Phylogenetic analysis of human group C rotavirus circulating in Brazil reveals a potential unique NSP4 genetic variant and high similarity with Asian strains

Group C rotaviruses (RVC) cause gastroenteritis in humans and animals worldwide, and the evidence for a possible zoonotic role has been recently provided. To gain information on the genetic diversity and relationships between human and animal RVC, we sequenced the VP4, VP7, and NSP4 genes of 12, 19, and 15 human strains, respectively, detected in São Paulo state during historical (1988 and 1993) and recent (2007 and 2008) Brazilian rotavirus surveillance. All RVC strains analyzed in the present study grouped into human genotype (G4-P[2]-E2), and did not show any evidence of animal ancestry. Phylogenetic analysis showed that RVC samples detected in 1988 and 1993 clustered together with strains from distinct continents, indicating that historical RVC strains circulating in São Paulo were closely related to those strains circulating worldwide. All three genes (VP7, VP4 and NSP4) of São Paulo RVC strains isolated in 2007-2008 exhibited close phylogenetic relationship with human RVC strains isolated in China and Japan, suggesting that they are genetically linked, and that a gene flow could be occurring between this Asian countries and Brazil. We identified two distinct clusters in the NSP4 phylogenetic tree. One cluster formed exclusively by human Brazilian strains detected in 1997 and 2003-2004 in Rio de Janeiro, Bahia, and Rio Grande do Sul states (Subgroup II) previously described in a different study, that displayed low sequence identities to other human strains formerly published, and to the Brazilian RVC strains (Subgroup I) characterized in the present study. These data suggests the circulation of two genetic profiles of the NSP4 gene in Brazil. High sequence diversity in NSP4 gene was previously reported in Asia, and additional diversity in NSP4 RVC strains spreading in the world should be expected. More in-depth molecular and epidemiological analysis of human RVC throughout the world will be needed to understand their diversity and clarify their evolution, as well as to develop classifications schemes.

Rotavirus genotypes and the indigenous children of Brazilian midwest in the vaccine era, 2008-2012: footprints of animal genome

World group A rotavirus (RVA) surveillance data provides useful estimates of the disease burden, however, indigenous population might require special consideration. The aim of this study was to describe the results of G­ and P­types from Brazilian native children ≤3 years. Furthermore, selected strains have been analyzed for the VP7, VP6, VP4, and NSP4 encoding genes in order to gain insight into genetic variability of Brazilian strains. A total of 149 samples, collected during 2008­2012, were tested for RVA using ELISA and PAGE, following by RT­PCR and sequencing. RVA infection was detected in 8.7% of samples (13/149). Genotype G2P[4] was detected in 2008 and 2010, G8P[6] in 2009, and G3P[8] in 2011. The phylogenetic analysis of the VP7 and VP4 genes grouped the Brazilian G2P[4] and G3P[8] strains within the lineages currently circulating in humans worldwide. However, the phylogenetic analysis of the VP6 and NSP4 from the Brazilian G2P[4] strains, and the VP7 and NSP4 from the Brazilian G3P[8] strains suggest a distant common ancestor with different animal strains (bovine, caprine, and porcine). The epidemiological and genetic information obtained in the present study is expected to provide an updated understanding of RVA genotypes circulating in the native infant population, and to formulate policies for the use of RVA vaccines in indigenous Brazilian people. Moreover, these results highlight the great diversity of human RVA strains circulating in Brazil, and an in­depth surveillance of human and animal RVA will lead to a better understanding of the complex dynamics of RVA evolution

Seroconversion of 2009 pandemic influenza A (H1N1) vaccination in kidney transplant patients and the influence of different risk factors.

BACKGROUND: Influenza may present a high morbidity and mortality in solid organ transplanted patients (SOTP). Annual influenza virus vaccine is recommended for SOTP. However, low levels of seroconversion in SOTP have been reported. The aim of this study was to evaluate the immunogenicity of 2009 pandemic influenza A (H1N1) - A(H1N1)pdm09--vaccine in kidney transplant patients and to analyze which features might affect seroconversion. METHODS: This study was conducted from March to August 2010 at the Renal Transplantation Unit of University of São Paulo, Brazil. A total of 85 renal transplant patients attending the outpatient unit received one 15-µg intramuscular dose of A(H1N1) pdm09 influenza vaccine (reassortant vaccine virus A/California/7/2009 [NYMC X-179A]). Blood samples were collected immediately before and 21 days after the vaccine was given. Antibody response was measured by the standard hemagglutination-inhibition (HI) assay. The primary immunogenicity endpoint for this study was seroconversion in previously seronegative patients (HI titers <1:40), and the secondary endpoint was the identification of features that could affect seroconversion in this population. RESULTS: Five (5.9%) patients presented HI titers prevaccination ≥ 1:40 and were excluded from further analysis. Seroconversion in previously negative patients occurred in 27 (34%) of 80 patients. Prevaccination HI titers geometrical mean was 5.8 and postvaccination 19.6 (ratio 3.4). Significant seroconversion rate factors were female gender, non-Caucasian ethnicity, and post-transplant time before vaccination. No impact was seen on seroconversion for age, donor type, tacrolimus and cyclosporine blood levels, renal function, or blood lymphocyte counts. Mycophenolate (MPA) showed a lower rate of seroconversion when compared with azathioprine. Tacrolimus and cyclosporine had similar seroconversion rates. Sirolimus use was associated with the highest rate of seroconversion, although these patient numbers were low. Immunosuppresssion containing MPA was considerably less effective in seroconversion than drug combinations with no MPA. Patients receiving sirolimus had more chance of seroconversion. HI titers geometric means pre/post vaccine were as follows: MPA (n = 56): 5.8/12.8; tacrolimus (n = 50): 5.9/16.2; cyclosporine (n = 18): 5.4/24.2; azathioprine (n = 19): 6.2/51.6; and sirolimus (n = 6): 8/80. By univariate analysis, being female and non-White were variables associated with 3.3 times more chance of seroconversion than being male and White. In the multivariate analysis, the variables remaining in the model showed similar hazard ratios. CONCLUSIONS: In this study, the monovalent A(H1N1)pdm09 influenza vaccine demonstrated low rates of seroconversion, particularly in patients on MPA, but with potentially higher response rates in patients on sirolimus.

Immunogenicity and safety of two doses of a non-adjuvanted influenza A H1N1/2009 vaccine in young autoimmune rheumatic diseases patients.

OBJECTIVES: The aim of this study was to evaluate the immunogenicity and safety of the influenza A H1N1/2009 vaccine in children under 9 years old with autoimmune rheumatic diseases (ARD). METHODS: Thirty-eight ARD patients and 11 healthy children received two doses of non-adjuvanted influenza A/California/7/2009 (H1N1) virus-like vaccine. Subjects were evaluated before and 21 days after vaccination. Seroprotection (SP) and seroconversion (SC) rates, geometric mean titers (GMT) and factor increases (FI) in GMT were calculated. RESULTS: Mean ages were comparable between patients and controls. Pre-vaccination SP and GMT were similar in patients and controls (p > 0.05). Three weeks after immunization, SP (81.6% vs. 81.8%, p = 1.0), SC (81.6% vs. 90.9%, p = 0.66), GMT (151.5 vs. 282.1, p = 0.26) and the FI in GMT (16.7 vs. 36.3, p = 0.23) were similar in patients and controls, with both groups achieving an adequate response, according to the European Medicines Agency and Food and Drug Administration standards. Analysis of the possible factors influencing SC showed no difference in demographic data, leukocyte/lymphocyte counts or immunosuppressant use between seroconverted and non-seroconverted patients (p > 0.05). The vaccine demonstrated a satisfactory safety profile in this population. CONCLUSIONS: Two doses of influenza A H1N1/2009 vaccination induced an effective antibody response and caused adverse events in rare instances, suggesting this vaccine is appropriate and can be recommended for this age group.

Effective seroconversion and safety following the pandemic influenza vaccination (anti-H1N1) in patients with juvenile idiopathic arthritis.

OBJECTIVES: To assess the vaccine response in juvenile idiopathic arthritis (JIA) as an extension of previous observation of immunogenicity and safety of a non-adjuvanted influenza A H1N1/2009 vaccine in a large population of juvenile rheumatic diseases. Moreover, to assess the possible influence of demographic data, disease subtypes, disease activity, and treatment on immunogenicity and the potential deleterious effect of the vaccine in the disease itself, particularly in the number of arthritis and inflammatory markers. METHODS: A total of 95 patients with JIA and 91 healthy controls were evaluated before and 21 days after vaccination, and serology for anti-H1N1 was performed by haemagglutination inhibition assay (HIA). Patient and physician visual analogue scales (VAS), Childhood Health Assessment Questionnaire (CHAQ), number of active joints, acute phase reactants, and treatments were evaluated before and after vaccination. Adverse events were also reported. RESULTS: JIA patients and controls were comparable regarding mean current age (14.9 ± 3.2 vs. 14.6 ± 3.7 years, p = 0.182). After vaccination, the seroconversion rate was significantly lower in JIA patients compared to controls (83.2% vs. 95.6%, p = 0.008), particularly in the polyarticular subtype (80% vs. 95.6%, p = 0.0098). Of note, JIA subtypes, number of active joints, acute phase reactants, CHAQ, patient and physician VAS, and use of disease-modifying anti-rheumatic drugs (DMARDs)/immunosuppressive drugs were similar between seroconverted and non-seroconverted patients (p > 0.05). Regarding vaccine safety, no deterioration was observed in the number of active joints and acute phase reactants during the study period. CONCLUSION: Influenza A H1N1/2009 vaccination in JIA induces a lower but effective protective antibody response probably independent of disease parameters and treatment with an adequate disease safety profile.

An outbreak of group C rotavirus gastroenteritis among adults living in Valentim Gentil, São Paulo State, Brazil.

An outbreak of gastroenteritis affecting adults and children occurred in the small city of Valentim Gentil, São Paulo, Brazil, in 1993. Nineteen faecal samples (from 10 cases and 9 contracts) were examined by direct electron microscopy (DEM), immune electron microscopy (IEM), polyacrylamide gel electrophoresis (PAGE), and enzyme-linked immunosorbent assay (ELISA) for group A and C rotaviruses. DEM detected rotavirus in 6 of the 10 cases and in none of the contacts. All of the samples were negative for group A rotavirus by ELISA. Analysis by PAGE showed an electrophoretic profile suggestive of group C rotavirus in two cases. Group C rotavirus was identified by IEM in 4 of the cases and in 1 of the contacts. All of the samples were submitted to ELISA for group C rotavirus. This resulted in a total of 10 positives-7 for diarrhoeal cases and 3 for contacts. This outbreak was strongly associated with group C rotavirus. The importance of combining different diagnostic methods is emphasised.

A novel human rotavirus serotype with dual G5-G11 specificity.

Rotavirus serotype G5 isolates were recently recovered from children with diarrhoea in Brazil. Like most human strains, they exhibited long electropherotypes and subgroup II and Wa-like VP4 specificity. We report the successful propagation and the molecular and antigenic characterization of one of these isolates (IAL-28). Cross-neutralization of IAL-28 and a single gene reassortant, UK x IAL-28, which contains the gene encoding the IAL-28 VP7 in the UK genomic background, with prototype G1 to G14 rotaviruses demonstrated that IAL-28 has antigenic determinants specific for both G5 and G11 serotypes. Sequence analysis of the gene encoding VP7 suggested that one or two amino acid substitutions at positions 96 and 100 on IAL-28 VP7 were possibly responsible for the additional G11 specificity. G5 rotaviruses are found in horses and predominate in piglets, whereas G11 has been identified exclusively as a swine pathogen.

[Gastroenteritis and acute respiratory infections among children up to 5 years old in an area of Southeastern Brazil, 1986-1987, II--Diarrhea]. / Gastroenterites e infecções respiratórias agudas em crianças menores de 5 anos, em área da região Sudeste do Brasil, 1986-1987. II--Diarréias.

INTRODUCTION: The decrease morbi-mortality gastroenteritis in is related to the factor responsible largely for the fall in infant mortality and mortality from communicable diseases in developing countries. Nevertheless, diarrhea is still a considerable public health problem in these countries, especially among children under 5 years old. OBJECTIVES: To describe some aspects of the of gastroenteritis epidemiology among children up to 5 years old, resident in areas of S. Paulo county. MATERIAL AND METHOD: A probabilistic sample of children up to 5 years old was studied (n = 468). The epidemiological survey was undertaken in five areas S. Paulo county (Brazil) from March 1986 to May 1987. Data were obtained through household interviews once a month over a year. RESULTS: During the follow-up 139 diarrhea episodes were registered, with a mean duration of 5.5 days. Twenty percent of the diarrhea events were followed by at least one other case in the household. The incidence of gastroenteritis was 2.78 episodes per 100 children/month. The highest incidence affected the children of up to 2 years of age. In 46.1% of the gastroenteritis episodes medical assistance was not sought, the children were treated by their mothers or not at all: 51.8% of the diarrhea events were attended to by the primary health care service, and only 2.1% were attended to by a hospital. No child died as a consequence of diarrhea. Of the therapeutical interventions used the most frequent were oral rehydration (25.2%) and oral rehydration with antibiotics (11.5%). Various socio-economic and personal background factors such as living conditions, water supply, sewarage, coverage; family income per capita and prior history of frequent gastroenteritis were associated with a higher incidence of gastroenteritis. DISCUSSION: The results seen to reflect the tendency of morbi-mortality by gastroenteritis to decrease in S. Paulo county during the 1980's when hospital treatment of diarrhea presented a considerable reduction. This tendency must be observed closely, because it will influence the changes to be mode in the kind of demand for medical care.

Outbreak of severe gastroenteritis in adults and children associated with type G2 rotavirus. Study Group on Diarrhea of the Instituto Adolfo Lutz.

An outbreak of severe gastroenteritis affecting 132 adults and children occurred in the small city of Mirassol, São Paulo, Brazil, in 1992. The outbreak of diarrhoeal disease had an abrupt onset and afflicted all age segments of the population. Group A rotavirus was the only pathogen associated with the epidemic. It was detected in 12 of the 27 (44%) stool specimens analyzed and was identified as serotype G2 rotavirus. Severe dehydration was common among adults and older children, and 35% of all the notified cases were hospitalized for parenteral rehydration. Contamination of the main water supply was the most likely source.

Survey of rotavirus G and P types associated with human gastroenteritis in São Paulo, Brazil, from 1986 to 1992.

Rotavirus strains causing gastroenteritis in Brazilian children were characterized by PCR-based typing assays. In addition to strains bearing the major human G and P types, large numbers of strains bearing P3 (M37-like), P6 (HCR3-like), untypeable P and G types, and complex mixtures of P and G types not previously recognized were present in the community.

Comparative analysis of VP8* sequences from rotaviruses possessing M37-like VP4 recovered from children with and without diarrhoea.

Rotavirus strains belonging to G types 1 to 4 and having a P3 genotype (M37-like VP4) were recovered from children with symptomatic and asymptomatic infections. Partial sequences of their VP4 genes were determined in an attempt to characterize these strains further. The genomic regions encoding VP8*, the connecting and putative fusion peptides and three other regions in VP5* were sequenced. The deduced amino acid sequences were compared with rotavirus strains belonging to different P genotypes that had been previously reported. High degrees of identity were found between the VP8* fragment of all human P3 strains (92.7 to 99.7%) suggesting that they belong to the same genotype, regardless of differences in their virulence. Furthermore, based on comparative sequence analysis, we did not identify any amino acid(s) that differ appreciably between symptomatic and asymptomatic strains and could therefore be associated with virulence. The results suggest that the P3 genotype, although frequently associated with asymptomatic infections, may not be the single determining factor in attenuation of symptoms.

Identification of Norwalk virus in artificially seeded shellfish and selected foods.

A rotavirus dsRNA purification protocol was adapted to extract Norwalk ssRNA from artificially contaminated shellfish, and a sensitive reverse transcription-polymerase chain reaction assay for Norwalk virus was devised to identify an estimated 20-200 genomic copies. The technique includes deproteinization with guanidinium isothiocyanate, adsorption of RNA to hydroxyapatite, and sequential precipitation with cetyltrimethylammonium bromide and ethanol. The protocol allows high recovery of viral RNA free of enzymatic inhibitors from oysters, clams, and a variety of food matrices. Norwalk virus sequences were copied and amplified by using primers selected from the polymerase gene. Digestion of the amplified products with restriction enzymes ensured the specificity of the test. This rapid and sensitive assay may significantly improve the prospect for the routine screening of the uncultivatable Norwalk virus in food stuffs.

VP4 typing of bovine and porcine group A rotaviruses by PCR.

A PCR typing assay was developed to identify rotavirus P types (VP4 specificity) of bovine NCDV, UK, and B223 strains and porcine OSU and Gottfried strains. Thirty-nine human and animal strains representing all known, and some undefined, rotavirus P types were used to develop and evaluate the specificity of the method. No cross-amplification was observed. The PCR results agreed with previous characterizations by monoclonal antibodies, sequence analysis, and hybridization assays, except for the Gottfried strain, which showed a P type distinct from the human asymptomatic strains. Analysis of a small number of field specimens suggested a high level of VP4 polymorphism among porcine strains. The assay should be of value in typing field isolates and tracing interspecies infections.

Identification of bovine and porcine rotavirus G types by PCR.

A new seminested PCR typing assay has been extended to identify the important veterinary rotavirus serotypes G5, G6, G10, and G11, as well as the rare human serotype G8. The specificity of the method was evaluated with 30 standard laboratory strains of the G1 to G6 and G8 to G11 types. Rotavirus strain types G6 and G8, not previously recognized in pigs, were identified in field specimens of porcine origin.

Rotavirus serotype G5 associated with diarrhea in Brazilian children.

Rotavirus serotype G5 in fecal specimens of 38 Brazilian children with diarrhea was identified by PCR and enzyme immunoassays. The strains exhibited long RNA electropherotypes and either subgroup II or nonsubgroup I-nonsubgroup II specificities. Serotype G5 has been found in piglets and horses but not yet in humans.

[Rotavirus, adenovirus, astrovirus, calicivirus and small round virus particles in feces of children with and without acute diarrhea, from 1987 to 1988, in the greater São Paulo]. / Rotavírus, adenovírus, astrovírus, calicivírus e "small round virus particles" em fezes de crianças, com e sem diarréia aguda, no período de 1987 a 1988, na grande São Paulo.

Between 1987 and 1988, 193 faecal specimens from children, with or without diarrhea, were submitted to enzyme immunoassay, polyacrylamide-gel electrophoresis and electronmicroscopy tests for virus detection. The positivity for Rotavirus, Adenovirus, Astrovirus, Calicivirus and Small Round Virus Particles (SRVP) was 11.3%, 3.1%, 2.1%, 1.0% and 4.1%, respectively, for the 97 children with acute diarrhea. Of the 96 children without diarrhea, 4.2% were positive for Rotavirus, 1.0% for Calicivirus and 7.3% for SRVP. Of 15 positive specimens for Rotavirus, 14 showed electrophoretic patterns proper to group A and 1 specimen of group C Rotavirus. The analysis of electrophorotypes demonstrated great heterogeneity of electrophoretic patterns and predominance of subgroup 2, "long". The association of virus, bacteria and parasites was present both in children with or without acute diarrhea.