Genome-Wide Evolutionary Analyses of G1P[8] Strains Isolated Before and After Rotavirus Vaccine Introduction.

Rotaviruses are the most important etiological agent of acute gastroenteritis in young children worldwide. Among the first countries to introduce rotavirus vaccines into their national immunization programs were Belgium (November 2006) and Australia (July 2007). Surveillance programs in Belgium (since 1999) and Australia (since 1989) offer the opportunity to perform a detailed comparison of rotavirus strains circulating pre- and postvaccine introduction. G1P[8] rotaviruses are the most prominent genotype in humans, and a total of 157 G1P[8] rotaviruses isolated between 1999 and 2011 were selected from Belgium and Australia and their complete genomes were sequenced. Phylogenetic analysis showed evidence of frequent reassortment among Belgian and Australian G1P[8] rotaviruses. Although many different phylogenetic subclusters were present before and after vaccine introduction, some unique clusters were only identified after vaccine introduction, which could be due to natural fluctuation or the first signs of vaccine-driven evolution. The times to the most recent common ancestors for the Belgian and Australian G1P[8] rotaviruses ranged from 1846 to 1955 depending on the gene segment, with VP7 and NSP4 resulting in the most recent estimates. We found no evidence that rotavirus population size was affected after vaccine introduction and only six amino acid sites in VP2, VP3, VP7, and NSP1 were identified to be under positive selective pressure. Continued surveillance of G1P[8] strains is needed to determine long-term effects of vaccine introductions, particularly now rotavirus vaccines are implemented in the national immunization programs of an increasing number of countries worldwide.

Characterization of G2P[4] rotavirus strains causing outbreaks of gastroenteritis in the Northern Territory, Australia, in 1999, 2004 and 2009.

Outbreaks of rotavirus diarrhea cause a large disease burden in the Alice Springs region of the Northern Territory, Australia. The introduction of the rotavirus vaccine Rotarix® has been associated with an increase in detection of G2P[4] strains in many countries. However, G2P[4] emergence has also been observed in vaccine-naive countries, suggesting a general global increase in the circulation of G2P[4] strains. A G2P[4] rotavirus outbreak occurred in 2009, 28 months after the introduction of the Rotarix® vaccine and 43 children were hospitalized. Pre-vaccine introduction, G2P[4] strains were observed associated with large outbreaks in 1999 and 2004. To determine the genetic relationship between these strains whole genome sequence analysis was conducted on representative strains from each of the G2P[4] outbreaks, in 1999, 2004 and 2009. Phylogenetic analysis revealed the majority of genes from 2009 outbreak strain clustered with contemporary global strains, while the VP7 gene clustered with contemporary and older strains and was antigenically distinct to the majority of contemporary global G2P[4] strains; suggesting the strain was an intragenogroup reassortant. The 1999 and 2009 strains appear to share similar evolutionary origins, and both had a high degree of genetic identity to previously identified Australian and global strains. Conversely, the 2004 outbreak strain was more divergent in comparison to Australian and global strains. The 1999 and 2004 outbreaks likely occurred due to the accumulation of immunologically naïve children in the population following low levels of G2P[4] rotavirus disease in the community in the years prior to each outbreak. The 2009 outbreak was associated with moderate vaccine coverage in the population and vaccine efficacy against the strain was low. The circulation of this unusual strain in the population combined with low vaccine coverage and diminished vaccine efficacy likely contributed to the outbreak occurring in this population.

Characterization of G2P[4] rotavirus strains associated with increased detection in Australian states using the RotaTeq® vaccine during the 2010-2011 surveillance period.

The introduction of rotavirus vaccines Rotarix® and RotaTeq® into the Australian National Immunisation Program in July 2007 has resulted in a dramatic decrease in the burden of rotavirus disease. G2P[4] strains became the dominant genotype Australia-wide during the 2010-2011 surveillance period and for the first time since vaccine introduction, a higher proportion were isolated in jurisdictions using RotaTeq® vaccine compared to locations using Rotarix®. Phylogenetic analysis of the VP7 gene of 32 G2P[4] strains identified six genetic clusters, these distinct clusters were also observed in the VP4 gene for a subset of 12 strains. The whole genome was determined for a representative strain of clusters; A (RVA/Human-wt/AUS/SA066/2010/G2P[4]), B (RVA/Human-wt/AUS/WAPC703/2010/G2P[4]), C (RVA/Human-wt/AUS/MON008/2010/G2P[4]) and E (RVA/Human-wt/AUS/RCH041/2010/G2P[4]). All of the strains possessed the archetypal DS-1 like genome constellation G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Three of the strains, SA066, MON008 and WAPC703 clustered together and were distinct to RCH041 for all 11 genes. The VP7 genes of 31/32 of the strains characterized in this study possessed five conserved amino acid substitutions when compared to the G2 VP7 gene present in the RotaTeq® vaccine. Three of the substitutions were in the VP7 antigenic regions A and C, the substitutions A87T, D96N and S213D have been reported in the majority of G2P[4] strains circulating globally over the previous decade. These changes may have improved the ability of strains to circulate in settings of high vaccine use.

Identification and characterization of antibody-binding epitopes on the norovirus GII.3 capsid.

Genotype II.3 (GII.3) noroviruses are a major cause of sporadic gastroenteritis, particularly in children. The greater incidence of GII.3 noroviruses in the pediatric population compared to the adult demographic suggests development of herd immunity to this genotype, possibly as a consequence of limited evolution of immune epitopes. This study aimed to identify and characterize immune epitopes on the GII.3 capsid protein and to determine the level of immune cross-reactivity within the genotype. A panel of seven GII.3 virus-like particles (VLPs), representing norovirus strains isolated during 1975 to 2008, was tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with human sera and a rabbit anti-GII.3 strain-specific polyclonal serum generated against the 2008 GII.3 VLP. Immunoprecipitation of protease-digested GII.3 VLPs and sequencing of bound peptides via mass spectrometry were used to locate epitopes on the capsid. Two epitopes were investigated further using Mimotopes technology. Serum binding studies demonstrated complete intragenotype GII.3 cross-reactivity using both human and rabbit serum. Six immunoreactive regions containing epitopes were located on the GII.3 capsid protein, two within each capsid domain. Epitopes in the S and P1 domains were highly conserved within GII.3 noroviruses. P2 domain epitopes were variable and contained evolutionarily important residues and histo-blood group antigen (HBGA) binding residues. In conclusion, anti-GII.3 antibody-binding epitopes are highly cross-reactive and mostly conserved within GII.3 strains. This may account for the limited GII.3 prevalence in adults and suggests that a GII.3 strain may be a valuable inclusion in a multivalent pediatric targeted VLP vaccine. Exploration of norovirus immune epitopes is vital for effective vaccine design. IMPORTANCE This study represents an important contribution to the understanding of norovirus immunology in a pediatric genotype. The high cross-reactivity and conservation of GII.3 epitopes suggest development of herd immunity against GII.3 and indicate that a GII.3 strain would be a valuable inclusion in a pediatric targeted multivalent vaccine. Immunological understanding of pediatric norovirus strains is important since norovirus vaccines will likely target high-risk groups such as the pediatric population.

Selection and evolutionary analysis in the nonstructural protein NSP2 of rotavirus A.

Rotavirus A is the leading cause of acute gastroenteritis in infants and young children worldwide. The nonstructural protein 2 (NSP2) plays essential roles in the replication cycle of rotavirus and may play a role in protective immunity against rotavirus disease. Using a Bayesian approach, we measured the mutation rate of genotype N1 NSP2 gene sequences. The N1 genotype is the main NSP2 genotype associated with rotavirus strains causing severe disease, and was found to have a high mutation rate (8.7 × 10(-4) substitutions/site/year) in comparison to the rotavirus VP4 gene and rates of mutation in other RNA viruses. NSP2 has traditionally been considered as a conserved rotavirus protein and selection analysis indicated that the NSP2 protein was under strong negative selection, suggesting that most nucleotide substitutions were synonymous. This conservation is likely a result of functional constraints of NSP2 in the rotavirus replication cycle. Four sites of positive selection were identified; two of these (positions 249 and 255) were located in a previously characterised antibody binding epitope. The remaining sites were not located in known functional regions, and the reason for variation at these sites remains to be elucidated.

Phylogenetic analysis of rotavirus A NSP2 gene sequences and evidence of intragenic recombination.

The rotavirus non-structural protein NSP2 is one of the earliest and most abundant viral proteins produced during infection. This protein has multiple essential roles in the replication cycle involving RNA binding, viroplasm formation, helicase and can hydrolyse the γ-phosphate of RNA and NTPs acting as an RTPase and NTPase. In studying sequences from rotavirus strains isolated in Australia between 1984 and 2009, the NSP2 gene was seen to be highly conserved and clustered with defined NSP2 genotypes N1 and N2 according to the full genome based rotavirus classification system. Phylogenetic analysis indicated that NSP2 gene sequences isolated from Australian rotavirus strains formed four distinct lineages. Temporal variation was observed in several clusters during the 26 year period, with lineage D identified throughout the entire study period and lineage A only detected since 1999. Phylogenetic analysis and dendrograms identified NSP2 genes that exhibited reassortment between different virus VP7 genotypes, as well as a sequence from a human strain that grouped closely with the NSP2 genes of bovine rotavirus strains. This study also identified a sequence that fell between lineages and exhibited evidence of recombination, the first time that intergenic recombination has been detected in a NSP2 gene sequence. This study increases the understanding of the evolution mechanisms of NSP2 in view of improved vaccine design.

Identification of an antibody-binding epitope on the rotavirus A non-structural protein NSP2 using phage display analysis.

The non-structural protein 2 (NSP2) of rotavirus has important roles in rotavirus replication associated with RNA binding, hydrolysis of NTPs and RNA, and helix destabilizing properties. A cell-culture assay using an NSP2-specific mAb and polyclonal antiserum to block virus replication showed a 73 and 96 % reduction in the amount of virus produced during replication, respectively. Phage display technology was used to identify the antibody-binding region on the NSP2 protein with the motif (244)T-(Y/F)-Ø-Ø-Ø-X-K-Ø-G(252), where Ø is a hydrophilic residue and X is any amino acid. This region was mapped to the three-dimensional NSP2 crystal structure to visualize the epitope. Analysis revealed identity to a region on NSP2 that mapped to a site exposed on the surface of the protein, which could possibly interfere with a functionally important region of the protein. Antibody binding to this region could disrupt the essential roles of NSP2, such as the formation of viroplasms with NSP5 or the interaction with viral RNA, thereby indicating a possible mechanism for the observed inhibition of virus replication. Genetic analysis of the putative binding region of NSP2 revealed a high level of conservation, suggesting that the region is under strict control.