Complete genome sequence of bluetongue virus serotype 9: implications for serotyping.

The second complete genome of bluetongue virus serotype 9 (BTV-9) is presented in this report. The sequence analysis points to continued circulation in India of a mixed topotype virus apparently belonging to the BTV-9 serotype, and it raises questions about approaches for serotyping bluetongue viruses.

Evidence of bluetongue virus serotype 21 (BTV-21) divergence.

Bluetongue virus serotype 21 (BTV-21) was originally isolated from Australia, but has now been reported from India, Indonesia, China and Japan. We report the isolation, and sequencing of BTV-21 from India. The complete ORF sequence of VP2 gene of this isolate showed that it is closely related to recent BTV-21 isolates from Japan (93-94% identity), and distantly related to BTV-21 reference strain (86% identity). Our results, along with the available sequences of Japanese isolates, suggest that the currently circulating BTV-21 strains from India and Japan are divergent from the original strain(s) from Australia and shed light on designing molecular diagnostics for the detection of BTV.

Genetic characterization of bluetongue virus serotype 9 isolates from India.

Recent incursions of bluetongue virus (BTV) into previously naive geographical areas have emphasised the need to better understand virus movement and epidemiology. Several bluetongue virus (BTV) serotypes are known to exist in India, and some serotype viruses have been isolated. However, the complete genome of not a single isolate is available to date. We report the complete genome sequence of one, and partial sequences of three other Indian isolates of BTV-9. Evolutionary relationships with segment-2 and -6 sequences of BTV isolates around the world, deduced using four different phylogenetic analyses and a similarity programme, show that BTV-9 (Eastern), BTV-9 (Western), and BTV-5 form a triad of equidistant, genetically distinct groups of viruses. The Indian BTV-9 isolates were closely related to Mediterranean and European BTV-9 isolates (Eastern topotype) based on segment-2 and -6 sequences. By contrast, segment-5 analyses clustered the Indian BTV-9 isolates with South African BTV-3 reference strain (98% identity), which belongs to one of the Western types. These results have implications on BTV origin and movement, genotyping, serotyping, and vaccine design.

Immunogenicity of a Candidate Ebola Hemorrhagic Fever Vaccine in Mice Based on Controlled In Vitro Expression of Ebolavirus Glycoprotein.

Ebolavirus (EBOV) is the etiology of Ebola hemorrhagic fever (EHF). A major EHF outbreak in 2014-2015 in West Africa claimed >11,000 lives. A licensed vaccine is not available for EHF, although several vaccines have undergone clinical trials. We developed a human adenovirus (Ad) serotype 5-based candidate EHF vaccine based on controlled expression of the EBOV (Makona strain) glycoprotein (GP) as the immunogen. Two clones, AdGP72 and AdGP75, and a control Ad515 vector, were generated and tested for protein expression in vitro and immunogenicity in mice. Eight groups of mice were immunized with three doses of buffer, Ad515, AdGP72, and AdGP75, by two different dose regimens. Three different antigens (AdGP75-infected Vero E6 cell extract and two baculovirus expressed EBOV GP antigens, namely, GP alone or GP with EBOV VP40) were used to evaluate the immune response. Expression studies indicated that full-length GP was cleaved into its component subunits when expressed in mammalian cells through the Ad vectors. Moreover, in coimmunoprecipitation studies, EBOV GP was found to be associated with VP40 when expressed in baculoviruses. The candidate vaccines were immunogenic in mice, as evaluated by enzyme-linked immunosorbent assay using mammalian- or baculovirus-derived antigens. Further characterization and development of the candidate vaccines are warranted.

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

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.

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.

Type-specific seroprevalence of bluetongue in Andhra Pradesh, India, during 2005-2009.

Bluetongue (BT) is an infectious, arthropod-borne viral disease of domestic and wild ruminants caused by bluetongue virus (BTV), which is a double-stranded segmented RNA virus. Of the 26 confirmed BTV serotypes, 23 were reported in India based on the detection of antibodies or virus. In order to assess the prevalence of different serotypes in Andhra Pradesh, serum samples which were positive for BTV by group-specific antibody ELISA were subjected to type-specific neutralization of BTV serotypes 1, 2, 9, 10, 21 and 23. Of the 52 samples tested, 50.0, 44.23, 21.15, 26.92, 0, and 15.38 % neutralized BTV serotypes 1, 2, 9, 10, 21 and 23, respectively. However, 32.69 % of the ELISA positive sera could not neutralize any of these serotypes, indicating that there could be other serotype viruses (e.g., BTV-3 and -16) circulating in the State. This method can be used for surveillance of the circulating serotypes as well as for assessing the level of herd immunity, and assist in determining the vaccine strains to be used in multivalent vaccines.

Deep sequencing as a method of typing bluetongue virus isolates.

Bluetongue (BT) is an economically important endemic disease of livestock in tropics and subtropics. In addition, its recent spread to temperate regions like North America and Northern Europe is of serious concern. Rapid serotyping and characterization of BT virus (BTV) is an essential step in the identification of origin of the virus and for controlling the disease. Serotyping of BTV is typically performed by serum neutralization, and of late by nucleotide sequencing. This report describes the near complete genome sequencing and typing of two isolates of BTV using Illumina next generation sequencing platform. Two of the BTV RNAs were multiplexed with ten other unknown samples. Viral RNA was isolated and fragmented, reverse transcribed, the cDNA ends were repaired and ligated with a multiplex oligo. The genome library was amplified using primers complementary to the ligated oligo and subjected to single and paired end sequencing. The raw reads were assembled using a de novo method and reference-based assembly was performed based on the contig data. Near complete sequences of all segments of BTV were obtained with more than 20× coverage, and single read sequencing method was sufficient to identify the genotype and serotype of the virus. The two viruses used in this study were typed as BTV-1 and BTV-9E.

Immunotherapy of viral infections.

Among the microorganisms that cause diseases of medical or veterinary importance, the only group that is entirely dependent on the host, and hence not easily amenable to therapy via pharmaceuticals, is the viruses. Since viruses are obligate intracellular pathogens, and therefore depend a great deal on cellular processes, direct therapy of viral infections is difficult. Thus, modifying or targeting nonspecific or specific immune responses is an important aspect of intervention of ongoing viral infections. However, as a result of the unavailability of effective vaccines and the extended duration of manifestation, chronic viral infections are the most suitable for immunotherapies. We present an overview of various immunological strategies that have been applied for treating viral infections after exposure to the infectious agent.