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Human norovirus (HuNoV) is the most predominant viral agents of acute gastroenteritis. Point‐of‐care testing (POCT) based on lateral flow immunochromatography (LIFC) has become an important tool for rapid diagnosis of HuNoVs. However, low sensitivity and lack of quantitation are the bottlenecks of traditional LIFC. Thus, we established a rapid and accurate technique that combined immunomagnetic enrichment (IM) with LFIC to identify GII HuNoVs in fecal specimens. Before preparing immunofluorescent nanomagnetic microspheres and achieving the effect of HuNoV enrichment in IM and fluorescent signal in LFIC, amino‐functionalized magnetic beads (MBs) and carboxylated quantum dots (QDs) were coupled at a mass ratio of 4:10. Anti‐HuNoV monoclonal antibody was then conjugated with QDs‐MB. The limit of detection was 1.56 × 104 copies/mL, and the quantitative detection range was 1.56 × 104 copies/mL–1 × 106 copies/mL under optimal circumstances. The common HuNoV genotypes GII.2, GII.3, GII.4, and GII.17 can be detected, there was no cross‐reaction with various enteric viruses, including rotavirus, astrovirus, enterovirus, and sapovirus. A comparison between IM‐LFIC and RT‐qPCR for the detection of 87 fecal specimens showed a high level of agreement (kappa = 0.799). This suggested that the method is rapid and sensitive, making it a promising option for point‐of‐care testing in the future. [ABSTRACT FROM AUTHOR]

Norovirus is the primary foodborne pathogenic agent causing viral acute gastroenteritis. It possesses broad genetic diversity and the prevalence of different genotypes varies substantially. However, the differences in RNA‐dependent RNA polymerase (RdRp) activity among different genotypes of noroviruses remain unclear. In this study, the molecular mechanism of RdRp activity difference between the epidemic strain GII.17[P17] and the non‐epidemic strain GII.8[P8] was characterized. By evaluating the evolutionary history of RdRp sequences with Markov Chain Monte Carlo method, the evolution rate of GII.17[P17] variants was higher than that of GII.8[P8] variants (1.22 × 10−3 nucleotide substitutions/site/year to 9.31 × 10−4 nucleotide substitutions/site/year, respectively). The enzyme catalytic reaction demonstrated that the Vmax value of GII.17[P17] RdRp was 2.5 times than that of GII.8[P8] RdRp. And the Km of GII.17[P17] and GII.8[P8] RdRp were 0.01 and 0.15 mmol/L, respectively. Then, GII.8[P8] RdRp fragment mutants (A−F) were designed, among which GII.8[P8]‐A/B containing the conserved motif G/F were found to have significant effects on improving RdRp activity. The Km values of GII.8[P8]‐A/B reached 0.07 and 0.06 mmol/L, respectively. And their Vmax values were 1.34 times than that of GII.8[P8] RdRp. In summary, our results suggested that RdRp activities were correlated with their epidemic characteristics. These findings will ultimately provide a better understanding in replication mechanism of noroviruses and development of antiviral drugs. [ABSTRACT FROM AUTHOR]

The emergence of the novel GII.17 Kawasaki 2014 norovirus variant raising the interest of the public, has replaced GII.4 as the predominant cause of noroviruses outbreaks in East Asia during 2014–2015. Antigenic variation of the capsid protein is considered as one of the key mechanisms of norovirus evolution. In this study, we screened a panel of GII.17 mutants. First, we produced norovirus P proteins using cell-free protein synthesis (CFPS) system, comparing the results to pure proteins expressed in a cell-based system. Next, we determined the binding capability of specific monoclonal antibody (mAb) 2D11 using a unique set of wild-type GII.17 strains. Results of the EIA involving a panel of mutant cell-free proteins indicated that Q298 was the key residue within loop 1. These data highlighted the essential residues in the linear antibody binding characteristics of novel GII.17. Furthermore, it supported the CFPS as a promising tool for rapidly screening mutants via the scalable expression of norovirus P proteins. [ABSTRACT FROM AUTHOR]

Human norovirus is a common cause of acute gastroenteritis worldwide, and oysters have been found to be the main carriers for its spread. The lack of efficient pre-treatment methods has been a major bottleneck limiting the detection of viruses in oysters. In this study, we established a novel immunomagnetic enrichment method using polyamidoamine (PAMAM) dendrimer/SA-biotin-mediated cascade amplification for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) detection. We compared the capture efficiency of traditional immunomagnetic enrichment, biotin-amplified immunomagnetic enrichment, and PAMAM dendrimer/SA-biotin-mediated cascade-amplification immunomagnetic enrichment. The optimal capture efficiency of the novel method was 44.26 ± 1.45%, which increased by 183.17% (P < 0.01) and 18.09% (P < 0.05) compared with the first two methods, respectively. Three methods were all applied in detecting norovirus in 44 retail oysters, the detection rate of the PAMAM dendrimer/SA-biotin-mediated method was 25.0%, which was higher than those of traditional IME (15.90%) and SA-biotin-amplified IME (18.80%) by 9.1 and 6.2%, respectively. In conclusion, the novel method can be applied for the rapid detection of norovirus in oysters, which can help reduce the cost and time of detection and improve detection rates. [ABSTRACT FROM AUTHOR]

Norovirus diarrhea is a great threat to public health worldwide. To characterize the prevalence of circulating noroviruses associated with sporadic gastroenteritis cases in Guangzhou, 215 stool specimens were collected during two consecutive cold seasons in 2013-2015. Noroviruses were detected in 25 (11.63 %) samples, and GII.4 (6/9) and GII.17 (10/16) were identified as the most predominant variants of each of those seasons. The remaining strains belonged to the genotypes GII.P12/GII.3, GII.2, and GI.Pb/GI.6. The phylogenetic relationships of the GII.17 strains were analyzed based on their capsid protein sequences. This study suggests a significant shift of predominant variants associated with sporadic gastroenteritis in Guangzhou. [ABSTRACT FROM AUTHOR]

Noroviruses are still the primary cause of non-bacterial acute gastroenteritis worldwide. Recently, a novel GII.17 norovirus variant emerged and caused an infection peak in the cold season of 2014/2015 in some Asian countries, including China. In this study, in order to understand the evolutionary advantage of the novel variant, complete genomic sequences of GII.17 NoV strains from South China were comprehensively analyzed. Pairwise alignments of new GII.17 genomes with representative sequences of each GII genotype were performed. Inconsistent homology was observed between different protein-encoding regions, of which VPg (NS5) and P2 were found to be the most conserved and variable ones, respectively. The differences between new sequences and other reported GII.17 genomes were also compared, and 84 mismatched nucleotides were found, resulting in 15 amino acid changes. Then, all capsid sequences of different GII.17 NoV variants were collected for multiple alignments, and a total of 87 spots were identified during their evolution process. Homology modeling of capsid proteins of four GII.17 variants was carried out based on comparison with GII.4 VA387 strain, and structural differences were mainly embodied in five extended loops. Furthermore, positions of potential conformational epitope regions of new GII.17 variants were found more similar or adjacent to those of GII.4 rather than those of the former GII.17 variants. In summary, nine GII.17 strains from South China were extensively characterized based on their complete genomes, and a different distribution pattern of epitope residues was predicted on the new GII.17 variant capsid from that of the former ones. [ABSTRACT FROM AUTHOR]

Human noroviruses (HuNoVs) have been found as the leading cause of acute gastroenteritis outbreaks in all age groups and are significantly correlated with the consumption of shellfish. In this study, the contamination of HuNoVs in shellfish was estimated through a systematic review and meta-analysis. Studies on the contamination of HuNoVs in shellfish were searched from PubMed, Web of Science, Embase, and Cochrane Library from January 2000 to August 2021. A total of 75 studies were included, and the pooled HuNoVs prevalence in shellfish was 29% (95% CI: 23–35) worldwide. As revealed by the results of the subgroup meta-analysis, the prevalence of dominant genogroup was variable, and 4% (95% CI: 3–6), 13% (95% CI: 10–17), with 7% (95% CI: 4–11) of the samples, respectively, contaminated by GI alone, GII alone, and GI&GII. The HuNoVs prevalence of shellfish in Europe, America, and Asia was 33% (95% CI: 24–43), 24% (95% CI: 7–47), and 27% (95% CI: 18–35), respectively, while only 10% (95% CI: 5–17) in Africa. Furthermore, the prevalence of HuNoVs in shellfish was the highest in spring (35%, 95% CI: 23–49) and winter (35%, 95% CI: 22–50), and the lowest in summer (11%, 95% CI: 5–18). Oysters, clams, and mussels had comparable HuNoVs prevalence of 28% (95% CI: 20–37), 27% (95% CI: 16–39) and 24% (95% CI: 17–32), respectively. The prevalence of HuNoVs in shellfish from harvest areas and markets was 30% (95% CI: 23–38) and 30% (95% CI: 19–41), respectively. The results of this study suggest a substantial burden of HuNoVs in shellfish worldwide, with GII.4 (92.86%) and GII.2 (46.43%) as the predominant genotypes. This study provides information regarding the contamination of HuNoVs in shellfish worldwide, which will contribute to the development of appropriate control measures to prevent shellfish-related HuNoVs gastroenteritis. • The pooled prevalence of human noroviruses in shellfish was found as 29% (95% CI: 23–35) worldwide. • GII.4 (92.86%, 26/28) was found as the most common genotype in shellfish. • Significant differences were found in the seasonal and geographical prevalence of human noroviruses in shellfish. [ABSTRACT FROM AUTHOR]

Oysters and mussels are important vectors for norovirus (NoV). An efficient pretreatment method for NoV detection in oysters based on ISO 15216-2:2019 was established in our previous work, but its effectiveness for other types of shellfish remains unknown. Therefore, this study systematically compared the differences between the standard and modified ISO methods in detecting NoV for oysters and mussels. Using the standard ISO method, the recovery rates of NoV in oysters (2.10 ± 0.80 %) and mussels (2.39 ± 0.56 %) were comparable (p > 0.05, unpaired t -test). In contrast, the virus recovery rates in oysters (19.83 ± 3.64 %) and mussels (46.96 ± 3.55 %) were both significantly improved by the modified method. Also, a significant difference was found between the virus recovery rates in two shellfish (p < 0.05, unpaired t -test), resulting in a 2.09-fold difference in their virus concentrations. Additionally, the limits of detection at 95 % probability of the modified ISO method for oysters and mussels could both reach 3.33 × 103 copies/g of digestive glands. Finally, the modified ISO method has been successfully applied in commercial oysters (14/27, 51.85 %) and mussels (15/23, 65.22 %), and the results indicated a significant difference in NoV recovery rates between two shellfish (p < 0.05, one-way analysis of variance). In summary, the modified ISO method showed higher virus recovery rates than the standard ISO method, which would be used as an essential tool for NoV detection in oysters and mussels. • We developed a new method that improves viral detection in oysters and mussels. • Our method provided a higher virus recovery rate than the standard ISO method. • There were significant differences in virus recovery between oysters and mussels using the new method. [ABSTRACT FROM AUTHOR]

[Display omitted] • A digital isothermal detection chip was established for norovirus detection. • The first proposal of digital RAA. • Absolute quantification achieved at a linear range of 1.02 × 10–2.04 × 103 copies/μL. • The new method time is only 20 min. • The new method has been successfully applied in clinical testing. As the rapid spread of norovirus is a major public health problem, rapid diagnostic assays are essential in assisting the implementation of appropriate control measures. Here, we presented a facile sample partitioning norovirus digital isothermal detection (NoV-DID) chip that enables the application of a fast and low-cost digital nucleic acid amplification test (dNAAT) to norovirus detection. Our microfluidic chip had 3 layers, a PDMS cover layer, a functional layer, and a supporting layer, and consisted of a total of 5120 microchambers, each of which was able to contain a volume of 1.2 nL. This chamber digital reverse transcription recombinase-aid amplification (cdRT-RAA) assay achieved equal volume partition of samples within 10 min, with the signal amplification being completed in approximately 20 min at 39 °C, without the need for a thermal cycle device. The cdRT-RAA assay showed a good degree of linearity in the range of 1.02 × 100 to 2.04 × 103 copies (R2 = 0.99) with the limit of detection being 1.02 × 100 copies/μL, demonstrating that its sensitivity was comparable to that of RT-ddPCR. The digital nucleic acid detection method based on the microfluidic chip exhibited advantages of high sensitivity and high robustness, and might provide an alternative method for the early detection of norovirus and the prevention and control of epidemics. [ABSTRACT FROM AUTHOR]