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Short Communication |
1 Instituto de Investigaciones en Ciencias de la Salud, Universidad Nacional de Asunción, Río de la Plata y Lagerenza, Asunción (2511), Paraguay
2 Sección Virología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
3 Laboratorio de Gastroenteritis Virales, Departamento de Virología, INEI-ANLIS ‘Dr Carlos G. Malbrán’, Buenos Aires, Argentina
Correspondence
Gabriel I. Parra
gabriel_parra{at}hotmail.com
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ABSTRACT |
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Present address: Center for Pediatric Research, Children's Hospital of The King's Daughters, Eastern Virginia Medical School, 855 West Brambleton Avenue, Norfolk, VA 23510, USA. 
An alignment of the nucleotide sequences of the VP7 gene of the 43 G4 rotaviruses used in the recombination analysis is available in JGV Online.
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). They are members of the Reoviridae, containing 11 segments of double-stranded RNA enclosed in a triple-layered capsid. The rotavirus outer layer is constituted by two proteins, VP7 (glycoprotein) and VP4 (haemagglutinin). Both molecules independently induce protective neutralizing antibodies, leading to the classification of rotavirus into G (VP7) and P (VP4) types (Estes, 2001), and are also the main targets for current vaccine-development strategies (Cunliffe et al., 2002). At least 14 G types have been described so far, G1–G4 being the most common types found in humans worldwide (Desselberger et al., 2001). However, from season to season the prevalent G types found in a geographical region are different, exemplified by the great increase in prevalence of G4 detected worldwide during the past decade (Gentsch et al., 1996). One of the highest prevalences of G4 was reported in Argentina during national rotavirus surveillance (Bok et al., 2001). Studies of intragenotype diversity led to subdivision of the genotype into several lineages and sublineages, distinctly identified by unique genomic as well as epidemiological features. The phylogenetic tree obtained from the analysis of genotype 4 VP7 gene nucleotide sequences distributed worldwide showed two major lineages, designated I and II. Lineage I was further subdivided into four sublineages, Ia–Id. Sublineages Ib and Ic were most commonly present in Argentina, and were also found to be associated with different characteristic rotavirus enterotoxins (NSP4) (K. Bok and others, unpublished data). It was noticeable that one strain, ArgRes1723, appeared to be an intermediate between sublineages Ib and Ic. Despite clustering with a high bootstrap value with sublineage Ib, it also presented the asparagine insertion (amino acid 76) characteristic of sublineage Ic (Bok et al., 2002). This interesting observation could be explained by two mechanisms: intragenic recombination between strains from sublineages Ib and Ic; or sequence convergence (homoplasy) due to point mutations. Although genetic reassortment and nucleotide substitution are considered the most important mechanisms of evolution for rotaviruses (Estes, 2001; Gouvea & Brantly, 1995), Suzuki et al. (1998) have reported an intragenic recombination event between rotavirus strains of different serotypes. Many studies show the detection of mixed infections with two different serotypes infecting one individual, therefore increasing the probability of reassortment and/or recombination (Bok et al., 2001; Gouvea & Brantly, 1995; Jain et al., 2001). However, the detection of mixed infections with strains belonging to the same serotype has never been reported, as genotyping techniques currently cannot discriminate between variants of the same genotype (Ahmed et al., 1991). This study presents evidence that the ArgRes1723 strain is the evolutionary result of a genome recombination between two sublineages of the same rotavirus genotype.
Thirty Argentine VP7 gene sequences corresponding to rotavirus G4 strains were analysed, together with 13 additional sequences from other worldwide locations, collected from the GenBank database (alignments are available as a supplementary figure). The complete alignment was performed using CLUSTAL_X version 1.8, and subsequently analysed using Kimura two-parameters as a method of substitution and neighbour-joining to reconstruct the phylogenetic tree (MEGA version 2.0) (Kumar et al., 2001). The statistical significance of the relationships obtained was estimated by bootstrap resampling analysis (1000 repetitions). The phylogenetic results were further confirmed by parsimony analysis using 100 bootstrap repetitions (PAUP*; Swofford, 1998). Recombination was evidenced using the SIMPLOT program version 3.2 (Lole et al., 1999), with similarity and bootscanning analysis (Salminen et al., 1995) and GENECONV, a computer algorithm based on Sawyer's test (Sawyer, 1989). The putative recombinant sequence (ArgRes1723) was compared with strains from the two proposed parental sublineages (20 from sublineage Ib and 8 from sublineage Ic), and one sequence of a known outgroup (Gottfried). Informative sites were identified where two sequences shared one specific nucleotide but two additional sequences shared a different nucleotide.
The similarity plots showed that the ArgRes1723 sequence was more similar to sublineage Ic in the first portion of the sequence (named region A; Fig. 1a), then drastically diverged to become very similar to sublineage Ib in the second portion of the sequence (region B). Bootscanning analysis (Fig. 1b) showed the break point, estimated to be between nucleotides 336 and 387, after mapping the informative sites.
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) were constructed for each of the genomic regions: A, nucleotides 76–336; B, nucleotides 337–866 (nt positions refer to the Gottfried strain). Phylogenetic trees showed the recombinant nature of ArgRes1723, with a bootstrap value of 56 % in region A and 99 % in region B. Despite the low bootstrap value (56 %) of region A in the branch node of sublineage Ic, an internal branch that included ArgRes1723 showed a bootstrap value of 93 %.
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From these results it was concluded that intragenic recombination occurred between sequences of sublineages Ic and Ib, with a crossover point between nucleotides 336 and 387. Although possible RT-PCR artefacts could have caused the recombination between sequences in samples with mixed infections, the amplification and sequencing assays were independently repeated to minimize this possibility. Sublineage Ib and Ic strains were the most prevalent variants circulating in Argentina during 1997–98, so if recombination between rotavirus strains was possible, it would be expected to occur between these two variants. However, the generated strain might not show improved fitness, limiting its dissemination and consequent detection, which was the case throughout the remainder of the national surveillance study.
This is the first study reporting evidence of intragenic recombination between rotavirus strains of the same genotype. In addition to point mutations, there is now evidence that genetic diversity within a rotavirus genotype is also generated by recombination. Intragenic recombination might be an important mechanism used by rotaviruses to generate escape mutants avoiding the host's immune system. This observation becomes especially meaningful if the recombination process occurred between strains of a sublineage (Ib) which was already considered established in the population, and another sublineage (Ic) which was a recent introduction in Argentina (Bok et al., 2002). These results raise the possibility that rotavirus recombination might not be an extraordinary event, but rather a phenomenon likely to occur frequently but to be detected rarely. The information presented here increases current knowledge about rotavirus evolution and the origin of divergence, and improves our understanding of the adaptation mechanisms used by rotaviruses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bok, K., Castagnaro, N., Borsa, A. & 8 other authors (2001). Surveillance for rotavirus in Argentina. J Med Virol 65, 190–198.[CrossRef][Medline]
Bok, K., Matson, D. O. & Gomez, J. A. (2002). Genetic variation of capsid protein VP7 in genotype G4 human rotavirus strains: simultaneous emergence and spread of different lineages in Argentina. J Clin Microbiol 40, 2016–2022.
Cunliffe, N. A., Bresee, J. S. & Hart, C. A. (2002). Rotavirus vaccines: development, current issues and future prospects. J Infect 45, 1–9.[CrossRef][Medline]
Desselberger, U., Iturriza-Gomara, M. & Gray, J. J. (2001). Rotavirus epidemiology and surveillance. Novartis Found Symp 238, 125–147; discussion 147–152.
Estes, M. (2001). Rotaviruses and their replication. In Fields Virology, 4th edn, pp. 1747–1786. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Gentsch, J. R., Woods, P. A., Ramachandran, M., Das, B. K., Leite, J. P., Alfieri, A., Kumar, R., Bhan, M. K. & Glass, R. I. (1996). Review of G and P typing results from a global collection of rotavirus strains: implications for vaccine development. J Infect Dis 174, S30–36.
Gouvea, V. & Brantly, M. (1995). Is rotavirus a population of reassortants? Trends Microbiol 3, 159–162.[CrossRef][Medline]
Jain, V., Das, B. K., Bhan, M. K., Glass, R. I. & Gentsch, J. R. (2001). Great diversity of group A rotavirus strains and high prevalence of mixed rotavirus infections in India. J Clin Microbiol 39, 3524–3529.
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 1244–1245.
Lole, K. S., Bollinger, R. C., Paranjape, R. S., Gadkari, D., Kulkarni, S. S., Novak, N. G., Ingersoll, R., Sheppard, H. W. & Ray, S. C. (1999). Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol 73, 152–160.
Salminen, M. O., Carr, J. K., Burke, D. S. & McCutchan, F. E. (1995). Identification of breakpoints in intergenotypic recombinants of HIV type 1 by bootscanning. AIDS Res Hum Retrovir 11, 1423–1425.[Medline]
Sawyer, S. (1989). Statistical tests for detecting gene conversion. Mol Biol Evol 6, 526–538.[Abstract]
Suzuki, Y., Gojobori, T. & Nakagomi, O. (1998). Intragenic recombinations in rotaviruses. FEBS Lett 427, 183–187.[CrossRef][Medline]
Swofford, D. L. (1998). PAUP* Phylogenetic Analysis Using Parsimony (*and other methods). Version 4. Sunderland, MA: Sinauer Associates.
Received 25 November 2003;
accepted 10 February 2004.
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