Less severe clinical symptoms of Norwalk virus 8fIIb inoculum compared to its precursor 8fIIa from human challenge studies.

Noroviruses (NoV) are a leading cause of epidemic gastroenteritis. Human challenge studies have been used to examine the infectivity, pathogenicity, and host immune response to NoV as well as vaccine efficacy. The goal of this study was to conduct a meta-analysis of data from five previously completed human challenge trials and compare the response to the secondary NV inoculum (8fIIb) to its precursor (8fIIa). We investigated a total of 158 subjects: 76 subjects were experimentally challenged with NV inoculum 8fIIa, and 82 subjects were challenged with 8fIIb. We compared demographic characteristics, infection, illness, mean severity score, blood types, and duration of viral shedding between the two groups of subjects. There were no statistically significant differences in overall infection and illness rates between subjects inoculated with 8fIIa and 8fIIb. However, individuals challenged with 8fIIa had significantly higher severity scores (5.05 vs. 3.22, p = .008) compared with those challenged with 8fIIb. We also observed that infection with 8fIIb was associated with significantly longer duration of viral shedding compared with 8fIIa (11.0 days vs. 5.0 days, p = .0005). These results have serious implications for the development of new NoV inocula for human challenge studies to test candidate vaccine efficacy-where illness severity and duration of viral shedding are important outcomes.

Determination of the 50% human infectious dose for Norwalk virus.

BACKGROUND: Noroviruses are the most common cause of gastroenteritis in the United States. An understanding of the infectious dose of these viruses is important for risk assessment studies. METHODS: Healthy adults were enrolled in a randomized, double-blind, placebo-controlled evaluation of different dosages of Norwalk virus. Eligible subjects were monitored for clinical gastroenteritis, and infection status was determined. The presence of virus in vomitus was also assessed. RESULTS: Fifty-seven persons were enrolled; 8 received placebo and an additional 8 persons were considered to be nonsusceptible on the basis of being secretor negative. Twenty-one persons were infected (all blood group O or A), and 67% of those infected developed viral gastroenteritis. The 50% human infectious dose was calculated to be 3.3 reverse transcription polymerase chain reaction units (approximately 1320 genomic equivalents [gEq]) for secretor-positive blood group O or A persons and 7.0 (approximately 2800 gEq) for all secretor-positive persons. The time of illness onset was inversely correlated with inoculum dose. The maximal concentration of virus shedding was higher for persons with gastroenteritis. Norwalk virus was identified in 15 of 27 (56%) vomitus samples at a median concentration of 41 000 gEq/mL. CONCLUSIONS: The 50% human infectious dose measured is higher than previous estimates and similar to that of other RNA viruses. Clinical Trials Registration NCT00138476.

Secretory pathway antagonism by calicivirus homologues of Norwalk virus nonstructural protein p22 is restricted to noroviruses.

BACKGROUND: Our previous report that the Norwalk virus nonstructural protein p22 is an antagonist of the cellular secretory pathway suggests a new aspect of norovirus/host interaction. To explore conservation of function of this highly divergent calicivirus protein, we examined the effects of p22 homologues from four human and two murine noroviruses, and feline calicivirus on the secretory pathway. FINDINGS: All human noroviruses examined induced Golgi disruption and inhibited protein secretion, with the genogroup II.4 Houston virus being the most potent antagonist. Genogroup II.6 viruses have a conserved mutation in the mimic of an Endoplasmic Reticulum export signal (MERES) motif that is highly conserved in human norovirus homologues of p22 and is critical for secretory pathway antagonism, and these viruses had reduced levels of Golgi disruption and inhibition of protein secretion. p22 homologues from both persistent and nonpersistent strains of murine norovirus induced Golgi disruption, but only mildly inhibited cellular protein secretion. Feline calicivirus p30 did not induce Golgi disruption or inhibit cellular protein secretion. CONCLUSIONS: These differences confirm a norovirus-specific effect on host cell secretory pathway antagonism by homologues of p22, which may affect viral replication and/or cellular pathogenesis.

Heterotypic humoral and cellular immune responses following Norwalk virus infection.

Norovirus immunity is poorly understood as the limited data available on protection after infection are often contradictory. In contrast to the more prominent GII noroviruses, GI norovirus infections are less frequent in outbreaks. The GI noroviruses display very complex patterns of heterotypic immune responses following infection, and many individuals are highly susceptible to reinfection. To study the immune responses and mechanisms of GI.1 persistence, we built structural models and recombinant virus-like particles (VLPs) of five GI strains: GI.1-1968, GI.1-2001, GI.2-1999, GI.3-1999, and GI.4-2000. Structural models of four GI genotype capsid P domain dimers suggested that intragenotype structural variation is limited, that the GI binding pocket is mostly preserved between genotypes, and that a conserved, surface-exposed epitope may allow for highly cross-reactive immune responses. GI VLPs bound to histo-blood group antigens (HBGAs) including fucose, Lewis, and A antigens. Volunteers infected with GI.1-1968 (n = 10) had significant increases between prechallenge and convalescent reactive IgG for all five GI VLPs measured by enzyme immunoassay. Potential cross-neutralization of GI VLPs was demonstrated by convalescent-phase serum cross-blockade of GI VLP-HBGA interaction. Although group responses were significant for all GI VLPs, each individual volunteer demonstrated a unique VLP blockade pattern. Further, peripheral blood mononuclear cells (PBMCs) were stimulated with each of the VLPs, and secretion of gamma interferon (IFN-gamma) was measured. As seen with blockade responses, IFN-gamma secretion responses differed by individual. Sixty percent responded to at least one GI VLP, with only two volunteers responding to GI.1 VLP. Importantly, four of five individuals with sufficient PBMCs for cross-reactivity studies responded more robustly to other GI VLPs. These data suggest that preexposure history and deceptive imprinting may complicate PBMC and B-cell immune responses in some GI.1-1968-challenged individuals and highlight a potential complication in the design of efficacious norovirus vaccines.

Norovirus infectivity in humans and persistence in water.

To examine the long-term infectivity of human norovirus in water, 13 study subjects were challenged at different time points with groundwater spiked with the prototype human norovirus, Norwalk virus. Norwalk virus spiked in groundwater remained infectious after storage at room temperature in the dark for 61 days (the last time point tested). The Norwalk virus-seeded groundwater was stored for 1,266 days and analyzed, after RNase treatment, by reverse transcription-quantitative PCR (RT-qPCR) to detect Norwalk virus RNA contained within intact capsids. Norwalk virus RNA within intact capsids was detected in groundwater for 1,266 days, with no significant log(10) reduction throughout 427 days and a significant 1.10-log(10) reduction by day 1266. Purified Norwalk virus RNA (extracted from Norwalk virus virions) persisted for 14 days in groundwater, tap water, and reagent-grade water. This study demonstrates that Norwalk virus in groundwater can remain detectable for over 3 years and can remain infectious for at least 61 days. (ClinicalTrials.gov identifier NCT00313404.).

Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples.

Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72 degrees C, 37 degrees C, and 19 degrees C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72 degrees C and 37 degrees C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19 degrees C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37 degrees C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.

Norwalk virus shedding after experimental human infection.

Noroviruses are the most common cause of viral gastroenteritis in the United States. To determine the magnitude and duration of virus shedding in feces, we evaluated persons who had been experimentally infected with Norwalk virus. Of 16 persons, clinical gastroenteritis (watery diarrhea and/or vomiting) developed in 11; symptomatic illness lasted 1-2 days. Virus shedding was first detected by reverse transcription-PCR (RT-PCR) 18 hours after participant inoculation and lasted a median of 28 days after inoculation (range 13-56 days). The median peak amount of virus shedding was 95 x 10(9) (range 0.5-1,640 x 10(9)) genomic copies/g feces as measured by quantitative RT-PCR. Virus shedding was first detected by antigen ELISA approximately 33 hours (median 42 hours) after inoculation and lasted 10 days (median 7 days) after inoculation. Understanding of the relevance of prolonged fecal norovirus excretion must await the development of sensitive methods to measure virus infectivity.

Norwalk virus: how infectious is it?

Noroviruses are major agents of viral gastroenteritis worldwide. The infectivity of Norwalk virus, the prototype norovirus, has been studied in susceptible human volunteers. A new variant of the hit theory model of microbial infection was developed to estimate the variation in Norwalk virus infectivity, as well as the degree of virus aggregation, consistent with independent (electron microscopic) observations. Explicit modeling of viral aggregation allows us to express virus infectivity per single infectious unit (particle). Comparison of a primary and a secondary inoculum showed that passage through a human host does not change Norwalk virus infectivity. We estimate the average probability of infection for a single Norwalk virus particle to be close to 0.5, exceeding that reported for any other virus studied to date. Infected subjects had a dose-dependent probability of becoming ill, ranging from 0.1 (at a dose of 10(3) NV genomes) to 0.7 (at 10(8) virus genomes). A norovirus dose response model is important for understanding its transmission and essential for development of a quantitative risk model. Norwalk virus is a valuable model system to study virulence because genetic factors are known for both complete and partial protection; the latter can be quantitatively described as heterogeneity in dose response models.

Virological investigation on aerosol from waste depuration plants.

Aerosol from activated mud decontamination plants used for the treatment of urban sewage can represent a vehicle for bacteria, virus and fungi. As a result, they become an infective hazard for plant personnel, the general population residing in the surrounding area and the occasional visitor. The present investigation focuses on the identification of enteric-type viruses in this kind of aerosol. The following methods were employed on 214 samples collected in the 1999-2000 period: cell culture (BGM, RD, Hep-2), electron microscopy, and polymerase chain reaction (PCR). Cytopathic effect was mild in 180 samples, and severe in 14, upon their first passage in culture. Virus identification was based on positivity to both electron microscopy (EM) and PCR. Thus, one positive sample was recognized to be of enteric-type virus and two positive samples were recognized as reovirus-type. All samples were negative for Norwalk-type virus or HAV. There was considerable discrepancy between electron microscopy and PCR concerning the number of enteric-type viruses recognized. A possible explanation is contamination with animal-type enterovirus.

Identification of Norwalk virus in artificially seeded shellfish and selected foods.

A rotavirus dsRNA purification protocol was adapted to extract Norwalk ssRNA from artificially contaminated shellfish, and a sensitive reverse transcription-polymerase chain reaction assay for Norwalk virus was devised to identify an estimated 20-200 genomic copies. The technique includes deproteinization with guanidinium isothiocyanate, adsorption of RNA to hydroxyapatite, and sequential precipitation with cetyltrimethylammonium bromide and ethanol. The protocol allows high recovery of viral RNA free of enzymatic inhibitors from oysters, clams, and a variety of food matrices. Norwalk virus sequences were copied and amplified by using primers selected from the polymerase gene. Digestion of the amplified products with restriction enzymes ensured the specificity of the test. This rapid and sensitive assay may significantly improve the prospect for the routine screening of the uncultivatable Norwalk virus in food stuffs.

Recovery and detection of enterovirus, hepatitis A virus and Norwalk virus in hardshell clams (Mercenaria mercenaria) by RT-PCR methods.

A method for recovery of enteric viruses from hardshell clams (Mercenaria mercenaria) has been developed and evaluated. Seeded 50-g samples of clam tissue homogenates were processed by adsorption elution precipitation, two fluorocarbon extractions and PEG precipitation. Clam concentrates were assayed by infectivity and by RT-PCR after guanidinium isothiocyanate (GIT) extraction and/or an indirect immunomagnetic capture (IC) of the virus using paramagnetic beads. GIT extraction removed PCR inhibitors and allowed a reliable RT-PCR detection of viral RNA. The detection sensitivity of GIT extraction-RT-PCR was < 1 PFU of poliovirus 1, < 10 PFU of HAV and 1-11 PCRU of Norwalk virus. IC was very effective for additional concentration and purification of enteric viruses from clam concentrates removing most RT-PCR inhibitors. The sensitivity of this method was comparable to the GIT extraction and the sample volume tolerance for PCR was increased about 10-fold. Both methods gave similar efficiency for virus detection in samples seeded with low virus levels. The procedure developed in this study is effective for enteric viruses detection in hardshell clams by RT-PCR.

Ultraviolet inactivation of feline calicivirus, human enteric viruses and coliphages.

Norwalk and Norwalk-like viruses (NLV) are major causes of food- and water-related disease in the United States. There is no host cell line in which the NLV can be tested for infectivity. Feline calicivirus (FCV) and NLV both belong to the family Caliciviridae. FCV can be assayed for infectivity in the Crandell Reese feline kidney cell line, so FCV serves as a surrogate for NLV. This study is the first report of UV inactivation of FCV and also of using the plaque technique, in contrast to the 50% tissue culture infectious dose end point technique, to determine the FCV infectivity titer. The infectivity titers (log10 plaque-forming units/mL) of UV-inactivated FCV, hepatitis A virus (HAV), poliovirus type 1 (PV1) and two small, round coliphages were plotted as a function of UV dose and analyzed by regression analysis and analysis of variance. These fitted straight-line curves represent exponential inactivation, so UV inactivation can be said to show "one-hit kinetics." The decimal inactivation doses of UV for FCV, HAV, PV1, MS2 and phiX174 were 47.85, 36.50, 24.10, 23.04 and 15.48 mW s/cm2, respectively. FCV appears to be the most UV resistant among the tested viruses.

Norwalk and "Norwalk-like viruses" in epidemic gastroenteritis.

Despite the lack of a cell culture or animal model system, the past decade has seen tremendous advances in our understanding of NLV. Prior to 1990, the only nucleotide sequence information for caliciviruses was from viruses isolated from animals. There are now sequences available for more than 100 NLV isolates and more are rapidly accumulating. Such information is being used for development of new and more sensitive diagnostic assays. The CDC, under the National Food Safety Initiative and in cooperation with state and local governments, is working toward implementing routine surveillance and outbreak responses to limit or prevent widespread illness from the same identified source. Such increased surveillance and continued epidemiologic studies are necessary and critical to assess the risks and contain food-borne and water-borne outbreaks caused by the NLVs.

Raw oyster consumption and outbreaks of viral gastroenteritis in New Zealand: evidence for risk to the public's health.

OBJECTIVE: To investigate the relationship between consumption of raw Pacific half shell oysters and outbreaks of Norwalk-like virus (NLV) gastroenteritis in Auckland in the last third of 1999. METHOD: Ten outbreaks were investigated as retrospective cohorts using standardised questionnaires relating to food and drink exposures. Trace back of oysters and site inspections of implicated commercial growing areas were performed. Virological analyses compared oysters linked to outbreaks and faecal samples from cases. RESULTS: Eighty-six cases were identified, of whom 32 (37.2%) were confirmed NLV positive on faecal analysis. The summary risk estimate for illness among oyster consumers for all outbreaks was RR 8.23 (95% CI 4.55-14.90; p< 0.001) and in five of seven outbreaks permitting statistical analysis, the risk for those consuming raw oysters was greater than five-fold that of non-consumers. NLVs were identified in two batches of oysters from different growing areas, implicated in four outbreaks. In both the strain (Genogroup II/3 'Mexico-like virus') from cases matched that in the oysters from the same harvest batch. CONCLUSION: The epidemiological and virological evidence implicates oysters as the source of a number of outbreaks of NLV gastroenteritis. This is the first time NLVs have been identified in commercially farmed Pacific oysters in New Zealand. Sewage effluent from recreational boats was the likely source of faecal contamination of growing waters in one site. IMPLICATIONS: Combined use of virological and epidemiological methods have proved invaluable in investigating NLV outbreaks. Further research is needed into enteric viral contamination of commercial oyster farms.

A novel "small round virus" inducing poult enteritis and mortality syndrome and associated immune alterations.

The role of a novel "small round virus" (SRV) isolated from poult enteritis and mortality syndrome (PEMS) cases in inducing PEMS and associated immune alterations was examined in this study. Specific-pathogen-free and conventional poults were orally challenged with SRV and/or turkey coronavirus and monitored for clinical signs. Intestines, thymus, bursa, and spleens were examined for SRV antigen at various days postinoculation (DPI). Peripheral blood lymphocytes (PBLs), thymocytes, and splenic lymphocytes from inoculated poults or lymphocytes isolated from healthy poults after incubation with SRV in vitro were examined for lymphoproliferative potential against concanavalin A (Con A). The incidence of lymphocyte subpopulations in the peripheral blood and thymic lymphocytes of SRV-challenged poults was examined by flow cytometry. The results of these studies showed that the SRV challenge induced diarrhea, growth suppression, and atrophy of thymus and bursa resembling those of PEMS in field and/or experimental infections. The SRV antigen was detected in intestinal tissues soon after infection (i.e., at 2 and 4 DPI), whereas lymphoid tissues such as thymus, bursa, and spleen were positive for SRV antigen starting at 4 DPI until 8 DPI, suggesting virus translocation to lymphoid organs. The responsiveness of PBLs to Con A at 2 DPI was significantly reduced in all virus challenge groups (e.g., 28% and 22% in the SRV-alone group in studies 1 and 2, respectively) below the uninfected group. However, this suppressed response was no longer evident in the SRV group by 7 DPI. The SRV incubation with normal thymocytes and splenocytes in vitro resulted in significantly reduced lymphoproliferative response against Con A (41.2% and 10.49% reductions at 1:50 SRV dilution vs. controls in thymocytes and splenocytes, respectively). Flow cytometry analysis revealed a sudden decline at 2 DPI in the numbers of CD4- CD8+ lymphocyte subset in PBLs of SRV-infected poults. However, by 8 DPI, SRV-challenged poults had relatively higher CD4- CD8+ lymphocytes in PBLs. On the contrary, thymocytes had higher percentages of CD4- CD8+ lymphocytes at 2 and 4 DPI and reached comparable levels at 8 DPI in controls and SRV-infected poults. No differences were observed in CD4+ CD8- lymphocyte numbers in controls vs. SRV-infected poults. The findings of these studies imply that SRV may be a promising primary etiologic agent of PEMS. Furthermore, the SRV infection may compromise the lymphocyte-mediated immune defenses by reducing lymphoproliferation and the CD4- CD8+ (presumably T-cytotoxic cells) lymphocytes during the acute stage of SRV infection.

Comparative reduction of Norwalk virus, poliovirus type 1, F+ RNA coliphage MS2 and Escherichia coli in miniature soil columns.

Norwalk-like viruses (NLVs) are important agents of waterborne illness and have been linked to several groundwater-related outbreaks. The presence of human enteric viruses, in particular the presence of NLVs, is difficult to detect in the environment. Consequently, surrogate organisms are typically used as indicators of viruses from faecal contamination. Whether traditional bacterial indicators are reliable indicators for viral pathogens remains uncertain. Few studies have directly compared mobility and reduction of bacterial indicators (e.g. coliforms, Escherichia coli) and other surrogate indicators (coliphages) with pathogenic human viruses in soil systems. In this study the mobility and comparative reduction of the prototype NLV, Norwalk Virus (NV), was compared to poliovirus 1 (PV1), a bacterial indicator (E coli, EC) and a viral indicator (coliphage MS2) through miniature soil columns. Replicate, 10 cm deep, miniature columns were prepared using three soils representing a range of soil textures (sand, organic muck, and clay). Columns were initially conditioned, then incubated at 10-14 degrees C, dosed twice weekly for 8 weeks with one column pore volume of virus-seeded groundwater per dose, followed by 8 weeks of dosing with one column pore volume per dose of unseeded, simulated rainwater. Columns were allowed to drain after each dosing until an effluent volume equivalent to an applied dose was collected. Column effluents and doses were assayed for all viruses and EC. Rapid mobility with minimal reduction was observed for all organisms in the sand. Similar reductions were observed in organic muck for most organisms but NV showed a greater reduction. No organisms were shown to pass through the clay columns. Elution of viruses, in particular PV1, from the columns was gradual. After cessation of microbe dosing, E. coli was less detectable than viruses in column effluents and, therefore, unreliable as a virus indicator.

A Norwalk-like virus outbreak on the Appalachian Trail.

In May and June 1999, an outbreak of acute gastrointestinal illness occurred among long-distance hikers on the Appalachian Trail between Catawba and Troutville, Virginia. An investigation found that 45 out of 70 hikers had become ill within two days of arriving in Catawba, Virginia. Water samples were collected from a general store frequented by the hikers and from several nearby buildings and a popular all-you-can-eat restaurant. Symptoms were consistent with those caused by Norwalk-like viruses, and laboratory diagnosis detected Norwalk-like viruses in stool and serum specimens. People who consumed food items prepared at the general store were almost twice as likely to become ill as persons who did not consume those foods. Environmental sampling of water from the taps inside and outside the general store and from several surrounding establishments in Catawba found contamination by fecal coliform bacteria but not by Norwalk-like virus. Since several hikers reported illness prior to arriving at Catawba, person-to-person transmission of a highly contagious agent such as Norwalk-like virus could not be ruled out. Poor sanitation, scarce water supplies, and crowding can increase the risk of gastrointestinal illness among long-distance hikers.

[Norwalk virus and Noro virus].

Norwalk virus and Noro virus are members of the Caliciviridae. These viruses are morphological similarity in each other and shows small round structure. These viruses also are well known as main pathogens of acute infectious gastroenteritis. Clinical features include an incubation period of 24 of 48 hours and illness period of 18 to 72 hours with vomiting and diarrhea in most patients and high secondary attack rates. Oral transmitted infection occurs contaminated water and foods. In our country, outbreak of Noro virus-related gastroenteritis are reported sometimes in hospital and nursing home from winter to early spring seasons. This article are described to the morphlogy, physical characteristics, epidemiology, and clinical manifestation relating to Norwalk virus and Noro virus.