Kim Y. Green

Replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-αβ receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.

Proposal for a unified norovirus nomenclature and genotyping

Noroviruses belong to a genus of genetically diverse viruses within the family Caliciviridae and cause acute gastroenteritis in humans and animals. They are subdivided into genogroups, each of which further segregates into genotypes. Until recently, a new genotype was based on a defined pairwise distance cutoff of complete VP1 sequences, but with the increasing number of available norovirus sequences, this cutoff is no longer accurate, and sequences in the public database have been misclassified. In this paper, we demonstrate that the pairwise distance cutoff method can no longer be used and outline a phylogenetic approach to classify noroviruses. Furthermore, we propose a dual nomenclature using both ORF1 and VP1 sequences, as recombination is common and recognizing recombinant viruses may be relevant. With the continuing emergence of new norovirus lineages, we propose to coordinate nomenclature of new norovirus genotypes through an international norovirus working group.

Diarrheal Disease during Operation Desert Shield

BACKGROUND Under combat conditions infectious disease can become a major threat to military forces. During Operation Desert Shield, there were numerous outbreaks of diarrhea among the U.S. forces. To evaluate the causes of and risk factors for diarrheal disease, we collected clinical and epidemiologic data from U.S. troops stationed in northeastern Saudi Arabia. METHODS Between September and December 1990, stool cultures for enteric pathogens were obtained from 432 military personnel who presented with diarrhea, cramps, vomiting, or hematochezia. In addition, a questionnaire was administered to 2022 soldiers in U.S. military units located in various regions of Saudi Arabia. RESULTS A bacterial enteric pathogen was identified in 49.5 percent of the troops with gastroenteritis. Enterotoxigenic Escherichia coli and Shigella sonnei were the most common bacterial pathogens. Of 125 E. coli infections, 39 percent were resistant to trimethoprim-sulfamethoxazole, 63 percent to tetracycline, and 48 percent to ampicillin. Of 113 shigella infections, 85 percent were resistant to trimethoprim-sulfamethoxazole, 68 percent to tetracycline, and 21 percent to ampicillin. All bacterial isolates were sensitive to norfloxacin and ciprofloxacin. After an average of two months in Saudi Arabia, 57 percent of the surveyed troops had at least one episode of diarrhea, and 20 percent reported that they were temporarily unable to carry out their duties because of diarrheal symptoms. Vomiting was infrequently reported as a primary symptom, but of 11 military personnel in whom vomiting was a major symptom, 9 (82 percent) had serologic evidence of infection with the Norwalk virus. CONCLUSIONS Gastroenteritis caused by enterotoxigenic E. coli and shigella resistant to a number of drugs was a major problem that frequently interfered with the duties of U.S. troops during Operation Desert Shield.

Norovirus Gastroenteritis in Immunocompromised Patients

Norovirus causes gastroenteritis in immunocompetent and immunocompromised hosts. It is the most common cause of gastroenteritis in U.S. adults seeking emergency department care. Genetic features and emerging treatments are discussed.

Evolutionary dynamics of GII.4 noroviruses over a 34-year period.

ABSTRACT Noroviruses are a major cause of epidemic gastroenteritis in children and adults, and GII.4 has been the predominant genotype since its first documented occurrence in 1987. This study examined the evolutionary dynamics of GII.4 noroviruses over more than three decades to investigate possible mechanisms by which these viruses have emerged to become predominant. Stool samples (n = 5,424) from children hospitalized at the Childrens Hospital in Washington, DC, between 1974 and 1991 were screened for the presence of noroviruses by a custom multiplex real-time reverse transcription-PCR. The complete genome sequences of five GII.4 noroviruses (three of which predate 1987 by more than a decade) in this archival collection were determined and compared to the sequences of contemporary strains. Evolutionary analysis determined that the GII.4 VP1 capsid gene evolved at a rate of 4.3 × 10−3 nucleotide substitutions/site/year. Only six sites in the VP1 capsid protein were found to evolve under positive selection, most of them located in the shell domain. No unique mutations were observed in or around the two histoblood group antigen (HBGA) binding sites in the P region, indicating that this site has been conserved since the 1970s. The VP1 proteins from the 1974 to 1977 noroviruses contained a unique sequence of four consecutive amino acids in the P2 region, which formed an exposed protrusion on the modeled capsid structure. This protrusion and other observed sequence variations did not affect the HBGA binding profiles of recombinant virus-like particles derived from representative 1974 and 1977 noroviruses compared with more recent noroviruses. Our analysis of archival GII.4 norovirus strains suggests that this genotype has been circulating for more than three decades and provides new ancestral strain sequences for the analysis of GII.4 evolution.

Correlation of patient immune responses with genetically characterized small round-structured viruses involved in outbreaks of nonbacterial acute gastroenteritis in the United States, 1990 to 1995

Small round‐structured viruses (SRSVs) are a genetically and antigenically diverse group of caliciviruses that are the most common cause of outbreaks of acute nonbacterial gastroenteritis. We have applied both molecular techniques to characterize SRSVs in fecal specimens and serologic assays using four different expressed SRSV antigens to examine the distribution of outbreak strains in the United States and determine if the immune responses of patients were strain specific. Strains from 23 outbreaks of SRSV gastroenteritis were characterized by reverse transcription‐PCR and nucleotide sequencing of a 277‐base region of the capsid gene. These strains segregated into two distinct genogroups, I and II, comprising four and six clusters of strains respectively, each representing a distinct phylogenetic lineage. Serum IgG responses in patients were measured by enzyme immunoassay using expressed capsid antigens of Norwalk virus (NV), Toronto virus (TV), Hawaii virus (HV), and Lordsdale virus (LV), representing four of the 10 clusters. While strains in genogroups I and II were antigenically distinct, within genogroups, the specificity of the immune response varied greatly. Patients infected with genogroup I strains which had as much as 38.5% aa divergence from NV demonstrated relatively homologous seroresponses to the single NV antigen. In contrast, in genogroup II, homologous seroresponses to TV and HV were only present when the infecting strains showed less than 6.5% aa divergence from these antigens. These results suggest that TV and HV represent not only separate genetic clusters in genogroup II but also separate antigenic groups, each of which is related but distinguishable. In addition, two genetically distinct SRSV strains were identified for which we have no homologous antigen. This study suggests that while current molecular diagnostics are capable of detecting the full range of SRSVs, additional expressed antigens will be required to detect an immune response to SRSV infection caused by all the antigenically diverse strains. J. Med. Virol. 53:372–383, 1997.

Cleavage Map and Proteolytic Processing of the Murine Norovirus Nonstructural Polyprotein in Infected Cells

ABSTRACT Murine norovirus (MNV) is presently the only member of the genus Norovirus in the Caliciviridae that can be propagated in cell culture. The goal of this study was to elucidate the proteolytic processing strategy of MNV during an authentic replication cycle in cells. A proteolytic cleavage map of the ORF1 polyprotein was generated, and the virus-encoded 3C-like (3CL) proteinase (Pro) mediated cleavage at five dipeptide cleavage sites, 341E/G342, Q705/N706, 870E/G871, 994E/A995, and 1177Q/G1178, that defined the borders of six proteins with the gene order p38.3 (Nterm)-p39.6 (NTPase)-p18.6-p14.3 (VPg)-p19.2 (Pro)-p57.5 (Pol). Bacterially expressed MNV 3CL Pro was sufficient to mediate trans cleavage of the ORF1 polyprotein containing the mutagenized Pro sequence into products identical to those observed during cotranslational processing of the authentic ORF1 polyprotein in vitro and to those observed in MNV-infected cells. Immunoprecipitation and Western blot analysis of proteins produced in virus-infected cells demonstrated efficient cleavage of the proteinase-polymerase precursor. Evidence for additional processing of the Nterm protein in MNV-infected cells by caspase 3 was obtained, and Nterm sequences 118DRPD121 and 128DAMD131 were mapped as caspase 3 cleavage sites by site-directed mutagenesis. The availability of the MNV nonstructural polyprotein cleavage map in concert with a permissive cell culture system should facilitate studies of norovirus replication.

A predominant role for Norwalk-like viruses as agents of epidemic gastroenteritis in Maryland nursing homes for the elderly.

Stool specimens from 156 Maryland nursing home residents, who became ill during 20 outbreaks of gastroenteritis from November 1987 through February 1988, were analyzed. All tested negative for astroviruses, enteroviruses, Group A rotaviruses, Sapporo-like caliciviruses, and enteric bacteria (i.e., Salmonella, Clostridium, and Shigella species). Eighty-two (52%) were positive for Norwalk-like viruses (NLVs), members of the family Caliciviridae. Six distinct genetic clusters within genogroups I and II of the NLVs were detected; a genogroup II (GII) virus closely related to the Camberwell virus in the NLV GII/4 genetic cluster was the predominant strain. Serologic evidence of infection with > or = 1 NLV was detected in 61 (56%) of 109 patients tested against 3 NLV antigens (i.e., Norwalk, Hawaii, and Toronto viruses). Sixteen (80%) outbreaks met the definition for an NLV outbreak. Taken together with a retrospective analysis of bacterial gastroenteritis in this same setting, these data support a major role for NLVs as etiologic agents of gastroenteritis in elderly persons.

Chimpanzees as an animal model for human norovirus infection and vaccine development

Noroviruses are global agents of acute gastroenteritis, but the development of control strategies has been hampered by the absence of a robust animal model. Studies in chimpanzees have played a key role in the characterization of several fastidious hepatitis viruses, and we investigated the feasibility of such studies for the noroviruses. Seronegative chimpanzees inoculated i.v. with the human norovirus strain Norwalk virus (NV) did not show clinical signs of gastroenteritis, but the onset and duration of virus shedding in stool and serum antibody responses were similar to that observed in humans. NV RNA was detected in intestinal and liver biopsies concurrent with the detection of viral shedding in stool, and NV antigen expression was observed in cells of the small intestinal lamina propria. Two infected chimpanzees rechallenged 4, 10, or 24 mo later with NV were resistant to reinfection, and the presence of NV-specific serum antibodies correlated with protection. We evaluated the immunogenicity and efficacy of virus-like particles (VLPs) derived from NV (genogroup I, GI) and MD145 (genogroup II, GII) noroviruses as vaccines. Chimpanzees vaccinated intramuscularly with GI VLPs were protected from NV infection when challenged 2 and 18 mo after vaccination, whereas chimpanzees that received GII VLPs vaccine or a placebo were not. This study establishes the chimpanzee as a viable animal model for the study of norovirus replication and immunity, and shows that NV VLP vaccines could induce protective homologous immunity even after extended periods of time.

Outbreak Management and Implications of a Nosocomial Norovirus Outbreak

BACKGROUND Noroviruses are enterically transmitted and are a frequent cause of gastroenteritis, affecting 23 million people annually in the United States. We describe a norovirus outbreak and its control in a tertiary care hospital during February-May 2004. METHODS Patients and health care workers met the case definition if they had new onset of vomiting and/or diarrhea during the outbreak period. Selected stool samples were tested for norovirus RNA. We also determined outbreak costs, including the estimated lost revenue associated with unit closures, sick leave, and cleaning expenses. RESULTS We identified 355 cases that affected 90 patients and 265 health care workers and that were clustered in the coronary care unit and psychiatry units. Attack rates were 5.3% (7 of 133) for patients and 29.9% (29 of 97) for health care workers in the coronary care unit and 16.7% (39 of 233) for patients and 38.0% (76 of 200) for health care workers in the psychiatry units. Thirteen affected health care workers (4.9%) required emergency department visits or hospitalization. Detected noroviruses had 98%-99% sequence identity with representatives of a new genogroup II.4 variant that emerged during 2002-2004 in the United States (e.g., Farmington Hills and other strains) and Europe. Aggressive infection-control measures, including closure of units and thorough disinfection using sodium hypochlorite, were required to terminate the outbreak. Costs associated with this outbreak were estimated to be