Mônica Simões Rocha Ferreira

Viral load and genotypes of noroviruses in symptomatic and asymptomatic children in Southeastern Brazil

BACKGROUND Noroviruses (NoVs) are a major etiological agent of sporadic acute gastroenteritis worldwide. OBJECTIVES To detect, quantify and characterize genogroups and genotypes of NoVs in children with and without gastrointestinal symptoms. STUDY DESIGN NoVs were investigated by RT-PCR in a total of 319 fecal specimens from children up to three years old with (n=229) and without (n=90) acute diarrhea, between February 2003 and June 2004 in the emergency room in Vitória, Southeastern Brazil. NoVs were quantified by real-time PCR and genotyped. RESULTS NoVs were detected in 17% (40/229) and 13% (12/90) of symptomatic and asymptomatic children, respectively. Six NoV-rotavirus A mixed infections were observed. Fifty-one strains were characterized as NoV GII and one as GI. Twenty strains were characterized as GII/4 (9/13), GII/3 (1/13), GII/6 (2/13) and GII/14 (1/13) in symptomatic and GII/3 (6/7) and GII/8 (1/7) in asymptomatic children. The median RNA viral loads were 8.39 and 7.15log(10)copies/g of fecal specimens for symptomatic and asymptomatic children, respectively (p=0.011). NoV load was lower when it was present in a mixed infection with rotavirus A (p=0.0005). CONCLUSIONS This study demonstrates a diversity of NoV strains circulating in this geographic area, and reports GII/8 and GII/14 in the American Continent for the first time. In addition, it confirms GII/4 as the most prevalent genotype in symptomatic children and identified GII/3 in an important frequency, especially in asymptomatic children. Furthermore, preliminary results show that symptomatic patients present a viral load that is significantly greater than asymptomatic children (p=0.011).

Surveillance of norovirus infections in the state of Rio De Janeiro, Brazil 2005-2008.

A 4‐year (2005–2008) norovirus (NoV) surveillance study was conducted in the state of Rio Janeiro, Brazil, to demonstrate the role of these viruses in outbreaks and sporadic cases of acute gastroenteritis. A cohort of 1,687 fecal samples was obtained from patients with gastroenteritis; 324 were rotavirus‐positive. Of the remainder 1,363 rotavirus‐negative samples, 1,087 samples were tested for NoV RNA in this study. The study enrolled 267 outpatients from Municipal Public Health Centers and 820 inpatients, whose samples were obtained by active surveillance in Public Hospitals. Fecal samples were tested by reverse transcription (RT) followed by polymerase chain reaction (PCR) using the MON 431–434 set of degenerate primers for NoV GI and GII detection, and there were 35.1% (381/1,087) positive samples for NoV, consisting of 30.2% (248/820) and 49.8% (133/267) from inpatient and outpatient, respectively. Children infected by NoV had significantly more frequent mucus in feces, vomiting and fever. No seasonal pattern in NoV infections was observed in patients admitted to hospital; however, two peaks of NoV infections were observed from ambulatory cases, suggesting that there was an occurrence of outbreaks in those time periods. Molecular characterization revealed GII to be the most prevalent genogroup, totaling 96.3% (104/108) of all sequences analyzed, and GII.4 was the genotype detected most frequently (80.7%), followed by GII.6, 3, 14, 7, and 8. Two GI strains, GI.2 and GI.3, were also observed. The number of outbreaks and sporadic cases described in this study highlights the need to implement diagnosis of NoV in surveillance laboratories. J. Med. Virol. 82:1442–1448, 2010.

Genetic diversity of noroviruses in Brazil

Norovirus (NoV) infections are a major cause of acute gastroenteritis outbreaks around the world. In Brazil, the surveillance system for acute diarrhoea does not include the diagnosis of NoV, precluding the ability to assess its impact on public health. The present study assessed the circulation of NoV genotypes in different Brazilian states by partial nucleotide sequencing analysis of the genomic region coding for the major capsid viral protein. NoV genogroup II genotype 4 (GII.4) was the prevalent (78%) followed by GII.6, GII.7, GII.12, GII.16 and GII.17, demonstrating the great diversity of NoV genotypes circulating in Brazil. Thus, this paper highlights the importance of a virological surveillance system to detect and characterize emerging strains of NoV and their spreading potential.

Rotavirus Strain Diversity in Rio de Janeiro, Brazil: Characterization of VP4 and VP7 Genotypes in Hospitalized Children

Rotavirus strains from 91 patients treated at a childrens hospital from 1996 to 1998 in Rio de Janeiro, Brazil, were characterized by electropherotyping, reverse transcription-PCR amplification for P and G genotypes, and Southern hybridization. Results obtained showed that following predominant [P],G type combination: P[4], G2 (21 per cent), P[8], G1 (17 per cent), P[8], G3 (13 per cent), which are prevalent throughout the world. However, an unexpected number of cases were associated with uncommon genotypes: P[8], G2 (13 per cent), P[8], G5 (11 per cent), P[8], G9 (7 per cent), P[8], G10 (4 per cent), P[6], G4 (3 per cent), P[6], G3 (1 per cent), P[4], G9 (1 per cent), and P[6], G9 (1 per cent). Mixed infections with more than one type were identified in only two cases and 16 per cent of the samples were not G and/or P typeable. A subset of G types was confirmed by Southern hybridization and chemiluminescent detection. Rotavirus seasonal distribution was observed between April and July. The contribution of the results obtained in the present investigation corroborates the required epidemiological surveillance for rotavirus infection in Brazil.

Detection of caliciviruses associated with acute infantile gastroenteritis in Salvador, an urban center in Northeast Brazil

Acute gastroenteritis caused by viruses is one of the leading causes of infantile morbidity. The aim of the present study was to investigate the presence of human caliciviruses of the genera norovirus and sapovirus in children up to 3 years of age with acute gastroenteritis from low-income communities in the city of Salvador, Brazil. This study is an extension of previous work carried out to establish the profile of the most prevalent enteric pathogens present in these communities. In this report, 139 fecal samples, collected from July 2001 to January 2002 were analyzed by RT-PCR and 13 (9%) were positive for human caliciviruses. By sequencing, seven isolates were characterized as norovirus genogroup GII and one as sapovirus genotype GII/1. Sequencing of the previously detected group-A rotaviruses and human astroviruses was also performed and revealed the circulation of rotavirus group A genotypes G1P[8] and G9P[8], and human astrovirus genotypes 6, 7, and 8. No mixed infection was observed. Community-based studies provide geographically representative information on disease burden. However, there are only a few reports in developing countries concerning the genotypes of the most important gastroenteric viruses detected in such communities. The present findings demonstrate the wide diversity of genotypes of the most important viruses responsible for acute gastroenteritis circulating in low-income communities.

Bayesian coalescent inference reveals high evolutionary rates and expansion of Norovirus populations

Noroviruses (NoV) are a leading cause of outbreaks of nonbacterial acute gastroenteritis in humans worldwide and have become an important cause of hospitalization of children in South America. NoV belong to the family Caliciviridae and are non-enveloped single stranded, positive sense, RNA viruses. NoV of genotype GII/4 have emerged worldwide, causing four epidemic seasons of viral gastroenteritis during which four novel variants emerged. Despite the importance of NoV outbreaks, little is known about the evolutionary rates, viral spread and population dynamics of NoV populations. In order to gain insight into these matters, a Bayesian Markov chain Monte Carlo (MCMC) approach was used to analyze region D or full-length VP1 gene sequences of GII/4 NoV populations isolated in Brazil or Japan, respectively. The results of these studies revealed that the expansion population growth model was the best to fit the data in both datasets. The dates of the most common recent ancestors revealed that these viruses can quickly emerge in a geographical location. A mean evolutionary rate of 1.21 x 10(-2) nucleotide substitution/site/year (s/s/y) was obtained for the VP1 gene using full-length sequences. This rate is higher than the rates reported for other rapidly evolving RNA. Roughly similar rates (1.44 x 10(-2)s/s/y) were found using region D sequences, revealing the suitability of this region for evolutionary studies, in agreement with previous reports. High evolutionary rates and fast population growth may have contributed to the vigorous initial transmission dynamics of the GII/4 NoV populations studied.

Molecular Characterization of Noroviruses and HBGA from Infected Quilombola Children in Espirito Santo State, Brazil

Noroviruses (NoV) are the main etiological agents of gastroenteritis outbreaks worldwide and susceptibility to NoV infection has been related to the histo-blood group antigen (HBGA). This study aimed to determine the prevalence of NoV strains and to evaluate the HBGA phenotype and genotype of children from semi-isolated Quilombola communities, descendents of black slaves in Brazil. A total of 397 children up to eleven years old, with and without diarrhea, from Quilombola Communities in the Espirito Santo State, Brazil, were investigated for the presence of NoV from August 2007 to September 2009. Feces were collected from all the children, and blood from the NoV positive children. NoV was screened by reverse transcription-PCR with primers for the RNA-dependent RNA polymerase region; genogroup was determined by PCR with primers for the C and D regions and genotyped by sequencing. HBGA phenotype was performed by gel-spinning and FUT2 and FUT3 were analyzed by PCR or sequencing analysis. NoV were detected in 9.2% (12/131) of diarrheic and 1.5% (4/266) of non-diarrheic children (p<0.05, Fisher’s exact test). GI and GII genogroups were present in 12.5% and 87.5% of the samples, respectively. The following genotypes were characterized: GII.4 (25%), GII.12 (25%), GII.6 (12.5%) and GI.1 (6.3%), GI.3 (12.5%) and GI.4 (6.3%). Children infected with NoV showed the A (n = 6), O (n = 6), and B (n = 2) HBGA phenotypes, and 13 of them were classified as secretors (Se) and one as a non secretor (se). Mutations of Se 40, 171,216,357,428,739,960 were found for the FUT2 gene and mutations of Le 59, 202, 314 for the FUT3 gene. The only se child was infected by NoV GI, whereas the Se children were indiscriminately infected by GI or GII. This study showed rates of NoV infection in symptomatic and asymptomatic Quilombola children consistent with other studies. However, children under 12 months were seven times more affected than those between 1 and 5 years old. GII.12 was as frequent as GII.4 and GI.1 and GI.4 were described for the first time in Brazil. Owing to the small number of cases studied, no clear pattern of susceptibility and/or HBGA resistance could be inferred.

Genotyping of gastroenteric viruses in hospitalised children: first report of norovirus GII.21 in Brazil.

This retrospective study (April-September 2003) was designed to investigate the roles of the main viruses responsible for cases of acute infantile gastroenteritis in hospitalised children up to two years of age. The viruses were identified in 64.7% (88/136) of the cases and the detection rates of rotavirus A (RVA), norovirus (NoV) and astrovirus were 41.9% (57/136), 30.3% (24/79) and 12.7% (7/55), respectively. RVA and NoV were detected in 20 of the 24 reported nosocomial infection cases. This study identified the first circulation of the genotype NoV GII.21 in Brazil and highlights the need to establish differential diagnoses through active laboratorial surveillance.

HCMV gB genotypes in cervical secretion and placenta tissues in the state of Espírito Santo, Southeastearn Brazil

Human cytomegalovirus (HCMV) displays genetic variability in several regions, supposed to be related with strain-specific tissue tropism and immunopathogenesis. Based on sequence variation in the UL55 gene that encodes gB glycoprotein, HCMV strains can be assigned to one of four genotypes. Previous studies have addressed gB genotyping mostly by investigating strains derived from immunosuppressed patients, sometimes without previous knowledge about genotype distribution in a geographic area. The present study verified the distribution of HCMV gB genotypes of strains obtained from immunocompetent women at Vitoria City, Espirito Santo State, Southeastern, Brazil. The HCMV genome was extracted from their cervical secretion, fetal and maternal placenta tissues (chorionic villous and decidua) from abortion cases and from white blood cells (WBCs). HCMV genotyping was performed by restriction fragment length polymorphism analyses of amplified product from the high variability site of the UL55 gene. All four genotypes were observed in both cervical secretion and placenta, whereas in WBCs a single gB1 genotype was detected. HCMV gB1 and gB2 genotypes were detected, respectively, in nine and in six of the 23 studied samples, while gB3 and gB4 were each found in four separate samples of the total. The differences in genotype frequency were not considered statistically significant. No mixed genotype infection was observed. The results indicated that the four gB HCMV genotypes had no particular tropism for placenta tissues and that all genotypes circulated within immunocompetent women at the time and in the region of study.