Male-specific single-stranded RNA (FRNA) coliphages belong to the family Leviviridae. They are classified into two genera (Levivirus and Allolevivirus), which can be subdivided into four genogroups (genogroups I and II in Levivirus and genogroups III and IV in Allolevivirus). Relatively few strains have been completely characterized, and hence, a detailed knowledge of this virus family is lacking. In this study, we sequenced and characterized the complete genomes of 19 FRNA strains (10 Levivirus strains and 9 Allolevivirus strains) and compared them to the 11 complete genome sequences available in GenBank. Nucleotide similarities among strains of Levivirus genogroups I and II were 75% to 99% and 83 to 94%, respectively, whereas similarities among strains of Allolevivirus genogroups III and IV ranged from 70 to 96% and 75 to 95%, respectively. Although genogroup I strain fr and genogroup III strains MX1 and M11 share only 70 to 78% sequence identity with strains in their respective genogroups, phylogenetic analyses of the complete genome and the individual genes suggest that strain fr should be grouped in Levivirus genogroup I and that the MX1 and M11 strains belong in Allolevivirus genogroup III. Strains within each genus share >50% sequence identity, whereas between the two genera, strains have <40% nucleotide sequence identity. Overall, amino acid composition, nucleotide similarities, and replicase catalytic domain location contributed to phylogenetic assignments. A conserved eight-nucleotide signature at the 3 end of the genome distinguishes leviviruses (5 ACCACCCA 3) from alloleviviruses (5 TCCTCCCA 3).
Human norovirus (NoV) is the leading cause of nonbacterial gastroenteritis worldwide (3). The Centers for Disease Control and Prevention (CDC) estimate that 23 million cases of acute gastroenteritis due to NoV occur each year, with symptoms including acute-onset vomiting, watery nonbloody diarrhea with abdominal cramps, and nausea (35). NoV outbreaks are pervasive for many reasons, but particularly because the virus is highly contagious and environmentally hardy (7). Additionally, infected individuals can excrete millions of viral particles in feces, leading to large numbers in sewage (16). Without proper removal or inactivation during wastewater treatment, the viruses can be released into recreational and shellfishharvesting water bodies. Complete inactivation of NoV during sewage treatment is rare, and even in areas with proper wastewater treatment, contamination of oyster beds has been reported (5,16,17,32,38). Because bivalve molluscan shellfish are believed to act as filters for viruses and other microbes and because NoV is extremely infectious (as little as one viral particle is required for disease), the disease risk for consumption of raw oysters is high (27,33,40).Human NoV genogroup I (GI) and GII have been detected in oyster samples harvested from bays and estuaries worldwide (5,10,20). Ueki et al. (42) detected NoV in both shellfish and the surrounding river water in Japan and concluded that NoV contamination was most likely due to sewage and treated wastewater input into the river; however, no study has yet been able to characterize how NoV may be naturally distributed in an estuarine system, including in water, adhered to particles (including plankton), and in shellfish. The limitations are due in part to a lack of adequate detection methods specifically adapted to different environmental-sample types (8). Using a newly developed detection and quantification protocol (21), this study aimed to examine the distribution of NoV genogroups across a range of sample types within an estuarine system with the goal of better characterizing possible circulation of viruses between water, plankton, and oysters. MATERIALS AND METHODS Controls. (i) NoV-positive controls.Three NoV-positive fecal samples, representing genotypes GI.4, GI.3b, and GII.4 Minerva, were provided as controls for this study by the CDC. Stool samples were diluted to obtain a 20% suspension in phosphate-buffered saline (PBS), vortexed, and centrifuged at 15,700 ϫ g for 2 min.(ii) Viral-RNA extraction from stool. For stool samples, viral RNA was extracted from the clarified PBS extracts using the MagMAX-96 Viral Isolation Kit (Ambion, Austin, TX) and the KingFisher Instrument (Thermo Electron Corporation, Waltham, MA), which automatically purifies viral RNA. The purified RNA was eluted into 55 l elution buffer, provided in the kit.(iii) RNA transcript standards. To enable quantification by real-time reverse transcription (RT)-PCR, we used GI and GII plasmids (1) to generate RNA runoff transcripts. Briefly, the norovirus 3-kb plasmids were pu...
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