Denmark coronavirus spreads to mink farms : new animal sources for human COVID‐19

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Updated severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) data in humans are provided in Table 1. This synopsis summarizes the latest findings on animal sources that could pose a risk for human SARS‐CoV‐2 infection and coronavirus disease‐2019 (COVID‐19).

The information provided may be important during xenotransplantation or for immunocompromised individuals who own or work with animals on a regular basis. It is widely accepted that coronavirus species can be identified in both humans and various animal species and are commonly associated with respiratory or gastrointestinal disease, or both. 1

With SARS‐CoV‐2 cases in humans continuously increasing on a daily basis, it is important to understand which animal species may potentially be susceptible to SARS‐CoV‐2 infection and hence may serve as a reservoir for human infections.

Table 1

Facts on high pathogenic human CoVs

VirusTime of circulationLaboratory confirmed casesDeathsCase fatality rate (%)Country distribution
SARS‐CoV a2002‐200380967749.626
MERS‐CoV b2012‐ongoing24948533527
SARS‐CoV‐2 c2019‐ongoing25 602 665852 7583.3Global pandemic

aSource: https://www.who.int/csr/sars/country/table2004_04_21/en/.bSource: https://www.who.int/emergencies/mers‐cov/en/.cSource: https://covid19.who.int (Accessed 2020/09/03).This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.

Previously, it has been determined that animals within the Felidae (domestic cats; captive tigers and lions), Canidae (pet dogs), and Mustelidae (farmed minks) families can become naturally infected with SARS‐CoV‐2. The majority, if not all cases previously reported, 2 were due to the close contact of pets or farmed animals with COVID‐19‐infected patients.

​Table 2 provides updated information for Felidae, Canidae and Mustelidae but also additional species investigated. Previous results have been further confirmed in a recent study conducted in Northern Italy where more than 500 companion animals were sampled at the time of frequent human SARS‐CoV2 infection. 3

While SARS‐CoV‐2 RNA was not found in any animal, 3.4% of all dogs and 3.9% of the cats investigated had measurable neutralizing antibody titers, furthermore, the presence of COVID‐19 in a household was identified as a risk factor. 3

In addition, under experimental conditions, ferrets were shown to be a suitable model to mimic SARS‐CoV‐2 replication in the respiratory tract of humans.

However, clinical signs or mortality are not always seen. 4 Moreover, a recent study indicated that SARS‐CoV‐2 is transmitted via direct contact within 1‐3 days between ferrets housed in the same cage, but also via air within 3‐7 days when housed in separate cages while sharing the same airflow. 5

The robust airborne transmission of SARS‐CoV‐2 shown in that study further confirms that physical distancing measures are important. 5

Table 2

Summary of findings in animals to date (Adapted from OIE Technical Factsheet, Infection with SARS‐CoV‐2 in animals)

FamilySpeciesType of infectionExperimental infection characteristicsSusceptibilityClinical signsTransmissionSerological surveillance
Animal# (Reference)RouteDose aNone, low, highPositive/total number testedReference
SuidaePigsExperimental9 65 4Intra‐nasal105 TCID50105 PFUNoneNoNo0/18726
PoultryChickensExperimental17 65 410 7Oculo‐oronasalIntra‐nasal105 TCID50104.5 PFU105.4 TCID50NoneNoNo0/15326
DuckExperimental5 410 7Intra‐nasalIntra‐choanal104.5 PFU106 TCID50NoneNoNo0/15326
TurkeysExperimental10 7Intra‐choanal105.4 TCID50NoneNoNoNA
Japanese quailExperimental10 7Intra‐choanal105.4 TCID50NoneNoNoNA
White Chinese geeseExperimental10 7Intra‐choanal106 TCID50NoneNoNoNA
RuminantsCattleNA0/10726
Sheep0/13326
GoatsNA
CaninaeDogsNatural and experimental5 4Intra‐nasal105 PFULowNo or mildNo8/1800/497326
FelidaeCats (domestic)Natural and experimental14 43 27Intra‐nasalNA105 PFUNAHighNo or mildYes6/600/87326
Tigers and lionsNaturalHighYesYes0/826
MusteliadaeFerretsExperimental10 69 4Intra‐nasal105 TCID50105 PFUHighNo or mildYes0/226
Minks (American minks, Neovison vison)NaturalHighYesYes, also mink‐human0/8126
PteropodidaeEgyptian fruit bats (Rousettus aegyptiacus)Experimental9 6Intra‐nasal105 TCID50HighNoYesNA
CricetidaeGolden Syrian hamstersExperimental4 59 1215 1113 13Intra‐nasal6 × 105 TCID508 × 104 TCID50105 PFU105 PFUHighNo or mildYesNA
Old word monkeysSubfamily CercopithecinesMacaques (Macaca fascicularis and Macaca mulatta)Experimental5 15Intra‐nasal106 TCID50HighYesYesNA
3 163 163 1610 18Intra‐nasal and intra‐tracheal1.1 × 106 PFU1.1 × 105 PFU1.1 × 104 PFU
Abbreviation: NA, not available.
aMedian tissue culture infectious dose (TCID50) per animal or plaque forming unit (PFU).
This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.

Fortunately, SARS‐CoV‐2 experimental infection trials in poultry using chickens and ducks demonstrated a lack of susceptibility of these species to the virus (Table ​(Table22). 4 , 6 A recently published study with a larger cohort, conducted in the USA, further confirmed these negative results by expanding the poultry species range tested by also including turkeys, quails, and geese (Table ​(Table22). 7

Since our last update, additional SARS‐CoV‐2‐infected mink farms have been discovered with a total of 25 farms in the Netherlands 8 , 9 (https://promedmail.org/promed‐post/?id=7588293), 3 farms in Denmark (https://www.oie.int/fileadmin/Home/MM/Update_1_Letter_to_OIE_about_the_COVID‐19_situation_in_Denmark.pdf), and one farm in Spain (https://promedmail.org/promed‐post/?id=7584560). Overall, one million Dutch minks and 100 000 Spanish minks have been culled so far (https://www.theguardian.com/world/2020/jul/17/spain‐to‐cull‐nearly‐100000‐mink‐in‐coronavirus‐outbreak).

In general, affected mink farms are considered spillover events from the human pandemic and the source of infection was likely infected humans entering the farm. 8 However, humans infected by minks have also been identified 10 and this may have happened 2‐6 times with the transmission route not entirely established (https://www.government.nl/latest/news/2020/05/19/new‐results‐from‐research‐into‐covid‐19‐on‐mink‐farms).

In addition to reports of more cases of naturally infected animals, new SARS‐CoV‐2 animal models have been reported (Table ​(Table2).2).

Recently, clinical and pathological manifestations of COVID‐19 have been reproduced in a golden Syrian hamster model. 11

Infected hamsters developed mild clinical signs and weight loss but eventually recovered and developed serum neutralizing antibodies 14 days post‐challenge. 11

Similar results were also obtained by another group which demonstrated SARS‐CoV‐2 antigen by immunohistochemistry in nasal mucosa and bronchial epithelial cells between 2 and 5 days post‐infection. 12

Since then, the golden Syrian hamster model has been used to show that surgical masks reduce the risk of SARS‐CoV‐2 contact transmission. 13

In fact, a surgical mask partition between challenged and naïve hamsters significantly reduced transmission to 25%. 13

Of note, mice, although members of the Cricetiadae family, are not susceptible to SARS‐CoV‐2 unless the virus is genetically adapted by serial passaging. 14

This perhaps indicates that a species cannot be categorized as susceptible or resistant due to their family.

Non‐human primates (rhesus macaques) were successfully infected with SARS‐CoV‐2, and characteristic respiratory signs were observed in both 3‐ to 5‐year‐old and 15‐year‐old rhesus macaques (Table ​(Table22). 15

Viral replication in the respiratory tract was more pronounced in older monkeys and lasted for 14 days. These results confirm that rhesus macaques can be infected by SARS‐CoV‐2. 15

In line with this research, a US group used the rhesus macaque SARS‐CoV‐2 model to test protective immunity after re‐exposure. 16

The rhesus macaques had high viral loads in the upper and lower respiratory tract and pathologic evidence of viral pneumonia after initial challenge.

Following re‐challenge, there was approximately a 5 log10 reduction in median SARS‐CoV‐2 viral loads in bronchoalveolar lavage and nasal mucosa samples when compared with viral loads after primary infection. 16

Similar results were also obtained by a Chinese group. 17 Furthermore, a SARS‐CoV‐2 DNA vaccine candidate was successfully tested in the rhesus macaque model indicating >3.1 log10 (bronchoalveolar lavage) and >3.7 log10 (nasal mucosa) reductions in median viral loads when compared to placebo controls. 18

Several scientific groups have used an alternative approach to identify possible SARS‐CoV‐2 susceptible animals. Rather than searching for naturally infected animals or performing experimental infection trials, the receptor angiotensin‐converting enzyme 2 (ACE2), which binds to the receptor binding domain (RBD) of the spike protein of SARS‐CoV‐2, essential for host cell entry and replication initialization, was investigated by comparing its structure across animal species.

Early virus infectivity studies used HeLa cells that either expressed ACE2 proteins from selected species or not to show that SARS‐CoV‐2 uses ACE2 proteins for cell entry in humans, Chinese horseshoe bats, civets, and pigs, but not in mice. 19

In a follow‐up study, X‐ray structures of human ACE2 bound to the RBD of SARS‐CoV‐2 were used to predict its binding to ACE2 orthologue proteins from different animals. 20 Of the 20 amino acids in ACE2 that make contact with the spike protein, only 13 are necessary for ACE2 to function as a SARS‐CoV‐2 receptor, possibly indicating a minimal species barrier.

Pigs and dogs were considered exceptions as they have low ACE2 expression in their respiratory tract. 20

Further, using flow cytometry to detect interactions of RBD‐Fc proteins with ACE2 orthologues expressed on the surface of 293T cells, and assays with pseudoviruses expressing the spike protein, species with an orthologue ACE2 receptor were identified: ruminants (camels, cattle, goats, sheep), horses, pigs, cats, and rabbits; this receptor also supports viral entry of SARS‐CoV‐1, a bat‐CoV (Bat‐CoV RaTG13), and Pangolin‐CoV. 21 Using a surface ACE2 binding assay with HeLa cells transduced with lentiviruses expressing ACE2 from different species, a different study investigated birds, reptiles (alligators, turtles, lizards), mammals, amphibians, coelacanths bone fish, and cartilaginous fish.

ACE2 orthologues were identified in 80 mammalian species, including pets, livestock, and animals commonly found in zoos and aquaria. 22 Overall, results so far indicate that many more mammalian species may potentially be susceptible to SARS‐CoV‐2 infection and replication, and can therefore also serve as possible reservoirs.

New information recently became available on the possible origin of SARS‐CoV‐2. Soon after the discovery of SARS‐CoV‐2, bats had been suggested as the most likely reservoir host. As expected, 7/9 fruit bats (Rousettus aegyptiacus) had a transient SARS‐CoV‐2 infection after experimental inoculation and 1/3 contact bats also became infected. 6

Recently, the pangolin species has been suggested as a natural reservoir of SARS‐CoV‐2. Pangolin‐associated coronaviruses belonging to two sub‐lineages of SARS‐CoV‐2‐related coronaviruses were identified in Malayan pangolins. 23 , 24

Specifically, five key amino acid residues of the RBD involved in the interaction with human ACE2 are consistent between Pangolin‐CoV and SARS‐CoV‐2 in contrast to only one out of the five key residues between SARS‐CoV‐2 and Bat‐CoV RaG13. 25

Moreover, at the whole genome level, Pangolin‐CoV is 91.0% identical to SARS‐CoV‐2 whereas RaTG13 and Pangolin‐CoV are only 90.6% identical. 25

In summary, since SARS‐CoV‐2 emerged in the human population toward the end of 2019, it has been spreading at a high rate and infection rates in humans continue to increase.

There is confirmed evidence that SARS‐CoV‐2 from COVID‐19‐infected humans can spillover to certain animal species within the families Mustelidae, Felinae, and Caninae. Commonly, infections in animal hosts are subclinical but occasionally clinical signs can be observed.

Moreover, cats, dogs, ferrets, Egyptian fruit bats, golden Syrian hamsters, and macaques have been experimentally infected and some of these species are now used for SARS‐CoV‐2 research.

There is however surprisingly little information on other species which are predicted to potentially serve as reservoirs for humans. Of note, the sample size of species that have been tested was low.

This lack of knowledge requires attention, in cases of xenotransplantation most organs or products of animal origin should be tested for the presence of SARS‐CoV‐2 prior to their use in patients.

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Outbreak investigation

Following initial detection of SARS-CoV-2 in mink on two farms on April 23rd and April 25th, respectively, as part of routine health monitoring done by the Royal GD Animal Health service and subsequent investigation by Wageningen Bioveterinary Research (WBVR), the national reference laboratory for notifiable animal diseases, a One Health outbreak investigation team was convened (39, 40).

Subsequently, respiratory signs and increased mortality in mink was made notifiable by the Dutch Ministry of Agriculture, Nature and Food Quality and the farms were quarantined (no movements of animals and manure and visitor restrictions).

On May 7th two other mink farms in the same region were confirmed to be infected.

By the end of May the Dutch minister of Agriculture decided that all mink on SARS-CoV-2 infected farms had to be culled. Moreover, as the clinical manifestation of the infection was highly variable within and between farms, including asymptomatic infections, weekly testing of dead animals for SARS-CoV-2 infections became compulsory for all mink farms in the Netherlands. Moreover, a nation-wide transport ban of mink and mink manure, and a strict hygiene and visitor protocol was implemented.

The first infected mink farms were culled from June 6th onwards. From the 10th infected farm (NB10) onwards, culling took place within 1-3 days after diagnosis. In this manuscript, the data up to June 26th, when a total of 16 mink farms in the Netherlands were found positive for SARS-CoV-2 infections, is presented.

Veterinary and human contact tracing

The Netherlands Food and Consumer Product Safety Authority (NVWA) traced animal related contacts with other mink farms. Backward and forward tracing of possible high-risk contacts was done in the framework of the standard epidemiological investigation by the NVWA (i.e.

focused on movement of vehicles, visitors such as veterinary practitioners, (temporary) workers, sharing of equipment between farms and transport and delivery of materials, such as feed, pelts, carcasses and manure).

Persons with possible exposure from this investigation, as well as farm owners and resident farm workers were asked to report health complaints to the municipal health service for testing and – in the case of confirmed infections – for health advice and further contact tracing.

Farm owners and workers on infected mink farms were informed of potential risks and were given advice on the importance and use of personal protective equipment and hygiene when handling animals (41).

The contact structure on the farms was assessed through in-depth interviews, to identify additional persons with possible exposure to mink. In order to provide an enhanced set of reference genome sequences, anonymized samples from patients that had been diagnosed with COVID-19 in the area of the same four-digits postal codes as farms NB1-NB4 in March and April 2020 were retrieved from clinical laboratories in the region.

SARS-CoV-2 diagnostics and sequencing

The presence of viral RNA in mink samples was determined using a RT-PCR targeting the E gene as previously described (42). For the human samples, diagnostic RT-PCR was performed for the E and the RdRp gene (42). In addition, serology was performed, using the Wantai Ig total and IgM ELISA, following the manufacturer’s instructions(43).

For all samples with a Ct value <32, sequencing was performed using a SARS-CoV-2 specific multiplex PCR for Nanopore sequencing, as previously described (3).

The libraries were generated using the native barcode kits from Nanopore (EXP-NBD104 and EXP-NBD114 and SQK-LSK109) and sequenced on a R9.4 flow cell multiplexing 24 samples per sequence run. Flow cells were washed and reused until less than 800 pores were active. The resulting raw sequence data was demultiplexed using

Porechop (https://github.com/rrwick/Porechop). Primers were trimmed after which a reference-based alignment was performed. The consensus genome was extracted and positions with a coverage <30 were replaced with an “N” as described previously (44).

Mutations in the genome compared to the GISAID sequence EPI_ISL_412973 were confirmed by manually checking the mapped reads and homopolymeric regions were manually checked and resolved by consulting reference genomes.

The average SNP difference was determined using snp-dists (https://github.com/tseemann/snp-dists). All sequences generated in this study are available on GISAID.

Phylogenetic analysis

All available near full-length Dutch SARS-CoV-2 genomes available on 1st of July were selected (n=1,775) and aligned with the sequences from this study using MUSCLE (45). Sequences with >10% “Ns” were excluded.

The alignment was manually checked for discrepancies after which IQ-TREE (46) was used to perform a maximum likelihood phylogenetic analysis under the GTR+F+I +G4 model as best predicted model using the ultrafast bootstrap option with 1,000 replicates.

The phylogenetic trees were visualized in Figtree (http://tree.bio.ed.ac.uk/software/figtree/). For clarity reasons all bootstrap values below 80 were removed.

To look at potential relationships with migrant workers, also all Polish sequences from GISAID (47) were included in the alignment (Supplementary table 1).

Mapping specific mutation patterns on mink farms and in mink farm employees

Amino acid coordinates are described in relation to the Genbank NC_045512.2 reference genome. Open reading frames were extracted from the genome alignment using the genome annotation as supplied with the reference genome. A custom R script was used to distinguish

synonymous from non-synonymous mutations and non-synonymous mutations were visualized using a tile map from the ggplot2 package (48).

Geographical overview of mink farms in the Netherlands and SARS-CoV-2 positive farms

To protect confidentiality, SARS-CoV-2 positive mink farms were aggregated at municipality level. The datasets “Landbouw; gewassen, dieren en grondgebruik naar gemeente” and “Wijk- en Buurtkaart 2019” from Statistics Netherlands (CBS) were used (49). Maps were created using R packages sp (50), raster (51) and rgdal (52) and ArcGIS 10.6 software by ESRI.

Results

SARS-CoV-2 was first diagnosed on two mink farms in the Netherlands on April 23rd (NB1) and April 25th (NB2), respectively. After the initial detection of SARS-CoV-2 on these farms an in- depth investigation was started to look for potential transmission routes and to perform an environmental and occupational risk assessment. Here, we describe the results of the outbreak investigation of the first 16 SARS-CoV-2 infected mink farms by combining SARS-CoV- 2 diagnostics, WGS and in-depth interviews.

Screening of farm workers and contacts

Farm owners of the 16 SARS-CoV-2 positive mink farms were contacted by the municipal health services to conduct contact investigation and samples were taken for RT-PCR-based and serological SARS-CoV-2 diagnostics. In total, 97 individuals were tested by either serological assays and/or RT-PCR. In total, 43 out of 88 (49%) upper-respiratory tract samples tested positive by RT-PCR while 38 out of 75 (51%) serum samples tested positive for SARS- CoV-2 specific antibodies. In total, 66 of 97 (67%) of the persons tested had evidence for SARS- CoV-2 infection (table 1).

Table 1. Overview of human sampling on SARS-CoV-2 positive mink farms.

Farm:First diagnosis in animals:Date(s) of sampling employees and family members:PCR positive (%)Serology positive (%)Employees and family members tested positive (PCR and/or serology)
NB124-04-202028-04-2020 – 11-05-20205/6 (83%)5/5 (100%)6/6 (100%)
NB225-04-202031-03-2020 – 30-04-20201/2 (50%)8/8 (100%)8/8 (100%)
NB307-05-202011-05-2020 – 26-05-20205/7 (71%)0/6 (0%)*5/7 (71%)
NB407-05-202008-05-20201/3 (33%)2/2 (100%)2/3 (66%)
NB531-05-202001-06-20202/7 (29%)3/6 (50%)3/7 (43%)
NB631-05-202001-06-20201/6 (17%)4/6 (66%)4/6 (66%)
NB731-05-202010-06-2020 – 01-07-20208/10 (80%)NA**8/10 (80%)
NB802-06-202003-06-20205/10 (50%)5/9 (56%)8/10 (80%)
NB904-06-202007-06-20201/7 (14%)1/7 (14%)2/7 (29%)
NB1008-06-202011-06-20201/8 (13%)3/8 (38%)4/8 (50%)
NB1108-06-202011-06-20201/3 (33%)0/2 (0%)1/3 (33%)
NB1209-06-202011-06-20206/9 (66%)2/8 (25%)7/9 (78%)
NB1314-06-202011-06-2020 – 18-06-20203/3 (33%)0/2 (0%)3/3 (33%)
NB1414-06-202014-06-20201/3 (100%)5/6 (83%)5/6 (83%)
NB1521-06-202010-06-2020 – 30-06-20202/2 (100%)NA**2/2 (100%)
NB1621-06-202023-06-20200/2 (0%)NA**0/2 (0%)
Total:  43/88 (49%)38/75 (51%)66/97 (68%)

* Serology was done approximately one week before the positive PCR test.

** No serology was performed

Anthropozoonotic transmission of SARS-CoV-2

During the interview on April 28th, four out of five employees from NB1 reported that they had experienced respiratory symptoms before the outbreak was detected in minks, but none of them had been tested for SARS-CoV-2.

The first day of symptoms of people working on NB1 ranged from April 1st to May 9th. For 16 of the mink, sampled on April 28th, and one farm employee, sampled on May 4th, a WGS was obtained (hCov- 19/Netherlands/NoordBrabant_177/2020).

The human sequence clusters within the mink sequences although it had 7 nucleotides difference with the closest mink sequence (Figure 1 and cluster A in figures 2 and 3). On farm NB2, SARS-CoV-2 was diagnosed on April 25th. Retrospective analysis showed that one employee from NB2 had been hospitalized with SARS- CoV-2 on March 31st.

All samples from the 8 employees taken on April 30th were negative by RT-PCR but tested positive for SARS-CoV-2 antibodies. The virus sequence obtained from animals was distinct from that of farm NB1, indicating a separate introduction (Figure 2 and 3, cluster B).




Figure 1: Zoom of the phylogenetic analysis of NB1. A maximum likelihood analysis was performed using all available SARS-CoV-2 Dutch sequences. Sequences from mink on NB1 are depicted in red and from the employee on NB1 in blue. The two sequences in black at the root of the cluster are the closest matching human genome sequences from the national SARS-CoV-2 sequence database. Scale bar represents units of substitutions per site.

Zoonotic transmission of SARS-CoV-2

On mink farm NB3 SARS-CoV-2 infection was diagnosed on May 7th. Initially all seven employees tested negative for SARS-CoV-2, but when retested between May 19th and May 26th after developing COVID-19 related symptoms, 5 out of 7 individuals working or living on the farm tested positive for SARS-CoV-2 RNA.

WGS were obtained from these five individuals and the clustering of these sequences with the sequences derived from mink from NB3, together with initial negative test result and the start of the symptoms, indicate that the employees were infected with SARS-CoV-2 after the mink on the farm got infected.

Also, an additional infection was observed based on contact-tracing: a close contact of one of the employees – who did not visit the farm – got infected with the SARS-CoV-2 strain found on NB3.

Animal and human sequences from farm NB3 were related to those from farm NB1, but were both part of cluster A.

Similarly, on mink farm NB7 zoonotic transmission from mink to human most likely occurred.

On this farm, SARS-CoV-2 infection in mink was diagnosed on May 31st and employees initially tested negative for SARS-CoV-2 but started to develop symptoms at a later stage. Samples were taken between June 10th and July 1st from 10 employees of which 8 tested positive for SARS-CoV-2 RNA.

From 2 samples WGS could be generated from the employees which clustered together with the sequences from the animals from this farm.

Comparison with national reference database and enhanced regional sampling

The sequences generated from mink farms and from mink farm employees were compared with the national database consisting of around 1,775 WGS. In addition, to discriminate between locally acquired infections and mink farm related SARS-CoV-2 infection, and to determine the potential risk for people living close to mink farms, WGS was also performed on 34 SARS-CoV-2 positive samples from individuals who live in the same four-digit postal code area compared to the first four mink farms.

These local sequences reflected the general diversity seen in the Netherlands and were not related to the clusters of mink sequences found on the mink farms, thereby also giving no indication of spill-over to people living in close proximity to mink farms (sequences shown in magenta, Figure 2).

The sequences from the mink farm investigation were also compared to sequences from Poland (n=65), since many of the mink farm workers were seasonal migrants from Poland, but the these were not related.

Figure 2: Maximum likelihood analysis of all SARS-CoV-2 Dutch sequences. The sequences derived from minks from different farms are indicated with different colors, human sequences related to the mink farms in blue and samples from similar 4-digit postal code are indicated in magenta. Scale bar represents units of substitutions per site.

Mink farm related sequence clusters

Phylogenetic analysis of the mink SARS-CoV-2 genomes showed that mink sequences of 16 farms grouped into 5 different clusters (Figure 2 and 3). Viruses from farms NB1, NB3, NB4,NB8, NB12, NB13 and NB16 belonged to cluster A, sequences from NB2 were a separate cluster (B), those from farms NB6, NB7, NB9 and NB14 grouped together in cluster C, NB5, NB8, NB10 and NB15 grouped to cluster D, and NB11 had sequences designated as cluster E.

On farm NB8, SARS-CoV-2 viruses could be found from both cluster A and cluster D.

A detailed inventory of possible common characteristics, like farm owner, shared personnel, feed supplier and veterinary service provider, was made. In some cases, a link was observed with the same owners of several farms, for instance for cluster A for NB1 and NB4, and for NB8 and NB12.

Although NB7, NB11 and NB15 were also linked to the same owner, viruses from these farms belonged to cluster C, D and E respectively. No common factor could be identified for most farms and clustering could also not be explained by geographic distances as multiple clusters were detected in different farms located close to each other (Table 2 and figure 4).

Table 2. Overview of the clusters detected on the different farms.

Farm:Date of diagnosis:Sequence cluster:Same owner:Feed supplier:Vet**:Number of sequences (human):Sequence diversity (average):Mink population size:Detection***:
NB124-04-20ANB1, NB41I17 (1)0-9 (3.9)75,711Notification
NB225-04-20B 1II80-8 (3.6)50,473Notification
NB307-05-20A 2III5 (5)0-2 (0.6)12,400Notification
NB407-05-20ANB1, NB41I1NA67,945Contact tracing NB1
NB531-05-20D 1IV1NA38,936EWS-Ser+PM- 1st
NB631-05-20C 3V90-12 (6.8)54,515EWS-Ser+PM- 1st
NB731-05-20CNB7, NB11, NB153II6 (2)0-4 (1.4)79,355EWS-PM-1st
NB802-06-20A/DNB8, NB12*3V6 (5)0-6 (2.6)39,144EWS-Ser+PM- 1st
NB904-06-20C 2V2 (1)0-3 (1.5)32,557EWS-Ser+PM- 2nd
NB1008-06-20D 3II40-3 (1.1)26,824EWS-Ser+PM- 2nd
NB1108-06-20ENB7, NB11, NB153II40-4 (2.2)38,745EWS-PM-2nd
NB1209-06-20ANB8, NB12*3II50-3 (1.2)55,352Notification
NB1314-06-20A 3V5 (3)0-5 (3.2)20,366EWS-PM-5th
NB1414-06-20C 3II5 (1)0-7 (3.7)28,375EWS-PM-5th
NB1521-06-20DNB7, NB11, NB153II50-2 (0.6)35,928EWS-PM-6th
NB1621-06-20A 3II50-4 (1.6)66,920EWS-PM-6th

* There was exchange of personnel in these two locations.

** Veterinarian II and V were both from the same veterinary practice.

*** Notification: based on reporting of clinical signs which was obligated from 26 April onwards; EWS-Ser- Detection based on a one-off nation-wide compulsory serological screening of all mink farms at the end of May/early June by GD Animal Health; EWS-PM-Detection based on the early warning monitoring system for which carcasses of animals that died of natural causes were submitted weekly for PCR testing by GD Animal Health from the end of May onwards in a weekly cycle (EWS-PM 1st to 6th post mortem screening).

In total 18 sequences from mink farm employees or close contacts were generated from seven different farms. In most cases, these human sequences were near-identical to the mink sequences from the same farm.

For NB1 the situation was different and the human sequence clusters deeply within the sequences derived from mink (Figure 1), with 7 nucleotides difference with the closest related mink sequence. This was also the case on farm NB14, with 4 nucleotides difference with the closest related mink sequence.

Employees sampled at mink farm NB8 clustered with animals from NB12 which can be explained by the exchange of personnel between these two farms.

Figure 3: Phylogenetic analysis of SARS-CoV-2 strains detected in the 5 mink farm clusters. The sequences derived from different farms are depicted in different colors. Scale bar represents units of substitutions per site.
Figure 4. Geographical overview of SARS-CoV-2 positive mink farms per municipality affected. The proportion of SARS-CoV-2 positive mink farms over the total number of mink farms (CBS, 2019) is indicated. Symbols for positive farms are colored by cluster and shapes indicate farms with a same owner.

Within farm diversity

SARS-CoV-2 was detected on mink farm NB1-NB4 after reports of respiratory symptoms and increased mortality in mink. The sequences from farm NB1 had between 0 and 9 nucleotides differences (average 3.9 nucleotides) and from NB2 between 0 and 8 nucleotides differences (average of 3.6), which is much more than what has been observed in outbreaks in human settings.

The sequences from NB3 had 0 to 2 nucleotides difference suggesting that the virus was recently introduced, in line with the observed disease in humans, which occurred in the weeks post diagnosis of the infection in mink. After the initial detection of SARS-CoV-2 on mink farms, farms were screened weekly.

The first, second, fifth and sixth weekly screening yielded new positives. The sequences of mink at NB6 had between 0 and 12 nucleotides differences, whereas diversity was lower for the subsequent farm sequences (Table 2).

Several non-synonymous mutations were identified among the mink sequences compared to the Wuhan reference sequence NC_045512.2. However, no particular amino acid substitutions were found in all mink samples (Figure 5).

Of note, three of the clusters had the position 614G variant (clusters A, C and E), and 2 had the original variant. There were no obvious differences in the presentation of disease in animals or humans between clusters based on the data available at this stage, but further data collection and analysis, also for cases after NB16, are ongoing to investigate this further.

The observed mutations can also be found in the general population and the same mutations also were found in human cases which were related to the mink farms.

Figure 5. Overview of the specific amino acid mutations found in mink farms. Above the x-axis the open reading frames (ORF) are indicated and on the x-axis the amino acid position within each ORF is indicated. On the y-axis the sequence names are indicated and on the right side of the
graph the cluster numbers and specific farm identifiers and the type of host are used to group the samples.

Discussion

Here we show ongoing SARS-CoV-2 transmission in mink farms and spill-over events to humans. To the best of our knowledge, these are the first animal to human SARS-CoV-2 transmission events documented. More research in minks and other mustelid species, to demonstrate if these species can be a true reservoir of SARS-CoV-2 although from our observations we consider this likely.

After the detection of SARS-CoV-2 on mink farms, 68% of the tested farm workers and/or relatives or contacts were shown to be infected with SARS- CoV-2, indicating that contact with SARS-CoV-2 infected mink is a risk factor for contracting COVID-19.

A high diversity in the sequences from some mink farms was observed which most likely can be explained by many generations of infected animals before an increase in mortality was observed.

The current estimates are that the substitution rate of SARS-CoV-2 is around 1.16*10^-3 substitutions/site/year (53), which corresponds to around one mutation per two weeks. This could mean that the virus was already circulating in mink farms for some time. However, there was also a relatively high sequence diversity observed in farms which still tested negative one week prior, hinting towards a faster evolution of the virus in the mink population.

This can indicate that the virus might replicate more efficiently in mink or might have acquired mutations which makes the virus more virulent. However, no specific mutations were found in all mink samples, making increased virulence less likely. In addition, mink farms have large populations of animals which could lead to very efficient virus transmission. Generation intervals for SARS-CoV-2 in humans have been estimated to be around 4-5

days(54), but with high dose exposure in a high-density farm could potentially be shorter. Recently, a specific mutation in the spike protein (D614G) was shown to result in an increased virulence in vitro (55), while it was not associated an increased growth rate for cluster nor an increased mortality (56).

This mutation was present in farm clusters A, C and E, but no obvious differences in clinical presentation, disease severity, or rate of transmission to humans was observed.

While we found sequences matching with the animal sequences on several farms, not all of these can be considered direct zoonotic transmissions.

For instance, the two employees from mink farm NB3 were most likely infected while working at the mink farm given the specific clustering in the phylogenetic tree and the timing of infection. Subsequent human infections may have originated from additional zoonotic infections, or from human to human transmission within their household.

Further proof that animals were the most likely source of infection was provided by the clear phylogenetic separation between farm related human cases and animal cases, from sequences from cases within the same 4-digit postal code area. Spill-back into the community living in the same 4-digit postal code area was not observed using sequence data, but cannot be entirely ruled out as the testing strategy during April and May was focusing on health care workers, persons with more severe symptoms, and persons at risk for complications, rather than monitoring community transmission and milder cases.

While the number of SARS-CoV-2 infected individuals was decreasing in the Netherlands in May and June, an increase in detection of SARS-CoV-2 in mink farms was observed. Based on WGS these sequences are part of multiple individual transmission chains linked to the mink farms and are not a reflection of the situation in the human population during this time.

In some cases, the farms had the same owner but in other cases no epidemiological link could be established. People coming to the different farms might be a

source but also semi-wild cats roaming around the farms or wildlife might play a role (27). So far, the investigation failed to identify common factors that might explain farm to farm spread. During interviews, it became clear that farms had occasionally hired temporary workers that had not been included in the testing and were lost to follow-up, stressing the need for vigorous biosecurity and occupational health guidance.

Since our observation, SARS-CoV-2 infections have also been described in mink farms in Denmark, Spain and the USA (5759), and mink farming is common in other regions of the world as well, also in China where around 26 million mink pelts are produced on a yearly basis (60). The population size and the structure of mink farms is such that it is conceivable that SARS-CoV-2 – once introduced – could continue to circulate. Therefore, continued monitoring and cooperation between human and animal health services is crucial to prevent the animals serving as a reservoir for continued infection in humans.

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SUPPLEMENT




Supplementary Figure 1: Number of mink farms per municipality in the Netherlands. Overview of the total number of mink farms per municipality (CBS, 2019). Municipalities with SARS-CoV-2 affected farms by June 21st 2020 are shown in red.

REFERENCE LINK: https://doi.org/10.1101/2020.09.01.277152 – This article is a preprint and has not been certified by peer review

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