African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a fatal hemorrhagic disease of domestic pigs and wild boar. The virus primarily infects macrophage and monocyte host cells, these do not grow in vitro. Many attempts have been made to establish sustainable ASFV-sensitive cell lines, but which supported only low viral replication levels of limited, mostly artificially attenuated strains of ASFV. Here, we examined the competence of a novel cell line of immortalized porcine kidney macrophages (IPKM) for ASFV infection. We demonstrated that IPKM cells can facilitate high levels (> 10^7.0 TCID₅₀/mL) of viral replication of ASFV, and hemadsorption reactions and cytopathic effects were observed as with porcine alveolar macrophages when inoculated with virulent field isolates: Armenia07, Kenya05/Tk-1, and Espana75. These results suggested that IPKM may be a valuable tool for the isolation, replication, and genetic manipulation of ASFV in both basic and applied ASF research.

African swine fever (ASF) and classical swine fever (CSF) are contagious swine diseases that are clinically indistinguishable from each other; hence, reliable test methods for accurate diagnosis and differentiation are highly demanded. By employing a buffer system suitable for crude extraction of nucleic acids together with an impurity-tolerant enzyme, we established a multiplex assay of real-time reverse-transcription polymerase chain reaction (rRT-PCR) for simultaneous detection of ASF virus (ASFV), CSF virus (CSFV) and swine internal control derived genes in a sample without the need for prior purification of viral nucleic acids. We applied this method to test serum and tissue samples of infected pigs and wild boars and compared the statistical sensitivities and specificities with those of standard molecular diagnostic methods. When a serum was used as a test material, the newly established assay showed 94.4% sensitivity for both and 97.9 and 91.9% specificity for ASFV and CSFV detection, respectively. In contrast, the results were 100% identical with those obtained by the standard methods when a crude tissue homogenate was used as a test material. The present data indicate that this new assay offers a practical, quick, and reliable technique for differential diagnosis of ASF and CSF where geographical occurrences are increasingly overlapping.

African swine fever (ASF) is an infectious Suidae disease caused by the ASF virus (ASFV). Adaptation to less susceptible, non-target host cells is one of the most common techniques used to attenuate virulent viruses. However, this may induce many mutations and large-scale rearrangements in the viral genome, resulting in immunostimulatory potential loss of the virus in vivo. This study continuously maintained the virulent ASFV strain, Armenia2007 (Arm07), to establish an attenuated ASFV strain with minimum genetic alteration in a susceptible host cell line, immortalized porcine kidney macrophage (IPKM). A mutant strain was successfully isolated via repeated plaque purification in combination with next-generation sequencing analysis. The isolated strain, Arm07ΔMGF, which was obtained from a viral fluid at a passage level of 20, lacked 11 genes in total in the MGF300 and MGF360 regions and showed marked reduction in virulence against pigs. Moreover, all the pigs survived the challenge with the parental strain when pigs were immunized twice with 10⁵ TCID₅₀ of Arm07ΔMGF, although viremia and fever were not completely prevented after the challenge infection. These findings suggest that this naturally attenuated, spontaneously occurring ASFV strain may provide a novel platform for ASF vaccine development.

African swine fever is a hemorrhagic viral disease with a mortality rate of nearly 100% in pigs. Hence, it is classified as a notifiable disease by the World Organization for Animal Health. Because no field-available vaccine exists, African swine fever virus (ASFV) control and eradication solely depend on good farm biosecurity management and rapid and accurate diagnosis. In this study, we developed a new indirect serological enzyme-linked immunosorbent assay (ELISA) using recombinant p11.5 protein from ASFV as a solid-phase target antigen. The cutoffs were determined by receiver operating curve analysis performed with serum samples obtained from naïve and infected pigs. Based on the results of a commercially available serological ELISA, the relative sensitivity and specificity of our assay were 93.4% and 94.4% (N = 166; area under the curve = 0.991; 95% confidence interval = 0.982–0.999), respectively. Furthermore, to compare the performance of the serological ELISAs, we conducted the assays on a panel of sera collected from pigs and boars experimentally infected with different ASFV isolates. The results indicated the greater sensitivity of the newly developed assay and its ability to detect anti-ASFV antibodies earlier after virus inoculation.

Immortalized porcine kidney macrophage (IPKM) cells are highly susceptible to major African swine fever virus (ASFV) isolates. To clarify the compatibility of this cell line for ASFV isolation from biomaterials, animal experiments and in vitro isolation were performed. Pork products seized at international airports were subjected to virus inoculation in pigs (in vivo) and IPKM cell cultures (in vitro) to examine the viability and virulence of the contaminating viruses. Moreover, the viruses isolated using IPKM cells were inoculated into pigs to assess the virulence shift from the original materials. All pigs that were inoculated with either homogenate samples of seized pork product or IPKM-isolated ASFVs developed typical symptoms of ASF and died (or were euthanized) within the term of the animal experiments. The success rate of virus isolation in IPKM cells was comparable to that observed in porcine primary alveolar macrophage (PAM) cells. The IPKM cell line would be an ideal tool for the isolation and propagation of live ASFVs with high efficiency and enhanced usability, such as immortal, proliferative, and adhesive properties. The isolated viruses retained biologically similar characteristics to those of the original ones during isolation in vitro.

Background/Objectives: African swine fever (ASF), a fatal febrile hemorrhagic disease in domestic pigs and Eurasian wild boars, is caused by ASF virus (ASFV). ASF continues to spread across the globe, causing a significant impact on the world’s pig industry. Recently, highly virulent chimeric ASFV (chASFV) strains with recombined genomes of the p72 genotype I and II viruses have been reported in China, Vietnam and Russia. Methods: In order to understand the propensity of ASFV genome for recombination, we attempted to experimentally generate chASFVs both in vitro and in vivo employing two distinct attenuated ASFV strains: OUR T88/3 (genotype I) and AQSΔB119L (genotype II). Results: When IPKM cells were co-infected with ASFV OUR T88/3 and AQSΔB119L strains, three genetically distinct chASFV emerged. When pigs were inoculated with the individual chASFV isolates, all pigs developed acute ASF. When four pigs were co-infected with ASFV OUR T88/3 and AQSΔB119L, all of them developed acute ASF and died or were euthanized. Three chASFV strains were successfully isolated from splenic homogenates from each pig. Conclusions: Our research indicates that genotype I and II chASFV with diverse genomes can be easily generated experimentally both in vitro and in vivo.