Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics.

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.

Temporal turnover of viral biodiversity and functional potential in intertidal wetlands.

As the central members of the microbiome networks, viruses regulate the composition of microbial communities and drive the nutrient cycles of ecosystems by lysing host cells. Therefore, uncovering the dynamic patterns and the underlying ecological mechanisms mediating the tiniest viral communities across space and through time in natural ecosystems is of crucial importance for better understanding the complex microbial world. Here, the temporal dynamics of intertidal viral communities were investigated via a time-series sampling effort. A total of 1911 viral operational taxonomic units were recovered from 36 bimonthly collected shotgun metagenomes. Functionally important auxiliary metabolic genes involved in carbohydrate, sulfur, and phosphorus metabolism were detected, some of which (e.g., cysH gene) were stably present within viral genomes over time. Over the sampling period, strong and comparable temporal turnovers were observed for intertidal viromes and their host microbes. Winter was determined as the pivotal point for the shifts in viral diversity patterns. Notably, the viral micro-diversity covaried with the macro-diversity, following similar temporal patterns. The relative abundances of viral taxa also covaried with their host prokaryotes. Meanwhile, the virus-host relationships at the whole community level were relatively stable. Further statistical analyses demonstrated that the dynamic patterns of viral communities were highly deterministic, for which temperature was the major driver. This study provided valuable mechanistic insights into the temporal turnover of viral communities in complex ecosystems such as intertidal wetlands.

Unveiling the hidden dangers: enteropathogens carried by flies in Pudong New Area.

BACKGROUND: Flies are acknowledged as vectors of diseases transmitted through mechanical means and represent a significant risk to human health. The study aimed to determine the prevalence of enteropathogens carried by flies in Pudong New Area to inform strategies for preventing and controlling flies. METHODS: Samples were collected from various locations in the area using cage trapping techniques between April and November 2021, encompassing various habitats such as parks, residential areas, restaurants, and farmers' markets. The main fly species were identified using cryomicrography and taxonomic enumeration, with 20 samples per tube collected from different habitats. Twenty-five enteropathogens were screened using GI_Trial v3 TaqManTM microbial arrays. RESULTS: A total of 3,875 flies were collected from 6,400 placements, resulting in an average fly density of 0.61 flies per cage. M. domestica were the most common species at 39.85%, followed by L. sericata at 16.57% and B. peregrina at 13.14%. Out of 189 samples, 93 tested positive for enteropathogens, with nine different pathogens being found. 12.70% of samples exclusively had parasites, a higher percentage than those with only bacteria or viruses. The study found that M. domestica had fewer enteropathogens than L. sericata and B. peregrina, which primarily harbored B. hominis instead of bacteria and viruses such as E. coli, Astrovirus, and Sapovirus. During spring testing, all three fly species exhibited low rates of detecting enteropathogens. M. domestica were found in residential areas with the highest number of pathogen species, totaling six. In contrast, L. sericata and B. peregrina were identified in farmers' markets with the highest number of pathogen species, totaling six and seven, respectively. CONCLUSIONS: Flies have the potential to serve as vectors for the transmission of enteropathogens, thereby posing a substantial risk to public health.

A Systematic Bioinformatics Approach for Mapping the Minimal Set of a Viral Peptidome.

Sequence changes in viral genomes generate protein sequence diversity that enables viruses to evade the host immune system, hindering the development of effective preventive and therapeutic interventions. The massive proliferation of sequence data provides unprecedented opportunities to study viral adaptation and evolution. An alignment-free approach removes various restrictions posed by an alignment-dependent approach for studying sequence diversity. The publicly available tool, UNIQmin, offers an alignment-free approach for studying viral sequence diversity at any given rank of taxonomy lineage and is big data ready. The tool performs an exhaustive search to determine the minimal set of sequences required to capture the peptidome diversity within a given dataset. This compression is possible through the removal of identical sequences and unique sequences that do not contribute effectively to the peptidome diversity pool. Herein, we describe a detailed four-part protocol utilizing UNIQmin to generate the minimal set for the purpose of viral diversity analyses, alignment-free at any rank of the taxonomy lineage, using the recent global public health threat Monkeypox virus (MPX) sequence data as a case study. The protocol enables a systematic bioinformatics approach to study sequence diversity across taxonomic lineages, which is crucial for our future preparedness against viral epidemics. This is particularly important when data are abundant, freely available, and alignment is not an option. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Tool installation and input file preparation Basic Protocol 2: Generation of a minimal set of sequences for a given dataset Basic Protocol 3: Comparative minimal set analysis across taxonomic lineage ranks Basic Protocol 4: Factors affecting the minimal set of sequences.

Hecatomb: an integrated software platform for viral metagenomics.

BACKGROUND: Modern sequencing technologies offer extraordinary opportunities for virus discovery and virome analysis. Annotation of viral sequences from metagenomic data requires a complex series of steps to ensure accurate annotation of individual reads and assembled contigs. In addition, varying study designs will require project-specific statistical analyses. FINDINGS: Here we introduce Hecatomb, a bioinformatic platform coordinating commonly used tasks required for virome analysis. Hecatomb means "a great sacrifice." In this setting, Hecatomb is "sacrificing" false-positive viral annotations using extensive quality control and tiered-database searches. Hecatomb processes metagenomic data obtained from both short- and long-read sequencing technologies, providing annotations to individual sequences and assembled contigs. Results are provided in commonly used data formats useful for downstream analysis. Here we demonstrate the functionality of Hecatomb through the reanalysis of a primate enteric and a novel coral reef virome. CONCLUSION: Hecatomb provides an integrated platform to manage many commonly used steps for virome characterization, including rigorous quality control, host removal, and both read- and contig-based analysis. Each step is managed using the Snakemake workflow manager with dependency management using Conda. Hecatomb outputs several tables properly formatted for immediate use within popular data analysis and visualization tools, enabling effective data interpretation for a variety of study designs. Hecatomb is hosted on GitHub (github.com/shandley/hecatomb) and is available for installation from Bioconda and PyPI.

Bioinformatic Approaches for Comparative Analysis of Viruses.

The field of viral genomic studies has experienced an unprecedented increase in data volume. New strains of known viruses are constantly being added to the GenBank database and so are completely new species with little or no resemblance to our databases of sequences. In addition to this, metagenomic techniques have the potential to further increase the number and rate of sequenced genomes. Besides, it is important to consider that viruses have a set of unique features that often break down molecular biology dogmas, e.g., the flux of information from RNA to DNA in retroviruses and the use of RNA molecules as genomes. As a result, extracting meaningful information from viral genomes remains a challenge and standard methods for comparing the unknown and our databases of characterized sequences may need adaptations. Thus, several bioinformatic approaches and tools have been created to address the challenge of analyzing viral data. This chapter offers descriptions and protocols of some of the most important bioinformatic techniques for comparative analysis of viruses. The authors also provide comments and discussion on how viruses' unique features can affect standard analyses and how to overcome some of the major sources of problems. Protocols and topics emphasize online tools (which are more accessible to users) and give the real experience of what most bioinformaticians do in day-by-day work with command-line pipelines. The topics discussed include (1) clustering related genomes, (2) whole genome multiple sequence alignments for small RNA viruses, (3) protein alignment for marker genes and species affiliation, (4) variant calling and annotation, and (5) virome analyses and pathogen identification.

Analytical and clinical validation of a novel, laboratory-developed, modular multiplex-PCR panel for fully automated high-throughput detection of 16 respiratory viruses.

BACKGROUND: Viral respiratory Infections pose a health risk, especially to vulnerable patient populations. Effective testing programs can detect and differentiate these infections at an early stage, which is particularly important for high-risk clinical departments. The objective of this study was to develop and validate a multiplex PCR-panel for 16 different respiratory viruses on a fully-automated high-throughput platform. METHODS: Three multiplex-PCR assays were designed to run on the cobas5800/6800/8800 systems, consolidating 16 viral targets: RESP1: SARS-CoV-2, influenza-A/B, RSV; RESP2: hMPV, hBoV, hAdV, rhino-/ENV; RESP3: HPIV-1-4, hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1. Analytic performance was evaluated using digital-PCR based standards and international reference material. Clinical performance was determined by comparing results from clinical samples with reference assays. RESULTS: Analytical sensitivity (i.e. lower limit of detection (LoD), 95 % probability of detection) was determined as follows: SARS-CoV-2: 29.3 IU/ml, influenza-A: 179.9 cp/ml, influenza-B: 333.9 cp/ml and RSV: 283.1 cp/ml. LoDs of other pathogens ranged between 9.4 cp/ml (hCoV-NL63) and 21,419 cp/ml (HPIV-2). Linearity was verified over 4-7 log-steps with pooled standard differentials (SD) ranging between 0.18-0.70ct. Inter-/intra-run variability (precision) was assessed for all targets over 3 days. SDs ranged between 0.13-0.74ct. Positive agreement in clinical samples was 99.4 % and 95 % for SARS-CoV-2 and influenza-A respectively. Other targets were in the 80-100 % range. Negative agreement varied between 96.3-100 %. DISCUSSION: Lab-developed tests are a key factor for effective clinical diagnostics. The multiplex panel presented in this study demonstrated high performance and provides an easily scalable high-throughput solution for respiratory virus testing, e.g. for testing in high-risk patient populations.

Prevalence of human respiratory pathogens and associated mucosal cytokine levels in young children and adults: a cross-sectional observational study in the Netherlands during the winter of 2012/2013.

Respiratory pathogens can cause severe disease and even death, especially in the very young and very old. Studies investigating their prevalence often focus on individuals presenting to healthcare providers with symptoms. However, the design of prevention strategies, e.g. which target groups to vaccinate, will benefit from knowledge on the prevalence of, risk factors for and host response to these pathogens in the general population. In this study, upper respiratory samples (n = 1311) were collected cross-sectionally during winter from 11- and 24-month old children, their parents, and adults ≥60 years of age that were recruited irrespective of seeking medical care. Almost all children, approximately two-thirds of parents and a quarter of older adults tested positive for at least one pathogen, often in the absence of symptoms. Viral interference was evident for the combination of rhinovirus and respiratory syncytial virus. Attending childcare facilities and having siblings associated with increased pathogen counts in children. On average, children showed increased levels of mucosal cytokines compared to parents and especially proinflammatory molecules associated with the presence of symptoms. These findings may guide further research into transmission patterns of respiratory pathogens and assist in determining the most appropriate strategies for the prediction and prevention of disease.

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

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

Genetic Diversity and Detection of Respiratory Viruses Excluding SARS-CoV-2 during the COVID-19 Pandemic in Gabon, 2020-2021.

Acute respiratory infections are a major global burden in resource-limited countries, including countries in Africa. Although COVID-19 has been well studied since the pandemic emerged in Gabon, Central Africa, less attention has been paid to other respiratory viral diseases, and very little data are available. Herein, we provide the first data on the genetic diversity and detection of 18 major respiratory viruses in Gabon during the COVID-19 pandemic. Of 582 nasopharyngeal swab specimens collected from March 2020 to July 2021, which were SARS-CoV-2 negative, 156 were positive (26%) for the following viruses: enterovirus (20.3%), human rhinovirus (HRV) (4.6%), human coronavirus OC43 (1.2%), human adenovirus (0.9%), human metapneumovirus (hMPV) (0.5%), influenza A virus (IAV) (0.3%), and human parainfluenza viruses (0.5%). To determine the genetic diversity and transmission route of the viruses, phylogenetic analyses were performed using genome sequences of the detected viruses. The IAV strain detected in this study was genetically similar to strains isolated in the USA, whereas the hMPV strain belonging to the A2b subtype formed a cluster with Kenyan strains. This study provides the first complete genomic sequences of HRV, IAV, and hMPV detected in Gabon, and provides insight into the circulation of respiratory viruses in the country.

Virus Quasispecies Rarefaction: Subsampling with or without Replacement?

In quasispecies diversity studies, the comparison of two samples of varying sizes is a common necessity. However, the sensitivity of certain diversity indices to sample size variations poses a challenge. To address this issue, rarefaction emerges as a crucial tool, serving to normalize and create fairly comparable samples. This study emphasizes the imperative nature of sample size normalization in quasispecies diversity studies using next-generation sequencing (NGS) data. We present a thorough examination of resampling schemes using various simple hypothetical cases of quasispecies showing different quasispecies structures in the sense of haplotype genomic composition, offering a comprehensive understanding of their implications in general cases. Despite the big numbers implied in this sort of study, often involving coverages exceeding 100,000 reads per sample and amplicon, the rarefaction process for normalization should be performed with repeated resampling without replacement, especially when rare haplotypes constitute a significant fraction of interest. However, it is noteworthy that different diversity indices exhibit distinct sensitivities to sample size. Consequently, some diversity indicators may be compared directly without normalization, or instead may be resampled safely with replacement.

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

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

Ten different viral agents infecting and co-infecting children with acute gastroenteritis in Southern Italy: Role of known pathogens and emerging viruses during and after COVID-19 pandemic.

Acute gastroenteritis (AGE) represents a world public health relevant problem especially in children. Enteric viruses are the pathogens mainly involved in the episodes of AGE, causing about 70.00% of the cases. Apart from well-known rotavirus (RVA), adenovirus (AdV) and norovirus (NoV), there are various emerging viral pathogens potentially associated with AGE episodes. In this study, the presence of ten different enteric viruses was investigated in 152 fecal samples collected from children hospitalized for gastroenteritis. Real time PCR results showed that 49.3% of them were positive for viral detection with the following prevalence: norovirus GII 19.7%, AdV 15.8%, RVA 10.5%, human parechovirus (HPeV) 5.3%, enterovirus (EV) 3.3%, sapovirus (SaV) 2.6%. Salivirus (SalV), norovirus GI and astrovirus (AstV) 1.3% each, aichivirus (AiV) found in only one patient. In 38.2% of feces only one virus was detected, while co-infections were identified in 11.8% of the cases. Among young patients, 105 were ≤5 years old and 56.0% tested positive for viral detection, while 47 were >5 years old with 40.0% of them infected. Results obtained confirm a complex plethora of viruses potentially implicated in gastroenteritis in children, with some of them previously known for other etiologies but detectable in fecal samples. Subsequent studies should investigate the role of these viruses in causing gastroenteritis and explore the possibility that other symptoms may be ascribed to multiple infections.

Epidemiology and Clinical impact of single and multi-viral respiratory infections in post-pandemic era.

Acute respiratory tract infections (ARI) are common diseases in children and adults and could cause severe infections in high-risk patients, like the immunocompromised and elderly, and are the leading cause of morbidity, hospitalization and mortality. This study aimed to explore the prevalence of respiratory viruses and the clinical impact of single- and multi-infection among hospitalized patients in various age groups. 3578 nasopharyngeal swabs (NPS) were analyzed for pathogen detection of acute respiratory tract infections. 930 out of 3578 NPS were diagnosed positive for at least one respiratory virus. The distribution of viral infections, prevalence and pathogen, differed significantly among age groups. Most RTI are observed in the age group over 65 years (50.6%) with a high SARS-CoV2 prevalence, following by group <5 years (25.6%), where the most frequently detected viruses were RSV, Rhinovirus, FluA-H3, MPV, and AdV. The co-infection rate also varies according to age and, in some cases, especially in older adults, could have severe clinical impact. This study emphasizes that it is important to know and analyze, in all age groups of hospitalized patients, the epidemiology of respiratory viruses, the prevalence of coinfections, and the clinical impact of various pathogens. Furthermore, in a clinical setting, the rapid diagnosis of respiratory infections by means of molecular tests is crucial not only to avoid hospital outbreaks, but also to allow early and optimal treatment to reduce morbidity and mortality.

Epidemiology and molecular characterization of respiratory viruses at the end of COVID-19 pandemic in Lombardy, Northern Italy.

The COVID-19 pandemic forced the adoption of non-pharmaceutical interventions (NPIs) which influenced the circulation of other respiratory pathogens, such as Influenza virus (FLU), Parainfluenza virus (PIV), Respiratory Syncytial virus (RSV), Rhinovirus (RV), Enterovirus (EV), Adenovirus (AdV), Human Metapneumovirus (hMPV), and Human Coronavirus (CoV). The aim of the current study was to investigate how, with the end of the pandemic, the withdrawal of the NPIs impacted on the circulation and distribution of common respiratory viruses. The analyzed samples were collected from June 2021 to March 2023 (post-pandemic period) and compared to ones from the pandemic period. Nucleic acid detection of all respiratory viruses was performed by multiplex real time Polymerase Chain Reaction (PCR) and sequencing was conducted by Next Generation Sequencing (NGS) technique. Our analysis shows that the NPIs adopted against SARS-CoV-2 were also effective in controlling the spread of other respiratory viruses. Moreover, we documented how RV/EVs were the most commonly identified species, with the more abundant strains represented by Coxsackievirus (CV)-A/B and RV-A/C. RV/EVs were also detected in some co-infection cases; in particular, the majority of co-infections concerned CV-B/RV-A, CV-B/ECHO. Given the pandemic potential of respiratory viruses, accurate molecular screening is essential for a proper surveillance and prevention strategy.

Loss to gain: pseudogenes in microorganisms, focusing on eubacteria, and their biological significance.

Pseudogenes are defined as "non-functional" copies of corresponding parent genes. The cognition of pseudogenes continues to be refreshed through accumulating and updating research findings. Previous studies have predominantly focused on mammals, but pseudogenes have received relatively less attention in the field of microbiology. Given the increasing recognition on the importance of pseudogenes, in this review, we focus on several aspects of microorganism pseudogenes, including their classification and characteristics, their generation and fate, their identification, their abundance and distribution, their impact on virulence, their ability to recombine with functional genes, the extent to which some pseudogenes are transcribed and translated, and the relationship between pseudogenes and viruses. By summarizing and organizing the latest research progress, this review will provide a comprehensive perspective and improved understanding on pseudogenes in microorganisms. KEY POINTS: • Concept, classification and characteristics, identification and databases, content, and distribution of microbial pseudogenes are presented. • How pseudogenization contribute to pathogen virulence is highlighted. • Pseudogenes with potential functions in microorganisms are discussed.

Viruses contribute to microbial diversification in the rumen ecosystem and are associated with certain animal production traits.

BACKGROUND: The rumen microbiome enables ruminants to digest otherwise indigestible feedstuffs, thereby facilitating the production of high-quality protein, albeit with suboptimal efficiency and producing methane. Despite extensive research delineating associations between the rumen microbiome and ruminant production traits, the functional roles of the pervasive and diverse rumen virome remain to be determined. RESULTS: Leveraging a recent comprehensive rumen virome database, this study analyzes virus-microbe linkages, at both species and strain levels, across 551 rumen metagenomes, elucidating patterns of microbial and viral diversity, co-occurrence, and virus-microbe interactions. Additionally, this study assesses the potential role of rumen viruses in microbial diversification by analyzing prophages found in rumen metagenome-assembled genomes. Employing CRISPR-Cas spacer-based matching and virus-microbe co-occurrence network analysis, this study suggests that the viruses in the rumen may regulate microbes at strain and community levels through both antagonistic and mutualistic interactions. Moreover, this study establishes that the rumen virome demonstrates responsiveness to dietary shifts and associations with key animal production traits, including feed efficiency, lactation performance, weight gain, and methane emissions. CONCLUSIONS: These findings provide a substantive framework for further investigations to unravel the functional roles of the virome in the rumen in shaping the microbiome and influencing overall animal production performance. Video Abstract.

Prevalence of Respiratory Viruses in Children With Acute Respiratory Infections in Shanghai, China, From 2013 to 2022.

BACKGROUND: A variety of viruses can cause acute respiratory infections (ARIs), resulting in a high disease burden worldwide. To explore the dominant viruses and their prevalence characteristics in children with ARIs, comprehensive surveillance was carried out in the Pudong New Area of Shanghai. METHODS: Between January 2013 and December 2022, the basic and clinical information, and respiratory tract specimens of 0-14 years old children with ARIs were collected in five sentinel hospitals in Shanghai Pudong. Each specimen was tested for eight respiratory viruses, and the positive rates of different age groups, case types (inpatient or outpatient) were analyzed. RESULTS: In our study, 30.67% (1294/4219) children with ARIs were positive for at least one virus. Influenza virus (IFV) was the most commonly detected respiratory virus (349/4219, 8.27%), followed by respiratory syncytial virus (RSV) (217/4219, 5.14%), para-influenza virus (PIV) (215/4219, 5.10%), and human coronavirus (HCoV, including 229E, OC43, NL63, and HKU1) (184/4219, 4.36%). IFV was the leading respiratory virus in outpatients aged 5-14 years (201/1673, 12.01%); RSV was the most prevalent respiratory virus in both inpatients (61/238, 25.63%) and outpatients (4/50, 8.00%) for ARI patients aged <6 months old. For PIV, HMPV, HCoV, and HRV, the risk of infection usually was higher among young children. Co-infection with more than two viruses was seen in 3.25% (137/4219). CONCLUSIONS: IFV and RSV played important roles in ARIs among children, but the risk populations were different. There are needs for targeted diagnosis and treatment and necessary immunization and non-pharmaceutical interventions.