Historically, mpox has been characterized as an endemic zoonotic disease that transmits through contact with the reservoir rodent host in West and Central Africa. However, in May 2022, human cases of mpox were detected spreading internationally beyond countries with known endemic reservoirs. When the first cases from 2022 were sequenced, they shared 42 nucleotide differences from the closest mpox virus (MPXV) previously sampled. Nearly all these mutations are characteristic of the action of APOBEC3 deaminases, host enzymes with antiviral function. Assuming APOBEC3 editing is characteristic of human MPXV infection, we developed a dual-process phylogenetic molecular clock that-inferring a rate of ~6 APOBEC3 mutations per year-estimates that MPXV has been circulating in humans since 2016. These observations of sustained MPXV transmission present a fundamental shift to the perceived paradigm of MPXV epidemiology as a zoonosis and highlight the need for revising public health messaging around MPXV as well as outbreak management and control.

Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages; however, there can be limitations due to sequence dropout when used to identify specific key mutations. Recently, ThermoFisher Scientific has developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants. Over a 6-week period during the months of April and May 2021, we set out to assess the ThermoFisher TaqMan mutation panel genotyping assay, initially for three mutations of concern and then for an additional two mutations of concern, against SARS-CoV-2-positive clinical samples and the corresponding COVID-19 Genomics UK Consortium (COG-UK) sequencing data. We demonstrate that genotyping is a powerful in-depth technique for identifying specific mutations, is an excellent complement to genome sequencing, and has real clinical health value potential, allowing laboratories to report and take action on variants of concern much more quickly.

We report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike ΔH69/V70 in multiple independent lineages, often occurring after acquisition of receptor binding motif replacements such as N439K and Y453F, known to increase binding affinity to the ACE2 receptor and confer antibody escape. In vitro, we show that, although ΔH69/V70 itself is not an antibody evasion mechanism, it increases infectivity associated with enhanced incorporation of cleaved spike into virions. ΔH69/V70 is able to partially rescue infectivity of spike proteins that have acquired N439K and Y453F escape mutations by increased spike incorporation. In addition, replacement of the H69 and V70 residues in the Alpha variant B.1.1.7 spike (where ΔH69/V70 occurs naturally) impairs spike incorporation and entry efficiency of the B.1.1.7 spike pseudotyped virus. Alpha variant B.1.1.7 spike mediates faster kinetics of cell-cell fusion than wild-type Wuhan-1 D614G, dependent on ΔH69/V70. Therefore, as ΔH69/V70 compensates for immune escape mutations that impair infectivity, continued surveillance for deletions with functional effects is warranted.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein¹ and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.