Final Report: Studies of the Infectivity of Norwalk and Norwalk-like Viruses

EPA Grant Number: R826139
Title: Studies of the Infectivity of Norwalk and Norwalk-like Viruses
Investigators: Moe, Christine L. , Stewart, Paul , Heizer, William , Frelinger, Jeffrey A.
Institution: University of North Carolina at Chapel Hill
EPA Project Officer: Hahn, Intaek
Project Period: January 16, 1998 through January 15, 2001 (Extended to January 15, 2002)
Project Amount: $587,842
RFA: Drinking Water (1997) RFA Text |  Recipients Lists
Research Category: Water , Drinking Water

Objective:

The overall objective of this research project was to develop our understanding of the risks associated with exposure to waterborne caliciviruses as a function of dose and host susceptibility factors. Specifically, our goal was to determine the infectious dose of two important human caliciviruses (HuCVs), a prototype Genogroup I virus (Norwalk virus (NV)), and a prototype Genogroup II virus (Snow Mountain Agent (SMV)), which are recognized as major waterborne pathogens. Evidence from outbreaks suggests that these viruses have a very low infectious dose. The results of the study are valuable for estimating the risk of NV and SMV infection and gastroenteritis associated with exposure to contaminated water and evaluating the adequacy of current microbiological standards for drinking water. The multifaceted study constitutes the second phase of a two-part strategy to define the dose-infectivity relationship of NV and a parallel study of the infectivity of SMV. The first phase was a pilot NV dose-ranging study that was supported by the U.S. Environmental Protection Agency (EPA) and completed in 1998. The second phase was conducted at the University of North Carolina-Chapel Hill (UNC). The specific objectives of the second phase were to: (1) identify the dose range of NV and SMV (ID10, ID50, and ID90) in human volunteers with various levels of preexisting antibodies; (2) determine the characteristics of volunteers that are susceptible to infection; and (3) evaluate the fit of several mathematical models of dose-infectivity to our data.

Approach

Three human challenge studies were conducted. The first study included 31 volunteers and examined 3 low doses of NV. The second study included 15 volunteers and examined 3 doses of SMA. The third study included two volunteers and one dose of Hawaii virus (another Genogroup II HuCV). The purpose of this third study was to determine if the Hawaii virus inoculum received from Dr. John Treanor was still infectious.

Healthy volunteers-men and women 18-51 years of age-were recruited from UNC and the surrounding community by advertisements on campus and in local newspapers. A telephone screening script was used to accurately and consistently explain the study rationale and requirements to each potential subject. A brief assessment of eligibility was conducted at the time of the telephone contact. Potential subjects were invited for an outpatient visit at UNC's General Clinical Research Center (GCRC) to meet with the Project Coordinator for further screening and determination of eligibility. During the outpatient visit, the study was explained in more detail. Eligible subjects were required to read, understand, and sign a consent form, pass a physical examination, and provide blood and urine samples for screening evaluation. The health of the volunteers was evaluated by a medical history, physical examination, and appropriate laboratory tests (urinalysis, blood cell counts, blood chemistry assays, and HIV antibody test). Subjects with laboratory evaluations within clinically acceptable values, as judged by the study physicians, were contacted to enter the dosing phase of the study. Each subject's normal diet and nutritional status was assessed during an interview with the GCRC nutritionist. Female volunteers of childbearing age were included only with a negative pregnancy test. We excluded volunteers with any of the following conditions: food handlers, persons in contact with child care or geriatric care centers, persons with young children or elderly relatives living with them, subjects with clinically significant conditions as judged by the study physicians, including any chronic condition (lupus, cancer, renal disease, immunocompromised, HIV-positive, etc.), subjects unable to understand the consent form or study procedures, subjects not able or willing to comply with enteric isolation procedures for the necessary duration or unable/unwilling to remain in the GCRC for 5 consecutive days/6 consecutive nights, subjects who had had any other enteric infections or symptoms in the previous month, persons with gastrointestinal difficulties or a baseline stool specimen from which an enteric pathogen was isolated.

The study was reviewed and approved by UNC's School of Medicine Committee for the Protection of Human Subjects.

Eligible volunteers were admitted to the GCRC for dosing and stayed for 5 days to be monitored for symptoms. Prior to dosing, prechallenge stool, serum, and saliva specimens were collected. Each volunteer then ingested 100 ml of 2 percent sodium bicarbonate solution 2 minutes before and 5 minutes after administration of the inoculum to neutralize stomach acidity. The inoculum was thawed immediately before dosing and diluted in 80 ml of deionized, sterile water.

After inoculation, study subjects were monitored for gastrointestinal symptoms and vital signs in the GCRC every 2 hours while awake during the first 48 hours postchallenge, and then every 8 hours on days 3-5 postchallenge. Study subjects were discharged from the GCRC at 5 days postchallenge and returned for followup visits on days 8, 14, and 21 postchallenge. At the time of discharge, subjects were instructed on the importance of hand washing and careful personal hygiene and given antimicrobial soap and stool collection containers. All stool and vomitus passed by the subjects during the first 7 days postchallenge were collected and stored at 5°C. Serum and saliva specimens were collected daily during the first 5 days postchallenge. Additional stool, serum, and saliva specimens were collected at each of the followup visits. Color, consistency, and weight of stool specimens were recorded at the GCRC.

Determination of Infection and Symptoms

Infection was defined as viral shedding (detection of NV RNA or SMV RNA in stool by reverse transcriptase-polymerase chain reaction (RT-PCR)) or seroconversion (fourfold or greater rise in NV or SMV-specific IgG). All fecal specimens were examined by RT-PCR using NV-specific primers located in the RNA-dependent RNA polymerase gene (SR33/SR48) (Ando, et al., 1995). Fecal specimens in the SMV study were tested with the SR33/SR46 primers. The sequences of these primers are shown in Table 1. Fecal specimens were stored at 4°C until testing. Fecal suspensions (1 percent) were prepared using a modified heat-release RT-PCR method (Schwab, et al., 1997). Positive stool controls, water, and reagent controls were included in each RT-PCR assay. PCR products were analyzed by 3 percent agarose gel electrophoresis, stained with ethidium bromide, and visualized with UV light. Stool samples were considered positive if a band of the correct size comigrated with that of the positive control. To avoid false negative results, all negative fecal specimens from the first 5 days postchallenge were reexamined using a PEG-CTAB (Jiang, et al., 1992) or Ultraspec (BioTecx) RNA extraction method on freon-extracted 20 percent stool suspensions. These methods are more time-consuming, but also are more effective at removing RT-PCR inhibitors than the heat release method (Tseng, et al., 1996). The extracted RNA was then tested by RT-PCR as described above. Vomitus specimens that were collected by study subjects also were tested for NV RNA or SMV RNA using the same methods as for stool specimens.

Symptoms considered in determining the occurrence of NV or SMV illness included diarrhea, vomiting, abdominal pain, myalgia, fatigue, and chills, but excluded fever and headache. The duration of illness was defined as the elapsed time from the onset of the first symptom to end of the last symptom. Diarrhea was defined as more than two unformed stools in 24 hours. The onset of a diarrhea episode was defined as the time of the first of the two or more unformed stools. Hours free of diarrhea were defined as those hours not in any 24-hour diarrhea-positive window of time. Combined duration of all diarrhea was defined as the elapsed time from the onset of the first episode to the termination of the last episode.

Sequence Analyses of Positive RT-PCR Products. Selected RT-PCR products of the correct size were purified for sequencing using the QIAquick™ column purification kit (QIAGEN, Chatsworth, CA). Sequences were generated using the ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit with Amplitaq DNA Polymerase FS (Perkin-Elmer, Applied Biosystems Division, Foster City, CA). Reactions were performed on an ABI 370 Automated Sequencer according to the manufacturer's protocol. Data collection and analyses were performed with the ABI 373 software. The sequencing primers were the same as those used for the RT-PCR reactions. Sequences were generated in both directions to determine a consensus sequence for each product. The sequences from each positive PCR product were compared to the NV sequence in GenBank (Accession #M87661) using GCG software [Wisconsin Package Version 9.1; Swofford, 1991].

Serological Evaluation. All sera from the NV-challenged volunteers was tested for anti-NV IgG by enzyme immunoassay (EIA) using recombinant NV capsid as antigen (from Dr. Mary Estes) (Monroe, et al., 1993). Seroconversion was defined as a greater than fourfold increase in prechallenge NV-specific IgG titer. All sera from each subject were tested on a single plate to minimize plate-to-plate variation. Subjects were considered to have preexisting anti-NV IgG if their baseline sera had net absorbance (P-N) greater than two times plate background.

Sera from the SMV-challenged volunteers were tested for anti-SMV IgG by EIA using recombinant SMV capsid expressed in the Venezuelan Equine Encephalitis (VEE) system by our collaborator Dr. Ralph Baric (Baric, et al., 2002). Sera from the HV-challenged volunteers were tested for anti-HV IgG by EIA using recombinant HV capsid expressed in the VEE system by Dr. Baric's laboratory.

Evaluation of Mucosal Immune Response. The salivary antibody response was evaluated by EIA developed in our laboratory. Polyvinyl microtiter plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated with either 75 µl of rNV antigen (4 µg/ml in 0.025 M tris-buffered saline (TBS) [pH 7.4]) (antigen-positive wells) or 0.025 M TBS alone (antigen-negative wells) and incubated at room temperature for 4 hours. Wells were washed twice with TBS with 0.05 percent Tween 20 (TBS-T) and blocked with 185 µl of 5 percent Blotto (Carnation nonfat milk) in TBS overnight at 4°C. After the wells were washed six times with TBS-T, duplicate dilutions of test saliva (1:16; 75 µl/well diluted in 1 percent Blotto-TBS) were added to each well and the plates were incubated for 2 hours at 37°C. After six washes with TBS-T, bound antibody was detected by the addition of alkaline phosphatase (ALP)-conjugated goat antihuman IgG or IgA (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) (1:1000 dilution in 1 percent Blotto-TBS, 75 µl/well) and incubated for 2 hours at 37°C. After six washes with TBS-T, 100 µl of p-nitrophenylphosphate substrate (Sigma®; St. Louis, MO) in diethanolamine buffer (pH 9.8) was added to each well. The plates were incubated in the dark at room temperature for 90 minutes before the optical density was determined as the ratio of the absorbance values at 405 and 630 nm (MR 5000 Microplate Reader; Dynatech Laboratories, Inc., Chantilly, VA).

Commercially available pooled human IgG3, kappa and human IgA (Sigma®) were used to generate standard curves to provide relative quantitative estimates of anti-NV immunoglobulins in the saliva samples. These products are purified from pooled sources (myeloma plasma and human colostrum, respectively). Adults are almost universally antibody-positive for NV, so these commercial products include anti-NV antibodies.

Secretor Phenotyping. To measure H antigen expression, Nunc-Immuno™ Plates were coated with saliva, blocked in 3 percent BSA/PBS for 1 hour at 37°C, and incubated with primary anticarbohydrate antibodies, diluted in PBS containing 5 percent defatted milk for 2 hours at 37°C. Alkaline phosphatase-conjugated antimouse Ig diluted in 5 percent milk/PBS, was added and reactions developed using p-nitrophenyl phosphate as substrate. The following primary mAbs were used: anti-A, 3-3A; anti-B, ED3; anti-H type 1, BG-4 (Signet Laboratories); anti-H types 1/3, LM-137; anti-Leb, 2-25LE; anti-Lea, 7-LE; anti-Ley, 12-4LE; anti-H type 2, 19-0LE.

Cellular Immunity in NV Challenged Volunteers. To characterize the cellular immune response in NV-challenged volunteers, peripheral blood mononuclear cells (PBMCs) from whole blood samples were collected using a Ficoll separation technique as described by the manufacturer (Pharmacia-Amersham) and cryo-preserved. Peptides, 15 AA/overlap 9, spanning the entire NV ORF 2 sequence were prepared and divided into 2 pools consisting of 34 peptides each, including a 16 peptide overlap. PBMCs were thawed, washed, and diluted to 4x106 cells/ml in R-10 medium and 200 µl of cell suspension added per assay well. PBMCs from each NV-challenged volunteer were then incubated for 18 hours with the nonspecific T-cell activators PMA (10ng/ml) and ionomycin (0.5 µg/ml), peptide pool 1 (amino acid 1-345, 5 µg/ml each), peptide pool 2 (amino acid 180-530, 5 µg/ml each), or medium as control. Culture supernatants were harvested and assayed for the Th1 cytokines delta-IFN, IL-2, and TNF-alpha and the Th2 cytokines IL-4, Il-5, and IL-10 using the BD PharMingen Cytometric Bead Array (CBA) Kit (BD #550749) as recommended by the manufacturer. The CBA kit combines capture beads for the six cytokines with flow cytometry, forming a multiplexed assay analogous to six separate cytokine EIAs, and measures cytokine concentrations in the range of pg/ml. Each kit also supplies cytokine reagent for construction of standard curves for comparison to unknowns. Advantages of this assay system include small sample volume and limited time because all six cytokines are measured in the same well. Each bead event is considered a replicate, and 1,800 beads are counted per sample.

Summary/Accomplishments (Outputs/Outcomes):

NV Study

Inoculum Titer. The inoculum was titered by RT-PCR six times in 1997 using various dilution series, and was examined by electron microscopy (see Table 2). Small aliquots of the inoculum were used for titration and preparation of doses to prevent repeated freezing and thawing of the inoculum, and loss of virus titer. Viral particle count by electron microscopy indicated a titer of approximately 8x106 particles/ml. However, the presence of protein debris and large aggregates of viral particles (approximately 60-80 particles) made it difficult to obtain an accurate estimate of the titer. The RT-PCR endpoint (last dilution that yielded a visible amplicon of the correct size) ranged from 7.8x10-6 to 1x10-5. We assumed that the RT-PCR endpoint contained the minimum amount of viral genome that could be amplified and detected by RT-PCR (i.e., at least one PCR detectable unit (PDU)). Based on the geometric mean titer from the six titration assays, we estimated the titer of the inoculum to be approximately 1x108 PDU per ml. A total of eight different doses, ranging from 1x10-6 µl to 100 µl, were examined over the six trials.

NV Subjects. The 76 volunteers in the NV study ranged in age from 19 to 51 years, with a mean age of 27.6 years. Of the 55 percent of the subjects that were male, 71 percent were Caucasian, 25 percent were African American, and 4 percent were Asian. About one third of the subjects were students (37 percent), and the others either worked on campus or lived in the community. One subject who was diagnosed with an asymptomatic shigellosis infection 24 hours after receiving the inoculum did not become infected with NV and was excluded from the analysis.

Incidence of Infection and Illness. A total of 25 subjects (33 percent) became infected with NV. Infected subjects were generally older than uninfected subjects (mean age of 32 versus 25 years) and were twice as likely to have NV-specific IgG in their baseline serum specimen (84 percent versus 42 percent). Infection was generally associated with mild illness: 18 subjects (72 percent) had typical gastroenteritis symptoms, and 7 had asymptomatic infections. The most common symptoms were nausea and vomiting (see Table 3) and affected 60 percent and 52 percent, respectively, of the infected subjects. Three uninfected subjects experienced nausea and/or vomiting. Despite repeated testing, no NV RNA could be detected in their stool or vomitus, and they did not seroconvert.

Onset and Duration. Symptoms typically started on Day 2 postchallenge and resolved within 24-48 hours. The incubation period for infected subjects ranged from 1 to 38 hours with a mean of 18 hours. The mean duration of symptoms was about 39 hours, and the range was 2 to 97 hours.

Virus Shedding and Seroconversion. Most infected subjects (70 percent) shed NV in their stool by Day 2 postchallenge. At the first followup visit, Day 8 postchallenge, 85 percent of the infected subjects were shedding NV in stool specimens collected Days 5-8 postchallenge. By the second followup visit at Day 14 postchallenge, 30 percent of the infected subjects had NV-positive stool samples, and NV RNA could still be detected in stool specimens collected at the final followup visit, 18-23 days post-challenge, from three subjects. The RT-PCR products from these last specimens were sequenced and confirmed to be NV. There was correlation between viral shedding and seroconversion. Two subjects who received low doses (10-6 to 10-5 µl) seroconverted, but had no detectable NV RNA in their stool specimens. Another subject had what appeared to be NV RNA in her stool on Days 3 and 4 postchallenge, but did not seroconvert. Subsequent attempts to sequence these amplicons were only partially successful in confirming NV RNA. NV RNA was detected in the vomitus of several subjects.

Salivary Immune Response. Fifteen of 19 (79 percent) infected subjects showed a qreater than or equal to4-fold increase in NV-specific salivary IgG and IgA antibody titers, when comparing prechallenge saliva to saliva collected postchallenge. The four other infected subjects had two- to four-fold rises in NV-specific salivary IgG and IgA antibody titers. None of the infected subjects had a qreater than or equal tofour-fold rise in NV-specific serum IgG by Day 4 postchallenge, but six infected volunteers had a qreater than or equal tofour-fold rise in NV-specific salivary IgA by Day 4. Of the 19 uninfected subjects, a qreater than or equal to4-fold increase in salivary NV-specific IgG was seen in only 2 subjects, and only 1 subject demonstrated a qreater than or equal to4-fold increase in salivary IgA. There were strong correlations between IgG seroconversion and qreater than or equal tofour-fold increases in NV-specific salivary IgA and IgG antibody titers.

When we examined the salivary IgA response in the first 5 days postchallenge, we observed that some volunteers who did not become infected had an early peak in anti-NV salivary IgA. We are conducting additional analyses of this salivary IgA response to determine if this is a protective immune response. Volunteers who became infected usually had a peak anti-NV salivary IgA response around 14 days postchallenge-similar to the anti-NV salivary IgG response.

Susceptibility Alleles and NV Infection. Our colleagues, Jacques La Pendu and Jason Jiang, have observed that NV specifically binds to gastrointestinal tissues of secretors, but not of non-secretors, indicating that the human histo-blood carbohydrate antigens (H antigens) are responsible for the binding and that it is controlled by polymorphism at the FUT2 locus (Marionneau, et al., 2002). If H antigens are important receptors for NV docking and entry, then individuals homozygous for FUT2 polymorphisms that prevent H antigen expression may be resistant to infection. Our preliminary analyses of the phenotype data on our subjects indicate that 41 of the 76 subjects were secretor positive (Se+) and presumably susceptible to infection. At least 17 subjects appear to be secretor negative (Se-) and may have been resistant to infection. The analyses on the remaining 17 subjects are in progress.

Cellular Immunity. T-cell responses were not evident in FUT2-/Se- volunteers' PBMCs challenged with NV at Day 8 postchallenge. As cytokine responses were evident following PMA-ionomycin treatment, these results were consistent with the hypothesis that these individuals were resistant to infection. In contrast, the productively infected volunteer PBMCs displayed evidence of a T-cell response to peptide pool 2 (AA 180-530), but not peptide pool 1 (AA 1-345), localizing one or more reactive epitopes to within AA 345-530. This volunteer secreted delta-IFN in response to AA 345-530, suggesting a Th1 or CTL response to the NV peptides. To determine whether heterotypic T-cell responses are elicited following NLV challenge, PBMCs from a SMV-challenged volunteer were incubated with NV peptide pool 1, pool 2, or SMV-VLPs (75 µg/ml). As compared to the medium control, several cytokines were induced by peptide pool 1 and SMV-VLPs. Importantly, NV peptide pool 1 is at the N-terminus of the ORF2 capsid protein, which is the most highly conserved domain among NLVs. These data suggest that: (1) NLV VLPs can be used to stimulate cytokine responses in PBMCs; (2) cross reactive T-cell epitopes exist among the GI and GII genogroups; and (3) T-cell responses may represent a component of the protective immunity following NLV infection in humans. To our knowledge, this is the first demonstration of a T-cell response following NV infection. Further analyses of the T-cell response are in progress.

Model-Based Estimation of NV Dose-Infectivity. Initial analysis of the dose-infectivity relationship, using a simple beta-Poisson model, revealed that the model could not adequately account for variation in infectivity among the subjects. We are currently working on other models that account for the presence of anti-NV serum IgG at baseline and secretor phenotype status of the volunteer. We also are developing models to reduce error in the measurement of the dose and variability in the dose due to viral aggregates. Finally, we will model the relationship between NV dose and illness.

SMV Study

Inoculum Titer. The inoculum was titered by RT-PCR endpoint titration as described in the Methods section using broadly reactive primers for Genogroup II Noroviruses. The endpoint was between 10-3 and 10-4. We estimate that the titer of the SMV inoculum is about 1x106 PDU per ml. Three doses from 0.01 µl to 100 µl were tested with five subjects per dose.

SMV Subjects. The 15 volunteers in the SMV study ranged in age from 21 to 54 years, with a mean age of 30.7 years. Of the 47 percent of the subjects that were male, 73 percent were Caucasian, and 27 percent were African-American.

Incidence of Infection and Illness. A total of nine subjects (60 percent) became infected with SMV. Infected subjects were generally older than uninfected subjects (mean age of 33.8 versus 26 years). Six of the nine infected subjects (67 percent) had SMV-specific IgG in their baseline serum compared to 83 percent of the uninfected subjects. Seven of the infected subjects (78 percent) had typical gastroenteritis symptoms, and two had asymptomatic infections. None of the uninfected subjects reported any symptoms. Further analyses of the symptoms are ongoing.

Onset and Duration. This analysis is in progress.

Virus Shedding and Seroconversion. All nine subjects who had SMV RNA detected in their stool by RT-PCR also seroconverted. Virus shedding was detected as early as Day 2 postchallenge, and as late as Day 8 postchallenge. Further analyses of the shedding patterns are in progress.

Salivary Immune Response. This analysis is in progress.

Dose-Response Relationship. These analyses are in progress. Our preliminary models indicate that SMV is not as infectious as NV. As with the NV subjects, we have tested these subjects for their secretor status. Our preliminary results indicate that all the subjects who became infected were secretor-positive. This suggests that secretor status may be a marker for susceptibility to SMV infection as well as for NV infection.

HV Study

An RT-PCR product could not be detected in the HV inoculum; it was not possible to determine the titer by RT-PCR. By electron microscopy, there were few visible virus particles so the electron microscopist could not estimate a titer based on particle count. Two volunteers received 5 ml of the HV inoculum in March 2001. HV RNA could be detected in fecal specimens from both volunteers by RT-PCR using broadly reactive primers for Genogroup II Noroviruses. Neither of these volunteers had anti-HV antibodies in their baseline serum, and both of them seroconverted. One volunteer had gastrointestinal symptoms. These results indicate that the inoculum is infectious and could be used in future challenge studies. However, to determine the dose-response relationship for HV and compare it to NV and SMV, it would be necessary to find better primers, or a better RNA extraction method to titer the HV inoculum.

Conclusions:

A. Specific Conclusions

1. Diagnosis of Norwalk Virus Infections:

  • RT-PCR detection of viral shedding.
      - Most infected volunteers shed virus for 8 days.
      - Few infected volunteers shed virus for 21 days postchallenge (longer than previous reports).
  • Consistency between IgG seroconversion and virus shedding.

2. Health Outcomes:

  • Mild illness
      - 50 percent of infected volunteers reported that they could not have worked during symptomatic illness.
  • Nausea and vomiting were the predominant symptoms and were more common than diarrhea in this population of infected volunteers.
  • Most of the illness had a short duration (24-48 hours).
  • Most infections were symptomatic.

3. Immune Response:

  • Anti-NV Serum IgG
      - 55 percent of subjects had anti-NV serum IgG prechallenge.
      - Anti-NV serum IgG was not protective. Infected subjects were more likely to have anti-NV IgG than uninfected subjects.
  • Anti-NV Salivary IgG.
      - Salivary IgG response mirrors serum IgG response.
      - Saliva antibody assays have potential as alternative noninvasive diagnostic tools.

4. Infectivity

  • NV is highly infectious. We observed infection at <1 PDU.
  • Virus aggregates in inoculum limited our ability to measure low doses. This may explain some of the variability in dose-response data.
  • Low infectious dose, mild or asymptomatic infection, and prolonged shedding will facilitate secondary transmission.

B. Overall Conclusions

This work is still in progress, and our understanding of the factors that affect human susceptibility and response to Norwalk virus infection is rapidly evolving. Clearly NV is a highly infectious virus, and our preliminary dose-response models suggest that it is one of the most infectious agents that has ever been described.

Genetic traits that may increase or decrease host susceptibility to enteric infection are recently beginning to be recognized. For some infectious diseases, such as malaria, a genetic basis for susceptibility has been well documented. However, little is known about genetic susceptibility to waterborne pathogens. Recent evidence suggests that persons with the immunogenetic marker HLA-B27 are at greater risk of developing reactive arthritis induced by enteric bacterial infections from Yersinia, Shigella, and Salmonella, but the mechanism for this increased susceptibility is not understood (Simonet, 1999). There is evidence that persons with type O blood group are at increased risk of severe infection with V. cholerae O1 (Swerdlow, et al., 1994; Glass, et al., 1985) and possibly increased risk of infection (Tacket, et al., 1995). Our human challenge studies with Norwalk virus and others (Graham, Johnson) have observed that a small proportion of volunteers do not become infected even at high doses. Hutson, et al. (2002) recently reported an association between ABO histo-blood group type and risk of NV infection and symptomatic disease postchallenge. Individuals of blood type O were more likely to be infected with NV, and persons with B histo-blood group antigen had a decreased risk of infection.

Marionneau, et al. (2002) demonstrated that NV recombinant virus-like particles (VLPs) bind to tissue sections from the gastroduodenal junction and to some human saliva. These findings suggest that attachment of NV may depend on the presence of H type 1 and/or H types 3/4 carbohydrates on gastroduodenal epithelial cells. Our lab has confirmed these findings by testing secretor status (presence of H type 1 antigen) in specimens from volunteers in two NV challenge studies (Lindesmith, et al., submitted). Volunteers who were secretor-negative did not become infected with NV at any of the doses tested and their saliva did not bind NV VLPs. We did not observe higher infection rates among individuals with blood type O. However, saliva from volunteers with B modified H type 1 antigen appeared to have less affinity for NV VLPs and these subjects seemed less likely to become infected. About 55 percent of secretor-positive volunteers became infected with NV postchallenge. Risk of infection in this susceptible population depended in part on the dose of the NV challenge and on the ability of the volunteer to mount an early mucosal IgA response. Overall, these observations indicate that host susceptibility to NV infection is affected by genetic determinants (the FUT2 gene that codes for the FUT2 enzyme that catalyzes modifications of H antigen precursors) and acquired immunity (a protective mucosal IgA memory response).

Differences in binding affinity to various histo-blood group antigens also may explain epidemiological trends in outbreaks from different human calicivirus (HuCV) strains. Our laboratory and other investigators (Jiang, et al., 2002, ICV abstract) have observed that HuCV VLPs from different strains have different binding affinities. VLPs from Lordsdale virus, a strain that has predominated HuCV outbreaks in the past 5-10 years, binds to several histo-blood group antigens, suggesting that a large portion of the population are susceptible to this strain of HuCV. In contrast, Snow Mountain Virus and Mexico virus VLPs bind to fewer histo-blood group antigens, suggesting that fewer individuals may have receptors for these viruses and be susceptible to these infections.

These recent observations have several implications for microbial risk assessment. They suggest that host susceptibility needs to be considered in risk assessment and that it is possible to collect data on individuals and populations that provide insight into host susceptibility. For example, 80 percent of the European and North American population are secretor-positive and probably susceptible to NV infection. Other ethnic groups have different proportions of secretor positive individuals. In our study, about 45 percent of the secretor-positives appeared to have acquired mucosal immunity to NV and not develop NV infection after exposure. This could be related to age and frequency of exposure to NV-contaminated vehicles. With increased understanding of susceptibility to HuCV infection, these factors may be included in risk assessment models to more accurately estimate risks of HuCV infection and/or illness in different populations.

 

Table 1. Sequences of NLV Primer Sets Used in the Study.

Name of primers
Sequence ( 5' to 3')
Polarity
Location on NV
NLV genogroups amplified
SR33
TGT CAC GAT CTC ATC ATC ACC
-
4868-4888
GI and GII
SR48
GTG AAC AGC ATA AAT CAC TGG
+
4766-4686
GI
SR46
TGG AAT TCC ATC GCC CAC TGG
+
4766-4686
GII


Table 2. Titration of 8FIIa Inoculum by RT-PCR and Electron Microscopy.

  Date Dilution Series RT-PCREndpoint PDU/ml*
Investigator 1 7/97 10-fold 1x10-5 1x108
  7/97 10-fold 1x10-5 1x108
  7/97 10-fold 1x10-5 1x108
Investigator 2 7/97 2-fold 1.5x10-5 6.4x107
  7/97 2-fold 1.5x10-5 6.4x107
  7/97 2-fold 7.8x10-6 1.3x108
EM particle count 7/97     8.1x106
*PCR detectable units per ml
Geometric mean = 9x107 PDU/ml


Table 3. Prevalence of Gastroenteritis Symptoms Among Infected and Uninfected NV Study Volunteers.

 
Infected (N=25) No. (%)
Uninfected (N=50) No. (%)
RR
P-value
Nausea1
15 (60)
2 (4)
15
<0.001
Vomiting2
13 (52)
3 (6)
8.7
<0.001
Abdominal pain3
12 (48)
1 (2)
24
<0.001
Diarrhea4
7 (28)
0 (0)
-
 
1Nausea: One or more reports of moderate-severe nausea OR 4 consecutive hours of mild nausea
2Vomiting: One or more reports of vomiting
3Abdominal pain: One or more reports of moderate-severe abdominal pain OR 4 consecutive hours of mild abdominal pain
4Diarrhea: More than two unformed stools in 24 hours, rolling 24 hour window


Table 4. Norwalk Virus and Snow Mountain Virus From This Study Given to Other Investigators.

Investigator Institution Material given Research Purpose
John Herrmann University of Massachusetts NV stool and sera NV cultivation, development of monoclonal antibodies to NV, development of NV IgM assay and NV monoclonal antibody
Ralph Baric UNC-Chapel Hill, Chapel Hill, NC NV, SMV stool and sera Cloning of NV and SMV capsid genes into VEE vector
Mark Sobsey UNC-Chapel Hill, Chapel Hill, NC NV, SMV stool Removal and inactivation of NV by water treatment processes, NV cultivation
Marian Koopmans RIVM, Bilthoven, Netherlands New NV inoculum Development of primate model of NV infection
Michele Hardy Montana State University, Bozeman, MT SMV stool Molecular studies of ORF1 polyprotein processing
Jack Odle NC State University, Raleigh, NC New NV inoculum Development of piglet model of NV infection
Lee-Ann Jaykus NC State University, Raleigh, NC NV stool Development of NV detection methods in foods
Arnie Sair NC State University, Raleigh, NC Saliva from NV volunteers Investigating salivary immune response
Jason Jiang University of Cincinnati, Cincinnati, OH Saliva from NV volunteers Investigating host susceptibility factors
Jacques LePendu INSERM, Institut de Biologie, Nantes, France Saliva from NV volunteers Investigating host susceptibility factors
Shermalyn Greene BioMerieux, RTP, NC NV stool and new inoculum Development of NASBA detection method for NV
Pending Collaborations
Ricardo DeLeon Metropolitan Water District of Southern California NV stool NV cultivation
Tamie Ando Viral Gastroenteritis Section, CDC NV stool NV cultivation
Kellogg Schwab Johns Hopkins University, Baltimore, MD NV inoculum, NV stool Studies of NV survival in water
Debra Huffman University of South Florida, Tampa, FL NV stool Development of gene chip technology to detect Norwalk-like viruses
Linda Saif Ohio State University, Columbus, OH SMV Development of piglet model of SMV infection


References:

Baric RS, Yount B, Lindesmith L, Harrington PR, Greene SR, Tseng F-C, Davis N, Johnston RE, Klapper DG, Moe CL. Expression and self-assembly of Norwalk virus capsid protein from Venezuelan Equine Encephalitis virus replicons. Journal of Virology 2002;76(6):3023-3030.

Glass RI, Holmgren J, Haley CE, et al. Predisposition for cholera of individuals with O blood group: possible evolutionary significance. American Journal of Epidemiology 1985;121:791-796.

Hutson AM, et al. Norwalk virus infection and disease is associated with ABO histo-blood group type. Journal of Infectious Diseases 2002:1335-1337.

Jiang X, Wang M, Graham DY, Estes MK. Detection of Norwalk virus in stool by polymerase chain reaction. Journal of Clinical Microbiology 1992;30:2529-2534.

Marionneau S, et al. Norwalk virus binds to histo-blood group antigens present on gastroduodenal epithelial cells of secretor individuals. Gastroenterology 2002(122):1967-1977.

Monroe SS, Stine SE, Jiang X, Estes MK, Glass RI. Detection of antibody to recombinant Norwalk virus antigen in specimens from outbreaks of gastroenteritis. Journal of Clinical Microbiology 1993;31:2866-2872.

Schwab KJ, Estes MK, Heill FH, Atmar RL. Use of heat release and an internal RNA standard control in reverse transcription-PCR detection of Norwalk virus from stool samples. Journal of Clinical Microbiology 1997;35:511-514.

Simonet ML. Enterobacteria in reactive arthritis: Yersinia, Shigella, and Salmonella. Review of Rheumatic English 1999;66:14S-18S, discussion 19S.

Swerdlow DL, Mintz ED, Rodriguez M, et al. Severe life-threatening cholera associated with blood group O in Peru: implications for the Latin American epidemic. Journal of Infectious Diseases 1994;170:468-472.

Tacket CO, Losonsky G, Nataro JP, et al. Extension of the volunteer challenge model to study South American cholera in a population of volunteers predominantly with blood group antigen O. Transactions of the Royal Society of Tropical Medicine and Hygiene 1995;89:75-77.

Tseng F, Moe C, Baric R. Comparison of three RNA extraction/RT-PCR methods to detect Norwalk and Norwalk-related viruses in fecal specimens. Presented at the 96th Annual Meeting of the American Society for Microbiology, New Orleans, LA, May 1996.


Journal Articles on this Report : 13 Displayed | Download in RIS Format

Other project views: All 38 publications 13 publications in selected types All 13 journal articles
Type Citation Project Document Sources
Journal Article Baric RS, Yount B, Lindesmith L, Harrington PR, Greene SR, Tseng F-C, Davis N, Johnston RE, Klapper DG, Moe CL. Expression and self-assembly of Norwalk virus capsid protein from Venezuelan equine encephalitis virus replicons. Journal of Virology 2002;76(6):3023-3030. R826139 (Final)
  • Abstract from PubMed
  • Full-text: JVI Full Text
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  • Other: JVI PDF
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  • Journal Article Brinker JP, Blacklow NR, Estes MK, Moe CL, Schwab KJ, Herrmann JE. Detection of Norwalk virus and other genogroup 1 human caliciviruses by a monoclonal antibody, recombinant-antigen-based immunoglobulin M capture enzyme immunoassay. Journal of Clinical Microbiology 1998;36(4):1064-1069. R826139 (Final)
  • Abstract from PubMed
  • Full-text: JCM Full Text
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  • Other: JCM PDF
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  • Journal Article Brinker JP, Blacklow NR, Jiang X, Estes MK, Moe CL, Herrmann JE. Immunoglobulin M antibody test to detect genogroup II Norwalk-like virus infection. Journal of Clinical Microbiology 1999;37(9):2983-2986. R826139 (Final)
  • Abstract from PubMed
  • Full-text: JCM Full Text
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  • Other: JCM PDF
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  • Journal Article Greene SR, Moe CL, Jaykus L-A, Cronin M, Grosso L, van Aarle P. Evaluation of the NucliSens® Basic Kit assay for detection of Norwalk virus RNA in stool specimens. Journal of Virological Methods 2003;108(1):123-131. R826139 (Final)
  • Abstract from PubMed
  • Abstract: Science Direct
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  • Journal Article Harrington PR, Lindesmith L, Yount B, Moe CL, Baric RS. Binding of Norwalk virus-like particles to ABH histo-blood group antigens is blocked by antisera from infected human volunteers or experimentally vaccinated mice. Journal of Virology 2002;76(23):12335-12343. R826139 (Final)
  • Abstract from PubMed
  • Full-text: JVI Full Text
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  • Journal Article Harrington PR, Yount B, Johnston RE, Davis N, Moe C, Baric RS. Systemic, mucosal, and heterotypic immune induction in mice inoculated with Venezuelan equine encephalitis replicons expressing Norwalk virus-like particles. Journal of Virology 2002;76(2):730-742. R826139 (Final)
  • Abstract from PubMed
  • Full-text: JVI Full Text
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  • Journal Article Harrington PR, Vinje J, Moe CL, Baric RS. Norovirus capture with histo-blood group antigens reveals novel virus-ligand interactions. Journal of Virology 2004;78(6):3035-3045. R826139 (Final)
  • Abstract from PubMed
  • Full-text: Journal of Virology
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  • Other: Journal of Virology PDF
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  • Journal Article Lindesmith L, Moe C, Marionneau S, Ruvoen N, Jiang X, Lindblad L, Stewart P, LePendu J, Baric R. Human susceptibility and resistance to Norwalk virus infection. Nature Medicine 2003;9(5):548-553. R826139 (Final)
  • Abstract from PubMed
  • Journal Article Lindesmith L, Moe C, LePendu J, Frelinger JA, Treanor J, Baric RS. Cellular and humoral immunity following Snow Mountain virus challenge. Journal of Virology 2005;79(5):2900-2909. R826139 (Final)
  • Abstract from PubMed
  • Full-text: Journal of Virology
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  • Other: Journal of Virology PDF
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  • Journal Article LoBue AD, Lindesmith L, Yount B, Harrington PR, Thompson JM, Johnston RE, Moe CL, Baric RS. Multivalent norovirus vaccines induce strong mucosal and systemic blocking antibodies against multiple strains. Vaccine 2006;24(24):5220-5234. R826139 (Final)
  • Abstract from PubMed
  • Journal Article Moe CL, Sair A, Lindesmith L, Estes MK, Jaykus L-A. Diagnosis of Norwalk virus infection by indirect enzyme immunoassay detection of salivary antibodies to recombinant Norwalk virus antigen. Clinical and Diagnostic Laboratory Immunology 2004;11(6):1028-1034. R826139 (Final)
  • Abstract from PubMed
  • Full-text: CVI Full Text
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  • Journal Article Sair AI, D'Souza DH, Moe CL, Jaykus L-A. Improved detection of human enteric viruses in foods by RT-PCR. Journal of Virological Methods 2002;100(1-2):57-69. R826139 (Final)
  • Abstract from PubMed
  • Journal Article Teunis PFM, Moe CL, Liu P, Miller SE, Lindesmith L, Baric RS, Le Pendu J, Calderon RL. Norwalk virus: how infectious is it? Journal of Medical Virology 2008;80(8):1468-1476. R826139 (Final)
  • Abstract from PubMed
  • Supplemental Keywords:

    drinking water, dose-response, risk assessment, epidemiology, pathogens, viruses, susceptibility, health effects, human health., RFA, Health, Scientific Discipline, Water, Environmental Chemistry, Chemistry, Epidemiology, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Analytical Chemistry, genetic susceptability, Drinking Water, microbial contamination, pathogens, public water systems, risk factors, sensitive populations, microbial risk assessment, human health effects, infants, Norwalk, waterborne disease, exposure and effects, chemical byproducts, disinfection byproducts (DPBs), dose response, exposure, children, community water system, calciviruses, gastroenteritis, immuno-compromised population, human exposure, susceptibility, treatment, immune system response, elderly, emerging pathogens, drinking water contaminants, infectivity, water treatment, age dependent response, drinking water system, toxics

    Progress and Final Reports:

    Original Abstract
  • 1998
  • 1999
  • 2000 Progress Report