Development and validation of four Real-Time quantitative RT-PCRs specific for the positive or negative strands of a bisegmented dsRNA viral genome

Abstract

Four tagged quantitative Real-Time RT-PCRs (qRT-PCRs) were developed to quantify the positive and negative strands of segments A and B of the bisegmented double-stranded RNA (dsRNA) genome of infectious bursal disease virus (IBDV, family Birnaviridae, genus Avibirnavirus). The qRT-PCRs were validated using single-stranded RNAs corresponding to each genomic strand (A+, B+, A-, B-). Specific quantitation proved possible from 5×10(7) to 5×10(2) copies of the template per reaction, with excellent reproducibility and linearity. The methods detected similar amounts of A+ and A- and of B+ and B- in a purified dsRNA viral genome preparation, thus corroborating the accuracy of quantitation. The qRT-PCRs were used to quantify the four strands in CsCl purified virus fractions and in samples collected during propagation of IBDV in cell culture. Purified virus fractions contained similar amounts of A- and B- strands, but also a large and unexplained excess of A+ and even more B+ strands. Results of the in vitro kinetic study showed an early accumulation of positive strands and a more delayed and lower accumulation of the A- and B- strands, both in similar amounts. These results suggest that minus strand synthesis occurs in IBDV after the equimolar packaging of A+ and B+ strands.