Weiming Zhong

Noroviruses bind to human ABO, Lewis, and secretor histo-blood group antigens: Identification of 4 distinct strain-specific patterns

We characterized the binding of 8 Noroviruses (NORs) to histo-blood group antigens (HBGAs) in human saliva using recombinant NOR (rNOR) capsid proteins. Among the 8 rNORs tested, 6 formed viruslike particles (VLPs) when the capsid proteins were expressed in insect cells, all of which revealed variable binding activities with saliva; the remaining 2 rNORs did not form VLPs, and the proteins did not bind, or bound weakly, to saliva. Four distinct binding patterns were associated with different histo-blood types, defined by Lewis, secretor, and ABO types. Three patterns (VA387, NV, and MOH) recognized secretors, and 1 pattern (VA207) recognized Lewis-positive nonsecretors. The 3 secretor-recognizing patterns were defined as A/B (MOH), A/O (NV), and A/B/O (VA387) binders. Oligosaccharides containing the Lewis and ABH antigenic epitopes were involved in binding. Our findings suggest that different strains of NORs may recognize different human HBGAs on intestinal epithelial cells as receptors for infection.

Norovirus and Histo-Blood Group Antigens: Demonstration of a Wide Spectrum of Strain Specificities and Classification of Two Major Binding Groups among Multiple Binding Patterns

ABSTRACT Noroviruses, an important cause of acute gastroenteritis, have been found to recognize human histo-blood group antigens (HBGAs) as receptors. Four strain-specific binding patterns to HBGAs have been described in our previous report. In this study, we have extended the binding patterns to seven based on 14 noroviruses examined. The oligosaccharide-based assays revealed additional epitopes that were not detected by the saliva-based assays. The seven patterns have been classified into two groups according to their interactions with three major epitopes (A/B, H, and Lewis) of human HBGAs: the A/B-binding group and the Lewis-binding group. Strains in the A/B binding group recognize the A and/or B and H antigens, but not the Lewis antigens, while strains in the Lewis-binding group react only to the Lewis and/or H antigens. This classification also resulted in a model of the norovirus/HBGA interaction. Phylogenetic analyses showed that strains with identical or closely related binding patterns tend to be clustered, but strains in both binding group can be found in both genogroups I and II. Our results suggest that noroviruses have a wide spectrum of host range and that human HBGAs play an important role in norovirus evolution. The high polymorphism of the human HBGA system, the involvement of multiple epitopes, and the typical protein/carbohydrate interaction between norovirus VLPs and HBGAs provide an explanation for the virus-ligand binding diversities.

Genetic diversity among sapoviruses

Summary.Norovirus and Sapovirus are two genera of the family Caliciviridae that contain viruses that can cause acute gastroenteritis in humans. Noroviruses (NOR) are genetically highly diverse but limited studies of the genetic diversity of sapoviruses (SAP) have been reported. In this study we characterized twenty-five SAP detected in our laboratory from outbreaks or sporadic cases of acute gastroenteritis in children from different geographical locations and in adults involved in a cruise ship outbreak investigation and a nursing home outbreak. Based on significant differences of partial RNA polymerase sequences (278–286 nt), the 25 strains were grouped into 12 genetic clusters, including 9 potential new clusters. Extended sequence analysis of the capsid gene of selected strains representing five potential new clusters supported this grouping. Four strains (Hou7-1181/90, Mex340/90, Cruise ship/00 and Argentina39) had <84% amino acid (aa) identity to each other and to the published sequences in the GenBank. Mex14917/00 was almost identical to Stockholm/97/SE whose RNA polymerase sequence was unknown. Phylogenetic and distance analyses of the capsid region of the four new strains showed that Hou7-1181/90 and Argentina39 represent two new genogroups and Mex340/90 and Cruise ship/00 belong to two new clusters within the London/92 genogroup. Thus, based on the capsid sequences we propose to classify the currently known SAP into nine genetic clusters within five genogroups, including one genogroup that is represented by an animal calicivirus, the porcine enteric calicivirus (PEC).

Mutations within the P2 Domain of Norovirus Capsid Affect Binding to Human Histo-Blood Group Antigens: Evidence for a Binding Pocket

ABSTRACT Noroviruses (NORs) are an important cause of acute gastroenteritis. Recent studies of NOR receptors showed that different NORs bind to different histo-blood group antigens (HBGAs), and at least four distinct binding patterns were observed. To determine the structure-function relationship for NORs and their receptors, two strains representing two of the four binding patterns were studied. Strain VA387 binds to HBGAs of A, B, and O secretors, whereas strain MOH binds to HBGAs of A and B secretors only. Using multiple sequence alignments, homology modeling, and structural analysis of NOR capsids, we identified a plausible “pocket” in the P2 domain that may be responsible for binding to HBGA receptors. This pocket consists of a conserved RGD/K motif surrounded by three strain-specific hot spots (N302, T337, and Q375 for VA387 and N302, N338, and E378 for MOH). Subsequent mutagenesis experiments demonstrated that all four sites played important roles in binding. A single amino acid mutation at T337 (to A) in VA387 or a double amino acid mutation at RN338 (to TT) in MOH abolished binding completely. Change of the entire RGD motif to SAS abolished binding in case of VA387, whereas single amino acid mutations in that motif did not have an apparent effect on binding to A and B antigens but decreased binding to H antigen. Multiple mutations at the RGK motif of MOH (SIRGK to TFRGD) completely knocked out the binding. Mutation of N302 or Q375 in VA387 affected binding to type O HBGA only, while switch mutants with three amino acid changes at either site from MOH to VA387 resulted in a weak binding to type O HBGAs. A further switch mutant with three amino acid changes at E378 from MOH to VA387 diminished the binding to type A HBGA only. Taken together, our data indicate that the binding pocket likely exists on NOR capsids. Direct evidence of this hypothesis requires crystallography studies.

Norovirus P Particle, a Novel Platform for Vaccine Development and Antibody Production

ABSTRACT The norovirus P particle is an octahedral nanoparticle formed by 24 copies of the protrusion (P) domain of the norovirus capsid protein. This P particle is easily produced in Escherichia coli, extremely stable, and highly immunogenic. There are three surface loops per P domain, making a total of 72 loops per particle, and these are potential sites for foreign antigen presentation for immune enhancement. To prove this concept, a small peptide (His tag, 7 amino acids [aa]) and a large antigen (rotavirus VP8, 159 aa) were inserted into one of the loops. Neither insertion affects P particle formation, while both antigens were presented well on the P particle surface. The immune-enhancement effect of the P particle was demonstrated by significantly increased antibody titers induced by the P particle-presented antigens compared to the titers induced by free antigens. In addition, the measured neutralization antibody titers and levels of protection against rotavirus shedding in mice immunized with the VP8 chimeric P particles were significantly higher than those of mice immunized with the free VP8 antigen. Sera from P particle-VP8 chimera-vaccinated animals also blocked norovirus virus-like particle (VLP) binding to the histo-blood group antigen (HBGA) receptors. From these data, the P particle appears to be an excellent vaccine platform for antigen presentation. The readily available three surface loops and the great capacity for foreign antigen insertion make this platform attractive for wide application in vaccine development and antibody production. The P particle-VP8 chimeras may serve as a dual vaccine against both rotavirus and norovirus.

Predicting Susceptibility to Norovirus GII.4 by Use of a Challenge Model Involving Humans

BACKGROUND GII.4 is the predominant norovirus genotype worldwide. Challenge models involving humans have shown the association of human histo-blood group antigens (HBGAs) and susceptibility to infection with Norwalk virus (GI.1 norovirus), but the association of HBGAs and infection with other noroviruses is based on results of epidemiological studies. We performed the first GII.4 challenge study involving humans and prospectively evaluated the relationship between HBGAs and norovirus infection and associated illness. METHODS Forty healthy adults (23 secretors and 17 nonsecretors of HBGAs) were challenged with 5 10(4) reverse-transcription polymerase chain reaction (RT-PCR) units of GII.4 norovirus. Subjects were assessed daily for clinical illness, and stool specimens were evaluated for norovirus by RT-PCR. Infection was defined by detection of norovirus and/or seroconversion to GII.4 antibody. RESULTS Of the 23 secretors, 16 (70%) were infected with norovirus, 13 (57%) became ill (characterized by vomiting and/or diarrhea), and 12 (52%) developed norovirus-associated illness. In contrast, only 1 nonsecretor (5.9%) became ill, and another nonsecretor shed virus for a single day (P < .001 for each variable, compared with secretors). Infection occurred in secretors regardless of ABO blood group. Illness was mild to moderate in severity and lasted 1-3 days. CONCLUSIONS Secretor status determined the susceptibility to norovirus GII.4 challenge. This human challenge model should be useful for evaluating norovirus vaccines and antiviral agents. Clinical trials registration. NCT01322503.

Seroprevalence of Noroviruses in Swine

ABSTRACT Noroviruses (NVs) are important human pathogens that cause acute gastroenteritis. Genetically related animal enteric NVs have also been described, but there is no evidence of interspecies transmission of NVs. In this study we characterized antibody prevalence among domestic pigs by using recombinant capsid antigens of two human NVs (Norwalk and Hawaii) and one swine NV (SW918) that is genetically related to GII human NVs. Recombinant SW918 capsid protein expressed in baculovirus self-assembled into virus-like particles (VLPs) that were detected by antibodies against GII (Hawaii and Mexico), but not GI (Norwalk and VA115), human NVs. NVs recognize human histo-blood group antigens as receptors, but SW918 VLPs did not bind to human saliva samples with major histo-blood group types. Seventy-eight of 110 (71%) pig serum samples from the United States and 95 of 266 (36%) pig serum samples from Japan possessed antibodies against SW918. Serum samples from pigs in the United States were also tested for antibodies against human NVs; 63% were positive for Norwalk virus (GI) and 52% for Hawaii virus (GII). These results indicate that NV infections are common among domestic pigs; the finding of antigenic relationships between SW918 and human NVs and the detection of antibodies against both GI and GII human NVs in domestic animals highlights the importance of further studies on NV gastroenteritis as a possible zoonotic disease.

Gastroenteritis in US Marines during Operation Iraqi Freedom

BACKGROUND Approximately 83,000 US Marines participated in the opening phase of Operation Iraqi Freedom in Spring 2003. A Navy Preventive Medicine laboratory was set up in Ad Diwaniyah, Iraq, to provide clinical diagnostic support for Marine medical units during a period of repositioning in south-central Iraq. METHODS Specimen collection boxes were sent to >30 primary care medical stations handling 500-900 personnel each. The laboratory had capability to detect many different disease agents, especially those causing febrile illness. Diarrheal stool diagnostic evaluation included plating and biochemical identification, antigen serologic testing, fluorescent antibody antigen detection, disk diffusion antimicrobial susceptibility testing, enzyme immunoassay, and reverse-transcriptase polymerase chain reaction for norovirus (NV). Confirmation and sequencing work for NV was done at Cincinnati Childrens Hospital Medical Center (Ohio). RESULTS By far the most common reason for infectious disease sick call visits was gastrointestinal illness; no other symptoms had equivalent impact. An enteropathogen was detected in 57 (44%) of 129 stool samples, with NV detected in 30 stool samples (23%) obtained from 14 different battalion or similar-sized units; next in frequency were Shigella flexneri and Shigella sonnei, which were isolated from 26 stool samples (20%) obtained from 15 units. Sequencing the NV RNA polymerase gene demonstrated that NV strains represented 7 genetic clusters, including 2 strains from genogroup I and 5 from genogroup II. Ciprofloxacin was effective in vitro against most bacterial agents, but neither doxycyline (which was taken daily as the antimalarial prophylaxis dose) nor trimethoprim-sulfamethoxazole were effective. CONCLUSIONS Multiple strains of Shigella species and NV predominated, probably because they do not require a large inoculum to cause infection. Otherwise, personnel remained free of infectious illness during this phase of the conflict, because other infectious agents were rare or absent.

Human Milk Contains Elements That Block Binding of Noroviruses to Human Histo—Blood Group Antigens in Saliva

Noroviruses (NVs) recognize human histo-blood group antigens (HBGAs) as receptors. We characterized the interaction of human milk samples with recombinant virus-like particles representing VA387, Norwalk, VA207, and MOH. Milk samples from 60 healthy women were tested for human HBGAs and for their ability to block the binding of NVs. Fifty-four women were secretors (Se+), and 6 were nonsecretors (Se-). No women had detectable A or B antigens in their milk samples. All 54 Se+ milk samples, but 0 of 6 Se- milk samples, blocked VA387 and Norwalk virus (Se+ binders) from binding to saliva samples. All 6 Lewis-positive Se- milk samples blocked binding to VA207, and variable blocking activities were exhibited by the Se+ milk samples. No milk samples blocked the binding of MOH to A and B antigens. Secretor and Lewis, but not A or B antigens, were present in human milk and were responsible for blocking NV binding to receptors and therefore are likely to be decoy receptors that protect breast-fed infants from NV infection.

Genetic and Phenotypic Characterization of GII-4 Noroviruses That Circulated during 1987 to 2008

ABSTRACT The predominance and continual emergence of new variants in GII-4 noroviruses (NVs) in recent years have raised questions about the role of host immunity and histo-blood group antigens (HBGAs) in NV evolution. To address these questions, we performed a genetic and phenotypic characterization of GII-4 variants circulating in the past decade (1998 to 2008). Ninety-three GII-4 sequences were analyzed, and of them, 16 strains representing 6 genetic clusters were selected for further characterization. The HBGA binding properties were determined by both saliva- and oligosaccharide-binding assays using P particles as a model of NV capsid. The antigenic properties were also examined by enzyme immunoassay (EIA), Western blot analysis, and receptor blocking assay, using P-particle-specific antibodies from immunized mice and GII-4 virus-infected patients. Our results showed that 15 of the 16 GII-4 viruses bound to saliva of all A, B, and O secretors. Oligosaccharide binding assays yielded largely consistent results, although the binding affinities to some oligosaccharides varied among some strains. The only nonbinder had a mutation in the binding site. While antigenic variations were detected among the 16 strains, significant cross-blocking on the HBGA binding was also noted. Sequence alignment revealed high conservation of HBGA binding interfaces with some variations in adjacent regions. Taken together, our data suggested that the ability of GII-4 to recognize different secretor HBGAs persisted over the past decade, which may explain the predominance of GII-4 over other genotypes. Our data also indicated that both the host immunity and HBGAs play a role in NV evolution. While host immunity may continue driving NV for antigenic change, the functional selection by the HBGAs tends to lock the architecture of the capsid/HBGA interfaces and allows only limited variations outside the HBGA binding sites. A potential outcome of such counterselection between theses two factors in NV evolution is discussed.