The development and implementation of genotype specific qRT-PCR's for the diagnosis of Avian Orthoreovirus (ARV)

Abstract

Avian Orthoreovirus (ARV) is a double-stranded RNA-virus and is the causative agent of viral tenosynovitis. The virus causes economic losses in broiler flocks due to high feed conversion, lameness, culling and low carcass weights. Clinical disease is seen among broilers despite vaccination of breeder flocks. To improve immunity in young chicks, circulating field ARV's need to be determined to be incorporated in new vaccines. However, nucleotide variation is high in the viral genome and thus many ARV-subtypes exist, which makes development of a sensitive qRT-PCR for the detection of ARV difficult. The main objective of this study was to develop a qRT-PCR suitable for routine diagnostic detection of ARV in field samples. In this study forty-one confirmed reovirus RNA-samples, collected from affected flocks, were subjected to a qRT-PCR with genotype specific primers and probes. Prior to assessment of the RNA-samples, testing conditions for qRT-PCR were optimized using a standard sample. qRT-PCR resulted in Ct-values for thirty-five RNA-samples. Six samples were without a Ct-value. Degenerate qRT-PCR performed on negative samples did not result in an increase in the amount of Ct-values. Results were confirmed by electrophoresis. New sequence information was obtained from the RNA-samples included in this study. qRT-PCR worked on the majority of cluster 1 RNA-samples, but results for the samples of cluster 2, 4 and 5 varied. Further optimizing of qRT-PCR conditions is necessary and/or new primers and probes will have to be developed in order to improve diagnostic results.