Canine Parvovirus Research Articles

In vivo Immunogenicity and Antigenicity of MAP-8 Peptides Derived from the Structural and Non-Structural Proteins of Canine Parvovirus Type 2

In vivo Immunogenicity and Antigenicity of MAP-8 Peptides Derived from the Structural and Non-Structural Proteins of Canine Parvovirus Type 2

Dynamin independent endocytosis is an alternative cell entry mechanism for multiple animal viruses.

Mammalian receptor-mediated endocytosis (RME) often involves at least one of three isoforms of the large GTPase dynamin (Dyn). Dyn pinches-off vesicles at the plasma membrane and mediates uptake of many viruses, although some viruses directly penetrate the plasma membrane. RME is classically interrogated by genetic and pharmacological interference, but this has been hampered by undesired effects. Here we studied virus entry in conditional genetic knock-out (KO) mouse embryonic fibroblasts lacking expression of all three dynamin isoforms (Dyn-KO-MEFs). The small canine parvovirus known to use a single receptor, transferrin receptor, strictly depended on dynamin. Larger viruses or viruses known to use multiple receptors, including alphaviruses, influenza, vesicular stomatitis, bunya, adeno, vaccinia, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and rhinoviruses infected Dyn-KO-MEFs, albeit at higher dosage than wild-type MEFs. In absence of the transmembrane protease serine subtype 2 (TMPRSS2), which normally activates the SARS-CoV-2 spike protein for plasma membrane fusion, SARS-CoV-2 infected angiotensin-converting enzyme 2 (ACE2)-expressing MEFs predominantly through dynamin- and actin-dependent endocytosis. In presence of TMPRSS2 the ancestral Wuhan-strain bypassed both dynamin-dependent and -independent endocytosis, and was less sensitive to endosome maturation inhibitors than the Omicron B1 and XBB variants, supporting the notion that the Omicron variants do not efficiently use TMPRSS2. Collectively, our study suggests that dynamin function at endocytic pits can be essential for infection with single-receptor viruses, while it is not essential but increases uptake and infection efficiency of multi-receptor viruses that otherwise rely on a functional actin network for infection.

Two novel sites determine genetic relationships between CPV-2 and FPV: an epidemiological survey of canine and feline parvoviruses in Changchun, China (2020)

Canine parvovirus (CPV-2) and feline parvovirus (FPV) cause severe hemorrhagic diarrhea disease in dogs, cats, and fur-bearing and wildlife carnivores worldwide, continuing to pose significant threats. In this study, 140 rectal swabs were collected from 70 domestic dogs and 70 cats with clinical diarrhea in veterinary clinics in Changchun during 2020. A total of 64.3% (45/70) of dogs and 55.7% (39/70) of cats tested positive for CPV-2 or FPV using colloidal gold strips. Amino acid (aa) sequence alignment of the VP2 protein from 39 CPV-2 and 36 FPV samples revealed that 79.5% (31/39) were CPV-2c, 17.9% (7/39) were a new CPV-2a, and 2.6% (1/39) were mink enteritis virus (MEV). and 8.3% (3/36) FPV from the cats was infected by CPV-2, which suggested that CPV-2c was the dominant variant in dogs and FPV was the major pathogen in cats in Changchun city. Phylogenetic relationships of VP2 genes showed that 26 parvoviruses were closely related to domestic strains previously published in China; however, 8 FPVs and CPV-JL-15/China/2020 were clustered in the lineage of South Asiatic and European countries, and 7 out of 8 FPVs were close to Italy. In addition to Q247H, I248Y, F544Y, and E/V545V/K, two novel site mutations of N23D or L630P in NS1 protein, associated with viral cross-species transmissions, were first found as a reminder of genetic relationships of CPV-2 variants and FPVs in the same branch. Thus, regular and massive virus surveillance of parvovirus is necessary to cope with its ongoing infection, circulation, mutations, and evolutions to new subtypes with strong survival abilities.

Genetic Diversity and Recombination Analysis of Canine Parvoviruses Prevalent in Central and Eastern China, from 2020 to 2023

Canine parvovirus type-2 (CPV-2), the primary causative agent of serious canine enteric diseases, is highly contagious and associated with high fatality rates worldwide. To comprehend the current emergence of CPV-2 in central and eastern China, 130 rectal swabs from domestic or stray dogs with gastroenteritis symptoms were collected during 2020–2023. A total of 118 positive samples were detected via polymerase chain reaction, and further used to amplify and sequence the VP2 gene. Sequence analysis of the deduced amino acids of VP2 protein indicated that CPV-2c was the most prevalent variant (n = 106, 89.83%), followed by the novel CPV-2a (n = 10, 8.47%) and CPV-2b (n = 2, 1.69%) variants. The VP2 protein from the obtained and reference strains showed 86.95% (AH2103 and HB2108) to 99.94% identity. Based on the nine predicted recombination events, some prevalent CPV-2c strains were highly similar to previously isolated strains, indicating their complex evolution and recombination. The predicted analysis suggested that mutations in the antigen epitope (Val219Ile, Phe267Tyr, and Asn426Glu) and other mutations (Met87Leu, Ile101Thr, and Ser297Ala) affect the tertiary structure of the VP2 protein. This research will help us understand the recent evolution and mutation of Chinese CPV-2 and provide suggestions for updating the CPV-2 vaccine.

Oxidative stress markers in canine parvovirus infection

Parvovirus infections are among the deadly viral diseases in dogs and sick puppies. Young dogs are more prone to the virus. Antioxidants are important components of the defense system which prevent cell damage by neutralizing reactive oxygen species. Malondialdehyde is a reliable and commonly used marker of oxidative stress. The present study was performed to determine the role of oxidative stress markers in canine parvovirus disease. Thirty Cane Corso male dogs with an average age of 3-6 months were enrolled in this study out of which 10 animals were spontaneously infected with the parvovirus and the other 10 were healthy. The activity of superoxide dismutase (SOD), total antioxidant capacity (TAC), and malondialdehyde were measured by colorimetric methods whereas the enzymatic activity of glutathione peroxidase (GPX) was measured. Catalase was measured by means of rapid spectrophotometry and chromography was used for obtaining the concentrations of vitamin D and vitamin E. The increase in malondialdehyde in infected dogs demonstrates that oxidative stress plays a role in the pathogenesis of the parvovirus. The significant elevation in TAC, and lack of important changes in superoxide dismutase and catalase are indicative of a good response by the antioxidant system against oxidative stress induced by the parvovirus. In conclusion, oxidative stress is involved in the pathogenesis of parvovirus. Thus, strengthening the antioxidant system can be effective in the prevention and treatment of canine parvovirus infection.

Generation of canine neutralizing antibodies against canine parvovirus by single B cell antibody technology.

Canine parvovirus (CPV) is a significant threat to canines and is widely distributed worldwide. While vaccination is currently the most effective preventive measure, existing vaccines are not able to offer comprehensive and dependable protection against CPV infection. Hence, there is a need to explore alternative or complementary strategies to tackle this problem. In this study, we present an approach for the efficient screening of canine antibodies targeting CPV using a single B cell antibody technique. We sorted single IgM- IgG+ CPV+ B cells from canine peripheral blood mononuclear cells using fluorescence-activated cell sorting (FACS) and obtained the variable region genes of heavy and light chains (VH and VL) by nested PCR amplification. Canine monoclonal antibodies were expressed in HEK293 cells, and a total of 60 antibodies were obtained, five of which demonstrated neutralizing activity against CPV. Those findings demonstrate the effectiveness of the method for obtaining canine monoclonal antibodies, which in turn aids in the identification and screening of neutralizing antibodies against various canine pathogens.

Transcriptome profiling of Canine Parvovirus 2 Nonstructural gene 1(CPV2.NS1) transfected 4T1 mice mammary tumor cells to elucidate its oncolytic effects

Oncolytic viral gene therapy is a directed approach to target cancer cells without affecting healthy cells of the body. Canine parvovirus (CPV2) is an oncolytic virus that precisely targets and destroys neoplastic cells by causing DNA damage, mitochondrial damage, and apoptosis. Non-structural gene 1 (NS1) of CPV, concerned with viral DNA replication is a key mediator of cytotoxicity of CPV and can specifically cause tumor cell lysis. In the present study, by using the transcriptomics approach, we tried to identify molecular pathways and key genes involved in CPV2.NS1 mediated 4T1 mice mammary tumor cell death. We identified necroptosis and mitochondrial damage-mediated apoptosis as major cell death pathways leading to CPV2.NS1 transfected 4T1 cancer cell death. Various DEGs identified in our study play an important role in pathways like the PI3K/AKT pathway, diverse metabolic pathways, MAPK signaling pathway, and FGF signaling pathway, whichare mostly dysregulated in cancerous conditions. Histone variant H2A.X genes, Capn2, and Mapk10/JNK are predicted as key genes that play a role in causing endoplasmic reticulum stress and mitochondrial damage, thereby leading to necroptosis and apoptosis. This study is a preliminary work done to identify key genes and molecular pathways involved in CPV2.NS1 mediated 4T1 cancer cells death which need to be further validated to establish this viral gene as a potent oncolytic agent.

Molecular detection and quantification of canine parvovirus 2 using a fast and sensitive SYBR® green-based quantitative polymerase chain reaction assay in dogs affected with gastroenteritis

Background and Aim: Viral gastroenteritis in canines is primarily caused by the canine parvovirus 2 (CPV-2). Infections by this virus can cause severe consequences in dogs, such as fever, vomiting, diarrhea, septicemia, systemic inflammation, and immunosuppression. Therefore, the mortality rate of persistent infections caused by this virus is significantly high. The capsid protein VP2 genome of canine parvovirus has undergone many changes, resulting in the emergence of different genotypes, including CPV-2a, CPV-2b, and CPV-2c. Diagnostic procedures often lack the necessary specificity for early infection diagnosis. Early detection of the infection enhances the likelihood of canine survival because the canine will receive prompt therapy. Hence, this study aimed to develop a quantitative polymerase chain reaction (qPCR)-based diagnostic technique using SYBR Green for the rapid and accurate detection and quantification of CPV-2. Materials and Methods: The assay was specifically designed to identify a portion of the conserved NS gene using primers that amplify a 125-bp fragment. The qPCR method was executed in the fast mode to expedite the process using Power up SYBR Green Master Mix reagent. A standard curve was constructed using the amplified and purified PCR product of the NS gene. Results: The limit of detection and quantification were determined in the one amplified-DNA copy. The standard curve showed an efficiency of 99.5% and inter- and intra-assay coefficients of variation of 0.387%–0.976% and 0.085%–0.430%, respectively. The assay was specific for the amplification of CPV-2, as no amplification was observed for other viral genomes (canine adenovirus II, canine distemper virus, canine coronavirus, and canine astrovirus) or from the negative controls. Inter- and intra-tests for repeatability showed low test variability around the run time. To validate the present assay, 200 samples of fezzes from canines with gastroenteritis and symptoms associated with enteric infection were tested using the qPCR protocol. From the analyzed samples, 136 were positive for CPV-2 by qPCR assay, of which 110 were before diagnostic positive for the virus by endpoint PCR, showing high sensitivity of the current assay. CPV-2 was detected in dogs over 2 weeks old up to dogs 9 years old, where the highest viral concentration found was 16429595 gene copies in dogs aged 2 weeks. Conclusion: In the present study, a rapid, specific, repeatable, and sensitive assay was developed for the detection and quantification of CPV-2. Furthermore, it was demonstrated that in the population of domestic dogs in Ecuador affected with gastrointestinal disease, the virus is presented in dogs of different ages and not only in young dogs. Keywords: canine parvovirus, gastroenteritis, quantitative polymerase chain reaction, SYBR green.

Molecular typing of Protoparvovirus carnivoran 1 in Egyptian cats diagnosed with feline panleukopenia

Feline panleukopenia (FPL) in cats is caused by either feline parvovirus (FPV) or canine parvovirus (CPV-2), which belong to the same species “Protoparvovirus carnivoran 1”. While FPV is widely recognized as the principal cause of FPL, CPV-2 has been detected at a higher rate than FPV in sick cats in a recent Egyptian study. To assess this conflict, the present study aimed to determine which Protoparvovirus carnivoran 1 is commonly associated with FPL in Egyptian cats. From Dec-2022 to Jan-2024, 43 cats presenting with acute gastroenteritis and testing positive for FPL using in-clinic assay, SNAP® parvo, were tested for Protoparvovirus carnivoran 1 DNA using conventional PCR. Typing of Protoparvovirus carnivoran 1 was conducted by partial VP2 gene sequencing. Additional epidemiological aspects of the disease were investigated, including seasonal pattern, case-fatality rate, median survival time to death, and the association between FPL outcomes and selected factors like age, sex, vaccination status, and clinical signs (vomiting and diarrhea). All cats tested positive for Protoparvovirus carnivoran 1 DNA and FPV was detected in all cats with strong PCR amplicons (n=39). The following seasonal pattern was recorded: cases emerging in autumn, peaking during winter, declining in spring, and disappearing in summer. The case-fatality rate was 41.6%, and the median time to death was two days. None of the studied factors affected FPL outcomes. In conclusion, FPL in Egyptian cat populations is primarily caused by FPV, not CPV-2, and is particularly prevalent in winter.

Antibody Response of Mice to Bali Isolate of Canine Parvovirus Propagated in Madin-Darby Canine Kidney Cell Culture

Canine parvovirus (CPV) infection is still common among dogs, leading to severe disease with high mortality. The potential of a local isolate of CPV as an effective vaccine to prevent the disease warrants investigation. This study aimed to determine the antibody response in mice against a Bali isolate of CPV propagated in the Madin-Darby Canine Kidney (MDCK) cell culture. The virus was purified using polyethylene glycol (PEG)-6000 and mixed with an Aluminum hydroxide adjuvant. Fifteen 7-week female mice were divided into three treatment groups: treatment group 1 (PEG-purified virus and Adjuvant), treatment group 2 (crude unpurified virus and adjuvant), and treatment group 3 (adjuvant without virus), with five replicates per group. The Bali isolate of CPV was successfully replicated in MDCK cells, achieving a titer of 210-211 hemagglutination (HA) units after eight serial passages through the cell culture. The virus was confirmed as CPV by immunocytochemistry test using a monoclonal antibody and hemagglutination inhibition (HI) test using chicken anti-CPV polyclonal antibody. Following the first immunization, the antibody endpoint titer in mice immunized with PEG-purified CPV (5.6) was significantly higher than those immunized with crude unpurified CPV (4.2) and adjuvant without CPV (1.4). Similarly, after the second immunization, the antibody endpoint titer in mice immunized with PEG-purified CPV (7.6) also remained significantly higher than those immunized with crude unpurified CPV (6.4) and adjuvant without CPV (0.8). Significant increases in antibody endpoint titer were observed after the second immunization in mice immunized with PEG-purified CPV and crude unpurified CPV, but not in those given adjuvant without CPV. The Bali isolate of CPV propagated in MDCK cell culture induced a robust antibody response in mice, suggesting it’s a potential as an alternative vaccine candidate for preventing CPV infection in dogs.

Toward establishing a rapid constant temperature detection method for canine parvovirus based on endonuclease activities.

The NMED method has been established in the laboratory and used for CPV detection. The method has several advantages, including simple sampling, high sensitivity, intuitive results, and no requirement for expensive equipment. The establishment of this method has commercial potential and offers a novel approach and concept for the future development of clinical detection of pathogenic microorganisms.

Production and Evaluation of an Inactivated Adjuvanted Vaccine against Canine Parvovirus in Morocco.

The study conducted in Morocco focused on addressing the challenges posed by canine parvovirus (CPV-2) through comprehensive research, vaccine development, and efficacy assessment. Through real-time PCR screening and genotyping, CPV-2 variants were identified circulating in the region. An inactivated vaccine, derived from a CPV-2 strain isolated from a symptomatic dog, was produced and evaluated for safety and efficacy. The vaccine, from the strain named "CaPV M/3-2022", demonstrated safety in vaccinated puppies, with no adverse reactions observed during the trial period. Efficacy trials showed that vaccinated puppies remained healthy and exhibited lower viral excretion post-challenge compared to unvaccinated controls. These results indicate that the vaccine effectively protects against illness related to CPV-2 and reduces viral shedding. The study provides valuable insights into CPV-2 epidemiology in Morocco, offers a promising vaccine solution, and underscores the importance of vaccination in controlling CPV-2 outbreaks and protecting canine health.

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.

Establishment of ELISA method for canine adenovirus type 1.

Canine adenovirus (CAdV) had a high prevalence in fox populations and induced fox encephalitis. No ELISA kits specifically for CAdV-1 antigen had been commercialized for foxes in China. It is crucial to develop a rapid and accurate ELISA method for detecting of CAdV-1. The monoclonal antibodies (mAbs, IgG1A) and HRP-labeled polyclonal antibodies (pAbs) were used to establish the ELISA method in this experiment. The results showed that the optimal concentration and coating time for the mAbs (IgG1A) were 2.15 μg/mL and overnight at 4°C, respectively. The dilution ratio of the HRP-labeled pAbs was 1:2000. Five percent skimmed milk was selected as the blocking agent. The optimal incubation times for blocking, CAdV-1, and HRP-labeled pAbs were all 1 h. The cut-off value for negative rectal swab was determined to be 0.366 ± 0.032. The maximum dilution ratio was 100 TCID50/mL. The ELISA method was positive to CAdV-1, and that was negative to CAdV-2, Canine Parvovirus (CPV) and Canine Distempervirus (CDV). The ELISA method showed good repeatability, sensitivity, and specificity. Compared with RT-PCR, the sensitivity, specificity, and coincidence rates of the ELISA method were 93.75, 90.9, and 92.86%, respectively. These results indicate that the established ELISA method can be used for the large-scale screening and epidemiology surveillance of CAdV-1 in foxes.

Development and Application of Colloidal Gold Test Strips for the Rapid Detection of Canine Brucellosis.

Brucellosis is a global problem, with the causative agent being the genus Brucella. B. canis can cause undulant fever in dogs, which is a zoonotic disease that can spread not only among dogs but also to humans. This poses a public health threat to society. In this study, a rapid and straightforward immune colloidal gold test strip was developed for the diagnosis of canine brucellosis through the detection of anti-LPS antibodies in serum samples. Rabbit anti-canine IgG conjugated with colloidal gold was employed as the colloidal gold-labeled antibody. The extracted high-purity R-LPS was employed as the capture antigen in the test line (T-line), while goat anti-rabbit IgG was utilized as the capture antibody in the control line (C-line). The colloidal gold strip exhibited high specificity in the detection of brucellosis, with no cross-reaction observed with the common clinical canine diseases caused by Canine coronavirus (CCV), Canine distemper virus (CDV), and Canine parvovirus (CPV). In comparison to the commercial iELISA kit, the sensitivity and specificity of the colloidal gold test strip were found to be 95.23% and 98.76%, respectively. The diagnostic coincidence rate was 98.47%. The findings of this study indicate that colloidal gold test strips may be employed as a straightforward, expeditious, sensitive, and specific diagnostic instrument for the identification of canine brucellosis, particularly in resource-limited regions.

Unveiling the Broad-Spectrum Virucidal Potential of Purified Clinoptilolite-Tuff.

Due to its remarkable surface properties, natural clinoptilolite-tuff interacts with a variety of biochemical, pharmaceutical, chemical, and microbiological entities, including human viruses. In the present work, the virucidal activity of purified clinoptilolite-tuff (PCT) was investigated using a variety of viruses, differing in their structure and composition. Influenza A virus, Herpes Simplex virus, Rhinovirus, and Parvovirus were chosen to represent enveloped and non-enveloped viruses with RNA and DNA genomes. Beside human viruses, Canine Parvovirus and bacteriophages T4 and MS2 were used to represent animal and bacterial viruses, respectively. The virucidal activity of PCT was quantified by examining the residual viral activity on susceptible cell lines upon incubation with PCT. A wide range of antiviral efficiencies was observed, ranging from up to 99% for Herpes Simplex virus to no activity for Rhinovirus and both bacteriophages. This study reveals that the virucidal potential of PCT is not universal and depends on a complex set of factors including virus structure and medium composition. The environmental and medical implications of this research are discussed for uses such as wastewater treatment or wound healing.

Identificación de genogrupos de protoparvovirus y sus variantes en leucocitos de gatos domésticos del Valle Central de Costa Rica

Carnivore protoparvovirus 1 (CPPV-1) includes feline parvovirus (or feline panleukopenia virus, FPV) and canine parvovirus 2 (CPV-2). Currently, although CPV-2 is not found in nature, its variants CPV-2a, CPV-2b, and CPV-2c are found and can infect cats as well. The objective of this research was to identify CPPV-1 and its variants as antibodies against CPPV-1 in the blood of domestic cats from the Central Valley of Costa Rica. Blood samples were collected from 155 cats, along with the following data: sex, age, breed, vaccination information, the environment where the animal lives (urban or rural), coexistence with other cats, health status, any chronic diseases, lifestyle (indoor or outdoor cat), and place of origin. Blood samples were analyzed by hemagglutination inhibition assay (HIA) and molecular techniques (polymerase chain reaction (PCR) and sequencing). Antibodies against CPPV-1 were detected using HIA in all cats; a total of 112 (72.3%) had protective titers (≥1:80). Using real-time PCR, CPPV-1 was detected in the blood of 42 (27.1%) cats, none of which presented anemia, leukopenia, or any chronic disease; five (11.9%) were vaccinated, and three (7.1%) had low body condition but showed appetite and had no other symptom or sign associated with CPPV-1. The group of qPCR-positive cats did not show significant differences compared to the qPCR-negative group regarding age, sex, lifestyle, coexistence with other cats, and antibody titers. Ten asymptomatic and healthy cats showed FPV (n=2) and CPV-2c (n=8) in blood with nucleotide similarities of 100% (GenBank M38246) and 99.8%-100% (GenBank AF401519), respectively.

Prognostic indicators for canine parvoviral enteritis in a Teaching Hospital in Italy: a retrospective study of 76 cases.

Potential prognostic indicators have been associated with decreased survival during canine parvoviral enteritis (CPE), such as body weight, sex, and clinicopathological parameters. Few studies reported the prognostic factors for CPE in Italy; therefore, the aim of this study was to identify prognostic factors associated with the survival of dogs admitted to the Veterinary Teaching Hospital of Perugia University, naturally infected with canine parvovirus. Seventy-six medical records of dogs with a definitive diagnosis of parvoviral infection admitted from 2017 to 2021 have been reviewed and included in the study. From medical records were extracted data on signalment, history, clinical examination, hematology, serum biochemistry, treatments, progression of clinical signs during hospitalization and outcome. The data have been subjected to univariate and multivariate statistical analysis. Our results showed winter season, male sex, dog ownership, small breed, normal sensory status, normal heart rate, normal hydration status, abdominal pain, increased capillary reperfusion time, and normal white blood cell count as positive prognostic factors. The survival model confirmed that parameters such as male sex, small breed, and ownership increased the survival rate during hospitalization. Data reported in the present study are partially in agreement with previous studies and added new information on the possible prognostic factors in dogs affected by CPE in Italy.

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.

In silico designing of multi-epitope vaccine against canine parvovirus using reverse vaccinology.

Canine parvovirus (CPV-2) is a highly contagious virus affecting dogs worldwide, posing a significant threat. The VP2 protein stands out as the predominant and highly immunogenic structural component of CPV-2. Soon after its emergence, CPV-2 was replaced by variants known as CPV-2a, 2b and 2c, marked by changes in amino acid residue 426 of VP2. Additional amino acid alterations have been identified within VP2, with certain modifications serving as signatures of emerging variants. In Brazil, CPV-2 outbreaks persist with diverse VP2 profiles. Vaccination is the main preventive measure against the virus. However, the emergence of substitutions presents challenges to conventional vaccine methods. Commercial vaccines are formulated with strains that usually do not match those currently circulating in the field. To address this, the study aimed to investigate CPV-2 variants in Brazil, predict epitopes, and design an in silico vaccine tailored to local variants employing reverse vaccinology. The methodology involved data collection, genetic sequence analysis, and amino acid comparison between field strains and vaccines, followed by the prediction of B and T cell epitope regions. The predicted epitopes were evaluated for antigenicity, allergenicity and toxicity. The final vaccine construct consisted of selected epitopes linked to an adjuvant and optimized for expression in Escherichia coli. Structural predictions confirmed the stability and antigenicity of the vaccine, while molecular docking demonstrated interaction with the canine toll-like receptor 4. Molecular dynamics simulations indicated a stable complex formation. In silico immune simulations demonstrated a progressive immune response post-vaccination, including increased antibody production and T-helper cell activity. The multi-epitope vaccine design targeted prevalent CPV-2 variants in Brazil and potentially other regions globally. However, experimental validation is essential to confirm our in silico findings.