Genome-wide characterization of structure variations in the Xiang pig for genetic resistance to African swine fever.

African swine fever (ASF) is a rapidly fatal viral haemorrhagic fever in Chinese domestic pigs. Although very high mortality is observed in pig farms after an ASF outbreak, clinically healthy and antibody-positive pigs are found in those farms, and viral detection is rare from these pigs. The ability of pigs to resist ASF viral infection may be modulated by host genetic variations. However, the genetic basis of the resistance of domestic pigs against ASF remains unclear. We generated a comprehensive set of structural variations (SVs) in a Chinese indigenous Xiang pig with ASF-resistant (Xiang-R) and ASF-susceptible (Xiang-S) phenotypes using whole-genome resequencing method. A total of 53,589 nonredundant SVs were identified, with an average of 25,656 SVs per individual in the Xiang pig genome, including insertion, deletion, inversion and duplication variations. The Xiang-R group harboured more SVs than the Xiang-S group. The F-statistics (FST) was carried out to reveal genetic differences between two populations using the resequencing data at each SV locus. We identified 2,414 population-stratified SVs and annotated 1,152 Ensembl genes (including 986 protein-coding genes), in which 1,326 SVs might disturb the structure and expression of the Ensembl genes. Those protein-coding genes were mainly enriched in the Wnt, Hippo, and calcium signalling pathways. Other important pathways associated with the ASF viral infection were also identified, such as the endocytosis, apoptosis, focal adhesion, Fc gamma R-mediated phagocytosis, junction, NOD-like receptor, PI3K-Akt, and c-type lectin receptor signalling pathways. Finally, we identified 135 candidate adaptive genes overlapping 166 SVs that were involved in the virus entry and virus-host cell interactions. The fact that some of population-stratified SVs regions detected as selective sweep signals gave another support for the genetic variations affecting pig resistance against ASF. The research indicates that SVs play an important role in the evolutionary processes of Xiang pig adaptation to ASF infection.

Development and optimization of sampling techniques for environmental samples from African swine fever virus-contaminated surfaces with no organic contaminants.

African swine fever (ASF) is a highly contagious diseases in domestic pigs and wild boars with up to 100% mortality. ASF virus (ASFV) is a causative agent responsible for ASF and highly resistant in environments, which creates a significant challenge for the control and eradication of the virus. Despite the geographical expansion of ASFV and international movement of products to sustain the swine production system, there is limited knowledge on the use of environmental samples to perform surveillance to prevent the introduction of ASFV into ASFV-free areas and for control of transmission in affected areas. Therefore, this study aimed to develop and optimize sampling techniques for environmental samples for ASFV detection. The stainless steel surfaces were contaminated with ASFV-infected blood, swabbed using different devices, and then processed through different techniques. The environmental samples were processed and tested using qPCR analysis. The results showed that the use of pre-moistened gauze surgical sponges, sweeping pads, and sponge sticks resulted in increased sensitivity, when compared to either dry sampling devices or Dacron swab. In particular, the combination of the sponge stick and the commercial nucleic acid preservative supported the best detection of ASFV DNA on the clean stainless steel surfaces evaluated. Pre-incubation for the short period of time and centrifugation at low speed were sufficient to provide satisfactory diagnostic sensitivity of ASFV detection using qPCR for environmental samples. Our findings contribute to the development of techniques for environmental samples for ASFV surveillance to prevent the introduction and dissemination of ASFV.

Development of plate-type and tubular chemiluminescence immunoassay against African swine fever virus p72.

African swine fever (ASF) is a highly contagious and fatal viral disease that has caused huge economic losses to the pig and related industries worldwide. At present, rapid, accurate, and sensitive laboratory detection technologies are important means of preventing and controlling ASF. However, because attenuated strains of African swine fever virus (ASFV) are constantly emerging, an ASFV antibody could be used more effectively to investigate the virus and control the disease on pig farms. The isolation of ASFV-specific antibodies is also essential for the diagnosis of ASF. Therefore, in this study, we developed two chemiluminescence immunoassays (CLIAs) to detect antibodies directed against ASFV p72: a traditional plate-type blocking CLIA (p72-CLIA) and an automatic tubular competitive CLIA based on magnetic particles (p72-MPCLIA). We compared the diagnostic performance of these two methods to provide a feasible new method for the effective prevention and control of ASF and the purification of ASFV. The cut-off value, diagnostic sensitivity (Dsn), and diagnostic specificity (Dsp) of p72-CLIA were 40%, 100%, and 99.6%, respectively, in known background serum, whereas those of p72-MPCLIA were 36%, 100%, and 99.6%, respectively. Thus, both methods show good Dsn, Dsp, and repeatability. However, when analytical sensitivity was evaluated, p72-MPCLIA was more sensitive than p72-CLIA or a commercial enzyme-linked immunosorbent assay. More importantly, p72-MPCLIA reduced the detection time to 15 min and allowed fully automated detection. In summary, p72-MPCLIA showed superior diagnostic performance and offered a new tool for detecting ASFV infections in the future. KEY POINTS: • Two chemiluminescence immunoassay (plate-type CLIA and tubular CLIA) methods based on p72 monoclonal antibody (mAb) were developed to detect ASFV antibody. • Both methods show good diagnostic performance (Dsn (100%), Dsp (99.6%), and good repeatability), and p72-MPCLIA detects antibodies against ASFV p72 with high efficiency in just 15 min.

[Effects of inhibitors of DNA repair enzymes OGG1 and MTH1 on the replication of African swine fever virus].

African swine fever virus (ASFV), as a contagious viral pathogen, is responsible for the occurrence of African swine fever (ASF), a rapidly spreading and highly lethal disease. Since ASFV was introduced into China in 2018, it has been quickly spread to many provinces, which brought great challenges to the pig industry in China. Due to the limited knowledge about the pathogenesis of ASFV, neither vaccines nor antiviral drugs are available. We have found that ASFV infection can induce oxidative stress responses in cells, and DNA repair enzymes play a key role in this process. This study employed RNA interference, RT-qPCR, Western blotting, Hemadsorption (HAD), and flow cytometry to investigate the effects of the inhibitors of DNA repair enzymes OGG1 and MTH1 on ASFV replication and evaluated the anti-ASFV effects of the inhibitors. This study provides reference for the development of anti-viral drugs.

A methodological framework to characterize the wildlife-livestock interface: The case of wild boar in mainland Spain.

BACKGROUND: The representation of wildlife-livestock interface (WLI) at an accurate spatial resolution poses several challenges. Furthermore, there is a lack of published material providing detailed descriptions of geospatial techniques for the purpose of producing visual results that are interpretable and contrastable for epidemiological analysis. OBJECTIVES: Our aim is to develop a standardized, applicable, and scalable methodological framework for describing and characterizing the WLI across a large spatial extent. Subsequently, we aim to employ this framework to depict specific WLI based on different epidemiological scenarios determined by the abundance of wild boar (Sus scrofa) and domestic ungulates as an illustrative case, specifically focusing on mainland Spain. METHODS: To establish a methodological framework, we merged data from both wild and domestic sources into a hexagonal grid. We utilized data on wild boar hunting and the locations of pig, cattle, sheep, and goat farms in mainland Spain. New variables were derived from this combined dataset to illustrate the overlapping abundance between wild boar and domestic species. Finally, a cluster analysis of the generated variables was carried out, with the aim of distinguishing and characterizing various scenarios of the wild boar-domestic ungulate interface in mainland Spain. RESULTS: The hexagonal grid proved appropriate to represent and evaluate the WLI at fine spatial resolution over such broad extent. Despite the inability to ascribe a dominant livestock type and production system to a specific region, we were able to identify fifteen main areas of interest in terms of overlap. As for extensive livestock, normally at the highest risk of interaction with wild boar, the primary regions in Spain were those with dehesa agroecosystem and the Atlantic areas. Certain scenarios were particularly relevant in terms of risk for interaction and subsequent transmission of disease, namely, the case of extensive pig production in south western Spain (dehesa agroecosystem), which is especially concerned about the potential introduction of African Swine fever (ASF) in the Country. DISCUSSION AND CONCLUSIONS: We provide a basis for visualizing and understanding of different WLI scenarios, which is extensible to other regions and interfaces, and automatable where precise source of data from wildlife and livestock are available. This spatial statistics framework enables the utilization of high-resolution data, ensuring consistency on uniform grids. This aligns with the needs of high-resolution disease dissemination models based on wildlife behaviour. Such aspects are crucial for developing risk assessment and improving strategies for the prevention, control, and eradication of shared priority emerging diseases at national and international levels, such as ASF.

Fangchinoline Inhibits African Swine Fever Virus Replication by Suppressing the AKT/mTOR/NF-κB Signaling Pathway in Porcine Alveolar Macrophages.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is one of the most important infectious diseases that cause high morbidity and mortality in pigs and substantial economic losses to the pork industry of affected countries due to the lack of effective vaccines. The need to develop alternative robust antiviral countermeasures, especially anti-ASFV agents, is of the utmost urgency. This study shows that fangchinoline (FAN), a bisbenzylisoquinoline alkaloid found in the roots of Stephania tetrandra of the family Menispermaceae, significantly inhibits ASFV replication in porcine alveolar macrophages (PAMs) at micromolar concentrations (IC50 = 1.66 µM). Mechanistically, the infection of ASFV triggers the AKT/mTOR/NF-κB signaling pathway. FAN significantly inhibits ASFV-induced activation of such pathways, thereby suppressing viral replication. Such a mechanism was confirmed using an AKT inhibitor MK2206 as it inhibited AKT phosphorylation and ASFV replication in PAMs. Altogether, the results suggest that the AKT/mTOR pathway could potentially serve as a treatment strategy for combating ASFV infection and that FAN could potentially emerge as an effective novel antiviral agent against ASFV infections and deserves further in vivo antiviral evaluations.

Development of microsatellite markers for the soft tick Ornithodoros phacochoerus.

BACKGROUND: Soft ticks of the genus Ornithodoros are responsible for the maintenance and transmission of the African swine fever (ASF) virus in the sylvatic and domestic viral cycles in Southern Africa. They are also the main vectors of the Borrelia species causing relapsing fevers. Currently, no genetic markers are available for Afrotropical Ornithodoros ticks. As ASF spreads globally, such markers are needed to assess the role of ticks in the emergence of new outbreaks. The aim of this study is to design microsatellite markers that could be used for ticks of the Ornithodoros moubata complex, particularly Ornithodoros phacochoerus, to assess population structure and tick movements in ASF endemic areas. METHODS: A total of 151 markers were designed using the O. moubata and O. porcinus genomes after elimination of repeated sequences in the genomes. All designed markers were tested on O. phacochoerus and O. porcinus DNA to select the best markers. RESULTS: A total of 24 microsatellite markers were genotyped on two populations of O. phacochoerus and on individuals from four other Ornithodoros species. Nineteen markers were selected to be as robust as possible for population genetic studies on O. phacochoerus. CONCLUSIONS: The microsatellite markers developed here represent the first genetic tool to study nidicolous populations of O. phacochoerus.

Comparative evaluation of disease dynamics in wild boar and domestic pigs experimentally inoculated intranasally with the European highly virulent African swine fever virus genotype II strain "Armenia 2007".

Since the reintroduction of African swine fever virus (ASFV) in Europe in 2007 and its subsequent spread to Asia, wild boar has played a crucial role in maintaining and disseminating the virus. There are significant gaps in the knowledge regarding infection dynamics and disease pathogenesis in domestic pigs and wild boar, particularly at the early infection stage. We aimed to compare domestic pigs and wild boar infected intranasally to mimic natural infection with one of the original highly virulent genotype II ASFV isolates (Armenia 2007). The study involved euthanising three domestic pigs and three wild boar on days 1, 2, 3, and 5 post-infection, while four domestic pigs and four wild boar were monitored until they reached a humane endpoint. The parameters assessed included clinical signs, macroscopic lesions, viremia levels, tissue viral load, and virus shedding in nasal and rectal swabs from day 1 post-infection. Compared with domestic pigs, wild boar were more susceptible to ASFV, with a shorter incubation period and earlier onset of clinical signs. While wild boar reached a humane endpoint earlier than domestic pigs did, the macroscopic lesions were comparatively less severe. In addition, wild boar had earlier viremia, and the virus was also detected earlier in tissues. The medial retropharyngeal lymph nodes were identified as key portals for ASFV infection in both subspecies. No viral genome was detected in nasal or rectal swabs until shortly before reaching the humane endpoint in both domestic pigs and wild boar, suggesting limited virus shedding in acute infections.

Stability Analysis of a Fractional-Order African Swine Fever Model with Saturation Incidence.

This article proposes and analyzes a fractional-order African Swine Fever model with saturation incidence. Firstly, the existence and uniqueness of a positive solution is proven. Secondly, the basic reproduction number and the sufficient conditions for the existence of two equilibriums are obtained. Thirdly, the local and global stability of disease-free equilibrium is studied using the LaSalle invariance principle. Next, some numerical simulations are conducted based on the Adams-type predictor-corrector method to verify the theoretical results, and sensitivity analysis is performed on some parameters. Finally, discussions and conclusions are presented. The theoretical results show that the value of the fractional derivative α will affect both the coordinates of the equilibriums and the speed at which the equilibriums move towards stabilization. When the value of α becomes larger or smaller, the stability of the equilibriums will be changed, which shows the difference between the fractional-order systems and the classical integer-order system.

Identification of Two Linear Epitopes on MGF_110-13L Protein of African Swine Fever Virus with Monoclonal Antibodies.

African swine fever caused by African swine fever virus (ASFV) is an acute, highly contagious swine disease with high mortality. To facilitate effective vaccine development and find more serodiagnostic targets, fully exploring the ASFV antigenic proteins is urgently needed. In this study, the MGF_110-13L was identified as an immunodominant antigen among the seven transmembrane proteins. The main outer-membrane domain of MGF_110-13L was expressed and purified. Two monoclonal antibodies (mAbs; 8C3, and 10E4) against MGF_110-13L were generated. The epitopes of two mAbs were preliminary mapped with the peptide fusion proteins after probing with mAbs by enzyme-linked immunosorbent assay (ELISA) and Western blot. And the two target epitopes were fine-mapped using further truncated peptide fusion protein strategy. Finally, the core sequences of mAbs 8C3 and 10E4 were identified as 48WDCQDGICKNKITESRFIDS67, and 122GDHQQLSIKQ131, respectively. The peptides of epitopes were synthesized and probed with ASFV antibody positive pig sera by a dot blot assay, and the results showed that epitope 10E4 was an antigenic epitope. The epitope 10E4 peptide was further evaluated as a potential antigen for detecting ASFV antibodies. To our knowledge, this is the first report of antigenic epitope information on the antigenic MGF_110-13L protein of ASFV.

A suitable sampling strategy for the detection of African swine fever virus in living and deceased pigs in the field: a retrospective study.

African swine fever (ASF) is a fatal disease that threatens the health status of the swine population and thus can impact the economic outcome of the global pig industry. Monitoring the ASF virus (ASFV) is of utmost concern to prevent and control its distribution. This study aims to identify a suitable sampling strategy for ASFV detection in living and deceased pigs under field conditions. A range of samples, comprising tissues obtained from deceased pigs, as well as serum and tonsil swab samples from live pigs, were gathered and subjected to detection using the qPCR method. The findings revealed that the mandibular lymph nodes demonstrated the highest viral loads among superficial tissues, thereby indicating their potential suitability for detecting ASFV in deceased pigs. Additionally, the correlations between virus loads in various tissues have demonstrated that tonsil swab samples are a viable specimen for monitoring live pigs, given the strong associations observed with other tissues. These findings indicated two dependable sample types for the detection of ASFV: mandibular lymph nodes for deceased pigs and tonsil swabs for live pigs, which supply some references for the development of efficacious preventive measures against ASFV.

Pathogenicity and virulence of African swine fever virus.

African swine fever (ASF) is a devastating disease with a high impact on the pork industry worldwide. ASF virus (ASFV) is a very complex pathogen, the sole member of the family Asfaviridae, which induces a state of immune suppression in the host through infection of myeloid cells and apoptosis of lymphocytes. Moreover, haemorrhages are the other main pathogenic effect of ASFV infection in pigs, related to the infection of endothelial cells, as well as the activation and structural changes of this cell population by proinflammatory cytokine upregulation within bystander monocytes and macrophages. There are still many gaps in the knowledge of the role of proteins produced by the ASFV, which is related to the difficulty in producing a safe and effective vaccine to combat the disease, although few candidates have been approved for use in Southeast Asia in the past couple of years.

The antibodies against the A137R protein drive antibody-dependent enhancement of African swine fever virus infection in porcine alveolar macrophages.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a highly contagious disease that can kill up to 100% of domestic pigs and wild boars. It has been shown that the pigs inoculated with some ASF vaccine candidates display more severe clinical signs and die earlier than do pigs not immunized. We hypothesize that antibody-dependent enhancement (ADE) of ASFV infection may be caused by the presence of some unidentified antibodies. In this study, we found that the ASFV-encoded structural protein A137R (pA137R) can be recognized by the anti-ASFV positive sera, indicating that the anti-pA137R antibodies are induced in the ASFV-infected pigs. Interestingly, our results demonstrated that the anti-pA137R antibodies produced in rabbits or pigs enhanced viral replication of different ASFV strains in primary porcine alveolar macrophages (PAMs), the target cells of ASFV. Mechanistic investigations revealed that anti-pA137R antibodies were able to promote the attachment of ASFV to PAMs and two types of Fc gamma receptors (FcγRs), FcγRII and FcγRIII, mediated the ADE of ASFV infection. Taken together, anti-pA137R antibodies are able to drive ASFV ADE in PAMs. These findings shed new light on the roles of anti-ASFV antibodies and have implications for the pathophysiology of the disease and the development of ASF vaccines.

Six adenoviral vectored African swine fever virus genes protect against fatal disease caused by genotype I challenge.

African swine fever virus causes a lethal hemorrhagic disease in domestic swine and wild boar for which currently licensed commercial vaccines are only available in Vietnam. Development of subunit vaccines is complicated by the lack of information on protective antigens as well as suitable delivery systems. Our previous work showed that a pool of eight African swine fever virus genes vectored using an adenovirus prime and modified vaccinia virus boost could prevent fatal disease after challenge with a virulent genotype I isolate of the virus. Here, we identify antigens within this pool of eight that are essential for the observed protection and demonstrate that adenovirus-prime followed by adenovirus-boost can also induce protective immune responses against genotype I African swine fever virus. Immunization with a pool of adenoviruses expressing individual African swine fever virus genes partially tailored to genotype II virus did not protect against challenge with genotype II Georgia 2007/1 strain, suggesting that different antigens may be required to induce cross-protection for genetically distinct viruses. IMPORTANCE: African swine fever virus causes a lethal hemorrhagic disease in domestic pigs and has killed millions of animals across Europe and Asia since 2007. Development of safe and effective subunit vaccines against African swine fever has been problematic due to the complexity of the virus and a poor understanding of protective immunity. In a previous study, we demonstrated that a complex combination of eight different virus genes delivered using two different viral vector vaccine platforms protected domestic pigs from fatal disease. In this study, we show that three of the eight genes are required for protection and that one viral vector is sufficient, significantly reducing the complexity of the vaccine. Unfortunately, this combination did not protect against the current outbreak strain of African swine fever virus, suggesting that more work to identify immunogenic and protective viral proteins is required to develop a truly effective African swine fever vaccine.

The Distal Promoter of the B438L Gene of African Swine Fever Virus Is Responsible for the Transcription of the Alternatively Spliced B169L.

The B169L protein (pB169L) of African swine fever virus (ASFV) is a structural protein with an unidentified function during the virus replication. The sequences of the B169L gene and the downstream B438L gene are separated by short intergenic regions. However, the regulatory mode of the gene transcription remains unknown. Here, we identified two distinct promoter regions and two transcription start sites (TSSs) located upstream of the open reading frame (ORF) of B438L. Using the promoter reporter system, we demonstrated that the cis activity of the ORF proximal promoter exhibited significantly higher levels compared with that of the distal promoter located in the B169L gene. Furthermore, transfection with the plasmids with two different promoters for B438L could initiate the transcription and expression of the B438L gene in HEK293T cells, and the cis activity of the ORF proximal promoter also displayed higher activities compared with the distal promoter. Interestingly, the B438L distal promoter also initiated the transcription of the alternatively spliced B169L mRNA (B169L mRNA2) encoding a truncated pB169L (tpB169L) (amino acids 92-169), and the gene transcription efficiency was increased upon mutation of the initiation codon located upstream of the alternatively spliced B169L gene. Taken together, we demonstrated that the distal promoter of B438L gene initiates the transcription of both the B438L mRNA and B169L mRNA2. Comprehensive analysis of the transcriptional regulatory mode of the B438L gene is beneficial for the understanding of the association of B438L protein and pB169L and the construction of the gene-deleted ASFV.

Google trends as an early indicator of African swine fever outbreaks in Southeast Asia.

African Swine Fever (ASF) is a reportable disease of swine that causes far-reaching losses to affected countries and regions. Early detection is critically important to contain and mitigate the impact of ASF outbreaks, for which timely available data is essential. This research examines the potential use of Google Trends data as an early indicator of ASF outbreaks in Southeast Asia, focusing on the three largest swine producing countries, namely, Vietnam, the Philippines, and Thailand. Cross-correlation and Kullback-Leibler (KL) divergence indicators were used to evaluate the association between Google search trends and the number of ASF outbreaks reported. Our analysis indicate strong and moderate correlations between Google search trends and number of ASF outbreaks reported in Vietnam and the Philippines, respectively. In contrast, Thailand, the country of this group in which outbreaks were reported last, exhibits the weakest correlation (KL = 2.64), highlighting variations in public awareness and disease dynamics. These findings suggest that Google search trends are valuable for early detection of ASF. As the disease becomes endemic, integrating trends with other epidemiological data may support the design and implementation of surveillance strategies for transboundary animal diseases in Southeast Asia.

Viroporin-like activity of the hairpin transmembrane domain of African swine fever virus B169L protein.

African swine fever virus (ASFV) is the causative agent of a contagious disease affecting wild and domestic swine. The function of B169L protein, as a potential integral structural membrane protein, remains to be experimentally characterized. Using state-of-the-art bioinformatics tools, we confirm here earlier predictions indicating the presence of an integral membrane helical hairpin, and further suggest anchoring of this protein to the ER membrane, with both terminal ends facing the lumen of the organelle. Our evolutionary analysis confirmed the importance of purifying selection in the preservation of the identified domains during the evolution of B169L in nature. Also, we address the possible function of this hairpin transmembrane domain (HTMD) as a class IIA viroporin. Expression of GFP fusion proteins in the absence of a signal peptide supported B169L insertion into the ER as a Type III membrane protein and the formation of oligomers therein. Overlapping peptides that spanned the B169L HTMD were reconstituted into ER-like membranes and the adopted structures analyzed by infrared spectroscopy. Consistent with the predictions, B169L transmembrane sequences adopted α-helical conformations in lipid bilayers. Moreover, single vesicle permeability assays demonstrated the assembly of lytic pores in ER-like membranes by B169L transmembrane helices, a capacity confirmed by ion-channel activity measurements in planar bilayers. Emphasizing the relevance of these observations, pore-forming activities were not observed in the case of transmembrane helices derived from EP84R, another ASFV protein predicted to anchor to membranes through a α-helical HTMD. Overall, our results support predictions of viroporin-like function for the B169L HTMD.IMPORTANCEAfrican swine fever (ASF), a devastating disease affecting domestic swine, is widely spread in Eurasia, producing significant economic problems in the pork industry. Approaches to prevent/cure the disease are mainly restricted to the limited information concerning the role of most of the genes encoded by the large (160-170 kba) virus genome. In this report, we present the experimental data on the functional characterization of the African swine fever virus (ASFV) gene B169L. Data presented here indicates that the B169L gene encodes for an essential membrane-associated protein with a viroporin function.

Bis-benzylisoquinoline alkaloids inhibit African swine fever virus internalization and replication by impairing late endosomal/lysosomal function.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly infectious disease afflicting domestic pigs and wild boars. It exhibits an alarming acute infection fatality rate of up to 100%. Regrettably, no commercial vaccines or specific drugs for combating this disease are currently available. This study evaluated the anti-ASFV activities in porcine alveolar macrophages, 3D4/21 cells, and PK-15 cells of four bis-benzylisoquinoline alkaloids (BBAs): cepharanthine (CEP), tetrandrine, fangchinoline, and iso-tetrandrine. Furthermore, we demonstrated that CEP, which exhibited the highest selectivity index (SI = 81.31), alkalized late endosomes/lysosomes, hindered ASFV endosomal transport, disrupted virus uncoating signals, and thereby inhibited ASFV internalization. Additionally, CEP disrupted ASFV DNA synthesis, leading to the inhibition of viral replication. Moreover, berbamine was labeled with NBD to synthesize a fluorescent probe to study the cellular location of these BBAs. By co-staining with Lyso-Tracker and lysosome-associated membrane protein 1, we demonstrated that BBAs target the endolysosomal compartments for the first time. Our data together indicated that BBAs are a class of natural products with significant inhibitory effects against ASFV infection. These findings suggest their potential efficacy as agents for the prevention and control of ASF, offering valuable references for the identification of potential drug targets.IMPORTANCEThe urgency and severity of African swine fever (ASF) underscore the critical need for effective interventions against this highly infectious disease, which poses a grave threat to domestic pigs and wild boars. Our study reveals the potent anti-African swine fever virus (ASFV) efficacy of bis-benzylisoquinoline alkaloids (BBAs), particularly evident in the absence of progeny virus production under a 5 µM concentration treatment. The structural similarity among cepharanthine, tetrandrine, fangchinoline, and iso-tetrandrine, coupled with their analogous inhibitory stages and comparable selectivity indexes, strongly suggests a shared antiviral mechanism within this drug category. Further investigation revealed that BBAs localize to lysosomes and inhibit the internalization and replication of ASFV by disrupting the endosomal/lysosomal function. These collective results have profound implications for ASF prevention and control, suggesting the potential of the investigated agents as prophylactic and therapeutic measures. Furthermore, our study offers crucial insights into identifying drug targets and laying the groundwork for innovative interventions.

Spatio-temporal clustering of African swine fever virus (Asfarviridae: Asfivirus) circulating in the Kaliningrad region based on three genome markers.

INTRODUCTION: The rapid spread of African swine fever in the Kaliningrad region makes it necessary to use the methods of molecular epidemiology to determine the dynamics and direction of ASF spread in this region of Russia. The aim of the study was to determine single nucleotide polymorphisms within molecular markers K145R, O174L and MGF 505-5R of ASFVs isolated in Kaliningrad region and to study the circulating of the pathogen in European countries by subgenotyping and spatio-temporal clustering analysis. MATERIALS AND METHODS: Blood samples from living domestic pigs and organs from dead domestic pigs and wild boars, collected in the Kaliningrad region between 2017 and 2022 were used. Virus isolation was carried out in porcine bone-marrow primary cell culture. Amplicons of genome markers were amplified by PCR with electrophoretic detection and subsequent extraction of fragments from agarose gel. Sequencing was performed using the Sanger method. RESULTS: The circulation of two genetic clusters of ASFV isolates on the territory of the Kaliningrad has been established: epidemic (K145R-III, MGF 505-5R-II, O174L-I - 94.3% of the studied isolates) and sporadic (K145R-II, MGF 505-5R-II, O174L-I - 5.7%). CONCLUSION: The broaden molecular genetic surveillance of ASFV isolates based on sequencing of genome markers is necessary in the countries of the Eurasian continent to perform a more detailed analysis of ASF spread between countries and within regions.

Establishment of a Suitable Diagnostic Workflow to Ensure Sensitive Detection of African Swine Fever Virus Genome in Porcine Semen.

The rapid spread of African swine fever virus (ASFV), causing severe and often lethal disease in domestic pigs and Eurasian wild boar, continues to be a threat to pig populations and dependent industries. Despite scientific achievements that have deepened our understanding of ASFV pathogenesis, alternative transmission routes for ASFV remain to be elucidated. We previously demonstrated the efficient transmission of ASFV from infected boars to naïve recipient gilts via artificial insemination, thereby highlighting the importance of surveillance of boar semen prior to its shipment. Since the accurate and reliable detection of even low amounts of ASFV in boar semen is key to disease prevention and control, we established a suitable diagnostic workflow to efficiently detect the ASFV genome in boar semen. Here, we assessed the sensitivity of various routine nucleic acid extraction kits as well as qPCR protocols in detecting the ASFV genome in the blood and semen of infected boars. The feasibility of the respective kits and methods for future use in boar studs was also considered. Variability in sensitivity mostly concerned samples with low to very low amounts of the ASFV genome. Ultimately, we defined a well-suited workflow for precisely detecting the ASFV genome in boar semen as early as 2 days post ASFV infection.