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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.

Knowledge about African swine fever virus (ASFV) transmission and its survival in the environment is mandatory to develop rational control strategies and combat this serious disease in pigs. In this study, the risk that environmental contamination poses for infection of naïve pigs was investigated. Naïve pigs were introduced as sentinels into contaminated pens kept at ambient temperature (about 18–22˚C) either on the same day or up to 3 days after ASFV-infected pigs were removed. Three experiments were carried out in which four to six pigs per pen were inoculated with virulent ASFV isolates OURT88/1 (genotype I), Georgia 2007/1, or POL/2015/Podlaskie (genotype II), respectively. The majority of the inoculated pigs developed acute disease but with no evident haemorrhagic lesions or haemorrhagic diarrhoea and were culled at the predefined humane endpoint. The levels of ASFV DNA detected in the blood of the infected animals reached 107−9 genome copies/ml before euthanasia. Environmental swabs were taken from different surfaces in the animal rooms, as well as from faeces and urine, close to the time of introduction of the naïve animals. Relatively low quantities of virus DNA were detected in the environmental samples, in the range of 103−7 genome copies per swab or per gram and ml of faeces and urine. No infectious virus was recovered from these environmental samples. Neither clinical signs nor virus genomes were detected in the blood of any of the sentinel pigs over a period of 2 to 3 weeks after exposure, indicating that transmission from the ASFV-contaminated environment did not occur. Interestingly, viral DNA was detected in nasal and oral swabs from some of the sentinel animals at early days of exposure (ranging between 103.7−5.8 genome copies per swab), though none of them developed ASF. The results indicate a relatively low risk of ASFV transmission from a contaminated environment under the conditions provided in these experimental studies and in the absence of b

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.