title: Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Microbiology, Department of Clinical Microbiology, The Hebrew University-Hadassah Medical School, Ein Karen, Jerusalem, Israel. hb@cc.huji.ac.il
authors.forename: Herve
authors.initials: H
authors.lastname: Bercovier
authors.forename: Yolanta
authors.initials: Y
authors.lastname: Fishman
authors.forename: Ronen
authors.initials: R
authors.lastname: Nahary
authors.forename: Sharon
authors.initials: S
authors.lastname: Sinai
authors.forename: Amir
authors.initials: A
authors.lastname: Zlotkin
authors.forename: Marina
authors.initials: M
authors.lastname: Eyngor
authors.forename: Oren
authors.initials: O
authors.lastname: Gilad
authors.forename: Avi
authors.initials: A
authors.lastname: Eldar
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Hedrick
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002347:Carps / Q000821:virology*; D003001:Cloning, Molecular; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology*; D006564:Herpesviridae / Q000201:enzymology / Q000235:genetics*; D007668:Kidney / Q000821:virology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D013937:Thymidine Kinase