Sylvia de Fátima dos Santos Guerra

Effectiveness of the monovalent G1P[8] human rotavirus vaccine against hospitalization for severe G2P[4] rotavirus gastroenteritis in Belém, Brazil.

Background: Brazil initiated universal immunization of infants with the G1P[8] human rotavirus (RV) vaccine in March 2006. This study evaluated vaccine effectiveness (VE) against severe rotavirus gastroenteritis (RVGE) hospitalizations. Methods: Matched case-control study conducted at 4 hospitals in Belém from May 2008 to May 2009. Cases were children hospitalized with RVGE age-eligible to have received 2 doses of the human RV vaccine (≥12 weeks of age and born after March 6, 2006). For each case, 1 neighborhood and 1 hospital control without gastroenteritis was selected, matching by birth date (±8 and ±6 weeks, respectively). Matched odds ratio of 2-dose RV vaccination in cases versus controls was used to estimate VE (1 − odds ratio × 100%). Results: Of 538 RVGE cases, 507 hospital controls and 346 neighborhood controls included, 54%, 61%, and 74% had received both RV vaccine doses. VE against RVGE hospitalization was 75.8% (95% confidence interval [CI]: 58.1–86.0) using neighborhood controls and 40.0% (95% CI: 14.2–58.1) using hospital controls. VE in children 3 to 11 months and ≥12 months of age was 95.7% (95% CI: 67.8–99.4) and 65.1% (95% CI: 37.2–80.6) using neighborhood controls, and 55.6% (95% CI: 12.3–77.5) and 32.1% (95% CI: −3.7–55.5) using hospital controls. G2P[4] accounted for 82.0% of RVGE hospitalizations. G2P[4]-specific VE was 75.4% (95% CI: 56.7–86.0) using neighborhood controls and 38.9% (95% CI: 11.1–58.0) using hospital controls. Conclusions: Although fully heterotypic G2P[4] was the predominant RV strain, good VE was demonstrated. VE was highest in children aged 3 to 11 months. However, protection in children ≥12 months of age, important for optimal public health impact, was significantly sustained based on estimates obtained using neighborhood controls.

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. J. Med. Virol. 86:1065–1072, 2014.

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. J. Med. Virol. 84:1993–2002, 2012.

Rotavirus strain surveillance for three years following the introduction of rotavirus vaccine into Belém, Brazil

The monovalent human rotavirus (RV) vaccine, RIX4414 (Rotarix™, GlaxoSmithKline Biologicals) was introduced into Brazils Expanded Program on Immunization in March 2006. One year after vaccine introduction, the G2P[4] strain was found to be predominant, with an apparent extinction of many non‐G2 strains. This study investigated the diversity of circulating strains in the three years following RIX4414 introduction. Between May 2008 and May 2011, stool samples were collected from children aged ≥12 weeks who were hospitalized for severe lab confirmed RV‐gastroenteritis (≥3 liquid or semi‐liquid motions over a 24‐h period for <14 days, requiring ≥1 overnight hospital stay and intravenous rehydration therapy) in Belém, Brazil. RV‐gastroenteritis was detected by ELISA and the G‐ and P‐types were determined by RT‐PCR assays. During the first year of surveillance nucleotide sequencing was used for typing those samples not previously typed by RT‐PCR. A total of 1,726 of 10,030 severe gastroentertis hospitalizations (17.2%) were due to severe RVGE. G2P[4] was detected in 57.2% of circulating strains over the whole study period, however it predominated during the first 20 months from May 2008 to January 2009. G1P[8] increased in the last part of the study period from May 2010 to May 2011 and represented 36.6% (112/306) of the circulating strains. G2P[4] was the predominant RV strain circulating during the first 20 months of the study, followed by G1P[8]. These findings probably reflect a natural fluctuation in RV strains over time, rather than a vaccine‐induced selective pressure. J. Med. Virol. 87:1303–1310, 2015.

Oral live attenuated human rotavirus vaccine (RotarixTM) offers sustained high protection against severe G9P[8] rotavirus gastroenteritis during the first two years of life in Brazilian children

In a large Phase III trial conducted in 10 Latin American countries, the safety and efficacy of the live attenuated monovalent rotavirus vaccine RIX4414 was evaluated in 15,183 healthy infants followed up during the first two years of life. Belém was the only site in Brazil included in this multicentre trial. The study in Belém included a subset of 653 infants who were followed up until 24 months of age for protection against severe rotavirus gastroenteritis. These subjects were randomly assigned in a 1:1 ratio to receive two doses of vaccine (n = 328) or two doses of placebo (n = 325) at approximately two and four months of age. Of the 653 enrolled infants, 23 dropped out during the study period. For the combined two-year period, the efficacy of RIX4414 was 72.3% [95% confidence interval (CI) 37.5-89.1%] against severe rotavirus-related gastroenteritis, reaching a protection rate of 81.8% (95% CI 36.4-96.6%) against circulating wild-type G9 rotavirus strains. It is concluded that two doses of RIX4414 are highly efficacious against severe rotavirus gastroenteritis in Belém during the first two years of life and provide high protection against the worldwide emergence and spread of G9P[8] strains.

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.

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.

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.

Caracterização molecular dos genótipos G e P de rotavírus, cepa G9, de crianças com gastrenterite aguda na Região Metropolitana de Belém, Estado do Pará, de 1999 a 2007

O rotavirus (RV) e o principal agente viral associado a gastrenterite, sendo responsavel por 39% de todas as internacoes hospitalares decorrentes de casos de diarreia, bem como por aproximadamente 520 mil mortes de criancas de ate cinco anos de idade. Pertencente a familia Reoviridae, genero Rotavirus, seu genoma e composto por 11 segmentos de RNA de cadeia dupla que codificam seis proteinas virais estruturais (VPs) e seis proteinas virais nao estruturais (NSPs). As proteinas VP4 e VP7 compoem o capsideo externo do RV e definem os genotipos P e G, respectivamente. Ate o momento, no minimo 23 genotipos de RV do tipo G e 31 do tipo P ja foram identificados. Ja foi registrada a emergencia global das cepas do sorotipo G9, as quais sao associadas a doenca mais grave em humanos. Ha tambem o registro de dupla especificidade, sendo que, na maioria das vezes, as cepas de G9 pertencem ao genotipo P[8]. Enquanto o genotipo G9 apresenta seis diferentes linhagens, quatro linhagens distintas sao reconhecidas como pertencentes ao genotipo P[8]. Este estudo teve como objetivo caracterizar os genes VP4 e VP7 dos genotipos G9 de RV que circularam na Regiao Metropolitana de Belem, Estado do Para, na Regiao Norte do Brasil, de 1999 a 2007. O dsRNA viral de 38 amostras foi extraido de suspensao fecal e analisado com o uso de eletroforese em gel de poliacrilamida para determinar os eletroferotipos, seguido de reacoes sequenciais. No total, 32 amostras de G9P[8] foram analisadas e todas elas apresentaram eletroferotipo longo. A analise filogenetica do gene VP7 mostrou que todas as cepas de G9 pertenciam a linhagem 3 com um certo grau de similaridade entre elas; apenas oito alteracoes nucleotidicas foram detectadas nesta linhagem. Entretanto, foram observadas apenas tres substituicoes de aminoacidos neste estudo, nas posicoes 43 (I®V), 66 (A®V) e 73 (Q®R). As substituicoes nas posicoes 43 e 73 foram observadas somente nas amostras de 2007. A analise filogenetica do gene VP4 revelou que todas as cepas de P[8] pertenciam a linhagem 3, com 15 alteracoes nucleotidicas correspondendo as quatro substituicoes de aminoacidos nas posicoes 108 (V®I), 172 (R®K), 173 (I®V) e 275 (K®R). As substituicoes de aminoacidos nas posicoes 172 e 275 foram observadas apenas em isolados de RV de 1999 a 2002. No total, as amostras de RV G9 demonstraram uma alta homologia por todo o periodo investigado. Curiosamente, as cepas isoladas em 2007 foram as mais divergentes no que diz respeito aos genes VP4 e VP7. Os resultados ressaltam a necessidade de uma vigilância continua de cepas de RV circulantes em nossa regiao para detectar a emergencia de novas variantes geneticas que representariam um desafio as estrategias atuais de imunizacao a RV. Esta vigilância deve incluir um monitoramento de perto da diversidade genetica das cepas emergentes de G9.Rotavirus (RV) is the major viral agent associated with gastroenteritis and is responsible for 39% of all diarrheal cases requiring hospitalization, as well as approximately 520 thousand deaths among children under five years of age. Rotavirus belongs to the Reoviridae family, genus Rotavirus and its genome consists of 11 segments of double-stranded RNA which encode six structural viral proteins (VPs) and six non-structural viral proteins (NSPs). VP4 and VP7 proteins make up the outer layer of the RV particle and define the two genotypes, P and G, respectively. To date, at least 23 G genotypes and 31 P genotypes have been identified. G9-type RV strains, which are associated with a more severe disease in humans, have emerged globally. Dual type-specificities have been reported, and G9 strains most often belong to the P[8] genotype. While G9 genotype includes six distinct lineages, four distinct lineages are currently recognized as belonging to the P[8] genotype. The aim of this study was to characterize the VP4 and VP7 genes of the RV G9 genotypes that circulated in the Metropolitan Region of Belem, Para, in northern Brazil from 1999 to 2007. The viral dsRNA of 38 samples was extracted from fecal suspensions and analyzed using polyacrylamide gel electrophoresis to determine electropherotypes, followed by sequencing reactions. Overall, 32 selected G9P[8] samples were analyzed, all of which possessed long electropherotype. Phylogenetic analysis of the VP7 gene showed that all G9 strains belonged to lineage 3 with a high degree of similarity between them; only eight nucleotide changes have been detected in this lineage. However, only three of these amino acid substitutions were observed in our study, occurring at positions 43 (I®V), 66 (A®V) and 73 (Q®R). Of interest, the substitutions at positions 43 and 73 were only found in samples from 2007. Phylogenetic analysis of the VP4 gene revealed that all of the P[8] strains belonged to lineage 3, with 15 nucleotide changes corresponding to four amino acid substitutions at positions 108 (V®I), 172 (R®K), 173 (I®V) and 275 (K®R). The amino acid substitutions at positions 172 and 275 were observed only in RV isolates from 1999 to 2002. Overall, the RV G9 samples showed high homology throughout the entire study period. Interestingly, strains isolated in 2007 were the most divergent with respect to both VP4 and VP7 genes. Our data underscore the need for continuous surveillance of circulating RV strains in our region to detect the emergence of new genetic variants that would pose a challenge to current RV immunization strategies. This should include close monitoring of the genetic diversity of emerging G9 strains.

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.