Viral Capsid, Antibody, and Receptor Interactions: Experimental Analysis of the Antibody Escape Evolution of Canine Parvovirus.

Canine parvovirus (CPV) is a small nonenveloped single-stranded DNA virus that causes serious diseases in dogs worldwide. The original strain of the virus (CPV-2) emerged in dogs during the late 1970s due to a host range switch of a virus similar to the feline panleukopenia virus that infected another host. The virus that emerged in dogs had altered capsid receptor and antibody binding sites, with some changes affecting both functions. Further receptor and antibody binding changes arose when the virus became better adapted to dogs or to other hosts. Here, we used in vitro selection and deep sequencing to reveal how two antibodies with known interactions select for escape mutations in CPV. The antibodies bound two distinct epitopes, and one largely overlapped the host receptor binding site. We also generated mutated antibody variants with altered binding structures. Viruses were passaged with wild-type (WT) or mutated antibodies, and their genomes were deep sequenced during the selective process. A small number of mutations were detected only within the capsid protein gene during the first few passages of selection, and most sites remained polymorphic or were slow to go to fixation. Mutations arose both within and outside the antibody binding footprints on the capsids, and all avoided the transferrin receptor type 1 binding footprint. Many selected mutations matched those that have arisen in the natural evolution of the virus. The patterns observed reveal the mechanisms by which these variants have been selected in nature and provide a better understanding of the interactions between antibody and receptor selections. IMPORTANCE Antibodies protect animals against infection by many different viruses and other pathogens, and we are gaining new information about the epitopes that induce antibody responses against viruses and the structures of the bound antibodies. However, less is known about the processes of antibody selection and antigenic escape and the constraints that apply in this system. Here, we used an in vitro model system and deep genome sequencing to reveal the mutations that arose in the virus genome during selection by each of two monoclonal antibodies or their mutated variants. High-resolution structures of each of the Fab:capsid complexes revealed their binding interactions. The wild-type antibodies or their mutated variants allowed us to examine how changes in antibody structure influence the mutational selection patterns seen in the virus. The results shed light on the processes of antibody binding, neutralization escape, and receptor binding, and they likely have parallels for many other viruses.

Development and characterization of a novel specific monoclonal antibody against mink enteritis virus and its antigen epitope analysis.

This study prepared a novel monoclonal antibody (MAb) against mink enteritis parvovirus (MEV) and identified its antigen epitope. The antibody subclass is identified as IgG1, the titers of the MAb is up to 1:1 × 106 and keeps stably after low-temperature storage for 9 months or 11 passages of the MAb cells. The MAb can specifically recognize MEV in the cells in IFA, but not Aleutian disease virus (ADV) or canine distemper virus (CDV). Its antigen epitope was identified as a polypeptide containing 5 key amino acids (378YAFGR382) and the homology in 20 MEV strains, 4 canine parvovirus strains, and 4 feline panleukopenia virus strains was 100%. This study supplies a biological material for developing new methods to detect MEV.

Development of a time-resolved fluorescence immunoassay kit for detecting canine coronavirus and parvovirus through double labeling.

OBJECTIVE: Canine enteric coronavirus (CCV) and canine parvovirus type 2 (CPV-2) are the main pathogens responsible for acute gastroenteritis in dogs, and both single and mixed infections are common. This study aimed to establish a double-labeling time-resolved fluorescence immunoassay (TRFIA) to test and distinguish CCV and CPV-2 diseases. METHODS: A sandwich double-labeling TRFIA method was established and optimized using europium(III) (Eu3+)/samarium(III) (Sm3+) chelates. CCV/CPV-2 antigens were first captured by the immobilized antibodies. Then, combined with Eu3+/Sm3+-labeled paired antibodies, the Eu3+/Sm3+ fluorescence values were detected after dissociation to calculate the CCV/CPV-2 ratios. The performance, clinical performance and methodology used for laboratory (sensitivity, specificity, accuracy and stability) testing were evaluated. RESULTS: A double-label TRFIA for CCV and CPV-2 detection was optimized and established. The sensitivity of this TRFIA kit was 0.51 ng/mL for CCV and 0.80 ng/mL for CPV-2, with high specificity for CCV and CPV-2. All the accuracy data were less than 10%, and the recovery ranged from 101.21 to 110.28%. The kits can be temporarily stored for 20 days at 4 °C and can be stored for 12 months at temperatures less than - 20 °C. Based on a methodology comparison of 137 clinically suspected patients, there was no statistically significant difference between the TRFIA kit and the PCR method. Additionally, for CCV detection, the clinical sensitivity was 95.74%, and the clinical specificity was 93.33%. For CPV-2 detection, the clinical sensitivity was 92.86%, and the clinical specificity was 96.97%. CONCLUSION: In this study, a double-label TRFIA kit was prepared for CCV and CPV-2 detection with high laboratory sensitivity, specificity, accuracy, stability, clinical sensitivity and specificity. This kit provides a new option for screening/distinguishing between CCV and CPV-2 and may help improve strategies to prevent and control animal infectious diseases in the future.

Canine bufavirus (Carnivore protoparvovirus-3) infection in dogs with respiratory disease.

Canine bufavirus (CBuV) or Carnivore protoparvovirus-3, a nonenveloped DNA virus belonging to the genus Protoparvovirus, family Parvoviridae, has been identified in dogs with respiratory and enteric diseases. Although CBuV detection has been reported in multiple countries, descriptions of pathologic findings associated with infection have not yet been provided. In this study, the authors necropsied 14 dogs (12 puppies and 2 adult dogs) from a breeding colony that died during multiple outbreaks of respiratory diseases. Postmortem investigations revealed extensive bronchointerstitial pneumonia with segmental type II pneumocyte hyperplasia in all necropsied puppies but less severe lesions in adults. With negative results of common pathogen detection by ancillary testing, CBuV DNA was identified in all investigated dogs using a polymerase chain reaction (PCR). Quantitative PCR demonstrated CBuV DNA in several tissues, and in situ hybridization (ISH) indicated CBuV tissue localization in the lung, tracheobronchial lymph node, and spinal cord, suggesting hematogenous spread. Dual CBuV ISH and cellular-specific immunohistochemistry were used to determine the cellular tropism of the virus in the lung and tracheobronchial lymph node, demonstrating viral localization in various cell types, including B-cells, macrophages, and type II pneumocytes, but not T-cells. Three complete CBuV sequences were successfully characterized and revealed that they clustered with the CBuV sequences obtained from dogs with respiratory disease in Hungary. No additional cases were identified in small numbers of healthy dogs. Although association of the bufavirus with enteric disease remains to be determined, a contributory role of CBuV in canine respiratory disease is possible.

High-resolution asymmetric structure of a Fab-virus complex reveals overlap with the receptor binding site.

Canine parvovirus is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Overlap on the surface of parvovirus capsids between the antigenic epitope and the receptor binding site has contributed to cross-species transmission, giving rise to closely related variants. It has been shown that Mab 14 strongly binds and neutralizes canine but not feline parvovirus, suggesting this antigenic site also controls species-specific receptor binding. To visualize the conformational epitope at high resolution, we solved the cryogenic electron microscopy (cryo-EM) structure of the Fab-virus complex. We also created custom software, Icosahedral Subparticle Extraction and Correlated Classification, to solve a Fab-virus complex with only a few Fab bound per capsid and visualize local structures of the Fab-bound and -unbound antigenic sites extracted from the same complex map. Our results identified the antigenic epitope that had significant overlap with the receptor binding site, and the structures revealed that binding of Fab induced conformational changes to the virus. We were also able to assign the order and position of attached Fabs to allow assessment of complementarity between the Fabs bound to different positions. This approach therefore provides a method for using cryo-EM to investigate complementarity of antibody binding.

Inhibition of canine parvovirus 2 (CPV-2) replication by TAT-scFv through targeting of the viral structural protein VP2 of CPV-2.

Canine parvovirus (CPV) causes severe infectious disease with a high mortality rate in dogs. CPV is still a major health issue of dogs in the clinic. Therefore, there is an urgent need to develop effective drugs to treat the disease. In this study, we fused the transactivating transcriptional activator peptide (TAT) with scFv. TAT-scFv was identified by Western blot. CCK8 kit was used to detect the toxicity of TAT-scFv to cells. The binding activity of TAT-scFv to CPV-2-VP2 was detected by DAS ELISA. The cell uptake rate of TAT-scFv was assessed by IFA. After infection with CPV-2, F81 cells were incubated by TAT-scFv. The replication of virus was measured to determine the neutralization effect of TAT-scFv on intracellular and extracellular viruses. Protein docking was used to predict the amino acid (AA) sites of VP2 binding to TAT-scFv. TAT-scFv was expressed in Escherichia coli and purified. The DAS ELISA showed that TAT-scFv could bind with CPV-2-VP2. We demonstrated that TAT-scFv entered cells in a dose-dependent and time-dependent manner and effectively inhibited the replication of CPV-2. Using protein docking, we determined the interaction pattern and found that the N-terminal region (AA 41-49) and the C-terminal region (AA 558) of VP2 interacted with the TAT-scFv. Taken together, these results suggest that, TAT-scFv may be a potential antiviral drug for inhibiting CPV-2 replication and controlling disease caused by CPV-2.

Whole genome sequence analysis of CPV-2 isolates from 1998 to 2020.

Canine parvovirus-2 (CPV-2) is a virus with worldwide spread causing canine gastroenteritis. New strains of this virus have unique characteristics and are resistant to some vaccine strains. Therefore, understanding the root causes of resistance has proven to be of increasing concern to many scientists. This study collected 126 whole genome sequences of CPV-2 subtypes with specific collection dates from the NCBI data bank. The whole genome sequences of CPV-2 collected from different countries were analyzed to detect the new substitutions and update these mutations. The result indicated 12, 7, and 10 mutations in NS1, VP1, and VP2, in that respective order. Moreover, the A5G and Q370R mutations of VP2 are the most common changes in the recent isolates of the CPV-2C subtype, and the new N93K residue of VP2 is speculated to be the cause of vaccine failure. To summarize, the observed mutations, which are increasing over time, causes several changes in viral characteristic. A comprehensive understanding of these mutations can lead us to control potential future epidemics associated with this virus more efficiently.

First isolation and molecular characterization of canine parvovirus-type 2b (CPV-2b) from red foxes (Vulpes vulpes) living in the wild habitat of Turkey.

BACKGROUND: The canine parvovirus, with its many variants, is responsible for a pivotal and common viral infection affecting millions of dogs and other carnivore species worldwide, particularly the wild ones, which are considered as the main reservoir hosts. To that end, this study investigated the presence of canine parvovirus (CPV) in red foxes (Vulpes vulpes) living in wild habitats of several regions of Turkey. METHODS: We randomly collected 630 archival fox stool specimens from rural areas of 22 provinces and used real-time PCR to detect CPV. RESULTS: Two of the 630 (0.3%) stool samples were positive for CPV-DNA, named Tr-Fox/128(Aydin) and Tr-Fox/159(Manisa). We attempted to isolate the virus in a MDCK cell line, and cytopathic effects were observed four days post-inoculation. Three regions corresponding to the CPV capsid protein VP2 gene from extracted DNA of positive samples were amplified by conventional PCR, and the products were visualised, purified, and Sanger sequenced. Three overlapping DNA raw sequence fragments, were read, assembled, and aligned to obtain approximately 1.5 kb-long regions that cover most of the VP2 gene, then deposited in GenBank. After comparing the isolates with parvovirus sequences data of domestic and wild carnivores by BLAST processing, our isolates' similarity rate with each other was 99.40%, with base differences in 9 nucleotide positions. They were classified as 2b variant closely related to isolates from dogs in Turkey, Egypt, Iraq, Italy, Thailand, and China. CONCLUSION: This study presents evidence of interspecies transmission of CPV, of which there are no reports on prevalence in wildlife carnivores of our country. Identification of CPV in red foxes threatens local and hunting dogs, which may contract the infection or disseminate it to other wild animal species or vice-versa.

Genetic diversity and relatedness of feline parvovirus in Vietnam and its potential implications for canine-feline transmission.

Feline panleukopenia, caused by feline parvovirus (FPV), has been studied worldwide, but there have been very few studies conducted in Vietnam. In this study, 19 rectal swab samples were collected from northern Vietnam in 2018-2019 and screened for the presence of FPV using PCR. Through sequence analysis of the full-length VP2 gene, it was found that the FPV strains detected in Vietnam were closely related to those obtained from dogs in Vietnam, Asia, Europe, and America. Moreover, the FPV strains found in Vietnam may constitute a distinct group, related to viruses sampled in China. Interestingly, most of the nucleotide changes identified were T-C substitutions.

Clinico-epidemiological survey of feline parvovirus circulating in three Egyptian provinces from 2020 to 2021.

Feline parvovirus infection, caused by feline parvovirus and canine parvovirus 2, is a highly contagious, life-threatening disease affecting cats. The available epidemiological data on parvovirus infection in cats in Egypt is limited. Therefore, the aim of the current study was to provide data concerning the epidemiological profile of cats infected with parvovirus, including the prevalence of parvovirus infection in cats in three Egyptian provinces (Sohag, Assiut, and Cairo) and the associated risk factors. Using rapid antigen tests of fecal samples and conventional PCR, the overall prevalence of parvovirus infection in cats was found to be 35% (35/100) and 43% (43/100), respectively. Anorexia, bloody diarrhea, severe dehydration, hypothermia, and vomiting were the most common clinical findings significantly associated with parvovirus-infected cats. The geographical location (Sohag) and the season (winter) were both statistically significant risk factors for parvovirus infection. These findings indicate that parvoviruses are circulating in different regions of Egypt. Our study provides baseline epidemiological data for future preventive and control measures against parvovirus infection, as well as highlighting the need for future genomic surveillance studies involving a large study population from various parts of Egypt in order to better shape the epidemiological picture of parvovirus infection.

A comprehensive molecular survey of viral pathogens associated with canine gastroenteritis.

Viral pathogens are the primary cause of canine gastroenteritis. However, few structured comprehensive studies on the viral etiology of canine gastroenteritis have been conducted. In this study, 475 rectal swabs collected over three years (2018-2021) from clinical canine gastroenteritis cases were screened for the presence of six major enteric viruses - canine parvovirus 2 (CPV-2), canine distemper virus (CDV), canine adenovirus 2 (CAdV-2), canine coronavirus (CCoV), canine astrovirus (CaAstV), and canine rotavirus (CRV) - by real-time PCR. The most frequently detected virus was CPV-2, which was present in 64.8% of the samples (subtype 2a, 21.1%; 2b, 77.4%; 2c, 1.5%), followed by CDV (8%), CaAstV (7.2%), CCoV (5.9%), and CAdV-2 (4.6%). Two to four of these viruses in different combinations were found in 16.8% of the samples, and CRV was not detected. The complete genome sequences of Indian isolates of CDV, CCoV, and CaAstV were determined for the first time, and phylogenetic analysis was performed. This study highlights the need for routine prophylactic vaccination with the appropriate vaccines. Notably, 70.3% of animals vaccinated with DHPPiL were found to be positive for at least one virus. Hence, regular molecular analysis of the prevalent viruses is crucial for addressing vaccination failures.

Investigation of canine chaphamaparvovirus, canine bufavirus, and canine adenovirus in dogs with diarrhea: First report of novel canine bufavirus in Turkey.

Viral enteritis is a significant cause of death among dogs younger than 6 months. In this study, the presence of canine chaphamaparvovirus (CaChPV), canine bufavirus (CBuV), and canine adenovirus (CAdV) was investigated in 62 diarrheal dogs previously tested for other viral pathogens (canine parvovirus type 2, canine coronavirus, and canine circovirus). CBuV was detected in two dogs (3.22%) and CaChPV in one dog (1.61%). One dog tested positive for three parvoviruses (CPV-2b, CBuV, and CaChPV). All dogs tested negative to CAdV-1/CAdV-2. A long genome fragment of one of the two identified CBuVs and of the CaChPV was obtained and analyzed. New Turkish CBuVs had high identity rates (96%-98% nt; 97%-98% aa) with some Italian CBuV strains (CaBuV/9AS/2005/ITA and CaBuV/35/2016/ITA). The phylogenetic analysis powerfully demonstrated that these viruses belonged to a novel genotype (genotype 2). A part of the genome ChPV-TR-2021-19 revealed high identity rates (> 98% nt and > 99% aa) with some Canadian CaChPV strains (NWT-W88 and NWT-W171) and the Italian CaChPV strain Te/37OVUD/2019/IT. This study is the first report on the detection of CBuV-2 and the concomitant presence of three canine parvoviruses in Turkey. The obtained data will contribute to the molecular epidemiology and the role in the etiology of enteric disease of new parvoviruses.

Biotechnological and virological analysis of canine parvovirus infections by C-reactive protein levels, serological, hematological and molecular techniques.

The study aims to approach Canine Parvovirus (CPV) diagnosis using multi-method biotechnological techniques including molecular, serological, and hematological analyses. CPVs are causing severe global viral diseases with high dog mortality. Samples were taken from 52 unvaccinated dogs exhibiting symptoms between 2020 and 2021. These included stool, blood, serum, and patient data. CPV genomic DNA was extracted from fresh stools, with DNA concentration and purity measured using Nano drop-spectrophotometry. CPV genomic DNA was detected via RT-PCR in 29 samples (55.8%), CPV IgM-Ab and IgG-Ab were detected in the sera through ELISA in 27 samples (51.9%), and Canine parvovirus antigens were identified in the stool samples by immunochromatography in 20 samples (38.5%). Utilizing canine-specific quantitative ELISA kits, the average level of serum C-reactive protein (CRP) was determined to be 4.66 g/L (with a range of 3.27 to 6.05 g/L). Hematological analysis revealed lymphopenia in 89.6%, leucopenia in 44.8%, anemia in 68.9%, and low hematocrit in 82.8%. All the dogs examined were under 1 year of age, among which 21 (72.4%) were up to 3 months old, and 8 (27.6%) were up to 6 months old, testing positive for CPV. The highest CPV positivity, at 93.1% (n=27), was observed among dogs with outdoor access. The results indicated that hematological parameters and CRP alone were not specific for CPV diagnosis, but provided valuable data for prognosis and differential diagnosis. No significant differences were observed in RT-PCR and ELISA results. However, a noticeable reduction in positivity rates was evident in lateral immunochromatographic viral antigen detection in stool.

Colloidal gold and fluorescent immunochromatographic test strips for canine parvovirus detection.

Canine parvovirus (CPV) is an acute and highly infectious virus causing disease in puppies and, thus, affecting the global dog industry. The current CPV detection methods are limited by their sensitivity and specificity. Hence, the current study sought to develop a rapid, sensitive, simple, and accurate immunochromatographic (ICS) test to detect and control the spread and prevalence of CPV infection. More specifically, 6A8, a monoclonal antibody (mAb) with high specificity and sensitivity, was obtained by preliminary screening. The 6A8 antibody was labelled with colloidal gold particles. Subsequently, 6A8 and goat anti-mouse antibodies were coated onto a nitrocellulose membrane (NC) as the test and control lines, respectively. Furthermore, 6A8 and rabbit IgG antibodies were labelled with fluorescent microspheres and evenly sprayed onto a glass fibre membrane. Both strips could be prepared in 15 min with no noticeable cross-reactivity with other common canine intestinal pathogens. The strips were simultaneously used to detect CPV in 60 clinical samples using real-time quantitative PCR, hemagglutination, and hemagglutination inhibition assays. The colloidal gold (fluorescent) ICS test strip was stable for 6 (7) and 4 (5) months at 4 °C and room temperature (18-25 °C). Both test strips were easy to prepare and rapidly detected CPV with high sensitivity and specificity. Moreover, the results were easily interpretable. This study establishes a simple method for two CPV diseases, colloidal gold and fluorescent immunochromatographic (ICS) test strips. KEY POINTS: • CPV test strips do not exhibit cross-reactivity with other canine intestinal pathogens. • The strips are stable for months at 4 °C and at room temperature (18-25 °C). • These strips are a promising approach for the timely diagnosis and treatment of CPV.

Canine parvovirus type 2 vaccines in Brazil: Viral load in commercial vaccine vials and phylogenetic analysis of the vaccine viruses.

Canine parvovirus type 2 (CPV-2) is the etiological agent of a highly contagious and frequently fatal disease in dogs. Live attenuated vaccines (LAV) are recommended to prevent and control this disease. Commercial vaccines are typically produced with CPV-2 strains adapted to cell culture and usually non-pathogenic. The present study aimed to determine the viral load of CPV-2 vaccines commercially available in Brazil and to characterize the vaccine virus by DNA analysis of its capsid gene. The results demonstrated that all vaccine strains presented high homology of the VP2 gene and they were all closely related to the original CPV-2 strains. However, vaccine strains presented several differences in comparison with field strains currently circulating in Brazil. Seventy-one vials contained viral loads ranging from 7.4E3 to 4.9E10 DNA copies/ml. Nine vials did not contain any detectable CPV-2 DNA. In conclusion, there are genetic and antigenic differences among CPV-2 vaccines and field strains. Additionally, some vaccines have been commercialized with low titers of CPV-2. It is important to improve the quality of the vaccines to prevent or reduce the spread of CPV-2 in Brazil.

Clinical, histopathological, and molecular characterization of canine pigmented viral plaques.

Canine pigmented viral plaques (PVPs) are proliferative epidermal lesions caused by canine papillomaviruses (CPVs). Although the lesions are benign, neoplastic transformation has been reported. Cases reported in the literature are few and mainly focused on genome sequencing. The aim of this study was to collect data on the epidemiology, clinicopathological features, and genotyping of PVPs. Fifty-five canine PVPs were retrospectively retrieved and histologically evaluated. Follow-up was available for 33 cases. The median age was 6.5 years and pugs were the most represented breed (25%). There were 4 clinical presentations: a single lesion (24%), multiple lesions (75%) in one (41%) or different sites (34%), and generalized lesions all over the body (24%). The abdomen and axillae were the most common sites. In single lesions, no recurrence was observed after conventional surgery, whereas different medical treatments reported for multiple lesions were not successful. Spontaneous regression was reported in 3 cases. Neoplasia in contiguity with PVPs was seen in 5 of 55 lesions (9%), and 1 dog was euthanized due to invasive squamous cell carcinoma (SCC). The most useful histopathological features for diagnosis were scalloped profile, epidermal spikes, hypergranulosis, and hyperpigmentation. L1 immunolabeling was present in 14 of 16 cases (87%). Sequencing revealed that 10 of 16 cases were associated with CPV-9 (71%), 2 cases were associated with CPV-4 (14%), and 2 cases were associated with CPV-8 (14%). In conclusion, this represents a large cohort study on canine PVPs reporting data on clinicopathological features, therapy, outcome, and the type of CPV involved for the first time in Italy.

Efficacy of ultrasonographic caudal vena cava to aorta ratios for quantifying canine parvoviral enteritis rehydration.

Quantifying changes in intravascular fluid volume is important for treatment planning and follow-up assessment in dogs with dehydration. Recently, it has been reported that current standard methods used to estimate intravascular fluid volume in dogs are inadequate, invasive, or have complications such as thrombosis. The ultrasonographic ratio of dimensions for the caudal vena cava relative to the aorta (CVC/Ao) has been previously described as a promising, noninvasive method for quantifying changes in blood volume in dogs. This prospective observational study aimed to describe ultrasonographic CVC/Ao values before and after fluid replacement in a sample of dogs with varying degrees of dehydration due to naturally-occurring canine parvoviral enteritis (CPE), test correlations between this measure and clinical dehydration scores and determine the clinical efficacy of this measure for fluid therapy follow-up. The clinical dehydration score of 30 dogs naturally infected with canine parvovirus was determined at the first admission using standard clinical scoring methods, and then CVC/Ao was measured ultrasonographically. Following initial fluid therapy, the clinical dehydration scores and ultrasonographic CVC/Ao values were remeasured. On the basis of receiver operating characteristic analyses, ultrasonographic CVC/Ao was found to be a more sensitive and specific indicator than physical examination-based methods for estimating intravascular fluid alterations in dogs with dehydration due to parvovirus and rehydration following fluid therapy. Findings supported the use of this measure for treatment planning and follow-up in future dogs presenting with dehydration.

Spillover of Canine Parvovirus Type 2 to Pigs, South Dakota, USA, 2020.

In 1978, canine parvovirus type 2 originated from spillover of a feline panleukopenia-like virus, causing a worldwide pandemic of enteritis and myocarditis among canids. In 2020, the virus was identified in pigs in South Dakota, USA, by PCR, sequencing, in situ hybridization, and serology. Genetic analysis suggests spillover from wildlife.

Genetic characterization and evolutionary analysis of canine parvovirus in Tangshan, China.

Canine parvovirus (CPV) is a major enteric virus of carnivores worldwide that poses a considerable threat to dogs. In this study, we investigated the genetic variation of CPV in Tangshan, China, and the relationships between CPV disease and the vaccination status, age, and gender of dogs. Seventy-seven fecal samples from dogs in Tangshan that tested positive for CPV were obtained for analysis. Twenty-two full-length VP2 gene sequences were successfully amplified. The 22 strains included 17 CPV-2c variants, four new CPV-2a variants, and one new CPV-2b variant. Phylogenetic analysis showed that all of the CPV-2c strains clustered together and were closely related to CPV-2c strains from Asia but distantly related to CPV-2c strains from Europe. Further amino acid sequence analysis showed that, relative to CPV-2c strains from Europe, most of the CPV-2c stains in this study had A5G, F267Y, Y324I, and Q370R mutations. These findings provide a more comprehensive understanding of the variants of CPV circulating in China.

First report of canine bufavirus in India.

Canine bufavirus (CBuV), a novel protoparvovirus of dogs that is associated with enteric and respiratory symptoms, has been reported only in Italy and China. The enteric prevalence of CBuV in India was investigated, and the nearly complete genome sequence (4292 bp) was amplified and reconstructed for one strain. A nucleotide sequence alignment indicated 93.42-98.81% identity to the other available CBuV sequences and 70.88-73.39% and 54.4-54.8% identity to human bufavirus and canine parvovirus 2 (CPV-2), respectively. The current strain is most closely related to Chinese CBuV strains, which together form an Asian lineage. This first report of the prevalence of CBuV in India emphasizes the need for further epidemiological surveillance.