MOLECULAR TYPING OF BLUETONGUE VIRUS ISOLATES
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Date
42660
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PVNR TVU
Abstract
Bluetongue (BT) is an arthropod-borne, non-contagious and economically important viral disease of domestic and wild ruminants caused by bluetongue virus (BTV). The clinical disease is seen mainly in sheep. BTV is a segmented double stranded RNA virus belonging to the Orbivirus genus of family Reoviridae and is transmitted by Culicoides sps. Out of the 27 serotypes prevalent in world, 24 have been reported in India.
The present study was taken up with the objective of typing BTV isolates obtained during outbreaks of 2015 from the states of Telangana and Andhra Pradesh by RT-PCR and confirmation of the results by Serum neutralization test. Blood samples were collected from BT suspected sheep showing symptoms of high fever, oral lesions, frothy salivation, facial edema and mucopurulent nasal discharges. The collected blood samples were processed in KC cells followed by BHK-21 cells. Extraction of RNA was done from CPE exhibiting infected BHK-21 cell culture supernatant and then subjected to 1% agarose gel electrophoresis for segmented pattern of RNA, one of the features of BTV. Upon
confirmation of segmented RNA pattern, the samples were confirmed as BTV by NS3 gene specific PCR. All the 50 samples were positive to BTV group specific NS3 conventional PCR and hence selected for molecular typing. Typing by RT-PCR was done using 10 serotype specific primers for BTV- 1, 2E, 4, 9, 10, 12, 16, 21, 23 and 24. Out of the 50 isolates, 23 isolates were positive for mixed BTV serotype infections {(06NLR/15-BTV-1,4); (13NLR/15-BTV-1,4,12); (18NLR/15-BTV-2E,4); (19NLR/15-BTV-1,12); (22NLR/15-BTV-1,4); (24NLR/15-BTV-4,12); (05PBR/15-BTV-1,4); (06PBR/15-BTV-1,4); (K4/15-BTV-4,12); (7GDK/15-BTV-2E,4,12); (1SHA/15-BTV-2E,9); (01ALG/15-BTV-4,12); (02ALG/15-BTV-1,4,24); (07ALG/15-BTV-4,16,24); (08ALG/15-BTV-16,24); (09ALG/15-BTV-1,4,16,24); (10ALG/15-BTV-12,16,24); (11ALG/15-BTV-12,16,24); (12ALG/15-BTV-1,12,16,24); (14ALG/15-BTV-12,24); (16ALG/15-BTV-12,16,24) (18ALG/15-BTV-12,16,24); (19ALG/15-BTV-1,12,16,24)} and 18 isolates were positive for single BTV serotype {(03NLR/15-BTV-1); (04NLR/15-BTV-1); (04PBR/15-BTV-1);(02NLR/15-BTV-2E); (2PKL/15-BTV-2E); (09NLR/15-BTV-4); (11NLR/15-BTV-4); (12NLR/15-BTV-4); (23NLR/15-BTV-4); (6PLR/15-BTV-4); (02PBR/15-BTV-4); (03PBR/15-BTV-4); (6PKL/15-BTV-4); (20NLR/15-BTV-12); (1MNL/15-BTV-12); (2MNL/15-BTV-12); (01NLR/15-BTV-24); (5PKL/15-BTV-24)}. Interestingly 9 isolates {(08NLR/15), (14NLR/15), (15NLR/15), (17NLR/15), (01PBR/15), (K3/15), (K9/15), (3PLR/15), (17ALG/15)} could not be typed with the primers specific to 10 serotypes of BTV, suggesting that these isolates belong to serotypes other than 1, 2E, 4, 9, 10, 12, 16, 21, 23 and 24. Out of the 50 BTV isolates, 8 molecular typed and 3 untyped isolates were also typed by SNT using reference sera of BTV-1, 2, 4, 9, 16. Of the 8 typed isolates, 5 and 3 were typed to be single serotype and mixed infections, respectively, by molecular assay. However, SNT could majorly be correlated to molecular typing for those isolates which were typed to be single serotype viruses, than mixed types.
The current study reveals circulation of several serotypes of BTV, both individually and in the form of mixed infection in the field. For effective control of the disease, circulating serotypes of the virus need to be incorporated in the vaccine. Molecular typing provides a rapid method for efficient typing of the viruses, especially in cases of mixed serotype infections.
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organic compounds, diseases, proteins, biological phenomena, lipids, aromatic compounds, esters, sowing, extraction, alcohols