In vitro Characterization of Fitness and Convalescent Antibody Neutralization of SARS-CoV-2 Cluster 5 Variant Emerging in Mink at Danish Farms

Abstract

In addition to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit to animals that include hamsters, cats, dogs, mink, ferrets, tigers, lions, cynomolgus macaques, rhesus macaques, and treeshrew. Among these, mink are particularly susceptible. Indeed, 10 countries in Europe and North America reported SARS-CoV-2 infection among mink on fur farms. In Denmark, SARS-CoV-2 spread rapidly among mink farms and spilled-over back into humans, acquiring mutations/deletions with unknown consequences for virulence and antigenicity. Here we describe a mink-associated SARS-CoV-2 variant (Cluster 5) characterized by 11 amino acid substitutions and four amino acid deletions relative to Wuhan-Hu-1. Temporal virus titration, together with genomic and subgenomic viral RNA quantitation, demonstrated a modest in vitro fitness attenuation of the Cluster 5 virus in the Vero-E6 cell line. Potential alterations in antigenicity conferred by amino acid changes in the spike protein that include three substitutions (Y453F, I692V, and M1229I) and a loss of two amino acid residues 69 and 70 (ΔH69/V70), were evaluated in a virus microneutralization assay. Compared to a reference strain, the Cluster 5 variant showed reduced neutralization in a proportion of convalescent human COVID-19 samples. The findings underscore the need for active surveillance SARS-CoV-2 infection and virus evolution in susceptible animal hosts.

Keywords: COVID-19; SARS-CoV-2; coronavirus; mink; virus neutralization.

FIGURE 1

The mink-associated mutations in the SARS-CoV-2 spike protein. (A) The combination and frequency of mink-associated spike changes detected in SARS-CoV-2 infected humans up until 30th October 2020. (B) Phylogenetic grouping of mink-associated variant transmission Clusters 1–5 (lineage B.1.1.298). (C) The crystal structure of a closed pre-fusion spike trimer [PDB: 6ZGE]. The positions of amino acid changes are indicated with red spheres. The receptor binding domain (RBD) is indicated in green, the N-terminal domain (NTD) in beige, and the S2 domain in gray. The regions encompassing the S1147L and M1229I substitutions are not within the crystal structure; however, their relative positions are indicated. (D) The location of the Y453F substitution in the receptor binding domain complexed with a host ACE2 receptor (blue) [PBD: 6LZG].

FIGURE 2

Genomic characterization and growth kinetics of the SARS-CoV-2 Cluster 5 variant in Vero-E6 cells. (A) Amino acid changes identified in the SARS-CoV-2 Cluster 5 variant and a Danish SARS-CoV-2 strain (H1) isolated in March 2020 relative to the reference strain Wuhan-Hu-1. (B) Temporal cytopathic effect of Vero-E6 cells following infection with the two virus strains at a multiplicity of infection (MOI) of 0.01. The arrow indicates the characteristic rounding of SARS-CoV-2 infected cells. (C) Temporal increase in H1 and Cluster 5 virus levels following inoculation of Vero-E6 cells at an MOI of 0.01. At the indicated time points, the virus titre was determined in a SARS-CoV-2-specific anti-nucleocapsid protein ELISA and the TCID₅₀/mL calculated using the Reed and Muench method. (D) Quantitation of SARS-CoV-2 E gene genomic and subgenomic RNA in infected Vero-E6 cells and cell culture supernatant. Cells were infected at a multiplicity of infection of 0.0125 for 1 h, washed three times to remove all inoculum, overlaid with culture media, and cells and culture supernatant harvested at the indicated time points.

FIGURE 3

Neutralization of the SARS-CoV-2 Cluster 5 virus relative to an early epidemic SARS-CoV-2 virus (H1). In all instances, neutralization was evaluated using live SARS-CoV-2 viruses isolated from clinical samples. (A) Neutralization activity of twofold serial diluted convalescent COVID-19 plasma was tested in triplicate with 300× TCID₅₀ Cluster 5 or H1 virus. Thick lines represent the four-parameter titration curve calculated from the mean of triplicate measurements; thin lines represent each individual replicate. (B) The relative neutralization titres measured at 24 h post-inoculation for plasma samples for the individuals in (A) calculated as the interpolation of the four-parameter titration curve with the 50% cut-off value and presented as the reciprocal serum dilution. Bars represent the mean titre of triplicate measurements with the standard deviation. (C) Neutralization titres determined for 44 convalescent plasma from non-hospitalized individuals with PCR confirmed SARS-CoV-2 infection. Each line represents an individual plasma sample ranked by titre against the early pandemic strain.