Reassortment of bluetongue virus vaccine serotypes in cattle

Abstract

Bluetongue (BT) is a non-contagious viral disease of domestic and wild ruminants. Bluetongue virus (BTV), an Orbivirus that belongs to the family Reoviridae, is the causing agent of the disease. The virus consists of a ten segmented double stranded (ds) RNA genome and currently 27 serotypes have been identified worldwide. The virus is transmitted by Culicoide biting midges (Diptera: Ceratoponidae) and the occurrence of the disease depends on the presence and abundance of competent vectors. In South Africa most European ovine breeds are more susceptible to the disease than indigenous sheep, while cattle and goats in general prove to be sub-clinically infected. During recent outbreaks in Europe (2008 2011) cattle showed severe clinical symptoms and mortality. The role of cattle in the epidemiology of the disease in South Africa is however poorly understood. Bluetongue virus has the ability to reassort its genome segments in vertebrate hosts or vectors which have been infected with more than one strain at the same time. This phenomenon has been reported previously. In 1987, reassortment was investigated in cattle between BTV serotypes 11 and 17 where six reassortants with unique genetic profiles were described. In 2008, in Europe, segment 5 of BTV serotype 16 was identical to the South African vaccine strain of BTV serotype 2. In India in 2013 studies showed that in some isolates obtained from an outbreak segment 6 of BTV serotype 21 were 97.6% identical to segment 6 of BTV serotype 16. Bluetongue disease is controlled by annual vaccination. In South Africa the freeze dried polyvalent BTV vaccine is mainly used to vaccinate sheep, and the vaccine consists of three bottles, each bottle includes five serotypes and each bottle is vaccinated at a three week interval, between August to October of each year. The vaccine proves to be effective in producing immunity against the disease but there are multiple side effects. The main concern is that vaccine virus can be detected during the viraemic period in inoculated sheep. The titre levels are also sufficient to be transferred to non-vaccinated animal hosts via Culicoides midges. The possibility of reassortment between genome segments of vaccine and wild type strain viruses when simultaneously infected therefore exists. This might result in the emergence of new virus serotypes with different phenotypic characteristics i.e. reversion of the live attenuated vaccine strain to a virulent strain. The aim of the project was to investigate the potential generation of genetic reassortant viruses between field and vaccine serotypes of BTV within cattle. Six cattle between the ages of six and twelve months were used. Before the onset of the project cattle were tested for antibodies specific to BTV using a commercial available cELISA and for viral nucleic acid with RT-PCR. Only animals showing negative results by both the tests were used in the trial. The animals were housed in vector-free stables for the duration of the trial. The cattle were divided into two groups; the first group was infected with BTV serotypes from Bottle B of the Onderstepoort Biological Products (OBP) vaccine (BTV serotype 3, 8, 9, 10 and 11), while the second group was infected with the same vaccine serotypes and simultaneously infected with a wild type BTV serotype 4. Blood samples were collected daily from the animals from Day 1 to Day 39 post inoculation. Viraemia was detected between day 2 to day 39 and in one of the animals viraemia could be detected until 39 days post inoculation using virus isolation. Buffy coats as well as first cell culture passages of buffy coats were used to isolate the virus using the plaque forming unit method. The vaccine parental strains were obtained from Bottle two of the vaccine using the plaque forming unit assay and the isolated viruses were serotyped using a serum neutralization assay. Plaques were isolated and amplified on Vero cells. BTV serotype 4 was isolated in the Department of Veterinary Tropical Diseases from a field sample. RNA was extracted from the isolated plaques as well as the six parental strains and compared using polyacrylamide gel electrophoresis (PAGE).