Complete Genome Sequences of Novel Rat Noroviruses in Hong Kong

Abstract

We report two genome sequences of novel noroviruses isolated from fecal swab specimens of brown rats in Hong Kong. The complete genome is approximately 7.5 kb in length and consists of 3 overlapping open reading frames encoding ORF1 polyprotein, VP1, and VP2, respectively. Sequence analysis suggested that these noroviruses should be classified in genogroup V, but they are distinct from other known rodent noroviruses and represent a novel cluster within the genogroup.

GENOME ANNOUNCEMENT

Noroviruses (NoVs) are a genus of genetically diverse single-stranded positive-sense RNA viruses in the family Caliciviridae. They are the most important viral agents responsible for human gastroenteritis worldwide, and their role in causing enteric disease among different animals has been increasingly recognized (1, 4). Transmission of NoVs can occur through various routes, including food, environment, and personal contact (2), resulting in gastroenteritis outbreaks as well as sporadic cases in non-outbreak settings (3).

Based on the VP1 amino acid sequence, NoVs can be classified into 5 genogroups (GI to GV), which are further divided into clusters and strains (11). This phylogenetic classification is correlated with the host specificity of NoVs among humans and other mammals (1). Importantly, the discovery of the murine NoVs greatly facilitated NoV research, as they are the only known NoVs capable of productive infection in cell cultures (9). Related NoVs have also been found in other rodents, including the wood mouse (Apodemus sylvaticus), the large Japanese field mouse (Apodemus speciosus), and the black rat (Rattus rattus), but not in the common brown rat (Rattus norvegicus) (5, 8).

As part of an ongoing surveillance study on animal viruses, we discovered a novel NoV strain in fecal swab specimens from two brown rats in Hong Kong. Complete genome sequences were obtained using previously described strategies for single-stranded RNA viruses (6, 7, 10). Viral RNA was extracted by the use of a QIAamp RNA blood minikit (Qiagen, Hilden, Germany). Reverse transcription was performed using a SuperScript III kit (Invitrogen, San Diego, CA). Initial PCR primers were designed based on the multiple alignment of other NoV genomes, while additional primers were designed based on results from earlier rounds of genome sequencing. Sequences from the ends of the viral genomes were obtained by random amplification of cDNA ends (RACE) using a SMARTer RACE cDNA amplification kit (Clontech, Mountain View, CA). PCR products were sequenced twice with an ABI Prism 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA) using PCR primers. Sequence assembly was performed manually using BioEdit 7.0.9.

The complete genome of the present NoV is approximately 7,541 bases long, with G+C content of 53.9%. The genomes of the two NoV isolates are similar, with nucleotide sequence identity of 91.4%. The genome organization is typical of other NoVs, with 3 overlapping open reading frames encoding ORF1 polyprotein (1,700 amino acids [aa]), VP1 capsid protein (548 aa), and VP2 minor structural protein (218 aa). The 5′ and 3′ untranslated regions are 10 bases and 143 bases long, respectively. Sequence analysis of the predicted VP1 protein showed the isolates to be most closely related to other GV NoVs, with highest sequence identities (63.1% to 63.3%) to a strain isolated from striped field mice (Apodemus agrarius) in Hungary. The level of sequence identity, as well as phylogenetic analysis of VP1 sequences, suggests that the present NoVs potentially belong to a novel cluster in GV (11).

Nucleotide sequence accession numbers.

One full-length genome and one nearly full-length genome of the novel NoV have been submitted to GenBank under accession no. JX486102 and JX486101.