Norovirus Sequences Research Articles

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.

Comprehensive full genome analysis of norovirus strains from eastern India, 2017–2021

BackgroundWorldwide, noroviruses are the leading cause of acute gastroenteritis (AGE) in people of all age groups. In India, norovirus rates between 1.4 to 44.4% have been reported. Only a very few complete norovirus genome sequences from India have been reported.ObjectiveTo perform full genome sequencing of noroviruses circulating in India during 2017–2021, identify circulating genotypes, assess evolution including detection of recombination events.MethodologyForty-five archived norovirus-positive samples collected between October 2017 to July 2021 from patients with AGE from two hospitals in Kolkata, India were processed for full genome sequencing. Phylogenetic analysis, recombination breakpoint analysis and comprehensive mutation analysis were also performed.ResultsFull genome analysis of norovirus sequences revealed that strains belonging to genogroup (G)I were genotyped as GI.3[P13]. Among the different norovirus capsid-polymerase combinations, GII.3[P16], GII.4 Sydney[P16], GII.4 Sydney[P31], GII.13[P16], GII.16[P16] and GII.17 were identified. Phylogenetic analysis confirmed phylogenetic relatedness with previously reported norovirus strains and all viruses were analyzed by Simplot. GII[P16] viruses with multiple residue mutations within the non-structural region were detected among circulating GII.4 and GII.3 strains. Comprehensive mutation analysis and selection pressure analysis of GII[P16] viruses showed positive as well as negative selection sites. A GII.17 strain (NICED-BCH-11889) had an untypeable polymerase type, closely related to GII[P38].ConclusionThis study highlights the circulation of diverse norovirus strains in eastern India. These findings are important for understanding norovirus epidemiology in India and may have implications for future vaccine development.

Norovirus evolves as one or more distinct clonal populations in immunocompromised hosts.

Noroviruses are an important cause of chronic diarrhea in patients with compromised immune systems. Presently, there are no effective therapies to clear the virus, which can persist for years in the intestinal tract. The goal of our study was to develop a better understanding of the norovirus strains that are associated with these long-term infections. With the remarkable diversity of norovirus strains detected in the immunocompromised patient cohort we studied, it appears that most, if not all, noroviruses circulating in nature may have the capacity to establish a chronic infection when a person is unable to mount an effective immune response. Our work is the most comprehensive genetic data set generated to date in which near full-length genomes from noroviruses associated with chronic infection were analyzed by high-resolution next-generation sequencing. Analysis of this data set led to our discovery that certain patients in our cohort were shedding noroviruses that could be subdivided into distinct haplotypes or populations of viruses that were co-evolving independently. The ability to track haplotypes of noroviruses during chronic infection will allow us to fine-tune our understanding of how the virus adapts and maintains itself in the human host, and how selective pressures such as antiviral drugs can affect these distinct populations.

Patterns and Temporal Dynamics of Natural Recombination in Noroviruses.

Noroviruses infect a wide range of mammals and are the major cause of gastroenteritis in humans. Recombination at the junction of ORF1 encoding nonstructural proteins and ORF2 encoding major capsid protein VP1 is a well-known feature of noroviruses. Using all available complete norovirus sequences, we systematically analyzed patterns of natural recombination in the genus Norovirus both throughout the genome and across the genogroups. Recombination events between nonstructural (ORF1) and structural genomic regions (ORF2 and ORF3) were found in all analyzed genogroups of noroviruses, although recombination was most prominent between members of GII, the most common genogroup that infects humans. The half-life times of recombinant forms (clades without evidence of recombination) of human GI and GII noroviruses were 10.4 and 8.4-11.3 years, respectively. There was evidence of many recent recombination events, and most noroviruses that differed by more than 18% of nucleotide sequence were recombinant relative to each other. However, there were no distinct recombination events between viruses that differed by over 42% in ORF2/3, consistent with the absence of systematic recombination between different genogroups. The few inter-genogroup recombination events most likely occurred between ancient viruses before they diverged into contemporary genogroups. The recombination events within ORF1 or between ORF2/3 were generally rare. Thus, noroviruses routinely exchange full structural and nonstructural blocks of the genome, providing a modular evolution.

Investigation of genotype diversity of 7,804 norovirus sequences in humans and animals of China.

Norovirus is a prominent enteric virus responsible for severe acute gastroenteritis disease burden worldwide. In our current study, we analyzed 7,804 norovirus sequences of human and animals in China which were detected from 1980 to 2020 from GenBank. The GenBank database was searched up to May 2021 with the following search terms: "norovirus" or "norwalk virus" and "China." The 7,804 norovirus sequences were collected and evaluated by phylogenetic analysis using MEGA X software package. The online typing tool (https://www.rivm.nl/mpf/typingtool/norovirus/) was used to confirm the genotypes. There were 36 norovirus genotypes prevailing in China. GII.4 was the most prevalent genotype, and GII.2, GII.3 and GII.17 also emerged during different time periods. Most sequences were detected in East China (41.72%, 3,256/7,804), but different norovirus genotypes were distributed widely across the country. A variety of norovirus genotypes, including GI, GII, GIII, GIV, GV, GVI, GVII and GX, were reported in different animals. Furthermore, a GI.3 sequence detected from animal had high identity with norovirus detected in human from the same region, indicating the potential norovirus zoonotic transmission in China. In conclusion, these results indicated that norovirus sequences with considerable genetic diversity distributed widely in China, with potential reverse zoonotic transmission from human to animals.

Development and Characterization of Synthetic Norovirus RNA for Use in Molecular Detection Methods.

Reference materials are essential for the quality assurance of molecular detection methods. We developed and characterized synthetic norovirus GI and GII RNA reference materials. Norovirus GI and GII RNA sequences including the ORF1-ORF2 junction region were designed based on 1,495 reported norovirus sequences and synthesized via plasmid preparation and in vitro transcription. The synthetic norovirus GI and GII RNAs were evaluated using six commercial norovirus detection kits used in Korea and subjected to homogeneity and stability analyses. A multicenter study involving five laboratories and using four commercial real-time PCR norovirus detection assays was conducted for synthetic norovirus RNA characterization and uncertainty measurements. The synthetic norovirus GI and GII RNAs were positively detected using the six commercial norovirus detection kits and were homogeneous and stable for one year when stored at -20°C or -70°C. All data from the five laboratories were within a range of 1.0 log copies/μL difference for each RNA, and the overall mean concentrations for norovirus GI and GII RNAs were 7.90 log copies/μL and 6.96 log copies/μL, respectively. The synthetic norovirus GI and GII RNAs are adequate for quality control based on commercial molecular detection reagents for noroviruses with high sequence variability. The synthetic RNAs can be used as reference materials in norovirus molecular detection methods.

Untangling an AGS Outbreak Caused by the Recombinant GII.12[P16] Norovirus With Nanopore Sequencing.

For a rapidly spreading virus such as NoV (norovirus), pathogen identification, genotype classification, and transmission tracing are urgent for epidemic control. Here, we applied the Nanopore metatranscriptomic sequencing to determine the causative pathogen of a community AGS (Acute gastroenteritis) outbreak. The results were also confirmed by RT-PCR. The NGS (Next Generation Sequencing) library was constructed within 8 hours and sequence analyses were carried out in real-time. NoV positive reads were detected in 13 of 17 collected samples, including two water samples from sewage treatment tank and cistern. A nearly complete viral genome and other genome fragments could be generated from metatranscriptomic sequencing of 13 samples. The NoV sequences from water samples and cases are identical suggesting the potential source of the outbreak. The sequencing results also indicated the outbreak was likely caused by an emerging recombinant GII.12[P16] virus, which was only identified in the United States and Canada in 2017–2018. This is the first report of this emerging variant in mainland China, following the large outbreaks caused by the recombinant GII.17[P17] and GII.2[P16] in 2014 and 2016, respectively. Closely monitoring of the prevalence of this recombinant strain is required. Our data also highlighted the importance of real-time sequencing in emerging pathogens’ surveillance.

Application of Next Generation Sequencing on Norovirus‐contaminated oyster samples

Bivalve molluscan shellfish (BMS) contamination with gastroenteritis viruses such as norovirus (NoV) is recognized as a significant public health risk worldwide. These foodborne epidemics are characterized by very low viral concentrations in the implicated foods, and by diverse viruses or multiple NoV strains originating from human sewage, resulting in different strains (co-)infecting the consumers. Next-generation sequencing (NGS) offers promising means to describe the diversity of strains present in BMS or to retrace transmission chains in outbreak settings, but their sensitivity and reproducibility remained to be assessed for this application. In this work, we evaluated the ability of three promising NGS methods to sequence diverse NoV in environmental or food BMS and human stool samples. Using laboratory-prepared samples of known NoV composition, we evaluated the sensitivity, reproducibility, repeatability and selectivity of metabarcoding, capture-based metagenomics and long amplicon sequencing, considering representative NoV strains from genogroup I and II, and the impact of the BMS matrix. The metabarcoding, with separate amplification of polymerase and capsid gene segments followed by Illumina sequencing, was the most sensitive method. It was applied to a selection of 212 BMS samples collected through the European Commission’s NoV baseline survey (BLS), demonstrating a high diversity of NoV sequences found in the BMS which reflect the diversity of NoV strains circulating in the European human population. Besides, a capture-based metagenomics with enrichment of vertebrate viruses was applied on 20 of these BLS samples as well as 20 BMS linked to outbreaks and 10 related human stool samples. In BMS, it yielded NoV sequences compatible with the genomes identified in stool samples, but they were too short to allow definitive confirmation of the infection source. The present report describes NGS methods, including the bioinformatic pipelines, applicable to molecular epidemiology of NoV in BMS, their current limitations and expected outcomes.

Global and regional circulation trends of norovirus genotypes and recombinants, 1995-2019: A comprehensive review of sequences from public databases.

Human noroviruses are the leading global cause of viral gastroenteritis. Attempts at developing effective vaccines and treatments against norovirus disease have been stymied by the extreme genetic diversity and rapid geographic distribution of these viruses. The emergence and replacement of predominantly circulating norovirus genotypes has primarily been attributed to mutations on the VP1 capsid protein leading to genetic drift, and more recently to recombination events between the ORF1/ORF2 junction. However, large‐scale research into the historical and geographic distribution of recombinant norovirus strains has been limited in the literature. We performed a comprehensive historical analysis on 30,810 human norovirus sequences submitted to public databases between the years 1995 and 2019. During this time, 37 capsid genotypes and 56 polymerase types were detected across 90 different countries, and 97 unique recombinant genomes were also identified. GII.4, both capsid and polymerase, was the predominately circulating type worldwide for the majority of this time span, save for a brief swell of GII.17 and GII.2 capsid genotypes and a near‐total eclipse by GII.P16, GII.P21 and GII.P31 beginning in 2013. Interestingly, an analysis of 4067 recombinants found that 50.2% (N = 2039) of all recorded sequences belonged to three recently emerged recombinant strains: GII.2[P16], GII.4[P31], and GII.4[P16]. This analysis should provide an important historical foundation for future studies that evaluate the emergence and distribution of noroviruses, as well as the design of cross‐protective vaccines.

Epidemiology of Astrovirus, Norovirus and Sapovirus in Greek pig farms indicates high prevalence of Mamastrovirus suggesting the potential need for systematic surveillance

BackroundAstrovirus, Norovirus and Sapovirus exhibit a wide distribution in swine pig herds worldwide. However, the association of porcine Astrovirus (PAstV), porcine Norovirus (PoNoV) and porcine Sapovirus (PoSaV) with disease in pigs remains uncertain. In this study, we investigated the prevalence of PAstV, PoNoV and PoSaV in Greek pig farms using both conventional RT-PCR and SYBR-Green Real-time RT-PCR in an effort to compare the sensitivity of the two methods. We examined 1400 stool samples of asymptomatic pigs originating from 28 swine farms throughout Greece in pools of five.ResultsPAstV was detected in all 28 swine farms examined, with an overall prevalence of 267/280 positive pools (95.4%). Porcine Caliciviruses prevalence was found at 36 and 57 out of the 280 examined samples, by the conventional and SYBR-Green Real time RT-PCR, respectively. Sequencing and phylogenetic analysis of the positive samples revealed that the detected PAstV sequences are clustered within PAstV1, 3 and 4 lineages, with PAstV3 being the predominant haplotype (91.2%). Interestingly, sequencing of the Calicivirus positive samples demonstrated the presence of non-target viruses, i.e. Sapovirus, Kobuvirus and Sapelovirus sequences and one sequence highly similar to bat Astrovirus, while no Norovirus sequence was detected.ConclusionsThe high prevalence of PAstV in Greek pig farms poses a necessity for further investigation of the pathogenicity of this virus and its inclusion in surveillance programs in case that it proves to be important. To our knowledge, this is the first epidemiological study of these viruses in pig farms in Greece.

First report of norovirus sequences isolated from raccoon dogs in mainland China

Noroviruses can infect humans and a wide variety of other mammalian hosts, causing varying degrees of diarrhea. In this study, two novel norovirus genomes were identified for the first time in farmed raccoon dogs, designated as raccoon dog noroviruses BUCT-K1 and BUCT-K4. Neither the farmers nor the raccoon dogs had symptoms (e.g., diarrhea) at the time of sample collection. We collected 14 stool samples from two farms, and 85.7% (12/14) of the samples were norovirus positive by RT-PCR. The two norovirus genomes have the highest identity to Dog/Z7/19/CH, suggesting that the norovirus might have been transmitted from dogs to raccoon dogs. Genomic and evolutionary analyses indicated that different directions of evolution occurred following the spread of the norovirus to the raccoon dogs. This study has increased knowledge of norovirus-infected animal species and has provided additional information on the norovirus family.

Next generation sequencing of near-full length genome of norovirus GII.4 from Botswana

Noroviruses are highly diverse, with genotype GII.4 causing most epidemics. This study aimed to investigate the evolutionary dynamics of norovirus genogroup GII strains among acutely infected children under 5 years in Botswana, between 2016 and 2018. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify the whole norovirus genome, followed by next-generation sequencing using Oxford Nanopore technology. Twelve samples were successfully analyzed, with 11 identified as norovirus GII.4 Sydney [P31] and one as GII.4 Sydney [P13]. This study generated the first near-full length norovirus sequences in Botswana (93–95% coverage). Our results show that the norovirus GII.4 strains circulating in Botswana are under evolution through recombination and antigenic drift. Recombination in the GII.4 Sydney [P31] and GII.4 Sydney [P13] strains occurred in the ORF1/ORF2 junction and within ORF1, respectively. This study provides the first description of the GII.4 Sydney [P13] recombinant. Amino acid variation in the immunogenic sites was analyzed. Mutations in epitope A correlate with the emergence of novel norovirus GII.4 strains with altered antigenicity. In this study, we identified 43 unique amino acid substitutions in the VP1 region, with six occurring in epitopes, A (G295N, and E368Q) and E (S40T, N412D, N412K and T413H). The shell subdomain of the GII.4 Sydney [P13] variant was closely related to norovirus GII.17. Lastly, we also observed several mutations in the T cell restricted epitopes of both strains. Our study has made a novel contribution to understanding the evolution of norovirus GII.4 in Botswana.

Development of a Tetraplex qPCR for the Molecular Identification and Quantification of Human Enteric Viruses, NoV and HAV, in Fish Samples.

Human enteric viruses such as norovirus (NoV) and hepatitis A virus (HAV) are some of the most important causes of foodborne infections worldwide. Usually, infection via fish consumption is not a concern regarding these viruses, since fish are mainly consumed cooked. However, in the last years, raw fish consumption has become increasingly common, especially involving the use of seabass and gilthead seabream in dishes like sushi, sashimi, poke, and carpaccio. Therefore, the risk for viral infection via the consumption of raw fish has also increased. In this study, a virologic screening was performed in 323 fish specimens captured along the Portuguese coast using a tetraplex qPCR optimised for two templates (plasmid and in vitro transcribed RNA) to detect and quantify NoV GI, NoV GII and HAV genomes. A difference of approximately 1-log was found between the use of plasmid or in vitro transcribed RNA for molecular-based quantifications, showing an underestimation of genome copy-number equivalents using plasmid standard-based curves. Additionally, the presence of NoV genomic RNA in a pool of seabass brains was identified, which was shown to cluster with a major group of human norovirus sequences from genogroup I (GI.1) by phylogenetic analysis. None of the analysed fish revealed the presence of NoV GII or HAV. This result corroborates the hypothesis that enteric viruses circulate in seawater or that fish were contaminated during their transportation/handling, representing a potential risk to humans through raw or undercooked fish consumption.

Human Calicivirus Typing tool: A web-based tool for genotyping human norovirus and sapovirus sequences.

The family Caliciviridae consists of a genetically diverse group of RNA viruses that infect a wide range of host species including noroviruses and sapoviruses which cause acute gastroenteritis in humans. Typing of these viruses relies on sequence-based approaches, and therefore there is a need for rapid and accurate web-based typing tools. To develop and evaluate a web-based tool for rapid and accurate genotyping of noroviruses and sapoviruses. The Human Calicivirus Typing (HuCaT) tool uses a set of curated reference sequences that are compared to query sequences using a k-mer (DNA substring) based algorithm. Outputs include alignments and phylogenetic trees of the 12 top matching reference sequences for each query. The HuCaT tool was validated with a set of 1310 norovirus and 239 sapovirus sequences covering all known human norovirus and sapovirus genotypes. HuCaT tool assigned genotypes to all queries with 100 % accuracy and was much faster (17 s) than BLAST (150 s) or phylogenetic analyses approaches. The web-based HuCaT tool supports rapid and accurate genotyping of human noroviruses and sapoviruses.

Progress on genotyping of norovirus

Norovirus are now recognized as one of the main pathogens causing acute gastroenteritis in both developed and developing countries. However, norovirus are easily mutated and recombined, and have many genotypes. In early studies, norovirus were amplified and identified by amino acid sequence of VP1 region. It was found that norovirus were easily mutated and recombined in or near the overlapping regions of polymerase and capsid. A two regions genotyping method was positively proposed internationally. Depending on the 2 times standard deviation standard method for two regions identification, norovirus polymerase regions can be divided into 10 gene groups and 76 genotypes including 2 tentative gene groups and 16 tentative genotypes. The VP1 region can be divided into 12 gene groups and 53 genotypes including 2 tentative gene groups and 5 tentative genotypes. However, the tentative gene groups and genotypes need to be further identified and reclassified. In this article, characteristics of norovirus sequences, principles of different genotyping methods, methods of sequence amplification, on-line genotyping tools and the latest studies in norovirus genotypes are reviewed and introduced.

Genome Sequence of a Human Norovirus GII.4 Hong Kong[P31] Variant in Hong Kong, China.

We report the nearly complete genome of a norovirus GII.4 Hong Kong[P31] variant (GII strain Hu/HK/2019/GII.4 Hong Kong[P31]/CUHK-NS-2200) that was detected in a patient with gastroenteritis in August 2019. The genome was sequenced by metagenomic next-generation sequencing and was found to have 92.8% nucleotide similarity to the closest GII.4 norovirus sequence in GenBank.

Characterization of Norovirus and Other Human Enteric Viruses in Sewage and Stool Samples Through Next-Generation Sequencing

This study aimed to optimize a method to identify human enteric viruses in sewage and stool samples using random primed next-generation sequencing. We tested three methods, two employed virus enrichment based on the binding properties of the viral capsid using pig-mucin capture or by selecting viral RNA prior to library preparation through a capture using the SureSelect target enrichment. The third method was based on a non-specific biophysical precipitation with polyethylene glycol. Full genomes of a number of common human enteric viruses including norovirus, rotavirus, husavirus, enterovirus and astrovirus were obtained. In stool samples full norovirus genome were detected as well as partial enterovirus genome. A variety of norovirus sequences was detected in sewage samples, with genogroup II being more prevalent. Interestingly, the pig-mucin capture enhanced not only the recovery of norovirus and rotavirus but also recovery of astrovirus, sapovirus and husavirus. Documenting sewage virome using these methods provides information for molecular epidemiology and may be useful in developing strategies to prevent further spread of viruses.

Should we pay attention to recombinant norovirus strain GII.P7/GII.6?

BackgroundRecombinant norovirus strain GII.P7/GII.6 has been circulating in Asia and around the world for at least 20 years, but has been responsible for relatively few outbreaks. MethodsWe used statistical analyses, real-time reverse transcription – PCR, and genome sequence analyses to investigate an outbreak of gastroenteritis, identifying the pathogen, the risk factors associated with the outbreak, and the molecular features of GII.P7/GII.6 strains. ResultsAn outbreak of gastroenteritis was reported at a school involving 12 students and lasting 6 days, from September 13 to September 18, 2017. Epidemiological studies suggested that norovirus was transmitted from person to person and not via contaminated food or drinking water in this outbreak. Using a sequence analysis of the junction region between open reading frames 1 and 2, the pathogen was identified as a recombinant norovirus (strain GII.P7/GII.6).The full-length genome of the outbreak strain shared 86%–97% identity with those of other GII.P7/GII.6 strains. Phylogenetic trees were constructed from partial open reading frame 1 (ORF1) and ORF2 sequences from the outbreak strain and GII.P7/GII.6 norovirus sequences available in GenBank. On the ORF1 tree, the partial sequences of ORF1 were grouped into cluster A (with GII.6), cluster B (with GII.7), and a separate cluster (C), based on the GII.6 and GII.7 reference strains. The ORF2 tree showed all GII.P7/GII.6 strains formed a cluster together with GII.6 strains. Amino-acid substitutions and insertions/deletions were common in the capsid protein, especially in it’s P2 and P1 domains. The outbreak was controlled within several days using appropriate measures. ConclusionsBecause it may play a prominent role in future outbreaks, recombinant norovirus strain GII.P7/GII.6 should be monitored with routine surveillance.

Genetic characterization of norovirus GII.4 variants circulating in Canada using a metagenomic technique

BackgroundHuman norovirus is the leading cause of viral gastroenteritis globally, and the GII.4 has been the most predominant genotype for decades. This genotype has numerous variants that have caused repeated epidemics worldwide. However, the molecular evolutionary signatures among the GII.4 variants have not been elucidated throughout the viral genome.MethodA metagenomic, next-generation sequencing method, based on Illumina RNA-Seq, was applied to determine norovirus sequences from clinical samples.ResultsHerein, the obtained deep-sequencing data was employed to analyze full-genomic sequences from GII.4 variants prevailing in Canada from 2012 to 2016. Phylogenetic analysis demonstrated that the majority of these sequences belong to New Orleans 2009 and Sydney 2012 strains, and a recombinant sequence was also identified. Genome-wide similarity analyses implied that while the capsid gene is highly diverse among the isolates, the viral protease and polymerase genes remain relatively conserved. Numerous amino acid substitutions were observed at each putative antigenic epitope of the VP1 protein, whereas few amino acid changes were identified in the polymerase protein. Co-infection with other enteric RNA viruses was investigated and the astrovirus genome was identified in one of the samples.ConclusionsOverall this study demonstrated the application of whole genome sequencing as an important tool in molecular characterization of noroviruses.

Analysis of frozen strawberries involved in a large norovirus gastroenteritis outbreak using next generation sequencing and digital PCR

Virus-contaminated frozen berries have been frequently identified as cause of foodborne disease outbreaks. To provide new tools for virus detection and characterization in berries, next generation sequencing (NGS) and reverse transcription-digital PCR (RT-dPCR) techniques were tested here with strawberries previously involved in a large-scale norovirus (NoV) gastroenteritis outbreak in Germany. By NGS, about 29 million sequence reads were generated, which mainly showed identities to sequences from the plant matrix and from the bacterial flora. Most abundant virus sequences originated from plant-specific viruses, whereas sequences with high identity to human viruses were rare. Only two sequence reads showed homologies to human NoV. They were identical to GII.P16/GII.13 NoV sequences from patients and a strawberry sample independently analyzed during the outbreak. Quantification of the GII NoV RNA of the berries using RT-dPCR confirmed a low mean virus amount of 185 copies/25 g, which is similar to independently assessed RT-qPCR results (257 copies/25 g). The study shows that identification of human-pathogenic viruses in naturally contaminated frozen berries is possible using NGS technologies. However, the method needs to be further optimized in order to enable convenient and reproducible detection of a low amount of human-pathogenic virus sequences in a background of highly abundant nucleic acids of other sources.