Volume 27, Number 1—January 2021
Detection of Norovirus Variant GII.4 Hong Kong in Asia and Europe, 2017−2019
We report a new norovirus GII.4 variant, GII.4 Hong Kong, with low-level circulation in 4 Eurasia countries since mid-2017. Amino acid substitutions in key residues on the virus capsid associated with the emergence of pandemic noroviruses suggest that GII.4 Hong Kong has the potential to become the next pandemic variant.
Noroviruses are a genetically diverse group of RNA viruses found in a plethora of terrestrial, aerial, and aquatic mammalian species, including humans, pigs, cows, sheep, rats, bats, sea lions, and harbor porpoises (1). These viruses are classified into 48 genotypes, including the pandemic GII.4 genotype. In humans, norovirus is the cause of almost one fifth of all cases of acute gastroenteritis globally and the leading cause in all age groups (2,3). The highest disease burden has been documented in young children (4,5), whereas, in developed countries, infections have been associated with increased deaths in the elderly (6).
Norovirus GII.4 viruses have caused most gastroenteric infections for >2 decades, and new variants have emerged every 2–4 years since 2002 (7). Six pandemic GII.4 variants have been named after the place of first reported sequence and year of predominance: US 95–96, Farmington Hills 2002, Hunter 2004, Den Haag 2006, New Orleans 2009, and Sydney 2012. Several norovirus vaccine candidates are in preclinical studies and clinical trials (8); however, it is currently unclear if these vaccine formulations could protect against newly emerging GII.4 viruses that are often associated with more norovirus outbreaks and hospitalizations.
Norovirus surveillance networks, such as NoroNet, CaliciNet U.S. and CaliciNet China, are tracking norovirus genotypes (9–11). Surveillance data from NoroNet has shown that GII.4 variants can be detected at low levels years before they cause a new pandemic (9). We report the detection of a new norovirus variant called GII.4 Hong Kong that was circulating sporadically in communities of 2 countries in Asia and 2 in Europe during 2017–2019. This new variant has sequence features of a potential pandemic GII.4 variant.
We recently reported the full genome of a new norovirus GII.4 variant called GII.4 Hong Kong, discovered from the local molecular surveillance of norovirus gastroenteritis in hospitalized patients in Hong Kong, China (12). The strain Hu/HK/2019/GII.4 Hong Kong[P31]/CUHK-NS-2200 was collected in August 2019. Preliminary analysis indicated that the full genome had 91.1% nucleotide identity to that of the prototype of the most recent pandemic GII.4 Sydney variant, Hu/GII.4/Sydney/NSW0514/2012/AU, which had predominated worldwide since 2012. The major capsid protein of GII.4 Hong Kong had 89.6% amino acid identity with GII.4 Sydney 2012; phylogenetic distance within this new variant had no overlap with distance between other known GII.4 variants, suggesting a new GII.4 variant (Appendix Figure). Coordinated through the international norovirus classification working group (1), the 2 most widely used norovirus and calicivirus typing tools hosted by the National Institute for Public Health (RIVM) of the Netherlands (https://www.rivm.nl/mpf/typingtool/norovirus) and the US Centers for Disease Control and Prevention (https://norovirus.ng.philab.cdc.gov) have been updated and synchronized to include the new norovirus to enable retrospective identification and prospective monitoring of GII.4 Hong Kong.
We subsequently identified and sequenced 4 additional GII.4 Hong Kong strains from archived stool samples from Hong Kong, the Philippines, the Netherlands, and the United Kingdom (Table 1); these strains were collected during May 2017–March 2019 (2 in 2017, 1 in 2018, and 1 in 2019). Prevalence of the new variant was <1% in each country, except the Philippines (Table 1). The youngest patient was an 11-month-old hospitalized infant and the oldest patient was a 90-year-old woman living in a nursing home.
We obtained full genomes from 2 patients using next-generation sequencing and a complete major capsid protein sequence from 1 patient using Sanger sequencing. We sequenced part of the polymerase-capsid junction region in another case but could not sequence the complete capsid because of low viral load. All consensus virus sequences have been deposited into GenBank (Table 1). The new GII.4 variant was not found in norovirus outbreaks in the United States.
We performed sequence analysis on the 4 available complete major capsid protein sequences of GII.4 Hong Kong. Sequence alignment showed that they shared a pairwise identity of 96.4%–99.3% at nucleotide level and 97.2%–99.4% at amino acid level. We performed maximum-likelihood phylogenetic inference using MEGA version 6.06 (http://megasoftware.net). The 4 GII.4 Hong Kong strains formed a monophyletic cluster that was distant from other pandemic GII.4 variants but was closest to GII.4 Osaka, which had no pandemic spread (Figure 1). We calculated root-to-tip distance that reflected phylogenetic relatedness using TempEst version 1.5.3 (http://tree.bio.ed.ac.uk/software/tempest). Sequences of GII.4 Hong Kong had the longest distance from the root at both nucleotide and amino acid levels (Figure 2), indicating the virus had evolved farthest from ancestral GII.4. Inclusion of the new variant preserved and extended the strong linear clock−like relationship of evolutionary distance with time (nucleotide R2 = 0.9691; amino acid R2 = 0.9362). R2 values of ≈1 indicate a nearly constant rate of accumulation of virus mutations over time, which has been characteristic in the molecular evolution of the major capsid protein of GII.4 variants since the 1970s (7). Of note, amino acid sequences of GII.4 Hong Kong occupied a distinct spatial-temporal area on the plot with no overlap with other GII.4 variants (Figure 2, panel B). A recent large-scale sequence analysis and antibody blockage confirmation study proposed a set of 5 residues on the major capsid protein that are influential in the emergence and replacement of pandemic GII.4 variants since 1995 (7). We observed that residues on 4 of these 5 positions on GII.4 Hong Kong have changed compared with the consensus of GII.4 Sydney 2012 and that 3 residues have changed compared with the consensus of GII.4 Osaka (Table 2).
The last GII.4 variant, GII.4 Sydney, which has been circulating since 2012, had only a few substitutions when a new recombinant, GII.4 Sydney[P16], emerged in 2015 (10). Our findings in 4 countries of low-level circulation (but probably not unique cases) of a new GII.4 variant over a 2-year period clearly indicates that the evolution of GII.4 has been ongoing, if mostly underreported. Phylogeny indicates that the major capsid protein of GII.4 Hong Kong may have evolved from the older GII.4 Osaka variant that was epidemic in 2007, rather than from the newer GII.4 Sydney 2012 variant. The cause of this finding and its implications into GII.4 evolution remain elusive. Although substitutions were found in most of key antigenic residues that have been suggested to be influential in the emergence of new pandemic GII.4 variants, GII.4 Hong Kong has yet to cause outbreaks. Previously, GII.4 variants have been detected at low levels for as long as 18 years before they became pandemic (13). GII.4 Hong Kong viruses may still need to further explore the antigenicity landscape to evade herd immunity in humans or to wait for waning of cross-reacting host immunity against GII.4 variants, ultimately allowing pandemic spread of the new variant (14). The mechanism for GII.4 viruses to become pandemic remains poorly understood; viral RNA polymerase may also contribute to virus fitness by influencing replication and shedding amount (15). Not all GII.4 variants have resulted in pandemics; those of GII.4 Asia, GII.4 Yerseke, and GII.4 Osaka did not.
In conclusion, we report a new norovirus GII.4 variant called GII.4 Hong Kong that has been circulating sporadically in Eurasia since mid-2017. Because some GII.4 variants do not become predominant pandemic strains, continued surveillance of both outbreaks and sporadic cases is important for monitoring emergent norovirus strains.
Dr. Chan is a senior scientific reviewer at the Research Office of the Food and Health Bureau of Hong Kong. At the time of this study, he was an assistant professor in the Department of Microbiology and Stanley Ho Centre for Emerging Infectious Diseases and a principal investigator in the Li Ka Shing Institute of Health Sciences of the Chinese University of Hong Kong. His research interest is intestinal and respiratory viral infections, especially those caused by noroviruses and influenza viruses.
We thank Amy Douglas for her support in collecting the metadata for the UK case and Gabriel Parra for his helpful advice and comments on the manuscript.
This work was supported in part by the commissioned Health and Medical Research Fund of the HKSAR Government (CU-15-C2 to M.C.-W.C.) and the Wellcome Trust (203268/Z/16/Z to J.B.). M.d.G was funded through METASTAVA (grant no. 773830).
Author contributions: M.C.-W.C. and M.d.G. conceived the study. M.C.-W.C., S.R., J.B., L.-Y.Z., J.C.M.C., C.C., M.A.I., S.-L.L., K.N.M., H.V., J.B., M.K. and M.d.G. coordinated data collection. P.C. prepared the Appendix Figure. J.V. and H.V. implemented data integration into virus typing tools. M.C.-W.C. performed initial data analysis and drafted the manuscript. All authors contributed to data analysis and interpretation and critically reviewed and commented on the manuscript before submission.
Members of NOROPATROL who contributed: Sunando Roy, Florencia Tettamanti Boshier, Helena Tutill, Ellen Fragaszy, Rachel Williams, Richard Goldstein, Judith Breuer, Andrew Hayward (University College London, UK); Shilpi Sheth, Daniel Kelly, Kathleen O’Reilly, David Allen, John Edmunds (London School of Hygiene and Tropical Medicine, UK); Sarah O’Brien (University of Liverpool, UK); Lisa Lindesmith, Ralph Baric (University of North Carolina, USA); Cristina Celma, Karen Fuentes, Stuart Beard (Public Health England, UK).
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TablesCite This Article
Original Publication Date: December 18, 2020
1Current affiliation: Research Office, Food and Health Bureau, Hong Kong, China.
2Members who contributed data are listed at the end of this article.