Development of an improved RT‐PCR for specific detection of spring viraemia of carp virus

Abstract

Spring viraemia of carp (SVC) is a rhabdovirus infection, which has a significant economic impact in pond cultures of carp in Europe and western Independent States of the former Soviet Union. The causative agent of SVC, spring viraemia of carp virus (SVCV), has been divided into four subgroups, Ia, Ib, Ic and Id, on the basis of glycoprotein (G) protein gene sequences. In this study, a new primer set was designed from a G gene sequence of SVCV to identify the four subtypes of SVCV by reverse transcription polymerase chain reaction (RT-PCR). The specific PCR products of 369 bp were amplified from 15 SVCV isolates of all four subtypes. However, pike fry rhabdovirus (PFRV), which is antigenically related to SVCV, and other viruses antigenically related to SVCV and PFRV were not amplified. The four subtypes of SVCV were specifically amplified by the RT-PCR. Furthermore, the detection limit of the RT-PCR was 7.1 × 10(2) copies/reaction, and it was not influenced by the addition of RNA extracted from fish tissues. The RT-PCR will be applied not only to RNA extracted from viral suspensions, but also from fish tissue. It will contribute to rapid identification of SVCV in fish with clinical signs of SVC.

Spring viraemia of carp virus: recent advances

“…These assays have clearly improved the specificity and sensitivity of detection (Kim, 2012). Although PCR is generally considered impractical for routine diagnostics in the field owing to the need for specialized instruments, skilled operators and isolation of RNA extracts, a recent report described an improved RT-PCR assay that was able to accurately detect SVCV directly from fish tissues, indicating the potential application of this technology for SVCV detection in infected fish in the field (Shimahara et al, 2015).…”

Cyprinid viral diseases and vaccine development

“…KHVD is Table 1 Cyprinid viral diseases. [84][85][86][87][88][89][90][91][92][93][94][95] seasonal, occurring when water temperature is between 18°C and 28°C. It is highly contagious and extremely virulent with mortality rate up to 80%-100%.…”

“…These assays have clearly improved the specificity and sensitivity of detection [88]. Although PCR is generally considered impractical for routine diagnostics in the field owing to the need for specialized instruments, skilled operators and isolation of RNA extracts, a recent report described an improved RT-PCR assay that is able to accurately detect SVCV directly from fish tissues, indicating the potential application of this technology for SVCV detection in infected fish in the field [89]. A loop-mediated isothermal amplification (LAMP) assay has been increasingly used for the detection of viruses [90][91][92][93].…”

“…A KHV vaccine patent is granted in the United States in 2018. A attenuated recombinant KHV comprising a genome in which ORF56 and ORF 57 are mutated, wherein the herpesvirus is capable of [84][85][86][87][88][89][90][91][92][93][94][95] replication, got the patent granted to protect common carp and koi against KHV indicating excellent immunoprotection capability [190]. KHV liposome oral vaccine can protect common carp from KHV infection [191].…”

Untitled

“…Moreover, IFA and ELISA appear to easy cross-reaction with other rhabdoviruses, leading to possible false-positive diagnoses (Way, 1991). During the last decades, various PCR-based assays have been applied to SVCV detection, e.g., reverse transcription combined with nested PCR (RT-PCR) (Koutna, Vesely, Psikal, & Hulova, 2003) multiplex real-time quantitative RT-PCR (Liu et al, 2008), and one-step TaqMan real-time quantitative (Yue et al, 2008) and improved RT-PCR (Shimahara et al, 2016). Although these assays have clearly improved the specificity and sensitivity for SVCV detection (Kim, 2012), the PCR assays are not widely applied on-site because of apparatus expense, the need for skilled operation and their long detection times.…”