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 Characterization of Enteric Viruses Causing Acute Gastroenteritis among Children under 5 Years Old in Distrito Central, Honduras.

INTRODUCTION: Diarrheal diseases constitute a significant public health problem in terms of mortality and morbidity. In Honduras and around the world, RVs have consistently emerged as the single most important etiologic agent in acute childhood diarrhea. However, other viruses, such as NoVs and HAstVs, have also been shown to be responsible for viral gastroenteritis. Unfortunately, the country has limited information concerning the etiologic role of these viral agents in acute gastroenteritis. This study investigated the frequency, genotypes, and epidemiological characteristics of RV-A, NoVs, and HAstVs among children under 5 years old in Distrito Central, Honduras. METHODS: Stool samples and their corresponding epidemiological data were collected from children with acute gastroenteritis in three healthcare centers in Distrito Central. All samples were screened by immunoassays for RV-A and HAstVs. RV-A-positive samples were molecularly characterized by RT-PCR and genotyping assays. RT-PCR was also applied to confirm HAstVs positivity and to detect NoVs, followed by nucleotide sequencing to assign their genotypes. RESULTS: Our results show that at least one viral agent was detected in 31% of the children. The frequency of RV-A, NoVs, and HAstVs was 14%, 13%, and 5%, respectively. The most frequent RV-A genotype was G2P[4], occurring in 93% of cases. 92.3% of NoVs-positive samples belonged to genogroup II, with GII.4 and GII.16 being the most common. HAstVs were clustered into three genotypes: HAstV-1, HAstV-2, and HAstV-8. Only one sample showed coinfection with NoVs and HAstVs. CONCLUSION: This comprehensive molecular and epidemiological characterization of enteric viruses demonstrates the vast diversity of these agents and describes for the first time NoVs and HAstVs as causative agents of acute childhood gastroenteritis in Distrito Central, Honduras. This suggests that further in-depth studies of the pediatric population are necessary to develop and implement effective preventive and control measures in the country.

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.

Human Enterovirus Diversity by Next-Generation Sequencing Analysis in Urban Sewage Samples From Buenos Aires Metropolitan Area, Argentina: A Retrospective Study.

Human Enteroviruses (hEVs) are responsible for a wide variety of human diseases. During hEVs infection, virions are excreted in human feces and the fecal-oral route is the primary pathway for person-to-person transmission. Sewage surveillance could help in monitoring hEVs circulation and describing their diversity in a specific population. In this study, sewage samples collected in Buenos Aires Metropolitan Area (Argentina) were retrospectively studied through an amplicon-deep sequencing approach and phylogenetic analyses to characterize hEVs spread. We identified 17 different hEVs types belonging to A, B, and C species. To the best of our knowledge, this is the first report in Buenos Aires for 7 identified hEV-C types. Phylogenetic analyses suggest several introductions of coxsackievirus B4, echovirus 1, and echovirus 9 in the country, along with the national spread reached by some variants. Besides, well-supported monophyletic groups of Argentine, Uruguayan, and Brazilian strains unveiled regional circulation patterns for some variants. These results extend our knowledge about hEVs circulation in Buenos Aires and might exhort authorities to implement more active sewage surveillance in the region.

Lactoferrin affects rhinovirus B-14 entry into H1-HeLa cells.

Lactoferrin is part of the innate immune system, with antiviral activity against numerous DNA and RNA viruses. Rhinoviruses, the leading cause of the common cold, are associated with exacerbation of respiratory illnesses such as asthma. Here, we explored the effect of bovine lactoferrin (BLf) on RV-B14 infectivity. Using different assays, we show that the effect of BLf is strongest during adhesion of the virus to the cell and entry. Tracking the internalisation of BLf and virus revealed a degree of colocalisation, although their interaction was only confirmed in vitro using empty viral particles, indicating a possible additional influence of BLf on other infection steps.

Environmental Surveillance through Next-Generation Sequencing to Unveil the Diversity of Human Enteroviruses beyond the Reported Clinical Cases.

The knowledge about circulation of Human Enteroviruses (EVs) obtained through medical diagnosis in Argentina is scarce. Wastewater samples monthly collected in Córdoba, Argentina during 2011-2012, and then in 2017-2018 were retrospectively studied to assess the diversity of EVs in the community. Partial VP1 gene was amplified by PCR from wastewater concentrates, and amplicons were subject of next-generation sequencing and genetic analyses. There were 41 EVs detected, from which ~50% had not been previously reported in Argentina. Most of the characterized EVs (60%) were detected at both sampling periods, with similar values of intratype nucleotide diversity. Exceptions were enterovirus A71, coxsackievirus B4, echovirus 14, and echovirus 30, which diversified in 2017-2018. There was a predominance of types from EV-C in 2017-2018, evidencing a common circulation of these types throughout the year in the community. Interestingly, high genetic similarity was evidenced among environmental strains of echovirus 30 circulating in 2011-2012 and co-temporal isolates obtained from patients suffering aseptic meningitis in different locations of Argentina. This study provides an updated insight about EVs circulating in an important region of South America, and suggests a valuable role of wastewater-based epidemiology in predicting outbreaks before the onset of cases in the community.

Epidemiology, Etiology and Clinical Aspects of Childhood Acute Encephalitis in a Tertiary Pediatric Hospital in Costa Rica.

BACKGROUND: Limited data are available on childhood encephalitis in Latin America. Our study aimed to increase insight on clinical presentation, etiology and outcome of children with acute encephalitis in Costa Rica. METHODS: We conducted a prospective, observational study during an 8-month period at the Hospital Nacional de Niños "Dr. Carlos Sáenz Herrera" in Costa Rica. Case definition was according to "International Encephalitis Consortium" in children <13 years. We analyzed demographic characteristics, clinical symptoms, neurologic imaging, etiology, treatment and mortality. RESULTS: Forty patients were identified. Mean age was 5 years and 57.5% were male. Most frequently neurologic symptoms were altered mental status (100.0%), headache (57.5%) and seizures (52.5%). Etiology was determined in 52.5% of cases. Probable or confirmed viral etiology was identified in 6 cases (15.0%) and bacterial etiology in also 6 cases (15.0%). A possible etiology was identified in 7 cases (17.5%). Autoimmune encephalitis was diagnosed in 2 patients (5.0%). Enterovirus and Streptococcus pneumoniae were the most common confirmed agents. No cases of herpes simplex virus were found. Etiology of 19 cases (47.5%) remained unknown. Sequelae were reported in 45.0% of patients. Mortality rate was 15.0% (6 cases), 3 caused by virus (adenovirus, human herpesvirus 6, enterovirus), 2 by bacteria (S. pneumoniae, Haemophilus influenzae type b) and 1 of unknown etiology. Diffuse cerebral edema was the most important mortality predictor (P < 0.001). CONCLUSIONS: Acute encephalitis in our study was associated with significant morbidity and mortality. Early and aggressive antiviral, antibiotic and anticerebral edema treatment is necessary when acute encephalitis is suspected.

Detection and identification of enteroviruses circulating in children with acute gastroenteritis in Pará State, Northern Brazil (2010-2011).

Although acute gastroenteritis (AGE) has been reported as a common infectious disease in children, there is scarce information about enterovirus (EV) circulating associated with AGE cases in Brazil. The purpose of the present study was to identify and characterize the enteroviruses associated with AGE in children in Belém, Brazil. A total of 175 stool samples were obtained from children hospitalized revealing the presence of EV in 26.3% (46/175) of infections. EV type was identified in 78.3% (36/46) and EV-B species (61.1%; 22/36) was the most prevalent EV-detected followed by EV-C (25%; 9/36) and EV-A (13.9%; 5/36). This study has provided important information about the enterovirus circulation in Pará state, Northern Brazil.

Enterovirus detection and serotyping of fecal material collected from three children living on the outskirts of Belém city, Amazon region, Brazil, during the first 3 years of life (1983-1986).

In the current investigation, fecal material was obtained during a community-based longitudinal study conducted from 1983 to 1986. This study consisted of 71 children aged newborn to 3 years. A total of 216 samples from three of these children were screened by real-time quantitative polymerase chain reaction (RT-qPCR) for the presence of enteroviruses, and positive samples were serotyped by VP1 and VP3 sequencing of the viral genome. Of these, 12 (5.6%) came from symptomatic cases, and the remaining asymptomatic cases were collected fortnightly during the 3 years of study. A positivity of 63.4% (137/216) was obtained by RT-qPCR, with 58.3% (7/12) in relation to the symptomatic group and 63.7% (130/204) in relation to the asymptomatic group. The 137 positive samples were inoculated into the RD, HEp2C, and L20B cell lines, and the cytopathic effect was observed in 37.2% (51/137) samples. It was also possible to identify 40.9% (56/137), between isolated (n = 46) and nonisolated (n = 10). Enterovirus serotype diversity (n = 25) was identified in this study, with the predominant species being B (80.3%), followed by C (16.1%) and A (3.6%). Cases of reinfection by different serotypes were also observed in the three children studied. Analyses involving different age groups of these minors confirmed that the most affected age was between 12 to 24 months, with a prevalence of 77.6% (52/67). The enterovirus (EV) circulated in the 3 years of research, showed peaks in some months, without defined seasonality. This study demonstrated a high circulation and serotype diversity of EV in fecal samples, collected over 30 years ago. This endorsed the evaluation of important points of the epidemiology of these viruses, such as the presence of coinfection and reinfection of the same individual by different circulating serotypes. Understanding the frequency and duration of EV infections is important in determining their association with persistent diarrhea.

Recombination in Enteroviruses, a Multi-Step Modular Evolutionary Process.

RNA recombination is a major driving force in the evolution and genetic architecture shaping of enteroviruses. In particular, intertypic recombination is implicated in the emergence of most pathogenic circulating vaccine-derived polioviruses, which have caused numerous outbreaks of paralytic poliomyelitis worldwide. Recent experimental studies that relied on recombination cellular systems mimicking natural genetic exchanges between enteroviruses provided new insights into the molecular mechanisms of enterovirus recombination and enabled to define a new model of genetic plasticity for enteroviruses. Homologous intertypic recombinant enteroviruses that were observed in nature would be the final products of a multi-step process, during which precursor nonhomologous recombinant genomes are generated through an initial inter-genomic RNA recombination event and can then evolve into a diversity of fitter homologous recombinant genomes over subsequent intra-genomic rearrangements. Moreover, these experimental studies demonstrated that the enterovirus genome could be defined as a combination of genomic modules that can be preferentially exchanged through recombination, and enabled defining the boundaries of these recombination modules. These results provided the first experimental evidence supporting the theoretical model of enterovirus modular evolution previously elaborated from phylogenetic studies of circulating enterovirus strains. This review summarizes our current knowledge regarding the mechanisms of recombination in enteroviruses and presents a new evolutionary process that may apply to other RNA viruses.

The rare enterovirus c99 and echovirus 29 strains in Brazil: potential risks associated to silent circulation.

Human enteroviruses (EVs) are associated with a wide spectrum of human diseases. Here we report the complete genome sequences of one EV-C99 strain and one E29 strain obtained from children suffering from acute gastroenteritis, without symptoms of enteroviral syndromes. This is the first report of EV-C99 in South America, and the second E29 genome described worldwide. Continuous surveillance on EVs is vital to provide further understanding of the circulation of new or rare EV serotypes in the country. The present study also highlights the capacity of EVs to remain in silent circulation in populations.

Identification and Phylogenetic Characterization of Human Enteroviruses Isolated from Cases of Aseptic Meningitis in Brazil, 2013-2017.

Aseptic meningitis is a common viral infection associated with human enteroviruses. The aim of the present study was to identify and characterize the enteroviruses associated with outbreaks and sporadic cases of aseptic meningitis that occurred in different regions of Brazil between 2013 and 2017. Cerebrospinal fluids obtained from patients admitted to public health facilities were analyzed. A total of 303 patients were positive for Human Enteroviruses (EV) by cell culture isolation with a median isolation rate throughout the year of 12%. We were able to identify enterovirus serotypes in 295 clinical specimens. Nineteen different serotypes were identified; the large majority corresponded to HEV-B species. Echovirus 30 (E-30) and Echovirus 6 (E-6) were the most prevalent genotypes (66.8%). Sequence analysis suggested that circulating E-30 was closely related to E-30 from other American countries; while E-6 was derived from Europe. Most of the patients consisted of children ≤ 15 years old. The temporal distribution of all aseptic meningitis and EV-positive cases showed an obvious seasonal pattern during autumn. Our results have provided valuable information about the enteroviral etiology of the aseptic meningitis cases in Brazil pointing to the importance of enterovirus surveillance in neurological diseases.

Surveillance of Enteric Viruses and Thermotolerant Coliforms in Surface Water and Bivalves from a Mangrove Estuary in Southeastern Brazil.

This study was conducted to evaluate the microbiological quality of a mangrove estuary in the Vitória Bay region, Espírito Santo, Brazil. We analyzed the presence and concentration of enteric viruses and thermotolerant coliforms in water, mussels (Mytella charruana and Mytella guyanensis), and oysters (Crassostrea rhizophorae), collected over a 13-month period. Human adenovirus, rotavirus A (RVA), and norovirus genogroup II were analyzed by quantitative PCR. The highest viral load was found in RVA-positive samples with a concentration of 3.0 × 104 genome copies (GC) L-1 in water samples and 1.3 × 105 GC g-1 in bivalves. RVA was the most prevalent virus in all matrices. Thermotolerant coliforms were quantified as colony-forming units (CFU) by the membrane filtration method. The concentration of these bacteria in water was in accordance with the Brazilian standard for recreational waters (< 250 CFU 100 mL-1) during most of the monitoring period (12 out of 13 months). However, thermotolerant coliform concentrations of 3.0, 3.1, and 2.6 log CFU 100 g-1 were detected in M. charruana, M. guyanensis, and C. rhizophorae, respectively. The presence of human-specific viruses in water and bivalves reflects the strong anthropogenic impact on the mangrove and serves as an early warning of waterborne and foodborne disease outbreaks resulting from the consumption of shellfish and the practice of water recreational activities in the region.

Detection and molecular characterisation of bovine Enterovirus in Brazil: four decades since the first report.

It is suggested that bovine enteroviruses (BEV) are involved in the aetiology of enteric infections, respiratory disease, reproductive disorders and infertility. In this study, bovine faecal samples collected in different Brazilian states were subjected to RNA extraction, reverse transcription-polymerase chain reaction analysis and partial sequencing of the 5'-terminal portion of BEV. One hundred and three samples were tested with an overall positivity of 14.5%. Phylogenetic analysis clustered these BEV Brazilian samples into the Enterovirus F clade. Our results bring an important update of the virus presence in Brazil and contribute to a better understanding of the distribution and characterisation of BEV in cattle.

Environmental Surveillance of Human Enteroviruses in Córdoba City, Argentina: Prevalence and Detection of Serotypes from 2009 to 2014.

Environmental surveillance is an effective approach to investigate the circulation of human enteroviruses (EVs) in the population. EVs excreted by patients who present diverse clinical syndromes can remain infectious in the environment for several weeks, and limited data on circulating environmental EVs are available. A 6-year (2009-2014) surveillance study was conducted to detect non-polio enteroviruses (NPEVs) in the urban sewage of Cordoba city, Argentina. Echovirus 6 (E-6) was the most prevalent (28%), followed by E-14 (17%), E-16 (14%), Coxsackievirus (CV) A9 (11%), E-20 (9%), and CVA24 (6%). Other minority serotypes (E-7, E-13, E-21, E-25, and CVB4) were found, which together represented 14% of the total. In the absence of a systematic EV disease surveillance system, the detection and characterization of sewage-borne NPEVs will help us better understand the changes in EV disease trends and the epidemic background of circulating EVs, which could help interpret the EV trends and warn of future outbreaks in this area.

Longitudinal survey of Teschovirus A, Sapelovirus A, and Enterovirus G fecal excretion in suckling and weaned pigs.

Fecal samples from 27 pigs were longitudinally analyzed for Teschovirus A (TV-A), Sapelovirus A (SV-A), and Enterovirus G (EV-G) RNA presence. Suckling piglet fecal samples were negative for the three enteric picornaviruses. However, these picornaviruses were detected in 22/27 weaned pig fecal samples. This study provides new data on TV-A, SV-A, and EV-G infection dynamics.

Acute Flaccid Myelitis Associated with Enterovirus D68 in Children, Argentina, 2016.

After a 2014 outbreak of severe respiratory illness caused by enterovirus D68 in the United States, sporadic cases of acute flaccid myelitis have been reported worldwide. We describe a cluster of acute flaccid myelitis cases in Argentina in 2016, adding data to the evidence of association between enterovirus D68 and this polio-like illness.

The rare enterovirus c99 and echovirus 29 strains in Brazil: potential risks associated to silent circulation

Human enteroviruses (EVs) are associated with a wide spectrum of human diseases. Here we report the complete genome sequences of one EV-C99 strain and one E29 strain obtained from children suffering from acute gastroenteritis, without symptoms of enteroviral syndromes. This is the first report of EV-C99 in South America, and the second E29 genome described worldwide. Continuous surveillance on EVs is vital to provide further understanding of the circulation of new or rare EV serotypes in the country. The present study also highlights the capacity of EVs to remain in silent circulation in populations.

Identification of enteric viruses circulating in a dog population with low vaccine coverage

ABSTRACT Although the use of vaccines has controlled enteric diseases in dogs in many developed countries, vaccine coverage is still under optimal situation in Brazil. There is a large population of nonimmunized dogs and few studies about the identification of the viruses associated with diarrhea. To address this situation, stool samples from 325 dogs were analyzed by polymerase chain reaction for the detection of common enteric viruses such as Canine adenovirus (CAdV), Canine coronavirus (CCoV), Canine distemper virus (CDV), Canine rotavirus (CRV) and Carnivorous protoparvovirus 1 (canine parvovirus 2; CPV-2). At least one of these species was detected in 56.6% (184/325) of the samples. The viruses detected most frequently in either diarrheic or nondiarrheic dog feces were CPV-2 (54.3% of the positive samples), CDV (45.1%) and CCoV (30.4%), followed by CRV (8.2%) and CAdV (4.9%). Only one agent was detected in the majority of the positive samples (63%), but co-infections were present in 37% of the positive samples and mainly included CDV and CPV-2. The data presented herein can improve the clinical knowledge in regions with low vaccine coverage and highlight the need to improve the methods used to control these infectious diseases in domestic dogs.

Hand, foot and mouth disease and herpangina caused by enterovirus A71 infections: a review of enterovirus A71 molecular epidemiology, pathogenesis, and current vaccine development.

Enterovirus A71 (EV-A71) infections are one of the main etiological agents of hand, foot and mouth disease (HFMD) and herpangina worldwide. EV-A71 infection is a life-threatening communicable disease and there is an urgent global need for the development of vaccines for its prevention and control. The morbidity rate of EV-A71 infection differs between countries. The pathogen's genetic lineages are undergoing rapid evolutionary changes. An association between the occurrence of EV-A71 infection and the circulation of different genetic strains of EV-A71 virus has been identified around the world. In this review, we present and discuss the molecular epidemiology and pathogenesis of the human disease caused by EV-A71 infection, as well as current prospects for the development of an EV-A71 vaccine.