José Paulo Gagliardi Leite

Rotavirus G and P types circulating in Brazil : Characterization by RT-PCR, probe hybridization, and sequence analysis

SummaryWe used reverse transcription-polymerase chain reaction (RT-PCR) to determine the P and G genotypes of 130 culture-adapted rotavirus strains isolated from 181 fecal specimens of children under 5 years of age from 9 states and the Federal District of Brazil. The 4 genotypes found most commonly worldwide were also common in Brazil and P[8]G1 was the most prevalent (43%), followed by P[4]G2 (12%), P[8]G3 (6%), and P[8]G4 (6%). However, unusual types P[8]G5, P[6]G2, P[9]G1, P[9]G3, and mixed infections were responsible for 12% and 21% of the cases, respectively. Genotype G5 strains were detected in specimens collected in all 9 areas surveyed from all 4 regions of Brazil. The unusual strain diversity in Brazil suggests that when tetravalent rotavirus vaccines currently being developed are introduced into Brazil, laboratory surveillance will be essential to monitor protection against unusual strains, particularly those of genotype 5, as well as emergence of novel reassortants that may evolve from the large pool of children with mixed infections.

Sequence analysis of NSP4 gene of human rotavirus allows classification into two main genetic groups

The rotavirus nonstructural glycoprotein NSP4 may represent the first identified viral enterotoxin. We have sequenced reverse transcription‐polymerase chain reaction (RT‐PCR)‐generated fragments of 16 NSP4 genes of human rotavirus (HRV) strains from six different countries, representing seven different G and P type combinations. Based on the amount of sequence divergence between these and 11 previously sequenced NSP4 genes of human and animal rotaviruses, three distinct genetic groups could be recognized. Most strains within a group were closely related to each other at the nucleotide (nt) and amino acid (aa) levels (usually <10% divergence) but more distantly related (maximum 30.0% nt divergence and 24.7% aa divergence) to members of the other groups. Intergroup variation occurred in two highly variable regions of NSP4 (aa 16–34 and aa 131–148). The NSP4 “toxic peptide” (aa 114–135) exhibited aa variation at its carboxy terminus both within and between genetic groups. The largest group (genetic group II) contained HRV strains of subgroup II specificity (including genotypes P[8]G1, P[8]G3, P[6]G3, and P[8]G5 and serotype P8[11]G9), and the smaller group (genetic group I) contained HRV strains of subgroup I specificity (genotype P[4]G2). The NSP4 sequence of the rhesus rotavirus vaccine strain was distinct from all other strains and formed the third group (genetic group III). The NSP4 genes of animal rotaviruses UK, NCDV, and SA11 (genetic group I) and YM (genetic group II) and two possible human‐animal rotavirus reassortant strains, Brazilian P[8]G5 and Indian P[11]G9 (genetic group II), could also be classified into one of these groups, suggesting a close evolutionary relationship between human and animal NSP4 genes. These results will facilitate studies of the host immune response to NSP4, which may be relevant to future HRV vaccine design. J. Med. Virol. 53:41–50, 1997. Published 1997 Wiley‐Liss, Inc.

Characterization of human rotavirus genotype P(8)G5 from Brazil by probe-hybridization and sequence

SummaryWe report the molecular characterization of rotavirus genotype P[8]G5 strains found in fecal specimens collected in four different regions of Brazil, using digoxigenin (dig)-labeled oligonucleotide probes, sequence analysis, and RNA-RNA hybridization. The closest sequence relationships of the neutralization antigens of these strains were to the VP4 protein of P1A[8]G1 strain KU (93.3% identity in amino acids 11 to 282) and to the VP7 protein of G serotype 5 strain OSU (87.6% identity in amino acids 8 to 232). Based on VP7 sequence differences, we designed dig-probes that allowed us to discriminate porcine OSU-like strains from G5 strains isolated from Brazilian infants. The genetic relationships of two P[8]G5 isolates to other rotavirus genogroups were analyzed by RNA-RNA hybridization with [32P]-GTP probes representative of serotypes P1A[8]G1 (Wa), P[8]G3 (AU17), and P9[7]G5 (OSU). The Brazilian P[8]G5 strains showed sequence homology with genes of Wa-like and OSU-like strains, suggesting that these two strains were naturally occurring reassortants between members of the Wa and porcine rotavirus genogroups. The identification of these strains in diverse geographic areas of Brazil underscores their stability and demonstrates the emergence of clinically important rotavirus diarrhea strains by reassortment.

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.

Detection of field isolates of human and animal group C rotavirus by reverse transcription-polymerase chain reaction and digoxigenin-labeled oligonucleotide probes.

Rotaviruses (RV) are important etiological agents of acute gastroenteritis in infants and young children, as well as the young of a variety of animals worldwide. These viruses belong to Reoviridae family and contain a genome of 11 segments of double-stranded RNA (dsRNA). Two major proteins, VP4 and VP7, encoded by genome segments 4 and 7, 8 or 9, respectively, evoke a neutralizing antibody response and form the basis for the current classification of group (gp) A rotavirus into P (VP4) and G (VP7) serotypes. Although much recent progress has been made on the molecular biology of gp C RV, routine methods to detect and discriminate human, porcine, and bovine strains are not available widely. In this study, a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) and digoxigenin-labeled (dig) oligonucleotide probes using chemiluminescence has been developed to detect and discriminate VP7 genes from culture-adapted and field isolates of human, porcine and bovine gp C RV. The multiplex RT-PCR and dig-probes were specific for the VP7 genes of human, porcine and bovine gp C RV and allowed detection and characterization of single and mixed infections of porcine gp C RV with porcine gp A or gp B rotaviruses. Detection rates for gp C RV were more than 50% when compared with polyacrylamide gel electrophoresis. These new diagnostic assays may help determine the epidemiological importance of these viruses in human and animal infections.

Molecular epidemiology of group a rotavirus among children admitted to hospital in Salto, Uruguay, 2011-2012: First detection of the emerging genotype G12

Group A rotavirus (RVA) is the most important etiologic agent of infant acute gastroenteritis (AGE) worldwide. Detection and molecular characterization of RVA in Salto department, Northwestern region of Uruguay, was conducted on 175 clinical samples, being 153 stool and 22 vomit samples, collected from hospitalized children with AGE, between 0–15 years old, from two hospitals of Salto city during 2011 and 2012. RVA was detected and genotyped by seminested multiplex RT‐PCR in order to determine G‐ and P‐genotypes. Positive samples were sequenced and phylogenetic analyses were carried out in order to determine lineages and sub‐lineages. RVA were detected in 64 (37%) of the samples and the G and P genotypes observed were: 6% G1P[8], 23% G2P[4]/G2P[X]/GXP[4], 23% G3P[8]/G3P[X], 14% G12P[8]/G12P[X], 16% GXP[8], 1,5% G12P[9], 3% G2P[4]/[8], and 16% non‐typeable. VP7 and VP4 genotypes related to DS‐1 like gene constellation were prevalent during 2011 and those VP7 and VP4 genotypes related to Wa‐like constellation were prevalent during 2012 (mainly represented by G3P[8]). Interestingly, RVA was detected in vomit samples in a high prevalence (41%). RVA was observed mainly in the age group between 1 and 5 years old (75% of the cases), and seasonality with a high detection rate in winter season was observed for the two consecutive years of surveillance. To our knowledge, this study represents the first detection and molecular characterization of RVA in Salto department, Northwestern region of Uruguay; and the first identification of the emerging genotype G12 in the country. J. Med. Virol. 87:754–763, 2015.

Rotavirus G serotypes and P[], G genotypes identified in cases of reinfection among children participating in a trial with rhesus-human reassortant tetravalent vaccine (RRV-TV) in Belém, Brazil

Group A rotaviruses are the most important agents of severe diarrhea in children and infants worldwide. The aim of present study was to identify rotavirus G serotypes and P[],G genotypes in cases of reinfection among children who participated in a vaccine trial with the tetravalent rhesus-human reassortant rotavirus vaccine (RRV-TV 4 x 10(4) pfu/dose) in Belém, Brazil. From July 1990 to June 1992, 540 children received, at their first, third and fifth months of life, oral doses of either vaccine or placebo. A total of 90 rotavirus diarrheal episodes among children who completed the three-dose vaccination schedule were recorded. We studied 11 reinfection rotavirus cases among five children (three female and two male). Fecal specimens were tested by using a enzyme immunoassay (IDEIA Rotavirus), followed by EIA with monoclonal antibodies to determine infecting serotypes Gl, G2, G3 and G4 and subgroups I and II. The viral dsRNA was extracted and electrophoresed through polyacrylamide gel and then subjected to reverse-transcription-polymerase chain reaction and nested-PCR for the determination of Gl, G2, G3, G4, G5 and G9 and P[4], P[6], P[8] and P[9] rotavirus genotypes. A total of 11 cases of reinfection (12 per cent) occurred among five children, three from the placebo group and two from the vaccine group. In four of the cases of reinfection G serotypes and P[],G genotypes were as follows: for the first and second infections, respectively: (1) G2/P[4],G2 and Gl/P[4],G1; (2) G2/P[4],G2 and G2/P[6],G5; (3) G2/P[4],G2 and G1/P[8],G1; and (4) G2/P[8],G1 and G1/P[8],G1. A fifth child had three successive infections caused by serotypes/genotypes G1/P[8],G1, in the first and second infections, and G2/P[4],G2 in the third infection. The common genotypes and unusual genotypes were detected in 8 (73 per cent) and 3 (27 per cent) of the isolates, respectively. With regards to the clinical severity, in two children a score indicated moderate/severe disease in both first and second infections. One child had three successive infections; the first episode was moderate/severe, the second very severe and the third was not available. In contrast, in two other children, the first episode was very severe, and the second episode was moderate/severe in one child and data was not available for the other child. The results obtained in the present investigation underscore the need to broaden our knowledge of the immunity in rotavirus reinfection. This should be useful regarding future rotavirus vaccination strategies in Brazil.

Molecular epidemiology of the human group A rotavirus in the Paraná State, Brazil

From January/2000 to December/2003, 550 diarrheic fecal samples from the children and adults were collected in several geographical regions of Parana State, Brazil. The enzyme immunoassay showed 120 (21.8%) samples positive for the group A rotaviruses. One hundred and fourteen samples were genotyped by multiplex-nested-PCR assay. The highest frequency (77.5%) of the positive samples (n=93) was observed in the children under 5 years old. Rotavirus diarrhea was more frequent in the cold and dry seasons of the four evaluated years. The most frequent genotypes were: G1 (50.9%), G4 (9.6%), G9 (7.0%), G2 (1.7%), G3 (0.9%), P[ 8] (71.9%), and P[ 4] (3.5%). The P[ 8] G1 (46.5%) and P[ 8] G4 (9.6%) were the main combinations found to P and G genotypes. The mixed infections, characterized by the rotaviruses with more than one genotype G or P, and nontypeable rotavirus were observed in 8.8, 3.5, and 16.7% of the samples, respectively. The identification of the G9 genotype in the rotavirus strains tested along the four years of studies ratifies the emergency of this genotype also in Parana State, South region of Brazil, as the worldwide.