key: cord-0270843-s6sp3rme authors: Oude Munnink, Bas B.; Sikkema, Reina S.; Nieuwenhuijse, David F.; Molenaar, Robert Jan; Munger, Emmanuelle; Molenkamp, Richard; van der Spek, Arco; Tolsma, Paulin; Rietveld, Ariene; Brouwer, Miranda; Bouwmeester-Vincken, Noortje; Harders, Frank; der Honing, Renate Hakze-van; Wegdam-Blans, Marjolein C.A.; Bouwstra, Ruth J.; GeurtsvanKessel, Corine; van der Eijk, Annemiek A.; Velkers, Francisca C.; Smit, Lidwien A.M.; Stegeman, Arjan; van der Poel, Wim H.M.; Koopmans, Marion P.G. title: Jumping back and forth: anthropozoonotic and zoonotic transmission of SARS-CoV-2 on mink farms date: 2020-09-01 journal: bioRxiv DOI: 10.1101/2020.09.01.277152 sha: 0ea505a6f2b000cda528a20a91829dba2b65dbb2 doc_id: 270843 cord_uid: s6sp3rme The zoonotic origin of the SARS-CoV-2 pandemic is still unknown. Animal experiments have shown that non-human primates, cats, ferrets, hamsters, rabbits and bats can be infected by SARS-CoV-2. In addition, SARS-CoV-2 RNA has been detected in felids, mink and dogs in the field. Here, we describe an in-depth investigation of outbreaks on 16 mink farms and humans living or working on these farms, using whole genome sequencing. We conclude that the virus was initially introduced from humans and has evolved, most likely reflecting widespread circulation among mink in the beginning of the infection period several weeks prior to detection. At the moment, despite enhanced biosecurity, early warning surveillance and immediate culling of infected farms, there is ongoing transmission between mink farms with three big transmission clusters with unknown modes of transmission. We also describe the first animal to human transmissions of SARS-CoV-2 in mink farms. One sentence summary SARS-CoV-2 transmission on mink farms. 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 A novel coronavirus from 374 patients with pneumonia in China An interactive web-based dashboard to track COVID-19 in 376 real time Rapid SARS-CoV-2 whole-genome sequencing and analysis for 393 informed public health decision-making in the Netherlands A new coronavirus associated with human respiratory disease in China A Novel Bat Coronavirus Closely Related to SARS-CoV-2 Contains 400 Natural Insertions at the S1/S2 Cleavage Site of the Spike Protein A pneumonia outbreak associated with a new coronavirus of 406 probable bat origin CoV-2 related coronaviruses in Malayan pangolins Pangolins Harbor SARS-CoV-2-Related Coronaviruses Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-417 coronavirus 2. Science (80-. ) SARS-CoV-2 in Domestic Cats Pathogenesis and 425 transmission of SARS-CoV-2 virus in golden Syrian hamsters SUBJECT AREAS Infectious 426 Simulation of the 430 clinical and pathological manifestations of Coronavirus Disease Syrian hamster model: implications for disease pathogenesis and 432 transmissibility Respiratory disease 436 in rhesus macaques inoculated with SARS-CoV-2. Nature Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman 445 primate model. Science (80-. ) Experimental Transmission Studies of SARS-CoV-2 in Fruit Bats, Ferrets, Pigs 461 and Chickens. SSRN Electron Infection 466 of dogs with SARS-CoV-2 First detection and genome sequencing of SARS-470 CoV-2 in an infected cat in France First Reported 474 Cases of SARS-CoV-2 Infection in Companion Animals Promed Post -ProMED-mail Is COVID-19 the first pandemic that evolves into a panzootic Hakze-485 van der Honing SARS-CoV-2 infection in farmed minks, the Clinical and Pathological Findings in SARS-CoV-2 Disease Outbreaks in Farmed 499 Mink (Neovison vison) Bedrijfsmatig gehouden dieren en SARS-CoV-2 | Nieuws en media | NVWA Genomic monitoring to understand the emergence and spread of Usutu virus in the 506 Rapid outbreak sequencing of Ebola virus 516 Sierra Leone identifies transmission chains linked to sporadic cases Genomic and epidemiological monitoring of yellow fever virus transmission 532 potential. Science (80-. ) Real-time, portable 548 genome sequencing for Ebola surveillance COVID-19 in health-care workers in three 555 hospitals in the south of the Netherlands: a cross-sectional study An automated genotyping tool for enteroviruses and noroviruses Detection of 2019 novel 569 coronavirus (2019-nCoV) by real-time RT-PCR An evaluation of COVID-19 serological assays informs future diagnostics and exposure 574 assessment Whole Genome Nanopore Sequencing, using Usutu Virus as an Example MUSCLE: multiple sequence alignment with high accuracy and high 579 throughput IQ-TREE: a fast and effective 581 stochastic algorithm for estimating maximum-likelihood phylogenies GISAID: Global initiative on sharing all influenza data -from vision 584 to reality Classes and Methods for Spatial Data: the sp Package. R News GitHub rspatial/raster: R raster package Evolution and epidemic spread of SARS-607 CoV-2 in Brazil. Science (80-. ) Estimating the 609 generation interval for coronavirus disease (COVID-19) based on symptom onset data SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the 618 COVID-19 Virus Promed Post -ProMED-mail COVID-19 hits U.S. mink farms after ripping through Europe. Science (80-. ) No Title) This work has received funding from the European Union's Horizon 2020 research and 631 innovation programme under grant agreement No 101003589 (RECoVER), from ZonMW (grant agreement No. 10150062010005), and from 633 the Netherlands Ministry of Agriculture All data, code and materials used described in this manuscript are publicly available.