Developments of an Isolation System and a Nested PCR Diagnosis Method with Superior Sensitivity for Koi Herpesvirus

摘要

Koi herpes virus (KHV) infection is an emerging, highly contagious fish disease that has imposed high mortality in common carp and koi. Previous studies have shown that the difficulty in viral identification might be due to the latent infection of KHVin Koi. In the present study, we reported the establishment of methodologies for KHV diagnosis. Taiwan koi fin (TKF-1) cells were found to be the only type of cell culture that KHV could be sustained and the cytopathogenic effects (CPE) could be induceddirectly by the inoculation of the homogenates of KHV-infected fishes tissues. Other cells cultures derived from koi, goldfish, fathead minnow, grouper, eel, tilapia and soft-shell turtle were proven not to be susceptible for the KHV. KHV infection could be further confirmed by the detection of the 409 bp fragments of KHV DMA using polymerase chain reaction in TKF-1 cells. Moreover, level of ORF 81 protein of KHV could be determined by indirect immunofluorescence assay 12 hours post infection. In addition, to increase the specificity and sensitivity in diagnosing KHV, we developed a nested PCR protocol capable of detecting as few as 17 KHV virions. This technology was verified with the suspected but asymptomatic koi and was proved to be a more sensitive tool for diagnosing KHV-infected koi during latent infection than others. This is the first report in Taiwan regarding the establishment of koi cell cultures for the propagations of locally isolated KHV and the development of a new diagnostic method with higher sensitivity for KHV infection.