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.

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.

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.

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.

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.

Genogroup I avian picobirnavirus detected in Brazilian broiler chickens: a molecular epidemiology study.

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 15.3 % (13/85) by PAGE and 49.4 % (42/85) by RT-PCR. Sequencing of these strains demonstrated a considerable RdRp gene heterogeneity that ranged from 56.1 to 100 % at the nucleotide level compared with prototypes of different species and water sewage, and from 50.3 to 100 % 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.

Diversity of rotavirus strains circulating in Northern Brazil after introduction of a rotavirus vaccine: high prevalence of G3P[6] genotype.

Rotavirus A (RVA) is the most common cause of severe acute gastroenteritis in infants and young children worldwide, causing 453,000 deaths annually. In Brazil, the most frequent genotype identified was G1 during almost three decades in the pre-vaccination period; however, after anti-rotavirus vaccine introduction, there was a predominance of G2 genotype. The aim of this study was to determine the G and P genotypes of rotaviruses isolated from children under 5 years of age with acute gastroenteritis in the Northern region of Brazil, and discuss the emergence of G3P[6] genotype. A total of 783 stool specimens were obtained between January 2011 and March 2012. RVA antigen was detected in 33% (272/783) of samples using a commercial enzyme-linked immunosorbent assay and type-specificity was determined by reverse-transcription polymerase chain reaction. The most common binary combination was G2P[4], representing 41% of cases, followed by G3P[6] (15%), G1P[8] (8%), G3P[8] (4%), G9P[8] (3%), and G12P[6] (2%). G3P[6] strains were analyzed further and phylogenetic analysis of VP7 gene showed that G3 strains clustered into lineage I and showed a high degree of amino acid identity with vaccine strain RV3 (95.1-95.6%). For VP4 sequences, G3P[6] clustered into lineage Ia. It was demonstrated by the first time the emergence of unusual genotype G3P[6] in the Amazon region of Brazil. This genotype shares neither VP7 nor VP4 specificity with the used vaccine and may represent a challenge to vaccination strategies. A continuous monitoring of circulating strains is therefore needed during the post-vaccine era in Brazil.

Genetic diversity of porcine sapoviruses in pigs from the Amazon region of Brazil.

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.

Detection, epidemiology and characterization of VP6 and VP7 genes of group D rotavirus in broiler chickens.

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.

Norovirus infection in children admitted to hospital for acute gastroenteritis in Belém, Pará, Northern Brazil.

Noroviruses are the leading cause of epidemic, non-bacterial outbreaks of acute gastroenteritis, and are also a major cause of sporadic acute gastroenteritis in infants. The aim of the present study was to identify norovirus infections in children not infected by rotavirus admitted to hospital for acute gastroenteritis in Belém. A total of 348 fecal specimens were obtained from children with diarrhea aged less than 5 years, all of whom had tested negative for rotavirus, between May 2008 and April 2010. Fecal samples were screened for norovirus antigen using enzyme-immunoassay (EIA). Specimens were subjected to reverse-transcription polymerase chain reaction (RT-PCR) using the primers Mon432/434-Mon431/433 for detection of the GI and GII norovirus strains, respectively. Based on both methods, the overall norovirus positivity rate was 36.5% (127/348). Of the 169 samples collected in the first year, 44.4% (n = 75) tested positive for norovirus using both methods, 35.5% (n = 60) by EIA and 40.8% (n = 69) by RT-PCR. Using RT-PCR as a reference standard, a sensitivity of 78.3%, specificity of 94%, and agreement of 87.6% were recorded. Genome sequencing was obtained for 22 (31.9%) of the 69 positive samples, of which 90.9% (20/22) were genotype GII.4d and 9.1% (2/22) were genotype GII.b. Norovirus infection was most frequent in children under 2 years of age (41.5%-115/277). The peak incidence (62.1%) of norovirus-related acute gastroenteritis in these patients (not infected by rotavirus) was observed in February 2010. These findings emphasize the importance of norovirus as a cause of severe acute gastroenteritis among children in Belém, Pará, Northern Brazil.

Detection of the pandemic norovirus variant GII.4 Sydney 2012 in Rio Branco, state of Acre, northern Brazil.

Noroviruses (NoVs) are important cause of gastroenteritis in humans worldwide. Genotype GII.4 is responsible for the majority of outbreaks reported to date. This study describes, for the first time in Brazil, the circulation of NoV GII.4 variant Sydney 2012 in faecal samples collected from children aged less than or equal to eight years in Rio Branco, state of Acre, northern Brazil, during July-September 2012.

Group A rotavirus and norovirus display sharply distinct seasonal profiles in Belém, northern Brazil.

Several viruses have been associated with acute gastroenteritis (AGE), and group A rotavirus (RVA) and norovirus (NoV) are the most prevalent. This study aimed to assess their prevalence among children hospitalised for diarrhoea during a three-year surveillance study. From May 2008-April 2011, overall positivity rates of 21.6% (628/2904) and 35.4% (171/483) were observed for RVA and NoV, respectively. The seasonality observed indicated distinct patterns when both viruses were compared. This finding may explain why hospitalisation for AGE remains constant throughout the year. Continuous AGE monitoring is needed to better assess the patterns of infection.

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.

Phylogenetic analysis of probable non-human genes of group A rotaviruses isolated from children with acute gastroenteritis in Belém, Brazil.

Rotaviruses (RVs) are the main cause of acute viral gastroenteritis in both humans and young animals of various species such as calves, horses, pigs, dogs, cats, and birds. The genetic diversity of RVs is related to a variety of evolutionary mechanisms, including point mutation, and genome reassortment. The objective of this study was to characterize molecularly genes that encode structural and nonstructural proteins in unusual RV strains. The clinical specimens selected for this study were obtained from children and newborn with RV gastroenteritis, who participated in research projects on viral gastroenteritis conducted at the Evandro Chagas Institute. Structural (VP1-VP4, VP6, and VP7) and nonstructural (NSP1-NSP6) genes were amplified from stool samples by the polymerase chain reaction and subsequently sequenced. Eight unusual RV strains isolated from children and newborn with gastroenteritis were studied. Reassortment between genes of animal origin were observed in 5/8 (62.5%) strains analyzed. These results demonstrate that, although rare, interspecies (animal-human) transmission of RVs occurs in nature, as observed in the present study in strains NB150, HSP034, HSP180, HST327, and RV10109. This study is the first to be conducted in the Amazon region and supports previous data showing a close relationship between genes of human and animal origin, representing a challenge to the large-scale introduction of RV vaccines in national immunization programs.

Identification of lineage III of G12 rotavirus strains in diarrheic children in the Northern Region of Brazil between 2008 and 2010.

This study reports on the surveillance for rotavirus genotypes and the identification of G12 human rotavirus in the Northern Region of Brazil. Rotavirus-positive samples were collected from children <5 years of age with acute diarrhea from January 2008 to October 2010. G2P[4] was the most prevalent genotype, accounting for 45.6% (126/303) of cases. Five rotavirus strains bearing G12P[6] genotype specificity were detected. Phylogenetic analysis of the VP7 gene showed that G12 strains clustered into lineage III. This is the first detection of G12 strains from lineage III in Latin America, broadening the current evidence for the worldwide emergence of this genotype.

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.

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.

Identification of two sublineages of genotype G2 rotavirus among diarrheic children in Parauapebas, Southern Pará State, Brazil.

On a world scale, group A human rotaviruses are the most common cause of severe acute gastroenteritis during infancy and childhood, including five (G1, G2, G3, G4, and G9) epidemiologically important genotypes. Among these, G2 denotes a different genogroup which appears to have a cyclic pattern of occurrence and yet little information is available about its genetic variability. The aim of this report was to characterize the emergence of G2 genotype in Paraupebas, Southern Pará State, Brazil, some of which detected after introduction of rotavirus vaccine. A total of 241 fecal specimens from young children with acute gastroenteritis were collected from the "Yutaka Takeda Hospital," a Municipality Hospital, and at the Parauapebas' Health Unit, Pará, from January to September 2006 and during March to November 2008. All samples were tested for rotavirus using immunochromatography, polyacrylamide gel electrophoresis (PAGE), and RT-PCR, yielding an overall positivity of 12.45% (30/241). Rotavirus G2P[4] was identified in 27 of 30 samples (90%), followed by G1P[8] (2/30, 6.67%) and G9P[8] (1/30, 3.33%). Phylogenetic analysis was performed in 15 of the G2 strains, all of which grouped into lineage II. Four of these strains clustered into sublineage II-a (year 2006) and 11 into one possible new sublineage named II-c (year 2008, except SAL-1920-C). The recent re-emergence of G2 genotype associated with lineage II in Brazil warrants the continuous monitoring of circulating rotavirus strains following the nationwide universal use of rotavirus vaccine.

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.