Molecular Characterization of Enteric Viruses Causing Acute Gastroenteritis among Children under 5 Years Old in Distrito Central, Honduras.

INTRODUCTION: Diarrheal diseases constitute a significant public health problem in terms of mortality and morbidity. In Honduras and around the world, RVs have consistently emerged as the single most important etiologic agent in acute childhood diarrhea. However, other viruses, such as NoVs and HAstVs, have also been shown to be responsible for viral gastroenteritis. Unfortunately, the country has limited information concerning the etiologic role of these viral agents in acute gastroenteritis. This study investigated the frequency, genotypes, and epidemiological characteristics of RV-A, NoVs, and HAstVs among children under 5 years old in Distrito Central, Honduras. METHODS: Stool samples and their corresponding epidemiological data were collected from children with acute gastroenteritis in three healthcare centers in Distrito Central. All samples were screened by immunoassays for RV-A and HAstVs. RV-A-positive samples were molecularly characterized by RT-PCR and genotyping assays. RT-PCR was also applied to confirm HAstVs positivity and to detect NoVs, followed by nucleotide sequencing to assign their genotypes. RESULTS: Our results show that at least one viral agent was detected in 31% of the children. The frequency of RV-A, NoVs, and HAstVs was 14%, 13%, and 5%, respectively. The most frequent RV-A genotype was G2P[4], occurring in 93% of cases. 92.3% of NoVs-positive samples belonged to genogroup II, with GII.4 and GII.16 being the most common. HAstVs were clustered into three genotypes: HAstV-1, HAstV-2, and HAstV-8. Only one sample showed coinfection with NoVs and HAstVs. CONCLUSION: This comprehensive molecular and epidemiological characterization of enteric viruses demonstrates the vast diversity of these agents and describes for the first time NoVs and HAstVs as causative agents of acute childhood gastroenteritis in Distrito Central, Honduras. This suggests that further in-depth studies of the pediatric population are necessary to develop and implement effective preventive and control measures in the country.

Genetic diversity of enteric viruses responsible of gastroenteritis in urban and rural Burkina Faso.

BACKGROUND: Viral gastrointestinal infections remain a major public health concern in developing countries. In Burkina Faso, there are very limited updated data on the circulating viruses and their genetic diversity. OBJECTIVES: This study investigates the detection rates and characteristics of rotavirus A (RVA), norovirus (NoV), sapovirus (SaV) and human astrovirus (HAstV) in patients of all ages with acute gastrointestinal infection in urban and rural areas. STUDY DESIGN & METHODS: From 2018 to 2021, stool samples from 1,295 patients with acute gastroenteritis were collected and screened for RVA, NoV, SaV and HAstV. Genotyping and phylogenetic analyses were performed on a subset of samples. RESULTS: At least one virus was detected in 34.1% of samples. NoV and SaV were predominant with detection rates of respectively 10.5 and 8.8%. We identified rare genotypes of NoV GII, RVA and HAstV, recombinant HAstV strains and a potential zoonotic RVA transmission event. CONCLUSIONS: We give an up-to-date epidemiological picture of enteric viruses in Burkina Faso, showing a decrease in prevalence but a high diversity of circulating strains. However, viral gastroenteritis remains a public health burden, particularly in pediatric settings. Our data advocate for the implementation of routine viral surveillance and updated management algorithms for diarrheal disease.

Genetic Diversity and Phylogenetic Relationship of Human Norovirus Sequences Derived from Municipalities within the Sverdlovsk Region of Russia.

Human noroviruses (HuNoVs) are highly contagious pathogens responsible of norovirus-associated acute gastroenteritis (AGE). GII.4 is the prevailing HuNoV genotype worldwide. Currently there are no studies on the molecular monitoring and phylogenetic analysis of HuNoVs in the territory of the Sverdlovsk region; therefore, it is not possible to objectively assess their genetic diversity. The aim of the study is to carry out genotyping and phylogenetic analysis of HuNoVs in the Sverdlovsk region from 2022 to 2023. Fecal samples (n = 510) were collected from children suffering from HuNoV-AGE in municipalities of the Sverdlovsk region and the capsid genotype was determined by amplifying the ORF1/ORF2 junction. Of the 196 HuNoVs typed, which represent 38% of the studied samples, the largest share of HuNoV genotypes belong to the GII genogroup-86%, followed by the GI genogroup-14%. Noroviruses GII.4 and GII.17 were the co-dominant capsid genotypes (33.2% each). Phylogenetic analysis demonstrates that the identified sequences on the territory of the Sverdlovsk region have the smallest genetic distance, which gives grounds for their unification into a common cluster. Routine monitoring and phylogenetic analysis of circulating norovirus pathogens spectrum will enable timely tracking of HuNoVs genetic diversity and evolutionary events. This will lead to the development of more effective anti-epidemic measures, ultimately reducing the burden of infectious diseases.

Transposase-Assisted RNA/DNA Hybrid Co-Tagmentation for Target Meta-Virome of Foodborne Viruses.

Foodborne diseases are major public health problems globally. Metagenomics has emerged as a widely used tool for pathogen screening. In this study, we conducted an updated Tn5 transposase-assisted RNA/DNA hybrid co-tagmentation (TRACE) library construction approach. To address the detection of prevalent known foodborne viruses and the discovery of unknown pathogens, we employed both specific primers and oligo-T primers during reverse transcription. The method was validated using clinical samples confirmed by RT-qPCR and compared with standard RNA-seq library construction methods. The mapping-based approach enabled the retrieval of nearly complete genomes (>95%) for the majority of virus genome segments (86 out of 88, 97.73%), with a mean coverage depth of 21,494.53× (ranging from 77.94× to 55,688.58×). Co-infection phenomena involving prevalent genotypes of Norovirus with Astrovirus and Human betaherpesvirus 6B were observed in two samples. The updated TRACE-seq exhibited superior performance in viral reads percentages compared to standard RNA-seq library preparation methods. This updated method has expanded its target pathogens beyond solely Norovirus to include other prevalent foodborne viruses. The feasibility and potential effectiveness of this approach were then evaluated as an alternative method for surveilling foodborne viruses, thus paving the way for further exploration into whole-genome sequencing of viruses.

Analysis of viral diversity in dogs with acute gastroenteritis from Brazilian Amazon.

Viral gastroenteritis is commonly reported in dogs and involves a great diversity of enteric viruses. In this research, viral diversity was investigated in dogs with diarrhea in Northern Brazil using shotgun metagenomics. Furthermore, the presence of norovirus (NoV) was investigated in 282 stool/rectal swabs of young/adult dogs with or without diarrhea from two public kennels, based on one-step reverse transcription polymerase chain reaction (RT-PCR) for genogroup VI and VII (GVI and GVII) and real-time RT-PCR for GI, GII, and GIV. Thirty-one viral families were identified, including bacteriophages. Phylogenetic analyses showed twelve complete or nearly complete genomes belonging to the species of Protoparvovirus carnivoran1, Mamastrovirus 5, Aichivirus A2, Alphacoronavirus 1, and Chipapillomavirus 1. This is the first description of the intestinal virome of dogs in Northern Brazil and the first detection of canine norovirus GVII in the country. These results are important for helping to understand the viral groups that circulate in the canine population.

Epidemics and diversity of norovirus variants with acute gastroenteritis outbreak in Hongshan District, Wuhan City, China, 2021-2023.

BACKGROUND: Norovirus is the predominant pathogen causing foodborne illnesses and acute gastroenteritis (AGE) outbreaks worldwide, imposing a significant disease burden. This study aimed to investigate the epidemiological characteristics and genotypic diversity of norovirus outbreaks in Hongshan District, Wuhan City. METHODS: A total of 463 AGE cases from 39 AGE-related outbreaks in Hongshan District between January 1, 2021, and June 30, 2023, were included in the study. Reverse transcription-polymerase chain reaction (RT-PCR) was used to identify norovirus types GI and GII in anal swab samples from all cases. Norovirus-positive samples were sequenced and analyzed for the open reading frame (ORF) 1/ORF2 hinge region. RESULTS: 26 norovirus infectious outbreaks were reported among 39 acute diarrheal outbreaks, including 14 outbreaks in kindergartens, 8 in elementary schools, and 4 in universities. Based on clinical symptoms and epidemiological investigations, a total of 1295 individuals were identified as having been exposed to norovirus, yielding an attack rate of 35.75 %. A higher proportion of outbreaks was observed during the winter and spring seasons (38.46 %). Additionally, norovirus-positive samples were subjected to sequencing and analysis of the open reading frame (ORF) 1/ORF2 hinge region. Genotypic data for norovirus was successfully obtained from 18 (69.23 %) of the infectious outbreaks, revealing 10 distinct recombinant genotypes. GII.4 Sydney 2012 [P31] and GII.17[P17] were the predominant strains in 2021 and 2022, GII.3 [P12] emerged as the dominant strain in 2023. CONCLUSION: Norovirus outbreaks in Hongshan District predominantly occurred in crowded educational institutions, with peaks in the cold season and a high attack rate in universities. GII.3 [P12] has become the locally predominant strain.

Diverse genotypes of norovirus genogroup I and II contamination in environmental water in Thailand during the COVID-19 outbreak from 2020 to 2022.

Noroviruses (NoVs) are the most significant viral pathogens associated with waterborne and foodborne outbreaks of nonbacterial acute gastroenteritis in humans worldwide. This study aimed to investigate the prevalence and diversity of NoVs contaminated in the environmental water in Chiang Mai, Thailand. A total of 600 environmental water samples were collected from ten sampling sites in Chiang Mai from July 2020 to December 2022. The presence of NoV genogroups I (GI), GII, and GIV were examined using real-time RT-PCR assay. The genotype of the virus was determined by nucleotide sequencing and phylogenetic analysis. The results showed that NoV GI and GII were detected at 8.5% (51/600) and 11.7% (70/600) of the samples tested, respectively. However, NoV GIV was not detected in this study. NoV circulated throughout the year, with a higher detection rate during the winter season. Six NoV GI genotypes (GI.1-GI.6) and eight NoV GII genotypes (GII.2, GII.3, GII.7, GII.8, GII.10, GII.13, GII.17, and GII.21) were identified. Among 121 NoV strains detected, GII.17 was the most predominant genotype (24.8%, 30 strains), followed by GII.2 (21.5%, 26 strains), GI.3 (17.4%, 21 strains), and GI.4 (16.5%, 20 strains). Notably, NoV GII.3, GII.7, GII.8, and GII.10 were detected for the first time in water samples in this area. This study provides insight into the occurrence and seasonal pattern of NoV along with novel findings of NoV strains in environmental water in Thailand during the COVID-19 outbreak. Our findings emphasize the importance of further surveillance studies to monitor viral contamination in environmental water.

Epidemiological Characteristics and Genotypic Features of Rotavirus and Norovirus in Jining City, 2021-2022.

Diarrhea, often caused by viruses like rotavirus (RV) and norovirus (NV), is a global health concern. This study focuses on RV and NV in Jining City from 2021 to 2022. Between 2021 and 2022, a total of 1052 diarrhea samples were collected. Real-Time Quantitative Fluorescent Reverse Transcriptase-PCR was used to detect RV-A, NV GI, and NV GII. For RV-A-positive samples, VP7 and VP4 genes were sequenced for genotype analysis, followed by the construction of evolutionary trees. Likewise, for NV-GII-positive samples, VP1 and RdRp genes were sequenced for genotypic analysis, and evolutionary trees were subsequently constructed. Between 2021 and 2022, Jining City showed varying detection ratios: RV-A alone (excluding co-infection of RV-A and NV GII) at 7.03%, NV GI at 0.10%, NV GII alone (excluding co-infection of RV-A and NV GII) at 5.42%, and co-infection of RV-A and NV GII at 1.14%. The highest RV-A ratios were shown in children ≤1 year and 2-5 years. Jining, Jinxiang County, and Liangshan County had notably high RV-A ratios at 24.37% (excluding co-infection of RV-A and NV GII) and 18.33% (excluding co-infection of RV-A and NV GII), respectively. Jining, Qufu, and Weishan had no RV-A positives. Weishan showed the highest NV GII ratios at 35.48% (excluding co-infection of RV-A and NV GII). Genotype analysis showed that, in 2021, G9P[8] and G2P[4] were dominant at 94.44% and 5.56%, respectively. In 2022, G8P[8], G9P[8], and G1P[8] were prominent at 75.86%, 13.79%, and 10.35%, respectively. In 2021, GII.3[P12], GII.4[P16], and GII.4[P31] constituted 71.42%, 14.29%, and 14.29%, respectively. In 2022, GII.3[P12] and GII.4[P16] accounted for 55.00% and 45.00%, respectively. RV-A and NV showed varying patterns for different time frames, age groups, and regions within Jining. Genotypic shifts were also observed in prevalent RV-A and NV GII strains in Jining City from 2021 to 2022. Ongoing monitoring of RV-A and NV is recommended for effective prevention and control.

Epidemiology of norovirus infection in Nigeria: a systematic review and meta-analysis.

Human norovirus (HuNoV) is responsible for most cases of gastroenteritis worldwide, but information about the prevalence and diversity of HuNoV infections in lower-income settings is lacking. In order to provide more information about the burden and distribution of norovirus in Nigeria, we systematically reviewed original published research articles on the prevalence of HuNoV in Nigeria by accessing databases, including PubMed, Web of Science, ScienceDirect, Google Scholar, and African Journals Online (AJOL). The protocol for the review was registered on PROSPERO (registration number CRD42022308857). Thirteen relevant articles were included in the review, and 10 of them were used for meta-analysis. The pooled prevalence of HuNoV-associated gastroenteritis among children below 5 years of age in Nigeria, determined using the random-effects model, was 10.9% (95% CI, 6.7-16.7%). Among children below the age of 5 presenting with HuNoV infections, the highest prevalence was in children ≤2 years old (n = 127, 83%). The prevalence of HuNoV infections was seen to decrease with increasing age. In addition, HuNoV was detected in asymptomatic food handlers, bats, and seafoods. A total of 85 sequences of HuNoV isolates from Nigeria have been determined, and based on those sequences, the most prevalent norovirus genogroup was GII (84%). Genotypes GII.4 and GI.3 were the most frequently identified genotypes, with GII.4 constituting 46% of all of the HuNoVs identified in Nigeria. These results suggest a risk associated with cocirculation of emerging variants with known genotypes because of their recombination potential. Larger molecular epidemiological studies are still needed to fully understand the extent and pattern of circulation of HuNoVs in Nigeria.

Genetic diversity and distribution of noroviruses among all age groups of patients with diarrhea in Amhara National Regional State, Ethiopia.

BACKGROUND: Norovirus (NoV) is the leading cause of diarrheal disease worldwide and the impact is high in developing countries, including Ethiopia. Moreover, there is a significant and fluctuating global genetic diversity that varies across diverse environments over time. Nevertheless, there is a scarcity of data on the genetic diversity of NoV in Ethiopia. OBJECTIVE: This study was aimed to assess the genetic diversity and distribution of NoVs circulating in the Amhara National Regional State, Ethiopia, by considering all age groups. METHODS: A total of 519 fecal samples were collected from diarrheal patients from May 01/2021 to November 30/ 2021. The fecal samples were screened for the presence of NoVs using real-time RT-PCR by targeting a portion of the major capsid protein coding region. The positive samples were further amplified using conventional RT-PCR, and sequenced. RESULTS: The positivity rate of NoV was (8.9%; 46/519). The detection rate of NoV genogroup II (GII) and genogroup I (GI) was 38 (82.6%) and 8 (17.4%), respectively. Overall, five distinct GII (GII.3, GII.6, GII.10, GII.17, and GII.21) and two GI (GI.3 and GI.5) genotypes were detected. Within the GII types, GII.3 was the predominant (34.2%) followed by GII.21 (15.8%), GII.17 (10.5%), GII.6 and GII.10 each (2.6%). Norovirus GII.21 is reported for the first time in Ethiopia. The genetic diversity and distribution of NoVs were significantly different across the four sampling sits and age groups. The phylogenetic analysis revealed close relatedness of the current strains with published strains from Ethiopia and elsewhere. CONCLUSION: The distribution and genetic diversity of NoV was considerably high, with predominance of non-GII.4 genotypes. The GII.21 genotype is a new add on the growing evidences on the genetic diversity of NoVs in Ethiopia. Future nationwide surveillance studies are necessary to gain comprehensive data in Ethiopia.

Seasonality of respiratory, enteric, and urinary viruses revealed by wastewater genomic surveillance.

Wastewater surveillance can reveal population-level infectious disease burden and emergent public health threats can be reliably assessed through wastewater surveillance. While molecular methods for wastewater monitoring of microorganisms have traditionally relied on PCR-based approaches, next-generation sequencing (NGS) can provide deeper insights via genomic analyses of multiple diverse pathogens. We conducted a year-long sequencing surveillance of 1,408 composite wastewater samples collected from 12 neighborhood-level access points in the greater Tempe area, Arizona, USA, and show that variation in wastewater viruses is driven by seasonal time and location. The temporal dynamics of viruses in wastewater were influenced cyclically, with the most dissimilarity between samples 23 weeks apart (i.e., winter vs summer, spring vs fall). We identified diverse urinary and enteric viruses including polyomaviruses, astroviruses, and noroviruses, and showed that their genotypes/subtypes shifted across seasons. We show that while wastewater data of certain respiratory viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strongly correlate with clinical case rates, laboratory-reported case incidences were discordant with surges of high viral load in wastewater for other viruses like human coronavirus 229E. These results demonstrate the utility of wastewater sequencing for informing decision-making in public health.IMPORTANCEWastewater surveillance can provide insights into the spread of pathogens in communities. Advances in next-generation sequencing (NGS) methodologies allow for more precise detection of viruses in wastewater. Long-term wastewater surveillance of viruses is an important tool for public health preparedness. This system can act as a public health observatory that gives real-time early warning for infectious disease outbreaks and improved response times.

Longitudinal analysis of the enteric virome in paediatric subjects from the Free State Province, South Africa, reveals early gut colonisation and temporal dynamics.

The gut of healthy neonates is devoid of viruses at birth, but rapidly becomes colonised by normal viral commensals that aid in important physiological functions like metabolism but can, in some instances, result in gastrointestinal illnesses. However, little is known about how this colonisation begins, its variability and factors shaping the gut virome composition. Thus, understanding the development, assembly, and progression of enteric viral communities over time is key. To explore early-life virome development, metagenomic sequencing was employed in faecal samples collected longitudinally from a cohort of 17 infants during their first six months of life. The gut virome analysis revealed a diverse and dynamic viral community, formed by a richness of different viruses infecting humans, non-human mammals, bacteria, and plants. Eukaryotic viruses were detected as early as one week of life, increasing in abundance and diversity over time. Most of the viruses detected are commonly associated with gastroenteritis and include members of the Caliciviridae, Picornaviridae, Astroviridae, Adenoviridae, and Sedoreoviridae families. The most common co-occurrences involved asymptomatic norovirus-parechovirus, norovirus-sapovirus, sapovirus-parechovirus, observed in at least 40 % of the samples. Majority of the plant-derived viruses detected in the infants' gut were from the Virgaviridae family. This study demonstrates the first longitudinal characterisation of the gastrointestinal virome in infants, from birth up to 6 months of age, in sub-Saharan Africa. Overall, the findings from this study delineate the composition and variability of the healthy infants' gut virome over time, which is a significant step towards understanding the dynamics and biogeography of viral communities in the infant gut.

Identifying SNP threshold from P2 sequences for investigating norovirus transmission.

Noroviruses are a group of non-enveloped single-stranded positive-sense RNA virus belonging to Caliciviridae family. They can be transmitted by the fecal-oral route from contaminated food and water and cause mainly acute gastroenteritis. Outbreaks of norovirus infections could be difficult to detect and investigate. In this study, we developed a simple threshold detection approach based on variations of the P2 domain of the capsid protein. We obtained sequences from the norovirus hypervariable P2 region using Sanger sequencing, including 582 pairs of epidemiologically-related strains from 35 norovirus outbreaks and 6402 pairs of epidemiologically-unrelated strains during the four epidemic seasons. Genetic distances were calculated and a threshold was performed by adopting ROC (Receiver Operating Characteristic) curve which identified transmission clusters in all tested outbreaks with 80 % sensitivity. In average, nucleotide diversity between outbreaks was 67.5 times greater than the diversity within outbreaks. Simple and accurate thresholds for detecting norovirus transmissions of three genotypes obtained here streamlines molecular investigation of norovirus outbreaks, thus enabling rapid and efficient responses for the control of norovirus.

Rotavirus vaccines in Africa and Norovirus genetic diversity in children aged 0 to 5 years old: a systematic review and meta-analysis : Rotavirus vaccines in Africa and Norovirus genetic diversity.

Noroviruses are the second leading cause of death in children under the age of 5 years old. They are responsible for 200 million cases of diarrhoea and 50,000 deaths in children through the word, mainly in low-income countries. The objective of this review was to assess how the prevalence and genetic diversity of noroviruses have been affected by the introduction of rotavirus vaccines in Africa. PubMed, Web of Science and Science Direct databases were searched for articles. All included studies were conducted in Africa in children aged 0 to 5 years old with gastroenteritis. STATA version 16.0 software was used to perform the meta-analysis. The method of Dersimonian and Laird, based on the random effects model, was used for the statistical analyses in order to estimate the pooled prevalence's at a 95% confidence interval (CI). Heterogeneity was assessed by Cochran's Q test using the I2 index. The funnel plot was used to assess study publication bias. A total of 521 studies were retrieved from the databases, and 19 were included in the meta-analysis. The pooled norovirus prevalence's for pre- and post-vaccination rotavirus studies were 15% (95 CI, 15-18) and 13% (95 CI, 09-17) respectively. GII was the predominant genogroup, with prevalence of 87.64% and 91.20% respectively for the pre- and post-vaccination studies. GII.4 was the most frequently detected genotype, with rates of 66.84% and 51.24% respectively for the pre- and post-vaccination studies. This meta-analysis indicates that rotavirus vaccination has not resulted in a decrease in norovirus infections in Africa.

Epidemiological Features of Human Norovirus Genotypes before and after COVID-19 Countermeasures in Osaka, Japan.

We investigated the molecular epidemiology of human norovirus (HuNoV) in all age groups using samples from April 2019 to March 2023, before and after the COVID-19 countermeasures were implemented. GII.2[P16] and GII.4[P31], the prevalent strains in Japan before COVID-19 countermeasures, remained prevalent during the COVID-19 pandemic, except from April to November 2020; in 2021, the prevalence of GII.2[P16] increased among children. Furthermore, there was an increase in the prevalence of GII.4[P16] after December 2022. Phylogenetic analysis of GII.P31 RdRp showed that some strains detected in 2022 belonged to a different cluster of other strains obtained during the present study period, suggesting that HuNoV strains will evolve differently even if they have the same type of RdRp. An analysis of the amino acid sequence of VP1 showed that some antigenic sites of GII.4[P16] were different from those of GII.4[P31]. The present study showed high infectivity of HuNoV despite the COVID-19 countermeasures and revealed changes in the prevalent genotypes and mutations of each genotype. In the future, we will investigate whether GII.4[P16] becomes more prevalent, providing new insights by comparing the new data with those analyzed in the present study.

Epidemiological and genetic characteristics of norovirus in Hangzhou, China, in the postepidemic era.

OBJECTIVE: Norovirus (NoV) is an important human pathogen that can cause severe gastroenteritis in vulnerable populations. This study aimed to analyze the epidemiological and genetic characteristics of 2021-2023 NoV in Hangzhou, China. METHODS: This study enrolled patients aged 0-18 years who underwent NoV RNA detection in the hospital between January 2021 and October 2023 and analyzed the epidemiological characteristics of NoV. Polymerase chain reaction (PCR) was used to detect NoV RNA. Subtype classification and whole-genome sequencing were performed. RESULTS: There was a high prevalence of NoV infection in 2023, with NoV-positive samples accounting for 63.10 % of the total number of positive samples collected during the three-year period. The prevalence was abnormally high in summer, and the number of positive samples accounted for 48.20 % of the total positive samples for the whole year, which was much greater than the level in the same period in previous years (2023, 48.20% vs 2021, 13.66% vs 2022, 15.21 %). The GⅡ.4 subtype played a leading role, followed by increased mixed infection with GⅠ.5 and GⅡ.4. Whole-genome sequencing results suggested that GII.P16-GⅡ.4 had R297H and D372N key locus mutations. The evolutionary rate was 4.29 × 10-3 for the RdRp gene and 4.84 × 10-3 for the VP1 gene. The RdRp gene and VP1 gene of NoV GII.P16-GⅡ.4 have undergone rapid population evolution during the COVID-19 epidemic. CONCLUSION: In the summer of 2023, an abnormally high incidence of NoV appeared in Hangzhou, China. The major epidemic strain GII.P16-GⅡ.4 showed a certain range of gene mutations and a fast evolutionary rate.

Whole-Genome Sequencing-Based Confirmatory Methods on RT-qPCR Results for the Detection of Foodborne Viruses in Frozen Berries.

Accurate detection, identification, and subsequent confirmation of pathogens causing foodborne illness are essential for the prevention and investigation of foodborne outbreaks. This is particularly true when the causative agent is an enteric virus that has a very low infectious dose and is likely to be present at or near the limit of detection. In this study, whole-genome sequencing (WGS) was combined with either of two non-targeted pre-amplification methods (SPIA and SISPA) to investigate their utility as a confirmatory method for RT-qPCR positive results of foods contaminated with enteric viruses. Frozen berries (raspberries, strawberries, and blackberries) were chosen as the food matrix of interest due to their association with numerous outbreaks of foodborne illness. The hepatitis A virus (HAV) and human norovirus (HuNoV) were used as the contaminating agents. The non-targeted WGS strategy employed in this study could detect and confirm HuNoV and HAV at genomic copy numbers in the single digit range, and in a few cases, identified viruses present in samples that had been found negative by RT-qPCR analyses. However, some RT-qPCR-positive samples could not be confirmed using the WGS method, and in cases with very high Ct values, only a few viral reads and short sequences were recovered from the samples. WGS techniques show great potential for confirmation and identification of virally contaminated food items. The approaches described here should be further optimized for routine application to confirm the viral contamination in berries.

Epidemiological and molecular surveillance of norovirus in the Brazilian Amazon: description of recombinant genotypes and improvement of evolutionary analysis.

Noroviruses are highly infectious, genetically diverse viruses. Global outbreaks occur frequently, making molecular surveillance important for infection monitoring. This cross-sectional descriptive study aimed to monitor cases of norovirus gastroenteritis in the Brazilian Amazon. Fecal samples were tested by immunoenzymatic assay, RT-PCR and genetic sequencing for the ORF1/ORF2 and protease regions. Bayesian inference with a molecular clock was employed to construct the phylogeny. The norovirus prevalence was 25.8%, with a higher positivity rate among children aged 0-24 months. Genogroup GII accounted for 98.1% of the sequenced samples, while GI accounted for 1.9% of them. The GII.P16/GII.4 genotype was the most prevalent, with an evolution rate of 2.87x10-3 and TMRCA estimated in 2012. This study demonstrates that norovirus is a primary causative agent of gastroenteritis and provides data on viral genetic diversity that may facilitate infection surveillance and vaccine development.

Antigenic Characterization of Novel Human Norovirus GII.4 Variants San Francisco 2017 and Hong Kong 2019.

Norovirus is a major cause of acute gastroenteritis; GII.4 is the predominant strain in humans. Recently, 2 new GII.4 variants, Hong Kong 2019 and San Francisco 2017, were reported. Characterization using GII.4 monoclonal antibodies and serum demonstrated different antigenic profiles for the new variants compared with historical variants.

Evaluation of Extraction Methods to Detect Noroviruses in Ready-to-Eat Raw Milk Minas Artisanal Cheese.

This study aimed to assess two homogenization methods to recover norovirus from Minas artisanal cheese (MAC) made with raw bovine milk obtained from four microregions of the Minas Gerais state, Brazil, with different ripening times and geographical and abiotic characteristics. For this purpose, 33 fiscal samples were artificially contaminated with norovirus GI and GII, and Mengovirus (MgV), used as an internal process control (IPC). TRIzol® reagent and Proteinase K homogenization methods were evaluated for all samples were then subjected to RNA extraction using viral magnetic beads and RT-qPCR Taqman® for viral detection/quantification. Proteinase K method showed better efficiency results for both norovirus GI and GII, with means recovery efficiency of 45.7% (95% CI 34.3-57.2%) and 41.4% (95% CI 29.1-53.6%), respectively, when compared to TRIzol method (16.6% GI, 95% CI 8.4-24.9%, and 12.3% GII, 95% CI 7.0-17.6%). The limits of detection for norovirus GI and GII for this method were 101GC/g and 103GC/g, respectively, independent of cheese origin. MgV was detected and revealed in 100% success rate in all types of cheese, with mean recovery efficiency of 25.6% for Proteinase K, and 3.8% for the TRIzol method. According to cheese origin, Triangulo Mineiro MAC had the highest mean recovery rates for the three viral targets surveyed (89% GI, 87% GII, and 51% MgV), while Serro MAC showed the lowest rates (p < 0.001). Those results indicate that the proteinase K adapted method is suitable for norovirus GI and GII detection in MAC and corroborated MgV as an applicable IPC to be used during the process.