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

Abstract

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.

Plaque Purification of Bluetongue Virus Serotype-4 (BTV-4)

“…Plaques can be seen as white areas. (Reddy et al, 2015) Identifying of plaques in the current study can be correlated with the observations of Howell et al, (1967) and Dulbecco (1952). The incubation time for plaque development is higher when compared with Cooper's (1962) study in which plaques were produced within 40 hrs of incubation.…”

“…Polymerase chain reaction for plaque purified virus was carried out with primers (IDT-DNA) specific for available bluetongue virus serotypes 2,4,9,10,12,16,21,23,24) (Reddy et al, 2015) along with positive and negative controls for each serotype.…”

“…serological and molecular confirmation. But, due to limitations of serological and molecular techniques, a combination of Reverse transcription polymerase chain reaction (RT-PCR) with Virus neutralization test (VNT) may provide better results for which plaque purification of the samples is necessary (Reddy et al, 2015). Hence, the current study was undertaken with an aim to plaque purify BTV4 isolated from BT outbreaks in Andhrapradesh during 2015.…”

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

“…Blood samples from sheep showing BT‐like symptoms were collected from several outbreaks from different parts of Andhra Pradesh and Telangana states in South India, and virus isolation was accomplished by passaging the RBC lysates in KC cells followed by BHK‐21 cells. All the isolates ( n = 108) were tested by RT–PCR with primers against eight serotypes of BTV (BTV‐1, BTV‐2, BTV‐9, BTV‐12, BTV‐16, BTV‐21, BTV‐23, BTV‐24) circulating in India during the last decade (Krishnajyothi et al., ; Reddy et al., ). Sequence generated from one of the previously untyped virus isolates using next‐generation sequencing platform revealed that the isolate was related to BTV‐4 (Acc.…”

“…Although serotyping of BTV is based on the neutralization of virus using homotypic sera, typing based on reverse transcription–PCR and sequencing of seg‐2 is gaining popularity. However, nucleic acid sequences of seg‐2 of BTV isolated from different geographical areas differ significantly and the primers designed based on the reference viruses generally fail to detect different seg‐2 topotypes of the same serotype (Maan et al., , ; Mertens et al., ; Reddy et al., ). Hence, it is important to identify different topotypes and subtopotypes circulating in different geographical regions.…”