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Characterization and Control of KHV, A New Herpes Viral Pathogen of Koi and Common Carp

In this project we proposed to characterize the virus genome and the structural virion polypeptides to allow development of improved diagnostic approaches and potential vaccination strategies. These goals have been mostly achieved and the corresponding data were published in three papers (see below) and three more manuscripts are in preparation. The virion polypeptides of KHV strains isolated from USA (KHV-U) and Israel (KHV-I) were found to be identical. Purified viral DNA analyzed with a total of 5 restriction enzymes demonstrated no fragment length polymorphism between KHV-I and KHV-U but both KHV isolates differed significantly from the cyprinid herpesvirus (CHV) and the ictalurid herpesvirus (channel catfish virus or CCV). Using newly obtained viral DNA sequences two different PCR assays were developed that need to be now further tested in the field. We determined by pulse field analysis that the size of KHV genome is around 280 kbp (1-1. Bercovier, unpublished results). Sequencing of the viral genome of KHV has reached the stage where 180 kbp are sequenced (twice and both strands). Four hypothetical genes were detected when DNA sequences were translated into amino acid sequences. The finding of a gene of real importance, the thymidine kinase (TK) led us to extend the study of this specific gene. Four other genes related to DNA synthesis were found. PCR assays based on defined sequences were developed. The PCR assay based on TK gene sequence has shown improved sensitivity in the detection of KHV DNA compared to regular PCR assays.  With the ability to induce experimental infections in koi with KHV under controlled laboratory conditions we have studied the progress and distribution of virus in host tissues, the development of immunity and the establishment of latent infections. Also, we have investigated the important role of water temperature on severity of infections and mortality of koi following infections with KHV. These initial studies need to be followed by an increased focus on long-term fate of the virus in survivors. This is essential in light of the current "controlled exposure program" used by farmers to produce KHV "naturally resistant fish" that may result in virus or DNA carriers.  The information gained from the research of this project was designed to allow implementation of control measures to prevent the spread of the virus both by improved diagnostic approaches and preventive measures. We have accomplished most of these goals but further studies are needed to establish even more reliable methods of prevention with increased emphases on improved diagnosis and a better understanding of the ecology of KHV.