Genus-specific detection of alphaviruses by a semi-nested reverse transcription-polymerase chain reaction

Abstract

A reverse transcription-polymerase chain reaction (RT-PCR) was developed for the genus-specific detection of alphaviruses. Based on the available published sequences, degenerate primers were designed to ensure hybridization to a conserved region within the nonstructural protein 1 gene of all alphavirus species. The expected 434-basepair (bp) cDNA fragment was amplified from all 27 alphavirus species by using RNA extracted from 200 microl of infected cell culture supernatant. In addition, eight strains of Venezuelan equine encephalitis (VEE) virus and 10 strains of Sindbis virus were amplified. The viral origin of the amplicons was confirmed by restriction enzyme analysis and comparison with the expected cleavage pattern based on published sequence data. The PCR products of alphavirus species with thus far unknown nucleotide composition were sequenced. About 120 nucleotides downstream of the forward primer, a region showing sufficient homology for the design of another forward primer was found and used in a semi-nested PCR. The expected 310-bp semi-nested fragment was demonstrated for all viruses investigated. The sensitivity of the RT-PCR was about 1,200 plaque-forming units (PFU) for VEE virus reference strain Trinidad donkey. The detection limit after the semi-nested PCR was 1.2 PFU. The sensitivity was not hampered by the presence of human serum, thus making this test suitable for an application in viremic individuals. Chikungunya virus RNA was amplified from infected mouse brain tissue by the described RT-PCR assay. Our data suggest that the semi-nested RT-PCR may be applied as a highly sensitive alternative to virus isolation in the rapid screening and diagnosis of alphavirus infections, including post-mortem diagnosis. Phylogenetic analysis of the amplicon sequence data identified six genotypes within the Alphavirus genus.