Sequence-specific nanoparticle barcode strategy for multiplex human enterovirus typing.

Human enteroviruses (HEV) can cause a range of diseases from mild to potentially life-threatening. Identification and genotyping of HEV are crucial for disease management. Existing typing methods, however, have inherent limitations. Developing alternative methods to detect HEV with more virus types, high accuracy, and sensitivity in an accessible manner presents a technological and analytical challenge. Here, a sequence-specific nanoparticle barcode (SSNB) method is presented for simultaneous detection of 10 HEV types. This method significantly increases sensitivity, enhancing detection by 10-106 times over the traditional multiplex hybrid genotyping (MHG) method, by resolving cross-interference between the multiple primer sets. Furthermore, the SSNB method demonstrates a 100% specificity in accurately distinguishing between 10 different HEV types and other prevalent clinical viruses. In an analysis of 70 clinical throat swab samples, the SSNB method shows slightly higher detection rate for positive samples (50%) compared to the RT-PCR method (48.6%). Additionally, further assessment of the typing accuracy for samples identified as positive by SSNB using sequencing method reveals a concordance rate of 100%. The combined high sensitivity and specificity level of the methodology, together with the capability for multiple type analysis and compatibility with clinical workflow, make this approach a promising tool for clinical settings.

Epidemiology and molecular detection of human adenovirus and non-polio enterovirus in fecal samples of children with acute gastroenteritis: A five-year surveillance in northern Brazil.

Acute gastroenteritis (AGE) is a common pediatric infection that remains a significant cause of childhood morbidity and mortality worldwide, especially in low-income regions. Thus, the objective of this study was to detect human adenovirus (HAdV) and non-polio enterovirus (NPEV) in fecal samples from the Gastroenteritis Surveillance Network, and to identify circulating strains by nucleotide sequencing. A total of 801 fecal samples were tested using qPCR/RT-qPCR, and 657 (82.0%) were inoculated into HEp-2C and RD cell lines. The HAdV and NPEV positivity rates obtained using qPCR/RT-qPCR were 31.7% (254/801) and 10.5% (84/801), respectively, with 5.4% (43/801) co-detection. Cytopathic effect was observed in 9.6% (63/657) of patients, 2.7% (18/657) associated with HAdV, and 6.2% (41/657) associated with NPEV after testing by ICC-PCR. A comparison of the two methodologies demonstrated an agreement of 93.5% for EVNP and 64.4% for HAdV. These two viruses were detected throughout the study period, with HAdV positivity rates ranging from 41% in Amapá to 18% in Pará. The NEPV varied from 18% in Pará/Rondônia to 3% in Acre. The most affected age group was over 60 months for both HAdV and NPEV. Samples previously positive for rotavirus and norovirus, which did not show a major difference in the presence or absence of diarrhea, fever, and vomiting, were excluded from the clinical analyses of these two viruses. These viruses circulated over five years, with a few months of absence, mainly during the months corresponding to the waves of SARS-CoV-2 infection in Brazil. Five HAdV species were identified (A, B, C, D, and F), with a greater predominance of HAdV-F41 (56.5%) followed by HAdV-C (15.2%). Three NPEV species (A, B, and C) were detected, with serotypes E14 (19.3%) and CVA-24 (16.1%) being the most prevalent. The present study revealed a high diversity of NPEV and HAdV types circulating in children with AGE symptoms in the northern region of Brazil.

Molecular Evolutionary Dynamics of Coxsackievirus A6 Causing Hand, Foot, and Mouth Disease From 2021 to 2023 in China: Genomic Epidemiology Study.

Background: Hand, foot, and mouth disease (HFMD) is a global public health concern, notably within the Asia-Pacific region. Recently, the primary pathogen causing HFMD outbreaks across numerous countries, including China, is coxsackievirus (CV) A6, one of the most prevalent enteroviruses in the world. It is a new variant that has undergone genetic recombination and evolution, which might not only induce modifications in the clinical manifestations of HFMD but also heighten its pathogenicity because of nucleotide mutation accumulation. Objective: The study assessed the epidemiological characteristics of HFMD in China and characterized the molecular epidemiology of the major pathogen (CV-A6) causing HFMD. We attempted to establish the association between disease progression and viral genetic evolution through a molecular epidemiological study. Methods: Surveillance data from the Chinese Center for Disease Control and Prevention from 2021 to 2023 were used to analyze the epidemiological seasons and peaks of HFMD in Henan, China, and capture the results of HFMD pathogen typing. We analyzed the evolutionary characteristics of all full-length CV-A6 sequences in the NCBI database and the isolated sequences in Henan. To characterize the molecular evolution of CV-A6, time-scaled tree and historical population dynamics regarding CV-A6 sequences were estimated. Additionally, we analyzed the isolated strains for mutated or missing amino acid sites compared to the prototype CV-A6 strain. Results: The 2021-2023 epidemic seasons for HFMD in Henan usually lasted from June to August, with peaks around June and July. The monthly case reporting rate during the peak period ranged from 20.7% (4854/23,440) to 35% (12,135/34,706) of the total annual number of cases. Analysis of the pathogen composition of 2850 laboratory-confirmed cases identified 8 enterovirus serotypes, among which CV-A6 accounted for the highest proportion (652/2850, 22.88%). CV-A6 emerged as the major pathogen for HFMD in 2022 (203/732, 27.73%) and 2023 (262/708, 37.01%). We analyzed all CV-A6 full-length sequences in the NCBI database and the evolutionary features of viruses isolated in Henan. In China, the D3 subtype gradually appeared from 2011, and by 2019, all CV-A6 virus strains belonged to the D3 subtype. The VP1 sequences analyzed in Henan showed that its subtypes were consistent with the national subtypes. Furthermore, we analyzed the molecular evolutionary features of CV-A6 using Bayesian phylogeny and found that the most recent common ancestor of CV-A6 D3 dates back to 2006 in China, earlier than the 2011 HFMD outbreak. Moreover, the strains isolated in 2023 had mutations at several amino acid sites compared to the original strain. Conclusions: The CV-A6 virus may have been introduced and circulating covertly within China prior to the large-scale HFMD outbreak. Our laboratory testing data confirmed the fluctuation and periodic patterns of CV-A6 prevalence. Our study provides valuable insights into understanding the evolutionary dynamics of CV-A6.

Molecular epidemiology and characterization of enteroviruses detected in cerebrospinal fluid and respiratory samples in Slovenia, 2014-2023.

Enterovirus (EV) infections have various symptoms and severe complications, including death. To determine EV prevalence and EV types in Slovenia, data on over 25 000 EV RNA tests for diagnostics and surveillance from 2014 to 2023 were analyzed. Altogether, 3733 cerebrospinal fluid (CSF) and 21 297 respiratory (sentinel and clinical) samples were tested for EV RNA. EV typing was performed on all residual EV-positive CSF samples and on subset of respiratory specimens. Altogether, 1238 samples tested positive for EV RNA: 238 (6.4%) CSF and 1000 (4.7%) respiratory samples. EV-positive patients were predominantly male (p < 0.001). Many EV-positive CSF samples were from infants under 3 months (33.1%), whereas most EV-positive respiratory samples were from children 1 to 2 years old (49.2%). Echovirus 30 (E-30) was most frequent in CSF (33.0%), followed by CV-B5 (13.8%) and E-6 (13.8%). CV-A6 was most frequent in respiratory samples (16.0%), followed by EV-D68 (7.6%) and CV-A5 (7.4%). EV types in CSF and respiratory samples show diverse dynamics, with some outbreaks indicated. A significant difference was found in the EV detection rate between CSF and respiratory samples by age. Various EV types were characterized, showing that some EV types are more neurotropic or cause more severe infections.

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.

[Epidemiological and etiological characteristics of herpes pharyngitis in 3 prefectures in Jiangsu Province].

Objective: To analyze the epidemiological and etiological characteristics of herpes pharyngitis (HA) in three prefectures of Jiangsu Province, and provide evidence for the prevention and control of HA in Jiangsu. Methods: Three surveillance sentinel hospitals in Wuxi, Suzhou and Yancheng were selected from May 2018 to December 2022, and information related to HA visits and hospitalized cases was regularly collected from the hospital inpatient management system by age groups. Enterovirus nucleic acid detection was performed by RT-PCR, and sequencing analysis, identification of genotype subtypes, and phylogenetic analysis were performed on the sequences of the gene encoding the coat protein VP1 of the main prevalent strains. Results: A total of 57 709 HA cases were recorded in the sentinel hospitals in in Wuxi, Suzhou and Yancheng, which was 1.76 times higher than the reported cases of hand, foot and mouth disease during the same period (57 709/32 831).The percentage of HA hospitalizations was 1.35% (781/57 709), and the percentage of hospitalizations showed an increasing trend from year to year (χ2=62.79, P<0.001 ).The incidence peak of HA was during May-July. The cases were mainly children aged 12-59 months (67.07%, 38 708/57 709), with the highest case number in age group 36-59 months (34.40%, 19 852/57 709). The HA positivity rate was 33.82% (644/1 904); enterovirus A was predominant (54.04%, 348/644); of these, Coxsackievirus (CV)A6 accounted for the highest percentage (52.59%, 183/348), while CVA16 and CVA4 accounted for 24.71% (86/348) and 15.23% (53/348), respectively. All 10 CVA4 HA endemic strains belonged to the C2 gene subtype, and all 6 CVA6 HA endemic strains belonged to the D3a gene subtype; and were genetically closer to and related to the strains in some areas of China (Fujian Province, Guangzhou City, Jiangxi Province, Yunnan Province, Tianjin City, etc.). Conclusions: The disease burden of HA was heavy in 3 areas in Jiangsu, children in age group 12-59-month were mainly affected, and the annual incidence peak of HA was during May-July. The pathogens causing HA varied, but predominated by enterovirus A and had low intra-typical differentiation, and no new evolutionary branches were found, suggesting that it is necessary to include HA in foot and mouth disease surveillance or regarded as a notifiable communicable disease.

Molecular Genotyping of Circulating Enterovirus in the Lazio Region from 2012 to 2023.

Enteroviruses (EVs) are ubiquitous viruses that circulate worldwide, causing sporadic or epidemic infections, typically during the summer and fall. They cause a broad spectrum of illnesses, ranging from an unspecified febrile clinical presentation to a severe illness. EVs are recognized to be the most frequent etiological agents of aseptic meningitis in children. However, as the infection is usually mild and self-limiting, it remains underestimated, and the epidemiology of EVs is poorly understood. To date, no vaccine or effective therapy for all types of enteroviruses is available, and EVs constitute a public health concern. Here, we investigated the molecular epidemiology of EV strains circulating in the Lazio region over a 10-year time span (2012-2023) by using a sequence-typing approach and phylogenetic analysis. The epidemiological trend of EV infection has undergone changes during the SARS-CoV-2 pandemic (2020-2021), which resulted in a modification in terms of the number of diagnosed cases and seasonality. From 2022, the circulation of EVs showed a behavior typical of the pre-pandemic period, although changes in predominantly circulating strains have been noted. Both epidemic and sporadic circulation events have been characterized in the Lazio region. Further analyses are needed to better characterize any strain with higher potential pathogenic power and to identify possible recombinant strains.

Exploration of Potential Broad-Spectrum Antiviral Targets in the Enterovirus Replication Element: Identification of Six Distinct 5' Cloverleaves.

Enterovirus genomic replication initiates at a predicted RNA cloverleaf (5'CL) at the 5' end of the RNA genome. The 5'CL contains one stem (SA) and three stem-loops (SLB, SLC, SLD). Here, we present an analysis of 5'CL conservation and divergence for 209 human health-related serotypes from the enterovirus genus, including enterovirus and rhinovirus species. Phylogenetic analysis indicates six distinct 5'CL serotypes that only partially correlate with the species definition. Additional findings include that 5'CL sequence conservation is higher between the EV species than between the RV species, the 5'CL of EVA and EVB are nearly identical, and RVC has the lowest 5'CL conservation. Regions of high conservation throughout all species include SA and the loop and nearby bases of SLB, which is consistent with known protein interactions at these sites. In addition to the known protein binding site for the Poly-C binding protein in the loop of SLB, other conserved consecutive cytosines in the stems of SLB and SLC provide additional potential interaction sites that have not yet been explored. Other sites of conservation, including the predicted bulge of SLD and other conserved stem, loop, and junction regions, are more difficult to explain and suggest additional interactions or structural requirements that are not yet fully understood. This more intricate understanding of sequence and structure conservation and variability in the 5'CL may assist in the development of broad-spectrum antivirals against a wide range of enteroviruses, while better defining the range of virus isotypes expected to be affected by a particular antiviral.

Epidemiology of Enterovirus Genotypes in Association with Human Diseases.

Enteroviruses (EVs) are well-known causes of a wide range of infectious diseases in infants and young children, ranging from mild illnesses to severe conditions, depending on the virus genotypes and the host's immunity. Recent advances in molecular surveillance and genotyping tools have identified over 116 different human EV genotypes from various types of clinical samples. However, the current knowledge about most of these genotypes, except for those of well-known genotypes like EV-A71 and EV-D68, is still limited due to a lack of comprehensive EV surveillance systems. This limited information makes it difficult to understand the true burden of EV-related diseases globally. Furthermore, the specific EV genotype associated with diseases varies according to country, population group, and study period. The same genotype can exhibit different epidemiological features in different areas. By integrating the data from established EV surveillance systems in the USA, Europe, Japan, and China, in combination with other EV infection studies, we can elaborate a better understanding of the distribution of prevalent EV genotypes and the diseases associated with EV. This review analyzed the data from various EV surveillance databases and explored the EV seroprevalence and the association of specific EV genotypes with human diseases.

A unique case of double meningitis with enterovirus and reactivated varicella-zoster virus in a male teenager.

This paper presents a unique case of double meningitis with enterovirus and reactivated varicella-zoster virus without shingles in an immunocompetent male teenager, a case that offers many important medical lessons, all "gravitating" around physiopathological reasoning of any clinical case in general.

First detection of multiple cases related to CV-A16 strain of B1c clade in Beijing in 2022.

Coxsackievirus A16 (CV-A16) is a significant etiologic agent of hand, foot, and mouth disease (HFMD) and herpangina (HA), with the capacity to progress to severe complications, including encephalitis, aseptic meningitis, acute flaccid paralysis, myocarditis, and other critical conditions. Beijing's epidemiological surveillance system, established in 2008, encompasses 29 hospitals and 16 district disease control centers. From 2019 to 2021, the circulation of CV-A16 was characterized by the co-circulation of B1a and B1b clades. Multiple cases of HFMD linked to clade B1c has not been reported in Beijing until 2022. This study enrolled 400 HFMD and 493 HA cases. Employing real-time RT-PCR, 368 enterovirus-positive cases were identified, with 180 selected for sequencing. CV-A16 was detected in 18.89% (34/180) of the cases, second only to CV-A6, identified in 63.33% (114/180). Full-length VP1 gene sequences were successfully amplified and sequenced in 22 cases, revealing the presence of clades B1a, B1b, and B1c in 14, 3, and 5 cases, respectively. A cluster of five B1c clade cases occurred between June 29 and July 17, 2022, within a 7-km diameter region in Shunyi District. Phylogenetic analysis of five complete VP1 gene sequences and two full-genome sequences revealed close clustering with the 2018 Indian strain (GenBank accession: MH780757.1) within the B1c India branch, with NCBI BLAST results showing over 98% similarity. Comparative sequence analysis identified three unique amino acid variations (P3S, V25A, and I235V). The 2022 Shunyi District HFMD cases represent the first instances of spatiotemporally correlated CV-A16 B1c clade infections in Beijing, underscoring the necessity for heightened surveillance of B1c clade CV-A16 in HFMD and HA in this region.

Viral metagenomics combined with non-targeted serum metabolomics reveals the role of enteroviruses in a mouse model of coronary heart disease.

BACKGROUND: Coronary heart disease (CHD) is a common cardiovascular disease that is associated with altered gut microbiota. Enteroviruses, an essential component of the gut microbiome, may play an important role in disease progression. However, the relationship between enteroviruses and CHD remains unclear. The development of high-throughput sequencing technologies has facilitated research on the interconnections between viruses and disease-related metabolites. METHODS AND RESULTS: Mice were fed a high-fat diet (CHD group) or chow diet (Sham group) for 12 weeks, and ligation of the left anterior descending coronary artery was performed at the end of week 8. After 4 weeks, all animals were euthanised. Subsequently, the animals were evaluated for basic haemato-biochemical parameters and cardiac function, and aorta staining was performed. Based on enteroviral metagenomics and serum UPLC-MS/MS metabolomics analyses, we evaluated the association between enteroviral groups and serum metabolites of CHD mouse model. A high-fat diet and coronary ligation enabled the establishment of the CHD mouse model. Notably, the enterovirus spectrum of the sham group was significantly different from that of the CHD group, with 24 viral communities of different family and species classification, such as Tsarbombavirus, Mingyongvirus, Claudivirus, and Firehammervirus, exhibiting significant differences. In addition, 731 Differential metabolites were detected in the serum of both groups of mice. Correlation network analysis revealed a close relationship between various metabolites related to lipid metabolism and different viruses, including Tsarbombavirus, Mingyongvirus, Claudivirus, and Firehammervirus. CONCLUSIONS: An animal model of CHD, characterised by lipid disturbance and myocardial ischaemia, was established using a high-fat diet and ligation of the left anterior descending branch of the coronary artery. Tsarbombavirus, Firehammervirus, Mingyongvirus, and Claudivirus were associated with metabolites in the lipid metabolism pathway. The results indicate that Tsarbombavirus may be the main genus interacting with CHD-related metabolites in mice. Conclusively, the findings of our study provide novel insights into the potential relationship enterovirus groups and metabolites associated with CHD.

Clinical features of hand, foot and mouth disease caused by Coxsackievirus A6 in Xi'an, China, 2013-2019: A multicenter observational study.

PURPOSE: To investigate the clinical features of hand, foot, and mouth disease (HFMD) caused by coxsackievirus A6 (CVA6) and this work may help early diagnose of atypical HFMD. METHODS: From January 2013 to December 2019, a total of 7,208 patients with a clinical diagnosis of HFMD in Xi'an Children's Hospital, Xi'an Central Hospital, and Xi'an Jiaotong University Second Affiliated Hospital, were included in this observational study. The clinical data, specimens and follow-up results were collected. Real-time RT‒PCR was performed for the detection and typing of enterovirus nucleic acids. RESULTS: Of the 7,208 clinically diagnosed HFMD patients, 5,622 were positive for enterovirus nucleic acids, and the positive proportions of CVA6, enterovirus 71 (EV-A71), coxsackievirus A16 (CVA16), and other enteroviruses were 31.0% (1,742/5,622), 27.0% (1,518/5,622), 35.0% (1,968/5,622), and 7.0% (394/5,622), respectively. Based on the etiology, patients were divided into CVA6 group, EV-A71group, and CVA16 group. The mean age at onset was significantly higher in the CVA6 group (4.62±2.13 years) than in the EV-A71 group and CVA16 group (3.45±2.25 years and 3.35±2.13 years, respectively; both P < 0.05). The male/female ratio was 1.45 (1,031/711) in the CVA6 group and was not significantly different from the other two groups. The incidence of fever was significantly higher in the CVA6 group [82.5% (1,437/1,742)] than in the EV-A71 group [51.3% (779/1,518)] and the CVA16 group [45.9% (903/1,968)] (P < 0.05). In the CVA6 group, the rashes were more frequently on the trunk and elbows/knees and were significantly different from the other two groups (P < 0.05). The number of patients with two or more rash morphologies was significantly higher in the CVA6 group than in the other two groups (P < 0.05). The incidence of bullous rash in the CVA6 group [20.2%; n = 352] was higher than in the EV-A71 group [0.33%; n = 5] and CVA16 group [0.66%; n = 13] (P < 0.05). The incidence of neurological complications was significantly higher in the EV-A71 group [52.1% (791/1,518)] than in the CVA16 group [5.1% (100/1,968)] and the CVA6 group [0.8% (14/1,742)] (P < 0.05). In the follow-up period, 160 patients (9.2%) with CVA6 HFMD experienced onychomadesis, but no onychomadesis was observed in the EV-A71 and CVA16 groups. The average WBC count was significantly higher in the CVA6 group than in the CVA16 group (P < 0.05). The number of patients with increased CRP was significantly larger in the CVA6 group than in the CVA16 group but was significantly smaller than that in the EV-A71 group (P < 0.05). CONCLUSIONS: CVA6 has become one of the main pathogens of HFMD in the Xi'an area during 2013-2019. The main clinical manifestations were slightly different from those of HFMD caused by EV-A71 or CVA16, with a higher frequency of fever, diverse morphologies and diffuse distribution of rashes, fewer neurological complications and some onychomadesis.

Molecular characteristics of the structure protein VP1 in Coxsackievirus A10 Isolates from China.

INTRODUCTION: Coxsackievirus A10 (CVA10) is a non-enveloped, positive-sense single-stranded RNA virus classified within the Enterovirus genus in the Picornaviridae family. It is among the pathogens that can cause hand, foot and mouth disease. This study aimed to analyze the temporal and spatial distribution of CVA10 in China to understand its epidemiological characteristics of CVA10. METHODOLOGY: We collected the VP1 sequences of CVA10 from January 1, 2004, to December 31, 2019, from the GenBank database and created the global map using MapChart. We selected 56 known CVA10 genotype sequences. Then, MEGA6.06 was used to construct a phylogenetic tree with the collected gene sequences and the known reference sequences for comparative analysis to assess the distribution of CVA10 genotypes in different countries between 2004 and 2019. RESULTS: CVA10 has been widely detected or reported globally. In China, the prevalent genotype of CVA10 was mainly genotype B before 2008 and genotype C after 2009. In other countries, the prevalence of genotype D was dominant, followed by genotypes C and F, and the prevalence of CVA10 varied from continent to continent. CONCLUSIONS: Monitoring CVA10 genotypes or evolutionary branches should be strengthened, and the study of epidemic genotype characteristics should be enhanced. This will serve as a basis for further research and development of monovalent CVA10 or polyvalent vaccines designed for effective disease prevention.

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.

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.

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.

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.