Metagenomic Surveillance of Viral Gastroenteritis in a Public Health Setting.

Norovirus is the primary cause of viral gastroenteritis (GE). To investigate norovirus epidemiology, there is a need for whole-genome sequencing and reference sets consisting of complete genomes. To investigate the potential of shotgun metagenomic sequencing on the Illumina platform for whole-genome sequencing, 71 reverse transcriptase quantitative PCR (RT-qPCR) norovirus positive-feces (threshold cycle [CT], <30) samples from norovirus surveillance within The Netherlands were subjected to metagenomic sequencing. Data were analyzed through an in-house next-generation sequencing (NGS) analysis workflow. Additionally, we assessed the potential of metagenomic sequencing for the surveillance of off-target viruses that are of importance for public health, e.g., sapovirus, rotavirus A, enterovirus, parechovirus, aichivirus, adenovirus, and bocaparvovirus. A total of 60 complete and 10 partial norovirus genomes were generated, representing 7 genogroup I capsid genotypes and 12 genogroup II capsid genotypes. In addition to the norovirus genomes, the metagenomic approach yielded partial or complete genomes of other viruses for 39% of samples from children and 6.7% of samples from adults, including adenovirus 41 (N = 1); aichivirus 1 (N = 1); coxsackievirus A2 (N = 2), A4 (N = 2), A5 (N = 1), and A16 (N = 1); bocaparvovirus 1 (N = 1) and 3 (N = 1); human parechovirus 1 (N = 2) and 3 (N = 1); Rotavirus A (N = 1); and a sapovirus GI.7 (N = 1). The sapovirus GI.7 was initially not detected through RT-qPCR and warranted an update of the primer and probe set. Metagenomic sequencing on the Illumina platform robustly determines complete norovirus genomes and may be used to broaden gastroenteritis surveillance by capturing off-target enteric viruses. IMPORTANCE Viral gastroenteritis results in significant morbidity and mortality in vulnerable individuals and is primarily caused by norovirus. To investigate norovirus epidemiology, there is a need for whole-genome sequencing and reference sets consisting of full genomes. Using surveillance samples sent to the Dutch National Institute for Public Health and the Environment (RIVM), we compared metagenomics against conventional techniques, such as RT-qPCR and Sanger-sequencing, with norovirus as the target pathogen. We determined that metagenomics is a robust method to generate complete norovirus genomes, in parallel to many off-target pathogenic enteric virus genomes, thereby broadening our surveillance efforts. Moreover, we detected a sapovirus that was not detected by our validated gastroenteritis RT-qPCR panel, which exemplifies the strength of metagenomics. Our study shows that metagenomics can be used for public health gastroenteritis surveillance, the generation of reference-sets for molecular epidemiology, and how it compares to current surveillance strategies.

Mpox outbreak in the Netherlands, 2022: public health response, characteristics of the first 1,000 cases and protection of the first-generation smallpox vaccine.

In early May 2022, a global outbreak of mpox started among persons without travel history to regions known to be enzootic for monkeypox virus (MPXV). On 8 August 2022, the Netherlands reported its 1,000th mpox case, representing a cumulative incidence of 55 per million population, one of the highest cumulative incidences worldwide. We describe characteristics of the first 1,000 mpox cases in the Netherlands, reported between 20 May and 8 August 2022, within the context of the public health response. These cases were predominantly men who have sex with men aged 31-45 years. The vast majority of infections were acquired through sexual contact with casual partners in private or recreational settings including LGBTQIA+ venues in the Netherlands. This indicates that, although some larger upsurges occurred from point-source and/or travel-related events, the outbreak was mainly characterised by sustained transmission within the Netherlands. In addition, we estimated the protective effect of first-generation smallpox vaccine against moderate/severe mpox and found a vaccine effectiveness of 58% (95% CI: 17-78%), suggesting moderate protection against moderate/severe mpox symptoms on top of any possible protection by this vaccine against MPXV infection and disease. Communication with and supporting the at-risk population in following mitigation measures remains essential.

Higher risk of SARS-CoV-2 Omicron BA.4/5 infection than of BA.2 infection after previous BA.1 infection, the Netherlands, 2 May to 24 July 2022.

BackgroundIn summer 2022, SARS-CoV-2 Omicron BA.5 became dominant in Europe. In vitro studies have shown a large reduction of antibody neutralisation for this variant.AimWe aimed to investigate differences in protection from previous infection and/or vaccination against infection with Omicron BA.4/5 vs BA.2.MethodsWe employed a case-only approach including positive PCR tests from community testing between 2 May and 24 July 2022 that were tested for S gene target failure (SGTF), which distinguishes BA.4/5 from BA.2 infection. Previous infections were categorised by variant using whole genome sequencing or SGTF. We estimated by logistic regression the association of SGTF with vaccination and/or previous infection, and of SGTF of the current infection with the variant of the previous infection, adjusting for testing week, age group and sex.ResultsThe percentage of registered previous SARS-CoV-2 infections was higher among 19,836 persons infected with Omicron BA.4/5 than among 7,052 persons infected with BA.2 (31.3% vs 20.0%). Adjusting for testing week, age group and sex, the adjusted odds ratio (aOR) was 1.4 (95% CI: 1.3-1.5). The distribution of vaccination status did not differ for BA.4/5 vs BA.2 infections (aOR = 1.1 for primary and booster vaccination). Among persons with a previous infection, those currently infected with BA4/5 had a shorter interval between infections, and the previous infection was more often caused by BA.1, compared with those currently infected with BA.2 (aOR = 1.9; 95% CI: 1.5-2.6).ConclusionOur results suggest immunity induced by BA.1 is less effective against BA.4/5 infection than against BA.2 infection.

Elevated risk of infection with SARS-CoV-2 Beta, Gamma, and Delta variants compared with Alpha variant in vaccinated individuals.

The extent to which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) break through infection- or vaccine-induced immunity is not well understood. We analyzed 28,578 sequenced SARS-CoV-2 samples from individuals with known immune status obtained through national community testing in the Netherlands from March to August 2021. We found evidence of an increased risk of infection by the Beta (B.1.351), Gamma (P.1), or Delta (B.1.617.2) variants compared with the Alpha (B.1.1.7) variant after vaccination. No clear differences were found between vaccines. However, the effect was larger in the first 14 to 59 days after complete vaccination compared with ≥60 days. In contrast to vaccine-induced immunity, there was no increased risk for reinfection with Beta, Gamma, or Delta variants relative to the Alpha variant in individuals with infection-induced immunity.

SARS-CoV-2 outbreaks in secondary school settings in the Netherlands during fall 2020; silent circulation.

BACKGROUND: In fall 2020 when schools in the Netherlands operated under a limited set of COVID-19 measures, we conducted outbreaks studies in four secondary schools to gain insight in the level of school transmission and the role of SARS-CoV-2 transmission via air and surfaces. METHODS: Outbreak studies were performed between 11 November and 15 December 2020 when the wild-type variant of SARS-CoV-2 was dominant. Clusters of SARS-CoV-2 infections within schools were identified through a prospective school surveillance study. All school contacts of cluster cases, irrespective of symptoms, were invited for PCR testing twice within 48 h and 4-7 days later. Combined NTS and saliva samples were collected at each time point along with data on recent exposure and symptoms. Surface and active air samples were collected in the school environment. All samples were PCR-tested and sequenced when possible. RESULTS: Out of 263 sampled school contacts, 24 tested SARS-CoV-2 positive (secondary attack rate 9.1%), of which 62% remained asymptomatic and 42% had a weakly positive test result. Phylogenetic analysis on 12 subjects from 2 schools indicated a cluster of 8 and 2 secondary cases, respectively, but also other distinct strains within outbreaks. Of 51 collected air and 53 surface samples, none were SARS-CoV-2 positive. CONCLUSION: Our study confirmed within school SARS-CoV-2 transmission and substantial silent circulation, but also multiple introductions in some cases. Absence of air or surface contamination suggests environmental contamination is not widespread during school outbreaks.

Regional importation and asymmetric within-country spread of SARS-CoV-2 variants of concern in the Netherlands.

Background: Variants of concern (VOCs) of SARS-CoV-2 have caused resurging waves of infections worldwide. In the Netherlands, the Alpha, Beta, Gamma, and Delta VOCs circulated widely between September 2020 and August 2021. We sought to elucidate how various control measures, including targeted flight restrictions, had impacted the introduction and spread of these VOCs in the Netherlands. Methods: We performed phylogenetic analyses on 39,844 SARS-CoV-2 genomes collected under the Dutch national surveillance program. Results: We found that all four VOCs were introduced before targeted flight restrictions were imposed on countries where the VOCs first emerged. Importantly, foreign introductions, predominantly from other European countries, continued during these restrictions. After their respective introductions into the Netherlands, the Alpha and Delta VOCs largely circulated within more populous regions of the country with international connections before asymmetric bidirectional transmissions occurred with the rest of the country and the VOC became the dominant circulating lineage. Conclusions: Our findings show that flight restrictions had limited effectiveness in deterring VOC introductions due to the strength of regional land travel importation risks. As countries consider scaling down SARS-CoV-2 surveillance efforts in the post-crisis phase of the pandemic, our results highlight that robust surveillance in regions of early spread is important for providing timely information for variant detection and outbreak control. Funding: None.

Antigenic cartography using sera from sequence-confirmed SARS-CoV-2 variants of concern infections reveals antigenic divergence of Omicron.

Large-scale vaccination campaigns have prevented countless hospitalizations and deaths due to COVID-19. However, the emergence of SARS-CoV-2 variants that escape from immunity challenges the effectiveness of current vaccines. Given this continuing evolution, an important question is when and how to update SARS-CoV-2 vaccines to antigenically match circulating variants, similarly to seasonal influenza viruses where antigenic drift necessitates periodic vaccine updates. Here, we studied SARS-CoV-2 antigenic drift by assessing neutralizing activity against variants of concern (VOCs) in a set of sera from patients infected with viral sequence-confirmed VOCs. Infections with D614G or Alpha strains induced the broadest immunity, whereas individuals infected with other VOCs had more strain-specific responses. Omicron BA.1 and BA.2 were substantially resistant to neutralization by sera elicited by all other variants. Antigenic cartography revealed that Omicron BA.1 and BA.2 were antigenically most distinct from D614G, associated with immune escape, and possibly will require vaccine updates to ensure vaccine effectiveness.

High prevalence of acute hepatitis E virus infection in pigs in Dutch slaughterhouses.

Hepatitis E is caused by hepatitis E virus (HEV), one of the causes of acute viral hepatitis. Domestic pigs are considered as the main reservoir of HEV-3. The recently reported high prevalence of HEV in liver- and meat products on the Dutch market warranted a cross-sectional prevalence study on HEV infection among 5-6 months old pigs slaughtered in the Netherlands (n = 250). For this, liver, caecum content and blood samples were analyzed for the presence of genomic HEV RNA by RT-PCR. In addition, a serological test was performed to detect HEV IgG. Background information was retrieved on the corresponding farms to evaluate potential risk factors for HEV at pig slaughter age. HEV IgG was detected in sera from 167 pigs (67.6 %). HEV RNA was detected in 64 (25.6 %) caecum content samples, in 40 (16.1 %) serum samples and in 25 (11.0 %) liver samples. The average level of viral contamination in positive samples was log10 4.6 genome copies (gc)/g (range 3.0-8.2) in caecum content, log10 3.3 gc/ml (range 2.4-5.9) in serum and log10 3.2 gc/0.1 g (range 1.7-6.2) in liver samples. Sequence analyses revealed HEV-3c only. Ten times an identical strain was detected in two or three samples obtained from the same pig. Each animal in this study however appeared to be infected with a unique strain. The presence of sows and gilts and welfare rating at the farm of origin had a significant effect (p < 0.05) on the distribution over the four groups representing different stages of HEV infection based on IgG or RNA in caecum and/or serum. The observed proportion of tested pigs with viremia (16 %) was higher than in other reported studies and was interestingly often observed in combination with a high number of HEV genome copies in liver and caecum content as detected by RT-qPCR. Data provided will be useful for risk assessment for food safety of pork products, will provide baseline data for future monitoring of HEV infections in pigs and new thoughts for mitigation strategies.

Regional importation and asymmetric within-country spread of SARS-CoV-2 variants of concern in the Netherlands.

Variants of concern (VOCs) of SARS-CoV-2 have caused resurging waves of infections worldwide. In the Netherlands, Alpha, Beta, Gamma and Delta variants circulated widely between September 2020 and August 2021. To understand how various control measures had impacted the spread of these VOCs, we analyzed 39,844 SARS-CoV-2 genomes collected under the Dutch national surveillance program. We found that all four VOCs were introduced before targeted flight restrictions were imposed on countries where the VOCs first emerged. Importantly, foreign introductions, predominantly from other European countries, continued during these restrictions. Our findings show that flight restrictions had limited effectiveness in deterring VOC introductions due to the strength of regional land travel importation risks. We also found that the Alpha and Delta variants largely circulated more populous regions with international connections after their respective introduction before asymmetric bidirectional transmissions occurred with the rest of the country and the variant dominated infections in the Netherlands. As countries consider scaling down SARS-CoV-2 surveillance efforts in the post-crisis phase of the pandemic, our results highlight that robust surveillance in regions of early spread is important for providing timely information for variant detection and outbreak control.

Increased risk of infection with SARS-CoV-2 Omicron BA.1 compared with Delta in vaccinated and previously infected individuals, the Netherlands, 22 November 2021 to 19 January 2022.

Infections with the Omicron SARS-CoV-2 variant are rapidly increasing worldwide. Among 174,349 SARS-CoV-2-infected individuals (≥ 12 years), we observed an increased risk of S gene target failure, predictive of the Omicron variant, in vaccinated (odds ratio (OR): 3.6; 95% confidence interval (CI): 3.4-3.7) and previously infected individuals (OR: 4.2; 95% CI: 3.8-4.7) compared with infected naïve individuals. This suggests vaccine- or infection-induced immunity against SARS-CoV-2 infections is less effective against the Omicron than the Delta variant.

Rapid reinfection with SARS-CoV-2 variant-of-concern Alpha detected in a nurse during an outbreak at a non-covid inpatient ward: lessons learned.

We describe the lessons learned during a SARS-CoV-2 variant-of-concern Alpha outbreak investigation at a normal care unit in a university hospital in Amsterdam in December 2020. The outbreak consisted of nine nurses and two roomed-in patient family members. (attack rate 18%). One nurse tested positive with a phylogenetically distinct variant, after a documented infection 83 days prior. Three key points were taken from this investigation. First, it was controlled by adherence to existing guidelines, despite increased transmissibility of the variant. Second, viral sequencing can inform transmission cluster inference, but the epidemiological context is essential to draw appropriate conclusions. Third, reinfections with Alpha variants can occur rapidly after primary infection.

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.

Author Correction: Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the Netherlands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the Netherlands.

In late December 2019, a cluster of cases of pneumonia of unknown etiology were reported linked to a market in Wuhan, China1. The causative agent was identified as the species Severe acute respiratory syndrome-related coronavirus and was named SARS-CoV-2 (ref. 2). By 16 April the virus had spread to 185 different countries, infected over 2,000,000 people and resulted in over 130,000 deaths3. In the Netherlands, the first case of SARS-CoV-2 was notified on 27 February. The outbreak started with several different introductory events from Italy, Austria, Germany and France followed by local amplification in, and later also outside, the south of the Netherlands. The combination of near to real-time whole-genome sequence analysis and epidemiology resulted in reliable assessments of the extent of SARS-CoV-2 transmission in the community, facilitating early decision-making to control local transmission of SARS-CoV-2 in the Netherlands. We demonstrate how these data were generated and analyzed, and how SARS-CoV-2 whole-genome sequencing, in combination with epidemiological data, was used to inform public health decision-making in the Netherlands.

ICTV Virus Taxonomy Profile: Caliciviridae.

The family Caliciviridae includes viruses with single-stranded, positive-sense RNA genomes of 7.4-8.3 kb. The most clinically important representatives are human noroviruses, which are a leading cause of acute gastroenteritis in humans. Virions are non-enveloped with icosahedral symmetry. Members of seven genera infect mammals (Lagovirus, Norovirus, Nebovirus, Recovirus, Sapovirus, Valovirus and Vesivirus), members of two genera infect birds (Bavovirus and Nacovirus), and members of two genera infect fish (Minovirus and Salovirus). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Caliciviridae, which is available at ictv.global/report/caliciviridae.

Updated classification of norovirus genogroups and genotypes.

Noroviruses are genetically diverse RNA viruses associated with acute gastroenteritis in mammalian hosts. Phylogenetically, they can be segregated into different genogroups as well as P (polymerase)-groups and further into genotypes and P-types based on amino acid diversity of the complete VP1 gene and nucleotide diversity of the RNA-dependent RNA polymerase (RdRp) region of ORF1, respectively. In recent years, several new noroviruses have been reported that warrant an update of the existing classification scheme. Using previously described 2× standard deviation (sd) criteria to group sequences into separate clusters, we expanded the number of genogroups to 10 (GI-GX) and the number of genotypes to 48 (9 GI, 27 GII, 3 GIII, 2 GIV, 2 GV, 2 GVI and 1 genotype each for GVII, GVIII, GIX [formerly GII.15] and GX). Viruses for which currently only one sequence is available in public databases were classified into tentative new genogroups (GNA1 and GNA2) and genotypes (GII.NA1, GII.NA2 and GIV.NA1) with their definitive assignment awaiting additional related sequences. Based on nucleotide diversity in the RdRp region, noroviruses can be divided into 60 P-types (14 GI, 37 GII, 2 GIII, 1 GIV, 2 GV, 2 GVI, 1 GVII and 1 GX), 2 tentative P-groups and 14 tentative P-types. Future classification and nomenclature updates will be based on complete genome sequences and will be coordinated and disseminated by the international norovirus classification-working group.

Improving preparedness to respond to cross-border hepatitis A outbreaks in the European Union/European Economic Area: towards comparable sequencing of hepatitis A virus.

IntroductionSequence-based typing of hepatitis A virus (HAV) is important for outbreak detection, investigation and surveillance. In 2013, sequencing was central to resolving a large European Union (EU)-wide outbreak related to frozen berries. However, as the sequenced HAV genome regions were only partly comparable between countries, results were not always conclusive.AimThe objective was to gather information on HAV surveillance and sequencing in EU/European Economic Area (EEA) countries to find ways to harmonise their procedures, for improvement of cross-border outbreak responses.MethodsIn 2014, the European Centre for Disease Prevention and Control (ECDC) conducted a survey on HAV surveillance practices in EU/EEA countries. The survey enquired whether a referral system for confirming primary diagnostics of hepatitis A existed as well as a central collection/storage of hepatitis A cases' samples for typing. Questions on HAV sequencing procedures were also asked. Based on the results, an expert consultation proposed harmonised procedures for cross-border outbreak response, in particular regarding sequencing. In 2016, a follow-up survey assessed uptake of suggested methods.ResultsOf 31 EU/EEA countries, 23 (2014) and 27 (2016) participated. Numbers of countries with central collection and storage of HAV positive samples and of those performing sequencing increased from 12 to 15 and 12 to 14 respectively in 2016, with all countries typing an overlapping fragment of 218 nt. However, variation existed in the sequenced genomic regions and their lengths.ConclusionsWhile HAV sequences in EU/EEA countries are comparable for surveillance, collaboration in sharing and comparing these can be further strengthened.

Highly sensitive parechovirus CODEHOP PCR amplification of the complete VP1 gene for typing directly from clinical specimens and correct typing based on phylogenetic clustering.

PURPOSE: Human parechoviruses (HPeVs), particularly type 3, can cause severe neurological disease and neonatal sepsis in infants. HPeV3 lacks the receptor-binding motif arginine-glycine aspartic acid (RGD), and is proposed to use a different receptor associated with severe disease. In contrast, HPeV1, which contains the RGD motif, is associated with mild disease. Rapid characterization of the presence/absence of this motif is essential for understanding their epidemiology and differential disease profiles. Current HPeV typing assays are based on partial capsid genes and often do not encompass the C-terminus where the RGD region is localized/absent. In addition, these assays lack sensitivity to enable characterization within low viral-load samples, such as cerebral spinal fluid. METHODOLOGY: We developed a highly sensitive HPeV CODEHOP PCR, which enables typing of parechoviruses directly from clinical samples while generating a complete VP1 gene, including the C-terminus. RESULTS: The assay was HPeV-specific and has a sensitivity of 6.3 TCID50 ml-1 for HPeV1 and 0.63 TCID50 ml-1 for HPeV3. Analysis of the complete VP1 gene in comparison to partial VP1 fragments generated by previously published PCRs showed homologous clustering for most types. However, phylogenetic analysis of partial VP1 fragments showed incongruent typing based on the 75  % homology classification rule. In particular, the strains designated as type 17 were found to be either type 3 or 4 when using the (near-) complete VP1 fragment. CONCLUSION: While enabling sensitive characterization of HPeVs directly from clinical samples, the HPeV CODEHOP PCR enables the characterization of RGD and non-RGD strains and correct HPeV typing based on the complete VP1.

Monitoring of pork liver and meat products on the Dutch market for the presence of HEV RNA.

The aim of the present study was to assess pork liver and meat products present on the Dutch market for the presence of hepatitis E virus (HEV) RNA. HEV RNA was detected in 27.3% of 521 products sampled from Dutch retail stores in 2016. 12.7% of livers were positive for HEV RNA (n = 79), 70.7% of liverwurst (n = 99), 68.9% of liver pate (n = 90), but in none of the pork chops (n = 98), fresh sausages (n = 103) or wild boar meat (n = 52). The highest level of HEV RNA contamination was observed in a liver (reaching up to 1 × 106 copies/g), followed by ready to eat liverwurst and liver pate (up to 3 × 104 copies/g and 7 × 104 copies/g respectively). Sequence analyses revealed mainly genotype 3c, but also some 3a, 3e and 3f strains. One strain derived from a liver sample was 100% (493 nt) identical with one isolated from a HEV case with onset of disease close in time and geography, although no direct epidemiological link could be established. Despite liverwurst and liver pate undergo heat treatment (information dd. Mid 2017) that may be sufficient to inactivate HEV, persons at risk, including Dutch transplant recipients, have been advised to avoid the consumption of raw liver as well as liverwurst and liver pate.