Complete Genome Sequence of a Mink Calicivirus in China

Abstract

We report the complete genome sequence of a novel calicivirus isolated from a diseased mink in China. The complete viral genome is approximately 8.4 kb in length and consists of three open reading frames. The availability of the complete genome sequence is helpful for further investigation into the molecular characteristics and epidemiology of calicivirus in mink.

GENOME ANNOUNCEMENT

Mink calicivirus (MCV), a member of genus Vesivirus in the Caliciviridae family, is a single-stranded positive-sense RNA virus first isolated in the United States from normal clinically fine mink in 1964 (1, 3). A new strain of MCV, MCV-DL, was isolated from a disease outbreak in mink in 2007 in Shenyang Province, northeastern China. Although MCV has previously been detected (2), the complete genome sequence has not yet been reported.

To provide more information about MCV and to aid researchers studying calicivirus taxonomy, we determined the entire genome sequence of the Chinese MCV isolate. Complete genome sequencing was performed using strategies reported for other positive-sense single-stranded RNA viruses (4–6). Genomic RNA was extracted from MCV-infected cell cultures and converted to cDNA by a combined random-priming and oligo(dT)-priming strategy. First-round PCR primers were designed from multiple genome alignments of available vesiviruses (4–6), and further sets of primers were designed on the basis of the partial genomic sequences obtained. Overlapping fragments covering the entire genome were generated and sequenced. PCR products were cloned into vector pMD18-T (TaKaRa) and sequenced by Invitrogen. The 5′ and 3′ ends of the viral genome were amplified by rapid amplification of cDNA ends. All fragments were sequenced in both directions. Overlapping consensus sequences were assembled and manually edited to produce the final genome sequence using the software program BioEdit 7.0.9. Phylogenetic trees were constructed using MEGA 4.0 (7).

The complete genome of MCV-DL was found to be 8,409 nucleotides (nt), excluding the polyadenylate tract, with a GC content of 45.6%. Three open reading frames (ORFs) were predicted from the nucleotide sequence with DNAStar (version 5.0; DNAStar Inc., Madison, WI) and by comparing the results with the genome organization and ORFs of other caliciviruses (2, 5). ORF1, stretching from nt 14 to nt 5851, encodes a large putative polyprotein of 1,946 amino acids (aa). ORF2 ranges from nt 5857 to nt 7899, and ORF3 ranges from nt 8130 to nt 8306, encoding proteins of 681 and 59 aa, respectively. The 5′ and 3′ untranslated regions are 13 and 103 nt long, respectively.

Phylogenetic analysis of the available genome sequences of representative vesiviruses showed that MCV-DL clustered with calicivirus 2117 (accession no. AY343325, unknown origin) and canine calicivirus (CaCV; accession no. NC_004542, isolated from a canine) rather than with the other vesiviruses. Across the whole genome, the highest sequence identity (60.0%) was with calicivirus 2117. Alignment of the sequences of the predicted gene products of MCV and other vesiviruses showed that the highest amino acid sequence identities were with calicivirus 2117 and CaCV. The proteins encoded by ORF1, ORF2, and ORF3 of MCV were found to have 63.5, 59.5, and 40.8% identity with calicivirus 2117 and 42.6, 49.2, and 50.8% amino acid sequence identity with CaCV.

This is the first report of a complete MCV genome sequence which is critical for further investigation of the molecular characteristics and should allow elucidation of its phylogenetic relationship with other vesiviruses.

Nucleotide sequence accession number.

The complete genome sequence of MCV strain MCV-DL/2007/CN has been deposited in GenBank under accession no. JX847605.