Unveiling genotypic diversity of Theileria orientalis in lethal outbreaks among bovines in Karnataka, India.

Theileria orientalis, the causal agent of oriental theileriosis, is known to cause mild disease in cattle and buffalo across the world. Recently, different genotypes of T. orientalis have emerged as pathogenic, causing high reported morbidity in cattle. This study focuses on investigating three suspected outbreaks of oriental theileriosis that resulted in fatalities among crossbred and indigenous bulls in Karnataka, India. Examination of blood smears revealed the presence of T. orientalis piroplasms within erythrocytes. The genetic characterization of T. orientalis was conducted by targeting specific markers, including the mpsp gene, p23 gene, and ribosomal DNA markers (18S rRNA gene, ITS-1, and ITS-2). Analysis based on the 18S rRNA gene unveiled the presence of both Type A and Type E genotypes of T. orientalis in the outbreaks. The mpsp gene-based analysis identified genotype 7 of T. orientalis in crossbred cows, whereas genotype 1 (Chitose B) was found to be present in indigenous bulls. Haplotype network analysis based on the mpsp gene revealed the presence of 39 distinct haplotypes within the 12 defined genotypes of T. orientalis with a high haplotype diversity of 0.9545 ± 0.017. Hematological and biochemical analysis revealed a decrease in calcium, hemoglobin levels, red blood cell counts, and phosphorus. This study constitutes the initial documentation of a clinical outbreak of oriental theileriosis in indigenous bulls with genotype 1 (Chitose 1B). Substantial epidemiological investigations are imperative to gain a comprehensive understanding of the geographical distribution of distinct genotypes and the diverse clinical manifestations of the disease across various hosts.

Post-Vaccination Sero-Monitoring of Peste des Petits Ruminants in Sheep and Goats in Karnataka: Progress towards PPR Eradication in India.

Peste des petits ruminants (PPR) presents economic challenges in enzootic countries impacting small ruminant productivity. The state of Karnataka, India, implemented a mass vaccination campaign in alignment with the PPR-Global Eradication Programme (GEP) and the National Strategic Plan for PPR eradication. This study was conducted from January to March 2023 to assess seroconversion in post-vaccinated goats and sheep at the epidemiological unit (epi-unit) level, aligning with the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) guidelines in the PPR Global Control and Eradication Strategy (GCES). Before vaccination, 3466 random serum samples were collected from small ruminants of three age groups (6-12 months, 1-2 years, and >2 years) across 116 epi-units, spanning 82 taluks in 28 districts. Post-vaccination sero-monitoring included 1102 serum samples collected from small ruminants of the 6-12-month age group only, across 111 epi-units covering 64 taluks in 23 districts. The PPRV antibody status was determined using an indigenous hemagglutinin (H) protein monoclonal antibody-based competitive ELISA kit. Pre-vaccination, the PPR seropositivity rates were 55%, 62%, and 66% in the age groups of 6-12 months, 1-2 years, and >2 years, respectively, with a 61% PPRV antibody prevalence across all the age groups. Notably, 41% of the epi-units exhibited antibody prevalence rates of ≥70%, indicating a substantial population immunity, possibly attributed to the previous vaccination program in the state since 2011. In contrast, only 17% of the epi-units had below 30% seroprevalence rates, emphasizing the need for intensified vaccination. Statistical analysis of the data revealed significant correlations (p < 0.05) between the presence of PPRV antibodies and host factors such as species, breed, and sex. Post-vaccination seroprevalence in the 6-12 months age group was found to be 73.4%, indicating the use of an efficacious vaccine. On the evaluation of vaccination immunity in the 6-12 months age group, it was revealed that over 69% of the epi-units achieved a response surpassing ≥70%, indicating a significant improvement from 42% of the epi-units in pre-vaccination. For active PPR eradication, a mass vaccination campaign (>95% coverage) targeting small ruminant populations aged >4 months is advocated, aiming to achieve the desired herd immunity of >80%. This study offers crucial insights into PPR baseline seroprevalence/immunity status and vaccine efficacy, guiding national strategies towards a PPR-free India and further supporting the global eradication initiative.

First evidence of lumpy skin disease in mithun (Bos frontalis) in India.

Lumpy skin disease (LSD) is a disease of cattle that is also known to cause mild infection in buffaloes. To date, there have been no reports of LSD in mithun (Bos frontalis), a bovine species distributed in Northeast India, Bangladesh, Myanmar, and parts of China. In the present study, the presence of typical clinical signs, virus isolation, PCR amplification, sequence analysis, and the demonstration of antibodies in serum by indirect enzyme-linked immunosorbent assay and serum neutralization test, confirmed the occurrence of LSD in mithun for the first time in India. Phylogenetic analysis based on the full-length RPO30 and P32 genes of LSD virus from mithun and cattle revealed 100% sequence identity, indicating circulation of the same strain in both species in India and the possibility of spillover between species.

Unravelling the genomic origins of lumpy skin disease virus in recent outbreaks.

Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus and family Poxviridae. LSDV was endemic in most of Africa, the Middle East and Turkey, but since 2015, several outbreaks have been reported in other countries. In this study, we used whole genome sequencing approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations compared to the Reference Neethling strain sequence and the previous field strains. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. Thirty-eight genes have more than 2 variations per gene, and these genes belong to viral-core proteins, viral binding proteins, replication, and RNA polymerase proteins. We highlight the importance of several SNPs in various genes, which may play an essential role in the pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues.

Epidemiological investigation of ASF outbreaks in Kerala (India): detection, source tracing and economic implications.

This study investigates suspected African swine fever (ASF) outbreaks in two villages of Kannur district in Kerala, India, with the aim of identifying the causative agent and its genotype, the source of infection, and estimating the economic losses due to the outbreaks. Clinically, the disease was acute with high mortality, while gross pathology was characterized by widespread haemorrhages in various organs, especially the spleen, which was dark, enlarged and had friable cut surfaces with diffuse haemorrhages. Notably, histopathological examination revealed multifocal, diffuse haemorrhages in the splenic parenchyma and lymphoid depletion accompanied by lymphoid cell necrosis. The clinico-pathological observations were suggestive of ASF, which was confirmed by PCR. The source of outbreak was identified as swill and it was a likely point source infection as revealed by epidemic curve analysis. The phylogenetic analysis of p72 gene identified the ASFV in the current outbreak as genotype-II and IGR II variant consistent with ASFVs detected in India thus far. However, the sequence analysis of the Central Variable Region (CVR) of the B602L gene showed that the ASFVs circulating in Kerala (South India) formed a separate clade along with those found in Mizoram (North East India), while ASFVs circulating in Arunachal Pradesh and Assam states of India grouped in to different clade. This study represents the first investigation of ASF outbreak in South India, establishing the genetic relatedness of the ASFV circulating in this region with that in other parts of the country. The study also underscores the utility of the CVR of the B602L gene in genetically characterizing highly similar Genotype II ASFVs to understand the spread of ASF within the country.

Lumpy Skin Disease (LSD) in Yak (Bos grunniens): An Evidence of Species Spillover from Cattle in India.

Lumpy skin disease (LSD), caused by the lumpy skin disease virus (LSDV), is a global concern that affects cattle and buffalo. Recently, the disease has been reported in new species such as the Indian Gazelle, Camel, Banteng, Gaur, and Giraffe from various parts of the world. This report provides an insight into the occurrence of LSD in Yak from Sikkim, a North-Eastern state of India. During the investigation, both cattle and yak exhibited typical clinical signs of LSD, including skin nodular lesions. The morbidity, mortality, and case fatality rates for cattle were 9.08%, 1.84%, and 20.24%, respectively. Similarly, the morbidity, mortality, and case fatality rates in yak were 7.57%, 1.24%, and 16.33%, respectively. The virus isolation and amplification of LSDV-specific genes confirmed the presence of LSDV in cattle, yak, and vectors. Further, demonstrated antibodies in randomly collected sera from naïve and unvaccinated cattle and yak using indirect Enzyme Linked Immuno-sorbent Assay (iELISA) and Serum Neutralisation test (SNT) from this region. Sequencing and phylogenetic analysis of P32, GPCR, and RPO30 genes revealed that the virus isolated from both species was 100% identical to each other and also closely related to the field LSDV isolates circulating in the Indian subcontinent. The study highlighted the emergence of LSDV in unconventional hosts and underscored the need to include other bovine species in national disease control programs, encompassing disease surveillance initiatives.

Diagnosis of rabies using reverse-transcription polymerase chain reaction on post-mortem skin tissue specimens of the nasolabial plate in a rabies suspected cow: a case study.

In India, rabies in cattle is under-reported. Religious sentiments hamper its diagnosis, discouraging post-mortem examination, particularly opening the cranium. Specimens of peripheral tissue innervated by the cranial nerves could potentially be used as alternative diagnostic specimens to the brain. Herein, we present a case study of a novel approach for diagnosing rabies in a cow suspected of having rabies, using skin tissue specimens of the nasolabial plate obtained post-mortem. Brain and nasolabial tissue specimens tested positive for rabies using conventional reverse-transcription polymerase chain reaction. This approach has been previously shown to have a high diagnostic sensitivity in animals. We encourage further studies with more nasolabial plate skin specimens for both post- and antemortem diagnosis of rabies in cattle.

Evaluation of the safety, immunogenicity and efficacy of a new live-attenuated lumpy skin disease vaccine in India.

Lumpy skin disease (LSD) was reported for the first time in India in 2019 and since then, it has become endemic. Since a homologous (LSD-virus based) vaccine was not available in the country, goatpox virus (GPV)-based heterologous vaccine was authorized for mass immunization to induce protection against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed by using an Indian field strain, isolated in 2019 from cattle. The virus was attenuated by continuous passage (P = 50) in Vero cells. The vaccine (50th LSDV passage in Vero cells, named as Lumpi-ProVacInd) did not induce any local or systemic reaction upon its experimental inoculation in calves (n = 10). At day 30 post-vaccination (pv), the vaccinated animals were shown to develop antibody- and cell-mediated immune responses and exhibited complete protection upon virulent LSDV challenge. A minimum Neethling response (0.018% animals; 5 out of 26,940 animals) of the vaccine was observed in the field trials conducted in 26,940 animals. There was no significant reduction in the milk yield in lactating animals (n = 10108), besides there was no abortion or any other reproductive disorder in the pregnant animals (n = 2889). Sero-conversion was observed in 85.18% animals in the field by day 30 pv.

Japanese Encephalitis Virus Genotype III Strains Detection and Genome Sequencing from Indian Pig and Mosquito Vector.

Japanese encephalitis viruses (JEVs) are globally prevalent as deadly pathogens in humans and animals, including pig, horse and cattle. Japanese encephalitis (JE) still remains an important cause of epidemic encephalitis worldwide and exists in a zoonotic transmission cycle. Assam is one of the highly endemic states for JE in India. In the present study, to understand the epidemiological status of JE circulating in pigs and mosquito, particularly in Assam, India, molecular detection of JEV and the genome sequencing of JEV isolates from pigs and mosquitoes was conducted. The genome analysis of two JEV isolates from pigs and mosquitoes revealed 7 and 20 numbers of unique points of polymorphism of nucleotide during alignment of the sequences with other available sequences, respectively. Phylogenetic analysis revealed that the isolates of the present investigation belong to genotype III and are closely related with the strains of neighboring country China. This study highlights the transboundary nature of the JEV genotype III circulation, which maintained the same genotype through mosquito-swine transmission cycles.

Multisectoral prioritization of zoonotic diseases in Haryana (India) using one health approach.

Zoonotic diseases have huge livestock and public health burden worldwide, including India. Prioritizing zoonotic diseases is one of the important tasks under 'One Health' as it facilitates effective policy making, proper allocation of resources and promotion of multisectoral collaboration. Although some efforts have been made to prioritizing zoonotic diseases at national level in India, it is important to identify priority diseases in regional settings due to wide variation in climate and demography of different states. Therefore, the present study aims to prioritize zoonotic diseases for the state of Haryana (India). One Health Zoonotic Disease Prioritization (OHZDP) tool was used in this study to prioritize zoonotic diseases. Based on literature review of the past 23 years (2000-2022) on prevalence, morbidity, and mortality of zoonotic diseases, twenty-three high-scoring zoonotic diseases in Haryana and neighboring states of India were initially shortlisted for prioritization. A three-day participatory workshop was conducted involving 17 experts representing the Health, Animal Husbandry and Wildlife departments of Haryana. The Analytical Hierarchy Process (AHP) was used to rank the criteria, which were used to score the selected diseases using the decision tree analysis. The participants selected the following 7 criteria along with their relative weights to score the diseases: (1) Severity of disease in humans, (2) Severity of disease in animals, (3) Presence of disease in the region, (4) Transmission and outbreak potential, (5) Socio-economic impact, (6) Availability of interventions, and (7) Existing inter-sectoral collaboration for surveillance and reporting. The top scoring eight diseases selected as priority zoonotic diseases for Haryana were rabies, Japanese encephalitis, bovine tuberculosis, leptospirosis, avian influenza (H5N1), brucellosis, glanders and Influenza A (H1N1). Sensitivity analysis did not reveal any significant variation in prioritization results by varying criteria weights. This is the first systemic attempt to prioritize zoonotic diseases in the state and this will help in formulating effective monitoring, prevention, and control strategies for zoonotic diseases in the regional settings.

S-adenosylmethionine-dependent methyltransferase inhibitor DZNep blocks transcription and translation of SARS-CoV-2 genome with a low tendency to select for drug-resistant viral variants.

We report the in vitro antiviral activity of DZNep (3-Deazaneplanocin A; an inhibitor of S-adenosylmethionine-dependent methyltransferase) against SARS-CoV-2, besides demonstrating its protective efficacy against lethal infection of infectious bronchitis virus (IBV, a member of the Coronaviridae family). DZNep treatment resulted in reduced synthesis of SARS-CoV-2 RNA and proteins without affecting other steps of viral life cycle. We demonstrated that deposition of N6-methyl adenosine (m6A) in SARS-CoV-2 RNA in the infected cells recruits heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), an RNA binding protein which serves as a m6A reader. DZNep inhibited the recruitment of hnRNPA1 at m6A-modified SARS-CoV-2 RNA which eventually suppressed the synthesis of the viral genome. In addition, m6A-marked RNA and hnRNPA1 interaction was also shown to regulate early translation to replication switch of SARS-CoV-2 genome. Furthermore, abrogation of methylation by DZNep also resulted in defective synthesis of the 5' cap of viral RNA, thereby resulting in its failure to interact with eIF4E (a cap-binding protein), eventually leading to a decreased synthesis of viral proteins. Most importantly, DZNep-resistant mutants could not be observed upon long-term sequential passage of SARS-CoV-2 in cell culture. In summary, we report the novel role of methylation in the life cycle of SARS-CoV-2 and propose that targeting the methylome using DZNep could be of significant therapeutic value against SARS-CoV-2 infection.

Sero-positivity of Japanese Encephalitis Virus in Equine Population of India Using IgG ELISA: Unraveling the Need for Vaccination.

Japanese encephalitis (JE) is a mosquito borne flaviviral zoonoses, causing fatal disease in equines and humans. JE is endemic in most of the states of India with occurrence of human cases every year. The horses are not vaccinated against JE in India and thus they are at more risk of acquiring the disease. Due to nonavailability of indigenously developed ELISA and high cost of imported kits, regular sero-surveillance is not being carried out to assess the true picture of JE virus in equine population of India. Therefore, a recombinant NS1 protein based indirect IgG ELISA was developed with the objective to assess the sero-positivity of JE virus in equine population of India. The diagnostic sensitivity and specificity of developed ELISA was 84.73% and 86.70%, respectively. The validation studies revealed good reproducibility of ELISA with kappa value ranging from 0.75 to 1 between the results of different laboratories. A total of 2,069 horse serum samples were screened using the developed ELISA and 401 samples were positive for IgG against JEV with an overall sero-positivity of 19.38% in equine population of India. A sero-positivity of 25.90% and 12.22% was recorded in Himachal Pradesh and Jammu-Kashmir, both hill states of North zone of India for the first time, revealing the spread of virus to the nonendemic parts of the country. The high sero-positivity of JE virus recorded in equine population warrants the need for initiation of vaccination of horses in India to prevent the morbidity and mortality.

Studies on Growth Characteristics and Cross-Neutralization of Wild-Type and Delta SARS-CoV-2 From Hisar (India).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly evolved to generate several antigenic variants. These variants have raised concerns whether pre-existing immunity to vaccination or prior infection would be able to protect against the newly emerging SARS-CoV-2 variants or not. We isolated SARS-CoV-2 from the coronavirus disease 2019 (COVID-19)-confirmed patients in the beginning of the first (April/May 2020) and second (April/May 2021) waves of COVID-19 in India (Hisar, Haryana). Upon complete nucleotide sequencing, the viruses were found to be genetically related with wild-type (WT) and Delta variants of SARS-CoV-2, respectively. The Delta variant of SARS-CoV-2 produced a rapid cytopathic effect (24-36 h as compared to 48-72 h in WT) and had bigger plaque size but a shorter life cycle (~6 h as compared to the ~8 h in WT). Furthermore, the Delta variant achieved peak viral titers within 24 h as compared to the 48 h in WT. These evidence suggested that the Delta variant replicates significantly faster than the WT SARS-CoV-2. The virus neutralization experiments indicated that antibodies elicited by vaccination are more efficacious in neutralizing the WT virus but significantly less potent against the Delta variant. Our findings have implications in devising suitable vaccination, diagnostic and therapeutic strategies, besides providing insights into understanding virus replication and transmission.

Emetine suppresses SARS-CoV-2 replication by inhibiting interaction of viral mRNA with eIF4E.

Emetine is a FDA-approved drug for the treatment of amebiasis. Previously we demonstrated the antiviral efficacy of emetine against some RNA and DNA viruses. In this study, we evaluated the in vitro antiviral efficacy of emetine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and found it to be a low nanomolar (nM) inhibitor. Interestingly, emetine exhibited protective efficacy against lethal challenge with infectious bronchitis virus (IBV; a chicken coronavirus) in the embryonated chicken egg infection model. Emetine treatment led to a decrease in viral RNA and protein synthesis without affecting other steps of viral life cycle such as attachment, entry and budding. In a chromatin immunoprecipitation (CHIP) assay, emetine was shown to disrupt the binding of SARS-CoV-2 mRNA with eIF4E (eukaryotic translation initiation factor 4E, a cellular cap-binding protein required for initiation of protein translation). Further, molecular docking and molecular dynamics simulation studies suggested that emetine may bind to the cap-binding pocket of eIF4E, in a similar conformation as m7-GTP binds. Additionally, SARS-CoV-2 was shown to exploit ERK/MNK1/eIF4E signalling pathway for its effective replication in the target cells. Collectively our results suggest that further detailed evaluation of emetine as a potential treatment for COVID-19 may be warranted.

Bovine brucellosis - a comprehensive review.

Brucellosis is a zoonotic disease of great animal welfare and economic implications worldwide known since ancient times. The emergence of brucellosis in new areas as well as transmission of brucellosis from wild and domestic animals is of great significance in terms of new epidemiological dimensions. Brucellosis poses a major public health threat by the consumption of non-pasteurized milk and milk products produced by unhygienic dairy farms in endemic areas. Regular and meticulous surveillance is essentially required to determine the true picture of brucellosis especially in areas with continuous high prevalence. Additionally, international migration of humans, animals and trade of animal products has created a challenge for disease spread and diagnosis in non-endemic areas. Isolation and identification remain the gold standard test, which requires expertise. The advancement in diagnostic strategies coupled with screening of newly introduced animals is warranted to control the disease. Of note, the diagnostic value of miRNAs for appropriate detection of B. abortus infection has been shown. The most widely used vaccine strains to protect against Brucella infection and related abortions in cattle are strain 19 and RB51. Moreover, it is very important to note that no vaccine, which is highly protective, safe and effective is available either for bovines or human beings. Research results encourage the use of bacteriophage lysates in treatment of bovine brucellosis. One Health approach can aid in control of this disease, both in animals and man.

Antiviral activity of Apigenin against buffalopox: Novel mechanistic insights and drug-resistance considerations.

We describe herein that Apigenin, which is a dietary flavonoid, exerts a strong in vitro and in ovo antiviral efficacy against buffalopox virus (BPXV). Apigenin treatment was shown to inhibit synthesis of viral DNA, mRNA and proteins, without affecting other steps of viral life cycle such as attachment, entry and budding. Although the major mode of antiviral action of Apigenin was shown to be mediated via targeting certain cellular factors, a modest inhibitory effect of Apigenin was also observed directly on viral polymerase. We also evaluated the selection of drug-resistant virus variants under long-term selection pressure of Apigenin. Wherein Apigenin-resistant mutants were not observed up to ~ P20 (passage 20), a significant resistance was observed to the antiviral action of Apigenin at ~ P30. However, a high degree resistance could not be observed even up to P60. To the best of our knowledge, this is the first report describing in vitro and in ovo antiviral efficacy of Apigenin against poxvirus infection. The study also provides mechanistic insights on the antiviral activity of Apigenin and selection of potential Apigenin-resistant mutants upon long-term culture.

Serological surveillance and clinical investigation of glanders among indigenous equines in India from 2015 to 2018.

Equine glanders is an infectious and notifiable bacterial disease caused by Burkholderia mallei. The disease has been reported in South American, African and Asian countries including India. Here, we present the outcome of glanders serosurveillance carried out between January 2015 and December 2018 to know the status of equine glanders among different states in India. A total of 102,071 equid sera from 299 districts of twenty-one states and one union territory were tested for glanders. Samples were screened with Hcp1 indirect ELISA followed by confirmatory diagnosis by CFT. During this four-year surveillance, a total of 932 glanders-positive cases were detected from 120 districts of 12 states. The study also revealed increasing trend of glanders from 2016 onwards with maximum occurrence in northern India. Overall seroprevalence ranged between 0.62% (95% CI, 0.52-0.72) and 1.145% (95% CI, 1.03-1.25). Seasonal shifting from winter to summer (March to June) coincided with highest number glanders incidence with corresponding seroprevalences of 1.2% (95% CI, 1.09-1.30). The present surveillance unveils territorial ingression of glanders to six states like Jammu & Kashmir, Gujarat, Rajasthan, Madhya Pradesh, Delhi and Tamil Nadu. In addition, re-emerging cases have been reported in Maharashtra, Haryana and Punjab after a gap of 10 years. Lack of awareness, little veterinary care and unrestricted movement of equids across state borders might have led to the introduction and establishment of the infection to these states. We believe that information from this study will provide a baseline data on glanders for devising surveillance and control strategies in India. Being a zoonotic disease, the persistence of glanders poses a potential threat to occupationally exposed humans especially equine handlers and veterinarians. Therefore, targeted surveillance of human population from each glanders outbreak is also recommended.

Latex agglutination test for rapid on-site serodiagnosis of Japanese encephalitis in pigs using recombinant NS1 antigen.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is a mosquitoe-borne viral zoonotic disease and globally around three billion people are at the risk of disease. The occurrence of JE cases has shown a rising trend during last decade in India. Pig is the amplifying host for JE virus and serves as a suitable sentinel model for the prediction of disease outbreak in humans. The development of a diagnostic test that is suitable for surveillance of JE in pigs is the need of the hour. The existing tests require elaborate laboratory facilities which make their application in rural settings difficult. Therefore, realizing the need for a rapid test, efforts were made to standardize a latex agglutination test (LAT) for serodiagnosis of JE in pigs. METHODS: Standardization of LAT by physical adsorption of recombinant NS1 (non-structural) protein of JE virus onto latex beads was done by altering six different variables, namely the antigen concentration, sensitization condition, surface blocking agent, blocking condition, particle concentration and reaction time. The standardized latex-protein complex was used for screening 246 pig serum samples under optimal conditions. RESULTS: The test was standardized with a diagnostic sensitivity and specificity of 82.24 and 87.83%, respectively. Screening of 246 field pig serum samples using standardized LAT showed a seropositivity of 50.4%. The results were available within 5 min after addition of test serum sample to the sensitized beads. INTERPRETATION & CONCLUSION: The findings of the study highlight the potential of LAT as a rapid on-site assay for JE diagnosis in pigs which would aid in predicting JE outbreaks in humans.

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.

Genetic characterization of equine herpesvirus 1 isolates from abortion outbreaks in India.

Equine herpesvirus 1 (EHV1) is a common pathogen of horses that causes upper respiratory tract disease, abortion, neonatal death and neurological disease. The neurological form of disease is called equine herpesvirus myeloencephalopathy (EHM). During the past decade, the incidence of EHM has been on the rise in Europe, North America, Australia and Asia. Some EHV1 isolates causing EHM exhibit a single-nucleotide polymorphism (SNP) in the DNA polymerase gene (ORF30) at position 2254 (A2254 to G2254). Further, based on polymorphism in the ORF68, EHV1 isolates have been classified into different groups. The aim of the present study was to estimate the genetic diversity of EHV1 and to determine the prevalence of the neuropathogenic genotype of EHV1 in India. Out of 133 clinical specimens from abortion cases in northern India, 56 were positive for EHV1 infection. Analysis of the A/G SNP by real-time PCR and sequence analysis revealed that 54 of 56 samples (96.43 %) were of the non-neuropathogenic genotype (A2254), while two (3.57 %) had the neuropathogenic marker (G2254). Sequence analysis of the polymorphic region of ORF68 of EHV1 isolates (n = 9) from India indicated that the Delhi/1998, Tohana-2/2013, Hisar-2/2014 and Hisar-15/1990 isolates belonged to group 4, while the Jind/1996, Rajasthan/1998, Delhi-3/2007 and Tohana-5/1996 isolates clustered within group 5. One isolate (Hisar-7/1990) exhibited SNPs at positions C710 and C713, forming a separate group. Here, we report for the first time the detection of neuropathogenic genotypes of EHV1 in India and show that Indian EHV1 isolates cluster within groups 4 and 5.