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.     

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.     

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.     

The VP8* Domain of Neonatal Rotavirus Strain G10P[11] Binds to Type II Precursor Glycans

Naturally occurring bovine-human reassortant rotaviruses with a P[11] VP4 genotype exhibit a tropism for neonates. Interaction of the VP8* domain of the spike protein VP4 with sialic acid was thought to be the key mediator for rotavirus infectivity. However, recent studies have indicated a role for nonsialylated glycoconjugates, including histo-blood group antigens (HBGAs), in the infectivity of human rotaviruses. We sought to determine if the bovine rotavirus-derived VP8* of a reassortant neonatal G10P[11] virus interacts with hitherto uncharacterized glycans. In an array screen of >600 glycans, VP8* P[11] showed specific binding to glycans with the Galβ1-4GlcNAc motif, which forms the core structure of type II glycans and is the precursor of H type II HBGA. The specificity of glycan binding was confirmed through hemagglutination assays; GST-VP8* P[11] hemagglutinates type O, A, and B red blood cells as well as pooled umbilical cord blood erythrocytes. Further, G10P[11] infectivity was significantly enhanced by the expression of H type II HBGA in CHO cells. The bovine-origin VP4 was confirmed to be essential for this increased infectivity, using laboratory-derived reassortant viruses generated from sialic acid binding rotavirus SA11-4F and a bovine G10P[11] rotavirus, B223. The binding to a core glycan unit has not been reported for any rotavirus VP4. Core glycan synthesis is constitutive in most cell types, and modification of these glycans is thought to be developmentally regulated. These studies provide the first molecular basis for understanding neonatal rotavirus infections, indicating that glycan modification during neonatal development may mediate the age-restricted infectivity of neonatal viruses.     

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.     

Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses

Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated via multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate the HMG shotgun glycan microarray (SGM). To investigate the potential role of HMGs as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains N155(G10P[11]) and RV3(G3P[6]) and a bovine strain, B223(G10P[11]). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using metadata-assisted glycan sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MSⁿ analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8* binding, many of which are novel HMGs, whose detailed structural assignments by MSⁿ are described in a companion report. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated that sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures.Human milk offers nutrition, innate immune protection, and other developmental benefits to infants (1, 2). In addition to essential nutrients and bioactive antibodies, human milk uniquely possesses a rich pool of free-reducing glycans (oligosaccharides), most of which are unique to human milk (3, 4). Depending on the blood group status and the lactation stage of an individual, the concentration of human milk glycans (HMGs)¹ larger than lactose varies between 5 and 15 g/l, making them the third largest component of human milk after lactose and lipids (5). Over the past decades, more than 100 structurally distinct HMGs have been identified (6–9). All of these glycans originate from a lactose that is extended by type 1 (Galβ1–3GlcNAc) or type 2 (Galβ1–4GlcNAc) N-acetyllactosamine in either linear or branch forms and further modified with α-linked fucose and/or N-acetylneuraminic acid. It has been shown that HMGs are only minimally digested in the upper gastrointestinal tract and are transported intact into the lower parts of intestine (10, 11). Additionally, ∼1% to 2% of HMGs are excreted via an infant''s urine and seem to appear in the circulation (12, 13).Accumulated evidence has indicated that HMGs play multiple biological roles. In addition to having well-known prebiotic effects that promote the growth of beneficial microflora in the intestine (14, 15), HMGs are suggested to competitively interfere with pathogen attachment to the host cell surface by acting as soluble decoy receptors (16–18), and such anti-adhesive effects are often glycan specific (19). For example, α1–2 fucosylated HMGs, which arise mainly from individuals that are Secretor(+), were observed to prevent the adherence of Campylobacter jejuni to epithelial cells (20) and were associated with protection against diarrhea caused by Campylobacter, caliciviruses, and Escherichia coli toxin in breastfed infants (21–23). Sialylated HMGs were exclusive receptors for influenza viruses (24–26) and showed a capacity to inhibit cholera toxin B (27), Vibrio cholera (28), enterotoxigenic E. coli, and uropathogenic E. coli strains (29, 30). It was also proposed that HMGs might serve as anti-inflammatory components and thus contribute to the lower incidence of necrotizing enterocolitis in breastfed infants. This idea is supported by the observations that the acidic fraction of HMG inhibits leukocyte rolling, adhesion, and activation (31) and disialyllacto-N-tetraose prevents necrotizing enterocolitis in neonatal rats (32). Furthermore, a variety of cytoprotective activities of HMGs have been reported against Clostridium difficile toxins (33), Helicobacter pylori (34, 35), Streptococcus pneumonia (36), Entamoeba histolytica (37), and HIV-1-gp120 (38). Although the numerous in vitro and in vivo data provide important information about the function of HMGs, these studies have typically used HMG fraction mixtures or a small panel of defined HMGs, and therefore the bioactive HMGs were not or poorly identified.In order to better understand the interactions of HMGs with various microorganisms, it is necessary to examine the entire milk metaglycome and identify the specific bioactive components, which is not possible via traditional methods that mainly focus on compositional analysis of HMGs (39). To find an efficient route for establishing the function–structure relationship of HMGs, we applied a “shotgun glycomics” approach to generate a shotgun glycan microarray (SGM) from isolated human milk glycans of a Lewis-positive, non-secretor individual (25, 40). The functional recognition studies, along with metadata-assisted glycan sequencing (MAGS), revealed novel epitopes/receptors for anti-TRA-1 antibodies, influenza viruses, and minute viruses of mice. Our work represented the first natural glycan microarray of HMGs containing over 100 glycans. Notably, the antibody binding data showed a lack of α1,2-fucosylated HMGs on this SGM, confirming that the donor was a non-secretor (41, 42).Here we describe our studies in which we prepared a SGM containing over 200 isolated HMG targets from pooled human milk of mixed Lewis and Secretor phenotypes and investigated the binding of rotavirus (RV) cell attachment protein to them. Human RVs are the leading cause of severe gastroenteritis in children, responsible for an estimated 453,000 deaths each year worldwide (43). As with many other pathogens, RV infection is initiated by the interaction with specific cellular glycans. The VP8* domain of the RV outer capsid spike protein VP4 mediates this process (44), but the identity of VP8* receptors is quite controversial. It was believed that VP8* recognized either terminal sialic acid or internal sialic acid, mainly based on crystallographic and NMR studies (45–48). However, recently a human strain (HAL1166) with a P[14] VP8* was found to bind to A-type histo-blood group antigen (49), a neonatal strain with a P[11] VP8* bound to type 2 precursor glycans (50), and several other P types recognized secretor-related antigens Lewis b and H type 1 (51). These studies indicate that sialic acid might not be required by all RVs and that the glycan receptors are genotype-dependent. The infectivity of a porcine RV was inhibited by sialyl HMGs in vitro (52); however, there are limited data on human RVs. Here, we demonstrate that the VP8* of two different human neonatal RVs and an additional bovine strain bound to HMGs independent of sialic acid and that each VP8* demonstrated a unique glycan-binding specificity.     

Structural Characterization by Multistage Mass Spectrometry (MSn) of Human Milk Glycans Recognized by Human Rotaviruses

We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.², describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.     

Diversity and relationships of cocirculating modern human rotaviruses revealed using large-scale comparative genomics

Group A rotaviruses (RVs) are 11-segmented, double-stranded RNA viruses and are primary causes of gastroenteritis in young children. Despite their medical relevance, the genetic diversity of modern human RVs is poorly understood, and the impact of vaccine use on circulating strains remains unknown. In this study, we report the complete genome sequence analysis of 58 RVs isolated from children with severe diarrhea and/or vomiting at Vanderbilt University Medical Center (VUMC) in Nashville, TN, during the years spanning community vaccine implementation (2005 to 2009). The RVs analyzed include 36 G1P[8], 18 G3P[8], and 4 G12P[8] Wa-like genogroup 1 strains with VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 genotype constellations of I1-R1-C1-M1-A1-N1-T1-E1-H1. By constructing phylogenetic trees, we identified 2 to 5 subgenotype alleles for each gene. The results show evidence of intragenogroup gene reassortment among the cocirculating strains. However, several isolates from different seasons maintained identical allele constellations, consistent with the notion that certain RV clades persisted in the community. By comparing the genes of VUMC RVs to those of other archival and contemporary RV strains for which sequences are available, we defined phylogenetic lineages and verified that the diversity of the strains analyzed in this study reflects that seen in other regions of the world. Importantly, the VP4 and VP7 proteins encoded by VUMC RVs and other contemporary strains show amino acid changes in or near neutralization domains, which might reflect antigenic drift of the virus. Thus, this large-scale, comparative genomic study of modern human RVs provides significant insight into how this pathogen evolves during its spread in the community.     

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.     

GII.3型诺如病毒GZ31597株变异情况及与受体组织血型抗原结合模式分析

诺如病毒(Noroviruses,NoVs)是引起非菌型胃肠炎暴发流行的主要病原体之一。为了解我国GII.3型NoVs毒株的变异以及受体结合模式,本研究对来自2015年一起中国广州NoVs胃肠炎暴发的GII.3型毒株GZ31597株进行聚合酶区和完整VP1区基因扩增、序列测定和序列分析,并表达VP1突出区蛋白(P蛋白),通过P蛋白与不同血型唾液样本的酶免疫分析法(EIA)测定实验确定其组织血型抗原(Histo-blood group antigens,HBGAs)结合模式。GZ31597株聚合酶和VP1基因系统进化分析表明,GZ31597株为GII.P12/GII.3-SubD基因型(聚合酶/衣壳区),该毒株较先前的GII.3毒株相比,在既是抗原表位又是HBGAs受体结合位点的氨基酸385残基发生了氨基酸转换。根据Western Blotting结果,证实P蛋白成功表达。唾液结合分析结果显示,该毒株P蛋白与A、B、AB、O型分泌型以及O型非分泌型唾液均可以结合,但结合值相对低。本研究表明该GII.P12/GII.3-SubD亚型的GII.3毒株在长期的流行过程中,通过氨基酸的转换,改变抗原性和受体结合活性,使GII.3型毒株在人群中继续流行。通过探索GII.3型NoVs在人群中长期广泛流行的原因,为GII.3型诺如病毒性胃肠炎的预防和控制提供重要依据。     

The Principal Genetic Determinants for Nasopharyngeal Carcinoma in China Involve the HLA Class I Antigen Recognition Groove

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy facilitated by Epstein-Barr Virus infection. Here we resolve the major genetic influences for NPC incidence using a genome-wide association study (GWAS), independent cohort replication, and high-resolution molecular HLA class I gene typing including 4,055 study participants from the Guangxi Zhuang Autonomous Region and Guangdong province of southern China. We detect and replicate strong association signals involving SNPs, HLA alleles, and amino acid (aa) variants across the major histocompatibility complex-HLA-A, HLA –B, and HLA -C class I genes (P_{HLA-A-aa-site-62} = 7.4×10⁻²⁹; P _{HLA-B-aa-site-116} = 6.5×10⁻¹⁹; P _{HLA-C-aa-site-156} = 6.8×10⁻⁸ respectively). Over 250 NPC-HLA associated variants within HLA were analyzed in concert to resolve separate and largely independent HLA-A, -B, and -C gene influences. Multivariate logistical regression analysis collapsed significant associations in adjacent genes spanning 500 kb (OR2H1, GABBR1, HLA-F, and HCG9) as proxies for peptide binding motifs carried by HLA- A*11:01. A similar analysis resolved an independent association signal driven by HLA-B*13:01, B*38:02, and B*55:02 alleles together. NPC resistance alleles carrying the strongly associated amino acid variants implicate specific class I peptide recognition motifs in HLA-A and -B peptide binding groove as conferring strong genetic influence on the development of NPC in China.     

The carbohydrate moiety and high molecular weight carrier of histo-blood group antigens are both required for norovirus-receptor recognition

Histo-blood group antigens (HBGAs) on human intestinal epithelium serve as receptors for noroviruses (NVs). These antigens also are expressed in milk and may act as decoy receptors to protect breast-fed infants and others against NV disease. In this study we demonstrated that human milk is highly variable in synthesis of HBGAs, which differs from that of saliva; a large quantity of small, soluble HBGAs are found in milk, but much less in saliva and are recognized by MAbs, but not by NVs. There is another group of HBGAs, of high MW, found in both milk and saliva, and recognized by both NVs and MAbs. These results suggest that the specificity of NVs and MAbs to HBGAs are different and the backbones in addition to the carbohydrate moiety are required for NV recognition. Further studies to define the structure and genetics of the high MW milk glycans are necessary.     

Epidemiology and Genetic Diversity of Rotavirus Strains in Children with Acute Gastroenteritis in Lahore,Pakistan

Pakistan harbors high disease burden of gastro-enteric infections with majority of these caused by rotavirus. Unfortunately, lack of proper surveillance programs and laboratory facilities have resulted in scarcity of available data on rotavirus associated disease burden and epidemiological information in the country. We investigated 1306 stool samples collected over two years (2008–2009) from hospitalized children under 5 years of age for the presence of rotavirus strains and its genotypic diversity in Lahore. The prevalence rate during 2008 and 2009 was found to be 34% (n = 447 out of 1306). No significant difference was found between different age groups positive for rotavirus (p>0.05). A subset of EIA positive samples was further screened for rotavirus RNA through RT-PCR and 44 (49.43%) samples, out of total 89 EIA positive samples, were found positive. G and P type prevalence was found as follows: G1P [4] = 3(6.81%); G1P [6] = 9(20.45%); G1P [8] = 1(2.27%); G2P [4] = 21(47.72%); G2P [8] = 1(2.27%); G9P [4] = 1(2.27%); G9P [6] = 1(2.27%) and G9P [8] = 7(15.90%). Phylogenetic analysis revealed that the VP7 and VP4 sequences clustered closely with the previously detected strains in the country as well as Belgian rotaviruses. Antigenic characterization was performed by analyzing major epitopes in the immunodominant VP7 and VP4 gene segments. Although the neutralization conferring motifs were found variable between the Pakistani strains and the two recommended vaccines strains (Rotarix™ and RotaTeq™), we validate the use of rotavirus vaccine in Pakistan based on the proven and recognized vaccine efficacy across the globe. Our findings constitute the first report on rotavirus’ genotype diversity, their phylogenetic relatedness and epidemiology during the pre-vaccination era in Lahore, Pakistan and support the immediate introduction of rotavirus vaccine in the routine immunization program of the country.     

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.     

A Metal-Based Inhibitor of NEDD8-Activating Enzyme

A cyclometallated rhodium(III) complex [Rh(ppy)₂(dppz)]⁺ (1) (where ppy = 2-phenylpyridine and dppz = dipyrido[3,2-a:2′,3′-c]phenazine dipyridophenazine) has been prepared and identified as an inhibitor of NEDD8-activating enzyme (NAE). The complex inhibited NAE activity in cell-free and cell-based assays, and suppressed the CRL-regulated substrate degradation and NF-κB activation in human cancer cells with potency comparable to known NAE inhibitor MLN4924. Molecular modeling analysis suggested that the overall binding mode of 1 within the binding pocket of the APPBP1/UBA3 heterodimer resembled that for MLN4924. Complex 1 is the first metal complex reported to suppress the NEDDylation pathway via inhibition of the NEDD8-activating enzyme.     

HLA Alleles Influence the Clinical Signature of Amoxicillin-Clavulanate Hepatotoxicity

Background and Aim

The genotype-phenotype interaction in drug-induced liver injury (DILI) is a subject of growing interest. Previous studies have linked amoxicillin-clavulanate (AC) hepatotoxicity susceptibility to specific HLA alleles. In this study we aimed to examine potential associations between HLA class I and II alleles and AC DILI with regards to phenotypic characteristics, severity and time to onset in Spanish AC hepatotoxicity cases.

Methods

High resolution genotyping of HLA loci A, B, C, DRB1 and DQB1 was performed in 75 AC DILI cases and 885 controls.

Results

The distributions of class I alleles A*3002 (P/Pc = 2.6E-6/5E-5, OR 6.7) and B*1801 (P/Pc = 0.008/0.22, OR 2.9) were more frequently found in hepatocellular injury cases compared to controls. In addition, the presence of the class II allele combination DRB1*1501-DQB1*0602 (P/Pc = 5.1E-4/0.014, OR 3.0) was significantly increased in cholestatic/mixed cases. The A*3002 and/or B*1801 carriers were found to be younger (54 vs 65 years, P = 0.019) and were more frequently hospitalized than the DRB1*1501-DQB1*0602 carriers. No additional alleles outside those associated with liver injury patterns were found to affect potential severity as measured by Hy’s Law criteria. The phenotype frequencies of B*1801 (P/Pc = 0.015/0.42, OR 5.2) and DRB1*0301-DQB1*0201 (P/Pc = 0.0026/0.07, OR 15) were increased in AC DILI cases with delayed onset compared to those corresponding to patients without delayed onset, while the opposite applied to DRB1*1302-DQB1*0604 (P/Pc = 0.005/0.13, OR 0.07).

Conclusions

HLA class I and II alleles influence the AC DILI signature with regards to phenotypic expression, latency presentation and severity in Spanish patients.     

Association of Increased Frequencies of HLA-DPB1*05∶01 with the Presence of Anti-Ro/SS-A and Anti-La/SS-B Antibodies in Japanese Rheumatoid Arthritis and Systemic Lupus Erythematosus Patients

Introduction

Autoantibodies to ribonucleoprotein are associated with a variety of autoimmune diseases, including rheumatoid arthritis (RA). Many studies on associations between human leukocyte antigen (HLA) alleles and RA have been reported, but few have been validated in RA subpopulations with anti-La/SS-B or anti-Ro/SS-A antibodies. Here, we investigated associations of HLA class II alleles with the presence of anti-Ro/SS-A or anti-La/SS-B antibodies in RA.

Methods

An association study was conducted for HLA-DRB1, DQB1, and DPB1 in Japanese RA and systemic lupus erythematosus (SLE) patients that were positive or negative for anti-Ro/SS-A and/or anti-La/SS-B antibodies.

Results

An increased prevalence of certain class II alleles was associated with the presence of anti-Ro/SS-A antibodies as follows: DRB1*08∶03 (Pc = 3.79×10⁻⁵, odds ratio [OR] 3.06, 95% confidence interval [CI] 1.98–4.73), DQB1*06∶01 (Pc = 0.0106, OR 1.70, 95%CI 1.26–2.31), and DPB1*05∶01 (Pc = 0.0040, OR 1.55, 95%CI 1.23–1.96). On the other hand, DRB1*15∶01 (Pc = 0.0470, OR 3.14, 95%CI 1.63–6.05), DQB1*06∶02 (Pc = 0.0252, OR 3.14, 95%CI 1.63–6.05), and DPB1*05∶01 (Pc = 0.0069, OR 2.27, 95% CI 1.44–3.57) were associated with anti-La/SS-B antibodies. The DPB1*05∶01 allele was associated with anti-Ro/SS-A (Pc = 0.0408, OR 1.69, 95% CI 1.19–2.41) and anti-La/SS-B antibodies (Pc = 2.48×10⁻⁵, OR 3.31, 95%CI 2.02–5.43) in SLE patients.

Conclusion

HLA-DPB1*05∶01 was the only allele associated with the presence of both anti-Ro/SS-A and anti-La/SS-B antibodies in Japanese RA and SLE patients.     

Interaction between HLA-DRB1-DQB1 Haplotypes in Sardinian Multiple Sclerosis Population

We performed a case-control study in 2,555 multiple sclerosis (MS) Sardinian patients and 1,365 healthy ethnically matched controls, analyzing the interactions between HLA-DRB1-DQB1 haplotypes and defining a rank of genotypes conferring a variable degree of risk to the disease. Four haplotypes were found to confer susceptibility (*13∶03-*03∶01 OR = 3.3, Pc 5.1×10⁻⁵, *04∶05-*03∶01 OR = 2.1, Pc 9.7×10⁻⁸, *15∶01-*06∶02 OR = 2.0, Pc = 9.1×10⁻³, *03∶01-*02∶01 OR = 1.7 Pc = 7.9×10⁻²²) and protection (*11, OR = 0.8, Pc = 2.7×10⁻², *16∶01-*05∶02 OR = 0.6, Pc = 4.8×10⁻¹⁶, *14∶01-4-*05∶031 = OR = 0.5, Pc = 9.8×10⁻⁴ and *15∶02-*06∶01 OR = 0.4, Pc = 5.1×10⁻⁴). The relative predispositional effect method confirms all the positively associated haplotypes and showed that also *08 and *04 haplotypes confers susceptibility, while the *11 was excluded as protective haplotype. Genotypic ORs highlighted two typologies of interaction between haplotypes: i) a neutral interaction, in which the global risk is coherent with the sum of the single haplotype risks; ii) a negative interaction, in which the genotypic OR observed is lower than the sum of the OR of the two haplotypes. The phylogenic tree of the MS-associated DRB1 alleles found in Sardinian patients revealed a cluster represented by *14∶01, *04∶05, *13∶03, *08∶01 and *03∶01 alleles. Sequence alignment analysis showed that amino acids near pocket P4 and pocket P9 differentiated protective from predisposing alleles under investigation. Furthermore, molecular dynamics simulation performed on alleles revealed that position 70 is crucial in binding of MBP 85–99 peptide. All together, these data suggest that propensity to MS observed in Sardinian population carried by the various HLA-DRB1-DQB1 molecules can be due to functional peculiarity in the antigen presentation mechanisms.     

Plasmodium vivax Population Structure and Transmission Dynamics in Sabah Malaysia

Despite significant progress in the control of malaria in Malaysia, the complex transmission dynamics of P. vivax continue to challenge national efforts to achieve elimination. To assess the impact of ongoing interventions on P. vivax transmission dynamics in Sabah, we genotyped 9 short tandem repeat markers in a total of 97 isolates (8 recurrences) from across Sabah, with a focus on two districts, Kota Marudu (KM, n = 24) and Kota Kinabalu (KK, n = 21), over a 2 year period. STRUCTURE analysis on the Sabah-wide dataset demonstrated multiple sub-populations. Significant differentiation (F _ST  = 0.243) was observed between KM and KK, located just 130 Km apart. Consistent with low endemic transmission, infection complexity was modest in both KM (mean MOI  = 1.38) and KK (mean MOI  = 1.19). However, population diversity remained moderate (H _E  = 0.583 in KM and H _E  = 0.667 in KK). Temporal trends revealed clonal expansions reflecting epidemic transmission dynamics. The haplotypes of these isolates declined in frequency over time, but persisted at low frequency throughout the study duration. A diverse array of low frequency isolates were detected in both KM and KK, some likely reflecting remnants of previous expansions. In accordance with clonal expansions, high levels of Linkage Disequilibrium (I _A ^S >0.5 [P<0.0001] in KK and KM) declined sharply when identical haplotypes were represented once (I _A ^S  = 0.07 [P = 0.0076] in KM, and I _A ^S = -0.003 [P = 0.606] in KK). All 8 recurrences, likely to be relapses, were homologous to the prior infection. These recurrences may promote the persistence of parasite lineages, sustaining local diversity. In summary, Sabah''s shrinking P. vivax population appears to have rendered this low endemic setting vulnerable to epidemic expansions. Migration may play an important role in the introduction of new parasite strains leading to epidemic expansions, with important implications for malaria elimination.     

Depression and Health Related Quality of Life in Adolescent Survivors of a Traumatic Brain Injury: A Pilot Study

Traumatic brain injury is (TBI) a leading cause of morbidity and mortality in youth. Adult survivors of a severe pediatric TBI are vulnerable to global impairments, including greater employment difficulties, poor quality of life (HRQoL) and increased risk of mental health problems. When estimating the health related quality of life in adolescents, the presence of anxiety and depression and the quality of social relationships are important considerations, because adolescents are entrenched in social development during this phase of maturation. The influence of anxiety, depression and loneliness on health related quality of life in adolescent survivors of TBI has not been documented. This pilot study aimed to identify and measure the relationship between anxiety, depression and loneliness and perceived health related quality of life in adolescent survivors of a TBI. Method: mixed method/cohort pilot study (11 adolescents, mild to severe TBI; 9 parents), using self-report and proxy-report measures of anxiety, depression, health related quality of life, loneliness and clinical psychiatric interviews (adolescent only). Results: Self-reported depression was significantly correlated with self-reported HRQoL (rs [11] = −0.88, p<0.001). Age at injury was significantly correlated with self-reported HRQoL (rs [11] = −0.68, p = 0.02). Self-reported depression predicted self-reported HRQoL (R² = 0.79, F [1, 10] = 33.48, p<0.001), but age at injury did not (R² = 0.19, F [1, 10] = 2.09, p = 0.18). Conclusions: Our results suggest that depression is a predictor of health related quality of life in youth post-TBI. The possibility of using targeted assessment and therapy for depression post-TBI to improve health related quality of life should be explored.