dogs. These findings indicate that CCoV type I and TGEV-like CCoV are already circulating in Japan, though no reports have been presented to date.     

Three-year serologic immunity against canine parvovirus type 2 and canine adenovirus type 2 in dogs vaccinated with a canine combination vaccine

A group of client-owned dogs and a group of dogs at a commercial kennel were evaluated for duration of antibody responses against canine parvovirus type 2 (CPV-2) and canine adenovirus type 1 (CAV-1) after receiving a combination vaccine containing recombinant canarypox-vectored canine distemper virus (CDV) and modified-live CPV-2, CAV-2, and canine parainfluenza virus, with (C6) or without (C4) two serovars of Leptospira (Recombitek C4 or C6, Merial). Duration of antibody, which correlates with protective immunity, was found to be at least 36 months in both groups. Recombitek combination vaccines can confidently be given every 3 years with assurance of protection in immunocompetent dogs against CPV-2 and CAV-1 as well as CDV. This allows this combination vaccine, like other, similar modified- live virus combination products containing CDV, CAV-2, and CPV-2, to be administered in accordance with the recommendations of the American Animal Hospital Association Canine Vaccine Task Force.     

Molecular investigation on the presence of canine parvovirus in Egypt

Canine parvovirus type 2 (CPV-2) causes a highly contagious gastroenteritis disease of dogs and wild canids. To investigate the CPV-2 prevalence in Dakahlia Governorate, Egypt, a total of 50 fecal swabs were collected from suspected diseased dogs during 2016–2017. Out of 50 collected samples, 35 samples (70 %) presented positive results for CPV-2 using immuno-chromatography (IC) as a rapid test. CPV-2DNA was detected in 42 samples (84 %) by using polymerase chain reaction (PCR). The frequencies of CPV-2 were significantly higher in German shepherd breed (46 %; 23/50) and in age groups less than 6 months (76%; 38/50). We evaluated the breed, age, sex, rapid test results and clinical signs as predictors for classification of animal status into infected and not infected. The best predictors for classification process were rapid test result and clinical signs. Both CPV-2b and CPV-2c subtypes were detected by CPV2-VP2 gene sequences analysis. Deduced amino acid sequences alignment showed substitutions at 3 sites (Arg453Pro, Ala574Glu and Gln457Leu). Further investigations are needed to reveal the genetic and antigenic relation between field and vaccinal strains of CPV-2 in Egypt.     

Peptide nucleic acid-based (PNA) array for the antigenic discrimination of canine parvovirus

A novel peptide nucleic acid (PNA)-based array was developed for use in ante-mortem antigenic typing discrimination in dogs with canine parvovirus (CPV). Cyclic benzothiazole-2-sulfonyl PNA monomers were synthesized that recognized GTA (CPV-2) and TAT (CPV-2a, -2b and -2c) at the nt 913–915 positions, and AAT (CPV-2 and CPV-2a), GAT (CPV-2b), and GAA (CPV-2c) at the nt 1276–1278 positions of the VP2 gene. The detection limits for aa 305 and aa 426 of the VP2 proteins belonging to the four CPV antigenic types were determined optically to be 40–2000 DNA copies, and the optimal cut-off fluorescence signaling value was fixed at 5000. The PNA array described here was developed from 135 field dog fecal specimens and had 89.8% (62/69) sensitivity and 90.4% (66/73) specificity compared with a real-time PCR using the TaqMan assay, a gold standard method. This CPV PNA array could be used together with MGB probe assays as an attractive novel tool for ante-mortem antigenic typing discrimination.     

广州地区犬细小病毒VP2基因的序列分析

犬细小病毒病是一种高度接触性传染病,临床上以急性出血性肠炎和心肌炎为特征。为研究广州地区犬细小病毒病的流行亚型,本试验采集2011—2012年间疑似病犬粪便,进行犬细小病毒（canine parvovirus,CPV）分离鉴定,提取病毒基因组进行VP2基因扩增和序列分析,与GenBank中登录的参考毒株进行比对,结果发现,分离的8株CPV中SCAU-5为CPV-2b 亚型,其余为CPV-2a亚型。结果表明,2011—2012年间广州地区的流行毒株以CPV-2a亚型为主。     

Antibodies to parvovirus, distemper virus and adenovirus conferred to household dogs using commercial combination vaccines containing Leptospira bacterin

To examine how the inclusion (+) or exclusion (-) of inactivated Leptospira antigens in a vaccine for canine parvovirus type 2 (CPV-2), canine distemper virus (CDV) and canine adenovirus type 2 (CAdV-2) affects antibody titres to CPV-2, CDV and CAdV-1 antigens, household dogs were vaccinated with commercially available vaccines from one of three manufacturers. CPV-2, CDV and CAdV-1 antibody titres were measured 11 to 13 months later and compared within three different age groups and three different bodyweight groups. There were significant differences between CPV-2 antibody titres in dogs vaccinated with (+) vaccine and those vaccinated with (-) vaccine for two products in the two-year-old group and for one product in the greater than seven-year-old group; no significant differences were seen that could be attributed to bodyweight. No differences in CDV antibody titres were observed within age groups, but a significant difference was seen in the 11 to 20 kg weight group for one product. Significant differences in CAdV-1 antibody titres were seen for one product in both the two-year-old group and the ≤10 kg weight group.     

Do two current canine parvovirus type 2 and 2b vaccines provide protection against the new type 2c variant?

Three groups (n=9 or 10) of 12-week-old canine parvovirus type 2 (CPV-2) antibody-negative puppies were vaccinated: one group with a product containing modified-live CPV-2b (Galaxy DA2PPv; Schering-Plough Animal Health), one group with a product containing modified-live CPV-2 (Continuum DAP, Intervet), and one group (controls) with sterile saline. All puppies receiving CPV-2 and CPV-2b vaccines developed antibody as determined by the hemagglutination inhibition assay. All groups of puppies were challenged with a combination of virulent CPV-2b and CPV-2c 5 weeks after vaccination. All puppies in the CPV-2 and CPV-2b vaccinated groups were protected from disease, whereas all control group puppies developed disease and 50% died or were euthanized. This study demonstrated that the CPV-2 and CPV-2b vaccine components of the Continuum DAP and Galaxy DA2PPv products, respectively, provided protection against the CPV-2b virus and also provided complete protection against the new CPV-2c variant.     

New approaches for the molecular characterization of canine parvovirus type 2 strains

Characterization of the canine parvovirus type 2 (CPV-2) is sometimes ambiguous, frequently requiring more than one technique for definitive prediction of the viral type. Taking into account the single-nucleotide polymorphisms encountered in the VP2-protein gene between types 2a and 2b and between type 2b and Glu-426 mutant (type 2c), two different minor groove binder (MGB) probe assays were developed for rapid identification of the CPV-2 variants. A total of 315 samples collected from dogs with diarrhoea were screened for CPV-2 by a real-time polymerase chain reaction (PCR) assay capable of detecting all CPV-2 types. In order to compare the type-specific assays with the traditional techniques [haemagglutination inhibition with monoclonal antibodies, PCR-restriction fragment-length polymorphism (RFLP), sequence analysis] for prediction of CPV-2 antigen specificity, the 203 samples tested CPV-2 positive were analysed using the different methods. The results showed a 100% concordance between the MGB probe assays and the combined conventional methods, with 116 samples characterized as type 2a, 32 as type 2b and 55 as type 2c. Therefore, the MGB probe assays represent a quick, reliable tool for prediction of CPV-2 antigen specificity, with regard to the more time-consuming assays currently used.     

Recent spreading of a divergent canine parvovirus type 2a (CPV-2a) strain in a CPV-2c homogenous population

Canine parvovirus type 2 (CPV-2), which causes acute hemorrhagic enteritis in dogs, is comprised of three antigenic variants (2a, 2b, and 2c) that are distributed worldwide with different frequencies. Variant prevalence was analyzed in 150 CPV-2-positive samples collected from the Uruguayan dog population in 2007-2010. Samples were analyzed with polymerase chain reaction, restriction fragment length polymorphism, and sequencing of the coding region for the largest and most variable loop of the VP2 capsid protein. CPV-2c was the only strain detected from 2007 to 2009. Uruguayan CPV-2c showed high homogeneity in both nucleotide and amino acid sequences, indicating a low level of genetic variability. In 2010, an unexpected epidemiological change occurred in Uruguay as a consequence of the appearance of a novel CPV-2a strain. This variant rapidly spread through the Uruguayan dog population and was detected in 20 of the 52 cases (38%) analyzed in 2010. CPV-2a sequences were identical in all field viruses analyzed, and in addition to the characteristic 426Asn residue, the sequences showed amino acid substitutions (267Tyr, 324Ile, and 440Ala) not observed in the Uruguayan CPV-2c. These data and the first detection in April 2010 suggest that the CPV-2a variant recently emerged in Uruguay and underwent clonal expansion. This observation is the first case in which a CPV-2a variant increased its frequency in a dog population where CPV-2c was prevalent. Our results emphasize the dynamic changes in CPV variants and highlight the importance of ongoing surveillance programs to provide a better understanding of virus epidemiology.     

First detection of canine parvovirus type 2c in pups with haemorrhagic enteritis in Spain

Canine parvovirus type 2 (CPV-2), the aetiological agent of haemorrhagic enteritis in dogs, includes three antigenic variants, types 2a, 2b and 2c. CPV-2c has been detected initially in Italy and subsequently in Vietnam. We report the first identification of this novel antigenic variant in Spain, where it caused an outbreak of fatal enteritis in basset hound pups in association with canine coronavirus type I and type II. We suggest that this new antigenic variant of CPV-2 could spread throughout Europe and that there is a subsequent need to update current CPV vaccines.     

Molecular characterization of canine parvovirus in Brazil by polymerase chain reaction assay

Canine parvovirus (CPV) was first isolated in 1978 in the USA. Analysis of CPV isolates by monoclonal antibodies and restriction enzymes have shown that after the first emergence of CPV (CPV-2) it evolved to give rise to new antigenic types, which were designated CPV type 2a and type 2b. These new types have replaced the original CPV type 2, although the proportions of each of the new antigenic types vary in different countries. In Brazil, CPV-like infections were first observed in 1979, however, there has been no information concerning the antigenic types of CPV prevailing in South America. In this study, we designed a PCR assay to type canine parvovirus strains in fecal samples collected from symptomatic dogs during 1980 through 1986 and 1990 through 1995. Our data showed that the CPV epizootic in Brazil followed the same pattern observed in the USA of emergence of CPV-2 followed by replacement by the variants CPV-2a and 2b. The predominant strain found during 1980 was CPV-2a, which was substantially replaced by CPV-2b from 1990 to 1995.     

Antibody titers for canine parvovirus type-2, canine distemper virus, and canine adenovirus type-1 in adult household dogs

Serum antibody titers for canine parvovirus type-2 (CPV-2), canine distemper virus (CDV) and canine adenovirus type-1 (CAV-1) were investigated in 1031 healthy adult household dogs (2 to 18 years old) given an annual inoculation in the previous 11 to 13 months. The number of dogs retaining significant titers of antibodies against CPV-2, CDV, and CAV-1 were 888 (86%), 744 (72%), and 732 (71%), respectively. There were no differences between males and females in antibody titers against the 3 viruses. Antibody titer for CPV-2 was significantly higher in younger dogs than in older dogs, CDV antibody was significantly higher in older dogs than in younger dogs, and CAV titer was not associated with age.     

2017-2021年成都市猫细小病毒的检测及其VP2基因的变异分析

本研究旨在通过对成都地区家养猫细小病毒(feline parvovirus,FPV)的分子检测,了解FPV在成都地区的流行及遗传变异情况.2017-2021年分别从四川成都地区采集168份家养猫的腹泻粪便样本,用PCR方法对168份样本进行FPV检测,并选取13份阳性样本进行VP2基因全长的克隆和测序,用Meg Ali...     

Occurrence of canine parvovirus type 2c in the United States.

Canine parvovirus (CPV) type 2 (CPV-2) emerged around 1978 as a major pathogen of dogs worldwide. In the mid-1980s, the original CPV-2 had evolved and was completely replaced by 2 variants, CPV-2a and CPV-2b. In 2000, a new variant of CPV (named CPV-2c) was detected in Italy and now cocirculates with types 2a and 2b in that country. The CPV-2c has also been reported from single outbreaks in Vietnam and Spain. This study was conducted to determine if CPV-2c occurs in the United States. Thirty-three fecal samples were collected from dogs in 16 states between April 2006 and April 2007 and were tested for CPV using real-time polymerase chain reaction (PCR). Positive samples were further tested using conventional PCR and minor-groove binding TaqMan PCR assays to determine the viral type and to differentiate vaccine strains from field strains. Twenty-seven samples were positive for CPV, 7 of which were CPV-2c from 5 states: Arizona, California, Georgia, Oklahoma, and Texas. Of the 7 isolates, 4 differed from European CPV-2c isolates by 2 additional single-nucleotide mutations at positions 4076 and 4104, the latter of which produces a ThrAla change at residue 440 located near a major antigenic site. The coast-to-coast geographic distribution of the states in which CPV-2c was detected strongly suggests that this new CPV variant is probably widespread in the United States. The continuous evolution of CPV requires that monoclonal antibody-based and nucleic acid-based diagnostic assays should be periodically checked for sensitivity on prevalent CPV strains.