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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.

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.

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 alphacoron-aviruses, 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.

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.

Atypical porcine pestivirus (APPV) was first discovered in North America in 2015 and was later shown to be associated with congenital tremor (CT) in piglets. CT is an occasional challenge in some Danish sow herds. Therefore, we initiated an observational case control study to clarify a possible relationship between CT and APPV in Danish pig production. Blood samples were collected from piglets affected by CT (n = 55) in ten different sow herds and from healthy piglets in five sow herds without a history of CT piglets (n = 25), as well as one sow herd with a sporadic occurrence of CT (n = 5). APPV was detected by RT-qPCR in all samples from piglets affected by CT and in three out of five samples from piglets in the herd with a sporadic occurrence of CT. In the herds without a history of CT, only one out of 25 piglets were positive for APPV. In addition, farmers or veterinarians in CT-affected herds were asked about their experience of the issue. CT is most often seen in gilt litters, and a substantial increase in pre-weaning mortality is only observed in severe cases. According to our investigations, APPV is a common finding in piglets suffering from CT in Denmark.

The European Commission requested EFSA to 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 ASF in wild boar and domestic pigs in the EU; ii) the ASF epidemiology in wild boar; iii) ASF virus (ASFV) survival in the environment and iv) ASF transmission by vectors. In this Scientific Opinion, the first research domain on ASF seasonality is addressed. Therefore, five 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 five research objectives, only two were prioritised and elaborated into a general protocol/study design research proposal, namely: 1) to monitor the herd incidence of ASF outbreaks in EU Member States (MS) and 2) to investigate potential (seasonal) risk factors for ASF incursion in domestic pig herds of different herd types and/or size. To monitor the incidence in different pig herd types, it is advised to collect, besides ASF surveillance data, pig population data describing at least the following parameters per farm from the first moment of incursion in an affected MS: the numbers of pigs (e.g. number of breeding pigs sows and boars, weaners and fatteners) and the location and the type of farm (including details on the level of biosecurity implemented on the farm and the outdoor/indoor production). We suggest collecting data from all ASF-affected MS through the SIGMA data model, which was developed for this purpose. To investigate potential risk factors for ASF incursion in domestic pig herds, we suggest a matched case–control design. Such a study design can be run either retrospectively or prospectively. The collected data on the pig herds and the ASF surveillance data in the SIGMA data model can be used to identify case and control farms.

Severe acute respiratory syndrome coronavirus 2 has caused a pandemic in humans. Farmed mink (Neovison vison) are also susceptible. In Denmark, this virus has spread rapidly among farmed mink, resulting in some respiratory disease. Full-length virus genome sequencing revealed novel virus variants in mink. These variants subsequently appeared within the local human community.

In June-November 2020, SARS-CoV-2-infected mink were detected in 290 of 1,147 Danish mink farms. In North Denmark Region, 30% (324/1,092) of people found connected to mink farms tested SARS-CoV- 2-PCR-positive and approximately 27% (95% confidence interval (CI): 25-30) of SARS-CoV-2-strains from humans in the community were mink-associated. Measures proved insufficient to mitigate spread. On 4 November, the government ordered culling of all Danish mink. Farmed mink constitute a potential virus reservoir challenging pandemic control.

SARS-CoV-2 infection is the cause of COVID-19 in humans. In April 2020, SARS-CoV-2 infection in farmed mink (Neovision vision) occurred in the Netherlands. The first outbreaks in Denmark were detected in June 2020 in three farms. A steep increase in the number of infected farms occurred from September and onwards. Here, we describe prevalence data collected from 215 infected mink farms to characterize spread and impact of disease in infected farms. In one third of the farms, no clinical signs were observed. In farms with clinical signs, decreased feed intake, increased mortality and respiratory symptoms were most frequently observed, during a limited time period (median of 11 days). In 65% and 69% of farms, virus and sero-conversion, respectively, were detected in 100% of sampled animals at the first sampling. SARS-CoV-2 was detected, at low levels, in air samples collected close to the mink, on mink fur, on flies, on the foot of a seagull, and in gutter water, but not in feed. Some dogs and cats from infected farms tested positive for the virus. Chickens, rabbits, and horses sampled on a few farms, and wildlife sampled in the vicinity of the infected farms did not test positive for SARS-CoV-2. Thus, mink are highly susceptible to infection by SARS-CoV-2, but routes of transmission between farms, other than by direct human contact, are unclear.

This report provides an update of the epidemiology of African swine fever (ASF) in the European Union during the period November 2018 to October 2019. In this period, ASF has been confirmed in Slovakia, whereas Czechia became officially ASF-free in March 2019, bringing the number of affected countries in the EU to nine. The report provides a narrative update of the situation in the different countries and an analysis of the temporal and spatial patterns of the disease. There has been no increase in the proportion of seropositive hunted wild boar in the affected areas. In hunted animals, the proportions of wild boar testing polymerase chain reaction-positive and enzyme-linked immunosorbent assay-positive has remained low (< 0.05). In addition to the obvious seasonal peak in summer in domestic pigs, seasonality of ASF in wild boar was statistically confirmed. A network analysis demonstrated that the median velocity of the natural propagation of the disease in wild boar populations was between 2.9 and 11.7 km/year. Human-mediated spread, both in pigs and wild boar, however, remains important. Several wild boar- and domestic pig-related risk factors for ASF occurrence in non-commercial farms in Romania were identified with a case–control study. This report also updates an extensive literature review on control measures to stop the spread of the disease in wild boar and on measures to separate wild boar populations. Several new studies have been identified in this reporting period, but these did not alter the conclusions of the previous reporting period. Field experience with the use of fences as part of the control strategy deployed in the Belgian focal outbreak of ASF in wild boar is described. So far, the measures have proven effective to keep ASF virus inside the affected area. This strategy included a combination of different measures, namely zoning, carcass removal, a complete feeding ban, specific hunting regulations and depopulation actions depending on the

Coronavirus inficerer mange forskellige pattedyrværter og fugle. Der er tale om en stor familie af forskellige virus, hvor visse coronavirus kan forårsage alvorlig sygdom hos dyr og mennesker. Zoonotisk transmission forekommer dog sjældent.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-borne virus of ruminants, emerged in livestock in northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of euros. Although the outbreak was successfully controlled through vaccination by early 2010, puzzlingly, a closely related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this long period without replication, we hypothesise that the second outbreak could have been initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence.

Following its introduction into Georgia in 2007, African swine fever virus (ASFV) has become widespread on the European continent and in Asia. In many cases, the exact route of introduction into domestic pig herds cannot be determined, but most introductions are attributed to indirect virus transmission. In this review, we describe knowledge gained about different matrices that may allow introduction of the virus into pig herds. These matrices include uncooked pig meat, processed pig-derived products, feed, matrices contaminated with the virus and blood-feeding invertebrates. Knowledge gaps still exist, and both field studies and laboratory research are needed to enhance understanding of the risks for ASFV introductions, especially via virus-contaminated materials, including bedding and feed, and via blood-feeding, flying insects. Knowledge obtained from such studies can be applied to epidemiological risk assessments for the different transmission routes. Such assessments can be utilized to help predict the most effective biosecurity and control strategies.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-borne virus of ruminants, emerged in livestock in northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of euros. Although the outbreak was successfully controlled through vaccination by early 2010, puzzlingly, a closely related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this long period without replication, we hypothesise that the second outbreak could have been initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence., SCOPUS: ar.j, info:eu-repo/semantics/published

The most significant knowledge gaps in the prevention and control of African swine fever (ASF) were identified by the EU Veterinary services and other stakeholders involved in pig production and wild boar management through an online survey. The respondents were asked to identify the major research needs in order to improve short-term ASF risk management. Four major gaps were identified: ‘wild boar’, ‘African swine fever virus (ASFV) survival and transmission’, ‘biosecurity’ and ‘surveillance’. In particular, the respondents stressed the need for better knowledge on wild boar management and surveillance, and improved knowledge on the possible mechanism for spread and persistence of ASF in wild boar populations. They indicated the need for research on ASFV survival and transmission from the environment, different products such as feed and feed materials, and potential arthropod vector transmission. In addition, several research topics on biosecurity were identified as significant knowledge gaps and the need to identify risk factors for ASFV entry into domestic pig holdings, to develop protocols to implement specific and appropriate biosecurity measures, and to improve the knowledge about the domestic pig–wild boar interface. Potential sources of ASFV introduction into unaffected countries need to be better understood by an in-depth analysis of the possible pathways of introduction of ASFV with the focus on food, feed, transport of live wild boars and human movements. Finally, research on communication methods to increase awareness among all players involved in the epidemiology of ASF (including truck drivers, hunters and tourists) and to increase compliance with existing control measures was also a topic mentioned by all stakeholders.

The European Commission requested EFSA to compare the reliability of wild boar density estimates across the EU and to provide guidance to improve data collection methods. Currently, the only EU‐wide available data are hunting data. Their collection methods should be harmonised to be comparable and to improve predictive models for wild boar density. These models could be validated by more precise density data, collected at local level e.g. by camera trapping. Based on practical and theoretical considerations, it is currently not possible to establish wild boar density thresholds that do not allow sustaining African swine fever (ASF). There are many drivers determining if ASF can be sustained or not, including heterogeneous population structures and human‐mediated spread and there are still unknowns on the importance of different transmission modes in the epidemiology. Based on extensive literature reviews and observations from affected Member States, the efficacy of different wild boar population reduction and separation methods is evaluated. Different wild boar management strategies at different stages of the epidemic are suggested. Preventive measures to reduce and stabilise wild boar density, before ASF introduction, will be beneficial both in reducing the probability of exposure of the population to ASF and the efforts needed for potential emergency actions (i.e. less carcass removal) if an ASF incursion were to occur. Passive surveillance is the most effective and efficient method of surveillance for early detection of ASF in free areas. Following focal ASF introduction, the wild boar populations should be kept undisturbed for a short period (e.g. hunting ban on all species, leave crops unharvested to provide food and shelter within the affected area) and drastic reduction of the wild boar population may be performed only ahead of the ASF advance front, in the free populations. Following the decline in the epidemic, as demonstrated through passive surveillance

This guidance defines the process for handling applications on new or modified stunning methods and the parameters that will be assessed by the EFSA Animal Health and Welfare (AHAW) Panel. The applications, received through the European Commission, should contain administrative information, a checklist of data to be submitted and a technical dossier. The dossier should include two or more studies (in laboratory and slaughterhouse conditions) reporting all parameters and methodological aspects that are indicated in the guidance. The applications will first be scrutinised by the EFSA's Applications Desk (APDESK) Unit for verification of the completeness of the data submitted for the risk assessment of the stunning method. If the application is considered not valid, additional information may be requested from the applicant. If considered valid, it will be subjected to assessment phase 1 where the data related to parameters for the scientific evaluation of the stunning method will be examined by the AHAW Panel. Such parameters focus on the stunning method and the outcomes of interest, i.e. immediate onset of unconsciousness or the absence of avoidable pain, distress and suffering until the loss of consciousness and duration of the unconsciousness (until death). The applicant should also propose methodologies and results to assess the equivalence with existing stunning methods in terms of welfare outcomes. Applications passing assessment phase 1 will be subjected to the following phase 2 which will be carried out by the AHAW Panel and focuses on the animal welfare risk assessment. In this phase, the Panel will assess the outcomes, conclusions and discussion proposed by the applicant. The results of the assessment will be published in a scientific opinion.

Batrachochytrium salamandrivorans (Bsal) is an emerging fungal pathogen of salamanders. Despite limited surveillance, Bsal was detected in kept salamanders populations in Belgium, Germany, Spain, the Netherlands and the United Kingdom, and in wild populations in some regions of Belgium, Germany and the Netherlands. According to niche modelling, at least part of the distribution range of every salamander species in Europe overlaps with the climate conditions predicted to be suitable for Bsal. Passive surveillance is considered the most suitable approach for detection of Bsal emergence in wild populations. Demonstration of Bsal absence is considered feasible only in closed populations of kept susceptible species. In the wild, Bsal can spread by both active (e.g. salamanders, anurans) and passive (e.g. birds, water) carriers; it is most likely maintained/spread in infected areas by contacts of salamanders or by interactions with anurans, whereas human activities most likely cause Bsal entry into new areas and populations. In kept amphibians, Bsal contamination via live silent carriers (wild birds and anurans) is considered extremely unlikely. The risk‐mitigation measures that were considered the most feasible and effective: (i) for ensuring safer international or intra‐EU trade of live salamanders, are: ban or restrictions on salamander imports, hygiene procedures and good practice manuals; (ii) for protecting kept salamanders from Bsal, are: identification and treatment of positive collections; (iii) for on‐site protection of wild salamanders, are: preventing translocation of wild amphibians and release/return to the wild of kept/temporarily housed wild salamanders, and setting up contact points/emergency teams for passive surveillance. Combining several risk‐mitigation measures improve the overall effectiveness. It is recommended to: introduce a harmonised protocol for Bsal detection throughout the EU; improve data acquisition on salamander abundance and dis

Porcine epidemic diarrhoea virus, strain CV777, was initially characterized in 1978 as the causative agent of a disease first identified in the UK in 1971. This coronavirus has been widely distributed among laboratories and has been passaged both within pigs and in cell culture. To determine the variability between different stocks of the PEDV strain CV777, sequencing of the full-length genome (ca. 28kb) has been performed in 6 different laboratories, using different protocols. Not surprisingly, each of the different full genome sequences were distinct from each other and from the reference sequence (Accession number AF353511) but they are >99% identical. Unique and shared differences between sequences were identified. The coding region for the surface-exposed spike protein showed the highest proportion of variability including both point mutations and small deletions. The predicted expression of the ORF3 gene product was more dramatically affected in three different variants of this virus through either loss of the initiation codon or gain of a premature termination codon. The genome of one isolate had a substantially rearranged 5´-terminal sequence. This rearrangement was validated through the analysis of sub-genomic mRNAs from infected cells. It is clearly important to know the features of the specific sample of CV777 being used for experimental studies.

Bat populations harbour a multitude of viruses; some of these are pathogenic or potentially pathogenic in other animals or humans. Therefore, it is important to monitor the populations and characterize these viruses. In this study, the presence of coronaviruses (CoVs) in different species of Danish bats was investigated using active surveillance at different geographical locations in Denmark. Faecal samples were screened for the presence of CoVs using pan-CoV real-time RT-PCR assays. The amplicons, obtained from five different species of bats, were sequenced. Phylogenetic analysis revealed a species-specific clustering with the samples from Myotis daubentonii, showing a close resemblance to coronavirus sequences obtained from the same species of bat in Germany and the United Kingdom. Our results show, for the first time, that multiple, distinct alphacoronaviruses are present in the Danish bat populations.

Et hegn kan være med til at begrænse risikoen for den katastrofale socioøkonomiske effekt, som en epidemi af afrikansk svinepest i Danmark kan få.

Within Eastern Europe, African swine fever virus (ASFV) has unexpectedly spread to farms with high biosecurity. In an attempt to explain this process, pigs were allowed to ingest flies that had fed on ASFV-spiked blood, which had a realistic titre for an infected pig. Some of the pigs became infected with the virus. Thus, ingestion of blood-sucking flies, having fed on ASFV-infected wild boar before entering stables, represents a potential route for disease transmission