Abstract

Channel catfish virus (CCV) is an unusual member of the Herpesviridae family and it causes lethal, hemorrhagic disease in channel catfish (Ictalurus punctatus) fingerlings. In severe epizootics, mortality can reach 100%. Despite having the genomic sequence of the type strain of CCV for seven years, very little is known about the structure and function of the viral proteins. The pathogenesis of CCV has been previously studied by isolating virus from various tissues of experimentally and naturally infected channel catfish. Nucleic acid probes, as well as PCR, have also been used to identify CCV sequences within different organ homogenates. In situ hybridization is a technique to precisely locate a specific segment of nucleic acid within a histologic section. The objective of this study was to identify the channel catfish cells, in vivo, that are infected with CCV by utilizing a negative-sense riboprobe generated from a putative CCV late gene. Open reading frame 46 (ORF 46) of the CCV genome was amplified by the PCR technique and cloned, in reverse orientation, into a plasmid vector immediately downstream of a bacteriophage T7 RNA polymerase binding site. This plasmid construct was purified and cleaved with a restriction enzyme in order to generate a negative-sense, digoxigenin-labeled, 555 base pair, runoff transcript using T7 RNA polymerase. Channel catfish, approximately 5 grams in size, were injected i.p. with approximately 10⁵ tissue culture infective dose 50 units of CCV and held at 26^oC. At 0, 2, 4, 8, 12, and 24 hours post-infection, two fish each were killed, fixed in 10% buffered formalin for 24 hours, and processed for histologic sectioning. In situ hybridization was carried out using the negative-sense ORF 46 riboprobe and hybridized probe was detected with alkaline phosphatase conjugated to an anti-digoxigenin antibody. By 48 hours post-infection all uncollected catfish had died. In sectioned fish, positive staining cells were first observed 8 hours post-infection with strong staining occurring at 16 and 24 hours post-infection. The predominant cells containing nucleic acid sequences complimentary to the ORF 46 probe were endothelial cells of the heart and liver. These findings suggest that in situ hybridization with CCV riboprobes is an effective method to directly study the pathogenesis of CCV in the tissues of experimentally infected fish. Further work is underway to use riboprobes generated from immediate early CCV genes in order to characterize the early events in channel catfish virus disease.