Following emergence of the SARS-CoV-2 variant Omicron in November 2021, the dominant BA.1 sub-lineage was replaced by the BA.2 sub-lineage in Denmark. We analysed the first 2,623 BA.2 cases from 29 November 2021 to 2 January 2022. No epidemiological or clinical differences were found between individuals infected with BA.1 versus BA.2. Phylogenetic analyses showed a geographic east-to-west transmission of BA.2 from the Capital Region with clusters expanding after the Christmas holidays. Mutational analysis shows distinct differences between BA.1 and BA.2.

Efforts to mitigate the COVID-19 pandemic include screening of existing antiviral molecules that could be re-purposed to treat SARS-CoV-2 infections. Although SARS-CoV-2 replicates and propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (nucs) often show decreased activity in these cells due to inefficient metabolization. SARS-CoV-2 exhibits low viability in human cells in culture. Here, serial passages of a SARS-CoV-2 isolate (original-SARS2) in the human hepatoma cell clone Huh7.5 led to the selection of a variant (adapted-SARS2) with significantly improved infectivity in human liver (Huh7 and Huh7.5) and lung cancer cells (unmodified Calu-1 and A549). The adapted virus exhibited mutations in the spike protein, including a 9 amino acid deletion and 3 amino acid changes (E484D, P812R, and Q954H). E484D also emerged in Vero E6 cultured viruses that became viable in A549 cells. Original and adapted viruses were susceptible to SR-B1 receptor blocking and adapted-SARS2 exhibited significantly less dependency of ACE2. Both variants were similarly neutralized by COVID-19 convalescent plasma but adapted-SARS2 exhibited increased susceptibility to exogenous type I interferon. Remdesivir inhibited original- and adapted-SARS2 similarly, demonstrating the utility of the system for the screening of nucs. Among the tested nucs, only remdesivir, molnupiravir and to a limited extent galidesivir, showed antiviral effect across human cell lines, whereas sofosbuvir, ribavirin, and favipiravir had no apparent activity. Analogously to the emergence of spike mutations in vivo, the spike protein is under intense adaptive selection pressure in cell culture. Our results indicate that the emergence of spike mutations will most likely not affect the activity of remdesivir.

Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance–associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1‐4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156‐RASs were not maintained. For genotypes 1 and 2, persistence of 156‐RASs depended on genome‐wide substitution networks, co‐selected under continued PI treatment and identified by next‐generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156‐RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre‐existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156‐RASs, we observed genome‐wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1‐6 revealed 156‐RASs as key determinants of high‐level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1‐3 156‐variants, which might pose a threat to clinically relevant combination treatments.