ABSTRACT-Three regions of koi herpesvirus (KHV) genomic DNA were compared for 34 samples from Japan, six from Indonesia, two from Taiwan, one from the Philippines, 13 from the Netherlands, one from the UK, one from the USA and one from Israel. The analyzed genomic regions included known PCRdetection targets (SphI-5, 9/5 and the thymidine kinase gene). The KHVs from Asian countries were very homogeneous, although two variants were noted based on a single nucleotide polymorphism. In contrast, seven variants were found in KHVs from outside of Asia, and although closely related to one another, they were clearly distinct from those from Asian. The results suggest that a clear genetic distinction exists between Asian and European (including each single isolate from the USA and Israel) types of KHV, and that unique types of KHV were independently introduced or emerged in the respective geographic locations.

No abstract

Phylogenetic analysis of nine strains of Edwardsiella ictaluri and eight strains of E. tarda (six typical motile strains and two atypical nonmotile strains) isolated from diseased fish was performed using the upstream region of the fimbrial gene cluster. Strains of E. ictaluri and E. tarda were significantly clustered into separate groups. Moreover, atypical E. tarda strains were clustered into a different group from the other strains. Three polymerase chain reaction (PCR) primer sets for differential detection of E. ictaluri as well as typical and atypical E. tarda were developed from the respective characteristic sequences. Strains of E. ictaluri, typical E. tarda, and atypical E. tarda were specifically detected by PCR using each primer set. No amplifications were observed after the use of these three primer sets with 25 other bacterial species, including fish pathogens. In addition, the three primer sets were able to detect the DNA of each target species from fish kidney and liver artificially infected with E. ictaluri or E. tarda.

An mRNA-specific reverse transcription (RT)-PCR primer set spanning the exon junction of a spliced putative terminase gene in the koi herpesvirus (KHV) was developed to detect the replicating stage of the virus. The proposed RT-PCR amplified a target gene from the RNA template, but not from a DNA template extracted from common carp brain (CCB) cells infected with KHV. In addition, the RT-PCR did not amplify the target gene of templates extracted from specific cell lines infected with either CyHV-1 or CyHV-2. RT-PCR detected mRNA from the scales of koi experimentally infected with KHV at 24 h post exposure (hpe). However, unlike conventional PCR, RT-PCR could not detect KHV DNA in fish at 0 hpe. The results indicate that the RT-PCR developed in this study is mRNA-specific and that the assay can detect the replicating stage of KHV from both fish and cultured cells infected with the virus.

ABSTRACT-Common carp Cyprinus carpio were experimentally exposed to koi herpesvirus (KHV) under water temperature regimes ranging from 16∞C to 28∞C. Fish maintained at 16∞C, 23∞C and 28∞C died due to infection at 21 -52, 5 -20 and 7 -14 days post-virus exposure (dpe), respectively. Cohabitation experiments in which KHVinfected common carp were kept with naive koi carp revealed that infectious virus was continuously shed from the infected common carp for 34 days (7-40 dpe) at 16∞C, for 14 days (1-14 dpe) at 23∞C and for 12 days (3-14 dpe) at 28∞C. The relatively extended duration of virus shedding observed at 16∞C suggests the potential risk of infection from fish maintained under low-temperature conditions as a source of viral inocula.Key words: koi herpesvirus, KHV, carp, water temperature, virus transmission, virus shedding tally exposed to KHV between 18∞C and 28∞C, but not at 13∞C. St-Hilaire et al. 7) reported that KHV-infected fish maintained at 12∞C exhibited no mortality for 30 weeks, but began dying after water temperature increased to 23∞C. In the present study, common carp were experimentally infected with KHV at water temperatures of 16∞C, 23∞C and 28∞C -the presumed permissive range of water temperatures for infection -and observed for mortality. We also examined the duration of virus shedding from the KHV-infected common carp under different water temperatures using cohabitation experiments. Materials and MethodsCommon carp brain (CCB) cells 8) were cultured in MEM supplemented with 5% fetal bovine serum. The KHV strain NRIA0308, isolated from diseased common carp in Japan in 2003, was used to inoculate CCB cells. After incubation at 20∞C for 14 days, the culture supernatant was used as a virus source for infecting common carp. The infective titer of the KHV virus stock determined using CCB cells was 10 3.6 TCID 50 /mL.KHV-free common carp (average 4.4 g in body weight, total 240 fish) from the National Research Institute of Aquaculture (NRIA) were divided into three groups consisting of 80 fish and acclimatized in 200 L aquaria adjusted to 16∞C, 23∞C or 28∞C (± 0.5∞C) over a 7-day period. Each group was separately infected with KHV by immersion in a 10 L aquarium containing 10 mL of the virus stock solution for 1 h, and 10 fish from each group were transferred to a 60 L aquarium adjusted to each water temperature and monitored for mortality for 60 days.For virus shedding experiments, common carp from each of the KHV-infected groups were cohabited with naive koi carp (average 3.7 g, total 81 fish). Twenty common carp from the KHV-infected group at 16∞C were transferred to a 28 L aquarium maintained at 16∞C (cohabitation aquarium), and three individual koi carp were introduced at intervals of 1, 3, 5, 7, 14, 21, 28, 40 and 60 days after the common carp were exposed to KHV. After cohabitation with infected common carp for 24 h, the koi carp were transferred to nine 10 L-aquaria adjusted to 23∞C and observed for mortality over a twoweek period. In order to maintain a constant number of fish in the...