Karam Pal Singh

Cell-mediated immune response and cross-protective efficacy of binary ethylenimine-inactivated bluetongue virus serotype-1 vaccine in sheep

Bluetongue is a serious hemorrhagic disease of sheep, cattle and other ruminants causing economic losses worldwide. Recent invasion of multiple bluetongue virus serotypes (BTV) in various countries warrants immediate development of efficacious vaccine that targets more than one serotype. In the present study, the cross-protective efficacy of binary ethylenimine (BEI)-inactivated BTV-1 vaccine was evaluated in Indian native sheep against virulent heterologous BTV-23 serotype challenge. BTV-1 vaccination induced significant cell-mediated immunity (CMI) as determined by lymphoproliferative responses, and increased CD8 T cell, IL-2 and IFN-gamma responses. Both naïve and immunized sheep also showed increased CD4 T cell, IL-12 and IFN-alpha responses. Collectively, these data suggested that inactivated BTV-1 vaccine induced appreciable CMI and greatly reduced the severity of heterologous BTV-23 infection.

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.

The Genome Sequence of Bluetongue Virus Type 2 from India: Evidence for Reassortment between Eastern and Western Topotype Field Strains

ABSTRACT Bluetongue virus type 2, isolated in India in 1982 (IND1982/01), was obtained from the Orbivirus Reference Collection at IAH Pirbright (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1982/01). Full genome sequencing and phylogenetic analyses show that IND1982/01 is a reassortant virus containing genome segments derived from both eastern and western topotypes. These data will help to identify further reassortment events involving this or other virus lineages in the subcontinent.

Molecular epidemiology studies of bluetongue virus

There are 24 distinct serotypes of bluetongue virus (BTV), whose identity is determined by the specificity of reactions between the proteins involved in cell attachment and penetration on the surface of the viral capsid, and the neutralizing antibodies that are generated during infection of the mammalian host. The BTV genes encoding these outer-capsid proteins show nucleotide sequence variations that correlate with both virus serotype and the geographical origin of the virus isolate. “Molecular epidemiology” studies can compare the RNA sequences of individual or multiple genome segments from novel BTV isolates, with those of existing strains from known locations and dates, identifying both virus serotype and topotype. Molecular epidemiology studies of BTV depend on the development of sequence databases for the RNA segments of individual virus isolates from defined locations with well-documented isolation dates and passage histories. Studies of BTV strains, which were generated by exchange (reassortment) of genome segments between two different BTV serotypes, have demonstrated that both of the BTV outer-capsid proteins VP2 and VP5 can influence the specificity of reactions between the virus particle and the neutralizing antibodies. Further sequencing studies will help to clarify relationships between the European field and vaccine strains of BTV.

Immune responses and protective efficacy of binary ethylenimine (BEI)-inactivated bluetongue virus vaccines in sheep

Abbreviations: Al(OH)3, aluminium hydroxide; BEI, binary ethylenimine; BTV, bluetongue virus; CMI, cellmediated immune response; CPE, cytopathic effect; CRI, clinical reaction index; d.p.i., days post inoculation; DTH, delayed-type hypersensitivity; EHDV, epizootic haemorrhagic disease virus of deer; FIA, Freund’s incomplete adjuvant; PBS, phosphate-buffered saline; pfu, plaque-forming unit; PHA, phytohaemagglutinin; RBC, red blood cell

Bluetongue in the Indian subcontinent

The Indian subcontinent is a geographically vast region located between 0–40N and 60–100E. It includes all of India, as well as Bangladesh, Bhutan, Nepal, Sri Lanka and Pakistan. Exotic breeds, cross-breeds and native breeds of sheep and cattle are all farmed in the region. In the Indian subcontinent. To date, 24 serotypes of bluetongue virus (BTV) have been recorded worldwide. Twenty-one serotypes have been reported from India, ten of these on the basis of virus isolation and eleven on the basis of neutralizing antibodies. The diagnosis of BT and identification of BTV is carried out in India using both serological- and nucleic acid-based diagnostic tests. Recurring outbreaks of clinical BT in sheep and production losses in other domestic ruminants have caused great economic loss in southern India. Native breeds of sheep that are particularly susceptible to clinical BT are reared mainly on a small scale, and therefore the disease is one of the major causes of economic loss to less-affluent farmers. The existence of 24 BTV serotypes, which largely fail to cross-protect, has made the goal of protective immunization against the disease particularly difficult to achieve in the region. Live attenuated or inactivated vaccines, based on local Indian strains of the virus, are not available, and there is no current BTV vaccination program within India. However, recent developments of inactivated or sub-unit vaccines may in the future help to control the disease in the subcontinent.

Apoptosis and immuno-suppression in sheep infected with bluetongue virus serotype-23.

The role of apoptosis in pathogenesis of bluetongue (BT) has been suggested from various in vitro studies. However, to date, no clear data are available regarding BTV-induced apoptosis and its consequences in natural host, sheep. In the present study, bluetongue virus (BTV)-induced apoptosis was studied in sheep blood and splenic mononuclear cells by analyzing annexin(+)-propidium iodide(-) early apoptotic cells, DNA ladder pattern, and caspase-3 gene expression. The onset of apoptosis and lymphocyte depletion in viraemic phase and IFN-alpha response indicated the involvement of BTV and IFN-alpha in the pathogenesis of BT. The development of Pasteurella pneumonia in 4 of 7 infected sheep during the experiment pointed to possible BTV-induced immuno-suppression and predisposition to secondary microbial infections. These results have significant implications not only in understanding immuno-pathological consequences but also in studying interactions of BTV with host cells.

A comparison of intradermal and intravenous inoculation of bluetongue virus serotype 23 in sheep for clinico-pathology, and viral and immune responses.

The pathogenesis of bluetongue (BT) could vary with route of inoculation. Using laboratory-passaged moderately virulent bluetongue virus serotype 23 (BTV-23), one of the most prevalent Indian serotype, we investigated the pathogenesis of BT in intradermally (ID) and intravenously (IV) inoculated native sheep. The ID inoculation resulted in relatively increased clinical signs and lesions in many organs as compared to IV inoculation. BTV-23 detection by real-time RT-PCR and isolation studies revealed that ID inoculation can be more efficient than IV ones in disseminating and spreading virus to systemic organs, including pre-scapular draining lymph node, spleen, lungs and pulmonary artery. Furthermore, the ID inoculation resulted in early onset and increased humoral response with significant increase (P<0.01) in antibody titre at various intervals. Taken together, these data suggest that ID inoculation can be more potent in reproducing many aspects of natural infection, including clinical disease, viral and immune responses, and may be useful route in setting up experimental infections for challenge or pathogenesis studies using laboratory passaged BTVs.

Complete Genome Sequence of an Isolate of Bluetongue Virus Serotype 2, Demonstrating Circulation of a Western Topotype in Southern India

ABSTRACT Bluetongue virus serotype 2 (IND2003/02) was isolated in Tiruneveli City, Tamil Nadu State, India, and is stored in the Orbivirus Reference Collection at the Institute for Animal Health, Pirbright, United Kingdom. The entire genome of this isolate was sequenced, showing that it is composed of a total of 19,203 bp (all 10 genome segments). This is the first report of the entire genome sequence of a western strain of BTV-2 isolated in India, indicating that this virus has been introduced and is circulating in the region. These data will aid in the development of diagnostics and molecular epidemiology studies of BTV-2 in the subcontinent.

Enhanced proinflammatory cytokine activity during experimental bluetongue virus-1 infection in Indian native sheep.

Bluetongue disease has been causing variable morbidity and mortality in sheep in India and other countries of the world. The experimental infection with Indian BTV-1 strain induced mild to sub-acute infection in native sheep. There were low induction of IL-12, IFN-γ and TNF-α cytokine mRNA expressions determined by real time RT-PCR in draining lymph nodes (DLN), spleen, and PBMCs during the initial stages of infection (8 days post inoculation, DPI) and higher around 15 DPI. The reduced pro-inflammatory cytokine (IFN-γ and TNF-α) responses during the initial stage of infection (8 DPI) was also accompanied by similarly decreased T-cell populations and overt clinical symptoms. Later up regulation of these cytokines and substantial increase in the proportion of CD8(+) T-cells occurred with reduction of clinical signs and disappearance of BTV-1 antigen from tissues as determined by immunohistochemistry and RT-PCR. Thus there is definite involvement of pro-inflammatory cytokines and CD8(+) T cell activity in disease induced by BTV-1 strain.