Increased pathogenicity and transmission of SARS-CoV-2 Omicron XBB.1.9 subvariants, including HK.3 and EG.5.1, relative to BA.2.

With the SARS-CoV-2 Omicron XBB.1.9 subvariants circulating worldwide, two XBB.1.9 variants, EG.5.1 and HK.3, spread rapidly and became dominant in mid-2023. However, the spike features, pathogenicity, and transmissibility of HK.3 are largely unknown. Here, we performed multiscale investigations to reveal the virological features of XBB.1.9 subvariants, including the newly emerging HK.3. HK.3 revealed high replication efficiency and enhanced TMPRSS2 utilization in vitro. The HK.3 spike exhibited enhanced processing, although its infectivity, fusogenicity, and human ACE2 (hACE2) binding affinity were comparable to those of the EG.5 and XBB.1 spikes. All XBB.1.9.1, EG.5.1, and HK.3 strains demonstrated efficient transmission in hamsters, although XBB.1.9.1 exhibited stronger fitness in the upper airways. XBB.1.9.1, EG.5.1, and HK.3 exhibited greater pathogenicity than BA.2 in H11-K18-hACE2 hamsters. Our studies provide insights into the newly emerging pathogens EG.5.1 and HK.3.IMPORTANCESARS-CoV-2 Omicron continues to circulate and evolve into novel lineages with indistinguishable pathogenicity and transmission. Ancestral Omicron lineages, such as BA.1 and BA.2, revealed attenuated pathogenicity and transmission, at least in animal models. However, on a previously reported Omicron-sensitive H11-K18-hACE2 hamster model, the infections of XBB.1.9 lineages, EG.5, and HK.3 led to faster lethality and more severe terminal bronchioles symptom than BA.2. They also revealed efficient transmission in a hamster model, which corresponds well with their prevalence in multiple countries. Our study highlights the importance of surveillance and virological studies on epidemic Omicron subvariants.