African swine fever virus (ASFV) is known to be very stable within a protein-rich environment and indirect virus transmission can be mediated via oral uptake of different materials. However, experimental studies in pigs have shown that infection by ASFV via the oral route can be difficult to establish. Currently, there is a lack of studies using strict oral inoculations of pigs with different doses of ASFV. Therefore, we aimed to determine the dose of a European genotype II ASFV that is required to establish infection of pigs by the oral route. In this study, 24 pigs were divided into four groups of six. Three of the groups were fed with a low, medium or high dose of the ASFV POL/2015/Podlaskie virus. The pigs in the fourth group served as positive controls and were inoculated intranasally, just once, using the low dose of the virus. All the pigs inoculated intranasally with ASFV succumbed to the infection, while only three of the six pigs that were fed the high dose of the virus became infected. None of the 12 pigs that were fed with either the medium or low dose of the virus became infected, despite receiving up to thirteen doses each. In two of the pigs infected by intranasal inoculation, the presence of a variant form of the ASFV genome was detected. The results obtained in this study underline that ASFV infection is more difficult to establish via the oral route when compared to the intranasal route. The high dose needed in order to establish oral infection could have implications for future strategies using baited vaccines containing infectious live-attenuated ASFV.

Vaccines against classical swine fever have proven very effective in protecting pigs from this deadly disease. However, little is known about how vaccination impacts the selective pressures acting on the classical swine fever virus (CSFV). Here we use high-throughput sequencing of viral genomes to investigate evolutionary changes in virus populations following the challenge of naïve and vaccinated pigs with the highly virulent CSFV strain “Koslov”. The challenge inoculum contained an ensemble of closely related viral sequences, with three major haplotypes being present, termed A, B, and C. After the challenge, the viral haplotype A was preferentially located within the tonsils of naïve animals but was highly prevalent in the sera of all vaccinated animals. We find that the viral population structure in naïve pigs after infection is very similar to that in the original inoculum. In contrast, the viral population in vaccinated pigs, which only underwent transient low-level viremia, displayed several distinct changes including the emergence of 16 unique non-synonymous single nucleotide polymorphisms (SNPs) that were not detectable in the challenge inoculum. Further analysis showed a significant loss of heterogeneity and an increasing positive selection acting on the virus populations in the vaccinated pigs. We conclude that vaccination imposes a strong selective pressure on viruses that subsequently replicate within the vaccinated animal.

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) 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.

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.

African swine fever virus (ASFV) is known to be very stable within a protein-rich environment and indirect virus transmission can be mediated via oral uptake of different materials. However, experimental studies in pigs have shown that infection by ASFV via the oral route can be difficult to establish. Currently, there is a lack of studies using strict oral inoculations of pigs with different doses of ASFV. Therefore, we aimed to determine the dose of a European genotype II ASFV that is required to establish infection of pigs by the oral route. In this study, 24 pigs were divided into four groups of six. Three of the groups were fed with a low, medium or high dose of the ASFV POL/2015/Podlaskie virus. Pigs in the fourth group served as positive controls and were inoculated intranasally, just once, using the low dose of the virus. All pigs inoculated intranasally with ASFV succumbed to the infection, while only three of the six pigs that were fed the high dose of the virus became infected. None of the 12 pigs that were fed either with the medium or low doses of the virus became infected, despite receiving up to thirteen doses each. In two of the pigs infected by intranasal inoculation, the presence of a variant form of the ASFV genome was detected. The results obtained in this study underline that ASFV infection is more difficult to establish via the oral route when compared to the intranasal route. The high dose needed in order to establish oral infection could have implications for future strategies using baited vaccines containing infectious live-attenuated ASFV.