Isolation and genetic characterization of parvoviruses from domestic cats reveals emergence of CPV-2c in India: A first report.

The objective of the present study was to isolate and characterize the VP2 gene of parvoviruses from domestic cats in India. For that, 38 fecal samples were screened by PCR with 36.84% positivity. Sequence analysis of those isolates showed canine parvovirus type-2c (CPV-2c) as the predominant variant, followed by feline panleukopenia virus (FPV) and 2a. Phylogenetic analysis of the CPV-2c sequences revealed clustering with Singaporean, South Korean, Mongolian and Bangladeshi dog 2c sequences. Phylogenetic analysis of the 2a isolate (MZC 2) was found to be clustered with Indian, Thai and Singaporean dog 2a isolates. Similarly, all the four FPV sequences were ancestrally related to Indian dog and cat FPV sequences hinting towards interspecies transmission between dogs and cats. Both synonymous and non-synonymous mutations were evident in CPV-2c, 2a and FPV sequences indicative of active evolution. In cell culture medium, CPV-2 showed cytopathogenic effects at the third passage level. In conclusion, the study provided the first report of CPV-2c in cats from India, which demands for extensive epidemiological surveillance to monitor interspecies spread and to shed more light on viral phylogenomics, their distribution in the country and in the Southeast Asian region and usage of current vaccines.

Whole-genome sequence analysis of canine parvovirus reveals replacement with a novel CPV-2c strain throughout India.

Canine parvovirus (CPV) infection causes severe gastroenteritis in canines, with high mortality in puppies. This virus evolved from feline panleukopenia virus by altering its transferrin receptor (TfR), followed by the emergence of CPV-2 variants in subsequent years with altered immunodominant amino acid residues in the VP2 protein. While previous studies have focused on the VP2 gene, there have been fewer studies on non-structural protein (NS1 and NS2) genes. In the present study, CPV genome sequences from clinical samples collected from canines throughout India in 2023, previous Indian CPV isolates from 2009-2019, and the current Indian CPV vaccine strain were compared. The study showed that the CPV-2c (N426E) variant had almost completely replaced the previously dominant CPV-2a variant (N426) in India. The Q370R mutation of VP2 was the most common change in the recent CPV-2c strain (CPV-2c 370Arg variant). Phylogenetic analysis showed the existence of three clades among the recent CPV-2c strains, and sequence analysis identified several new sites of positive selection in the VP1 (N-terminus), VP2, NS1, and NS2 protein-encoding genes in recent CPV strains, indicating the emergence of new CPV-2c variants with varied antigenic and replication properties. The predominant 'CPV-2c 370Arg variants' were grouped with the Chinese and Nigerian CPV-2c strains but were separate from the CPV vaccine strain and earlier isolates from our repository. VP2 epitope analysis predicted nine amino acid variations (including two new variations) in four potential linear B-cell epitopes in the CPV-2c 370Arg variants that might make vaccine failure more likely. This pan-Indian study lays the foundation for further research concerning the dynamics of virus evolution and understanding genetic mutations.

The first evidence of Asian-like CPV-2b in Slovakia in a vaccinated dog with an acute fatal course of parvovirus infection: a case report.

This study provides a comprehensive description of the clinical course of a fatal parvovirus infection in a vaccinated dachshund puppy, along with the first identification of a new CPV-2 variant in Slovakia, elucidated through molecular amino acid analysis of the VP2 gene. The dog exhibited clinical signs such as apathy, vomiting, and bloody diarrhea. After confirming CPV-2 infection with a commercial snap test, intensive therapy was initiated. The dog succumbed within 48 h of admission. A rectal swab sample was collected, CPV-2 was examined using the PCR method, and sequenced. The virus detected in the patient was related to strains of CPV-2c of Asian origin and unrelated to European CPV-2b strains. The sequence had genetic signatures typical of Asian strains (VP2: 5Gly, 267Tyr, 324Ile, 370Arg, and 440Thr). Phylogenetic analysis classified this strain as similar to Asian strains of CPV-2c. It is believed to be derived from an Asian strain similar to CPV-2c that acquired the 426Asp mutation. With this finding, we present the first evidence of an Asian-like CPV-2b strain in the territory of Slovakia.

A LAMP-CRISPR/Cas12b rapid detection platform for canine parvovirus detection.

Canine parvovirus (CPV) is one of the main pathogens causing toxic diarrhea in Chinese dogs, is the cause of large-scale epidemic of dogs, and poses a great threat to the dog industry in China. Rapid, sensitive, and specific CPV testing facilitates the timely diagnosis and treatment of sick dogs. The aim of this study was to build a LAMP-CRISPR/Cas12b platform for CPV detection. The loop mediated isothermal amplification (LAMP) technique was combined with CRISPR-Cas12b analysis to establish a "two-step" and "one-tube" CRISPR/Cas12b rapid CPV method, respectively. The detection system was constructed with specific LAMP primers and single guide RNA (sgRNA) for the highly conserved short fragment of the CPV gene, which could be detected within 1 h without cross-reaction with the other viruses causing canine diarrhea. The detection limits of both "two-step" and "one-tube" CRISPR/Cas12b reactions were 10-1 copies per µL, which was 100 times more sensitive than qPCR and LAMP. In order to achieve point-of-care testing (POCT) of CPV, a one-tube LAMP-CRISPR/Cas12b nucleic acid extraction and detection platform based on magnetic nanoparticle enrichment technology was established to achieve "sample in-result out". The results of this method for simulated samples were compared with those of quantitative real-time PCR; the results showed 100% consistency, and the time was shorter, which could be used to detect the diseased dogs earlier and provide a basis for clinical diagnosis. The LAMP-CRISPR/Cas12b method established in this study provides a sensitive and specific method for rapid detection of CPV, and provides technical support for rapid diagnosis of CPV.

Viral pathogens in domestic cats in southern Italy: A retrospective analysis in Sicily, 2020-2022.

A retrospective study was carried out on selected feline viral pathogens detected in domestic cat in Sicily, southern Italy. Samples from 64 cats, collected from 2020 to 2022, were analysed for the presence of feline panleukopenia virus, canine parvovirus type 2 (CPV-2), feline coronavirus (FCoV), feline calicivirus (FCV), feline herpesvirus type 1, norovirus (NoV), and rotavirus (RoV). Single (45 %) or mixed (38 %) viral infections were detected. FPV, related with other Italian FPV strains, remains the main viral cause of infection (66 %). CPV-2c Asian lineage strains (3 %) were detected for the first time in domestic cats in Europe. FCoV (29.6 %), either enteric or systemic, and systemic FCV (18.7 %) infections were detected in positive cats. Less commonly reported viruses (GIV.2/GVI.2 NoVs, RoV), potentially related to the animal/human interface, were detected at lower rates as well (5 %). The present epidemiological data suggest the need to improve disease prevention, immunization, and biosecurity strategies.

Detection of canine parvovirus type 2c (CPV-2c) in Palestine.

INTRODUCTION: The objective of the present study was to report, for the first time, the presence of canine parvovirus type 2c (CPV-2c) in domesticated dogs with acute gastroenteritis and to characterize the antigenic variants circulating in Palestine. METHODOLOGY: A veterinary clinical-based epidemiological study was carried out between December 2022 and April 2023. Fifty fecal samples were collected from dogs with gastroenteritis and screened for CPV-2 infection by polymerase chain reaction. The distribution of positive cases according to various epidemiological factors was studied. Partial sequencing of the viral protein 2 (VP2) gene was performed for the analysis of CPV-2 variants. RESULTS: Most of the investigated samples (60%; n = 50) during the study period were found positive for CPV-2 infection. There was no difference in the distribution of positive cases of CPV-2 infection based on age group, gender, location, and vaccination status. The analysis of nucleotide and amino acid sequences from amplified products, as well as phylogenetic analysis, revealed the presence of CPV-2c clustered with Asian CPV-2c variants. CONCLUSIONS: In summary, this study represents the initial genetic analysis of CPV-2 present in Palestinian dogs with gastroenteritis and provides evidence that confirms the existence of the CPV-2c variants. To determine the prevailing CPV-2 variant associated with the infection, it is crucial to conduct further sequence analysis using large populations of both domestic and wild canines.

Molecular characterization of full-length VP2 gene of canine parvovirus type 2 strains circulating in Egypt 2019-2021.

Canine parvovirus type 2 (CPV-2) is a major cause of fatal gastroenteritis and myocarditis in puppies of domestic and wild carnivores. CPV-2 has accumulated changes over time lead to the emergence of three antigenic variants CPV-2a, CPV-2b, and CPV-2c. VP2 is the major capsid protein that determines virus antigenicity, and host range. Although the three CPV-2 variants were previously identified in Egypt, most reports covered a restricted geographic region and/or time period, and only analyzed partial fragments of VP2 gene. Therefore, this study was designed to test 100 rectal swabs collected from 7 Egyptian governorates between 2019 and 2021 for CPV-2 using PCR. A total of 65 positive samples were identified, mostly in pure dog breeds of young age. The three variants co-circulated in 2019, while CPV-2b was not detected in 2020 and 2021. The frequency of CPV-2b and CPV-2c was higher in 2019 and 2021, respectively. Analysis of CPV-2 full-length VP2 gene sequence from 19/65 positive samples has identified four common amino acid substitutions F267Y, S297A, A300G, Y324I, which are characteristic for the new CPV-2 variants currently circulating worldwide. Unique substitutions including A5G, G36R, V38E, Q370R, and G392V were recognized in certain samples, and appears to have distinct effect on receptor binding, nuclear translocation, and inter-species transmission. Phylogenetic analysis showed separation of CPV-2 strains into two clades. All strains of this study were classified in clade I with Asian strains. In conclusion, this study provides updated comprehensive molecular analysis of CPV-2 variants in Egypt.

Detection and genetic characterization of circulating canine parvovirus from stray dogs in Shanghai, China.

Canine parvovirus (CPV) is the main cause of viral diarrhea in dogs. CPV became a global disease in 1978 and was endemic all over the world. CPV-2 was the first strain to be identified, but with genetic mutations, new genotypes such as CPV-2a/2b/2c/new-2a/new-2b have emerged. In this study, 128 fecal samples of stray dogs suspected of CPV-2 infection were collected from January to March 2021 in Shanghai, China. All samples were screened by PCR and further analyzed by VP2 gene. The positive rate of CPV-2 was 9.4% (12/128), of which 6 CPV-2 isolates were successfully isolated. Phylogenetic tree analysis showed that 4 isolates were CPV-2c genotype and 2 were new-CPV-2b genotype. VP-2 is a key protein that determines the antigenic properties, host range and receptor binding of cpv-2. The results of VP2 amino acid sequence analysis in this study showed that the CPV-2c isolated strain was the same as the previous strains reported in China, including F267Y, Y324I, Q370R and A5G mutations in addition to the typical N426E mutations. Similarly, in addition to the conventional N426D, S297A, F267Y and Y324I mutations, the new CPV-2b isolate also had a new mutation of T440A. This study further confirmed the prevalence of CPV-2c and new-CPV-2b in Shanghai, and also found a new mutation site of new-CPV-2c, which provided a theoretical basis for further enriching the epidemiological data of CPV-2 in Shanghai, as well as the development of vaccines and the prevention and control of the disease.

Molecular characteristics and genetic evolutionary analyses of circulating parvoviruses derived from cats in Beijing.

BACKGROUND: Feline parvovirus (FPV) is a member of the family Parvoviridae, which is a major enteric pathogen of cats worldwide. This study aimed to investigate the prevalence of feline parvovirus in Beijing of China and analyze the genetic features of detected viruses. RESULTS: In this study, a total of 60 (8.5%) parvovirus-positive samples were detected from 702 cat fecal samples using parvovirus-specific PCR. The complete VP2 genes were amplified from all these samples. Among them, 55 (91.7%) sequences were characterized as FPV, and the other five (8.3%) were typed as canine parvovirus type 2 (CPV-2) variants, comprised of four CPV-2c and a new CPV-2b strain. In order to investigate the origin of CPV-2 variants in cats, we amplified full-length VP2 genes from seven fecal samples of dogs infected with CPV-2, which were further classified as CPV-2c. The sequences of new CPV-2b/MT270586 and CPV-2c/MT270587 detected from feline samples shared 100% identity with previous canine isolates KT156833 and MF467242 respectively, suggesting the CPV-2 variants circulating in cats might be derived from dogs. Sequence analysis indicated new mutations, Ala91Ser and Ser192Phe, in the FPV sequences, while obtained CPV-2c carried mutations reported in Asian CPV variants, showing they share a common evolutionary pattern with the Asian 2c strains. Interestingly, the FPV sequence (MT270571), displaying four CPV-specific residues, was found to be a putative recombinant sequence between CPV-2c and FPV. Phylogenetic analysis of the VP2 gene showed that amino acid and nucleotide mutations promoted the evolution of FPV and CPV lineages. CONCLUSIONS: Our findings will be helpful to further understand the circulation and evolution of feline and canine parvovirus in Beijing.

High Prevalence of Antibodies against Canine Parvovirus and Canine Distemper Virus among Coyotes and Foxes from Pennsylvania: Implications for the Intersection of Companion Animals and Wildlife.

Canine distemper virus (CDV) and Canine parvovirus (CPV) can cause deadly infections in wildlife and companion animals. In this report, we screened serum from free-ranging eastern coyotes (Canis latrans; N = 268), red foxes (Vulpes vulpes; N = 63), and gray foxes (Urocyon cinereoargenteus; N = 16) from Pennsylvania, USA, for antibodies (Abs) to CDV and CPV. This comprehensive screening was achieved using a commercially available enzyme-linked immunosorbent assay (ELISA)-based colorimetric assay. Abs to CDV and CPV were detected in 25.4% and 45.5% of coyotes, 36.5% and 52.4% of red foxes, and 12.5% and 68.8% of gray foxes, respectively. Abs to both viruses were detected in 9.7% of coyotes, 19.1% of red foxes, and 12.5% of gray foxes. This study demonstrates significant wildlife exposure in a northeastern state to CDV and CPV. As wildlife species continue to urbanize, the probability of spillover between domestic animals and wildlife will increase. Ongoing surveillance of wildlife for CDV and CPV exposure is warranted. IMPORTANCECanine distemper virus (CDV) and Canine parvovirus (CPV) are significant health threats to domestic dogs (Canis familiaris) and wildlife. CDV and CPV have been identified in diverse vertebrates, including endangered wildlife species. Susceptibility to these viral pathogens varies significantly among geographic regions and between host species. High morbidity and mortality have been reported with infection by either virus in susceptible species, including dogs. As humans and companion animals encroach on wildlife habitat, and as wildlife becomes increasingly urbanized, the potential for transmission between species increases. This study assessed CPV and CDV Ab prevalence in wild canids (eastern coyotes, red foxes, and gray foxes) harvested in Pennsylvania between 2015 and 2020. High Ab prevalence was demonstrated for both viruses in each species. Ongoing monitoring of CPV and CDV in wildlife and increased efforts to vaccinate dogs and prevent spillover events are essential.

Novel Cyclovirus Species in Dogs with Hemorrhagic Gastroenteritis.

Nested PCRs with circovirus/cyclovirus pan-rep (replicase gene) primers detected eukaryotic circular Rep-encoding single-stranded DNA (CRESS DNA) viruses in three (samples CN9E, CN16E and CN34) of 18 canine parvovirus-2-positive fecal samples from household dogs with hemorrhagic gastroenteritis on the Caribbean island of Nevis. The complete genomes of CRESS DNA virus CN9E, CN16E and CN34 were determined by inverse nested PCRs. Based on (i) genome organization, (ii) location of the putative origin of replication, (iii) pairwise genome-wide sequence identities, (iv) the presence of conserved motifs in the putative replication-associated protein (Rep) and the arginine-rich region in the amino terminus of the putative capsid protein (Cp) and (v) a phylogenetic analysis, CN9E, CN16E and CN34 were classified as cycloviruses. Canine-associated cycloviruses CN16E and CN34 were closely related to each other and shared low genome-wide nucleotide (59.642-59.704%), deduced Rep (35.018-35.379%) and Cp (26.601%) amino acid sequence identities with CN9E. All the three canine-associated cycloviruses shared < 80% genome-wide pairwise nucleotide sequence identities with cycloviruses from other animals/environmental samples, constituting two novel species (CN9E and CN16E/34) within the genus Cyclovirus. Considering the feeding habits of dogs, we could not determine whether the cycloviruses were of dietary origin or infected the host. Interestingly, the CN9E putative Rep-encoding open reading frame was found to use the invertebrate mitochondrial genetic code with an alternative initiation codon (ATA) for translation, corroborating the hypothesis that cycloviruses are actually arthropod-infecting viruses. To our knowledge, this is the first report on the detection and complete genome analysis of cycloviruses from domestic dogs.

Phylogenetic Characteristics of Canine Parvovirus Type 2c Variant Endemic in Shanghai, China.

Canine parvovirus type 2 (CPV-2) has spread and mutated globally over the past 40 years. In the present study, 206 samples from dogs suspected of CPV-2 infection were collected from five veterinary clinics in Shanghai city, China. The average positive rate for CPV-2 was detected to be 40.78% using the PCR method. Using an F81 cell (feline kidney cell) culture, the isolates of three CPV-2c strains were obtained. The near full-length genome sequences of the isolates were determined and submitted to GenBank: CPV-SH2001 (MW650830), CPV-SH2002 (MW811188), and CPV-SH2003 (MW811189). By comparing the amino acid sequences of 12 CPV strains with those of 48 related strains retrieved from GenBank, all of the CPV strains from Shanghai were typed as belonging to a relatively new CPV-2c variant spreading in Asia, with typical amino acid residues (5Gly, 267Tyr, 324Ile, and 370Arg) in the VP2 protein. The divergence time of this new CPV-2c clade was estimated by the phylogenetic tree using the maximum likelihood and RelTime with Dated Tips (RTDT) approaches. Our results indicate that the 426 and 324 VP2 amino acid residues are under strong selection pressure with a posterior probability of 0.966 and 0.943, respectively. Therefore, this study provides insight into the phylogenetic characteristics of the current CPV-2c variant in Shanghai city, China.

Predominance of canine parvovirus 2b in Japan: an epidemiological study during 2014-2019.

Canine parvovirus 2 (CPV-2) is an important pathogen of domestic dogs and wild canids. In Japan, CPV-2 infection is one of the most common infectious diseases of dogs. We analyzed samples collected between 2014 and 2019 to identify antigenic variants of CPV-2 in dogs in Japan. Our results demonstrated that the CPV-2b variant was predominant. The CPV-2c variant was not found among our samples. Our findings demonstrate that the distribution of CPV-2 antigenic variants in Japan was more similar to that in Australia than to that in neighboring countries in Asia.

A novel one-step amplification refractory mutation system PCR (ARMS-PCR) for differentiation of canine parvovirus-2 variants.

Enteritis caused by CPV-2 antigenic variants (CPV-2a, 2b, and 2c) is frequently reported in dogs worldwide leading to significant morbidity and mortality. Here, we describe about a simple, single-step, ARMS-PCR strategy targeting the mutant 426 amino acid of VP2 to differentiate CPV-2 antigenic types. A total of 150 fecal samples were subjected to ARMS-PCR of which 18 were typed as CPV-2a, 79 were typed as CPV-2b, and 6 were typed as CPV-2c. The ARMS-PCR results were validated by randomly sequencing partial VP2 gene of 14 samples. Phylogenetic analysis of partial VP2 gene sequencing of each of the CPV-2 variants revealed that CPV-2a and CPV-2b isolates formed a separate clade of Indian lineage, while CPV-2c shared common evolutionary origin with Asian lineage. The developed technique is first of its kind, one-step, rapid, sequencing independent method for typing of CPV-2 antigenic variants.

Serial measurement of thyroid hormones in hospitalised dogs with canine parvoviral enteritis: Incidence of non-thyroidal illness syndrome and its association with outcome and systemic inflammatory response syndrome.

The aim of this study was to serially evaluate the serum concentrations of total thyroxine (tT4), free thyroxine (fT4) and thyroid-stimulating hormone (TSH) in dogs with canine parvoviral enteritis (CPVE) during a 5-day hospitalisation period and assess the association of these hormone concentrations with the outcome and the development of systemic inflammatory response syndrome (SIRS). Dogs with confirmed CPVE that were hospitalised for at least 5 days were included. The thyroid hormones concentrations were measured on days 1, 3 and 5 of hospitalisation. Twenty-eight dogs were included. All (28/28, 100%), 19/28 (69.7%) and 23/28 (82.1%) dogs had a low serum tT4, fT4 and TSH concentration, respectively, on at least 1 day during the hospitalisation period. Overall, 11/28 (39.3%) dogs were diagnosed with SIRS on at least 1 day. In survivors, serum tT4 concentration was significantly higher on day 5 (median, range: 11.8 nmol/L, <6.4-32.2 nmol/L) compared to those on days 1 (<6.4 nmol/L, <6.4-20.1 nmol/L; P = 0.010) or 3 (7.6 nmol/L, <6.4-25.2 nmol/L; P = 0.019). Survivors had a significantly higher tT4 concentration (median, range: 11.8 nmol/L, <6.4-32.2 nmol/L) on day 5 compared to non-survivors (<6.4 nmol/L, <6.4-7.2 nmol/L; P = 0.002). Regardless of the day of hospitalisation, dogs with SIRS had significantly lower tT4 (<6.4 nmol/L, <6.4-16.3 nmol/L) compared to dogs without SIRS (8.6 nmol/L, <6.4-32.2 nmol/L; P = 0.006). A significant difference was also found in fT4 between dogs with SIRS (<3.9 pmol/L, <3.9-16.2 pmol/L) and dogs without SIRS (15.1 pmol/L, <3.9-59.2; pmol/L; P < 0.001). Non-thyroidal illness syndrome was frequently observed in dogs with CPVE, and a negative association between tT4 and fT4 concentrations and SIRS was noted. Serial measurements of tT4 concentrations appeared to have prognostic value.

The detection of canine parvovirus type 2c of Asian origin in dogs in Romania evidenced its progressive worldwide diffusion.

BACKGROUND: Canine parvovirus (CPV) is one of the most important pathogens of dogs. Despite vaccination, CPV infections are still ubiquitous in dogs, and the three antigenic variants 2a, 2b and 2c are variously distributed in the canine population worldwide. To date, no information is available on CPV variants circulating in some European countries. The aim of this study was to genetically characterise the CPV detected in ten dogs with clinical signs of acute gastroenteritis in Romania. The presence of Carnivore protoparvovirus 1 DNA was investigated in faecal samples using an end-point PCR targeting the complete VP2 gene and positive amplicons were sequenced and analysed. RESULTS: All ten dogs with acute gastroenteritis tested positive to Carnivore protoparvovirus 1 DNA in faecal samples. The identified viruses belonged to CPV-2c type, showed identical sequences of the VP2 gene and were characterised by distinctive amino acid residues in the deduced VP2 protein: 5-glicine (5Gly), 267-tirosine (267Tyr), 324-isoleucine (324Ile) and 370-arginine (370Arg). These distinctive amino acid residues have already been reported in CPV-2c widespread in Asia and occasionally detected in Italy and Nigeria. CONCLUSIONS: Since CPV-2c with VP2 amino acid residues 5Gly, 267Tyr, 324Ile and 370Arg were never reported before 2013, it can be assumed that this virus is progressively expanding its spread in the world dog population. This study adds new data about the presence of this new virus in Europe and underline worrying questions about its potential impact on the health of the canine population.

Parvovirus-induced encephalitis in a juvenile raccoon.

A juvenile raccoon was euthanized because of severe neurologic signs. At postmortem examination, no significant gross lesions were present. Histologic evaluation demonstrated nonsuppurative encephalitis in thalamus, brainstem, and hippocampus, cerebellar Purkinje cell loss, as well as poliomyelitis and demyelination of the spinal cord. Parvovirus antigen-specific immunohistochemistry revealed immunopositive neurons in the brainstem, cerebral cortex, and hippocampus. A few Purkinje cells were also immunopositive. DNA extracted from formalin-fixed, paraffin-embedded brain tissue (thalamus, hippocampus, cerebral cortex) yielded a positive signal using PCR targeting both feline and canine parvovirus. Sequencing analyses from a fragment of the NS1 gene and a portion of the VP2 gene confirmed the presence of DNA of a recent canine parvovirus variant (CPV-2a-like virus) in the cerebellum. Our case provides evidence that a recent canine parvovirus (CPV) strain (Carnivore protoparvovirus 1) can infect cerebral and diencephalic neurons and cause encephalitis in an otherwise healthy raccoon. Parvovirus-induced encephalitis is a differential diagnosis of rabies and canine distemper in raccoons with neurologic signs.

Determination of parvovirus antigen in the vaccine using time-resolved fluorescence immunoassay.

As a highly contagious and potentially fatal disease of dogs, canine parvovirus type 2 (CPV-2) usually causes severe myocarditis and gastroenteritis, while vaccine injection has greatly reduced the incidence of CPV-2 diseases. However, there is currently a lack of simple and effective method for quantitative detection of CPV-2 in vaccine. Therefore, this study aims to prepare an accurate method to determine the CPV-2 antigen (CPV-2-Ag) in vaccine. Here, a sandwich time-resolved fluorescence immunoassay (TRFIA) was established and optimized. Anti-CPV-2 antibodies were immobilized on 96-well plates to capture CPV-2-Ag, and then bound together with the detection antibodies labeled with Europium(III) (Eu3+ ) chelates; finally, time-resolved fluorometry was employed to measure the fluorescence intensity. Vaccination was performed to evaluate the relationship between CPV-2-Ag concentration and antibody titer. The sensitivity is 1.15 mEU/mL (LogY = 1.524 + 0.8667 × LogX, R2  = 0.9933), and the average recovery is among 91.00% to 106.39% without cross-reactions with the other canine viral antigen. The correlation between ELISA assay and this method is up to 0.9861. And, there is high correlation between the CPV-2-Ag concentration and antibody titers (R2  = 0.9234). This immunoassay established has high sensitivity, accuracy, and specificity, which indicate that this method could be suitable for quantitative detection of CPV-2-Ag in vaccine evaluation.

First detection of canine parvovirus 2b DNA in a crab-eating fox pup (Cerdocyon thous, Linnaeus, 1766).

The crab-eating fox (Cerdocyon thous) is a small wild mammal present in all Brazilian biomes and in some countries of South America. This study aimed to verify the involvement of viral infectious agents in the death of a wild crab-eating fox pup (Cerdocyon thous) in Brazil. The Center for Medicine and Research of Wild Animals of the Universidade Estadual Paulista received a free-living crab-eating fox aged approximately 21 days and apparently healthy. After 13 days, the animal presented anorexia, diarrhea, fever, prostration, and neurological signs progressing to death with an inconclusive diagnosis. In a retrospective study, tissue fragments stored at - 80 °C were used to identify nucleic acids from major canine viruses, such as canine parvovirus-2 (CPV-2), canine adenovirus A types 1 and 2, canid alphaherpesvirus 1, and canine distemper virus. The amplified product with the expected length for CPV-2 was obtained from the heart fragment. After performing nucleotide (nt) sequencing of the amplicon, it was possible to demonstrate that the crab-eating fox strain exhibited high (99.8%) nt identity with the CPV-2b prototype (CPV-39 strain). Additionally, deduced amino acid (aa) sequence analysis showed the GAT codon for the aa Asp (D) at position 426 of the CPV-2 viral protein VP2, which characterizes the subtype 2b. To the best of the authors' knowledge, this report describes the first detection of CPV-2b DNA in tissue fragments from a crab-eating fox.

A systematic literature review and meta-analysis of characterization of canine parvoviruses 2 prevalent in mainland China.

BACKGROUND: Canine parvovirus 2 (CPV-2) is a pathogenic virus that infects dogs, causing a highly infectious disease. Monitoring CPV-2 spread is an important part of prevention; however, the prevalence and epidemiological characteristics of CPV-2 have not been systematically evaluated and analyzed in mainland China. Therefore, a systematic review and meta-analysis were performed to assess prevalence and epidemiological characteristics of CPV-2 in domestic dogs in mainland China. METHODS: In this study, Chinese and English literature on CPV-2 epidemiology published between January 2006 and December 2019 was evaluated. Regarding meta-analysis, the random-effect model was employed by forest plot with 95% of confidence interval. The number of CPV-2 infections was identified and the pooled prevalence of infection, as well as the epidemiological characteristics, was calculated using meta-analysis. RESULTS: A total of 39 studies (data from 137,844 dogs) met the evaluation criteria and were used in our study. The pooled prevalence of CPV-2 infection in mainland China was 36%. CPV-2 infection were associated with age, breed, sampling season and immunization status, but not with gender, publication time and diagnostic methods. CONCLUSIONS: Our results indicated that CPV-2 is prevalent among dogs in China. It is therefore necessary to carry out continuous surveillance and epidemiological studies of CPV-2. In addition, accordingly, effective measures should be taken to prevent the transmission and spread of CPV-2 among the Chinese dog population.