The Impact of Rotavirus Vaccines on Genotype Diversity: A Comprehensive Analysis of 2 Decades of Australian Surveillance Data

The Impact of Rotavirus Vaccines on Genotype Diversity: A Comprehensive Analysis of 2 Decades of... Abstract Background Introduction of rotavirus vaccines into national immunization programs (NIPs) could result in strain selection due to vaccine-induced selective pressure. This study describes the distribution and diversity of rotavirus genotypes before and after rotavirus vaccine introduction into the Australian NIP. State-based vaccine selection facilitated a unique comparison of diversity in RotaTeq and Rotarix vaccine states. Methods From 1995 to 2015, the Australian Rotavirus Surveillance Program conducted genotypic analysis on 13051 rotavirus-positive samples from children <5 years of age, hospitalized with acute gastroenteritis. Rotavirus G and P genotypes were determined using serological and heminested multiplex reverse-transcription polymerase chain reaction assays. Results G1P[8] was the dominant genotype nationally in the prevaccine era (1995–2006). Following vaccine introduction (2007–2015), greater genotype diversity was observed with fluctuating genotype dominance. Genotype distribution varied based on the vaccine implemented, with G12P[8] dominant in states using RotaTeq, and equine-like G3P[8] and G2P[4] dominant in states and territories using Rotarix. Conclusions The increased diversity and differences in genotype dominance observed in states using RotaTeq (G12P[8]), and in states and territories using Rotarix (equine-like G3P[8] and G2P[4]), suggest that these vaccines exert different immunological pressures that influence the diversity of rotavirus strains circulating in Australia. rotavirus, genotype, RotaTeq, Rotarix, selective pressure Rotavirus vaccines have been successfully implemented in the routine immunization programs of 93 countries [1]. The globally licensed rotavirus vaccines, RotaTeq (Merck, West Point, Pennsylvania) and Rotarix (GSK Biologics, Rixensart, Belgium), have been associated with a significant reduction in rotavirus hospitalizations and all-cause gastroenteritis deaths in all settings where they have been introduced [2–4]. However, these vaccines have different genome compositions and antigenic characteristics. Rotarix is a single attenuated human G1P[8] rotavirus strain, administered in a 2-dose oral regimen at 2 and 4 months of age [5]. RotaTeq combines 5 separate bovine-human reassortant strains, each containing a human gene encoding the outer capsid VP7 or VP4 protein (G1, G2, G3, G4, or P[8]) within a backbone of a bovine rotavirus strain [6]. RotaTeq is administered in a 3-dose oral regimen at 2, 4, and 6 months of age. The outer capsid proteins, VP7 and VP4, elicit neutralizing antibodies implicated in genotype-specific and genotype cross-reactive protection [7, 8]. Both vaccines have been reported to protect against severe disease caused by the 5 common VP7 genotypes (G1, G2, G3, G4, and G9) and VP4 genotypes (P[4], P[6], and P[8]); however, they also provide broad heterotypic protection against other, less common genotypes [9]. The mechanism and extent to which cross-protection occurs is not well elucidated, nor whether this could impact vaccine escape. Rotavirus vaccination was introduced into the Australian national immunization program (NIP) for all infants on 1 July 2007 [10]. Unlike other countries and regions, Australia allowed each state and territory to independently decide which vaccine to implement in the NIP through a competitive tender process. As a result, Queensland, South Australia, and Victoria selected to use RotaTeq, and the Australian Capital Territory, New South Wales, Northern Territory, and Tasmania selected to use Rotarix (Figure 1). Western Australia initially selected Rotarix, before changing to RotaTeq in 2009. Rotavirus vaccine coverage for 2 or 3 doses (depending on vaccine) in Australia is high, reported at 86.1% by the end of 2015 [11]. Rotavirus vaccines have reduced rotavirus-related hospitalizations in Australia, in both the age group eligible for rotavirus vaccination and nonvaccinated children born prior to vaccine introduction, suggestive of an indirect benefit of rotavirus vaccination [12]. RotaTeq vaccine effectiveness in Queensland is estimated at 89.3% for rotavirus hospitalizations, and in New South Wales, >77% reduction in acute gastroenteritis (AGE) emergency presentations has been observed following Rotarix introduction [2, 13]. The state-based vaccine tender has provided a unique opportunity to observe the impact of RotaTeq and Rotarix introduction on genotype diversity, and to compare genotype distribution between states and territories Australia-wide. Figure 1. View largeDownload slide Pattern of state-based vaccine use within the national immunization program in Australia. Queensland, South Australia, and Victoria implemented RotaTeq (light gray); Australian Capital Territory, New South Wales, Northern Territory, and Tasmania implemented Rotarix (dark gray). Western Australia initially used Rotarix but switched to RotaTeq in 2009. Circles represent locations of collaborating centers contributing samples to the Australian Rotavirus Surveillance Program. Figure 1. View largeDownload slide Pattern of state-based vaccine use within the national immunization program in Australia. Queensland, South Australia, and Victoria implemented RotaTeq (light gray); Australian Capital Territory, New South Wales, Northern Territory, and Tasmania implemented Rotarix (dark gray). Western Australia initially used Rotarix but switched to RotaTeq in 2009. Circles represent locations of collaborating centers contributing samples to the Australian Rotavirus Surveillance Program. Variation in circulating rotavirus strains has been observed in the years prior to the induction of rotavirus vaccines. Molecular epidemiological studies worldwide identified >60 G/P combinations in humans, although 90% of strains belonged to 1 of 5 common genotypes (G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]) [14, 15]. The impact of rotavirus vaccines on the natural pattern of circulating rotavirus strains is not known. Documenting changes in rotavirus genotype prevalence, recognizing emergence of rare or uncommon genotypes, and identifying potential vaccine escape strains will inform future vaccination strategies and the development of new rotavirus vaccines. Mass introduction of other non rotavirus live vaccines, such as hepatitis B vaccine, have previously been shown to affect the diversity of circulating strains within a population, where strain replacement, emergence of novel strains, and vaccine escape strains have been observed [16]. The Australian Rotavirus Surveillance Program has monitored the rotavirus genotypes present in children hospitalized with rotavirus-associated AGE in Australia since 1989 [17]. Nineteen collaborating laboratories across Australia collect rotavirus-positive specimens from children <5 years of age hospitalized with AGE. In the current study, we investigated the temporal diversity and geographic trends of rotavirus genotypes observed in children <5 years of age, hospitalized with AGE in Australia from 1995 to 2015. The specific aims were to (1) compare rotavirus strain diversity before and after vaccine introduction, and (2) determine whether differences in genotype diversity and distribution exist between states, based on the vaccine implemented. This state-by-state analysis provides a unique opportunity to compare and contrast the effect of both RotaTeq and Rotarix on the distribution and diversity of wild-type rotavirus strains. METHODS Study Design This study represents a longitudinal analysis of the rotavirus genotypes detected in faecal samples collected from children <5 years of age, who were hospitalized with AGE in Australia. The study period comprises the 12 years prior to rotavirus vaccine introduction into the NIP in Australia (1995–2006), and the 9 years following vaccine introduction (2007–2015). Rotavirus-positive samples were sent to the National Rotavirus Reference Centre (NRRC) laboratory at the Murdoch Children’s Research Institute (MCRI) by 19 laboratories and hospitals collaborating in the Australian Rotavirus Surveillance Program (Figure 1 and Supplementary Table 1). Collaborating centers analyzed fecal samples for the presence of rotavirus using enzyme immunoassay (EIA) or latex agglutination. Rotavirus-positive specimens were then stored at –20°C to –80°C, and the de-identified samples were forwarded at regular intervals each year to the NRRC laboratory, together with metadata including date of collection, date of birth, sex, and postcode. Upon receipt, samples were allocated a unique laboratory code and entered into the NRRC sample-tracking database (Excel and REDCap). Samples were then stored at –80°C until analyzed. The presence of rotavirus antigen was confirmed using ProSpecT Rotavirus Test, a commercial rotavirus EIA (Thermo Fisher, Australia), as per the manufacturer’s instructions. Samples confirmed as rotavirus positive underwent genotyping analysis, whereas unconfirmed specimens (EIA negative) were not processed further. Rotavirus Genotyping Specimens processed prior to 2007 were serotyped using an in-house monoclonal antibody–based serotyping EIA [18]. This comprised of a panel of monoclonal antibodies specific to the major outer capsid protein (VP7) of group A human rotavirus serotypes G1, G2, G3, G4, and G9 [18]. P-genotyping and reverse-transcription polymerase chain reaction (RT-PCR) were not routinely performed prior to 2007. For samples that could not be assigned a serotype or were processed after 2007, rotavirus G and P genotypes were determined using a heminested multiplex RT-PCR assay [19]. Viral RNA was extracted from 10%–20% (w/v) fecal extracts using the QIAamp Viral RNA mini extraction kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The first-round RT-PCR reactions were performed using the QIAGEN 1-step RT-PCR kit, using VP7 primers VP7F and VP7R, or VP4 primers VP4F and VP4R [20, 21]. The second-round genotyping PCR reactions were conducted using the AmpliTaq DNA Polymerase with Buffer II (Applied Biosystems, Foster City, California), together with specific oligonucleotide primers for G types (G1, G2, G3, G4, G8, G9, and G12) or P types (P[4], P[6], P[8], P[9], P[10], and P[11]), as previously described [22]. The G and P genotype of each sample was assigned using agarose gel analysis of second-round PCR products. The VP7 nucleotide sequence from PCR-nontypeable samples was determined by Sanger sequencing, as the primers used in the current G-typing protocol could not assign a genotype to equine-like G3, G12, and unusual or uncommon rotavirus strains. Suspect vaccine excretion cases from RotaTeq states that could not be P-typed, or G1P[8] strains from infants within the age range of recent vaccination in Rotarix states, were also sequenced. First-round VP7 or VP4 amplicons were purified for sequencing using commercial kits as previously described [22]. Purified DNA together with oligonucleotide primers (VP7F/R or VP4F/R) were sent to the Australian Genome Research Facility (Melbourne) and sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Reaction Kit (Applied Biosystems) in an Applied Biosystems 3730xl DNA Analyzer. Sequences were edited with Sequencher version 4.10.1. Genotype assignment was determined using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and RotaC version 2.0 (http://rotac.regatools.be) [23]. Data Analysis Samples were excluded if patient age was unknown or >5 years, or if a duplicate sample was provided. Samples containing a rotavirus vaccine strain were also excluded (Figure 2). According to the date of collection, samples were divided into prevaccine era (1995–2006) and vaccine era (2007–2015). Vaccine group was assigned according to the state or territory where the sample was collected (RotaTeq group: Queensland, South Australia, Victoria, and Western Australia [before 1 May 2009]; Rotarix group: Australian Capital Territory, New South Wales, Northern Territory, and Western Australia [1 May 2009 or later]). Data are presented as the proportion (percentage) of a specific genotype compared to the total number of strains collected for that era, per vaccine group, and for each state and territory. Figure 2. View largeDownload slide Stool sample flowchart. Abbreviation: WA, Western Australia. Figure 2. View largeDownload slide Stool sample flowchart. Abbreviation: WA, Western Australia. RESULTS During the period 1995–2015, a total of 21545 rotavirus-positive fecal specimens were received from collaborating laboratories in the 8 states and territories across Australia. Of these, 13051 were confirmed as rotavirus positive and included in the final analysis (Figure 2). Reasons for exclusion included insufficient sample (n = 760), missing or not processed (n = 468), duplicate (n = 327), age >5 years (n = 2280), age unknown (n = 1064), negative EIA result when analyzed at MCRI (n = 3439), and identification of vaccine strain (n = 156) (Figure 2). In the prevaccine era (1995–2006), 9301 samples were analyzed; 3318 samples from states that subsequently implemented the RotaTeq vaccine, and 5983 samples from states and territories that subsequently implemented the Rotarix vaccine. Following vaccine introduction (2007–2015), 3750 samples were analyzed; 2179 from children in states that had implemented RotaTeq vaccine and 1571 in states and territories that had implemented Rotarix vaccine. Increased Genotypic Diversity and Unusual Strains Identified Following Vaccine Introduction The diversity of rotavirus genotypes observed was greater in the period following rotavirus vaccine introduction (Figure 3). In the prevaccine era, the common circulating genotypes were G1, G2, G3, G4, and G9, which represented 82.9% (n = 7714 of 9301) of all samples genotyped in that period. G1 strains were the predominant genotype, representing 53.4% (4966/9301) of all samples genotyped, followed by G9 (1349/9301 [14.5%]), G3 (599/9301 [6.4%]), G2 (434/9301 [4.7%]), and G4 (366/9301 [3.9%]). In contrast, following vaccine introduction (2007–2015), these 5 common genotypes were identified in only 63% (2365/3750) of all samples genotyped (Figure 3). The most common genotypes identified in the vaccine era were G1P[8] (983/3750 [26.2%]), G2P[4] (780/3750 [20.8%]), and G12P[8] (683/3750 [18.2%]) (Figure 3). Other genotypes identified following vaccine introduction included a novel equine-like G3P[8] (343/3750 [9.1%]) [7], G3P[8] (312/3750 [8.3%]), G9P[8] (226/3750 [6%]), and G4P[8] (64/3750 [1.7%]) (Figure 3). G12P[8], only identified in a single outbreak involving 10 patients in the prevaccine era, emerged as a dominant genotype from 2012 onward; identified in 683 samples between 2012 and 2015. Previously uncommon VP7 genotypes such as G8, G10, and other animal-reassortant strains (excluding equine-like G3P[8] strains), were also detected more frequently in the vaccine era (n = 63) compared to the prevaccine era (n = 17) (Supplementary Table 2). This increase, together with the emergence of the equine-like G3P[8] strain and G12P[8] strains, suggests that a more diverse population of genotypes was circulating following vaccine introduction. Figure 3. View largeDownload slide Comparison of genotype distribution (% of all rotavirus strains) before and after rotavirus vaccine introduction to the Australian national immunization program. Colors represent specific rotavirus genotypes. Samples with an unusual genotype are listed in Supplementary Table 2. Data are presented as the proportion (%) of a specific genotype compared to the total strains collected. Figure 3. View largeDownload slide Comparison of genotype distribution (% of all rotavirus strains) before and after rotavirus vaccine introduction to the Australian national immunization program. Colors represent specific rotavirus genotypes. Samples with an unusual genotype are listed in Supplementary Table 2. Data are presented as the proportion (%) of a specific genotype compared to the total strains collected. Differences in the Geographical Distribution of Genotypes According to Vaccine Use The pattern of rotavirus genotypes causing hospitalization in children <5 years of age was similar across states and territories in the prevaccine era, with G1P[8] the dominant genotype detected (Figure 4A and 4B). The higher proportion of G9P[8] and G3P[8] strains identified in states and territories that would later introduce Rotarix may have been influenced by outbreaks in the Northern Territory due to G9P[8] in 2001–2003, and G3P[8] in 2003–2006, which spread to other states (Supplementary Figure 1A and 1B) [8]. Figure 4. View largeDownload slide Comparison of rotavirus G and P type (% of all rotavirus strains) based on vaccine used within the state-based national immunization program (NIP). A, Genotype distribution prior to vaccine introduction in Australia categorized by the vaccine to be introduced into the state-based immunization program. B, Genotype distribution remained consistent across all states and territories in the prevaccine era. C, Genotype distribution following vaccine introduction in Australia, categorized according to the vaccine implemented in the state-based NIP. D, Within vaccine groups, genotype distribution remained consistent across all states and territories following vaccine introduction. Samples with an unusual genotype are listed in Supplementary Table 2. Colors represent specific rotavirus genotypes. Abbreviations: ACT, Australian Capital Territory; NSW, New South Wales; NT, Northern Territory; QLD, Queensland; SA, South Australia; Tas, Tasmania; Vic, Victoria; WA, Western Australia. Figure 4. View largeDownload slide Comparison of rotavirus G and P type (% of all rotavirus strains) based on vaccine used within the state-based national immunization program (NIP). A, Genotype distribution prior to vaccine introduction in Australia categorized by the vaccine to be introduced into the state-based immunization program. B, Genotype distribution remained consistent across all states and territories in the prevaccine era. C, Genotype distribution following vaccine introduction in Australia, categorized according to the vaccine implemented in the state-based NIP. D, Within vaccine groups, genotype distribution remained consistent across all states and territories following vaccine introduction. Samples with an unusual genotype are listed in Supplementary Table 2. Colors represent specific rotavirus genotypes. Abbreviations: ACT, Australian Capital Territory; NSW, New South Wales; NT, Northern Territory; QLD, Queensland; SA, South Australia; Tas, Tasmania; Vic, Victoria; WA, Western Australia. In the first 5 years following vaccine introduction, no single genotype was dominant Australia-wide (Supplementary Figure 1). In 2012–2015, G12P[8] emerged as the dominant genotype in all states that had introduced the RotaTeq vaccine (Supplementary Figure 1A), representing 30.5% (665/2179) of all strains genotyped (Figure 4C). In contrast, G12P[8] strains were only identified in 1.1% (18/1571) of all strains in the states and territories that introduced the Rotarix vaccine (Figure 4C and D). G2P[4] became a dominant genotype in states that implemented Rotarix, identified in 26.7% (419/1571) of samples, compared to 16.6% (361/2179) of samples from states using RotaTeq (Figure 4C and 4D). Equine-like G3P[8] strains emerged across 3 consecutive years (2013–2015), and was observed in 83% (117/141) of samples collected in Rotarix states and territories in 2013, 23.1% (24/104) in 2014, and 49.7% (74/149) in 2015 (Supplementary Figure 1B). Equine-like G3P[8] and G9P[8] strains were identified at a higher frequency in states and territories using Rotarix (equine-like G3P[8], 13.7% [215/1571]; G9P[8], 11.8% [185/1571]) compared to states using RotaTeq (equine-like G3P[8], 5.9% [128/2179]; G9P[8], 1.9% [41/2179]). When analyzed by state, these patterns of dominance remained consistent within vaccine groups (Figure 4D). In this study, Western Australia was analyzed separately as the vaccine in the NIP changed in May 2009 from Rotarix to RotaTeq (Supplementary Figure 1C). For the period up to 2009, the circulating genotype patterns were similar to other states using Rotarix (Supplementary Figure 1B and 1C). However, from 2010 onward, the genotype distribution changed to reflect patterns observed within RotaTeq states (Supplementary Figure 1A and 1C). One exception was the large proportion of equine-like G3P[8] samples identified in 2013, which was similar to states and territories using Rotarix. DISCUSSION This study demonstrates that the introduction of rotavirus vaccines into the NIP in Australia altered the diversity and distribution of circulating rotavirus genotypes in children aged <5 years who were hospitalized with rotavirus gastroenteritis. Historically, rotavirus genotype distribution has shown temporal and geographic movement across Australia, where strains spread across the country over a period of 1–2 years (eg, genotype G9P[8]) [17]. This geographic and temporal pattern has been disrupted in the vaccine era, with adjacent states using different vaccines exhibiting varied patterns of genotype diversity maintained over many years. This analysis highlighted the importance of sustaining rotavirus surveillance programs, as critical differences in genotype distribution between vaccine locations were not obvious within the first 5 years of rotavirus vaccine introduction. Rotavirus diversity is generated by genetic drift, genetic shift, and zoonotic transmission [24]. Vaccine-induced immune pressure may be an additional selective pressure that leads to the evolution of rotavirus strains capable of causing severe disease in vaccinated children. In this study, sustained dominance of G12P[8] was observed in states using RotaTeq, while dominance of G2P[4] and equine-like G3P[8] was observed in states and territories using Rotarix. This is highly suggestive of vaccine-specific selection pressure, as these observations occurred within a country with high vaccine coverage and demonstrated vaccine effectiveness. The differences observed in adjacent states and territories using either RotaTeq or Rotarix cannot be explained by geographical or demographic differences (Figure 1). However, these differences could be due to vaccine composition and antigenic characteristics [25]. Rotarix contains a monovalent human G1P[8] strain, and has been shown to elicit heterotypic protection against non-G1P[8] strains, whereas the pentavalent human-bovine reassortant vaccine, RotaTeq, generates homotypic protection against each of the G1, G2, G3, G4, and P[8] strains contained in the vaccine and has been shown to elicit heterotypic protection. The ability of each vaccine to generate broader heterotypic protection is not well defined [9]. This study reports an increased detection of previously rare or novel genotypes emerging in the vaccine era, supporting the hypothesis that widespread vaccine use may increase strain diversity. However, emerging genotypes have also been detected in countries with low coverage or no vaccination programs [26, 27]. For example, the novel equine-like G3P[8] strain has been detected in countries with a range of vaccine coverage [7, 26, 28–30]. The first report of this strain was from Japan, where rotavirus vaccine coverage was estimated to be 40%–45% [31]. Since then, this strain has been detected in countries with high vaccine coverage (Australia, Brazil, and the United States), moderate vaccine coverage (Germany, Taiwan, and Spain), and no vaccine coverage within the NIP (Hungary, Thailand, and Turkey) [29]. Multiple amino acid differences have been observed in the antigenic regions of the VP7 gene between human wild-type G3P[8] strains and the equine-like VP7 gene in Australia, suggesting that this equine-like strain is antigenically distinct from human strains [7]. The global emergence of this strain, irrespective of vaccine coverage, suggests that vaccines may not be highly effective against this strain. Global movement of rotavirus strains occurs, reflected by the increasing incidence of G12P[8] in countries with and without vaccine coverage [32–35]. In this study, G12P[8] strains were primarily found in states that use RotaTeq. This is difficult to attribute to natural fluctuations or demographic differences. Similar observations were reported in Finland, where reported vaccine (RotaTeq) coverage is >90% [35]. In Finland, greater genotype diversity was observed in the vaccine era, over 2 rotavirus seasons [35]. Of particular interest is that G12P[8] strains emerged during this period and were detected more often in vaccinated children (13.8%) compared with unvaccinated children (6.5%) [35]. The question remains why G12P[8] and equine-like G3P[8] are responsible for a substantial burden of disease in populations with high vaccine coverage such as Australia and Finland [35]. It has been proposed that the VP7 and VP4 antigenic regions of the equine-like G3P[8] and G12P[8] strains could be distinct to that of the Rotarix and RotaTeq vaccines [7, 36, 37], resulting in lower vaccine effectiveness against these strains. G2P[4] strains have increased in circulation in Australia in the vaccine era, particularly in Rotarix states and territories. Despite Rotarix vaccine introduction in the Northern Territory, Australia, an outbreak associated with G2P[4] strains occurred in 2009 [38]. The average age of patients was of vaccine age (5.5 months); however, there was only approximately 50% vaccine coverage, and vaccine effectiveness against rotavirus hospitalizations was only 19% [38, 39]. A waning heterotypic response afforded by Rotarix, combined with low circulating numbers of G2P[4] in the preceding years, could have contributed to a decrease in herd immunity to this genotype [38]. Furthermore, prior exposure to G2P[4] strains may not have generated effective homotypic protection against the outbreak strain, as it exhibited changes in the VP7 gene antigenic region, compared to other contemporary G2P[4] strains [38]. Excluding this outbreak, G2P[4] strains are still a prominent cause of disease in Rotarix states, possibly reflecting limited or short-lasting heterotypic immunity. An increased proportion of G2P[4] strains have also been reported in Brazil and Belgium after introduction of the Rotarix vaccine [40, 41]. Prelicensure efficacy studies could not consistently demonstrate comparable levels of enduring protection for heterotypic strains such as G2P[4] [9]. Despite reporting vaccine effectiveness of 85% against G2P[4], a study from Belgium described higher incidence rates of G2P[4] in vaccinated hospitalized cases compared with unvaccinated hospitalized cases [40]. It has been hypothesized that a lower efficacy against heterotypic strains such as G2P[4] could drive an alteration in circulating genotypes [38, 40, 42]. In this study, G2P[4] strains account for 16.6% of strains genotyped in states utilizing the RotaTeq vaccine. Comparison of the RotaTeq component strain and G2P[4] strains circulating in RotaTeq states revealed substitutions in antigenic regions that have also been reported in the majority of G2P[4] strains circulating globally over the last decade [43]. The G2 RotaTeq component strain was isolated in 1992; therefore, if contemporary strains continue to accumulate mutations in antigenic regions, the vaccine strains may need to be updated. After vaccine introduction, the dominance of G1P[8] strains has significantly reduced globally [44]. Several studies have revealed that contemporary strains exhibit numerous mutations in antigenic regions compared to the original vaccine strains [25, 36, 45, 46]. A multicountry phylogenetic analysis revealed unique subclusters of strains identified after Rotarix introduction in Belgium and RotaTeq introduction in Victoria, Australia [45, 46]. Furthermore, the increase in G1P[8] detected in 2010–2012 in our study was attributed to the emergence of a G1P[8] outbreak strain that appeared to evade protection from Rotarix in Alice Springs, Northern Territory [21]. These findings, together with the ongoing presence of G1P[8] in a global context, suggest that rotavirus vaccines may need to be reviewed to meet the evolutionary changes that may challenge the success of vaccination programs. This longitudinal, nationwide analysis of rotavirus genotypes was conducted over a 21-year period prior to and following rotavirus vaccine introduction, reporting increases in genotype diversity and detection of unusual rotavirus strains. Differences in genotype dominance and diversity were apparent between states using the RotaTeq vaccine, compared with states and territories using the Rotarix vaccine. This suggests that vaccine-related immune selection pressure occurs and this phenomenon differs between vaccines, supporting our contention that vaccine pressure drives strain selection. Rotavirus vaccines have been shown to be highly efficacious in reducing severe disease; however, rotavirus strains continue to evolve and novel strains continue to emerge. Ongoing surveillance and the use of phylogenetic analysis will be important to provide further insight on the impact of rotavirus vaccines on strain diversity. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Author contributions. S. R.-F. coordinated the Australian Rotavirus Surveillance Program from 2010 to 2018; during this period, S. R.-F. collated samples and conducted experiments. S. R.-F. performed the literature search, data collection, analysis, and interpretation; contributed to the study design; and wrote the first draft of the manuscript. N. B.-S., C. M. D., and K. B. performed data collection, data analysis, and data interpretation, and contributed to the study design. C. D. K., G. L. B., R. F. B., D. C., and J. E. B. designed and directed the study. These authors provided conceptual and technical guidance for all aspects of the project including data interpretation and revision of the manuscript. Australian Rotavirus Surveillance Group. R. Alexander, C. Bletchly, S. Bradbury, H. Cook, J. De Boer, C. Farrar, G. Gilmore, R. Givney, G. Harnett, J. Hennessy, G. Higgins, M. Karimi, A. Kesson, D. Kotsanas, M. Lahra, J. Lang, S. Lambert, A. Lawrence, M. Leung, A. Levy, K. Lindsay, M. Lyon, E. Malinksy, J. McLeod, J. McMahon, C. McIver, J. Merif, C. Moffat, F. Moore, F. Morey, G. Nimmo, M. Nissen, T. Olma, W. Rawlinson, H. Reed, K. Ross, S. Schepetiuk, Tilson, L, V. Sintchenko, D. Smith, P. Southwell, I. Tam, and B. Truscott. Acknowledgments. We thank H. Tran for providing technical support, and all collaborating laboratories for their effort in collecting and providing specimens. Previous members of the Enteric Virus Group who contributed to the Australian Rotavirus Surveillance Program include H. Bugg, D. Cannan, R. Clark, P. Masendycz, and E. Palombo. Financial support. The Australian Rotavirus Surveillance Program is supported by research grants from the vaccine companies Commonwealth Serum Laboratories (CSL) and GlaxoSmithKline (ID116120), as well as the Australian Government Department of Health (RFQ1-2015 ARSP). The Murdoch Children’s Research Institute is supported by the Victorian Government’s Operational Infrastructure Support program. C. M. D. is supported through the Australian National Health and Medical Research Council with an Early Career Fellowship (1113269). Potential conflicts of interest. J. E. B. is Director of the Australian Rotavirus Surveillance Program, World Health Organization (WHO) Collaborating Centre for Child Health, and WHO Asia-Pacific Rotavirus Regional reference laboratory; the Research Lead of the RV3-BB rotavirus vaccine program at MCRI; and Chair of a clinical events committee for a trial conducted by the University of Maryland for which MCRI receives payment to compensate for her time. C. D. K. is currently employed as Senior Program Officer, Enteric and Diarrheal Disease, Bill & Melinda Gates Foundation. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Presented in part: 12th International Rotavirus Symposium, Melbourne, Australia, September 2016. References 1. World Health Organization. Vaccine in national immunization programme update. Available at: www.who.int/immunization/monitoring_surveillance/VaccineIntroStatus.pptx?ua=1. Accessed 9 February 2018. 2. Macartney KK, Porwal M, Dalton Det al.   Decline in rotavirus hospitalisations following introduction of Australia’s national rotavirus immunisation programme. J Paediatr Child Health  2011; 47: 266– 70. Google Scholar CrossRef Search ADS PubMed  3. Richardson V, Parashar U, Patel M. Childhood diarrhea deaths after rotavirus vaccination in Mexico. N Engl J Med  2011; 365: 772– 3. Google Scholar CrossRef Search ADS PubMed  4. Rha B, Tate JE, Payne DCet al.   Effectiveness and impact of rotavirus vaccines in the United States—2006–2012. Expert Rev Vaccines  2014; 13: 365– 76. Google Scholar CrossRef Search ADS PubMed  5. Ruiz-Palacios GM, Pérez-Schael I, Velázquez FRet al.   Human Rotavirus Vaccine Study Group. Safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis. N Engl J Med  2006; 354: 11– 22. Google Scholar CrossRef Search ADS PubMed  6. Vesikari T, Matson DO, Dennehy Pet al.   Rotavirus Efficacy and Safety Trial (REST) Study Team. Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine. N Engl J Med  2006; 354: 23– 33. Google Scholar CrossRef Search ADS PubMed  7. Cowley D, Donato CM, Roczo-Farkas S, Kirkwood CD. Emergence of a novel equine-like G3P[8] inter-genogroup reassortant rotavirus strain associated with gastroenteritis in Australian children. J Gen Virol  2016; 97: 403– 10. Google Scholar CrossRef Search ADS PubMed  8. Donato CM, Cannan D, Bogdanovic-Sakran N, Snelling TL, Kirkwood CD. Characterisation of a G9P[8] rotavirus strain identified during a gastroenteritis outbreak in Alice Springs, Australia post Rotarix vaccine introduction. Vaccine  2012; 30( Suppl 1): A152– 8. Google Scholar CrossRef Search ADS PubMed  9. Clarke E, Desselberger U. Correlates of protection against human rotavirus disease and the factors influencing protection in low-income settings. Mucosal Immunol  2015; 8: 1– 17. Google Scholar CrossRef Search ADS PubMed  10. Kirkwood CD, Boniface K, Barnes GL, Bishop RF. Distribution of rotavirus genotypes after introduction of rotavirus vaccines, Rotarix and RotaTeq, into the National Immunization Program of Australia. Pediatr Infect Dis J  2011; 30: S48– 53. Google Scholar CrossRef Search ADS PubMed  11. Hull BP, Hendry AJ, Dey A, Beard FH, Brotherton JM, McIntyre PB. Annual immunisation coverage annual report, 2015 . Available at: http://www.ncirs.edu.au/assets/surveillance/coverage/Annual-Immunisation-Coverage-Report-2015.pdf. Accessed 19 January 2018. 12. Buttery JP, Lambert SB, Grimwood Ket al.   Reduction in rotavirus-associated acute gastroenteritis following introduction of rotavirus vaccine into Australia’s National Childhood vaccine schedule. Pediatr Infect Dis J  2011; 30: S25– 9. Google Scholar CrossRef Search ADS PubMed  13. Lambert SB, Faux CE, Hall Let al.   Early evidence for direct and indirect effects of the infant rotavirus vaccine program in Queensland. Med J Aust  2009; 191: 157– 60. Google Scholar PubMed  14. Gentsch JR, Laird AR, Bielfelt Bet al.   Serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs. J Infect Dis  2005; 192( Suppl 1): S146– 59. Google Scholar CrossRef Search ADS PubMed  15. 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. Google Scholar CrossRef Search ADS PubMed  16. Romanò L, Paladini S, Galli C, Raimondo G, Pollicino T, Zanetti AR. Hepatitis B vaccination. Hum Vaccin Immunother  2015; 11: 53– 7. Google Scholar CrossRef Search ADS PubMed  17. Kirkwood CD, Boniface K, Bogdanovic-Sakran N, Masendycz P, Barnes GL, Bishop RF. Rotavirus strain surveillance—an Australian perspective of strains causing disease in hospitalised children from 1997 to 2007. Vaccine  2009; 27( Suppl 5): F102– 7. Google Scholar CrossRef Search ADS PubMed  18. Coulson BS, Unicomb LE, Pitson GA, Bishop RF. Simple and specific enzyme immunoassay using monoclonal antibodies for serotyping human rotaviruses. J Clin Microbiol  1987; 25: 509– 15. Google Scholar PubMed  19. Gouvea V, Glass RI, Woods Pet al.   Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol  1990; 28: 276– 82. Google Scholar PubMed  20. Gómara MI, Cubitt D, Desselberger U, Gray J. Amino acid substitution within the VP7 protein of G2 rotavirus strains associated with failure to serotype. J Clin Microbiol  2001; 39: 3796– 8. Google Scholar CrossRef Search ADS PubMed  21. Simmonds MK, Armah G, Asmah Ret al.   New oligonucleotide primers for P-typing of rotavirus strains: strategies for typing previously untypeable strains. J Clin Virol  2008; 42: 368– 73. Google Scholar CrossRef Search ADS PubMed  22. Kirkwood CD, Roczo-Farkas S. Australian Rotavirus Surveillance Program annual report, 2013. Commun Dis Intell Q Rep  2014; 38: E334– 42. Google Scholar PubMed  23. Maes P, Matthijnssens J, Rahman M, Van Ranst M. RotaC: a web-based tool for the complete genome classification of group A rotaviruses. BMC Microbiol  2009; 9: 238. Google Scholar CrossRef Search ADS PubMed  24. Matthijnssens J, Bilcke J, Ciarlet Met al.   Rotavirus disease and vaccination: impact on genotype diversity. Future Microbiol  2009; 4: 1303– 16. Google Scholar CrossRef Search ADS PubMed  25. Matthijnssens J, Joelsson DB, Warakomski DJet al.   Molecular and biological characterization of the 5 human-bovine rotavirus (WC3)-based reassortant strains of the pentavalent rotavirus vaccine, RotaTeq. Virology  2010; 403: 111– 27. Google Scholar CrossRef Search ADS PubMed  26. Jain S, Vashistt J, Changotra H. Rotaviruses: is their surveillance needed? Vaccine  2014; 32: 3367– 78. Google Scholar CrossRef Search ADS PubMed  27. Dóró R, László B, Martella Vet al.   Review of global rotavirus strain prevalence data from six years post vaccine licensure surveillance: is there evidence of strain selection from vaccine pressure? Infect Genet Evol  2014; 28: 446– 61. Google Scholar CrossRef Search ADS PubMed  28. Arana A, Montes M, Jere KC, Alkorta M, Iturriza-Gómara M, Cilla G. Emergence and spread of G3P[8] rotaviruses possessing an equine-like VP7 and a DS-1-like genetic backbone in the Basque Country (North of Spain), 2015. Infect Genet Evol  2016; 44: 137– 44. Google Scholar CrossRef Search ADS PubMed  29. Dóró R, Marton S, Bartókné AHet al.   Equine-like G3 rotavirus in Hungary, 2015: is it a novel intergenogroup reassortant pandemic strain? Acta Microbiol Immunol Hung  2016; 63: 243– 55. Google Scholar CrossRef Search ADS PubMed  30. Luchs A, Timenetsky Mdo C. Group A rotavirus gastroenteritis: post-vaccine era, genotypes and zoonotic transmission. Einstein  2016; 14: 278– 87. Google Scholar CrossRef Search ADS PubMed  31. Malasao R, Saito M, Suzuki Aet al.   Human G3P[4] rotavirus obtained in Japan, 2013, possibly emerged through a human-equine rotavirus reassortment event. Virus Genes  2015; 50: 129– 33. Google Scholar CrossRef Search ADS PubMed  32. Delogu R, Ianiro G, Camilloni B, Fiore L, Ruggeri FM. Unexpected spreading of G12P[8] rotavirus strains among young children in a small area of central Italy. J Med Virol  2015; 87: 1292– 302. Google Scholar CrossRef Search ADS PubMed  33. Wylie KM, Weinstock GM, Storch GA. Emergence of rotavirus G12P[8] in St. Louis during the 2012–2013 rotavirus season. J Pediatric Infect Dis Soc  2015; 4: e84– 9. Google Scholar CrossRef Search ADS PubMed  34. Neves MA, Pinheiro HH, Silva RSet al.   High prevalence of G12P[8] rotavirus strains in Rio Branco, Acre, Western Amazon, in the post-rotavirus vaccine introduction period. J Med Virol  2016; 88: 782– 9. Google Scholar CrossRef Search ADS PubMed  35. Markkula J, Hemming-Harlo M, Salminen MTet al.   Rotavirus epidemiology 5-6 years after universal rotavirus vaccination: persistent rotavirus activity in older children and elderly. Infect Dis  2017; 49: 388– 95. Google Scholar CrossRef Search ADS   36. Zeller M, Patton JT, Heylen Eet 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– 76. Google Scholar CrossRef Search ADS PubMed  37. Matthijnssens J, Heylen E, Zeller M, Rahman M, Lemey P, Van Ranst M. Phylodynamic analyses of rotavirus genotypes G9 and G12 underscore their potential for swift global spread. Mol Biol Evol  2010; 27: 2431– 6. Google Scholar CrossRef Search ADS PubMed  38. Donato CM, Cowley D, Donker NC, Bogdanovic-Sakran N, Snelling TL, Kirkwood CD. Characterization of G2P[4] rotavirus strains causing outbreaks of gastroenteritis in the Northern Territory, Australia, in 1999, 2004 and 2009. Infect Genet Evol  2014; 28: 434– 45. Google Scholar CrossRef Search ADS PubMed  39. Snelling TL, Andrews RM, Kirkwood CD, Culvenor S, Carapetis JR. Case-control evaluation of the effectiveness of the G1P[8] human rotavirus vaccine during an outbreak of rotavirus G2P[4] infection in central Australia. Clin Infect Dis  2011; 52: 191– 9. Google Scholar CrossRef Search ADS PubMed  40. Matthijnssens J, Zeller M, Heylen Eet al.   RotaBel Study Group. Higher proportion of G2P[4] rotaviruses in vaccinated hospitalized cases compared with unvaccinated hospitalized cases, despite high vaccine effectiveness against heterotypic G2P[4] rotaviruses. Clin Microbiol Infect  2014; 20: O702– 10. Google Scholar CrossRef Search ADS PubMed  41. Luchs A, Cilli A, Morillo SG, Carmona Rde C, Timenetsky Mdo C. Rotavirus genotypes circulating in Brazil, 2007–2012: implications for the vaccine program. Rev Inst Med Trop Sao Paulo  2015; 57: 305– 13. Google Scholar CrossRef Search ADS PubMed  42. Pitzer VE, Bilcke J, Heylen Eet al.   Did large-scale vaccination drive changes in the circulating rotavirus population in Belgium? Sci Rep  2015; 5: 18585. Google Scholar CrossRef Search ADS PubMed  43. Donato CM, Zhang ZA, Donker NC, Kirkwood CD. Characterization of G2P[4] rotavirus strains associated with increased detection in Australian states using the RotaTeq(R) vaccine during the 2010–2011 surveillance period. Infect Genet Evol  2014; 28: 398– 412. Google Scholar CrossRef Search ADS PubMed  44. Leshem E, Lopman B, Glass Ret al.   Distribution of rotavirus strains and strain-specific effectiveness of the rotavirus vaccine after its introduction: a systematic review and meta-analysis. Lancet Infect Dis  2014; 14: 847– 56. Google Scholar CrossRef Search ADS PubMed  45. Zeller M, Donato C, Trovão NSet al.   Genome-wide evolutionary analyses of G1P[8] strains isolated before and after rotavirus vaccine introduction. Genome Biol Evol  2015; 7: 2473– 83. Google Scholar CrossRef Search ADS PubMed  46. Zeller M, Heylen E, Tamim Set al.   Comparative analysis of the Rotarix™ vaccine strain and G1P[8] rotaviruses detected before and after vaccine introduction in Belgium. PeerJ  2017; 5: e2733. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

The Impact of Rotavirus Vaccines on Genotype Diversity: A Comprehensive Analysis of 2 Decades of Australian Surveillance Data

Loading next page...
 
/lp/ou_press/the-impact-of-rotavirus-vaccines-on-genotype-diversity-a-comprehensive-dxIHeA8KtM
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
ISSN
0022-1899
eISSN
1537-6613
D.O.I.
10.1093/infdis/jiy197
Publisher site
See Article on Publisher Site

Abstract

Abstract Background Introduction of rotavirus vaccines into national immunization programs (NIPs) could result in strain selection due to vaccine-induced selective pressure. This study describes the distribution and diversity of rotavirus genotypes before and after rotavirus vaccine introduction into the Australian NIP. State-based vaccine selection facilitated a unique comparison of diversity in RotaTeq and Rotarix vaccine states. Methods From 1995 to 2015, the Australian Rotavirus Surveillance Program conducted genotypic analysis on 13051 rotavirus-positive samples from children <5 years of age, hospitalized with acute gastroenteritis. Rotavirus G and P genotypes were determined using serological and heminested multiplex reverse-transcription polymerase chain reaction assays. Results G1P[8] was the dominant genotype nationally in the prevaccine era (1995–2006). Following vaccine introduction (2007–2015), greater genotype diversity was observed with fluctuating genotype dominance. Genotype distribution varied based on the vaccine implemented, with G12P[8] dominant in states using RotaTeq, and equine-like G3P[8] and G2P[4] dominant in states and territories using Rotarix. Conclusions The increased diversity and differences in genotype dominance observed in states using RotaTeq (G12P[8]), and in states and territories using Rotarix (equine-like G3P[8] and G2P[4]), suggest that these vaccines exert different immunological pressures that influence the diversity of rotavirus strains circulating in Australia. rotavirus, genotype, RotaTeq, Rotarix, selective pressure Rotavirus vaccines have been successfully implemented in the routine immunization programs of 93 countries [1]. The globally licensed rotavirus vaccines, RotaTeq (Merck, West Point, Pennsylvania) and Rotarix (GSK Biologics, Rixensart, Belgium), have been associated with a significant reduction in rotavirus hospitalizations and all-cause gastroenteritis deaths in all settings where they have been introduced [2–4]. However, these vaccines have different genome compositions and antigenic characteristics. Rotarix is a single attenuated human G1P[8] rotavirus strain, administered in a 2-dose oral regimen at 2 and 4 months of age [5]. RotaTeq combines 5 separate bovine-human reassortant strains, each containing a human gene encoding the outer capsid VP7 or VP4 protein (G1, G2, G3, G4, or P[8]) within a backbone of a bovine rotavirus strain [6]. RotaTeq is administered in a 3-dose oral regimen at 2, 4, and 6 months of age. The outer capsid proteins, VP7 and VP4, elicit neutralizing antibodies implicated in genotype-specific and genotype cross-reactive protection [7, 8]. Both vaccines have been reported to protect against severe disease caused by the 5 common VP7 genotypes (G1, G2, G3, G4, and G9) and VP4 genotypes (P[4], P[6], and P[8]); however, they also provide broad heterotypic protection against other, less common genotypes [9]. The mechanism and extent to which cross-protection occurs is not well elucidated, nor whether this could impact vaccine escape. Rotavirus vaccination was introduced into the Australian national immunization program (NIP) for all infants on 1 July 2007 [10]. Unlike other countries and regions, Australia allowed each state and territory to independently decide which vaccine to implement in the NIP through a competitive tender process. As a result, Queensland, South Australia, and Victoria selected to use RotaTeq, and the Australian Capital Territory, New South Wales, Northern Territory, and Tasmania selected to use Rotarix (Figure 1). Western Australia initially selected Rotarix, before changing to RotaTeq in 2009. Rotavirus vaccine coverage for 2 or 3 doses (depending on vaccine) in Australia is high, reported at 86.1% by the end of 2015 [11]. Rotavirus vaccines have reduced rotavirus-related hospitalizations in Australia, in both the age group eligible for rotavirus vaccination and nonvaccinated children born prior to vaccine introduction, suggestive of an indirect benefit of rotavirus vaccination [12]. RotaTeq vaccine effectiveness in Queensland is estimated at 89.3% for rotavirus hospitalizations, and in New South Wales, >77% reduction in acute gastroenteritis (AGE) emergency presentations has been observed following Rotarix introduction [2, 13]. The state-based vaccine tender has provided a unique opportunity to observe the impact of RotaTeq and Rotarix introduction on genotype diversity, and to compare genotype distribution between states and territories Australia-wide. Figure 1. View largeDownload slide Pattern of state-based vaccine use within the national immunization program in Australia. Queensland, South Australia, and Victoria implemented RotaTeq (light gray); Australian Capital Territory, New South Wales, Northern Territory, and Tasmania implemented Rotarix (dark gray). Western Australia initially used Rotarix but switched to RotaTeq in 2009. Circles represent locations of collaborating centers contributing samples to the Australian Rotavirus Surveillance Program. Figure 1. View largeDownload slide Pattern of state-based vaccine use within the national immunization program in Australia. Queensland, South Australia, and Victoria implemented RotaTeq (light gray); Australian Capital Territory, New South Wales, Northern Territory, and Tasmania implemented Rotarix (dark gray). Western Australia initially used Rotarix but switched to RotaTeq in 2009. Circles represent locations of collaborating centers contributing samples to the Australian Rotavirus Surveillance Program. Variation in circulating rotavirus strains has been observed in the years prior to the induction of rotavirus vaccines. Molecular epidemiological studies worldwide identified >60 G/P combinations in humans, although 90% of strains belonged to 1 of 5 common genotypes (G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]) [14, 15]. The impact of rotavirus vaccines on the natural pattern of circulating rotavirus strains is not known. Documenting changes in rotavirus genotype prevalence, recognizing emergence of rare or uncommon genotypes, and identifying potential vaccine escape strains will inform future vaccination strategies and the development of new rotavirus vaccines. Mass introduction of other non rotavirus live vaccines, such as hepatitis B vaccine, have previously been shown to affect the diversity of circulating strains within a population, where strain replacement, emergence of novel strains, and vaccine escape strains have been observed [16]. The Australian Rotavirus Surveillance Program has monitored the rotavirus genotypes present in children hospitalized with rotavirus-associated AGE in Australia since 1989 [17]. Nineteen collaborating laboratories across Australia collect rotavirus-positive specimens from children <5 years of age hospitalized with AGE. In the current study, we investigated the temporal diversity and geographic trends of rotavirus genotypes observed in children <5 years of age, hospitalized with AGE in Australia from 1995 to 2015. The specific aims were to (1) compare rotavirus strain diversity before and after vaccine introduction, and (2) determine whether differences in genotype diversity and distribution exist between states, based on the vaccine implemented. This state-by-state analysis provides a unique opportunity to compare and contrast the effect of both RotaTeq and Rotarix on the distribution and diversity of wild-type rotavirus strains. METHODS Study Design This study represents a longitudinal analysis of the rotavirus genotypes detected in faecal samples collected from children <5 years of age, who were hospitalized with AGE in Australia. The study period comprises the 12 years prior to rotavirus vaccine introduction into the NIP in Australia (1995–2006), and the 9 years following vaccine introduction (2007–2015). Rotavirus-positive samples were sent to the National Rotavirus Reference Centre (NRRC) laboratory at the Murdoch Children’s Research Institute (MCRI) by 19 laboratories and hospitals collaborating in the Australian Rotavirus Surveillance Program (Figure 1 and Supplementary Table 1). Collaborating centers analyzed fecal samples for the presence of rotavirus using enzyme immunoassay (EIA) or latex agglutination. Rotavirus-positive specimens were then stored at –20°C to –80°C, and the de-identified samples were forwarded at regular intervals each year to the NRRC laboratory, together with metadata including date of collection, date of birth, sex, and postcode. Upon receipt, samples were allocated a unique laboratory code and entered into the NRRC sample-tracking database (Excel and REDCap). Samples were then stored at –80°C until analyzed. The presence of rotavirus antigen was confirmed using ProSpecT Rotavirus Test, a commercial rotavirus EIA (Thermo Fisher, Australia), as per the manufacturer’s instructions. Samples confirmed as rotavirus positive underwent genotyping analysis, whereas unconfirmed specimens (EIA negative) were not processed further. Rotavirus Genotyping Specimens processed prior to 2007 were serotyped using an in-house monoclonal antibody–based serotyping EIA [18]. This comprised of a panel of monoclonal antibodies specific to the major outer capsid protein (VP7) of group A human rotavirus serotypes G1, G2, G3, G4, and G9 [18]. P-genotyping and reverse-transcription polymerase chain reaction (RT-PCR) were not routinely performed prior to 2007. For samples that could not be assigned a serotype or were processed after 2007, rotavirus G and P genotypes were determined using a heminested multiplex RT-PCR assay [19]. Viral RNA was extracted from 10%–20% (w/v) fecal extracts using the QIAamp Viral RNA mini extraction kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The first-round RT-PCR reactions were performed using the QIAGEN 1-step RT-PCR kit, using VP7 primers VP7F and VP7R, or VP4 primers VP4F and VP4R [20, 21]. The second-round genotyping PCR reactions were conducted using the AmpliTaq DNA Polymerase with Buffer II (Applied Biosystems, Foster City, California), together with specific oligonucleotide primers for G types (G1, G2, G3, G4, G8, G9, and G12) or P types (P[4], P[6], P[8], P[9], P[10], and P[11]), as previously described [22]. The G and P genotype of each sample was assigned using agarose gel analysis of second-round PCR products. The VP7 nucleotide sequence from PCR-nontypeable samples was determined by Sanger sequencing, as the primers used in the current G-typing protocol could not assign a genotype to equine-like G3, G12, and unusual or uncommon rotavirus strains. Suspect vaccine excretion cases from RotaTeq states that could not be P-typed, or G1P[8] strains from infants within the age range of recent vaccination in Rotarix states, were also sequenced. First-round VP7 or VP4 amplicons were purified for sequencing using commercial kits as previously described [22]. Purified DNA together with oligonucleotide primers (VP7F/R or VP4F/R) were sent to the Australian Genome Research Facility (Melbourne) and sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Reaction Kit (Applied Biosystems) in an Applied Biosystems 3730xl DNA Analyzer. Sequences were edited with Sequencher version 4.10.1. Genotype assignment was determined using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and RotaC version 2.0 (http://rotac.regatools.be) [23]. Data Analysis Samples were excluded if patient age was unknown or >5 years, or if a duplicate sample was provided. Samples containing a rotavirus vaccine strain were also excluded (Figure 2). According to the date of collection, samples were divided into prevaccine era (1995–2006) and vaccine era (2007–2015). Vaccine group was assigned according to the state or territory where the sample was collected (RotaTeq group: Queensland, South Australia, Victoria, and Western Australia [before 1 May 2009]; Rotarix group: Australian Capital Territory, New South Wales, Northern Territory, and Western Australia [1 May 2009 or later]). Data are presented as the proportion (percentage) of a specific genotype compared to the total number of strains collected for that era, per vaccine group, and for each state and territory. Figure 2. View largeDownload slide Stool sample flowchart. Abbreviation: WA, Western Australia. Figure 2. View largeDownload slide Stool sample flowchart. Abbreviation: WA, Western Australia. RESULTS During the period 1995–2015, a total of 21545 rotavirus-positive fecal specimens were received from collaborating laboratories in the 8 states and territories across Australia. Of these, 13051 were confirmed as rotavirus positive and included in the final analysis (Figure 2). Reasons for exclusion included insufficient sample (n = 760), missing or not processed (n = 468), duplicate (n = 327), age >5 years (n = 2280), age unknown (n = 1064), negative EIA result when analyzed at MCRI (n = 3439), and identification of vaccine strain (n = 156) (Figure 2). In the prevaccine era (1995–2006), 9301 samples were analyzed; 3318 samples from states that subsequently implemented the RotaTeq vaccine, and 5983 samples from states and territories that subsequently implemented the Rotarix vaccine. Following vaccine introduction (2007–2015), 3750 samples were analyzed; 2179 from children in states that had implemented RotaTeq vaccine and 1571 in states and territories that had implemented Rotarix vaccine. Increased Genotypic Diversity and Unusual Strains Identified Following Vaccine Introduction The diversity of rotavirus genotypes observed was greater in the period following rotavirus vaccine introduction (Figure 3). In the prevaccine era, the common circulating genotypes were G1, G2, G3, G4, and G9, which represented 82.9% (n = 7714 of 9301) of all samples genotyped in that period. G1 strains were the predominant genotype, representing 53.4% (4966/9301) of all samples genotyped, followed by G9 (1349/9301 [14.5%]), G3 (599/9301 [6.4%]), G2 (434/9301 [4.7%]), and G4 (366/9301 [3.9%]). In contrast, following vaccine introduction (2007–2015), these 5 common genotypes were identified in only 63% (2365/3750) of all samples genotyped (Figure 3). The most common genotypes identified in the vaccine era were G1P[8] (983/3750 [26.2%]), G2P[4] (780/3750 [20.8%]), and G12P[8] (683/3750 [18.2%]) (Figure 3). Other genotypes identified following vaccine introduction included a novel equine-like G3P[8] (343/3750 [9.1%]) [7], G3P[8] (312/3750 [8.3%]), G9P[8] (226/3750 [6%]), and G4P[8] (64/3750 [1.7%]) (Figure 3). G12P[8], only identified in a single outbreak involving 10 patients in the prevaccine era, emerged as a dominant genotype from 2012 onward; identified in 683 samples between 2012 and 2015. Previously uncommon VP7 genotypes such as G8, G10, and other animal-reassortant strains (excluding equine-like G3P[8] strains), were also detected more frequently in the vaccine era (n = 63) compared to the prevaccine era (n = 17) (Supplementary Table 2). This increase, together with the emergence of the equine-like G3P[8] strain and G12P[8] strains, suggests that a more diverse population of genotypes was circulating following vaccine introduction. Figure 3. View largeDownload slide Comparison of genotype distribution (% of all rotavirus strains) before and after rotavirus vaccine introduction to the Australian national immunization program. Colors represent specific rotavirus genotypes. Samples with an unusual genotype are listed in Supplementary Table 2. Data are presented as the proportion (%) of a specific genotype compared to the total strains collected. Figure 3. View largeDownload slide Comparison of genotype distribution (% of all rotavirus strains) before and after rotavirus vaccine introduction to the Australian national immunization program. Colors represent specific rotavirus genotypes. Samples with an unusual genotype are listed in Supplementary Table 2. Data are presented as the proportion (%) of a specific genotype compared to the total strains collected. Differences in the Geographical Distribution of Genotypes According to Vaccine Use The pattern of rotavirus genotypes causing hospitalization in children <5 years of age was similar across states and territories in the prevaccine era, with G1P[8] the dominant genotype detected (Figure 4A and 4B). The higher proportion of G9P[8] and G3P[8] strains identified in states and territories that would later introduce Rotarix may have been influenced by outbreaks in the Northern Territory due to G9P[8] in 2001–2003, and G3P[8] in 2003–2006, which spread to other states (Supplementary Figure 1A and 1B) [8]. Figure 4. View largeDownload slide Comparison of rotavirus G and P type (% of all rotavirus strains) based on vaccine used within the state-based national immunization program (NIP). A, Genotype distribution prior to vaccine introduction in Australia categorized by the vaccine to be introduced into the state-based immunization program. B, Genotype distribution remained consistent across all states and territories in the prevaccine era. C, Genotype distribution following vaccine introduction in Australia, categorized according to the vaccine implemented in the state-based NIP. D, Within vaccine groups, genotype distribution remained consistent across all states and territories following vaccine introduction. Samples with an unusual genotype are listed in Supplementary Table 2. Colors represent specific rotavirus genotypes. Abbreviations: ACT, Australian Capital Territory; NSW, New South Wales; NT, Northern Territory; QLD, Queensland; SA, South Australia; Tas, Tasmania; Vic, Victoria; WA, Western Australia. Figure 4. View largeDownload slide Comparison of rotavirus G and P type (% of all rotavirus strains) based on vaccine used within the state-based national immunization program (NIP). A, Genotype distribution prior to vaccine introduction in Australia categorized by the vaccine to be introduced into the state-based immunization program. B, Genotype distribution remained consistent across all states and territories in the prevaccine era. C, Genotype distribution following vaccine introduction in Australia, categorized according to the vaccine implemented in the state-based NIP. D, Within vaccine groups, genotype distribution remained consistent across all states and territories following vaccine introduction. Samples with an unusual genotype are listed in Supplementary Table 2. Colors represent specific rotavirus genotypes. Abbreviations: ACT, Australian Capital Territory; NSW, New South Wales; NT, Northern Territory; QLD, Queensland; SA, South Australia; Tas, Tasmania; Vic, Victoria; WA, Western Australia. In the first 5 years following vaccine introduction, no single genotype was dominant Australia-wide (Supplementary Figure 1). In 2012–2015, G12P[8] emerged as the dominant genotype in all states that had introduced the RotaTeq vaccine (Supplementary Figure 1A), representing 30.5% (665/2179) of all strains genotyped (Figure 4C). In contrast, G12P[8] strains were only identified in 1.1% (18/1571) of all strains in the states and territories that introduced the Rotarix vaccine (Figure 4C and D). G2P[4] became a dominant genotype in states that implemented Rotarix, identified in 26.7% (419/1571) of samples, compared to 16.6% (361/2179) of samples from states using RotaTeq (Figure 4C and 4D). Equine-like G3P[8] strains emerged across 3 consecutive years (2013–2015), and was observed in 83% (117/141) of samples collected in Rotarix states and territories in 2013, 23.1% (24/104) in 2014, and 49.7% (74/149) in 2015 (Supplementary Figure 1B). Equine-like G3P[8] and G9P[8] strains were identified at a higher frequency in states and territories using Rotarix (equine-like G3P[8], 13.7% [215/1571]; G9P[8], 11.8% [185/1571]) compared to states using RotaTeq (equine-like G3P[8], 5.9% [128/2179]; G9P[8], 1.9% [41/2179]). When analyzed by state, these patterns of dominance remained consistent within vaccine groups (Figure 4D). In this study, Western Australia was analyzed separately as the vaccine in the NIP changed in May 2009 from Rotarix to RotaTeq (Supplementary Figure 1C). For the period up to 2009, the circulating genotype patterns were similar to other states using Rotarix (Supplementary Figure 1B and 1C). However, from 2010 onward, the genotype distribution changed to reflect patterns observed within RotaTeq states (Supplementary Figure 1A and 1C). One exception was the large proportion of equine-like G3P[8] samples identified in 2013, which was similar to states and territories using Rotarix. DISCUSSION This study demonstrates that the introduction of rotavirus vaccines into the NIP in Australia altered the diversity and distribution of circulating rotavirus genotypes in children aged <5 years who were hospitalized with rotavirus gastroenteritis. Historically, rotavirus genotype distribution has shown temporal and geographic movement across Australia, where strains spread across the country over a period of 1–2 years (eg, genotype G9P[8]) [17]. This geographic and temporal pattern has been disrupted in the vaccine era, with adjacent states using different vaccines exhibiting varied patterns of genotype diversity maintained over many years. This analysis highlighted the importance of sustaining rotavirus surveillance programs, as critical differences in genotype distribution between vaccine locations were not obvious within the first 5 years of rotavirus vaccine introduction. Rotavirus diversity is generated by genetic drift, genetic shift, and zoonotic transmission [24]. Vaccine-induced immune pressure may be an additional selective pressure that leads to the evolution of rotavirus strains capable of causing severe disease in vaccinated children. In this study, sustained dominance of G12P[8] was observed in states using RotaTeq, while dominance of G2P[4] and equine-like G3P[8] was observed in states and territories using Rotarix. This is highly suggestive of vaccine-specific selection pressure, as these observations occurred within a country with high vaccine coverage and demonstrated vaccine effectiveness. The differences observed in adjacent states and territories using either RotaTeq or Rotarix cannot be explained by geographical or demographic differences (Figure 1). However, these differences could be due to vaccine composition and antigenic characteristics [25]. Rotarix contains a monovalent human G1P[8] strain, and has been shown to elicit heterotypic protection against non-G1P[8] strains, whereas the pentavalent human-bovine reassortant vaccine, RotaTeq, generates homotypic protection against each of the G1, G2, G3, G4, and P[8] strains contained in the vaccine and has been shown to elicit heterotypic protection. The ability of each vaccine to generate broader heterotypic protection is not well defined [9]. This study reports an increased detection of previously rare or novel genotypes emerging in the vaccine era, supporting the hypothesis that widespread vaccine use may increase strain diversity. However, emerging genotypes have also been detected in countries with low coverage or no vaccination programs [26, 27]. For example, the novel equine-like G3P[8] strain has been detected in countries with a range of vaccine coverage [7, 26, 28–30]. The first report of this strain was from Japan, where rotavirus vaccine coverage was estimated to be 40%–45% [31]. Since then, this strain has been detected in countries with high vaccine coverage (Australia, Brazil, and the United States), moderate vaccine coverage (Germany, Taiwan, and Spain), and no vaccine coverage within the NIP (Hungary, Thailand, and Turkey) [29]. Multiple amino acid differences have been observed in the antigenic regions of the VP7 gene between human wild-type G3P[8] strains and the equine-like VP7 gene in Australia, suggesting that this equine-like strain is antigenically distinct from human strains [7]. The global emergence of this strain, irrespective of vaccine coverage, suggests that vaccines may not be highly effective against this strain. Global movement of rotavirus strains occurs, reflected by the increasing incidence of G12P[8] in countries with and without vaccine coverage [32–35]. In this study, G12P[8] strains were primarily found in states that use RotaTeq. This is difficult to attribute to natural fluctuations or demographic differences. Similar observations were reported in Finland, where reported vaccine (RotaTeq) coverage is >90% [35]. In Finland, greater genotype diversity was observed in the vaccine era, over 2 rotavirus seasons [35]. Of particular interest is that G12P[8] strains emerged during this period and were detected more often in vaccinated children (13.8%) compared with unvaccinated children (6.5%) [35]. The question remains why G12P[8] and equine-like G3P[8] are responsible for a substantial burden of disease in populations with high vaccine coverage such as Australia and Finland [35]. It has been proposed that the VP7 and VP4 antigenic regions of the equine-like G3P[8] and G12P[8] strains could be distinct to that of the Rotarix and RotaTeq vaccines [7, 36, 37], resulting in lower vaccine effectiveness against these strains. G2P[4] strains have increased in circulation in Australia in the vaccine era, particularly in Rotarix states and territories. Despite Rotarix vaccine introduction in the Northern Territory, Australia, an outbreak associated with G2P[4] strains occurred in 2009 [38]. The average age of patients was of vaccine age (5.5 months); however, there was only approximately 50% vaccine coverage, and vaccine effectiveness against rotavirus hospitalizations was only 19% [38, 39]. A waning heterotypic response afforded by Rotarix, combined with low circulating numbers of G2P[4] in the preceding years, could have contributed to a decrease in herd immunity to this genotype [38]. Furthermore, prior exposure to G2P[4] strains may not have generated effective homotypic protection against the outbreak strain, as it exhibited changes in the VP7 gene antigenic region, compared to other contemporary G2P[4] strains [38]. Excluding this outbreak, G2P[4] strains are still a prominent cause of disease in Rotarix states, possibly reflecting limited or short-lasting heterotypic immunity. An increased proportion of G2P[4] strains have also been reported in Brazil and Belgium after introduction of the Rotarix vaccine [40, 41]. Prelicensure efficacy studies could not consistently demonstrate comparable levels of enduring protection for heterotypic strains such as G2P[4] [9]. Despite reporting vaccine effectiveness of 85% against G2P[4], a study from Belgium described higher incidence rates of G2P[4] in vaccinated hospitalized cases compared with unvaccinated hospitalized cases [40]. It has been hypothesized that a lower efficacy against heterotypic strains such as G2P[4] could drive an alteration in circulating genotypes [38, 40, 42]. In this study, G2P[4] strains account for 16.6% of strains genotyped in states utilizing the RotaTeq vaccine. Comparison of the RotaTeq component strain and G2P[4] strains circulating in RotaTeq states revealed substitutions in antigenic regions that have also been reported in the majority of G2P[4] strains circulating globally over the last decade [43]. The G2 RotaTeq component strain was isolated in 1992; therefore, if contemporary strains continue to accumulate mutations in antigenic regions, the vaccine strains may need to be updated. After vaccine introduction, the dominance of G1P[8] strains has significantly reduced globally [44]. Several studies have revealed that contemporary strains exhibit numerous mutations in antigenic regions compared to the original vaccine strains [25, 36, 45, 46]. A multicountry phylogenetic analysis revealed unique subclusters of strains identified after Rotarix introduction in Belgium and RotaTeq introduction in Victoria, Australia [45, 46]. Furthermore, the increase in G1P[8] detected in 2010–2012 in our study was attributed to the emergence of a G1P[8] outbreak strain that appeared to evade protection from Rotarix in Alice Springs, Northern Territory [21]. These findings, together with the ongoing presence of G1P[8] in a global context, suggest that rotavirus vaccines may need to be reviewed to meet the evolutionary changes that may challenge the success of vaccination programs. This longitudinal, nationwide analysis of rotavirus genotypes was conducted over a 21-year period prior to and following rotavirus vaccine introduction, reporting increases in genotype diversity and detection of unusual rotavirus strains. Differences in genotype dominance and diversity were apparent between states using the RotaTeq vaccine, compared with states and territories using the Rotarix vaccine. This suggests that vaccine-related immune selection pressure occurs and this phenomenon differs between vaccines, supporting our contention that vaccine pressure drives strain selection. Rotavirus vaccines have been shown to be highly efficacious in reducing severe disease; however, rotavirus strains continue to evolve and novel strains continue to emerge. Ongoing surveillance and the use of phylogenetic analysis will be important to provide further insight on the impact of rotavirus vaccines on strain diversity. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Author contributions. S. R.-F. coordinated the Australian Rotavirus Surveillance Program from 2010 to 2018; during this period, S. R.-F. collated samples and conducted experiments. S. R.-F. performed the literature search, data collection, analysis, and interpretation; contributed to the study design; and wrote the first draft of the manuscript. N. B.-S., C. M. D., and K. B. performed data collection, data analysis, and data interpretation, and contributed to the study design. C. D. K., G. L. B., R. F. B., D. C., and J. E. B. designed and directed the study. These authors provided conceptual and technical guidance for all aspects of the project including data interpretation and revision of the manuscript. Australian Rotavirus Surveillance Group. R. Alexander, C. Bletchly, S. Bradbury, H. Cook, J. De Boer, C. Farrar, G. Gilmore, R. Givney, G. Harnett, J. Hennessy, G. Higgins, M. Karimi, A. Kesson, D. Kotsanas, M. Lahra, J. Lang, S. Lambert, A. Lawrence, M. Leung, A. Levy, K. Lindsay, M. Lyon, E. Malinksy, J. McLeod, J. McMahon, C. McIver, J. Merif, C. Moffat, F. Moore, F. Morey, G. Nimmo, M. Nissen, T. Olma, W. Rawlinson, H. Reed, K. Ross, S. Schepetiuk, Tilson, L, V. Sintchenko, D. Smith, P. Southwell, I. Tam, and B. Truscott. Acknowledgments. We thank H. Tran for providing technical support, and all collaborating laboratories for their effort in collecting and providing specimens. Previous members of the Enteric Virus Group who contributed to the Australian Rotavirus Surveillance Program include H. Bugg, D. Cannan, R. Clark, P. Masendycz, and E. Palombo. Financial support. The Australian Rotavirus Surveillance Program is supported by research grants from the vaccine companies Commonwealth Serum Laboratories (CSL) and GlaxoSmithKline (ID116120), as well as the Australian Government Department of Health (RFQ1-2015 ARSP). The Murdoch Children’s Research Institute is supported by the Victorian Government’s Operational Infrastructure Support program. C. M. D. is supported through the Australian National Health and Medical Research Council with an Early Career Fellowship (1113269). Potential conflicts of interest. J. E. B. is Director of the Australian Rotavirus Surveillance Program, World Health Organization (WHO) Collaborating Centre for Child Health, and WHO Asia-Pacific Rotavirus Regional reference laboratory; the Research Lead of the RV3-BB rotavirus vaccine program at MCRI; and Chair of a clinical events committee for a trial conducted by the University of Maryland for which MCRI receives payment to compensate for her time. C. D. K. is currently employed as Senior Program Officer, Enteric and Diarrheal Disease, Bill & Melinda Gates Foundation. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Presented in part: 12th International Rotavirus Symposium, Melbourne, Australia, September 2016. References 1. World Health Organization. Vaccine in national immunization programme update. Available at: www.who.int/immunization/monitoring_surveillance/VaccineIntroStatus.pptx?ua=1. Accessed 9 February 2018. 2. Macartney KK, Porwal M, Dalton Det al.   Decline in rotavirus hospitalisations following introduction of Australia’s national rotavirus immunisation programme. J Paediatr Child Health  2011; 47: 266– 70. Google Scholar CrossRef Search ADS PubMed  3. Richardson V, Parashar U, Patel M. Childhood diarrhea deaths after rotavirus vaccination in Mexico. N Engl J Med  2011; 365: 772– 3. Google Scholar CrossRef Search ADS PubMed  4. Rha B, Tate JE, Payne DCet al.   Effectiveness and impact of rotavirus vaccines in the United States—2006–2012. Expert Rev Vaccines  2014; 13: 365– 76. Google Scholar CrossRef Search ADS PubMed  5. Ruiz-Palacios GM, Pérez-Schael I, Velázquez FRet al.   Human Rotavirus Vaccine Study Group. Safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis. N Engl J Med  2006; 354: 11– 22. Google Scholar CrossRef Search ADS PubMed  6. Vesikari T, Matson DO, Dennehy Pet al.   Rotavirus Efficacy and Safety Trial (REST) Study Team. Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine. N Engl J Med  2006; 354: 23– 33. Google Scholar CrossRef Search ADS PubMed  7. Cowley D, Donato CM, Roczo-Farkas S, Kirkwood CD. Emergence of a novel equine-like G3P[8] inter-genogroup reassortant rotavirus strain associated with gastroenteritis in Australian children. J Gen Virol  2016; 97: 403– 10. Google Scholar CrossRef Search ADS PubMed  8. Donato CM, Cannan D, Bogdanovic-Sakran N, Snelling TL, Kirkwood CD. Characterisation of a G9P[8] rotavirus strain identified during a gastroenteritis outbreak in Alice Springs, Australia post Rotarix vaccine introduction. Vaccine  2012; 30( Suppl 1): A152– 8. Google Scholar CrossRef Search ADS PubMed  9. Clarke E, Desselberger U. Correlates of protection against human rotavirus disease and the factors influencing protection in low-income settings. Mucosal Immunol  2015; 8: 1– 17. Google Scholar CrossRef Search ADS PubMed  10. Kirkwood CD, Boniface K, Barnes GL, Bishop RF. Distribution of rotavirus genotypes after introduction of rotavirus vaccines, Rotarix and RotaTeq, into the National Immunization Program of Australia. Pediatr Infect Dis J  2011; 30: S48– 53. Google Scholar CrossRef Search ADS PubMed  11. Hull BP, Hendry AJ, Dey A, Beard FH, Brotherton JM, McIntyre PB. Annual immunisation coverage annual report, 2015 . Available at: http://www.ncirs.edu.au/assets/surveillance/coverage/Annual-Immunisation-Coverage-Report-2015.pdf. Accessed 19 January 2018. 12. Buttery JP, Lambert SB, Grimwood Ket al.   Reduction in rotavirus-associated acute gastroenteritis following introduction of rotavirus vaccine into Australia’s National Childhood vaccine schedule. Pediatr Infect Dis J  2011; 30: S25– 9. Google Scholar CrossRef Search ADS PubMed  13. Lambert SB, Faux CE, Hall Let al.   Early evidence for direct and indirect effects of the infant rotavirus vaccine program in Queensland. Med J Aust  2009; 191: 157– 60. Google Scholar PubMed  14. Gentsch JR, Laird AR, Bielfelt Bet al.   Serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs. J Infect Dis  2005; 192( Suppl 1): S146– 59. Google Scholar CrossRef Search ADS PubMed  15. 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. Google Scholar CrossRef Search ADS PubMed  16. Romanò L, Paladini S, Galli C, Raimondo G, Pollicino T, Zanetti AR. Hepatitis B vaccination. Hum Vaccin Immunother  2015; 11: 53– 7. Google Scholar CrossRef Search ADS PubMed  17. Kirkwood CD, Boniface K, Bogdanovic-Sakran N, Masendycz P, Barnes GL, Bishop RF. Rotavirus strain surveillance—an Australian perspective of strains causing disease in hospitalised children from 1997 to 2007. Vaccine  2009; 27( Suppl 5): F102– 7. Google Scholar CrossRef Search ADS PubMed  18. Coulson BS, Unicomb LE, Pitson GA, Bishop RF. Simple and specific enzyme immunoassay using monoclonal antibodies for serotyping human rotaviruses. J Clin Microbiol  1987; 25: 509– 15. Google Scholar PubMed  19. Gouvea V, Glass RI, Woods Pet al.   Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol  1990; 28: 276– 82. Google Scholar PubMed  20. Gómara MI, Cubitt D, Desselberger U, Gray J. Amino acid substitution within the VP7 protein of G2 rotavirus strains associated with failure to serotype. J Clin Microbiol  2001; 39: 3796– 8. Google Scholar CrossRef Search ADS PubMed  21. Simmonds MK, Armah G, Asmah Ret al.   New oligonucleotide primers for P-typing of rotavirus strains: strategies for typing previously untypeable strains. J Clin Virol  2008; 42: 368– 73. Google Scholar CrossRef Search ADS PubMed  22. Kirkwood CD, Roczo-Farkas S. Australian Rotavirus Surveillance Program annual report, 2013. Commun Dis Intell Q Rep  2014; 38: E334– 42. Google Scholar PubMed  23. Maes P, Matthijnssens J, Rahman M, Van Ranst M. RotaC: a web-based tool for the complete genome classification of group A rotaviruses. BMC Microbiol  2009; 9: 238. Google Scholar CrossRef Search ADS PubMed  24. Matthijnssens J, Bilcke J, Ciarlet Met al.   Rotavirus disease and vaccination: impact on genotype diversity. Future Microbiol  2009; 4: 1303– 16. Google Scholar CrossRef Search ADS PubMed  25. Matthijnssens J, Joelsson DB, Warakomski DJet al.   Molecular and biological characterization of the 5 human-bovine rotavirus (WC3)-based reassortant strains of the pentavalent rotavirus vaccine, RotaTeq. Virology  2010; 403: 111– 27. Google Scholar CrossRef Search ADS PubMed  26. Jain S, Vashistt J, Changotra H. Rotaviruses: is their surveillance needed? Vaccine  2014; 32: 3367– 78. Google Scholar CrossRef Search ADS PubMed  27. Dóró R, László B, Martella Vet al.   Review of global rotavirus strain prevalence data from six years post vaccine licensure surveillance: is there evidence of strain selection from vaccine pressure? Infect Genet Evol  2014; 28: 446– 61. Google Scholar CrossRef Search ADS PubMed  28. Arana A, Montes M, Jere KC, Alkorta M, Iturriza-Gómara M, Cilla G. Emergence and spread of G3P[8] rotaviruses possessing an equine-like VP7 and a DS-1-like genetic backbone in the Basque Country (North of Spain), 2015. Infect Genet Evol  2016; 44: 137– 44. Google Scholar CrossRef Search ADS PubMed  29. Dóró R, Marton S, Bartókné AHet al.   Equine-like G3 rotavirus in Hungary, 2015: is it a novel intergenogroup reassortant pandemic strain? Acta Microbiol Immunol Hung  2016; 63: 243– 55. Google Scholar CrossRef Search ADS PubMed  30. Luchs A, Timenetsky Mdo C. Group A rotavirus gastroenteritis: post-vaccine era, genotypes and zoonotic transmission. Einstein  2016; 14: 278– 87. Google Scholar CrossRef Search ADS PubMed  31. Malasao R, Saito M, Suzuki Aet al.   Human G3P[4] rotavirus obtained in Japan, 2013, possibly emerged through a human-equine rotavirus reassortment event. Virus Genes  2015; 50: 129– 33. Google Scholar CrossRef Search ADS PubMed  32. Delogu R, Ianiro G, Camilloni B, Fiore L, Ruggeri FM. Unexpected spreading of G12P[8] rotavirus strains among young children in a small area of central Italy. J Med Virol  2015; 87: 1292– 302. Google Scholar CrossRef Search ADS PubMed  33. Wylie KM, Weinstock GM, Storch GA. Emergence of rotavirus G12P[8] in St. Louis during the 2012–2013 rotavirus season. J Pediatric Infect Dis Soc  2015; 4: e84– 9. Google Scholar CrossRef Search ADS PubMed  34. Neves MA, Pinheiro HH, Silva RSet al.   High prevalence of G12P[8] rotavirus strains in Rio Branco, Acre, Western Amazon, in the post-rotavirus vaccine introduction period. J Med Virol  2016; 88: 782– 9. Google Scholar CrossRef Search ADS PubMed  35. Markkula J, Hemming-Harlo M, Salminen MTet al.   Rotavirus epidemiology 5-6 years after universal rotavirus vaccination: persistent rotavirus activity in older children and elderly. Infect Dis  2017; 49: 388– 95. Google Scholar CrossRef Search ADS   36. Zeller M, Patton JT, Heylen Eet 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– 76. Google Scholar CrossRef Search ADS PubMed  37. Matthijnssens J, Heylen E, Zeller M, Rahman M, Lemey P, Van Ranst M. Phylodynamic analyses of rotavirus genotypes G9 and G12 underscore their potential for swift global spread. Mol Biol Evol  2010; 27: 2431– 6. Google Scholar CrossRef Search ADS PubMed  38. Donato CM, Cowley D, Donker NC, Bogdanovic-Sakran N, Snelling TL, Kirkwood CD. Characterization of G2P[4] rotavirus strains causing outbreaks of gastroenteritis in the Northern Territory, Australia, in 1999, 2004 and 2009. Infect Genet Evol  2014; 28: 434– 45. Google Scholar CrossRef Search ADS PubMed  39. Snelling TL, Andrews RM, Kirkwood CD, Culvenor S, Carapetis JR. Case-control evaluation of the effectiveness of the G1P[8] human rotavirus vaccine during an outbreak of rotavirus G2P[4] infection in central Australia. Clin Infect Dis  2011; 52: 191– 9. Google Scholar CrossRef Search ADS PubMed  40. Matthijnssens J, Zeller M, Heylen Eet al.   RotaBel Study Group. Higher proportion of G2P[4] rotaviruses in vaccinated hospitalized cases compared with unvaccinated hospitalized cases, despite high vaccine effectiveness against heterotypic G2P[4] rotaviruses. Clin Microbiol Infect  2014; 20: O702– 10. Google Scholar CrossRef Search ADS PubMed  41. Luchs A, Cilli A, Morillo SG, Carmona Rde C, Timenetsky Mdo C. Rotavirus genotypes circulating in Brazil, 2007–2012: implications for the vaccine program. Rev Inst Med Trop Sao Paulo  2015; 57: 305– 13. Google Scholar CrossRef Search ADS PubMed  42. Pitzer VE, Bilcke J, Heylen Eet al.   Did large-scale vaccination drive changes in the circulating rotavirus population in Belgium? Sci Rep  2015; 5: 18585. Google Scholar CrossRef Search ADS PubMed  43. Donato CM, Zhang ZA, Donker NC, Kirkwood CD. Characterization of G2P[4] rotavirus strains associated with increased detection in Australian states using the RotaTeq(R) vaccine during the 2010–2011 surveillance period. Infect Genet Evol  2014; 28: 398– 412. Google Scholar CrossRef Search ADS PubMed  44. Leshem E, Lopman B, Glass Ret al.   Distribution of rotavirus strains and strain-specific effectiveness of the rotavirus vaccine after its introduction: a systematic review and meta-analysis. Lancet Infect Dis  2014; 14: 847– 56. Google Scholar CrossRef Search ADS PubMed  45. Zeller M, Donato C, Trovão NSet al.   Genome-wide evolutionary analyses of G1P[8] strains isolated before and after rotavirus vaccine introduction. Genome Biol Evol  2015; 7: 2473– 83. Google Scholar CrossRef Search ADS PubMed  46. Zeller M, Heylen E, Tamim Set al.   Comparative analysis of the Rotarix™ vaccine strain and G1P[8] rotaviruses detected before and after vaccine introduction in Belgium. PeerJ  2017; 5: e2733. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

The Journal of Infectious DiseasesOxford University Press

Published: May 22, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off