Emergence and spread of SARS-CoV-2 variants from farmed mink to humans and back during the epidemic in Denmark, June-November 2020.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) not only caused the COVID-19 pandemic but also had a major impact on farmed mink production in several European countries. In Denmark, the entire population of farmed mink (over 15 million animals) was culled in late 2020. During the period of June to November 2020, mink on 290 farms (out of about 1100 in the country) were shown to be infected with SARS-CoV-2. Genome sequencing identified changes in the virus within the mink and it is estimated that about 4000 people in Denmark became infected with these mink virus variants. However, the routes of transmission of the virus to, and from, the mink have been unclear. Phylogenetic analysis revealed the generation of multiple clusters of the virus within the mink. Detailed analysis of changes in the virus during replication in mink and, in parallel, in the human population in Denmark, during the same time period, has been performed here. The majority of cases in mink involved variants with the Y453F substitution and the H69/V70 deletion within the Spike (S) protein; these changes emerged early in the outbreak. However, further introductions of the virus, by variants lacking these changes, from the human population into mink also occurred. Based on phylogenetic analysis of viral genome data, we estimate, using a conservative approach, that about 17 separate examples of mink to human transmission occurred in Denmark but up to 59 such events (90% credible interval: (39-77)) were identified using parsimony to count cross-species jumps on transmission trees inferred using Bayesian methods. Using the latter approach, 136 jumps (90% credible interval: (117-164)) from humans to mink were found, which may underlie the farm-to-farm spread. Thus, transmission of SARS-CoV-2 from humans to mink, mink to mink, from mink to humans and between humans were all observed.

Porcine respiratory coronavirus genome sequences; comparisons and relationships to transmissible gastroenteritis viruses.

Porcine respiratory coronavirus (PRCV) was initially detected in Europe, and later in the United States of America (US), in the 1980s. In this study we obtained and compared PRCV sequences from Europe and the US, and investigated how these are related to transmissible gastroenteritis virus (TGEV) sequences. The whole genome sequences of Danish (1/90-DK), Italian (PRCV15087/12 III NPTV Parma), and Belgian PRCV (91V44) strains are presented. These sequences were aligned with nine other PRCV sequences from Europe and the US, and 43 TGEV sequences. Following alignment of the PRCV sequences, it was apparent that multiple amino acid variations in the structural proteins were distinct between the European and US strains. The alignments were used to build phylogenetic trees to infer the evolutionary relationships between the strains. In these trees, the European PRCV strains clustered as a separate group, whereas the US strains of PRCV all clustered with TGEVs.

A Deep Sequencing Strategy for Investigation of Virus Variants within African Swine Fever Virus-Infected Pigs.

African swine fever virus (ASFV) is the causative agent of African swine fever, an economically important disease of pigs, often with a high case fatality rate. ASFV has demonstrated low genetic diversity among isolates collected within Eurasia. To explore the influence of viral variants on clinical outcomes and infection dynamics in pigs experimentally infected with ASFV, we have designed a deep sequencing strategy. The variant analysis revealed unique SNPs at <10% frequency in several infected pigs as well as some SNPs that were found in more than one pig. In addition, a deletion of 10,487 bp (resulting in the complete loss of 21 genes) was present at a nearly 100% frequency in the ASFV DNA from one pig at position 6362-16849. This deletion was also found to be present at low levels in the virus inoculum and in two other infected pigs. The current methodology can be used for the currently circulating Eurasian ASFVs and also adapted to other ASFV strains and genotypes. Comprehensive deep sequencing is critical for following ASFV molecular evolution, especially for the identification of modifications that affect virus virulence.

Increased Presence of Circulating Cell-Free, Fragmented, Host DNA in Pigs Infected with Virulent African Swine Fever Virus.

African swine fever virus (ASFV) causes severe hemorrhagic disease in domestic pigs and wild boar, often with high case fatality rates. The virus replicates in the circulating cells of the monocyte-macrophage lineage and within lymphoid tissues. The infection leads to high fever and a variety of clinical signs. In this study, it was observed that ASFV infection in pigs resulted in a >1000-fold increase in the level of circulating cell-free DNA (cfDNA), derived from the nuclei of host cells in the serum. This change occurred in parallel with the increase in circulating ASFV DNA. In addition, elevated levels (about 30-fold higher) of host mitochondrial DNA (mtDNA) were detected in the serum from ASFV-infected pigs. For comparison, the release of the cellular enzyme, lactate dehydrogenase (LDH), a commonly used marker of cellular damage, was also found to be elevated during ASFV infection, but later and less consistently. The sera from pigs infected with classical swine fever virus (CSFV), which causes a clinically similar disease to ASFV, were also tested but, surprisingly, this infection did not result in the release of cfDNA, mtDNA, or LDH. It was concluded that the level of cfDNA in the serum is a sensitive host marker of virulent ASFV infection.

Multiplex Real-Time RT-PCR Assays for Detection and Differentiation of Porcine Enteric Coronaviruses.

It is important to be able to detect and differentiate between distinct porcine enteric coronaviruses that can cause similar diseases. However, the existence of naturally occurring recombinant coronaviruses such as swine enteric coronavirus (SeCoV) can give misleading results with currently used diagnostic methods. Therefore, we have developed and validated three duplex real-time quantitative RT-PCR assays for the simultaneous detection of, and differentiation between, porcine epidemic diarrhea virus (PEDV) and SeCoV. Transmissible gastroenteritis virus (TGEV) is also detected by two out of these three assays. In addition, a novel triplex assay was set up that was able to detect and differentiate between these alphacoronaviruses and the porcine deltacoronavirus (PDCoV). The validated assays have low limits of detection, close to 100% efficiency, and were able to correctly identify the presence of PEDV and SeCoV in 55 field samples, whereas 20 samples of other pathogens did not give a positive result. Implementing one or more of these multiplex assays into the routine diagnostic surveillance for PEDV will ensure that the presence of SeCoV, TGEV, and PDCoV will not go unnoticed.

Detection of African Swine Fever Virus and Blood Meals of Porcine Origin in Hematophagous Insects Collected Adjacent to a High-Biosecurity Pig Farm in Lithuania; A Smoking Gun?

A seasonal trend of African swine fever (ASF) outbreaks in domestic pig farms has been observed in affected regions of Eastern Europe. Most outbreaks have been observed during the warmer summer months, coinciding with the seasonal activity pattern of blood-feeding insects. These insects may offer a route for introduction of the ASF virus (ASFV) into domestic pig herds. In this study, insects (hematophagous flies) collected outside the buildings of a domestic pig farm, without ASFV-infected pigs, were analyzed for the presence of the virus. Using qPCR, ASFV DNA was detected in six insect pools; in four of these pools, DNA from suid blood was also identified. This detection coincided with ASFV being reported in the wild boar population within a 10 km radius of the pig farm. These findings show that blood from ASFV-infected suids was present within hematophagous flies on the premises of a pig farm without infected animals and support the hypothesis that blood-feeding insects can potentially transport the virus from wild boars into domestic pig farms.

Experimental Infection of Pigs with Recent European Porcine Epidemic Diarrhea Viruses.

Porcine epidemic diarrhea virus (PEDV), belonging to the genus Alphacoronavirus, can cause serious disease in pigs of all ages, especially in suckling pigs. Differences in virulence have been observed between various strains of this virus. In this study, four pigs were inoculated with PEDV from Germany (intestine/intestinal content collected from pigs in 2016) and four pigs with PEDV from Italy (intestine/intestinal material collected from pigs in 2016). The pigs were re-inoculated with the same virus on multiple occasions to create a more robust infection and enhance the antibody responses. The clinical signs and pathological changes observed were generally mild. Two distinct peaks of virus excretion were seen in the group of pigs inoculated with the PEDV from Germany, while only one strong peak was seen for the group of pigs that received the virus from Italy. Seroconversion was seen by days 18 and 10 post-inoculation with PEDV in all surviving pigs from the groups that received the inoculums from Germany and Italy, respectively. Attempts to infect pigs with a swine enteric coronavirus (SeCoV) from Slovakia were unsuccessful, and no signs of infection were observed in the inoculated animals.

Estimating transmission dynamics of African swine fever virus from experimental studies.

African swine fever virus (ASFV) continues to spread across the world, and currently, there are no treatments or vaccines available to combat this virus. Reliable estimates of transmission parameters for ASFV are therefore needed to establish effective contingency plans. This study used data from controlled ASFV inoculations of pigs to assess the transmission parameters. Three models were developed with (binary, piecewise-linear and exponential) time-dependent levels of infectiousness based on latency periods of 3-5 days derived from the analysis of 294 ethylenediamine tetraacetic acid-stabilized blood samples originating from 16 pigs with direct and 10 pigs with indirect contact to 8 inoculated pigs. The models were evaluated for three different discrete latency periods of infection. The likelihood ratio test showed that a binary model had an equally good fit for a latency period of 4 or 5 days as the piecewise-linear and exponential model. However, for a latency period of 3 days, the piecewise-linear and exponential models had the best fit. The modelling was done in discrete time as testing was conducted on specific days. The main contribution of this study is the estimation of ASFV genotype II transmission through the air in a confined space. The estimated transmission parameters via air are not much lower than for direct contact between pigs. The estimated parameters should be useful for future simulations of control measures against ASFV.

Influence of African Swine Fever Virus on Host Gene Transcription within Peripheral Blood Mononuclear Cells from Infected Pigs.

African swine fever virus (ASFV) has become a global threat to the pig production industry and has caused enormous economic losses in many countries in recent years. Peripheral blood mononuclear cells (PBMCs) from pigs infected with ASFV not only express ASFV genes (almost 200 in number) but have altered patterns of host gene expression as well. Both up- and down-regulation of host cell gene expression can be followed using RNAseq on poly(A)+ mRNAs harvested from the PBMCs of pigs collected at different times post-infection. Consistent with the time course of changes in viral gene expression, only few and limited changes in host gene expression were detected at 3 days post-infection (dpi), but by 6 dpi, marked changes in the expression of over 1300 host genes were apparent. This was co-incident with the major increase in viral gene expression. The majority of the changes in host gene expression were up-regulation, but many down-regulated genes were also identified. The patterns of changes in gene expression within the PBMCs detected by RNAseq were similar in each of the four infected pigs. Furthermore, changes in the expression of about twenty selected host genes, known to be important in host defence and inflammatory responses, were confirmed using high-throughput microfluidic qPCR assays.

Experimental Infections of Pigs with African Swine Fever Virus (Genotype II); Studies in Young Animals and Pregnant Sows.

African swine fever is an important viral disease of wild and domestic pigs. To gain further knowledge of the properties of the currently circulating African swine fever virus (ASFV), experimental infections of young pigs (approximately 8 weeks of age) and pregnant sows (infected at about 100 days of gestation) with the genotype II ASFV Georgia/2007 were performed. The inoculated young pigs developed typical clinical signs of the disease and the infection was transmitted (usually within 3-4 days) to all of the "in contact" animals that shared the same pen. Furthermore, typical pathogical lesions for ASFV infection were found at necropsy. Inoculation of pregnant sows with the same virus also produced rapid onset of disease from post-infection day three; two of the three sows died suddenly on post-infection day five, while the third was euthanized on the same day for animal welfare reasons. Following necropsy, the presence of ASFV DNA was detected in tonsils, spleen and lymph nodes of some of the fetuses, but the levels of viral DNA were much lower than in these tissues from the sows. Thus, only limited transplacental transmission occurred during the course of this experiment. These studies contribute towards further understanding about the spread of this important viral disease in domestic pigs.

A Multi-Laboratory Comparison of Methods for Detection and Quantification of African Swine Fever Virus.

African swine fever is a viral disease of the family Suidae. Methods to detect and quantify African swine fever virus (ASFV) include qPCR and virus infectivity assays. Individual laboratories often use in-house procedures for these assays, which can hamper the comparison of results. The objective of this study was to estimate the probability of ASFV detection using these assays, and to determine the inter-test correlations between results. This was achieved by testing a panel of 80 samples at three reference laboratories. Samples were analysed using nucleic acid extraction and qPCR, as well as virus infectivity assays. For qPCR, a very high probability (ranging from 0.96 to 1.0) of detecting ASFV DNA was observed for all tested systems. For virus infectivity assays in cells, the probability of detecting infectious ASFV varied from 0.68 to 0.90 and was highest using pulmonary alveolar macrophages, followed by MARC145 cells, peripheral blood monocytes, and finally wild boar lung cells. Intraclass correlation coefficient estimates of 0.97 (0.96-0.98) between qPCR methods, 0.80 (0.74-0.85) to 0.94 (0.92-0.96) between virus infectivity assays, and 0.77 (0.68-0.83) to 0.95 (0.93-0.96) between qPCR methods and virus infectivity assays were obtained. These findings show that qPCR gives the highest probability for the detection of ASFV.

Uptake and Survival of African Swine Fever Virus in Mealworm (Tenebrio molitor) and Black Soldier Fly (Hermetia illucens) Larvae.

Insect production offers a sustainable source of nutrients for livestock. This comes with a risk for transmission of pathogens from the insects into the livestock sector, including viruses causing serious diseases, such as African swine fever virus (ASFV), classical swine fever virus and foot-and-mouth disease virus. ASFV is known to survive for a long time within animal meat and byproducts. Therefore, we conducted experimental exposure studies of insects to ASFV using larvae of two key insect species produced for food and feed, the mealworm; Tenebrio molitor, and the black soldier fly, Hermetia illucens. The larvae were exposed to ASFV POL/2015/Podlaskie, via oral uptake of serum or spleen material from ASFV-infected pigs. Using qPCR, the amounts of viral DNA present immediately after exposure varied from ~104.7 to 107.2 genome copies per insect. ASFV DNA was detectable in the larvae of H. illucens for up to 3 days post exposure and in T. molitor larvae for up to 9 days post exposure. To assess the presence of infectious virus within the larvae and with this, the risk of virus transmission via oral consumption, pigs were fed cakes containing larvae exposed to ASFV. Pigs that consumed 50 T. molitor or 50 H. illucens virus-exposed larvae did not become infected with ASFV. Thus, it appears, that in our experimental setting, the risk of ASFV transmission via consumption of unprocessed insect larvae, used as feed, is low.

Identification of African Swine Fever Virus Transcription within Peripheral Blood Mononuclear Cells of Acutely Infected Pigs.

African swine fever virus (ASFV) has become widespread in Europe, Asia and elsewhere, thereby causing extensive economic losses. The viral genome includes nearly 200 genes, but their expression within infected pigs has not been well characterized previously. In this study, four pigs were infected with a genotype II strain (ASFV POL/2015/Podlaskie); blood samples were collected before inoculation and at both 3 and 6 days later. During this period, a range of clinical signs of infection became apparent in the pigs. From the blood, peripheral blood mononuclear cells (PBMCs) were isolated. The transcription of the ASFV genes was determined using RNAseq on poly(A)+ mRNAs isolated from these cells. Only very low levels of virus transcription were detected in the PBMCs at 3 days post-inoculation (dpi) but, at 6 dpi, extensive transcription was apparent. This was co-incident with a large increase in the level of ASFV DNA within these cells. The pattern of the virus gene expression was very reproducible between the individual pigs. Many highly expressed genes have undefined roles. Surprisingly, some genes with key roles in virus replication were expressed at only low levels. As the functions of individual genes are identified, information about their expression becomes important for understanding their contribution to virus biology.

Infection, recovery and re-infection of farmed mink with SARS-CoV-2.

Mink, on a farm with about 15,000 animals, became infected with SARS-CoV-2. Over 75% of tested animals were positive for SARS-CoV-2 RNA in throat swabs and 100% of tested animals were seropositive. The virus responsible had a deletion of nucleotides encoding residues H69 and V70 within the spike protein gene as well as the A22920T mutation, resulting in the Y453F substitution within this protein, seen previously in mink. The infected mink recovered and after free-testing of 300 mink (a level giving 93% confidence of detecting a 1% prevalence), the animals remained seropositive. During further follow-up studies, after a period of more than 2 months without any virus detection, over 75% of tested animals again scored positive for SARS-CoV-2 RNA. Whole genome sequencing showed that the viruses circulating during this re-infection were most closely related to those identified in the first outbreak on this farm but additional sequence changes had occurred. Animals had much higher levels of anti-SARS-CoV-2 antibodies in serum samples after the second round of infection than at free-testing or during recovery from initial infection, consistent with a boosted immune response. Thus, it was concluded that following recovery from an initial infection, seropositive mink were readily re-infected by SARS-CoV-2.

Research priorities to fill knowledge gaps in wild boar management measures that could improve the control of African swine fever in wild boar populations.

The European Commission asked EFSA to provide study designs for the investigation of four research domains (RDs) according to major gaps in knowledge identified by EFSA in a report published in 2019: (RD 1) African swine fever (ASF) epidemiology in wild boar; (RD 2) ASF transmission by vectors; (RD 3) African swine fever virus (ASFV) survival in the environment, and (RD 4) the patterns of seasonality of ASF in wild boar and domestic pigs in the EU. In this Scientific Opinion, the second RD on ASF epidemiology in wild boar is addressed. Twenty-nine research objectives were proposed by the working group and broader ASF expert networks and 23 of these research objectives met a prespecified inclusion criterion. Fourteen of these 23 research objectives met the predefined threshold for selection and so were prioritised based on the following set of criteria: (1) the impact on ASF management; (2) the feasibility or practicality to carry out the study; (3) the potential implementation of study results in practice; (4) a possible short time-frame study (< 1 year); (5) the novelty of the study; and (6) if it was a priority for risk managers. Finally, after further elimination of three of the proposed research objectives due to overlapping scope of studies published during the development of this opinion, 11 research priorities were elaborated into short research proposals, considering the potential impact on ASF management and the period of one year for the research activities.

In vitro Characterization of Fitness and Convalescent Antibody Neutralization of SARS-CoV-2 Cluster 5 Variant Emerging in Mink at Danish Farms.

In addition to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit to animals that include hamsters, cats, dogs, mink, ferrets, tigers, lions, cynomolgus macaques, rhesus macaques, and treeshrew. Among these, mink are particularly susceptible. Indeed, 10 countries in Europe and North America reported SARS-CoV-2 infection among mink on fur farms. In Denmark, SARS-CoV-2 spread rapidly among mink farms and spilled-over back into humans, acquiring mutations/deletions with unknown consequences for virulence and antigenicity. Here we describe a mink-associated SARS-CoV-2 variant (Cluster 5) characterized by 11 amino acid substitutions and four amino acid deletions relative to Wuhan-Hu-1. Temporal virus titration, together with genomic and subgenomic viral RNA quantitation, demonstrated a modest in vitro fitness attenuation of the Cluster 5 virus in the Vero-E6 cell line. Potential alterations in antigenicity conferred by amino acid changes in the spike protein that include three substitutions (Y453F, I692V, and M1229I) and a loss of two amino acid residues 69 and 70 (ΔH69/V70), were evaluated in a virus microneutralization assay. Compared to a reference strain, the Cluster 5 variant showed reduced neutralization in a proportion of convalescent human COVID-19 samples. The findings underscore the need for active surveillance SARS-CoV-2 infection and virus evolution in susceptible animal hosts.

Full-Genome Sequences of Alphacoronaviruses and Astroviruses from Myotis and Pipistrelle Bats in Denmark.

Bat species worldwide are receiving increased attention for the discovery of emerging viruses, cross-species transmission, and zoonoses, as well as for characterizing virus infections specific to bats. In a previous study, we investigated the presence of coronaviruses in faecal samples from bats at different locations in Denmark, and made phylogenies based on short, partial ORF1b sequences. In this study, selected samples containing bat coronaviruses from three different bat species were analysed, using a non-targeted approach of next-generation sequencing. From the resulting metagenomics data, we assembled full-genome sequences of seven distinct alphacoronaviruses, three astroviruses, and a polyomavirus, as well as partial genome sequences of rotavirus H and caliciviruses, from the different bat species. Comparisons to published sequences indicate that the bat alphacoronaviruses belong to three different subgenera-i.e., Pedacovirus, Nyctacovirus, and Myotacovirus-that the astroviruses may be new species in the genus Mamastrovirus, and that the polyomavirus could also be a new species, but unassigned to a genus. Furthermore, several viruses of invertebrates-including two Rhopalosiphum padi (aphid) viruses and a Kadipiro virus-present in the faecal material were assembled. Interestingly, this is the first detection in Europe of a Kadipiro virus.

Research objectives to fill knowledge gaps in African swine fever virus survival in the environment and carcasses, which could improve the control of African swine fever virus in wild boar populations.

The European Commission requested that EFSA provide study designs for the investigation of four research domains according to major gaps in knowledge identified by EFSA in a report published in 2019: i) the patterns of seasonality of African Swine Fever (ASF) in wild boar and domestic pigs in the EU; ii) the epidemiology of ASF in wild boar; iii) survival of ASF virus (ASFV) in the environment and iv) transmission of ASFV by vectors. In this Scientific Opinion, the third research domain on ASFV survival is addressed. Nine research objectives were proposed by the working group and broader ASF expert networks, such as ASF stop, ENETWILD, VectorNet, AHAW network and the AHAW Panel Experts. Of the nine research objectives, only one was prioritised and elaborated into a general protocol/study design research proposal, pertaining ASFV survival in feed and bedding. To investigate the survival of ASFV in feed, bedding and roughage, laboratory survival studies are proposed. To investigate possible risk mitigation measures, proof-of-concept approaches should be investigated.

Research priorities to fill knowledge gaps in the control of African swine fever: possible transmission of African swine fever virus by vectors.

The European Commission requested that EFSA provide study designs for the investigation of four research domains according to major gaps in knowledge identified by EFSA in a report published in 2019: (i) the patterns of seasonality of African Swine Fever (ASF) in wild boar and domestic pigs in the EU; (ii) the epidemiology of ASF in wild boar; (iii) survival of ASF virus (ASFV) in the environment and (iv) transmission of ASFV by vectors. In this Scientific Opinion, the fourth research domain on ASFV transmission by vectors is addressed. Eleven research objectives were proposed by the EFSA working group and broader ASF expert networks, such as ASF stop, ENETWILD, VectorNet, AHAW network and the AHAW Panel Experts. Of the 11 research objectives, six were prioritised based on the following set of criteria: (1) the impact on ASF management; (2) the feasibility or practicality to carry out the study; (3) the potential implementation of study results in practice; (4) a possible short time-frame study (< 1 year); (5) the novelty of the study and (6) if it was a priority for risk managers. The prioritised research objectives were: (I) Studies on the potential vector fauna at the pig-wild boar interface and the feeding preference of blood-feeding potential vectors in ASF-affected areas; (II) Assessment of the efficacy of insect screens on indoor/outdoor pig holdings to prevent the entry of blood-sucking vectors (i.e. Stomoxys) in ASF endemic areas; (III) Assess the role of mechanical vectors in the virus transmission in ASF-affected areas; (IV) Distribution of the potential mechanical transmission vectors in ASF-affected areas of the EU; (V) ASFV transmission by synanthropic birds; and (VI) Assessment on the presence/absence of the soft tick Ornithodoros erraticus in ASF-affected areas in Europe. For each of the selected research objectives, a research protocol has been proposed considering the potential impact on ASF management and the period of 1 year for the research activities.

Ability of different matrices to transmit African swine fever virus.

This opinion assesses the risk posed by different matrices to introduce African swine fever virus (ASFV) to non-affected regions of the EU. Matrices assessed are feed materials, enrichment/bedding materials and empty live pigs transport vehicles returning from affected areas. Although the risk from feed is considered to be lower than several other pathways (e.g. contact with infected live animals and swill feeding), it cannot be ruled out that matrices assessed in this opinion pose a risk. Evidence on survival of ASFV in different matrices from literature and a public consultation was used in an Expert Knowledge Elicitation (EKE) on the possible contamination of products and traded or imported product volumes used on pig farms. The EKE results were used in a model that provided a risk-rank for each product's contamination likelihood ('q'), its trade or import volume from affected EU or Eurasian areas (N) and the modelled number of potentially infected pig farms (N × q). The products ranking higher regardless of origin or destination were mash and pelleted compound feed, feed additives and cereals. Bedding/enrichment materials, hydrolysed proteins and blood products ranked lowest regardless of origin or destination. Empty vehicles ranked lower than compound feed but higher than non-compound feed or bedding/enrichment material. It is very likely (95-99% certainty) that compound feed and cereals rank higher than feed materials, which rank higher than bedding/enrichment material and forage. As this is an assessment based on several parameters including the contamination and delivery to a pig farm, all of which have the same impact on the final ranking, risk managers should consider how the relative rank of each product may change with an effective storage period or a virus inactivation step.