Development of in situ hybridization and RT-PCR assay for the detection of a nodavirus (PvNV) that causes muscle necrosis in Penaeus vannamei

Abstract

A nodavirus (tentatively named PvNV, Penaeus vannamei nodavirus) that causes muscle necrosis in P. vannamei was found in Belize in 2004. From 2004 to 2006, shrimp samples collected from Belize exhibited clinical signs, white, opaque lesions in the tails and histopathology similar to those of shrimps infected by infectious myonecrosis virus (IMNV). Histological examination revealed multifocal necrosis and hemocytic fibrosis in the skeletal muscle. In addition, basophilic, cytoplasmic inclusions were found in striated muscle, lymphoid organ and connective tissues. However, IMNV was not detected in these shrimps by either RT-PCR or in situ hybridization, suggesting that these lesions may be caused by another RNA virus. Thus, a cDNA library was constructed from total RNA extracted from hemolymph collected from infected shrimp. One clone (designated PvNV-4) with a 928 bp insert was sequenced and found to be similar (69% similarity when comparing the translated amino acid sequences) to the capsid protein gene of MrNV (Macrobrachium rosenbergii nodavirus). The insert of PvNV-4 was labeled with digoxigenin-11-deoxyuridine triphosphate (dUTP) and hybridized to tissue sections of P. vannamei with muscle necrosis collected in Belize and from laboratory bioassays. The samples were positive for PvNV infection. Positively reacting tissues included skeletal muscle, connective tissues, the lymphoid organ, and hemocytes in the heart and gills. In addition, we experimentally infected both P. vannamei and P. monodon with PvNV prepared from Belize samples. A nested RT-PCR assay developed from the PvNV-4 cloned sequence showed that both species are susceptible to PvNV infection.