ABSTRACT:The first outbreaks of a disease connected with high mortality of common carp and koi carp caused by koi herpesvirus (KHV) were reported in 1998 in Israel and in the United States. Since then, several cases have been confirmed all over the world. At present, this viral disease is considered to be one of the most risky factors affecting populations of common carp and koi carp. Affected fish become disoriented and swim erratically with high breathing frequency, swollen gills and partially local skin lesions. The virus was isolated from the tissues of fish showing signs of the disease and subsequently cultured on koi fin (KF-1) cells. Electron microscopic examinations revealed morphological signs identical with viruses of the family Herpesviridae. Analysis of virion polypeptides and gene DNA showed the differences between KHV and the well-known herpesvirus of cyprinids, Herpesvirus cyprini (CHV), and Channel catfish virus (CCV). Water temperature is a factor influencing the onset and severity of disease. Fish seem most susceptible at water temperatures of 18-28°C, no morbidities occur at 13°C and 30°C. At present, diagnosis of KHV is mainly based on detection of viral DNA by PCR method.

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Cyprinid herpesvirus-3 (CyHV-3, previously described as koi herpesvirus) is a causative agent of the lethal disease in koi and common carp that can adversely affect common and koi carp breeds. Outbreaks with mass mortality have occurred world wide since 1998. At present, several different polymerase chain reaction (PCR) tests are in use to detect CyHV-3 in koi and common carp. In this study, we compared the sensitivity of the single-round PCR assays of Gray, Gilad, and Bercovier, and further the Gilad and Bercovier-nested PCR assays by testing tissue samples with known amounts of CyHV-3 DNA and by testing the panel of field tissue samples (106) of koi and common carp. The sensitivity of all the PCR protocols used in this study was similar within testing the spiked homogenate sample with a known amount of CyHV-3 DNA (ca. 12 DNA copies), with the exception of Gray PCR (25 copies). Detection levels of the individual PCR methods in field samples varied. The single-round Bercovier method was the most sensitive in comparison with the following two single-round methods, based on the number of positive field samples (34%). The improvement of sensitivity and confirmation of specifity were sufficient when the two-round Bercovier method was used with a detection of 44.3% of positive samples in total.

Rapid antigen detection enzyme-linked immunosorbent assay (ELISA) testing of cell cultures with organ homogenate from fish, collected from farms with a predominance of common carp or in natural aquaculture in the Czech Republic between 1995 and 2008, identified piscine vesiculovirus in 27 of 178 samples. Using reverse transcription semi-nested PCR, targeting a 550 nucleotide region of the glycoprotein (G) gene, piscine vesiculovirus was confirmed in 23 of the 27 organ samples diagnosed by ELISA as infected. PCR products were amplified and sequenced from 18 isolates from common carp Cyprinus carpio (family Cyprinidae), 2 isolates from northern pike Esox lucius (family Esocidae), and 1 isolate each from Siberian sturgeon Acipenser baerii (family Acipenseridae), common barbel Barbus barbus (family Cyprinidae), and koi carp Cyprinus carpio koi (family Cyprinidae). The sequences (based on 401 nucleotides) clustered into 2 genogroups. The majority of isolates (n = 22), including those from sturgeon and pike, grouped with the spring viraemia of carp virus (SVCV) Genogroup I and Subgroup Id. The 22 isolates could be further subdivided into 2 groups: Id1 (n = 20) and Id2 (n = 2). A marker (a non-conservative nucleotide substitution) for the Id1 SVCV group was identified. It was specifically found in all sequences of Id1 isolates when testing SVCV originating from different countries. The remaining isolate from barbel, was classified in the pike fry-like rhabdovirus Genogroup IV. This is the first confirmation of natural SVCV infection in sturgeon and pike, and pike fry-like rhabdovirus infection in barbel. In the case of the pike fry-like rhabdovirus, this is also its first identification in the Czech Republic. According to the presence/absence of evident clinical signs of rhabdoviral disease in the 3 infected hosts, only the sturgeon seemed to be susceptible to the monitored rhabdovirus.

Monoclonal antibodies (MAbs) against spring viraemia of carp virus (SVCV), a severe disease in cyprinid fish, were prepared. Nine MAbs were characterised using Western blotting (WB) where all reacted with glycoprotein G, except for MAb 2E1, which showed no reactivity in WB. All nine MAbs showed specificity in an immunoperoxidase test. In ELISA assays, their titres ranged between 1:32 000 and 1:128 000. A panel of SVCV isolates from different European regions were set up and examined by sandwich ELISA assay using the MAbs at a concentration of 15 µg/ml. Only MAb 4C12/3C8 showed low sensitivity in most of the isolates at an absorbance of A 450 . Of the other MAbs, even the lowest absorbance value measured exceeded cut-off for evaluation of the whole reaction. No cross-reaction with the infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV) or infectious pancreatic necrosis virus (IPNV) was demonstrated. 2E1 did not show cross-reactivity with PFRV classified in genogroup III-IV and reacted with a Czech SVCV isolate; its identity was confirmed by means of RT PCR assay. The others MAbs reacted positively with PFRV F4 reference strain, isolated from Esox lucius L. (genogroup III).