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.

First complete genome sequence of lumpy skin disease virus directly from a clinical sample in South India.

Lumpy skin disease (LSD), a notifiable disease listed by the World Organization for Animal Health and a fast fast-moving transboundary viral disease infecting cattle and buffaloes, was reported in India in 2019 and has since rapidly spread across the country. This study reports the first complete genome sequence and analysis of a pathogenic LSD virus (LSDV) from India (LSDV/208/PVNRTVU/2020) obtained by direct sequencing of a suspected clinical sample using Illumina and Nanopore sequencing technologies. The complete genome sequence of LSDV/208/PVNRTVU/2020 is 150445 bp long, codes for 156 putative genes and carries identical 2254 bp inverted terminal repeats at either ends. The unique features reported in the LSDV isolates from the recent outbreaks in Asia, namely, the insertions of 12 nucleotides in the viral G-protein coupled receptor (GPCR) and 27 nucleotides leading to duplication of 9 aminoacids in the extracellular enveloped virus-specific (EEV) genes were also conserved in LSDV/208/PVNRTVU/2020. Phylogenetic analysis of the complete genome sequence of LSDV/208/PVNRTVU/2020 revealed its close relation with Kenyan strains and clustered away from vaccine strains. Further analysis showed evidence of strong purifying selection without any recombination events. The data presented in this study could be useful for designing effective strategies such as developing rapid diagnostics and vaccines to control LSD.

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.

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.

VP2 Gene-Based Molecular Evolutionary Patterns of Major Circulating Bluetongue Virus Serotypes Isolated during 2014-2018 from Telangana and Andhra Pradesh States of India.

INTRODUCTION: Bluetongue disease is an economically important viral disease of livestock caused by bluetongue virus (BTV) having multiple serotypes. It belongs to the genus Orbivirus of family Reoviridae and subfamily Sedoreovirinae. The genome of BTV is 10 segmented dsRNA that codes for 7 structural and 4 nonstructural proteins, of which VP2 was reported to be serotype-specific and a major antigenic determinant. OBJECTIVE: It is important to know the circulating serotypes in a particular geographical location for effective control of the disease. The present study unravels the molecular evolution of the circulating BTV serotypes during 2014-2018 in Telangana and Andhra Pradesh states of India. METHODS: Multiple sequence alignment with available BTV serotypes in GenBank and phylogenetic analysis were performed for the partial VP2 sequences of major circulating BTV serotypes during the study period. RESULTS: The multiple sequence alignment of circulating serotypes with respective reference isolates revealed variations in antigenic VP2. The phylogenetic analysis revealed that the major circulating serotypes were grouped into eastern topotypes (BTV-1, BTV-2, BTV-4, and BTV-16) and Western topotypes (BTV-5, BTV-12, and BTV-24). CONCLUSION: Our study strengthens the need for development of an effective vaccine, which can induce the immune response for a range of serotypes within and in between topotypes.

E3 Gene-Based Genetic Characterization of Canine Adenovirus-2 Isolated from Cases of Canine Gastroenteritis in India Revealed a Novel Group of the Virus.

Canine adenovirus (CAV) circulates as two distinct serotypes, CAV-1 and CAV-2, which are antigenically related but differ in their clinical manifestations. CAV is one of the important viral agents in the etiology of canine gastroenteritis. Here, we report the molecular surveillance and genetic characterization of CAV from clinical cases of canine gastroenteritis. A total of 302 fecal/rectal swabs were collected from dogs presented with gastroenteritis at various clinics in and around Hyderabad, India during 2018-19. These samples were tested for CAV using polymerase chain reaction with primers designed for the CAV E3 gene and the virus was isolated from positive samples. CAV-2 nucleic acid was present in 4.9% of the test samples. The partial sequence analyses of the E3 gene of the CAV-2 isolates revealed a frameshift mutation by insertion of nucleotide "G" at 1077 position of E3 gene, which resulted in an extension of the polypeptide chain by eleven amino acids. As a result, isolates from the current study formed a novel group, and the virus that was previously subdivided into two groups worldwide is now categorized under three. The study identifies a novel group of CAV-2 circulating in India providing an updated information regarding CAV-2.

Mutation in the VP2 gene of P1-2A capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype O.

Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37 °C for 75 days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV "O" 100 GPID50 challenge virus.