Inactivation of Human Norovirus GII.4's Infectivity in Fresh Oysters (Crassostrea gigas) through Thermal Treatment in Association with Propidium Monoazide.

The current study investigated the effects of heat treatment (85 °C or 100 °C for 5-20 min) on human norovirus (HuNoV) GII.4's capsid stability in fresh oysters. In addition, propidium monoazide (PMA) was used in viral samples to distinguish infectious viruses and evaluated using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Further, we explored the effect of the heat treatment on oyster quality (Hunter color and hardness). The titer of HuNoV for oysters significantly (p < 0.05) decreased to 0.39-1.32 and 0.93-2.27 log10 copy number/µL in the non-PMA and PMA-treated groups, respectively, after heat treatment. HuNoV in oysters not treated with PMA showed a decrease of <1.5 - log10, whereas in PMA-treated oysters, a decrease of >1 - log10 was observed after treatment at 85 °C for 10 min. Treatments for both 15 min and 20 min at 100 °C showed a >99% log10 reduction using PMA/RT-qPCR. In the Hunter color, an increase in heat temperature and duration was associated with a significant decrease in 'L' (brightness+, darkness-) and an increase in 'a' (redness+, greenness-) and 'b' (yellowness+, blueness-) (p < 0.05). Our findings confirmed that the hardness of oyster meat significantly increased with increasing temperature and time (p < 0.05). This study demonstrated that PMA/RT-qPCR was effective in distinguishing HuNoV viability in heat-treated oysters. The optimal heat treatment for oysters was 10 min at 85 °C and 5 min at 100 °C.

Application of High-Pressure Processing (or High Hydrostatic Pressure) for the Inactivation of Human Norovirus in Korean Traditionally Preserved Raw Crab.

Human norovirus (HuNoV) is a common cause of outbreaks linked to food. In this study, the effectiveness of a non-thermal method known as high-pressure processing (HPP) on the viable reduction of an HuNoV GII.4 strain on raw crabs was evaluated at three different pressures (200, 400, and 600 MPa). HuNoV viability in raw crabs was investigated by using propidium monoazide/sarkosyl (PMA) as a nucleic acid intercalating dye prior to performing a real-time reverse transcription-polymerase chain reaction (RT-qPCR). The effect of the HPP exposure on pH, sensory, and Hunter colors were also assessed. HuNoV was reduced in raw crabs compared with control to HPP (0.15-1.91 log) in non-PMA and (0.67-2.23 log) in PMA. HuNoV genomic titer reduction was <2 log copy number/µL) when HPP was treated for 5 min without PMA pretreatment, but it was reduced to >2 log copy number/µL after PMA. The pH and Hunter colors of the untreated and HPP-treated raw crabs were significantly different (p < 0.05), but sensory attributes were not significant. The findings indicate that PMA/RT-qPCR could be used to detect HuNoV infectivity without altering the quality of raw crabs after a 5 min treatment with HPP. Therefore, HuNoV GII.4 could be reduced up to 2.23 log in food at a commercially acceptable pressure duration of 600 MPa for 5 min.

Inactivation of Human Norovirus GII.4 and Vibrio parahaemolyticus in the Sea Squirt (Halocynthia roretzi) by Floating Electrode-Dielectric Barrier Discharge Plasma.

Human norovirus (HNoV) GII.4 and Vibrio parahaemolyticus may be found in sea squirts. Antimicrobial effects of floating electrode-dielectric barrier discharge (FE-DBD) plasma (5-75 min, N2 1.5 m/s, 1.1 kV, 43 kHz) treatment were examined. HNoV GII.4 decreased by 0.11-1.29 log copy/µL with increasing duration of treatment time, and further by 0.34 log copy/µL when propidium monoazide (PMA) treatment was added to distinguish infectious viruses. The decimal reduction time (D1) of non-PMA and PMA-treated HNoV GII.4 by first-order kinetics were 61.7 (R2 = 0.97) and 58.8 (R2 = 0.92) min, respectively. V. parahaemolyticus decreased by 0.16-1.5 log CFU/g as treatment duration increased. The D1 for V. parahaemolyticus by first-order kinetics was 65.36 (R2 = 0.90) min. Volatile basic nitrogen showed no significant difference from the control until 15 min of FE-DBD plasma treatment, increasing after 30 min. The pH did not differ significantly from the control by 45-60 min, and Hunter color in "L" (lightness), "a" (redness), and "b" (yellowness) values reduced significantly as treatment duration increased. Textures appeared to be individual differences but were not changed by treatment. Therefore, this study suggests that FE-DBD plasma has the potential to serve as a new antimicrobial to foster safer consumption of raw sea squirts.

Emerging norovirus GII.4 Sydney[P31] causing acute gastroenteritis outbreak in children in Japan, during COVID-19, 2021.

INTRODUCTION: Norovirus (NoV) is the most common agent causing outbreaks and sporadic cases of acute gastroenteritis among all ages, especially children under 5 years old. During the coronavirus disease 2019 (COVID-19) pandemic, NoV infection has decreased drastically in Japan due to school closures and no outbreak related to NoV infection had been reported. METHOD: In mid-September 2021, NoV outbreak occurred in kindergarten and nursery schools in Maizuru, Kyoto prefecture, Japan. Twenty-six stool samples collected from patients who were diagnosed of NoV gastroenteritis from the outbreak by an immunochromatographic (IC) kit at a pediatric outpatient clinic in Maizuru city during 3 weeks from September 13 to October 8, 2021 were examined for the presence of NoV GII by reverse transcriptase-polymerase chain reaction (RT-PCR), genome sequencing, and phylogenetic analysis. RESULT: All 26 samples were confirmed positive to NoV GII and their genotypes were identified as GII.4 Sydney[P31]. The amino acid substitutions in open reading frame1 (ORF1) and ORF2 genes were found when compared with previously detected sporadic NoV GII.4 Sydney[P31] strains isolated in Japan. The clinical characterization of infected children was described. Most of the children were mild cases and vomiting was the most frequent clinical symptom. CONCLUSION: This study reported a recent emergence of NoV GII.4 Sydney[P31] causing acute gastroenteritis outbreak in children in Japan during the COVID-19 pandemic and suggests a need for further monitoring of NoV GII.4 variants.

Assessment of potential infectivity of human norovirus in the traditional Korean salted clam product "Jogaejeotgal" by floating electrode-dielectric barrier discharge plasma.

This study investigated the antiviral effects of floating electrode-dielectric barrier discharge (FE-DBD) plasma treatment (1.1 kV, 43 kHz, N2 1.5 m/s, 5-30 min) against human norovirus (HuNoV) GII.4 in Jogaejeotgal Infectivity was assessed using real-time quantitative-PCR (RT-qPCR) following treatment of samples with propidium monoazide (PMA) and sodium lauroyl sarcosinate (Sarkosyl). This study also investigated the effects of FE-DBD plasma treatment on Jogaejeotgal quality (assessed using pH value and Hunter colors). Following inoculation, the average titers of HuNoV GII.4 in Jogaejeotgal significantly (P < 0.05) decreased with increases in the FE-DBD plasma treatment time in both the non-PMA-treated and PMA + Sarkosyl-treated samples; in the non-PMA and PMA + Sarkosyl treated Jogaejeotgal, HuNoV GII.4 titers (log10 copy number/µL) were to: 3.16 and 2.95 (5 min), 2.90 and 2.48 (10 min), 2.82 and 2.40 (15 min), 2.58 and 2.26 (20 min), 2.48 and 2.06 (25 min), and 2.23 and 1.91 (30 min), respectively. The average titers of HuNoV demonstrated significant (P < 0.05) reductions of 0.35 log10 (55.3%) in PMA + Sarkosyl-treated samples compared with the non-PMA treated samples following exposure to 5-30 min of FE-DBD plasma. Reductions of >1-log for HuNoV in PMA + Sarkosyl- treated Jogaejeotgal required treatments of FE-DBD of 5-30 min. Using the first order kinetic model (R2 = 0.95), GII.4 decimal reduction time (D-value) resulting from FE-DBD plasma was 23.75 min. The pH and Hunter colors ("L", "a", and "b") were not significantly different (P > 0.05) between the untreated and FE-DBD plasma-treated Jogaejeotgal. Based on these results, the PMA + Sarkosyl/RT-qPCR method could be assessing HuNoV viability following 5-30 min treatment of FE-DBD plasma. Furthermore, may be an optimal treatment for Jogaejeotgal without altering the food quality (color and pH).

Detection of Norovirus Variant GII.4 Hong Kong in Asia and Europe, 2017-2019.

We report a new norovirus GII.4 variant, GII.4 Hong Kong, with low-level circulation in 4 Eurasia countries since mid-2017. Amino acid substitutions in key residues on the virus capsid associated with the emergence of pandemic noroviruses suggest that GII.4 Hong Kong has the potential to become the next pandemic variant.

A Heterodimeric Antibody Fragment for Passive Immunotherapy Against Norovirus Infection.

Human noroviruses cause an estimated 685 million infections and 200 000 deaths annually worldwide. Although vaccines against GII.4 and GI.1 genotypes are under development, no information is available regarding vaccines or monoclonal antibodies to other noroviral genotypes. Here, we developed 2 variable-domain llama heavy-chain antibody fragment (VHHs) clones, 7C6 and 1E4, against GII.4 and GII.17 human noroviruses, respectively. Although 7C6 cross-reacted with virus-like particles (VLPs) of GII.17, GII.6, GII.3, and GII.4, it neutralized only GII.4 norovirus. In contrast, 1E4 reacted with and neutralized only GII.17 VLPs. Both VHHs blocked VLP binding to human induced pluripotent stem cell-derived intestinal epithelial cells and carbohydrate attachment factors. Using these 2 VHHs, we produced a heterodimeric VHH fragment that neutralized both GII.4 and GII.17 noroviruses. Because VHH fragments are heat- and acid-stable recombinant monoclonal antibodies, the heterodimer likely will be useful for oral immunotherapy and prophylaxis against GII.4 and GII.17 noroviruses in young, elderly, or immunocompromised persons.

Nucleotide composition and synonymous codon usage of open reading frames in Norovirus GII.4 variants.

Norovirus GII.4 variants, a genotype in genogroup II belonging to the genus Norovirus, is a single-strand positive sense RNA containing three open reading frames (ORF1, ORF2 and ORF3) and is the most important pathogen causing nonbacterial gastroenteritis outbreaks. By using bioinformatic softwares such as Codon W, SPSS and so on, a total of 292 strains of the viruses isolated from 1974 to 2016 were analyzed for nucleotide composition and synonymous codon usage in each ORF. The result shows that it is enriched for A over the other bases in nucleotide composition, G behind the other bases in the 3rd site of all synonymous codons in the three ORFs. The patterns of nucleotide composition and codon bias of ORF2 are similar to those of ORF3 and different from those of ORF1. There are generally UpA motif and CpG motif in the codons with the lowest proportion. Correspondence analysis indicates that the codon usage may be changing over a certain time period for ORF1 in 2006 and 2012, ORF2 in 2012, and ORF3 in 2013. ENC (effective number of codons) plot and other analyses indicate that both natural selection and mutational pressure play partly roles in the ORFs, but natural selection is more important for ORF2 and ORF3. Besides, we also found all optimal codons in the ORFs. The study provides a basic understanding of the mechanism for norovirus GII.4 codon usage bias. AbbreviationsORFOpen Reading FrameENCEffective Number of CodonsCOAcorrespondence analysisRSCURelative Synonymous Codon UsageCAICodon Adaptation IndexCBICodon Bias IndexFopfrequency of optimal codonsL_symnumber of synonymous codonsL_aalength amino acidsGRAVYgrand average of hydropathicityAromaaromaticityCommunicated by Ramaswamy H. Sarma.

Phylogenetic characterization of norovirus strains detected from sporadic gastroenteritis in Seoul during 2014-2016.

BACKGROUND: Phylogenetic analysis of norovirus (NoV) is efficient for tracking NoV transmission. To determine the widespread NoV strains in Seoul, we conducted an extensive phylogenetic characterization of NoV-positives from 1659 diarrheal specimens collected in 2014-2016 for the Seoul NoV-surveillance. RESULTS: When the large numbers of NoV partial VP1 genome sequences were analyzed in acute gastroenteritis patients along with the phylogenetic characterization, we could identify molecular epidemiologic patterns based on the genetic characteristics of sporadic NoV strains circulating in Seoul, which could provide a detailed description of the genome-wide and community-wide NoV evolution in each genotype. The average NoV detection rate in our study period was 16.34% that was increased by 7.44% from 13.17% in 2014 to 20.61% in 2016. Prevalence of NoV GI and GII was 4.43% and 93.36%, respectively, and the GII.4, GII.17, and GII.3 were found to be the major type among 17 genotypes of NoV. The most prevalent one was GII.4 (50.92%) that was followed by GII.17 (18.08%) and GII.3 (9.96%). According to an extensive phylogenetic analysis based on partial VP1 sequences of 1008 NoV (276 sporadic, 518 outbreak and 214 reference), pandemic strains of GII.17, GII.4 and GII.3 have emerged in succession during the 2014-2016 Seoul NoV-surveillance. GII.17 emerged as GII.17|Kawasaki323 in 2014, and became the predominant genotype in 2015 with GII.17|2014_Kawasaki lineages (CUHK-NS-616/Kawasaki308). The formerly predominant GII.4 remained high-level with GII.4|2012_Sydney in 2014 and internally replaced to GII.4|2016_Kawasaki194 lineage (NOR-2565/NOR-2558/OH16002) that caused the sporadic NoV explosion since December 2015. Sporadically prevalent GII.3|Hu/Aichio334-13/2013 failed to develop any outbreaks, whereas sporadic GII.3|Hu/3-28/2015/HNZZ/CHN caused heavy outbreaks in Seoul without preparation time since November 2016. CONCLUSIONS: This is the first extensive phylogenetic study revealing the important events of NoV strains circulating in Seoul. Particularly, our study period from 2014 to 2016 was very dynamic with the emergences of the three main NoV strains (GII.17|2014_Kawasaki, GII.4|2016_Kawasaki194 and GII.3|Hu/3-28/2015/HNZZ/CHN) every year. We are sure that it is hard to detect above findings by simple conventional analysis. Our present study reports a future paradigm of the NoV molecular epidemiology, which might be highly valuable to track new strains and predict oncoming outbreaks.

Epidemiology and molecular characteristics of norovirus GII.4 Sydney outbreaks in Taiwan, January 2012-December 2013.

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.

Induction of homologous and cross-reactive GII.4-specific blocking antibodies in children after GII.4 New Orleans norovirus infection.

Noroviruses (NoVs) are major causative agents of acute gastroenteritis (AGE) in children worldwide and the most common viral cause of AGE in countries where rotavirus incidence has been eliminated by vaccination. Previous infections with the dominant GII.4 NoV genotype confer only partial protection against evolving immune escape variants that emerge every few years. The objective of this work was to investigate GII.4-specific homologous and cross-reactive antibody responses in young children after NoV GII.4-2009 New Orleans (NO) infection. Virus-like particles (VLPs) representing GII.4-1999, GII.4-2009 NO, and GII.4-2012 Sydney genotypes were used in ELISA and histo-blood group antigen blocking assays to examine acute and convalescent sera of five children <2 years of age infected with GII.4-2009 NO. GII.4-2009 NO infection induced IgG seroconversion to all three tested NoV GII.4 variants. Homologous blocking antibodies to GII.4-2009 NO were detected in each convalescent sera. Fourfold increase in cross-blocking antibodies to GII.4-2012 Sydney was observed in 4/5 subjects, but no child developed cross-blocking antibodies to GII.4-1999. In conclusion, antibodies induced in young children after norovirus GII.4 infection are targeted against the causative variant and may cross-protect against strains that are closely related, but not with more distinct and earlier GII.4 genotypes.