Norovirus in symptomatic and asymptomatic individuals: cytokines and viral shedding.

Noroviruses (NoV) are the most common cause of epidemic gastroenteritis world-wide. NoV infections are often asymptomatic, although individuals still shed large amounts of NoV in their stool. Understanding the differences between asymptomatic and symptomatic individuals would help in elucidating mechanisms of NoV pathogenesis. Our goal was to compare the serum cytokine responses and faecal viral RNA titres of asymptomatic and symptomatic NoV-infected individuals. We tested serum samples from infected subjects (n = 26; 19 symptomatic, seven asymptomatic) from two human challenge studies of GI.1 NoV for 16 cytokines. Samples from prechallenge and days 1-4 post-challenge were tested for these cytokines. Cytokine levels were compared to stool NoV RNA titres quantified previously by reverse transcription-polymerase chain reaction (RT-qPCR). While both symptomatic and asymptomatic groups had similar patterns of cytokine responses, the symptomatic group generally exhibited a greater elevation of T helper type 1 (Th1) and Th2 cytokines and IL-8 post-challenge compared to the asymptomatic group (all P < 0·01). Daily viral RNA titre was associated positively with daily IL-6 concentration and negatively with daily IL-12p40 concentration (all P < 0·05). Symptoms were not associated significantly with daily viral RNA titre, duration of viral shedding or cumulative shedding. Symptomatic individuals, compared to asymptomatic, have greater immune system activation, as measured by serum cytokines, but they do not have greater viral burden, as measured by titre and shedding, suggesting that symptoms may be immune-mediated in NoV infection.

Human norovirus infection and the acute serum cytokine response.

Noroviruses (NoV) are the most common cause of epidemic gastroenteritis worldwide. The acute immune response to NoV in humans is poorly understood, hindering research on prevention and treatment. To elucidate the acute immune response and test for cytokine predictors of susceptibility to infection, serum samples from two human NoV challenge studies were tested for 16 cytokines. Subjects who became infected (n = 26) were age-matched with subjects who remained uninfected following NoV challenge (n = 26). Samples were tested from prechallenge and days 1-4 post-challenge. Cytokine responses were compared between infected and uninfected groups. Overall, infected individuals exhibited an elevation in T helper type 1 (Th1) and Th2 cytokines, as well as chemokines interleukin (IL)-8 and monocyte chemoattractant protein (MCP-1), compared to uninfected individuals (all P < 0.05). Most cytokines peaked on day 2 post-challenge in infected subjects, and tumour necrosis factor (TNF)-α, IL-8, and IL-10 remained elevated to day 3. The only cytokine elevated significantly among infected subjects to day 4 post-challenge was IL-10 (P = 0.021). Prechallenge cytokine concentrations were not predictive of infection status post-challenge. There were no significant changes in serum cytokines among NoV-challenged subjects who remained uninfected. These results suggest that NoV infection elicits a Th1-type response, with some Th2 activation. Persistent elevation of IL-10 among infected subjects is consistent with activation of adaptive immune responses, such as B cell expansion, as well as down-regulation of Th1 cytokines. This study presents the first comprehensive description of the acute cytokine response to GI.1 NoV in humans.

Disease course and viral shedding in experimental Norwalk virus and Snow Mountain virus infection.

Norovirus is the most common cause of acute infectious gastroenteritis, causing approximately 21 million cases annually in the USA. The virus is highly contagious and resistant to decontamination, making outbreaks difficult to control. To facilitate the development of better control methods, this study characterized the viral shedding patterns in stools from subjects experimentally infected with genogroup I or II norovirus. Viral stool titers were determined by quantitative real-time RT-PCR for all stools produced in the first 7 days post-challenge and representative stools through day 35 post-challenge. The shedding titers and disease course were analyzed with respect to virus type, illness, and subject demographics. Infection with GII.2 Snow Mountain (SMV) resulted in more symptoms and a higher frequency of painful symptoms compared to GI.1 Norwalk (NV) infection. However, NV infection produced stool viral titers approximately 2 logs higher than those seen in SMV infections. Both NV and SMV were shed in stools for up to 3 weeks after the resolution of symptoms, but long shedding durations were more common in NV infections. For each challenge virus, shedding titers and patterns were not correlated with subject demographics or clinical course. This is the first study to report shedding dynamics in experimental GII norovirus infection.

Heterogeneity in norovirus shedding duration affects community risk.

Norovirus is a common cause of gastroenteritis in all ages. Typical infections cause viral shedding periods of days to weeks, but some individuals can shed for months or years. Most norovirus risk models do not include these long-shedding individuals, and may therefore underestimate risk. We reviewed the literature for norovirus-shedding duration data and stratified these data into two distributions: regular shedding (mean 14-16 days) and long shedding (mean 105-136 days). These distributions were used to inform a norovirus transmission model that predicts the impact of long shedders. Our transmission model predicts that this subpopulation increases the outbreak potential (measured by the reproductive number) by 50-80%, the probability of an outbreak by 33%, the severity of transmission (measured by the attack rate) by 20%, and transmission duration by 100%. Characterizing and understanding shedding duration heterogeneity can provide insights into community transmission that can be useful in mitigating norovirus risk.

The epidemiology of published norovirus outbreaks: a review of risk factors associated with attack rate and genogroup.

The purpose of this study was to examine global epidemiological trends in human norovirus (NoV) outbreaks by transmission route and setting, and describe relationships between these characteristics, viral attack rates, and the occurrence of genogroup I (GI) or genogroup II (GII) strains in outbreaks. We analysed data from 902 reverse transcriptase-polymerase chain reaction-confirmed, human NoV outbreaks abstracted from a systematic review of articles published from 1993 to 2011 and indexed under the terms 'norovirus' and 'outbreak'. Multivariate regression analyses demonstrated that foodservice and winter outbreaks were significantly associated with higher attack rates. Foodborne and waterborne outbreaks were associated with multiple strains (GI+GII). Waterborne outbreaks were significantly associated with GI strains, while healthcare-related and winter outbreaks were associated with GII strains. These results identify important trends for epidemic NoV detection, prevention, and control.

Enteric virus infection risk from intrusion of sewage into a drinking water distribution network.

Contaminants from the soil surrounding drinking water distribution systems are thought to not enter the drinking water when sufficient internal pressure is maintained. Pressure transients may cause short intervals of negative pressure, and the soil near drinking water pipes often contains fecal material due to the proximity of sewage lines, so that a pressure event may cause intrusion of pathogens. This paper presents a risk model for predicting intrusion and dilution of viruses and their transport to consumers. Random entry and dilution of virus was simulated by embedding the hydraulic model into a Monte Carlo simulation. Special attention was given to adjusting for the coincidence of virus presence and use of tap water, as independently occurring short-term events within the longer interval that the virus is predicted to travel in any branch of the distribution system. The probability that a consumer drinks water contaminated with virus is small, but when this happens the virus concentration tends to be high and the risk of infection may be considerable. The spatial distribution of infection risk is highly heterogeneous. The presence of a chlorine residual reduces the infection risk.

Molecular epidemiology of norovirus gastroenteritis outbreaks in North Carolina, United States: 1995-2000.

Noroviruses (NoVs) are the most common cause of acute non-bacterial gastroenteritis outbreaks in the US. We investigated 16 gastroenteritis outbreaks in North Carolina (NC), from 1995 to 2000, to further characterize the epidemiology of NoV using RT-PCR on stool and ELISA on sera. NoV were identified in 14 outbreaks by RT-PCR. Sequence analyses of the amplicons indicated the outbreak strains belonged to the following clusters: five GII/4, three GI/3, one GI/4, one GII/2, one GII/5, one GII/7, and one GII/13 (prototype strain). We detected NoV in stool samples from one outbreak but could not determine its specific cluster within the GII genogroup based on polymerase sequence analysis. The five GII/4 strains were classified as the "95/96 US common strain" and occurred throughout the 5-year period. In contrast to national trends, the majority (86%) of NoV outbreaks identified in North Carolina were foodborne. Of the 12 food-related NoV outbreaks, we were able to document transmission by food handlers in two outbreaks. Person-to-person transmission from primary cases was suggested in three outbreaks. Our results indicate that NoVs are important agents of viral gastroenteritis outbreaks in NC.

Cohort studies of health effects among people exposed to estuarine waters: North Carolina, Virginia, and Maryland.

A variety of human symptoms have been associated with exposure to the dinoflagellate Pfiesteria and have been grouped together into a syndrome termed "possible estuary-associated syndrome." Prospective cohort studies of health effects associated with exposure to estuarine waters that may contain Pfiesteria spp. and related organisms are in progress in North Carolina, Virginia, and Maryland. The three studies recruited cohorts of 118-238 subjects who work or engaged in recreation in estuary waters. Baseline health and neuropsychological evaluations are conducted, and study subjects are followed prospectively for 2-5 years with periodic assessments of health and performance on a battery of neuropsychological tests. Health symptoms and estuary water exposure are recorded by telephone interviews or diaries every 1-2 weeks. Water quality information, including measurements of Pfiesteria spp., is collected in the areas where the subjects are working. Because it is not possible to measure individual exposure to Pfiesteria or a toxin produced by this organism, these studies examine surrogate exposure measures (e.g., time spent in estuary waters, in a fish kill area, or in waters where Pfiesteria DNA was detected by molecular amplification). Preliminary analyses of the first 2 years (1998-2000) of data indicate that none of the three ongoing cohorts have detected adverse health effects. However, there have not been any reported fish kills associated with Pfiesteria since the studies began, so it is possible that none of the study subjects have been exposed to toxin-producing Pfiesteria spp.

Outbreaks of acute gastroenteritis associated with Norwalk-like viruses in campus settings.

Norwalk-like viruses (NLVs) are transmitted by fecally contaminated food, water, fomites, and person-to-person contact. They are a leading cause of acute gastroenteritis epidemics in industrialized countries. NLV outbreaks are characterized by a 12- to 48-hour incubation period; nausea, vomiting, and diarrhea for 24 to 72 hours; and high secondary attack rates. NLV infections spread rapidly on college and university campuses because of close living quarters, shared bathrooms and common rooms, many food handlers, popular self-service salad bars in dining halls, and person-to-person contact through sports and recreational activities. The illness is generally mild and self-limited but an outbreak can strain the resources of campus health services and cause high absenteeism among both students and staff. Treatment is primarily through antiemetic medication and oral rehydration. Prevention and control of NLV outbreaks rests on promoting hand washing; enforcement of strict hygiene in all food preparation areas; and prompt, rigorous cleaning of potentially contaminated areas where someone has been ill.

Transmission of Norwalk virus during a football game.

BACKGROUND: During a college football game in Florida, diarrhea and vomiting developed in many of the members of a North Carolina team. The next day, similar symptoms developed in some of the players on the opposing team. METHODS: We interviewed those who ate the five meals served to the North Carolina team before the game and some of the players on the opposing team who became ill. Patients with primary cases were members or staff of the team who had vomiting or diarrhea at least 10 hours after but no more than 50 hours after eating a box lunch served the day before the game. Patients with secondary cases had a later onset of symptoms or had symptoms without having eaten the box lunch. Stool samples were examined by electron microscopy and by a reverse-transcription-polymerase-chain-reaction (RT-PCR) assay. RESULTS: The two football teams shared no food or beverages and had no contact off the playing field. Of five meals served to the North Carolina team before the game, only the box lunch was associated with a significant risk of illness (relative risk of illness, 4.1; 95 percent confidence interval, 1.6 to 10.0). The rate of attack among those who ate the box lunch was 62 percent. There were 11 secondary cases among the members and staff of the North Carolina team and 11 such cases among the Florida players. All four stool samples obtained from North Carolina patients were positive for Norwalk-like virus on electron microscopy. All four samples as well as one of two stool samples from players on the Florida team were positive for a Norwalk-like virus of genogroup I on RT-PCR assay; the RT-PCR products had identical sequences. CONCLUSIONS: This investigation documents person-to-person transmission of Norwalk virus among players during a football game. Persons with acute gastroenteritis should be excluded from playing contact sports.

Seroprevalence and seroincidence of Norwalk-like virus infection among Brazilian infants and children.

To determine the importance of Norwalk-like viruses (NLVs) as pediatric pathogens in a developing country, the seroprevalence and seroincidence of this group of viruses in a cohort of children less than 4 years of age in an urban shantytown in northeastern Brazil was examined. Serum samples were collected approximately every 6 months from 135 children who were surveyed three times each week for diarrhea and vomiting. NLV IgG was measured by an enzyme immunosorbent assay (EIA) with recombinant Norwalk virus capsid protein. Overall NLV seroprevalence was 71%, and the overall NLV seroconversion rate was 0.7 seroconversions per child-year. The highest age-specific NLV seroconversion rate (0.8 seroconversions per child-year) was observed in the 13-24-month age group. For all study children, the incidence of diarrhea and vomiting was significantly greater (P < 0.01) during time periods spanned by serum pairs that indicated NLV seroconversion compared with time periods without NLV seroconversion. However, NLV seroconversion was not associated with gastrointestinal symptoms during the first year of life.

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.

Genomic diversity of "Norwalk like viruses" (NLVs): pediatric infections in a Brazilian shantytown.

"Norwalk-like viruses" (NLVs) are a common cause of epidemic gastroenteritis in adults and children in developed countries. However, little is known about the role of NLVs in endemic pediatric gastroenteritis in developing countries. We sequenced Genogroup I and II NLV reverse transcription-polymerase chain reaction (RT-PCR) products from an 81-nucleotide region of the viral RNA polymerase gene to examine the molecular epidemiology of NLV infection in children younger than 5 years of age in Forteleza, Ceará, Brazil. NLV-positive PCR products were obtained from stool specimens collected over a 16-month period (1990-1991) from diarrhea cases and controls in a cohort of 120 children in an urban shantytown and from a study in the same city of hospitalized children with persistent diarrhea. Eight unique strains were detected in 15 specimens from 10 cohort children and in two hospital specimens. Nucleotide identity between the strains (5 Genogroup I, 3 Genogroup II) ranged from 63% to 88%. We designated these strains BraV1-8, for Brazil virus 1-8. The degree of genomic diversity of NLV strains we identified in this cohort during a short time period suggests multiple foci of infection within the community. Furthermore, sequence analysis of strains from two children with multiple symptomatic NLV infections indicates that infection with one strain was not protective against subsequent infection with a different strain in the same genogroup. These findings have implications for vaccine development and the prevention of pediatric gastroenteritis in developing countries.

Astrovirus infection in association with acute, persistent and nosocomial diarrhea in Bangladesh.

BACKGROUND: Diarrhea is an important public health concern in developing countries such as Bangladesh. Diarrhea in children that persists for 14 days or more occurs in 7% of patients in Bangladesh and frequently results in death. Astrovirus has been demonstrated as a cause of acute and nosocomial diarrhea and can be excreted for prolonged periods, yet its importance as a cause of diarrhea among children in a developing country like Bangladesh has not been investigated. METHODS: We tested 629 stool specimens from patients with acute diarrhea, 153 from patients with persistent diarrhea, 175 specimens from 76 patients hospitalized for diarrhea who were sampled repeatedly to detect nosocomial infection and 428 from nonhospitalized healthy children (controls). All children enrolled in the study were <5 years of age. Astrovirus was detected by enzyme immunoassay and other enteropathogens were detected by standard techniques. RESULTS: The detection of astrovirus increased significantly with the duration of diarrhea. Astrovirus was found in 23 (15%) specimens from patients with persistent diarrhea, 26 (4%) patients with acute diarrhea, but only 8 (2%) healthy controls. This trend remained when we limited our analysis to infants <12 months of age and to episodes in which astrovirus was the sole pathogen. Among patients with nosocomial diarrhea, 16% of postadmission specimens were positive for astrovirus when the admission specimen was negative. CONCLUSION: The observation that astrovirus is detected more frequently with diarrhea of increasing duration suggests the need for further studies to determine whether astrovirus plays a causative role in persistent diarrhea or is a secondary agent.

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.

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.

Application of PCR to detect Norwalk virus in fecal specimens from outbreaks of gastroenteritis.

Norwalk virus (NV) and other small round-structured viruses (SRSVs) are frequent causes of gastroenteritis outbreaks. The recent cloning and sequencing of the NV genome has made it possible to detect NV and Norwalk-related viruses from fecal specimens by reverse transcription (RT)-PCR. We applied this technique to the examination of a total of 139 fecal specimens from 19 outbreaks characterized by NV serology, including 56 samples from 7 NV outbreaks, 36 from 6 Norwalk-related virus outbreaks, and 47 from 6 outbreaks with SRSVs visualized by electron microscopy that were serologically unrelated to NV. Three primer pairs were evaluated: two pairs in the polymerase region of NV and one pair near the 3' end of the genome. When one set of primers (primer pair 51-3) from the polymerase region was used, 40% of all samples were positive by RT-PCR and specimens from the NV outbreaks were more likely to be positive (64%) than those from outbreaks associated with Norwalk-related viruses (44%) or SRSVs (8%). To determine the relationship of the outbreak strains to NV, we compared the sequences of a 145-base portion of the polymerase gene from 10 specimens obtained from five different outbreaks characterized as NV by serology. No two outbreak strains had the same sequence in this 145-base portion of the polymerase gene, and the identities of the nucleotide and amino acid sequences of these products compared with the sequences of the corresponding region of NV ranged from 62 to 79% and 69 to 90%, respectively. Because of sequence diversity in the polymerase region, the successful application of RT-PCR to investigations of outbreaks of suspected NV-associated gastroenteritis will depend on the use of either multiple primer pairs or primers made against regions of the genome that are more conserved.

Norwalk virus-associated gastroenteritis traced to ice consumption aboard a cruise ship in Hawaii: comparison and application of molecular method-based assays.

Investigation of an outbreak of acute nonbacterial gastroenteritis on a cruise ship provided an opportunity to assess new molecular method-based diagnostic methods for Norwalk virus (NV) and the antibody response to NV infection. The outbreak began within 36 h of embarkation and affected 30% of 672 passengers and crew. No single meal, seating, or food item was implicated in the transmission of NV, but a passenger's risk of illness was associated with the amount of ice (but not water) consumed (chi-square for trend, P = 0.009). Of 19 fecal specimens examined, 7 were found to contain 27-nm NV-like particles by electron microscopy and 16 were positive by PCR with very sensitive NV-specific primers, but only 5 were positive by a new highly specific antigen enzyme immunoassay for NV. Ten of 12 serum specimen pairs demonstrated a fourfold or greater rise in antibody titer to recombinant baculovirus-expressed NV antigen. The amplified PCR band shared only 81% nucleotide sequence homology with the reference NV strain, which may explain the lack of utility of the fecal specimen enzyme immunoassay. This report, the first to document the use of these molecular method-based assays for investigation of an outbreak, demonstrates the importance of highly sensitive viral diagnostics such as PCR and serodiagnosis for the epidemiologic investigation of NV gastroenteritis.

Detection of astrovirus in pediatric stool samples by immunoassay and RNA probe.

Two new astrovirus assays, a rapid biotin-avidin enzyme immunoassay (EIA) and RNA probe hybridization, were developed and compared with an established astrovirus assay, an indirect EIA, and immune electron microscopy. Sensitivity and specificity were evaluated by using a screening panel of 22 astrovirus-positive and 305 astrovirus-negative fecal specimens. The biotin-avidin assay was equivalent in performance to the reference indirect assay, and both could detect about 10 ng of viral protein. Although the probe was more sensitive than either EIA and could detect higher dilutions of virus in tissue culture and stool specimens, it did not detect more astrovirus-positive fecal specimens. Of the 22 astrovirus-positive specimens detected by the EIAs, 20 were confirmed by immune electron microscopy with hyperimmune rabbit antiserum. To determine the usefulness of EIAs for large epidemiologic studies, EIAs were used to screen 1,289 stool specimens from three studies of children with and without diarrhea. Astrovirus was detected in 3.5% of specimens from children with diarrhea and 1.9% of specimens from those without diarrhea. Our results indicate that the biotin-avidin EIA is an efficient, sensitive, and specific method for routinely screening large numbers of fecal samples and that its application in epidemiologic studies may yield higher rates of astrovirus infection than have been found previously by other methods.