Epidemiologic and Clinical Characteristics of Human Bocavirus Infection in Children with or without Acute Gastroenteritis in Acre, Northern Brazil.

Human bocavirus (HBoV) is an emerging virus detected around the world that may be associated with cases of acute gastroenteritis (AGE). However, its contribution to AGE has not been elucidated. This study aimed to describe the frequency, clinical features, and HBoV species circulation in children up to 5 years with or without AGE symptoms in Acre, Northern Brazil. A total of 480 stool samples were collected between January and December 2012. Fecal samples were used for extraction, nested PCR amplification, and sequencing for genotyping. Statistical analysis was applied to verify the association between epidemiological and clinical characteristics. Overall, HBoV-positivity was 10% (48/480), with HBoV-positive rates of 8.4% (19/226) and 11.4% (29/254) recorded in diarrheic and non-diarrheic children, respectively. The most affected children were in the age group ranging between 7 and 24 months (50%). HBoV infection was more frequent in children who live in urban areas (85.4%), use water from public networks (56.2%), and live with adequate sewage facilities (50%). Co-detection with other enteric viruses was 16.7% (8/48) and the most prevalent coinfection was RVA+ HBoV (50%, 4/8). HBoV-1 was the most frequent species detected in diarrheic and non-diarrheic children, responsible for 43.8% (21/48) of cases, followed by HBoV-3 (29.2%, 14/48) and HBoV-2 (25%, 12/48). In this study, HBoV infection was not always associated with AGE, as most HBoV cases belonged to the non-diarrheal group. Future studies are warranted in order to determine the role of HBoV in causing acute diarrhea disease.

Arboviruses in Free-Ranging Birds and Hematophagous Arthropods (Diptera, Nematocera) from Forest Remnants and Urbanized Areas of an Environmental Protection Area in the Amazon Biome.

The rapid and disorderly urbanization in the Amazon has resulted in the insertion of forest fragments into cities, causing the circulation of arboviruses, which can involve hematophagous arthropods and free-ranging birds in the transmission cycles in urban environments. This study aimed to evaluate the circulation of arboviruses in free-ranging birds and hematophagous arthropods captured in an Environmental Protection Area in the Belem metropolitan area, Brazil. Birds were captured using mist nets, and hematophagous arthropods were collected using a human protected attraction technique and light traps. The birds' sera were subjected to a hemagglutination inhibition test to detect antibodies against 29 arbovirus antigens. Arthropod macerates were inoculated into C6/36 and VERO cell cultures to attempt viral isolation and were tested using indirect immunofluorescence, subsequent genetic sequencing and submitted for phylogenetic analysis. Four bird sera were positive for arbovirus, and one batch of Psorophora ferox was positive for Flavivirus on viral isolation and indirect immunofluorescence. In addition, the Ilheus virus was detected in the sequencing and phylogenetic analysis. The presence of antibodies in sera from free-ranging birds and the isolation of Ilheus virus in Psorophora ferox indicate the circulation of arboviruses in forest remnants in the urban center of Belem.

Detection of a novel species A, DS-1-like, G4P[6] rotavirus strain from a Brazilian child with gastroenteritis.

Rotaviruses belonging to species A (RVA) remain among the most common causes of severe gastroenteritis in children aged <5 years, leading to substantial morbidity and mortality worldwide. Genome reassortment events between two human strains or human and animal strains represent one of the mechanisms which appear to generate the broad genetic variability of circulating. According to a nucleotide, sequence-based classification system, RVA strains are currently classified into three genotype constellations including Wa-like (genogroup I), DS-1-like (genogroup II), and AU-like (genogroup III). The present study reports the detection of an unusual RVA G4P[6] strain (coded as strain HSE005), which might have originated from a natural reassortment event between human and animal RVA strains. Molecular characterization of this isolate showed that it belonged to genogroup II, genotype G4P[6]. In addition, two genes (VP3 and NSP4) of this strain denoted evidence of reassortment events involving strains of distinct zoonotic evolutionary origins. Therefore, we propose that a new G4P[6] strain was identified, highlighting a possible first zoonotic transmission including a reassortment event that involved the VP3 gene.

Ocurrence of rotavirus and picobirnavirus in wild and exotic avian from amazon forest.

The present study reports the occurrence of rotavirus A (RVA), rotavirus D (RVD), rotavirus F (RVF), rotavirus G (RVG), and picobirnavirus (PBV) in fecal specimens of wild (n = 22), and exotic birds (n = 1) from different cities of Pará state. These animals were hospitalized at Veterinary Hospital of the Federal University of Pará, Brazil, in a period from January 2018 to June 2019. The animals exhibited different clinical signs, such as diarrhea, malnutrition, dehydration, and fractures. The results showed 39.1% (9/23) of positivity for RVA by RT-qPCR. Among these, one sample (1/9) for the NSP3 gene of T2 genotype was characterized. About 88.9% (8/9) for the VP7 gene belonging to G1, G3 equine like and G6 genotypes, and 55.5% (5/9) for the VP4 gene of P[2] genotype were obtained. In the current study, approximately 4.5% of the samples (1/23) revealed coinfection for the RVA, RVD and RVF groups. Furthermore, picobirnavirus (PBV) was detected in one of the 23 samples tested, and was classified in the Genogroup I. The findings represent the first report of RVA, RVD, RVF, RVG, and PBV genotypes in wild birds in Brazil, and due to wide distribution it can implies potential impacts of RVs, and PBVs on avian health, and other animals contributing to construction of new knowledge, and care perspectives.

Rotavirus antigenemia as a common event among children hospitalised for severe, acute gastroenteritis in Belém, northern Brazil.

BACKGROUND: Rotavirus antigenemia and RNAemia (the presence of rotavirus RNA in serum) have been commonly identified among paediatric patients with acute gastroenteritis. In this study we examined the association between rotavirus antigenemia and clinical features, and sought to determine the genotypes of rotaviruses detected in paired stool and serum samples. METHODS: Paired stool and serum samples were obtained from children hospitalised for acute gastroenteritis in Belém, Brazil, between June 2012 and June 2015. The 20-point Vesikari scoring system was used to assess the disease severity upon a retrospective medical record review. Stool and serum samples were primarily screened for the presence of rotavirus antigen using a commercial ELISA assay. The rotavirus isolates from stool and serum samples were genotyped by using the classical reverse-transcriptase polymerase chain reaction (RT-PCR) and/or through nucleotide sequencing of VP4 and VP7 genes. Viral load was estimated using real-time RT-PCR. RESULTS: In total rotavirus antigen was detected in 109 (24.2%) stool samples from 451 children, whereas antigenemia occurred in 38.5% (42/109) of these patients. We demonstrated that patients positive for rotavirus RNA in paired stool and serum samples were more likely to have a higher frequency of vomiting episodes in a 24-h period (p = 0.0035). Our findings also suggested that children not vaccinated against rotavirus are more likely to develop antigenemia, as compared to those given at least one vaccine dose (p = 0.0151). G12P [8] and G2P [4] genotypes were predominant throughout the study period, accounting for 52.3% (57/109) and 27.5% (30/109) of the typed isolates, respectively. Ten stool-serum pairs could be typed for VP4 and VP7 genes. Seven of these pairs showed concordant results with G2P [4] genotype being detected in stool and serum samples, whereas discrepancies between genotypes (G2P [4]/G2P[NT] and G12P [8]/G2P[NT]) were seen in three pairs. CONCLUSIONS: Rotavirus antigenemia and RNAemia occur in a significant number of children hospitalised for acute gastroenteritis in Belém, Brazil, and may contribute to a greater disease severity, particularly translated into a greater number of vomiting episodes. This study documented a high concordance of genotypes detected in a subgroup of paired stool and serum samples.

Emergence of G12P[6] rotavirus strains among hospitalised children with acute gastroenteritis in Belém, Northern Brazil, following introduction of a rotavirus vaccine.

Species A rotavirus still remains a major cause of acute gastroenteritis in infants and young children. Globally, six genotypes (G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] and G12P[8]) account for >90% of circulating strains; however, genotype G12 in combination with P[6] or P[9] has been detected at increasing rates. We sought to broaden our knowledge about the rotavirus strains circulating during the early post-vaccine-introduction period. Stool samples were obtained from children hospitalised for acute gastroenteritis in Belém, Northern Brazil, from May 2008 to May 2011 and examined by reverse transcription polymerase chain reaction and nucleotide sequencing. A total of 122 out of the original 1076 rotavirus strains were judged to be non-typeable in the first analysis and were therefore re-examined. G2P[4] was the most prevalent genotype (58.0%), followed by G1P[8] (16.9%), and G12P[6] (7.5%). G12P[6] strains were identified at similar rates during the first (2.5%) and second (3.9%) years, and the rate jumped to 15.6% in the third year. Analysis of VP7 sequences of the G12P[6] strains showed that they belonged to lineage III. In addition, co-circulating G12P[6] strains displaying long and short RNA patterns were found to belong to the Wa-like and DS-1-like constellation, respectively. Additional unusual circulating strains G12P[9] and G3P[9] were also identified. This hospital-based study showed a high prevalence of G12P[6] strains in the third year of surveillance. Our results highlight the need for continuous longitudinal monitoring of circulating rotavirus strains after introduction of rotavirus vaccines in Brazil and elsewhere.

First Complete Genome Sequence of a Feline Alphacoronavirus 1 Strain from Brazil.

We identified a strain of Alphacoronavirus 1, FCoV-SB22, from a pool of fecal samples from domestic cats from a rural settlement in the municipality of Santa Bárbara, Pará, Brazil. The nucleotide identity with feline coronavirus was 91.5%. The present study reports the first complete genome sequence of a feline coronavirus from Brazil.

Proposed New Strain of Canine Kobuvirus from Fecal Samples of Brazilian Domestic Dogs.

A proposed new strain of canine Kobuvirus was identified in fecal samples of domestic dogs from a rural community located in the municipality of Peixe-Boi, Pará, Brazil. The nucleotide identity was 92.3% similar to other representatives of the family Picornaviridae, genus Kobuvirus, and species Aichivirus A, which suggests that this is possibly a new strain within this species.

Correction: Rotavirus A in wild and domestic animals from areas with environmental degradation in the Brazilian Amazon.

[This corrects the article DOI: 10.1371/journal.pone.0209005.].

Rotavirus A in wild and domestic animals from areas with environmental degradation in the Brazilian Amazon.

Acute gastroenteritis is one of the main causes of mortality in humans and young animals. Domestic and mainly wild animals such as bats, small rodents and birds are highly diversified animals in relation to their habitats and ecological niches and are widely distributed geographically in environments of forest fragmentation in some areas of the Amazon, being considered important sources for viruses that affect humans and other animals. Due to the anthropical activities, these animals changed their natural habitat and adapted to urbanized environments, thus representing risks to human and animal health. Although the knowledge of the global diversity of enteric viruses is scarce, there are reports demonstrating the detection of rotavirus in domestic animals and animals of productive systems, such as bovines and pigs. The present study investigated the prevalence of Rotavirus A in 648 fecal samples of different animal species from the northeastern mesoregion of the state of Pará, Brazil, which is characterized as an urbanized area with forest fragments. The fecal specimens were collected from October 2014 to April 2016 and subjected to a Qualitative Real-Time Polymerase Chain Reaction (RT-qPCR), using the NSP3 gene as a target. It was observed that 27.5% (178/648) of the samples presented positive results for RVA, with 178 samples distributed in birds (23.6%), canines (21.35%), chiropterans (17.98%), bovines (14.6%), horses (8.43%), small rodents (6.74%), pigs (3.93%) and felines (3.37%), demonstrating the circulation of RVA in domestic animals and suggesting that such proximity could cause transmissions between different species and the occurrence of rearrangements in the genome of RVA as already described in the literature, associated to the traces of environmental degradation in the studied areas.

First Whole-Genome Characterization of Avian Nephritis Virus 2 of Broiler Chicken from Pará, Brazil.

Our results show the first full-genome characterization of avian nephritis virus 2 recovered from stools of broiler chickens at a commercial farm located in Benevides, Pará, Brazil. Nucleotide analyses of whole-genome sequences showed the isolate to be a strain of Avastrovirus 2 in the family Astroviridae.

A Proposed New Strain of Avian Picornavirus in Broiler Chicken from Brazil.

A new strain of avian picornavirus was identified in fecal samples from broiler chickens in a commercial farm in the municipality of Benevides, Pará, Brazil. Genomic analysis showed it to have a nucleotide identity of 78.4% with the family Picornaviridae, genus Avisivirus, and species Avisivirus A, suggesting that this is a possible new strain within this species.

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.

Norovirus genogroups I and II in environmental water samples from Belém city, Northern Brazil.

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.

Detection of a novel equine-like G3 rotavirus associated with acute gastroenteritis in Brazil.

Genotype G3P[8] of rotavirus A (RVA) is detected worldwide, usually associated with Wa-like constellation and exhibiting a long RNA migration pattern. More recently, a novel inter-genogroup, G3P[8] reassortant variant with a short electropherotype, has emerged in Asia, Oceania and Europe, denoting an overall potential of unusual rotavirus strains. During a RVA surveillance in Brazil, G3P[8] strains were found displaying a short electropherotype pattern, which had not been detected before in this region. This study aims to characterize the complete genome of 10 G3P[8] strains detected in the northern region of Brazil. All G3P[8] samples were subjected to partial sequencing, and the whole-genome phylogenetic analysis demonstrated that all strains possessed I2-R2-C2-M2-A2-N1-T2-E2-H2 genotype background, representing reassortants with an equine-like G3 VP7 and amino acid changes in VP4 and VP7 antigenic regions as compared to vaccine strains. Phylogenetic analysis demonstrated high nucleotide identity in almost all RNA segments of G3P[8] DS-1 samples detected in Asia, Oceania and Europe as well as G3P[4] strains in Japan. This study reports a novel, equine-like G3P[8] strain circulating in Brazil and isolated from children hospitalized for severe gastroenteritis, and highlights the complex dynamics of RVA molecular epidemiology. Our findings point to a novel RVA strain emerging in this region, and studies should be done to detect whether this may represent a challenge to current vaccine strategies.

Analysis of uncommon norovirus recombinants from Manaus, Amazon region, Brazil: GII.P22/GII.5, GII.P7/GII.6 and GII.Pg/GII.1.

Norovirus (NoV) is responsible for outbreaks and sporadic cases of nonbacterial acute gastroenteritis in humans worldwide. The virus consists of small round particles containing a single-stranded RNA genome that is divided into three Open Reading Frames. NoV evolves via mechanisms of antigenic drift and recombination, which lead to the emergence of new strains that are capable of causing global epidemics. Recombination usually occurs in the ORF1/ORF2 overlapping region and generates strains with different genotypes in the polymerase and capsid region. The primary objective of this study was to analyze recombination in positive-NoV samples. Specimens were collected during 2011, 2012 and 2014, from children under two years of age presenting gastrointestinal symptoms such as vomiting and diarrhea. The partial polymerase (B region), capsid (D region) genes and the ORF1-ORF2 overlap regions were sequenced in each sample. The recombinant analyses were performed in the Simplot software v.3.5.1 and RDP4 Beta v. 4.6 program. These analyses showed that GII.Pg/GII.1, GII.P7/GII.6, and GII.P22/GII.5 were recombinant strains. To our knowledge, this is the first time that the GII.P22/GII.5 and GII.Pg/GII.1 strains were described in South America and the GII.P7/GII.6 was detected in Northern of Brazil.

Clinical Severity and Rotavirus Vaccination among Children Hospitalized for Acute Gastroenteritis in Belém, Northern Brazil.

In March 2006, Brazil introduced the monovalent rotavirus (RV) vaccine (Rotarix™) into the public sector. This study assessed the severity of rotavirus gastroenteritis (RVGE) according to the vaccination status among hospitalized children. We identified 1023 RVGE episodes among not vaccinated (n = 252), partially vaccinated (n = 156) and fully vaccinated (n = 615) children. Very severe gastroenteritis (scored ≥ 15) was reported in 16.7, 17.9 and 13.5% of not vaccinated, partially vaccinated and fully vaccinated children, respectively. There was a trend for a shorter duration of RV diarrhoea among vaccinated children than in not vaccinated children (p = 0.07). A protective effect of vaccination was noted when mean duration of symptoms and hospital stay are analysed, comparing unvaccinated, partially vaccinated and fully vaccinated children (p < 0.05). We showed a vaccination dose effect trend, with fully vaccinated children having less-severe RVGE than not vaccinated and partially vaccinated children.

Phylogenetic analysis of human group C rotavirus in hospitalized children with gastroenteritis in Belém, Brazil.

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.

High prevalence of G12P[8] rotavirus strains in Rio Branco, Acre, Western Amazon, in the post-rotavirus vaccine introduction period.

The present study aimed to provide a molecular characterization of circulating rotavirus (RVA) strains in Rio Branco, Acre, in the post-rotavirus vaccination period, particularly with regard to the emerging, increasingly prevalent G12P[8] genotype. A total of 488 fecal specimens from diarrheic and non-diarrheic children were obtained between January and December 2012. RVA detection was initially performed using enzyme-linked immunosorbent assay (ELISA) method, followed by reverse-transcription polymerase chain reaction (RT-PCR) using specific primers. RVA was detected in 18.3% (44/241) of the children with acute diarrhea and in 1.2% (3/247) of the non-diarrheic children (P < 0.001), with overall RVA-positivity of 9.6% (47/488). The most common genotype was G2P[4] with 43.2% (19/44) of the diarrheic cases, followed by G12P[8] (27.3%, 12/44), G3P[6] (18.2%, 8/44), G3P[8] (4.5%, 2/44), and G12P[6] (2.3%, 1/44). G12 samples belonged to lineage III and were from children aged 4-52 months. All of these children had acute diarrhea associated with fever (83.3%, 10/12) and vomiting (66.7%, 8/12). Most of the cases occurred in August (58.3%, 7/12), 75% (9/12) of which having received the full vaccination scheme with Rotarix™. For the first time G12 was reported at relative high prevalence in Brazil. Our findings warrant further monitoring studies on the molecular characterization of circulating RVA strains after rotavirus vaccine introduction in Brazil and elsewhere, since the occurrence of either unusual our emerging genotypes may pose a challenge to vaccination strategies.

Occurrence of Norovirus GIV in Environmental Water Samples from Belém City, Amazon Region, Brazil.

Noroviruses are the major cause of non-bacterial acute gastroenteritis outbreaks in humans, with few reports about the occurrence of the norovirus GIV strain. We investigated the presence of norovirus GIV in surface water (river, bay, and stream) and untreated sewage, and we determined a positivity rate of 9.4% (9/96). The strains genotyped were GIV.1. To our knowledge, this is the first report of GIV in Brazil.