Molecular analysis of spring viraemia of carp virus in China: a fatal aquatic viral disease that might spread in East Asian

Abstract

Spring viraemia of carp (SVC) is a fatal viral disease for cyprinid fish, which is caused by spring viraemia of carp virus (SVCV). To date, no SVC outbreak has been reported in China. Between 1998 and 2002, outbreaks of SVC were reported in ornamental and wild fish in Europe and America, imported from multiple sources including China. Based on phylogenetic analysis, the viral strain isolated from America was shown to be originated from Asia. These outbreaks not only resulted in huge economic losses, but also raise an interesting question as to whether SVCV really exists in China and if so, is it responsible for SVC outbreaks? From 2002 to 2006, we screened 6700 samples from ornamental fish farms using the cell culture method of the Office International des Epizooties (OIE), and further verified the presence of SVCV by ELISA and real-time quantitative RT-PCR. Two infected samples were found and the complete genome of SVCV was sequenced from one of the isolates, termed SVCV-C1. Several unique hallmarks of SVCV-C1 were identified, including six amino acid (KSLANA) insertion in the viral RNA-dependent RNA polymerase (L) protein and ten nucleotide insertion in the region between glycoprotein (G) and L genes in European SVCV strains. Phylogenetic tree analysis of the full-length G protein of selected SVCV isolates from the United Kingdom and United States revealed that G proteins could be classified into Ia and Id sub genogroups. The Ia sub genogroup can be further divided into newly defined sub genogroups Ia-A and Ia-B. The isolates derived from the United States and China including the SVCV-C1 belongs to in the Ia-A sub genogroup. The SVCV-C1 G protein shares more than 99% homology with the G proteins of the SVCV strains from England and the United States, making it difficult to compare their pathogenicity. Comparison of the predicted three-dimensional structure based on the published G protein sequences from five SVCV strains revealed that the main differences were in the loops of the pleckstrin homology domains. Since SVCV is highly pathogenic, we speculate that SVC may therefore pose a serious threat to farmed cyprinid fish in China.

Figure 1. Molecular analysis of the SVCV-C1 genome.

A, alignment of the nucleotide sequences in the G/L gene junction of the SVCV-C1 isolate, European reference viruses (Bjorklund) and US isolates. The stop codon of G gene and start codon of L gene are in bold. The dashes (–) indicate gaps not seen in isolate SVCV-A1. The conservative polyadenylation signal TATG[A]7 and transcription initiation sequence AACAG are underlined. B, significant diversity of protein sequences in the whole genomes of five SVCV strains. SVCV-C1 and SVCV-A1 were isolated from Asian strain and Fijan strain; Bjorklund strain and VR-1390 strain were isolated from European (comment: the three SVCV reference full length sequences used in the comparison were simply submitted by different researchers for the same virus). The different amino acids in proteins P, G and L were labeled above SVCV-C1. Six amino acids (KSLANA) inserted in the L protein of SVCV-C1 strain and dots represent deletion in other strains. The accession numbers used for analysis are: SVCV-C1, EU177782; SVCV-A1, DQ097384; Fijan strain, AJ318079; Bjorklund strain, NC_002803; VR-1390 strain, U18101; five US isolates, DQ227500, DQ227501, DQ227502, DQ227503, DQ227504.

Figure 2. The phylogenetic tree generated by Neighbor-Joining method based on the G proteins from all SVCV isolates.

Two genogroups, Ia and Id were differentiated by using the p-distance substitution model with bootstrap test of 1,000 replicates. SVCV G proteins isolated in China belong to genogroup Ia which can be further divided into Ia-A and Ia-B sub genogroups. SVCV-C1 belongs to Ia-A subgroup. Country origins of the isolates are shown in the brackets. RV represents the reference virus.

Figure 3. The 3D structure and alignment of SVCV G proteins (domain III).

A, Four domains are colored: red (domain I: 19–35aa, 328–401aa), blue (domain II: 36–53aa, 277–327aa, 402–428aa), orange (domain III: 54–68aa, 199–276aa), yellow (domain IV: 69–198aa, yellow). Variations of amino acids between SVCV-C1 and other four strains are colored with green. B, The 3D structure of domain III in G protein showing the locations of amino acid variations. C, Multiple alignment of domain III of G protein. Protein secondary structures (α helices and β sheets) are indicated above the amino acid sequences. *GenBank accession numbers used are: SVCV-C1, EU177782; SVCV-A1, DQ097384; Fijan strain, AJ318079; Bjorklund strain, NC_002803; VR-1390 strain, U18101.

Figure 4. Optimization of real-time quantitative RT- PCR assay.

A, optimization of primer and probe concentration. B, optimization of Mg2+ concentration. C, comparison of the sensitivity between real time RT-PCR assay and cell culture method.