Detection and differentiation of Schmallenberg, Akabane and Aino viruses by one-step multiplex reverse-transcriptase quantitative PCR assay.

Abstract

BACKGROUND: Schmallenberg virus (SBV), Akabane virus (AKAV) and Aino virus (AINV) are members of the Simbu serogroup within the genus Orthobunyavirus, family Bunyaviridae, which can cause reproductive disorders including abortion, stillbirth and congenital malformation in ruminants. Because, the clinical signs are similar, confirmatory diagnosis requires viral detection to differentiate infection between these three viruses. METHODS: In this study, a one-step multiplex reverse-transcriptase quantitative PCR (one-step mRT-qPCR) was developed for the simultaneous detection and differentiation of SBV, AKAV and AINV. RESULTS: The detection limit of the one-step mRT-qPCR for SBV, AKAV and AINV were 2.4 copies (10 (0.6) TCID 50/ml), 96.2 copies (10 (1.5) TCID 50/ml) and 52.3 copies (10 (1.2) TCID 50/ml), respectively. Various field samples such as bovine serum, bovine whole blood, bovine brain, goat serum and Culicoides were analyzed using the one-step mRT-qPCR and compared with previously published RT-qPCRs. The test results of the field samples were identical for the one-step mRT-qPCR and RT-qPCRs, which showed all samples to be negative for SBV, AKAV and AINV, except for one bovine brain sample (1/123) that was positive for AKAV. CONCLUSION: The one-step mRT-qPCR allows for the simultaneous detection of three viral pathogens (SBV, AKAV and AINV) that cause reproductive failure.