Acute gastroenteritis outbreak associated with multiple and rare norovirus genotypes after storm events in Santa Catarina, Brazil.

Norovirus is a major cause of acute diarrheal disease (ADD) outbreaks worldwide. In the present study, we investigated an ADD outbreak caused by norovirus in several municipalities of Santa Catarina state during the summer season, southern Brazil in 2023. As of the 10th epidemiological week of 2023, approximately 87 000 ADD cases were reported, with the capital, Florianópolis, recording the highest number of cases throughout the weeks. By using RT-qPCR and sequencing, we detected 10 different genotypes, from both genogroups (G) I and II. Some rare genotypes were also identified. Additionally, rotavirus and human adenovirus were sporadically detected among the ADD cases. Several features of the outbreak suggest that sewage-contaminated water could played a role in the surge of ADD cases. Storm events in Santa Catarina state that preceded the outbreak likely increased the discharge of contaminated wastewater and stormwater into water bodies, such as rivers and beaches during a high touristic season in the state. Climate change-induced extreme weather events, including intensified rainfall and frequent floods, can disturb healthcare and sanitation systems. Implementing public policies for effective sanitation, particularly during peak times, is crucial to maintain environmental equilibrium and counter marine pollution.

Simultaneous enterovirus EV-D68 and CVA6 infections causing acute respiratory distress syndrome and hand, foot and mouth disease.

BACKGROUND: Although most enterovirus (EV) infections can be asymptomatic, these viral agents can cause serious conditions associated with central nervous system, respiratory disease and uncommon manifestations of hand, foot and mouth disease (HFMD). EV-coinfections have been rarely reported with development of complications and severe clinical outcome. An atypical case of a child presenting HFMD and severe acute respiratory syndrome, co-infected with EV-D68 and CVA6, is reported herein. CASE PRESENTATION: A 3-year-old boy was admitted in the emergency department unit showing fever, abdominal pain and tachycardia. Twenty-four hours after hospitalization the child developed severe clinical symptoms associated with HFMD and was discharged after recovery. Two days later, the child was readmitted with fever, cough and respiratory distress. RT-PCR and Sanger sequencing confirmed positivity for EV-D68 and CVA6 in oro and nasopharynges swabs and vesicles fluid, respectively. Phylogenetic analysis based on VP1 gene sequences suggested that CVA6 was closely related with HFMD viruses circulating in Turkey, while EV-D68 was genetically related to a Chinese strain. CONCLUSIONS: To the best of our knowledge, this case is the first report of a double infection caused by CVA6 and EV-D68, which shed light on the pathogenesis of enterovirus infections. Further studies must be conducted to ascertain the role and clinical significance of EV co-infections, as well as a potential synergistic pathway between these viruses.

Re-emergence of a coxsackievirus A24 variant causing acute hemorrhagic conjunctivitis in Brazil from 2017 to 2018.

A large outbreak (over 200,000 cases) of acute hemorrhagic conjunctivitis (AHC) took place in Brazil during the summer of 2017/2018, seven years after a nationwide epidemic, which occurred in 2011. To identify the etiological agent, 80 conjunctival swabs from patients with a clinical presentation suggestive of AHC were analyzed at the national enterovirus laboratory. Real-time RT-PCR for human enteroviruses was performed, and enterovirus RNA was detected in 91.25% (73/80) of the specimens. Twenty-nine swab fluids were used to inoculate cell cultures (RD and Hep2C), and 72.4% (21/29) yielded a cytopathic effect. Genotype IV coxsackievirus A24v (CV-A24v) was identified as the causative agent of the outbreak. Phylogenetic analysis based on the VP1 gene revealed that Brazilian isolates were genetically related to strains that caused an outbreak in French Guiana in 2017. Our results show the re-emergence of CV-A24v causing AHC outbreaks in Brazil between the end of 2017 and the beginning of 2018.

Rare natural type 3/type 2 intertypic capsid recombinant vaccine-related poliovirus isolated from a case of acute flaccid paralysis in Brazil, 2015.

A natural type 3/type 2 intertypic capsid recombinant vaccine-related poliovirus was isolated from an acute flaccid paralytic case in Brazil. Genome sequencing revealed the uncommon location of the crossover site in the VP1 coding region (nucleotides 3251-3258 of Sabin 3 genome). The Sabin 2 donor sequence replaced the last 118 nt of VP1, resulting in the substitution of the complete antigenic site IIIa by PV2-specific amino acids. The low overall number of nucleotide substitutions in P1 region indicated that the predicted replication time of the isolate was about 8-9 weeks. Two of the principal determinants of attenuation in Sabin 3 genomes were mutated (U472C and C2493U), but the temperature-sensitive phenotype of the isolate was preserved. Our results support the theory that there exists a PV3/PV2 recombination hotspot site in the tail region of the VP1 capsid protein and that the recombination may occur soon after oral poliovirus vaccine administration.

Assessment of Gastroenteric Viruses in Marketed Bivalve Mollusks in the Tourist Cities of Rio de Janeiro, Brazil, 2022.

This study investigated the prevalence and genetic diversity of gastroenteric viruses in mussels and oysters in Rio de Janeiro, Brazil. One hundred and thirty-four marketed bivalve samples were obtained between January and December 2022. The viral analysis was performed according to ISO/TS 15216, and the screening revealed the detection of norovirus GII/GI (40.3%), sapovirus (SaV; 12.7%), human mastadenovirus (7.5%), and rotavirus A (RVA; 5.9%). In total, 44.8% (60) of shellfish samples tested positive for one or more viruses, 46.7% (28/60) of the positive samples tested positive for a single viral agent, 26.7% (16) tested positive for two viral agents, 8.3% (5) for three viral agents, and 13.3% (8) for four viral agents. Additionally, three mussel samples were contaminated with the five investigated viruses (5%, 3/60). Norovirus GII showed the highest mean viral load (3.4 × 105 GC/g), followed by SaV (1.4 × 104 GC/g), RVA (1.1 × 104 GC/g), human mastadenovirus (3.9 × 103 GC/g), and norovirus GI (6.7 × 102 GC/g). Molecular characterization revealed that the recovered norovirus strains belonged to genotypes GII.2, GII.6, GII.9, GII.17, and GII.27; SaV belonged to genotypes GI.1 and GIV.1; RVA to genotypes G6, G8, P[8]-III, and human mastadenovirus to types F40 and F41. The GII.27 norovirus characterized in this study is the only strain of this genotype reported in Brazil. This study highlights the dissemination and diversity of gastroenteric viruses present in commercialized bivalves in a touristic area, indicating the potential risk to human health and the contribution of bivalves in the propagation of emerging pathogens.

Norovirus Epidemiology and Genotype Circulation during the COVID-19 Pandemic in Brazil, 2019-2022.

Norovirus stands out as a leading cause of acute gastroenteritis (AGE) worldwide, affecting all age groups. In the present study, we investigated fecal samples from medically attended AGE patients received from nine Brazilian states, from 2019 to 2022, including the COVID-19 pandemic period. Norovirus GI and GII were detected and quantified using RT-qPCR, and norovirus-positive samples underwent genotyping through sequencing the ORF1/2 junction region. During the four-year period, norovirus prevalence was 37.2%, varying from 20.1% in 2020 to 55.4% in 2021. GII genotypes dominated, being detected in 92.9% of samples. GII-infected patients had significantly higher viral concentrations compared to GI-infected patients (median of 3.8 × 107 GC/g and 6.7 × 105 GC/g, respectively); and patients aged >12-24 months showed a higher median viral load (8 × 107 GC/g) compared to other age groups. Norovirus sequencing revealed 20 genotypes by phylogenetic analysis of RdRp and VP1 partial regions. GII.4 Sydney[P16] was the dominant genotype (57.3%), especially in 2019 and 2021, followed by GII.2[P16] (14.8%) and GII.6[P7] (6.3%). The intergenogroup recombinant genotype, GIX.1[GII.P15], was detected in five samples. Our study is the first to explore norovirus epidemiology and genotype distribution in Brazil during COVID-19, and contributes to understanding the epidemiological dynamics of norovirus and highlighting the importance of continuing to follow norovirus surveillance programs in Brazil.

Enterovirus detection in stool samples from Mozambican children with acute gastroenteritis.

Enteroviruses (EV) are predominantly enteric viruses, present in all parts of the world causing disease in humans with a broad spectrum of clinical presentations. The purpose of this study was to identify non-polio enteroviruses (NPEV) in stool samples collected from children with acute gastroenteritis (AGE) symptoms of unknown etiology in four provinces (Maputo, Nampula, Sofala and Zambézia) of Mozambique. From June 2014 to March 2018, 327 stool samples were collected from children hospitalized with AGE in health care units. NPEVs were detected in 52 samples (52/327; 15.9%) and were more frequent in children under 5 years of age. The age group from 12 to 23 months was the most affected and showed more severity of disease. We also identified 26 different EV-types with the following detection pattern EV-B>EV-C>EV-A. The major EV-types were EV-A119 (9/52; 17.3%) and EV-C99 (8/52; 15.4%), accounting for 32.7% of the total. In addition to EV-A119, other uncommon EV-types were also identified, such as EV-B75, EV-B97 and EV-C113. The current study shows a high heterogeneity of EV types circulating in children with AGE in Mozambique as well as the identification of rarely described enteroviruses.

A RT-PCR method for selective amplification and phenotypic characterization of all three serotypes of Sabin-related polioviruses from viral mixtures.

Outbreaks caused by vaccine-derived polioviruses are challenging the final eradication of paralytic poliomyelitis. Therefore, the surveillance of the acute flaccid paralysis cases based on poliovirus isolation and characterization remains an essential activity. Due to the use of trivalent oral poliovirus vaccine (OPV), mixtures containing more than one serotype of Sabin-related polioviruses are frequently isolated from clinical samples. Because each poliovirus isolate needs to be individually analyzed, we designed polymerase chain reaction primers that can selectively distinguish and amplify a genomic segment of the three Sabin-related poliovirus serotypes present in mixtures, thus, optimizing the diagnosis and providing prompt information to support epidemiologic actions.

Analysis of Coxsackievirus B5 Infections in the Central Nervous System in Brazil: Insights into Molecular Epidemiology and Genetic Diversity.

Coxsackievirus B5 (CVB5) is one of the most prevalent enteroviruses types in humans and causes annual epidemics worldwide. In the present study, we explored viral genetic diversity, molecular and epidemiological aspects of CVB5 obtained from cerebrospinal fluid and stool samples of patients with aseptic meningitis or acute flaccid paralysis, information that is still scarce in Brazil. From 2005 to 2018, 57 isolates of CVB5 were identified in the scope of the Brazilian Poliomyelitis Surveillance Program. Phylogenetic analyses of VP1 sequences revealed the circulation of two CVB5 genogroups, with genogroup B circulating until 2017, further replaced by genogroup A. Network analysis based on deduced amino acid sequences showed important substitutions in residues known to play critical roles in viral host tropism, cell entry, and viral antigenicity. Amino acid substitutions were investigated by the Protein Variation Effect Analyzer (PROVEAN) tool, which revealed two deleterious substitutions: T130N and T130A. To the best of our knowledge, this is the first report to use in silico approaches to determine the putative impact of amino acid substitutions on the CVB5 capsid structure. This work provides valuable information on CVB5 diversity associated with central nervous system (CNS) infections, highlighting the importance of evaluating the biological impact of certain amino acids substitutions associated with epidemiological and structural analyses.

Environmental Monitoring for Enteroviruses in Maputo, Mozambique-2018.

Due to the possibility of wild poliovirus importation from endemic regions and the high circulation of vaccine-derived poliovirus type 2 in the African region, Mozambique implemented a surveillance program to monitor the circulation of enteroviruses in the environment. From January to November 2018, a period that immediately preceded the cVDPV outbreak in Africa, 63 wastewater samples were collected from different areas in Maputo city. A total of 25 samples (39.7%) were positive based on cell culture isolation. Non-polio enteroviruses were found in 24 samples (24/25; 96%), whereas 1 Sabin-related poliovirus was isolated. Neither wild nor vaccine-derived poliovirus was detected. High circulation of EVB species was detected. Environmental surveillance in the One Health approach, if effectively applied as support to acute flaccid paralysis, can be a powerful aid to the public health system to monitor poliovirus besides non-polio enteroviruses in polio-free areas.

Poliovirus 3C proteinase inhibition by organotelluranes.

The 3C proteinase, essential for human poliovirus (PV) replication, has unique characteristics as its three-dimensional structure resembles chymotrypsin, but its catalytic nucleophile is a cysteine SH group rather than the OH group of serine. Here, we describe the use of tellurium compounds as inhibitors of PV3C proteinase. A rapid, stoichiometric and covalent inactivation of PV3C was observed with both a chloro-telluroxetane and a bis-vinylic organotellurane. These compounds also inhibit human cathepsins B, L, S, and K with second order rate constants higher than those obtained for PV3C. Chloro-telluroxetane inhibits replication of PV in human embryonic rhabdomyosarcoma cells in the low micromolar range and below the toxic level for the host cells. Bis-vinylic organotellurane is more effective as antiviral agent but reduces the cell viability by 20% at 10 µm, a concentration almost completely inhibiting virus growth. This is the first description of inhibition of viral 3C proteinase with antiviral property by this class of compounds.

Serologic evidence of the recent circulation of Saint Louis encephalitis virus and high prevalence of equine encephalitis viruses in horses in the Nhecolândia sub-region in South Pantanal, Central-West Brazil.

As in humans, sub-clinical infection by arboviruses in domestic animals is common; however, its detection only occurs during epizootics and the silent circulation of some arboviruses may remain undetected. The objective of the present paper was to assess the current circulation of arboviruses in the Nhecolândia sub-region of South Pantanal, Brazil. Sera from a total of 135 horses, of which 75 were immunized with bivalent vaccine composed of inactive Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus(WEEV) and 60 were unvaccinated, were submitted to thorough viral isolation, reverse transcriptase polymerase chain reaction (RT-PCR) and neutralization tests for Saint Louis encephalitis virus (SLEV), EEEV, WEEV and Mayaro virus (MAYV). No virus was isolated and viral nucleic-acid detection by RT-PCR was also negative. Nevertheless, the prevalence of neutralizing antibodies in horses older than seven months was 43.7% for SLEV in equines regardless of vaccine status, and 36.4% for WEEV and 47.7% for EEEV in unvaccinated horses. There was no evidence of MAYV infections. The serologic evidence of circulation of arboviruses responsible for equine and human encephalitis, without recent official reports of clinical infections in the area, suggests that the Nhecolândia sub-region in South Pantanal is an important area for detection of silent activity of arboviruses in Brazil.

Detection and characterization of a coxsackievirus B2 strain associated with acute meningoencephalitis, Brazil, 2018.

Although different etiological agents can cause acute meningoencephalitis, this syndrome is usually associated with viruses. Among these, enteroviruses play a significant role. Here, we describe a fatal case of meningoencephalitis in a previously healthy teenager. Real-time RT-PCR and cell culture assays were performed with serum and cerebrospinal fluid (CSF) from a clinically diagnosed meningoencephalitis case that occurred in Rio de Janeiro State, Brazil. Coxsackievirus B2 (CVB2) was identified. Phylogenetic analysis revealed that the identified CVB2 was genetically related to strains known to cause neurological diseases. This case highlights the importance of continuous laboratory surveillance of central nervous system infections.

Molecular characterization and epidemiological aspects of non-polio enteroviruses isolated from acute flaccid paralysis in Brazil: a historical series (2005-2017).

Due to the advanced stage of polio eradication, the possible role of non-polio enteroviruses (NPEVs) associated to acute flaccid paralysis (AFP) cases has been highlighted. In this study, we described epidemiological aspects of NPEVs infections associated to AFP and explore the viral genetic diversity, information still scarce in Brazil. From 2005 to 2017, 6707 stool samples were collected in the scope of the Brazilian Poliomyelitis Surveillance Program. NPEVs were isolated in 359 samples (5.3%) and 341 (94.9%) were genotyped. About 46 different NPEV types were identified with the following detection pattern EV-B > EV-A > EV-C. The major EV-types were CVA2, CV4, EV-A71, CVB3, CVB5, E6, E7, E11, CVA13 and EV-C99, which corresponds to 51.6% of the total. Uncommon types, such as CVA12, EV-90 and CVA11, were also identified. Different E6 genogroups were observed, prevailing the GenIII, despite periods of co-circulation, and replacement of genogroups along time. CVA2 sequences were classified as genotype C and data suggested its dispersion in South-American countries. CVA13 viruses belonged to cluster B and Venezuelan viruses composed a new putative cluster. This study provides extensive information on enterovirus diversity associated with AFP, reinforcing the need of tailoring current surveillance strategies to timely monitor emergence/re-emergence of NPEVs.

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.

Hepatitis E virus is not detected in association with neurological disorders among Brazilian children.

Hepatitis E virus is increasingly being associated with idiopathic neurological disease. We tested 325 stool samples from Brazilian children presenting acute flaccid paralysis or Guillain-Barré syndrome using a broadly reactive and sensitive Reverse-transcription Polymerase chain reaction. Hepatitis E genome was not detected in any of the samples tested. Our results suggest that hepatitis E virus does not seem to be associated as the etiologic agent of acute flaccid paralysis and Guillain-Barré syndrome cases occurred in Brazilian children during the period of investigation (2010-2012).

Enterovirus B74 associated with hand, foot and mouth disease.

Enterovirus 74 (EV-B74) has been associated with cases of acute flaccid paralysis (AFP) but it is not a commonly found enterovirus. In this work, we present the characterization of an EV-B74 detected from the serum sample of a one-year-old boy presenting with signs and symptoms clinically compatible with hand, foot and mouth disease (HFMD). This is the first report of EV-B74 in Brazil.

An Environmental Surveillance in Uruguay Reveals the Presence of Highly Divergent Types of Human Enterovirus Species C and a High Frequency of Species A and B Types.

Information about Human Enterovirus circulation in Uruguay is scarce. The aim of this study was to generate the first description about their circulation in the country through the study of sewage samples collected before and after the switch from Oral Poliovirus Vaccine to Inactivated Poliovirus Vaccine. Viruses were concentrated by an adsorption-elution to a negatively charged membrane, and real-time quantitative PCR and qualitative PCR methods were used to detect, quantify, and characterize enteroviruses. Positive samples were inoculated in RD cells and two passages were performed. Additionally, RD+ samples were subsequently passed onto L20B cells. Human Enteroviruses were detected in 67.6% of the samples, with concentrations between 4.9 and 6.6 Log10 genomic copies per liter. 10% of positive samples replicated in RD cells, of which none in L20B cells. Molecular characterization of Human Enterovirus strains directly detected from sewage sample concentrates allowed the identification of highly divergent members of species C such as Enterovirus C99 and Coxsackievirus A13, as well as the frequent detection of species A and B members (particularly Coxsackievirus A16 and Echovirus 6, respectively). Other detected types were Coxsackievirus A2, A22, B1, B5, Echovirus 5, and 9. The characterization of viruses isolated in cell culture revealed the presence of Echovirus 6 and Coxsackievirus B3. Despite the absence of poliovirus, a wide circulation of different enterovirus types was evidenced in Uruguayan sewage samples, highlighting that the local populations are exposed to different kinds of diseases originated by several human enterovirus.

Acute flaccid paralysis laboratorial surveillance in a polio-free country: Brazil, 2005-2014.

The last case of paralytic poliomyelitis caused by wild poliovirus in Brazil occurred in 1989. The interruption of the indigenous poliovirus transmission was obtained through mass immunization campaigns to eligible children and an active epidemiological and laboratorial surveillance of all cases of acute flaccid paralysis (AFP) among children under 15 y of age. This paper describes and evaluates the performance of the AFP surveillance system in different geographic areas of Brazil between 2005 and 2014, using indicators recommended by WHO. AFP surveillance indicators as well as virological investigation of polio and non-polio enteroviruses in stool samples received in the laboratory were assessed from 2005-2014. During the period, 5463 cases of AFP were investigated. Of these, 55% were males and 45% were females. Those under 5 y of age represented 48% of all cases reported and investigated. AFP notification rate was within the acceptable values with mean value of 1.3 (North), 1.4 (Northeast), 1.1 (Southern), 1.0 (Southeast) and 1.4 (Midwest) cases of AFP per 100.000 population aged 15 y as well as the adequacy of fecal specimens received in the laboratory. Sabin- related polioviruses accounted for 1.7% of the isolates while, 6.7% were non-polio enterovirus with the values ranging from 5.0% to 8.9 %. No wild-type polio was found. The AFP epidemiological and laboratorial surveillance activities have been kept at appropriate levels in Brazil. These data are a very strong indication, which supports the status of country free of polio.

Detection and characterization of a coxsackievirus B2 strain associated with acute meningoencephalitis, Brazil, 2018

Abstract Although different etiological agents can cause acute meningoencephalitis, this syndrome is usually associated with viruses. Among these, enteroviruses play a significant role. Here, we describe a fatal case of meningoencephalitis in a previously healthy teenager. Real-time RT-PCR and cell culture assays were performed with serum and cerebrospinal fluid (CSF) from a clinically diagnosed meningoencephalitis case that occurred in Rio de Janeiro State, Brazil. Coxsackievirus B2 (CVB2) was identified. Phylogenetic analysis revealed that the identified CVB2 was genetically related to strains known to cause neurological diseases. This case highlights the importance of continuous laboratory surveillance of central nervous system infections.