Complete genome analysis of African swine fever virus isolated from domestic pigs during the first ASF outbreaks in India.

African swine fever (ASF), considered as the most dreadful swine disease due to its very high mortality, emerged in India in 2020. The complete genome analysis of ASF viruses isolated during the first outbreaks in India showed a few unique non-synonymous mutations in MGF 369-11L, MGF 505-4R, K205R and B263R genes. Frame shifts in the protein coding sequences were observed in DP60R, ASFV-G_ACD 00190, MGF 110-10-L-MGF110-14L fusion, MGF 360-14L and I267L genes of Indian ASF viruses as compared to ASFV/Georgia/2007. Complete genome based phylogenetic analysis of p72-genotype-II viruses showed the clustering of Indian isolates with ASFV/Wuhan/2019 in a separate clade. Phylogenetic analysis of concatenated sequences of 14 open reading frames (ORF) having single nucleotide polymorphisms (SNP) showed distinct grouping of Indian ASFVs with other Asian ASFVs. This is the first complete genome characterization of ASF viruses isolated from domestic pigs in India. The results indicate that number of Tandem Repeat Sequence (TRS) in the intergenic region between I73R and I329L genes, and the 14 ORFs with SNP reported in this study could be the genetic determinants to differentiate the closely related p72-genotype II viruses circulating in Asia.

First complete genome characterization of duck plague virus from India.

In this study, we report the complete genome sequencing of the Duck plague virus from India for the first time. The sequencing was done on the MinION nanopore sequencer from Oxford Nanopore Technologies. The closest relative is the European strain 2085v, with 99.98 and 99.8% identity at the amino acid and nucleotide level respectively. Moreover, 72 out of 77 ORFs are completely conserved between the 2 strains. The high similarity with the European strain over the only three other pathogenic strains reported from China points to the circulation of European strain in India. The fly pathways of migratory birds and co-habitation with native species being a probable reason. More complete genome data from diverse sampling locations are needed to characterize the genomic features, develop diagnostics, vaccines, and understand the evolution of the virus.

Evidence of Transmission of Goatpox between Domestic Goats and Wild Himalayan Goral (Naemorhedus goral) in Arunachal Pradesh, India.

Examination of carcasses of Himalayan goral (Naemorhedus goral) revealed nodular, pox-like eruptions in the skin. Similar disease was also seen in domestic goats (Capra aegagrus hircus) in the same area. Goatpox virus was identified as the etiology of the disease in both cases, with probable transmission between the species.

First complete genome characterization of swinepox virus directly from a clinical sample indicates divergence of a Eurasian-lineage virus.

In this study, we report the complete genome sequence of swinepox virus from a clinical sample from a naturally occurring infection in India. The sequencing was done on a Nanopore MinION sequencer from Oxford Nanopore Technologies. Two new annotations were added to the genome. Three of the genes were found to have frameshifts, which might be of importance in relation to infection. When compared to the only other reported whole genome sequence of swinepox virus, which was obtained from an isolate from America in 1999, our sequence is only 98.19% identical at the nucleotide level. The average amino acid sequence identity of the viral proteins, based on the common 149 annotations, is also 98.19%, demonstrating that these viruses are distinctly divergent. Owing to the fact that swinepox virus infects only swine, it could not have entered America until the introduction of swine in the 16th century from Europe. The swinepox viruses in both continents have continued to evolve independently. The sequence divergence identified here indicates a Eurasian-lineage virus that is geographically distinct from the American-lineage swinepox virus.

Genetic characterization of African swine fever virus from domestic pigs in India.

African swine fever (ASF) is the most dreaded disease of pigs, which can cause mortality of up to 100%. Following disease outbreaks with high mortality in pigs in two states of north-east India, namely Arunachal Pradesh and Assam in early 2020, we confirmed the first occurrence of African swine fever (ASF) in domestic pigs in India by real-time PCR, virus isolation and nucleotide sequencing. Genetic analyses in three independent genomic regions (B646L gene encoding the p72 protein, E183L gene encoding the p54 protein and the central variable region (CVR) of B602L gene) showed that the Indian ASF viruses are similar to the post-2007-p72-genotype II viruses reported from Asia and Europe, suggesting the transboundary expansion of ongoing ASF outbreaks in the region.

Detection of coinfection of a divergent subgroup of genotype I Japanese encephalitis virus in multiple classical swine fever virus outbreaks in pigs of Assam, India.

A retrospective investigation of pig tissue samples from different classical swine fever virus (CSFV) outbreaks was undertaken employing RT-PCR for possible coinfection with other swine viruses. Four samples from three different outbreaks were found to be coinfected with Japanese encephalitis virus (JEV). Phylogenetic analysis was done based on complete E gene sequenced from all four coinfected samples. This revealed a new introduction of a divergent subgroup of JEV genotype I in India. This is the first report of detection of coinfection of JEV and CSFV in pigs and the first incidence of JEV genotype I in pigs in India.

Pathodynamics of Circulating Strains of Duck Enteritis Virus: A Step Forward to Understand Its Pathogenesis.

Duck enteritis virus (DEV) causes an acute and contagious infection in duck. The present study was carried out to evaluate the pathogenicity and pathodynamics of DEV isolates from different natural outbreaks in the Assam Province of India. A total of six wild-type isolates of DEV were revived in ducklings to determine its biologic characterization. Postmortem examination of infected ducklings revealed DEV-specific gross lesions in different organs. The presence of DEV was confirmed by its genome amplification and the presence of viral antigens from collected tissue samples by indirect fluorescent antibody test. All the isolates revived in ducklings were further propagated in duck embryo fibroblast cells. Highly virulent and low virulent isolates of DEV were selected for further study based on median duck infectivity dose (DID50) and median tissue culture infectivity dose (TCID50). The highly virulent isolate of DEV had values of 102 DID50/ml and 106.33 TCID50/ml, whereas the low virulent strain had titers of 10 DID50/ml and 104.83 TCID50/ml in the cell culture. Our results showed replication of DEV in ducks with the highest and lowest viral titers in the thymus and bursa of Fabricius, respectively. In addition, microscopic analysis revealed necrosis and degeneration of submucosal esophageal glands and glandular epithelium. The study will be useful to understand the organ tropism and pathologic alteration among the virulent DEV isolates.

Patodinámica de las cepas circulantes del virus de la enteritis del pato: Un paso adelante para comprender su patogenia. El virus de la enteritis del pato (DEV) causa una infección aguda y contagiosa en el pato. El presente estudio se llevó a cabo para evaluar la patogenicidad y la patodinámica de los aislamientos del virus de la enteritis del pato de diferentes brotes naturales en la provincia de Assam en la India. Se replicaron un total de seis aislamientos del virus de la enteritis del pato de tipo silvestre en patitos para su caracterización biológica. El examen post mortem de los patitos infectados reveló lesiones macroscópicas específicas de la enteritis viral en diferentes órganos. La presencia del virus de la enteritis viral de pato fue confirmada por su amplificación del genoma y por la presencia de antígenos virales mediante la prueba indirecta de anticuerpos fluorescentes con muestras de tejido recolectadas. Todos los aislamientos replicados en patitos se propagaron adicionalmente en células de fibroblastos de embriones de pato. Se seleccionaron aislamientos del virus de la enteritis del pato altamente virulentos y poco virulentos para un estudio adicional basado en la dosis de infectividad en el pato (DID50) y la dosis de infectividad de cultivo de tejidos (TCID50). El aislado altamente virulento del virus de la enteritis del pato mostró valores de 102 DID50/ml y 106.33 TCID50/ml, mientras que la cepa virulenta baja tenía títulos de 10 DID50/ml y 104.83 TCID50/ml en cultivo celular. Nuestros resultados mostraron la replicación del virus de la enteritis viral en patos con los títulos virales más altos y más bajos en el timo y en la bolsa de Fabricio, respectivamente. Además, el análisis microscópico reveló necrosis y degeneración de las glándulas esofágicas submucosas y del epitelio glandular. El estudio será útil para comprender el tropismo de los órganos y la alteración patológica entre los aislados virulentos del virus de la enteritis viral del pato.

Evaluation of surface glycoproteins of classical swine fever virus as immunogens and reagents for serological diagnosis of infections in pigs: a recombinant Newcastle disease virus approach.

Classical swine fever (CSF) is an important viral disease of domestic pigs and wild boar. The structural proteins E2 and Erns of classical swine fever virus (CSFV), which participate in the attachment of the virion to the host cell surface and its subsequent entry, are immunogenic. The E2 and Erns proteins are used for diagnosis and the development of vaccines against CSFV infection in swine. Newcastle disease virus (NDV) has been successfully used as a viral vector to express heterologous proteins. In the present study, the E2 and Erns proteins of CSFV were expressed in cell culture as well as embryonated chicken eggs, using recombinant NDV (rNDV). Rescued rNDV expressing the E2 and Erns proteins induced the production of CSFV-neutralizing antibodies upon intranasal vaccination of pigs. Serum samples from vaccinated animals were found to neutralize both homologous and heterologous CSFV strains. Furthermore, rNDV expressing the E2 and Erns proteins of CSFV was used to develop an indirect ELISA, which was used to measure the the antibody titers of randomly collected serum samples. The results suggested that the ELISA based on rNDV-expressed E2 and Erns proteins could be used to screen for CSFV infections. This study shows that rNDV-based expression of CSFV antigens is potentially applicable for development of vaccines and diagnostic tests for CSFV infection. This approach could be an economically favorable alternative to the existing vaccine and diagnostics for CSFV in pigs.

Lentiviral-mediated delivery of classical swine fever virus Erns gene into porcine kidney-15 cells for production of recombinant ELISA diagnostic antigen.

Classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family causes contagious fatal disease in swine. Antibodies against E2, Erns and NS3 proteins of virus can be detected in infected animals. Development of an ELISA coating antigen to improve the sensitivity of detecting Erns-specific antibodies in pig sera is always desirable for diagnosis as well as for differentiation of infected from vaccinated animals. In present study, a lentivirus-based gene delivery system was used to develop a stable PK-15 cell line expressing Erns (PK-Erns) for production of diagnostic antigen. The Lenti-Erns virus was purified from the supernatant of co-transfected 293LTV cells and used to transduce PK-15 cells. The homogenous PK-Erns cell line was produced by single cell cloning by monitoring eGFP expression. The Erns gene in the genomic DNA and RNA transcripts in total RNA isolated from PK-Erns cells were detected by PCR and RT-PCR, respectively. Expression of 45 kDa Erns glycoprotein was detected in western blot using CSFV-specific hyperimmune sera. The use of PK-Erns cell lysate as antigen in serial dilution and single dilution ELISAs with known positive and negative pig sera was investigated. The PK-Erns ELISA revealed sensitivity equivalent to commercial HerdChek ELISA kit. The sensitivity, specificity and accuracy of the PK-Erns ELISA was 95%, 100% and 96.66%, respectively compared to ELISA using purified CSFV as coating antigen. When field pig sera (n = 69) were tested in PK-Erns ELISA, a significant correlation between the titers from serial dilution and single dilution ELISA was observed. This indicated that PK-Erns cell line can serve as continuous source of ELISA diagnostic antigen for detection of CSFV-specific antibodies in pig sera.

Identification of swinepox virus from natural outbreaks in pig population of Assam.

Outbreaks of swinepox [caused by a swinepox virus (SWPV)] in pigs were investigated in 3 districts of Assam, a north eastern state of India. Diagnosis of the disease was carried out employing both standard virological as well as molecular methods. Three representative isolates from different places were selected for inoculation into confluent monolayers of Porcine Kidney-15 (PK-15) cell line. The cytopathic effects were characterized by cell rounding, nuclear vacuolation, cell fusion, granulation of cells and finally detachment from third blind passage onwards. The three genes viz., SPV18-20 and P42 of SWPV was targeted for confirmation of the virus. Swinepox virus was successfully adapted to the PK-15 cell line from seventh passage onwards. The isolated viruses were characterized by sequencing and phylogenetic analysis of P42 gene (extracellular envelope protein), a homologue of vaccinia virus F13L gene. In India, studies on swine pox are very limited. This is the first report on successful isolation of swinepox virus from north eastern region of India. Assam and the other north-eastern states of India being a hub for pig husbandry, isolation of swinepox virus will help in developing and formulating control strategies against the disease.

Mosquito abundance and pig seropositivity as a correlate of Japanese encephalitis in human population in Assam, India.

BACKGROUND & OBJECTIVES: Assam is the most vulnerable state for Japanese encephalitis (JE) in India. The situation warrants characterization of epidemiological patterns of JE in vectors, pigs and human population. This investigation was aimed to determine the relative abundance of mosquito species and seroprevalence of JE in pigs in order to draw an epidemiological association with reported human JE cases in Assam. METHODS: Pig sera and mosquitoes from selected farms in Sivasagar and Kamrup districts of Assam were collected fortnightly for one year during June 2015-May 2016. Pig sera were tested for JE antibodies by haemagglutination and virus neurtralization tests. Mosquito species were identified microscopically following the taxonomic keys. The results were analyzed with data on confirmed human JE cases in the selected districts. RESULTS: Culex gelidus (26.07%) and Cx. tritaeniorhynchus (24.07%) were the most abundant species in collected mosquitoes (n = 997). A total of 22.99% of pigs (n = 335) were JEV seropositive and 45.65% of human acute encephalitis syndrome cases (n = 230) were positive for JE virus (JEV) infection. Relative mosquito abundance, pig positivity and human cases were highest during monsoon (June-September) and least during winter (December-February). Rise in mosquito population was observed during pre-monsoon season (March-May) and concurrently higher number of human cases and pig seropositivity were recorded. A good correlation was observed between mosquito number and JEV positivity in pigs/human, and between pigs and human cases (p < 0.05). Human population in Sivasagar was at higher risk for JE infection (OR: 6.46, p < 0.0001) than in Kamrup rural district. INTERPRETATION & CONCLUSION: This study indicates that a seasonal correlation exists between mosquito abundance and JEV seroconversion in pigs with concurrent human JEV outbreaks under field conditions in Sivasagar and Kamrup rural districts of Assam and that monitoring mosquito abundance/density and pig JEV seropositivity may help in predicting JEV outbreak in human population in the region.

Incidence of elephant endotheliotropic herpesvirus in Asian elephants in India.

Elephant endotheliotropic herpesviruses (EEHVs) are the cause of acute hemorrhagic disease in endangered Asian and African elephants. In the present study, we report the incidence of EEHV infection and associated mortality in the captive elephant of Assam, India. Our result showed the gross morphology and histopathological changes of EEHV infection in the elephant. Moreover, the phylogenetic analysis of the polymerase, helicase, and GPCR genes from the infected tissue samples suggested the presence of EEHV1A virus.

Molecular characterization of classical swine fever virus isolates from India during 2012-14.

Classical swine fever is a highly contagious and economically important viral disease of pigs. Outbreaks of classical swine fever virus (CSFV) were recorded in different places in the Kamrup district of Assam in India between the years 2012 and 2014. The nucleotide sequences of the 10 CSFV isolates were analyzed based on the partial nucleotide sequences of the E2, 5'NTR and NS5B genes. Phylogenetic analysis indicated the dominance of subgroup 2.2 along with 2.1 strains in the northeast part of India. Variation in the nucleotide sequences of E2, 5'NTR and 3'NS5B genes of CSFV allows tracking changes in the virus population over time. The study will provide epidemiological information useful for assessing CSFV circulating genogroups in India.

Development of single dilution immunoassay to detect E2 protein specific classical swine fever virus antibody.

Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease in swine. The disease is endemic in different parts of the world and vaccination is the only way to protect pigs from CSFV infection. The virus surface protein E2 is the major immunogenic protein eliciting protective immunity against CSFV infection in swine. The whole virus antigen cannot differentiate CSFV from other pestiviruses as it cross reacts with border disease and bovine viral diarrhoea viruses. Commercial available ELISA is based on the whole CSFV particle and can lead to false positive results. Moreover, the available commercial ELISA is not cost effective. In the present study, a recombinant E2 protein based single serum dilution ELISA was developed which showed enhanced sensitivity, specificity and accuracy as compared to commercial CSFV detection ELISA. The recombinant E2 protein based ELISA could be an alternate to existing diagnostics against CSFV infection in pigs.

Molecular characterization of Newcastle disease virus strains isolated from different outbreaks in Northeast India during 2014-15.

Newcastle disease virus (NDV) isolates recovered from different outbreaks in chicken flocks in Assam during 2014-15 were genotypically and pathotypically characterized. Nucleotide sequence analysis of fusion (F) and hemagglutinin protein genes showed a close similarity with genotype XIII strains of NDV. Amino acid sequence of F protein showed a virulent cleavage site (112)R-R-Q-K-R-F(117). Furthermore, pathogenicity test in one-day-old chicks and embryonated chicken eggs showed a virulent pathotype of the isolated NDV strains. The study will help us to understand the biology of circulating strains of NDV in Northeastern part of India.

Molecular characterization of E2 glycoprotein of classical swine fever virus: adaptation and propagation in porcine kidney cells.

Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease, hog cholera in pigs. The disease is endemic in many parts of the world, and vaccination is the only way to protect the animals from CSFV infection. The lapinized vaccine strains are occasionally not protective because of animal to animal passage, inadequate vaccination strategy, suboptimal vaccine dose, and emergence of new variants. The surface glycoprotein E2 of CSFV is a major antigenic determinant and can modulate the disease outcome in pigs. In the present study, we characterized the CSFV in porcine kidney cells. The CSFV vaccine strains showed enhanced replication following 15 passages in porcine kidney cells. Nucleotide sequence analysis of the E2 protein gene of the cell culture-adapted vaccine strain of CSFV showed a mutation in putative amino acid sequences that are identical to its virulent counterpart. The study suggests the possibility of exaltation in vaccine strains following its adaptation in host cells and paves the way for a further exploration of the biology of its outbreak.

Experimental infection of pigs with group A rotavirus and enterotoxigenic Escherichia coli in India: gross, histopathological and immunopathological study.

The authors describe a detailed study conducted in Assam, India, of gross, histopathological and immunopathological alterations in pigs experimentally infected with rotavirus and enterotoxigenic Escherichia coli (ETEC) expressing K88 pili. A total of 30 Caesarean derived piglets were infected experimentally with rotavirus alone or in combination with ETEC to study the gross and histopathological alterations and the distribution pattern of different B- and T cell subsets in the gut. Villus atrophy, especially in the jejunum and ileum, was the consistent lesion in piglets infected with rotavirus, while in piglets simultaneously infected with rotavirus and ETEC, severe necrosis of the intestinal villi was observed. Ultrastructural studies revealed similar pathological alterations in the ileum of the infected piglets. A morphometric study of the intestinal villi and crypts showed a reduction in the ratio between the average villus height and crypt depth (VH:CD ratio) in the group infected with rotavirus (5.95±0.33) and those infected with rotavirus and ETEC (7.90±0.16). A higher (p<0.01) reduction in the VH:CD ratio was observed in the jejunum (8.83±0.79) and ileum (8.46±0.78) compared that in the duodenum (10.03±0.50) of the infected pigs. Piglets infected with rotavirus and sacrificed on day 6 post infection revealed the presence of lymphocytes containing cytoplasmic IgA+ (cIgA+) cells in the villus lamina propria and intra-epithelial CD8+ T cells in the villus epithelia. Rotavirus infection of young piglets in association with ETEC was more severe than rotavirus infection alone. Such infection resulted in marked clinico-pathological and immunological alterations in the infected piglets.