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.

Infection kinetics and antibody responses in Deccani sheep during experimental infection and superinfection with bluetongue virus serotypes 4 and 16.

AIM: The current study was designed to understand the infection kinetics and antibody responses of major circulating serotypes of bluetongue virus (BTV) in India, i.e., BTV-4 and BTV-16 through experimental infection and superinfection of Deccani sheep, a popular breed of sheep found in the southern states of India. MATERIALS AND METHODS: Experimental infection with 106 TCID50/ml BTV-4 was followed by superinfection with BTV-16 and vice versa. Along with observing for clinical signs and immunological responses in the experimentally infected sheep, the effect of infection of one specific serotype on the outcome of superinfection with a different serotype was also studied. RESULTS: Certain interesting findings have been made in the course of experimental infection, such as prominent signs of infection in BTV-4 infection, mild or no clinical signs in BTV-16-infected and superinfected animals, and non-seroconversion of one of the BTV-16-superinfected animals. In addition, BTV was isolated from infected sheep in all the experimental conditions except BTV-16 superinfection. Furthermore, it was observed that immune response in the form of type-specific antibodies was slower with BTV-16 superinfection. CONCLUSION: Superinfection of a sheep with more than one serotype of BTV is a common phenomenon in BT endemic countries like India. Such situation was replicated in an experimental infection in the current study, and the findings to our knowledge are first of a kind and are likely to aid in unfolding the newer aspects of BTV pathogenesis and virulence.

Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus.

AIM: The present study was designed to standardize real-time polymerase chain reaction (PCR) for detecting the bluetongue virus from blood samples of sheep collected during outbreaks of bluetongue disease in the year 2014 in Andhra Pradesh and Telangana states of India. MATERIALS AND METHODS: A 10-fold serial dilution of Plasmid PUC59 with bluetongue virus (BTV) NS3 insert was used to plot the standard curve. BHK-21 and KC cells were used for in vitro propagation of virus BTV-9 at a TCID50/ml of 105 ml and RNA was isolated by the Trizol method. Both reverse transcription-PCR and real-time PCR using TaqMan probe were carried out with RNA extracted from virus-spiked culture medium and blood to compare the sensitivity by means of finding out the limit of detection (LoD). The results were verified by inoculating the detected and undetected dilutions onto cell cultures with further cytological (cytopathic effect) and molecular confirmation (by BTV-NS1 group-specific PCR). The standardized technique was then applied to field samples (blood) for detecting BTV. RESULTS: The slope of the standard curve obtained was -3.23, and the efficiency was 103%. The LoD with RT-PCR was 8.269E×103 number of copies of plasmid, whereas it was 13 with real-time PCR for plasmid dilutions. Similarly, LoD was determined for virus-spiked culture medium, and blood with both the types of PCR and the values were 103 TCID 50/ml and 104 TCID 50/ml with RT-PCR and 10° TCID 50/ml and 102 TCID 50/ml with real-time PCR, respectively. The standardized technique was applied to blood samples collected from BTV suspected animals; 10 among 20 samples were found positive with Cq values ranging from 27 to 39. The Cq value exhibiting samples were further processed in cell cultures and were confirmed to be BT positive. Likewise, Cq undetected samples on processing in cell cultures turned out to be BTV negative. CONCLUSION: Real-time PCR was found to be a very sensitive as well as reliable method to detect BTV present in different types of samples, including blood samples collected from BTV-infected sheep, compared to RT-PCR. The LoD of BTV is likely influenced by sample type, possibly by the interference by the other components present in the sample.

Isolation and evolutionary analysis of Australasian topotype of bluetongue virus serotype 4 from India.

Bluetongue (BT) is a Culicoides-borne disease caused by several serotypes of bluetongue virus (BTV). Similar to other insect-borne viral diseases, distribution of BT is limited to distribution of Culicoides species competent to transmit BTV. In the tropics, vector activity is almost year long, and hence, the disease is endemic, with the circulation of several serotypes of BTV, whereas in temperate areas, seasonal incursions of a limited number of serotypes of BTV from neighbouring tropical areas are observed. Although BTV is endemic in all the three major tropical regions (parts of Africa, America and Asia) of the world, the distribution of serotypes is not alike. Apart from serological diversity, geography-based diversity of BTV genome has been observed, and this is the basis for proposal of topotypes. However, evolution of these topotypes is not well understood. In this study, we report the isolation and characterization of several BTV-4 isolates from India. These isolates are distinct from BTV-4 isolates from other geographical regions. Analysis of available BTV seg-2 sequences indicated that the Australasian BTV-4 diverged from African viruses around 3,500 years ago, whereas the American viruses diverged relatively recently (1,684 CE). Unlike Australasia and America, BTV-4 strains of the Mediterranean area evolved through several independent incursions. We speculate that independent evolution of BTV in different geographical areas over long periods of time might have led to the diversity observed in the current virus population.

Epidemiology of Bluetongue in India.

Bluetongue (BT) is an insectborne endemic disease in India. Although infections are observed in domestic and wild ruminants, the clinical disease and mortality are observed only in sheep, especially in the southern states of the country. The difference in disease patterns in different parts of the country could be due to varied climatic conditions, sheep population density and susceptibility of the sheep breeds to BT. Over the five decades after the first report of BT in 1964, most of the known serotypes of bluetongue virus (BTV) have been reported from India either by virus isolation or by detection of serotype-specific antibodies. There have been no structured longitudinal studies to identify the circulating serotypes throughout the country. At least ten serotypes were isolated between 1967 and 2000 (BTV-1-4, 6, 9, 16-18, 23). Since 2001, the All-India Network Programme on Bluetongue and other laboratories have isolated eight different serotypes (BTV-1-3, 9, 10, 12, 16, 21). Genetic analysis of these viruses has revealed that some of them vary substantially from reference viruses, and some show high sequence identity with modified live virus vaccines used in different parts of the world. These observations have highlighted the need to develop diagnostic capabilities, especially as BT outbreaks are still declared based on clinical signs. Although virus isolation and serotyping are the gold standards, rapid methods based on the detection of viral nucleic acid may be more suitable for India. The epidemiological investigations also have implications for vaccine design. Although only a handful serotypes may be involved in causing outbreaks every year, the combination of serotypes may change from year to year. For effective control of BT in India, it may be pertinent to introduce sentinel and vector traps systems for identification of the circulating serotypes and to evaluate herd immunity against different serotypes, so that relevant strains can be included in vaccine formulations.

Molecular Typing of Bluetongue Viruses Isolated Over a Decade in South India.

Bluetongue (BT) is an arthropod-borne viral disease mostly of sheep. Bluetongue virus (BTV) is a segmented double-stranded RNA virus belonging to the genus Orbivirus of family Reoviridae and is transmitted by midges belonging to Culicoides spp. The disease is endemic in the tropics and subtropics, and the incidence is high in southern India. Twenty-six serotypes of BTV have been reported worldwide. Although most of the serotypes have been reported in India, information regarding currently circulating serotypes is essential to develop control programs. Both serological assays and nucleic acid-based assays have been used for typing BTV. Segment 2, which codes for the outer capsid protein VP2, is the target for PCR-based typing; however, the VP2 sequence diversity among viruses belonging to the same serotype but isolated from different geographical areas makes it essential to develop geographical based reagents. In this study, reverse transcription PCR was developed based on sequences of Indian isolates of BTV (serotypes 1, 2, 9, 10, 12, 16, 21 and 23), and this was applied to type 52 isolates obtained during the last decade. It was found that multiple serotypes circulate, with involvement of more than one serotype infecting individual animals and herds over a period in a given area. Detection of circulating serotypes and estimation of herd immunity against different serotypes of BTV may provide important information for predicting the distribution of these serotypes and inclusion of serotypes in vaccines.

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.

Sequences of genes encoding type-specific and group-specific antigens of an Indian isolate of bluetongue virus serotype 10 (BTV-10) and implications for their origin.

Bluetongue, a transboundary disease, is endemic in several tropical countries and is caused by bluetongue virus (BTV). The origin and movement of BTV can be predicted by comparing nucleotide sequences of its segmented RNA genome. Such analyses have been useful in evaluating the source of the virus responsible for recent incursion of BTV into previously unreported areas. Besides several serotypes, genetically related BTV strains circulate in each endemic area, but such clusters of strains have been reported to be distinct from one geographical region to another. We obtained partial or complete sequences of the open reading frames encoded by VP2, VP6, VP7, NS1 and NS2 genes of a BTV-10 isolate of India and compared them with other BTV-10 sequences available in public database. Sequences of all the five genes showed >99% identity to BTV-10 prototype, vaccine strain and vaccine-like virus isolates from the USA. Our results suggest that Indian BTV-10 virus analysed in this study possibly originated from the United States.