Pandemic GII Research Articles

Human Monoclonal Antibodies That Neutralize Pandemic GII.4 Noroviruses

Human noroviruses are responsible for approximately 200,000 deaths worldwide each year. In 2012, the GII.4 Sydney strain emerged and became the major circulating norovirus strain associated with human disease. Our understanding of the human norovirus-specific antibody response is limited because few human monoclonal antibodies (mAbs) to noroviruses have been described, and there are no functional assays to measure virus neutralization. We studied the antibody-mediated response to the genogroup (G) II.4 strain by isolating mAbs to GII.4 from infected patients and developing virus neutralization assays. We used a robust human hybridoma technique to isolate mAbs from patients previously infected with norovirus and identified mAbs that blocked virus binding to cell receptors, using virus-like particles to test blockade ability. We tested the ability of select mAbs to neutralize live human noroviruses using stem cell-derived human enteroids. We isolated a panel of 25 IgG or IgA human mAbs that recognized norovirus GII.4 Sydney 2012 and determined their potential to block virus binding to cell receptors. In competition binding studies, most antibodies recognized 3 major antigenic sites on the GII.4 Sydney 2012 protruding (P) domain. We isolated and characterized human mAbs that neutralize live human norovirus GII.4 Sydney 2012-the predominant strain responsible for recent outbreaks. Analyses of these antibodies identified neutralizing epitopes; further studies will provide insight into the human immune response to this deadly virus.

Higher Viral Load of Emerging Norovirus GII.P16-GII.2 than Pandemic GII.4 and Epidemic GII.17, Hong Kong, China.

We compared viral load of emerging recombinant norovirus GII.P16-GII.2 with those for pandemic GII.Pe-GII.4 and epidemic GII.P17-GII.17 genotypes among inpatients in Hong Kong. Viral load of GII.P16-GII.2 was higher than those for other genotypes in different age groups. GII.P16-GII.2 is as replication competent as the pandemic genotype, explaining its high transmissibility and widespread circulation.

Increased Detection of Emergent Recombinant Norovirus GII.P16-GII.2 Strains in Young Adults, Hong Kong, China, 2016–2017

A new recombinant norovirus GII.P16-GII.2 outnumbered pandemic GII.4 as the predominant GII genotype in the winter of 2016–2017 in Hong Kong, China. Half of hospitalized case-patients were older children and adults, including 13 young adults. This emergent norovirus targets a wider age population compared with circulating pandemic GII.4 strains.

The emerging GII.P16-GII.4 Sydney 2012 norovirus lineage is circulating worldwide, arose by late-2014 and contains polymerase changes that may increase virus transmission.

Noroviruses are a leading cause of human gastroenteritis worldwide. The norovirus genotype GII.4 is the most prevalent genotype in the human population and has caused six pandemics since 1995. A novel norovirus lineage containing the GII.P16 polymerase and pandemic GII.4 Sydney 2012 capsid was recently detected in Asia and Germany. We demonstrate that this lineage is also circulating within the UK and USA and has been circulating since October 2014 or earlier. While the lineage does not contain unique substitutions in the capsid, it does contain polymerase substitutions close to positions known to influence polymerase function and virus transmission. These polymerase substitutions are shared with a GII.P16-GII.2 virus that dominated outbreaks in Germany in Winter 2016. We suggest that the substitutions in the polymerase may have resulted in a more transmissible virus and the combination of this polymerase and the pandemic GII.4 capsid may result in a highly transmissible virus. Further surveillance efforts will be required to determine whether the GII.P16-GII.4 Sydney 2012 lineage increases in frequency over the coming months.

Human Sera Collected between 1979 and 2010 Possess Blocking-Antibody Titers to Pandemic GII.4 Noroviruses Isolated over Three Decades.

The emergence of pandemic GII.4 norovirus (NoV) strains has been proposed to occur due to changes in receptor usage and thereby to lead to immune evasion. To address this hypothesis, we measured the ability of human sera collected between 1979 and 2010 to block glycan binding of four pandemic GII.4 noroviruses isolated in the last 4 decades. In total, 268 sera were investigated for 50% blocking titer (BT50) values of virus-like particles (VLPs) against pig gastric mucin (PGM) using 4 VLPs that represent different GII.4 norovirus variants identified between 1987 and 2012. Pre- and postpandemic sera (sera collected before and after isolation of the reference NoV strain) efficiently prevented binding of VLP strains MD145 (1987), Grimsby (1995), and Houston (2002), but not the Sydney (2012) strain, to PGM. No statistically significant difference in virus-blocking titers was observed between pre- and postpandemic sera. Moreover, paired sera showed that blocking titers of ≥160 were maintained over a 6-year period against MD145, Grimsby, and Houston VLPs. Significantly higher serum blocking titers (geometric mean titer [GMT], 1,704) were found among IgA-deficient individuals than among healthy blood donors (GMT, 90.9) (P < 0.0001). The observation that prepandemic sera possess robust blocking capacity for viruses identified decades later suggests a common attachment factor, at least until 2002. Our results indicate that serum IgG possesses antibody-blocking capacity and that blocking titers can be maintained for at least 6 years against 3 decades of pandemic GII.4 NoV.IMPORTANCE Human noroviruses (NoVs) are the major cause of acute gastroenteritis worldwide. Histo-blood group antigens (HBGAs) in saliva and gut recognize NoV and are the proposed ligands that facilitate infection. Polymorphisms in HBGA genes, and in particular a nonsense mutation in FUT2 (G428A), result in resistance to global dominating GII.4 NoV. The emergence of new pandemic GII.4 strains occurs at intervals of several years and is proposed to be attributable to epochal evolution, including amino acid changes and immune evasion. However, it remains unclear whether exposure to a previous pandemic strain stimulates immunity to a pandemic strain identified decades later. We found that prepandemic sera possess robust virus-blocking capacity against viruses identified several decades later. We also show that serum lacking IgA antibodies is sufficient to block NoV VLP binding to HBGAs. This is essential, considering that 1 in every 600 Caucasian children is IgA deficient.

Human norovirus inhibition by a human milk oligosaccharide

Human noroviruses are the leading cause of outbreaks of acute gastroenteritis. Norovirus interactions with histo-blood group antigens (HBGAs) are known to be important for an infection. In this study, we identified the HBGA binding pocket for an emerging GII genotype 17 (GII.17) variant using X-ray crystallography. The GII.17 variant bound the HBGA with an equivalent set of residues as the leading pandemic GII.4 variants. These structural data highlights the conserved nature of HBGA binding site between prevalent GII noroviruses. Noroviruses also interact with human milk oligosaccharides (HMOs), which mimic HBGAs and may function as receptor decoys. We previously showed that HMOs inhibited the binding of rarely detected GII.10 norovirus to HBGAs. We now found that an HMO, 2′-fucosyllactose (2′FL), additionally blocked both the GI.1 and GII.17 noroviruses from binding to HBGAs. Together, these findings provide evidence that 2′FL might function as a broadly reactive antiviral against multiple norovirus genogroups.

Evolutionary Constraints on the Norovirus Pandemic Variant GII.4_2006b over the Five-Year Persistence in Japan.

Norovirus GII.4 is a major cause of global outbreaks of viral gastroenteritis in humans, and has evolved by antigenic changes under the constantly changing human herd immunity. Major shift in the pandemic GII.4 strain periodically occurs concomitant with changes in the antigenic capsid protein VP1. However, how the newly emerged strain evolves after the onset of pandemic remains unclear. To address this issue, we examined molecular evolution of a pandemic lineage, termed the GII.4_2006b, by using the full-length viral genome and VP1 sequences (n = 317) from stools collected at 20 sites in Japan between 2006 and 2011. Phylogenetic tree showed a radial diversification of the genome sequences of GII.4_2006b, suggesting a rapid genetic diversification of the GII.4_2006b population from a few ancestral variants. Impressively, amino acid sequences of the variable VP1 in given seasons remained as homogeneous as those of viral enzymes under annual increase in the nucleotide diversity in the VP1 coding region. The Hamming distances between the earliest and subsequent variants indicate strong constraints on amino acid changes even for the highly variable P2 subdomain. These results show the presence of evolutionary constraints on the VP1 protein and viral enzymes, and suggest that these proteins gain near maximal levels of fitness benefits in humans around the onset of the outbreaks. These findings have implications for our understanding of molecular evolution, mechanisms of the periodic shifts in the pandemic NoV GII.4 strains, and control of the NoV GII.4 pandemic strain.

Intergenotype Recombination among New Norovirus GII.4 Variants in the Complete Genome

GII.4 noroviruses (NoVs) are a major cause of acute gastroenteritis in humans. A new variant of GII.4, the Sydney variant, has recently become more prevalent on a global scale. Intragenotype recombinations are widespread within the pandemic NoV GII.4 lineage, and are likely to be important forces driving the evolution and emergence of novel GII.4 viruses. In this study, we sought to examine the role that intergenotype recombination has played in the emergence of GII.4 Sydney 2012 variants. The results show that the GII.4 Sydney 2012 variants, Kawasaki194 and CA3477, were intergenotype recombination NoV strains with a GII.4 capsid and a GII.P16 polymerase gene. It has been reported for the first time that GII.4 new variant recombinants come from intergenotype recombination of GII.P16 and GII.4 strains in the complete genome.

Characterization of novel intragenotype recombination events among norovirus pandemic GII.4 variants

Recently, there has been an increase in the number of children hospitalized due to norovirus infection in Brazil. This is due both to the occurrence of more severe norovirus-related gastroenteritis cases after the introduction of the rotavirus vaccine and an increase in the tools for the detection of the disease. This pathogen is transmitted by the fecal-oral route, and the illness is characterized by diarrhea, vomiting, nausea and abdominal cramps. The genome of the virus is organized into three open reading frames showing strong mutation rates. Additionally, homologous recombination events, which can increase the virulence of the virus and lead to genotyping mistakes in molecular epidemiological studies, frequently occur. The purpose of this study was to describe two recombination events among different GII.4 variants that infected children who were hospitalized for severe acute gastroenteritis during distinct periods of time in Belém, Brazil. The recombination among the variants US95_96/Kaiso_2003 and Den Haag_2006b/Yerseke_2006a were observed in May 2003 and February 2009, respectively. In both cases, the association between the dominant variant at that point in time and another that was circulating at a low frequency in the population of Belém was demonstrated. Interestingly, the position of the breakpoint of the recombination event in the genome was the polymerase gene and was located at the nucleotide positions 4.834 and 5.002, which is an unusual location for the occurrence of recombination as other studies have previously reported the junction region as a breakpoint. In this study, both recombinant variant strains were related to severe cases of diarrhea that lead to hospitalization, demonstrating the viral evolution of GII.4 in response to selective pressures, which ultimately lead to the emergence of novel viral types in the pediatric population. The cases discussed here reinforce the need for continuous norovirus surveillance. To our knowledge, these two GII.4 variant recombinations have not yet been previously described.

A Multi-Site Study of Norovirus Molecular Epidemiology in Australia and New Zealand, 2013-2014.

BackgroundNorovirus (NoV) is the major cause of acute gastroenteritis across all age groups. In particular, variants of genogroup II, genotype 4 (GII.4) have been associated with epidemics globally, occurring approximately every three years. The pandemic GII.4 variant, Sydney 2012, was first reported in early 2012 and soon became the predominant circulating NoV strain globally. Despite its broad impact, both clinically and economically, our understanding of the fundamental diversity and mechanisms by which new NoV strains emerge remains limited. In this study, we describe the molecular epidemiological trends of NoV-associated acute gastroenteritis in Australia and New Zealand between January 2013 and June 2014.MethodologyOverall, 647 NoV-positive clinical faecal samples from 409 outbreaks and 238 unlinked cases of acute gastroenteritis were examined by RT-PCR and sequencing. Phylogenetic analysis was then performed to identify NoV capsid genotypes and to establish the temporal dominance of circulating pandemic GII.4 variants. Recombinant viruses were also identified based on analysis of the ORF1/2 overlapping region.FindingsPeaks in NoV activity were observed, however the timing of these epidemics varied between different regions. Overall, GII.4 NoVs were the dominant cause of both outbreaks and cases of NoV-associated acute gastroenteritis (63.1%, n = 408/647), with Sydney 2012 being the most common GII.4 variant identified (98.8%, n = 403/408). Of the 409 reported NoV outbreaks, aged-care facilities were the most common setting in both Western Australia (87%, n = 20/23) and New Zealand (58.1%, n = 200/344) while most of the NoV outbreaks were reported from hospitals (38%, n = 16/42) in New South Wales, Australia. An analysis of a subset of non-GII.4 viruses from all locations (125/239) showed the majority (56.8%, n = 71/125) were inter-genotype recombinants. These recombinants were surprisingly diverse and could be classified into 18 distinct recombinant types, with GII.P16/GII.13 (24% of recombinants) the most common.ConclusionThis study revealed that following its emergence in 2012, GII.4 Sydney 2012 variant continued to be the predominant cause of NoV-associated acute gastroenteritis in Australia and New Zealand between 2013 and 2014.

Molecular identification of emergent GII.P17-GII.17 norovirus genotype, Romania, 2015.

The novel GII.P17-GII.17 norovirus genotype has been reported as cause of gastroenteritis outbreaks in China and Japan since the winter season 2014/15, replacing the pandemic strain GII.4 Sydney 2012. These emergent strains have also been sporadically reported on other continents than Asia. GII.P17-GII.17 isolates, similar to Kawasaki308 2015, were identified in three patients during a large outbreak of acute gastroenteritis affecting 328 people in Romania, in neighbouring localities, in 2015.

Identification of the novel Kawasaki 2014 GII.17 human norovirus strain in Italy, 2015.

Surveillance of noroviruses in Italy identified the novel GII.17 human norovirus strain, Kawasaki 2014, in February 2015. This novel strain emerged as a major cause of gastroenteritis in Asia during 2014/15, replacing the pandemic GII.4 norovirus strain Sydney 2012, but being reported only sporadically elsewhere. This novel strain is undergoing fast diversification and continuous monitoring is important to understand the evolution of noroviruses and to implement the future strategies on norovirus vaccines.

Detection and analysis of recombination in GII.4 norovirus strains causing gastroenteritis outbreaks in Alberta

Recombination is an important mechanism generating genetic diversity in norovirus (NoV) that occurs commonly at the NoV polymerase-capsid (ORF1/2) junction. The genotyping method based on partial ORF2 sequences currently used to characterize circulating NoV strains in gastroenteritis outbreaks in Alberta cannot detect such recombination events and provides only limited information on NoV genetic evolution. The objective of this study was to determine whether any NoV GII.4 strains causing outbreaks in Alberta are recombinants. Twenty stool samples collected during outbreaks occurring between July 2004 and January 2012 were selected to include the GII.4 variants Farmington Hills 2002, Hunter 2004, Yerseke 2006a, Den Haag 2006b, Apeldoorn 2007, New Orleans 2009, and Sydney 2012 based on previous NoV ORF2-genotyping results. Near full-length NoV genome sequences were obtained, aligned with reference sequences from GenBank and analyzed with RDPv4.13. Two sequences corresponding to Apeldoorn 2007, and Sydney 2012 were identified as recombinants with breakpoints near the ORF1/2 junction and putative parental strains as previously reported. We also identified, for the first time, a non-recombinant sequence resembling the ORF2–3 parent of the recombinant cluster Sydney 2012 responsible for the most recent pandemic. Our results confirmed the presence of recombinant NoV GII.4 strains in Alberta, and highlight the importance of including additional genomic regions in surveillance studies to trace the evolution of pandemic NoV GII.4 strains.

113 Epidemics of Acute Gastroenteritis Caused by Viruses and Vaccine Development in China

The leading etiological agents of viral gastroenteritis are group A rotaviruses, noroviruses, adenoviruses (type 40, 41) and astroviruses in China in the last 20 years. The prevailing serotypes of rotaviruses are G type 1, 2, 3, 4 and 9, while G8 has been emerged recently in China. Accordingly, trivalent and hexavalent vaccines are under development by the two Institutes under CNBG. The trivalent oral vaccines are reassortants of VP4 and VP7 genes of human strains and other backbone genes of Lanzhou lamb rotavirus (LLR). The safety of lamb rotavirus vaccine strain has been proved since licensing in 2000 in China. The phase 3 clinical trials of the trivalent vaccines are in the progress in Henan province, China. The hexavalent oral vaccines are reassortants of VP7 genes of human strains and the other genes of UK strain, developed by and collaborated with NIH, USA, and PATH. The preclinical studies at Wuhan Institute of Biological Product have been completed and the hexavalent vaccine is ready for phase 1 clinical study. The novoviruses are increasingly detected in epidemics of acute viral gastroenteritis and spontaneous cases of people of all ages in China, including the rapid spreading of the newly emerged pandemic GII.4 variant (Sydney strain 2012) to China. The new Sydney strains were isolated in different areas and analysis of complete sequence demonstrated the rapid evolution. The surveillances and epidemic studies of these emerging viruses support the development of vaccines against the infections caused by noroviruses. The new variant co-circulated with other genotypes of GI and GII genogroups in human and multiple valent vaccines may needed to be developed.

Analysis of early strains of the norovirus pandemic variant GII.4 Sydney 2012 identifies mutations in adaptive sites of the capsid protein

Global surveillance for norovirus identified in 2012 the emergence of a novel pandemic GII.4 variant, termed Sydney 2012. In Italy, the novel pandemic variant was identified as early as November 2011 but became predominant only in the winter season 2012–2013. Upon sequencing and comparison with strains of global origin, the early Sydney 2012 strains were found to differ from those spreading in 2012–2013 in the capsid (ORF2) putative epitopes B, C and D, segregating into a distinct phylogenetic clade. At least three residues (333, 340 and 393, in epitopes B, C and D, respectively) of the VP1 varied among Sydney 2012 strains of different clades. These findings suggest that the spread of the pandemic variant in Italy during the winter season 2012–2013 was due to the introduction of strains distinct from those circulating at low frequency in the former winter season and that similar strains were also circulating elsewhere worldwide.

Detection of the pandemic norovirus variant GII.4 Sydney 2012 in Rio Branco, state of Acre, northern Brazil

Noroviruses (NoVs) are important cause of gastroenteritis in humans worldwide. Genotype GII.4 is responsible for the majority of outbreaks reported to date. This study describes, for the first time in Brazil, the circulation of NoV GII.4 variant Sydney 2012 in faecal samples collected from children aged less than or equal to eight years in Rio Branco, state of Acre, northern Brazil, during July-September 2012.

Evidence for Recombination between Pandemic GII.4 Norovirus Strains New Orleans 2009 and Sydney 2012

During 2012, a novel pandemic GII.4 norovirus variant, Sydney 2012, emerged worldwide. A signature of the variant was a GII.Pe ORF1, in association with GII.4 Apeldoorn 2008-like ORF2-ORF3 genes. We report the detection of recombinant GII.4 Sydney 2012 strains, possessing the ORF1 gene of the former pandemic variant New Orleans 2009.

Emergence of New Pandemic GII.4 Sydney Norovirus Strain Correlates With Escape From Herd Immunity

GII.4 noroviruses are a significant source of acute gastroenteritis worldwide, causing the majority of human norovirus outbreaks. Evolution of the GII.4 major capsid protein occurs rapidly, resulting in the emergence of new strains that produce successive waves of pandemic disease. A new pandemic isolate, GII.4 2012 Sydney, largely replaced previously circulating strains in late 2012. We compare the antigenic properties of GII.4 2012 Sydney with previously circulating strains. To determine whether GII.4-2012 Sydney is antigenically different from recently circulating strains GII.4-2006 Minerva and GII.4-2009 New Orleans in previously identified blockade epitopes, we compared reactivity and blockade profiles of GII.4-2006, GII.4-2009, and GII.4-2012 virus-like particles in surrogate neutralization/blockade assays using monoclonal antibodies and human polyclonal sera. Using monoclonal antibodies that map to known blockade epitopes in GII.4-2006 and GII.4-2009 and human outbreak polyclonal sera, we demonstrate either complete loss or significantly reduced reactivity and blockade of GII.4.2012 compared to GII.4-2006 and GII.4-2009. GII.4-2012 Sydney is antigenically different from GII.4-2006 Minerva and GII.4-2009 New Orleans in at least 2 key blockade epitopes. Viral evolution in key potential neutralization epitopes likely allowed GII.4-2012 to escape from human herd immunity and emerge as the new predominant strain.

Recombination within the pandemic norovirus GII.4 lineage.

Norovirus (NoV) is the leading cause of viral gastroenteritis globally. Since 1996, NoV variants of a single genetic lineage, GII.4, have been associated with at least six pandemics of acute gastroenteritis and caused between 62 and 80% of all NoV outbreaks. The emergence of these novel GII.4 variants has been attributed to rapid evolution and antigenic variation in response to herd immunity; however, the contribution of recombination as a mechanism facilitating emergence is increasingly evident. In this study, we sought to examine the role that intragenotype recombination has played in the emergence of GII.4 variants. Using a genome-wide approach including 25 complete genome sequences generated as part of this study, 11 breakpoints were identified within the NoV GII.4 lineage. The breakpoints were located at three recombination hot spots: near the open reading frame 1/2 (ORF1/2) and ORF2/3 overlaps, as well as within ORF2, which encodes the viral capsid, at the junction of the shell and protruding domains. Importantly, we show that recombination contributed to the emergence of the recent pandemic GII.4 variant, New Orleans 2009, and a newly identified GII.4 variant, termed Sydney 2012. Reconstructing the evolutionary history of the GII.4 lineage reveals the widespread impact of both inter- and intragenotype recombination on the emergence of many GII.4 variants. Lastly, this study highlights the many challenges in the identification of true recombination events and proposes that guidelines be applied for identifying NoV recombinants.

Emergence of New Pandemic GII.4 Sydney Norovirus Strain Correlates With Escape From Herd Immunity

Emergence of New Pandemic GII.4 Sydney Norovirus Strain Correlates With Escape From Herd Immunity