RESUMO
AIMS: The present study aimed to use a conventional and metagenomic approach to investigate the microbiological diversity of water bodies in a network of drainage channels and rivers located in the central area of the city of Belém, northern Brazil, which is considered one of the largest cities in the Brazilian Amazon. METHODS AND RESULTS: In eight of the analyzed points, both bacterial and viral microbiological indicators of environmental contamination-physical-chemical and metals-were assessed. The bacterial resistance genes, drug resistance mechanisms, and viral viability in the environment were also assessed. A total of 473 families of bacteria and 83 families of viruses were identified. Based on the analysis of metals, the levels of three metals (Cd, Fe, and Mn) were found to be above the recommended acceptable level by local legislation. The levels of the following three physicochemical parameters were also higher than recommended: biochemical oxygen demand, dissolved oxygen, and turbidity. Sixty-three bacterial resistance genes that conferred resistance to 13 different classes of antimicrobials were identified. Further, five mechanisms of antimicrobial resistance were identified and viral viability in the environment was confirmed. CONCLUSIONS: Intense human actions combined with a lack of public policies and poor environmental education of the population cause environmental degradation, especially in water bodies. Thus, urgent interventions are warranted to restore the quality of this precious and scarce asset worldwide.
Assuntos
Bactérias , Metagenômica , Microbiologia da Água , Brasil , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/classificação , Bactérias/efeitos dos fármacos , Saúde Ambiental , Rios/microbiologia , Rios/virologia , Vírus/genética , Vírus/isolamento & purificação , Monitoramento Ambiental , Farmacorresistência Bacteriana/genética , Humanos , Cidades , Metais/farmacologiaRESUMO
In this investigation, fecal specimens from children with diarrhea were collected from four community studies conducted between 1982 and 2019 in Belém, Brazilian Amazon. A total of 234 samples were tested by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to detect infections by picornaviruses of the Enterovirus (EV), Parechovirus (HPeV), Cosavirus (HCoSV), Kobuvirus (Aichivirus - AiV) and Salivirus (SalV) genera. The positive samples were subjected to different amplification protocols of the VP1 region of the genome, such as nested PCR or snPCR, and were subsequently genotyped by sequencing VP1 and VP3 of the viral genome. Positivity was observed in 76.5% (179/234) of the samples tested using RT-qPCR for at least one virus, and co-infection was observed in 37.4% (67/179) of the cases. EV was detected in 50.8% (119/234), HPeV in 29.9% (70/234), HCoSV in 27.3% (64/234), and AiV/SalV in 2.1% (5/234) of the specimens tested by RT-qPCR. Using nested PCR and/or snPCR techniques, the positivity rates were 94.11% (112/119) for EV, 72.85% (51/70) for HPeV, and 20.31% (13/64) for HCoSV. It was not possible to amplify the samples that were positive for AiV/SalV. Sequencing revealed 67.2% (80/119) EV, 51.4% (36/70) HPeV, and 20.31% (13/64) HCoSV. Forty-five different types of EV were found among species A, B, and C; HCoSV identified five species, including a possible recombinant strain; all HPeV were identified as belonging to species A, in two samples a possible recombination involving three different strains was verified. This study demonstrated the high circulation and diversity of different types of picornaviruses in fecal samples, including those collected more than 30 years ago. This endorsed the evaluation of important points in the epidemiology of these viruses, such as the presence of co-infection and the possibility of knowing more about these agents, considering that some were recently described; therefore, their detection in older samples can provide more data about their ancestry.
Assuntos
Coinfecção , Infecções por Enterovirus , Enterovirus , Infecções por Picornaviridae , Picornaviridae , Vírus , Criança , Humanos , Idoso , Picornaviridae/genética , Coinfecção/epidemiologia , Brasil/epidemiologia , Infecções por Enterovirus/epidemiologia , Enterovirus/genética , Diarreia/epidemiologiaRESUMO
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.
Assuntos
Infecções por Enterovirus/virologia , Enterovirus/genética , Fezes/virologia , Gastroenterite/virologia , Brasil/epidemiologia , Pré-Escolar , Enterovirus/classificação , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/epidemiologia , Gastroenterite/epidemiologia , Humanos , Lactente , Recém-Nascido , Estudos Longitudinais , Filogenia , Sorogrupo , SorotipagemRESUMO
Enteric adenovirus (AdV), sapovirus (SaV), and human astrovirus (HAstV) are important pathogens involved in the gastroenteritis etiology. In this study, a total of 219 fecal samples and sera were collected from children hospitalized for acute gastroenteritis (AGE) in two large pediatric hospitals in Belém, from March 2012 to April 2015. The samples were analyzed by polymerase chain reaction (PCR) for AdV and HAstV (astrovirus) detection, and Nested-PCR and qPCR for SaV detection. AdV was detected in 50.2% (110/219) of the cases, with 42.7% (47/110) being sequenced and classified as: species F (63.9% - 30/47), A (4.2% - 2/47), B (6.4% - 3/47), C (17.1% - 8/47), D (4.2% - 2/47), and E (4.2% - 2/47). Of the 110 AdV-positive feces samples, 80 paired sera presented sufficient amounts and were also tested for this virus, of which 51 (63.7%) showed positive results and 26 (70.3%) pairs (feces plus sera) presented concordant results after sequencing being classified as: species F (21/26; 80.8%), A (1/26; 3.8%), B (1/26; 3.8%), and C (3/26; 11.5%). Overall, HAstV rate in the feces samples was 1.8% (4/219), including both HAstV-1a (2/4; 50%) and HAstV-2c (2/4; 50%). SaV was detected in 4.6% (10/219) of the fecal samples, out of which 50% (5/10) of the positive samples were characterized into the genogroups GI.1 (1), GI.2 (2), and GII.4 (2). These findings highlighted the important contributions of AdV, HAstV, and SaV in the enteric virus spectrum in our region and showed the high genetic diversity of AdV. In addition, it demonstrated for the first time in Brazil, the circulation of AdV in the serum of hospitalized children with AGE.
Assuntos
Infecções por Adenoviridae/epidemiologia , Gastroenterite/virologia , Variação Genética , Viremia/epidemiologia , Doença Aguda/epidemiologia , Adenoviridae/genética , Infecções por Adenoviridae/virologia , Infecções por Astroviridae/epidemiologia , Infecções por Astroviridae/virologia , Brasil/epidemiologia , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Criança , Pré-Escolar , Diarreia/epidemiologia , Diarreia/virologia , Feminino , Gastroenterite/epidemiologia , Genótipo , Hospitalização , Humanos , Lactente , Recém-Nascido , Masculino , Mamastrovirus/genética , Filogenia , Sapovirus/genéticaRESUMO
Norovirus (NoV) is a major cause of nonbacterial acute gastroenteritis (AGE) outbreaks worldwide, with infections reported in semiclosed environments, particularly in hospitals and nursing homes. Astrovirus (HAstV) is prevalent worldwide, especially in developing countries. We aimed to determine the prevalence, spatial distribution, and genetic diversity of NoV and HAstV in children under 5 years of age in Rio Branco city, Acre State, Amazon Region, Brazil. Stool samples from children with (n = 240) and without (n = 248) AGE were collected from January to December 2012 from seven neighborhoods. The overall NoV prevalence was 12.3% (60 of 488); representing 15.8% (38 of 240) of the symptomatic samples and 8.9% (22 of 248) of the controls. HAstVs infection was observed in 4.7% (23 of 488) of the samples tested, 6.2% (15 of 240) of AGE cases, and 2.4% (6 of 248) of the controls (plus two without information about feces consistency). Infections were found in all age groups with higher frequency in children less than two years of age, for both viruses. NoV was detected in all neighborhoods, with a higher concentration in the fourth (30%; 18 of 60). NoV nucleotide sequencing performed in 86.7% (52 of 60) of the positive samples showed the circulation of the strains GII.4 (57.7%; 30 of 52), GIIPe/GII.4 (19.2%; 10 of 52), GII.7, GII.Pg/GII.1, and GII.Pc (3.8%; 2 of 52 for each), GII.6 and GII.Pg (1.9%; 1 of 52 for each), and GI.3 (7.7%; 4 of 52). Three GII.4 variants were detected: Den Haag_2006b (n = 1), New Orleans_2009 (n = 1), and Sydney_2012 (n = 14). HAstV types HAstV-1a (81.8%; 9 of 11) and HAstV-2c (18.2%; 2 of 11) were observed in the 47.8% (11 of 23) of characterized samples. This is the first data obtained in Acre State regarding the prevalence of these viruses and provides epidemiological and molecular information for a better understanding of their role among children with and without AGE.
Assuntos
Infecções por Astroviridae/epidemiologia , Astroviridae/genética , Infecções por Caliciviridae/epidemiologia , Gastroenterite/virologia , Variação Genética , Norovirus/genética , Adolescente , Adulto , Brasil/epidemiologia , Criança , Pré-Escolar , Fezes/virologia , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Filogenia , Prevalência , RNA Viral/genética , Estudos Retrospectivos , Adulto JovemRESUMO
BACKGROUND: Currently, norovirus (NoV) is associated with one-fifth of all acute gastroenteritis (AGE) cases worldwide. The NoV GII.17_2014 variant has been associated with gastroenteritis outbreaks in several Asian countries, replacing the previously dominant Sydney 2012 variant. There is limited data about circulation of this new strain in Brazil. This study aimed to describe the phylogenetic and evolutionary characteristics of the GII.17_2014 strains in the Northern region of Brazil. METHODS: NoV was detected by enzyme immunoassay (EIA) in 645 stool samples of AGE cases that were reported in Pará and Amazonas states during 2015-2016. All positive samples were tested for NoV GI and GII by reverse transcription polymerase chain reaction (RT-PCR) and the amplicons were subjected to genome sequencing. The GII.17-positive samples were retested by PCR using different sets of designed primers, which target a highly conserved capsid gene region. Next, the amplicons were sequenced and phylogenetically analyzed using Bayesian inferences. RESULTS: Of the 645 samples tested, 208 (32.2%) tested were positive for NoV by EIA, among which 95 (45.7%) were genotyped. Among the genotyped samples, 12 (12.6%) were characterized as GII.17_2014 with the first case detected in November 2015 (1/30, 3.3%) and the others in 2016 (11/65, 16.9%). All strains found in our study were clustered in clade D (epidemic strain). The uncorrelated log-normal model estimations calculated the rate of evolution for GII-17 strains as 1.95 × 10- 3 (1.28 × 10- 3-2.63 × 10- 3). In total, 36 nucleotide changes were observed after analyzing the VP1 sequence, among which 28 occurred in the P2 region. CONCLUSIONS: These data demonstrate the evolutionary dynamics in NoV GII.17_2014 strains, which indicated high mutation rates with nucleotide substitutions and indels that are related to the elevated levels of antigenic diversity. This partly explains the increase in viral prevalence.
Assuntos
Infecções por Caliciviridae/virologia , Evolução Molecular , Gastroenterite/virologia , Tipagem Molecular , Norovirus/classificação , Norovirus/genética , Brasil/epidemiologia , Infecções por Caliciviridae/epidemiologia , Proteínas do Capsídeo/genética , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/virologia , Surtos de Doenças , Epidemias , Fezes/virologia , Gastroenterite/epidemiologia , Genótipo , Humanos , Tipagem Molecular/métodos , Filogenia , Prevalência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Virologia/métodosRESUMO
This study aimed to investigate the presence of norovirus (NoV) in recreational waters of four estuarine beaches located in Mosqueiro Island, Belém city, Brazilian Amazon, during two years of monitoring (2012 and 2013). NoV particles were concentrated on filtering membrane by the adsorption-elution method and detected by semi-nested RT-PCR (reverse transcription polymerase chain reaction) and sequencing. NoV positivity was observed in 37.5% (39/104) of the surface water samples, with genogroup GI (69.2%) occurring at a higher frequency than GII (25.7%), with a cocirculation of both genogroups in two samples (5.1%). This virus was detected in all sampling points analyzed, showing the highest detection rate at the Paraíso Beach (46.2%). Statistically, there was a dependence relationship between tide levels and positive detection, with a higher frequency at high tide (46.7%) than at low tide (25%) periods. Months with the highest detection rates (April 2012 and April/May 2013) were preceded by periods of higher precipitation (March 2012 and February/March 2013). Phylogenetic analysis showed the circulation of the old pandemic variant (GII.4-US_95-96) and GI.8. The NoV detection demonstrated viral contamination on the beaches and evidenced the health risk to bathers, mainly through recreational activities such as bathing, and highlighted the importance of including enteric viruses research in the recreational water quality monitoring.
Assuntos
Norovirus , Microbiologia da Água , Brasil , Cidades , Monitoramento Ambiental , Genótipo , Filogenia , Recreação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Globally, Norovirus (NoV) is considered the most common cause of diarrheal episodes across all age groups. Despite its wide genetic diversity, the GII.4 strain is the most predominant and has been associated with epidemics worldwide. In this study, we characterized sporadic cases of diarrhea from NoV-positive children, during a five-year period (2010-2014). METHODS: A total of 250 NoV-positive samples identified by an enzyme immunoassay (EIA) were subjected to RT-PCR and partial nucleotide sequencing for polymerase and capsid genes. Phylogenetic analysis was performed to identify NoV genotypes using the binary classification. In addition, sequences from the P2 subdomain (capsid) gene of GII-4 variants were characterized by evolutionary analyses, using the MCMC method implemented in the BEAST package. A 3D structure was built using protein modeling. RESULTS: Phylogenetic analysis demonstrated a predominance of genotype GII.4 (52.4% - 99/189), variants New Orleans_2009 and Sydney_2012 followed by GII.P7/GII.6 with 6.3% (12/189). Amino acid analyses of the GII.4 strains showed several important amino acid changes. A higher evolutionary rate was found, 7.7 × 10- 3 in the Sydney variant and 6.3 × 10- 3 in the New Orleans. Based in evolutionary analysis the time to the most recent common ancestor (TMRCA) has been calculated as estimates of the population divergence time. Thus, TMRCA for New Orleans and Sydney variant were 2008.7 and 2010.7, respectively. Also, we observed a lineage of transition between New Orleans and Sydney. CONCLUSION: This study describes the different strains of norovirus isolated from Amazonas state in Brazil during a five-year period. Considering that NoV are capable of changing their antigenic epitopes rapidly, a continuous surveillance is important to monitor the occurrence and changes of the NoV in the community through epidemiological studies. These results contribute to the understanding of NoV molecular epidemiology and its evolutionary dynamics in Amazonas state, Brazil.
Assuntos
Infecções por Caliciviridae/complicações , Infecções por Caliciviridae/epidemiologia , Gastroenterite/epidemiologia , Gastroenterite/virologia , Norovirus/genética , Brasil/epidemiologia , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/genética , Criança , Pré-Escolar , Epidemias , Deriva Genética , Variação Genética , Genótipo , Humanos , Lactente , Epidemiologia Molecular , Norovirus/isolamento & purificação , Filogenia , Fatores de TempoRESUMO
Fecal specimens were collected during a longitudinal, community-based study in the city of Belém, North Brazil, that was conducted over 3 years (October 1982 to March 1986), in which 20 children were included from birth to 3 years of age. A total of 229 fecal samples were screened by real time RT-PCR targeting the junction region (ORF 1/2) of the norovirus (NoV) genome. NoV-positive samples were subjected to PCR and sequencing of the viral polymerase (ORF1) and viral protein 1 (VP1) genes (ORF2). The junction region was also sequenced to assess for recombination when ORF1 and ORF2 genotyping results were dissimilar. Samples classified as GII.P4/GII.4 were further characterized by sequencing the P2 subdomain of the viral capsid to determine possible alterations. An overall positivity of 16.1% (37/229) was observed, including GI (16.2%-6/37) and GII (83.8%-31/37) genogroups. Cases of NoV reinfection in at least 2-month intervals were observed, and 12 children developed at least one case of asymptomatic NoV infection. In total, 48.6% (18/37) NoV-positive samples were subjected to nucleotide sequencing analysis targeting the following polymerase genes: GI.P3 (n = 1), GII.Pa (n = 1), GII.Pc (n = 1), GII.P4 (n = 5), GII.P6 (n = 5), GII.P7 (n = 3), GII.P12 (n = 1), and GII.P22 (n = 1). For the VP1 gene, characterization was performed in 14 (77.8%) samples: GI.3 (n = 1), GII.2 (n = 1), GII.4 (n = 4), GII.6 (n = 4), GII.7 (n = 1), GII.12 (n = 1), GII.14 (n = 1), and GII.23 (n = 1). Recombination events were confirmed in three cases (GII.P12/GII.2, GII.P7/GII.14, and GII.Pa/GII.12), and four samples genotyped as GII.P4/GII.4 were analyzed to identify variants. None had contemporary counterparts. Three children developed consecutive NoV infections by different genotypes. The present report documents the importance of NoV as a cause of childhood infection during a longitudinal study conducted more than 30 years ago.
Assuntos
Infecções por Caliciviridae/virologia , Fezes/virologia , Gastroenterite/virologia , Norovirus/genética , Brasil/epidemiologia , Infecções por Caliciviridae/epidemiologia , Proteínas do Capsídeo/genética , Pré-Escolar , Feminino , Gastroenterite/epidemiologia , Variação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Estudos Longitudinais , Masculino , Norovirus/classificação , Norovirus/isolamento & purificação , Fases de Leitura Aberta , Filogenia , Saúde Pública , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Recombinação GenéticaRESUMO
Fecal specimens were collected during a longitudinal, community-based study in the city of Belém, North Brazil, that was conducted over 3 years (October 1982 to March 1986), in which 20 children were included from birth to 3 years of age. A total of 229 fecal samples were screened by real time RT-PCR targeting the junction region (ORF 1/2) of the norovirus (NoV) genome. NoV-positive samples were subjected to PCR and sequencing of the viral polymerase (ORF1) and viral protein 1 (VP1) genes (ORF2). The junction region was also sequenced to assess for recombination when ORF1 and ORF2 genotyping results were dissimilar. Samples classified as GII.P4/GII.4 were further characterized by sequencing the P2 subdomain of the viral capsid to determine possible alterations. An overall positivity of 16.1% (37/229) was observed, including GI (16.2%-6/37) and GII (83.8%-31/37) genogroups. Cases of NoV reinfection in at least 2-month intervals were observed, and 12 children developed at least one case of asymptomatic NoV infection. In total, 48.6% (18/37) NoV-positive samples were subjected to nucleotide sequencing analysis targeting the following polymerase genes: GI.P3 (n = 1), GII.Pa (n = 1), GII.Pc (n = 1), GII.P4 (n = 5), GII.P6 (n = 5), GII.P7 (n = 3), GII.P12 (n = 1), and GII.P22 (n = 1). For the VP1 gene, characterization was performed in 14 (77.8%) samples: GI.3 (n = 1), GII.2 (n = 1), GII.4 (n = 4), GII.6 (n = 4), GII.7 (n = 1), GII.12 (n = 1), GII.14 (n = 1), and GII.23 (n = 1). Recombination events were confirmed in three cases (GII.P12/GII.2, GII.P7/GII.14, and GII.Pa/GII.12), and four samples genotyped as GII.P4/GII.4 were analyzed to identify variants. None had contemporary counterparts. Three children developed consecutive NoV infections by different genotypes. The present report documents the importance of NoV as a cause of childhood infection during a longitudinal study conducted more than 30 years ago.
Assuntos
Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Gastroenterite/epidemiologia , Gastroenterite/virologia , Genótipo , Norovirus/classificação , Norovirus/genética , Brasil/epidemiologia , Pré-Escolar , Fezes/virologia , Feminino , Técnicas de Genotipagem , Humanos , Lactente , Recém-Nascido , Estudos Longitudinais , Masculino , Epidemiologia Molecular , Norovirus/isolamento & purificação , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Proteínas Virais/genéticaRESUMO
This study investigated the presence of norovirus (NoV) GI and GII in environmental samples from the northern region of Brazil. Water samples were collected monthly (November 2008/October 2010) from different sources and sewage and concentrated by the adsorption-elution method. The NoV investigation used molecular methods followed by sequencing reactions. The general positivity for NoV was 33.9% (57/168). Considering the results obtained only in the semi-nested RT-PCR (reverse transcription polymerase chain reaction) and only in the TaqMan® real-time PCR, the rates were 26.8% (45/168) and 27.4% (46/168), respectively, being for NoV GI 22.2% (10/45) and 19.6% (9/46); for GII 17.8% (8/45) and 15.2% (7/46); and for GI + GII 60% (27/45) and 65.2% (30/46), respectively. Different GI (GI.1, GI.4, GI.7 and GI.8) and GII (GII.4, GII.6, GII.9, GII.12 and GII.14) genotypes were detected. These results demonstrated the NoV was disseminated in the waters of Belém city due to a lack of sanitation that allowed the discharge of contaminated effluents into these aquatic ecosystems.
Assuntos
Monitoramento Ambiental/métodos , Água Doce/microbiologia , Norovirus/isolamento & purificação , Esgotos/microbiologia , Brasil , Genótipo , Norovirus/genética , Fases de Leitura Aberta , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNARESUMO
BACKGROUND: Norovirus (NoV) is a major cause of acute gastroenteritis (AGE) worldwide, especially in children under five years. Studies involving the detection and molecular characterisation of NoV have been performed in Brazil, demonstrating its importance as an etiological agent of AGE. OBJECTIVES: The objectives of this study were to investigate the frequency of human NoV and to genotype the strains isolated from 0-14-year-old patients of AGE in Manaus, Brazil, over a period of two years. METHODS: A total of 426 faecal samples were collected between January 2010 and December 2011. All samples were tested for the presence of NoV antigens using a commercial enzyme immunoassay kit. RNA was extracted from all faecal suspensions and reverse transcription-polymerase chain reaction (RT-PCR) for the NoV-polymerase partial region was performed as a trial test. Positive samples were then subjected to PCR with specific primers for partial capsid genes, which were then sequenced. FINDINGS: NoV was detected in 150 (35.2%) faecal samples, for at least one of the two techniques used. NoV was detected in children from all age groups, with the highest positivity observed among the group of 1-2 years old. Clinically, fever was verified in 43% of the positive cases and 46.3% of the negative cases, and vomiting was observed in 75.8% and 70.8% cases in these groups, respectively. Monthly distribution showed that the highest positivity was observed in January 2010 (81.2%), followed by February and April 2010 and March 2011, when the positivity rate reached almost 50%. Phylogenetic analyses performed with 65 positive strains demonstrated that 58 (89.2%) cases of NoV belonged to genotype GII.4, five (7.7%) to GII.6, and one (1.5%) each to GII.7 and GII.3. MAIN CONCLUSIONS: This research revealed a high circulation of NoV GII.4 in Manaus and contributed to the understanding of the importance of this virus in the aetiology of AGE cases, especially in a region with such few studies available.
Assuntos
Infecções por Caliciviridae/virologia , Gastroenterite/virologia , Norovirus/genética , Norovirus/isolamento & purificação , Doença Aguda , Adolescente , Brasil/epidemiologia , Infecções por Caliciviridae/epidemiologia , Criança , Pré-Escolar , Fezes/virologia , Feminino , Gastroenterite/epidemiologia , Variação Genética , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Prevalência , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Group C rotavirus (RVC) is potentially an important pathogen associated with acute gastroenteritis (AG), especially in outbreaks. This study aims to detect and molecularly characterize RVC in hospitalized children with AG in Belém, Brazil. From May 2008 to April 2011, 279 stools were subjected to reverse-transcription polymerase chain reaction targeting VP7, VP6, VP4, and NSP4 genes. RVC positivity rate was 2.1% (6/279) and phylogenetic analysis of positive samples yields genotype G4-P[2]-I2-E2. No evidence of zoonotic transmission and VP7 gene demonstrated close relationship with Asian strains. RVC surveillance is worth to expand information on evolutionary and epidemiological features of this virus.
Assuntos
Gastroenterite/virologia , Variação Genética , Genótipo , Filogenia , Infecções por Rotavirus/virologia , Rotavirus/classificação , Rotavirus/genética , Brasil/epidemiologia , Criança Hospitalizada , Pré-Escolar , Fezes/virologia , Feminino , Gastroenterite/epidemiologia , Humanos , Lactente , Masculino , Epidemiologia Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rotavirus/isolamento & purificação , Infecções por Rotavirus/epidemiologia , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
Although viruses are well-established causes of acute gastroenteritis, few data on the circulation of these pathogens in Porto Velho, state of Rondônia, Brazil, are available. Thus, faecal samples from hospitalised diarrhoeic children, under six years of age, were collected and tested for the presence of norovirus (NoV), adenovirus (AdV) and astrovirus (AstV) from February 2010-February 2012. Specimens were screened by reverse-transcription polymerase chain reaction and viruses were found in 10.7% (63/591) of the cases. NoV, AdV and AstV were detected in 7.8%, 2% and 0.8% of the samples, respectively. NoV infection was observed at all ages and was most prevalent in zero-18-month-old children (84.7%; p = 0.002). A higher incidence of NoV was detected from February-April 2010, when it was found in 52.2% of the cases. Co-infections involving these viruses, rotavirus and enteropathogenic bacteria were detected in 44.4% (28/63) of the children with viral diarrhoea. Nosocomial infections were demonstrated in 28.6% (18/63) of the cases in which viruses were detected. The present paper reports, for the first time, the circulation of NoV and AstV among the paediatric population of Porto Velho and it contributes to our understanding of the roles of these pathogens in gastrointestinal infections.
Assuntos
Infecções por Adenovirus Humanos/epidemiologia , Infecções por Astroviridae/epidemiologia , Infecções por Caliciviridae/epidemiologia , Diarreia/virologia , Gastroenterite/epidemiologia , Doença Aguda , Adenoviridae/isolamento & purificação , Brasil/epidemiologia , Criança , Pré-Escolar , Infecção Hospitalar/epidemiologia , Fezes/virologia , Hospitalização , Humanos , Lactente , Recém-Nascido , Mamastrovirus/isolamento & purificação , Norovirus/isolamento & purificação , Reação em Cadeia da Polimerase , Prevalência , Floresta Úmida , Estações do AnoRESUMO
Picobirnavirus (PBV) belongs to the family Picobirnaviridae. Picobirnaviruses contain a bisegmented dsRNA genome that is non-enveloped. A total of 85 pooled faecal samples were collected from the poultry of 37 farms from the Metropolitan Mesoregion of Belém (MMB), Pará state, Brazil. The viral RNA from each sample was analysed by PAGE and reverse transcriptase PCR (RT-PCR). For each county affected, at least one positive sample was selected, cloned and sequenced. The samples showed a positivity of 15.3â% (13/85) by PAGE and 49.4â% (42/85) by RT-PCR. Sequencing of these strains demonstrated a considerable RdRp gene heterogeneity that ranged from 56.1 to 100â% at the nucleotide level compared with prototypes of different species and water sewage, and from 50.3 to 100â% among themselves. Avian picobirnavirus (AvPBV) was detected in MMB broiler farms and showed a heterogeneous relationship with the prototypes used. This report includes what is believed to be the first gene sequencing of AvPBV in Brazilian broiler chickens.
Assuntos
Picobirnavirus/genética , Picobirnavirus/isolamento & purificação , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/virologia , Infecções por Vírus de RNA/veterinária , Animais , Brasil/epidemiologia , Galinhas , Fezes/virologia , Genoma Viral , Genótipo , Epidemiologia Molecular , Filogenia , Picobirnavirus/classificação , Infecções por Vírus de RNA/epidemiologia , Infecções por Vírus de RNA/virologiaRESUMO
Sapoviruses (SaVs) belong to the family Caliciviridae and are related to gastroenteritis viruses of humans and animals. These agents have been reported from several countries of the world and represent an important cause of economic loss. The Amazon area has a high degree of diversity of animals and plants, is located in the Northern Region of Brazil and accounts for a large part of the Brazilian territory. In this study, stool samples were collected from pigs during the phase of nursing (less than 28 days of age) and post-weaning (29 to 56 days of age) from January 2008 to February 2009. A total of 169 specimens (108 nursing and 61 post-weaning pigs) were tested by reverse transcription polymerase chain reaction (RT-PCR) using the primers p289/p290 for the detection of caliciviruses (CVs), i.e., SaVs and noroviruses (NoVs). Positive sequences were analyzed using BioEdit software (v. 7.1.3.0) and compared with other sequences registered in the GenBank database. A positive frequency of 12.4 % (21/169) was observed, and all of the viruses found were identified as SaVs, with 15 belonging to genogroup GIII (71.4 %), three to GVII-1 (14.3 %) and three to GVIII-2 (14.3 %). No NoVs were detected. The frequency of SaV infections was significantly higher in nursing pigs (17.6 %-19/108) than in post-weaning pigs (3.3 %-2/61). Considering the consistency of the samples, 14.7 % of the samples were classified as diarrheic, but statistical analysis demonstrated that there was no significant difference compared to normal specimens (p = 0.5795). For the first time, we have demonstrated the circulation of SaVs in pigs from the Amazon.
Assuntos
Infecções por Caliciviridae/veterinária , Gastroenterite/veterinária , Sapovirus/genética , Doenças dos Suínos/virologia , Animais , Brasil/epidemiologia , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Gastroenterite/epidemiologia , Gastroenterite/virologia , Filogenia , Suínos , Doenças dos Suínos/epidemiologiaRESUMO
Rotaviruses infect humans and animals and are classified into eight groups (A to H). Group D rotavirus (RVD) has been described in birds, although relatively few reports are available. The present study focused on RVD, including epidemiological and molecular aspects of samples collected from broiler chickens in the state of Pará, Brazil. A total of 85 faecal samples were collected between 2008 and 2011 from 37 chicken farms located in eight different municipalities. The viral double-stranded RNA was extracted from faecal suspensions and analysed using polyacrylamide gel electrophoresis (PAGE), followed by reverse transcriptase-polymerase chain reaction (RT-PCR) and nucleotide sequencing of the VP6 and VP7 genes. Comparing the positive results, 16.5% (14/85) were obtained by PAGE and 35.3% (30/85) by RT-PCR. Samples from seven of eight municipalities were positive for RVD and infections were recorded in 17 (45.9%) of 37 chicken farms. The RVD infection rate was significantly higher in the 16-day to 30-day age group (62.2%; 23/37) compared with other ages. No consistent relationship was found between the infection rate and either the population density in poultry houses or the climatic conditions. The nucleotide sequences of the VP6 gene were 89.9 to 90.9% similar to the prototype strain 05V0049 and were 88.3 to 100% similar among themselves; VP7 gene nucleotide sequences were 84.3 to 85.4% similar to the prototype strain 05V0049 and 93.8 to 100% similar among themselves. Overall, this study provides new insights into the epidemiology and genome characterization of group D rotaviruses.
Assuntos
Antígenos Virais/genética , Proteínas do Capsídeo/genética , Galinhas/virologia , Genoma Viral/genética , Infecções por Rotavirus/epidemiologia , Rotavirus/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Brasil/epidemiologia , Fezes/virologia , Dados de Sequência Molecular , Filogenia , RNA de Cadeia Dupla/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Rotavirus/genética , Infecções por Rotavirus/virologia , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA/veterináriaRESUMO
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.
Assuntos
Adenovírus Humanos , Enterovirus , Fezes , Gastroenterite , Humanos , Gastroenterite/virologia , Gastroenterite/epidemiologia , Brasil/epidemiologia , Fezes/virologia , Pré-Escolar , Lactente , Adenovírus Humanos/genética , Adenovírus Humanos/isolamento & purificação , Adenovírus Humanos/classificação , Masculino , Enterovirus/genética , Enterovirus/isolamento & purificação , Feminino , Criança , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/virologia , Infecções por Enterovirus/diagnóstico , Doença Aguda , Infecções por Adenovirus Humanos/epidemiologia , Infecções por Adenovirus Humanos/virologia , FilogeniaRESUMO
INTRODUCTION: Metagenomic sequencing is a powerful tool that is widely used in laboratories worldwide for taxonomic characterization of microorganisms in clinical and environmental samples. In this study, we utilized metagenomics to investigate comprehensively the microbial diversity in fecal samples of children over a four-year period. Our methods were carefully designed to ensure accurate and reliable results. MATERIAL AND METHODS: Validated and analyzed were metagenomic data obtained from sequencing 27 fecal samples from children under 10 years old with gastroenteritis over a four-year period (2012-2016). The fecal specimens were collected from patients who received care at public health facilities in the northern region of Brazil. Sequencing libraries were prepared from cDNA and sequenced on the Illumina HiSeq. Kraken-2 was utilized to classify bacterial taxonomy based on the 16S rRNA gene, using the Silva rRNA database. Additionally, the Diamond program was used for mapping to the non-redundant protein database (NR database). Phylogenomic analyses were conducted using Geneious R10 and MEGA X software, and Bayesian estimation of phylogeny was performed using the MrBayes program. The results indicate significant heterogeneity among norovirus strains, with evidence of recombination and point mutations. This study presents the first complete genome of parechovirus 8 in the region. Additionally, it describes the bacterial populations and bacteriophages present in feces, with a high abundance of Firmicutes and Proteobacteria, including an increased proportion of the Enterobacteriaceae family. The presented data demonstrate the genetic diversity of microbial populations and provide a comprehensive report on viral molecular characterization. These findings are relevant for genomic studies in gastrointestinal infections. The metagenomic approach is a powerful tool for investigating microbial diversity in children with gastroenteritis. However, further studies are imperative to conduct genomic analysis of identified bacterial strains and thoroughly analyze antimicrobial resistance genes.
RESUMO
Viral gastroenteritis is commonly reported in dogs and involves a great diversity of enteric viruses. In this research, viral diversity was investigated in dogs with diarrhea in Northern Brazil using shotgun metagenomics. Furthermore, the presence of norovirus (NoV) was investigated in 282 stool/rectal swabs of young/adult dogs with or without diarrhea from two public kennels, based on one-step reverse transcription polymerase chain reaction (RT-PCR) for genogroup VI and VII (GVI and GVII) and real-time RT-PCR for GI, GII, and GIV. Thirty-one viral families were identified, including bacteriophages. Phylogenetic analyses showed twelve complete or nearly complete genomes belonging to the species of Protoparvovirus carnivoran1, Mamastrovirus 5, Aichivirus A2, Alphacoronavirus 1, and Chipapillomavirus 1. This is the first description of the intestinal virome of dogs in Northern Brazil and the first detection of canine norovirus GVII in the country. These results are important for helping to understand the viral groups that circulate in the canine population.