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Vera Gouvea

Bio: Vera Gouvea is an academic researcher from Federal University of Rio de Janeiro. The author has contributed to research in topics: Rotavirus & Serotype. The author has an hindex of 26, co-authored 47 publications receiving 5243 citations. Previous affiliations of Vera Gouvea include Centers for Disease Control and Prevention & Oswaldo Cruz Foundation.
Topics: Rotavirus, Serotype, Diarrhea, Gene, Reoviridae

Papers
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Journal ArticleDOI
TL;DR: A PCR typing method was devised in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels, which provided a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.
Abstract: The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR method provides a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.

1,524 citations

Journal ArticleDOI
TL;DR: The results suggest that gene 4 typing will be useful in providing more a complete characterization of HRV strains of epidemiologic or vaccine-related interest.
Abstract: Five genetically distinct human rotavirus (HRV) gene 4 groups have been described on the basis of comparative nucleotide sequencing and the predicted amino acid sequences, and at least four of them represent distinct VP4 antigenic types. To identify each gene 4 type and investigate its distribution in HRV isolates from patients with diarrhea, we developed a polymerase chain reaction (PCR) typing method using sequence information available for four genetically distinct gene 4 types. Rotavirus double-stranded RNAs (dsRNAs) isolated from stool samples were first reverse transcribed and amplified by PCR by using two oligonucleotide primers that correspond to regions that are highly conserved among all known HRV gene 4 types. The 876-bp dsDNA products were then reamplified by PCR in the presence of a cocktail containing one conserved plus-sense primer and four type-specific minus-sense primers (selected from the hypervariable region of gene 4), resulting in products of 345, 483, 267, and 391 bp corresponding to gene 4 types 1, 2, 3, and 4, respectively. This method reliably identified the gene 4 types of 16 well-characterized HRV isolates. Our results were independently confirmed for all 16 strains by reverse transcription and PCR amplification of HRV dsRNA in the presence of alternate type-specific primer pairs. For direct gene 4 typing of HRV in stool samples, we developed a method to extract rotavirus dsRNA from stool specimens by using glass powder. Our results suggest that gene 4 typing will be useful in providing more a complete characterization of HRV strains of epidemiologic or vaccine-related interest.

1,343 citations

Journal ArticleDOI
TL;DR: 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, as a result of finding them in field specimens of porcine origin.
Abstract: 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. Images

296 citations

Journal ArticleDOI
TL;DR: Rotavirus serotype G5 in fecal specimens of 38 Brazilian children with diarrhea was identified by PCR and enzyme immunoassays and exhibited long RNA electropherotypes and either subgroup II or nonsubgroup I-nonsubgroup II specificities.
Abstract: 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 Images

210 citations

Journal ArticleDOI
TL;DR: Data suggest a possible relationship between group C rotavirus and extrahepatic biliary atresia in the 10 patients in whom virus RNA was detected.
Abstract: The purpose of this retrospective study was to examine liver tissue from patients with cholestatic disease for the presence of group C rotavirus RNA. The reverse transcriptase-polymerase chain reaction (PCR) for genes 5 and 6 was used, and the PCR products were subjected to liquid hybridization with a 32P-labeled probe. A second amplification with nested primers was also used. Samples from 32 subjects (20 with biliary atresia or choledochal cyst and 12 controls) were tested. Ten of 20 biliary atresia patients were positive for group C rotavirus RNA; no controls were positive (P 1 sample that was positive. These data suggest a possible relationship between group C rotavirus and extrahepatic biliary atresia in the 10 patients in whom virus RNA was detected.

209 citations


Cited by
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Journal ArticleDOI
TL;DR: A PCR typing method was devised in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels, which provided a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.
Abstract: The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR method provides a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.

1,524 citations

Journal ArticleDOI
TL;DR: The results suggest that gene 4 typing will be useful in providing more a complete characterization of HRV strains of epidemiologic or vaccine-related interest.
Abstract: Five genetically distinct human rotavirus (HRV) gene 4 groups have been described on the basis of comparative nucleotide sequencing and the predicted amino acid sequences, and at least four of them represent distinct VP4 antigenic types. To identify each gene 4 type and investigate its distribution in HRV isolates from patients with diarrhea, we developed a polymerase chain reaction (PCR) typing method using sequence information available for four genetically distinct gene 4 types. Rotavirus double-stranded RNAs (dsRNAs) isolated from stool samples were first reverse transcribed and amplified by PCR by using two oligonucleotide primers that correspond to regions that are highly conserved among all known HRV gene 4 types. The 876-bp dsDNA products were then reamplified by PCR in the presence of a cocktail containing one conserved plus-sense primer and four type-specific minus-sense primers (selected from the hypervariable region of gene 4), resulting in products of 345, 483, 267, and 391 bp corresponding to gene 4 types 1, 2, 3, and 4, respectively. This method reliably identified the gene 4 types of 16 well-characterized HRV isolates. Our results were independently confirmed for all 16 strains by reverse transcription and PCR amplification of HRV dsRNA in the presence of alternate type-specific primer pairs. For direct gene 4 typing of HRV in stool samples, we developed a method to extract rotavirus dsRNA from stool specimens by using glass powder. Our results suggest that gene 4 typing will be useful in providing more a complete characterization of HRV strains of epidemiologic or vaccine-related interest.

1,343 citations

Journal ArticleDOI
TL;DR: The temporal and geographical distribution of human rotavirus G and P types was reviewed by analysing a total of 45571 strains collected globally from 124 studies reported from 52 countries on five continents published between 1989 and 2004 and revealed several characteristic features.
Abstract: A safe and effective rotavirus vaccine is urgently needed, particularly in developing countries. Critical to vaccine development and implementation is a knowledge base concerning the epidemiology of rotavirus G and P serotypes/genotypes throughout the world. The temporal and geographical distribution of human rotavirus G and P types was reviewed by analysing a total of 45571 strains collected globally from 124 studies reported from 52 countries on five continents published between 1989 and 2004. Four common G types (G1, G2, G3 and G4) in conjunction with P[8] or P[4] represented over 88% of the strains analysed worldwide. In addition, serotype G9 viruses associated with P[8] or P[6] were shown to have emerged as the fourth globally important G type with the relative frequency of 4.1%. When the global G and/or P type distributions were divided into five continents/subcontinents, several characteristic features emerged. For example, the P[8]G1 represented over 70% of rotavirus infections in North America, Europe and Australia, but only about 30% of the infections in South America and Asia, and 23% in Africa. In addition, in Africa (i) the relative frequency of G8 was as high as that of the globally common G3 or G4, (ii) P[6] represented almost one-third of all P types identified and (iii) 27% of the infections were associated with rotavirus strains bearing unusual combinations such as P[6]G8 or P[4]G8. Furthermore, in South America, uncommon G5 virus appeared to increase its epidemiological importance among children with diarrhea. Such findings have (i) confirmed the importance of continued active rotavirus strain surveillance in a variety of geographical settings and (ii) provided important considerations for the development and implementation of an effective rotavirus vaccine (e.g. a geographical P-G type adjustment in the formulation of next generation multivalent vaccines).

1,218 citations

Journal ArticleDOI
TL;DR: In infants, natural rotavirus infection confers protection against subsequent infection, which increases with each new infection and reduces the severity of the diarrhea.
Abstract: Background Rotavirus is the leading cause of severe diarrhea in infants. To provide a base line for assessing the efficacy of rotavirus vaccines, we evaluated the protection that is conferred by natural rotavirus infection. Methods We monitored 200 Mexican infants from birth to two years of age by weekly home visits and stool collections. A physician assessed the severity of any episodes of diarrhea and collected additional stool specimens for testing by enzyme immunoassay and typing of strains. Serum collected during the first week of life and every four months thereafter was tested for antirotavirus IgA and IgG. Results A total of 316 rotavirus infections were detected on the basis of the fecal excretion of virus (56 percent) or a serologic response (77 percent), of which 52 percent were first and 48 percent repeated infections. Children with one, two, or three previous infections had progressively lower risks of both subsequent rotavirus infection (adjusted relative risk, 0.62, 0.40, and 0.34, respecti...

860 citations

Journal ArticleDOI
TL;DR: Strain surveillance helps to determine whether the most prevalent local strains are likely to be covered by the serotype antigens found in current vaccines, and identified globally (G9) or regionally (G5, G8, and P2A[6]) common serotypes not cover by the reassortant vaccines that have undergone efficacy trials.
Abstract: The development of rotavirus vaccines that are based on heterotypic or serotype-specific immunity has prompted many countries to establish programs to assess the disease burden associated with rotavirus infection and the distribution of rotavirus strains. Strain surveillance helps to determine whether the most prevalent local strains are likely to be covered by the serotype antigens found in current vaccines. After introduction of a vaccine, this surveillance could detect which strains might not be covered by the vaccine. Almost 2 decades ago, studies demonstrated that 4 globally common rotavirus serotypes (G1-G4) represent >90% of the rotavirus strains in circulation. Subsequently, these 4 serotypes were used in the development of reassortant vaccines predicated on serotype-specific immunity. More recently, the application of reverse-transcription polymerase chain reaction genotyping, nucleotide sequencing, and antigenic characterization methods has confirmed the importance of the 4 globally common types, but a much greater strain diversity has also been identified (we now recognize strains with at least 42 P-G combinations). These studies also identified globally (G9) or regionally (G5, G8, and P2A[6]) common serotype antigens not covered by the reassortant vaccines that have undergone efficacy trials. The enormous diversity and capacity of human rotaviruses for change suggest that rotavirus vaccines must provide good heterotypic protection to be optimally effective.

598 citations