- Short report
- Open Access
Nucleotide mismatches between the VP7 gene and the primer are associated with genotyping failure of a specific lineage from G1 rotavirus strains
Virology Journal volume 3, Article number: 35 (2006)
In recent years it was reported that the accumulation of point mutations in VP4 and VP7 genes of rotavirus strains was the main cause of the failure of the G or P-typing. Failures in the correct genotyping of G1, G2, G8, G9 and G10 rotavirus strains were reported in the most commonly used reverse transcription (RT)-PCR strategies. Collecting VP7 gene sequences of G1 rotavirus strains from databases we found that 74 (61.2 %) out of 121 G1 strains from lineage I showed the four specific mismatches at the 5' end of the 9T1-1 primer, previously associated with the failure of G1-typing. Thus, a great percentage of the G1 strains from lineage I worldwide reported could not have been typed if the Das's RT-PCR strategy were used. This analysis shows that the failure on the detection of the G1 strains could be due to the diversification of rotavirus strains in phylogenetic lineages. Therefore, the use of different RT-PCR strategies with different primer binding locations on the VP7 gene or new typing methodologies -like microarrays procedures- could be a better option to avoid the failure of the G-typing of rotavirus strains detected during surveillance programs.
Causing more than 450,000 deaths per year, group A rotaviruses are the most important cause of acute diarrhea in children throughout the world .
Based on the antigenicity and amino acid differences from the two outermost proteins, VP4 and VP7 respectively, group A rotaviruses are classified into P and G-types . At the time, at least 26 P and 15 G-types were detected [2–4], most of them showing a high degree of intragenic diversification due to point mutations, insertions and/or deletions [5–12]. Although the most frequent human G-types of a given geographical region change from season to season, the genotype G1 is considered the most prevalent worldwide .
Since the vaccination against rotavirus may induce selective effects on the diversity of strains, vaccine-escape mutants could emerge. In order to evaluate the vaccine efficiency, the surveillance programs should detect the diversity of rotavirus strains before, during and after the introduction of a rotavirus vaccine .
Since the introduction of reverse transcription (RT)-PCR for rotavirus genotyping, many epidemiological surveillances have been conducted and new data has been collected to understand this complex epidemiology . However, in recent years it was reported that the accumulation of point mutations in VP4 and VP7 genes was the main cause of the failure of the G or P-typing of rotavirus strains [7, 8, 12, 16–21].
At least, there are four multiplex RT-PCR strategies commonly used for rotavirus G-typing [17, 22–24] and one for P-typing . In the one developed by Gouvea et al , it was reported failures in the correct genotyping of G2, G8, G9 and G10 rotavirus strains [7, 12, 17, 20, 21], and recently it was suggested the use of modified or degenerated primers to avoid the mismatches between the primer and the VP7 gene [17, 20].
In a recent paper published by Rahman et al , it was reported the failure of the Das's RT-PCR strategy to detect most (75%) of the G1 human rotavirus isolated in Bangladesh during the surveillance in 2002. They argue that this failure was due to four mismatches found at the 5' end of the primer binding site. Although two G1 strains correctly typed had a 100% identity with the untypeable strains, they concluded that the remainder 25% could be typed because the 3' end of the primer binding site had a perfect match.
In order to evaluate how many G1 strains included in the GenBank database have these four specific mismatches and its clustering within specific lineages, we collected 173 sequences of the VP7 gene from G1 strains from the GenBank database Release 151, December 2005 (alignments are available from the authors on request). A phylogenetic tree was constructed from aligned coding sequences, using Neighbor-joining or parsimony methods with Kimura 2-parameter as a model of nucleotide substitution with the MEGA 3.1  and Phylip v3.65 softwares. The statistical significance of the tree was preformed by bootstrapping, using 1000 pseudo-replicates data sets.
All the strains grouped within one of the four lineages previously reported by Jin et al  (Fig. 1). The typeable and untypeable G1 strains reported by Rahman et al , grouped within the lineage I with a high bootstrap value (95 %) (data not shown). Seventy four (61.2 %) out of 121 G1 strains from lineage I showed the four specific mismatches at the 5' end of the 9T1-1 primer. This was associated with the failure of genotyping of G1 strains from Bangladesh. Two of them showed more than four nucleotide mismatches. Forty two (34.7 %) showed only 3 out of the four nucleotide mismatches and the remainder 4.1% showed 3 out of the four nucleotide mismatches plus others mismatches at the 9T1-1 primer binding site. The strains grouped within lineage II, III and IV, showed no more than two nucleotide mismatches, except for one sample from lineage III (Brz-2; GenBank number: U26362) that showed three out of the four nucleotide mismatches (Fig. 1).
Interestingly, when we compared the 173 sequences of G1 strains with the aBT1 primer binding site, we found that 170 strains showed one or two nucleotide mismatches and 3 showed three mismatches, suggesting that Gouvea's RT-PCR strategy could type these strains correctly (data not shown).
Thus, taking into account that 75% of the G1 strains with four nucleotide mismatches were not detected during the surveillance in Bangladesh , probably a high percentage out of the 121 strains from lineage I, included in our analysis, could not be typed by the Das's RT-PCR strategy. It is noteworthy that G1 strains from lineage I were the most reported worldwide.
It was suggested to use modified or degenerated primers, or change the priming binding site, in order to avoid the mismatches between the primer and the VP7 gene [7, 17, 20]. However, the failure of the G-typing of rotavirus strains, detected during surveillance programs, could be avoided through different RT-PCR strategies that use different primer binding locations on the VP7 gene [7, 19, 27] or by new typing methodologies, like microarrays procedures [28, 29].
This analysis shows that the failure on the detection of the G1 strains could be due to the diversification of rotavirus strains in phylogenetic lineages, as it was showed by Santos et al  in G9 strains when they used the Gouvea's RT-PCR strategy.
Parashar UD, Gibson CJ, Bresee JS, Glass RI: Rotavirus and severe childhood diarrhea. Emerg Infect Dis 2006, 12: 304-306.
Estes M: Rotaviruses and their replication. In Fields virology. 4th edition. Edited by: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B and Straus SE. Philadelphia, Pa, Lippincott/Williams & Wilkins; 2001:1747-1785.
Martella V, Ciarlet M, Baselga R, Arista S, Elia G, Lorusso E, Banyai K, Terio V, Madio A, Ruggeri FM, Falcone E, Camero M, Decaro N, Buonavoglia C: Sequence analysis of the VP7 and VP4 genes identifies a novel VP7 gene allele of porcine rotaviruses, sharing a common evolutionary origin with human G2 rotaviruses. Virology 2005, 337: 111-123. 10.1016/j.virol.2005.03.031
Martella V, Ciarlet M, Banyai K, Lorusso E, Cavalli A, Corrente M, Elia G, Arista S, Camero M, Desario C, Decaro N, Lavazza A, Buonavoglia C: Identification of a novel VP4 genotype carried by a serotype G5 porcine rotavirus strain. Virology 2005, Dec 16 (Epub ahead of print).
Jin Q, Ward RL, Knowlton DR, Gabbay YB, Linhares AC, Rappaport R, Woods PA, Glass RI, Gentsch JR: Divergence of VP7 genes of G1 rotaviruses isolated from infants vaccinated with reassortant rhesus rotaviruses. Arch Virol 1996, 141: 2057-2076. 10.1007/BF01718215
Diwakarla CS, Palombo EA: Genetic and antigenic variation of capsid protein VP7 of serotype G1 human rotavirus isolates. J Gen Virol 1999, 80: 341-344.
Santos N, Volotao EM, Soares CC, Albuquerque MC, da Silva FM, Chizhikov V, Hoshino Y: VP7 gene polymorphism of serotype G9 rotavirus strains and its impact on G genotype determination by PCR. Virus Res 2003, 93: 127-138. 10.1016/S0168-1702(02)00318-0
Iturriza-Gomara M, Green J, Brown DW, Desselberger U, Gray JJ: Diversity within the VP4 gene of rotavirus P strains: implications for reverse transcription-PCR genotyping. J Clin Microbiol 2000, 38: 898-901.
Banyai K, Martella V, Jakab F, Melegh B, Szucs G: Sequencing and phylogenetic analysis of human genotype P rotavirus strains detected in Hungary provides evidence for genetic heterogeneity within the P VP4 gene. J Clin Microbiol 2004, 42: 4338-4343. 10.1128/JCM.42.9.4338-4343.2004
Bok K, Matson DO, Gomez JA: Genetic variation of capsid protein VP7 in genotype g4 human rotavirus strains: simultaneous emergence and spread of different lineages in Argentina. J Clin Microbiol 2002, 40: 2016-2022. 10.1128/JCM.40.6.2016-2022.2002
Gouvea V, Lima RC, Linhares RE, Clark HF, Nosawa CM, Santos N: Identification of two lineages (WA-like and F45-like) within the major rotavirus genotype P. Virus Res 1999, 59: 141-147. 10.1016/S0168-1702(98)00124-5
Page NA, Steele AD: Antigenic and genetic characterization of serotype G2 human rotavirus strains from South Africa from 1984 to 1998. J Med Virol 2004, 72: 320-327. 10.1002/jmv.10571
Santos N, Hoshino Y: Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine. Rev Med Virol 2005, 15: 29-56. 10.1002/rmv.448
Palombo EA: Genetic and antigenic diversity of human rotaviruses: potential impact on the success of candidate vaccines. FEMS Microbiol Lett 1999, 181: 1-8.
Desselberger U, Iturriza-Gomara M, Gray JJ: Rotavirus epidemiology and surveillance. Novartis Found Symp 2001, 238: 125-147.
Arista S, Giammanco GM, De Grazia S, Colomba C, Martella V: Genetic variability among serotype G4 Italian human rotaviruses. J Clin Microbiol 2005, 43: 1420-1425. 10.1128/JCM.43.3.1420-1425.2005
Iturriza-Gomara M, Kang G, Gray J: Rotavirus genotyping: keeping up with an evolving population of human rotaviruses. J Clin Virol 2004, 31: 259-265. 10.1016/j.jcv.2004.04.009
Das S, Varghese V, Chaudhuri S, Barman P, Kojima K, Dutta P, Bhattacharya SK, Krishnan T, Kobayashi N, Naik TN: Genetic variability of human rotavirus strains isolated from Eastern and Northern India. J Med Virol 2004, 72: 156-161. 10.1002/jmv.10542
Rahman M, Sultana R, Podder G, Faruque AS, Matthijnssens J, Zaman K, Breiman RF, Sack DA, Van Ranst M, Azim T: Typing of human rotaviruses: nucleotide mismatches between the VP7 gene and primer are associated with genotyping failure. Virol J 2005, 2: 24. 10.1186/1743-422X-2-24
Martella V, Terio V, Arista S, Elia G, Corrente M, Madio A, Pratelli A, Tempesta M, Cirani A, Buonavoglia C: Nucleotide variation in the VP7 gene affects PCR genotyping of G9 rotaviruses identified in Italy. J Med Virol 2004, 72: 143-148. 10.1002/jmv.10563
Adah MI, Rohwedder A, Olaleyle OD, Werchau H: Nigerian rotavirus serotype G8 could not be typed by PCR due to nucleotide mutation at the 3' end of the primer binding site. Arch Virol 1997, 142: 1881-1887. 10.1007/s007050050206
Taniguchi K, Wakasugi F, Pongsuwanna Y, Urasawa T, Ukae S, Chiba S, Urasawa S: Identification of human and bovine rotavirus serotypes by polymerase chain reaction. Epidemiol Infect 1992, 109: 303-312.
Das BK, Gentsch JR, Cicirello HG, Woods PA, Gupta A, Ramachandran M, Kumar R, Bhan MK, Glass RI: Characterization of rotavirus strains from newborns in New Delhi, India. J Clin Microbiol 1994, 32: 1820-1822.
Gouvea V, Glass RI, Woods P, Taniguchi K, Clark HF, Forrester B, Fang ZY: Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol 1990, 28: 276-282.
Gentsch JR, Glass RI, Woods P, Gouvea V, Gorziglia M, Flores J, Das BK, Bhan MK: Identification of group A rotavirus gene 4 types by polymerase chain reaction. J Clin Microbiol 1992, 30: 1365-1373.
Kumar S, Tamura K, Nei M: MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 2004, 5: 150-163. 10.1093/bib/5.2.150
Santos N, Volotao EM, Soares CC, Campos GS, Sardi SI, Hoshino Y: Predominance of rotavirus genotype G9 during the 1999, 2000, and 2002 seasons among hospitalized children in the city of Salvador, Bahia, Brazil: implications for future vaccine strategies. J Clin Microbiol 2005, 43: 4064-4069. 10.1128/JCM.43.8.4064-4069.2005
Chizhikov V, Wagner M, Ivshina A, Hoshino Y, Kapikian AZ, Chumakov K: Detection and genotyping of human group A rotaviruses by oligonucleotide microarray hybridization. J Clin Microbiol 2002, 40: 2398-2407. 10.1128/JCM.40.7.2398-2407.2002
Lovmar L, Fock C, Espinoza F, Bucardo F, Syvanen AC, Bondeson K: Microarrays for genotyping human group a rotavirus by multiplex capture and type-specific primer extension. J Clin Microbiol 2003, 41: 5153-5158. 10.1128/JCM.41.11.5153-5158.2003
We are grateful to Natalia Gudiño for the language corrections of the manuscript, and Juan Stupka and the three anonymous referees for critical corrections.
GIP performed the sequences analysis, phylogenetic analysis and wrote the manuscript; EEE download the sequences from database, performed the sequences analysis and critically revised the manuscript.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
Rights and permissions
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
About this article
Cite this article
Parra, G.I., Espinola, E.E. Nucleotide mismatches between the VP7 gene and the primer are associated with genotyping failure of a specific lineage from G1 rotavirus strains. Virol J 3, 35 (2006). https://doi.org/10.1186/1743-422X-3-35
- Acute Diarrhea
- Nucleotide Mismatch
- Correct Genotyping
- Typing Methodology
- Microarrays Procedure