- Open Access
Typing of human rotaviruses: Nucleotide mismatches between the VP7 gene and primer are associated with genotyping failure
© Rahman et al; licensee BioMed Central Ltd. 2005
- Received: 06 March 2005
- Accepted: 24 March 2005
- Published: 24 March 2005
Rotavirus genotyping is performed by using reverse transcription PCR with type-specific-primers. Because the high rotavirus mutation rate generates an extensive genomic variation, different G-type-specific primer sets are applied in different geographical locations. In Bangladesh, a significant proportion (36.9%) of the rotavirus strains isolated in 2002 could not be G-typed using the routinely used primer set. To investigate the reason why the strains were untypeable, nucleotide sequencing of the VP7 genes was performed.
Four nucleotide substitutions at the G1 primer-binding site of the VP7 gene of Bangladeshi G1 rotaviruses rendered a major proportion of circulating strains untypeable using the routine primer set. Using an alternative primer set, we could identify G1 rotaviruses as the most prevalent genotype (44.8%), followed by G9 (21.7%), G2 (15.0%) and G4 (13.8%).
Because of the natural variation in the rotaviral gene sequences, close monitoring of rotavirus genotyping methods is important.
- Stool Specimen
- Hyperimmune Antiserum
- Untypeable Strain
- Sandwich Type Enzyme Immunoassay
- Dhaka Hospital
Rotaviruses remain the most common cause of acute gastroenteritis worldwide and cause an estimated 600,000 deaths in children less than 5 years of age . The high disease burden motivated major efforts to develop a suitable rotavirus vaccine. However, the vaccine efficacy is being challenged by the extensive strain diversity of the rotaviruses [3, 7–9, 13, 14].
Rotaviruses belong to the Reoviridae, and their genome consists of 11 segments of double stranded RNA. The gene segment coding for the VP7 glycoprotein is the basis for genotyping group A rotaviruses into at least 15 G-genotypes. Among them, G1, G2, G3, G4 and G9 are the most common G-types in humans [5, 15, 16, 19, 21, 23]. The importance of type-specific immunological protection against rotavirus disease is still under discussion .
G-genotyping is performed using type-specific-primer-based RT-PCR. Two common primer sets introduced by Gouvea et al.  and Das et al.  are currently used in rotavirus G-typing surveillance programs . A failure to genotype or mistyping has already been reported from different parts of the world. These reports showed that nucleotide sequence differences between the target region of the respective genes and the primer sequences used for typing led to the genotyping failure [1, 10, 11, 17].
The Dhaka hospital of ICDDR,B, situated in the central Bangladesh, and the Matlab hospital, located 45 km south east of Dhaka respectively treat about 100,000 and 15,000 diarrhoeal patients each year. A hospital surveillance system has been established in these hospitals by ICDDR,B to collect information on clinical, epidemiological and demographic characteristics of the patients attending the hospital since 1978. In Bangladesh, rotavirus strains have previously been typed using a variety of techniques. Serotyping was introduced by Ward et al.  with specimens collected during 1985–1986 in Dhaka using neutralization with hyperimmune antisera against prototype rotavirus strains G1, G2, G3 and G4. They concluded that epitopic variations between rotavirus strains influenced the sensitivity of serotyping. Fun et al.  detected the major rotavirus types (G1 to G4) by RNA hybridization with serotype-specific synthetic oligonucleotide probes, but this method could not type 33.3% of the Bangladeshi rotaviruses. Likewise, RT-PCR depending on type-specific oligonucleotide primers failed to type a significant portion of rotaviruses. Rotavirus surveillance studies in Bangladesh between 1987 and 1997 reported that 1,095 (43.7%) samples out of 2,515 were G-untypeable [25–27].
In this study, we characterized rotavirus positive stool specimens collected in the Dhaka and Matlab hospitals during 2002 by using RT-PCR based on the primer set described by Das et al. . We found that a major proportion of the specimens were untypeable. Nucleotide sequences of VP7 genes were performed to investigate the reason why they were untypeable with the routine primer set. The untypeable specimens were further characterized by using a different primer set described by Gouvea et al. .
Detection of rotavirus strains
In 2002, a total of 3,803 patients with history of watery diarrhea were included in the hospital surveillance system. In Dhaka and Matlab, 535 (27.2%) and 358 (19.4%) specimens were positive for group A rotavirus antigens by enzyme immunoassay.
G typing using the Das primer set
Rotavirus G-typing was carried out for all rotavirus positive specimens from Matlab and for every fourth of the rotavirus-positive specimens from Dhaka. Some samples were excluded from this study due to unavailability of sufficient amount of stool specimens for testing. G-typing was performed on 433 rotavirus ELISA-positive stool samples by RT-PCR using the primer set described by Das et al. , which was routinely used in our laboratory. The most prevalent G types were G9 (20.5%); G2 (14.6%), and G4 (13.8%). G1 comprised only 11.6% of the isolates and 36.9% of the rotavirus-positive samples were untypeable.
VP7 gene sequence analysis
Distribution of G types using Gouvea primer set
Because of the natural variation in the rotaviral gene sequences, close monitoring of rotavirus genotyping methods is important. The findings described in this paper will be important for genotyping strategies in the rotavirus surveillance studies.
Stool specimens were collected from patients who presented with diarrhea to the Dhaka and Matlab hospitals of ICDDR,B in 2002. In the Dhaka hospital, stool specimens are routinely collected from every 50th patient and in Matlab hospital, every patient with diarrhea submits a stool specimen for testing.
Rotavirus antigen detection
Rotavirus antigens (group A-specific VP6 proteins) were detected in the stool specimen using a solid phase sandwich type enzyme immunoassay modelled after Dakopatts commercial kit incorporating rabbit hyperimmune antisera produced at ICDDR,B and an anti-human rotavirus-horseradish peroxidase conjugate (Dakopatts, Copenhagen, Denmark) using the same criteria for determination of positivity as those used by the Dakopatts kit .
The QIAamp Viral RNA mini kit (Qiagen/Westburg, Leusden, The Netherlands) was used according to the manufacturer's instructions for the extraction of rotavirus RNA from the stool samples.
A reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out using the Qiagen OneStep RT-PCR Kit (Qiagen/Westburg) as previously described by Das et al.  and Gouvea et al.  for rotavirus G-types (G1, G2, G3, G4 and G9) using type-specific oligonucleotide primers. The reaction was carried out with an initial reverse transcription step at 45°C for 30 min, followed by 35 cycles of amplification (30 sec at 94°C, 30 sec at 50°C, 1 min at 72°C), and a final extension of 7 min at 72°C in a thermal cycler (Eppendorf, Hamburg, AG). PCR products were run on a 2% agarose gel, and stained with ethidium bromide. Specific segment sizes for the different G types were visualized under UV-light.
The amplified PCR products were purified with the QIA quick PCR purification kit (Qiagen/Westburg), and sequenced in both directions using the dideoxy-nucleotide chain termination method with the ABI PRISM® BigDye Terminator Cycle Sequencing Reaction kit (Perkin-Elmer Applied Biosystems, Foster City, California) on an automated sequencer (ABI PRISM™ 3100). The Beg9 and End9 RT-PCR primers were used as sequencing primers.
The chromatogram sequencing files were inspected using Chromas 2.2 (Technelysium, Queensland, Australia), and consensus sequences were prepared using SeqMan II (DNASTAR, Madison, WI). Multiple sequence alignments were performed using CLUSTALX 1.81 . Sequences were manually edited in the GeneDoc version 2.6.002 alignment editor .
The nucleotide sequence data were deposited in GenBank using the National Center for Biotechnology Information (NCBI, Bethesda, MD) Sequin 5.15 submission tool http://www3.ncbi.nlm.nih.gov/ under accession numbers AY631049-AY631055.
This study was funded by the Program for Appropriate Technology in Health (PATH), grant number GAT 770-790-01451-SPS. ICDDR,B acknowledges with gratitude the commitment of PATH to the Centre's research efforts.
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