Sequence and Genome Organization of a Human Small Round-Structured (Norwalk-Like) Virus

Lambden, Paul R.; Caul, E.O.; Cr, Ashley; Clarke, Ian N.

doi:10.1126/science.8380940

Cited by 343 publications

(242 citation statements)

References 17 publications

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“…In addition, in all published SLV strains except a human strain, London/92 and a porcine strain, PEC/Cowden [5,15,18,27,29,40], an additional ORF has been predicted in +1 frame, overlapping the N terminus of the capsid gene (capsid overlap). To date, the sequence of a ∼3-kb region extending from the RNA polymerase gene to the 3 poly(A) tail is available in GenBank for 9 NLV strains, of which 6 have had their entire genome sequenced: Norwalk/68 [17], Southampton/91 [22], Lordsdale/93 [9], Camberwell/94 [3,36], Chiba/87 (GenBank accession number No. AB042808) and Jena/80 [26].…”

Section: Introductionmentioning

confidence: 99%

Genetic classification of "Sapporo-like viruses"

Schuffenecker

Ando

Thouvenot

et al. 2001

Archives of Virology

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Section: Introductionmentioning

confidence: 99%

Genetic classification of "Sapporo-like viruses"

Schuffenecker

Ando

Thouvenot

et al. 2001

Archives of Virology

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“…Since the initial cloning and sequencing of the NV genome (4, 10), many other norovirus sequences have been reported (11,12). Despite the availability of cloned genomes, molecular studies of NV replication and development of antiviral drugs have been hampered by the lack of a suitable in vitro culture system and an animal model.…”

mentioning

confidence: 99%

“…Expression of VP1 in insect cells infected with baculovirus recombinants results in the self-assembly of empty recombinant virus-like particles (VLPs) (6, 7). VP2 is also incorporated into VLPs when VP2 is coexpressed with VP1 (8), and its presence increases the yield and stability of VLPs (9).Since the initial cloning and sequencing of the NV genome (4, 10), many other norovirus sequences have been reported (11,12). Despite the availability of cloned genomes, molecular studies of NV replication and development of antiviral drugs have been hampered by the lack of a suitable in vitro culture system and an animal model.…”

mentioning

confidence: 99%

Replication and packaging of Norwalk virus RNA in cultured mammalian cells

Asanaka

Atmar

Ruvolo

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

101

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Human noroviruses, the most common cause of nonbacterial gastroenteritis, are characterized by high infectivity rate, low infectious dose, and unusually high stability outside the host. However, human norovirus research is hindered by the lack of a cell culture system and a small animal model of infection. Norwalk virus (NV) is the prototype strain of human noroviruses. We report here replication of NV viral RNA and its packaging into virus particles in mammalian cells by intracellular expression of native forms of NV viral RNA devoid of extraneous nucleotide sequences derived from the expression vector by the use of replication-deficient vaccinia virus MVA encoding the bacteriophage T7 RNA polymerase (MVA͞ T7). Expressed genomic RNA was found to replicate; NV subgenomic RNA was transcribed from genomic RNA by use of NV nonstructural proteins expressed from genomic RNA and was subsequently translated into NV capsid protein VP1. Viral genomic RNA was packaged into virus particles generated in mammalian cells. The cesium chloride (CsCl) density gradient profile of virus particles containing genomic RNA was similar to that of NV purified from stool. These observations indicate that the NV cDNA constructed here is a biologically infectious clone, and that mammalian cells have the ability to replicate NV genomic RNA. This work establishes a mammalian cell-based system for analysis of human norovirus replication and, thus, makes it feasible to investigate antiviral agents in mammalian cells.human norovirus ͉ norovirus RNA replication N oncultivatable human norovirus belongs to the Norovirus genus of the family Caliciviridae and is classified as a Group B biodefense pathogen. Human noroviruses are responsible for almost all outbreaks (Ͼ95%) of nonbacterial gastroenteritis in the United States and Europe (1, 2). Incidents of the rapid spread of norovirus-related illness in the cruise ship industry have led to an increased recognition of the impact of norovirus infections on public health (3).Norwalk virus (NV), the prototype strain of human norovirus, is a nonenveloped virus that contains a Ϸ7.7-kb positive-sense single-stranded RNA genome (4). The NV genome is polyadenylated at its 3Ј end and encodes three primary ORFs (Fig. 1A) (4). ORF1 encodes a nonstructural polyprotein containing the following genes from the 5Ј end to the 3Ј end, respectively: p48, nucleotide triphosphatase (NTPase), p22, VPg, protease (Pro), and RNA polymerase (RNA Pol) (Fig. 1 A) (5). ORF2 and ORF3 encode structural proteins, the major capsid protein (VP1) and a minor structural protein (VP2), respectively ( Fig. 1 A) (4). Expression of VP1 in insect cells infected with baculovirus recombinants results in the self-assembly of empty recombinant virus-like particles (VLPs) (6, 7). VP2 is also incorporated into VLPs when VP2 is coexpressed with VP1 (8), and its presence increases the yield and stability of VLPs (9).Since the initial cloning and sequencing of the NV genome (4, 10), many other norovirus sequences have been reported (11,12). Despite ...

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“…NV causes outbreaks of acute non-bacterial gastroenteritis in humans, and the genome of NV is approximately 7.6 kb in size. The genome organization shows three open reading frames (ORFs) encoding non-structural proteins, a capsid protein and a protein whose function is not known (9,15). The capsid protein of NV has an apparent molecular mass of 60 kDa.…”

mentioning

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Characterization of Monoclonal Antibodies Generated against Norwalk virus Gil Capsid Protein Expressed in Escherichia coli

Yoda

Terano

Suzuki

et al. 2000

Microbiology and Immunology

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The Norwalk virus (NV) causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. The capsid protein of NV36 (genogroup II, Mexico virus type) was expressed in an Escherichia coli system and ten monoclonal antibodies (MAbs) were generated against it. The reactivity of these MAbs was characterized using enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis towards 20 overlapping fragments of the NV36 capsid protein expressed in E. coli. All of the MAbs recognized sequential (continuous) epitopes on the three antigenic regions. Six of the 10 MAbs recognized fragment 2 (equivalent residues 31-70), three MAbs recognized fragment 13 (residues 361-403) and one MAb recognized fragment 7 (residues 181-220), suggesting that the Nterminal domain (residues 1-220) may contain more antigenic epitopes than the C-terminal domain (residues 210-548). Furthermore, two MAbs (1B4 and 1F6) reacted in WB with three purified NV strains (genogroup II) derived from patients' stool samples. It was also found that genogroup I recombinant NV96-908 (genogroup I, KY89 type) could be detected as sensitively as recombinant NV36 (genogroup II) by ELISA with a set of the MAbs produced here.

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Sequence and Genome Organization of a Human Small Round-Structured (Norwalk-Like) Virus

Cited by 343 publications

References 17 publications

Genetic classification of "Sapporo-like viruses"

Genetic classification of "Sapporo-like viruses"

Replication and packaging of Norwalk virus RNA in cultured mammalian cells

Characterization of Monoclonal Antibodies Generated against Norwalk virus Gil Capsid Protein Expressed in Escherichia coli

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