Initial characteristics of koi herpesvirus and development of a polymerase chain reaction assay to detect the virus in koi, Cyprinus carpio koi

Abstract

Since 1998, episodes of mass mortality have occurred in populations of common carp Cyprinus carpio carpio in Israel and in populations of koi Cyprinus carpio koi in Israel and the USA. A herpesvirus isolated from infected fish has been shown in experimental studies to induce disease and mortality similar to those observed in outbreaks at infected farms. Initial characteristics of the virus show that it is clearly different from Herpesvirus cyprini (CHV), the most commonly known herpesvirus from cyprinid fish. The koi herpesvirus (KHV) has 31 virion polypeptides. Twelve of the virion polypeptides of KHV have similar molecular weights to those of CHV and 10 are similar to those of channel catfish virus (CCV). Both virion polypeptide and restriction fragment length polymorphism analyses of genomic DNA showed that the first KHV isolates from Israel and the USA were identical. In contrast, the genomic DNA restriction fragments clearly distinguish KHV from CHV and CCV. A polymerase chain reaction (PCR) assay to detect the virus in koi tissues was developed with sequences obtained from 1 restriction fragment of KHV DNA. The PCR assay effectively detected a 484 base pair sequence from KHV but did not amplify genomic DNA from either CHV or CCV. The PCR assay detected as little as 1 pg of KHV DNA mixed with 100 ng of host DNA. Viral sequences were amplified from koi obtained from field collections and from koi that were experimentally exposed to 10 2 TCID 50 ml -1 of KHV via the waterborne route. All KHV exposed fish dying of infection between 8 and 10 d post exposure or surviving to 14 d post exposure were found to be positive by PCR, while unexposed control koi were all negative. The assay also showed the presence of KHV DNA in tissues of koi obtained from farms in Israel. The PCR assay should assist virus isolation procedures and histologic and electron microscopic analyses now commonly used to detect KHV infection. Current studies are examining the possibility of using the PCR to detect KHV DNA in live fish and the relative sensitivity and specificity of the KHV PCR assay compared with other diagnostic tests.

Koi Herpes Virus: Do Acipenserid Restitution Programs Pose a Threat to Carp Farms in the Disease-Free Zones?

“…All samples investigated according to the method of Gilad et al (2002) were confirmed under the same conditions by nested PCR using 2 µL of Gilad's PCR product and the primer pair KHV 1Fn-1Rn (Bergmann et al 2006). As positive control DNA obtained from CCB cells replicated KHV-I (Gilad et al 2002) was used. Channel catfish herpesvirus (CCV), carp pox virus (CyHV-1), goldfish haematopoietic necrosis herpesvirus (CyHV-2), and herpesvirus anguillae (HVA) were used as heterologous virus controls (negative controls).…”

“…PCRs for detection of KHV fragments were done according to assays published by Gilad et al (2002), Bergmann et al (2006), and Bercovier et al (2005) (Table 1). All samples investigated according to the method of Gilad et al (2002) were confirmed under the same conditions by nested PCR using 2 µL of Gilad's PCR product and the primer pair KHV 1Fn-1Rn (Bergmann et al 2006). As positive control DNA obtained from CCB cells replicated KHV-I (Gilad et al 2002) was used.…”

“…DNA was sequenced using the Prism BigDye Terminator v1.1 Cycle Sequencing Kit on the DNA sequencer "3130 Genetic Analyzer" (both Applied Biosystems). Sequencing reactions were performed using the primers pairs according to Gilad et al (2002) and KHV-1Fn and 1Rn (Bergmann et al 2006). Sequence data were assembled and edited with GCG, Version 11.1 (Accelrys Inc.).…”

Koi Herpes Virus: A Review and Risk Assessment of Indian Aquaculture

“…Several diagnostic tests based on PCR [6,25,29,89], nested PCR [7,20], real-time PCR [27] and loop-mediated isothermal amplification (LAMP) [31,75,87,88] have been developed for detection of KHV. A highly sensitive PCR method for detection of TK gene of KHV is able to detect 10 fg of KHV DNA [6].…”

“…A highly sensitive PCR method for detection of TK gene of KHV is able to detect 10 fg of KHV DNA [6]. The sensitivity of this method is *10-1,000 times greater than other PCR methods [25,29]. PCR primers targeting DNA polymerase gene and the major envelope protein gene of KHV have a sensitivity of 100 femtogram (fg) and 1,000 fg of KHV DNA, respectively in infected gills [41].…”