Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis.

BACKGROUND: Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary. RESULTS: A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection. CONCLUSION: The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.

Genome sequences of three koi herpesvirus isolates representing the expanding distribution of an emerging disease threatening koi and common carp worldwide.

Since the mid-1990s, lethal infections of koi herpesvirus (KHV) have been spreading, threatening the worldwide production of common carp and koi (both Cyprinus carpio). The complete genome sequences of three KHV strains from Japan, the United States, and Israel revealed a 295-kbp genome containing a 22-kbp terminal direct repeat. The finding that 15 KHV genes have clear homologs in the distantly related channel catfish virus (ictalurid herpesvirus 1) confirms the proposed place of KHV in the family Herpesviridae, specifically in the branch with fish and amphibian hosts. KHV thus has the largest genome reported to date for this family. The three strains were interpreted as having arisen from a wild-type parent encoding 156 unique protein-coding genes, 8 of which are duplicated in the terminal repeat. In each strain, four to seven genes from among a set of nine are fragmented by frameshifts likely to render the encoded proteins nonfunctional. Six of the affected genes encode predicted membrane glycoproteins. Frameshifts or other mutations close to the 3' ends of coding sequences were identified in a further six genes. The conclusion that at least some of these mutations occurred in vivo prompts the hypothesis that loss of gene functions might be associated with emergence of the disease and provides a basis for further investigations into the molecular epidemiology of the virus.

Immunogenicity of a 16.7 kDa Mycobacterium paratuberculosis antigen.

Mycobacterium paratuberculosis (MPT), the agent of paratuberculosis is a slow growing mycobacteria that causes important economic losses mainly due to lower weight gains and drastic decrease in milk production. Existing paratuberculosis vaccines are not completely protective and induce antibodies/delayed type hypersensitivity (DTH) reaction that cannot be differentiated from those of naturally infected animals. New potent acellular vaccines that allow discrimination between infected and vaccinated animals are needed to improve the control of this disease. We have identified, expressed and purified a hypothetical thiol peroxidase of MPT (MPT-TP) in mice. We also characterized the immunogenicity of this antigen in mice. The recombinant MPT-TP (rMPT-TP) antigen induced a high production of IFNgamma, IL-6, and NO and a low production of IL-10 by spleen cells of immunized mice. Addition of Ribi adjuvant to rMPT-TP resulted in lower IFNgamma secretion and higher NO production in spleen cells. A similar level of proliferation of spleen cells exposed to rMPT-TP was found in immunized groups (rMPT-TP and rMPT-TP emulsified in Ribi). DTH responses in mice footpads were observed only in mice immunized with rMPT-TP emulsified in Ribi. Addition of Ribi adjuvant clearly induced a significantly higher anti-rMPT-TP antibody production of all classes tested and decreased the IgG1/IgG2a ratio. MPT-TP demonstrated antigenic characteristics that make this antigen a potential component in the development of a future subunit vaccine against paratuberculosis.