Analysis of Archival Sera from Norovirus-Infected Individuals Demonstrates that Cross-Blocking of Emerging Viruses is Genotype-Specific.

BACKGROUND: Rapidly evolving RNA viruses, such as human norovirus, generate extraordinary sequence diversity, posing a significant challenge to vaccine design. This diversity coupled with short-lasting natural immunity leads to re-infection throughout one's lifetime. How re-exposure shapes humoral immunity to future norovirus strains remains incompletely understood. METHODS: We profiled the antibody responses following two community gastroenteritis outbreaks with GII.2 and GII.6 noroviruses in 1971. Using diverse VLPs, ELISA, and carbohydrate-blocking assays (surrogate for neutralization), we examined the antibody response at acute and convalescent timepoints following GII.6 infection. RESULTS: Convalescent sera displayed strong homologous blocking, demonstrating a 5-fold increase in GII.6 carbohydrate-blockade over acute samples, and broad blocking of diverse archival and modern GII.6 noroviruses. Convalescent sera displayed limited carbohydrate-blocking of heterotypic VLPs, despite high ELISA binding titers. Select individuals developed broad cross-genotype blockade, but this response was established before the second outbreak. Finally, we applied a novel competitive carbohydrate-blocking assay to demonstrate the epitope-specificity and discrete compartments of the neutralizing response. CONCLUSIONS: Our data show that infection generates narrow, focused immunity directed towards the infecting genotype. We did detect broad cross-blocking in specific individuals, but these responses could be attributed to diverse, genotype-specific antibodies pre-dating GII.6 infection.

Antigenic Characterization of Novel Human Norovirus GII.4 Variants San Francisco 2017 and Hong Kong 2019.

Norovirus is a major cause of acute gastroenteritis; GII.4 is the predominant strain in humans. Recently, 2 new GII.4 variants, Hong Kong 2019 and San Francisco 2017, were reported. Characterization using GII.4 monoclonal antibodies and serum demonstrated different antigenic profiles for the new variants compared with historical variants.

Minimal Antigenic Evolution after a Decade of Norovirus GII.4 Sydney_2012 Circulation in Humans.

Norovirus is a major human pathogen that can cause severe gastroenteritis in vulnerable populations. The extensive viral diversity presented by human noroviruses constitutes a major roadblock for the development of effective vaccines. In addition to the large number of genotypes, antigenically distinct variants of GII.4 noroviruses have chronologically emerged over the last 3 decades. The last variant to emerge, Sydney_2012, has been circulating at high incidence worldwide for over a decade. We analyzed 1449 capsid sequences from GII.4 Sydney_2012 viruses to determine genetic changes indicative of antigenic diversification. Phylogenetic analyses show that Sydney_2012 viruses scattered within the tree topology with no single cluster dominating during a given year or geographical location. Fourteen residues presented high variability, 7 of which mapped to 4 antigenic sites. Notably, ~52% of viruses presented mutations at 2 or more antigenic sites. Mutational patterns showed that residues 297 and 372, which map to antigenic site A, changed over time. Virus-like particles (VLPs) developed from wild-type Sydney_2012 viruses and engineered to display all mutations detected at antigenic sites were tested against polyclonal sera and monoclonal antibodies raised against Sydney_2012 and Farmington_Hills_2002 VLPs. Minimal changes in reactivity were detected with polyclonal sera and only 4 MAbs lost binding, with all mapping to antigenic site A. Notably, reversion of residues from Sydney_2012 reconstituted epitopes from ancestral GII.4 variants. Overall, this study demonstrates that, despite circulating for over a decade, Sydney_2012 viruses present minimal antigenic diversification and provides novel insights on the diversification of GII.4 noroviruses that could inform vaccine design. IMPORTANCE GII.4 noroviruses are the major cause of acute gastroenteritis in all age groups. This predominance has been attributed to the continued emergence of phylogenetically discrete variants that escape immune responses to previous infections. The last GII.4 variant to emerge, Sydney_2012, has been circulating at high incidence for over a decade, raising the question of whether this variant is undergoing antigenic diversification without presenting a major distinction at the phylogenetic level. Sequence analyses that include >1400 capsid sequences from GII.4 Sydney_2012 showed changes in 4 out of the 6 major antigenic sites. Notably, while changes were detected in one of the most immunodominant sites over time, these resulted in minimal changes in the antigenic profile of these viruses. This study provides new insights on the mechanism governing the antigenic diversification of GII.4 norovirus that could help in the development of cross-protective vaccines to human noroviruses.

Antigenic cartography reveals complexities of genetic determinants that lead to antigenic differences among pandemic GII.4 noroviruses.

Noroviruses are the predominant cause of acute gastroenteritis, with a single genotype (GII.4) responsible for the majority of infections. This prevalence is characterized by the periodic emergence of new variants that present substitutions at antigenic sites of the major structural protein (VP1), facilitating escape from herd immunity. Notably, the contribution of intravariant mutations to changes in antigenic properties is unknown. We performed a comprehensive antigenic analysis on a virus-like particle panel representing major chronological GII.4 variants to investigate diversification at the inter- and intravariant level. Immunoassays, neutralization data, and cartography analyses showed antigenic similarities between phylogenetically related variants, with major switches to antigenic properties observed over the evolution of GII.4 variants. Genetic analysis indicated that multiple coevolving amino acid changes-primarily at antigenic sites-are associated with the antigenic diversification of GII.4 variants. These data highlight complexities of the genetic determinants and provide a framework for the antigenic characterization of emerging GII.4 noroviruses.

Genome-wide analyses of human noroviruses provide insights on evolutionary dynamics and evidence of coexisting viral populations evolving under recombination constraints.

Norovirus is a major cause of acute gastroenteritis worldwide. Over 30 different genotypes, mostly from genogroup I (GI) and II (GII), have been shown to infect humans. Despite three decades of genome sequencing, our understanding of the role of genomic diversification across continents and time is incomplete. To close the spatiotemporal gap of genomic information of human noroviruses, we conducted a large-scale genome-wide analyses that included the nearly full-length sequencing of 281 archival viruses circulating since the 1970s in over 10 countries from four continents, with a major emphasis on norovirus genotypes that are currently underrepresented in public genome databases. We provided new genome information for 24 distinct genotypes, including the oldest genome information from 12 norovirus genotypes. Analyses of this new genomic information, together with those publicly available, showed that (i) noroviruses evolve at similar rates across genomic regions and genotypes; (ii) emerging viruses evolved from transiently-circulating intermediate viruses; (iii) diversifying selection on the VP1 protein was recorded in genotypes with multiple variants; (iv) non-structural proteins showed a similar branching on their phylogenetic trees; and (v) contrary to the current understanding, there are restrictions on the ability to recombine different genomic regions, which results in co-circulating populations of viruses evolving independently in human communities. This study provides a comprehensive genetic analysis of diverse norovirus genotypes and the role of non-structural proteins on viral diversification, shedding new light on the mechanisms of norovirus evolution and transmission.

Global and regional circulation trends of norovirus genotypes and recombinants, 1995-2019: A comprehensive review of sequences from public databases.

Human noroviruses are the leading global cause of viral gastroenteritis. Attempts at developing effective vaccines and treatments against norovirus disease have been stymied by the extreme genetic diversity and rapid geographic distribution of these viruses. The emergence and replacement of predominantly circulating norovirus genotypes has primarily been attributed to mutations on the VP1 capsid protein leading to genetic drift, and more recently to recombination events between the ORF1/ORF2 junction. However, large-scale research into the historical and geographic distribution of recombinant norovirus strains has been limited in the literature. We performed a comprehensive historical analysis on 30,810 human norovirus sequences submitted to public databases between the years 1995 and 2019. During this time, 37 capsid genotypes and 56 polymerase types were detected across 90 different countries, and 97 unique recombinant genomes were also identified. GII.4, both capsid and polymerase, was the predominately circulating type worldwide for the majority of this time span, save for a brief swell of GII.17 and GII.2 capsid genotypes and a near-total eclipse by GII.P16, GII.P21 and GII.P31 beginning in 2013. Interestingly, an analysis of 4067 recombinants found that 50.2% (N = 2039) of all recorded sequences belonged to three recently emerged recombinant strains: GII.2[P16], GII.4[P31], and GII.4[P16]. This analysis should provide an important historical foundation for future studies that evaluate the emergence and distribution of noroviruses, as well as the design of cross-protective vaccines.

Genotype-Specific Neutralization of Norovirus Is Mediated by Antibodies Against the Protruding Domain of the Major Capsid Protein.

Human noroviruses are the most common viral agents of acute gastroenteritis. Recently, human intestinal enteroids were shown to be permissive for norovirus infection. We tested their suitability as a system to study norovirus neutralization. Hyperimmune sera raised against virus-like particles (VLPs) representing different genotypes showed highly specific neutralization activity against GII.4 and GII.6 noroviruses. Carbohydrate blocking assays and neutralization exhibited similar patterns in antibody responses. Notably, sera produced against chimeric VLPs that presented swapped structural shell and protruding (P) domains, from different genotypes showed that neutralization is primarily mediated by antibodies mapping to the P domain of the norovirus capsid protein. This study provides empirical information on the antigenic differences among genotypes as measured by neutralization, which could guide vaccine design.

Recombinant Nontypeable Genotype II Human Noroviruses in the Americas.

We report multiple nontypeable genotype II noroviruses circulating in South America; nucleotides differed by >25% from those of other genotypes. These viruses have been circulating in the Americas for ≈20 years and show recombination with other genotypes. Clues to norovirus natural history can guide development of treatment and prevention plans.

Neutralizing Antibody Responses to Homologous and Heterologous H1 and H3 Influenza A Strains After Vaccination With Inactivated Trivalent Influenza Vaccine Vary With Age and Prior-year Vaccination.

BACKGROUND: Prior influenza immunity influences the homologous neutralizing antibody responses elicited by inactivated influenza vaccines (IIV), but neutralizing antibody responses to heterologous strains have not been extensively characterized. METHODS: We analyzed neutralizing antibody titers in individuals aged 1-88 who received the 2009-2010 season IIV before infection by or vaccination against the 2009 pandemic H1N1 virus. Neutralization titers to homologous and heterologous past, recent, and advanced H1 and H3 strains, as well as H2, H5, and H7 strains, were measured using influenza hemagglutinin pseudoviruses. We performed exploratory analyses based on age, prior-year IIV, and prevaccination titer, without controlling for Type I errors. RESULTS: IIV elicited neutralizing antibodies to past and advanced H1 and H3 strains, as well as to an H2 strain in individuals who were likely infected early in life. The neutralization of avian subtype viruses was rare, and there was no imprinting of neutralization responses to novel avian subtype viruses based on the influenza group. Compared to adults, children had higher seroresponse rates to homologous and heterologous strains, and their sera generated larger antigenic distances among strains. Seroresponse rates to homologous and heterologous strains were lower in subjects vaccinated with prior-year IIV, though postimmunization titers were generally high. CONCLUSIONS: IIV elicited neutralizing antibodies to heterologous H1 and H3 strains in all ages groups, but titers and seroresponse rates were usually higher in children. Prior-year vaccination with the same strains tended to blunt IIV neutralization responses to all strains in young and old age groups, yet postimmunization titers were high.

Updated classification of norovirus genogroups and genotypes.

Noroviruses are genetically diverse RNA viruses associated with acute gastroenteritis in mammalian hosts. Phylogenetically, they can be segregated into different genogroups as well as P (polymerase)-groups and further into genotypes and P-types based on amino acid diversity of the complete VP1 gene and nucleotide diversity of the RNA-dependent RNA polymerase (RdRp) region of ORF1, respectively. In recent years, several new noroviruses have been reported that warrant an update of the existing classification scheme. Using previously described 2× standard deviation (sd) criteria to group sequences into separate clusters, we expanded the number of genogroups to 10 (GI-GX) and the number of genotypes to 48 (9 GI, 27 GII, 3 GIII, 2 GIV, 2 GV, 2 GVI and 1 genotype each for GVII, GVIII, GIX [formerly GII.15] and GX). Viruses for which currently only one sequence is available in public databases were classified into tentative new genogroups (GNA1 and GNA2) and genotypes (GII.NA1, GII.NA2 and GIV.NA1) with their definitive assignment awaiting additional related sequences. Based on nucleotide diversity in the RdRp region, noroviruses can be divided into 60 P-types (14 GI, 37 GII, 2 GIII, 1 GIV, 2 GV, 2 GVI, 1 GVII and 1 GX), 2 tentative P-groups and 14 tentative P-types. Future classification and nomenclature updates will be based on complete genome sequences and will be coordinated and disseminated by the international norovirus classification-working group.

Static and Evolving Norovirus Genotypes: Implications for Epidemiology and Immunity.

Noroviruses are major pathogens associated with acute gastroenteritis worldwide. Their RNA genomes are diverse, with two major genogroups (GI and GII) comprised of at least 28 genotypes associated with human disease. To elucidate mechanisms underlying norovirus diversity and evolution, we used a large-scale genomics approach to analyze human norovirus sequences. Comparison of over 2000 nearly full-length ORF2 sequences representing most of the known GI and GII genotypes infecting humans showed a limited number (≤5) of distinct intra-genotypic variants within each genotype, with the exception of GII.4. The non-GII.4 genotypes were comprised of one or more intra-genotypic variants, with each variant containing strains that differed by only a few residues over several decades (remaining "static") and that have co-circulated with no clear epidemiologic pattern. In contrast, the GII.4 genotype presented the largest number of variants (>10) that have evolved over time with a clear pattern of periodic variant replacement. To expand our understanding of these two patterns of diversification ("static" versus "evolving"), we analyzed using NGS the nearly full-length norovirus genome in healthy individuals infected with GII.4, GII.6 or GII.17 viruses in different outbreak settings. The GII.4 viruses accumulated mutations rapidly within and between hosts, while the GII.6 and GII.17 viruses remained relatively stable, consistent with their diversification patterns. Further analysis of genetic relationships and natural history patterns identified groupings of certain genotypes into larger related clusters designated here as "immunotypes". We propose that "immunotypes" and their evolutionary patterns influence the prevalence of a particular norovirus genotype in the human population.

Evolutionary dynamics of non-GII genotype 4 (GII.4) noroviruses reveal limited and independent diversification of variants.

Noroviruses are extremely diverse, with ≥30 genotypes infecting humans. GII genotype 4 (GII.4) noroviruses, the most prevalent genotype, present a constant accumulation of mutations on the major capsid protein (VP1), resulting in the chronological emergence of new variants every 2-8 years. On the other hand, non-GII.4 noroviruses present a limited number of changes on the capsid protein over time. Despite limited diversification, non-GII.4 viruses can also be associated with large outbreaks. To gain insights into the evolutionary dynamics of non-GII.4 viruses, we performed variant-specific phylogenetic analyses on a comprehensive dataset of 13 genotypes. Although the genotypes with a single variant presented a linear (clock-like) evolution, maximum-likelihood analyses revealed a lack of clock-like signals for the genotypes with ≥3 variants: GI.3, GII.6 and GII.17. Notably, the evolutionary pattern of non-GII.4 viruses showed clock-like signals when each variant was analysed separately. A minimal impact on the long-term clock-like evolution of VP1 was detected due to the exchange (recombination) of the polymerase types. The linear evolution, without replacement among variants, is explained by minimal changes at the protein level due to the higher ratio of synonymous compared to non-synonymous substitutions in their evolution. Taken together, these data indicate that (i) the variants of non-GII.4 noroviruses evolve and persist in the population independently, probably due to strong evolutionary constraints on VP1, and (ii) variant-specific analyses with robust sequence databases that cover long periods of surveillance are needed to limit the potential for misinterpretation of the evolutionary dynamics of non-GII.4 noroviruses.

Genetic linkage of capsid protein-encoding RNA segments in group A equine rotaviruses.

Rotavirus virions are formed by three concentric protein layers that enclose the 11 dsRNA genome segments and the viral proteins VP1 and VP3. Interactions amongst the capsid proteins (VP2, VP6, VP7 and VP4) have been described to play a major role in viral fitness, whilst restricting the reassortment of the genomic segments during co-infection with different rotavirus strains. In this work we describe and characterize the linkage between VP6 and VP7 proteins based on structural and genomic analyses of group A rotavirus strains circulating in Argentinean horses. Strains with the VP7 genotype G3 showed a strong association with the VP6 genotype I6, whilst strains with G14 were associated with the I2 genotype. Most of the differences on the VP6 and VP7 proteins were observed in interactive regions between the two proteins, suggesting that VP6 : VP7 interactions may drive the co-evolution and co-segregation of their respective gene segments.

Genome of Emerging Norovirus GII.17, United States, 2014.

To determine whether the norovirus strain GII.17 recently detected in Maryland, USA, (Hu/GII.17/Gaithersburg/2014/US) is spreading globally, we characterized the genome. High similarity with the norovirus GII.17 that caused recent outbreaks in Asia indicates that the same strain was present in the United States during the 2014-15 norovirus season (winter).

Sequential gastroenteritis episodes caused by 2 norovirus genotypes.

We investigated sequential episodes of acute norovirus gastroenteritis in a young child within an 11-month period. The infections were caused by 2 distinct genotypes (GII.4 and GII.6). Failure to achieve cross-protective immunity was linked to absence of an enduring and cross-reactive mucosal immune response, a critical consideration for vaccine design.

The feline calicivirus leader of the capsid protein is associated with cytopathic effect.

Open reading frame 2 (ORF2) of the feline calicivirus (FCV) genome encodes a capsid precursor that is posttranslationally processed to release the mature capsid protein (VP1) and a small protein of 124 amino acids, designated the leader of the capsid (LC). To investigate the role of the LC protein in the virus life cycle, mutations and deletions were introduced into the LC coding region of an infectious FCV cDNA clone. Three cysteine residues that are conserved among all vesivirus LC sequences were found to be critical for the recovery of FCV with a characteristic cytopathic effect in feline kidney cells. A cell-rounding phenotype associated with the transient expression of wild-type and mutagenized forms of the LC correlated with the cytopathic and growth properties of the corresponding engineered viruses. The host cellular protein annexin A2 was identified as a binding partner of the LC protein, consistent with a role for the LC in mediating host cell interactions that alter the integrity of the cell and enable virus spread.

Development of Norwalk virus-specific monoclonal antibodies with therapeutic potential for the treatment of Norwalk virus gastroenteritis.

Passive immunoprophylaxis or immunotherapy with norovirus-neutralizing monoclonal antibodies (MAbs) could be a useful treatment for high-risk populations, including infants and young children, the elderly, and certain patients who are debilitated or immunocompromised. In order to obtain antinorovirus MAbs with therapeutic potential, we stimulated a strong adaptive immune response in chimpanzees to the prototype norovirus strain Norwalk virus (NV) (genogroup I.1). A combinatorial phage Fab display library derived from mRNA of the chimpanzees' bone marrow was prepared, and four distinct Fabs reactive with Norwalk recombinant virus-like particles (rVLPs) were recovered, with estimated binding affinities in the subnanomolar range. Mapping studies showed that the four Fabs recognized three different conformational epitopes in the protruding (P) domain of NV VP1, the major capsid protein. The epitope of one of the Fabs, G4, was further mapped to a specific site involving a key amino acid residue, Gly365. One additional specific Fab (F11) was recovered months later from immortalized memory B cells and partially characterized. The anti-NV Fabs were converted into full-length IgG (MAbs) with human γ1 heavy chain constant regions. The anti-NV MAbs were tested in the two available surrogate assays for Norwalk virus neutralization, which showed that the MAbs could block carbohydrate binding and inhibit hemagglutination by NV rVLP. By mixing a single MAb with live Norwalk virus prior to challenge, MAbs D8 and B7 neutralized the virus and prevented infection in a chimpanzee. Because chimpanzee immunoglobulins are virtually identical to human immunoglobulins, these chimpanzee anticapsid MAbs may have a clinical application.

Genetic characterization of feline calicivirus strains associated with varying disease manifestations during an outbreak season in Missouri (1995-1996).

Feline calicivirus (FCV) is a common cause of mild to severe upper respiratory tract disease (URTD) in cats. FCV strain 21223 was isolated from a kitten with severe pneumonia in a disease outbreak with unusually high mortality (35 %) that occurred in a Missouri feline colony in 1995-1996. Phylogenetic analysis of the genome sequence of strain 21223 indicated the emergence of a new FCV strain. Analysis of the full-length genome sequence of a closely related (99.5 % nucleotide identity) strain, 3786, obtained from an asymptomatic animal in the same colony four months later, showed the presence of seven amino acid substitutions, with six of them located in the VP1 capsid sequence encoded by ORF2. Comparative analysis of the E-region sequences (426-521 aa ORF2) presumably involved in virus-host cell receptor interactions did not identify amino acid substitutions unique to the virulent strain. We determined the complete genome sequences of four virus isolates that were collected in regional catteries in the months following the outbreak that were associated with different manifestations of the disease (URTD, chronic stomatitis, and gingivitis). We show that genetically distinct FCV strains were cocirculating in the area, and no apparent correlation could be made between overall sequence and observed disease.

Chimpanzees as an animal model for human norovirus infection and vaccine development.

Noroviruses are global agents of acute gastroenteritis, but the development of control strategies has been hampered by the absence of a robust animal model. Studies in chimpanzees have played a key role in the characterization of several fastidious hepatitis viruses, and we investigated the feasibility of such studies for the noroviruses. Seronegative chimpanzees inoculated i.v. with the human norovirus strain Norwalk virus (NV) did not show clinical signs of gastroenteritis, but the onset and duration of virus shedding in stool and serum antibody responses were similar to that observed in humans. NV RNA was detected in intestinal and liver biopsies concurrent with the detection of viral shedding in stool, and NV antigen expression was observed in cells of the small intestinal lamina propria. Two infected chimpanzees rechallenged 4, 10, or 24 mo later with NV were resistant to reinfection, and the presence of NV-specific serum antibodies correlated with protection. We evaluated the immunogenicity and efficacy of virus-like particles (VLPs) derived from NV (genogroup I, GI) and MD145 (genogroup II, GII) noroviruses as vaccines. Chimpanzees vaccinated intramuscularly with GI VLPs were protected from NV infection when challenged 2 and 18 mo after vaccination, whereas chimpanzees that received GII VLPs vaccine or a placebo were not. This study establishes the chimpanzee as a viable animal model for the study of norovirus replication and immunity, and shows that NV VLP vaccines could induce protective homologous immunity even after extended periods of time.