Simone Guadagnucci Morillo

Human Rotavirus Serotype G9, São Paulo, Brazil, 1996-2003

Diverse rotavirus strains are present, and frequency of G9 is high.

Rare G3P(3) rotavirus strain detected in Brazil: Possible human-canine interspecies transmission

BACKGROUND An unusual strain of human rotavirus G3P[3] (R2638 strain) was detected from a 1-year-old child patient during the epidemiological survey of rotavirus in the state of São Paulo, Brazil in 2011. OBJECTIVE The aim of this study was to carry out sequence analyses of the two outer capsid proteins (VP4 and VP7) of the R2638 strain detected in order to obtain further information of the genetic relationships between human and animal rotaviruses. STUDY DESIGN Rotavirus G3P[3] was detected using a commercial immunoenzymatic assay, SDS-PAGE, and genotyped by RT-PCR. The analysis of the genetic relationship between human and animal rotaviruses was carried out by sequencing the VP7 and VP4 genes. RESULTS The VP7 gene of the R2638 strain displayed the highest nucleotide identity to the canine strains A79-10 (96.6%) and CU-1 (96.2%) isolated in USA. The VP4 sequence showed the highest nucleotide identity to P[3] canine rotavirus strain RV52/96 isolated in Italy at 94.1%. Furthermore, the VP4 genes of P[3] strains could be discriminated into two phylogentically distinct clusters. CONCLUSION The present study reinforces the hypothesis that animals rotaviruses might be able to cross the species barriers, and the lack of systematic surveillance of rotavirus infection in small animals hinders the ability to establish firm epidemiologic connections. Moreover, in 2006 rotavirus vaccine was included in the Brazilian Immunization Program, and selective vaccine pressure could increase the circulation of uncommon strains. This is the first report of G3P[3] in over 20-year period of monitoring in Brazil.

Norovirus 3rd Generation kit: an improvement for rapid diagnosis of sporadic gastroenteritis cases and valuable for outbreak detection.

The aim of this study was to evaluate the sensitivity and specificity of the Ridascreen(®) Norovirus 3rd Generation kit compared to the RT-PCR. A retrospective, descriptive study was conducted with 245 specimens from sporadic cases and outbreak surveillance samples of gastroenteritis in Brazil from 2006 to 2009. Overall, the kit showed a sensitivity of 61.8% and a specificity of 92.5%. The sensitivity for outbreaks diagnosis was 87.9% and specificity 83.8%. The Ridascreen(®) 3rd Generation could detect specimen containing genogroup (G) II with high sensitivity. However, GI and mixed infections (GI/GII) were unlikely to be detected by the kit. ELISA for Norovirus (NoV) detection provides a rapid, technically simple assay system that can be used to increase the surveillance of gastroenteritis outbreaks, especially in Public Health Laboratories with high sample throughput. This assay is useful for the detection of NoV outbreaks and is an improvement as compared to previous ELISAs; however, due to its sensitivity, RT-PCR in still required for routine NoV detection in sporadic cases investigation.

Characterization of rotavirus strains from day care centers: pre- and post-rotavirus vaccine era

OBJECTIVES In 2006 the rotavirus vaccine was included in the Brazilian Immunization Program. The aim of this study was to report the results of a 5-year surveillance study of rotavirus strains in children < 5 years with acute gastroenteritis from day care centers in the state of São Paulo, Brazil. METHODS This retrospective study was conducted with 30 day care centers from 2004 to 2008 with convenient surveillance fecal specimens, investigated by ELISA, SDS-PAGE, RT-PCR and gene sequencing to genotype characterization. RESULTS Rotavirus infection was detected in 28.3% of samples (38/134). The most frequent genotypes detected were G9P[8] and G1P[8] in 2004; G1P[8] in 2005; GNTP[NT] in 2006; G2P[4] in 2007; and there were no cases in 2008. Mixed infections were not observed. Detection rate declined from 65.7% (23/35) in 2004 to 50% (9/18) in 2007. CONCLUSIONS Genotype distribution varied according to collection year, accompanied by a reduction in detection rate. Use of rotavirus vaccine requires implementation of post-marketing surveillance to monitor rotavirus strain diversity and its efficacy against possible new emerging genotypes.

Rotavirus in adults, Brazil, 2004–2011: G2P[4] dominance and potential impact on vaccination

OBJECTIVES The aim of this study was to monitor rotavirus (RV) infections in adults >18 years with acute gastroenteritis during 2004-2011 national Brazilian RV surveillance. In addition, to characterize the RV group A (RVA) strains in order to gain insight into the supposed vaccine selective pressure imposed to Brazilian children population. METHODS A total of 2102 convenient fecal specimens were investigated by ELISA, PAGE, and RT-PCR. RESULTS RV was detected in 203 (9.6%) of 2102 specimens, and showed a marked peak of detection in September. RVA infection was detected in 9.4% (197/2102) and RV group C (RVC) in 0.3% (6/2102). The most frequent genotypes detected in 2004 and 2005 were G9P[8] (38.5%; 5/13) and G1P[8] (54.5%; 6/11), respectively. The dominant genotype identified from 2006 to 2011 was G2P[4] (64.4%; 116/180). Detection rate varied during the 8-year period of the study from 0.7% to 12.9%. CONCLUSION The high detection rate of G2P[4] in adults provides further evidence that its dominance reflects the seasonality of RVA strains instead of the supposed selective advantage created by vaccination program. It also can be suggested that adult infections may serve as a reservoir to maintain RVA strains in childhood gastroenteritis. Considering the detection rate, the evident reduction of RVA frequency observed in children after vaccine introduction was not present in adults.

Caracterização molecular de cepas de rotavírus e norovírus: um estudo de 6 anos (2004-2009)

OBJECTIVE To monitor rotavirus (RV) and norovirus (NoV) infections in hospitalized children ≤ 5 years with acute gastroenteritis in the state of São Paulo, Brazil, during a 6-year period (2004- 2009). METHODS This retrospective study was conducted with 61 medical centers with convenient surveillance fecal specimens, investigated by enzyme-linked immunosorbent assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, reverse polymerase chain reaction and sequencing to genotype characterization. RESULTS RV and NoV infections were detected in 29.6% (144/487) and 29.2% (26/89) of the samples, respectively. The most frequent RV genotypes detected were G9P[8] in 2004; G1P[8] in 2005; G9P[8] in 2006; and G2P[4] during 2007, 2008, and 2009. Detection rate declined from 36.3% (33/91) in 2004 to 4.2% (4/95) in 2009. NoV genogroup GII was found in 61.6% (16/26) of the samples, and GI in 11.5% (3/26). Mixed NoV-RV infections were observed in 2.2% (2/89) of the samples, involving GI+G9P[8] and GI+G2P[4] strains. CONCLUSIONS Genotype distribution varied according to collection year, accompanied by a reduction in detection rate. Use of RV vaccine requires implementation of post-marketing surveillance to monitor RV strain diversity and its efficacy against possible new emerging genotypes. NoVs have been increasingly identified as relevant etiological agents among hospitalized children and play an important role in the viral etiology of pediatric acute gastroenteritis in the state of São Paulo.

Identification and Molecular Characterization of Norovirus in São Paulo State, Brazil

Norovirus (NoV), previously called Norwalk-like virus, represents an important group of human pathogens associated with outbreaks of nonbacterial gastroenteritis. Epidemiological surveys of outbreaks have shown that the most important routes of transmission are person-to-person contacts and contaminated food and water, with the virus affecting adults and children. NoV is classified into genogroups, being genogroups GI, GII and GIV found in humans. In view of the high genetic diversity of NoV and the lack of information about this virus in Brazil, the aim of the present study was the molecular characterization of NoV isolated from diarrheic stool samples of patients from São Paulo State. In this study, 204 stool specimens collected during diarrhea outbreaks were analyzed by RT-PCR, and 12 were sequenced for genogroup confirmation. One-step PCR was performed in order to amplify the B region of ORF 1 using the MON 431, 432, 433 and 434 primer pool. From total, 32 (15.7%) stool specimens were positive for NoV genogroup GII. Comparison of the sequences of the PCR products to GenBank sequences showed 88.8% to 98.8% identity, suggesting the presence of genogroup GII in gastroenteritis outbreaks in São Paulo State.

ROTAVIRUS GENOTYPES CIRCULATING IN BRAZIL, 2007-2012: IMPLICATIONS FOR THE VACCINE PROGRAM

SUMMARY Regarding public health in Brazil, a new scenario emerged with the establishment of universal rotavirus (RV) vaccination programs. Herein, the data from the five years of surveillance (2007-2012) of G- and P-type RV strains isolated from individuals with acute gastroenteritis in Brazil are reported. A total of 6,196 fecal specimens were investigated by ELISA and RT-PCR. RVs were detected in 19.1% (1,181/6,196). The peak of RV incidence moved from June-August to September. RV was detected less frequently (19.5%) among children ≤ 5 years than in older children and adolescents (6-18 years) (40.6%). Genotype distribution showed a different profile for each year: G2P[4] strains were most prevalent during 2007-2010, G9P[8] in 2011, and G12P[8] in 2012. Mixed infections (G1+G2P[4], G2+G3P[4]+P[8], G2+G12P[8]), unusual combinations (G1P[4], G2P[6]), and rare strains (G3P[3]) were also identified throughout the study period. Widespread vaccination may alter the RV seasonal pattern. The finding of RV disease affecting older children and adolescents after vaccine implementation has been reported worldwide. G2P[4] emergence most likely follows a global trend seemingly unrelated to vaccination, and G12, apparently, is emerging in the Brazilian population. The rapidly changing RV genotype patterns detected during this study illustrate a dynamic population of co-circulating wildtype RVs in Brazil.

Monitoring of group C rotavirus in children with acute gastroenteritis in Brazil: an emergent epidemiological issue after rotavirus vaccine?

Group C rotavirus (GpCRV) has a worldwide distribution; however, its epidemiology and ecology are still unclear. Evidence for a possible zoonotic role has been postulated recently for Brazilian children strains. The aim of this study was to monitor GpCRV in children ≤15 years with acute gastroenteritis during the 2007–2010 national Brazilian rotavirus surveillance, and to undertake the molecular characterization of the major VP6 capsid protein. A total of 3,019 fecal samples were first screened for Group A rotavirus (GpARV). A total of 2,205 GpARV ELISA negative samples were tested further for the presence of GpCRV by SDS–PAGE, electronic microscopy, and RT‐PCR for the VP6 gene. The genetic diversity of GpCRV was carried out by sequencing the VP6 gene. GpARV and GpCRV infections were detected in 24.6% (742/3,019) and 0.3% (8/3,019), respectively. The GpCRV detection rate increased from 0.2% (1/422) in 2007 to 1% (7/708) in 2008, and GpCRV cases were not detected in 2009 and 2010. The phylogenetic analysis indicated that the strains belonged to the human lineage, and showed a genetic relationship with the GpCRV strain from Japan isolated in 2009. None of the study sequences was related closely to animal GpCRV strains. This study provides further evidence that GpCRV is a minor cause of acute childhood gastroenteritis in Brazil, and does not suggest that GpCRV may assume epidemiological importance in the future, even after the introduction of a GpARV vaccine. In addition, the molecular analyses of the GpCRV samples in this study do not support the zoonotic hypothesis. J. Med. Virol. 83:1631–1636, 2011.

Detection of the emerging rotavirus G12P[8] genotype at high frequency in brazil in 2014: Successive replacement of predominant strains after vaccine introduction.

The continuum characterization of rotavirus (RVA) genotypes is essential to understand how vaccine introduction could impact virus epidemiology. In the present study, an unexpected rapid changing pattern of RVA genotypes distribution in Brazilian population during three followed seasons is described. From January/2012 to December/2014, a total of 3441 fecal specimens were collected from collaborating centers across Southern, Southeastern and Midwest of Brazil. All specimens were screened for RVA using ELISA, and genotyped by RT-PCR. Differences in proportions were tested using Chi-Squares. A p-value of less than 0.05 was considered statistically significant. RVA was detected in 19.7% (677/3441). Among RVA positive cases (n=677), a total of 652 (96.3%) samples were successfully amplified by RT-PCR. G3P[8] remained prevalent in 2012 (37.6%, 69/185) and 2013 (40.1%, 74/186) (χ(2)=0.107, p=0.743), but declined markedly in 2014 (3.5%, 10/281) (χ(2)=71.770, p=0.000). G12P[8] was second highest strain in 2012 (22.7%, 42/185), decrease rapidly in 2013 (2.7%, 5/186) (χ(2)=26.224, p=0.000) and re-emerged as the predominant genotype in 2014 (86.6%, 243/281) (χ(2)=118.299, p=0.000). From July/2014, G12P[8] was the single genotype detected in all regions studied. The sudden emergence, spread and predominance of G12P[8] strain in Brazil, raised the hypothesis of a possible G12 outbreak being in progress. Nationally, the long term decline in gastroenteritis hospitalization observed in the country after RVA vaccine introduction was confirmed. Nevertheless, the sharp increase in diarrhea hospitalization prevalence from 2013 to 2014 observed in Southern and Southeastern regions is consistent with what appears to be an outbreak of G12P[8]. Continued surveillance is needed to verify the effectiveness of the RotarixTM vaccine in Brazil together with potential emergence of unusual genotypes.