Characterization of a Novel P[25],G11 Human Group A Rotavirus
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微生物临床杂志 2005年第7期
ICDDR,B, Centre for Health and Population Research, Mohakhali, Dhaka-1212, Bangladesh
Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research, University of Leuven, B-3000 Leuven, Belgium
ABSTRACT
A novel rotavirus strain (Dhaka6) isolated from a 21-year-old Bangladeshi male patient was characterized by sequence analysis of its VP7 and VP4 gene segments. Phylogenetic analysis of the VP7 gene of the Dhaka6 strain revealed a common evolutionary lineage with porcine G11 rotavirus strains. This isolate is the first reported G11 rotavirus strain infecting a human host. Comparison of the VP4 gene sequences with all currently recognized 24 different P genotypes revealed only low nucleotide (54 to 71%) and amino acid (52 to 76%) sequence identities. This lack of high sequence similarity in the VP4 gene indicates that the Dhaka6 isolate represents a new group A rotavirus P genotype, to which we propose assignment of the designation P[25].
INTRODUCTION
Rotaviruses belong to the Reoviridae and contain 11 segments of double-stranded RNA encoding six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural viral proteins (NSP1 to NSP6). Group A rotaviruses are a common cause of acute infectious diarrhea in humans and in a wide variety of mammalian and avian species. A dual system of classification of group A rotaviruses is based on two outer capsid proteins, i.e., VP4 (P types) and VP7 (G types). Rotaviruses can be serotyped by neutralization assays with panels of antisera and genotyped by sequence analysis of different gene segments (8, 11). Although there is a close relationship between the two classification systems, it has recently been proposed that the term serotype should be reserved for serological analysis and that the term genotype should be used for genetic classification and comparative sequence analysis. Strains that have more than 89% amino acid identity are considered to be of the same genotype (7, 9, 38).
So far, strains of at least 15 G genotypes and 24 P genotypes have been isolated from humans and a variety of mammal and avian species (20, 23, 24, 24a, 29, 32, 38). The major human G types are G1, G2, G3, G4, and G9, combined with the P types P[8] and P[4] (1, 9, 16, 17, 19, 36). In Bangladesh in 2002, G1 (44.8%) was the most prevalent human rotavirus genotype, followed by G9 (21.7%), G2 (15.0%), and G4 (13.8%). A G11 rotavirus (strain YM, P[7],G11) was first isolated from pigs in several regions of Mexico in 1983 (34) and was subsequently identified in the United States and Venezuela (3, 33). It was never found in humans or any other host. The other commonly identified G types in pigs are G5, G3, and G4 (4, 5, 6, 12) and are combined with two major P types, P[6] and P[7] (2, 18, 22, 35, 37).
Although most rotaviruses appear to be host restricted, interspecies transmission of rotaviruses between animals and humans has been described (20). Rotaviruses are excreted and dispersed in the environment after replication in the gastrointestinal tracts of humans or animals. They were reportedly found to survive in water sources, although the presence of uncoated rotavirus segments in the environment has not been proven. Whole viruses can be transferred from animals to humans and vice versa, but they rarely produce severe clinical manifestations in a new host unless they go through a unique event for segmented viruses called reassortment. Serious infections in human caused by reassortant rotavirus strains have been reported by many investigators. Reassortment events are facilitated by the occurrence of mixed infections in a host where they exchange one or more segments during their replication, resulting in a new and diverse progeny population of rotaviruses (25, 26, 27, 37).
In this report, we present the complete nucleotide sequence of the coding region of the VP7 gene and the partial nucleotide sequence of the VP4 gene of the Dhaka6 rotavirus isolate and compare them with those of the corresponding genes of human and animal rotavirus strains. This is, to our knowledge, the first reported case of a G11 rotavirus infection in a human patient. This is also the first description of a novel VP4 gene specificity distinct from any of the known rotavirus P types.
MATERIALS AND METHODS
Enzyme immunoassay for the detection of rotavirus antigen. Group A-specific VP6 antigen was detected in stool specimens by a solid-phase sandwich-type enzyme immunoassay (40), using rabbit hyperimmune antisera produced at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh, and an anti-human rotavirus-horseradish peroxidase conjugate (Dakopatts, Copenhagen, Denmark) (40).
RNA extraction. Viral RNA was extracted from the fecal sample by use of a QIAamp viral RNA mini kit (QIAGEN/Westburg, Leusden, The Netherlands), according to the manufacturer's instructions.
RT-PCR. The extracted RNA was denatured at 97°C for 5 min. A multiplex reverse transcriptase PCR (RT-PCR) was carried out with a OneStep RT-PCR kit (QIAGEN/Westburg) for different rotavirus G genotypes (G1, G2, G3, G4, and G9), as previously described by Gouvea et al. (13). Type-specific forward primers derived from distinct regions of the VP7 gene for G1 (primer aBT1; 5'-CAAGTACTCAAATCAATGATGG-3'), G2 (primer aCT2; 5'-CAATGATATTAACACATTTTCTGTG-3'), G3 (primer aET3; 5'-CGTTTGAAGA AGTTGCAACAG-3'), G4 (primer aDT4; 5'-CGTTTCTGGTGAGGAGTTG-3'), and G9 (primer aFT9; 5'-CTAGATGTAACTACAACTAC-3') were combined with reverse primer RVG9 (5'-GGTCACATCATACAATTCT-3'). This primer mix allows the amplification of fragments with G-type-specific segment sizes visible by polyacrylamide gel electrophoresis. The reaction was carried out with an initial reverse transcription step at 45°C for 30 min, followed by PCR activation at 95°C for 15 min, 35 cycles of amplification (30 s at 94°C, 45 s at 50°C, 1 min at 72°C), and a final extension of 7 min at 72° in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA). PCR products were run on a polyacrylamide gel, stained with ethidium bromide, and visualized under UV light.
As the multiplex PCR with type-specific primers could not detect the G genotype of strain Dhaka6, we amplified a 1,062-bp fragment of the VP7 gene with the consensus forward primer Beg9 (5'-GGCTTTAAAAGAGAGAATTTCCGTCTGG-3') and the reverse primer End9 (5'-GGTCACATCATACAATTCTAATCTAAG-3') (13). The consensus primers Con3 (5'-TGGCTTCGCCATTTTATAGACA-3') and Con2 (5'-ATTTCGGACCATTTATAACC-3') (10) failed to amplify the VP4 gene segments of our strain. Therefore, we designed new primers which were derived from the nucleotide sequence alignment of the VP4 genes available in GenBank. Forward primer VP4-1-17F (5'-GGCTATAAAATGGCTTCGC-3'; nucleotides [nt] 1 to 17) and reverse primer VP4-1083-1099R2 (5'-GCTTGTGAATCATCCCA-3'; nt 1083 to 1099) were designed for amplification of the first 1,099 bp of nucleotides of the VP4 gene. For the amplification of the 3' part of VP4, we used forward primer VP4-986-1004F (5'-TGGTGTTAATGATTTCAGC-3'; nt 986 to 1004) and reverse primer VP4-1919-1937R (5'-GTACWGCNGCTGAAATATC-3'; nt 1919 to 1937).
Nucleotide sequencing. The PCR amplicons were purified with a QIAquick PCR purification kit (QIAGEN/Westburg) and sequenced in both directions by the dideoxy-nucleotide chain termination method with the ABI PRISM BigDye Terminator Cycle Sequencing Reaction kit (Applied Biosystems) on an automated sequencer (ABI PRISM 3100). The same primers used in the first RT-PCR were used as sequencing primers.
DNA and protein sequence analysis. The chromatogram sequencing files were inspected by using Chromas 2.3 (Technelysium, Queensland, Australia), and consensus sequences were prepared by using SeqMan II (DNASTAR, Madison, WI). Multiple-sequence alignments were calculated by using CLUSTALX 1.81 (39). Sequences were manually edited in the GeneDoc alignment editor. Nucleotide and protein sequence similarity searches were performed by using GeneDoc alignment editor (version 2.6.002) (28).
Phylogenetic analysis. Phylogenetic and molecular evolutionary analyses were conducted by using the MEGA version 2.1 software package (21), based on the different G and P rotavirus sequences available in GenBank version 143.0. Genetic distances were calculated by the Kimura two-parameter method. The dendrograms were constructed by the neighbor-joining method.
Nucleotide sequence accession numbers. The nucleotide sequence data reported in this paper were submitted to GenBank by using the National Center for Biotechnology Information (Bethesda, MD) Sequin Application version 5.26 submission tool (http://www.ncbi.nlm.nih.gov) and can be found under GenBank accession numbers AY773003 (for the VP7 gene) and AY773004 (for the VP4 gene).
RESULTS
Case history and rotavirus antigen detection. A 21-year-old Bangladeshi male patient was admitted to the hospital of ICDDR,B in 2001 with severe diarrhea and abdominal pain. No vomiting or fever was recorded. The patient worked as a laborer in a water pump station in central Dhaka. No travel history was recorded, but it is very unlikely that he had traveled outside Bangladesh. Visits to his village outside Dhaka were possible. The stool frequency had been more than 16 times during the previous 24 h. After 5 days of passing watery stools, progressive dehydration necessitated hospitalization, and oral rehydration solution was given to the patient. The patient absconded from the hospital 3 h after admission. Further investigation was not possible because the patient was not available at his address. The stool specimen was found to be positive for rotavirus antigen by a VP6-specific enzyme-linked immunosorbent assay and was negative for the common enteric pathogens Vibrio, Shigella, and Salmonella. A routine multiplex VP7 RT-PCR method (13) with G1, G2, G3, G4, and G9 type-specific primers could not identify the G genotype; and further characterization by nucleotide sequencing was needed.
Nucleotide sequencing of VP7 and VP4 genes. The complete open reading frame (978 bp) and deduced amino acid sequence of the VP7-encoding gene of the Dhaka6 strain was determined (GenBank accession number AY773003) and compared with the VP7 sequences of prototype strains belonging to G1 to G15 (Table 1). Sequence comparison indicated that the VP7 sequence of strain Dhaka6 was most closely related to the VP7 sequence of G11 porcine rotavirus strains (87 to 91% nucleotide identity and 95% amino acid identity) and showed the highest similarity (91% nucleotide identity and 95% amino acid identity) to Mexican porcine rotavirus strain YM (P[7],G11). It was also observed that another porcine strain, OSU (P[7],G5), was closely related to our G11 strain (83% nucleotide identity and 90% amino acid identity). Rotavirus strains representing other G types exhibited much lower nucleotide and amino acid identities (62 to 78% nucleotide identity and 56 to 86% amino acid identity) with our strain. A dendrogram that included the sequences of all 15 known VP7 G types of rotaviruses in the GenBank database (release 143.0) was constructed (Fig. 1). Phylogenetic analysis confirmed that our Dhaka6 strain clustered with the G11 porcine rotavirus prototype strain in a monophyletic branch.
The amino acid sequences of three intragenotype-conserved important antigenic regions of 15 established rotavirus G genotypes were aligned with the respective sequences of the Dhaka6 strain (Fig. 2). The Dhaka6 strain showed a close relationship with the G11 porcine rotaviruses in these regions, with a few amino acid changes. Only one amino acid change in antigenic region A, no changes in antigenic region B, and two amino acid changes in antigenic region C were observed when the Dhaka6 amino acid sequence was compared with that of G11 porcine strain YM. The sequences of the antigenic regions of Dhaka6 strain showed more than 30% divergence compared with the sequences of antigenic regions of strains of genotypes other than G11.
We could not amplify the VP4 gene of the Dhaka6 strain with the general VP4-specific primers Con2 and Con3, and a modified primer set was designed for sequencing of a partial 1,894-bp fragment of the VP4-encoding gene, which was compared with those of other reference P genotypes available in GenBank (Table 2). The VP4 nucleotide and amino acid sequence similarities of Dhaka6 (GenBank accession number AY773004) to the sequences of all currently recognized P types were very low (54 to 71% nucleotide identity and 52 to 75% amino acid identity). Since the VP4 amino acid sequence identity of the Dhaka6 strain to all recognized P types is less than the cutoff of 89%, the Dhaka6 isolate represents a new group A rotavirus P genotype, for which we propose the name P[25]. A more detailed phylogenetic analysis (Fig. 3) that included the sequences of all known rotavirus P genotypes confirmed that the Dhaka6 strain is located in a separate branch that is only distantly related to the other P genotypes.
DISCUSSION
In Bangladesh, pigs are uncommon farm animals, and no genotyping studies of rotavirus isolates from pigs or other animals have been conducted thus far. Therefore, the identification of the Dhaka6 strain with a G11 VP7 specificity in a human patient raises the question whether this strain was of animal origin. The source of infection remained unresolved, as the patient absconded from the hospital and further interviewing was not possible. About 10 million people live in Dhaka City; and more than 50% of them, mostly people with low incomes, do not have a permanent address. The patient informed us that his monthly income was only about $US50 and that he was temporarily living in a house. Upon further investigation of the home address of the patient, he could not be located or contacted. No evidence of pig farming was found at the home address of the patient. Since the patient was infected in July, which is the rainy monsoon season in Bangladesh, the chance of contact with animal feces is higher due to flooding. The Dhaka6 strain is the first G11 strain which has been found to infect a human host. Thus, the capacity of this strain for human-to-human transmission is doubtful.
The VP7 gene has three regions that exhibit a high degree of sequence divergence among different rotavirus serotypes but that are highly conserved within the same predicted serotype. Rotaviruses of the same serotype exhibit only one or two amino acid changes in these regions (14, 15). The sequences of the Dhaka6 strain in these regions were most similar to those of porcine G11 strains YM and A253, with only a few amino acid changes (Fig. 2). In contrast, high sequence divergence (more than 30%) was observed when the Dhaka6 antigenic region sequences were compared with the antigenic region sequences of strains of serotypes other than G11. These findings suggest that the VP7 segment of the Dhaka6 strain may have originated from G11 porcine rotaviruses.
Figure 1 shows that G11 and G5 strains may have originated from a common ancestor. Interestingly, these strains also contain a common VP4 specificity, P[7]. Although the Dhaka6 strain shares a common VP7 evolutionary lineage with G11 and G5 strains, it differs from the P[7] VP4 specificity carried by them (Fig. 3). The Dhaka6 strain might therefore be the result of a natural reassortment event where a G11 strain may have been the donor of the VP7 gene.
Genotyping based on nucleotide sequence analysis has currently surpassed serotyping as a more rapid and accurate system for the classification of rotaviruses (9, 41). Strains with more than 89% amino acid sequence identity belong to the same VP4 genotype. The VP4 gene of the Dhaka6 strain showed less than 76% amino acid identity with the VP4 sequences of established P types. This suggests that the VP4 of the Dhaka6 strain is very different from any other P types reported thus far and belongs to a novel P genotype, for which we propose the name P[25].
Since the Dhaka6 strain contains a novel VP4 genotype, it is very difficult to speculate about the origin of this gene. The VP4 protein of most human rotavirus strains contains 775 amino acids, while the VP4 proteins of animal rotaviruses contain 776 amino acids, with an additional amino acid between positions 134 and 136 of the VP8 fragment of the VP4 protein (7). Like the animal rotaviruses, the VP4 protein of the Dhaka6 strain contains an extra amino acid at position 135, which suggests that the Dhaka6 VP4 might be of animal origin.
The discovery of a novel P[25],G11 rotavirus is an illustration of the enormous diversity among the circulating rotavirus strains, especially in developing countries. Complete genome sequencing of all 11 segments of this novel strain may reveal the origins of the genes other than VP4 and VP7. Although animal-to-human transmission of rotaviruses is likely to be a rather infrequent event, such zoonotic transmissions and the ensuing reassortment events are a driving force in the evolution of rotaviruses. G9 is an example of such a reassortant strain, which took less than 20 years to go from a sporadically isolated genotype to a globally present dominant rotavirus (26, 30, 31). The rapid emergence of novel rotavirus genotypes has important implications for future vaccine strategies. Epidemiological rotavirus surveillance may need to be expanded to include domestic animals, as they may serve as an important reservoir of novel rotavirus strains.
ACKNOWLEDGMENTS
This research study was funded by the ICDDR,B, Centre for Health and Population Research, and the Flemish Fonds voor Wetenschappelijk Onderzoek, grant G.0288.01.
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Laboratory of Clinical and Epidemiological Virology, Rega Institute for Medical Research, University of Leuven, B-3000 Leuven, Belgium
ABSTRACT
A novel rotavirus strain (Dhaka6) isolated from a 21-year-old Bangladeshi male patient was characterized by sequence analysis of its VP7 and VP4 gene segments. Phylogenetic analysis of the VP7 gene of the Dhaka6 strain revealed a common evolutionary lineage with porcine G11 rotavirus strains. This isolate is the first reported G11 rotavirus strain infecting a human host. Comparison of the VP4 gene sequences with all currently recognized 24 different P genotypes revealed only low nucleotide (54 to 71%) and amino acid (52 to 76%) sequence identities. This lack of high sequence similarity in the VP4 gene indicates that the Dhaka6 isolate represents a new group A rotavirus P genotype, to which we propose assignment of the designation P[25].
INTRODUCTION
Rotaviruses belong to the Reoviridae and contain 11 segments of double-stranded RNA encoding six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural viral proteins (NSP1 to NSP6). Group A rotaviruses are a common cause of acute infectious diarrhea in humans and in a wide variety of mammalian and avian species. A dual system of classification of group A rotaviruses is based on two outer capsid proteins, i.e., VP4 (P types) and VP7 (G types). Rotaviruses can be serotyped by neutralization assays with panels of antisera and genotyped by sequence analysis of different gene segments (8, 11). Although there is a close relationship between the two classification systems, it has recently been proposed that the term serotype should be reserved for serological analysis and that the term genotype should be used for genetic classification and comparative sequence analysis. Strains that have more than 89% amino acid identity are considered to be of the same genotype (7, 9, 38).
So far, strains of at least 15 G genotypes and 24 P genotypes have been isolated from humans and a variety of mammal and avian species (20, 23, 24, 24a, 29, 32, 38). The major human G types are G1, G2, G3, G4, and G9, combined with the P types P[8] and P[4] (1, 9, 16, 17, 19, 36). In Bangladesh in 2002, G1 (44.8%) was the most prevalent human rotavirus genotype, followed by G9 (21.7%), G2 (15.0%), and G4 (13.8%). A G11 rotavirus (strain YM, P[7],G11) was first isolated from pigs in several regions of Mexico in 1983 (34) and was subsequently identified in the United States and Venezuela (3, 33). It was never found in humans or any other host. The other commonly identified G types in pigs are G5, G3, and G4 (4, 5, 6, 12) and are combined with two major P types, P[6] and P[7] (2, 18, 22, 35, 37).
Although most rotaviruses appear to be host restricted, interspecies transmission of rotaviruses between animals and humans has been described (20). Rotaviruses are excreted and dispersed in the environment after replication in the gastrointestinal tracts of humans or animals. They were reportedly found to survive in water sources, although the presence of uncoated rotavirus segments in the environment has not been proven. Whole viruses can be transferred from animals to humans and vice versa, but they rarely produce severe clinical manifestations in a new host unless they go through a unique event for segmented viruses called reassortment. Serious infections in human caused by reassortant rotavirus strains have been reported by many investigators. Reassortment events are facilitated by the occurrence of mixed infections in a host where they exchange one or more segments during their replication, resulting in a new and diverse progeny population of rotaviruses (25, 26, 27, 37).
In this report, we present the complete nucleotide sequence of the coding region of the VP7 gene and the partial nucleotide sequence of the VP4 gene of the Dhaka6 rotavirus isolate and compare them with those of the corresponding genes of human and animal rotavirus strains. This is, to our knowledge, the first reported case of a G11 rotavirus infection in a human patient. This is also the first description of a novel VP4 gene specificity distinct from any of the known rotavirus P types.
MATERIALS AND METHODS
Enzyme immunoassay for the detection of rotavirus antigen. Group A-specific VP6 antigen was detected in stool specimens by a solid-phase sandwich-type enzyme immunoassay (40), using rabbit hyperimmune antisera produced at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh, and an anti-human rotavirus-horseradish peroxidase conjugate (Dakopatts, Copenhagen, Denmark) (40).
RNA extraction. Viral RNA was extracted from the fecal sample by use of a QIAamp viral RNA mini kit (QIAGEN/Westburg, Leusden, The Netherlands), according to the manufacturer's instructions.
RT-PCR. The extracted RNA was denatured at 97°C for 5 min. A multiplex reverse transcriptase PCR (RT-PCR) was carried out with a OneStep RT-PCR kit (QIAGEN/Westburg) for different rotavirus G genotypes (G1, G2, G3, G4, and G9), as previously described by Gouvea et al. (13). Type-specific forward primers derived from distinct regions of the VP7 gene for G1 (primer aBT1; 5'-CAAGTACTCAAATCAATGATGG-3'), G2 (primer aCT2; 5'-CAATGATATTAACACATTTTCTGTG-3'), G3 (primer aET3; 5'-CGTTTGAAGA AGTTGCAACAG-3'), G4 (primer aDT4; 5'-CGTTTCTGGTGAGGAGTTG-3'), and G9 (primer aFT9; 5'-CTAGATGTAACTACAACTAC-3') were combined with reverse primer RVG9 (5'-GGTCACATCATACAATTCT-3'). This primer mix allows the amplification of fragments with G-type-specific segment sizes visible by polyacrylamide gel electrophoresis. The reaction was carried out with an initial reverse transcription step at 45°C for 30 min, followed by PCR activation at 95°C for 15 min, 35 cycles of amplification (30 s at 94°C, 45 s at 50°C, 1 min at 72°C), and a final extension of 7 min at 72° in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA). PCR products were run on a polyacrylamide gel, stained with ethidium bromide, and visualized under UV light.
As the multiplex PCR with type-specific primers could not detect the G genotype of strain Dhaka6, we amplified a 1,062-bp fragment of the VP7 gene with the consensus forward primer Beg9 (5'-GGCTTTAAAAGAGAGAATTTCCGTCTGG-3') and the reverse primer End9 (5'-GGTCACATCATACAATTCTAATCTAAG-3') (13). The consensus primers Con3 (5'-TGGCTTCGCCATTTTATAGACA-3') and Con2 (5'-ATTTCGGACCATTTATAACC-3') (10) failed to amplify the VP4 gene segments of our strain. Therefore, we designed new primers which were derived from the nucleotide sequence alignment of the VP4 genes available in GenBank. Forward primer VP4-1-17F (5'-GGCTATAAAATGGCTTCGC-3'; nucleotides [nt] 1 to 17) and reverse primer VP4-1083-1099R2 (5'-GCTTGTGAATCATCCCA-3'; nt 1083 to 1099) were designed for amplification of the first 1,099 bp of nucleotides of the VP4 gene. For the amplification of the 3' part of VP4, we used forward primer VP4-986-1004F (5'-TGGTGTTAATGATTTCAGC-3'; nt 986 to 1004) and reverse primer VP4-1919-1937R (5'-GTACWGCNGCTGAAATATC-3'; nt 1919 to 1937).
Nucleotide sequencing. The PCR amplicons were purified with a QIAquick PCR purification kit (QIAGEN/Westburg) and sequenced in both directions by the dideoxy-nucleotide chain termination method with the ABI PRISM BigDye Terminator Cycle Sequencing Reaction kit (Applied Biosystems) on an automated sequencer (ABI PRISM 3100). The same primers used in the first RT-PCR were used as sequencing primers.
DNA and protein sequence analysis. The chromatogram sequencing files were inspected by using Chromas 2.3 (Technelysium, Queensland, Australia), and consensus sequences were prepared by using SeqMan II (DNASTAR, Madison, WI). Multiple-sequence alignments were calculated by using CLUSTALX 1.81 (39). Sequences were manually edited in the GeneDoc alignment editor. Nucleotide and protein sequence similarity searches were performed by using GeneDoc alignment editor (version 2.6.002) (28).
Phylogenetic analysis. Phylogenetic and molecular evolutionary analyses were conducted by using the MEGA version 2.1 software package (21), based on the different G and P rotavirus sequences available in GenBank version 143.0. Genetic distances were calculated by the Kimura two-parameter method. The dendrograms were constructed by the neighbor-joining method.
Nucleotide sequence accession numbers. The nucleotide sequence data reported in this paper were submitted to GenBank by using the National Center for Biotechnology Information (Bethesda, MD) Sequin Application version 5.26 submission tool (http://www.ncbi.nlm.nih.gov) and can be found under GenBank accession numbers AY773003 (for the VP7 gene) and AY773004 (for the VP4 gene).
RESULTS
Case history and rotavirus antigen detection. A 21-year-old Bangladeshi male patient was admitted to the hospital of ICDDR,B in 2001 with severe diarrhea and abdominal pain. No vomiting or fever was recorded. The patient worked as a laborer in a water pump station in central Dhaka. No travel history was recorded, but it is very unlikely that he had traveled outside Bangladesh. Visits to his village outside Dhaka were possible. The stool frequency had been more than 16 times during the previous 24 h. After 5 days of passing watery stools, progressive dehydration necessitated hospitalization, and oral rehydration solution was given to the patient. The patient absconded from the hospital 3 h after admission. Further investigation was not possible because the patient was not available at his address. The stool specimen was found to be positive for rotavirus antigen by a VP6-specific enzyme-linked immunosorbent assay and was negative for the common enteric pathogens Vibrio, Shigella, and Salmonella. A routine multiplex VP7 RT-PCR method (13) with G1, G2, G3, G4, and G9 type-specific primers could not identify the G genotype; and further characterization by nucleotide sequencing was needed.
Nucleotide sequencing of VP7 and VP4 genes. The complete open reading frame (978 bp) and deduced amino acid sequence of the VP7-encoding gene of the Dhaka6 strain was determined (GenBank accession number AY773003) and compared with the VP7 sequences of prototype strains belonging to G1 to G15 (Table 1). Sequence comparison indicated that the VP7 sequence of strain Dhaka6 was most closely related to the VP7 sequence of G11 porcine rotavirus strains (87 to 91% nucleotide identity and 95% amino acid identity) and showed the highest similarity (91% nucleotide identity and 95% amino acid identity) to Mexican porcine rotavirus strain YM (P[7],G11). It was also observed that another porcine strain, OSU (P[7],G5), was closely related to our G11 strain (83% nucleotide identity and 90% amino acid identity). Rotavirus strains representing other G types exhibited much lower nucleotide and amino acid identities (62 to 78% nucleotide identity and 56 to 86% amino acid identity) with our strain. A dendrogram that included the sequences of all 15 known VP7 G types of rotaviruses in the GenBank database (release 143.0) was constructed (Fig. 1). Phylogenetic analysis confirmed that our Dhaka6 strain clustered with the G11 porcine rotavirus prototype strain in a monophyletic branch.
The amino acid sequences of three intragenotype-conserved important antigenic regions of 15 established rotavirus G genotypes were aligned with the respective sequences of the Dhaka6 strain (Fig. 2). The Dhaka6 strain showed a close relationship with the G11 porcine rotaviruses in these regions, with a few amino acid changes. Only one amino acid change in antigenic region A, no changes in antigenic region B, and two amino acid changes in antigenic region C were observed when the Dhaka6 amino acid sequence was compared with that of G11 porcine strain YM. The sequences of the antigenic regions of Dhaka6 strain showed more than 30% divergence compared with the sequences of antigenic regions of strains of genotypes other than G11.
We could not amplify the VP4 gene of the Dhaka6 strain with the general VP4-specific primers Con2 and Con3, and a modified primer set was designed for sequencing of a partial 1,894-bp fragment of the VP4-encoding gene, which was compared with those of other reference P genotypes available in GenBank (Table 2). The VP4 nucleotide and amino acid sequence similarities of Dhaka6 (GenBank accession number AY773004) to the sequences of all currently recognized P types were very low (54 to 71% nucleotide identity and 52 to 75% amino acid identity). Since the VP4 amino acid sequence identity of the Dhaka6 strain to all recognized P types is less than the cutoff of 89%, the Dhaka6 isolate represents a new group A rotavirus P genotype, for which we propose the name P[25]. A more detailed phylogenetic analysis (Fig. 3) that included the sequences of all known rotavirus P genotypes confirmed that the Dhaka6 strain is located in a separate branch that is only distantly related to the other P genotypes.
DISCUSSION
In Bangladesh, pigs are uncommon farm animals, and no genotyping studies of rotavirus isolates from pigs or other animals have been conducted thus far. Therefore, the identification of the Dhaka6 strain with a G11 VP7 specificity in a human patient raises the question whether this strain was of animal origin. The source of infection remained unresolved, as the patient absconded from the hospital and further interviewing was not possible. About 10 million people live in Dhaka City; and more than 50% of them, mostly people with low incomes, do not have a permanent address. The patient informed us that his monthly income was only about $US50 and that he was temporarily living in a house. Upon further investigation of the home address of the patient, he could not be located or contacted. No evidence of pig farming was found at the home address of the patient. Since the patient was infected in July, which is the rainy monsoon season in Bangladesh, the chance of contact with animal feces is higher due to flooding. The Dhaka6 strain is the first G11 strain which has been found to infect a human host. Thus, the capacity of this strain for human-to-human transmission is doubtful.
The VP7 gene has three regions that exhibit a high degree of sequence divergence among different rotavirus serotypes but that are highly conserved within the same predicted serotype. Rotaviruses of the same serotype exhibit only one or two amino acid changes in these regions (14, 15). The sequences of the Dhaka6 strain in these regions were most similar to those of porcine G11 strains YM and A253, with only a few amino acid changes (Fig. 2). In contrast, high sequence divergence (more than 30%) was observed when the Dhaka6 antigenic region sequences were compared with the antigenic region sequences of strains of serotypes other than G11. These findings suggest that the VP7 segment of the Dhaka6 strain may have originated from G11 porcine rotaviruses.
Figure 1 shows that G11 and G5 strains may have originated from a common ancestor. Interestingly, these strains also contain a common VP4 specificity, P[7]. Although the Dhaka6 strain shares a common VP7 evolutionary lineage with G11 and G5 strains, it differs from the P[7] VP4 specificity carried by them (Fig. 3). The Dhaka6 strain might therefore be the result of a natural reassortment event where a G11 strain may have been the donor of the VP7 gene.
Genotyping based on nucleotide sequence analysis has currently surpassed serotyping as a more rapid and accurate system for the classification of rotaviruses (9, 41). Strains with more than 89% amino acid sequence identity belong to the same VP4 genotype. The VP4 gene of the Dhaka6 strain showed less than 76% amino acid identity with the VP4 sequences of established P types. This suggests that the VP4 of the Dhaka6 strain is very different from any other P types reported thus far and belongs to a novel P genotype, for which we propose the name P[25].
Since the Dhaka6 strain contains a novel VP4 genotype, it is very difficult to speculate about the origin of this gene. The VP4 protein of most human rotavirus strains contains 775 amino acids, while the VP4 proteins of animal rotaviruses contain 776 amino acids, with an additional amino acid between positions 134 and 136 of the VP8 fragment of the VP4 protein (7). Like the animal rotaviruses, the VP4 protein of the Dhaka6 strain contains an extra amino acid at position 135, which suggests that the Dhaka6 VP4 might be of animal origin.
The discovery of a novel P[25],G11 rotavirus is an illustration of the enormous diversity among the circulating rotavirus strains, especially in developing countries. Complete genome sequencing of all 11 segments of this novel strain may reveal the origins of the genes other than VP4 and VP7. Although animal-to-human transmission of rotaviruses is likely to be a rather infrequent event, such zoonotic transmissions and the ensuing reassortment events are a driving force in the evolution of rotaviruses. G9 is an example of such a reassortant strain, which took less than 20 years to go from a sporadically isolated genotype to a globally present dominant rotavirus (26, 30, 31). The rapid emergence of novel rotavirus genotypes has important implications for future vaccine strategies. Epidemiological rotavirus surveillance may need to be expanded to include domestic animals, as they may serve as an important reservoir of novel rotavirus strains.
ACKNOWLEDGMENTS
This research study was funded by the ICDDR,B, Centre for Health and Population Research, and the Flemish Fonds voor Wetenschappelijk Onderzoek, grant G.0288.01.
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