Analysis of viral diversity in dogs with acute gastroenteritis from Brazilian Amazon.

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.

Epidemiological and molecular surveillance of norovirus in the Brazilian Amazon: description of recombinant genotypes and improvement of evolutionary analysis.

Noroviruses are highly infectious, genetically diverse viruses. Global outbreaks occur frequently, making molecular surveillance important for infection monitoring. This cross-sectional descriptive study aimed to monitor cases of norovirus gastroenteritis in the Brazilian Amazon. Fecal samples were tested by immunoenzymatic assay, RT-PCR and genetic sequencing for the ORF1/ORF2 and protease regions. Bayesian inference with a molecular clock was employed to construct the phylogeny. The norovirus prevalence was 25.8%, with a higher positivity rate among children aged 0-24 months. Genogroup GII accounted for 98.1% of the sequenced samples, while GI accounted for 1.9% of them. The GII.P16/GII.4 genotype was the most prevalent, with an evolution rate of 2.87x10-3 and TMRCA estimated in 2012. This study demonstrates that norovirus is a primary causative agent of gastroenteritis and provides data on viral genetic diversity that may facilitate infection surveillance and vaccine development.

Environmental health of water bodies from a Brazilian Amazon Metropolis based on a conventional and metagenomic approach.

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.

Microbial Diversity in Children with Gastroenteritis in the Amazon Region of Brazil: Development and Validation of a Molecular Method for Complete Sequencing of Viral Genomes.

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.

Epidemiological and molecular surveillance of norovirus in the Brazilian Amazon: description of recombinant genotypes and improvement of evolutionary analysis

ABSTRACT Noroviruses are highly infectious, genetically diverse viruses. Global outbreaks occur frequently, making molecular surveillance important for infection monitoring. This cross-sectional descriptive study aimed to monitor cases of norovirus gastroenteritis in the Brazilian Amazon. Fecal samples were tested by immunoenzymatic assay, RT-PCR and genetic sequencing for the ORF1/ORF2 and protease regions. Bayesian inference with a molecular clock was employed to construct the phylogeny. The norovirus prevalence was 25.8%, with a higher positivity rate among children aged 0-24 months. Genogroup GII accounted for 98.1% of the sequenced samples, while GI accounted for 1.9% of them. The GII.P16/GII.4 genotype was the most prevalent, with an evolution rate of 2.87x10−3 and TMRCA estimated in 2012. This study demonstrates that norovirus is a primary causative agent of gastroenteritis and provides data on viral genetic diversity that may facilitate infection surveillance and vaccine development.

Molecular analysis of canine circovirus in dogs from animal shelters in Belém, Pará, northern Brazil: first detection at the amazon region.

The canine circovirus (CanCV) is a single-stranded DNA virus that has become an important emerging virus associated with gastroenteritis in dogs worldwide. In the present study, the CanCV was detected by PCR in 15% (22/147) of dogs from animal shelters in Belém, between 2019 and 2020. We observed an association between the CanCV infection and the presence of diarrhea in animals younger than one year of age (p > 0.01). The Brazilian strains were grouped in Chinese genotypes, with 99.54 to 100% nucleotilde homology. The GMRF Bayesian Skyride used the molecular clock model, which was the best suited technique to plot the dataset. The most recent common ancestor (TMRCA) was estimated in 2017, with the evolution rate of 1.6 x 10-3 s/s/y. The viral family diversity was also investigated, with emphasis on the families of the enteric pathogenic viruses Parvoviridae, Picornaviridae and Astroviridae, which were detected in the CanCV positive pooled samples. This study highlights the importance of the CanCV as an emergent virus that causes diarrhea in Brazilian dogs. The results found herein contribute to the understanding of the role of CanCV in enteric diseases and in the evolutionary molecular characterization of the circulating genotypes. Furthermore, we increased the understanding of the fecal virome in dogs with diarrhea, providing data for the monitoring and prevention viral gastroenteric diseases in domestic animals.

O circovírus canino (CanCV) é um vírus de DNA de fita simples que se tornou um importante vírus emergente associado à gastroenterite em cães em todo o mundo. No presente estudo, o CanCV foi detectado por PCR em 15% (22/147) dos cães de abrigos de animais em Belém, entre 2019 e 2020. Observamos uma associação entre a infecção pelo CanCV e a presença de diarreia em animais menores de um ano de idade (p > 0,01). As linhagens brasileiras foram agrupadas em genótipos chineses, com 99,54 a 100% de homologia nucleotídica. O GMRF Bayesian Skyride foi o modelo de relógio molecular utilizado, sendo o método mais adequado para o conjunto de dados. O ancestral comum mais recente (TMRCA) foi estimado em 2017, com taxa de evolução de 1,6 x 10-3 s/s/ano. A diversidade de famílias virais também foi investigada, com destaque para as famílias dos vírus patogênicos entéricos Parvoviridae, Picornaviridae e Astroviridae, que foram detectadas nos pools de amostras positivas para CanCV. Este estudo destaca a importância do CanCV como um vírus emergente que causa diarreia em cães brasileiros. Os resultados aqui encontrados contribuem para a compreensão do papel do CanCV nas doenças entéricas e na caracterização molecular evolutiva dos genótipos circulantes. Além disso, aumentamos a compreensão do viroma fecal em cães com diarreia, fornecendo dados para o monitoramento e prevenção de doenças gastroentéricas virais em animais domésticos.

Circulation of adenovirus and other viruses in urban drainage channels: an environmental surveillance in Belém, Amazon region, Brazil.

Urban channels in amazon cities are very polluted, with garbage and sewage disposal in these aquatic environments, favoring the high dissemination of waterborne viruses such as human adenovirus (HAdV). The aim of this study was to perform the detection and molecular characterization of adenovirus in urban channels and in a wastewater treatment plant located in a metropolitan city in the Amazon. Additionally, metagenomic analyses were performed to assess viral diversity. Samples were concentrated by organic flocculation, analyzed by quantitative real time PCR (qPCR) and sequenced (Sanger e next generation sequencing). Cell culture was performed to verify the viability of HAdV particles. A total of 104 samples were collected, being the HAdV positivity of 76% (79/104). Among the positive samples, 29.1% (23/79) were characterized as HAdV-F40 (87%, 20/23), HAdV-F41 (8.7%, 2/23), and HAdV-B (4.3%, 1/23). Average precipitation rates ranged from 163 to 614 mm, while the pH ranged from 6.9 to 7.6. Eight positive samples were inoculated into A549 cells and in 4 of these, was observed changes in the structure of the cell monolayer, alteration in the structure of the cell monolayer was observed, but without amplification when analyzed by PCR. The metagenomic data demonstrated the presence of 14 viral families, being the most abundant: Myoviridae (41% of available reads), Siphoviridae (24.5%), Podoviridae (14.1%), and Autographiviridae (6.9%) with more than 85% of the total number of identified reads. This study reinforcing that continuous surveillance may contribute to monitoring viral diversity in aquatic environments and provide early warning of potential outbreaks.

Detection of G3 human-like rotavirus in institutionalized dogs from Brazil.

Viral gastroenteritis is a common clinical problem in dogs and group A rotavirus (RVA) is one of the agents involved in this etiology. It mainly affects dogs in the first 6 months of life, and these animals are considered an important reservoir and potential transmitters of the virus to other susceptible hosts, such as humans. Among the different types of RVA, G3 is the most detected in dogs, and this genotype is also involved in infections in other animals, including humans. Thus, the present study aims to investigate the presence of RVA in samples of dogs from a public kennel. A total of 64 fecal samples from dogs with diarrhea were analyzed, collected from April 2019 to March 2020, from the kennel of the Zoonosis Control Center, located in Belém, a city in the North of Brazil. The extracted genetic material was subjected to reverse transcription followed by real-time PCR (RT-qPCR); the positives were tested by RT-PCR with a specific primer for the RVA VP7 gene, after nucleotide sequencing and phylogenetic analysis. One sample was subjected to high-performance sequencing. A positivity of 7.8% (5/64) was observed for RVA, all characterized as G3, grouping in the G3-III lineage, with greater similarity to human samples. Different regions of the RVA genome fragments were found. These results emphasize the need for animal health surveillance to better understand the global strain dispersion of RVA and elucidate possible interspecies transmission events, monitoring the genetic diversity of this pathogen.

Nearly Complete Genome Sequences of Two Canine Mamastrovirus 5 Strains from Latin America.

We report the nearly complete genome sequences of CAstV-PK01 and CAstV-PK03, two canine astrovirus strains belonging to the species Mamastrovirus 5, which were detected in fecal swab samples collected from puppies with diarrhea from two different kennels in the Brazilian Amazon.

Molecular characterization of the gastrointestinal eukaryotic virome in elderly people in Belem, Para, Brazil.

Acute gastroenteritis is one of the main causes of mortality and morbidity worldwide, affecting mainly children, the immunocompromised and elderly people. Enteric viruses, especially rotavirus A, are considered important etiological agents, while long-term care facilities are considered favorable environments for the occurrence of sporadic cases and outbreaks of acute gastroenteritis. Therefore, it is important to monitor the viral agents present in nursing homes, especially because studies involving the elderly population in Brazil are scarce, resulting in a lack of available virological data. As a result, the causative agent remains unidentified in a large number of reported acute gastroenteritis cases. However, the advent of next-generation sequencing provides new opportunities for viral detection and discovery. The aim of this study was to identify the viruses that circulate among elderly people with and without acute gastroenteritis, living in residential care homes in Belém, Pará, Brazil, between 2017 and 2019. Ninety-three samples were collected and screened by immunochromatography and qPCR. After, the samples were analyzed individually or in pools by next generation sequencing to identify the viruses circulating in this population. In 26 sequenced samples, members of 13 eukaryotic virus families were identified. The most abundantly present virus families were Parvoviridae, Genomoviridae and Smacoviridae. Contigs displaying similarity to pegiviruses were also detected. Furthermore, a near-complete rotavirus A genome was obtained and could be classified as G3P[8] genotype with the equine DS-1-like genetic background. Complete sequences of the VP4 and VP7 genes of a rotavirus C were also detected, belonging to G4P[2]. This study demonstrates the first characterization of the gastrointestinal virome in elderly in Northern Brazil. A diversity of viruses was found to be present in patients with and without diarrhea, reinforcing the need to monitor elderly people residing in long-term care facilities, especially in cases of acute gastroenteritis.

Retrospective molecular analysis of norovirus recombinant strains in the amazon region, Brazil.

BACKGROUND: Noroviruses are enteric viruses that cause acute gastroenteritis worldwide. Over two decades, GII.4 genotype was responsible for most cases. However, recombinant strains have emerged and changed the epidemiological context of these infections. OBJECTIVES: The aim of this study was to identify the recombinant genetic strains of norovirus causing gastroenteritis in Brazilian children from the Amazon region. METHODS: We analyzed 534 cases of gastroenteritis between 2015 and 2016. Genotypic characterization was performed by partial sequencing of ORF1 and ORF2. Evolutionary history was inferred by Bayesian inference using MrBayes. Recombinant strains were confirmed by Simplot and RDP4 analysis. FINDINGS: We performed viral detection tests and identified a norovirus frequency of 31.8% (175/534). Based on viral RdRp and VP1 genes, nine genotypes were identified: GIIP31/GII.4, GII·P16/GII.4, GII·P7/GII.6, GII·P21/GII.13, GII·P33/GII.1, GII·P17/GII.17, GI·P7/GI.7, GII·P4/NT, and GII.7/NT. The phylogenetic tree showed evolutionary relationships among the genotypes, including the recombinant strains. This is the first description of GII·P33/GII.1 and GII·P21/GII.13 genotypes in Brazil. CONCLUSION: Norovirus evolution has been characterized by the continuous replacement of variants that have new antigenic properties. In recent years, recombinant strains have displaced GII.4, improving the viral fitness and influencing the viral transmissibility and pathogenicity.

Evolutionary and Molecular Analysis of Complete Genome Sequences of Norovirus From Brazil: Emerging Recombinant Strain GII.P16/GII.4.

Noroviruses (NoVs) are enteric viruses that cause acute gastroenteritis, and the pandemic GII.4 genotype is spreading and evolving rapidly. The recombinant GII.P16/GII.4_Sydney strain emerged in 2016, replacing GII.P31/GII.4_Sydney (GII.P31 formerly known as GII.Pe) in some countries. We analyzed the complete genome of 20 NoV strains (17 GII.P31/GII.4_ Sydney and 3 GII.P16/GII.4_Sydney) from Belém and Manaus, Brazil, collected from 2012 to 2016. Phylogenetic trees were constructed by maximum likelihood method from 191 full NoV-VP1 sequences, demonstrated segregation of the Sydney lineage in two larger clades, suggesting that GII.4 strains associated with GII.P16 already have modifications compared with GII.P31/GII.4. Additionally, the Bayesian Markov Chain Monte Carlo method was used to reconstruct a time-scaled phylogenetic tree formed by GII.P16 ORF1 sequences (n = 117) and three complete GII.P16 sequences from Belém. The phylogenetic tree indicated the presence of six clades classified into different capsid genotypes and locations. Evolutionary rates of the ORF1 gene of GII.P16 strains was estimated at 2.01 × 10-3 substitutions/site/year, and the most recent common ancestors were estimated in 2011 (2011-2012, 95% HPD). Comparing the amino acid (AA) sequence coding for ORF1 with the prototype strain GII.P16/GII.4, 36 AA changes were observed, mainly in the non-structural proteins p48, p22, and RdRp. GII.P16/GII.4 strains of this study presented changes in amino acids 310, 333, 373, and 393 of the antigenic sites in the P2 subdomain, and ML tree indicating the division within the Sydney lineage according to the GII.P16 and GII.P31 polymerases. Notably, as noroviruses have high recombination rates and the GII.4 genotype was prevalent for a long time in several locations, additional and continuous evolutionary analyses of this new genotype should be needed in the future.

Molecular and evolutionary characterization of norovirus GII.17 in the northern region of Brazil.

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.

Epidemiological and molecular investigation of norovirus and astrovirus infections in Rio Branco, Acre, Northern Brazil: A retrospective study.

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.

Detection and genotyping of enteric viruses in hospitalized children with acute gastroenteritis in Belém, Brazil: Occurrence of adenovirus viremia by species F, types 40/41.

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.

Norovirus RNA in serum associated with increased fecal viral load in children: Detection, quantification and molecular analysis.

Worldwide, norovirus (NoV) is a major cause of acute gastroenteritis (AGE) responsible for pandemics every ~3 years, and over 200,000 deaths per year, with the majority in children from developing countries. We investigate the incidence of NoV in children hospitalized with AGE from Belém, Pará, Brazil, and also correlated viral RNA levels in their blood and stool with clinical severity. For this purpose, paired stool and serum samples were collected from 445 pediatric patients, ≤9 years between March 2012 and June 2015. Enzyme-linked immunosorbent assay (EIA) was used to detect NoV in stool and reverse transcription quantitative PCR (RT-qPCR) used to quantify NoV RNA levels in sera (RNAemia) and in the positive stool. Positives samples were characterized by the partial ORF1/2 region sequence of viral genome. NoV antigen was detected in 24.3% (108/445) of stool samples, with RNAemia also present in 20.4% (22/108). RNAemia and a high stool viral load (>107 genome copies/gram of faeces) were associated with longer hospitalizations. The prevalent genotypes were GII.4 Sydney_2012 (71.6%-58/81) and New Orleans_2009 (6.2%-5/81) variants. Eight other genotypes belonging to GII were detected and four of them were recombinant strains. All sera were characterized as GII.4 and shared 100% similarity with their stool. The results suggest that the dissemination of NoV to the blood stream is not uncommon and may be related to increased faecal viral loads and disease severity.

Molecular epidemiology and temporal evolution of norovirus associated with acute gastroenteritis in Amazonas state, Brazil.

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.

Astrovirus infection in hospitalized children: Molecular, clinical and epidemiological features.

BACKGROUND: Astrovirus (HAstV) is a common viral pathogen that causes gastroenteritis worldwide. It is classified into eight classical human types (HAstV-1/8) and seven other less prevalent types, described as HAstV VA1, VA2, VA3, VA4, MLB-1, MLB-2 and MLB-3. During outbreaks, the elderly and children are the most affected, and the spread of the virus is associated with person-to-person contact, food ingestion and contaminated water. OBJECTIVES: The aim of the present study was to investigate the prevalence of infection and genetic diversity of HAstV strains. Samples were collected from children with acute gastroenteritis admitted to a large pediatric hospital during a surveillance period of three years (2008-2011) in Belém city, Pará State, Amazon Region, Northern Brazil. STUDY DESIGN: Screening and genotyping tests were conducted using RT-PCR to detect the classical and non-classical HAstV types using specific primers. A semi-nested RT-PCR protocol was developed to improve viral detection in samples with a low viral load. RESULTS: The overall positivity observed in this study was 3.9% (19/483). The age distribution showed a high prevalence of positive cases in children under one year old (5.3%). We found vomiting associated with 75% of the positive cases, fever with 82.3%, and dehydration with 76.9%. Most patients with positive cases demonstrated two to five days of diarrhea, two to three episodes of vomiting during hospitalization, and three bowel movements per day. Co-infection with HAstV and norovirus was observed in three cases (15.8%), and no pattern of seasonality or any relationship between the HAstV positivity rate and climate variables was observed. Eighteen positive samples (94.7%-18/19) were genotyped based on the ORF 2 region, and the greatest prevalence was of HAstV-1a (66.6%-12/18), followed by HAstV-2 (22.2%-4/18, comprising two type-2b and two type-2c genotypes), HAstV-3c (5.6%-1/18) and HAstV-4c (5.6%-1/18). No non-classical types were detected in the clinical samples analyzed. CONCLUSIONS: The present study showed that although HAstV infections occur at low frequency, they are involved in severe pediatric cases of acute gastroenteritis presenting with a high diversity of strains, including the lineages 3c and 4c, which were never before detected in Brazil.

High prevalence of norovirus in children with sporadic acute gastroenteritis in Manaus, Amazon Region, northern Brazil.

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.

High prevalence of norovirus in children with sporadic acute gastroenteritis in Manaus, Amazon Region, northern Brazil

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.