Kanisht Batra

Full-Genome Sequencing as a Basis for Molecular Epidemiology Studies of Bluetongue Virus in India.

Since 1998 there have been significant changes in the global distribution of bluetongue virus (BTV). Ten previously exotic BTV serotypes have been detected in Europe, causing severe disease outbreaks in naïve ruminant populations. Previously exotic BTV serotypes were also identified in the USA, Israel, Australia and India. BTV is transmitted by biting midges (Culicoides spp.) and changes in the distribution of vector species, climate change, increased international travel and trade are thought to have contributed to these events. Thirteen BTV serotypes have been isolated in India since first reports of the disease in the country during 1964. Efficient methods for preparation of viral dsRNA and cDNA synthesis, have facilitated full-genome sequencing of BTV strains from the region. These studies introduce a new approach for BTV characterization, based on full-genome sequencing and phylogenetic analyses, facilitating the identification of BTV serotype, topotype and reassortant strains. Phylogenetic analyses show that most of the equivalent genome-segments of Indian BTV strains are closely related, clustering within a major eastern BTV ‘topotype’. However, genome-segment 5 (Seg-5) encoding NS1, from multiple post 1982 Indian isolates, originated from a western BTV topotype. All ten genome-segments of BTV-2 isolates (IND2003/01, IND2003/02 and IND2003/03) are closely related (>99% identity) to a South African BTV-2 vaccine-strain (western topotype). Similarly BTV-10 isolates (IND2003/06; IND2005/04) show >99% identity in all genome segments, to the prototype BTV-10 (CA-8) strain from the USA. These data suggest repeated introductions of western BTV field and/or vaccine-strains into India, potentially linked to animal or vector-insect movements, or unauthorised use of ‘live’ South African or American BTV-vaccines in the country. The data presented will help improve nucleic acid based diagnostics for Indian serotypes/topotypes, as part of control strategies.

Development and evaluation of real time RT-PCR assays for detection and typing of Bluetongue virus

Bluetongue virus is the type species of the genus Orbivirus, family Reoviridae. Bluetongue viruses (BTV) are transmitted between their vertebrate hosts primarily by biting midges (Culicoides spp.) in which they also replicate. Consequently BTV distribution is dependent on the activity, geographic distribution, and seasonal abundance of Culicoides spp. The virus can also be transmitted vertically in vertebrate hosts, and some strains/serotypes can be transmitted horizontally in the absence of insect vectors. The BTV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in order of decreasing size (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable BTV protein and the primary target for neutralising antibodies. Consequently VP2 (and Seg-2) determine the identity of the twenty seven serotypes and two additional putative BTV serotypes that have been recognised so far. Current BTV vaccines are serotype specific and typing of outbreak strains is required in order to deploy appropriate vaccines. We report development and evaluation of multiple ‘TaqMan’ fluorescence-probe based quantitative real-time type-specific RT-PCR assays targeting Seg-2 of the 27+1 BTV types. The assays were evaluated using orbivirus isolates from the ‘Orbivirus Reference Collection’ (ORC) held at The Pirbright Institute. The assays are BTV-type specific and can be used for rapid, sensitive and reliable detection / identification (typing) of BTV RNA from samples of infected blood, tissues, homogenised Culicoides, or tissue culture supernatants. None of the assays amplified cDNAs from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures.

Development and evaluation of loop-mediated isothermal amplification assay for rapid detection of Capripoxvirus

Aim: The present study was undertaken to develop a nucleic acid-based diagnostic assay loop-mediated isothermal amplification assay (LAMP) targeting highly conserved genomic regions of Capripoxvirus (CaPVs) and its comparative evaluation with real-time polymerase chain reaction (PCR). Material and Methods: Lyophilized vaccine strain of sheeppox virus (SPPV) was used for optimization of LAMP assay. The LAMP assay was designed using envelope immunogenic protein (P32) coding gene targeting highly conserved genomic regions of CaPV responsible for causing sheep pox, goat pox, and lumpy skin disease in sheep, goat and cattle respectively. Serial tenfold dilution of SPPV recombinant plasmid DNA was used for a calculating limit of detection. Analytical sensitivity and specificity were performed. Results: The test described is quick (30 min), sensitive and specific for detection of CaPVs. The described assay did not show any cross-reactivity to other related viruses that cause apparently similar clinical signs. It was found to be ten times more sensitive than conventional PCR however, 100 times less sensitive than quantitative PCR (qPCR). LAMP assay results were monitored by color change method using picogreen dye and agarose gel electrophoresis. Conclusion: LAMP assay can be a very good alternative for CaPV detection to other molecular techniques requiring sophisticated equipments.

Genome Sequence of Bluetongue Virus Type 2 from India: Evidence for Reassortment between Outer Capsid Protein Genes

ABSTRACT Southern Indian isolate IND1994/01 of bluetongue virus serotype 2 (BTV-2), from the Orbivirus Reference Collection at the Pirbright Institute (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1994/01), was sequenced. Its genome segment 6 (Seg-6) [encoding VP5(OCP2)] is identical to that of the Indian BTV-1 isolate (IND2003/05), while Seg-5 and Seg-9 are closely related to isolates from South Africa and the United States, respectively.

Concurrent infection of bluetongue and peste-des-petits-ruminants virus in small ruminants in Haryana State of India

Bluetongue (BT) and peste-des-petits-ruminants (PPR) are major transboundary diseases of small ruminant, which are endemic in India. Testing of bluetongue virus (BTV) and peste-des-petits-ruminants virus (PPRV) from recent outbreaks (2015-2016) in different regions of Haryana State of India revealed that 27.5% of the samples showed the presence of dual infection of BTV and PPRV. Analysis of Seg-2 of BTV (the serotype-determining protein) showed the presence of BTV-12w in several isolates. However, analysis of N gene fragment amplicons showed that viruses belong to lineage IV were most closely related to a pathogenic strain of PPRV from Delhi. This is the first report of co-circulation of PPRV lineage IV and bluetongue virus serotype 12 in the state.

Isolation and molecular characterization of contagious pustular dermatitis virus from Rajasthan, India

This article describes the isolation and identification of contagious pustular dermatitis virus/orf virus (ORFV) from an outbreak of contagious pustular dermatitis (orf) in flocks of goats, in the north western region of India (Rajasthan). The virus was isolated in Vero cell cultures from scab and swab suspensions and has been identified using GIF/IL-2 and B2L gene specific primers in PCR and sequencing. The virus showed high nucleotide identity with previously reported Chinese, far eastern, Brazilian and Indian isolates. This report described the use of molecular tools for fast, reliable and confirmatory diagnosis of ORFV infection.

Nutrigenomic evaluation of garlic (Allium sativum) and holy basil (Ocimum sanctum) leaf powder supplementation on growth performance and immune characteristics in broilers

AIM In this study, a planned research work was conducted to investigate the nutrigenomic aspects of supplementation of Allium sativum (garlic) and Ocimum sanctum (holy basil) leaf powder on the growth performance and immune characteristics of broilers. MATERIALS AND METHODS A 6 weeks feeding trial was conducted with 280-day-old Ven Cobb broilers, distributed randomly into seven experimental groups. Each treatment had 4 replicates with 10 birds each. The birds of the control group (T1) were fed a basal diet formulated as per BIS standards. The broilers of treatment groups T2 and T3 were fed basal diet supplemented with the commercially available garlic powder (GP) at levels of 0.5% and 1.0% of the feed, respectively, while broilers in T4 and T5 were fed basal diet supplemented with commercial grade holy basil leaf powder (HBLP) at levels 0.5% and 1.0% of the feed, respectively. Birds in the T6 were fed with 0.5% GP and 0.5% HBLP, whereas T7 was fed with 1.0% GP and 1.0% HBLP. At the end of the feeding trial (6th week), blood samples were collected and analyzed for relative mRNA expression of toll-like receptors (TLR) 2, TLR 4 and TLR 7 using real-time polymerase chain reaction. RESULTS The mean body weight gain and feed conversion efficiency were improved (p<0.05) in broilers fed the GP and HBLP incorporated diets compared with the control group. The relative mRNA expression levels of TLR 2, TLR 4 and TLR 7 in the peripheral blood of the broilers were found to be increased (p<0.05) in the birds supplemented with graded levels of the GP and HBLP as compared to the untreated group. CONCLUSION The present work concludes that the inclusion of GP and HBLP could enhance the production performance and immune status of birds by augmenting the T-cell mediated immune response and thereby protects them from disease without decreasing growth traits as a possible substitution to conventional antimicrobials.

Full-Genome Sequence Analysis of a Reassortant Strain of Bluetongue virus Serotype 16 from Southern India.

ABSTRACT The complete genome sequence of a reassortant field strain (IND2014/01) of Bluetongue virus (BTV) serotype 16, isolated from sheep from southern India in 2014, was sequenced. The total genome size was 19,186 bp. Sequence comparisons of all genome segments, except segment 5 (Seg-5), showed that IND2014/01 belonged to the major eastern topotype of BTV.

Recombinant interferon stimulated protein 15 (rISG15) as a molecular marker for detection of early pregnancy in Bubalus bubalis

Early and accurate diagnosis of pregnancy in animals is important for improving the reproductive management of livestock. The buffalo (Bubalus bubalis) is the most important dairy animal in India, but there are reproductive problems resulting from extended calving interval and ovulation occurring in the absence of behavioral estrus. The lack of simple methods for early pregnancy diagnosis intensifies these problems. The present study, therefore, was conducted to ascertain the role of the interferon-stimulated gene, (ISG), 15 in pregnancy detection. The anti-ISG15 Mab based ELISA was developed that could be used for detecting pregnancy at 18 to 20 days after artificial insemination (AI). The ISG15 protein was isolated from a pregnant buffalo and was amplified, and cloned in Escherichia coli by using coding region primers. The ISG15 gene was expressed in the host Escherichia coli BL21 (DE3), and the protocol was standardized for optimum gene expression. Using immortal hybridoma (fused myeloma and B cells) cells, a highly specific and sensitive antibody, anti-ISG15 mAb, for detecting ISG15 (protein) in the serum of pregnant buffaloes was obtained. A blocking ELISA was developed using the anti-ISG15 mAb to detect pregnancy in buffalo within 18 to 21 days after AI. The ISG15 gene was upregulated (P <  0.05) in pregnant buffalo at 18 to 21 days of pregnancy. This assay has an overall diagnostic accuracy of 75.0%. It, therefore, is concluded that recombinant ISG15 retains the potential for detecting pregnancy in B. bubalis and may have applications in ELISA kits for pregnancy detection in closely related species.

A comprehensive study on seroprevalence of bluetongue virus in Haryana state of India

Aim: The aim of present study was to determine seroprevalence of bluetongue virus (BTV) in Haryana state of India. Materials and Methods: A total of 803 serum samples, 408 of cattle and 395 of buffalo origin, respectively, were collected from different villages of Haryana. Sampling was done randomly to obtain unbiased results. The samples were evaluated by a competitive enzyme-linked immunosorbent assay for the presence of BTV antibodies. Results: Overall seroprevalence of BTV antibody in cattle and buffaloes for all 21 districts of Haryana state was found to be 75.49% and 92.91%, respectively. The prevalence of BTV in different agroclimatic zones ranged between 72-77% and 90-94% for cattle and buffalo, respectively. In buffaloes, the BTV seroprevalence was comparatively higher than in cattle. Conclusion: The study showed that BTV is circulating in cattle and buffalo populations in the Northern part of India.