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.

Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway.

“…However, Y453F in particular, negatively impacts replication kinetics of SARS-CoV-2 in human cells, potentially explaining why the Danish mink-origin outbreaks did not propagate further following the culling of the mink. Furthermore, in line with other studies 16,17,31 , we found none of these mutations had a large antigenic impact, so vaccination is likely to remain effective against mustelid-adapted strains. Finally, we have shown that several VOC strains, or VOC-associated mutations, partially adapt SARS-CoV-2 spike to mustelid ACE2.…”

“…The D614G containing strain, SARS-CoV-2/England/IC19/2020, was used as previously described 41 . The Cluster 5 isolate -SARS-CoV-2/hu/DK/CL-5/1was isolated as previously described 16 and was kindly provided by Kevin Bewley at Public Health England. All viral stocks used in this study were grown in Vero cells in OptiPRO SFM containing 2x GlutaMAX.…”

“…This large-scale outbreak resulted in an estimated 4000 mink-associated human cases and promoted the Danish government to cull all 17 million farmed mink in the country and several countries imposed total travel bans on the affected regions 15 . Of particular concern was the increased acquisition of spike mutations in mink-associated SARS-CoV-2 viruses, as demonstrated by the Cluster 5 variant identified in September 2020 which had several additional changes in the spike glycoprotein including, Δ69-70 in the N-terminal domain (NTD), and I692V and M1229I in S2 16 . Early data indicated a possible change in antigenicity whereby Cluster 5 variant virus might be less readily neutralised by antibodies in convalescent sera from individuals infected by earlier variants 16,17 .…”

Natural and Experimental SARS-CoV-2 Infection in Domestic and Wild Animals

“…Importantly, phylogenetic evidence indicated that zoonotic mink-to-human transmission of SARS-CoV-2 occurred at both Dutch and Danish farms [ 94 , 95 , 96 , 98 , 182 , 183 ]. It was determined that between June and November 2020, 214 human cases of COVID-19 were identified in Denmark with SARS-CoV-2 variants associated with farmed mink [ 98 , 181 ].…”

Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway

“…The D614G containing strain, SARS-CoV-2/England/IC19/2020, was used as previously described 41 . The Cluster 5 isolate -SARS-CoV-2/hu/DK/CL-5/1 -was isolated as previously described 16 and was kindly provided by Kevin Bewley at Public Health England. All viral stocks used in this study were grown in Vero cells in OptiPRO SFM containing 2x GlutaMAX.…”