Genotypes, Recombinant Forms, and Variants of Norovirus GII.4 in Gipuzkoa (Basque Country, Spain), 2009-2012

Background: Noroviruses (NoVs) are genetically diverse, with genogroup II-and within it-genotype 4 (GII.4) being the most prevalent cause of acute gastroenteritis worldwide. The aim of this study was to characterize genogroup II NoV causing acute gastroenteritis in the Basque Country (northern Spain) from 2009-2012. Methods: The presence of NoV RNA was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) in stool specimens from children younger than 15 years old with community-acquired acute gastroenteritis, and from hospitalized adults or elderly residents of nursing homes with acute gastroenteritis. For genotyping, the open reading frames ORF1 (encoding the polymerase) and ORF2 (encoding the major capsid protein) were partially amplified and sequenced. Recombinant strains were confirmed by PCR of the ORF1/ORF2 junction region. Results: NoV was detected in 16.0% (453/2826) of acute gastroenteritis episodes in children younger than 2 years, 9.9% (139/1407) in children from 2 to 14 years, and 35.8% (122/341) in adults. Of 317 NoVs characterized, 313 were genogroup II and four were genogroup I. The GII.4 variants Den Haag-2006b and New Orleans-2009 predominated in 2009 and 2010-2011, respectively. In 2012, the New Orleans-2009 variant was partially replaced by the Sydney-2012 variant (GII.Pe/GII.4) and New Orleans-2009/Sydney-2012 recombinant strains. The predominant capsid genotype in all age groups was GII.4, which was the only genotype detected in outbreaks. The second most frequent genotype was GII.3 (including the recently described recombination GII.P16/GII.3), which was detected almost exclusively in children. Conclusion: Nine different genotypes of NoV genogroup II were detected; among these, intergenotype recombinant strains represented an important part, highlighting the role of recombination in the evolution of NoVs. Detection of new NoV strains, not only GII.4 strains, shortly after their first detection in other parts of the world shows that many NoV strains can spread rapidly.

Adsorption and aggregation properties of norovirus GI and GII virus-like particles demonstrate differing responses to solution chemistry

The transport properties (adsorption and aggregation behavior) of virus-like particles (VLPs) of two strains of norovirus ("Norwalk" GI.1 and "Houston" GII.4) were studied in a variety of solution chemistries. GI.1 and GII.4 VLPs were found to be stable against aggregation at pH 4.0-8.0. At pH 9.0, GI.1 VLPs rapidly disintegrated. The attachment efficiencies (a) of GI.1 and GII.4 VLPs to silica increased with increasing ionic strength in NaCl solutions at pH 8.0. The attachment efficiency of GI.1 VLPs decreased as pH was increased above the isoelectric point (pH 5.0), whereas at and below the isoelectric point, the attachment efficiency was erratic. Ca(2+) and Mg(2+) dramatically increased the attachment efficiencies of GI.1 and GII.4 VLPs, which may be due to specific interactions with the VLP capsids. Bicarbonate decreased attachment efficiencies for both GI.1 and GII.4 VLPs, whereas phosphate decreased the attachment efficiency of GI.1, while increasing GII.4 attachment efficiency. The observed differences in GI.1 and GII.4 VLP attachment efficiencies in response to solution chemistry may be attributed to differential responses of the unique arrangement of exposed amino acid residues on the capsid surface of each VLP strain.

Serological correlate of protection against norovirus-induced gastroenteritis

Norovirus infection is the leading cause of acute nonbacterial gastroenteritis. Histoblood group antigens (HBGAs) are host susceptibility determinants for Norwalk virus (NV) infection. We hypothesized that antibodies that block NV-HBGA binding are associated with protection from clinical illness following NV exposure.

Seroepidemiology of norovirus-associated travelers' diarrhea

Background: Noroviruses (NoVs) are the most common cause of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and were recently identified as a leading cause of travelers' diarrhea (TD) in US and European travelers to Mexico, Guatemala, and India.

Methods: Serum and diarrheic stool samples were collected from 75 US student travelers to Cuernavaca, Mexico, who developed TD. NoV RNA was detected in acute diarrheic stool samples using reverse transcription-polymerase chain reaction (RT-PCR). Serology assays were performed using GI.1 Norwalk virus (NV) and GII.4 Houston virus (HOV) virus-like particles (VLPs) to measure serum levels of immunoglobulin A (IgA) and IgG by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA); serum IgM was measured by capture enzyme-linked immunosorbent assay (ELISA), and the 50% antibody-blocking titer (BT50 ) was determined by a carbohydrate-blocking assay.

Results: NoV infection was identified in 12 (16%; 9 GI-NoV and 3 GII-NoV) of 75 travelers by either RT-PCR or fourfold or more rise in antibody titer. Significantly more individuals had detectable preexisting IgA antibodies against HOV (62/75, 83%) than against NV (49/75, 65%) (p = 0.025) VLPs. A significant difference was observed between NV- and HOV-specific preexisting IgA antibody levels (p = 0.0037), IgG (p = 0.003), and BT50 (p = <0.0001). None of the NoV-infected TD travelers had BT50  > 200, a level that has been described previously as a possible correlate of protection.

Conclusion: We found that GI-NoVs are commonly associated with TD cases identified in US adults traveling to Mexico, and seroprevalence rates and geometric mean antibody levels to a GI-NoV were lower than to a GII-NoV strain.

Quantifying transmission of norovirus during an outbreak

Healthcare workers are thought to play a role in nosocomial transmission of norovirus, but the level and direction of norovirus transmission between patients and healthcare workers in sustaining transmission during an outbreak have not been quantified.

Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages.

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-alphabeta receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.

Brote de toxiinfección alimentaria por Norovirus: estudio epidemiológico y medidas sanitarias aplicadas para evitar su recurrencia

Objetivo: Debido a que las Toxiinfecciones alimentarias (TIA) originan daños para la salud, pérdidas económicas y requieren la intervención directa sobre la fuente de contaminación, el objetivo fue realizar un estudio clínico, epidemiológico e higiénico sanitario de un brote de TIA, sin posibilidad de análisis microbiológico. Diseño: Estudio descriptivo y de casos-controles. Emplazamiento: Comunidad de Munera. Albacete (España). Participantes: Población compuesta por las personas asistentes a tres comidas de celebración de comunión. Mediciones Principales: Se realizó una encuesta telefónica epidemiológica y se calcularon odds ratio (OR) crudas y ajustadas con intervalos de confianza al 95 %. Se realizó un modelo de regresión logística multivariante e inspección sanitaria del restaurante. Resultados: Se expusieron 204 personas y fueron encuestadas 62 (32 afectados y 30 sanos). La tasa global de ataque fue de 51,6 % y el periodo de incubación de 33 horas. Los síntomas principales: dolor abdominal, vómitos y diarreas. El tiempo de duración de la enfermedad fue 38 horas. Se observó asociación con la "Ensalada" (OR-cruda: 7,5; IC95: 1,48-37,91), resultando al borde de la significación estadística la relación con "Sepia" (ORc: 4,15; IC95: 1,00-17,18) y "Marisco" (ORc: 4,57; IC95: 0,87-24,08). La OR-ajustada para la "Ensalada" fue 5,41 (IC95: 1,00-29,23). La inspección sanitaria comprobó la existencia de deficiencias de estructura, limpieza, desinfección y formación de manipuladores. Conclusiones: La Ensalada fue el único alimento asociado estadísticamente, pero no podemos descartar la Sepia y Mariscos; mediante la inspección sanitaria y los criterios de Kaplan, es posible clasificarla como una infección por Norovirus. Se aplicaron medidas especiales de funcionamiento en el restaurante para evitar reincidencia del brote.

Wide Field-of-view Microscopes and Endoscopes for Time-lapse Imaging and High-throughput Screening

Wide field-of-view (FOV) microscopy is of high importance to biological research and clinical diagnosis where a high-throughput screening of samples is needed. This thesis presents the development of several novel wide FOV imaging technologies and demonstrates their capabilities in longitudinal imaging of living organisms, on the scale of viral plaques to live cells and tissues.

The ePetri Dish is a wide FOV on-chip bright-field microscope. Here we applied an ePetri platform for plaque analysis of murine norovirus 1 (MNV-1). The ePetri offers the ability to dynamically track plaques at the individual cell death event level over a wide FOV of 6 mm × 4 mm at 30 min intervals. A density-based clustering algorithm is used to analyze the spatial-temporal distribution of cell death events to identify plaques at their earliest stages. We also demonstrate the capabilities of the ePetri in viral titer count and dynamically monitoring plaque formation, growth, and the influence of antiviral drugs.

We developed another wide FOV imaging technique, the Talbot microscope, for the fluorescence imaging of live cells. The Talbot microscope takes advantage of the Talbot effect and can generate a focal spot array to scan the fluorescence samples directly on-chip. It has a resolution of 1.2 μm and a FOV of ~13 mm². We further upgraded the Talbot microscope for the long-term time-lapse fluorescence imaging of live cell cultures, and analyzed the cells’ dynamic response to an anticancer drug.

We present two wide FOV endoscopes for tissue imaging, named the AnCam and the PanCam. The AnCam is based on the contact image sensor (CIS) technology, and can scan the whole anal canal within 10 seconds with a resolution of 89 μm, a maximum FOV of 100 mm × 120 mm, and a depth-of-field (DOF) of 0.65 mm. We also demonstrate the performance of the AnCam in whole anal canal imaging in both animal models and real patients. In addition to this, the PanCam is based on a smartphone platform integrated with a panoramic annular lens (PAL), and can capture a FOV of 18 mm × 120 mm in a single shot with a resolution of 100─140 μm. In this work we demonstrate the PanCam’s performance in imaging a stained tissue sample.

Características clínicas e frequência de diarreia por norovírus em crianças hospitalizadas, vacinadas e não vacinadas contra rotavírus Rio de Janeiro, Brasil, 2004-2009

Os norovírus (NV) são uma importante causa de hospitalização infantil. Crianças internadas por gastroenterite por NV (GENV) são consideradas portadoras de diarreia grave. O objetivo desse estudo, realizado na cidade do Rio de Janeiro, Brasil, é descrever as características clínicas e a frequência da diarreia por NV em crianças hospitalizadas, comparando as taxas de detecção de NV em crianças vacinadas e não vacinadas contra rotavírus (Rotarix). Foram coletadas 659 amostras de fezes de igual número de crianças e encaminhadas para análise pela reação em cadeia pela polimerase, precedida de transcrição reversa no período de janeiro de 2004 a dezembro de 2009. O percentual de amostras positivas para os NV foi de 27,3% nesse período. Das 180 amostras positivas para NV, 55% tiveram origem na comunidade (aqCo) e 45% foram de aquisição nosocomial (aqNo). O percentual de GENV nos dois anos anteriores (2004 e 2005) à introdução da vacina Rotarix foi de 28,3%, sendo 11,3% o percentual de amostras aqCo. Nos dois anos posteriores (2008 e 2009), a GENV significou 24,4%, e as amostras aqCo foram 14,9% (p<0,05). Em 647 crianças, 494 não receberam a vacina Rotarix, enquanto 151 crianças receberam, pelo menos, uma dose. O percentual de GENV foi de 23,8% e 39,7%, respectivamente (p<0,05). Apesar do comportamento sazonal dos casos de GENV aqCo, esse fato não teve significância estatística. Das 180 crianças, 61,6% tinham peso ≤ p10 do NCHS, 82,2% tinham idade ≤ 5anos. As crianças com idade ≤ 2 anos foram mais acometidas nos casos de aqCo do que àquelas de aqNo (p<0,05). Foram observados em 82 crianças: vômitos (73,2%), febre (54,9%), tosse (20,7%), coriza (2,2%), sangue nas fezes (8,5%), erupção cutânea (4,9%) e broncoespasmo (7,3%). Houve significância estatística com relação à frequência maior de febre, coriza, tosse e broncoespasmo nas crianças com GENV de aqCo do que naquelas de aqNo (p<0,05). De 69 crianças, 73,9% apresentaram desidratação e, dessas, 76,5% necessitaram de hidratação venosa. Esses dados tiveram significância estatística, representada por maiores percentuais nas crianças com GENV de aqCo do que naquelas de aqNo (p<0,05). Esse estudo demonstra que os NV foram um importante agente etiológico nos casos de gastroenterites em crianças hospitalizadas e responsável por altas taxas de infecções nosocomiais. Estatisticamente, não foi comprovada uma tendência de aumento dos casos de GENV no período do estudo, como também do aumento da frequência de GENV nos anos posteriores em relação aos anos anteriores à introdução da vacina Rotarix no Brasil em 2006. No entanto, houve significância estatística quando foi avaliado o percentual de GENV em crianças hospitalizadas vacinadas e não vacinadas contra RV. Um aumento dos casos de GENV em crianças poderá vir a acontecer nos próximos anos, quando é esperado que um número maior de crianças será vacinado contra RV. Tosse, coriza e broncoespasmo são sintomas que devem ser mais detalhadamente investigados. Estratégias de prevenção contra a disseminação dos NV são condutas importantes em unidades de internação. Uma vacina eficaz contra norovírus pode ser um benefício significativo para reduzir o percentual de crianças hospitalizadas por diarreia.

Development and clinical validation of multiplex TaqMan® assays for rapid diagnosis of viral gastroenteritis.

There is a need to provide rapid, sensitive, and often high throughput detection of pathogens in diagnostic virology. Viral gastroenteritis is a serious health issue often leading to hospitalization in the young, the immunocompromised and the elderly. The common causes of viral gastroenteritis include rotavirus, norovirus (genogroups I and II), astrovirus, and group F adenoviruses (serotypes 40 and 41). This article describes the work-up of two internally controlled multiplex, probe-based PCR assays and reports on the clinical validation over a 3-year period, March 2007 to February 2010. Multiplex assays were developed using a combination of TaqMan™ and minor groove binder (MGB™) hydrolysis probes. The assays were validated using a panel of 137 specimens, previously positive via a nested gel-based assay. The assays had improved sensitivity for adenovirus, rotavirus, and norovirus (97.3% vs. 86.1%, 100% vs. 87.8%, and 95.1% vs. 79.5%, respectively) and also more specific for targets adenovirus, rotavirus, and norovirus (99% vs. 95.2%, 100% vs. 93.6%, and 97.9% vs. 92.3%, respectively). For the specimens tested, both assays had equal sensitivity and specificity for astrovirus (100%). Overall the probe-based assays detected 16 more positive specimens than the nested gel-based assay. Post-introduction to the routine diagnostic service, a total of 9,846 specimens were processed with multiplex 1 and 2 (7,053 pediatric, 2,793 adult) over the 3-year study period. This clinically validated, probe-based multiplex testing algorithm allows highly sensitive and timely diagnosis of the four most prominent causes of viral gastroenteritis.

Atmospheric pressure, nonthermal plasma inactivation of MS2 bacteriophage: effect of oxygen concentration on virucidal activity

AimsThe main aim of this study was to determine the virucidal inactivation efficacy of an in-house-designed atmospheric pressure, nonthermal plasma jet operated at varying helium/oxygen feed gas concentrations against MS2 bacteriophage, widely employed as a convenient surrogate for human norovirus.

Methods and ResultsThe effect of variation of percentage oxygen concentration in the helium (He) carrier gas was studied and found to positively correlate with MS2 inactivation rate, indicating a role for reactive oxygen species (ROS) in viral inactivation. The inactivation rate constant increased with increasing oxygen concentrations up to 075% O-2. 3 log(10) (999%) reductions in MS2 viability were achieved after 3min of exposure to the plasma source operated in a helium/oxygen (9925%:075%) gas mixture, with >7 log(10) reduction after 9min exposure.

ConclusionsAtmospheric pressure, nonthermal plasmas may have utility in the rapid disinfection of virally contaminated surfaces for infection control applications.

Significance and Impact of StudyThe atmospheric pressure, nonthermal plasma jet employed in this study exhibits rapid virucidal activity against a norovirus surrogate virus, the MS2 bacteriophage, which is superior to previously published inactivation rates for chemical disinfectants.

The risk of viral dispersal and aerosolization by different hand-drying methods

Background World Health Organization hand hygiene guidelines state that if electric hand dryers are used, they should not aerosolize pathogens. Previous studies have investigated the dispersal by different hand-drying devices of chemical indicators, fungi and bacteria on the hands. This study assessed the aerosolization and dispersal of virus on the hands to determine any differences between hand-drying devices in their potential to contaminate other occupants of public washrooms and the washroom environment. Methods A suspension of MS2, an Escherichia coli bacteriophage virus, was used to artificially contaminate the hands of participants prior to using three different handdrying devices: jet air dryer, warm air dryer, paper towel dispenser. Virus was detected by plaque formation on agar plates layered with the host bacterium. Vertical dispersal of virus was assessed at a fixed distance (0.4 m) and over a range of different heights (0.0 – 1.8 m) from the floor. Horizontal dispersal was assessed at different distances of up to three metres from the hand-drying devices. Virus aerosolization and dispersal was also assessed at different times up to 15 minutes after use by means of air sampling at two distances (0.1 and 1.0 m) and at a distance behind and offset from each of the hand-drying devices. Results Over a range of heights, the jet air dryer was shown to produce over 60 times greater vertical dispersal of virus from the hands than a warm air dryer and over 1300 times greater than paper towels; the maximum being detected between 0.6 and 1.2 metres from the floor. Horizontal dispersal of virus by the jet air dryer was over 20 times greater than a warm air dryer and over 190 times greater than paper towels; virus being detected at distances of up to three metres. Air sampling at three different positions from the hand-drying devices 15 minutes after use showed that the jet air dryer produced over 50-times greater viral contamination of the air than a warm air dryer and over 110-times greater than paper towels. Conclusions Due to their high air speed, jet air dryers aerosolize and disperse more virus over a range of heights, greater distances, and for longer times than other hand drying devices. If hands are inadequately washed, they have a greater potential to contaminate other occupants of a public washroom and the washroom environment. Main messages: Jet air dryers with claimed air speeds of over 600 kph have a greater potential than warm air dryers or paper towels to aerosolize and disperse viruses on the hands of users. The choice of hand-drying device should be carefully considered. Jet air dryers may increase the risk of transmission of human viruses, such as norovirus, particularly if hand washing is inadequate.