African Swine Fever Virus Outbreaks Research Articles

Immunoinformatics-guided approach for designing a pan-proteome multi-epitope subunit vaccine against African swine fever virus

Despite being identified over a hundred years ago, there is still no commercially available vaccine for the highly contagious and deadly African swine fever virus (ASFV). This study used immunoinformatics for the rapid and inexpensive designing of a safe and effective multi-epitope subunit vaccine for ASFV. A total of 18,858 proteins from 100 well-annotated ASFV proteomes were screened using various computational tools to identify potential epitopes, or peptides capable of triggering an immune response in swine. Proteins from genotypes I and II were prioritized for their involvement in the recent global ASFV outbreaks. The screened epitopes exhibited promising qualities that positioned them as effective components of the ASFV vaccine. They demonstrated antigenicity, immunogenicity, and cytokine-inducing properties indicating their ability to induce potent immune responses. They have strong binding affinities to multiple swine allele receptors suggesting a high likelihood of yielding more amplified responses. Moreover, they were non-allergenic and non-toxic, a crucial prerequisite for ensuring safety and minimizing any potential adverse effects when the vaccine is processed within the host. Integrated with an immunogenic 50S ribosomal protein adjuvant and linkers, the epitopes formed a 364-amino acid multi-epitope subunit vaccine. The ASFV vaccine construct exhibited notable immunogenicity in immune simulation and molecular docking analyses, and stable profiles in secondary and tertiary structure assessments. Moreover, this study designed an optimized codon for efficient translation of the ASFV vaccine construct into the Escherichia coli K-12 expression system using the pET28a(+) vector. Overall, both sequence and structural evaluations suggested the potential of the ASFV vaccine construct as a candidate for controlling and eradicating outbreaks caused by the pathogen.

Construction of and evaluation of the immune response to two recombinant pseudorabies viruses expressing the B119L and EP364R proteins of African swine fever virus.

African swine fever (ASF) is an infectious disease caused by ASF virus (ASFV), which is characterized by high infectivity, rapid onset of disease, and a high mortality rate. Outbreaks of ASFV have caused great economic losses to the global pig industry, and there is a need to develop safe and effective vaccines. In this study, two recombinant pseudorabies virus (PRV) strains, rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L, expressing the EP364R and B119L protein, respectively, of ASFV, were constructed by homologous recombination technology. Western blotting and immunofluorescence analysis showed that these foreign proteins were expressed in cells infected with the recombinant strains. The strains showed good genetic stability and proliferative characteristics for 20 passages in BHK-21 cells. Both of these strains were immunogenic in mice, inducing the production of specific antibodies against the expressed ASFV proteins while providing protection against lethal challenge with PRV. Thus, the recombinant strains rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L could be used as candidate vaccines for both ASFV and PRV. In addition, our study identifies two potential target genes for the development of safe and efficient ASFV vaccines, provides a reference for the construction of bivalent ASFV and PRV vaccines, and demonstrates the feasibility of developing a live ASFV vector vaccine.

The Effect of Temperature on the Stability of African Swine Fever Virus BA71V Isolate in Environmental Water Samples.

African swine fever virus (ASFV) is known to be very stable and can remain infectious over long periods of time especially at low temperatures and within different matrices, particularly those containing animal-derived organic material. However, there are some gaps in our knowledge pertaining to the survivability and infectivity of ASFV in groundwater. This study aims to determine the stability and infectivity of the cell culture-adapted ASFV strain BA71V by plaque assay after incubation of the virus within river water samples at three different environmentally relevant temperatures (4 °C, 15 °C, and 21 °C) over the course of 42 days. The results from this study indicate that ASFV can remain stable and infectious when maintained at 4 °C in river water for more than 42 days, but as incubation temperatures are increased, the stability is reduced, and the virus is no longer able to form plaques after 28 days and 14 days, respectively, when stored at 15 °C and 21 °C. Characterizing the survivability of ASFV in groundwater can allow us to develop more appropriate inactivation and disinfection methods to support disease control and mitigate ASFV outbreaks.

Polygalic acid inhibits african swine fever virus polymerase activity: findings from machine learning and in vitro testing.

African swine fever virus (ASFV), an extremely contagious virus with high mortality rates, causes severe hemorrhagic viral disease in both domestic and wild pigs. Fortunately, ASFV cannot be transmitted from pigs to humans. However, ongoing ASFV outbreaks could have severe economic consequences for global food security. Although ASFV was discovered several years ago, no vaccines or treatments are commercially available yet; therefore, the identification of new anti-ASFV drugs is urgently warranted. Using molecular docking and machine learning, we have previously identified pentagastrin, cangrelor, and fostamatinib as potential antiviral drugs against ASFV. Here, using machine learning combined with docking simulations, we identified natural products with a high affinity for AsfvPolX proteins. We selected five natural products (NPs) that are located close in chemical space to the six known natural flavonoids that possess anti-ASFV activity. Polygalic acid markedly reduced AsfvPolX polymerase activity in a dose-dependent manner. We propose an efficient protocol for identifying NPs as potential antiviral drugs by identifying chemical spaces containing high-affinity binders against ASFV in NP databases.

Rapid Visible Detection of African Swine Fever Virus Using Hybridization Chain Reaction-Sensitized Magnetic Nanoclusters and Affinity Chromatography.

African swine fever virus (ASFV) is a severe and persistent threat to the global swine industry. As there are no vaccines against ASFV, there is an immense need to develop easy-to-use, cost-effective, and rapid point-of-care (POC) diagnostic platforms to detect and prevent ASFV outbreaks. Here, a novel POC diagnostic system based on affinity column chromatography for the optical detection of ASFV is presented. This system employs an on-particle hairpin chain reaction to sensitize magnetic nanoclusters with long DNA strands in a target-selective manner, which is subsequently fed into a column chromatography device to produce quantitatively readable and colorimetric signals. The detection approach does not require expensive analytical apparatus or immobile instrumentation. The system can detect five genes constituting the ASFV whole genome with a detection limit of ≈19.8 pm in swine serum within 30min at laboratory room temperature. With an additional pre-amplification step using polymerase chain reaction (PCR), the assay is successfully applied to detect the presence of ASFV in 30 suspected swine samples with 100% sensitivity and specificity, similar to quantitative PCR. Thus, this simple, inexpensive, portable, robust, and customizable platform for the early detection of ASFV can facilitate the timely surveillance and implementation of control measures.

In vitro and in vivo antiviral activity of nucleoside analogue cHPMPC against African swine fever virus replication

African swine fever virus (ASFV) causes a haemorrhagic disease affecting wild boar and domestic pigs which can result in morbidity and fatality rates of up to 100%. ASFV is a large double-stranded DNA virus which replicates predominantly in the cell cytoplasm and codes for its replication and transcription machinery. No vaccine is widely available and control depends on early detection, culling of infected herds and adherence to biosecurity measures. In this study the small molecule nucleoside analogue, cyclic cidofovir (cHPMPC), was evaluated for its ability to inhibit replication of four different ASFV genotypes in primary porcine macrophages. Time of addition studies demonstrated that cHPMPC effectively inhibits ASFV replication and late gene expression when added pre-infection or early post-infection but not when added at late times, suggesting the drug target may be the virus DNA polymerase, or the RNA polymerase involved in late transcription. Oral administration of cHPMPC delayed onset of clinical signs and significantly reduced viral titres in blood and tissues of treated pigs. These results indicate that cHPMPC is a promising compound for further development to control ASFV outbreaks.

Thermal Inactivation of African Swine Fever Virus in Swill.

The indirect transmission of the African swine fever virus (ASFV) is through contaminated fomite, feed ingredients, pork- and pig-derived products, including swill, as ASFV is highly stable within suitable organic material. Some previous studies have indicated that ASFV outbreaks were associated with swill feeding, particularly in smallholder pig farms. These outbreaks emphasize the significance of the appropriate heat treatment of swill to eliminate ASFV residual titer. The World Organization for Animal Health (OIE) recommended the heat treatment of swill at a temperature of at least 90°C for at least 60 min, with continuous stirring, while the Food and Agriculture Organization (FAO) recommended heat treatment at 70°C for 30 min. The lack of scientific evidence regarding ASFV inactivation by heat treatment of swill leads to such inconsistent recommendations. Therefore, the objectives of this study were to assess the thermal inactivation of ASFV in three swill formulae and to develop a DT model to predict DT at some other inactivation temperatures. The significant reduction of ASFV in swill occurred at temperatures as low as 60°C. DT or decimal reduction time (DRT) is defined as the time required to reduce the virus titer by 1 log, and this was also used as a comparative index of heat resistance. The mean D60, D70, D75, and D80 of ASFV in three swill formulae were in the ranges 23.21–33.47, 5.83–10.91, 2.15–2.22, and 1.36–1.47 min, respectively. These DT could be widely used for any nutritive composition of swill other than the three swill formulae in this study since there was no statistical difference of all DT of ASFV across three swill formulae. Based on D70 and the predicted D90 from the DT model in this study, including the highest ASFV titer in pork products, the calculated inactivation times at 70 and 90°C were 119 and 4 min, respectively.

Development of a p72 trimer-based colloidal gold strip for detection of antibodies against African swine fever virus.

African swine fever virus (ASFV) causes a highly contagious and often lethal swine viral disease, and leads to tremendous economic losses to the swine industry. Unfortunately, there are no vaccines and effective antiviral agents available to prevent and control ASFV outbreaks. Therefore, it is necessary to develop simple and rapid strategies to monitor ASFV-infected pigs to restrain its spread. In the current study, ASFV capsid protein p72 was expressed along with its chaperone pB602L to form trimers in human embryonic kidney 293 (HEK293) cells. The p72 trimers were subsequently labeled with colloidal gold to develop a immunochromatographic strip. The strip showed high specificity to ASFV-positive serum and no cross-reactivity to other swine virus positive sera. Importantly, the strip showed a higher sensitivity of detecting ASFV antibodies in both positive standard serum and clinical serum samples than a commercial enzyme-linked immunosorbent assay (ELISA) kit. Taken together, these results demonstrate the strip as a reliable diagnostic tool against ASFV infection, which will be appropriate for application in prevention and control of ASFV.Key points• ASFV p72 trimers were successfully generated.• A colloidal gold strip was developed based on ASFV p72 trimers.• The strip is appropriate for detecting ASFV antibodies in the field.

Development and application of a duplex real-time PCR assay for differentiation of genotypes I and II African swine fever viruses.

Genotype II African swine fever virus (ASFV) has been plaguing Chinese pig industry and caused severe morbidity and mortality of pigs resulting in huge economic losses since its first report in August 2018. Most recently, two genotype I ASFVs with low virulence but efficient transmissibility in pigs were reported in China, which makes the diagnosis and control of this lethal disease more challenging. Therefore, it is prerequisite and important to differentiate genotype I from genotype II upon ASFV outbreaks before making any stringent control procedures. In this study, a duplex real-time PCR assay based on ASFV E296R gene was established which could simultaneously detect genotypes I and II ASFVs with two pairs of primers and two probes. Plasmid containing ASFV genes was used to test the sensitivity, repeatability, and reproducibility. DNA or cDNA samples of ASFV and other swine viruses were used to test the specificity. The results showed that the established duplex real-time PCR assay has satisfied specificity, sensitivity, repeatability, and reproducibility. In addition, the assay was applied to differentiate 84 ASFV positive clinical samples including lymph nodes, spleen, kidney, lung, liver, blood, nasal swab, and environmental swab samples which were sent to National ASF Reference Laboratory from April 2020 to September 2021. The results showed that all these ASFV positive samples belong to genotype II ASFV. The established duplex real-time PCR in this study provides a powerful tool for rapid detection and differentiation between genotypes I and II ASFVs and will facilitate efficient control of ASFV in China.

African Swine Fever: Prevalence, Farm Characteristics, Farmer’s Insight and Attitude toward Reporting of African Swine Fever Cases in the Northwest, West, Littoral and Southwest Regions of Cameroon

The African swine fever virus (ASFV) has been circulating in and ravaging the swine industry in Cameroon for decades. Annual ASFV epidemics occur in Cameroon from April to August. With the absence of vaccines and antiviral drugs against this virus, biosecurity has been the only effective control tool available. When properly applied, biosecurity measures allow control of the spread of ASFV and the eventual eradication of this virus. Many outbreak investigations by PCR were effected in Cameroon, with ASFV prevalences ranging from 15.23% to 42.80%. Considering that pre-outbreak studies are not available for Cameroon, the present study aimed at assessing the status of the animals before an outbreak. A two-stage cluster sampling study was conducted from January to March 2020. In this study, the primary unit was the farm and the secondary unit was the individual animals. In all, 97 farms were visited and 277 samples were collected. Pre-outbreak ASFV prevalence, as assessed by PCR, was 9.75%. In parallel, data were collected using a survey of farm characteristics, awareness and attitude of the farmers toward ASF. The survey results showed that 34.1% of the farms were backyard cemented piggeries with the majority having less than 10 pigs (54.1%). The majority of farmers (90.6%) had heard of and knew about the disease caused by ASF. Though 69.4% of the farmers were uninformed on the clinical signs of ASF, 73.6% of them did treat sick pigs presenting similar clinical signs to ASF (with no risk of mortality) with antibiotics, while 79.4% did not treat but sold the pigs presenting clinical signs similar to ASF with an increased risk of mortality. Sixty-three percent of the farmers had reported a case of ASF in the past and believed that reporting was useful and had no negative consequences on other farmers or third parties. We established that poor implementation of biosecurity measures in addition to poor training are contributing factors to the enzootic nature of ASFV in Cameroon and, thus, the spread of ASFV. Hence, pig farmers in Cameroon must be properly trained in ASFV awareness and the impact thereof on pig production. Furthermore, training will also facilitate the successful implementation of biosecurity measures to contain ASFV outbreaks.

A Deeper Insight into Evolutionary Patterns and Phylogenetic History of ASFV Epidemics in Sardinia (Italy) through Extensive Genomic Sequencing.

African swine fever virus (ASFV) is the etiological agent of the devastating disease African swine fever (ASF), for which there is currently no licensed vaccine or treatment available. ASF is defined as one of the most serious animal diseases identified to date, due to its global spread in regions of Africa, Europe and Asia, causing massive economic losses. On the Italian island of Sardinia, the disease has been endemic since 1978, although the last control measures put in place achieved a significant reduction in ASF, and the virus has been absent from circulation since April 2019. Like many large DNA viruses, ASFV mutates at a relatively slow rate. However, the limited availability of whole-genome sequences from spatial-localized outbreaks makes it difficult to explore the small-scale genetic structure of these ASFV outbreaks. It is also unclear if the genetic variability within outbreaks can be captured in a handful of sequences, or if larger sequencing efforts can improve phylogenetic reconstruction and evolutionary or epidemiological inference. The aim of this study was to investigate the phylogenetic patterns of ASFV outbreaks between 1978 and 2018 in Sardinia, in order to characterize the epidemiological dynamics of the viral strains circulating in this Mediterranean island. To reach this goal, 58 new whole genomes of ASFV isolates were obtained, which represents the largest ASFV whole-genome sequencing effort to date. We provided a complete description of the genomic diversity of ASFV in terms of nucleotide mutations and small and large indels among the isolates collected during the outbreaks. The new sequences capture more than twice the genomic and phylogenetic diversity of all the previously published Sardinian sequences. The extra genomic diversity increases the resolution of the phylogenetic reconstruction, enabling us to dissect, for the first time, the genetic substructure of the outbreak. We found multiple ASFV subclusters within the phylogeny of the Sardinian epidemic, some of which coexisted in space and time.

The impact of African swine fever virus on smallholder village pig production: An outbreak investigation in Lao PDR.

African swine fever virus (ASFV) causes a deadly disease of pigs which spread through southeast Asia in 2019. We investigated one of the first outbreaks of ASFV in Lao People's Democratic Republic amongst smallholder villages of Thapangtong District, Savannakhet Province. In this study, two ASFV affected villages were compared to two unaffected villages. Evidence of ASFV‐like clinical signs appeared in pig herds as early as May 2019, with median epidemic days on 1 and 18 June in the two villages, respectively. Using participatory epidemiology mapping techniques, we found statistically significant spatial clustering in both outbreaks (p < 0.001). Villagers reported known risk factors for ASFV transmission – such as free‐ranging management systems and wild boar access – in all four villages. The villagers reported increased pig trader activity from Vietnam before the outbreaks; however, the survey did not determine a single outbreak source. The outbreak caused substantial household financial losses with an average of nine pigs lost to the disease, and Monte Carlo analysis estimated this to be USD 215 per household. ASFV poses a significant threat to food and financial security in smallholder communities such as Thapangtong, where 40.6% of the district's population are affected by poverty. This study shows ASFV management in the region will require increased local government resources, knowledge of informal trader activity and wild boar monitoring alongside education and support to address intra‐village risk factors such as free‐ranging, incorrect waste disposal and swill feeding.

Arthropods as potential vectors of African swine fever virus outbreaks in pig farms in the Republic of Korea.

The seasonality of African swine fever (ASF) in the summers and outbreaks in farms with high biosecurity levels suggest that the ASF virus (ASFV) may be transmitted by arthropod vectors. Arthropods were collected in this study from 14 pig farms with ASF outbreaks in Korea in 2019 to explore the role of arthropods as potential ASFV vectors. A total of 28,729 arthropods, including 28,508 (99.2%) Diptera, were collected using blacklight traps, insect nets and yellow sticky strips. All arthropod samples were negative for ASFV genomic DNA according to laboratory tests using real‐time polymerase chain reaction. This result may reflect the effects of immediate control measures following the detection of farms with ASF outbreaks in the early phase of infection in Korea.

The contribution of insects to African swine fever virus dispersal: data from domestic pig farms in Lithuania.

Outbreaks of African Swine Fever (ASF) in domestic pig farms in Lithuania typically begin in June and are detected through October, suggesting that insects might be involved in the transmission of the virus. Entomological collecting was performed to obtain two data sets: from farms with ASF outbreaks, and from farms without ASF outbreaks but in an ASF-infected area. Target insects from the families Muscidae, Calliphoridae and Tabanidae were analysed for the presence of ASF Virus (ASFV) DNA. Musca domestica L., Calliphoridae flies and Stomoxys calcitrans (L.) collected by entomological net during ASF outbreaks were confirmed to be ASFV positive. Viral DNA detected in insects collected by Nzi traps from farms with no ASFV outbreaks indicate that Culex, Lucilia, M. domestica and S. calcitrans are likely to play a role in spreading the ASFV mechanically. This finding could suggest contamination from outside of the farms: from infected wild boar or their carcasses. The role of Stomoxys flies as mechanical vectors could be accidental, because we did not find a significant correlation between the activity of S. calcitrans and the number of ASF outbreaks in pig farms, whereas temperatures positively correlated to the number of ASF outbreaks during 2018-2019.

Structural Insight into African Swine Fever Virus dUTPase Reveals a Novel Folding Pattern in the dUTPase Family.

The African swine fever virus (ASFV) is the deadly pathogen of African swine fever (ASF) that induces high mortality, approaching 100% in domestic pigs, causes enormous losses to the global pig industry, and threatens food security. Currently, there is no effective treatment or preventive countermeasure. dUTPases (deoxyuridine 5'-triphosphate pyrophosphatases) are ubiquitous enzymes that are essential for the hydrolysis of dUTP and prevent the misincorporation of dUTP into newly synthesized DNA. Here, we present the crystal structures of the ASFV dUTPase in complex with the product dUMP and cofactor Mg2+ at a resolution of 2.2 Å. We observed that a unique "turning point" at G125 plays an unexpected critical role in the swapping region of the C-terminal segment, which is further stabilized by the interactions of the last C-terminal β strand with the β1 and β2 strands, thereby positioning the catalytic motif 5 into the active site of its own subunit instead of into a third subunit. Therefore, the ASFV dUTPase employs a novel two-subunit active site that is different than the classic trimeric dUTPase active site, which is composed of all three subunits. Meanwhile, further results confirmed that the configuration of motifs 1 to 5 has high structural homology with and a catalytic mechanism similar to that of the known trimeric dUTPases. In general, our study expands the information not only on the structural diversity of the conserved dUTPase family but also on the details needed to utilize this dUTPase as a novel target in the treatment of ASF.IMPORTANCE African swine fever virus (AFSV), a large enveloped double-stranded DNA virus, causes a deadly infection in domestic pigs. In addition to Africa, Europe, and South America, countries in Asia, such as China, Vietnam, and Mongolia, have suffered the hazards posed by ASFV outbreaks in recent years. Until now, there has been no vaccine for protection from ASFV infection or effective treatments to cure ASF. Here, we solved the crystal structure of the ASFV dUTPase-dUMP-Mg2+ complex. The ASFV dUTPase displays a noncanonical folding pattern that differs from that of the classic homotrimeric dUTPase, in which the active site is composed of two subunits. In addition, several nonconserved residues within the 3-fold axis channel play a vital role in ASFV dUTPase homotrimer stability. Our finding on these unique structural features of the ASFV dUTPase could be explored for the design of potential specific inhibitors that target this unique enzyme.

Host genome depletion to determine the evolution, genetic diversity and transmission patterns of full genome sequences of African swine fever genotype IX from Uganda

African swine fever (ASF) is a highly contagious viral haemorrhagic disease of domestic pigs, wild boar and feral swine. It is caused by the only DNA arbovirus, African swine fever virus (ASFV), which is the only member of the family Asfarviridae and is transmitted by the soft ticks in the genus Ornithodoros. ASF results in mortalities of up to 100% and exhibits different epidemiological patterns in Africa, Europe and Asia. This ASFV has recently spread to other countries in Europe and is also now present in Asia where it has spread like wildfire, resulting into high economic losses. The lack of a treatment or vaccine against this disease makes its control and prevention extremely difficult. This is partly due to the large size of the ASFV genome (170-190 kbp) and the many genes (187–190) that it encodes. There are currently 24 genotypes, with genotype IX predominating in Uganda and Kenya. Understanding the genetic characteristics of ASFV have focused mainly on partial fragments of selected genes, thus missing the opportunity to investigate the evolution, genetic diversity and patterns of transmission at the whole genomic level. Using the host-genome depletion approach we show an increase in the amount of ASFV DNA recovered directly from clinical material and generate genotype IX complete genomes on an NGS platform. We further show the inter-epidemic relationships of ASFV outbreaks in Uganda. The data contribute towards understanding the patterns and dynamics of transmission and maintenance of ASFV in a country in which ASF is endemic.

JOURNEY OF AFRICAN SWINE FEVER VIRUS IN EASTERN EU STATES

INTRODUCTION: In 2007, African swine fever virus (ASFv) broken its well-known boundaries. This was the reference year for the first report of African swine fever (ASF) in Georgia. Subsequently, the virus reached pigs and boars in Armenia and Russia. From the Caucasus area, ASFv jumped in all directions, between the Black Sea and the Caspian Sea, in relation to the density of backyard pigs and their trade. In the next ten years there have been notifications and registrations of ASFv outbreaks in Russia, Azerbaijan, Ukraine, Belarus, Lithuania, Poland, Estonia, Latvia, Moldova and the Czech Republic. Romania faced the first ASFv outbreak at the end of July 2017, in backyard pigs." in stead "density of backyard pigs and their trade. In the next ten years there have been notifications and registrations of ASFv outbreaks in Russia, Azerbaijan, Ukraine, Belarus, Lithuania, Poland, Estonia, Latvia, Moldova and the Czech Republic. Romania faced the first ASFv outbreak at the end of July 2017, in backyard pigs.OBJECTIVES: The aim of study is to analyse the ways ASFv spread from and into different regions recorded by Eastern European states.METHODS: The immediate notifications on ASFv to the World Organisation for Animal Health (OIE) were analysed from the Eastern-European states between 2007 and 2017. The analysis took into consideration the first occurrence of the disease under scrutiny in the country and the follow-up reports, in relation with the geospatial distribution of the outbreaks.RESULTS: The main route of ASFv introduction into local pig populations indicated by the Member States of the European Union was the trans-boundary circulation of boars. However, the spread of ASFv through both, wild and domestic pigs and also by the human alimentary customs/traditions in the affected areas shouldn’t be ignored. Three cycles of ASFv transmission have been identified and described by the epidemiologists: the domestic cycle, the sylvatic cycle and the tick-pig cycle.CONCLUSION: None of the ways to disseminate the ASFv should be excluded, and the origin of the first outbreaks remains unknown or inconclusive in Eastern EU states.

Survival and localization of African swine fever virus in stable flies (Stomoxys calcitrans) after feeding on viremic blood using a membrane feeder

Since 2014, African swine fever virus (ASFV) has been spreading within Eastern Europe. Within affected regions, the virus has infected some farms with high biosecurity and a marked seasonality of outbreaks in domestic pigs has been observed. ASFV transmission from stable flies, Stomoxys calcitrans, has previously been shown both mechanically and via ingestion of whole flies. Hence, blood-feeding flies may offer one explanation for the introductions into high biosecurity farms and for the observed seasonality. The aim of this study was to further elucidate the potential role of stable flies in ASFV transmission. Different parts of flies were analyzed for the presence of viral DNA and infectious virus at different time points following in vitro feeding of the flies on blood from an ASFV-infected pig.Using qPCR, ASFV DNA was detectable in mouth parts of flies for at least 12 h and remained in head and body samples from the flies for up to three days following feeding. Infectious virus was detected in fly body samples prepared at 3 h and 12 h after feeding.The presence of infectious ASFV in stable flies following feeding on viremic blood means that such flies are capable of transporting infectious virus. The detection of ASFV DNA in the flies for up to three days following feeding suggests that qPCR analysis of blood-feeding flies during ASFV outbreaks could be a useful method to elucidate the role of these flies in ASFV transmission under field conditions.

Inferring within-herd transmission parameters for African swine fever virus using mortality data from outbreaks in the Russian Federation.

SummaryMortality data are routinely collected for many livestock and poultry species, and they are often used for epidemiological purposes, including estimating transmission parameters. In this study, we infer transmission rates for African swine fever virus (ASFV), an important transboundary disease of swine, using mortality data collected from nine pig herds in the Russian Federation with confirmed outbreaks of ASFV. Parameters in a stochastic model for the transmission of ASFV within a herd were estimated using approximate Bayesian computation. Estimates for the basic reproduction number varied amongst herds, ranging from 4.4 to 17.3. This was primarily a consequence of differences in transmission rate (range: 0.7–2.2), but also differences in the mean infectious period (range: 4.5–8.3 days). We also found differences amongst herds in the mean latent period (range: 5.8–9.7 days). Furthermore, our results suggest that ASFV could be circulating in a herd for several weeks before a substantial increase in mortality is observed in a herd, limiting the usefulness of mortality data as a means of early detection of an outbreak. However, our results also show that mortality data are a potential source of data from which to infer transmission parameters, at least for diseases which cause high mortality.

Adaptation of an Invader ® assay for the detection of African swine fever virus DNA

A closed tube isothermal Invader ® assay (Third Wave Technologies Inc., Madison, Wisconsin, USA) was adapted for the detection of African swine fever virus (ASFV) DNA. Several ASFV Invader ® assays were designed successfully and tested on a real-time PCR instrument (iCycler™, BioRad). The assay exhibiting the lowest signal/noise ratio (VP73 ASFV Invader ® Assay) was analysed further using serial 10-fold dilutions of Lisbon 60 ASFV viral genome. The assay sensitivity was determined to be in the order of 2500 copies of ASFV DNA and showed a dynamic range of 4 logs, from 2.5 × 10 6 to 2500 copies. The high specificity of the test was demonstrated by the lack of cross-reactivity to the clinically similar but heterologous virus, classical swine fever virus. The sensitivity of the Invader ® assay is sufficient for the testing of acutely infected viremic animals in which the viral load will be high. The robustness and ease of use of the ASFV Invader ® assay, combined with the possibility to run and read the assay using simple and relatively inexpensive equipment, makes it suitable for laboratories lacking containment facilities and/or real-time PCR instrumentation or on a regional basis for on-site diagnosis close to putative sites of ASFV outbreaks.