Norwalklike viruses

The prototype virus for this genus is the Norwalk virus (NV), the first human virus to be described with an aetiological association with non-bacterial gastroenteritis (Kapikian et al 1972). The NV was the cause of an outbreak of gastroenteritis amongst adults and children in an elementary school in Norwalk, Ohio, USA. NV was visualized in human stool samples by immune electron microscopy using convalescent serum from a volunteer who had been experimentally infected with a faecal filtrate from the original outbreak. Subsequently other morphologically indistinguishable viruses have been described from similar outbreaks. The low numbers of virus particles shed during infection and the lack of a cell culture system has made it very difficult to characterize the Norwalk and related viruses. Early studies relied on the use of volunteers to produce enough virus for biochemical analysis. The NV has a buoyant density of 1.38—1.41 g/cm3 and a single capsid protein of 60 kDa (Greenberg et al 1981). Major advances came with the description of the NV genome (Jiang et al 1990) and the sequence of some small genome fragments (Jiang et al 1993, Matsui et al 1991). This work showed that the NV genome is comprised of positive-sense, single-stranded RNA approximately 7.5 kb in length with a 3' polyadenylated tail. Complete genome sequences of NV and another related virus, Southampton virus (SV) soon followed (Jiang et al 1993, Lambden et al 1993). The genome organisation of these viruses (5' region encoding a large non-structural polyprotein, preceding a single viral capsid protein ORF and a small ORF encoding a basic protein at the 3' terminus of the genome) confirmed that the NLVs should be classified within the Caliciviridae. The availability of these genome sequences made it possible to develop primer sets for the further investigation of the nature and extent of sequence diversity amongst the NLVs. Sequence comparisons showed that the viruses could be divided further into two genetic groupings or genogroups (Lew et al 1994) with SV and NV belonging to genogroup 1. Complete genome sequences are available for the following genogroup 2 viruses, Lordsdale (LV; Dingle et al 1995), Camberwell (CV; Seah et al 1999) and Hawaii virus (K. Green, personal communication). A generalized genome map for the NLVs is shown in Fig. 2.

OR-Fljpolyprotein processing

Alignment of the nucleotide sequences from the 5' region of ORF1 showed significant divergence between viruses from the two genogroups (Dingle et al 1995). Whilst the initiation codon for ORF1 has not been defined in the NLVs, comparison of the 'N-terminal' 150 amino acids also showed little amino acid sequence identity. Beginning at the first in frame AUG, ORF1 encodes a polyprotein of approximately 200 kDa containing motifs for 2C-like NTPase, 3C-like protease and 3D-like RNA-dependent RNA polymerase (Lambden & Clarke 1995). Because NLVs cannot be grown in cell culture, studies of polyprotein processing have been based on transcription/translation of RNA generated from cDNA clones, expression of the polyprotein in Escherichia coli and transfection of mammalian cells with cDNA. In these systems genogroup 1 (SV) and genogroup 2 (CV and LV) polyproteins undergo similar proteolytic cleavages (Liu et al 1996, 1999a, Seah et al 1999). Cleavage by the 3C-like protease occurs in cis at five specific sites liberating six defined polypeptides. The kinetics and order of cleavage have yet to be determined although preliminary work also suggests that the SV 3C-like protease has activity in trans. By analogy with animal caliciviruses further assignment of the biological functions of the cleavage products has been possible: the 3B fragment is thought to be the viral genome linked protein (VPg).

Immunoprecipitation of radiolabelled gradient-purified NV with acute and convalescent sera indicated that these viruses were comprised of a single major capsid protein with a molecular weight of 59 kDa (Greenberg et al 1981). Later studies with the Snow Mountain agent confirmed this observation (Madore et al 1986). A major breakthrough in studying the NLVs came with the discovery that expression of NV ORF2 in insect cells using a recombinant baculovirus led to the export of the capsid protein to the cell culture supernatant where it self-assembles to form virus-like particles (VLPs) (Jiang et al 1995). The VLPs appeared as 'empty' virions when viewed by negative-stain electron microscopy but were

Genus:- Norwalk - like viruses

(Norwalk strain)

Genus: - Sapporo - like viruses

(Manchester strain)


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