Comparison of Quantitative RT‐PCR with Cell Culture to Detect Viral Hemorrhagic Septicemia Virus (VHSV) IVb Infections in the Great Lakes

Abstract

Viral hemorrhagic septicemia virus (VHSV) is an important pathogen of cultured and wild fish in marine and freshwater environments. A new genotype, VHSV IVb, was isolated from a fish collected from the Great Lakes in 2003. Since the first isolation, VHSV IVb has been confirmed in 28 species, signaling the early invasion and continued spread of this Office International des Epizooties-reportable agent. For surveillance of this virus in both wild and experimental settings, we have developed a rapid and sensitive one-step quantitative real-time polymerase chain reaction (qRT-PCR) assay that amplifies a 100-base-pair conserved segment from both the genomic negative strand and the mRNA positive strand of the nucleoprotein (N) gene of VHSV IVb. This assay is linear over seven orders of magnitude, with an analytical capability of detecting a single copy of viral RNA and reproducibility at 100 copies. The assay is approximately linear with RNA input from 50 to 1000 ng per assay and works equally well with RNA prepared from a column-based or phenol-chloroform-based method. In wild-caught fish, 97% of the cases were found to be more than three orders of magnitude more sensitive using qRT-PCR than using cell culture. Of the 1,428 fish from the Great Lakes region tested in 2006 and 2007, 24% were positive by qRT-PCR whereas only 5% were positive by cell culture. All of the fish that were positive by cell culture were also positive by qRT-PCR. Importantly, qRT-PCR sensitivity is comparable to that of cell culture detection when comparing VHSV viral RNA levels with viral titer stocks, confirming that the high qRT-PCR signals obtained with diagnostic samples are due to the accumulation of N gene mRNA by transcriptional attenuation. The qRT-PCR assay is particularly valuable for rapid and high-throughput prescreening of fish before confirmatory testing by cell culture or sequencing tissue-derived amplicons and especially in detecting infection in fish that do not show clinical signs of VHS.

Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus

“…Detection of VHSV in such populations may not be readily detected using cell culture yet are detected via RT-qPCR, thereby resulting in higher sensitivities for RT-qPCR than by virus isolation. Such increased sensitivity of RT-qPCR over virus isolation has been reported by Hope et al (2010). Alternative validation conditions may also warrant the use of other modeling approa ches, such as latent class, to evaluate DSe and DSp as utilized by Nerette et al (2008).…”

“…Over the past several years multiple publications have demonstrated the use of qPCR to detect VHSV (Chico et al 2006, Matejusova et al 2008, Cutrin et al 2009, Hope et al 2010. One challenge posed by the use of these qPCR technologies for diagnostic purposes lies in their ability to detect all genetic variants of VHSV with a single assay.…”

“…Upon infection of the host, the 6 genes are transcribed progressively from the 3' to 5' end of the template virus RNA and in decreasing molar abundances (Kurath & Leong 1985). Thus, because of the primary position of the N-gene on the VHSV genome, N transcripts are most abundant during viral infection and therefore have been the target of several molecular assays used to detect VHSV (Batts et al 1993, Chico et al 2006, Lopez-Vazquez et al 2006, Matejusova et al 2008, Cutrin et al 2009, Hope et al 2010. The glycoprotein gene has also been a target of PCR-based detection methods (Bruchhof et al 1995, Miller et al 1998, Chico et al 2006, Cutrin et al 2009).…”

Immunohistochemistry and pathology of multiple Great Lakes fish from mortality events associated with viral hemorrhagic septicemia virus type IVb

“…It is debatable whether the molecular diagnosis of VHSV necessarily equates to the cause of mortality, and correlation of the presence of the virus in combination with gross and microscopic lesions is critical to make that link. Since VHSV has become very topical in the Great Lakes basin and the molecular tests used are very sensitive (Hope et al 2010), there is a possibility that every mortality event from which VHSV nucleic acid or virus is obtained will be ascribed to the virus. Immunohistochemistry (IHC) is routinely used to localize a pathogen to a lesion pattern and has been used previously for other genotypes of VHSV (Evensen & Olesen 1997, Brudeseth et al 2005, Dale et al 2009).…”

Viral load of various tissues of rainbow trout challenged with viral haemorrhagic septicaemia virus at various stages of disease

“…Neither method is currently the recommended method to detect VHSV for statutory purposes. However, real-time RT-PCR has recently been recommended to be applied in settings where VHSV is considered endemic and may be more sensitive than cell culture (Hope et al 2010).…”