Construction of an infectious Macrobrachium rosenbergii nodavirus from cDNA clones in Sf9 cells and improved recovery of viral RNA with AZT treatment

Abstract

Macrobrachium rosenbergii nodavirus (MrNV) is usually accompanied by extra small virus (XSV) in natural outbreaks of white tail disease (WTD) in the giant river prawn Macrobrachium rosenbergii. Testing the virulence of MrNV alone has been problematic due to the difficulty in completely separating XSV from MrNV by viral purification steps from naturally infected shrimp. However, based on reports of natural M. rosenbergii specimens from WTD outbreak ponds that were positive for MrNV but negative for XSV led us to hypothesize that MrNV alone might cause WTD. To test this hypothesis, we prepared the two, complete genomic RNA fragments (RNA1 and RNA2) of MrNV from cDNA clones and used these to transfect Sf9 cells that subsequently showed cellular changes, including cell swelling, syncytial cell formation, and development of cytoplasmic inclusions within 72h post-transfection. Replication of RNA1 and RNA2 increased in the transfected cells and transmission electron microscopy of the cell lysates revealed the presence of icosahedral viral-like particles that were 40–50nm in diameter. When naïve Sf9 cells were inoculated with the cell lysate, the newly infected cells showed cellular changes and produced strong immunoreactivity against MrNV capsid protein indicating the infectious nature of the cell lysate. When the lysates were injected into the whiteleg shrimp Penaeus vannamei, MrNV RNA replication in the shrimp was followed by morality accompanied by typical MrNV lesions that gave possible positive immunohistochemical reactions for the MrNV capsid protein. Treatment of the Sf9 cells with azidothymidine triphosphate (AZT) prior to transfection significantly increased viral RNA synthesis and pathogenicity when compared with untreated, transfected cells. Using this model to produce infectious MrNV without XSV contamination proves that MrNV alone can be lethal to shrimp and it opens the way to further investigate the molecular basis of MrNV pathogenesis, and to develop antiviral strategy to control white tail disease.