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Journal of Clinical Microbiology, September 2005, p. 4460-4465, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4460-4465.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Detection and Characterization of Human Group C Rotaviruses in Bangladesh

Mustafizur Rahman,^1,2* Sukalyani Banik,¹ Abu S. G. Faruque,¹ Koki Taniguchi,³ David A. Sack,¹ Marc Van Ranst,² and Tasnim Azim¹

ICDDR,B, Centre for Health and Population Research, Mohakhali, Dhaka 1212, Bangladesh,¹ Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research, University of Leuven, B-3000, Leuven, Belgium,² Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan³

Received 7 January 2005/ Returned for modification 28 May 2005/ Accepted 25 June 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Group C rotaviruses were detected by reverse transcription-PCR in 14 (2.3%) of 611 group A rotavirus-negative stool specimens from the patients admitted to the International Centre for Diarrhoeal Disease Research, Bangladesh hospital, Dhaka, Bangladesh, during July to December 2003. The low rate of detection suggested that infection with group C rotaviruses was an uncommon cause of hospitalization due to gastroenteritis. In addition, coinfections with pathogenic enteric bacteria were frequently observed in group C rotavirus-infected patients. Nucleotide sequence comparison of the VP4, VP6, and VP7 genes revealed that the Bangladeshi group C rotaviruses were most similar to Nigerian group C rotavirus strains. Phylogenetic analysis suggested that all human group C rotaviruses, including the strains isolated in our study, clustered in a monophyletic branch, which was distantly related to the branch comprised of animal group C rotaviruses.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rotaviruses are classified into seven antigenically distinct groups (A to G) on the basis of a common group antigen, the inner capsid protein (VP6). Groups A, B, and C are associated with acute gastroenteritis in humans and animals while groups D, E, F, and G only infect animals (11, 32, 33). In contrast to group A rotaviruses, which are the most common viral agents causing diarrheal infections in children younger than 3 years, group C rotaviruses cause sporadic cases of acute diarrhea or outbreaks of illness in children older than 3 years and adults (25, 27, 42). Seroepidemiological investigations have demonstrated that antibodies to group C rotaviruses are present in as much as 3 to 45% of the population tested. The viral detection rate in humans has remained low (5, 19, 30, 40). Therefore, the role of group C rotaviruses in the global picture of diarrheal illness has remained unresolved.

Group C rotaviruses were first isolated in piglets in 1980 (34) and in humans in 1982 (31). Since then, they have been recognized in humans and animals, both in industrialized countries including Australia, United States, United Kingdom, Finland, and Japan and in developing countries or regions such as India, China, Malaysia, and Latin America (3, 4, 5, 8, 10, 18, 22, 26, 29, 41). Thus, group C rotavirus strains are globally distributed and are thought to be one of the emerging pathogens in humans. However, no group C rotavirus infection either in animals or in humans has been reported from Bangladesh thus far.

Like group A rotaviruses, group C rotaviruses contain 11 segments of double-stranded RNA, but their RNA migration pattern in polyacrylamide gel electrophoresis (PAGE) is different (4-3-2-2) from that of group A rotaviruses (4-2-3-2). Serotyping of group C rotaviruses remains complicated due to the difficulties in adapting human group C rotaviruses in cell culture (13, 28). Sequence comparison suggests that genetic diversity exists among group C rotaviruses, although on a much narrower scale than among group A rotaviruses. Genotyping for group C rotaviruses has been proposed; however, no formal classification system based on VP4 (P type) and VP7 (G type) has been established (12, 16, 20, 39). Porcine Cowden and bovine Shintoku strains were proposed as different G serotypes, and the existence of a third G serotype for porcine HF strain was suggested (39). All human group C rotaviruses analyzed so far belong to a fourth G serotype, and a high degree of conservation exists among them (1, 6, 16, 21). Evolutionary studies also indicate that human group C rotaviruses evolved quite recently and possibly belong to a single globally distributed genotype (7, 16, 21). Jiang and colleagues (20) proposed three P genotypes, where human, porcine, and bovine group C rotaviruses constituted the different groups.

The International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) hospital in Dhaka in central Bangladesh treats about 100,000 diarrheal patients a year. In a systematic surveillance system (37), stool samples are collected from 2% of the patients attending the hospital for laboratory determination of the presence of bacterial and viral enteric pathogens. As part of this surveillance study, we determined the prevalence of group C rotaviruses among hospitalized patients who were negative for group A rotaviruses. We sequenced the VP4, VP6, and VP7 genes of the local group C rotavirus strains. Their genetic relationship with other group C rotavirus strains circulating all over the world was also examined.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study population and data collection. During July to December 2003, about 50,000 patients were admitted to the ICDDR,B Dhaka hospital with a history of watery diarrhea. As part of the surveillance, the stool specimens from every 50th patient (2% of sample; n = 956) were tested for various common enteric pathogens, which included group A rotaviruses and Vibrio, Shigella, and Salmonella species. In the present study, the stool specimens negative for group A rotaviruses (n = 611) were tested for group C rotaviruses.

Pooling of the samples. To decrease the costs of testing we have performed a two-step testing procedure, whereby specimens were first tested in pools of 10 samples in a single test by reverse transcription-PCR (RT-PCR), followed by a second PCR. It was considered that all samples in the negative pools were negative. Samples from positive pools were retested individually to determine which sample in the pool was positive. Pooling of 10 samples with the expected prevalence of group C rotaviruses (1 to 5%) resulted in a reduction of 40 to 80% of the laboratory costs.

Electropherotyping. One-hundred microliters of 2% stool suspensions in phosphate-buffered saline was treated with sodium acetate and extracted with an equal volume of phenol:chloroform:isoamylalcohol (25:24:1) mixture. The extracted RNA was tested for electropherotype (E-type) by polyacrylamide gel electrophoresis (PAGE) as described by Herring et al. (17).

Reverse transcription-PCR. RNA was extracted from the stool suspension using the QIAamp Viral RNA mini kit (QIAGEN/Westburg, Leusden, The Netherlands) according to the manufacturer's instructions. RT-PCR was carried out using the OneStep RT-PCR kit (QIAGEN/Westburg). The primers specific for the group C rotavirus VP6 gene were used as described by Gouvea et al. (15). The reaction was carried out with an initial reverse transcription step at 45°C for 30 min, followed by 40 cycles of amplification (30 s at 94°C, 30 s at 50°C, 1 min at 72°C) and a final extension of 7 min at 72°C in a thermal cycler. PCR products were run on a 1.5% ethidium bromide-stained agarose gel and visualized under UV light. A specific segment size (356 bp) for a group C rotavirus DNA product was observed on stained gels. A second PCR was also carried out with the first PCR product as a template using the same primer pairs.

Nucleotide sequencing. The PCR amplicons were purified with the QIAquick 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, Calif.) on an automated sequencer (ABI PRISM 310). The consensus primers GrC VP7-20F (5'-GCTGTCTGACAAACTGGTC-3'; strain Jajeri, accession number AF323982, nucleotide [nt] 20 to 38) and GrC VP7-1062R (5'-GCCACATGATCTTGTTTACGC-3'; strain Jajeri, nt 1042 to 1062) were used for VP7 gene sequencing. For the VP4 gene, consensus primers GrC VP4-1F (5'-GGCTTAAAAAGTAGAGATCG-3'; strain Jajeri, accession number AF323981, nt 1 to 20) and GrC VP4-1243R (5'-CCAGGATATGATCCTACAGG-3', strain Jajeri, nt 1224 to 1243) were used. The VP6 primers used in the RT-PCR were employed for VP6 gene sequencing (15).

DNA and protein sequence analysis. The chromatogram sequencing files were inspected using Chromas 2.23 (Technelysium, Queensland, Australia), and contigs were prepared using SeqMan II (DNASTAR, Madison, WI). Nucleotide and amino acid sequence similarity searches were performed using the National Center for Biotechnology Information (NCBI; National Institutes of Health, Bethesda, MD) BLAST (basic local alignment search tool) server on the GenBank database, release 145.0 (2). Multiple sequence alignments were calculated using CLUSTALX 1.81 (38). Sequences were manually edited in the GeneDoc, version 2.6.002, alignment editor.

Phylogenetic analysis. Phylogenetic and molecular evolutionary analyses were conducted using the MEGA version 2.1 software package (24). Genetic distances were calculated using the Kimura two-parameter model. The dendrograms were constructed using the neighbor-joining method.

Nucleotide sequence accession numbers. The nucleotide sequences reported in this paper were submitted to GenBank using National Center for Biotechnology Information (NCBI; Bethesda, MD) Sequin, version 5.00, under accession numbers AY754824 to AY754827.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Detection of group C rotaviruses. A total of 611 diarrheal stool specimens which were negative for group A rotaviruses were investigated for the presence of group C rotaviruses by PAGE and RT-PCR. Group C rotaviruses were detected in 14 samples (2.3% of the subset tested and 1.5% of the overall study) using one-step RT-PCR followed by a second PCR. PAGE could detect only one group C rotavirus strain with the characteristic 4-3-2-2 RNA migration pattern (Fig. 1).

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FIG. 1. Gel electrophoresis showing the characteristic genomic double-stranded RNA electrophoresis patterns of group A (lane A), group B (lane B), and group C (DhakaC13) (lane C) rotaviruses. The viral RNAs were analyzed by electrophoresis in a 10% polyacrylamide gel and visualized by staining with silver nitrate.

Clinical features of the group C rotavirus-infected patients. Clinical data of the 14 patients infected with group C rotaviruses are listed in Table 1. We found that eight group C rotavirus-infected patients (57.1%) had a variety of bacterial coinfections: six with Vibrio cholerae and two with Shigella flexneri. As the number of cases without coinfection was low (n = 6), it was difficult to determine whether the patient's clinical features were related to group C rotavirus and/or to the other pathogens.

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TABLE 1. Clinical features of the patients infected with group C rotaviruses

Sequence analysis of the VP6 gene. The partial VP6 sequences (nt 1020 to 1329, corresponding to the VP6 gene of the Jajeri strain) were determined for four group C rotavirus strains (DhakaC2, DhakaC4, DhakaC12, and DhakaC13) from the stool specimens with the most starting materials. They were almost identical to each other (99.3% nucleotide and 100% amino acid identities). Strain DhakaC13 was chosen as a representative of Bangladeshi group C rotaviruses. Sequence comparison revealed that the DhakaC13 strain had the greatest identity with the Jajeri strain, isolated in Nigeria, and with strain 208, isolated in China (98% nucleotide and 100% amino acid identities for both strains) (Table 2). Animal strains exhibited much less nucleotide and amino acid similarities with our DhakaC13 strain (82 to 84% nucleotide and 90 to 95% amino acid identities). A phylogenetic tree (Fig. 2) was constructed which included the partial nucleotide sequences of the published strains and the Bangladeshi strains (DhakaC2 and DhakaC13). Bangladeshi strains belonged to the human cluster, which was again distantly related to the animal clusters. A bovine strain (WD534tc) that has been suggested to be of porcine origin was found to belong to the porcine cluster as expected.

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TABLE 2. Nucleotide and amino acid sequence similarity between DhakaC13 and other group C rotavirus strains based on partial VP6 sequencesa

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FIG. 2. Neighbor-joining phylogenetic tree based on partial nucleotide sequences (nt 1020 to 1329) of the VP6 genes for DhakaC13 and other established group C rotavirus strains. Bo, bovine; Hu, human; Po, porcine. The numbers adjacent to the nodes represent the percentage of bootstrap support (of 1,000 replicates) for the clusters to the right of the node. Bootstrap values lower than 75% are not shown.

Sequence analysis of the VP4 gene. The partial VP4 gene sequence (nt 21 to 682) was determined for DhakaC13 and was compared with those of group C rotavirus strains available in the GenBank database (Table 3). The VP4 gene of DhakaC13 was closely related to the Nigerian Jajeri strain (98% identity at nucleotide and amino acid levels). Similarities with other human group C rotaviruses were also very high (96 to 97% at nucleotide levels and 94 to 96% at amino acid levels), whereas similarities with porcine and bovine group C rotaviruses were low (65 to 70% at the nucleotide level and 63 to 65% at the amino acid level). The phylogenetic tree revealed that three clusters were comprised of different host-specific strains (Fig. 3). DhakaC13 belonged to the human branch and was closely related to the Jajeri and Moduganari strains isolated from Nigeria. All human strains were distantly related to bovine and porcine clusters.

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TABLE 3. Nucleotide and amino acid sequence similarity between DhakaC13 and other group C rotavirus strains based on partial VP4 sequencesa

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FIG. 3. Neighbor-joining phylogenetic tree based on the nucleotide sequences (nt 21 to 682) of partial VP8* fragments of the VP4 genes for DhakaC13 and other established group C rotavirus strains. Bo, bovine; Hu, human; Po, porcine. The numbers adjacent to the nodes represent the percentage of bootstrap support (of 1,000 replicates) for the clusters to the right of the node. Bootstrap values lower than 75% are not shown.

Sequence analysis of the VP7 gene. The complete coding nucleotide sequence (nt 49 to 1054) and deduced amino acid sequence of the VP7 gene of the DhakaC13 strain was determined and compared with VP7 gene sequences of other group C rotavirus strains available in the GenBank database. Table 4 shows the similarity of the published group C rotavirus strains with the DhakaC13 strain. Sequence comparison indicated that the VP7 sequence of the DhakaC13 strain was also most closely related to the Jajeri strain (97% identity at nucleotide and 99% identity at amino acid level). The VP7 sequences of all human rotavirus C strains were 95 to 99% identical at the nucleotide level and 96 to 99% at the amino acid level. The strains isolated from animals exhibited much less similarity to our DhakaC13 strain (73 to 82% nucleotide and 71 to 87% amino acid identities). Three conserved sites (amino acid positions 67 to 69, 152 to 154, and 225 to 227) that are potential N-glycosylation sites (Asn-X-Ser/Thr) were found in the VP7 deduced amino acid sequence of the DhakaC13 strain. A more detailed phylogenetic analysis that included all VP7 gene sequences of group C rotaviruses (Fig. 4) confirmed that our DhakaC13 strain belonged to the human group C rotavirus strains which clustered in a monophyletic branch, distantly related to the animal branches.

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TABLE 4. Nucleotide and amino acid sequence similarity between DhakaC13 and other group C rotavirus strains based on partial VP7 sequencesa

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FIG. 4. Neighbor-joining phylogenetic tree based on nucleotide sequences (nt 49 to 1054) of the encoding regions of the VP7 gene for DhakaC13 and other established group C rotavirus. Bo, bovine; Hu, human; Po, porcine. The numbers adjacent to the nodes represent the percentage of bootstrap support (of 1,000 replicates) for the clusters to the right of the node. Bootstrap values lower than 75% are not shown.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study is the first report of group C rotaviruses in Bangladesh. The low rate of detection suggests that group C rotavirus is an uncommon cause of gastroenteritis in hospitalized patients in Bangladesh. Moreover, the frequent association of group C rotavirus with other pathogens casts doubt on the pathogenic role of group C rotaviruses in these patients. Coinfections with pathogenic enteric bacteria were frequently observed in group C rotavirus-infected patients (57.1%) compared to the patients infected by group A rotaviruses (6.9%, n = 345; data not shown) during the same period.

The importance of group C rotaviruses in humans has been considered insignificant due to their sporadic nature, but the distribution of group C rotaviruses has recently been shown to be more common than previously believed according to a number of seroepidemiological surveys (9, 23, 25, 27, 30, 35, 36, 43). The true burden of group C rotaviruses might be underestimated due to difficulty in detecting them. In our study, PAGE was seen to be relatively insensitive. We could detect only one out of 14 PCR-confirmed group C rotavirus-positive stool specimens by this method. Likewise, pooling of the samples for RT-PCR resulted in a 1:10 dilution of our samples. For this reason, it is possible that we failed to detect some of the positive group C rotaviruses in the diluted specimens. Therefore, it is required that a less expensive enzyme immunoassay method be developed to detect and differentiate group C rotavirus G serotypes from a large-scale epidemiological study.

A formal classification system for group C rotaviruses has not yet been established, although at least four G types have been proposed by many investigators using sequence analysis of different human and animal group C rotavirus strains. For group A rotaviruses, it was observed that strains having more than 89% amino-acid-identical VP7 sequences belonged to the same G serotype (14). We studied the similarities between 45 complete VP7 amino acid sequences of all group C rotavirus strains available in GenBank (Table 5). The lineages were confirmed by the VP7 phylogenetic tree (Fig. 4). We found that the group C rotavirus strains could be clustered into four different groups if we consider 89% amino acid identity as a cutoff for grouping them. The porcine strain HF isolated in the United States and the bovine Shintoku strain isolated in Japan belonged to lineage 1 and lineage 2, respectively. The other two porcine strains, WH and Cowden, comprised a third lineage (lineage 3). All human strains clustered in a monophyletic branch (lineage 4).

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TABLE 5. Amino acid sequence similarity matrix of VP7 proteins of group C rotavirus strains of four different lineages derived from the VP7 phylogenetic tree

Three P types were proposed by Jiang et al. (20) for group C rotavirus strains, which can be supported by the phylogenetic tree described in this paper (Fig. 2). However, since only eight VP4 gene sequences of human group C rotaviruses are available in GenBank, more sequences would be needed to better describe the P types of the group C rotavirus strains. We found that all human group C rotavirus strains clustered together in the same branch of the VP4 tree, which indicated one common ancestor of these strains.

Sequence analyses of the VP4, VP6, and VP7 genes of group C rotavirus strains revealed that the gene sequences are conserved and host restricted. These findings are supported by other group C rotavirus evolutionary studies (16, 21, 39). Further studies on the gene segments other than the VP4, VP6, and VP7 genes will be necessary to confirm whether the diversity of group C rotaviruses is really very low compared to group A rotaviruses.

Because this study is a hospital-based study, it is difficult to extrapolate our data to community rates of group C rotavirus infection, because only patients with significant dehydration were admitted. Moreover, older patients who are more frequently infected by group C rotaviruses usually do not go to hospitals unless the disease becomes extremely severe. Therefore, a community-based study would be required to investigate the true prevalence and burden of disease of group C rotaviruses. At the same time, it will be necessary to develop more sensitive and easy-to-use diagnostic methods specific for human group C rotaviruses for large-scale epidemiological studies.

 
This research study was funded by the ICDDR,B Centre for Health and Population Research and Flemish Fonds voor Wetenschappelijk, grant number G.0288.01.

 
* Corresponding author. Mailing address: Senior Research Officer, Virology Laboratory, Laboratory Sciences Division, ICDDR,B, Centre for Health and Population Research, Mohakhali, Dhaka 1212, Bangladesh. Phone: 8802-8811751-60, ext. 2409. Fax: 8802-881-2529. E-mail: mustafizur.rahman{at}uz.kuleuven.ac.be.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, September 2005, p. 4460-4465, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4460-4465.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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