Integrating Fréchet distance and AI reveals the evolutionary trajectory and origin of SARS-CoV-2.

A genome, composed of a precisely ordered sequence of four nucleotides (ATCG), encompasses a multitude of specific genome features like AAA motif. Mutations occurring within a genome disrupt the sequential order and composition of these features, thereby influencing the evolutionary trajectories and yielding variants. The evolutionary relatedness between a variant and its ancestor can be estimated by assessing evolutionary distances across a spectrum of genome features. This study develops a novel, alignment-free algorithm that considers both the sequential order and composition of genome features, enabling computation of the Fréchet distance (Fr) across multiple genome features to quantify the evolutionary status of a variant. Integrating this algorithm with an artificial recurrent neural network (RNN) reveals the quantitative evolutionary trajectory and origin of SARS-CoV-2, a puzzle unsolved by alignment-based phylogenetics. The RNN generates the evolutionary trajectory from Fr data at two levels: genome sequence mutations and organism variants. At the genome sequence level, SARS-CoV-2 evolutionarily shortens its genome to enhance its infectious capacity. Mutating signature features, such as TTA and GCT, increases its infectious potential and drives its evolution. At the organism level, variants mutating a single biomarker possess low infectious potential. However, mutating multiple markers dramatically increases their infectious capacity, propelling the COVID-19 pandemic. SARS-CoV-2 likely originates from mink coronavirus variants, with its origin trajectory traced as follows: mink, cat, tiger, mouse, hamster, dog, lion, gorilla, leopard, bat, and pangolin. Together, mutating multiple signature features and biomarkers delineates the evolutionary trajectory of mink-origin SARS-CoV-2, leading to the COVID-19 pandemic.

Sequence signatures within the genome of SARS-CoV-2 can be used to predict host source.

We conducted an in silico analysis to better understand the potential factors impacting host adaptation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in white-tailed deer, humans, and mink due to the strong evidence of sustained transmission within these hosts. Classification models trained on single nucleotide and amino acid differences between samples effectively identified white-tailed deer-, human-, and mink-derived SARS-CoV-2. For example, the balanced accuracy score of Extremely Randomized Trees classifiers was 0.984 ± 0.006. Eighty-eight commonly identified predictive mutations are found at sites under strong positive and negative selective pressure. A large fraction of sites under selection (86.9%) or identified by machine learning (87.1%) are found in genes other than the spike. Some locations encoded by these gene regions are predicted to be B- and T-cell epitopes or are implicated in modulating the immune response suggesting that host adaptation may involve the evasion of the host immune system, modulation of the class-I major-histocompatibility complex, and the diminished recognition of immune epitopes by CD8+ T cells. Our selection and machine learning analysis also identified that silent mutations, such as C7303T and C9430T, play an important role in discriminating deer-derived samples across multiple clades. Finally, our investigation into the origin of the B.1.641 lineage from white-tailed deer in Canada discovered an additional human sequence from Michigan related to the B.1.641 lineage sampled near the emergence of this lineage. These findings demonstrate that machine-learning approaches can be used in combination with evolutionary genomics to identify factors possibly involved in the cross-species transmission of viruses and the emergence of novel viral lineages.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible virus capable of infecting and establishing itself in human and wildlife populations, such as white-tailed deer. This fact highlights the importance of developing novel ways to identify genetic factors that contribute to its spread and adaptation to new host species. This is especially important since these populations can serve as reservoirs that potentially facilitate the re-introduction of new variants into human populations. In this study, we apply machine learning and phylogenetic methods to uncover biomarkers of SARS-CoV-2 adaptation in mink and white-tailed deer. We find evidence demonstrating that both non-synonymous and silent mutations can be used to differentiate animal-derived sequences from human-derived ones and each other. This evidence also suggests that host adaptation involves the evasion of the immune system and the suppression of antigen presentation. Finally, the methods developed here are general and can be used to investigate host adaptation in viruses other than SARS-CoV-2.

Serum protein electrophoresis in European mink (Mustela lutreola): reference intervals and comparison of agarose gel electrophoresis and capillary zone electrophoresis.

BACKGROUND: Knowledge of reference intervals for blood analytes, including serum protein fractions, is of great importance for the identification of infectious and inflammatory diseases and is often lacking in wild animal species. MATERIAL AND METHODS: Serum samples were obtained from European minks enrolled in the breeding program (n = 55). Agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE) were used to separate and identify protein fractions. Albumin, α1, α2, ß, and γ-globulins fractions were identified in all mink sera by both electrophoresis methods. Reference intervals (90% CI) were determined following the 2008 guidelines of the Clinical Laboratory Standard Institute. The methods were compared using Passing-Bablok regression, Bland-Altman analysis, and Lin's concordance correlation. RESULTS: A significant bias was found between methods for α1, α2, and γ-globulin. Lin's concordance correlation was considered unacceptable for α1, α2, and ß-globulins. Differences for gender between methods were found for albumin and α2-globuins, which were higher for males than females. γ-globulins were higher for adults than young minks using both methods; however, α1 and α2-globulins were lower. CONCLUSION: Both methods are adequate for identifying serum protein disorders, but the AGE and CZE methods are not equivalent. Therefore, reference intervals for each technique are required.

Detection of vesivirus in minks (Neovison vison), Italy 2021.

Vesiviruses are important animal pathogens with a broad host range, and they have also been involved in accidental contamination of cells used for the production of drugs for rare and life-threatening human diseases. A vesivirus (family Caliciviridae) was detected in minks (Neovison vison) with respiratory and neurological signs, during syndromic surveillance for SARS-CoV-2 conducted in Italy. The complete genome (8,397 nucleotides in length) of the vesivirus strain ITA/2021/mink/TE (OR130287) was obtained by combining NGS approach with 5' and 3' RACE protocols. The virus was seemingly more related (95.9-97.2% nt identity in the partial RNA-dependent RNA polymerase) to American vesivirus isolates 9/1980/US, 12/1980/US, and 20/1980/US dating back to the early 1980s than to recent mink strains. These results highlight the importance of gathering information on the virome of animals.

Strong selection signatures for Aleutian disease tolerance acting on novel candidate genes linked to immune and cellular responses in American mink (Neogale vison).

Aleutian disease (AD) is a multi-systemic infectious disease in American mink (Neogale vison) caused by Aleutian mink disease virus (AMDV). This study aimed to identify candidate regions and genes underlying selection for response against AMDV using whole-genome sequence (WGS) data. Three case-control selection signatures studies were conducted between animals (N = 85) producing high versus low antibody levels against AMDV, grouped by counter immunoelectrophoresis (CIEP) test and two enzyme-linked immunosorbent assays (ELISA). Within each study, selection signals were detected using fixation index (FST) and nucleotide diversity (θπ ratios), and validated by cross-population extended haplotype homozygosity (XP-EHH) test. Within- and between-studies overlapping results were then evaluated. Within-studies overlapping results indicated novel candidate genes related to immune and cellular responses (e.g., TAP2, RAB32), respiratory system function (e.g., SPEF2, R3HCC1L), and reproduction system function (e.g., HSF2, CFAP206) in other species. Between-studies overlapping results identified three large segments under strong selection pressure, including two on chromosome 1 (chr1:88,770-98,281 kb and chr1:114,133-120,473) and one on chromosome 6 (chr6:37,953-44,279 kb). Within regions with strong signals, we found novel candidate genes involved in immune and cellular responses (e.g., homologous MHC class II genes, ITPR3, VPS52) in other species. Our study brings new insights into candidate regions and genes controlling AD response.

A new species of Versteria (Cestoda: Taeniidae) parasitizing Neogale vison and Lontra canadensis (Carnivora: Mustelidae) from Western Canada.

Via molecular and morphological analyses, we describe adult specimens of a new species of Versteria (Cestoda: Taeniidae) infecting mink and river otter (Carnivora: Mustelidae) in Western Canada, as well as larval forms from muskrat and mink. These sequences closely matched those reported from adult specimens from Colorado and Oregon, as well as larval infections in humans and a captive orangutan. We describe here a new species from British Columbia and Alberta (Canada), Versteria rafei n. sp., based upon morphological diagnostic characteristics and genetic distance and phylogeny. Versteria rafei n. sp. differs from the three other described species of the genus in the smaller scolex and cirrus sac. It also differs from V. mustelae (Eurasia) and V. cuja (South America) by having an armed cirrus, which is covered in hair-like bristles, and in the shape of its hooks, with a long thorn-like blade, and short or long handle (vs. a short sharply curved blade and no difference in handle size in previously described species). The poorly known V. brachyacantha (Central Africa) also has an armed cirrus and similarly shaped hooks. However, it differs from the new species in the number and size of hooks. Phylogenetic analysis of the cox1 and nad1 mitochondrial regions showed that our specimens clustered with isolates from undescribed adults and larval infections in North America, and separate from V. cuja, confirming them to be a distinct species from the American Clade.

Genome-wide association studies for economically important traits in mink using copy number variation.

Copy number variations (CNVs) are structural variants consisting of duplications and deletions of DNA segments, which are known to play important roles in the genetics of complex traits in livestock species. However, CNV-based genome-wide association studies (GWAS) have remained unexplored in American mink. Therefore, the purpose of the current study was to investigate the association between CNVs and complex traits in American mink. A CNV-based GWAS was performed with the ParseCNV2 software program using deregressed estimated breeding values of 27 traits as pseudophenotypes, categorized into traits of growth and feed efficiency, reproduction, pelt quality, and Aleutian disease tests. The study identified a total of 10,137 CNVs (6968 duplications and 3169 deletions) using the Affymetrix Mink 70K single nucleotide polymorphism (SNP) array in 2986 American mink. The association analyses identified 250 CNV regions (CNVRs) associated with at least one of the studied traits. These CNVRs overlapped with a total of 320 potential candidate genes, and among them, several genes have been known to be related to the traits such as ARID1B, APPL1, TOX, and GPC5 (growth and feed efficiency traits); GRM1, RNASE10, WNT3, WNT3A, and WNT9B (reproduction traits); MYO10, and LIMS1 (pelt quality traits); and IFNGR2, APEX1, UBE3A, and STX11 (Aleutian disease tests). Overall, the results of the study provide potential candidate genes that may regulate economically important traits and therefore may be used as genetic markers in mink genomic breeding programs.

SARS-CoV-2 Outbreaks on Mink Farms-A Review of Current Knowledge on Virus Infection, Spread, Spillover, and Containment.

Many studies have been conducted to explore outbreaks of SARS-CoV-2 in farmed mink and their intra-/inter-species spread and spillover to provide data to the scientific community, protecting human and animal health. Studies report anthropozoonotic introduction, which was initially documented in April 2020 in the Netherlands, and subsequent inter-/intra-species spread of SARS-CoV-2 in farmed mink, likely due to SARS-CoV-2 host tropism capable of establishing efficient interactions with host ACE2 and the mink hosts' ability to enhance swift viral transmission due to their density, housing status, and occupational contacts. Despite the rigorous prevention and control measures adopted, transmission of the virus within and between animal species was efficient, resulting in the development of mink-associated strains able to jump back and forth among the mink hosts and other animal/human contacts. Current knowledge recognizes the mink as a highly susceptible animal host harboring the virus with or without clinical manifestations, furthering infection transmission as a hidden animal reservoir. A One Health approach is, thus, recommended in SARS-CoV-2 surveillance and monitoring on mink farms and of their susceptible contact animals to identify and better understand these potential animal hosts.

Risk assessment of SARS-CoV-2 replicating and evolving in animals.

The retransmissions of SARS-CoV-2 from several mammals - primarily mink and white-tailed deer - to humans have raised concerns for the emergence of a new animal-derived SARS-CoV-2 variant to worsen the pandemic. Here, we discuss animal species that are susceptible to natural or experimental infection with SARS-CoV-2 and can transmit the virus to mates or humans. We describe cutting-edge techniques to assess the impact of a mutation in the viral spike (S) protein on its receptor and on antibody binding. Our review of spike sequences of animal-derived viruses identified nine unique amino acid exchanges in the receptor-binding domain (RBD) that are not present in any variant of concern (VOC). These mutations are present in SARS-CoV-2 found in companion animals such as dogs and cats, and they exhibit a higher frequency in SARS-CoV-2 found in mink and white-tailed deer, suggesting that sustained transmissions may contribute to maintaining novel mutations. Four of these exchanges, such as Leu452Met, could undermine acquired immune protection in humans while maintaining high affinity for the human angiotensin-converting enzyme 2 (ACE2) receptor. Finally, we discuss important avenues of future research into animal-derived viruses with public health risks.

High seroprevalence for SARS-CoV-2 infection in dogs: Age as risk factor for infection in shelter and foster home animals.

SARS-CoV-2 has caused 775 outbreaks in 29 animal species across 36 countries, including dogs, cats, ferrets, minks, non-human primates, white-tailed deer, and lions. Although transmission from owners to dogs has been extensively described, no study to date has also compared sheltered, foster home and owner dogs and associated risk factors. This study aimed to identify SARS-CoV-2 infection and anti-SARS-CoV-2 antibodies from sheltered, fostered, and owned dogs, associated with environmental and management risk factors. Serum samples and swabs were collected from each dog, and an epidemiological questionnaire was completed by the shelter manager, foster care, and owner. A total of 111 dogs, including 222 oropharyngeal and rectal swabs, tested negative by RT-qPCR. Overall, 18/89 (20.22%) dogs presented IgG antibodies against the N protein of SARS-CoV-2 by magnetic ELISA, while none showed a reaction to the Spike protein. SARS-CoV-2 antibodies showed an age-related association, with 4.16 chance of positivity in adult dogs when compared with young ones. High population density among dogs and humans, coupled with repeated COVID-19 exposure, emerged as potential risk factors in canine virus epidemiology. Dogs exhibited higher seropositivity rates in these contexts. Thus, we propose expanded seroepidemiological and molecular studies across species and scenarios, including shelter dogs.

Lack of detection of SARS-CoV-2 in British wildlife 2020-21 and first description of a stoat (Mustela erminea) Minacovirus.

Repeat spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into new hosts has highlighted the critical role of cross-species transmission of coronaviruses and establishment of new reservoirs of virus in pandemic and epizootic spread of coronaviruses. Species particularly susceptible to SARS-CoV-2 spillover include Mustelidae (mink, ferrets and related animals), cricetid rodents (hamsters and related animals), felids (domestic cats and related animals) and white-tailed deer. These predispositions led us to screen British wildlife with sarbecovirus-specific quantitative PCR and pan coronavirus PCR assays for SARS-CoV-2 using samples collected during the human pandemic to establish if widespread spillover was occurring. Fourteen wildlife species (n=402) were tested, including: two red foxes (Vulpes vulpes), 101 badgers (Meles meles), two wild American mink (Neogale vison), 41 pine marten (Martes martes), two weasels (Mustela nivalis), seven stoats (Mustela erminea), 108 water voles (Arvicola amphibius), 39 bank voles (Myodes glareolous), 10 field voles (Microtus agrestis), 15 wood mice (Apodemus sylvaticus), one common shrew (Sorex aranaeus), two pygmy shrews (Sorex minutus), two hedgehogs (Erinaceus europaeus) and 75 Eurasian otters (Lutra lutra). No cases of SARS-CoV-2 were detected in any animals, but a novel minacovirus related to mink and ferret alphacoronaviruses was detected in stoats recently introduced to the Orkney Islands. This group of viruses is of interest due to pathogenicity in ferrets. The impact of this virus on the health of stoat populations remains to be established.

Epidemiologic and Genomic Evidence for Zoonotic Transmission of SARS-CoV-2 among People and Animals on a Michigan Mink Farm, United States, 2020.

Farmed mink are one of few animals in which infection with SARS-CoV-2 has resulted in sustained transmission among a population and spillback from mink to people. In September 2020, mink on a Michigan farm exhibited increased morbidity and mortality rates due to confirmed SARS-CoV-2 infection. We conducted an epidemiologic investigation to identify the source of initial mink exposure, assess the degree of spread within the facility's overall mink population, and evaluate the risk of further viral spread on the farm and in surrounding wildlife habitats. Three farm employees reported symptoms consistent with COVID-19 the same day that increased mortality rates were observed among the mink herd. One of these individuals, and another asymptomatic employee, tested positive for SARS-CoV-2 by real-time reverse transcription PCR (RT-qPCR) 9 days later. All but one mink sampled on the farm were positive for SARS-CoV-2 based on nucleic acid detection from at least one oral, nasal, or rectal swab tested by RT-qPCR (99%). Sequence analysis showed high degrees of similarity between sequences from mink and the two positive farm employees. Epidemiologic and genomic data, including the presence of F486L and N501T mutations believed to arise through mink adaptation, support the hypothesis that the two employees with SARS-CoV-2 nucleic acid detection contracted COVID-19 from mink. However, the specific source of virus introduction onto the farm was not identified. Three companion animals living with mink farm employees and 31 wild animals of six species sampled in the surrounding area were negative for SARS-CoV-2 by RT-qPCR. Results from this investigation support the necessity of a One Health approach to manage the zoonotic spread of SARS-CoV-2 and underscores the critical need for multifaceted public health approaches to prevent the introduction and spread of respiratory viruses on mink farms.

The possible impact of escaped captive American mink (Neogale vison) on the population of feral mink in Denmark.

The Danish feral mink (Neogale vison) population can be divided into wild-born and captive-born mink newly escaped from a farm. The impact of the captive-born mink on the population of feral population is unknown. Captive-born mink has lately been estimated to make up 25-30% of the feral mink population. In December 2020 Danish mink farms were decommissioned until January 2023. The aim of this study was to: 1) Examine whether a supplementation of farmed mink into the feral mink population influence the population growth and extinction rate of the feral mink, 2) Investigate which parameters affect the viability in feral mink populations the most. The age and mortality of 351 mink and the reproduction of 85 adult females culled by hunters from 2019-2022 were determined in three distinct geographic areas of Denmark (Jutland, Zealand, and Bornholm). VORTEX simulations were used to model the population viability and to simulate the impact of a supplementation of captive-born mink into the wild population. Simulations show that changes by 30% in the parameters: fecundity, percentages of breeding females and mortality influenced population size influenced the final population size significantly. The initial population size and inbreeding did not affect the population growth significantly. The simulations showed that the mink population in the regions of Jutland and Zealand could go extinct within 15 to 30 years without any supplementation of captive-born mink to the feral population. The population on Bornholm will however remain stable with current levels of mortality and reproduction even without supplementation of captive mink.

A Bayesian inference method to estimate transmission trees with multiple introductions; applied to SARS-CoV-2 in Dutch mink farms.

Knowledge of who infected whom during an outbreak of an infectious disease is important to determine risk factors for transmission and to design effective control measures. Both whole-genome sequencing of pathogens and epidemiological data provide useful information about the transmission events and underlying processes. Existing models to infer transmission trees usually assume that the pathogen is introduced only once from outside into the population of interest. However, this is not always true. For instance, SARS-CoV-2 is suggested to be introduced multiple times in mink farms in the Netherlands from the SARS-CoV-2 pandemic among humans. Here, we developed a Bayesian inference method combining whole-genome sequencing data and epidemiological data, allowing for multiple introductions of the pathogen in the population. Our method does not a priori split the outbreak into multiple phylogenetic clusters, nor does it break the dependency between the processes of mutation, within-host dynamics, transmission, and observation. We implemented our method as an additional feature in the R-package phybreak. On simulated data, our method correctly identifies the number of introductions, with an accuracy depending on the proportion of all observed cases that are introductions. Moreover, when a single introduction was simulated, our method produced similar estimates of parameters and transmission trees as the existing package. When applied to data from a SARS-CoV-2 outbreak in Dutch mink farms, the method provides strong evidence for independent introductions of the pathogen at 13 farms, infecting a total of 63 farms. Using the new feature of the phybreak package, transmission routes of a more complex class of infectious disease outbreaks can be inferred which will aid infection control in future outbreaks.

Investigation of the SARS-CoV-2 post-vaccination antibody response in Canadian farmed mink.

Currently, SARS-CoV-2 have been detected in farmed mink in 13 different countries. Due to the high susceptibility and transmissibility among mink, great concerns of mink serving as a reservoir to generate novel variants with unknown virulence and antigenic properties arose. These concerns have consequently resulted in entire mink productions being culled and banned. This study investigates the post-vaccination antibody response in the Canadian farmed mink vaccinated with a commercial Index spike protein-based vaccine, approved for use in cats, and compares the antibody response to that observed post infection in Danish farmed mink. Blood samples were obtained from 50 mink at the Canadian Centre for Fur Animal Research (CCFAR), Dalhousie University (Truro, Canada). The sera were initially analyzed for antibodies by enzyme-linked immunosorbent assay (ELISA), and selected sera was subsequently tested in a virus neutralization tests. The levels of neutralizing antibodies were evaluated for an ancestral D614G strain and a recent circulating SARS-CoV-2 variant of concern (Omicron BA.4). The results revealed that the vaccine induced a strong antibody response in mink by reaching antibody titer levels of up to 1:12800 in the ELISA. Moreover, high levels of neutralizing antibodies were obtained, and despite the great level of genetic differences between the ancestral and Omicron BA.4 strains, the vaccinated mink showed high levels of cross-reacting neutralizing antibodies. Interestingly, the antibody levels towards SARS-CoV-2 in the Canadian vaccinated mink were significantly higher than observed in recently SARS-CoV-2 infected Danish mink and equal to anamnestic responses following re-infection. In conclusion, the vaccine used in the Canadian farmed mink was able to induce a strong and broad-reacting antibody response in mink, which could limit the spread of SARS-CoV-2 in farmed mink and thereby reduce the risk of mink serving as a SARS-CoV-2 reservoir for human infections.

Structural basis of the American mink ACE2 binding by Y453F trimeric spike glycoproteins of SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters the host cell by binding to angiotensin-converting enzyme 2 (ACE2). While evolutionarily conserved, ACE2 receptors differ across various species and differential interactions with Spike (S) glycoproteins of SARS-CoV-2 viruses impact species specificity. Reverse zoonoses led to SARS-CoV-2 outbreaks on multiple American mink (Mustela vison) farms during the pandemic and gave rise to mink-associated S substitutions known for transmissibility between mink and zoonotic transmission to humans. In this study, we used bio-layer interferometry (BLI) to discern the differences in binding affinity between multiple human and mink-derived S glycoproteins of SARS-CoV-2 and their respective ACE2 receptors. Further, we conducted a structural analysis of a mink variant S glycoprotein and American mink ACE2 (mvACE2) using cryo-electron microscopy (cryo-EM), revealing four distinct conformations. We discovered a novel intermediary conformation where the mvACE2 receptor is bound to the receptor-binding domain (RBD) of the S glycoprotein in a "down" position, approximately 34° lower than previously reported "up" RBD. Finally, we compared residue interactions in the S-ACE2 complex interface of S glycoprotein conformations with varying RBD orientations. These findings provide valuable insights into the molecular mechanisms of SARS-CoV-2 entry.

Prioritizing Endangered Species in Genome Sequencing: Conservation Genomics in Action with the First Platinum-Standard Reference-Quality Genome of the Critically Endangered European Mink Mustela lutreola L., 1761.

The European mink Mustela lutreola (Mustelidae) ranks among the most endangered mammalian species globally, experiencing a rapid and severe decline in population size, density, and distribution. Given the critical need for effective conservation strategies, understanding its genomic characteristics becomes paramount. To address this challenge, the platinum-quality, chromosome-level reference genome assembly for the European mink was successfully generated under the project of the European Mink Centre consortium. Leveraging PacBio HiFi long reads, we obtained a 2586.3 Mbp genome comprising 25 scaffolds, with an N50 length of 154.1 Mbp. Through Hi-C data, we clustered and ordered the majority of the assembly (>99.9%) into 20 chromosomal pseudomolecules, including heterosomes, ranging from 6.8 to 290.1 Mbp. The newly sequenced genome displays a GC base content of 41.9%. Additionally, we successfully assembled the complete mitochondrial genome, spanning 16.6 kbp in length. The assembly achieved a BUSCO (Benchmarking Universal Single-Copy Orthologs) completeness score of 98.2%. This high-quality reference genome serves as a valuable genomic resource for future population genomics studies concerning the European mink and related taxa. Furthermore, the newly assembled genome holds significant potential in addressing key conservation challenges faced by M. lutreola. Its applications encompass potential revision of management units, assessment of captive breeding impacts, resolution of phylogeographic questions, and facilitation of monitoring and evaluating the efficiency and effectiveness of dedicated conservation strategies for the European mink. This species serves as an example that highlights the paramount importance of prioritizing endangered species in genome sequencing projects due to the race against time, which necessitates the comprehensive exploration and characterization of their genomic resources before their populations face extinction.

Metabolomic Profiling of Female Mink Serum during Early to Mid-Pregnancy to Reveal Metabolite Changes.

Mink embryos enter a period of diapause after the embryo develops into the blastocyst, and its reactivation is mainly caused by an increase in polyamine. The specific process of embryo diapause regulation and reactivation remains largely unexamined. This study aimed to identify changes in metabolites in the early pregnancy of mink by comparing and analyzing in serum metabolites up to twenty-nine days after mating. Blood samples were taken on the first day of mating, once a week until the fifth week. Metabolomic profiles of the serum samples taken during this period were analyzed by ultra-performance liquid chromatography/mass spectrometry. Multivariate statistical analyses identified differential metabolite expression at different time points in both positive and negative ion modes. The levels of dopamine, tyramine, L-phenylalanine, L-tyrosine, tyrosine, L-kynurenine, L-lysine, L-arginine, D-ornithine, and leucine changed significantly. These metabolites may be associated with the process of embryo diapause and subsequent reactivation.

Genomic characterization and phylogenetic analysis of Aleutian mink disease virus identified in a sudden death mink case.

Aleutian mink disease (AMD) is one of the most serious diseases in minks worldwide, it brings tremendous financial losses in mink farming. AMD virus (AMDV) has unusually high genetic diversity, its genomic structure remains unclear. In 2014, sudden death of breeding minks was occurred in northeast China. After clinical signs evaluation and virus isolation, AMDV was identified in all sudden death minks, we investigated the complete genomic sequence of AMDV-LM isolated from the sudden death case. The full-genome sequence of AMDV-LM was 7 nucleotides (nts) or 8 nts longer than isolates AMDV-BJ and AMDV-G. AMDV-LM contained two unique nucleotide changes in VP2 (G79T, T710C), which led to two amino acid changes G27W and L237S. For NS1, some unique point mutations, such as A374C, A428C, A463C, and T476A were found and resulted in four unique amino acid mutations at N24V, H125P, V143P, K155Q, and V159N, respectively. The predicted secondary structure of the 5' terminal of AMDV-LM formed a large bubble formation near the 5' end, which affected the stability of the U-shaped hairpin. Phylogenetic analysis demonstrated that AMDV-LM was closely related to Chinese isolates and confirmed that AMDV strains circulating in China had different origins of ancestors. This study was first to investigate the association of sudden death of adult breeding minks with AMDV infection. Our findings provide useful suggestions for evaluation of the pathogenic potential of AMDV, additional details on AMDV genome characterization were also presented. Future work should focus on the importance of AMDV-LM strain in mink infection.

Investigations into SARS-CoV-2 and other coronaviruses on mink farms in France late in the first year of the COVID-19 pandemic.

Soon after the beginning of the COVID-19 pandemic in early 2020, the Betacoronavirus SARS-CoV-2 infection of several mink farms breeding American minks (Neovison vison) for fur was detected in various European countries. The risk of a new reservoir being formed and of a reverse zoonosis from minks quickly became a major concern. The aim of this study was to investigate the four French mink farms to see whether SARS-CoV-2 was circulating there in late 2020. The investigations took place during the slaughtering period, thus facilitating different types of sampling (swabs and blood). On one of the four mink farms, 96.6% of serum samples were positive when tested with a SARS-CoV-2 ELISA coated with purified N protein recombinant antigen, and 54 out of 162 (33%) pharyngo-tracheal swabs were positive by RT-qPCR. The genetic variability among 12 SARS-CoV-2 genomes sequenced from this farm indicated the co-circulation of several lineages at the time of sampling. All the SARS-CoV-2 genomes detected were nested within the 20A clade (Nextclade), together with SARS-CoV-2 genomes from humans sampled during the same period. The percentage of SARS-CoV-2 seropositivity by ELISA varied between 0.3 and 1.1% on the other three farms. Interestingly, among these three farms, 11 pharyngo-tracheal swabs and 3 fecal pools from two farms were positive by end-point RT-PCR for an Alphacoronavirus very similar to a mink coronavirus sequence observed on Danish farms in 2015. In addition, a mink Caliciviridae was identified on one of the two farms positive for Alphacoronavirus. The clinical impact of these inapparent viral infections is not known. The co-infection of SARS-CoV-2 with other viruses on mink farms could help explain the diversity of clinical symptoms noted on different infected farms in Europe. In addition, the co-circulation of an Alphacoronavirus and SARS-CoV-2 on a mink farm would potentially increase the risk of viral recombination between alpha and betacoronaviruses as already suggested in wild and domestic animals, as well as in humans.