Yadav, Sarita (2018) Isolation and molecular characterization of bluetongue virus from Southern India. PhD thesis. https://theses.gla.ac.uk/30807/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] UNIVERSITY OF GLASGOW Isolation and Molecular characterization of Bluetongue Virus from Southern India by Sarita Yadav A thesis submitted in fulfilment for the degree of Doctor of Philosophy in Virology College of Medical, Veterinary and Life Sciences May 2018 0 Abstract Abstract BT is endemic in India, particularly in the South and BTV strains belonging to twelve BTV-serotypes (BTV-1, 2, 3, 4, 5, 9, 10, 12, 16, 21, 23 and 24) have been isolated since 2001. A pentavalent inactivated BTV vaccine containing serotypes 1, 2, 10, 16 and 23, is currently being used in the South. This study focusses on the isolation and the typing of BTV strains from Southern India, 2014-15, then generation of full length sequence data, primarily for BTV genome Seg-2 to provide molecular epidemiology information concerning the strains circulating in the region. Suspected field outbreaks of BTV from Telangana, Andhra Pradesh, Karnataka and Tamil Nadu states in Southern India during 2014-2015 were attended to collect well documented blood and necropsy clinical samples from mixed population of small ruminants and cattle. Of the 447 field samples (EDTA blood, spleen, lymph node and saliva), 236 gave positive results for BTV RNA by serogroup-specific real-time RT-PCR targeting Seg-9 and Seg-10 for 2014 and 2015 samples respectively. A total of 141 BTV virus isolations were made from 141 serogroup-specific qRT-PCR positive samples, by infecting KC and BHK cells. The isolated 63 BTVs were serotyped by qRT-PCR assay targeting Seg-2 of the BTV-1 to BTV-24, BTV-26, BTV-27 and BTV-29, confirming the current co-circulation of 7 BTV serotypes, including BTV-1, BTV-2, BTV-4, BTV-5, BTV-9, BTV-10 and BTV-12 in 62 BTV isolates. Dual and triple BTV co-infections with these serotypes were detected in 22 and 4 BTV isolates, respectively. None of the serotypes present in the pentavalent vaccine were identified in any of the 22 BTV isolates from vaccinated animals, suggesting that the vaccine was indeed effective against the homologous serotype field strains. Among the 7 serotypes identified by Seg-2 based ORF sequence analysis of 46 BTV isolates (BTV-1, BTV-2, BTV-4, BTV-5, BTV-9, BTV-10 and BTV-12), 4 serotypes BTV-1, BTV-2, BTV-4 and BTV-9, belonged to eastern topotypes, whereas 3 serotypes, BTV-5, BTV-10 and BTV-12 were identified as western topotypes. For 4 BTV isolates, the serotype detected by qRT-PCR was different of that detected by NGS. The full genome sequencing and analyses is still ongoing in Glasgow. This data will assist in understand the epidemiology of the BT in India and implementation of effective BTV control to ensure the use of appropriate BTV serotype and strains in the available BT vaccine in India. i Abbreviations Abbreviations µL Microlitre aa Amino acid AGE Agarose gel electrophoresis AGID Agar gel immuno-diffusion AHSV African horse sickness virus AMV Avian myeloblastosis virus ATCC American Type Culture Collection BHK Baby hamster kidney BHQ Black Hole Quencher bp Base pair BT Bluetongue BTV Bluetongue virus cDNA Complementary DNA CF Complement fixation CPE Cytopathic effects Ct Cycle threshold dd NTP Dideoxy nucleotides triphosphates DMEM Dulbecco's modified Eagle's medium DMSO Dimethyl sulphoxide ds Double-stranded EDTA Ethylene diamine tetra acetic acid EEV Equine Encephalosis Virus EHDV Epizootic Hemorrhagic Disease Virus ELISA Enzyme linked immune sorbent assay FAM 6-Carboxyfluorescein FBS Foetal bovine serum FLAC Full-length amplification of cDNA FMD foot and mouth disease FRET Fluorescence resonance energy transfer ii Abbreviations GTPV Goat Pox Virus HPLC High performance liquid chromatography ICAR Indian Council for Agriculture Research ICTV International Committee for the Taxonomy of Viruses ID Identification IND India ISVP Infectious subviral particles IVRI Indian Veterinary Research Institute Kb Kilobase Kbp Kilo base pair kDa Kilo Dalton LiCl Lithium chloride mL Millilitre MLV Modified live virus NCR Noncoding region NFW Nuclease free water NGS Next generation sequencing NIVEDI National Institute of Veterinary Epidemiology and Disease Informatics nM Nanomolar NPM Nextera PCR master mix NS Non-structural nt Nucleotide ᵒC Degree Celsius OIE Office International des Epizooties ORC Orbivirus Reference Collection ORF Open reading frame ORFV Orf Virus P Phosphorus PAGE Polyacrylamide gel electrophoresis pM Picomolar PPR Peste des Petite Ruminant iii Abbreviations PPRV Peste des Petite Ruminant Virus q Quantitative RGD Arginine-glycine-aspartate RNA Ribonucleic acid rpm Revolutions per minute RSB Resuspension buffer RT Reverse transcriptase RT-PCR Reverse transcription polymerase chain reaction S Sulphur SBL Sequencing by ligation SBS Sequencing by synthesis Seg Segment SMRT Single molecule real-time sequencing SNT Serum neutralization tests SPAT Single primer amplification technique spp Species SPPV Sheep Pox Virus ss Single stranded TAE Tris acetate EDTA TC Transcriptase complex UV Ultraviolet v/v Volume by volume VIBs Viral inclusion bodies VLP Virus-like particles VNT Virus neutralization tests iv Table of contents Table of contents Abstract…………………………………………………………….i Abbreviations………………………………………………………ii Table of contents……………………………………………………v List of Tables………………………………………………………..xv List of Figures……………………………………………………….xviii Acknowledgements…………………………………………………..xxii Declaration of Authorship……………………………………………xxiv Chapter 1: General Introduction ....................................................................... 1 1.1 Introduction ................................................................................................. 1 1.2 Taxonomy ................................................................................................... 2 1.2.1 dsRNA viruses ......................................................................................... 2 1.2.2 The family Reoviridae ............................................................................. 3 1.3 The genus Orbivirus .................................................................................... 3 1.4 The structure of the Bluetongue virus particle ............................................ 5 1.5 The BTV genome ........................................................................................ 7 1.6 BTV structural proteins ............................................................................... 8 1.6.1 Outer capsid proteins (VP2 and VP5) ...................................................... 8 1.6.2 Major core proteins (VP3 and VP7) ........................................................ 9 1.6.3 Minor enzymatic core proteins (VP1, VP4 and VP6) ........................... 10 1.7 BTV non-structural proteins (NS1, NS2, NS3, NS4 and NS5) ................ 11 1.8 The BTV replication cycle ........................................................................ 13 1.8.1 Attachment and entry ............................................................................. 13 v Table of contents 1.8.2 Uncoating ............................................................................................... 14 1.8.3 Transcription .......................................................................................... 14 1.8.4 Replication ............................................................................................. 15 1.8.5 Assembly and release ............................................................................. 15 1.9 BTV pathogenesis ..................................................................................... 16 1.9.1 Clinical signs .......................................................................................... 16 1.9.2 Pathogenesis ........................................................................................... 18 1.10 Evolution and molecular epidemiology of BTV ..................................... 20 1.10.1 BTV serogroup, serotypes and topotype .............................................. 21 1.11 DNA sequencing ..................................................................................... 23 1.11.1 The Maxam and Gilbert method .......................................................... 23 1.11.2 Sanger’s dideoxy sequencing technology ............................................ 23 1.11.3 Next generation sequencing ................................................................. 24 1.11.3.1 Illumina (MiSeq, NextSeq 500, and HiSeq) ..................................... 24 1.11.3.2 Ion Torrent (Life Technologies) ....................................................... 25 1.11.3.3 Roche pyrosequencing platforms ...................................................... 25 1.11.3.4 SOLiD sequencing ............................................................................ 25 1.11.3.5 Third generation sequencing ............................................................