Molecular epidemiology of enteroviruses from Guatemalan wastewater isolated from human lung fibroblasts.

The Global Specialized Polio Laboratory at CDC supports the Global Poliovirus Laboratory Network with environmental surveillance (ES) to detect the presence of vaccine strain polioviruses, vaccine-derived polioviruses, and wild polioviruses in high-risk countries. Environmental sampling provides valuable supplementary information, particularly in areas with gaps in surveillance of acute flaccid paralysis (AFP) mainly in children less than 15 years. In collaboration with Guatemala's National Health Laboratory (Laboratorio Nacional de Salud Guatemala), monthly sewage collections allowed screening enterovirus (EV) presence without incurring additional costs for sample collection, transport, or concentration. Murine recombinant fibroblast L-cells (L20B) and human rhabdomyosarcoma (RD) cells are used for the isolation of polioviruses following a standard detection algorithm. Though non-polio-Enteroviruses (NPEV) can be isolated, the algorithm is optimized for the detection of polioviruses. To explore if other EV's are present in sewage not found through standard methods, five additional cell lines were piloted in a small-scale experiment, and next-generation sequencing (NGS) was used for the identification of any EV types. Human lung fibroblast cells (HLF) were selected based on their ability to isolate EV-A genus. Sewage concentrates collected between 2020-2021 were isolated in HLF cells and any cytopathic effect positive isolates used for NGS. A large variety of EVs, including echoviruses 1, 3, 6, 7, 11, 13, 18, 19, 25, 29; coxsackievirus A13, B2, and B5, EV-C99, EVB, and polioviruses (Sabin 1 and 3) were identified through genomic typing in NGS. When the EV genotypes were compared by phylogenetic analysis, it showed many EV's were genomically like viruses previously isolated from ES collected in Haiti. Enterovirus occurrence did not follow a seasonality, but more diverse EV types were found in ES collection sites with lower populations. Using the additional cell line in the existing poliovirus ES algorithm may add value by providing data about EV circulation, without additional sample collection or processing. Next-generation sequencing closed gaps in knowledge providing molecular epidemiological information on multiple EV types and full genome sequences of EVs present in wastewater in Guatemala.

Production of norovirus-, rotavirus-, and enterovirus-like particles in insect cells is simplified by plasmid-based expression.

Insect cells have long been the main expression host of many virus-like particles (VLP). VLPs resemble the respective viruses but are non-infectious. They are important in vaccine development and serve as safe model systems in virus research. Commonly, baculovirus expression vector system (BEVS) is used for VLP production. Here, we present an alternative, plasmid-based system for VLP expression, which offers distinct advantages: in contrast to BEVS, it avoids contamination by baculoviral particles and proteins, can maintain cell viability over the whole process, production of alphanodaviral particles will not be induced, and optimization of expression vectors and their ratios is simple. We compared the production of noro-, rota- and entero-VLP in the plasmid-based system to the standard process in BEVS. For noro- and entero-VLPs, similar yields could be achieved, whereas production of rota-VLP requires some further optimization. Nevertheless, in all cases, particles were formed, the expression process was simplified compared to BEVS and potential for the plasmid-based system was validated. This study demonstrates that plasmid-based transfection offers a viable option for production of noro-, rota- and entero-VLPs in insect cells.

Characterization of enteroviruses circulating among farm animals and children in Central African Republic.

To characterize enteroviruses (EVs) circulating in farm animals in Central African Republic (CAR), we screened 192 stools of animals under 12 months belonging to family farms located in or near Bangui. To assess whether EV exchanges exist between these animals and humans, we also screened 195 stools of children who lived in contact with farm animals, as well as control stools of 358 children with no contact with farm animals. EVs were typed based on their capsid sequences.In children, all EVs belonged to species A, B and C, with EV-Cs accounting for 60%. Some EV-Cs shared recent common ancestors with lineages of vaccine-derived poliovirus that emerged in the country in 2019-2020. In animals, we identified EV-Gs that belonged to 10 different types, including a previously unknown one that we named EV-G28, while no EV-E or EV-F were observed. The CAR EV-Gs were genetically closely related to specimens sampled in other continents and some of them harboured the torovirus-derived insertion already reported in some EV-Gs. The worldwide circulation of EV-Gs is likely due the massive international trade of live animals. Besides, two human EV-Cs (coxsackievirus A17 and coxsackievirus A24) were detected in pigs, suggesting that these viruses could cross the species barrier. Our work provides original data on the epidemiology and ecology of EVs circulating among herd animals in Africa.

Structure and dynamics of enterovirus genotype networks.

Like all biological populations, viral populations exist as networks of genotypes connected through mutation. Mapping the topology of these networks and quantifying population dynamics across them is crucial to understanding how populations adapt to changes in their selective environment. The influence of mutational networks is especially profound in viral populations that rapidly explore their mutational neighborhoods via high mutation rates. Using a single-cell sequencing method, scRNA-seq-enabled acquisition of mRNA and consensus haplotypes linking individual genotypes and host transcriptomes (SEARCHLIGHT), we captured and assembled viral haplotypes from hundreds of individual infected cells, revealing the complexity of viral population structures. We obtained these genotypes in parallel with host cell transcriptome information, enabling us to link host cell transcriptional phenotypes to the genetic structures underlying virus adaptation. Our examination of these structures reveals the common evolutionary dynamics of enterovirus populations and illustrates how viral populations reach through mutational "tunnels" to span evolutionary landscapes and maintain connection with multiple adaptive genotypes simultaneously.

Capsid Integrity Detection of Enteric Viruses in Reclaimed Waters.

Climate change, unpredictable weather patterns, and droughts are depleting water resources in some parts of the globe, where recycling and reusing wastewater is a strategy for different purposes. To counteract this, the EU regulation for water reuse sets minimum requirements for the use of reclaimed water for agricultural irrigation, including a reduction in human enteric viruses. In the present study, the occurrence of several human enteric viruses, including the human norovirus genogroup I (HuNoV GI), HuNoV GII, and rotavirus (RV), along with viral fecal contamination indicator crAssphage was monitored by using (RT)-qPCR methods on influent wastewater and reclaimed water samples. Moreover, the level of somatic coliphages was also determined as a culturable viral indicator. To assess the potential viral infectivity, an optimization of a capsid integrity PMAxx-RT-qPCR method was performed on sewage samples. Somatic coliphages were present in 60% of the reclaimed water samples, indicating inefficient virus inactivation. Following PMAxx-RT-qPCR optimization, 66% of the samples tested positive for at least one of the analyzed enteric viruses, with concentrations ranging from 2.79 to 7.30 Log10 genome copies (gc)/L. Overall, most of the analyzed reclaimed water samples did not comply with current EU legislation and contained potential infectious viral particles.

Unveiling of the Co-Infection of Peste des Petits Ruminants Virus and Caprine Enterovirus in Goat Herds with Severe Diarrhea in China.

Here, we report the discovery of two viruses associated with a disease characterized by severe diarrhea on a large-scale goat farm in Jilin province. Electron Microscopy observations revealed two kinds of virus particles with the sizes of 150-210 nm and 20-30 nm, respectively. Detection of 276 fecal specimens from the diseased herds showed the extensive infection of peste des petits ruminants virus (63.77%, 176/276) and caprine enterovirus (76.81%, 212/276), with a co-infection rate of 57.97% (160/276). These results were partially validated with RT-PCR, where all five PPRV-positive and CEV-positive specimens yielded the expected size of fragments, respectively, while no fragments were amplified from PPRV-negative and CEV-negative specimens. Moreover, corresponding PPRV and CEV fragments were amplified in PPRV and CEV double-positive specimens. Histopathological examinations revealed severe microscopic lesions such as degeneration, necrosis, and detachment of epithelial cells in the bronchioles and intestine. An immunohistochemistry assay detected PPRV antigens in bronchioles, cartilage tissue, intestine, and lymph nodes. Simultaneously, caprine enterovirus antigens were detected in lung, kidney, and intestinal tissues from the goats infected by the peste des petits ruminants virus. These results demonstrated the co-infection of peste des petits ruminants virus with caprine enterovirus in goats, revealing the tissue tropism for these two viruses, thus laying a basis for the future diagnosis, prevention, and epidemiological survey for these two virus infections.

Detection of encephalitis-causing viruses reveals predominance of chikungunya virus in the state of Bahia, Brazil.

OBJECTIVES: Encephalitis is a severe neurological syndrome for which herpesvirus and enteroviruses are the most common etiological agents. Arboviruses, a wildly diverse group of pathogens, are also critical epidemiological agents associated with encephalitis. In Brazil, little is known about the causative agents of encephalitis. METHODS: We conducted a hospital surveillance for encephalitis between 2020 and 2022. Molecular (RT-PCR and qPCR) and serological (virus-specific IgM and viral antigens) techniques were performed in cerebrospinal fluid and serum samples obtained from study participants. RESULTS: In the 43 participants evaluated, the etiologic agent or the presence of IgM was detected in 16 (37.2%). Nine (20.9%) cases were positive for chikungunya virus (CHIKV), three (7.0%) for dengue virus, two (4.7%) for human adenovirus, one (2.3%) for varicella-zoster virus, and one (2.3%) for enterovirus. Whole-genome sequencing revealed that the CHIKV identified belongs to the East/Central/South African lineage. CONCLUSION: Herein, CHIKV is a common pathogen identified in encephalitis cases. Our results reinforce previous evidence that chikungunya represents a significant cause of encephalitis during CHIKV outbreaks and epidemics and add to existing information on the epidemiology of encephalitis in Brazil.

Evolution, recombination and geographic spreading of global Coxsackievirus A6.

BACKGROUND: The increasing incidence of hand, foot, and mouth disease (HFMD) associated with Coxsackievirus A6 (CVA6) has become a very significant public health problem. The aim of this study is to investigate the recombination, geographic transmission, and evolutionary characteristics of the global CVA6. METHODS: From 2019 to 2022, 73 full-length CVA6 sequences were obtained from HFMD patients in China and analyzed in combination with 1032 published whole genome sequences. Based on this dataset, the phylogenetic features, recombinant diversity, Bayesian phylodynamic characteristics, and key amino acid variations in CVA6 were analyzed. RESULTS: The four genotypes of CVA6, A, D, E, and F, are divided into 24 recombinant forms (RFs, RF-A - RF-X) based on differences in the P3 coding region. The eastern China region plays a key role in the dissemination of CVA6 in China. VP1-137 and VP1-138 are located in the DE loop on the surface of the CVA6 VP1 protein, with the former being a highly variable site and the latter having more non-synonymous substitutions. CONCLUSIONS: Based on whole genome sequences, this study contributes to the CVA6 monitoring, early warning, and the pathogenic mechanism by studying recombination diversity, geographical transmission characteristics, and the variation of important amino acid sites.

A multiplex SNaPshot assay for the detection of single nucleotide polymorphism associated with clinical severity of enterovirus infections.

Non-polio enterovirus infections are known to cause a variety of diseases and neurological complications. It is also known that the severity of these diseases largely differs among individuals with different genotypes and alleles. The Single Nucleotide Polymorphisms (SNPs) within specific genes have a considerable effect on the immune response to enteroviruses and on the outcome of disease, leading to variations in complications and infection susceptibility. Knowing the distribution of such SNPs can be valuable for individual case management and studying epidemiological parameters of enterovirus infections. In this feasibility study, a multiplex version of the primer extension-based technique called the SNaPshot Assay has been developed to examine SNPs in various relevant genes for predicting the clinical severity of enterovirus infections. It is already established that this technique is precise, consistent, scalable, and likely to exhibit high throughput. The multiplex SNaPshot can investigate multiple genetic susceptibility markers simultaneously, and the assay can be used to identify vulnerable populations, understand the epidemiology of infections, and manage the outbreaks of enteroviruses. Based on the literature, 15 SNPs were identified which are suspected for higher susceptibility to the worst outcomes after enterovirus infection and the assay was developed. Blood samples of 100 healthy volunteers were collected and tested for assay feasibility as well as to know the proportions of 15 selected SNPs. After the analysis, seven SNPs have been identified and suggested to be considered for future assays. Based on the pilot test results, it appears that positivity for any three out of the identified seven SNPs might indicate a higher risk, and future studies correlated with clinical studies among patients with and without severe diseases utilizing this assay will provide robust parameters to determine at-risk individuals more accurately.

Epidemiological and etiological investigations of hand, foot, and mouth disease in Jiashan, northeastern Zhejiang Province, China, during 2016 to 2022.

Background: Hand, foot, and mouth disease (HFMD) is a common infectious disease in children. Enterovirus A71 (EV71) and coxsackievirus A16 (CA16) have been identified as the predominant pathogens for several decades. In recent years, coxsackievirus A6 (CA6) and coxsackievirus A10 (CA10) have played increasingly important roles in a series of HFMD outbreaks. We performed a retrospective analysis of the epidemiology of HFMD and the spectrum of different viral serotypes, to elucidate the genetic and phylogenetic characteristics of the main serotypes in the Jiashan area during 2016 to 2022. Methods: Descriptive epidemiological methods were used to analyze the time and population distribution of HFMD in Jiashan during 2016 to 2022 based on surveillance data. Molecular diagnostic methods were performed to identify the viral serotypes and etiological characteristics of HFMD. Phylogenetic analyses was based on VP1 region of CA16 and CA6. Results: The average annual incidence rate of HFMD fluctuated from 2016 to 2022. Children aged 1-5 years accounted for 81.65% of cases and boys were more frequently affected than girls. Except when HFMD was affected by the COVID-19 epidemic in 2020 and 2022, epidemics usually peak in June to July, followed by a small secondary peak from October to December and a decline in February. Urban areas had a high average incidence and rural areas had the lowest. Among 560 sample collected in Jiashan, 472 (84.29%) were positive for enterovirus. The most frequently identified serotypes were CA6 (296, 52.86%), CA16 (102, 18.21%), EV71 (16, 2.86%), CA10 (14, 2.50%) and other enteroviruses (44, 7.86%). There were 71 and 142 VP1 sequences from CA16 and CA6, respectively. Substitution of N218D, A220L and V251I was detected in CA16 and may have been related to viral infectivity. Phylogenetic analysis showed that CA16 could be assigned to two genogroups, B1a and B1b, while all the CA6 sequences belonged to the D3a genogroup. Conclusion: CA6 and CA16 were the two major serotypes of enteroviruses circulating in the Jiashan area during 2016 to 2022. Continuous and comprehensive surveillance for HFMD is needed to better understand and evaluate the prevalence and evolution of the associated pathogens.

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.

Genomic epidemiology of CVA10 in Guangdong, China, 2013-2021.

Hand, Foot and Mouth Disease (HFMD) is a highly contagious viral illness primarily affecting children globally. A significant epidemiological transition has been noted in mainland China, characterized by a substantial increase in HFMD cases caused by non-Enterovirus A71 (EV-A71) and non-Coxsackievirus A16 (CVA16) enteroviruses (EVs). Our study conducts a retrospective examination of 36,461 EV-positive specimens collected from Guangdong, China, from 2013 to 2021. Epidemiological trends suggest that, following 2013, Coxsackievirus A6 (CVA6) and Coxsackievirus A10 (CVA10) have emerged as the primary etiological agents for HFMD. In stark contrast, the incidence of EV-A71 has sharply declined, nearing extinction after 2018. Notably, cases of CVA10 infection were considerably younger, with a median age of 1.8 years, compared to 2.3 years for those with EV-A71 infections, possibly indicating accumulated EV-A71-specific herd immunity among young children. Through extensive genomic sequencing and analysis, we identified the N136D mutation in the 2 A protein, contributing to a predominant subcluster within genogroup C of CVA10 circulating in Guangdong since 2017. Additionally, a high frequency of recombination events was observed in genogroup F of CVA10, suggesting that the prevalence of this lineage might be underrecognized. The dynamic landscape of EV genotypes, along with their potential to cause outbreaks, underscores the need to broaden surveillance efforts to include a more diverse spectrum of EV genotypes. Moreover, given the shifting dominance of EV genotypes, it may be prudent to re-evaluate and optimize existing vaccination strategies, which are currently focused primarily target EV-A71.

Analysis of mild and severe neonatal enterovirus infections in a Chinese neonatal tertiary center: a retrospective case-control study.

PURPOSE: To compare the clinical characteristics, virus serotype, and outcome in cases of mild and severe enteroviral infection at a tertiary neonatal intensive care unit in China. METHODS: A retrospective analysis of cases hospitalized between June and August 2019. Samples (stool or throat swabs) were examined using reverse transcription polymerase chain reaction. Positive cases were divided into two groups: mild infection and severe infection. RESULTS: A total of 149 cases were assigned to one of two groups: mild infection (n = 104) and severe infection (n = 45). There were no significant differences between the groups in terms of sex, gestational age, birth weight, mode of delivery, and onset within 7 days. Clinical symptoms in both groups mostly resembled sepsis (fever, rash, poor feeding, and lethargy); however, there were significant variations in concomitant symptoms such as hepatitis, thrombocytopenia, encephalitis, coagulopathy, and myocarditis. Severe cases were more likely to have abnormal complete blood counts, biochemical parameters, and cerebrospinal fluid markers. The predominant serotypes implicated in neonatal enterovirus infections were echoviruses and Coxsackievirus B. Invasive ventilation, intravenous immunoglobulin, vasoactive medications, and blood product transfusions were often required, with high mortality rates among severe cases. CONCLUSION: We found significant differences between mild and severe cases of neonatal enterovirus infection with respect to complications, laboratory findings, and enterovirus serotypes. It is crucial to exercise caution when newborns exhibit symptoms of sepsis, during an enterovirus outbreak. Anemia, thrombocytopenia, abnormal liver function, and coagulation dysfunction should be monitored closely as they could indicate the presence of a severe enteroviral infection.

Global landscape of coxsackieviruses in human health.

Coxsackieviruses-induced infections, particularly in infants and young children, are one of the most important public health issues in low- and middle-income countries, where the surveillance system varies substantially, and these manifestations have been disregarded. They are widespread throughout the world and are responsible for a broad spectrum of human diseases, from mildly symptomatic conditions to severe acute and chronic disorders. Coxsackieviruses (CV) have been found to have 27 identified genotypes, with overlaps in clinical phenotypes between genotypes. In this review, we present a concise overview of the most recent studies and findings of coxsackieviruses-associated disorders, along with epidemiological data that provides comprehensive details on the distribution, variability, and clinical manifestations of different CV types. We also highlight the significant roles that CV infections play in the emergence of neurodegenerative illnesses and their effects on neurocognition. The current role of CVs in oncolytic virotherapy is also mentioned. This review provides readers with a better understanding of coxsackieviruses-associated disorders and pointing the impact that CV infections can have on different organs with variable pathogenicity. A deeper knowledge of these infections could have implications in designing current surveillance and prevention strategies related to severe CVs-caused infections, as well as encourage studies to identify the emergence of more pathogenic types and the etiology of the most common and most severe disorders associated with coxsackievirus infection.

Poliomyelitis and COVID-19: a repeated history.

The measurement of the enterovirus and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in sewage water is relevant in the early detection of the introduction or disappearance of these viruses in the ecosystem. We evaluated the co-circulation of the enteroviruses and SARS-CoV-2 in 81 sewage water samples collected between September 2021 and April 2023 from different regions of north and southeast Romania, at the border with Ukraine. We used, for the molecular detection of the pathogens, the multiplex real-time polymerase chain reaction (PCR) assay produced for respiratory samples and the Respiratory 2.1 Plus panel Biofire Film array. The isolation of enteroviruses was performed on cell culture lines, in accordance with the World Health Organization (WHO) recommendations. By molecular investigations, we detected the SARS-CoV-2 in 22 (27%) samples, and the human rhinovirus/enterovirus in 64 (79%) samples. By isolation on cell culture lines, 27 samples (33,33%) were positive for non-polio enteroviruses, and no poliovirus strains were isolated, proving the maintenance of the polio-free status in Romania. In an emergency situation, the molecular detection of the pathogens in sewage water using a PCR system integrating sample preparation, amplification, detection, and analysis in 1 h could be implemented.

Increased circulation of echovirus 11 in the general population and hospital patients as elicited by the non-polio enterovirus laboratory-based sentinel surveillance in northern Italy, 2023.

OBJECTIVES: Following the alert of echovirus 11 (E-11) infection in neonates in EU/EEA Member States, we conducted an investigation of E-11 circulation by gathering data from community and hospital surveillance of enterovirus (EV) in northern Italy from 01 August 2021 to 30 June 2023. METHODS: Virological results of EVs were obtained from the regional sentinel surveillance database for influenza-like illness (ILI) in outpatients, and from the laboratory database of ten hospitals for inpatients with either respiratory or neurological symptoms. Molecular characterization of EVs was performed by sequence analysis of the VP1 gene. RESULTS: In our ILI series, the rate of EV-positive specimens showed an upward trend from the end of May 2023, culminating at the end of June, coinciding with an increase in EV-positive hospital cases. The E-11 identified belonged to the D5 genogroup and the majority (83%) were closely associated with the novel E-11 variant, first identified in severe neonatal infections in France since 2022. E-11 was identified sporadically in community cases until February 2023, when it was also found in hospitalized cases with a range of clinical manifestations. All E-11 cases were children, with 14 out of 24 cases identified through hospital surveillance. Of these cases, 60% were neonates, and 71% had severe clinical manifestations. CONCLUSION: Baseline epidemiological data collected since 2021 through EV laboratory-based surveillance have rapidly tracked the E-11 variant since November 2022, alongside its transmission during the late spring of 2023.

Validation of improved automated nucleic acid extraction methods for direct detection of polioviruses for global polio eradication.

Polioviruses (PV), the main causative agent of acute flaccid paralysis (AFP), are positive-sense single-stranded RNA viruses of the family Picornaviridae. As we approach polio eradication, accurate and timely detection of poliovirus in stool from AFP cases becomes vital to success for the eradication efforts. Direct detection of PV from clinical diagnostic samples using nucleic acid (NA) extraction and real-time reverse transcriptase polymerase chain reaction (rRT-PCR) instead of the current standard method of virus isolation in culture, eliminates the long turn-around time to diagnosis and the need for high viral titer amplification in laboratories. An essential component of direct detection of PV from AFP surveillance samples is the efficient extraction of NA. Potential supply chain issues and lack of vendor presence in certain areas of the world necessitates the validation of multiple NA extraction methods. Using retrospective PV-positive surveillance samples (n=104), two extraction kits were compared to the previously validated Zymo Research Quick-RNA™ Viral Kit. The Roche High Pure Viral RNA Kit, a column-based manual extraction method, and the MagMaX™ Pathogen RNA/DNA kit used in the automated Kingfisher Flex system were both non-inferior to the Zymo kit, with similar rates of PV detection in pivotal rRT-PCR assays, such as pan-poliovirus (PanPV), poliovirus serotype 2 (PV2), and wild poliovirus serotype 1 (WPV1). These important assays allow the identification and differentiation of PV genotypes and serotypes and are fundamental to the GPLN program. Validation of two additional kits provides feasible alternatives to the current piloted method of NA extraction for poliovirus rRT-PCR assays.

Construction of an infectious clone for enterovirus A89 and mutagenesis analysis of viral infection and cell binding.

Enterovirus A89 (EV-A89) is an unconventional strain belonging to the Enterovirus A species. Limited research has been conducted on EV-A89, leaving its biological and pathogenic properties unclear. Developing reverse genetic tools for EV-A89 would help to unravel its infection mechanisms and aid in the development of vaccines and anti-viral drugs. In this study, an infectious clone for EV-A89 was successfully constructed and recombinant enterovirus A89 (rEV-A89) was generated. The rEV-A89 exhibited similar characteristics such as growth curve, plaque morphology, and dsRNA expression with parental strain. Four amino acid substitutions were identified in the EV-A89 capsid, which were found to enhance viral infection. Mechanistic studies revealed that these substitutions increased the virus's cell-binding ability. Establishing reverse genetic tools for EV-A89 will significantly contribute to understanding viral infection and developing anti-viral strategies.IMPORTANCEEnterovirus A species contain many human pathogens and have been classified into conventional cluster and unconventional cluster. Most of the research focuses on various conventional members, while understanding of the life cycle and infection characteristics of unconventional viruses is still very limited. In our study, we constructed the infectious cDNA clone and single-round infectious particles for the unconventional EV-A89, allowing us to investigate the biological properties of recombinant viruses. Moreover, we identified key amino acids residues that facilitate EV-A89 infection and elucidate their roles in enhancing viral binding to host cells. The establishment of the reverse genetics system will greatly facilitate future study on the life cycle of EV-A89 and contribute to the development of prophylactic vaccines and anti-viral drugs.

Emergence and circulation of enterovirus B species in infants in southern China: A multicenter retrospective analysis.

BACKGROUND: Enteroviruses (EV) are common and can cause severe diseases, particularly in young children. However, the information of EV infection in infants in China is limited due to the vast population size and extensive geographical area of the country. Here, we conducted a retrospective multicenter analysis of available EV data to assess the current epidemiological situation in the infant population in southern China. METHODS: The study enrolled infants with suspected EV infection from 34 hospitals across 12 cities in southern China between 2019 to 2022, and the confirmation of EV was done using RT-PCR and VP1 gene sequencing. RESULTS: Out of 1221 infants enrolled, 330 (27.03%) were confirmed as EV-infected. Of these, 260 (78.79%) were newborns aged 0-28 days. The EV belonged to three species: EV-B (80.61%), EV-A (11.82%), and human rhinovirus (7.58%). Newborns were more susceptible to EV-B than older infants (p < 0.001). Within EV-B, we identified 15 types, with coxsackievirus (CV) B3 (20.91%), echovirus (E) 11 (19.70%), and E18 (16.97%) being the most common. The predominant EV types changed across different years. EV infection in infants followed a seasonal pattern, with a higher incidence from May to August. Furthermore, perinatal mother-to-child EV transmission in 12 mother-newborn pairs were observed. CONCLUSION: Our study is the first to demonstrate the emergence and widespread circulation of EV-B species, mainly CVB3, E11, and E18, in southern China, primarily affecting young infants. This research provides valuable insights for future epidemic assessment, prediction, as well as the elimination of mother-to-child transmission.

Identification of fangchinoline as a broad-spectrum enterovirus inhibitor through reporter virus based high-content screening.

Hand, foot, and mouth disease (HFMD) is a common pediatric illness mainly caused by enteroviruses, which are important human pathogens. Currently, there are no available antiviral agents for the therapy of enterovirus infection. In this study, an excellent high-content antiviral screening system utilizing the EV-A71-eGFP reporter virus was developed. Using this screening system, we screened a drug library containing 1042 natural compounds to identify potential EV-A71 inhibitors. Fangchinoline (FAN), a bis-benzylisoquinoline alkaloid, exhibits potential inhibitory effects against various enteroviruses that cause HFMD, such as EV-A71, CV-A10, CV-B3 and CV-A16. Further investigations revealed that FAN targets the early stage of the enterovirus life cycle. Through the selection of FAN-resistant EV-A71 viruses, we demonstrated that the VP1 protein could be a potential target of FAN, as two mutations in VP1 (E145G and V258I) resulted in viral resistance to FAN. Our research suggests that FAN is an efficient inhibitor of EV-A71 and has the potential to be a broad-spectrum antiviral drug against human enteroviruses.