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Variation in Severity of Symptoms Associated With Two Snow Mountain Virus Inocula.

Snow Mountain Virus (SMV), the prototype of genogroup II and genotype II Norovirus (NoV), was used in human challenge studies to examine the infectivity, pathogenicity, and immune response to NoV. Clinical and laboratory data from two previously completed SMV human challenge trials using two different inocula (primary and secondary) were analyzed to compare the infectivity, illness, viral shedding, and serum IgG conversion. The primary and secondary SMV inocula were sequenced for detecting single nucleotide mutations. Of 15 subjects challenged with the primary inoculum between 2000 and 2002, nine were infected, and seven presented with acute gastroenteritis. Of 33 subjects challenged with the secondary inoculum between 2016 and 2018, 25 were infected, and nine presented with acute gastroenteritis. There were no statistically significant differences in overall infection and illness rates between subjects challenged with the primary inoculum versus the secondary inoculum. However, subjects infected with the primary inoculum experienced more severe clinical symptoms of acute gastroenteritis, showing higher severity scores (6.00 vs. 2.94, p = 0.003) compared with those infected with the secondary inoculum. We also observed that infection with the secondary inoculum resulted in longer viral shedding compared with the primary inoculum. Partial sequencing of the SMV genome identified three mutations in both inocula. Understanding the differences between these two SMV inocula is critical for NoV vaccine evaluation and using a less pathogenic inoculum for a vaccine trial will require more participants to meet the target reduction in illness when evaluating the efficacy of candidate vaccines.

Humans↗

Snow Mountain virus genome sequence and virus-like particle assembly.

Snow Mountain virus (SMV) belongs to the Norovirus genus of the Caliciviridae family. SMV is a genogroup II (GII) reference strain of human enteric caliciviruses associated with epidemic gastroenteritis. In this study, the positive sense RNA genome sequence of SMV was determined to be 7,537 nucleotides in length excluding the 3' polyadenylated tract. The genome is organized into three open reading frames typical of caliciviruses in the Norovirus genus. Pairwise sequence alignments showed SMV ORF1 is highly conserved with other genogroup II noroviruses, and most closely related to GII strains Melksham and Hawaii virus. In addition, comparative sequence analyses indicated that SMV is likely a recombinant norovirus. VP1/VP2 proteins self-assembled into virus-like particles (VLPs) when expressed in insect cells by a recombinant baculovirus. Characterization of one clone that expressed VP1, but failed to assemble into VLPs, identified histidine residue 91 as important for particle assembly under standard conditions of expression.

Caliciviridae↗

Cellular and humoral immunity following Snow Mountain virus challenge.

Little is known about the immune response to noroviruses. To elucidate the immunobiology of norovirus infection in humans, 15 volunteers were challenged with Snow Mountain virus (SMV), a genogroup 2 norovirus. We assessed the cellular and humoral immune response and infection by analyzing stool, serum, saliva, and peripheral blood mononuclear cell (PBMC) responses pre- and postchallenge. In contrast to Norwalk virus (NV), SMV infection was not dependent upon blood group secretor status. Nine of 15 volunteers were infected and showed a >/=4-fold increase over the prechallenge anti-SMV serum immunoglobulin G (IgG) titer, mostly subclass IgG1. Although serum IgG elicited by SMV infection was cross-reactive with Hawaii virus (HV), another genogroup 2 norovirus, salivary IgA was less cross-reactive. Neither SMV-elicited serum IgG nor salivary IgA cross-reacted with NV, a genogroup 1 norovirus. Significant increases in serum gamma interferon (IFN-gamma) and IL-2, but not IL-6 or IL-10, were noted on day 2 postchallenge. For the majority of volunteers, both infected and uninfected, PBMCs stimulated with norovirus virus-like particles secreted IFN-gamma and other Th1 cytokines, suggesting previous norovirus exposure in most volunteers. Like the IgG antibodies, the SMV-activated T cells were cross-reactive with HV but not NV. IFN-gamma production was dependent upon CD4(+) cells, consistent with a predominant, but not exclusive, Th1 response. To our knowledge, this is the first report characterizing T-cell and cytokine responses following live norovirus challenge.

Adult↗

Viruses that multiply in the gut and cause endemic and epidemic gastroenteritis.

BACKGROUND: Acute infectious diarrhea in young children is a leading cause of morbidity and mortality in developing countries. Even in developed countries, infectious enteritis is second only to respiratory infections as a cause of morbidity in early childhood. OBJECTIVE: To nominate the various viral agents that cause enteritis, discuss the pathogenesis, clinical features, epidemiology and diagnostic procedures employed. STUDY DESIGN: Pertinent literature was reviewed and the findings of investigations carried out on viral enteritis by various colleagues recalled. RESULTS: The viruses causing gastroenteritis include: Rotaviruses; Adenoviruses-especially Ad 31, Ad 40 and Ad 41; members of the Caliciviridae, e.g. Norwalk virus, Hawaii virus, Snow Mountain virus, Taunton virus, Southampton virus, Toronto virus (formerly mini-reovirus) and others; Astrovirus; Coronavirus; Torovirus; Cytomegalovirus (CMV) and possibly Picobirnavirus. Enteritis-producing viruses replicate in columar epithelial cells in the distal parts of villi of the small intestine. Two mechanisms are addressed to explain why diarrhea occurs. Clinically, the main expression of illness is a watery diarrhea that lasts 24 h to about 7 days. Vomiting is of shorter duration and may not always accompany the diarrhea. Fever is generally 38.5 degrees C. Virus is shed in the stool for about 3-7 days. Diagnostic procedures employ electron microscopy (EM), immune electron microscopy (IEM), enzyme-linked immunosorbent assay (ELISA), time-resolved fluoroimmunoassay (TR-FIA), latex agglutination, polyacrylamide gel electrophoresis (PAGE) and the polymerase chain reaction (PCR). CONCLUSION: In developed countries viral enteritis among young children may be up to three times more common than bacterial gut disease. With the exception of CMV enteric involvement, the stool is characteristically not bloody and white blood cells are not found. Patient management may involve the employment of IV replacement therapy to counter dehydration and electrolyte imbalance. Milder cases may be managed with oral rehydration.

Journal Article↗

Diagnosis of astrovirus gastroenteritis by antigen detection with monoclonal antibodies.

An enzyme-linked immunosorbent assay (ELISA), based on monoclonal antibodies to the astrovirus group antigen, was designed for the detection of astroviruses in stools of patients with gastroenteritis. Compared to immune electron microscopy used as the standard test, the sensitivity of the astrovirus ELISA was 91% (31/34) and the specificity was 96% (54/56). All five of the known astrovirus serotypes could be detected in 16 samples on which serotyping was done. In tests on 155 stools containing other enteric viruses, including adenoviruses, rotaviruses, caliciviruses, Hawaii virus, Snow Mountain virus, and Norwalk virus (30, 20, 70, 24, 4, and 7 samples, respectively), only 3 were positive in the astrovirus ELISA. The combined specificity for all astrovirus immune electron microscopy-negative samples was 98% (206/211). The results demonstrate that the new ELISA provides a sensitive and specific means for the diagnosis of astrovirus gastroenteritis.

Antibodies, Monoclonal↗

Epitopes in the P2 domain of norovirus VP1 recognized by monoclonal antibodies that block cell interactions.

Noroviruses cause the majority of epidemic outbreaks of acute viral gastroenteritis worldwide. Human norovirus strains do not grow in cell culture, but recent carbohydrate binding, sequence and structural analyses have begun to define functional domains in the norovirus capsid that may be conserved among multiple antigenic types. The purpose of this study was to localize domains and define sequences in the major capsid protein VP1 that are important for cell interactions. Monoclonal antibodies to genogroups GI.1 and GII.2 reference strains Norwalk virus and Snow Mountain virus, respectively, were generated that blocked binding of recombinant virus-like particles to Caco-2 intestinal cells and inhibited haemagglutination. Peptides that mimicked the mAb binding epitopes were selected from a phage-displayed random nonapeptide library. Anti-recombinant Norwalk virus mAb 54.6 and anti-recombinant Snow Mountain virus mAb 61.21 recognized epitopes located in the protruding P2 domain of VP1. The epitope recognized by mAb 61.21 contained amino acids that are completely conserved among norovirus strains across genogroups, including strains isolated from swine, bovine and murine species. This study identifies the first epitope involved in inhibition of norovirus-cell interactions and supports increasing evidence that interactions between noroviruses and host cells rely on structures in the P2 domain of VP1.

Agglutination↗

Multivalent norovirus vaccines induce strong mucosal and systemic blocking antibodies against multiple strains.

Noroviruses are important agents of human gastroenteritis characterized by extensive sequence variation in the major capsid structural protein that likely encodes critical antigenic determinants of protective immunity. The lack of an infection model has limited detailed characterizations of viral antigenic relationships and identification of the essential components for protective immunity. This information would contribute to efficacious vaccine design against a broad array of norovirus strains. To understand the extent of heterotypic norovirus antibody specificity to inter- and intra-genogroup strains and its applicability to vaccine design, we collected sera from humans infected with different norovirus strains and from mice inoculated with alphavirus vectors expressing strain-specific recombinant norovirus-like particles (VLPs). We used VLPs that were assembled from Norwalk virus (NV), Hawaii virus (HV), Snow Mountain virus (SM) and Lordsdale virus (LV) as antigens to define and compare heterotypic antibody responses in humans and mice. We also examined if these heterotypic antibodies could block specific binding of ABH histo-blood group antigens, putative receptors for norovirus binding and entry, to norovirus VLPs. Furthermore, we examined the effect of multivalent inocula on the specificity, titer, and ligand blockade properties of systemic and mucosal norovirus-specific antibodies in mice. Our studies suggest that infection with one of several different genogroup I (GI) strains in humans induces heterotypic antibodies that block NV binding to ABH antigens, although comparable findings were not evident following infection with genogroup (GII) strains. Additionally, inoculating mice with vaccine cocktails encoding multiple norovirus VLPs enhances heterotypic and ligand attachment-blocking antibody responses against the LV strain not included in the cocktail. These data suggest that multivalent vaccination may provide better protection from a broader range of noroviruses than monovalent vaccination.

Amino Acid Sequence↗

Immunoglobulin M antibody test to detect genogroup II Norwalk-like virus infection.

Sera obtained from adult volunteers inoculated with genogroup II Norwalk-like viruses (NLVs), Hawaii virus, and Snow Mountain virus and from patients involved in outbreaks of gastroenteritis were tested for genogroup II NLV Mexico virus-specific immunoglobulin M (IgM) by use of a monoclonal antibody, recombinant Mexico virus antigen (rMXV)-based IgM capture enzyme-linked immunosorbent assay (ELISA). Sera from genogroup I Norwalk virus (NV)-inoculated volunteers and from patients involved in a genogroup I NLV outbreak were also tested. In sera from those infected with genogroup I NV or NLVs in volunteer and outbreak studies, only 3 of 25 were rMXV IgM positive; in contrast, 24 of 25 were IgM positive for recombinant NV (rNV). In sera from those infected with genogroup II NLVs in volunteer and outbreak studies, 28 of 47 were rMXV IgM positive and none were IgM positive for rNV, showing the specificity of each IgM test for its respective genogroup. In an outbreak of gastroenteritis not characterized as being of viral etiology but suspected to be due to NV, 7 of 13 persons had IgM responses to rMXV, whereas none had IgM responses to rNV, thus establishing the diagnosis as genogroup II NLV infection. The rMXV-based IgM capture ELISA developed is specific for the diagnosis of genogroup II NLV infections.

Adolescent↗

Subclass-specific serum antibody responses to recombinant Norwalk virus capsid antigen (rNV) in adults infected with Norwalk, Snow Mountain, or Hawaii virus.

Subclass-specific antibody responses to the Norwalk virus capsid protein in adults challenged with Norwalk, Snow Mountain, or Hawaii virus were evaluated by solid-phase enzyme immunoassay using recombinant Norwalk virus capsid antigen (rNV). Fourfold or greater serum immunoglobulin G (IgG) antibody responses to rNV were detected in 15 of 20 volunteers challenged with Norwalk virus, and serum IgA and IgM antibody responses to rNV were seen in almost all subjects who had rNV IgG responses. Serum rNV IgG antibody responses also were detected in 6 of 15 volunteers challenged with Snow Mountain virus and 2 of 12 volunteers challenged with the Hawaii virus. However, the magnitude of antibody response and the geometric mean postchallenge rNV IgG antibody titers were lower in subjects challenged with Snow Mountain or Hawaii virus, and serum IgA and IgM responses generally did not occur.

Adult↗

Detection of Norwalk virus and other genogroup 1 human caliciviruses by a monoclonal antibody, recombinant-antigen-based immunoglobulin M capture enzyme immunoassay.

Sera obtained from two groups of adult volunteers infected with Norwalk virus (NV) and two groups of patients involved in two natural outbreaks were tested for NV-reactive immunoglobulin M (IgM) by use of a monoclonal antibody, recombinant-antigen-based IgM capture enzyme immunoassay (EIA). No NV-reactive IgM was detected in the preinoculation sera of 15 volunteers, and 14 of 15 showed NV-reactive antibodies postinfection with NV. All of the volunteers showed IgG seroconversion to NV. In the outbreak studies, all 9 persons in one outbreak and 19 of 24 in another outbreak had NV-reactive IgM. In the first outbreak, only three of nine seroconverted to NV, which was likely due to late collection of acute-phase sera. In the second outbreak, 21 of 24 showed IgG seroconversion to NV. Sequencing of viruses isolated from five stool samples selected from those in the second outbreak showed that they were human calicivirus (HuCV) genogroup 1 viruses related, but not identical, to NV. In the volunteer studies, NV-reactive IgM was first detected 8 days postinoculation. The time of development of NV-reactive IgM antibodies in natural outbreaks was estimated to be similar to that found in the volunteer studies. Sera from three Hawaii virus-infected volunteers, four Snow Mountain virus patients, and 80 healthy individuals were negative for NV-reactive IgM, indicating test specificity for HuCV genogroup I infections. This capture IgM EIA is suitable for diagnosis of NV and other HuCV genogroup I infections and is especially useful when sera and fecal samples have not been collected early in the course of an outbreak.

Antibodies, Monoclonal↗

Limitations of molecular biological techniques for assessing the virological safety of foods.

Enteric viruses, including hepatitis A, Norwalk, and Snow Mountain viruses, Hawaii agent, and rotaviruses have been associated with outbreaks of foodborne illness. Classical culturing procedures are available for poliovirus; however, hepatitis A, Norwalk, and many of the other viruses and agents cannot be propagated in cell culture, therefore, molecular biological tools have emerged as a possible means to detect enteric viruses in foods and environmental samples. There are limitations however in the application of polymerase chain reaction and reverse transcription polymerase chain reaction that restrict their usefulness for measuring the virological safety of foods. The most serious limitation is that molecular techniques fail to discriminate between viable and inactivated viruses even though inactivated viruses pose no threat to the consumer and may be present at levels substantially higher than the virulent forms. Other disadvantages include a lack of assay sensitivity and specificity, high assay costs, and a level of technical expertise not available in most food-testing laboratories. Overall, scientific advances in the development of molecular biological tools have outpaced the demonstration of their validity in assessing the virological safety of foods.

Food Microbiology↗

Development of an ELISA to detect MX virus, a human calicivirus in the snow Mountain agent genogroup.

MX virus is a Snow Mountain agent (SMA) genogroup human calicivirus (HuCV) identified in a Mexican child with diarrhoea. An ELISA using hyperimmune antisera to the recombinant MX virus (rMX) capsid was developed to detect SMA genogroup HuCVs in stool specimens. The rMX ELISA detected the prototype MX virus, SMA, and Hawaii agent (HA), but not Norwalk virus (NV) or Sapporo virus. Twenty-three diarrhoea stool specimens from children attending day care centres in Norfolk, Virginia, were positive by the rMX ELISA and results were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Eight of 20 diarrhoea stool specimens from children in the United Kingdom previously shown to contain small round structured viruses (SRSVs) or HuCVs by electron microscopy were also positive by the rMX ELISA and RT-PCR. Sequence analysis of the RT-PCR products showed that all the rMX ELISA-positive viruses belong to the SMA genogroup. These data also showed that the SMA genogroup can be further divided into two subgroups: subgroup 1 includes prototypes SMA and HA, and subgroup 2 includes MX virus, minireovirus, Oth-25 and Bristol virus.

Animals↗

Detection, quantitation, and phylogenetic analysis of noroviruses in Japanese oysters.

Noroviruses (NVs) cause many cases of oyster- or clam-associated gastroenteritis in various countries. We collected 191 samples from Japanese oysters intended for raw consumption that had been harvested from the sea in two different areas between December 2001 and February 2002. To detect, quantitate, and phylogenetically analyze the NV genome in purified concentrates from the stomachs and digestive diverticula of these oysters, we amplified the NV capsid gene by reverse transcription-PCR. Phylogenetic analysis was performed by using the neighbor-joining method. We detected the NV genome in 17 of 191 oysters (9%). Phylogenetic analysis indicated genogroup I (Norwalk virus type) in 3 of the 17 oysters and genogroup II (Snow Mountain virus type) in the other 14. Both genogroups showed wide genetic diversity. To quantify the NV capsid gene in these oysters, we performed real-time PCR using genogroup-specific probes. More than 10(2) copies of the NV genome were detected in 11 of 17 oysters. The results suggested that about 10% of Japanese oysters intended for raw consumption harbored NVs, and more than 50% of those oysters in which NVs were detected had a large amount.

Animals↗

Molecular detection and sequence analysis of human caliciviruses from acute gastroenteritis outbreaks in Hungary.

Three viral gastroenteritis (VGE) outbreaks that occurred in 1998-1999, in Hungary were investigated for the presence of human caliciviruses (HuCVs). HuCVs in stool specimens were detected by reverse transcription-polymerase chain reaction (RT-PCR) using primer pair 289/290, which was designed based on the RNA-dependent RNA polymerase (RdRp) sequence. RT-PCR results were confirmed by sequencing showing that all three outbreak strains belonged to genogroup II of "Norwalk-like viruses" (NLVs). Two strains had high sequence identity with strains in known genetic clusters (Hawaii and Lordsdale clusters). The third strain (MOH) had distinct RdRp sequence, sharing 77/86% (nt/aa) identity with Snow Mountain virus (SMV), the closest genogroup II virus. To characterize MOH further, we cloned, sequenced, and expressed in baculovirus its capsid gene. It had 75/79% (nt/aa) identity with SMV, but 97/98% (nt/aa) identity with NLV/Hillingdon/90/UK, a recently identified genetic cluster of HuCVs. The recombinant MOH (rMOH) capsid protein self-assembled into virus-like particles (VLPs), which is antigenically distinct from other recombinant HuCV capsid antigens available in our laboratory. Further study of this VLP will have important applications in antigenic characterization and diagnosis of HuCVs.

Acute Disease↗

The role of human caliciviruses in epidemic gastroenteritis.

Members of the Caliciviridae family of small, positive-sense RNA viruses exhibit a broad host range. The Norwalk and Norwalk-like caliciviruses in this family are major etiologic agents of epidemic gastroenteritis in humans. This illness characteristically lasts 24-48 h and often occurs in group settings such as families, schools, institutions, or communities. The spread of the human caliciviruses is considered to be predominantly by person-to-person contact via the fecal-oral-route. However, the ingestion of calicivirus-contaminated food or water can result in large-scale common-source outbreaks. Many basic features concerning the biology and replication of the human caliciviruses are not known because they have not yet been grown in cell culture and the virus does not appear to replicate in animal models other than the chimpanzee. Sequence analysis of RT-PCR-generated DNA fragments derived from serotypically distinct reference strains (such as the Norwalk, Hawaii, and Snow Mountain viruses) and other circulating strains associated with gastroenteritis has provided evidence for marked genetic diversity among these viruses. Moreover, analysis of the antigenic relationships among these viruses using paired sera from individuals infected with well-characterized reference strains or from animals immunized with recombinant "virus-like particles" (VLPs) suggests that several serotypes of these viruses are circulating worldwide. The availability of molecular techniques for the detection of these fastidious viruses has enabled epidemiologic studies that have strengthened the association of human caliciviruses with acute gastroenteritis and has demonstrated a potential role for antigenic diversity in the natural history of these pathogens.

Animals↗

Detection of Norwalk virus in stool specimens by reverse transcriptase-polymerase chain reaction and nonradioactive oligoprobes.

A reverse transcriptase (RT)-polymerase chain reaction (PCR)-oligoprobe (OP), or RT-PCR-OP, method was developed for the detection of the Norwalk virus, which causes acute, epidemic gastroenteritis, in stool specimens. The Norwalk virus genome regions encoding the following two proteins were amplified by RT-PCR: the RNA polymerase (260-bp product) and a putative immunogenic protein (224-bp product). The resulting DNA fragments (amplicons) were hybridized to a digoxigenin-labeled internal OP specific to each amplicon. The detection limit of Norwalk virus, as determined by the endpoint of RT-PCR amplification for serially diluted, positive stool specimens, was similar to the actual virion titer as estimated by electron microscopy and at least 100-fold greater than the titer determined by radioimmunoassay (RIA). The RT-PCR-OP assay was specific for Norwalk virus and negative for other enteric viruses, including human and animal caliciviruses, hepatitis E virus, Snow Mountain agent, astroviruses, 16 human enteroviruses, and 5 human rotaviruses. Components of fecal specimens that interfere with RT-PCR were removed successfully by Sephadex G-200 gel chromatography. Of 20 stool specimens from human volunteers that were positive for Norwalk virus by RIA, a specific RT-PCR-OP result was obtained in 95% (19 of 20) of the samples by using the immunogenic protein primers and 75% (15 of 20) by using the polymerase primers. Twenty-six stool specimens from asymptomatic children and adults were negative by the Norwalk virus RT-PCR-OP. RT-PCR-OP detected Norwalk virus in the 4 of 21 coded fecal specimens that were also positive by enzyme immunoassay. Two samples that were positive by RIA or enzyme immunoassay were negative by RT-PCR, perhaps because viral RNA was not present or RT-PCR inhibitors were not adequately removed.

Adult↗

Norovirus capture with histo-blood group antigens reveals novel virus-ligand interactions.

Noroviruses are genetically diverse, uncultivable, positive-sense RNA viruses and are the most common cause of epidemic acute gastroenteritis in humans in the United States. Recent studies of norovirus attachment in vitro by using recombinant virus-like particles (VLPs) suggest that various norovirus strains exhibit different patterns of attachment to ABH histo-blood group antigens, which are carbohydrate epitopes present in high concentrations on mucosal cell surfaces of the gut. However, attachment of live norovirus strains to histo-blood group antigens has not been investigated to date. Utilizing a newly designed magnetic bead-virus capture method, we characterized histo-blood group antigen attachment properties of various norovirus strains obtained from clinical stool specimens to compare the attachment properties of wild-type virus and VLPs and to further map norovirus attachment. Consistent with previous reports using VLPs, various strains of noroviruses exhibited different patterns of attachment to histo- blood group antigens. Norwalk virus bound specifically to H type 1, H type 3, and Le(b). Two genogroup II noroviruses, one representing the Toronto genotype and the other from a novel genotype, bound specifically to Le(b). A Desert Shield-like strain did not attach to H types 1, 2, or 3, H type 1 and 3 precursors, Le(a), or Le(b). Surprisingly, wild-type Snow Mountain virus (SMV) attached specifically to H type 3, which contradicted previous findings with SMV VLPs. On further investigation, we found that stool components promote this attachment, providing the first known observation that one or more components of human feces could promote and enhance norovirus attachment to histo-blood group antigens.

ABO Blood-Group System↗

Sequence analysis of the gene encoding the capsid protein of the Snow Mountain human calicivirus.

Snow Mountain virus (SMV) is the reference strain for serotype 3 as determined by immune electron microscopy of the human caliciviruses that are associated with epidemic gastroenteritis. In order to establish the genetic relationship of its capsid protein with those from other human caliciviriuses, the sequence of the open reading frame 2 (ORF2) encoding the SMV capsid protein was determined. The SMV ORF2 sequence was 1626 nucleotides in length and the deduced protein of 542 amino acids had a calculated molecular weight of 59.2 kD. The SMV capsid sequence showed approximately 48 and 77% amino acid sequence identity with the capsid proteins of the Norwalk (serotype 1) and Hawaii (serotype 2) human calicivirus reference strains, respectively, a finding consistent with its serotypic distinctiveness. Furthermore, the predicted amino acid sequence of the SMV capsid was found to share highest sequence identity (98%) with the Melksham human calicivirus in database searches.

Caliciviridae↗