Detection of the first recombinant African swine fever virus (genotypes I and II) in domestic pigs in Russia.

INTRODUCTION: African swine fever (ASF) is a contagious viral disease that affects pigs and wild boars, with a mortality rate of up to 100% in susceptible animals. The virus has been circulating in Europe and Asia since its introduction in 2007. Initially, all studied isolates were identified as genotype II, but in 2021 genotype I was reported in China. Later in 2023, the first recombinant virus of genotype I and II was identified in China, with an isolate dating back to 2021, this was followed by the detection of 6 recombinant isolates in Vietnam. METHODS: In this study, an ASFV isolate from the Primorsky Region of Russia obtained from a domestic pig was analyzed by sequencing several genome markers as well as the full genome. Eight pigs were infected with the isolate to assess its virulence. RESULTS: Virus replication in cell culture showed hemadsorption, while sequencing of genome markers clustered the isolate into both genotype I and genotype II. The whole-genome sequence showed that the Russian isolate shared a 99.99% identity with recombinant isolates described earlier in China. Experimental animals developed ASF disease after the introduction of a low dose of the virus (10 HAU50) and died within 7 days post-infection, presenting an acute form of the disease. CONCLUSION: This is the first report on recombinant ASFV in Russia's territory. The results once again confirm the transboundary nature of the disease, demonstrating the vulnerability of the global pig industry underscoring the need for developing new ASF vaccines effective against recombinant strains and emphasizing the importance of continuous molecular monitoring to detect emerging threats promptly.

Molecular characterization of emerging recombinant African swine fever virus of genotype I and II in Vietnam, 2023.

African swine fever virus (ASFV) recombinant strains pose new challenges for diagnosis and control. This study characterizes genotype I and II recombinant ASFV strains identified in northern Vietnam in 2023 through whole-genome sequencing and comparative genomic analysis. Seven ASFV-positive samples from six provinces were analyzed, with recombinant strains detected in Bac Giang, Phu Tho, and Vinh Phuc provinces. Isolates showed hemadsorption positivity despite having genotype I B646L, indicating their recombinant nature. Genome-wide analysis revealed 19 recombination breakpoints consistent with Chinese recombinant strains. Vietnamese isolates shared 99.86-99.98% nucleotide identity with Chinese recombinants, forming a distinct monophyletic group. Comparative analysis identified 50 SNPs and INDELs, with 39 variations found across Vietnamese strains, distinguishing them from Chinese isolates. Unique genetic markers in C962R, I329L, and MGF 505-11L genes distinguished Vietnamese recombinants from Chinese counterparts, while mutations in C122R and NP1450L differentiated all recombinants from parental genotypes. The central variable region (CVR) of the B602L gene showed diversity among Vietnamese isolates, while the I73R-I329L intergenic regions were recognized as in the IGR2 group. This study enhances understanding of recombinant ASFV evolution through homologous recombination and identifies new genetic markers for improved detection and characterization. The observed genetic diversity highlights challenges for existing diagnostic methods and vaccine development, emphasizing the need for continued surveillance and research into the functional implications of these genetic variations on ASFV pathogenicity and transmissibility.

Rapid detection of African swine fever virus via SERS probe-modified sandwich hybridization assay.

Rapid and reliable detection method for African swine fever virus (ASFV) is proposed by surface-enhanced Raman spectroscopy (SERS). The ASFV target DNA can be specifically captured by sandwich hybridization between nanomagnetic beads and a SERS probe. Experimental results show that the significant Raman signal of the SERS probe with gold nanoparticles and a molecular reporter DTNB (5,5'-dimercapto-bis (2-nitrobenzoic acid)) can be adopted for detecting the hybridization chain reaction of ASFV DNA. The advantage of the SERS sandwich hybridization assay is the large response range from the single molecule level to 108 copies per mL, which not only can overcome the tedious time required for the amplification reaction but also provides a comparative method to polymerase chain reaction. Furthermore, real samples of African swine fever virus were detected from different subjects of swine fever virus including porcine reproductive respiratory syndrome virus and Japanese encephalitis virus. The proposed biosensor method can rapidly detect ASFV correctly within 15 min as a simple, convenient, low-cost detection approach. The biosensor can be used as a platform for the determination in biological, food, and environmental analytical fields.

Validation of a direct multiplex real-time reverse transcription PCR assay for rapid detection of African swine fever virus using swine field samples in Vietnam.

OBJECTIVE: This study validates a direct multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay which was previously established for enabling rapid and simultaneous detection of African swine fever (ASF) virus (ASFV) and classical swine fever virus. The assay eliminates the need for viral nucleic acid purification using a buffer system for crude extraction and an impurity-tolerant enzyme. However, the assay had not yet been validated using field samples of ASFV-infected pigs. Therefore, to address this gap, we tested 101 samples collected from pigs in Vietnam during 2018 and 2021 for validation. RESULTS: The rRT-PCR assay demonstrated a diagnostic sensitivity of 98.8% and a specificity of 100%. Remarkably, crude samples yielded results comparable to those of purified samples, indicating the feasibility of using crude samples without compromising accuracy in ASFV detection. Our findings emphasize the effectiveness of the rRT-PCR assay for the prompt and accurate diagnosis of both swine fever viruses, which is essential for effective disease prevention and control in swine populations.

Multigenic family 110 (1 L-5-6 L) of African swine fever virus modulate cytokine genes expression in vitro.

BACKGROUND: African swine fever (ASF) is a viral disease that affects pigs and wild boars providing economic burden in swine industry. METHODS AND RESULTS: In this study, we investigated the effect of deleting the ASFV multigene family 110 (MGF110) fragment (1 L-5-6 L) on apoptosis modulation and the expression of proinflammatory cytokines. Gene expression in swine peripheral blood macrophages infected with either the parental "Volgograd/14c" strain or the gene-deleted "Volgograd/D(1L-5-6L) MGF110" strain was analyzed. Caspase-3 activity was 1.15 times higher in macrophages infected with the parental ASFV strain compared to the gene-deleted strain. Gene expression analysis of Caspase-3 (Cas-3), Interferon-A (IFN-A), Tumor Necrosis Factor A (TNF-A), B-cell Lymphoma-2 (Bcl-2), Nuclear Factor Kappa B (NF-kB), Interleukin-12 (IL-12), and Heat Shock Protein-70 (HSP-70) using RT-qPCR at various time points after infection revealed significant differences in expression profiles between the strains. The peak expression of cytokines (except NF-kB) occurred at 24 h post-infection with the "Volgograd/D(1L-5-6L) MGF110" strain. In samples infected with the ASFV "Volgograd/14c" strain, the most intense expression was observed at 72 and 96 h, except for Bcl-2 and NF-kB, which peaked at 6 h post-infection. The cytokine expression trend for the "Volgograd/D(1L-5-6L) MGF110" strain was more stable with higher expression values. CONCLUSION: The expression trend for the parental strain increased over time, reaching maximum values at 72 and 96 h post-infection, but the overall expression level was lower than that of the gene-deleted strain. These findings suggest that deleting the multigene family 110 members (1 L-5-6 L) contributes to ASFV attenuation without affecting virus replication kinetics.

Cryo-EM structure of DNA polymerase of African swine fever virus.

African swine fever virus (ASFV) is one of the most important causative agents of animal diseases and can cause highly fatal diseases in swine. ASFV DNA polymerase (DNAPol) is responsible for genome replication and highly conserved in all viral genotypes showing an ideal target for drug development. Here, we systematically determined the structures of ASFV DNAPol in apo, replicating and editing states. Structural analysis revealed that ASFV DNAPol had a classical right-handed structure and showed the highest similarity to the structure of human polymerase delta. Intriguingly, ASFV DNAPol has a much longer fingers subdomain, and the thumb and palm subdomain form a unique interaction that has never been seen. Mutagenesis work revealed that the loss of this unique interaction decreased the enzymatic activity. We also found that the ß-hairpin of ASFV DNAPol is located below the template strand in the editing state, which is different from the editing structures of other known B family DNAPols with the ß-hairpin above the template strand. It suggests that B family DNAPols have evolved two ways to facilitate the dsDNA unwinding during the transition from replicating into editing state. These findings figured out the working mechanism of ASFV DNAPol and will provide a critical structural basis for the development of antiviral drugs.

A novel live DNA tagging system for African swine fever virus shows that bisbenzimide Hoechst 33342 can effectively block its replication.

African swine fever virus (ASFV) infection causes a frequently fatal disease in domestic swine that has affected more than 50 countries worldwide since 2021, with a major impact on animal welfare and economy. The development of effective vaccines or antivirals against this disease are urgently required for its effective control. Live detection of viral replication has been used as a tool for the screening and characterization of antiviral compounds in other dsDNA genome containing viruses. Here, we have adapted the ANCHOR fluorescent DNA labelling system to ASFV by constructing and characterizing a novel recombinant virus. We show that this virus is viable and effectively tags viral DNA replication sites, which can be detected and quantified in real time. Further, we have used high content cell microscopy to test the antiviral activity of bisbenzimide compounds and show that Hoechst 33342 has specific anti-ASFV activity. We expect this novel tool to be useful both in the further study of ASFV replication as in the screening of new specific antiviral compounds.

Multiplexed CRISPR-Cas system targeting ASFV genes in vivo: solution lies within.

The emergence and spread of the African swine fever virus (ASFV) posed a significant threat to the global swine breeding industry, calling for innovative approaches benefiting viral containment and control. A recent study (Z. Zheng, L. Xu, H. Dou, Y. Zhou, X., et al., Microbiol Spectr 12: e02164-23, 2024, https://doi.org/10.1128/spectrum.02164-23) established a multiplexed CRISPR-Cas system targeting the genome of ASFV and tested the consequent antiviral activity both in vitro and in vivo. Application of this system showed a significant reduction of viral replication in vitro, while the germline-edited pigs expressing this system exhibited normal growth with continuous guide RNA expression. Although no survival advantage was observed upon ASFV challenge compared with nonengineered pigs, this marks the first attempt of germline editing to pursue ASFV resistance and paves the way for future disease-resistant animal breeding approaches utilizing CRISPR-Cas technology.

Analysis of the Unique Historical Isolate of African Swine Fever Virus Isolate Spencer from Outbreaks in 1951.

African swine fever (ASF) is a deadly hemorrhagic disease of domestic and wild swine that was first described in the early 20th century after the introduction of European pigs to Kenya. The etiological agent, the African swine fever virus (ASFV), is a large DNA virus within the Asfarviridae family that is broadly categorized epidemiologically into genotypes based on the nucleotide sequence of B646L, the gene encoding the major capsid protein p72. ASF outbreaks in Africa have been linked historically to 25 genotypes by p72 nucleotide analysis and, recently, to 6 genotypes by amino acid comparison, whereas global outbreaks of ASF outside of Africa have only been linked to 2 genotypes: genotype I, which led to an outbreak in Europe during the 1960s that later spread to South America, and genotype II, responsible for the current pandemic that began in Georgia in 2007 and has since spread to Europe, Asia, and Hispaniola. Here, we present an analysis of the genome of ASFV Spencer, an isolate that was collected in 1951 near Johannesburg, South Africa. While nucleotide analysis of Spencer indicates the p72 coding sequence is unique, differentiating from the closest reference by five nucleotides, the predicted amino acid sequence indicates that it is 100% homologous to contemporary genotype 1. Full genome analysis reveals it is more similar to Mkuzi1979 and encodes genes that share similarity with either genotype 1 or genotype 2 outbreak strains.

Genetic Characterization of African Swine Fever Italian Clusters in the 2022-2023 Epidemic Wave by a Multi-Gene Approach.

The first report of African swine fever virus (ASFV) genotype II in Italy in 2022 marked the beginning of a significant invasion in at least eight Italian regions with different infection clusters. In this study, we used the multi-gene approach to investigate the epidemiological associations between ASFV strains causing cases and outbreaks in wild boar and pigs in Italy from January 2022 to the end of 2023. Our results confirm that all the tested ASFV-positive Italian samples belonged to genotype II and show high homology with genotype II ASFV sequences previously collected in Eurasian countries. Molecular characterization revealed the presence of four genetic groups in Italy. The majority of African swine fever (ASF) samples analyzed in the current study (72%) belonged to genetic group 3, which was the most representative in Europe. The results also provide evidence of the prevalence of genetic group 19 (15.9%). In addition, we identified new putative genetic groups, genetic group 25 (9.1%) and genetic group 26 (3.0%), which have never been described before. This is the first detailed report on the molecular characterization of more than 130 ASFV strains circulating in Italy.

Identification and Characterization of a Novel B Cell Epitope of ASFV Virulence Protein B125R Monoclonal Antibody.

The African swine fever virus (ASFV) is an ancient, structurally complex, double-stranded DNA virus that causes African swine fever. Since its discovery in Kenya and Africa in 1921, no effective vaccine or antiviral strategy has been developed. Therefore, the selection of more suitable vaccines or antiviral targets is the top priority to solve the African swine fever virus problem. B125R, one of the virulence genes of ASFV, encodes a non-structural protein (pB125R), which is important in ASFV infection. However, the epitope of pB125R is not well characterized at present. We observed that pB125R is specifically recognized by inactivated ASFV-positive sera, suggesting that it has the potential to act as a protective antigen against ASFV infection. Elucidation of the antigenic epitope within pB125R could facilitate the development of an epitope-based vaccine targeting ASFV. In this study, two strains of monoclonal antibodies (mAbs) against pB125R were produced by using the B cell hybridoma technique, named 9G11 and 15A9. The antigenic epitope recognized by mAb 9G11 was precisely located by using a series of truncated ASFV pB125R. The 52DPLASQRDIYY62 (epitope on ASFV pB125R) was the smallest epitope recognized by mAb 9G11 and this epitope was highly conserved among different strains. The key amino acid sites were identified as D52, Q57, R58, and Y62 by the single-point mutation of 11 amino acids of the epitope by alanine scanning. In addition, the immunological effects of the epitope (pB125R-DY) against 9G11 were evaluated in mice, and the results showed that both full-length pB125R and the epitope pB125R-DY could induce effective humoral and cellular immune responses in mice. The mAbs obtained in this study reacted with the eukaryotic-expressed antigen proteins and the PAM cell samples infected with ASFV, indicating that the mAb can be used as a good tool for the detection of ASFV antigen infection. The B cell epitopes identified in this study provide a fundamental basis for the research and development of epitope-based vaccines against ASFV.

A Retrospective Analysis Reveals That the 2021 Outbreaks of African Swine Fever Virus in Ghana Were Caused by Two Distinct Genotypes.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a highly infectious and lethal disease of domesticated swine. Outbreaks of ASF have been mostly restricted to the continent of Africa. The outbreaks that have occurred outside of Africa were controlled by extensive depopulation of the domesticated pig population. However, in 2007, an outbreak occurred in the country of Georgia, where ASFV infected wild pigs and quickly spread across eastern Europe. Since the reintroduction of ASF into Europe, variants of the current pandemic strain, ASFV Georgia 2007/01 (ASFV-G), which is classified as Genotype 2 based on p72 sequencing, have been reported in countries within western Europe, Asia, and the island of Hispaniola. Additionally, isolates collected in 2020 confirmed the presence of variants of ASFV-G in Nigeria. Recently, we reported similar variants of ASFV-G collected from domestic pigs suspected of dying of ASF in Ghana in 2022. Here, we retroactively report, based on full-length sequencing, that similar variants were present in Ghana in 2021. The SNP analysis revealed derivatives of ASFV with distinct genetic markers. Furthermore, we identified three full-length ASFV genomes as Genotype 1, indicating that there were two genotypes circulating in proximity during the 2021 ASF outbreaks in Ghana.

A Standardized Pipeline for Assembly and Annotation of African Swine Fever Virus Genome.

Obtaining a complete good-quality sequence and annotation for the long double-stranded DNA genome of the African swine fever virus (ASFV) from next-generation sequencing (NGS) technology has proven difficult, despite the increasing availability of reference genome sequences and the increasing affordability of NGS. A gap analysis conducted by the global African swine fever research alliance (GARA) partners identified that a standardized, automatic pipeline for NGS analysis was urgently needed, particularly for new outbreak strains. Whilst there are several diagnostic and research labs worldwide that collect isolates of the ASFV from outbreaks, many do not have the capability to analyze, annotate, and format NGS data from outbreaks for submission to NCBI, and some publicly available ASFV genomes have missing or incorrect annotations. We developed an automated, standardized pipeline for the analysis of NGS reads that directly provides users with assemblies and annotations formatted for their submission to NCBI. This pipeline is freely available on GitHub and has been tested through the GARA partners by examining two previously sequenced ASFV genomes; this study also aimed to assess the accuracy and limitations of two strategies present within the pipeline: reference-based (Illumina reads) and de novo assembly (Illumina and Nanopore reads) strategies.

Spleen Swabs for Sensitive and High-Throughput Detection of African Swine Fever Virus by Real-Time PCR.

African swine fever (ASF) continues to spread in Africa, Europe, Asia and the island of Hispaniola, increasing the need to develop more streamlined and highly efficient surveillance and diagnostic capabilities. One way to achieve this is by further optimization of already established standard operating procedures to remove bottlenecks for high-throughput screening. Real-time polymerase chain reaction (real-time PCR) is the most sensitive and specific assay available for the early detection of the ASF virus (ASFV) genome, but it requires high-quality nucleic acid extracted from the samples. Whole blood from live pigs and spleen tissue from dead pigs are the preferred samples for real-time PCR. Whole blood can be used as is in nucleic acid extractions, but spleen tissues require an additional homogenization step. In this study, we compared the homogenates and swabs prepared from 52 spleen samples collected from pigs experimentally inoculated with highly and moderately virulent ASF virus strains. The results show that not only are the spleen swabs more sensitive when executed with a low-cell-count nucleic acid extraction procedure followed by real-time PCR assays but they also increase the ability to isolate ASFV from positive spleen samples. Swabbing is a convenient, simpler and less time-consuming alternative to tissue homogenization. Hence, we recommend spleen swabs over tissue homogenates for high-throughput detection of ASFV by real-time PCR.

A Non-Hemadsorbing Live-Attenuated Virus Vaccine Candidate Protects Pigs against the Contemporary Pandemic Genotype II African Swine Fever Virus.

African swine fever (ASF) is a highly contagious and severe hemorrhagic transboundary swine viral disease with up to a 100% mortality rate, which leads to a tremendous socio-economic loss worldwide. The lack of safe and efficacious ASF vaccines is the greatest challenge in the prevention and control of ASF. In this study, we generated a safe and effective live-attenuated virus (LAV) vaccine candidate VNUA-ASFV-LAVL3 by serially passaging a virulent genotype II strain (VNUA-ASFV-L2) in an immortalized porcine alveolar macrophage cell line (3D4/21, 50 passages). VNUA-ASFV-LAVL3 lost its hemadsorption ability but maintained comparable growth kinetics in 3D4/21 cells to that of the parental strain. Notably, it exhibited significant attenuation of virulence in pigs across different doses (103, 104, and 105 TCID50). All vaccinated pigs remained healthy with no clinical signs of African swine fever virus (ASFV) infection throughout the 28-day observation period of immunization. VNUA-ASFV-LAVL3 was efficiently cleared from the blood at 14-17 days post-infection, even at the highest dose (105 TCID50). Importantly, the attenuation observed in vivo did not compromise the ability of VNUA-ASFV-LAVL3 to induce protective immunity. Vaccination with VNUA-ASFV-LAVL3 elicited robust humoral and cellular immune responses in pigs, achieving 100% protection against a lethal wild-type ASFV (genotype II) challenge at all tested doses (103, 104, and 105 TCID50). Furthermore, a single vaccination (104 TCID50) provided protection for up to 2 months. These findings suggest that VNUA-ASFV-LAVL3 can be utilized as a promising safe and efficacious LAV candidate against the contemporary pandemic genotype II ASFV.

A Low-Cost Handheld Centrifugal Microfluidic System for Multiplexed Visual Detection Based on Isothermal Amplification.

A low-cost, handheld centrifugal microfluidic system for multiplexed visual detection based on recombinase polymerase amplification (RPA) was developed. A concise centrifugal microfluidic chip featuring four reaction units was developed to run multiplexed RPA amplification in parallel. Additionally, a significantly shrunk-size and cost-effective handheld companion device was developed, incorporating heating, optical, rotation, and sensing modules, to perform multiplexed amplification and visual detection. After one-time sample loading, the metered sample was equally distributed into four separate reactors with high-speed centrifugation. Non-contact heating was adopted for isothermal amplification. A tiny DC motor on top of the chip was used to drive steel beads inside reactors for active mixing. Another small DC motor, which was controlled by an elaborate locking strategy based on magnetic sensing, was adopted for centrifugation and positioning. Visual fluorescence detection was optimized from different sides, including material, surface properties, excitation light, and optical filters. With fluorescence intensity-based visual detection, the detection results could be directly observed through the eyes or with a smartphone. As a proof of concept, the handheld device could detect multiple targets, e.g., different genes of African swine fever virus (ASFV) with the comparable LOD (limit of detection) of 75 copies/test compared to the tube-based RPA.

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.

Structures of African swine fever virus topoisomerase complex and their implications.

African swine fever virus (ASFV) is the causal agent of African swine fever (ASF), which is contagious and highly lethal to domestic pigs and wild boars. The genome of ASFV encodes many proteins important for ASFV life cycle. The functional importance of topoisomerase AsfvTopII has been confirmed by in vivo and in vitro assays, but the structure of AsfvTopII is poorly studied. Here, we report four AsfvTopII complex structures. The ATPase domain structures reveal the detailed basis for ATP binding and hydrolysis, which is shared by AsfvTopII and eukaryotic TopIIs. The DNA-bound structures show that AsfvTopII follows conserved mechanism in G-DNA binding and cleavage. Besides G-DNA, a T-DNA fragment is also captured in one AsfvTopII structure. Mutagenesis and in vitro assays confirm that Pro852 and the T-DNA-binding residue Tyr744 are important for the function of AsfvTopII. Our study not only advances the understanding on the biological function of AsfvTopII, but also provides a solid basis for the development of AsfvTopII-specific inhibitors.

The role of gastropods in African swine fever virus ecology.

The spread of the African swine fever virus (ASF virus) genotype ii in the Eurasian region has been very successful and often inexplicable. The virus spreads rapidly and persists in areas with wild boar populations, but areas without feral pig populations are also affected. The virus has shown the ability to survive for a long time in the environment without a population of susceptible hosts, both pigs and Ornithodoros soft ticks. Published data indicated that ASF viruses persist significantly longer in an environment with some freshwater snails (especially Pomacea bridgesii, Tarebia granifera, Asolene spixii, Melanoides tuberculate, and Physa fontinalis), compared to freshwater without snails. Data obtained in this study suggest that gastropods theoretically can be the hosts of the ASF virus. Also, we have proven the possibility of long-term existence of an infectious virus when infected in vitro.

Assessment of reference genes for qRT-PCR normalization to elucidate host response to African swine fever infection.

Viral infection disrupts the normal regulation of the host gene's expression. In order to normalise the expression of dysregulated host genes upon virus infection, analysis of stable reference housekeeping genes using quantitative real-time-PCR (qRT-PCR) is necessary. In the present study, healthy and African swine fever virus (ASFV) infected porcine tissues were assessed for the expression stability of five widely used housekeeping genes (HPRT1, B2M, 18 S rRNA, PGK1 and H3F3A) as reference genes using standard algorithm. Total RNA from each tissue sample (lymph node, spleen, kidney, heart and liver) from healthy and ASFV-infected pigs was extracted and subsequently cDNA was synthesized, and subjected to qRT-PCR. Stability analysis of reference genes expression was performed using the Comparative delta CT, geNorm, BestKeeper and NormFinder algorithm available at RefFinder for the different groups. Direct Cycle threshold (CT) values of samples were used as an input for the web-based tool RefFinder. HPRT1 in spleen, 18 S rRNA in liver and kidney and H3F3A in heart and lymph nodes were found to be stable in the individual healthy tissue group (group A). The majority of the ASFV-infected organs (liver, kidney, heart, lymph node) exhibited H3F3A as stable reference gene with the exception of the ASFV-infected spleen, where HPRT1 was found to be the stable gene (group B). HPRT1 was found to be stable in all combinations of all CT values of both healthy and ASFV-infected porcine tissues (group C). Of five different reference genes investigated for their stability in qPCR analysis, the present study revealed that the 18 S rRNA, H3F3A and HPRT1 genes were optimal reference genes in healthy and ASFV-infected different porcine tissue samples. The study revealed the stable reference genes found in healthy as well as ASF-infected pigs and these reference genes identified through this study will form the baseline data which will be very useful in future investigations on gene expression in ASFV-infected pigs.