Genetic analysis of G12P[8] rotaviruses detected in the largest U.S. G12 genotype outbreak on record
Introduction
Before widespread use of the 2 currently licensed rotavirus vaccines, Group A rotaviruses (RVA) caused approximately half a million deaths yearly among children younger than 5 years globally (Parashar et al., 2009). In the U.S., deaths were not common but RVA was a significant cause of morbidity in young children (Payne et al., 2008). RVA, members of the Reoviridae family, possess a genome of 11 double-stranded RNA (dsRNA) segments that encode 6 viral structural proteins (VP1–VP4, VP6 and VP7) and 6 nonstructural proteins (NSP1–NSP6) (Estes and Kapikian, 2007). Surrounding the dsRNA are 3 protein layers, a central core (VP2), a middle protein layer (VP6), and an outer capsid layer composed of VP7 and VP4 proteins (Estes and Kapikian, 2007). The traditional dual classification of RVA is based upon serotype specificities and the sequence diversity of the 2 outer capsid proteins, VP7 (glycosylated, G-type) and VP4 (protease-sensitive, P-type) (Estes, 1996). At least 27 G genotypes and 37 P genotypes have been identified (Matthijnssens et al., 2011, Trojnar et al., 2013). Of all possible combinations, 5 strains (G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]) are associated with 80–90% of the RVA disease burden (Patel et al., 2011). However, 3 G12 strains, G12P[6], G12P[8], and G12P[9] have been emerging in many areas of the world (Iturriza-Gomara et al., 2011, Matthijnssens et al., 2010).
After RVA genotype G12 was first reported in 1987 in the Philippines (Taniguchi et al., 1990), it was detected only sporadically for more than 13 years (Das et al., 2003, Griffin et al., 2002, Pongsuwanna et al., 2002, Samajdar et al., 2006). Recently, it has re-emerged worldwide, becoming the sixth most prevalent RVA VP7 genotype (Rahman et al., 2007). Based on phylogenetic and phylodynamic analyses, G12 genotype strains have been subdivided into 4 lineages (Matthijnssens et al., 2010, Rahman et al., 2007). Lineage I and IV each contain only one strain, the prototype G12 strain and a porcine G12P[7] strain, respectively. Lineage II consists of G12P[9] strains from South America and Asia. Lineage III contains the majority of the currently known G12 strains, which are associated with P types P[6] or P[8].
In 2009, the World Health Organization (WHO) recommended 2 RVA vaccines, Rotarix® and RotaTeq®, for routine immunization of all infants (2009). The monovalent attenuated human RVA vaccine, Rotarix® (GlaxoSmithKline Biologicals, Rixensart, Belgium), contains the single most prevalent genotype (G1P[8]) and was developed to induce heterotypic immunity against a number of epidemiologically and clinically important strains (Ruiz-Palacios et al., 2006). In contrast, the pentavalent bovine-human reassortant vaccine, RotaTeq® (Merck & Co, Inc., Whitehouse Station, NJ) was developed to provide serotype-specific immunity against 4 common G types (G1–G4) and one common P type (P[8]) (Vesikari et al., 2006). Based on large phase III trials and post marketing surveillance studies, both the monovalent Rotarix® and the pentavalent RotaTeq® vaccines provide protection against severe RVA disease from a wide variety of circulating strains in industrialized and developing countries (Patel et al., 2011).
The Centers for Disease Control and Prevention (CDC) currently supports RVA surveillance in the U.S. through the New Vaccine Surveillance Network (NVSN) (Payne et al., 2008). During the 2006–07 RVA surveillance season, the G12P[8] strain was detected in 69% (77 of 111) of RVA-positive samples from the NVSN surveillance site in Rochester, NY. Currently this RVA outbreak is the largest in U.S. history associated with genotype G12 (Payne et al., 2009). This overwhelming prevalence of G12P[8] RVA during that season was very unusual, especially because the G12 genotype was not detected in Rochester during the previous 2005–06 or following 2007–08 RVA seasons (Payne et al., 2009). In addition, the hospitalization rates in Rochester during the 2006–07 season increased 3-fold for RVA disease when compared to the 2005–06 season (Payne et al., 2009). The aim of this study was to characterize the G12P[8] strains detected in Rochester during the 2006–07 outbreak at the genetic and protein structure level and to compare them to U.S. and worldwide wild-type G12 strains to better understand the evolution of this emerging genotype. In addition, we wanted to compare the antigenic regions of the outer capsid proteins of these G12P[8] strains against the cognate protein sequences of current RVA vaccines.
Section snippets
Samples and molecular methods
As a part of the NVSN surveillance network, active RVA surveillance was carried out in Rochester N.Y. USA during the 2006–2007 RVA season (Payne et al., 2009). Stool samples were screened for RVA antigen by enzyme immunoassay using the Premier™ Rotaclone® Rotavirus enzyme immunoassay kit (Meridian Diagnostics, Inc., Cincinnati, OH). RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR) based genotyping of the outer surface proteins VP7 and VP4 gene, and sequencing were
VP7 sequence analysis
The standard G-specific genotyping RT-PCR primer pool used in this study (Hull et al., 2011) lacked a G12 -specific primer, resulting in high number of G non-typeable strains by RT-PCR based genotyping. Thus, we sequenced all VP7 genes. The 77 VP7 sequences corresponded to nt 88–890 of the VP7 gene of reference strain Wa and are represented by 2 unique sequences, RVA/Human-wt/USA/Ro4426/2007/G12P[8] and RVA/Human-wt/USA/Ro4434/2007/G12P[8]. High nucleotide and amino acid sequence similarities
Discussion
In this study, selection analysis of the VP8∗ region and the VP7 gene, large regions of purifying selection were identified for Rochester G12P[8] strains. The data suggest a conservation of structure and function in those regions of the proteins between all the strains used in this study. In the VP8∗ region two surface amino acids, 79 and 218, showed conservative amino acid changes in strains Ro4487 and Ro4500, respectively. As these sites were under strong purifying selection, we can assume,
Conclusion
In conclusion, genetic characterization of G12P[8] strains from a large outbreak associated with severe disease found that the Rochester G12P[8] VP7 and VP4 genes appear to be similar to worldwide human G12-III lineage strains. Selection analyses identified one positively selected site in VP7 and strong purifying selection at multiple sites within VP7 and VP4. The mechanisms behind the evolutionary changes in G12P[8] strains identified in this study, and their ramifications for viral
Conflict of interest statement
The authors of this study declare that they have no conflict of interest, financial or otherwise, related to this article.
Disclaimer
The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Names of specific vendors, manufacturers, or products are included for public health and informational purposes; inclusion does not imply endorsement of the vendors, manufacturers, or products by the Centers for Disease Control and Prevention or the US Department of Health and Human Services.
Acknowledgment
We wish to thank Mary McCauley for editorial assistance.
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