In JoVE (1)

Other Publications (109)

Articles by Jan Vinjé in JoVE

Other articles by Jan Vinjé on PubMed

Foodborne Viruses

FEMS Microbiology Reviews. Jun, 2002  |  Pubmed ID: 12069883

Foodborne and waterborne viral infections are increasingly recognized as causes of illness in humans. This increase is partly explained by changes in food processing and consumption patterns that lead to the worldwide availability of high-risk food. As a result, vast outbreaks may occur due to contamination of food by a single foodhandler or at a single source. Although there are numerous fecal-orally transmitted viruses, most reports of foodborne transmission describe infections with Norwalk-like caliciviruses (NLV) and hepatitis A virus (HAV), suggesting that these viruses are associated with the greatest risk of foodborne transmission. NLV and HAV can be transmitted from person to person, or indirectly via food, water, or fomites contaminated with virus-containing feces or vomit. People can be infected without showing symptoms. The high frequency of secondary cases of NLV illness and - to a lesser extent - of hepatitis A following a foodborne outbreak results in amplification of the problem. The burden of illness is highest in the elderly, and therefore is likely to increase due to the aging population. For HAV, the burden of illness may increase following hygienic control measures, due to a decreasing population of naturally immune individuals and a concurrent increase in the population at risk. Recent advances in the research of NLV and HAV have led to the development of molecular methods which can be used for molecular tracing of virus strains. These methods can be and have been used for the detection of common source outbreaks. While traditionally certain foods have been implicated in virus outbreaks, it is clear that almost any food item can be involved, provided it has been handled by an infected person. There are no established methods for detection of viruses in foods other than shellfish. Little information is available on disinfection and preventive measures specifically for these viruses. Studies addressing this issue are hampered by the lack of culture systems. As currently available routine monitoring systems exclusively focus on bacterial pathogens, efforts should be made to combine epidemiological and virological information for a combined laboratory-based rapid detection system for foodborne viruses. With better surveillance, including typing information, outbreaks of foodborne infections could be reported faster to prevent further spread.

Natural History of Human Calicivirus Infection: a Prospective Cohort Study

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. Aug, 2002  |  Pubmed ID: 12115089

We investigated the natural history of human Calicivirus infection in the community. Clinical information was obtained from 99 subjects infected with Norwalk-like viruses (NLV) and 40 subjects infected with Sapporo-like viruses (SLV) in a prospective, community-based cohort study. NLV infection was common in all age groups, whereas SLV infection was mainly restricted to children aged <5 years. Symptoms lasted for a median of 5 and 6 days for NLV and SLV infections, respectively. Disease was characterized by diarrhea during the first 5 days (87% of patients with NLV infection and 95% of patients with SLV infection) and vomiting on the first day (74% for NLV and 60% for SLV). Vomiting was less common in children aged <1 year (59% for NLV and 44% for SLV) than it was among children aged >/=1 year (>75% for NLV and >67% for SLV). Overall, NLV was detected in 26% of patients up to 3 weeks after the onset of illness. This proportion was highest (38%) for children aged <1 year. SLV shedding subsided after 14 days. These data show that the durations of disease and viral shedding of caliciviruses are longer than has been described elsewhere. Therefore, the impact of these infections may have been underestimated.

A Waterborne Outbreak of Norwalk-like Virus Among Snowmobilers-Wyoming, 2001

The Journal of Infectious Diseases. Jan, 2003  |  Pubmed ID: 12552455

In February 2001, episodes of acute gastroenteritis were reported to the Wyoming Department of Health from persons who had recently vacationed at a snowmobile lodge in Wyoming. A retrospective cohort study found a significant association between water consumption and illness, and testing identified Norwalk-like virus (NLV) in 8 of 13 stool samples and 1 well. Nucleotide sequences from the positive well-water specimen and 6 of the positive stool samples were identical. This multistrain NLV outbreak investigation illustrates the importance of NLV as a cause of waterborne illness and should encourage monitoring for NLVs in drinking water.

Epidemiology of Norwalk-like Virus Infections in Cattle in The Netherlands

Veterinary Microbiology. Apr, 2003  |  Pubmed ID: 12554100

"Norwalk-like viruses" (NLVs) are the most common cause of acute non-bacterial gastroenteritis in humans. Cattle may be a reservoir of NLVs although never bovine NLVs have been found in humans. To gain more insight into the epidemiology of NLV, infections in cattle in The Netherlands were studied. Individual faecal samples from a large dairy herd and 243 pooled samples from veal calf farms were analysed for NLV by RT-PCR. Calves under 3 months of age in the dairy herd were sampled three to five times with 3-week intervals, whereas dairy cattle were sampled twice with a 2-month interval. In 31.6% (77/243) of the veal calf farm samples and in 4.2% (13/312) of the individual dairy cattle samples NLV was detected. The mean age of virus positive dairy cattle was 2.5 months. The highest numbers of NLV positive veal calf farms in The Netherlands were found in the regions with the highest number of veal calf farms. NLV infected veal calf farms were detected in every month throughout the study period. Cattle appeared to be hosts of NLVs, and virus shedding was weakly associated with diarrhoea. Complete ORF2 sequences were obtained from two calf NLVs and phylogenetic analyses suggested that these strains belong to a distinct cluster (GGIII/2) in between GGI and GGII NLVs of humans. Overall, genetic variation between strains as determined by sequence analysis of the P1/P2 capsid region was limited to 14.6%. Our data shows that NLV is endemic in the cattle population in The Netherlands and genetically distinct from NLVs in humans.

International Collaborative Study to Compare Reverse Transcriptase PCR Assays for Detection and Genotyping of Noroviruses

Journal of Clinical Microbiology. Apr, 2003  |  Pubmed ID: 12682125

To allow more rapid and internationally standardized assessment of the spread of noroviruses (previously called Norwalk-like viruses [NLVs]) as important food-borne pathogens, harmonization of methods for their detection is needed. Diagnosis of NLVs in clinical diagnostic laboratories is usually performed by reverse transciptase PCR (RT-PCR) assays. In the present study, the performance of five different RT-PCR assays for the detection of NLVs was evaluated in an international collaborative study by five laboratories in five countries with a coded panel of 91 fecal specimens. The assays were tested for their sensitivity, detection limit, and ease of standardization. In total, NLVs could be detected by at least one RT-PCR assay in 69 (84%) of the samples that originally tested positive. Sensitivity ranged from 52 to 73% overall and from 54 to 100% and 58 to 85% for genogroup I and II viruses, respectively. In all, 64% of the false-negative results were obtained with a set of diluted stools (n = 20) that may have lost quality upon storage. Sensitivity was improved when these samples were excluded from analysis. No one single assay stood out as the best, although the p1 assay demonstrated the most satisfactory overall performance. To promote comparability of data, this assay will be recommended for newly starting groups in future collaborative studies.

Development and Application of a Capsid VP1 (region D) Based Reverse Transcription PCR Assay for Genotyping of Genogroup I and II Noroviruses

Journal of Virological Methods. Mar, 2004  |  Pubmed ID: 14738976

Noroviruses (NoV), previously called "Norwalk-like viruses", have emerged as the single most important cause of acute gastroenteritis worldwide. Most diagnostic reverse transcription-polymerase chain reaction (RT-PCR) assays target the viral RNA-dependent RNA polymerase; however, the major capsid protein (VP1) is the reference genomic region for establishing genotypes. In this study, we analyzed complete NoV VP1 sequences (n=100) and determined a region (region D) that was most suitable to differentiate between genotypes. Within region D, we designed two genogroup specific, broadly reactive, degenerate primer sets (GI and GII). The region D primers were evaluated in a single-tube one-step RT-PCR assay using a panel of 81 (31 GI, 50 GII) NoV strains from both outbreaks and sporadic cases. In total, 95% of the samples tested positive using the new region D primer sets. Phylogenetic analysis of region D sequences (36 deduced amino acids for GI, 56 deduced amino acids for GII), revealed 19 clusters (7 within GI and 12 within GII) including three new genetically distinct clusters, two of which were unresolved using region A sequences. Phylogenetic analysis of the complete VP1 sequences revealed identical grouping of strains and confirmed the newly identified clusters using region D. In summary, we successfully developed and evaluated a broadly reactive RT-PCR assay for reliable genotyping of GI and GII noroviruses.

Norovirus Capture with Histo-blood Group Antigens Reveals Novel Virus-ligand Interactions

Journal of Virology. Mar, 2004  |  Pubmed ID: 14990722

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

Isolation and Characterization of Circulating Type 1 Vaccine-derived Poliovirus from Sewage and Stream Waters in Hispaniola

The Journal of Infectious Diseases. Apr, 2004  |  Pubmed ID: 15031784

Twenty-one cases of acute flaccid paralysis (AFP) were reported on the island of Hispaniola in 2000. Laboratory analysis confirmed the presence of circulating vaccine-derived poliovirus (cVDPV) type 1 in stool samples obtained from patients. As a complement to the active search for cases of AFP, environmental sampling was conducted during November and December 2000, to test for cVDPV in sewage, streams, canals, and public latrines. Fifty-five environmental samples were obtained and analyzed for the presence of polioviruses by use of cell culture followed by neutralization and reverse-transcription polymerase chain reaction. Of the 23 positive samples, 10 tested positive for poliovirus type 1, 7 tested positive for poliovirus type 2, 5 tested positive for poliovirus type 3, and 1 tested positive for both poliovirus type 2 and type 3. By sequence analysis of the complete viral capsid gene 1 (VP1), a 2.1%-3.7% genetic sequence difference between 7 type 1 strains and Sabin type 1 vaccine strain was found. Phylogenetic analysis showed that these viruses are highly related to cVDPV isolated from clinical cases and form distinct subclusters related to geographic region. Our findings demonstrate a useful role for environmental surveillance of neurovirulent polioviruses in the overall polio eradication program.

Molecular Detection and Genotyping of Male-specific Coliphages by Reverse Transcription-PCR and Reverse Line Blot Hybridization

Applied and Environmental Microbiology. Oct, 2004  |  Pubmed ID: 15466543

In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Q beta, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies.

Assessment of Areas at Increased Risk for Poliovirus Circulation in Ecuador

Epidemiology and Infection. Oct, 2004  |  Pubmed ID: 15473140

To assess areas at risk for poliovirus circulation in Ecuador, we first selected provinces at highest risk based on low immunization coverage with three doses of oral poliovirus vaccine, and a low number of reported cases of acute flaccid paralysis (AFP). Subsequently, we reviewed discharge data for the period 1996--2000 for diagnoses compatible with AFP in the only two national referral hospitals in Quito, and at least two main hospitals in each of the six selected provinces. Environmental samples from one or two cities/towns in each selected province were tested for poliovirus. Of the 14 identified AFP-compatible cases, 8 (57%) had been previously reported and investigated. We visited four out of the six unreported cases; none of those four had sequelae compatible with poliomyelitis. From the 14 environmental samples taken, we identified Sabin viruses in six of the samples; no vaccine-derived polioviruses were isolated. Using this methodology, we found no evidence of undetected poliovirus circulation in Ecuador.

Detection of Serum Antibodies to Bovine Norovirus in Veterinarians and the General Population in the Netherlands

Journal of Medical Virology. May, 2005  |  Pubmed ID: 15779045

The close genetic relationship of human and animal strains of norovirus has raised the possibility of transmission of noroviruses from animals to humans and may explain the emergence of certain norovirus strains. To assess if exposure to bovine noroviruses (NoV) might result in infection in humans, an enzyme immunoassay (EIA) was designed and validated in order to detect antibodies against bovine norovirus. This and two other EIAs were used to test sera from 210 veterinarians and 630 matched population controls for IgG and IgA antibodies to recombinant capsid protein of bovine NoV (rBoV), Norwalk virus (rNV), and Lordsdale virus (rLDV). Of 840 participants, IgG reactivity to rBoV was found in 185 (22%), to rNV in 638 (76%) and to rLDV in 760 (90%). IgG reactivity to rBoV was more common in veterinarians (58/210: 28%) than in controls (127/630: 20% [P = 0.03]). IgA reactivity to rBoV was similar in both veterinarians and controls. Cross-reactivity of IgA and IgG antibodies to rBoV and rNV was seen, but 26% of all specimens positive rBoV antibodies showed high IgG reactivity to rBoV but low reactivity to rNV, suggesting a specific response to bovine antigen. No evidence of overall cross-reactivity of antibodies to rBoV and rLDV was seen. Among veterinarians, youth spent on farm (Odds Ratio [OR] = 1.8) and membership of the bovine practitioners' society (OR = 2.7) were significantly associated with IgG seroreactivity to rBoV. These data indicate that bovine strains of NoV may infect humans though less frequently than human strains.

Rapid and Sensitive Detection of Noroviruses by Using TaqMan-based One-step Reverse Transcription-PCR Assays and Application to Naturally Contaminated Shellfish Samples

Applied and Environmental Microbiology. Apr, 2005  |  Pubmed ID: 15812014

Noroviruses (NoV), which are members of the family Caliciviridae, are the most important cause of outbreaks of acute gastroenteritis worldwide and are commonly found in shellfish grown in polluted waters. In the present study, we developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII NoV in fecal samples, as well as shellfish samples. The specificity and sensitivity of all steps of the assays were systematically evaluated, and in the final format, the monoplex assays were validated by using RNA extracted from a panel of 84 stool specimens, which included NoV strains representing 19 different genotypes (7 GI, 11 GII, and 1 GIV strains). The assays were further validated with 38 shellfish cDNA extracts previously tested by nested PCR. Comparison with a recently described real-time assay showed that our assay had significantly higher sensitivity and was at least as sensitive as the nested PCR. For stool specimens, a one-step duplex TaqMan RT-PCR assay performed as well as individual genogroup-specific monoplex assays. All other enteric viruses examined were negative, and no cross-reaction between genogroups was observed. These TaqMan RT-PCR assays provide rapid (less than 90 min), sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical and shellfish samples.

Lessons Learned from a Norovirus Outbreak in a Locked Pediatric Inpatient Psychiatric Unit

Infection Control and Hospital Epidemiology. Oct, 2005  |  Pubmed ID: 16276961

We report an outbreak of norovirus in a locked pediatric inpatient psychiatric unit with attack rates of 75% among 4 patients and 26% among 38 staff. Factors contributing to the outbreak included environmental contamination, close staff-patient contact including sharing meals, and inability to confine the index patient with the use of contact precautions.

Sequence Variation Among Group III F-specific RNA Coliphages from Water Samples and Swine Lagoons

Applied and Environmental Microbiology. Feb, 2006  |  Pubmed ID: 16461670

Typing of F-specific RNA (FRNA) coliphages has been proposed as a useful method for distinguishing human from animal fecal contamination in environmental samples. Group II and III FRNA coliphages are generally associated with human wastes, but several exceptions have been noted. In the present study, we have genotyped and partially sequenced group III FRNA coliphage field isolates from swine lagoons in North Carolina (NC) and South Carolina (SC), along with isolates from surface waters and municipal wastewaters. Phylogenetic analysis of a region of the 5' end of the maturation protein gene revealed two genetically different group III FRNA subclusters with 36.6% sequence variation. The SC swine lagoon isolates were more closely related to group III prototype virus M11, whereas the isolates from a swine lagoon in NC, surface waters, and wastewaters grouped with prototype virus Q-beta. These results suggest that refining phage genotyping systems to discriminate M11-like phages from Q-beta-like phages would not necessarily provide greater discriminatory power in distinguishing human from animal sources of pollution. Within the group III subclusters, nucleotide sequence diversity ranged from 0% to 6.9% for M11-like strains and from 0% to 8.7% for Q-beta-like strains. It is demonstrated here that nucleotide sequencing of closely related FRNA strains can be used to help track sources of contamination in surface waters. A similar use of phage genomic sequence information to track fecal pollution promises more reliable results than phage typing by nucleic acid hybridization and may hold more potential for field applications.

Genetic Diversity of Norovirus Among Children with Gastroenteritis in São Paulo State, Brazil

Journal of Clinical Microbiology. Nov, 2006  |  Pubmed ID: 16943348

Norovirus (NoV) is one of the most common causes of acute gastroenteritis in children and adults. To study the prevalence and genetic variability of NoV in children with acute gastroenteritis in São Paulo State, Brazil, we examined 234 stool samples from children with or without gastroenteritis during a 5-year period (1995 to 1999). NoV RNA was detected by reverse transcription-PCR and confirmed by DNA sequence analysis. We used two different oligonucleotide primer sets targeting the 3' end of the RNA polymerase gene (region B), as well a partial capsid region at the 3' end of the VP1 gene (region D). A total of 78 (33.3%) of the samples tested positive for NoV, and in region B, of the 66 strains sequenced, 4 (6.1%) belonged to GI, 52 (78.7%) belonged to GII, and five samples (7.6%) contained a mixture of the GI and GII genotypes. Phylogenetic analysis showed that the majority (40 of 66 [60.6%]) of the strains belonged to genotype GII.4. The nucleotide sequence identity of three strains was lower than 77.9% compared to a region B reference sequence database but showed 85.3 to 88.8% identity with GII.2 Melksham strain in region D, indicating the circulation of a possible recombinant NoV strain. One sample (GII.3) was sequenced only in region D. In conclusion, we have a total of 67 sequenced strains. This is the first report that describes the predominance of GII.4 NoV strains in children visiting the ambulatory of different hospitals in São Paulo State, Brazil, and we show that mixtures of different strains can be found in individual samples, including some possible new recombinant strains.

Surrogates for the Study of Norovirus Stability and Inactivation in the Environment: AA Comparison of Murine Norovirus and Feline Calicivirus

Journal of Food Protection. Nov, 2006  |  Pubmed ID: 17133824

Human noroviruses (NoVs) are the leading cause of food- and waterborne outbreaks of acute nonbacterial gastroenteritis worldwide. As a result of the lack of a mammalian cell culture model for these viruses, studies on persistence, inactivation, and transmission have been limited to cultivable viruses, including feline calicivirus (FCV). Recently, reports of the successful cell culture of murine norovirus 1 (MNV-1) have provided investigators with an alternative surrogate for human NoVs. In this study, we compared the inactivation profiles of MNV-1 to FCV in an effort to establish the relevance of MNV-1 as a surrogate virus. Specifically, we evaluated (i) stability upon exposure to pH extremes; (ii) stability upon exposure to organic solvents; (iii) thermal inactivation; and (iv) surface persistence under wet and dry conditions. MNV-1 was stable across the entire pH range tested (pH 2 to 10) with less than 1 log reduction in infectivity at pH 2, whereas FCV was inactivated rapidly at pH values < 3 and > 9. FCV was more stable than MNV-1 at 56 degrees C, but both viruses exhibited similar inactivation at 63 and 72 degrees C. Long-term persistence of both viruses suspended in a fecal matrix and inoculated onto stainless steel coupons were similar at 4 degrees C, but at room temperature in solution, MNV-1 was more stable than FCV. The genetic relatedness of MNV-1 to human NoVs combined with its ability to survive under gastric pH levels makes this virus a promising and relevant surrogate for studying environmental survival of human NoVs.

Mechanisms of GII.4 Norovirus Persistence in Human Populations

PLoS Medicine. Feb, 2008  |  Pubmed ID: 18271619

Noroviruses are the leading cause of viral acute gastroenteritis in humans, noted for causing epidemic outbreaks in communities, the military, cruise ships, hospitals, and assisted living communities. The evolutionary mechanisms governing the persistence and emergence of new norovirus strains in human populations are unknown. Primarily organized by sequence homology into two major human genogroups defined by multiple genoclusters, the majority of norovirus outbreaks are caused by viruses from the GII.4 genocluster, which was first recognized as the major epidemic strain in the mid-1990s. Previous studies by our laboratory and others indicate that some noroviruses readily infect individuals who carry a gene encoding a functional alpha-1,2-fucosyltransferase (FUT2) and are designated "secretor-positive" to indicate that they express ABH histo-blood group antigens (HBGAs), a highly heterogeneous group of related carbohydrates on mucosal surfaces. Individuals with defects in the FUT2 gene are termed secretor-negative, do not express the appropriate HBGA necessary for docking, and are resistant to Norwalk infection. These data argue that FUT2 and other genes encoding enzymes that regulate processing of the HBGA carbohydrates function as susceptibility alleles. However, secretor-negative individuals can be infected with other norovirus strains, and reinfection with the GII.4 strains is common in human populations. In this article, we analyze molecular mechanisms governing GII.4 epidemiology, susceptibility, and persistence in human populations.

Passenger Behaviors During Norovirus Outbreaks on Cruise Ships

Journal of Travel Medicine. May-Jun, 2008  |  Pubmed ID: 18494694

Norovirus causes a majority of outbreaks of gastrointestinal (GI) illness on cruise ships calling on the United States. Control measures include patient isolation, hand washing, and facility closure. Little is known about the behaviors and practices of people who have become ill with norovirus GI illness compared to those who remained well during an outbreak.

Detection of a Novel Intergenogroup Recombinant Norovirus from Kolkata, India

Virology. Jul, 2008  |  Pubmed ID: 18555887

Mutation and recombination are recognized as important driving forces of evolution among RNA viruses. An intergenogroup recombinant norovirus strain [Hu/Kol/NLV/L8775/AB290150/2006/India] was detected in the faecal specimen of a 17 year old male, who had suffered from acute watery diarrhea and severe dehydration. Sequence analysis confirmed that this novel recombinant strain had a polymerase gene fragment that closely resembled a Norovirus (NoV) genogroup-I genotype-3 virus (HuCV/NLV/GI.3/VA98115/AY038598/1998/USA) and a capsid gene resembling NoV genogroup-II genotype-4 virus (NoV/Hu/GII.4/Terneuzen70/EF126964/2006/NL). The crossing over and recombination was observed at nucleotide (nt) 790 of NoV GI VA98115 strain and nt808 of NoV GII Terneuzen70 strain. In both parent strains conserved nucleotide sequence and hairpin structure (DNA secondary structure) were reported at the junction point of ORF1 and ORF2, exhibiting the mechanism of recombination in these viruses. Thus this novel recombinant NoV is another step in evolution among NoVs, indicating that constant surveillance is important to successfully monitor emergence of these strains.

Systematic Literature Review of Role of Noroviruses in Sporadic Gastroenteritis

Emerging Infectious Diseases. Aug, 2008  |  Pubmed ID: 18680645

We conducted a systematic review of studies that used reverse transcription-PCR to diagnose norovirus (NoV) infections in patients with mild or moderate (outpatient) and severe (hospitalized) diarrhea. NoVs accounted for 12%(95% confidence interval [CI] 10%-15%) of severe gastroenteritis cases among children <5 years of age and 12% (95% CI 9%-15%) of mild and moderate diarrhea cases among persons of all ages. Of 19 studies among children <5 years of age, 7 were in developing countries where pooled prevalence of severe NoV disease (12%) was comparable to that for industrialized countries (12%). We estimate that each year NoVs cause 64,000 episodes of diarrhea requiring hospitalization and 900,000 clinic visits among children in industrialized countries, and up to 200,000 deaths of children <5 years of age in developing countries. Future efforts should focus on developing targeted strategies, possibly even vaccines, for preventing NoV disease and better documenting their impact among children living in developing countries, where >95% of the deaths from diarrhea occur.

Histo-blood Group Antigen Assay for Detecting Noroviruses in Water

Applied and Environmental Microbiology. Nov, 2008  |  Pubmed ID: 18776025

We evaluated a novel, magnetic-bead-based histo-blood group antigen assay for the recovery of low numbers of norovirus particles. Using this assay, with Norwalk virus seeded in environmental waters as a model, we were able to recover 30 to 300 genomic copies of the virus.

Self-assembly of the Recombinant Capsid Protein of a Swine Norovirus into Virus-like Particles and Evaluation of Monoclonal Antibodies Cross-reactive with a Human Strain from Genogroup II

Journal of Clinical Microbiology. Dec, 2008  |  Pubmed ID: 18842943

Noroviruses (NoVs) are responsible for the majority of gastroenteritis outbreaks in humans. Recently, NoV strains which are genetically closely related to human genogroup II (GII) NoVs have been detected in fecal specimens from swine. These findings have raised concern about the possible role of pigs as reservoirs for NoVs that could infect humans. To better understand the epidemiology of swine NoVs in both the swine and the human populations, rapid immunoassays are needed. In this study, baculovirus recombinants were generated to express the capsid gene of a swine NoV GII genotype 11 (GII.11) strain which self-assembled into virus-like particles (VLPs). Subsequently, the purified VLPs were used to evoke monoclonal antibodies (MAbs) in mice. A panel of eight promising MAbs was obtained and evaluated for their ability to bind to heterologous VLPs, denaturated antigens, and truncated capsid proteins. The MAbs could be classified into two groups: two MAbs that recognized linear epitopes located at the amino-terminal half (shell domain) of the swine NoV GII.11 VLPs and that cross-reacted with human GII.4 NoV VLPs. The other six MAbs bound to conformational epitopes and did not cross-react with the human GII.4 VLPs. To our knowledge, this is the first report on the characterization of MAbs against swine NoVs. The swine NoV VLPs and the MAbs described here may be further used for the design of diagnostic reagents that could help increase our knowledge of the prevalence of NoV infections in pigs and the possible role of pigs as reservoirs for NoVs.

Prospective Study of Etiologic Agents of Acute Gastroenteritis Outbreaks in Child Care Centers

The Journal of Pediatrics. Feb, 2009  |  Pubmed ID: 18783794

To investigate the etiology of outbreaks of acute gastroenteritis (AGE) in children attending childcare centers (CCCs) in North Carolina between October 2005 and March 2007.

Outbreak of Norovirus Infection Among River Rafters Associated with Packaged Delicatessen Meat, Grand Canyon, 2005

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. Jan, 2009  |  Pubmed ID: 19025489

Norovirus is often transmitted by infected food handlers at the point of service, whereas reports of food contamination before wholesale distribution are rare. In September 2005, we investigated reports of gastroenteritis among rafters who went on unrelated trips on the Colorado River.

A Rapid and Efficient Method for Quantitation of Genogroups I and II Norovirus from Oysters and Application in Other Complex Environmental Samples

Journal of Virological Methods. Mar, 2009  |  Pubmed ID: 19041894

The human health risk associated with the consumption of molluscan shellfish grown in sewage-contaminated waters is well established. Noroviruses are the principal agent of shellfish-related illness. This study describes the evaluation of two silica-based viral RNA extraction protocols as well as two real time RT-PCR assays for norovirus detection in shellfish and plankton. Using a GII RNA transcript, the Qiagen RNeasy method was able to recover 80%, 1.85%, and 0.14% of the RNA copies in seeded oyster, small plankton (63-200microm), and large plankton (>200microm) samples, respectively, whereas a silica-bead based method was able to recover only 0.175%, 0.0044%, and 0.0006% in the same seeded samples. The detection limit of two published TaqMan RT-PCR assays (A and B) evaluated with RNA run-off transcripts established RT-PCR assay A was more sensitive for detecting low copies of GI.3 RNA whereas RT-PCR assay B was more sensitive for detecting GI.4 and GII.4; however, only assay A was able to detect GI and GII in naturally contaminated shellfish whereas only assay B was able to detect GI and GII in naturally contaminated plankton. The combination of a rapid RNA extraction method followed by both TaqMan RT-PCR assays offers significant advantages for development of routine assays for norovirus detection in bivalve shellfish and shows promise for detection in other high inhibitor environmental sources, such as plankton.

Noroviruses: a Comprehensive Review

Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology. Jan, 2009  |  Pubmed ID: 19084472

Herd Immunity to GII.4 Noroviruses is Supported by Outbreak Patient Sera

Journal of Virology. Jun, 2009  |  Pubmed ID: 19297483

Noroviruses (NoVs) of genogroup II, cluster 4 (GII.4), are the most common cause of outbreaks of acute gastroenteritis worldwide. During the past 13 years, GII.4 NoVs caused four seasons of widespread activity globally, each associated with the emergence of a new strain. In this report, we characterized the most recent epidemic strain, GII.4-2006 Minerva, by comparing virus-like particle (VLP) antigenic relationships and histo-blood group antigen (HBGA) binding profiles with strains isolated earlier. We also investigated the seroprevalence and specificity of GII.4 antibody in the years prior to, during, and following the GII.4 pandemic of 1995 and 1996 using a large collection of acute- and convalescent-phase serum pairs (n = 298) collected from 34 outbreaks. In a surrogate neutralization assay, we measured the blockade of HBGA binding using a panel of GII.4 VLPs representing strains isolated in 1987, 1997, 2002, and 2006 and a GII.3 VLP representing a strain isolated in the mid-1990s. Serum titers required for 50% HBGA blockade were compared between populations. In general, blockade of GII.4 VLP-HBGA binding was greater with convalescent-phase outbreak sera collected near the time of origin of the VLP strain. Heterotypic genotypes did not contribute to herd immunity against GII.4 NoVs based on their inability to block GII.4 VLP binding to HBGA. However, previous exposure to GII.4 NoV followed by infection by GII.3 NoV appeared to evoke an immune response to GII.4 NoV. These results support the hypothesis that herd immunity is a driving force for GII.4 evolution in the U.S. population. The data also suggest that complex patterns of cross-protection may exist across NoV genotypes in humans.

Norovirus Distribution Within an Estuarine Environment

Applied and Environmental Microbiology. Sep, 2009  |  Pubmed ID: 19581478

Human norovirus (NoV) has been studied extensively as an important cause of gastroenteritis outbreaks worldwide. While oysters are a primary vehicle for infection, few studies have examined the wider distribution of NoV in the estuarine environment. Active shellfish-harvesting areas in Georgia were examined for the prevalence, genotype diversity, and concentrations of NoV in a variety of estuarine sample types over the course of 1 year. Of the 225 samples (9 oyster, 72 water, 72 63- to 200-microm plankton, and 72 >200-microm plankton) collected from 12 stations across two estuaries, 21 samples (9.3%) tested positive for NoV. By sample type, 55.0% (5/9) of oysters, 8.3% (6/72) of water samples, 11.1% (8/72) of 63- to 200-microm plankton samples, and 2.8% (2/72) of >200-microm plankton samples were positive for human NoV. The two NoV-positive >200-microm plankton samples, which contained mainly zooplankton, had the greatest quantity of NoV genomes (3.5 x 10(13) and 1.7 x 10(15) genomes g(-1)) of any sample tested. The majority, 90.5% (19/21), of the samples tested positive for genogroup I NoV, and only 9.5% (2/21) of the samples tested positive for genogroup II. The high concentrations of NoV in plankton samples compared to water and oyster samples were unexpected and provide new insights into the presence and distribution of human NoV in the water environment.

Norovirus Illness is a Global Problem: Emergence and Spread of Norovirus GII.4 Variants, 2001-2007

The Journal of Infectious Diseases. Sep, 2009  |  Pubmed ID: 19627248

Noroviruses (NoVs) are the most common cause of viral gastroenteritis. Their high incidence and importance in health care facilities result in a great impact on public health. Studies from around the world describing increasing prevalence have been difficult to compare because of differing nomenclatures for variants of the dominant genotype, GII.4. We studied the global patterns of GII.4 epidemiology in relation to its genetic diversity.

Identification of a Novel Astrovirus (astrovirus VA1) Associated with an Outbreak of Acute Gastroenteritis

Journal of Virology. Oct, 2009  |  Pubmed ID: 19706703

The etiology of a large proportion of gastrointestinal illness is unknown. In this study, random Sanger sequencing and pyrosequencing approaches were used to analyze fecal specimens from a gastroenteritis outbreak of unknown etiology in a child care center. Multiple sequences with limited identity to known astroviruses were identified. Assembly of the sequences and subsequent reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends generated a complete genome of 6,586 nucleotides. Phylogenetic analysis demonstrated that this virus, named astrovirus VA1 (AstV-VA1), is highly divergent from all previously described astroviruses. Based on RT-PCR, specimens from multiple patients in this outbreak were unequivocally positive for Ast-VA1.

Gene Mapping and Phylogenetic Analysis of the Complete Genome from 30 Single-stranded RNA Male-specific Coliphages (family Leviviridae)

Journal of Virology. Nov, 2009  |  Pubmed ID: 19710143

Male-specific single-stranded RNA (FRNA) coliphages belong to the family Leviviridae. They are classified into two genera (Levivirus and Allolevivirus), which can be subdivided into four genogroups (genogroups I and II in Levivirus and genogroups III and IV in Allolevivirus). Relatively few strains have been completely characterized, and hence, a detailed knowledge of this virus family is lacking. In this study, we sequenced and characterized the complete genomes of 19 FRNA strains (10 Levivirus strains and 9 Allolevivirus strains) and compared them to the 11 complete genome sequences available in GenBank. Nucleotide similarities among strains of Levivirus genogroups I and II were 75% to 99% and 83 to 94%, respectively, whereas similarities among strains of Allolevivirus genogroups III and IV ranged from 70 to 96% and 75 to 95%, respectively. Although genogroup I strain fr and genogroup III strains MX1 and M11 share only 70 to 78% sequence identity with strains in their respective genogroups, phylogenetic analyses of the complete genome and the individual genes suggest that strain fr should be grouped in Levivirus genogroup I and that the MX1 and M11 strains belong in Allolevivirus genogroup III. Strains within each genus share >50% sequence identity, whereas between the two genera, strains have <40% nucleotide sequence identity. Overall, amino acid composition, nucleotide similarities, and replicase catalytic domain location contributed to phylogenetic assignments. A conserved eight-nucleotide signature at the 3' end of the genome distinguishes leviviruses (5' ACCACCCA 3') from alloleviviruses (5' TCCTCCCA 3').

Multicenter Comparison of Two Norovirus ORF2-based Genotyping Protocols

Journal of Clinical Microbiology. Dec, 2009  |  Pubmed ID: 19846650

Point source norovirus outbreaks can be difficult to track due to high background levels of the virus in the environment and the limited strain variation in some genotyping regions. However, rapid and accurate source identification can limit the spread of a foodborne outbreak and reduce the number of cases. Harmonization of genotyping assays is critical for enabling the rapid exchange of sequence data nationally and internationally. Several regions of the genome have been proposed for this purpose, but no consensus has been reached. In the present study, two standardized genotyping protocols (region C and region D) were evaluated by nine laboratories in Canada and the United States, using a coded panel of 96 fecal specimens representing 22 different norovirus genotypes. Overall, region C typing had a success rate of 78% compared to 52% for region D; however, region D provides greater nucleotide sequence diversity for identifying new GII.4 variant strains. Significant differences in the genotyping success rate were observed among the nine participating laboratories (10% to 100%) and among the different genotypes (6% to 100%). For several genogroup II strains, reduced region D amplification correlated directly with mismatches between primer sequences and the template. Based on overall performance, we recommend the region C protocol for routine genotyping of noroviruses, while the region D protocol may be useful for identifying new GII.4 variants. Standardized genotyping protocols will enable rapid exchange of outbreak and sequence data through electronic norovirus surveillance networks.

Molecular Epidemiology of Genogroup II-genotype 4 Noroviruses in the United States Between 1994 and 2006

Journal of Clinical Microbiology. Jan, 2010  |  Pubmed ID: 19864482

Human noroviruses (NoVs) of genogroup II, genotype 4 (GII.4) are the most common strains detected in outbreaks of acute gastroenteritis worldwide. To gain insight into the epidemiology and genetic variation of GII.4 strains, we analyzed 773 NoV outbreaks reported to the CDC from 1994 to 2006. Of these NoV outbreaks, 629 (81.4%) were caused by GII viruses and 342 (44.2%) were caused by GII.4 strains. The proportion of GII.4 outbreaks increased from 5% in 1994 to 85% in 2006, but distinct annual differences were noted, including sharp increases in 1996, 2003, and 2006 each associated with newly emerging GII.4 strains. Sequence analysis of the full-length VP1 gene of GII.4 strains identified in this study and from GenBank segregated these viruses into at least 9 distinct subclusters which had 1.3 to 3.2% amino acid variation between strains in different subclusters. We propose that GII.4 subclusters be defined as having >5% sequence variation between strains. Our data confirm other studies on the rapid emergence and displacement of highly virulent GII.4 strains.

Novel Norovirus in Dogs with Diarrhea

Emerging Infectious Diseases. Jun, 2010  |  Pubmed ID: 20507751

To identify the prevalence and genetic variability of noroviruses in dogs, we tested fecal samples by using reverse transcription-PCR. We found canine norovirus in 40% and 9% of dogs with and without diarrhea, respectively. The virus was genetically unrelated to other noroviruses and constitutes a tentative new genogroup.

Diagnostic Accuracy and Analytical Sensitivity of IDEIA Norovirus Assay for Routine Screening of Human Norovirus

Journal of Clinical Microbiology. Aug, 2010  |  Pubmed ID: 20554813

Noroviruses (NoVs) are recognized as the leading cause of epidemic and sporadic acute gastroenteritis. Early detection of NoV is crucial to control the spread of the disease. In this study, we evaluated the diagnostic accuracy, analytical sensitivity, and analytical reactivity of the IDEIA Norovirus assay (an enzyme immunoassay [EIA]) in a prospective and retrospective study design. A total of 557 prospectively collected fecal samples and a panel of 97 archived fecal samples, including 21 different GI and GII genotypes, were tested by conventional reverse transcription-PCR (RT-PCR)/bidirectional sequencing, real-time RT-PCR, and electron microscopy. The sensitivity and specificity of the EIA were 57.6% and 91.9%, respectively. The sensitivity for detecting NoV in fecal samples from outbreaks improved from 44.1% when three samples were tested to 76.9% when five samples per outbreak were tested. The EIA was able to detect strains from 7 GI and 11 GII genotypes. The analytical sensitivity of the EIA was 3.1 x 10(6) and 1.6 x 10(7) virus particles g(-1) of fecal sample for NoV GI and GII strains, respectively. Most GII samples positive by EIA had a threshold cycle (C(T)) of <26.5, and 50% of the GII samples negative by EIA had a C(T) of >25.6, suggesting that, although strains from genotypes GI.8, GII.10, and GII.16 were not detected, the low sensitivity of the EIA is primarily caused by low virus concentration. In conclusion, the current EIA may be of use as a rapid screening test during a norovirus outbreak investigation when multiple fecal samples are available; however, sporadic samples should be tested by molecular methods.

A Norovirus Vaccine on the Horizon?

The Journal of Infectious Diseases. Dec, 2010  |  Pubmed ID: 20979457

Redefining Outcome of First Seizures by Acute Illness

Pediatrics. Dec, 2010  |  Pubmed ID: 21098153

Seizures are common in children, but the causes and recurrence risk for children with a nonfebrile first seizure remain poorly understood.

Comparative Efficacy of Seven Hand Sanitizers Against Murine Norovirus, Feline Calicivirus, and GII.4 Norovirus

Journal of Food Protection. Dec, 2010  |  Pubmed ID: 21219741

Contaminated hands or inanimate surfaces can act as a source of infection during outbreaks of human norovirus infection. We evaluated the virucidal efficacy of seven hand sanitizers containing various active ingredients, such as ethanol, triclosan, and chlorhexidine, and compared their effectiveness against feline calicivirus (FCV), murine norovirus (MNV), and a GII.4 norovirus fecal extract. We also tested the efficacy of 50, 70, and 90% of ethanol and isopropanol. Reduction of viral infectivity was measured by plaque assay, and the number of genomic copies was determined with a TaqMan real-time reverse transcription PCR assay. Based on the results of a quantitative suspension test, only one ethanol-based product (72% ethanol, pH 2.9) and one triclosan-based product (0.1% triclosan, pH 3.0) reduced the infectivity of both MNV and FCV (by >2.6 and ≥3.4 log units, respectively). Four of the seven products were effective against either MNV or FCV, whereas chlorhexidine was ineffective against both viruses. For these hand sanitizers, no correlation was found between reduced infectivity and decline of viral RNA. Ethanol and isopropanol concentrations ≥70% reduced the infectivity of MNV by ≥2.6 log units, whereas 50 and 70% ethanol reduced the infectivity of FCV by ≥2.2 log units after exposure for 5 min. The susceptibility of FCV to low pH and the relative high susceptibility of MNV to alcohols suggest that both surrogate viruses should be considered for in vitro testing of hand sanitizers.

Development and Evaluation of Novel One-step TaqMan Realtime RT-PCR Assays for the Detection and Direct Genotyping of Genogroup I and II Noroviruses

Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology. Mar, 2011  |  Pubmed ID: 21195660

Current detection and genotyping methods of genogroup (G) I and II noroviruses (NoVs) consist of a 2-step approach including detection of viral RNA by TaqMan realtime RT-PCR (RT-qPCR) followed by conventional RT-PCR and sequencing of partial regions of ORF1 or ORF2.

Incidence of Acute Gastroenteritis and Role of Norovirus, Georgia, USA, 2004-2005

Emerging Infectious Diseases. Aug, 2011  |  Pubmed ID: 21801613

Approximately 179 million cases of acute gastroenteritis (AGE) occur annually in the United States. However, lack of routine clinical testing for viruses limits understanding of their role among persons seeking medical care. Fecal specimens submitted for routine bacterial culture through a health maintenance organization in Georgia, USA, were tested with molecular diagnostic assays for norovirus, rotavirus, astrovirus, sapovirus, and adenovirus. Incidence was estimated by using national health care utilization rates. Routine clinical diagnostics identified a pathogen in 42 (7.3%) of 572 specimens; inclusion of molecular viral testing increased pathogen detection to 15.7%. Community AGE incidence was 41,000 cases/100,000 person-years and outpatient incidence was 5,400/100,000 person-years. Norovirus was the most common pathogen, accounting for 6,500 (16%) and 640 (12%) per 100,000 person-years of community and outpatient AGE episodes, respectively. This study demonstrates that noroviruses are leading causes of AGE among persons seeking medical care.

Novel Surveillance Network for Norovirus Gastroenteritis Outbreaks, United States

Emerging Infectious Diseases. Aug, 2011  |  Pubmed ID: 21801614

CaliciNet, the outbreak surveillance network for noroviruses in the United States, was launched in March 2009. As of January 2011, twenty state and local health laboratories had been certified to submit norovirus sequences and epidemiologic outbreak data to CaliciNet. During the network's first year, 552 outbreaks were submitted to CaliciNet, of which 78 (14%) were associated with foodborne transmission. A total of 395 (72%) outbreaks were typed as GII.4, of which 298 (75%) belonged to a new variant, GII.4 New Orleans, which first emerged in October 2009. Analysis of the complete capsid and P2 region sequences confirmed that GII.4 New Orleans is distinct from previous GII.4 variants, including GII.4 Minerva (2006b).

Novel GII.12 Norovirus Strain, United States, 2009-2010

Emerging Infectious Diseases. Aug, 2011  |  Pubmed ID: 21801639

In October 2009, a novel GII.12 norovirus strain emerged in the United States and caused 16% of all reported norovirus outbreaks during the winter season. Sequence analysis demonstrated a recombinant virus with a P2 region that was largely conserved compared with previously sequenced GII.12 strains.

Impact of an Emergent Norovirus Variant in 2009 on Norovirus Outbreak Activity in the United States

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. Sep, 2011  |  Pubmed ID: 21832262

In October 2009, a new genogroup II, type 4 (GII.4) norovirus variant was identified in the United States. We collected norovirus outbreak data from 30 states to assess whether this new strain was associated with increased acute gastroenteritis activity. No increase in norovirus outbreaks was observed during the 2009-2010 winter.

Monoclonal Antibody-based Antigenic Mapping of Norovirus GII.4-2002

Journal of Virology. Jan, 2012  |  Pubmed ID: 22090098

Noroviruses are the primary cause of epidemic gastroenteritis in humans, and GII.4 strains cause ∼80% of the overall disease burden. Surrogate neutralization assays using sera and mouse monoclonal antibodies (MAbs) suggest that antigenic variation maintains GII.4 persistence in the face of herd immunity, as the emergence of new pandemic strains is accompanied by newly evolved neutralization epitopes. To potentially identify specific blockade epitopes that are likely neutralizing and evolving between pandemic strains, mice were hyperimmunized with GII.4-2002 virus-like particles (VLPs) and the resulting MAbs were characterized by biochemical and immunologic assays. All of the MAbs but one recognized GII.4 VLPs representing strains circulating from 1987 to 2009. One MAb weakly recognized GII.4-1987 and -1997 while strongly interacting with 2002 VLPs. This antibody was highly selective and effective at blocking only GII.4-2002-ligand binding. Using bioinformatic analyses, we predicted an evolving GII.4 surface epitope composed of amino acids 407, 412, and 413 and subsequently built mutant VLPs to test the impact of the epitope on MAb binding and blockade potential. Replacement of the 2002 epitope with the epitopes found in 1987 or 2006 strains either reduced or ablated enzyme immunoassay recognition by the GII.4-2002-specific blockade MAb. These data identify a novel, evolving blockade epitope that may be associated with protective immunity, providing further support for the hypotheses that GII.4 norovirus evolution results in antigenic variation that allows the virus to escape from protective herd immunity, resulting in new epidemic strains.

Detection and Molecular Characterization of Noroviruses and Sapoviruses in Children Admitted to Hospital with Acute Gastroenteritis in Vietnam

Journal of Medical Virology. Feb, 2012  |  Pubmed ID: 22170550

Noroviruses (NoV) and sapoviruses (SaV) are recognized as important causes of acute gastroenteritis in children worldwide. In this study, the prevalence and genetic variability of NoV and SaV were determined in hospitalized children <5 years of age with acute gastroenteritis in Hanoi, Vietnam. A total of 501 fecal specimens collected between November-2007 and October-2008, that previously had been tested for rotavirus (RV), were tested for NoV and SaV by realtime RT-PCR. Positive samples were genotyped by conventional RT-PCR followed by sequencing. GII NoV was detected in 180 (36%) and SaV in 7 (1.4%) of the samples. NoV was detected year-round ranging from 9.5% in April to 81.5% in September among RV negative samples. NoV GII.4 Minerva (2006b) was the dominant genotype (93%) with a few other genotypes detected including GII.3 (4.4%), GII.13 (1.7%), and GII.2 (0.6%) but no GI strains. Only GI and GII SaV strains were detected in this study. No difference in NoV prevalence between age groups was noted. Frequency of vomiting or fever was similar between children with NoV and RV infection, yet, NoV caused diarrhea with longer duration. In conclusion, NoV is the second most frequent cause of diarrhea in hospitalized children in North Vietnam.

Epidemiologic and Clinical Features of Other Enteric Viruses Associated with Acute Gastroenteritis in American Indian Infants

The Journal of Pediatrics. Jul, 2012  |  Pubmed ID: 22336577

To investigate the viral etiology, through the use of molecular methods, of acute gastroenteritis (AGE), which is a considerable public health burden in Native American infants.

Environmental Transmission of Norovirus Gastroenteritis

Current Opinion in Virology. Feb, 2012  |  Pubmed ID: 22440972

The advent of molecular techniques and their increasingly widespread use in public health laboratories and research studies has transformed the understanding of the burden of norovirus. Norovirus is the most common cause of community-acquired diarrheal disease across all ages, the most common cause of outbreaks of gastroenteritis, and the most common cause of foodborne disease in the United States. They are a diverse group of single-stranded RNA viruses that are highly infectious and stable in the environment; both symptomatic and asymptomatic infections are common. Through shedding in feces and vomit, norovirus can be transmitted directly through an array of routes: person-to-person, food or the environment. The relative importance of environmental transmission of virus is yet to be fully quantified but is likely to be substantial and is an important feature that complicates control.

The Etiology of Severe Acute Gastroenteritis Among Adults Visiting Emergency Departments in the United States

The Journal of Infectious Diseases. May, 2012  |  Pubmed ID: 22454468

Acute gastroenteritis (AGE) remains a common cause of clinic visits and hospitalizations in the United States, but the etiology is rarely determined.

Sapovirus Outbreaks in Long-term Care Facilities, Oregon and Minnesota, USA, 2002-2009

Emerging Infectious Diseases. May, 2012  |  Pubmed ID: 22516204

We tested fecal samples from 93 norovirus-negative gastroenteritis outbreaks; 21 outbreaks were caused by sapovirus. Of these, 71% were caused by sapovirus genogroup IV and 66% occurred in long-term care facilities. Future investigation of gastroenteritis outbreaks should include multi-organism testing.

Experimental Inoculation of Juvenile Rhesus Macaques with Primate Enteric Caliciviruses

PloS One. 2012  |  Pubmed ID: 22666426

Tissue culture-adapted Tulane virus (TV), a GI.1 rhesus enteric calicivirus (ReCV), and a mixture of GII.2 and GII.4 human norovirus (NoV)-containing stool sample were used to intrastomacheally inoculate juvenile rhesus macaques (Macaca mulatta) in order to evaluate infection caused by these viruses. METHODOLOGY & FINDINGS: Two of the three TV-inoculated macaques developed diarrhea, fever, virus-shedding in stools, inflammation of duodenum and 16-fold increase of TV-neutralizing (VN) serum antibodies but no vomiting or viremia. No VN-antibody responses could be detected against a GI.2 ReCV strain FT285, suggesting that TV and FT285 represent different ReCV serotypes. Both NoV-inoculated macaques remained asymptomatic but with demonstrable virus shedding in one animal. Examination of duodenum biopsies of the TV-inoculated macaques showed lymphocytic infiltration of the lamina propria and villous blunting. TV antigen-positive (TV+) cells were detected in the lamina propria. In most of the TV+ cells TV co-localized perinuclearly with calnexin--an endoplasmic reticulum protein. A few CD20+TV+ double-positive B cells were also identified in duodenum. To corroborate the authenticity of CD20+TV+ B cells, in vitro cultures of peripheral blood mononuclear cells (PBMCs) from healthy macaques were inoculated with TV. Multicolor flow cytometry confirmed the presence of TV antigen-containing B cells of predominantly CD20+HLA-DR+ phenotype. A 2-log increase of viral RNA by 6 days post inoculation (p<0.05) suggested active TV replication in cultured lymphocytes.

Norovirus Outbreak of Probable Waterborne Transmission with High Attack Rate in a Guatemalan Resort

Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology. Sep, 2012  |  Pubmed ID: 22776162

In February 2009, a group of Guatemalan school children developed acute gastroenteritis (AGE) after participating in a school excursion.

Risk Factors for Death Among Children Less Than 5 Years Old Hospitalized with Diarrhea in Rural Western Kenya, 2005-2007: a Cohort Study

PLoS Medicine. 2012  |  Pubmed ID: 22802736

Diarrhea is a leading cause of childhood morbidity and mortality in sub-Saharan Africa. Data on risk factors for mortality are limited. We conducted hospital-based surveillance to characterize the etiology of diarrhea and identify risk factors for death among children hospitalized with diarrhea in rural western Kenya.

Antiviral Activity of Nucleoside Analogues Against Norovirus

Antiviral Therapy. 2012  |  Pubmed ID: 22910194

Norovirus (NoV) is the leading cause of epidemic gastroenteritis worldwide. The lack of a cell culture has significantly hampered the development of effective therapies against human NoV. Clinically approved nucleoside and non-nucleoside analogues have been used successfully against RNA viruses.

Sapovirus Gastroenteritis in Preschool Center, Puerto Rico, 2011

Emerging Infectious Diseases. Jan, 2013  |  Pubmed ID: 23260219

Emergence of a Norovirus GII.4 Strain Correlates with Changes in Evolving Blockade Epitopes

Journal of Virology. Mar, 2013  |  Pubmed ID: 23269783

The major capsid protein of norovirus GII.4 strains is evolving rapidly, resulting in epidemic strains with altered antigenicity. GII.4.2006 Minerva strains circulated at pandemic levels in 2006 and persisted at lower levels until 2009. In 2009, a new GII.4 variant, GII.4.2009 New Orleans, emerged and since then has become the predominant strain circulating in human populations. To determine whether changes in evolving blockade epitopes correlate with the emergence of the GII.4.2009 New Orleans strains, we compared the antibody reactivity of a panel of mouse monoclonal antibodies (MAbs) against GII.4.2006 and GII.4.2009 virus-like particles (VLPs). Both anti-GII.4.2006 and GII.4.2009 MAbs effectively differentiated the two strains by VLP-carbohydrate ligand blockade assay. Most of the GII.4.2006 MAbs preferentially blocked GII.4.2006, while all of the GII.4.2009 MAbs preferentially blocked GII.4.2009, although 8 of 12 tested blockade MAbs blocked both VLPs. Using mutant VLPs designed to alter predicted antigenic epitopes, binding of seven of the blockade MAbs was impacted by alterations in epitope A, identifying residues 294, 296, 297, 298, 368, and 372 as important antigenic sites in these strains. Convalescent-phase serum collected from a GII.4.2009 outbreak confirmed the immunodominance of epitope A, since alterations of epitope A affected serum reactivity by 40%. These data indicate that the GII.4.2009 New Orleans variant has evolved a key blockade epitope, possibly allowing for at least partial escape from protective herd immunity and provide epidemiological support for the utility of monitoring changes in epitope A in emergent strain surveillance.

Norovirus and Medically Attended Gastroenteritis in U.S. Children

The New England Journal of Medicine. Mar, 2013  |  Pubmed ID: 23514289

Cases of rotavirus-associated acute gastroenteritis have declined since the introduction of rotavirus vaccines, but the burden of norovirus-associated acute gastroenteritis in children remains to be assessed.

Prevalence and Genetic Diversity of Norovirus Among Patients with Acute Diarrhea in Guatemala

Journal of Medical Virology. Jul, 2013  |  Pubmed ID: 23595770

Noroviruses (NoVs) are a leading cause of acute gastroenteritis outbreaks and sporadic cases of diarrhea in industrialized countries. To study the prevalence and genetic diversity of NoVs in Guatemala, stool specimens were collected from hospitalized and ambulatory patients presenting with diarrhea (≥3 loose or liquid stools in a 24-hr period) who were enrolled in a prospective surveillance system in the Departments of Santa Rosa (October 2007 to August 2010) and Quetzaltenango (August 2009 to August 2010), Guatemala. Specimens were tested for rotavirus, enteric bacteria, and parasites by routine methods and for genogroups I and II NoV by real-time reverse transcription-PCR. A total of 2,403 stool specimens were collected from hospitalized (n = 528) and ambulatory patients (n = 1,875). Overall, 341 (14%) samples tested positive for NoVs including 114 (22%) hospitalized and 227 (12%) ambulatory patients. NoVs disease peaked during the winter (November-January) months. Among the 341 NoVs-positive patients, 32 (9%) were also positive for rotavirus, 32 (9%) for bacteria, and 9 (3%) for protozoa. Nucleotide sequences were obtained from 84 samples collected from hospitalized children aged <5 years of age, which could be grouped into nine GII and three GI genotypes with GII.4 (74%) and GI.8 (10%) being the most common. This is the first study on the prevalence of NoVs among hospitalized and ambulatory patients with diarrhea in Guatemala. The findings highlight the need to implement laboratory diagnostics for NoVs to improve appropriate clinical management of diarrheal diseases and guide vaccine development.

Proposal for a Unified Norovirus Nomenclature and Genotyping

Archives of Virology. Oct, 2013  |  Pubmed ID: 23615870

Noroviruses belong to a genus of genetically diverse viruses within the family Caliciviridae and cause acute gastroenteritis in humans and animals. They are subdivided into genogroups, each of which further segregates into genotypes. Until recently, a new genotype was based on a defined pairwise distance cutoff of complete VP1 sequences, but with the increasing number of available norovirus sequences, this cutoff is no longer accurate, and sequences in the public database have been misclassified. In this paper, we demonstrate that the pairwise distance cutoff method can no longer be used and outline a phylogenetic approach to classify noroviruses. Furthermore, we propose a dual nomenclature using both ORF1 and VP1 sequences, as recombination is common and recognizing recombinant viruses may be relevant. With the continuing emergence of new norovirus lineages, we propose to coordinate nomenclature of new norovirus genotypes through an international norovirus working group.

Presence of Antibodies Against Genogroup VI Norovirus in Humans

Virology Journal. Jun, 2013  |  Pubmed ID: 23735311

Noroviruses are important enteric pathogens in humans and animals. Recently, we reported a novel canine norovirus (CaNoV) in dogs with diarrhea belonging to a new genogroup (GVI). No data are available on exposure of humans to this virus.

Etiology of Viral Gastroenteritis in Children <5 Years of Age in the United States, 2008-2009

The Journal of Infectious Diseases. Sep, 2013  |  Pubmed ID: 23757337

Although rotavirus and norovirus cause nearly 40% of severe endemic acute gastroenteritis (AGE) in children <5 years of age in the United States, there are limited data on the etiologic role of other enteric viruses in this age group.

Genotype GI.6 Norovirus, United States, 2010-2012

Emerging Infectious Diseases. Aug, 2013  |  Pubmed ID: 23876252

We report an increase in the proportion of genotype GI.6 norovirus outbreaks in the United States from 1.4% in 2010 to 7.7% in 2012 (p<0.001). Compared with non-GI.6 outbreaks, GI.6 outbreaks were characterized by summer seasonality, foodborne transmission, and non-health care settings.

Norovirus Disease in the United States

Emerging Infectious Diseases. Aug, 2013  |  Pubmed ID: 23876403

Although recognized as the leading cause of epidemic acute gastroenteritis across all age groups, norovirus has remained poorly characterized with respect to its endemic disease incidence. Use of different methods, including attributable proportion extrapolation, population-based surveillance, and indirect modeling, in several recent studies has considerably improved norovirus disease incidence estimates for the United States. Norovirus causes an average of 570-800 deaths, 56,000-71,000 hospitalizations, 400,000 emergency department visits, 1.7-1.9 million outpatient visits, and 19-21 million total illnesses per year. Persons >65 years of age are at greatest risk for norovirus-associated death, and children <5 years of age have the highest rates of norovirus-associated medical care visits. Endemic norovirus disease occurs year round but exhibits a pronounced winter peak and increases by ≤ 50% during years in which pandemic strains emerge. These findings support continued development and targeting of appropriate interventions, including vaccines, for norovirus disease.

Effects and Clinical Significance of GII.4 Sydney Norovirus, United States, 2012-2013

Emerging Infectious Diseases. Aug, 2013  |  Pubmed ID: 23886013

During 2012, global detection of a new norovirus (NoV) strain, GII.4 Sydney, raised concerns about its potential effect in the United States. We analyzed data from NoV outbreaks in 5 states and emergency department visits for gastrointestinal illness in 1 state during the 2012-13 season and compared the data with those of previous seasons. During August 2012-April 2013, a total of 637 NoV outbreaks were reported compared with 536 and 432 in 2011-2012 and 2010-2011 during the same period. The proportion of outbreaks attributed to GII.4 Sydney increased from 8% in September 2012 to 82% in March 2013. The increase in emergency department visits for gastrointestinal illness during the 2012-13 season was similar to that of previous seasons. GII.4 Sydney has become the predominant US NoV outbreak strain during the 2012-13 season, but its emergence did not cause outbreak activity to substantially increase from that of previous seasons.

Emergence of New Pandemic GII.4 Sydney Norovirus Strain Correlates with Escape from Herd Immunity

The Journal of Infectious Diseases. Dec, 2013  |  Pubmed ID: 23908476

GII.4 noroviruses are a significant source of acute gastroenteritis worldwide, causing the majority of human norovirus outbreaks. Evolution of the GII.4 major capsid protein occurs rapidly, resulting in the emergence of new strains that produce successive waves of pandemic disease. A new pandemic isolate, GII.4 2012 Sydney, largely replaced previously circulating strains in late 2012. We compare the antigenic properties of GII.4 2012 Sydney with previously circulating strains.

Clinical Profile of Children with Norovirus Disease in Rotavirus Vaccine Era

Emerging Infectious Diseases. Oct, 2013  |  Pubmed ID: 24047618

Diagnostic Performance of Rectal Swab Versus Bulk Stool Specimens for the Detection of Rotavirus and Norovirus: Implications for Outbreak Investigations

Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology. Dec, 2013  |  Pubmed ID: 24139675

In January of 2008, during the peak of the rotavirus season in Guatemala, a gastroenteritis outbreak with high mortality among infants was reported in Guatemala. Despite extensive efforts, the investigation was limited by the lack of bulk stool specimens collected, particularly from the more severely dehydrated or deceased children.

Human Norovirus Detection and Production, Quantification, and Storage of Virus-like Particles

Current Protocols in Microbiology. Nov, 2013  |  Pubmed ID: 24510290

Human noroviruses constitute a significant worldwide disease burden. Each year, noroviruses cause over 267 million infections, deaths in over 200,000 children under the age of five, and over 50% of U.S. food-borne illness. Due to the absence of a tissue culture model or small animal model to study human norovirus, virus-like particles (VLPs) and ELISA-based biological assays have been used to answer questions about norovirus evolution and immunity as well to provide a potential vaccine platform. This chapter outlines the protocols for norovirus detection in stool, as well as norovirus VLP design, production, purification, and storage using a Venezuelan equine encephalitis virus (VEE)-based virus replicon particle (VRP) expression system.

Challenges of Culturing Human Norovirus in Three-dimensional Organoid Intestinal Cell Culture Models

PloS One. 2014  |  Pubmed ID: 23755105

Human noroviruses are the most common cause of acute gastroenteritis worldwide. Recently, cell culture systems have been described using either human embryonic intestinal epithelial cells (Int-407) or human epithelial colorectal adenocarcinoma cells (Caco-2) growing on collagen-I porous micro carrier beads in a rotating bioreactor under conditions of physiological fluid shear. Here, we describe the efforts from two independent laboratories to implement this three dimensional (3D) cell culture system for the replication of norovirus. Int-407 and Caco-2 were grown in a rotating bioreactor for up to 28 days. Prior to infection, cells were screened for the presence of microvilli by electron microscopy and stained for junction proteins (zonula occludens-1, claudin-1, and β-catenin). Differentiated 3D cells were transferred to 24-well plates and infected with bacteria-free filtrates of various norovirus genotypes (GI.1, GI.3, GI.8, GII.2, GII.4, GII.7, and GII.8). At 12 h, 24 h, and 48 h post inoculation, viral RNA from both cells and supernatants were collected and analyzed for norovirus RNA by real-time reverse transcription PCR. Despite observations of high expression of junction proteins and microvilli development in stained thin sections, our data suggest no significant increase in viral titer based on norovirus RNA copy number during the first 48 h after inoculation for the different samples and virus culture conditions tested. Our combined efforts demonstrate that 3D cell culture models using Int-407 or Caco-2 cells do not support norovirus replication and highlight the complexity and difficulty of developing a reproducible in vitro cell culture system for human norovirus.

Genotypic and Epidemiologic Trends of Norovirus Outbreaks in the United States, 2009 to 2013

Journal of Clinical Microbiology. Jan, 2014  |  Pubmed ID: 24172151

Noroviruses are the leading cause of epidemic acute gastroenteritis in the United States. From September 2009 through August 2013, 3,960 norovirus outbreaks were reported to CaliciNet. Of the 2,895 outbreaks with a known transmission route, person-to-person and food-borne transmissions were reported for 2,425 (83.7%) and 465 (16.1%) of the outbreaks, respectively. A total of 2,475 outbreaks (62.5%) occurred in long-term care facilities (LTCF), 389 (9.8%) in restaurants, and 227 (5.7%) in schools. A total of 435 outbreaks (11%) were typed as genogroup I (GI) and 3,525 (89%) as GII noroviruses. GII.4 viruses caused 2,853 (72%) of all outbreaks, of which 94% typed as either GII.4 New Orleans or GII.4 Sydney. In addition, three non-GII.4 viruses, i.e., GII.12, GII.1, and GI.6, caused 528 (13%) of all outbreaks. Several non-GII.4 genotypes (GI.3, GI.6, GI.7, GII.3, GII.6, and GII.12) were significantly more associated with food-borne transmission (odds ratio, 1.9 to 7.1; P < 0.05). Patients in LTCF and people ≥65 years of age were at higher risk for GII.4 infections than those in other settings and with other genotypes (P < 0.05). Phylogeographic analysis identified three major dispersions from two geographic locations that were responsible for the GI.6 outbreaks from 2011 to 2013. In conclusion, our data demonstrate the cyclic emergence of new (non-GII.4) norovirus strains, and several genotypes are more often associated with food-borne outbreaks. These surveillance data can be used to improve viral food-borne surveillance and to help guide studies to develop and evaluate targeted prevention methods such as norovirus vaccines, antivirals, and environmental decontamination methods.

Epidemiologic Implications of Asymptomatic Reinfection: a Mathematical Modeling Study of Norovirus

American Journal of Epidemiology. Feb, 2014  |  Pubmed ID: 24305574

The pathogenicity of norovirus is definitively established. However, norovirus is frequently detected in the stool of healthy individuals. To gain understanding of the apparent high prevalence of asymptomatic infection, we analyzed a dynamic transmission model of norovirus infection, disease, and immunity. We simulated norovirus epidemiology in low- and high-transmission settings by varying the basic reproduction number (R0). We predicted annual disease incidence values in children aged 0-4 years of 25% with a low R0 and 29% with a high R0. However, the point prevalence of asymptomatic infection rose sharply from 3% to 48% from the low to high R0 settings. Among older children and adults, the models projected that incidence of disease would rise from 6% to 16% from the low to high R0 settings, whereas asymptomatic infection prevalence was lower in this age group. Asymptomatic prevalence of norovirus can change dramatically with small changes in R0. The ratio of prevalence in cases to controls could be high in a developed country and close to or even less than 1 in a high-exposure setting, despite similar disease incidence. These findings highlight an important limitation of case-control studies for pathogens for which there is suboptimal diagnostic specificity.

Seroprevalence of Canine Norovirus in 14 European Countries

Clinical and Vaccine Immunology : CVI. Jun, 2014  |  Pubmed ID: 24671552

To investigate the prevalence of the recently described genogroup VI canine noroviruses (CNVs) in dogs in Europe, we tested 510 serum samples from dogs in 14 European countries for anti-IgG CNV antibodies. Seropositive dogs were found throughout Europe. Dogs with antibodies against human noroviruses were also found.

Feline Fecal Virome Reveals Novel and Prevalent Enteric Viruses

Veterinary Microbiology. Jun, 2014  |  Pubmed ID: 24793097

Humans keep more than 80 million cats worldwide, ensuring frequent exposure to their viruses. Despite such interactions the enteric virome of cats remains poorly understood. We analyzed a fecal sample from a single healthy cat from Portugal using viral metagenomics and detected five eukaryotic viral genomes. These viruses included a novel picornavirus (proposed genus "Sakobuvirus") and bocavirus (feline bocavirus 2), a variant of feline astrovirus 2 and sequence fragments of a highly divergent feline rotavirus and picobirnavirus. Feline sakobuvirus A represents the prototype species of a proposed new genus in the Picornaviridae family, distantly related to human salivirus and kobuvirus. Feline astroviruses (mamastrovirus 2) are the closest known relatives of the classic human astroviruses (mamastrovirus 1), suggestive of past cross-species transmission. Presence of these viruses by PCR among Portuguese cats was detected in 13% (rotavirus), 7% (astrovirus), 6% (bocavirus), 4% (sakobuvirus), and 4% (picobirnavirus) of 55 feline fecal samples. Co-infections were frequent with 40% (4/10) of infected cats shedding more than one of these five viruses. Our study provides an initial description of the feline fecal virome indicating a high level of asymptomatic infections. Availability of the genome sequences of these viruses will facilitate future tropism and feline disease association studies.

Divergent Picobirnaviruses in Human Feces

Genome Announcements. May, 2014  |  Pubmed ID: 24831146

The near-complete genomes of two picobirnaviruses (PBVs) in diarrheal stool samples, human picobirnaviruses D and E (HuPBV-D and -E), were genetically characterized. Their RNA-dependent RNA polymerase (RdRp) protein sequences had <66% identities to known PBVs. Due to a single nucleotide insertion, the open reading frame 2 (ORF2) in segment 1 of HuPBV-D was interrupted by a stop codon. A small stem-loop structure overlying the stop codon may result in translational readthrough into the rest of ORF2.

Etiology of Childhood Diarrhea After Rotavirus Vaccine Introduction: a Prospective, Population-based Study in Nicaragua

The Pediatric Infectious Disease Journal. Nov, 2014  |  Pubmed ID: 24879131

Nicaragua was the first developing nation to implement routine immunization with the pentavalent rotavirus vaccine (RV5). In this RV5-immunized population, understanding infectious etiologies of childhood diarrhea is necessary to direct diarrhea treatment and prevention efforts.

Comprehensive Comparison of Cultivable Norovirus Surrogates in Response to Different Inactivation and Disinfection Treatments

Applied and Environmental Microbiology. Sep, 2014  |  Pubmed ID: 25015883

Human norovirus is the leading cause of epidemic and sporadic acute gastroenteritis. Since no cell culture method for human norovirus exists, cultivable surrogate viruses (CSV), including feline calicivirus (FCV), murine norovirus (MNV), porcine enteric calicivirus (PEC), and Tulane virus (TuV), have been used to study responses to inactivation and disinfection methods. We compared the levels of reduction in infectivities of CSV and Aichi virus (AiV) after exposure to extreme pHs, 56°C heating, alcohols, chlorine on surfaces, and high hydrostatic pressure (HHP), using the same matrix and identical test parameters for all viruses, as well as the reduction of human norovirus RNA levels under these conditions. At pH 2, FCV was inactivated by 6 log10 units, whereas MNV, TuV, and AiV were resistant. All CSV were completely inactivated at 56°C within 20 min. MNV was inactivated 5 log10 units by alcohols, in contrast to 2 and 3 log10 units for FCV and PEC, respectively. TuV and AiV were relatively insensitive to alcohols. FCV was reduced 5 log10 units by 1,000 ppm chlorine, in contrast to 1 log10 unit for the other CSV. All CSV except FCV, when dried on stainless steel surfaces, were insensitive to 200 ppm chlorine. HHP completely inactivated FCV, MNV, and PEC at ≥300 MPa, and TuV at 600 MPa, while AiV was completely resistant to HHP up to 800 MPa. By reverse transcription-quantitative PCR (RT-qPCR), genogroup I (GI) noroviruses were more sensitive than GII noroviruses to alcohols, chlorine, and HHP. Although inactivation profiles were variable for each treatment, TuV and MNV were the most resistant CSV overall and therefore are the best candidates for studying the public health outcomes of norovirus infections.

Fluorinated TiO₂ As an Ambient Light-activated Virucidal Surface Coating Material for the Control of Human Norovirus

Journal of Photochemistry and Photobiology. B, Biology. Nov, 2014  |  Pubmed ID: 25222145

We evaluated the virucidal efficacy of light-activated fluorinated TiO₂ surface coatings on human norovirus and several surrogates (bacteriophage MS2, feline calcivirus (FCV), and murine norovirus (MNV)). Inactivation of viruses on surfaces exposed to a common fluorescent lamp was monitored and the effects of UVA intensity, temperature, and fluoride content were assessed. Destruction of RNA and capsid oxidation were evaluated for human norovirus inocula on the F-TiO₂ surfaces, while contact with the F-TiO₂ surface and exposure to residual UVA radiation of 10 μW cm(-2) for 60 min resulted in infectivity reductions for the norovirus surrogates of 2-3 log₁₀. Infectivity reductions on pristine TiO₂ surfaces in identical conditions were over 2 orders of magnitude lower. Under realistic room lighting conditions, MS2 infectivity declined below the lower detection limit after 12h. Reductions in RNA were generally low, with the exception of GII.4, while capsid protein oxidation likely played a larger role in infectivity loss. Inactivation of norovirus surrogates occurred significantly faster on F-TiO₂ compared to pristine TiO₂ surfaces. The material demonstrated antiviral action against human norovirus surrogates and was shown to effectively inhibit MS2 when exposed to residual UVA present in fluorescent room lighting conditions in a laboratory setting.

RNA Populations in Immunocompromised Patients As Reservoirs for Novel Norovirus Variants

Journal of Virology. Dec, 2014  |  Pubmed ID: 25275120

Noroviruses are the leading cause of acute gastroenteritis outbreaks worldwide. The majority of norovirus outbreaks are caused by genogroup II.4 (GII.4). Novel GII.4 strains emerge every 2 to 4 years and replace older variants as the dominant norovirus. Novel variants emerge through a combination of recombination, genetic drift, and selection driven by population immunity, but the exact mechanism of how or where is not known. We detected two previously unknown novel GII.4 variants, termed GII.4 UNK1 and GII.4 UNK2, and a diverse norovirus population in fecal specimens from immunocompromised individuals with diarrhea after they had undergone bone marrow transplantation. We hypothesized that immunocompromised individuals can serve as reservoirs for novel norovirus variants. To test our hypothesis, metagenomic analysis of viral RNA populations was combined with a full-genome bioinformatic analysis of publicly available GII.4 norovirus sequences from 1974 to 2014 to identify converging sites. Variable sites were proportionally more likely to be within two amino acids (P < 0.05) of positively selected sites. Further analysis using a hypergeometric distribution indicated that polymorphic site distribution was random and its proximity to positively selected sites was dependent on the size of the norovirus genome and the number of positively selected sites.In conclusion, random mutations may have a positive impact on driving norovirus evolution, and immunocompromised individuals could serve as potential reservoirs for novel GII.4 strains.

Enteric Bacteria Promote Human and Mouse Norovirus Infection of B Cells

Science (New York, N.Y.). Nov, 2014  |  Pubmed ID: 25378626

The cell tropism of human noroviruses and the development of an in vitro infection model remain elusive. Although susceptibility to individual human norovirus strains correlates with an individual's histo-blood group antigen (HBGA) profile, the biological basis of this restriction is unknown. We demonstrate that human and mouse noroviruses infected B cells in vitro and likely in vivo. Human norovirus infection of B cells required the presence of HBGA-expressing enteric bacteria. Furthermore, mouse norovirus replication was reduced in vivo when the intestinal microbiota was depleted by means of oral antibiotic administration. Thus, we have identified B cells as a cellular target of noroviruses and enteric bacteria as a stimulatory factor for norovirus infection, leading to the development of an in vitro infection model for human noroviruses.

Viral Gastroenteritis in Rotavirus Negative Hospitalized Children <5 Years of Age from the Independent States of the Former Soviet Union

Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases. Dec, 2014  |  Pubmed ID: 25460823

Rotavirus causes nearly 40% of all hospitalizations for AGE among children <5 years of age in the NIS of the former Soviet Union. The etiologic role of other established gastroenteritis viruses in this age group is unknown.

Advances in Laboratory Methods for Detection and Typing of Norovirus

Journal of Clinical Microbiology. Feb, 2015  |  Pubmed ID: 24989606

Human noroviruses are the leading cause of epidemic and sporadic gastroenteritis across all age groups. Although the disease is usually self-limiting, in the United States norovirus gastroenteritis causes an estimated 56,000 to 71,000 hospitalizations and 570 to 800 deaths each year. This minireview describes the latest data on laboratory methods (molecular, immunological) for norovirus detection, including real-time reverse transcription-quantitative PCR (RT-qPCR) and commercially available immunological assays as well as the latest FDA-cleared multi-gastrointestinal-pathogen platforms. In addition, an overview is provided on the latest nomenclature and molecular epidemiology of human noroviruses.

Norovirus Vaccine Against Experimental Human GII.4 Virus Illness: a Challenge Study in Healthy Adults

The Journal of Infectious Diseases. Mar, 2015  |  Pubmed ID: 25210140

Vaccines against norovirus, the leading cause of acute gastroenteritis, should protect against medically significant illness and reduce transmission.

Norovirus Infection and Disease in an Ecuadorian Birth Cohort: Association of Certain Norovirus Genotypes With Host FUT2 Secretor Status

The Journal of Infectious Diseases. Jun, 2015  |  Pubmed ID: 25505295

Although norovirus is the most common cause of gastroenteritis, there are few data on the community incidence of infection/disease or the patterns of acquired immunity or innate resistance to norovirus.

Noroviruses: Epidemiology, Immunity and Prospects for Prevention

Future Microbiology. 2015  |  Pubmed ID: 25598337

In recent years, noroviruses have become recognized as an important cause of both sporadic and epidemic acute gastroenteritis (AGE), largely due to the improved availability of broadly reactive real-time RT-PCR (TaqMan-based RT-PCR) assays. While there is substantial diversity among noroviruses, one specific genotype, GII.4, is the most common etiology in sporadic and epidemic AGE. Outbreaks of norovirus AGE most commonly occur in healthcare facilities and restaurants and result in significant morbidity and mortality and substantial healthcare costs. Norovirus vaccine development is progressing, and Phase I and II human trials have shown proof-of-principle that norovirus vaccines can reduce illness and infection.

Innate Susceptibility to Norovirus Infections Influenced by FUT2 Genotype in a United States Pediatric Population

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. Jun, 2015  |  Pubmed ID: 25744498

Norovirus is a leading cause of acute gastroenteritis (AGE). Noroviruses bind to gut histo-blood group antigens (HBGAs), but only 70%-80% of individuals have a functional copy of the FUT2 ("secretor") gene required for gut HBGA expression; these individuals are known as "secretors." Susceptibility to some noroviruses depends on FUT2 secretor status, but the population impact of this association is not established.

Norovirus Genotype Profiles Associated with Foodborne Transmission, 1999-2012

Emerging Infectious Diseases. Apr, 2015  |  Pubmed ID: 25811368

Worldwide, noroviruses are a leading cause of gastroenteritis. They can be transmitted from person to person directly or indirectly through contaminated food, water, or environments. To estimate the proportion of foodborne infections caused by noroviruses on a global scale, we used norovirus transmission and genotyping information from multiple international outbreak surveillance systems (Noronet, CaliciNet, EpiSurv) and from a systematic review of peer-reviewed literature. The proportion of outbreaks caused by food was determined by genotype and/or genogroup. Analysis resulted in the following final global profiles: foodborne transmission is attributed to 10% (range 9%%-11%) of all genotype GII.4 outbreaks, 27% (25%-30%) of outbreaks caused by all other single genotypes, and 37% (24%%-52%) of outbreaks caused by mixtures of GII.4 and other noroviruses. When these profiles are applied to global outbreak surveillance data, results indicate that ≈14% of all norovirus outbreaks are attributed to food.

Antimicrobial Activity of Bismuth Subsalicylate on Clostridium Difficile, Escherichia Coli O157:H7, Norovirus, and Other Common Enteric Pathogens

Gut Microbes. 2015  |  Pubmed ID: 25901890

Previous studies have shown bismuth subsalicylate (BSS) has antimicrobial properties, but few studies have addressed the mechanism of action. Furthermore, following BSS ingestion other bismuth salts form throughout the gastrointestinal tract including bismuth oxychloride (BiOCl) that also act upon enteric pathogens. To further understand the antimicrobial activity of bismuth in infectious diarrhea, the antimicrobial effect of BSS and BiOCl on Clostridium difficile, Salmonella, Shigella, Shiga toxin-producing Escherichia coli strains and norovirus (NoV) were measured. Bacterial enteric pathogens in pure culture or in human fecal material were exposed to 35mg/ml BSS or BiOCl with or without a vehicle suspension. BSS and BiOCl treated samples were quantified and visualized by transmission electron microscopy. To measure the effect on NoV, reduction of infectious murine NoV (MNV), a surrogate for human NoV, and Norwalk virus RNA levels were measured by viral plaque assay and RT-qPCR, respectively. BSS and BiOCl reduced bacterial growth by 3-9 logs in all strains with majority resulting in populations of <10 cfu/ml within 24 h. Similar results were found when fecal material was included. Microscopy images detected bismuth on bacterial membranes and within the bacterial organisms at 30 min post-treatment. At 8.8mg/ml BSS and BiOCl reduced infectivity of MNV significantly by 2.7 and 2.0 log after 24 h of exposure. In addition, both BSS and BiOCl slightly reduced the level of Norwalk replicon-bearing cells suggesting that bismuth may inhibit NoV in vivo. Collectively, our results confirm and build on existing data that BSS has antimicrobial properties against a wide-range of diarrhea-causing pathogens.

Epidemiology and Molecular Characteristics of Norovirus GII.4 Sydney Outbreaks in Taiwan, January 2012-December 2013

Journal of Medical Virology. Sep, 2015  |  Pubmed ID: 25946552

In 2012, a new norovirus GII.4 variant (GII.4 Sydney) emerged and caused the majority of the acute gastroenteritis outbreaks in Australia, Asia, Europe, and North America. We examined the epidemiologic and molecular virologic characteristics of reported acute gastroenteritis outbreaks determined to be caused by norovirus in Taiwan from January 2012 to December 2013. A total of 253 (45.7%) of 552 reported acute gastroenteritis outbreaks tested positive for norovirus, of which 165 (65.5%) were typed as GII.4 Sydney. GII.4 Sydney outbreaks were reported from all geographic areas of Taiwan and occurred most frequently in schools (35.8%) and long-term care facilities (24.2%). Person-to-person transmission was identified in 116 (70.3%) of the outbreaks. Phylogenetic analyses of full-length ORF2 of eight specimens indicated that GII.4 Sydney strains detected in Taiwan were closely related to strains detected globally. Continued outbreak surveillance and strain typing are needed to provide information on epidemiologic and virologic trends of novel norovirus strains.

Incidence of Medically-Attended Norovirus-Associated Acute Gastroenteritis in Four Veteran's Affairs Medical Center Populations in the United States, 2011-2012

PloS One. 2015  |  Pubmed ID: 25996826

An estimated 179 million acute gastroenteritis (AGE) illnesses occur annually in the United States. The role of noroviruses in hospital-related AGE has not been well-documented in the U. S. We estimated the population incidence of community- acquired outpatient and inpatient norovirus AGE encounters, as well as hospital-acquired inpatient norovirus AGE among inpatients at four Veterans Affairs (VA) Medical Centers (VAMCs). Fifty (4%) of 1,160 stool specimens collected ≤7 days from symptom onset tested positive for norovirus. During a one year period, the estimated incidence of outpatient, community- and hospital-acquired inpatient norovirus AGE was 188 cases, 11 cases, and 54 cases/ 100,000 patients, respectively. This study demonstrates the incidence of outpatient and community- and hospital-acquired inpatient norovirus AGE among the VA population seeking care at these four VAMCs.

Development of a Nucleic Acid Extraction Procedure for Simultaneous Recovery of DNA and RNA from Diverse Microbes in Water

Pathogens (Basel, Switzerland). May, 2015  |  Pubmed ID: 26016775

Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters.

Serological Correlates of Protection Against a GII.4 Norovirus

Clinical and Vaccine Immunology : CVI. Aug, 2015  |  Pubmed ID: 26041041

Noroviruses are the leading cause of acute gastroenteritis worldwide, and norovirus vaccine prevention strategies are under evaluation. The immunogenicity of two doses of bivalent genogroup 1 genotype 1 (GI.1)/GII.4 (50 μg of virus-like particles [VLPs] of each strain adjuvanted with aluminum hydroxide and 3-O-desacyl-4'monophosphoryl lipid A [MPL]) norovirus vaccine administered to healthy adults in a phase 1/2 double-blind placebo-controlled trial was determined using virus-specific serum total antibody enzyme-linked immunosorbent assay (ELISA), IgG, IgA, and histoblood group antigen (HBGA)-blocking assays. Trial participants subsequently received an oral live virus challenge with a GII.4 strain, and the vaccine efficacy results were reported previously (D. I. Bernstein et al., J Infect Dis 211:870-878, 2014, doi:10.1093/infdis/jiu497). This report assesses the impact of prechallenge serum antibody levels on infection and illness outcomes. Serum antibody responses were observed in vaccine recipients by all antibody assays, with first-dose seroresponse frequencies ranging from 88 to 100% for the GI.1 antigen and from 69 to 84% for the GII.4 antigen. There was little increase in antibody levels after the second vaccine dose. Among the subjects receiving the placebo, higher prechallenge serum anti-GII.4 HBGA-blocking and IgA antibody levels, but not IgG or total antibody levels, were associated with a lower frequency of virus infection and associated illness. Notably, some placebo subjects without measurable serum antibody levels prechallenge did not become infected after norovirus challenge. In vaccinees, anti-GII.4 HBGA-blocking antibody levels of >1:500 were associated with a lower frequency of moderate-to-severe vomiting or diarrheal illness. In this study, prechallenge serum HBGA antibody titers correlated with protection in subjects receiving the placebo; however, other factors may impact the likelihood of infection and illness after virus exposure. (This study is registered at under registration number NCT1609257.).

Transmission of Norovirus Within Households in Quininde, Ecuador

The Pediatric Infectious Disease Journal. Sep, 2015  |  Pubmed ID: 26090575

We studied the transmission of norovirus infection in households in Quininde, Ecuador. Among household contacts of norovirus positive children with diarrhea, norovirus negative children with diarrhea and asymptomatic controls, infection attack rates were 33%, 8% and 18%, respectively (N = 45, 36, 83). Infection attack rates were higher when index children had a higher viral load.

Differences in Norovirus-Associated Hospital Visits Between Jewish and Bedouin Children in Southern Israel

The Pediatric Infectious Disease Journal. Sep, 2015  |  Pubmed ID: 26107344

Population-based surveillance during 2006-2013 showed that norovirus hospitalization rates among Bedouin (low-middle income settings) children <5 years old were 13.9/10,000 person-years compared with 7.1/10,000 among Jewish (high-income settings) children who were <5 years (rate ratio: 2.0, 95% confidence interval: 1.6-2.3). Differences were most prominent among infants (59.7 vs. 19.7/10,000, respectively; rate ratio: 3.0, 95% confidence interval: 2.5-3.8). GII.3 and GII.4 strains dominated (67%) in both populations.

Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces

Applied and Environmental Microbiology. Sep, 2015  |  Pubmed ID: 26116675

Inanimate surfaces are regarded as key vehicles for the spread of human norovirus during outbreaks. ISO method 15216 involves the use of cotton swabs for environmental sampling from food surfaces and fomites for the detection of norovirus genogroup I (GI) and GII. We evaluated the effects of the virus drying time (1, 8, 24, or 48 h), swab material (cotton, polyester, rayon, macrofoam, or an antistatic wipe), surface (stainless steel or a toilet seat), and area of the swabbed surface (25.8 cm(2) to 645.0 cm(2)) on the recovery of human norovirus. Macrofoam swabs produced the highest rate of recovery of norovirus from surfaces as large as 645 cm(2). The rates of recovery ranged from 2.2 to 36.0% for virus seeded on stainless-steel coupons (645.0 cm(2)) to 1.2 to 33.6% for toilet seat surfaces (700 cm(2)), with detection limits of 3.5 log10 and 4.0 log10 RNA copies. We used macrofoam swabs to collect environmental samples from several case cabins and common areas of a cruise ship where passengers had reported viral gastroenteritis symptoms. Seventeen (18.5%) of 92 samples tested positive for norovirus GII, and 4 samples could be sequenced and had identical GII.1 sequences. The viral loads of the swab samples from the cabins of the sick passengers ranged from 80 to 31,217 RNA copies, compared with 16 to 113 RNA copies for swab samples from public spaces. In conclusion, our swab protocol for norovirus may be a useful tool for outbreak investigations when no clinical samples are available to confirm the etiology.

Human Norovirus Culture in B Cells

Nature Protocols. Dec, 2015  |  Pubmed ID: 26513671

Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RT-qPCR, requires ∼6 h.

A Diverse Group of Small Circular SsDNA Viral Genomes in Human and Non-human Primate Stools

Virus Evolution. 2015  |  Pubmed ID: 27774288

Viral metagenomics sequencing of fecal samples from outbreaks of acute gastroenteritis from the US revealed the presence of small circular ssDNA viral genomes encoding a replication initiator protein (Rep). Viral genomes were ∼2.5 kb in length, with bi-directionally oriented Rep and capsid (Cap) encoding genes and a stem loop structure downstream of Rep. Several genomes showed evidence of recombination. By digital screening of an in-house virome database (1.04 billion reads) using BLAST, we identified closely related sequences from cases of unexplained diarrhea in France. Deep sequencing and PCR detected such genomes in 7 of 25 US (28 percent) and 14 of 21 French outbreaks (67 percent). One of eighty-five sporadic diarrhea cases in the Gambia was positive by PCR. Twenty-two complete genomes were characterized showing that viruses from patients in the same outbreaks were closely related suggesting common origins. Similar genomes were also characterized from the stools of captive chimpanzees, a gorilla, a black howler monkey, and a lemur that were more diverse than the human stool-associated genomes. The name smacovirus is proposed for this monophyletic viral clade. Possible tropism include mammalian enteric cells or ingested food components such as infected plants. No evidence of viral amplification was found in immunodeficient mice orally inoculated with smacovirus-positive stool supernatants. A role for smacoviruses in diarrhea, if any, remains to be demonstrated.

Genetic Characterization of Norovirus Strains in Hospitalized Children from Pakistan

Journal of Medical Virology. Feb, 2016  |  Pubmed ID: 26175018

Norovirus is one of the most common causes of acute gastroenteritis among children in developing countries. No data on the prevalence and genetic variability of norovirus are available for Pakistan, where early childhood mortality due to acute gastroenteritis is common. We tested 255 fecal specimens from children under 5 years of age hospitalized between April 2006 and March 2008 with severe acute gastroenteritis in five hospitals in the four largest cities in Pakistan for norovirus by real-time RT-PCR. Positive samples were further genotyped by conventional RT-PCR targeting the 5'-end of the capsid gene followed by sequencing of the positive PCR products. Overall, 41 (16.1%) samples tested positive for norovirus with an equal frequency in rotavirus-positive and rotavirus-negative samples. Nine (22%) samples were genogroup (G)I positive, 30 (73%) GII positive and two (5%) samples contained a mixture of GI and GII viruses. Sequence analyses demonstrated co-circulation of 14 norovirus genotypes including four GI genotypes (GI.3, GI.5, GI.7, GI.8) and 10 GII genotypes (GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.9, GII.13, GII.16, and GII.21). The most prevalent genotypes were GI.7 and GII.4 both causing 12.2% of the infections. This report confirms the presence of multiple norovirus genotypes in hospitalized children with acute gastroenteritis in Pakistan and a lack of clear predominance of GII.4 viruses.

Epidemiologic, Virologic, and Host Genetic Factors of Norovirus Outbreaks in Long-term Care Facilities

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. Jan, 2016  |  Pubmed ID: 26508509

In the Unites States, long-term care facilities (LTCFs) are the most common setting for norovirus outbreaks. These outbreaks provide a unique opportunity to better characterize the viral and host characteristics of norovirus disease.

Multicenter Evaluation of the Xpert Norovirus Assay for Detection of Norovirus Genogroups I and II in Fecal Specimens

Journal of Clinical Microbiology. Jan, 2016  |  Pubmed ID: 26560532

Norovirus is the most common cause of sporadic gastroenteritis and outbreaks worldwide. The rapid identification of norovirus has important implications for infection prevention measures and may reduce the need for additional diagnostic testing. The Xpert Norovirus assay recently received FDA clearance for the detection and differentiation of norovirus genogroups I and II (GI and GII), which account for the vast majority of infections. In this study, we evaluated the performance of the Xpert Norovirus assay with both fresh, prospectively collected (n = 914) and frozen, archived (n = 489) fecal specimens. A Centers for Disease Control and Prevention (CDC) composite reference method was used as the gold standard for comparison. For both prospective and frozen specimens, the Xpert Norovirus assay showed positive percent agreement (PPA) and negative percent agreement (NPA) values of 98.3% and 98.1% for GI and of 99.4% and 98.2% for GII, respectively. Norovirus prevalence in the prospective specimens (collected from March to May of 2014) was 9.9% (n = 90), with the majority of positives caused by genogroup II (82%, n = 74). The positive predictive value (PPV) of the Xpert Norovirus assay was 75% for GI-positive specimens, whereas it was 86.5% for GII-positive specimens. The negative predictive values (NPV) for GI and GII were 100% and 99.9%, respectively.

Characterization of a Salivirus (Picornaviridae) from a Diarrheal Child in Guatemala

Genome Announcements. Feb, 2016  |  Pubmed ID: 26893429

The complete genome sequence of a salivirus was identified in a stool sample from a Guatemalan child with acute gastroenteritis during a 2009 norovirus outbreak. This genome (genotype A1 strain GUT/2009/A-1746) shares 82% to 94% genome-wide nucleotide identity with saliviruses from the United States, China, Germany, and Nigeria, representing the first salivirus sequence from Central America.

The Effect of Diarrheal Disease on Bivalent Oral Polio Vaccine (bOPV) Immune Response in Infants in Nepal

Vaccine. May, 2016  |  Pubmed ID: 27085172

A globally-coordinated phase out of all type 2 containing oral polio vaccine (OPV) is planned for April 2016 during which bivalent 1+3 OPV (bOPV) will replace trivalent OPV (tOPV) in routine immunization schedules and campaigns. Diarrhea impairs the immune response to tOPV, but the effect of diarrhea on bOPV is unknown.

Population-Based Incidence Rates of Diarrheal Disease Associated with Norovirus, Sapovirus, and Astrovirus in Kenya

PloS One. 2016  |  Pubmed ID: 27116458

Diarrheal diseases remain a major cause of mortality in Africa and worldwide. While the burden of rotavirus is well described, population-based rates of disease caused by norovirus, sapovirus, and astrovirus are lacking, particularly in developing countries.

Norovirus in a United States Virgin Islands Resort: Outbreak Investigation, Response, and Costs

Journal of Travel Medicine. May, 2016  |  Pubmed ID: 27296584

During 8-20 April 2012, an outbreak of gastrointestinal illness occurred among guests and employees of a resort hotel in St. Thomas, US Virgin Islands. We describe outbreak characteristics, and estimate indirect (non-medical) costs to travellers.

Using Multiplex Molecular Testing to Determine the Etiology of Acute Gastroenteritis in Children

The Journal of Pediatrics. Sep, 2016  |  Pubmed ID: 27329497

To detect the etiologic agents of acute gastroenteritis (AGE) in children using broad molecular-based techniques, and compare clinical presentations among etiologies.

Strain-Specific Virolysis Patterns of Human Noroviruses in Response to Alcohols

PloS One. 2016  |  Pubmed ID: 27337036

Alcohol-based hand sanitizers are widely used to disinfect hands to prevent the spread of pathogens including noroviruses. Alcohols inactivate norovirus by destruction of the viral capsid, resulting in the leakage of viral RNA (virolysis). Since conflicting results have been reported on the susceptibility of human noroviruses against alcohols, we exposed a panel of 30 human norovirus strains (14 GI and 16 GII strains) to different concentrations (50%, 70%, 90%) of ethanol and isopropanol and tested the viral RNA titer by RT-qPCR. Viral RNA titers of 10 (71.4%), 14 (100%), 3 (21.4%) and 7 (50%) of the 14 GI strains were reduced by > 1 log10 RNA copies/ml after exposure to 70% and 90% ethanol, and 70% and 90% isopropanol, respectively. RNA titers of 6 of the 7 non-GII 4 strains remained unaffected after alcohol exposure. Compared to GII strains, GI strains were more susceptible to ethanol than to isopropanol. At 90%, both alcohols reduced RNA titers of 8 of the 9 GII.4 strains by ≥ 1 log10 RNA copies/ml. After exposure to 70% ethanol, RNA titers of GII.4 Den Haag and Sydney strains decreased by ≥ 1.9 log10, whereas RNA reductions for GII.4 New Orleans strains were < 0.5 log10. To explain these differences, we sequenced the complete capsid gene of the 9 GII.4 strains and identified 17 amino acid substitutions in the P2 region among the 3 GII.4 variant viruses. When comparing with an additional set of 200 GII.4 VP1 sequences, only S310 and P396 were present in all GII.4 New Orleans viruses but not in the ethanol-sensitive GII.4 Sydney and GII.4 Den Haag viruses Our data demonstrate that alcohol susceptibility patterns between different norovirus genotypes vary widely and that virolysis data for a single strain or genotype are not representative for all noroviruses.

Comparison of Norovirus Genogroup I, II, and IV Seroprevalence Among Children in the Netherlands, 1963, 1983, and 2006

The Journal of General Virology. Jun, 2016  |  Pubmed ID: 27365054

Noroviruses are a major cause of acute gastroenteritis worldwide and are a genetic diverse group of viruses. Since 2002, an increasing number of norovirus outbreaks have been reported globally, but it is not clear whether this increase has been caused by a higher awareness or reflects emergence of new genogroup II genotype 4 (GII.4) variants. In this study the hypothesis is tested that the norovirus prevalence has increased post 2002 and related to the emergence of GII.4. Sera from children aged <5 years of three Dutch cross-sectional population based cohorts collected in 1963, 1983, and 2006/2007 (respectively n=143, n=130, and n=376) were tested for specific serum IgG by protein array using antigens to GII.4 and a range of other antigens representing norovirus GI,G II, and GIV genotypes. The protein array was validated by paired sera of norovirus infected patients and supernatants of B-cell cultures with single epitope specificity. Evidence for norovirus infection was found to be common among Dutch children in each cohort, but the prevalence towards different genotypes changed over time. At the genogroup level, GI seroprevalence decreased significantly between 1963 and 2006/2007, while a significant increase of GII and particularly genotype GII.4 specific antibodies was detected in the 2006/2007 cohort. There were no children with solely GII.4 antibodies in the 1963 cohort. This study shows that the high GII.4 norovirus incidence in very young children is a recent phenomenon. These findings are of importance for vaccine development and trials that are currently focussing mostly on GII.4 viruses.

Synthesis and Evaluation of Biotinylated Bivalent HistoBlood Group Antigens for Capturing Human Noroviruses

Bioconjugate Chemistry. Aug, 2016  |  Pubmed ID: 27383368

A panel of biotinylated bivalent H-type glycans that have been reported as binding ligands for human noroviruses were synthesized using a modular synthetic strategy. These glycoconjugates were attached to streptavidin-coated magnetic beads and used to recover human norovirus from fecal samples using a magnetic bead-based assay. The biotinylated bivalent glycans synthesized for this study exhibited similar or better capturing ability when compared to commercial biotinylated glycopolymers.

Detection of Human Norovirus in Intestinal Biopsies from Immunocompromised Transplant Patients

The Journal of General Virology. Sep, 2016  |  Pubmed ID: 27412790

Human noroviruses (HuNoVs) can often cause chronic infections in solid organ and haematopoietic stem cell transplant (HSCT) patients. Based on histopathological changes observed during HuNoV infections, the intestine is the presumed site of virus replication in patients; however, the cell types infected by HuNoVs remain unknown. The objective of this study was to characterize histopathological changes during HuNoV infection and to determine the cell types that may be permissive for HuNoV replication in transplant patients. We analysed biopsies from HuNoV-infected and non-infected (control) transplant patients to assess histopathological changes in conjunction with detection of HuNoV antigens to identify the infected cell types. HuNoV infection in immunocompromised patients was associated with histopathological changes such as disorganization and flattening of the intestinal epithelium. The HuNoV major capsid protein, VP1, was detected in all segments of the small intestine, in areas of biopsies that showed histopathological changes. Specifically, VP1 was detected in enterocytes, macrophages, T cells and dendritic cells. HuNoV replication was investigated by detecting the non-structural proteins, RdRp and VPg. We detected RdRp and VPg along with VP1 in duodenal and jejunal enterocytes. These results provide critical insights into histological changes due to HuNoV infection in immunocompromised patients and propose human enterocytes as a physiologically relevant cell type for HuNoV cultivation.

Complete Genome Sequence of Human Norovirus Strain GII.P7-GII.6 Detected in a Patient in the United States in 2014

Genome Announcements. Oct, 2016  |  Pubmed ID: 27795291

Homologous recombination is one of the driving forces contributing to the genetic variation of human norovirus, which is an important cause of sporadic and epidemic acute gastroenteritis globally. We report the near-complete genome of the novel recombinant norovirus strain GII.P7-GII.6, detected in an adult with norovirus gastroenteritis in the United States.

Norovirus and Sapovirus Epidemiology and Strain Characteristics Among Navajo and Apache Infants

PloS One. 2017  |  Pubmed ID: 28046108

Norovirus and sapovirus are important causes of acute gastroenteritis (AGE) among American Indian infants. We investigated the prevalence and molecular epidemiology of norovirus and sapovirus in American Indian infants who have historically experienced a high burden of AGE compared to other US populations. Stool samples were collected from 241 children with AGE (cases) and from 343 infants without AGE (controls) ≤9 months of age from 2002-2004. Cases experienced forceful vomiting and/or 3 or more watery or looser-than-normal stools in 24 hours. Stools were tested by real-time RT-PCR for norovirus GI, GII and GIV and sapovirus GI, GII, GIV and GV. Positive samples were genotyped after sequencing conventional RT-PCR products. Norovirus was identified in 76 (31.5%) of the cases and 70 (20.4%) of the controls (p<0.001). GII.3 and GII.4 Farmington Hills were the most frequently identified genotypes in 14.5% and 30.3% of cases and 17.1% and 27.1% of controls, respectively. Sapovirus GI and GII genotypes were identified in 8 (3.3%) of cases and 8 (2.3%) of controls and a single GIV virus was detected in a control. The same norovirus and sapovirus genotypes were circulating in the general U.S. population in the same time period. The high detection rate of norovirus in healthy controls suggests significant asymptomatic transmission in young infants in these communities.

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