Bramhadev Pattnaik

Isolation and pathotyping of H9N2 avian influenza viruses in Indian poultry.

A total of 1246 faecal and tissue samples collected/received from 119 farms located in various states of India were processed for isolation of avian influenza viruses (AIV) during 2003-2004 as part of a program to monitor AIV infection in Indian poultry population. Avian influenza virus was isolated for the first time in India from poultry farms with history of drop in egg production, respiratory illness and increased mortality in Haryana state. A total of 29 H9N2 AIV isolates were obtained from the states of Punjab, Haryana, Uttar Pradesh, Gujarat, and Orissa and Union Territory Delhi. Subtyping was done by HI, RT-PCR and neuraminidase inhibition assay. Pathotyping of six representative isolates by intravenous pathogenicity index (0.0/3.0) in 6-8 weeks old chicken, trypsin dependency in cell culture and HA cleavage site analysis (335RSSR*GLF341) confirmed that these isolates are low pathogenic. Nucleotide sequence analysis of the HA gene showed that the Indian isolates are very closely related (95.0-99.6%) and shared a homology of 92-96% with H9N2 isolates from Germany and Asian regions other than that of mainland China. Deduced amino acid sequences showed the presence of L226 (234 in H9 numbering) which indicates a preference to binding of alpha (2-6) sialic acid receptors. Two of the six isolates had 7 glycosylation sites in the HA1 cleaved protein and the remaining four had 5 sites. Phylogenetic analysis showed that they share a common ancestor Qa/HK/G1/97 isolate which had contributed internal genes of H5N1 virus circulating in Vietnam. Further characterization of Indian H9N2 isolates is required to understand their nature and evolution.

Multiple origins of foot-and-mouth disease virus serotype Asia 1 outbreaks, 2003-2007.

Viruses in 6 genetic groups have caused recent outbreaks in Asia.Emerging outbreaks of zoonotic diseases are affecting humans at an alarming rate. Until the ecological factors associated with zoonoses are better understood, disease emergence will continue. For Lyme disease, disease suppression has been demonstrated by a dilution effect, whereby increasing species diversity decreases disease prevalence in host populations. To test the dilution effect in another disease, we examined 17 ecological variables associated with prevalence of the directly transmitted Sin Nombre virus (genus Hantavirus, etiologic agent of hantavirus pulmonary syndrome) in its wildlife host, the deer mouse (Peromyscus maniculatus). Only species diversity was statistically linked to infection prevalence: as species diversity decreased, infection prevalence increased. The increase was moderate, but prevalence increased exponentially at low levels of diversity, a phenomenon described as zoonotic release. The results suggest that species diversity affects disease emergence.

Genetic analysis of H9N2 avian influenza viruses isolated from India

H9N2 avian influenza viruses are endemic in domestic poultry in Asia and are grouped into three major sublineages represented by their prototype strains A/Duck/Hong Kong/Y280/97 (Y280-like), A/Quail/Hong Kong/G1/97 (G1-like) and A/Chicken/Korea/38349-p96323/96 (Korean-like). To understand the genetic relationship of Indian viruses, we determined the partial nucleotide sequence of five H9N2 avian influenza viruses isolated from chicken in India during 2003–2004 and compared them with H9N2 sequences available in GenBank. Deduced amino acid sequence analysis revealed that four isolates shared an R–S–S–R/G motif at the cleavage site of HA, representing low pathogenicity in chickens, while one virus harbors an R–S–N–R/G motif at the same position. All the viruses maintained the human-like motif 226Lysine (H3 numbering) at the HA receptor binding site. Phylogenetic analysis showed that 50% of the genes (HA, NA, NP and M) were similar to G1-like viruses, whereas the remaining genes of the Indian isolates formed a separate, not yet defined, sublineage in the Eurasian lineage. Our finding provides evidence of a novel reassortant H9N2 genotype of G1-like viruses circulating in India.

Status of foot-and-mouth disease in India.

Foot-and-mouth disease (FMD) is endemic in India and causes severe economic loss. Status of FMD in the country for five fiscal years is presented. Outbreaks were more in number in 2007-2008 than 2010-2011. Three serotypes of FMD virus (O, A and Asia1) are prevalent. Serotype O was responsible for 80% of the confirmed outbreaks/cases, whereas Asia1 and A caused 12% and 8%, respectively. Geographical region-wise assessment indicated varying prevalence rate in different regions viz; 43% in Eastern region, 31.5% in Southern region, 11.6% in North-eastern region, 5% Central region, 4.4% Western region and 4% in Northern region. Highest number of outbreaks/cases was recorded in the month of September and lowest in June. Emergence and re-emergence of different genotypes/lineages within the serotypes were evident in real-time investigation carried out from time to time. Continues antigenic divergence in serotype A resulted in change in the vaccine strain in 2009. As on date, all genetic diversity within the serotypes is well tolerated by the vaccine strains. Unrestricted animal movements in the country play a major role in the spread of FMD.

Recombinant non-structural polyprotein 3AB-based serodiagnostic strategy for FMD surveillance in bovines irrespective of vaccination

In India, the proportion of bovines vaccinated against foot-and-mouth disease (FMD) is increasing since the implementation of the Government supported FMD Control Programme, and non-structural protein (NSP)-based serological assays for discriminating between antibodies induced by infection or vaccination (DIVA) could be useful. The FMD virus NSP 3AB was expressed in a prokaryotic system and an indirect ELISA (r3AB(3) I-ELISA) was developed and validated as a screening assay for detecting virus in vaccinated bovines. The diagnostic sensitivity of the assay was estimated to be 96%, while the diagnostic specificity varied between the naïve and vaccinates as 99.1% and 96.4%, respectively. This assay could detect antibodies to 3AB (3AB-Ab) from 10 to as late as 900 days post-infection in cattle infected experimentally. The in-house assay demonstrated higher sensitivity than a commercial 3ABC ELISA kit particularly with samples obtained from the late stages of infection. Transient post-vaccinal 3AB-Ab response could be detected in one of the three commercial vaccines during the six-month vaccination regimen, which emphasizes the fact that for a DIVA-compatible diagnostic strategy to be a realistic option, all vaccines need to be quality checked for the NSP content.

Genetic heterogeneity of Indian field isolates of foot-and-mouth disease virus serotype O as revealed by partial sequencing of 1D gene

The sequence of 165 nucleotides at the 3 end of the 1D gene, determined from RT PCR amplified cDNA fragments, of 25 type O strains isolated from different parts/regions of India during 1987 1995 and the vaccine strain (R2/75) currently in use in India were subjected to phylogenetic analysis. One isolate from the neighbouring country Nepal was also included in the study. The virus/ field strains showed high degree of genetic heterogeneity among themselves with % divergence in nucleotide sequence ranging from 1.2 to 19.4%. The Indian strains were much away (13.3 20.6%) from the exotic type O strains of O1BFS, O1K, and O1Campos. The type O strains analyzed were classified into three genotypes basing on level of divergence observed in nucleotide sequence. The type O vaccine virus (R2/75) was > 71% divergent (7.3-15.2%) from the field strains which revealed significant ( > 5%) genetic heterogeneity between the two. The phylogenetic analysis identified three distinct lineages, viz., (i) lineage 1 represented by the exotic strains, (ii) lineage 2 represented by 25 of the field strains which clustered into seven subgroups/sublines (2a-2g), and (iii) lineage 3 represented by a unique field isolate which shared the branching/origin with the vaccine strain. The lineage 2 which comprised of 25 of the 26 type O field strains analyzed, was placed almost at equidistance from the lineages 1 and 3 in the phylogenetic tree. The vaccine strain was closer to the viruses in lineage 2. Though there was no specific distribution pattern of sequences in different geographical regions of India, the viruses/ sequences in subgroup 2f appeared to be restricted to the southern states. Comparison of deduced amino acid sequence in the immunodominant regions 133-160 and 200-208 of the 1D gene product (VP1) showed that the two viruses in lineage 3 had unique amino acid residues at the positions 138 (D), 139 (G), 144 (I), and 158 (A) compared to rest of the strains including the exotic ones. Comparison of amino acid residues at critical positions 144, 148, 149, 151, 153, 154, and 208 revealed similarity between the type O strains analyzed. The virus strains showed variation (V/L/I) at position 144. One field strain showed replacement from Q149-->E and another from P208-->L. Thus, the study revealed that the type O FMD virus populations circulating in India and causing disease outbreaks are genetically much heterogeneous but related at the immunodominant region of VP1 polypeptide, and there are more than one genetically distinct virus populations in almost every region of the country which is possible due to unrestricted animal movement in the country. The involvement of vaccine virus in disease outbreaks was ruled out as the field strains (excluding the one in lineage 3) were phylogenetically distinct from it.

Foot-and-mouth Disease: Global Status and Future Road Map for Control and Prevention in India

Foot-and-mouth disease (FMD) is a transboundary, economically devastating and highly contagious viral disease of livestock, most importantly cattle, buffalo and pig. The disease also affects goats, sheep, wild ruminant species and elephants. The causative FMD virus is antigenically diverse having seven distinct serotypes and many variants within them. Being a single stranded RNA virus, it confirms the quasispecies nature with emergences and reemergences of different genetic lineages with altered antigenicity within the serotypes, making vaccination based control programme a high cost effective, time consuming and difficult to achieve. As per the OIE and FAO, the disease is a major threat to food security of the world, and particularly the countries having the disease are more prone to food insecurity. Further, FMD free status is an indicator of development, and all developed countries are free from it. The disease is endemic in India and three serotypes of the virus viz; O, A and Asia1 are circulating. Annual direct loss due to FMD in India has been estimated at Rs. 20,000xa0crores. Many countries in the world are now free from FMD with or without vaccination and presence of the disease in other neighboring countries is a major threat to them. Countries having FMD face trade barrier posed by FMD free countries, causing heavy economic loss to the livestock industry. Progressive control pathway has been developed by FAO for global eradication of FMD. Vaccination based FMD control programme is in operation in India which involves biannual vaccinations of all cattle and buffaloes in selected areas, regular active surveillance and antibody monitoring in vaccinated population with the objective of creating FMD free zones. At present, the disease occurrence, severity of the clinical disease and number of outbreaks have progressively and substantially declined in the control zones as a result of last 10 rounds of vaccination with an oil adjuvanted trivalent inactivated vaccine. In this review, FMD scenario in India and in the world is briefed. Besides, the measures taken for the control and eradication of this devastating disease is presented. Besides, the initial success achieved through the FMD control programme in India, a road map for the control and eradication of FMD at national level is discussed.

Comparative genomics of serotype Asia 1 foot-and-mouth disease virus isolates from India sampled over the last two decades

This study deals with a comparative analysis of complete genome sequences of twenty-one serotype Asia 1 foot-and-mouth disease (FMD) field viruses isolated over a period of two decades from India, two vaccine strains and seven exotic sequences. The Indian viruses could be grouped in to three distinct lineages at the entire coding region, evolving independently probably under differential selection pressure as evident from the lineage-specific signatures identified. This comparison revealed 80% of amino acids at the polyprotein region to be invariant. Twenty-one residues in L, 3A and P1 region were identified to be under positive selection of which some are antigenically critical. Analysis at functionally crucial motifs, receptor contact residues, polyprotein cleavage sites and at putative T-cell epitopes expands the knowledge beyond other serotypes. Antigenic site II in betaB-betaC loop of VP2 was highly unstable suggesting its exposure to extreme immune pressure. A single cross-over at the L proteinase region in an isolate from buffalo, also featuring an extensive deletion at the 5 untranslated region (UTR), reflects the role of intraserotypic genetic recombination in natural evolution. The likely biological relevance of deletions/insertions observed at UTRs, VP1 and 3A could not be deduced. Altogether, a substantial amount of data raised on full length genomes of type Asia 1 virus adds value to the FMD virus genomics.

Phylogenetic structure of serotype A foot-and-mouth disease virus: global diversity and the Indian perspective.

Global epidemiological analysis is vital for implementing progressive regional foot-and-mouth disease control programmes. Here, we have generated VP1 region sequences for 55 Indian type A outbreak strains and have included complete VP1 sequences from 46 other countries to obtain a comprehensive global phylogeographical impression. A total of 26 regional genotypes within three continental topotypes, based on a 15% nucleotide divergence cut-off criterion, could be identified. These genotypes correlated with distinct evolutionary lineages in the maximum-likelihood phylogeny. During the last decade, ten genotypes have been in circulation the world over and it was evident that no type A strain has transgressed the continental barriers during this period. A single genotype (genotype 18) within the Asia topotype has been circulating in India with neither any incursion nor any long distance movement of virus out of the country during the last ten years, although close genetic and epidemiological links between viruses from Bhutan and India were revealed.

Differential expression of pro-inflammatory cytokines in endometrial tissue of buffaloes with clinical and sub-clinical endometritis

The objective of this study was to investigate the endometrial expression of pro-inflammatory cytokines (IL1β, IL6, IL8 and TNFα) along with TLR4 and CD14 in normal and endometritic buffaloes. The genitalia were collected in the abattoir and divided into three groups as normal (gr. I=12), clinical endometritis (CE, gr. II=12) based on positive color reaction to white side test of uterine discharge and sub-clinical endometritis (SCE, gr. III=12) based on endometrial cytology (presence of ≥5% PMNs) and histopathology. The equal numbers of genitalia were grouped into follicular and luteal stage in each group. Endometrial tissue scrapings were used for total RNA extraction and cDNA was transcribed and amplified by Real time PCR. The results showed several fold higher expression of all cytokine transcripts in CE (gr. II), whereas significant up-regulation of CD14 (1 to 2-fold), IL6 (15 to 36-fold), IL8 (8 to 14-fold) and TNFα (10 to 11-fold) mRNA was observed in SCE. This indicates that the evaluation of expression patterns of certain cytokines gene holds promise to diagnose the severity and degree of uterine inflammation.