Virus Surveillance Research Articles

Epidemiology and Management of Plant Viruses Under a Changing Climate.

Plant viruses are an ever-present threat to agricultural production and provide a wide array of symptoms resulting in economic losses throughout the world. Diseases can be transmitted by insect vectors, as well as through pollen, seed, and other means. With the increased globalization of agriculture, the introduction of new viruses from exotic locations and their establishment in new production regions and even new crops is a growing concern. Advancing knowledge of the epidemiology of plant viruses including development of new diagnostic methods, virus surveillance, and modeling, virus ecology and evolution, virus interactions with insect vectors, and other factors are important toward reducing the spread of plant viruses and managing virus diseases.

A strategy for entomo-virological surveillance of yellow fever, dengue, zika, and chikungunya viruses in field-collected mosquitoes

Arboviruses transmitted by Culicidae insects are significant threats to human health, presenting dynamic transmission cycles and involving different vectors and hosts. The surveillance and characterization of the vectors involved in these cycles are crucial for understanding and preventing potential outbreaks. Therefore, we propose a strategy that we used for entomological surveillance of urban, rural, and sylvatic mosquitoes and to characterize natural infection by four major arboviruses.•Immature and adult mosquitoes were collected intra, peri and extradomicilie of urban and rural households, using different collection methodologies.•Mosquitoes were pooled or separated in head-thorax and abdomen, according to the species.•A multiplex nested RT-PCR (Reverse transcription polymerase chain reaction) method was used for the simultaneous detection of dengue virus (DENV), zika virus (ZIKV), chikungunya virus (CHIKV), and yellow fever virus (YFV).Overall, this strategy proved helpful for vectors surveillance at different ecosystems, as well as for implementing a low-cost molecular surveillance system that allows the early detection of potential outbreaks, and identify other potential vectors involved in viral transmission.

Rapidly developed, optimized, and applied wastewater surveillance system for real-time monitoring of low-incidence, high-impact MPOX outbreak.

Recent MPOX viral resurgences have mobilized public health agencies around the world. Recognizing the significant risk of MPOX outbreaks, large-scale human testing, and immunization campaigns have been initiated by local, national, and global public health authorities. Recently, traditional clinical surveillance campaigns for MPOX have been complemented with wastewater surveillance (WWS), building on the effectiveness of existing wastewater programs that were built to monitor SARS-CoV-2 and recently expanded to include influenza and respiratory syncytial virus surveillance in wastewaters. In the present study, we demonstrate and further support the finding that MPOX viral fragments agglomerate in the wastewater solids fraction. Furthermore, this study demonstrates that the current, most commonly used MPOX assays are equally effective at detecting low titers of MPOX viral signal in wastewaters. Finally, MPOX WWS is shown to be more effective at passively tracking outbreaks and/or resurgences of the disease than clinical testing alone in smaller communities with low human clinical case counts of MPOX.

Navigating the Landscape: A Comprehensive Review of Current Virus Databases.

Viruses are abundant and diverse entities that have important roles in public health, ecology, and agriculture. The identification and surveillance of viruses rely on an understanding of their genome organization, sequences, and replication strategy. Despite technological advancements in sequencing methods, our current understanding of virus diversity remains incomplete, highlighting the need to explore undiscovered viruses. Virus databases play a crucial role in providing access to sequences, annotations and other metadata, and analysis tools for studying viruses. However, there has not been a comprehensive review of virus databases in the last five years. This study aimed to fill this gap by identifying 24 active virus databases and included an extensive evaluation of their content, functionality and compliance with the FAIR principles. In this study, we thoroughly assessed the search capabilities of five database catalogs, which serve as comprehensive repositories housing a diverse array of databases and offering essential metadata. Moreover, we conducted a comprehensive review of different types of errors, encompassing taxonomy, names, missing information, sequences, sequence orientation, and chimeric sequences, with the intention of empowering users to effectively tackle these challenges. We expect this review to aid users in selecting suitable virus databases and other resources, and to help databases in error management and improve their adherence to the FAIR principles. The databases listed here represent the current knowledge of viruses and will help aid users find databases of interest based on content, functionality, and scope. The use of virus databases is integral to gaining new insights into the biology, evolution, and transmission of viruses, and developing new strategies to manage virus outbreaks and preserve global health.

Molecular surveillance of arboviruses in Nigeria

Arboviral infections are fast becoming a global public health concern as a result of its high fatality rate and sporadic spread. From the outbreak of Zika virus in the Americas, the endemicity of Yellow fever in West Africa and South America, outbreaks of West Nile virus in South Africa to the year-round and national risk of Dengue fever in Mainland China and India. The war against emerging and re-emerging viral infection could probably lead to the next pandemic. To be above the pending possible arboviral pandemic, consistent surveillance of these pathogens is necessary in every society. This study was aimed at conducting a surveillance for Yellow fever virus, Zika virus, Chikungunya virus, Dengue virus and Rift Valley fever virus in four states in Nigeria using molecular techniques. A cross-sectional study involving 1600 blood samples collected from febrile patients in Lagos, Kwara, Ondo and Delta States between 2018 and 2021 was conducted using Real time polymerase chain reaction for detection of the pathogens. Extraction and purification of viral RNA were done using Qiagen Viral RNA Mini Kit. Samples were analyzed using One Step PrimeScript III RT-PCR mix (Takara Bio) alongside optimized primers and probes designed in-house. Positive samples were sequenced on MinION platform (Nanopore technologies). Bioinformatic and phylogenetic analysis were performed with DNASTAR Lasergene 17.3. All the RNA extracted from samples collected from the four states were negative for ZIKV RNA, RVFV RNA, CHIKV RNA and DENV RNA. However, twelve of the samples (2%) tested positive for YFV RNA. Three full genomes of sizes 10,751 bp, 10,500 bp and 10,715 bp were generated and deposited in GenBank with accession numbers: ON323052, ON323053 and ON323054 respectively. Phylogenetic analysis shows clustering within lineage 3 of West African genotype. This result shows an active spread of Yellow fever in Delta State, Nigeria. However, there is no emergence of a new genotype There is a need for an intense surveillance of Yellow fever virus in Nigeria to avert a major outbreak.

Waterborne viruses in urban groundwater environments

The proportion of global population using urban aquifers as drinking water sources increases every year and indeed the groundwater quality is not monitored adequately. Although norovirus has been identified as the first cause of groundwater-related outbreaks, the surveillance of waterborne viruses has been rather neglected. From ageing or disrupted sewer systems, occasional sewer discharges (e.g. combined sewer overflows, storm runoff), to poorly managed reclaimed water infiltration practices, multiple are the pathways that cause groundwater quality deterioration. This study revises the main viral contamination sources and the factors affecting viral contamination of groundwater bodies in terms of transport, inactivation, and survival of the viral particles. It also summarizes the methods used for those reporting the presence of human viruses in urban groundwaters. A total of 36 articles have been included in the method survey spanning a period of 24 years (1999–2022). There is a need of systematic monitoring considering representative set of waterborne pathogens. The evaluation of the presence of human adenovirus seems a useful tool to predict the presence of other waterborne pathogens in groundwater. Large volume sampling methods, but also new passive sampling methodologies applied to groundwater, coupled to target massive sequencing approaches may elucidate the range of pathogens capable of contaminating urban groundwaters for further evaluation of risk.

Seasonal respiratory virus trends in pediatric patients during the COVID-19 pandemic in Brazil.

Acute respiratory infections are a constant public health problem causing childhood morbidity and mortality worldwide. Reported cases of major respiratory infections decreased in 2020 after restrictive measures were adopted to contain the COVID-19 pandemic, but there is little data on the impact after these measures were relaxed in the subsequent years. This study conducted molecular analysis to identify rhinovirus, respiratory syncytial virus, influenza A virus, and adenovirus in SARS-CoV-2-negative samples taken from symptomatic pediatric patients during 2021 and 2022 to ascertain the impact of pandemic response measures within the broader epidemiological scenario. The positivity rates found were 28.3% and 50.8%, in 2021 and 2022, respectively, representing a significant increase (1.8 times) in the circulation of non-SARS-CoV-2 viruses after the reduction of non-pharmacological measures to contain the COVID-19 pandemic. Within the positive samples, rhinovirus and respiratory syncytial virus were most frequent (44.4 and 18% in 2021; 44.5 and 22.5% in 2022), whereas influenza A and adenovirus were found in lower frequency (12.5 and 5.5% in 2021; 13.4 and 4.9% in 2022, respectively). Because these different respiratory virus diseases produce similar symptoms, diagnosis based on clinical condition alone can be inaccurate, and more reliable testing is required to select the best therapeutic approach for each case. The loosening of restrictive measures to contain the COVID-19 pandemic led to higher numbers of other respiratory infections in pediatric patients. Ongoing surveillance and differential diagnosis of respiratory viruses are required to better understand their seasonal patterns after the COVID-19 pandemic to guide prevention and control strategies.

Continued dominance of dengue virus serotype 2 during the recent Central India outbreaks (2019-2021) with evidence of genetic divergence

ABSTRACT Central India faced major dengue outbreaks in 2019 and 2021. In the present study, we aimed to identify the dengue virus serotypes and genotypes circulating in Central India during the COVID pre-pandemic year (2019) and ongoing-pandemic year (2021). For this purpose, the suspected cases were first tested by serological assays. Sero-positive samples were then subjected to molecular diagnosis by RT-PCR and semi-nested PCR. The serotypes obtained were confirmed by nucleotide sequencing. A phylogenetic analysis of serotypes was performed to identify the circulating genotypes. All four DENV serotypes were detected during 2019 and 2021, with the predominance of DENV2. Cases with multiple DENV serotype infections were also identified, involving DENV-2 in all the coinfections. Genotyping revealed that DENV-1 (Genotype V, American/African), DENV-2 (Genotype IV, Cosmopolitan), DENV-3 (Genotype III, Cosmopolitan), and DENV-4 (Genotype I) were involved during both outbreaks. DENV-2 detected in 2019 and 2021 has diverged from the previous strains detected in Central India (2016 and 2018), which may account for the higher transmission of DENV-2 during these outbreaks. The detection of heterologous DENV serotypes with high transmission efficiency calls for continuous viral monitoring and surveillance, which will contribute to a better understanding of changing viral dynamics and transmission patterns.

Evaluation of Viral Respiratory Pathogens Among Patients Initially Tested Negative for SARS-CoV-2

Background: A number of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses commonly circulating among vertebrates, such as influenza H1N1, respiratory syncytial virus (RSV), adenoviruses, and human coronavirus (HCoV)-229E, cause symptoms similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These viruses are important causes of cold, pneumonia, and shortness of breath in humans, which have been overlooked during the coronavirus disease 2019 (COVID-19) pandemic. Furthermore, the diagnosis of infection with these viruses mostly relies on physical examination and clinical history, despite the fact that accurate molecular diagnosis is available. Objectives: This study aimed to evaluate the presence of respiratory viruses in patients who were suspected to be infected with COVID-19 yet initially tested negative for SARS-CoV-2, as it could be beneficial in developing effective control measures and more reliable testing and surveillance of such viruses. Methods: In this study, laboratory samples of 123 patients referred to Ghaem Hospital of Mashhad, Iran, were evaluated that tested negative for SARS-CoV-2 in the initial assessment while showing the clinical symptoms of COVID-19. Initial testing for SARS-CoV-2 was carried out by the TaqMan real-time polymerase chain reaction (PCR) method using a kit approved by the Ministry of Health (Pishtaz Teb, Iran). Further analysis for the presence of 17 respiratory viruses was carried out using Genova kits based on the virus genome conserved sequences of influenza H1N1, influenza B, influenza A, SARS-CoV-2, HCoV-HKU1, HCoV-OC43, HCoV-NL63, HCoV-229E, metapneumovirus, RSV, human bocavirus 1, 2, 3, parainfluenza 1, 2, 3, and adenovirus. Results: According to the results of the present evaluations, out of 123 samples that were acquired using nasal and throat swabs and that initially tested negative for SARS-CoV-2, 8 cases of influenza A (47.1%), 1 case of parainfluenza (5.9%), 1 case of HKU1/OC-43 (5.9%), 4 cases of RSV (23.5%), 1 case of HCoV-NL63/HCoV-229-E (5.9%), and 2 cases of SARS-CoV-2 (11.8%) were detected. Conclusions: Based on the results of real-time PCR tests obtained from patients who had clinical symptoms of SARS-CoV-2 infections, it can be mentioned that due to the similar symptoms of patients with respiratory viral infections, individuals with respiratory symptoms could be examined for other viral infections in addition to SARS-CoV-2 infection, and a suitable basis for their prevalence in the community could be provided.

Characterization of A/H7 influenza virus global antigenic diversity and key determinants in the hemagglutinin globular head mediating A/H7N9 antigenic evolution.

A/H7 avian influenza viruses cause outbreaks in poultry globally, resulting in outbreaks with significant socio-economical impact and zoonotic risks. Occasionally, poultry vaccination programs have been implemented to reduce the burden of these viruses, which might result in an increased immune pressure accelerating antigenic evolution. In fact, evidence for antigenic diversification of A/H7 influenza viruses exists, posing challenges to pandemic preparedness and the design of vaccination strategies efficacious against drifted variants. Here, we performed a comprehensive analysis of the global antigenic diversity of A/H7 influenza viruses and identified the main substitutions in the hemagglutinin responsible for antigenic evolution in A/H7N9 viruses isolated between 2013 and 2019. The A/H7 antigenic map and knowledge of the molecular determinants of their antigenic evolution add value to A/H7 influenza virus surveillance programs, the design of vaccines and vaccination strategies, and pandemic preparedness.

SARS-CoV-2 in digital era: Diagnostic techniques and importance of nucleic acid quantification with digital PCRs.

Studies related to clinical diagnosis and research of SARS-CoV-2 are important in the current pandemic era. Although molecular biology has emphasised the importance of qualitative analysis, quantitative analysis with nucleic acids in relation to SARS-CoV-2 needs to be clearly emphasised, which can provide perspective for viral dynamic studies of SARS-CoV-2. In this regard, the requirement and utilization of digital PCR in COVID-19 research has substantially increased during the pandemic, necessitating the aggregation of its cardinal applications and future scopes. Hence, this meta-review comprehensively addresses and emphasises the importance of nucleic acid quantification of SARS-CoV-2 RNA with digital PCR (dPCR). Various quantitative techniques of clinical significance like immunological, proteomic and nucleic acid-based diagnosis and quantification, have been comparatively discussed. Furthermore, the core part of the article focusses on the working principle and advantages of digital PCR, along with its applications in COVID-19 research. Several important applications like viral load quantitation, environmental surveillance and assay validation have been extensively investigated and discussed. Certain key future scopes of clinical importance, like mortality prediction, viral/variant-symbiosis, and antiviral studies were also identified, suggesting several possible digital PCR applications in COVID-19 research.

CRISPR in Public Health: The Health Equity Implications and Role of Community in Gene-Editing Research and Applications.

CRISPR (clustered regularly interspaced short palindromic repeats) is a Nobel Prize-winning technology that holds significant promise for revolutionizing the prevention and treatment of human disease through gene editing. However, CRISPR's public health implications remain relatively uncertain and underdiscussed because (1) targeting genetic factors alone will have limited influence on population health, and (2) minority populations (racial/ethnic, sexual and gender)-who bear the nation's greatest health burdens-historically suffer unequal benefits from emerging health care innovations and tools. This article introduces CRISPR and its potential public health benefits (e.g., improving virus surveillance, curing genetic diseases that pose public health problems such as sickle cell anemia) while outlining several major ethical and practical threats to health equity. This includes minorities' grave underrepresentation in genomics research, which may lead to less effective and accepted CRISPR tools and therapies for these groups, and their anticipated unequal access to these tools and therapies in health care. Informed by the principles of fairness, justice, and equitable access, ensuring gene editing promotes rather than diminishes health equity will require the meaningful centering and engagement of minority patients and populations in gene-editing research using community-based participatory research approaches. (Am J Public Health. 2023;113(8):874-882. https://doi.org/10.2105/AJPH.2023.307315).

The impact of COVID-19 pandemic on surveillance of influenza and influenza-like viruses: a single center experience

Aims: Influenza and the agents responsible for influenza-like illness (ILI) are a significant cause of upper and lower respiratory tract infections, with a notable pattern of seasonal incidence. The surveillance of influenza and agents causing ILI is important for the developmentof a prophylaxis approach and infection control. In the present study, we utilize the Reverse transcription-polymerase chain reaction (RT-PCR) method to identify the presence of influenza virus and other agents associated with ILI in nasopharyngeal smears obtained from patients exhibiting symptoms of influenza and ILI. The study sample included patients who were admitted to multiple clinics and intensive care units (ICU) of the Health Sciences University Ankara Training and Research Hospital in the period preceding, and in the initial months and first year following the coronavirus disease 2019 (COVID-19) outbreak. Throughthe comparisons made in the study, the intention is to gain a better understanding of the influence of the COVID-19 pandemic on other viral infectious agents transmitted via the respiratory tract.
 Methods: Included in the study were 257 admitted to different wards and ICUs of our hospital due to symptoms of upper or lower respiratory tract infection and ILI between 2015 and 2021 (excluding data from 2017–2018). The study was conducted prospectively within the scope of the Global Influenza Hospital Surveillance Network project. Using sterile swabs, nasopharyngeal swab samples were collected from inpatients who provided informed consent for their participation in the study, and the samples were placed in a viral transport medium. The presence of influenza viruses (Influenza A, subtypes of Influenza A and Influenza B) and agents causing ILI (i.e. respiratory syncytial virus, coronavirus) was investigated with an RT-PCR approach.
 Results: The most common symptoms among the 257 patients included in the study with pre-diagnoses of influenza and ILI were cough (82.2%), fever (67.7%), shortness of breath (66.1%) and myalgia (40%). The RT-PCR detected a viral agent in 60 (23.3%) of the 257 patients, whereas no agent could be detected in 197 (76.6%) patients. Furthermore, 51 (18.5%) tested positive for influenza virus, five (1.9%) for respiratory syncytial virus (RSV), and four (1.5%) for SARS-CoV-2. An analysis of the results within two distinct time frames, namely prior to the COVID-19 pandemic (2016–2017, 2019–prior to December 10, 2020) and during the COVID-19 pandemic (between December 11, 2020 and 2021), influenza viruses (influenza A H1N1 and influenza A H3N2), RSV and influenza type B were identified as the dominant viruses before the COVID-19 pandemic, while the predominant viruses were a single influenza strain and four SARS-CoV-2 variants during the COVID-19 pandemic.A significant difference was noted in the distribution of viruses between the two time frames – prior to the pandemic and during the pandemic. Of the patients, 199 (77.4%) were discharged with full recovery while 58 patients died (22.6%). Of the 58 patients that died, 25 were female (25/131 females) and 33 were male (33/126 males), 11 had tested positive for influenza virus andone for RSV, while no infectious agent could be identified in 46 patients. 
 Conclusion: The implementation of molecular testing methods for the identification of viral infectious agents among inpatients during influenza and ILI outbreaks, administering antiviral and prophylactic treatments targeting influenza, RSV and SARS-CoV-2 infections, and adopting infection control measures, could significantly decrease mortality and morbidity rates while mitigating the complications associated with these infectious agents.

Genetic and Biological Characterization of H3N2 Avian Influenza Viruses Isolated from Poultry Farms in China between 2019 and 2021

H3N2 influenza viruses not only cause seasonal epidemics in humans but also circulate widely in animals, posing a threat to both animal and human health. Our previous studies indicate that H3N2 avian influenza viruses (AIVs) are readily detected in live poultry markets (LPMs); however, the evolution and biological characteristics of the H3N2 viruses in poultry farms in China are unclear. In this study, we performed active surveillance and collected 49,135 samples from poultry farms. In total, 21 H3N2 AIVs were isolated and their genetics, receptor-binding properties, and replication in mice were evaluated. Phylogenetic analysis indicated that H3N2 AIVs harbor complicated gene constellations and have undergone extensive reassortment; the viruses derived their genes from 12 different hemagglutinin subtypes of duck viruses, including H1, H2, H4, H5, H6, H7, H8, H9, H10, H11, H12, and H14. The complicated gene constellations indicated that H3N2 viruses may have been introduced into poultry farms from different sources, but none have become dominant in poultry farms. Although the H3N2 AIVs possessed avian-type receptor-binding preference, most of the isolates could replicate without preadaptation and some of H3N2 viruses caused weight loss in mice. Notably, two H3N2 viruses acquired the PB2 627K mutation after a single round of replication in mice, suggesting similar mutations could occur if they replicated in humans. Overall, our study demonstrates that the H3N2 AIVs pose a potential threat to the public health and emphasizes the need for continued surveillance of H3N2 viruses in the both LPMs and poultry farms.

A collaborative approach to improving representation in viral genomic surveillance.

The lack of routine viral genomic surveillance delayed the initial detection of SARS-CoV-2, allowing the virus to spread unfettered at the outset of the U.S. epidemic. Over subsequent months, poor surveillance enabled variants to emerge unnoticed. Against this backdrop, long-standing social and racial inequities have contributed to a greater burden of cases and deaths among minority groups. To begin to address these problems, we developed a new variant surveillance model geared toward building 'next generation' genome sequencing capacity at universities in or near rural areas and engaging the participation of their local communities. The resulting genomic surveillance network has generated more than 1,000 SARS-CoV-2 genomes to date, including the first confirmed case in northeast Louisiana of Omicron, and the first and sixth confirmed cases in Georgia of the emergent BA.2.75 and BQ.1.1 variants, respectively. In agreement with other studies, significantly higher viral gene copy numbers were observed in Delta variant samples compared to those from Omicron BA.1 variant infections, and lower copy numbers were seen in asymptomatic infections relative to symptomatic ones. Collectively, the results and outcomes from our collaborative work demonstrate that establishing genomic surveillance capacity at smaller academic institutions in rural areas and fostering relationships between academic teams and local health clinics represent a robust pathway to improve pandemic readiness.

Subtype specific virus enrichment with immunomagnetic separation method followed by NGS unravels the mixture of H5 and H9 avian influenza virus

Wild bird avian influenza type A virus (AIV) surveillance is important for the early detection of highly pathogenic AIVs and for providing early warnings to the poultry industry and veterinary services to implement more effective control measures against these viruses. Some field samples are often found to contain more than two kinds of AIV. Correct determination of the HA/NA subtype and complete nucleotide sequences of the component viruses in those samples are often critical for timely and accurate understanding of the field situation, but it is not easy to define the genomic structure of the constituent viruses unambiguously because AIV has eight segmented genomes. In this study, with immunomagnetic beads incorporating polyclonal antibodies of chicken for subtype-specific viral enrichment, we could selectively decrease the density of one of the two constituent viruses in a sample of different subtypes, H5 and H9, artificially generated; this was represented in the changes of Ct values with subtype specific real-time RT-PCR. Following this, with NGS, we could recover nearly complete genomic sequences and arrange the consensus sequences of gene segments of the constituent viruses confidently with the quantitative variable like genome coverage, linked along the gene segments and associated with the number of viral copies in a sample.

Influenza transmission during COVID-19 measures downscaling in Greece, August 2022: evidence for the need of continuous integrated surveillance of respiratory viruses.

After the near absence of influenza and other respiratory viruses during the first 2 years of the COVID-19 pandemic, an increased activity of mainly influenza A(H3N2) was detected at the beginning of August 2022 in Greece on three islands. Of 33 cases with respiratory symptoms testing negative for SARS-CoV-2 with rapid antigen tests, 24 were positive for influenza: 20 as A(H3N2) subtype and four as A(H1N1)pdm09 subtype. Phylogenetic analysis of selected samples from both subtypes was performed and they fell into clusters within subclades that included the 2022/23 vaccine strains. Our data suggest that influenza can be transmitted even in the presence of another highly infectious pathogen, such as SARS-CoV-2, with a similar transmission mode. We highlight the need for implementing changes in the current influenza surveillance and suggest a move from seasonal to continuous surveillance, especially in areas with a high number of tourists. Year-round surveillance would allow for a timelier start of vaccination campaigns and antiviral drugs procurement processes.

Antigen test swabs are comparable to nasopharyngeal swabs for sequencing of SARS-CoV-2

Viral genomic surveillance has been integral in the global response to the SARS-CoV-2 pandemic. Surveillance efforts rely on the availability of representative clinical specimens from ongoing testing activities. However, testing practices have recently shifted due to the widespread availability and use of rapid antigen tests, which could lead to gaps in future monitoring efforts. As such, genomic surveillance strategies must adapt to include laboratory workflows that are robust to sample type. To that end, we compare the results of RT-qPCR and viral genome sequencing using samples from positive BinaxNOW COVID-19 Antigen Card swabs (N = 555) to those obtained from nasopharyngeal (NP) swabs used for nucleic acid amplification testing (N = 135). We show that swabs obtained from antigen cards are comparable in performance to samples from NP swabs, providing a viable alternative and allowing for the potential expansion of viral genomic surveillance to outpatient clinic as well as other settings where rapid antigen tests are often used.

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.

Surveillance and Genomic Characterization of Influenza A and D Viruses in Swine, Belgium and the Netherlands, 2019-2021.

During 2019-2021, we isolated 62 swine influenza A viruses in Belgium and the Netherlands. We also detected influenza D in pigs in the Netherlands. The ever-changing diversity of influenza viruses and the identification of influenza D emphasize the need for more virus surveillance.