Background:The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. Aim: We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Methods: Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. Results: The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive -Global (EVAg), a European Union infrastructure project. Conclusion: The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.

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Noroviruses are the causative agents of the majority of viral gastroenteritis outbreaks in humans. During the past 15 years, noroviruses of genotype GGII.4 have caused four epidemic seasons of viral gastroenteritis, during which four novel variants (termed epidemic variants) emerged and displaced the resident viruses. In order to understand the mechanisms and biological advantages of these epidemic variants, we studied the genetic changes in the capsid proteins of GGII.4 strains over this period. A representative sample was drawn from 574 GGII.4 outbreak strains collected over 15 years of systematic surveillance in The Netherlands, and capsid genes were sequenced for a total of 26 strains. The three-dimensional structure was predicted by homology modeling, using the Norwalk virus (Hu/NoV/GGI.1/Norwalk/1968/US) capsid as a reference. The highly significant preferential accumulation and fixation of mutations (nucleotide and amino acid) in the protruding part of the capsid protein provided strong evidence for the occurrence of genetic drift and selection. Although subsequent new epidemic variants differed by up to 25 amino acid mutations, consistent changes were observed in only five positions. Phylogenetic analyses showed that each variant descended from its chronologic predecessor, with the exception of the 2006b variant, which is more closely related to the 2002 variant than to the 2004 variant. The consistent association between the observed genetic findings and changes in epidemiology leads to the conclusion that population immunity plays a role in the epochal evolution of GGII.4 norovirus strains.Since the beginning of viral gastroenteritis outbreak surveillance in the early 1990s, noroviruses have become recognized as the major cause of reported outbreaks of acute viral gastroenteritis worldwide. Noroviruses form a genus within the family Caliciviridae and are genetically and antigenically highly variable. Currently, five distinct genogroups (GGs) are recognized. Strains belonging to GGI, GGII, and GGIV are known to cause infections in humans. The GGs have been subdivided further into genotypes, defined by a minimum amino acid sequence identity over the complete capsid sequence of 80% (1).The strains most commonly identified as the cause of outbreaks belong to genotype GGII.4. In The Netherlands, this was the case for 68% of all norovirus outbreaks that were characterized during 12 years of surveillance and for up to 81% of all health care-related outbreaks. Since their first detection in The Netherlands in January 1995, the GGII.4 strains have consistently been present in the Dutch population (46). These observations are in agreement with those of other surveillance studies worldwide (3,4,15,17,29,36,55).During the past 15 years, four epidemic norovirus seasons have occurred, in the winters of 1995-1996, 2002-2003, 2004-2005, and 2006-2007. These worldwide epidemics were invariantly caused by the predominant genotype, GGII.4, and were attributed to the emergence of new variant lineages of this genotype (4,3...

Acute gastroenteritis is one of the most common diseases worldwide. In developed countries, viruses, particularly noroviruses, are recognized as the leading cause. In The Netherlands, the surveillance of gastroenteritis outbreaks with suspected viral etiologies (as determined by Kaplan criteria) was established by the National Institute for Public Health and the Environment in 1994. This paper presents an overview of viral gastroenteritis outbreaks reported from 1994 through 2005. A minimum epidemiological data set consisting of the associated setting(s), the probable transmission mode, the date of the first illness and the date of sampling, the number of persons affected, and the number of hospitalizations was requested for each reported outbreak. Stool samples were tested for the presence of norovirus, sapovirus, rotavirus, astrovirus, adenovirus, and Aichi virus by electron microscopy, enzyme-linked immunosorbent assay, and/or reverse transcription-PCR. A total of 6,707 stool samples from 941 gastroenteritis outbreaks were investigated. Noroviruses were detected as the causative agent in 735 (78.1%) of the outbreaks, and rotaviruses, adenoviruses, and astroviruses were found to be responsible for 46 (4.9%), 9 (1.0%), and 5 (0.5%) outbreaks, respectively. Among the gastroenteritis outbreaks in which a mode of transmission was identified, most outbreaks (38.1%) were associated with person-to-person transmission, and the majority (54.9%) of the outbreaks investigated were reported by residential institutions. Since 2002, the total number of outbreaks reported and the number of unexplained outbreaks have increased. Furthermore, the number of rotavirus-associated outbreaks has increased, especially in nursing homes. Despite thorough testing, 115 (12.2%) outbreaks suspected of having viral etiologies remain unexplained. Increases in numbers of reported outbreaks may indicate undefined changes in the criteria for reporting or the emergence of new pathogens.

, a cluster of cases of pneumonia of unknown etiology were reported linked to a market in Wuhan, China 1. 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 deaths 3. 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. Whole-genome sequencing (WGS) is a powerful tool to understand the transmission dynamics of outbreaks and inform outbreak control decisions 4-7. Evidence of this was seen during the 2014-2016 West African Ebola outbreak when real-time WGS was used to help public health decision-making, a strategy dubbed 'precision public health pathogen genomics' 8,9. Immediate sharing and analysis of data during outbreaks is now recommended as an integral part of outbreak response 10-12. Feasibility of real-time WGS requires access to sequence platforms that provide reliable sequences, access to metadata for interpretation, and data analysis at high speed and low cost. Therefore, WGS for outbreak support is an active area of research. Nanopore sequencing has been employed in recent outbreaks of Usutu, Ebola, Zika and yellow fever virus owing to the ease of use and relatively low start-up cost 4-7. The robustness of this method has recently been validated using Usutu virus 13,14. In the Netherlands, the first COVID-19 case was confirmed on 27 February and WGS was performed in near to real-time using an amplicon-based sequencing approach. From 22 January, symptomatic travelers from countries where SARS-CoV-2 was known to circulate were routinely tested. The first case of SARS-CoV-2 infection in the Netherlands was identified on 27 February in a person with recent travel history to Italy and an additional case was identified one day later, also in a person with recent travel history to Italy. The genomes of these first two positive samples were generated and analyzed by 29 February. These two viruses clustered differently in the phylogenetic tree, confirming separate introductions (Fig. 1a). The advice to test hospitalized patients with serious respiratory infections was issued on 24 February and subsequent attempts to identify possible local transmission chains triggered testing for SARS-CoV-2 on a large scale in h...

Sapoviruses (SaVs) belong to the Caliciviridae family and can cause gastroenteritis in humans and swine. Despite extensive testing, human sapoviruses have been found only in sporadic cases and in one mixed outbreak in children between 1994 and 2007 in the Netherlands. Here we describe a change in sapovirus epidemiology in the Netherlands resulting in sapovirus outbreaks and infections in adults. From November 2007 to January 2009, 478 outbreaks of acute gastroenteritis were reported to the National Institute for Public Health and the Environment in the Netherlands as a part of ongoing surveillance. Sapoviruses were found to be the most likely cause of 19 outbreaks (4%). During the same 2-year period, sapovirus infections were reported in Sweden, Slovenia, and Hungary. In the Netherlands, further characterization of outbreak strains showed that 12 (63%) sapovirus outbreaks were caused by genotype I.2 viruses. Most patients were adults older than 60 years (range, 1 to 100 years). Phylogenetic analysis using all presently available SaV sequences showed high homology between genotype I.2 strains detected in different geographical regions (Sweden, Slovenia, Taiwan, Japan, and Russia) since 2007. These first reported outbreaks of sapovirus infections in adults in the Netherlands were remarkable. Detection of identical genotypes in many samples might suggest that these viruses have the same origin, and since the infection is spreading fast, the prevalence of sapovirus infection may be increasing. The incidence of sapovirus infections in these countries suggests that a substantial part of Europe is affected by this virus.

Hepatitis E virus (HEV) infections in developed countries are recognized as an imported disease related to travel to endemic regions. However, increasing evidence suggests that HEV infection may also occur in the developed countries and that swine may act as a possible reservoir. To investigate the indigenous transmission of HEV in the Netherlands, sera from 50 blood donors and 1027 sera from patients with acute hepatitis were screened with an ELISA for HEV-specific IgG and IgM. Because the Netherlands is considered a nonendemic region, all positive ELISA results were confirmed by immunoblot to exclude false-positive results. Evidence of recent HEV infection was detected in 0% of the blood donors and 4.4% of the cases, based on combined positive IgM and IgG responses. The serodiagnosis was confirmed by a positive polymerase chain reaction (PCR) in 24 patients with hepatitis (2.3% overall, 51% of confirmed IgM+/IgG+ cases). IgG antibodies alone were detected in 4.2% of patients. We found related sequences to virus strains detected in Dutch pigs (genotype 3, 91-97% homology) in 89% of PCR-confirmed HEV patients. The detection of unique swine-like HEV sequences in 16 indigenous hepatitis patients without a recent travel history suggests that HEV is endemic in the Netherlands. We recommend including HEV tests in unexplained acute hepatitis patients, despite their travel history.

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