Comparison of Antigenic Dominants of VP7 in G9 and G1 Rotavirus Strains Circulating in La Rioja,Argentina,with the Vaccine Strains

Abstract

A massive vaccination in Argentina was implemented recently. The antigenic dominants of VP7 in G9 and G1 rotavirus strains, circulating in La Rioja, Argentina with strain vaccines were compared. From 2000 to 2010 in several attention centers of La Rioja, at northwestern Argentina, 418 stool samples from children younger than 5 years old were collected. Ninety were positive by immunochromatography and 51 were genotyped by reverse transcriptase–polymerase chain reaction followed by nested-multiplex polymerase chain reaction (PCR) with type-specific primers. Six G9 strains and four G1 strains were sequenced by MACROGEN Korea. The phylogenetic analysis was conducted in MEGA 6.0. The 940 bp were aligned using CLUSTALW and the tree was inferred using the UPGMA method. The antigenic dominants of VP7, 7-1a, 7-1b, and 7-2 were studied using BioEdit, 7.2.5. In the comparison between G9-lineage III d rotavirus (RV) strains circulating in La Rioja with ROTAVAC vaccine strain, three differences were detected corresponding to 100, 211, and 145 positions. In the comparison between G1-Lineage 1 strains and G1 Rotarix and G1 RotaTeq, three differences were observed in 94, 123, and 217 positions. All these positions were important for the escape to neutralization for study with monoclonal antibody. In conclusion, the differences between the G1 strains in La Rioja, Argentina and the G1 components of the RotaTeq and Rotarix vaccine strains are few, but important for the escape immunologic, and need to be monitored for appropriate evaluation of long-term impact of vaccine used in Argentina. Nevertheless, the VP7 antigenic regions of G9 RV strains circulating in La Rioja and ROTAVAC vaccine strains are different to other zones of Argentina and could play an important role in the failure of vaccine response in these regions and Argentina.

Introduction

Group A rotaviruses (RVs) are the leading cause of sporadic acute diarrhea in young children worldwide. It is estimated that RVs are associated with more than 450,000 deaths in that age group, most of them are in developing countries (17). Rotavirus genome comprises 11 segments of double-stranded RNA, which are surrounded by a triple-layered capsid protein. The outermost proteins of the capsid (VP7 and VP4) are responsible for eliciting neutralizing antibodies, and therefore, the major targets for vaccine development. Genetic and antigenic differences within the VP7 and VP4 have been used to classify rotavirus into G and P types, respectively (8). Three rotavirus vaccines, Rotarix^® [RV1; monovalent G1P (8); GlaxoSmithKline Biologicals]; RotaTeq^® [RV5; pentavalent G1, G2, G3, G4,P(8); Merck Vaccines] and ROTAVAC^® [RV9; monovalent G9P(11); Bharat Biotech], are commercially available. Specifically, Rotarix and RotaTeq have shown high efficacy in developed countries although efficacy is lower in developing countries (11), but these vaccines reduce greater number of severe rotavirus gastroenteritis. Rotavirus G1 strains are the most common genotype that causes diarrhea in humans and were incorporated into both rotavirus licensed vaccines (monovalent and multivalent) (2). The G9 genotype has emerged worldwide and is now considered fifth major human RV genotype (12,13). A massive vaccination in Argentina as per the WHO's recommendation was implemented in 2015. During the past decade in Argentina, the Rotavirus Strains G1P (8), G4P (8), G2P (4), G9P (8), G3P (8), and G3P (6) were sporadically detected (7). The genotype G9 RV, since 1980, in a different city of the country (5,3,17) and G1 genotype were detected (4,6). The antigenic dominants of VP7 rotavirus strains, defined by Aoki, S T (1), as well as 7-1a, 7-1b, and 7-2, and their comparison are important to know the relationship effective with vaccine strains. In La Rioja, Argentina, 13% of the viral diarrheas were detected and rotavirus (10) is the first agent. At the moment, unknown are the genotypes or antigenic dominants of circulating RV strain. The aim of this study was to analyze the antigenic dominants of rotavirus strains VP7 that are circulating in La Rioja, Argentina, and their comparison with three RVA vaccines available.

A total of 418 stool samples from children younger than 5 years old, hospitalized and outpatient, for viral diagnostic were collected from January 2000 through December 2010 in Hospital Dr. Vera Barros and in primary attention centers of La Rioja. The studied hospitalized children had less than 5 days of evolution from the beginning of the clinical manifestations and the samples were obtained during the first 48 h of hospitalization. The studied ambulatory children have less than 5 days of evolution from the beginning of clinical manifestations. The project and consent were evaluated and approved by Ethics Committee of Foundation Barceló HA (Resolution HCS N°: 4312/11). Ninety (21, 53%) were positive by immunochromatography (Vikia^® Rota-Adeno, bioMerieux). The RNA extraction from positive samples was done by alcohol precipitation (14). For visualization of the products amplified, reverse transcriptase–polymerase chain reaction (RT-PCR) was run in polyacrylamide gel with subsequent silver nitrate staining. Six genotype G rotavirus strains (G1, G2, G3, G4, G8, and G9) using primers specific for each were determined. From total positive, only 51 stool samples were genotyped by RT-PCR followed by nested-multiplex PCR with type-specific primers previously described (9). The low amount of genotypes obtained could be due to low amount of virus in samples or the presence of genotypes not studied. Several genotypes in some stool were identified and a total of 86 genotypes (date not shown) were detected because infection multiples were produced. The G1 and G9 RV strains were selected because these have a greater quantity in La Rioja and a major circulation worldwide during period studied, respectively. Both genotypes were amplified and submitted to MACROGEN Korea for the sequencing. The sequences were aligned using CLUSTALW input MEGA 6.0 software (Molecular Evolutionary Genetics Analysis). The accession numbers of G9 strains in GenBank are LR A1 (GenBank: ID: KT948645); LR B5 (GenBank: ID: KT948646); LR C6 (GenBank: ID: KT948647); LR D3 (GenBank: ID: KT948648); LR E7 (GenBank: ID: KT948649); and LR F9 (GenBank: ID: KT948650). The accession numbers of GenBank of G1 strains are LR G9 (GenBank: ID: KT948651); LR H9 (GenBank: ID: KT948652); LR I9 (GenBank: ID: KT948653); and LR J9 (GenBank: ID: KT948653). The evolutionary history was inferred using the UPGMA method. The optimal tree with the sum of branch length = 35.41016264 is shown. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. There were 827 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 6.0. The lineage designations G9 and G1 RVs were based on reported studies and the strains are described as follows, the strains/lineage (GenBank: accession). For lineage designations G9, the following strains were used: ITA-MARI 2005/Lineage III d (GenBank:EF150338); CIT254RV/Lineage III c (GenBank:AF281044); MC 345/Lineage III a (GenBank:D38055); 608VN/Lineage III b (GenBank:AB091777); 97S237/Lineage IV (GenBank:AF260959); OM 67/Lineage V (GenBank:AJ491179); 116E/Lineage II (GenBank:14072); and AU32/Lineage I (GenBank:AB045372). For the lineage designations G1 RV, the following strains Thai-804/Lineage 1(GenBank: DQ512979), Cos-70/Lineage 2 (GenBank:U26370), WA/Lineage 3 (GenBank:K02033), Kor-64/Lineage 4 (GenBank: U26378), PA5/90/Lineage 5 (GenBank DQ377573), AU19/Lineage 6(GenBank AB018697), Ban-59/Lineage 7 (GenBank:U2366), Egypt-7/Lineage 8 (GenBank:U26373), PA32/90/Lineage 9 (GenBank: DQ377574), SW20/21/Lineage 10 (GenBank: AF426162), and C60/Lineage 11 (GenBank: L24164) were used. The dominants antigenic of VP7, 7-1a, 7-1b, and 7-2 of G9 and G1 RVs circulating in La Rioja and vaccine strains were studied using the amino acid sequence obtained by BioEdit, 7.2.5. The comparison of dominants was carried out using strains available in GenBank. The strains were denominated with strain name (GenBank: number accession). The strains are Rotarix A41CB052A/G1P(8) strains (GenBank:JN849114); RotaTeq W179-9/G1P(8) strains (GenBank:GU565057); and ROTAVAC IN116E strains (GenBank:FJ361209).

The results showed that fourteen G9 RV strains were detected and correspond to 1 outpatient child during 2003 and 13 hospitalized children during 2008/2009 who were detected (dates not shown). Six samples from children hospitalized in 2008/2009 were sequenced. A total of 24 G1 RVs were detected. Fourteen G1 RVs from hospitalized children and 10 G1 RVs from outpatient children in 2008/2009 (dates not shown) were detected. Only four samples of children hospitalized in 2008/2009 were sequenced. From 1063 sequenced that were amplified, 940 bp were aligned. The Figure 1 shows the phylogenetic tree of the G9 and G1 strains of rotavirus circulating in La Rioja, Argentina. All G9 strains were lineage III d and all G1 strains show the lineage 1. Phylogenetic analyses have revealed that a single sublineage of G9 RV strains was responsible for this worldwide spread. The genotype G9P (8) was most frequently detected in 2008 (54.1%), and their VP7 gene as G9 lineages III was grouped by the phylogenetic analysis (5,17). In Argentina, G9 RV during the 1980–1988 and 1997–2003 period shows relatively low rates in the center (Cordoba) (3) and upto 40% in the south (Ushuaia) (5) was detected. Globally, G1P (8), G2P (4), G3P (8), G4P (8), and G9P (8) genotype combinations of rotavirus strains are the most common cause of human infections (2). Of these, G1P (8) strains are most predominant (37.7%) (11). In Argentina, the intragenotype diversity and the circulation of G1 lineage IV and V strains of G1 RV in Córdoba, Argentina, over a 27-year period (1980–2006), were detected. The cocirculation of lineage I and II strains in the 1990s and 2000–2006 was observed (4). In La Rioja, 16.28% of the stools of children are G9 RV strains, including lineage III d, suggesting their importance in Argentina. Moreover, the lineage 1 in all G1 strain rotavirus circulating in La Rioja, Argentina without cocirculation was detected, suggesting that the epidemiological surveillance of these strains in small cities could be important along with the vaccine implementation. The Figure 2 shows the alignments of amino acid corresponding to antigenic dominants of VP7. The Rotarix and RotaTeq vaccine strains, G9 and G1 RVs of strains circulating in La Rioja, Argentina, were compared. In G9 RV, two intragroup differences (100 and 211 positions) were observed. In position 100, D and N amino acids were detected, a common change to other strains of the world (11,18). In position 211, K was not observed in other strains of Argentina or available strains in GenBank (4,5). In the comparison between G9 RV strains circulating in La Rioja, Argentina with ROTOVAC vaccine strain, three differences were detected corresponding to 100, 211, and 145 positions. All these positions could be important for the escape to neutralization for monoclonal antibody (13,18). In G1 RV, intragroup differences were not observed and only three differences with vaccine strains, in 94, 123, and 217 positions, were detected, similar to strain circulation in Belgium during 2008–2009 (15,16). Two amino acid changes, 94 and 217 positions, have been shown to escape neutralization with monoclonal antibodies (13,18).

FIG. 1.

The tree phylogenetic of the nucleotide sequences of G1 and G9 rotavirus strains circulating in La Rioja, Argentina and strains with defined lineage. The evolutionary history was inferred using the UPGMA method. The optimal tree with the sum of branch length = 35.41016264 is shown. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. There were 827 positions in the final dataset. Phylogenetic analyses were conducted in MEGA 6.0.

FIG. 2.

The alignments of 29 amino acids corresponding to dominants antigenic of VP7, 7-1a, 7-1b and 7-2 of G9 and G1 RVs circulating in La Rioja and vaccine strains were studied using the amino acid sequence obtained by BioEdit, 7.2.5. Amino acids that differ between studied are indicated in the shaded boxes. RV, rotavirus.

Conclusions

In conclusion, the differences between the G1 strains in La Rioja, Argentina and the G1 components of the RotaTeq and Rotarix vaccine strains are few, but important for the escape immunologic and need to be monitored for appropriate evaluation of long-term impact of vaccine use in Argentina. Nevertheless, the VP7 antigenic regions of G9 RV strains circulating in La Rioja and ROTAVAC vaccine strains are different to other zones of Argentina and could play an important role in the failure of vaccine response in these regions and Argentina. More studies of other viral proteins are needed to know the importance of these differences in the selection of strain G9 RV for the pressure immunological of vaccine strains and their molecular evolution in Argentina. Continuous rotavirus surveillance is necessary to understand rotavirus evolution and to measure how genetic and antigenic changes might affect the effectiveness of vaccines in the future.

Footnotes

Acknowledgments

The authors are thankful to Dra. Silvia Nates for contributing to the standardization of methodologies in our laboratory and to Dra. Viviana Re for assisting in computer basic methodology for analysis of sequences. Foundation Barceló H.A. funds this work under the Project Resolution HCSN°: 4312/11 and supported the doctoral fellowship of Valeria Cuffia (ref.: 4312/11/LR/BD).

Author Disclosure Statement

The authors declare no conflicts of interest.

References

Aoki

, Trask Sh

, Coulson

, et al. Cross-linking of rotavirus outer capsid protein VP7 by antibodies or disulfides inhibits viral entry. J Virol, 2011; 85:10509–10517.

Banyai

, Laszlo

, Duque

, et al. Systematic review of regional and temporal trends in global rotavirus strain diversity in the pre-rotavirus vaccine era: insights for understanding the impact of rotavirus vaccination programs. Vaccine, 2012; 30S:A122–A130.

Barril

1, Martínez

, Giordano

, et al. Detection of group a human rotavirus G9 genotype circulating in Córdoba, Argentina, as early as 1980. J Med Virol, 2006; 78:1113–1118.

Barril

, Martínez

, Giordano

, et al. Genetic and antigenic evolution profiles of G1 rotaviruses in Córdoba, Argentina, during a 27-year period (1980–2006). J Med Virol, 2012; 85:363–369.

Bok

, Palacios

, Sijvarger

, et al. Emergence of G9P [6] human rotaviruses in Argentina: phylogenetic relationships among G9 strains. J Clin Microbio, 2001; 39:4020–4025.

Castello

, Argüelles

, Rota

, et al. Molecular epidemiology of group A rotavirus diarrhea among children in Buenos Aires, Argentina, from 1999 to 2003 and emergence of the infrequent genotype G12. J Clin Microbiol, 2006; 44:2046–2050.

Degiuseppe

, Giovacchini

, Stupka

. The Argentinean National Rotavirus Surveillance Network Rotavirus epidemiology and surveillance in Argentina: 2009–2011. Arch Arg Pediatr, 2013:111:148–154.

Estes

, Kapikian

. Rotaviruses. In: Knipe

, Howley

, Griffin

, Lamb

, Martin

, Roizman

, Straus

, eds. Fields Virology. Philadelphia, PA: Kluwer/Lippincott; Williams and Wilkins, 2007:1917–1974.

Gouvea

, Glass

, Woods

, et al. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol, 1990; 28:276–282.

10.

Guerra

, Cuffia

, Maldonado

, et al. Caracterización de diarreas virales en La Rioja. Congreso Argentino de Virología. Septiembre 2005. Asociación Argentina de Microbiología. Sociedad Argentina de virología. Rev Ar Microbio, 2005; 37:67.

11.

Kulkarni

, Arora

, et al. Sequence analysis of VP7 and VP4 genes of G1P [8] rotaviruses circulating among diarrheic children in Pune, India: a comparison with Rotarix and RotaTeq vaccine strains. Vaccine, 2014; 32S:A75–A83.

12.

Matthijnssens

, Nakagomi

, Kirkwood

, et al. Group A rotavirus universal mass vaccination: how and to what extent will selective pressure influence prevalence of rotavirus genotypes?. Expert Rev Vaccines, 2012; 11:1347–1354

13.

McDonald

1, Matthijnssens

, McAllen

, et al. Evolutionary dynamics of human rotaviruses: balancing reassortment with preferred genome constellations. PLoS Pathog, 2009; 5:e1000634.

14.

Perry

, La Torre

, Kelley

, et al. On the lability of poly (A) sequences during extraction of messenger RNA from polyribosomes. Biochim Biophys Acta, 1972; 262:220–226.

15.

Rahman

, Matthijnssens

, Goegebuer

, et al. Predominance of rotavirus G9 genotype in children hospitalized for rotavirus gastroenteritis in Belgium during 1999–2003. J Clin Virol, 2005; 33:11–16.

16.

Stupka

, Parra

, Gomez

, et al. Detection of human rotavirus G9P [8] strains circulating in Argentina: phylogenetic analysis of VP7 and NSP4 genes. J Med Virol, 2007; 79:838–842.

17.

Tate

, Burton

, Boschi-Pinto

, et al. WHO-coordinated global rotavirus surveillance network. WHO coordinated global rotavirus surveillance network 2008 estimate of worldwide rotavirus-associated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis. Lancet Infect Di, 2012; 12:136–141.

18.

Zeller

, Patton

, Heylen

, et al. Genetic analyses reveal differences in the VP7 and VP4 antigenic epitopes between human rotaviruses circulating in Belgium and rotaviruses in Rotarix and RotaTeq. J Clin Microbiol, 2012; 50:966–976.