Identification of spring viraemia of carp virus (SVCV) by combined RT-PCR and nested PCR.
A combination of single-tube reverse transcription (RT)-PCR and nested PCR was used to identify spring viraemia of carp rhabdovirus (SVCV) in infected cell cultures and fish tissues. Two pairs of specific primers (external and internal) were selected from the glycoprotein gene sequence. A specific product of 470 bp was amplified from RNA derived from 34 SVCV isolates including the reference strain (Fijan), using RT-PCR with the external primers. The subsequent PCR using the internal primers yielded a specific product of 141 bp in all cases. No PCR product was obtained following attempts to amplify RNA derived from other fish viruses including pike fry rhabdovirus (PFRV), or from non-infected cells. The identity of the cDNA was confirmed by direct sequencing. PCR sensitivity in the cell-culture system was assessed as about 10(-1) TCID50 ml(-1). PCR was further used for the detection of SVCV in 14 clinical samples. Nested PCR allowed us to diagnose the infection in all clinical samples in which SVCV infection was demonstrated by electron microscopy and ELISA. PCR amplification of the SVCV glycoprotein (G) gene is a potential method for rapid diagnosis of spring viraemia of carp; however, it is necessary to verify the method in a higher number of clinical tissue samples. PCR can now be added to current confirmatory diagnostic methods, for determination of SVCV in cell culture. Sequencing of RT-PCR products performed for 7 SVCV isolates (4 Czech, 2 Hungarian, and 1 isolate of unknown origin) revealed a high degree of homogeneity of the G gene region with that of the previously sequenced Fijan strain. The highest nucleotide variability (97.4 to 98.1% nucleotide similarity) was found between the Hungarian and the other isolates. Knowledge of genetic differences among SVCV isolates will be useful in the development of diagnostic methods and elaboration of vaccination programmes.
D000818:Animals; D002347:Carps / Q000821:virology*; D002478:Cells, Cultured; D005393:Fish Diseases / Q000235:genetics / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D012209:Rhabdoviridae / Q000302:isolation & purification; D018353:Rhabdoviridae Infections / Q000175:diagnosis / Q000235:genetics / Q000662:veterinary* / Q000821:virology; D012621:Seasons; D018116:Vesiculovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D014766:Viremia / Q000235:genetics / Q000662:veterinary* / Q000821:virology