Delana Andreza Melo Bezerra

Detection of avian group D rotavirus using the polymerase chain reaction for the VP6 gene

Group D rotaviruses (RVs-D) have been documented in birds and, while they may be common in these animals, few molecular studies are available for this specific group. In this study, specific primers for the gene that encodes for the RVs-D VP6 protein were designed and used in a reverse transcription polymerase chain reaction (RT-PCR). Thirty pools of samples were tested by polyacrylamide gel electrophoresis (PAGE) yielding a 30% (9/30) positivity. These pools were subjected subsequently to RT-PCR, with a 53% (16/30) positivity rate. The sensitivity of the PCR assay was demonstrated up to a dilution of 5 × 10(-4)ng/μL (0.5 pg/μL) of the cloned VP6 gene. The four samples were sequenced and showed 90.8-91.1% similarity with regards to the RVs-D VP6 gene. To assess for specificity our RT-PCR was applied to nine samples known to contain enteric viral agents other than group D rotaviruses including picobirnavirus, rotavirus group A, and reovirus with negative results. Overall, the data confirm the specificity of the primers used for detecting the RVs-D by RT-PCR, suggesting that this assay can be used for diagnostic purposes.

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.

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.

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.

Analysis of a genotype G3P[9] rotavirus a strain that shows evidence of multiple reassortment events between animal and human rotaviruses†

The species A rotaviruses (RVA) are important gastroenteric pathogens that infect humans and animals. RVA genotype G3P[9] has been described in human‐animal reassortment events, and the complexity of its hosts motivates the genetic investigation of this strain. Therefore, the aim of this study is to analyse a G3P[9] sample that was detected in a child with acute gastroenteritis. The 1A3739 sample featured the constellation G3P[9]‐I18‐R3‐C3‐Mx‐A19‐N3‐T3‐E3‐H6. The sequence for VP3 gene was not obtained. The phylogeny showed a closer relationship among genes VP7, VP1, NSP3, NSP4, and NSP5 with genes of animal origin, such as chiropter, alpaca, equine, and simian. In addition, the genes VP6 and NSP1 belong to the new genotypes I18 and A19, respectively. The emergence of strains such as these can interfere with the effectiveness of the RVA vaccine, and continuous monitoring is therefore important. Additional studies are needed to determine the evolutionary source and to identify a possible reservoir of RVA in nature.

Molecular epidemiology of avian rotavirus in fecal samples of broiler chickens in Amazon Region, Brazil, from August 2008 to May 2011

Enteric viruses cause avian diseases that result in economic losses. Avian Rotavirus (AvRV) is the most important virus associated with enteritis in poultry. The main goals of this study were to determine the prevalence of AvRV using molecular tests in broiler chickens (Gallus gallus) from the metropolitan mesoregion of Belem (Para State, Brazil), to provide epidemiological information, and to compare the rotaviruses detected in this study with reference to strains by phylogenetic analysis. Pooled fecal samples were collected from 37 poultry farms. The samples were tested for the NSP4 gene using reverse transcription polymerase chain reaction (RT-PCR). In total, 35 (41.2%) of the 85 fecal samples were positive for NSP4. There were 19 (51.4%) farms with at least one poultry house positive for AvRV. Considering the distribution of positive samples by age, the chicks aged 31-45 days comprised 18 (51.4%) of the 35 rotavirus-positive samples. Analyzing the data by density population, the houses with more than 9 birds/m2 had 25 (86.2%) positive samples, showing that higher infection rates occurred in higher density houses. To confirm the RT-PCR results and perform phylogenetic analysis, 20 positive samples were selected for sequencing. The rotaviruses detected in our study were clustered in a single group and had 93.5 to 100% sequence identity at the nucleotide level. The most affected age group included broiler chickens older than 15 days. Climatic conditions and high population densities favored the spread of AvRV and supported its uniform maintenance between seasons.

DETECTION AND MOLECULAR EPIDEMIOLOGY OF HUMAN BOCAVIRUS IN CHILDREN WITH ACUTE GASTROENTERITIS FROM BRAZIL

Human Bocavirus (HBoV) is a recently discovered virus and was first detected in the nasopharyngeal aspirate samples and after in stool samples, suggesting that HBoV may be a causative agent for human enteric infections. Due to absence of treatment options, there is a need to understand the disease-causing mechanism of these viruses. The aim of this was to demonstrate the prevalence of HBoV from children less than 10 years with acute gastroenteritis in Brazil, during November 2011 to November 2012. Stool samples from hospitalized children ≤ 10 years who presented symptoms of acute gastroenteritis were analyzed for the presence of HBoV DNA by nested-PCR. HBoV- positivity was detected in 24.0% (54/225) of samples. Two peaks of HBoV detection were observed, during November 2011 and July to September 2012. Co-infections between HBoV and rotavirus A were identified in 50.0% (27/54) of specimens. Phylogenetic analysis identified the presence of HBoV-1 (94.8%), HBoV-2 (2.6%) and HBoV-3 (2.6%) species, with only minor variations among them. Further investigations are necessary to improve the knowledge on the role of HBoV in gastrointestinal infections.

Epidemiología molecular del rotavirus aviario en heces de pollos de corte en la Región Amazónica, Brasil, de agosto de 2008 a mayo de 2011

Enteric viruses cause avian diseases that result in economic losses. Avian Rotavirus (AvRV) is the most important virus associated with enteritis in poultry. The main goals of this study were to determine the prevalence of AvRV using molecular tests in broiler chickens (Gallus gallus) from the metropolitan mesoregion of Belem (Para State, Brazil), to provide epidemiological information, and to compare the rotaviruses detected in this study with reference to strains by phylogenetic analysis. Pooled fecal samples were collected from 37 poultry farms. The samples were tested for the NSP4 gene using reverse transcription polymerase chain reaction (RT-PCR). In total, 35 (41.2%) of the 85 fecal samples were positive for NSP4. There were 19 (51.4%) farms with at least one poultry house positive for AvRV. Considering the distribution of positive samples by age, the chicks aged 31-45 days comprised 18 (51.4%) of the 35 rotavirus-positive samples. Analyzing the data by density population, the houses with more than 9 birds/m2 had 25 (86.2%) positive samples, showing that higher infection rates occurred in higher density houses. To confirm the RT-PCR results and perform phylogenetic analysis, 20 positive samples were selected for sequencing. The rotaviruses detected in our study were clustered in a single group and had 93.5 to 100% sequence identity at the nucleotide level. The most affected age group included broiler chickens older than 15 days. Climatic conditions and high population densities favored the spread of AvRV and supported its uniform maintenance between seasons.

Epidemiologia molecular do rotavirus aviário em fezes de frangos de corte na Região Amazônica, Brasil, de agosto de 2008 a maio de 2011

Enteric viruses cause avian diseases that result in economic losses. Avian Rotavirus (AvRV) is the most important virus associated with enteritis in poultry. The main goals of this study were to determine the prevalence of AvRV using molecular tests in broiler chickens (Gallus gallus) from the metropolitan mesoregion of Belem (Para State, Brazil), to provide epidemiological information, and to compare the rotaviruses detected in this study with reference to strains by phylogenetic analysis. Pooled fecal samples were collected from 37 poultry farms. The samples were tested for the NSP4 gene using reverse transcription polymerase chain reaction (RT-PCR). In total, 35 (41.2%) of the 85 fecal samples were positive for NSP4. There were 19 (51.4%) farms with at least one poultry house positive for AvRV. Considering the distribution of positive samples by age, the chicks aged 31-45 days comprised 18 (51.4%) of the 35 rotavirus-positive samples. Analyzing the data by density population, the houses with more than 9 birds/m2 had 25 (86.2%) positive samples, showing that higher infection rates occurred in higher density houses. To confirm the RT-PCR results and perform phylogenetic analysis, 20 positive samples were selected for sequencing. The rotaviruses detected in our study were clustered in a single group and had 93.5 to 100% sequence identity at the nucleotide level. The most affected age group included broiler chickens older than 15 days. Climatic conditions and high population densities favored the spread of AvRV and supported its uniform maintenance between seasons.