Evolutionary Dynamics of Human Rotaviruses: Balancing Reassortment with Preferred Genome Constellations

Group A human rotaviruses (RVs) are a major cause of severe gastroenteritis in infants and young children. Yet, aside from the genes encoding serotype antigens (VP7; G-type and VP4; P-type), little is known about the genetic make-up of emerging and endemic human RV strains. To gain insight into the diversity and evolution of RVs circulating at a single location over a period of time, we sequenced the eleven-segmented, double-stranded RNA genomes of fifty-one G3P[8] strains collected from 1974 to 1991 at Children''s Hospital National Medical Center, Washington, D. C. During this period, G1P[8] strains typically dominated, comprising on average 56% of RV infections each year in hospitalized children. A notable exception was in the 1976 and 1991 winter seasons when the incidence of G1P[8] infections decreased dramatically, a trend that correlated with a significant increase in G3P[8] infections. Our sequence analysis indicates that the 1976 season was characterized by the presence of several genetically distinct, co-circulating clades of G3P[8] viruses, which contained minor but significant differences in their encoded proteins. These 1976 lineages did not readily exchange gene segments with each other, but instead remained stable over the course of the season. In contrast, the 1991 season contained a single major clade, whose genome constellation was similar to one of the 1976 clades. The 1991 clade may have gained a fitness advantage after reassorting with as of yet unidentified RV strain(s). This study reveals for the first time that genetically distinct RV clades of the same G/P-type can co-circulate and cause disease. The findings from this study also suggest that, although gene segment exchange occurs, most reassortant strains are replaced over time by lineages with preferred genome constellations. Elucidation of the selective pressures that favor maintenance of RVs with certain sets of genes may be necessary to anticipate future vaccine needs.     

Simian Rotaviruses Possess Divergent Gene Constellations That Originated from Interspecies Transmission and Reassortment

Although few simian rotaviruses (RVs) have been isolated, such strains have been important for basic research and vaccine development. To explore the origins of simian RVs, the complete genome sequences of strains PTRV (G8P[1]), RRV (G3P[3]), and TUCH (G3P[24]) were determined. These data allowed the genotype constellations of each virus to be determined and the phylogenetic relationships of the simian strains with each other and with nonsimian RVs to be elucidated. The results indicate that PTRV was likely transmitted from a bovine or other ruminant into pig-tailed macaques (its host of origin), since its genes have genotypes and encode outer-capsid proteins similar to those of bovine RVs. In contrast, most of the genes of rhesus-macaque strains, RRV and TUCH, have genotypes more typical of canine-feline RVs. However, the sequences of the canine and/or feline (canine/feline)-like genes of RRV and TUCH are only distantly related to those of modern canine/feline RVs, indicating that any potential transmission of a progenitor of these viruses from a canine/feline host to a simian host was not recent. The remaining genes of RRV and TUCH appear to have originated through reassortment with bovine, human, or other RV strains. Finally, comparison of PTRV, RRV, and TUCH genes with those of the vervet-monkey RV SA11-H96 (G3P[2]) indicates that SA11-H96 shares little genetic similarity to other simian strains and likely has evolved independently. Collectively, our data indicate that simian RVs are of diverse ancestry with genome constellations that originated largely by interspecies transmission and reassortment with nonhuman animal RVs.Group A rotaviruses (RVs) are a major cause of acute dehydrating diarrhea in infants and children under the age of 5 years worldwide. These infections lead to approximately 527,000 deaths each year, the vast majority occurring in developing countries (33). RVs are also responsible for gastroenteritis in many other animal species, notably mammals and birds (16, 38). RVs are members of the family Reoviridae and possess a genome consisting of 11 segments of double-stranded RNA (dsRNA). The prototypic genome of a group A RV encodes six structural proteins (VP) and six nonstructural proteins (NSP) (5). The mature RV virion is a nonenveloped triple-layered icosahedral particle. The inner most protein layer is formed by the core lattice protein VP2. Attached to the interior surface of the VP2 layer near the fivefold axes are complexes of the viral RNA-dependent RNA polymerase VP1 and the RNA capping enzyme VP3. Collectively, VP1, VP2, VP3, and the dsRNA genome form the core of the virion (5, 11). The core is surrounded by VP6, the sole constituent of the intermediate protein layer of the virion. The antigenic properties of VP6 are used in classifying RV isolates into groups. The outer protein layer of the virion is composed of trimers of the VP7 glycoprotein penetrated by spikes of the VP4 attachment protein (50). The properties of VP7 and VP4 form the basis of a dual classification system defining RV G types (glycosylated) and P types (protease sensitive), respectively. At present, 23 G genotypes and 31 P genotypes have been recognized in the literature based on sequence analyses (17, 39, 42, 45, 47). Recently, a comprehensive sequence-based classification system was established for the RVs which, together with a uniform nomenclature, allows each genome segment of the virus to be assigned to a particular genotype. In the comprehensive classification system, the acronym Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx defines the genotypes of VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 encoding genome segments (17, 18).Several years ago, Nakagomi et al. provided evidence by RNA-RNA hybridization assays that RVs originating from different animal species could be resolved into genogroups based upon the existence of unique species-specific genome constellations (29-31). More recently, the concept that RVs preferentially retain certain species-related genome constellations has been further supported by whole-genome sequencing (8, 24). For human RVs, two major genogroups (Wa-like genogroup 1 and DS-1-like genogroup 2) and one minor genogroup (AU-1-like genogroup 3) have been described (8, 17, 30). Although these genogroups are generally species specific, it is believed that the human AU-1 genogroup is of feline origin (31) and that the human Wa and DS-1 genogroups share common ancestor with porcine and bovine RVs, respectively (17). Another recent study based on full genome sequence data has indicated that the rarely seen human G3P[3] RVs are of feline or canine origin (46). Two additional sequence-based studies have indicated that human RVs with P[14] specificity may have originated after interspecies transmission from rabbit RVs and RVs from hosts belonging to the order Artiodactyla (i.e., hoofed mammals with even toes, including ruminants and pigs) (19, 20). These examples indicate that interspecies transmission of entire RV gene constellations from one host species to another may contribute significantly to viral evolution. In addition to interspecies transmission, complete genome sequencing of RVs have revealed multiple examples of naturally occurring inter- and intragenogroup reassortment (17, 19, 21-23, 37, 41).The simian RV strains, notably RRV and the SA11 derivatives (e.g., SA11-Cl3 and SA11-4F), have been used extensively as models in the study of all aspects of RV biology, including characterizing genome replication and virion assembly, delineating high-resolution structures of viral proteins and the virion capsid, and describing the functions of viral proteins. Moreover, the RRV strain was used to create a set of human-simian reassortant viruses that formed the basis of the first commercially licensed RV vaccine (Rotashield; Wyeth Laboratories) (10). Serological analyses have indicated that simian RVs are probably endemic in wild nonhuman primate (NHP) species in Africa (32). However, whether or not unique genogroups or preferred genome constellation exist for the simian RVs has not been determined, because of the lack of comprehensive genetic data. Most simian RVs isolated to date (e.g., rhesus macaque viruses RRV [43] and TUCH [25] and the pig-tailed macaque virus PTRV [9]) have been recovered from monkeys kept in captivity in the United States. An important exception is the SA11 isolate, which was recovered from a vervet monkey in South Africa (15). Simian RV infections occur mostly in young monkeys, similar to human RV infections in children (32, 40).To gain further insight into the origins and properties of simian RVs, we sequenced and contrasted the genomes of PTRV, RRV, and TUCH with other RVs, including SA11-H96 (G3P[2]), the only previously fully sequenced simian RV (41). Our results reveal that these four simian RVs are of divergent ancestry and have evolved by combinations of interspecies transmission and reassortment with RVs naturally occurring in other animal species. Thus, the simian RVs do not possess a common genome constellation nor define a unique genogroup. Although frequently used as disease models, the simian RVs show limited genetic similarity with the human RVs (genogroups 1 and 2) responsible for most human disease.     

Rotavirus VP8*: Phylogeny, Host Range, and Interaction with Histo-Blood Group Antigens

The distal portion of rotavirus (RV) VP4 spike protein (VP8*) is implicated in binding to cellular receptors, thereby facilitating viral attachment and entry. While VP8* of some animal RVs engage sialic acid, human RVs often attach to and enter cells in a sialic acid-independent manner. A recent study demonstrated that the major human RVs (P[4], P[6], and P[8]) recognize human histo-blood group antigens (HBGAs). In this study, we performed a phylogenetic analysis of RVs and showed further variations of RV interaction with HBGAs. On the basis of the VP8* sequences, RVs are grouped into five P genogroups (P[I] to P[V]), of which P[I], P[IV], and P[V] mainly infect animals, P[II] infects humans, and P[III] infects both animals and humans. The sialic acid-dependent RVs (P[1], P[2], P[3], and P[7]) form a subcluster within P[I], while all three major P genotypes of human RVs (P[4], P[6], and P[8]) are clustered in P[II]. We then characterized three human RVs (P[9], P[14], and P[25]) in P[III] and observed a new pattern of binding to the type A antigen which is distinct from that of the P[II] RVs. The binding was demonstrated by hemagglutination and saliva binding assay using recombinant VP8* and native RVs. Homology modeling and mutagenesis study showed that the locations of the carbohydrate binding interfaces are shared with the sialic acid-dependent RVs, although different amino acids are involved. The P[III] VP8* proteins also bind the A antigens of the porcine and bovine mucins, suggesting the A antigen as a possible factor for cross-species transmission of RVs. Our study suggests that HBGAs play an important role in RV infection and evolution.     

Spike protein VP8* of human rotavirus recognizes histo-blood group antigens in a type-specific manner

Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Le(b)) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Le(b) and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells.     

Binding Patterns of Rotavirus Genotypes P[4], P[6], and P[8] in China with Histo-Blood Group Antigens

Rotaviruses (RVs) are an important cause of severe gastroenteritis in children. It has been found that RV may recognize the histo-blood group antigens (HBGAs) as ligands or receptors and bind HBGAs in a type-dependent manner. In this study, we investigated the binding specificity of VP8* proteins from human rotaviruses (RV) that are prevalent in China including genotypes P[4], P[6], and P[8]. Through the saliva- and oligosaccharide-based binding assays, we found that the VP8* proteins of P[4] and P[8] RV showed similar reactivity with the Le^b and H type 1 antigens, while P[6] RV weakly bound the Le^b antigen. These findings may facilitate further research into RV host specificity and vaccine development.     

Reassortment of Human and Animal Rotavirus Gene Segments in Emerging DS-1-Like G1P[8] Rotavirus Strains

The emergence and rapid spread of novel DS-1-like G1P[8] human rotaviruses in Japan were recently reported. More recently, such intergenogroup reassortant strains were identified in Thailand, implying the ongoing spread of unusual rotavirus strains in Asia. During rotavirus surveillance in Thailand, three DS-1-like intergenogroup reassortant strains having G3P[8] (RVA/Human-wt/THA/SKT-281/2013/G3P[8] and RVA/Human-wt/THA/SKT-289/2013/G3P[8]) and G2P[8] (RVA/Human-wt/THA/LS-04/2013/G2P[8]) genotypes were identified in fecal samples from hospitalized children with acute gastroenteritis. In this study, we sequenced and characterized the complete genomes of strains SKT-281, SKT-289, and LS-04. On whole genomic analysis, all three strains exhibited unique genotype constellations including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strains SKT-281 and SKT-289, and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strain LS-04. Except for the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P[8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P[8] strains, while the remaining VP7 and NSP4 genes appeared to be of equine and bovine origin, respectively. Thus, strains SKT-281 and SKT-289 appeared to be reassortant strains as to DS-1-like G1P[8], animal-derived human, and/or animal rotaviruses. On the other hand, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses, while three genes (VP4, VP2, and NSP1) were assumed to be derived from DS-1-like G1P[8] strains. Notably, the remaining NSP2 gene of strain LS-04 appeared to be of bovine origin. Thus, strain LS-04 was assumed to be a multiple reassortment strain as to DS-1-like G1P[8], locally circulating DS-1-like G2P[4], bovine-like human, and/or bovine rotaviruses. Overall, the great genomic diversity among the DS-1-like G1P[8] strains seemed to have been generated through reassortment involving human and animal strains. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like intergenogroup reassortant strains having G3P[8] and G2P[8] genotypes that have emerged in Thailand. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] strains and related reassortant ones.     

Distribution of human rotaviruses, especially G9 strains, in Japan from 1996 to 2000

A 4-year (1996-2000) survey of rotavirus infection involving 2,218 diarrheal fecal specimens of children collected from five regions of Japan was conducted. A total of 642 (28.9%) specimens were found to be rotavirus positive. A changed prevalence pattern of rotavirus G serotype was found with an increase of G9 and G2 and a decrease of G1, although G1 remained the prevailing serotype. Serotype G9 was unexpectedly determined to be the prevailing serotype in Sapporo (62.5%) and Tokyo (52.9%) in 1998-1999, and in Saga (78.4%) in 1999-2000. G9 strains isolated from 1998-1999 belonged to the P[8]-NSP4-Wa-group with long RNA pattern, while, G9 strains isolated from 1999-2000 belonged to three groups, the P[8]-NSP4-Wa-group with long RNA pattern, the P[4]-NSP4-KUN-group with short RNA pattern and a mixed-type group (P[4]/P[8]-NSP4-KUN/Wa-group with long RNA pattern). Both sequence and immunological analysis of VP7 revealed that the G9 strains from 1999-2000 were much more closely related to the G9 strains isolated worldwide in the 1990s, including G9 strains found in Thailand in 1997. However, the G9 strains from 1998-1999 were distinct from these and more closely related to the G9 prototype strains F45, AU32 and WI61 discovered in Japan and the US in the 1980s. Thus the G9 strains isolated in 1998-1999 had progenitors common to the G9 prototype strains, while the strains isolated in 1999-2000 did not directly evolve from them but were related to global G9 strains that have emerged in recent years. These data supported our previous report that G9 rotavirus might exist as two or more subtypes with diverse RNA patterns, P-genotype and NSP4 genogroup combinations (Y.M. Zhou et al., J. Med. Virol. 65: 619-628, 2001) and suggested that G9 rotavirus prevalent in Japan during two successive years belonged to different subtypes. The nucleotide sequences presented in this paper were submitted to DDBJ, EMBL and GenBank nucleotide sequence databases. The accession numbers are: 00-Ad2863VP7 (AB091746), 00-OS2986VP7 (AB091747), 00-SG2509VP7 (AB091748), 00-SG2518VP7 (AB091749), 00-SG2541 (AB091750), 00-SG2864 (AB091751), 00-SP2737VP7 (AB091752), 99-SP1542VP7 (AB091753), 99-SP1904VP7 (AB091754), 99-TK2082VP7 (AB091755) and 99-TK2091VP7 (AB091756).     

Structural Basis of Glycan Recognition in Globally Predominant Human P[8] Rotavirus

Rotavirus(RV)causes acute gastroenteritis in infants and children worldwide.Recent studies showed that glycans such as histo-blood group antigens(HBGAs)function as cell attachment factors affecting RV host susceptibility and prevalence.P[8]is the predominant RV genotype in humans,but the structural basis of how P[8]RVs interact with glycan ligands remains elusive.In this study,we characterized the interactions between P[8]VP8~*s and glycans which showed that VP8~*,the RV glycan binding domain,recognized both mucin core 2 and H type 1 antigens according to the ELISA-based oligosaccharide binding assays.Importantly,we determined the structural basis of P[8]RV-glycans interaction from the crystal structures of a Rotateq P[8]VP8~*in complex with core 2 and H type 1 glycans at 1.82.3 ?,respectively,revealing a common binding pocket and similar binding mode.Structural and sequence analysis demonstrated that the glycan binding site is conserved among RVs in the P[Ⅱ]genogroup,while genotype-specific amino acid variations determined different glycan binding preference.Our data elucidated the detailed structural basis of the interactions between human P[8]RVs and different host glycan factors,shedding light on RV infection,epidemiology,and development of anti-viral agents.     

Evidence of the co‐circulation of enteric viruses in sewage and in the population of Greater Cairo

Aims: To characterize major enteric viruses (enterovirus, rotavirus, norovirus, astrovirus and adenovirus) in the sewage of Greater Cairo and to compare the results with clinical data collected during the same period. Methods and Results: Seventy‐two sewage samples from two waste water treatment plants were collected from April 2006 through February 2007. Enteroviruses, noroviruses (NoVs) and rotaviruses (RVs) were detected by RT‐PCR in 22%, 18% and 8·3% of the samples, respectively. No adenovirus and astrovirus was detected. G2P[8], G9P[8], G1P[8], G2P[4] and rare G12 RV isolates were detected in the environment as well as a bovine RV. The environmental NoV strains mostly belonged to genogroup I (84%). Rotaviruses and some of the NoVs were similar to those found in the clinical samples at the same time. Conclusions: The comparison of environmental and clinical data suggests that similar RV and NoV isolates were circulating in the environment and in the population during the same period. Significance and Impact of the Study: Few studies have investigated the prevalence and the epidemiology of RVs and NoVs in Cairo. This work is the first to establish a correlation between viral gastroenteritis and the concomitant presence of enteric viruses in the environment for Greater Cairo where combined environmental and clinical surveys should help to prevent infections caused by these major pathogens.     

Frequent reassortments may explain the genetic heterogeneity of rotaviruses: analysis of Finnish rotavirus strains

The predominant rotavirus electropherotypes (e-types) during 17 epidemic seasons (1980 through 1997) in Finland were established, and representative virus isolates were studied by nucleotide sequencing and phylogenetic analysis. The virus isolates were either P[8]G1 or P[8]G4 types. The G1 and G4 strains formed one G1 lineage (VP7-G1-1) and one G4 lineage, respectively. Otherwise, they belonged to two P[8] lineages (VP4-P[8]-1 and -2) unrelated to their G types. Phylogenetic analysis of partial sequences of all 11 RNA segments obtained from the strains also revealed genetic diversity among gene segments other than those defining P and G types. With the exception of segments 1, 3, and 10, the sequences of the other segments could be assigned to 2 to 4 different genetic clusters. The results of this study suggest that, in addition to the RNA segments encoding VP4 and VP7, the other RNA segments may segregate independently as well. In total, the 9 predominant e-types represented 7 different RNA segment combinations when the phylogenetic clusters of their 11 genes were determined. The extensive genetic diversity and number of e-types among rotaviruses are best explained by frequent genetic reassortment.     

Generation of recombinant rotavirus with an antigenic mosaic of cross-reactive neutralization epitopes on VP4

Recombinant rotavirus (RV) with cDNA-derived chimeric VP4 was generated using recently developed reverse genetics for RV. The rescued virus, KU//rVP4(SA11)-II(DS-1), contains SA11 (simian RV strain, G3P[2])-based VP4, in which a cross-reactive neutralization epitope (amino acids 381 to 401) on VP5* is replaced by the corresponding sequence of a different P-type DS-1 (human RV strain, G2P[4]). Serological analyses with a panel of anti-VP4- and -VP7-neutralizing monoclonal antibodies revealed that the rescued virus carries a novel antigenic mosaic of cross-reactive neutralization epitopes on its VP4 surface. This is the first report of the generation of a recombinant RV with artificial amino acid substitutions.     

Cross-Serotype Immunity Induced by Immunization with a Conserved Rhinovirus Capsid Protein

Human rhinovirus (RV) infections are the principle cause of common colds and precipitate asthma and COPD exacerbations. There is currently no RV vaccine, largely due to the existence of ∼150 strains. We aimed to define highly conserved areas of the RV proteome and test their usefulness as candidate antigens for a broadly cross-reactive vaccine, using a mouse infection model. Regions of the VP0 (VP4+VP2) capsid protein were identified as having high homology across RVs. Immunization with a recombinant VP0 combined with a Th1 promoting adjuvant induced systemic, antigen specific, cross-serotype, cellular and humoral immune responses. Similar cross-reactive responses were observed in the lungs of immunized mice after infection with heterologous RV strains. Immunization enhanced the generation of heterosubtypic neutralizing antibodies and lung memory T cells, and caused more rapid virus clearance. Conserved domains of the RV capsid therefore induce cross-reactive immune responses and represent candidates for a subunit RV vaccine.     

Reassortant DS‐1‐like G1P[4] Rotavirus A strains generated from co‐circulating strains in Vietnam, 2012/2013

One major mechanism by which Rotavirus A (RVA) evolves is genetic reassortment between strains with different genotype constellations. However, the parental strains of the reassortants generated have seldom been identified. Here, the whole genome of two suspected reassortants, RVA/Human‐wt/VNM/SP127/2013/G1P[4] and RVA/Human‐wt/VNM/SP193/2013/G1P[4], with short RNA electropherotypes were examined by Illumina MiSeq sequencing and their ancestral phylogenies reconstructed. Their genotype constellation, G1‐P[4]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2, indicated that they were G1 VP7 mono‐reassortants possessing DS‐1‐like genetic backbones. The two strains were ≧99.7% identical across the genome. While their VP7 genes were ≧99.7 identical to that of a Wa‐like strain RVA/Human‐wt/VNM/SP110/2012/G1P[8] which co‐circulated during the 2012/2013 season, 10 genes were ≧99.8% identical to that of the DS‐1‐like strains RVA/Human‐wt/VNM/SP015/2012/G2P[4] (and SP108) that co‐circulated during the season. The identities were consistent with the phylogenetic relationships observed between the genes of the reassortants and those of the afore‐mentioned strains. Consequently, the G1P[4] strains appear to have been generated by genetic reassortment between SP110‐like and SP015‐like strains. In conclusion, this study provides robust molecular evidence for the first time that G1P[4] strains detected in Hanoi Vietnam were generated by inter‐genogroup reassortment between co‐circulating G1P[8] and G2P[4] strains within the same place and season.

     

Misdirected antibody responses against an N-terminal epitope on human rhinovirus VP1 as explanation for recurrent RV infections

Rhinoviruses (RVs) are the primary cause of upper respiratory tract infections, generally known as the common cold. Moreover, RV infections can trigger severe exacerbations of asthma and chronic obstructive pulmonary disease (COPD). We expressed the 4 major RV capsid proteins, VP1-VP4, in Escherichia coli and used these proteins as well as recombinant and synthetic VP1 fragments to study and map antibody responses in RV-infected humans. VP1, which on infection binds to ICAM 1, was identified as a major target for the memory immune response, residing in the IgG1 subclass and IgA class. Interestingly, this response was mainly directed against an N-terminal 20 mer peptide in VP1, P1a, which becomes exposed on intact RV only when it docks to its receptor ICAM 1. Molecular modeling using the 3-dimensional RV capsid structures revealed that P1a was localized inside the capsid and outside the areas involved in receptor binding or RV neutralization. Our results suggest misdirection of antibody responses against a nonprotective epitope as a mechanism how RV escapes immunity and causes recurrent infections. Based on these findings, it may be possible to design vaccines against RV infections and RV-induced respiratory diseases.     

Heterogeneity and temporal dynamics of evolution of G1 human rotaviruses in a settled population

A rotavirus sample collection from 19 consecutive years was used to investigate the heterogeneity and the dynamics of evolution of G1 rotavirus strains in a geographically defined population. Phylogenetic analysis of the VP7 gene sequences of G1P[8] human rotavirus strains showed the circulation of a heterogeneous population comprising three lineages and seven sublineages. Increases in the circulation of G1 rotaviruses were apparently associated with the introduction of novel G1 strains that exhibited multiple amino acid changes in antigenic regions involved in rotavirus neutralization compared to the strains circulating in the previous years. The emergence and/or introduction of G1 antigenic variants might be responsible for the continuous circulation of G1 rotaviruses in the local population, with the various lineages and sublineages appearing, disappearing, or cocirculating in an alternate fashion under the influence of immune-pressure mechanisms. Sequence analysis of VP4-encoding genes of the G1 strains revealed that the older strains were associated with a unique VP4 lineage, while a novel VP4 lineage emerged after 1995. The introduction of human rotavirus vaccines might alter the forces and balances that drive rotavirus evolution and determine the spread of novel strains that are antigenically different from those included in the vaccine formulations. The continuous emergence of VP7-VP4 gene combinations in human rotavirus strains should be taken into consideration when devising vaccination strategies.     

Initial interaction of rotavirus strains with N-acetylneuraminic (sialic) acid residues on the cell surface correlates with VP4 genotype, not species of origin

We examined 41 human and animal rotavirus strains representative of all known P genotypes for their dependency on cellular N-acetylneuraminic (sialic) acid (SA) residues for infectivity. Our results showed that all rotaviruses studied, whether of animal or human origin, belonging to P genotypes [1], [2], [3], and [7] depended on SA residues on the cell surface for efficient infectivity but that all human and animal rotavirus strains representative of the remaining known P genotypes were SA independent. The SA residue requirement for efficient infectivity did not change for reassortant rotavirus strains with altered VP4-VP7 combinations. The initial interaction of rotavirus strains with SA residues on the cell surface correlated with VP4 genotype specificity, not with species of origin or VP7 G serotype specificity (P = 0.001; r2 = 1.00, Pearson's correlation coefficient). In addition to being a requirement for infectivity, the presence of SA residues on the cell surface is a requirement for efficient growth in cell culture; recognition of the association of specific P genotypes with the binding of rotavirus to SA residues will facilitate our understanding of the molecular basis of the early events of rotavirus-cell interactions in cell culture models and of pathogenicity in vivo.     

Poly-LacNAc as an Age-Specific Ligand for Rotavirus P[11] in Neonates and Infants

Rotavirus (RV) P[11] is an unique genotype that infects neonates. The mechanism of such age-specific host restriction remains unknown. In this study, we explored host mucosal glycans as a potential age-specific factor for attachment of P[11] RVs. Using in vitro binding assays, we demonstrated that VP8* of a P[11] RV (N155) could bind saliva of infants (60.3%, N = 151) but not of adults (0%, N = 48), with a significantly negative correlation between binding of VP8* and ages of infants (P<0.01). Recognition to the infant saliva did not correlate with the ABO, secretor and Lewis histo-blood group antigens (HBGAs) but with the binding of the lectin Lycopersicon esculentum (LEA) that is known to recognize the oligomers of N-acetyllactosamine (LacNAc), a precursor of human HBGAs. Direct evidence of LacNAc involvement in P[11] binding was obtained from specific binding of VP8* with homopolymers of LacNAc in variable lengths through a glycan array analysis of 611 glycans. These results were confirmed by strong binding of VP8* to the Lec2 cell line that expresses LacNAc oligomers but not to the Lec8 cell line lacking the LacNAc. In addition, N155 VP8* and authentic P[11] RVs (human 116E and bovine B223) hemagglutinated human red blood cells that are known to express poly-LacNAc. The potential role of poly-LacNAc in host attachment and infection of RVs has been obtained by abrogation of 116E replication by the PAA-conjugated poly-LacNAc, human milk, and LEA positive infant saliva. Overall, our results suggested that the poly-LacNAc could serve as an age-specific receptor for P[11] RVs and well explained the epidemiology that P[11] RVs mainly infect neonates and young children.