Multiplex RT-PCR detection and sequence comparison of viruses MrNV and XSV associated with white tail disease in Macrobrachium rosenbergii

A multiplex reverse transcriptase polymerase chain reaction (MRT-PCR) assay was developed and applied to the detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) associated with field and hatchery outbreaks of white tail disease (WTD) in M. rosenbergii. Primers targeted regions of the capsid protein genes of the two viruses and produced amplicons of 1136 and 447 bp, for MrNV and XSV, respectively, with sensitivity to the level of 1 fg of total RNA for both the viruses. Assays could be completed within 7 to 9 h after samples were received. This is of great importance for epidemiological analysis and screening of large numbers of samples. MRT-PCR assay on samples collected from different hatcheries and farms of Nellore, India revealed the presence of both the viruses in all. Experimental infections produced typical clinical signs of white muscle in juveniles and mortality up to 20%. MrNV could be detected up to 1 month in surviving prawns, both by histology and RT-PCR. Sequence analysis of capsid protein gene amplicons of XSV revealed minor variations. By contrast, sequence analysis of MrNV amplicons showed no variation in amino acid sequence of the capsid protein gene.