Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR.
A quantitative real-time RT-PCR (Q-RT-PCR) was developed to detect and determine the amount of viral hemorrhagic septicemia virus (VHSV) in organs of experimentally infected rainbow trout. Primers and TaqMan probes targeting the glycoprotein (G) and the nucleoprotein (N) genes of the virus were designed. The efficiency, linear range and detection limit of the Q-RT-PCR were assessed on cell cultured virus samples. VHSV N gene amplification was more efficient and more sensitive than the VHSV G amplicon. On cell culture grown virus, samples could be accurately assayed over a range of seven logs of infectious particles per reaction. To demonstrate the utility of Q-RT-PCR in vivo, bath infection trials were carried out and samples from fish spleen, kidney, liver and blood were harvested and tested for VHSV. Q-RT-PCR was a more reliable method than either conventional RT-PCR or the cell culture assay for virus diagnosis. Results of VHSV RNA detection in fish shortly after infection as well as on asymptomatic fish several weeks after experimental challenge are presented here. This is the first report showing the utility of Q-RT-PCR for VHSV detection and quantitation both in vitro and in vivo. The suitability of this method to test the efficacy of antiviral treatments is also discussed.
Chico
Veronica
V
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche, Spain.
Gomez
Noemi
N
Estepa
Amparo
A
Perez
Luis
L
D000818:Animals; D001769:Blood / Q000821:virology; D002347:Carps; D002460:Cell Line; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005393:Fish Diseases / Q000175:diagnosis / Q000821:virology*; D007668:Kidney / Q000821:virology; D008099:Liver / Q000821:virology; D029141:Novirhabdovirus / Q000235:genetics / Q000302:isolation & purification*; D009698:Nucleoproteins / Q000235:genetics; D017686:Oncorhynchus mykiss / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D018353:Rhabdoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012680:Sensitivity and Specificity; D013154:Spleen / Q000821:virology; D014759:Viral Envelope Proteins / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics; D014776:Virus Cultivation
D017931:DNA Primers; C096089:G protein, viral hemorrhagic septicemia virus; D009698:Nucleoproteins; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins