Molecular Evolution and Epidemiology of Norovirus GII.4 Viruses in the United States

Noroviruses are the leading cause of acute gastroenteritis worldwide with GII.4 Sydney viruses responsible for the majority of infections until 2023. To study the evolutionary dynamics of GII.4 noroviruses in the US (2011-2023), we sequenced and analyzed 406 VP1 and 335 RdRp sequences submitted to CaliciNet. Time-scale analysis showed the average evolutionary rate of GII.4 strains was 5.56 x 10-3 substitutions/site/year and the emergence of a new cluster within GII.4 Sydney every 4 years starting with GII.4 Sydney[P31] from 2011-2015 followed by GII.4 Sydney[P16] from 2016-2020, and the most recent GII.4 Sydney[P16]-2020 from 2021-to date. Since 2017, based on amino acids in VP1, we observed the emergence of three novel GII.4 clusters (GII.4 San Francisco, GII.4 Allegany and GII.4 Wichita). GII.4 Sydney was identified with 4 P-types (P4, P12, P16, and P31). GII.4 San Francisco and GII.4 Allegany had a P31 RdRp, whereas GII.4 Wichita strains had P4. GII.4 Allegany and GII.4 Wichita exhibited major amino acid substitutions in epitopes A-E, G, and H, while GII.4 San Francisco viruses have an alanine insertion in epitope A. Both GII.4 Allegany and GII.4 Wichita VLPs bound porcine gastric mucin at a similar level as GII.4 New Orleans and GII.4 Sydney. However, blocking of binding to VLPs by human serum pools demonstrated their antigenicity was significantly different. We identified three new emerging GII.4 noroviruses co-circulating with GII.4 Sydney. Early detection of new strains will aid in tracking their spread and assessing their pandemic potential.