The zoonotic origin of the SARS-CoV-2 pandemic is still unknown. Animal experiments have 28
shown that non-human primates, cats, ferrets, hamsters, rabbits and bats can be infected by 29 SARS-CoV-2. In addition, SARS-CoV-2 RNA has been detected in felids, mink and dogs in the 30 field. Here, we describe an in-depth investigation of outbreaks on 16 mink farms and humans 31 living or working on these farms, using whole genome sequencing. We conclude that the virus 32 was initially introduced from humans and has evolved, most likely reflecting widespread including fish, shellfish, poultry, wild birds and exotic animals. The finding of cases with onset 50 of illness well before the period observed in the cluster, however, suggests the possibility of 51 other sources (4). Although closely related coronaviruses in bats (5, 6) and pangolins (7, 8) 52
have most sequence identity to SARS-CoV-2, the most likely diversion date of SARS-CoV-2 53 from the most closely related bat sequence is estimated to date back to somewhere between 54 1948-1982 (9). Therefore, the animal reservoir(s) of SARS-CoV-2 is (are) yet to be identified. 55
Experimental infections in dogs (10), cats (10, 11), ferrets (10, 12), hamsters (13, 14), 56 rhesus macaques (15), tree shrew (16), cynomolgus macaques (17), grivets (18), common 57 marmosets (19), rabbits (20), and fruit bats (21) have shown that these species are susceptible 58 to SARS-CoV-2, and experimentally infected cats, tree shrews, hamsters and ferrets could 59 transmit the virus. In contrast, experimental infection of pigs and several poultry species with 60 SARS-CoV-2 proved to be unsuccessful (10, 21, 22) . SARS-CoV-2 has also sporadically been 61 identified in naturally infected animals. In the USA and in Hong Kong, SARS-CoV-2 RNA has 62 been detected in dogs (23). In the Netherlands, France, Hong Kong, Belgium and the USA, cats 63
Porechop (https://github.com/rrwick/Porechop). Primers were trimmed after which a 131 reference-based alignment was performed. The consensus genome was extracted and 132 positions with a coverage <30 were replaced with an "N" as described previously (44) . 133
Mutations in the genome compared to the GISAID sequence EPI_ISL_412973 were confirmed 134 by manually checking the mapped reads and homopolymeric regions were manually checked 135 and resolved by consulting reference genomes. The average SNP difference was determined 136 using snp-dists (https://github.com/tseemann/snp-dists). All sequences generated in this 137 study are available on GISAID. 138 139
All available near full-length Dutch SARS-CoV-2 genomes available on 1 st of July were selected 141 (n=1,775) and aligned with the sequences from this study using MUSCLE (45). Sequences with 142 >10% "Ns" were excluded. The alignment was manually checked for discrepancies after which 143 IQ-TREE (46) was used to perform a maximum likelihood phylogenetic analysis under the 144 GTR+F+I +G4 model as best predicted model using the ultrafast bootstrap option with 1,000 145
replicates.
The 
SARS-CoV-2 was first diagnosed on two mink farms in the Netherlands on April 23 rd (NB1) and 165
April 25 th (NB2), respectively. After the initial detection of SARS-CoV-2 on these farms an in-166 depth investigation was started to look for potential transmission routes and to perform an 167 environmental and occupational risk assessment. Here, we describe the results of the 24-04-2020 28-04-2020 -11-05-2020 5/6 (83%) 5/5 (100%) 6/6 (100%)
25-04-2020 31-03-2020 -30-04-2020 1/2 (50%) 8/8 (100%) 8/8 (100%)
07-05-2020 11-05-2020 -26-05-2020 5/7 (71%) 0/6 (0%)* 5/7 (71%)
07-05-2020 08-05-2020 1/3 (33%) 2/2 (100%) 2/3 (66%)
31-05-2020 01-06-2020 2/7 (29%) 3/6 (50%) 3/7 (43%)
31-05-2020 01-06-2020 1/6 (17%) 4/6 (66%) 4/6 (66%) * Serology was done approximately one week before the positive PCR test. 
During the interview on April 28 th , four out of five employees from NB1 reported that they 186 had experienced respiratory symptoms before the outbreak was detected in minks, but none 187 of them had been tested for SARS-CoV-2. The first day of symptoms of people working on NB1 188 31-05-2020 10-06-2020 -01-07-2020 8/10 (80%) NA** 8/10 (80%)
02-06-2020 03-06-2020 5/10 (50%) 5/9 (56%) 8/10 (80%)
04-06-2020 07-06-2020 1/7 (14%) 1/7 (14%) 2/7 (29%)
NB10 08-06-2020 11-06-2020 1/8 (13%) 3/8 (38%) 4/8 (50%)
08-06-2020 11-06-2020 1/3 (33%) 0/2 (0%) 1/3 (33%)
09-06-2020 11-06-2020 6/9 (66%) 2/8 (25%) 7/9 (78%)
14-06-2020 11-06-2020 -18-06-2020 3/3 (33%) 0/2 (0%) 3/3 (33%)
14-06-2020 14-06-2020 1/3 (100%) 5/6 (83%) 5/6 (83%)
21-06-2020 10-06-2020 -30-06-2020 2/2 (100%) NA** 2/2 (100%) 
On mink farm NB3 SARS-CoV-2 infection was diagnosed on May 7 th . Initially all seven 208 employees tested negative for SARS-CoV-2, but when retested between May 19 th and May 209 26 th after developing COVID-19 related symptoms, 5 out of 7 individuals working or living on 210 the farm tested positive for SARS-CoV-2 RNA. WGS were obtained from these five individuals 211 and the clustering of these sequences with the sequences derived from mink from NB3, 212 together with initial negative test result and the start of the symptoms, indicate that the 213 employees were infected with SARS-CoV-2 after the mink on the farm got infected. Also, an 214 additional infection was observed based on contact-tracing: a close contact of one of the 215 employees -who did not visit the farm -got infected with the SARS-CoV-2 strain found on 216 
Phylogenetic analysis of the mink SARS-CoV-2 genomes showed that mink sequences of 16 246 farms grouped into 5 different clusters (Figure 2 and 3) . Viruses from farms NB1, NB3, NB4, 247
NB8, NB12, NB13 and NB16 belonged to cluster A, sequences from NB2 were a separate 248 cluster (B), those from farms NB6, NB7, NB9 and NB14 grouped together in cluster C, NB5, 249 NB8, NB10 and NB15 grouped to cluster D, and NB11 had sequences designated as cluster E. 250
On farm NB8, SARS-CoV-2 viruses could be found from both cluster A and cluster D. A detailed 251 inventory of possible common characteristics, like farm owner, shared personnel, feed 252 supplier and veterinary service provider, was made. In some cases, a link was observed with 253 the same owners of several farms, for instance for cluster A for NB1 and NB4, and for NB8 and 254 NB12. Although NB7, NB11 and NB15 were also linked to the same owner, viruses from these 255 farms belonged to cluster C, D and E respectively. No common factor could be identified for 256 most farms and clustering could also not be explained by geographic distances as multiple 257 clusters were detected in different farms located close to each other (Table 2 and figure 4) . 258 259 
In total 18 sequences from mink farm employees or close contacts were generated from seven 269 different farms. In most cases, these human sequences were near-identical to the mink 270 sequences from the same farm. For NB1 the situation was different and the human sequence 271 clusters deeply within the sequences derived from mink (Figure 1) 
increased mortality in mink. The sequences from farm NB1 had between 0 and 9 nucleotides 289 differences (average 3.9 nucleotides) and from NB2 between 0 and 8 nucleotides differences 290 (average of 3.6), which is much more than what has been observed in outbreaks in human 291 settings. The sequences from NB3 had 0 to 2 nucleotides difference suggesting that the virus 292 was recently introduced, in line with the observed disease in humans, which occurred in the 293 weeks post diagnosis of the infection in mink. After the initial detection of SARS-CoV-2 on 294 mink farms, farms were screened weekly. The first, second, fifth and sixth weekly screening 295 yielded new positives. The sequences of mink at NB6 had between 0 and 12 nucleotides 296 differences, whereas diversity was lower for the subsequent farm sequences (Table 2) . 297
Several non-synonymous mutations were identified among the mink sequences 298 compared to the Wuhan reference sequence NC_045512.2. However, no particular amino 299 acid substitutions were found in all mink samples (Figure 5) . Of note, three of the clusters had 300 the position 614G variant (clusters A, C and E), and 2 had the original variant. There were no 301 obvious differences in the presentation of disease in animals or humans between clusters 302 based on the data available at this stage, but further data collection and analysis, also for cases 303 after NB16, are ongoing to investigate this further. The observed mutations can also be found 304 in the general population and the same mutations also were found in human cases which were 305 related to the mink farms. 306 3  88  117  4  80  185  194  210  221  341  379  399  95  352  372  398  405  445  498  672  728  996  1096  1113  1126  1204  1236  1246  1352  1568  1588  1921  1957  1997  2001  2210  2508  2584  2706  2769  3063  3170  3202  3338  3379  3432  3522  3606  3615  3716  3829  3874  4177  4197  4308  11  187  314  794  1003  1092  1179  1181  1315  1369  1637  1681  1882  2025  2143  2222  2367  2491  32  34  55  57  182  207  219  224  229  258  38  45  25  62  115  6  153  176  177  257  261  367  453  486  501  614  832  942 
Here we show ongoing SARS-CoV-2 transmission in mink farms and spill-over events to 315 humans. To the best of our knowledge, these are the first animal to human SARS-CoV-2 316 transmission events documented. More research in minks and other mustelid species, to 317 demonstrate if these species can be a true reservoir of SARS-CoV-2 although from our 318 observations we consider this likely. After the detection of SARS-CoV-2 on mink farms, 68% of 319 the tested farm workers and/or relatives or contacts were shown to be infected with SARS-320
CoV-2, indicating that contact with SARS-CoV-2 infected mink is a risk factor for contracting 321
A high diversity in the sequences from some mink farms was observed which most 323 likely can be explained by many generations of infected animals before an increase in 324 mortality was observed. The current estimates are that the substitution rate of SARS-CoV-2 is 325 around 1.16*10^-3 substitutions/site/year (53), which corresponds to around one mutation 326 per two weeks. This could mean that the virus was already circulating in mink farms for some 327 time. However, there was also a relatively high sequence diversity observed in farms which 328 still tested negative one week prior, hinting towards a faster evolution of the virus in the mink 329 population. This can indicate that the virus might replicate more efficiently in mink or might 330 have acquired mutations which makes the virus more virulent. However, no specific mutations 331
were found in all mink samples, making increased virulence less likely. In addition, mink farms 332 have large populations of animals which could lead to very efficient virus transmission. 333
Generation intervals for SARS-CoV-2 in humans have been estimated to be around 4-5 334 days(54), but with high dose exposure in a high-density farm could potentially be shorter. 335
Recently, a specific mutation in the spike protein (D614G) was shown to result in an increased 336 virulence in vitro (55), while it was not associated an increased growth rate for cluster nor an 337 increased mortality (56). This mutation was present in farm clusters A, C and E, but no obvious 338 differences in clinical presentation, disease severity, or rate of transmission to humans was 339 observed. 340
While we found sequences matching with the animal sequences on several farms, not 341 all of these can be considered direct zoonotic transmissions. For instance, the two employees 342 from mink farm NB3 were most likely infected while working at the mink farm given the 343 specific clustering in the phylogenetic tree and the timing of infection. Subsequent human 344
infections may have originated from additional zoonotic infections, or from human to human 345 transmission within their household. Further proof that animals were the most likely source 346 of infection was provided by the clear phylogenetic separation between farm related human 347 cases and animal cases, from sequences from cases within the same 4-digit postal code area. 348
Spill-back into the community living in the same 4-digit postal code area was not observed 349 using sequence data, but cannot be entirely ruled out as the testing strategy during April and 350
May was focusing on health care workers, persons with more severe symptoms, and persons 351 at risk for complications, rather than monitoring community transmission and milder cases. During interviews, it became clear that farms had occasionally hired temporary workers that 361 had not been included in the testing and were lost to follow-up, stressing the need for vigorous 362 biosecurity and occupational health guidance. Since our observation, SARS-CoV-2 infections 363 have also been described in mink farms in Denmark, Spain and the USA (57-59), and mink 364 farming is common in other regions of the world as well, also in China where around 26 million 365 mink pelts are produced on a yearly basis (60). The population size and the structure of mink 366 farms is such that it is conceivable that SARS-CoV-2 -once introduced -could continue to 367 circulate. Therefore, continued monitoring and cooperation between human and animal 368 health services is crucial to prevent the animals serving as a reservoir for continued infection 369 in humans. 370 371