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. (C) 2011 Wiley-Liss, Inc.

Human group C rotavirus in children with diarrhea in the Federal District, Brazil

Group C rotaviruses are fastidious in their in vitro cell culture requirements. Recent serosurveys indicate that antibody to group C rotavirus is present in 3-45% of the human population in certain geographic locations, suggesting that rotavirus group C infection is more prevalent than previously believed and that the low rate of detection of these agents is probably due to the lack of sensitive diagnostic assays. From March to December 1994, 406 fecal specimens were collected from children under five years of age who were outpatients at the emergency services of nine public hospitals in Brasília, Federal District, Brazil. In addition to the samples from children, one public outpatient unit requested virological investigation of a stool sample from an HIV-seropositive adult male with diarrhea of sudden onset. All samples were analyzed by enzyme immunoassay for group A rotavirus and adenovirus (EIARA) and by polyacrylamide gel electrophoresis (PAGE). One hundred and seven (26%) were positive for group A rotavirus. Four samples from children and the sample from the HIV-seropositive patient, although negative by EIARA, showed a group C rotavirus profile by PAGE and were positive for rotavirus by electron microscopy. Using specific VP6 and VP7 primers for group C rotavirus, a reverse transcriptase-polymerase chain reaction (RT-PCR) was performed and products were detected by agarose gel electrophoresis and ethidium bromide staining. These products were confirmed to be specific for group C rotavirus by using digoxigenin-oligonucleotide probes, Southern hybridization and chemiluminescent detection. The five positive group C rotavirus samples were detected in August (3 samples) and September (2 samples). To the best of our knowledge, this is the first report of group C rotavirus detected in the Federal District, Brazil and in an HIV-seropositive patient with acute gastroenteritis.

Diarrhea outbreaks in suckling piglets due to rotavirus group C single and mixed (rotavirus groups A and B) infections

Porcine group A rotavirus (PoRVA) is a major cause of neonatal diarrhea in suckling and recently weaned piglets worldwide. The involvement of non-group A rotavirus in cases of neonatal diarrhea in piglets are sporadic. In Brazil there are no reports of the porcine rotavirus group C (PoRVC) as etiologic agent of the diarrhea outbreaks in piglets. The aim of this study was to describe the identification of rotavirus group C in single and in mixed infection with rotavirus groups A and B in three neonatal diarrhea outbreaks in suckling (<21-day-old) piglets, with 70% to 80% and 20% to 25% of morbidity and lethality rates, respectively, in three pig herds located in the state of Santa Catarina, Brazil. The diagnosis of PoRV in the diarrheic fecal samples was performed using polyacrylamide gel electrophoresis (PAGE) to identify the presence of porcine rotavirus groups A, B (PoRVB), and C, and by RT-PCR (PoRVA and PoRVC) and semi-nested (SN)-PCR (PoRVB) to partially amplify the VP4 (VP8*)-VP7, NSP2, and VP6 genes of PoRVA, PoRVB, and PoRVC, respectively. One RT-PCR (PoRVA and PoRVC) and SN-PCR (PoRVB) product of each group of rotavirus of each diarrhea outbreak was submitted to nucleotide (nt) sequence analysis. Based on the PAGE technique, 4 (25%) and 1 (6.25%) of the 16 diarrheic fecal samples evaluated in the first outbreak presented PoRVA and PoRVC electropherotype, respectively, and 11 (68.75%) were negative. In the second outbreak, 3 (42.85%) of the 7 fecal samples evaluated presented PoRVA electropherotype, and in 3 (42.85%) and in 1 (14.3%) fecal samples were detected inconclusive and negative results, respectively. Three (30%) of the 10 fecal samples of the third outbreak presented PoRVC electropherotype; 5 (50%) and 2 (20%) samples showed negative and inconclusive results, respectively. Based on the RT-PCR and SN-PCR assays in the first neonatal diarrhea outbreak, PoRVC was detected in 13 (81.2%) of the 16 diarrheic fecal samples evaluated. PoRVC single infection was identified in 4 (25%) of these samples and mixed infections with PoRVA and PoRVB in 9 (56.2%) fecal samples. All of the seven diarrheic fecal samples evaluated from the second neonatal diarrhea outbreak were positive for PoRVC, whereas its mixed infection with other PoRV groups was detected in 4 (57.2%) samples. In the third outbreak, PoRVC in single infection was detected in all of the 10 diarrheic fecal samples analyzed. In the nt sequence analysis, the PoRVA strains of the first and second outbreaks demonstrated higher nt identity with G4P[6] and G9P[23] genotypes, respectively. The PoRVB strains (first and second outbreaks) and the PoRVC strains (first, second, and third outbreaks) showed higher nt identity and clustered in the phylogenetic tree with PoRVB and PoRVC strains that belong to the N4 and I1 genotypes, respectively. This is the first description in Brazil of the involvement of PoRVC in the etiology of diarrhea outbreaks in suckling piglets. The results of this study demonstrated that PoRVC, in both single and mixed infections, is an important enteropathogen involved in neonatal diarrhea outbreaks in piglets and that the use of more sensitive diagnostic techniques allows the identification of mixed infections involving two or even three groups of PoRV, which may be more common than previously reported.

Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

Neonatal calf diarrhea is a multi-etiology syndrome of cattle and direct detection of the two major agents of the syndrome, group A rotavirus and Bovine coronavirus (BCoV) is hampered by their fastidious growth in cell culture. This study aimed at developing a multiplex semi-nested RT-PCR for simultaneous detection of BCoV (N gene) and group A rotavirus (VP1 gene) with the addition of an internal control (mRNA ND5). The assay was tested in 75 bovine feces samples tested previously for rotavirus using PAGE and for BCoV using nested RT-PCR targeted to RdRp gene. Agreement with reference tests was optimal for BCoV (kappa = 0.833) and substantial for rotavirus detection (kappa = 0.648). the internal control, ND5 mRNA, was detected successfully in all reactions. Results demonstrated that this multiplex semi-nested RT-PCR was effective in the detection of BCoV and rotavirus, with high sensitivity and specificity for simultaneous detection of both viruses at a lower cost, providing an important tool for studies on the etiology of diarrhea in cattle. (C) 2010 Elsevier B.V. All rights reserved.

Molecular characterization of the NSP4 gene of human group A rotavirus samples from the West Central region of Brazil

Nonstructural protein 4 (NSP4), encoded by group A rotavirus genome segment 10, is a multifunctional protein and the first recognized virus-encoded enterotoxin. The NSP4 gene has been sequenced, and five distinct genetic groups have been described: genotypes A-E. NSP4 genotypes A, B, and C have been detected in humans. In this study, the NSP4-encoding gene of human rotavirus strains of different G and P genotypes collected from children between 1987 and 2003 in three cities of West Central region of Brazil was characterized. NSP4 gene of 153 rotavirus-positive fecal samples was amplified by reverse transcriptase-polymerase chain reaction and then sequenced. For phylogenetic analysis, NSP4 nucleotide sequences of these samples were compared to nucleotide sequences of reference strains available in GenBank. Two distinct NSP4 genotypes could be identified: 141 (92.2%) sequences clustered with NSP4 genotype B, and 12 sequences (7.8%) clustered with NSP4 genotype A. These results reinforce that further investigations are needed to assess the validity of NSP4 as a suitable target for epidemiologic surveillance of rotavirus infections and vaccine development.

Group A rotavirus genotypes and the ongoing Brazilian experience: a review

Brazil was the first Latin American country to introduce universal group A rotavirus (RV-A) vaccination in March 2006, resulting in a unique epidemiological scenario. Since RV-A first identification in Brazil, 2,691 RV-A-positive stool samples, collected between 1982- 2007, were typed by independent research groups throughout the country. In the pre-vaccination era, 2,492 RV-A-positive samples collected from 1982-2005 were successfully typed, while 199 samples were analyzed from 2006-2007. According to the reviewed studies, there were two important times in the pre-vaccination era: (i) the period from 1982-1995, during which the detection of G5P[8] RV-A, in addition to the classical genotypes G1-4, challenged vaccine development programs; and (ii) the period from 1996-2005, during which genotype G9P[8] emerged, following a global trend. The rate of G2P[4] RV-A detection decreased from 26% (173/653) during 1982-1995 to 2% (43/1,839) during 1996-2005. The overall detection rate of RV-A genotypes from 1982-2005 was as follows: 43% (n = 1,079) G1P[8]/G1P[not typed (NT)]; 20% (n = 488) G9P[8]/G9P[NT]; 9% (n = 216) G2P[4]/G2P[NT]; 6% (n = 151) G3P[8]/G3P[NT]; 4% (n = 103) G4P[8]/G4P[NT]; and 4% (n = 94) G5P[8]/G5P[NT]. Mixed infections accounted for 189 (7%) of the positive samples, while atypical G/P combinations or other genotypes, including G6, G8, G10 and G12, were identified in 172 (7%) samples. The initial surveillance studies carried out in several Brazilian states with RV-A-positive samples collected in 2006 and 2007 show a predominance of G2P[4] strains (148/199 or 74%). Herein, we review RV-A typing studies carried out since the 1980s in Brazil, highlighting the dynamics of RV-A strain circulation profiles before and early after universal use of RV-A vaccine in Brazil.

Development of a Real-time PCR test for porcine group A rotavirus diagnosis

Group A Rotavirus (RVA) is one of the most common causes of diarrhea in humans and several animal species. A SYBR-Green Real-Time polymerase chain reaction (PCR) was developed to diagnose RVA from porcine fecal samples, targeting amplification of a 137-bp fragment of nonstructural protein 5 (NSP5) gene using mRNA of bovine NADH-desidrogenase-5 as exogenous internal control. Sixty-five samples were tested (25 tested positive for conventional PCR and genetic sequencing). The overall agreement (kappa) was 0.843, indicating 'very good' concordance between tests, presenting 100% of relative sensitivity (25+ Real Time PCR/25+ Conventional PCR) and 87.5% of relative sensitivity (35- Real Time PCR/40- Conventional PCR). The results also demonstrated high intra- and inter-assay reproducibility (coefficient of variation ≤1.42%); thus, this method proved to be a fast and sensitive approach for the diagnosis of RVA in pigs.

Genotyping of group A rotavirus samples from Brazilian children by probe hybridization

The G genotyping of 74 group A rotavirus samples was done by RNA-DNA hybridization (dot-blot) using oligonucleotide probes for the VP7 gene region of the human rotavirus serotypes/genotypes 1, 2, 3 and 4. Thirty-one samples could be genotyped by dot-blot showing the following results: G1 = 16, G4 = 6, G3 = 5, and G2 = 4. The data show circulation of genotypes G1-G4 and the predominance of G1. The knowledge of genotypes provides important information concerning rotavirus circulation in Central Brazil.

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

Group B rotaviruses (RV-B) were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8) were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

First description of group A rotavirus from fecal samples of ostriches (Struthio camelus)

This study investigated the occurrence of rotavirus infections in ostriches (Struthio camelus) reared in Northern Parana, Brazil. Fecal (n = 66) and serum (n = 182) samples from nine farms located in four different cities were analyzed by silver stained-polyacrylamide gel electrophoresis (ss-PAGE), RT-PCR assay, virus isolation, and counterimmunoelectroosmophoresis (CIE). Rotavirus group A seropositivity occurred in 5.49% (10/182) of serum samples of ostriches originated from two farms. Only 9.09% (6/66) of fecal samples from ostriches with diarrhea maintained in one farm were positive by ss-PAGE, RT-PCR, and virus isolation. The G (VP7) and P (VP4) genotypes of rotavirus wild strains isolated in cell culture were determined by multiplex-nested PCR. The genotyping identified two rotavirus strains: G6P[1] and G10P[1]. In three rotavirus strains it was only possible to identify the P type; one strain being P[1] and two strains that presented the combination of P[1] + P[7]. These findings might represent the first characterization of rotavirus in ostriches, and the finding of porcine and bovine-like rotavirus genotypes in ostriches might suggest virus reassortment and possible interspecies transmission. (C) 2011 Elsevier Ltd. All rights reserved.

Human disease in ribeira valley, brazil caused by caraparu, a group c arbovirus - Report of a case

The clinical and laboratory data of a disease in a resident of Ribeira Valley, São Paulo State, southeastern Brazil, caused by an agent close or identical to Caraparu, a Group C arbovirus, was described. Although there is evidence of an intensive circulation of several arboviruses in the area, no diagnosis of human disease by these agents has been made, except the encephalitis cases caused by Rocio virus during an epidemic in 1975-1977. An antigenic difference between Caraparu strains isolated in São Paulo and in Pará States and a close antigenic relationship between Caraparu strain from São Paulo and Bruconha virus were suggested by the serological tests.

Molecular characterization of group A rotavirus before and after the introduction of vaccines in Brazil

ABSTRACTINTRODUCTION:In this study, the molecular characteristics of group A rotavirus (RVA) were compared in samples obtained before and after RVA vaccine-introduction in Brazil.METHODS:Eighty samples were screened for the presence of RVA. Positive samples were molecularly analyzed.RESULTS:RVA positivity was 16.9%, with a predominance of G2P[4]. Periods: pre-vaccination: predominance of IId (G1), IId (G2) lineages, and I1 and E1 genotypes; post-vaccination: predominance of Ib (G1), IIa, and IIc (G2) lineages and I2 and E2 genotypes.CONCLUSIONS:Although changes in RVA-circulation pattern were observed in the post-vaccination period, it could not be attributed to vaccination process.

Use of Existing Meningococcal Group C Vaccine - Students in Higher Education (PDF 20KB)

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Detection and molecular characterization of group A rotavirus from hospitalized children in Rio de Janeiro, Brazil, 2004

Rotavirus is a major cause of infantile acute diarrhea, causing about 440,000 deaths per year, mainly in developing countries. The World Health Organization has been recommending the assessment of rotavirus burden and strain characterization as part of the strategies of immunization programs against this pathogen. In this context, a prospective study was made on a sample of 134 children with acute diarrhea and severe dehydration admitted to venous fluid therapy in two state hospitals in Rio de Janeiro, Brazil, from February to September 2004. Rotavirus where detected by polyacrylamide gel electrophoresis (PAGE) and by an enzyme-linked immunoassay to rotavirus and adenovirus (EIARA) in 48% of the children. Positive samples for group A rotavirus (n = 65) were analyzed by reverse transcription/heminested multiplex polymerase chain reaction to determine the frequency of G and [P] genotypes and, from these, 64 samples could be typed. The most frequent G genotype was G1 (58%) followed by G9 (40%). One mixed infection (G1/G9) was detected. The only [P] genotype identified was [8]. In order to estimate the rotavirus infection frequency in children who acquired diarrhea as hospital infection in those hospitals, we studied 24 patients, detecting the pathogen in 41% of them. This data suggest that genotype G9 is an important genotype in Rio de Janeiro, with implications to the future strategies of vaccination against rotavirus, reinforcing the need of continuous monitoring of circulating strains of the pathogen, in a surveillance context.