In 8 week-old fingerlings, the infection did not cause mortality. Behaviorally, affected fish often remain near the surface, swim lethargically and may exhibit respiratory distress and uncoordinated swimming. Het is slechts een methode om een KHV-uitbraak een halt toe te roepen en sterfte te beperken. Newly amplified sequences of glycoprotein genes of Korean CyHV-3 were deposited in the GenBank nucleotide sequence database with the following accession numbers: JQ308816 for ORF25, JQ308817 for ORF65, and JQ308819 for ORF116. Total cellular RNA was extracted from CCB cells by using an EZ-RNA kit (Biological Industries, Kibbutz Beit Haemek, Israel) according to the manufacturer’s instructions. The molecular structure of the recombinants and the absence of contamination between strains was also controlled by PCR (Figure 5 ) and sequencing of the regions used to target recombination (data not shown). A monolayer of EK-1 cells (approximately 80% confluent) in a 150-cm2 flask was washed once with GM and infected with 5 50% tissue culture infective doses (TCID50) of AngHV1 per cell in a total of 25 ml GM at 26°C for 30 min.
The United States strain of CyHV-3 (KHV-U) was a gift from Ronald Hedrick. Postmortem inspection of CyHV-2 infected goldfish revealed severe necrosis of gills and deterioration of the kidney and liver (Jeffery et al. We observed a moderate and late (day 6 and day 8 post-infection) up-regulation of TNFα1 and TNFα2. The PCR reactions contained 0.2 μM each of primers, 0.2 mM of dNTP mix, 1× PCR buffer, 2.5 U of Taq DNA polymerase (Sangon, Shanghai, China), 5 μL DNA template, and sterile ddH2O in a final volume of 25 μL. After incubation, bacteria were pelleted, resuspended in 40 ml 0.1 mg/ml lysozyme in 20 mM Tris-HCl, 500 mM NaCl and incubated for 30 min at room temperature (RT) to break down the peptidoglycan layer. The only currently acceptable measure for the control of CyHV-3 outbreaks is depopulation (eradication of the infected and exposed fish) and disinfection of all materials and systems that have been in contact with infected fish. In the present study, we investigated the ability of CyHV-3 to infect common carp during the early stages of its development (from embryos to fingerlings) after inoculation by immersion in water containing the virus.
Sequencing was performed by “DNA walking” using internal primers derived from previously sequenced fragments (see supplementary data, Appendix 5), with purified viral DNA as templates, and carried out by the dideoxynucleotide terminator cycle sequencing method employing a Prism BigDye Ready Reaction Terminator cycle sequencing kit (PE Applied Biosystems). Figure 5 Survival rates of carp infected with the CyHV-3 LUC strain. J. Mucus was first clarified by centrifugation (2000 g for 10 min at 4°C). Cells and viruses.C. CyHV-3 has a 295-kb dsDNA genome containing 156 ORFs (Aoki et al., 2007). A set of four LAMP primers (JF3 and JB3, JBIP and JFIP) recognizing six distinct regions in the viral putative DNA helicase gene sequence (GenBank accession KC245087) was designed by using the Primer Explorer version 4 (http://primerexplorer.jp/elamp4.0.0/index.html).
The sampling sites are indicated by closed circles. This disadvantage, however, can be overcome by a reverse transcription (RT)-PCR analysis that targets expressed viral genes, whereby in situ viral activities can be identified. Nor is there evidence that the virus even replicates in any of the 14 species of fish, two species each of amphibians and reptiles, and single species of bird, mammal and invertebrate tested . Purified L capsids showed 4 clear bands, the U capsids showed an additional fifth protein band of low molecular weight not present in complete virus particles (Figure 1a ). Molecular tools such as polymerase chain reaction (PCR) and quantitative real-time PCR have been established for the detection and quantification of CyHV-2 [1–7]. The CyHV-2 has been classified as a member of the genus Cyprinivirus, the family Alloherpesviridae in the Herpesvirales order, and its complete genome has been sequenced (GenBank accession JQ815364). Hedrick for technical assistance.
DNA microarray hybridization analysis, Northern blotting and stem-loop RT-qPCR were then used to definitively confirm that CyHV-3 expresses two pre-miRNAs during infection in vitro. There was no significant difference in the expression of host TNFα-1 and MHC-II genes during all phases of CyHV-3 infection. In each strain, four to seven genes from among a set of nine are fragmented by frameshifts likely to render the encoded proteins nonfunctional. Haenen O.L.M., Way K., Bergmann S.M., Ariel E. Received December 1, 2014. carassius) from European and Asian countries. However, due to the limited susceptibility of cells used for propagation, it is not always possible to successfully isolate CyHV-3 even from tissue samples that have high virus titres.
Cumulative mortalities of the 15, 20, 25 and 30°C groups were 10, 90, 90 and 60%, respectively. It is caused by a herpes virus. The virus contains at least 40 structural proteins, of which few have been characterised with respect to their immunogenicity.