key: cord-0994869-bd314v7x authors: Rasmussen, Thomas Bruun; Fonager, Jannik; Sværke Jørgensen, Charlotte; Lassaunière, Ria; Hammer, Anne Sofie; Quaade, Michelle Lauge; Boklund, Anette; Lohse, Louise; Strandbygaard, Bertel; Rasmussen, Morten; Michaelsen, Thomas Yssing; Mortensen, Sten; Fomsgaard, Anders; Belsham, Graham J.; Bøtner, Anette title: Infection, recovery and re-infection of farmed mink with SARS-CoV-2 date: 2021-05-07 journal: bioRxiv DOI: 10.1101/2021.05.07.443055 sha: 4fb622983e26932580518d3eadeff1e0bd4a013e doc_id: 994869 cord_uid: bd314v7x Mink, on a farm with about 15,000 animals, became infected with SARS-CoV-2. Over 75% of tested animals were positive for SARS-CoV-2 RNA in throat swabs and 100% of tested animals were seropositive. The virus responsible had a deletion of nucleotides encoding residues H69 and V70 within the spike protein gene. The infected mink recovered and after free-testing of the mink, the animals remained seropositive. During follow-up studies, after a period of more than 2 months without virus detection, over 75% of tested animals scored positive again for SARS-CoV-2 RNA. Whole genome sequencing showed that the virus circulating during this re-infection was most closely related to the virus identified in the first outbreak on this farm but additional sequence changes had occurred. Animals had much higher levels of anti-SARS-CoV-2 antibodies after re-infection than at free-testing. Thus, following recovery from an initial infection, seropositive mink rapidly became susceptible to re-infection by SARS-CoV-2. Article Summary Line Following widespread infection with SARS-CoV-2 of mink on a farm, all tested animals had seroconverted and the farm was then tested free of infection; however, less than 3 months later, a further round of infection affected more than 75% of tested animals. 3 were closely related to each other and also to the viruses present in August on Farm 4, but the 1 3 3 latter viruses had some additional changes (e.g. the deletion of residues H69 and V70 in the spike protein, see Table 2 ), which persisted throughout the rest of the outbreaks in farmed mink. Thus, Reserved space. Do not place any text in this section. Include the mandatory author checklist or your manuscript will be returned. Use continuous line numbering in your manuscript. those first detected on Farm 4. As indicated above, the November viruses from Farm 4 had the 1 3 7 A22920T mutation and the deletions in the S and ORF1a coding sequences. However, the 1 3 8 November viruses had additional changes across the genome, both within and outside of the S 1 3 9 gene, compared to the viruses in Farms 2 and 3 (Table 2 ). It is noteworthy that the Farm 4 1 4 0 sequences in November had changes at nt 10448 (encoding the substitution P3395S in ORF1a) 1 4 1 and 20756 (encoding S2430I in ORF1b) that had only been seen in a subset of the August 1 4 2 sequences from Farm 4 (samples Farm4_18_13-08-2020 and Farm4_19_13-08-2020, see Figure 1 4 3 2). These changes act as a fingerprint and strongly suggest that it was not an entirely new November also all shared changes at nt 3792 (resulting in A1176V), 5167, 10887 (resulting in 1 4 6 G3541E), 21727 and 23815 (the latter two silent changes are in the S gene) that were not present 1 4 7 in any of the Farm 4 sequences in August (Table 2 ). The presence of these additional sequence between August and November but does not prove that the virus has continued to replicate in 1 5 0 mink during this time. Phylogenetic analysis clearly showed that all viruses from Farm 4 were very closely 1 5 2 related to each other, including the viruses from both August and November ( Figure 2 ). As 1 5 3 described above, two of the early Farm 4 viruses (Farm4_18_13-08-2020 and Farm4_19_13-08-1 5 4 2020) shared additional changes at nt 10448 and 20756 (see Table 2 ) and the November viruses sequence changes (Table 2) . Reserved space. Do not place any text in this section. Include the mandatory author checklist or your manuscript will be returned. Use continuous line numbering in your manuscript. SARS-CoV-2 can readily infect humans and mink. In addition, certain other species, e.g. cats, dogs and ferrets, can also be infected following direct inoculation under experimental 1 6 0 conditions (10, 11). Furthermore, some cases of transmission from infected people to their cats 1 6 1 and dogs have occurred but it does not seem to happen more generally. Both cellular and 1 6 2 humoral immune responses occur within SARS-CoV-2-infected people and animals (12, 13) and 1 6 3 it is common for both humans and animals to be both seropositive and RT-qPCR positive simultaneously (see (2, 14) ). However, as people and animals recover, the levels of virus subside 1 6 5 but antibody levels persist, or increase, at least for some time. Farm 4 was the only Danish mink farm, where the animals were allowed to recover and Reserved space. Do not place any text in this section. Include the mandatory author checklist or your manuscript will be returned. Use continuous line numbering in your manuscript. immune pressure via RNA editing systems (e.g. by APOBEC) rather than selection for increased 2 0 5 transmissibility in particular hosts (15, 16, 17) . However, this process of RNA editing is not 2 0 6 relevant to the key mutation in the S gene (A22920T), which seems to be an adaptation that The sampling of the mink on Farm 4 tested, at most, 300 animals on any particular date, potentially decline more rapidly than antibodies generated from the infection in each animal. However, it seems difficult to reconcile this with the fact that >80% of throat swabs from mink 2 2 1 kits tested clearly positive by RT-qPCR in August, which indicated a high level of infection 2 2 2 amongst the kits in the first wave also. The measurements of antibody responses were made using an ELISA that targets the protein were present in up to 100% of the infected mink. The antibody titres, measured in this 2 2 6 assay, increased to very high levels during the period of re-infection (see Figure 1A ). In studies Reserved space. Do not place any text in this section. Include the mandatory author checklist or your manuscript will be returned. Use continuous line numbering in your manuscript. on human sera, samples testing clearly positive (10 x cut-off) in this ELISA all had neutralizing virus neutralization tests indicated a high correspondence between these two types of assay. November, after reinfection, had much higher levels of anti-SARS-CoV-2 antibodies as 2 3 2 measured in each assay ( Figure 1B) . The most plausible conclusion is that infection of farmed mink with SARS-CoV-2 does be a rapid increase in virus production and a strong challenge to neighbouring animals. Perhaps Reserved space. Do not place any text in this section. Include the mandatory author checklist or your manuscript will be returned. Use continuous line numbering in your manuscript. Reserved space. Do not place any text in this section. Include the mandatory author checklist or your manuscript will be returned. Use continuous line numbering in your manuscript. are plotted using the same colour scheme. T T T C T T T G + T G T T F a r m 4 _ 6 T T T C T T T G + T G T T G 4 8 8 A F a r m 4 _ 8 T T T C T T T G + T G T T Δ 2 1 9 8 4 -2 1 9 9 5 F a r m 4 _ 1 8 T T T T T T T T + T G T T F a r m 4 _ 1 9 T T T T T T T T + T G T T F a r m 4 _ 2 1 T T T C T T T G + T G T T A 6 5 2 C ( K 1 2 9 N ) 1 F a r m 4 _ 3 5 T T T C T T T G + T G T T Δ 2 7 9 8 2 -2 8 0 3 0 F a r m 4 _ 3 7 T T T C T T T G + T G T T T 1 8 7 3 C , G 2 0 3 5 T ( L 5 9 0 F ) N o v 2 0 2 0 F a r m 4 _ 1 T T T T T T T T + T G T T C 1 9 1 3 T ( R 5 5 0 C ) , C 3 7 9 2 T ( A 1 1 7 6 V ) , C 5 1 6 7 T , G 1 0 8 8 7 A ( G 3 5 4 1 E ) , C 2 1 7 2 7 T , T 2 3 8 1 5 C F a r m 4 _ 1 4 T T T T T T T T + T G T T A 3 3 0 3 G , C 3 7 9 2 T ( A 1 1 7 6 V ) , C 5 1 6 7 T , G 1 0 8 8 7 A ( G 3 5 4 1 E ) , C 2 1 7 2 7 T , T 2 3 8 1 5 C F a r m 4 _ 1 5 T T T T T T T T + T G T T A 3 3 0 3 G , C 3 7 9 2 T ( A 1 1 7 6 V ) , C 5 1 6 7 T , G 1 0 8 8 7 A ( G 3 5 4 1 E ) , C 2 1 7 2 7 T , T 2 3 8 1 5 C A A c h a n g e ---P 3 3 9 5 S -P 3 1 4 L T 7 3 0 I S 2 4 3 0 I Δ H 6 Rampant C→U hypermutation in the genomes of SARS-CoV-2 and other All the mink viruses, together with the EPI_ISL455326 clade 20B representative sequence, shown here were from clade 20B and had G28881A, G28882A and G28883C changes compared to the Wuhan strain. In addition, the mink viruses from Farm 4 also lacked nt 517-519 and nt 6510-6512. Other nt changes from the Wuhan reference sequence are highlighted in yellow while nt changes from the representative clade 20B virus are shown in red type