Multiple Novel Human Norovirus Recombinants Identified in Wastewater in Pretoria, South Africa by Next-Generation Sequencing.

The genogroup II genotype 4 (GII.4) noroviruses are a major cause of viral gastroenteritis. Since the emergence of the Sydney_2012 variant, no novel norovirus GII.4 variants have been reported. The high diversity of noroviruses and periodic emergence of novel strains necessitates continuous global surveillance. The aim of this study was to assess the diversity of noroviruses in selected wastewater samples from Pretoria, South Africa (SA) using amplicon-based next-generation sequencing (NGS). Between June 2018 and August 2020, 200 raw sewage and final effluent samples were collected fortnightly from two wastewater treatment plants in Pretoria. Viruses were recovered using skimmed milk flocculation and glass wool adsorption-elution virus recovery methods and screened for noroviruses using a one-step real-time reverse-transcription PCR (RT-PCR). The norovirus BC genotyping region (570-579 bp) was amplified from detected norovirus strains and subjected to Illumina MiSeq NGS. Noroviruses were detected in 81% (162/200) of samples. The majority (89%, 89/100) of raw sewage samples were positive for at least one norovirus, compared with 73% (73/100) of final effluent samples. Overall, a total of 89 different GI and GII RdRp-capsid combinations were identified, including 51 putative novel recombinants, 34 previously reported RdRp-capsid combinations, one emerging novel recombinant and three Sanger-sequencing confirmed novel recombinants.

Human rotavirus VP4 and VP7 genetic diversity and detection of GII norovirus in Ibadan as Nigeria introduces rotavirus vaccine.

OBJECTIVE: This cross-sectional study investigated the circulating strains of rotavirus and screened for noravirus in Ibadan, Nigeria as the country introduces the rotavirus vaccine into its national immunization program. METHODS: Sixty-five stool samples were collected from children younger than 5 years with clinically diagnosed diarrhea and screened for the presence of rotavirus and norovirus using RT-PCR. Rotavirus-positive samples were further analyzed to determine the G and P genotypes using semi-nested multiplex PCR. RESULTS: The rates of rotavirus and norovirus positivity were 30.8% and 10.8%, respectively, whereas the rate of rotavirus and norovirus mixed infection was 4.6%. G1 was the predominant VP7 genotype, followed by G2, G9, and G1G2G9, whereas the predominant VP4 genotype was P[4], followed by P[6], P[8], and P[9]. The mixed P types P[4]P[8] and P[4]P[6] were also detected. G1P[4] was the most common VP4 and VP7 combination, followed by G2P[4], G1[P6], G1P[8], G2P[6], G2P[9], G9P[6], G2G9P[4], G2P[4]P[6], G1P[4]P[8], G2G9P[8], G1G2G9P[8], and G1[non-typable] P[non-typable], which were detected in at least 5% of the samples. Four samples had a combination of non-typable G and P types. CONCLUSIONS: It is essential to monitor the circulation of virus strains prior to and during the implementation of the immunization program.

Identification of a novel norovirus species in fox.

A novel Norovirus (NoV) was identified by viral metagenomic analysis in fox fecal samples from the Xinjiang Uygur Autonomous Region of China. The virus exhibited typical genomic characteristics of NoVs. It was closely related to the canine NoV GVII strains with 86.0-86.2% and 91.9% amino acid identities in the capsid protein VP1 and RNA-dependent RNA polymerase (RdRp), respectively. The fox NoV clustered phylogenetically with the two canine NoV GVII strains, and it was distant from other NoVs. According to the new classification criteria of NoVs, the new fox NoV belongs to the same genotype as GVII, similar to canine GVII NoVs. Moreover, key amino acid residues in the Histo-blood group antigen (HBGA) binding sites and the HBGA binding pattern of the fox NoV differed significantly from those of human and canine GVII NoVs. This study identified a new GVII norovirus from wild foxes in China. These findings enrich our understanding of the diversity of NoVs and provide further evidence regarding the genetic heterogeneity of NoVs in carnivores.

Two-step purification of tag-free norovirus-like particles from silkworm larvae (Bombyx mori).

Recombinantly expressed VP1 of norovirus self-assembled and formed norovirus-like particles (NoV-LPs). This native VP1 was expressed using the Bombyx mori nucleopolyhedrovirus (BmNPV) expression system in silkworm larva. NoV-LPs were collected from silkworm fat body lysate by density gradient centrifugation. To improve the purity of the NoV-LP, the proteins were further purified using immobilized metal affinity chromatography based on the surface exposed side chain of histidine residues. The additional purification led to a highly purified virus-like particle (VLP). The morphology and size of the purified VLPs were examined using a transmission electron microscope, and dynamic light scattering revealed a monodispersed spherical morphology with a diameter of 34 nm. The purified product had a purity of >90% with a recovery yield of 48.7% (equivalent to 930 µg) from crude lysate, obtained from seven silkworm larvae. In addition, the purified VLP could be recognized by antibodies against GII norovirus in sandwich enzyme-linked immunosorbent assay, which indicated that the silkworm-derived VLP is biologically functional as a NoV-LP in its native state, is structurally correct, and exerts its biological function. Our results suggest that the silkworm-derived NoV-LP may be useful for subsequent applications, such as in a vaccine platform. Moreover, the silkworm-based expression system is known for its robustness, facile up-scalability, and relatively low expense compared to insect cell systems.

Molecular epidemiology of norovirus infections in children with acute gastroenteritis in 2017-2019 in Tianjin, China.

Norovirus (NoV) is the leading cause of acute gastroenteritis (AGE) worldwide. Globally, the GII.4 Sydney 2012 strain has predominated since 2012, although GII.4 variant strains have caused AGE outbreaks in China. Recent patterns of NoV genotype distributions in 6011 children with AGE in Tianjin, China were investigated. NoV was detected using real-time reverse-transcriptase polymerase chain reaction and sequencing of partial sequences of the viral capsid gene. NoV genotypes were determined, and phylogenetic analysis was conducted. Epidemiological and clinical data were compared between children infected with different NoV genotypes. NoV was detected in 27.6% of the specimens tested. GII.4 strains comprised 49.4% infections, followed by GII.3 at 39.9%. Genotypes GII.2, GII.13, GII.17, GII.1, GII.6, and GII.14 were also detected. NoV was detected during most of the year, with a peak season of cases in the winter. Diarrhea, vomiting, fever, abdominal pain, and dehydration were present in patients with NoV infection. The main genotypes were GII.4 and GII.3, with a slight increase in GII.2, beginning in March 2017. Among the GII.4 strains, GII.4 Sydney 2012 was the only epidemic strain in Tianjin. Patients with GII.4 genotypes were more likely to present with diarrhea and vomiting than those with GII.3. Children with GII. Others were prone to suffered from dehydration and abdominal pain than those with GII.3. NoV GII has become the main cause of viral AGE in Tianjin, China. The predominant genotypes of NoV were GII.4 and GII.3. Identification of emerging genotypes is crucial for the prevention and control of NoV-caused AGE.

Factors associated with the detection of norovirus among asymptomatic adults.

OBJECTIVES: Little is known about asymptomatic norovirus infection and its risk factors in healthy adults. This study investigated detection of norovirus in stool and its associated factors among asymptomatic healthy adults in a high-income country. METHODS: This prospective cross-sectional study-conducted between February 2016 and January 2017 at a teaching hospital in Japan-included apparently healthy adults aged ≥18 years who underwent voluntary health check-ups. Our primary outcome was detection of norovirus in stool specimens confirmed by real-time RT-PCR. We evaluated descriptive statistics and associated factors, including demographics, social habits, and clinical parameters. RESULTS: Among 15 532 participants, 4536 (29.2%, mean age 58.0 (standard deviation 11.8) years, male 44.6%) were enrolled, and 112 (2.5%, GI 57, GII 54, GI + GII 1) were norovirus-positive. Monthly prevalence rates of the GI norovirus were consistent throughout the year, while those of GII were high in May. Participants aged <40 and ≥ 80 years had higher rates of GII norovirus detection. Participants who occasionally consume alcohol, especially wine (odds ratio (OR) 0.17, 95% confidence interval (CI) 0.04-0.68), had lower norovirus detection rates than abstainers. Participants with untreated dyslipidaemia and a low high-density lipoprotein (HDL) cholesterol level had higher detection rates than those with treated dyslipidaemia (OR 1.48, 95%CI 1.07-2.05) and a normal HDL cholesterol level (OR 2.60, 95%CI 1.46-4.61). Some gastrointestinal and female genital diseases were associated with norovirus detection. CONCLUSIONS: The norovirus detection rate in asymptomatic adults was 2.5%. Participants with specific lifestyles or medical histories may have higher risks of asymptomatic norovirus infection.

Secretor Status Strongly Influences the Incidence of Symptomatic Norovirus Infection in a Genotype-Dependent Manner in a Nicaraguan Birth Cohort.

BACKGROUND: The role of histo-blood group on the burden and severity of norovirus gastroenteritis in young infants has not been well documented. METHODS: Norovirus gastroenteritis was assessed in 443 Nicaraguan children followed from birth until 3 years of age. Stool samples were tested for norovirus by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and histo-blood group antigens (HBGAs) were determined by phenotyping of saliva and blood. Hazard ratios and predictors of norovirus acute gastroenteritis (AGE) outcome stratified by HBGA were estimated using Cox proportional hazards models. RESULTS: Of 1353 AGE episodes experienced by children, 229 (17%) tested positive for norovirus with an overall incidence of 21.9/100 child-years. Secretor children were infected as early as 2 months of age and had a higher incidence of norovirus GII compared to nonsecretor children (15.4 vs 4.1/100 child-years, P = .006). Furthermore, all GII.4 AGE episodes occurred in secretor children. Children infected with GI (adjusted odds ratio [aOR], 0.09 [95% confidence interval {CI}, .02-.33]) or non-GII.4 viruses (aOR, 0.2 [95% CI, .07-.6]) were less likely to have severe AGE compared to GII.4-infected children. CONCLUSIONS: Secretor status in children strongly influences the incidence of symptomatic norovirus infection in a genogroup or genotype-dependent manner and provides evidence that clinical severity in children depends on norovirus genotypes.

Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry.

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Design of a High-Sensitivity Dimeric G-Quadruplex/Hemin DNAzyme Biosensor for Norovirus Detection.

G-quadruplexes can bind with hemin to form peroxidase-like DNAzymes that are widely used in the design of biosensors. However, the catalytic activity of G-quadruplex/hemin DNAzyme is relatively low compared with natural peroxidase, which hampers its sensitivity and, thus, its application in the detection of nucleic acids. In this study, we developed a high-sensitivity biosensor targeting norovirus nucleic acids through rationally introducing a dimeric G-quadruplex structure into the DNAzyme. In this strategy, two separate molecular beacons each having a G-quadruplex-forming sequence embedded in the stem structure are brought together through hybridization with a target DNA strand, and thus forms a three-way junction architecture and allows a dimeric G-quadruplex to form, which, upon binding with hemin, has a synergistic enhancement of catalytic activities. This provides a high-sensitivity colorimetric readout by the catalyzing H2O2-mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline -6-sulfonic acid) diammonium salt (ABTS). Up to 10 nM of target DNA can be detected through colorimetric observation with the naked eye using our strategy. Hence, our approach provides a non-amplifying, non-labeling, simple-operating, cost-effective colorimetric biosensing method for target nucleic acids, such as norovirus-conserved sequence detection, and highlights the further implication of higher-order multimerized G-quadruplex structures in the design of high-sensitivity biosensors.

Determination of murine norovirus aerosol concentration during toilet flushing.

Murine norovirus (MNV) was used as a surrogate for human viral pathogens (e.g., norovirus) to determine if toilet flushing resulted in the aerosolization of virus. A flushometer type toilet was seeded with a viral solution of 105 and 106 PFU mL-1 of MNV and then flushed. Upon flushing, two bioaerosol samplers were activated to collect aerosolized MNV. Prior to the experiment, two optical particle counters monitored particle size and number distribution of aerosol produced from flushing a toilet across height, position, and side. The location with the highest mean particle concentration, was behind the toilet and 0.15 m above the toilet bowl rim, which is where bioaerosol sampling occurred. Bioaerosol and toilet water samples were collected, extracted and then quantified using RT-ddPCR. The concentration of MNV collected after seeding the toilet water ranged from 2.18 × 105 to 9.65 × 106 total copies of MNV. Positive samples of airborne MNV were detected with collected concentrations ranging from 383 to 684 RNA copies/m3 of air. This study provides evidence that viral pathogens may be aerosolized when a toilet is flushed. Furthermore, the MNV used in this study is a model organism for human norovirus and may be generalizable to other viral pathogens (e.g., coronavirus). This study suggests that virus is aerosolized from toilet flushing and may contribute to human exposure to viral pathogens.

Norovirus outbreaks in long-term care facilities in Catalonia from 2017 to 2018.

Norovirus is the leading cause of outbreaks of acute viral gastroenteritis. We carried out this study to investigate outbreaks in long-term care facilities reported in 2017 and 2018 in Catalonia (Spain). The characteristics of the centers, exposed persons and the genogroups responsible were analyzed. Viral loads were estimated. The attack rate (AR) of the outbreaks studied, and the rate ratio (RR) and the odds ratio (OR) and their 95% confidence intervals as measures of association were calculated. The mean cycle thresholds were compared using the t-test for independent means. We included 30 outbreaks (4631 exposed people). The global AR was 25.93%. The RR of residents vs. staff was 2.28 (95% CI 2.0-2.6). The RR between AR in residents with total or severe dependence vs. residents with moderate, low or no-dependence was 1.23 (95% CI 1.05-1.45). The AR were higher in smaller centers than in larger ones (38.47% vs. 19.25% and RR 2; 95% CI 1.82-2.2). GII was responsible for 70% of outbreaks. No association was found between the genogroup and presenting symptoms (OR 0.96; 95% CI 0.41-2.26). Viral loads were higher in symptomatic than in asymptomatic patients (p = 0.001).

From a case-control survey to a diagnostic viral gastroenteritis panel for testing of general practitioners' patients.

OBJECTIVE: To evaluate the pathogenicity of a broad range of 11 possible gastroenteritis viruses, by means of statistical relationships with cases vs. controls, or Ct-values, in order to establish the most appropriate diagnostic panel for our general practitioner (GP) patients in the Netherlands (2010-2012). METHODS: Archived stool samples from 1340 cases and 1100 controls were retested using internally controlled multiplex real-time PCRs for putative pathogenic gastroenteritis viruses: adenovirus, astrovirus, bocavirus, enterovirus, norovirus GI and GII, human parechovirus, rotavirus, salivirus, sapovirus, and torovirus. RESULTS: The prevalence of any virus in symptomatic cases and asymptomatic controls was 16.6% (223/1340) and 10.2% (112/1100), respectively. Prevalence of astrovirus (adjusted odds ratio (aOR) 10.37; 95% confidence interval (CI) 1.34-80.06) and norovirus GII (aOR 3.10; CI 1.62-5.92) was significantly higher in cases versus controls. Rotavirus was encountered only in cases. We did not find torovirus and there was no statistically significant relationship with cases for salivirus (aOR 1,67; (CI) 0.43-6.54)), adenovirus non-group F (aOR 1.20; CI 0.75-1.91), bocavirus (aOR 0.85; CI 0.05-13.64), enterovirus (aOR 0.83; CI 0.50-1.37), human parechovirus (aOR 1.61; CI 0.54-4.77) and sapovirus (aOR 1.15; CI 0.67-1.98). Though adenovirus group F (aOR 6.37; CI 0.80-50.92) and norovirus GI (aOR 2.22, CI: 0.79-6.23) are known enteropathogenic viruses and were more prevalent in cases than in controls, this did not reach significance in this study. The Ct value did not discriminate between carriage and disease in PCR-positive subjects. CONCLUSIONS: In our population, diagnostic gastroenteritis tests should screen for adenovirus group F, astrovirus, noroviruses GI and GII, and rotavirus. Case-control studies as ours are lacking and should also be carried out in populations from other epidemiological backgrounds.

CRISPR-Cas12a combined with reverse transcription recombinase polymerase amplification for sensitive and specific detection of human norovirus genotype GII.4.

Human norovirus (NOV) is a common and serious virus that accounts for sporadic cases and outbreaks of gastroenteritis. This study aimed to develop rapid, reliable and portable detection systems by coupling reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR-Cas12a (RT-RPA-Cas12a) for NOV genotype GII.4. Here, three primers for RNA-dependent RNA polymerase gene of NOV were designed and screened. Then, RT-RPA products were detected using CRISPR-Cas12a system by combing with fluorescence or lateral flow (LF). RT-RPA-Cas12a-based fluorescence or LF assay can be completed within 40 min, with the detection limit of up to 9.65 × 102copies/mL and no cross-reactivity with metapneumovirus, bocavirus, seoul virus, and respiratory syncytial virus. Furthermore, the detection coincidence rates of RT-RPA-Cas12a-based fluorescence and LF with qRT-PCR were 98.3%. Therefore, the present study suggests that both RT-RPA-Cas12a-based fluorescence and LF are promising sensitive, specific and alternative method for diagnosis of NOV genotype GII.4 without ancillary equipment.

Improved diagnosis of gastrointestinal infections using a semi-automated multiplex real-time PCR for detection of enteropathogens.

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis.Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination.Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures.Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013-2018) included stool culture, microscopy and immunochromatography.Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013-2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis.Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

Human Coronaviruses Do Not Transfer Efficiently between Surfaces in the Absence of Organic Materials.

Human coronaviruses, including SARS-CoV-2, are known to spread mainly via close contact and respiratory droplets. However, other potential means of transmission may be present. Fomite-mediated transmission occurs when viruses are deposited onto a surface and then transfer to a subsequent individual. Surfaces can become contaminated directly from respiratory droplets or from a contaminated hand. Due to mask mandates in many countries around the world, the former is less likely. Hands can become contaminated if respiratory droplets are deposited on them (i.e., coughing or sneezing) or through contact with fecal material where human coronaviruses (HCoVs) can be shed. The focus of this paper is on whether human coronaviruses can transfer efficiently from contaminated hands to food or food contact surfaces. The surfaces chosen were: stainless steel, plastic, cucumber and apple. Transfer was first tested with cellular maintenance media and three viruses: two human coronaviruses, 229E and OC43, and murine norovirus-1, as a surrogate for human norovirus. There was no transfer for either of the human coronaviruses to any of the surfaces. Murine norovirus-1 did transfer to stainless steel, cucumber and apple, with transfer efficiencies of 9.19%, 5.95% and 0.329%, respectively. Human coronavirus OC43 transfer was then tested in the presence of fecal material, and transfer was observed for stainless steel (0.52%), cucumber (19.82%) and apple (15.51%) but not plastic. This study indicates that human coronaviruses do not transfer effectively from contaminated hands to contact surfaces without the presence of fecal material.

Differential impact of the COVID-19 pandemic on laboratory reporting of norovirus and Campylobacter in England: A modelling approach.

BACKGROUND: The COVID-19 pandemic has impacted surveillance activities for multiple pathogens. Since March 2020, there was a decline in the number of reports of norovirus and Campylobacter recorded by England's national laboratory surveillance system. The aim is to estimate and compare the impact of the COVID-19 pandemic on norovirus and Campylobacter surveillance data in England. METHODS: We utilised two quasi-experimental approaches based on a generalised linear model for sequential count data. The first approach estimates overall impact and the second approach focuses on the impact of specific elements of the pandemic response (COVID-19 diagnostic testing and control measures). The following time series (27, 2015-43, 2020) were used: weekly laboratory-confirmed norovirus and Campylobacter reports, air temperature, conducted Sars-CoV-2 tests and Index of COVID-19 control measures stringency. RESULTS: The period of Sars-CoV-2 emergence and subsequent sustained transmission was associated with persistent reductions in norovirus laboratory reports (p = 0.001), whereas the reductions were more pronounced during pandemic emergence and later recovered for Campylobacter (p = 0.075). The total estimated reduction was 47% - 79% for norovirus (12-43, 2020). The total reduction varied by time for Campylobacter, e.g. 19% - 33% in April, 1% - 7% in August. CONCLUSION: Laboratory reporting of norovirus was more adversely impacted than Campylobacter by the COVID-19 pandemic. This may be partially explained by a comparatively stronger effect of behavioural interventions on norovirus transmission and a relatively greater reduction in norovirus testing capacity. Our study underlines the differential impact a pandemic may have on surveillance of gastrointestinal infectious diseases.

Molecular epidemiology and genetic diversity of norovirus among hospitalized children with acute gastroenteritis in Tianjin, China, 2018-2020.

BACKGROUND: Norovirus (NoV) is a major cause of viral acute gastroenteritis (AGE) in children worldwide. Epidemiological analysis with respect to the virus strains is limited in China. This study aimed to investigate the prevalence, patterns, and molecular characteristics of NoV infection among children with AGE in China. METHODS: A total 4848 stool samples were collected from children who were admitted with AGE in Tianjin Children's Hospital from August 2018 to July 2020. NoV was preliminarily detected using real-time reverse transcription polymerase chain reaction (RT-PCR). Partial sequences of the RNA-dependent RNA polymerase (RdRp) and capsid genes of positive samples were amplified by conventional RT-PCR and then sequenced. The NoV genotype was determined by online Norovirus Typing Tool Version 2.0, and phylogenetic analysis was conducted using MEGA 6.0. RESULTS: The prevalence of NoV was 26.4% (1280/4848). NoV was detected in all age groups, with the 7-12 months group having the highest detection rate (655/2014, 32.5%). NoV was detected during most part of the year with higher frequency in winter than other seasons. Based on the genetic analysis of RdRp, GII. Pe was the most predominant genotype detected at 70.7% (381/539) followed by GII.P12 at 25.4% (137/539). GII.4 was the most predominant capsid genotype detected at 65.3% (338/518) followed by GII.3 at 26.8% (139/518). Based on the genetic analysis of RdRp and capsid sequences, the strains were clustered into 10 RdRp-capsid genotypes: GII.Pe-GII.4 Sydney 2012 (65.5%), GII.P12-GII.3 (27.2%), GII.P16-GII.2 (1.8%), GII.P12-GII.2 (0.2%), GII.P17-GII.17 (1.1%), GII.Pe-GII.3 (1.8%), GII.Pe-GII.2 (1.1%), GII.Pe-GII.1 (0.4%), GII.16-GII.4 Sydney 2012 (0.7%), and GII.P7-GII.6 (0.2%). The predominant NoV genotypes changed from GII.Pe-GII.4 Sydney 2012 and GII.P12-GII.3 between August 2018 and July 2019 to GII.Pe-GII.4 Sydney 2012 and GII.P16-GII.2 between August 2019 and July 2020. The patients with GII.Pe-GII.4 Sydney 2012 genotype were more likely to suffer from vomiting symptom than those with GII.P12-GII.3. CONCLUSIONS: NoV is an important pathogen responsible for viral AGE among children in China. GII.Pe-GII.4 Sydney 2012 and GII.P12-GII.3 were major recombinant genotypes. Knowledge of circulating genotypes and seasonal trends is of great importance for disease prevention and surveillance.

Comparative study of the geographical spread of genogroup II porcine norovirus and human norovirus.

Livestock pigs and porcine norovirus could be candidate tools for future studies on the geographic isolation of norovirus. In this study, we provide the first evidence for geographic isolation of the host as a determinant of the distribution of subgenotypes of the porcine norovirus genogroup II (GII) genotype 11. Environmental water samples were collected from peri-urban streams and estuaries in South Korea between 2014 and 2020. In total, 488 GII region C sequences of norovirus open reading frame 2 were isolated. A total of 14 genotypes were detected, two of which (GII.11 and GII.18) corresponded to porcine norovirus. Five human norovirus genotypes (GII.2, GII.3, GII.4, GII.6, and GII.17) and one porcine norovirus genotype (GII.11) comprised the subgenotypes. Integrated analysis of seasonal and geographical factors revealed that the possibility of the co-emergence of different GII.11 subgenotypes in the same province was lower than that of human norovirus subgenotypes in the same province. Additional algorithms designed to eliminate potential biases further supported the estimated restricted geographical spread of the GII.11 subgenotypes. Fecal contamination source tracking revealed low detection rates of porcine norovirus in the absence of upstream pig farms. These results suggest that a one-sided viral transmission route, mainly dependent on indirect contact owing to the limited chance of direct contact between geographically separated livestock pig populations, may be responsible for the restricted geographical spread of the GII.11 subgenotypes.

Diagnostic Accuracy of Immunochromatographic Tests for the Detection of Norovirus in Stool Specimens: a Systematic Review and Meta-Analysis.

Noroviruses are the leading cause of acute gastroenteritis in all age groups and constitute a major health and economic burden worldwide. This systematic review and meta-analysis aimed to determine the diagnostic accuracy of immunochromatographic tests (ICTs) for the detection of norovirus in stool specimens, which has not been performed previously. In this systematic review and meta-analysis (registered on PROSPERO, CRD42020186911), we searched Medline/PubMed, Embase, Cochrane Library, and Web of Science for all studies published up to 16 May 2020. The values for sensitivity, specificity, positive likelihood ratio (LR+), negative likelihood ratio (LR-), and diagnostic odds ratio (DOR) of ICTs with 95% confidence interval (CI) were pooled using a bivariate random-effects model. The summary receiver operating characteristic curve and area under the curve were used to summarize overall test accuracy. We included 43 studies describing 7,428 samples. The overall estimates of sensitivity, specificity, LR+, LR-, DOR, and accuracy of ICT for diagnosing norovirus were 0.61 (95% CI, 0.54 to 0.67), 0.97 (95% CI, 0.95 to 0.98), 17.08 (95% CI, 11.15 to 26.18), 0.40 (95% CI, 0.34 to 0.46), 53.9 (95% CI, 31.32 to 92.78), and 0.928, respectively. Significant differences in pooled sensitivities were noted between age groups and in pooled DOR and LR+ between genogroups of included samples. ICT provides low sensitivity but high specificity and accuracy for detecting norovirus. Thus, an ICT for norovirus can be a rapid and convenient way for identifying patients early; however, a negative result cannot rule out norovirus infection and should be confirmed by a reference test.

Molybdenum Trioxide Quantum Dot-Encapsulated Nanogels for Virus Detection by Surface-Enhanced Raman Scattering on a 2D Substrate.

The use of nanogels (NGs) to modulate surface-enhanced Raman scattering (SERS) activities is introduced as an innovative strategy to address certain critical issues with SERS-based immunoassays. This includes the chemical deformation of SERS nanotags, as well as their nonspecific interactions and effective "hotspots" formation. Herein, the polymeric cocoon and stimuli-responsive properties of NGs were used to encapsulate SERS nanotags containing plasmonic molybdenum trioxide quantum dots (MoO3-QDs). The pH-controlled release of the encapsulated nanotags and their subsequent localization by maleimide-functionalized magnetic nanoparticles facilitated the creation of "hotspots" regions with catalyzed SERS activities. This approach resulted in developing a biosensing platform for the ultrasensitive immunoassays of hepatitis E virus (HEV) or norovirus (NoV). The immunoassays were optimized using the corresponding virus-like particles to attain limits of detection of 6.5 and 8.2 fg/mL for HEV-LPs and NoV-LPs, respectively. The SERS-based technique achieved a signal enhancement factor of up to ∼108 due to the combined electromagnetic and chemical mechanisms of the employed dual-SERS substrate of MoO3-QDs/2D hexagonal boron nitride nanosheets. The highlight and validation of the developed SERS-based immunoassays was the detection of NoV in infected patients' fecal specimen and clinical HEV G7 subtype. Importantly, this system can be used to maintain the stability of SERS nanotags and improve their reliability in immunoassays.