Hepatopancreatic parvovirus of penaeid shrimp (HPV): Partial cloning and genome characterization, in situ hybridizationat the ultrastructural level, geographic diversity and non-invasive detection

Abstract

The genome of a Korean isolate of Hepatopancreatic parvovirus (HPV) was partially cloned, sequenced and characterized. Random PCR amplification of viral DNA was combined with conventional cloning methods to generate three clones named HPV8 (2,136 bp insert), HPV3 (1,560 bp insert), and CP1139 (413 bp insert). DNA sequencing demonstrated overlapping regions between HPV8 and HPV3 and between HPV3 and CPII39. The combined sequence of these three clones encompass approximately 3,350 bp of the total 5,000 bp estimated for the HPV genome. A large open reading frame (1,692 bp) was found within clones HPV3/CPII39 encoding a polypeptide of 549 residues (∼60 kDa) whose amino terminus shows 100% homology with the first 12 residues sequenced from an apparently single 54 kDa (by SDS-PAGE) structural protein found in a Korean isolate of HPV. Two new gene probes EC.592 (592 bp) and EC.350 (350 bp) were developed by PCR amplification of previously constructed HPV (Korean) clones and labeled with DIG11-dUTP. These probes recognize different regions of the HPV genome. The specificity of both probes was confirmed by in situ hybridization using HPV-infected Penaeus chinensis (Korean) as a positive control and specific-pathogen free P. vannamei and IHHNV-infected P. stylirostris, as negative controls. Both probes were used in in situ hybridization to compare their reaction to HPV-type lesions detected by conventional H&E histology in 7 species of HPV-infected shrimp, and one of freshwater prawn, from 13 countries. The results of this comparison strongly suggest the existence of genomic differences among these geographic isolates. A post-embedding in situ hybridization assay at the electron microscope level was developed to detect HPV nucleic acids on HPV-infected hepatopancreata from P. chinensis and P. monodon . Hybridized probe (EC.592) was detected with an anti-DIG sheep antibody conjugated to 10 nm gold particles and subsequent silver enhancement. Hybridization signal was observed within HPV-infected hepatopancreatic cells, which was specifically located within intranuclear viral inclusions, cytoplasm, microvillous border, and associated to necrotic debris within the lumen of hepatopancreatic tubules. A non-destructive method, based on the PCR, was developed to detect HPV by examination of crude fecal samples from HPV-infected shrimp.