scispace - formally typeset
Search or ask a question
Journal ArticleDOI

Identification of group A rotavirus gene 4 types by polymerase chain reaction.

01 Jun 1992-Journal of Clinical Microbiology (American Society for Microbiology)-Vol. 30, Iss: 6, pp 1365-1373
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.
Citations
More filters
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


Cites methods from "Identification of group A rotavirus..."

  • ...The 2 outer capsid proteins carry rotavirus serotype (neutralization)– specific antigens and are encoded by segments 4 (VP4 protease- sensitive protein and P serotype antigen) and by segments 7, 8, or 9 (VP7 glycoprotein and G serotype antigen)....

    [...]

  • ...In the case of strain 116E, the VP4 gene is highly related to the bovine serotype P8[11]G10 VP4 gene, whereas the remaining 10 genes are related to typical HRV strains of the Wa genogroup (e.g., P1A[8]G1, SG II, long electrophero- type) [121]; strain I321 (P[11]G10) contains 2 genes from HRVs of the Wa genogroup, and its remaining genes are from a bovine rotavirus [152]....

    [...]

  • ...Because the VP4 and VP7 proteins are encoded by separate gene segments, rotaviruses can generate new P-G serotype antigen combina- tions through reassortment after dual infection of single cells....

    [...]

  • ...Because identification of P serotypes is technically difficult, usually, only the corresponding VP4 genes are identified by genotyping methods in surveillance stud- ies....

    [...]

  • ...Nucleotide sequencing studies provided strong evidence that reassortment occurs between circulating genotype P[8] strains with G1, G3, G4, or G9 specificity when it was found that phylogenetically distinct VP4 genes of such strains can seg- regate with VP7 genes of 11 G serotype [138, 139]....

    [...]

Journal ArticleDOI
TL;DR: Between 1986 and 1993, 72% of rotavirus strains isolated from newborns at five hospitals in New Delhi, India, had long electropherotypes, subgroup II VP6 antigens, and G and P genotypes identical to those of prototype strain 116E.
Abstract: Between 1986 and 1993, 72% of rotavirus strains isolated from newborns at five hospitals in New Delhi, India, had long electropherotypes, subgroup II VP6 antigens, and G and P genotypes (G9P11) identical to those of prototype strain 116E A novel strain with a G9P6 genotype, representing 13% of the isolates, was identified These results demonstrate that G9P11 and G9P6 rotavirus strains are common in nurseries in New Delhi

459 citations


Cites background or methods from "Identification of group A rotavirus..."

  • ...amplification were identical to those described previously, except that 40 PCR cycles were used and the extension time was 3 min at 720C (8)....

    [...]

  • ...As a result, nucleic acid-based (genotyping) methods that detect genetically distinct VP4 genes and accurately predict P serotypes have been developed (8)....

    [...]

  • ...Rotavirus doublestranded RNA was extracted from cell lysates or fecal specimens, and 5 ,ul of the eluate was analyzed (8)....

    [...]

Journal ArticleDOI
TL;DR: Methods and oligonucleotide primers are described to overcome failures to type G9, G 10 and P[11] rotavirus strains, and cross-reactivity identified between G10 and G3 rotaviruses.

411 citations


Cites background or methods from "Identification of group A rotavirus..."

  • ...Similarly, a 876 bp fragment of the gene encoding VP4 was amplified and sequenced using primers Con2 and Con3 (Gentsch et al., 1992) from 10 rotavirus strains that failed to P-type....

    [...]

  • ...2nd round P[4] CTA TTG TTA GAG GTT AGA GTC (nt 474–494) 483 Gentsch et al. (1992) P[6] TGT TGA TTA GTT GGA TTC AA (nt 259–278) 267 Gentsch et al. (1992)...

    [...]

  • ...(2000b) P[9] TGA GAC ATG CAA TTG GAC (nt 385–402) 391 Gentsch et al. (1992) P[10] ATC ATA GTT AGT AGT CGG (nt 575–594) 583 Gentsch et al....

    [...]

  • ...(2000b) P[9] TGA GAC ATG CAA TTG GAC (nt 385–402) 391 Gentsch et al. (1992) P[10] ATC ATA GTT AGT AGT CGG (nt 575–594) 583 Gentsch et al. (1992) P[11] GTA AAC ATC CAG AAT GTG (nt 305–323) 312 This paper Con-3 As above...

    [...]

  • ...Reverse transcription with random primers and G and P typing semi-nested PCRs were performed as described previously ( Gentsch et al., 1992; Gouvea et al., 1990; Iturriza-Gómara et al., 1999)....

    [...]

Journal ArticleDOI
TL;DR: These studies indicate that while rotavirus strains have limited diversity in many settings, reassortment between common and uncommon serotypes or animal strains can arise in some settings and, thus, lead to unusual diversity.
Abstract: Candidate rotavirus vaccines have been prepared with reassortant strains specifically to protect against the 4 major rotavirus G serotypes (G1 -4). Many studies using P (VP4) genotyping methods have indicated that, worldwide, rotavirus strains of the 4 common G serotypes are each associated with 1 P genotype: GI, G3, and G4 are associated with P[8], and G2 is associated with P[4]. In contrast, G and P genotyping of rotavirus in specimens from India revealed that a high percentage of the childhood diarrhea strains belong to genotype P[6], and the most common strain had an unusual G serotype, G9. Similarly, in all regions surveyed in Brazil, apparent reassortants of genotype P[8], G5 were found in children with gastroenteritis. These studies indicate that while rotavirus strains have limited diversity in many settings, reassortment between common and uncommon serotypes or animal strains can arise in some settings and, thus, lead to unusual diversity.

401 citations


Cites methods from "Identification of group A rotavirus..."

  • ...Genotyping was done by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by probe hybridization confirmation of PCR products [39-41, 59, 60]....

    [...]

  • ...Genotyping methods have been reported for 6 of 7 HRV P serotypes, for 9 of 10 HRV G serotypes, and for several P and G serotypes identified only in animals [27, 39-44]....

    [...]

Journal ArticleDOI
TL;DR: The future development of a safe and highly effective vaccine against rotavirus could prevent, at least, cases of severe diarrhea and reduce mortality from this disease.

360 citations

References
More filters
Journal ArticleDOI
15 Aug 1970-Nature
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.

232,912 citations


"Identification of group A rotavirus..." refers methods in this paper

  • ...The concentration of rotavirus dsRNA from cell lysates was estimated by comparison with known amounts of dsRNA from purified virions that were analyzed by polyacrylamide gel electrophoresis (PAGE) and silver staining (16, 26)....

    [...]

Journal Article
01 Jan 1970-Nature
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products as mentioned in this paper.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.

203,017 citations

Journal ArticleDOI
TL;DR: A group of programs that will interact with each other has been developed for the Digital Equipment Corporation VAX computer using the VMS operating system.
Abstract: The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis and publication of biological sequence data. A group of programs that will interact with each other has been developed for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions for transfer are described.

14,575 citations


"Identification of group A rotavirus..." refers methods in this paper

  • ...1) was chosen for the first amplification because the primers correspond to regions that are highly conserved among HRV strains from VP4 genetic groups 1 to 4, as determined by best fit analysis with the University of Wisconsin Genetics Computer Group sequence analysis program (3) (using sequences found in the GenEMBL data bank)....

    [...]

Journal ArticleDOI

2,699 citations


"Identification of group A rotavirus..." refers background in this paper

  • ...events during amplification of complex nucleic acids (4)....

    [...]

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

Related Papers (5)