Norwalk Virus Antigen Research Articles

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.

Diagnosis of Norwalk Virus Infection by Indirect Enzyme Immunoassay Detection of Salivary Antibodies to Recombinant Norwalk Virus Antigen

Simple diagnostic tests are needed for the detection of norovirus (NoV) outbreaks. Salivary antibody assays provide an attractive alternative to collecting and testing serum or stool samples. Antibodies to Norwalk virus (NV) in oral fluid samples were compared with NV antibodies in serum collected from 38 volunteers challenged with NV inoculum. Pre- and postchallenge (day 4, 8, 14, and 21) saliva and serum samples were examined by enzyme immunoassay (EIA) using recombinant NV antigen. Of 18 infected subjects (those who shed NV in stool or who demonstrated immunoglobulin G [IgG] seroconversion), 15 (83%) had > or =4-fold increases in NV-specific salivary IgA and 15 (83%) had > or =4-fold increases in NV-specific salivary IgG when prechallenge and postchallenge saliva samples were compared. When the results of the IgA and IgG assays were combined, all 18 infected subjects showed > or =4-fold increases in NV-specific salivary IgG or IgA postchallenge titers compared to their prechallenge titers. One of 19 uninfected subjects had a > or =4-fold increase in NV-specific salivary IgG. The sensitivity of the combined assay results was 100%, and the specificity was 95%. NV-specific salivary IgA titers peaked around 14 days postchallenge. NV-specific salivary IgG and serum IgG titers continued to rise through 21 days postchallenge. The application of this EIA to an elementary school outbreak indicated that 67% of the subjects with confirmed infections had >4-fold rises in anti-NoV IgA when an antigen in the same genetic cluster as the outbreak virus was used. This is the first documented mucosal antibody response to NoV in children. This EIA provides a useful approach for diagnosing NoV outbreaks.

Diagnosis of Norwalk Virus Infection by Indirect Enzyme Immunoassay Detection of Salivary Antibodies to Recombinant Norwalk Virus Antigen

Diagnosis of Norwalk Virus Infection by Indirect Enzyme Immunoassay Detection of Salivary Antibodies to Recombinant Norwalk Virus Antigen

Prevalence of human calicivirus infections in Kenya as determined by enzyme immunoassays for three genogroups of the virus.

An epidemiological survey on human calicivirus (HuCV) infections and associated gastroenteritis in infants was conducted to clarify the prevalence of HuCV infections in infants and adults in Kenya. Enzyme immunoassays (EIAs) for three genogroups of HuCVs, Norwalk virus (NV), Mexico virus (MXV), and Sapporo virus (SV), were used to detect antigen or antibody. We tested 1,431 stool samples obtained from children younger than 6 years old with acute gastroenteritis who visited outpatient clinics in three districts in Kenya from August 1991 to July 1994. Thirty-two (2.2%) of these stool samples were positive for SV antigen. Only one (0.1%) of 1,186 samples was positive for NV antigen and none of 246 samples was positive for MXV antigen. One hundred ninety-three serum samples were tested for antibodies to NV and MXV, and 64 of them were examined for antibody to SV. The pattern of the age-related prevalence of serum antibody to NV was different from that of antibodies to MXV and SV. The acquisition of serum antibodies to HuCVs in the three genogroups appeared in early childhood, at about 1 to 2 years of age. The prevalence of serum antibody to NV was low (about 60%) throughout adulthood compared with a high prevalence of antibody (approximately 80 to 90%) to MXV and SV. These data indicate that infections with viruses in the three genogroups of HuCVs are common in Kenya, and immunological responses to NV may be different from those to MXV and SV. The EIAs for the detection of NV and MXV antigens appear to be quite specific for prototype NV and MXV strains, respectively, so that they can detect only a few strains of HuCVs related to them. Alternatively, NV and MXV caused less severe infections that did not bring children to the outpatient clinics for gastroenteritis in Kenya.

Norwalk virus infection in Venezuela

The presence of antibodies against Norwalk virus (NV) was studied in sera from different Venezuelan populations, using an enzyme immuno-assay (EIA) based on recombinant NV protein. Antibodies to NV were found in 47%-53% of urban subjects from Caracas, 83% of rural subjects from the west of the country, and 73%-93% of Amerindian subjects. The prevalences found in the rural and Amerindian groups were significantly higher than that in the urban group. Although about 50% of the children studied were seropositive for NV by the age of 5 years, only four (0.4%) of 1120 faecal samples from children with diarrhoea which were tested for the presence of NV antigen by sandwich EIA were found positive. An increase of at least 4-fold in the titre of anti-NV IgA was found in three (5%) of 61 pairs of sera taken during and 1 month after an acute episode of diarrhoea not due to rotavirus. NV was therefore not a predominant aetiological cause of gastro-enteritis in young children in Venezuela between 1993 and 1995, although it can be the cause of diarrhoea in infants.

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.

Monoclonal antibodies for detection of Norwalk virus antigen in stools.

Monoclonal antibodies against the prototype 8FIIa strain of Norwalk virus were prepared and applied to an enzyme immunoassay (EIA) for detecting Norwalk virus in stool specimens. The monoclonal antibodies immunoprecipitated a 58-kDa protein which had been produced by in vitro transcription-translation of Norwalk virus cloned cDNA, and they reacted by EIA with recombinant Norwalk virus capsid protein at a sensitivity level of 1 ng/ml. The EIA detected virus in all tested samples from 15 different Norwalk virus-infected volunteers. No cross-reactions were seen in stools containing other caliciviruses or in stools containing rotaviruses, astroviruses, or enteric adenoviruses.

Viral Shedding And Fecal Iga Response After Norwalk Virus Infection

Protection is not conferred by preexposure to Norwalk virus (NV). By use of an ELISA with baculovirus-expressed recombinant NV (rNV) capsid protein, the pattern of NV fecal shedding and the protective effect of rNV-specific fecal IgA (flgA) were investigated in volunteers who were repeatedly challenged with NV. After the first challenge, ill volunteers were significantly more likely than well volunteers to have NV antigen in their stool (P < .05). After challenge, antigen shedding was detected on days 1-13; ill volunteers shed the antigen longer (P = .02). A higher prechallenge rNV-specific flgA geometric mean titer was found in ill compared with well volunteers (P < .05) and in infected versus noninfected volunteers (P < .05). NV shedding was common after infection and was present up to 2 weeks after challenge. Preexisting rNV-specific flgA, like serum IgG, is not protective and may be a marker for symptomatic disease.

Detection of immunoglobulin M (IgM), IgA, and IgG Norwalk virus-specific antibodies by indirect enzyme-linked immunosorbent assay with baculovirus-expressed Norwalk virus capsid antigen in adult volunteers challenged with Norwalk virus

Pre- and postexposure sera collected from 17 adult volunteers challenged with Norwalk virus as described previously (D. Y. Graham, X. Jiang, T. Tanaka, A. Opekun, P. Madore, and M. K. Estes, J. Infect. Dis. 170:34-43, 1994) were examined for Norwalk virus-specific immunoglobulin M (IgM), IgA, and IgG by indirect enzyme-linked immunosorbent assays with recombinant Norwalk virus antigen bound to the solid phase. Sixteen of the 17 volunteers had evidence of past infection, all presenting with preexisting IgG antibody of high avidity; only one volunteer had no evidence of previous infection. Virus infection was detected in 14 of the 16 volunteers with evidence of past infection, and 9 of the infected volunteers had symptomatic illness. A significant rise in both virus-specific IgA and IgG titers was detected after challenge in all of the volunteers who became ill. Five of the asymptomatic volunteers who were infected had rising titers of virus-specific IgG, but only two of the five had a concomitant rise in their virus-specific IgA antibody titers. Antibody rises were detectable in eight of nine ill volunteers 8 to 11 days after challenge but in the asymptomatic volunteers only after more than 15 days had elapsed. Virus-specific IgM was detected after challenge in all 14 infected volunteers. Between symptomatic and asymptomatic volunteers there were no significant differences in titers of virus-specific IgG and IgA in serum before challenge; however, there were significantly higher titers in symptomatic volunteers between 8 and > 90 days after challenge for virus-specific IgG and 8 and 24 days after challenge for virus-specific IgA.

Sequence similarity of human caliciviruses and small round structured viruses

The application of reverse transcription-polymerase chain reaction (RT-PCR) using primers directed to the RNA dependent RNA polymerase region within ORF1 of Norwalk virus (NV) showed that 31 percent of morphologically typical human caliciviruses (HuCV) and 57% of small round structured viruses (SRSVs) produced a product of 470 bp similar to the NV control, NV 8FIIa/68/US. Alignment of the amino acid sequences of morphologically typical HuCVs with previously published sequences for SRSVs, NV, and Snow Mountain agent (SMA) showed a high degree of homology (90-92%) with SMA and a lesser extent of homology with NV (60-61%). The amino acid sequence of two strains of HuCV, HuCV/3C/92/UK, and HuCV/5C/92/UK differed by only one or two amino acids respectively in the RNA dependent RNA polymerase region from that of two strains of SRSV obtained from children in the United Kingdom, SRSV/4S/90/UK and Japan, SRSV/OTH-25/89/J which were found to have identical amino acid sequences. The use of an EIA for detection of NV antigen employing antisera raised to recombinant NV protein indicated that HuCVs and SRSVs obtained from children and adults in the United Kingdom were antigenically distinct from the prototype Norwalk virus, NV/8fIIa/68/US.

Prevalence of Antibodies to Norwalk Virus among Amerindlans In Isolated Amazonian Communities

The seroepidemiology of Norwalk virus infections was examined among Amerindians belonging to eight relatively isolated communities in the Amazon region by means of a new enzyme immunoassay using recombinant Norwalk virus antigen. The seroprevalence of antibodies to Norwalk virus ranged from 39% in the Maiogong to 100% in the Kubenkrankrein. The distribution of antibody levels varied greatly among groups; five of the eight communities had an antibody prevalence greater than 90% with many high values (> 100 units), while three had both a low seroprevalence and a preponderance of low values (< 100 units). While few children less than 5 years of age were sampled, no significant differences in antibody prevalence were noted among age groups, and the prevalence of antibody among children 5-10 years of age approached that of the older age groups. The low prevalence of titers of antibodies to Norwalk virus in several tribes living in these isolated Indian communities suggests that Norwalk virus may have been only recently introduced.

Characterization of a variant strain of Norwalk virus from a food-borne outbreak of gastroenteritis on a cruise ship in Hawaii

A gastroenteritis outbreak affecting at least 217 (41%) of 527 passengers on a cruise ship was caused by a variant strain of Norwalk virus (NV) that is related to but distinct from the prototype NV strain. Consumption of fresh-cut fruit served at two buffets was significantly associated with illness (P < or = 0.01), and a significant dose-response relationship was evident between illness and the number of various fresh-cut fruit items eaten. Seven (58%) of 12 paired serum specimens from ill persons demonstrated at least fourfold rises in antibody response to recombinant NV capsid antigen. A 32-nm small round-structured virus was visualized by electron microscopy in 4 (29%) of 14 fecal specimens, but none of the 8 specimens that were examined by an enzyme immunoassay for NV antigen demonstrated antigen. Four (40%) of 10 fecal specimens were positive by reverse transcriptase-PCR by using primer pairs selected from the polymerase region of NV. In a 145-bp region, the PCR product shared only 72% nucleotide sequence identity with the reference NV strain and 77% nucleotide sequence identity with Southampton virus but shared 95% nucleotide sequence identity with UK2 virus, a United Kingdom reference virus strain. In addition, the outbreak virus was serotyped as UK2 virus by solid-phase immune electron microscopy. The genetic and antigenic divergence of the outbreak strain from the reference NV strain highlights the need for more broadly reactive diagnostic assays and for improved understanding of the relatedness of the NV group of agents.

Epidemiological study of Norwalk virus infections in Japan and Southeast Asia by enzyme-linked immunosorbent assays with Norwalk virus capsid protein produced by the baculovirus expression system

In this study, we investigated Norwalk virus (NV) antigen and antibody to recombinant NV (rNV) in human populations in Japan and Southeast Asia by enzyme-linked immunosorbent assays (ELISAs). Baculovirus-expressed recombinant NV (rNV) capsid protein was used for preparing antisera to rNV or used as an antigen for detecting antibody to rNV. The ELISAs were specific for NV and had sensitivities equivalent to or higher than those of the previously developed radioimmunoassays. In 159 stool samples obtained from children, mainly younger than 10 years old, with acute gastroenteritis due to small round structured viruses in Japan, only 1 was positive for NV antigen. The pattern of acquisition of antibody to rNV was quite different from those of antibodies to group A rotavirus and human calicivirus Sapporo (HuCV-Sa) strain. The prevalence of antibody to rNV remained at a low level throughout childhood and then showed a steep rise during school age and early adulthood in Japan. A high prevalence of antibody was observed in samples collected from healthy adults in Japan and Southeast Asia. These results suggested that NV infection is common in adults in Japan and Southeast Asia but may be rare in infants in Japan. The HuCV-Sa strain was negative by the ELISA, and no serological relationship between NV and the HuCV-Sa strain was found. NV may be quite different from the HuCV-Sa strain, although both viruses are classified in the family Caliciviridae.

Efficacy of a recombinant Norwalk virus protein enzyme immunoassay for the diagnosis of infections with Norwalk virus and other human "candidate" caliciviruses.

A recombinant Norwalk virus antigen enzyme immunoassay (rNV EIA) for the detection fo Norwalk virus (NV) antibodies was evaluated using sera from volunteers and patients infected with Norwalk, Hawaii, Snow Mountain Agent, small round structured viruses (SRSV), and human calicivirus (HuCV) strains. The results for NV antibodies correlated well with previous results obtained using a NV radioimmunoassay. The test rNV EIA was found to be highly specific for NV. The results of tests on sera obtained from patients involved in confirmed outbreaks of SRSV in the UK showed that only 1/12 was due to Norwalk virus. Further evidence is presented that SRSV strain UK2 is antigenically related to NV.

ROLE OF NORWALK VIRUS IN TWO FOODBORNE OUTBREAKS OF GASTROENTERITIS: DEFINITIVE VIRUS ASSOCIATION

Two separate food-associated outbreaks of gastroenteritis occurred among Erie County, New York residents in June 1986. In one outbreak, cases of illness were estimated to have occurred in 50% of the approximately 700 persons in 13 groups who ate at an out-of-county restaurant during a seven-day period, and, in the second outbreak, illness occurred in 26 (30%) of 87 persons who attended a graduation party held in a private home. Laboratory investigation included serology (blocking radioimmunoassay) to determine seroconversion to Norwalk virus and an enzyme immunoassay for detection of Norwalk virus antigen in stools, which the investigators have found to be more specific for Norwalk virus than serology. Seroconversion to Norwalk virus occurred in 11 (79%) of 14 restaurant-related cases and seven (100%) of seven graduation party cases. Seroconversion to Norwalk virus antigen was also found in four (40%) of 10 food handlers at the restaurant and in two (100%) of two food handlers at the graduation party. Antigen was detected in the stools of three (20%) of 15 restaurant-related cases and four (67%) of six graduation party cases. No stools for viral analyses were available for testing from food handlers. All seven of the patients with Norwalk virus-positive stools were also positive by seroconversion. Widespread availability of reagents for stool antigen detection would result in confirmation of more outbreaks due to Norwalk virus and in a more timely manner.

Norwalk virus antigen and antibody response in an adult volunteer study.

To better define the optimum timing of specimen collection and identify alternate ways to diagnose Norwalk virus outbreaks, we looked at the timing of the antibody response and virus excretion in a human volunteer study. The Norwalk virus antibody titers and antigen in stool specimens were examined by biotin-avidin immunoassay. Our data suggest that in epidemic situations, convalescent-phase sera could be collected as soon as 13 days after the onset of illness and acute-phase sera could be collected as late as 5 days after onset. Our data also suggest that if sufficient serum samples are collected, convalescent-phase case and control serum samples can be used to identify Norwalk virus outbreaks. Antigen detection was much less sensitive than seroconversion for detecting infection.

Detection of Norwalk virus in stools by enzyme immunoassay.

The development of a solid-phase microtiter enzyme immunoassay (EIA) for detection of Norwalk virus antigen in stool samples is described. The EIA was compared with a previously developed radioimmunoassay (RIA) for detection of Norwalk virus antigen in stools obtained from 30 volunteers who received Norwalk virus. The EIA detected viral antigen in stools from 17 of the volunteers and the RIA detected viral antigen in 15. Seroconversion was a more sensitive indicator of infection in some patients. However, two samples from volunteers who were clinically ill but did not show seroconversion to Norwalk virus were positive for Norwalk virus antigen by both immunoassays. This indicates that antigen detection may be important for use in epidemiological studies. Neither of the immunoassays gave positive reactions for stools known to contain enteric adenovirus, rotavirus, or Hawaii virus, or in stools from patients with acute diarrhea of unknown cause. The stability of the EIA reagents and ease of use should provide a means for more extensive testing for Norwalk virus in outbreaks of gastroenteritis.