Development of visual detection of African swine fever virus using CRISPR/LwCas13a lateral flow strip based on structural protein gene D117L.

African swine fever virus (ASFV) is a large double stranded DNA arbovirus that is highly contagious and seriously endangers domestic and wild pigs. In the past decade, African swine fever (ASF) has spread in many countries in the Caucasus, Russian Federation, Eastern Europe and Asia, causing significant losses to the pig industry. At present, there is a lack of effective vaccine and treatment for ASF. Therefore, the rapid and accurate detection is crucial for ASF prevention and control. In this study, we have developed a portable lateral flow strip (LFS) detection mediated by recombinase polymerase amplification (RPA) and CRISPR/LwCas13a, which is performed at 37 ℃ and visualized by eyes without the need for complex instruments. This RPA-LwCas13a-LFS is based on the ASFV structural protein p17 gene (D117L), with a detection sensitivity up to 2 gene copies. This method is highly specific and has no cross reactivity to 7 other pig viruses. In the detection of two batches of 100 clinical samples, the p17 (D117L) RPA-LwCas13a-LFS had 100% coincidence with conventional quantitative PCR (qPCR). These findings demonstrate the potential of this simple, rapid, sensitive, and specific ASFV detection method for on-site ASFV detection.

Development of a Recombinase-aided Amplification Combined With Lateral Flow Dipstick Assay for the Rapid Detection of the African Swine Fever Virus.

OBJECTIVE: To establish a sensitive, simple and rapid detection method for African swine fever virus (ASFV) B646L gene. METHODS: A recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay was developed in this study. Recombinase-aided amplification (RAA) is used to amplify template DNA, and lateral flow dipstick (LFD) is used to interpret the results after the amplification is completed. The lower limits of detection and specificity of the RAA assay were verified using recombinant plasmid and pathogenic nucleic acid. In addition, 30 clinical samples were tested to evaluate the performance of the RAA assay. RESULTS: The RAA-LFD assay was completed within 15 min at 37 °C, including 10 min for nucleic acid amplification and 5 minutes for LFD reading results. The detection limit of this assay was found to be 200 copies per reaction. And there was no cross-reactivity with other swine viruses. CONCLUSION: A highly sensitive, specific, and simple RAA-LFD method was developed for the rapid detection of the ASFV.

An immortalized porcine macrophage cell line competent for the isolation of African swine fever virus.

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 (> 107.0 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.

Stability of African Swine Fever Virus in Carcasses of Domestic Pigs and Wild Boar Experimentally Infected with the ASFV "Estonia 2014" Isolate.

Europe is currently experiencing a long-lasting African swine fever (ASF) epidemic, both in domestic pigs and wild boar. There is great concern that carcasses of infected wild boar may act as long-term virus reservoirs in the environment. We evaluated the tenacity of ASF virus (ASFV) in tissues and body fluids from experimentally infected domestic pigs and wild boar, which were stored on different matrices and at different temperatures. Samples were analysed at regular intervals for viral genome and infectious virus. ASFV was most stable in spleen or muscles stored at -20 °C and in blood stored at 4 °C. In bones stored at -20 °C, infectious virus was detected for up to three months, and at 4 °C for up to one month, while at room temperature (RT), no infectious virus could be recovered after one week. Skin stored at -20 °C, 4 °C and RT remained infectious for up to three, six and three months, respectively. In urine and faeces, no infectious virus was recovered after one week, irrespective of the matrix. In conclusion, tissues and organs from decomposing carcasses that persist in the environment for a long time can be a source of infection for several months, especially at low temperatures.

X69R Is a Non-Essential Gene That, When Deleted from African Swine Fever, Does Not Affect Virulence in Swine.

African swine fever virus (ASFV) is currently causing devastating outbreaks in Asia and Europe, and the ASFV strain Georgia (ASFV-G) is responsible for these outbreaks. ASFV-G is highly virulent and continues to be maintained in these outbreak areas, apparently without suffering significant genomic or phenotypic changes. When comparing the genome of ASFV-G to other isolates, a thus-far uncharacterized gene, X69R, is highly conserved and, interestingly, is similar to another ASFV uncharacterized gene, J64R. All sequenced ASFV isolates have one or both of these genes, X69R or J64R, suggesting that the presence of at least one of these genes may be necessary for ASFV replication and or virulence. The X69R gene is present in the ASFV-G genome while J64R is absent. To assess the importance of X69R in ASFV-G functionality, we developed a recombinant virus by deleting the X69R gene from the ASFV-G genome (ASFV-G-ΔX69R). ASFV-G-ΔX69R had the same replication kinetics in primary swine macrophage cultures as the parental ASFV-G, indicating that the X69R gene is not essential for ASFV-G viability or efficient replication in the main target cell during in vivo infection. In addition, swine intramuscularly inoculated with a low dose (102 HAD50) of ASFV-G-ΔX69R developed a clinical disease indistinguishable from that induced by the same dose of the virulent parental ASFV-G isolate. Viremia values of ASFV-G-ΔX69R did not significantly differ from those detected in animals infected with parental virus. Therefore, deletion of the X69R gene from ASFV-G does not affect virus replication or virulence in swine.

Identification of a Continuously Stable and Commercially Available Cell Line for the Identification of Infectious African Swine Fever Virus in Clinical Samples.

African swine fever virus (ASFV) is causing outbreaks both in domestic pigs and wild boar in Europe and Asia. In 2018, the largest pig producing country, China, reported its first outbreak of African swine fever (ASF). Since then, the disease has quickly spread to all provinces in China and to other countries in southeast Asia, and most recently to India. Outbreaks of the disease occur in Europe as far west as Poland, and one isolated outbreak has been reported in Belgium. The current outbreak strain is highly contagious and can cause a high degree of lethality in domestic pigs, leading to widespread and costly losses to the industry. Currently, detection of infectious ASFV in field clinical samples requires accessibility to primary swine macrophage cultures, which are infrequently available in most regional veterinary diagnostic laboratories. Here, we report the identification of a commercially available cell line, MA-104, as a suitable substrate for virus isolation of African swine fever virus.

A porcine macrophage cell line that supports high levels of replication of OURT88/3, an attenuated strain of African swine fever virus.

The main target cells for African swine fever virus (ASFV) replication in pigs are of monocyte macrophage lineage and express markers typical of the intermediate to late stages of differentiation. The lack of a porcine cell line, which accurately represents these target cells, limits research on virus host interactions and the development of live-attenuated vaccine strains. We show here that the continuously growing, growth factor dependent ZMAC-4 porcine macrophage cell line is susceptible to infection with eight different field isolates of ASFV. Replication in ZMAC-4 cells occurred with similar kinetics and to similar high titres as in primary porcine bone marrow cells. In addition we showed that twelve passages of an attenuated strain of ASFV, OURT88/3, in ZMAC-4 cells did not reduce the ability of this virus to induce protection against challenge with virulent virus. Thus, the ZMAC-4 cells provide an alternative to primary cells for ASFV replication.

Evaluation in Swine of a Recombinant Georgia 2010 African Swine Fever Virus Lacking the I8L Gene.

African swine fever virus (ASFV) is the causative agent of African swine fever, a disease currently causing significant economic losses in Europe and Asia. Specifically, the highly virulent ASFV strain Georgia 2010 (ASFV-G) is producing disease outbreaks in this large geographical region. The ASFV genome encodes for over 150 genes, most of which are still not experimentally characterized. I8L is a highly conserved gene that has not been studied beyond its initial description as a virus ORF. Transcriptional analysis of swine macrophages infected with ASFV-G demonstrated that the I8L gene is transcribed early during the virus replication cycle. To assess the importance of I8L during ASFV-G replication in vitro and in vivo, as well as its role in virus virulence in domestic swine, we developed a recombinant virus lacking the I8L gene (ASFV-G-ΔI8L). Replication of ASFV-G-ΔI8L was similar to parental ASFV-G replication in primary swine macrophage cultures, suggesting that the I8L gene is not essential for ASFV-G replication in vitro. Similarly, replication of ASFV-G-ΔI8L in swine intramuscularly inoculated with 102 HAD50 displayed replication kinetics similar to ASFV-G. In addition, animals inoculated with ASFV-G-ΔI8L presented with a clinical disease indistinguishable from that induced by the same dose of the virulent parental ASFV-G isolate. We conclude that deletion of the I8L gene from ASFV-G does not affect virus replication in vitro or in vivo, nor changes the disease outcome in swine.

Pathogenesis of African swine fever in domestic pigs and European wild boar - Lessons learned from recent animal trials.

Over the last decade, African swine fever (ASF) has changed from an exotic disease of Sub-Saharan Africa to a considerable and serious threat to pig industry in Central Europe and Asia. With the introduction of genotype II strains into the European Union in 2014, the disease has apparently found a fertile breeding ground in the abundant wild boar population. Upon infection with highly virulent ASF virus (ASFV), a haemorrhagic fever like illness with high lethality is seen in naïve domestic pigs and wild boar. Despite intensive research, virulence factors, host-virus interactions and pathogenesis are still far from being understood, and neither vaccines nor treatment exist. However, to better understand the disease, and to work towards a safe and efficacious vaccine, this information is needed. The presented review targets the knowledge gained over the last five years with regard to ASF pathogenesis in the broader sense but with a focus on the pandemic genotype II strains. In this way, it is designed as an update and supplement to existing review articles on the same topic.

Interaction of porcine monocyte-derived dendritic cells with African swine fever viruses of diverse virulence.

African swine fever (ASF) is a devastating disease for which there is no vaccine. The ASF virus (ASFV) can infect dendritic cell (DC), but despite the critical role these cells play in induction of adaptive immunity, few studies investigated their response to ASFV infection. We characterized the in vitro interactions of porcine monocyte-derived DCs (moDC) with a virulent (22653/14), a low virulent (NH/P68) and an avirulent (BA71V) ASFV strain. At a high multiplicity of infection (MOI = 1), all three strains infected immature moDC. Maturation of moDC, with IFN-α/TNF-α, increased susceptibility to infection with 22653/14 and other virulent strains, but reduced susceptibility to NH/P68 and BA71V. The reduced moDC susceptibility to BA71V/NH/P68 was IFN-α mediated, whereas increased susceptibility to 22653/14 was induced by TNF-α. Using an MOI of 0.01, we observed that BA71V replicated less efficiently in moDC compared to the other isolates and we detected increased replication of NH/P68 compared to 22653/14. We observed that BA71V and NH/P68, but not 22653/14, downregulated expression of MHC class I on infected cells. All three strains decreased CD16 expression on moDC, whereas ASFV infection resulted in CD80/86 down-regulation and MHC class II DR up-regulation on mature moDC. None of the tested strains induced a strong cytokine response to ASFV and only modest IL-1α was released after BA71V infection. Overall our results revealed differences between strains and suggest that ASFV has evolved mechanisms to replicate covertly in inflammatory DC, which likely impairs the induction of an effective immune response.

Genetic Characterization of Circulating African Swine Fever Viruses in Nigeria (2007-2015).

Sequencing and analysis of three discrete genome regions of African swine fever viruses (ASFV) from archival samples collected in 2007-2011 and active and passive surveillance between 2012 and 2015 in Nigeria were carried out. Analysis was conducted by genotyping of three single-copy African swine fever (ASF) genes. The E183L and B646L genes that encode structural proteins p54 and p72, respectively, were utilized to delineate genotypes before intragenotypic resolution by characterization of the tetrameric amino acid repeat region within the hypervariable central variable region of the B602L gene. The results showed no variation in the p72 and p54 gene regions sequenced. Phylogeny of p72 sequences revealed that all the Nigerian isolates belonged to genotype I, while that of the p54 recovered the Ia genotype. Analysis of B602L gene revealed the differences in the number of tetrameric repeats. Four new variants (Tet-15, Tet-17a, Tet-17b and Tet-48) were recovered, while a fifth variant (Tet-20) was the most widely distributed in the country displacing Tet-36 reported previously in 2003-2006. The viruses responsible for ASF outbreaks in Nigeria are from very closely related but mutated variants of the virus that have been circulating since 1997. A practical implication of the genetic variability of the Nigerian viral isolates in this study is the need for continuous sampling and analysis of circulating viruses, which will provide epidemiological information on the evolution of ASFV in the field versus new incursion for informed strategic control of the disease in the country.

African Swine Fever Virus Georgia Isolate Harboring Deletions of MGF360 and MGF505 Genes Is Attenuated in Swine and Confers Protection against Challenge with Virulent Parental Virus.

UNLABELLED: African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene family 360 (MGF360) and MGF505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-ΔMGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360 or MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, and MGF505-3R. ASFV-G-ΔMGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-ΔMGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 10(2) or 10(4) 50% hemadsorbing doses (HAD50) remained healthy, without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed, although a proportion of these animals harbored the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-ΔMGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G. IMPORTANCE: The main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence led to the production of genetically modified recombinant viruses that, while attenuated, are able to confer protection in pigs challenged with homologous viruses. Here we have produced an attenuated recombinant ASFV derived from highly virulent ASFV strain Georgia (ASFV-G) lacking only six of the multigene family 360 (MGF360) and MGF505 genes (ASFV-G-ΔMGF). It is demonstrated, by first time, that deleting specific MGF genes alone can completely attenuate a highly virulent field ASFV isolate. Recombinant virus ASFV-G-ΔMGF effectively confers protection in pigs against challenge with ASFV-G when delivered once via the intramuscular (i.m.) route. The protection against ASFV-G is highly effective by 28 days postvaccination. This is the first report of an experimental vaccine that induces solid protection against virulent ASFV-G.

Increase in chemokines CXCL10 and CCL2 in blood from pigs infected with high compared to low virulence African swine fever virus isolates.

Modulation of the expression of chemokines and chemokine receptors in whole blood was compared following infection of pigs with high and low virulence isolates of African swine fever virus. Levels of mRNAs for CCL2, CCL3L1, CCL4, CXCL10, CCR1 and CCR5 were significantly increased in at least one time point following infection in two experiments and CCL5, CCR9 and CXCR4 mRNA were significantly increased in one of the experiments. The results showed that greatest fold increases in mRNAs for CXCL10 and CCL2 were observed following infection of pigs. CXCL10 mRNA was increased by up to 15 fold in infected compared to uninfected pigs. CXCL10 protein was also detected in serum from pigs infected with the high virulence Benin 97/1 isolate. Levels of CCL2 mRNA were increased in pigs infected with high virulence Benin 97/1 isolate compared to low virulence OURT88/3 isolate and this correlated with an increase of greater than 30 fold in levels of CCL2 protein detected in serum from pigs infected with this isolate. An increase in overall chemotaxis active compounds in defibrinated plasma samples from Benin 97/1 infected pigs was observed at 3 days post-infection (dpi) and a decrease by 7 dpi as measured by chemotaxis assay using normal pig leucocytes in vitro. Increased levels of CXCL10 may either contribute to the activation of lymphocyte priming toward the Th1 phenotype or induction of T lymphocyte apoptosis. Increased levels of CCL2, a chemoattractant for macrophages, may result in increased recruitment of monocytes from bone marrow thus increasing the pool of cells susceptible to infection.

The effect of preprocessing by sequence-independent, single-primer amplification (SISPA) on metagenomic detection of viruses.

Compared to routine diagnostics, screening for pathogens in outbreak situations, with or without intentional release, poses demands on the detection technology to not only indicate the presence of already known causative agents but also novel and unexpected pathogens. The metagenomic approach to detecting viral pathogens, using unbiased high-throughput sequencing (HTS), is a well-established methodology with a broad detection range and wide applicability on different sample matrices. To prepare a sample for HTS, the common presequencing steps include homogenization, enrichment, separation (eg, magnetic separation), and amplification. In this initial study, we explored the benefits and drawbacks of preprocessing by sequence-independent, single-primer amplification (SISPA) of nucleic acids by applying the methodology to artificial samples. More specifically, a synthetic metagenome was divided into 2 samples, 1 unamplified and 1 diluted, and amplified by SISPA. Subsequently, both samples were sequenced using the Ion Torrent Personal Genome Machine (PGM), and the resulting datasets were analyzed by using bioinformatics, short read mapping, de novo assembly, BLAST-based taxonomic classification, and visualization. The results indicate that even though SISPA introduces a strong amplification bias, which makes it unsuitable for whole-genome sequencing, it is still useful for detecting and identifying viruses.

Modulation of chemokine and chemokine receptor expression following infection of porcine macrophages with African swine fever virus.

African swine fever virus (ASFV) is the only member of the Asfarviridae, a large DNA virus family which replicates predominantly in the cytoplasm. Most isolates cause a fatal haemorrhagic disease in domestic pigs, although some low virulence isolates cause little or no mortality. The modulation of chemokine responses following infection of porcine macrophages with low and high virulence isolates was studied to indicate how this may be involved in the induction of pathogenesis and of effective immune responses. Infection with both low and high virulence isolates resulted in down-regulation of mRNA levels for chemokines CCL2, CCL3L, CXCL2 and chemokine receptors CCR1, CCR5, CXCR3, CXCR4 and up-regulation in expression of mRNAs for CCL4, CXCL10 and chemokine receptor CCR7. Levels of CCL4, CXCL8, CXCL10 mRNAs were higher in macrophages infected with low virulence isolate OURT88/3 compared to high virulence isolate Benin 97/1. Levels of CXCL8 and CCL2 protein were significantly reduced in supernatants from macrophages infected with Benin 97/1 isolate compared to OURT88/3 and mock-infected macrophages. There was also a decreased chemotactic response of donor cells exposed to supernatants from Benin 97/1 infected macrophages compared to those from OURT88/3 and mock-infected macrophages. The data show that infection of macrophages with the low virulence strain OURT88/3 induces higher expression of key inflammatory chemokines compared to infection with high virulence strain Benin 97/1. This may be important for the induction of effective protective immunity that has been observed in pigs immunised with the OURT88/3 isolate.

Methods for growing and titrating African swine fever virus: field and laboratory samples.

Growing African swine fever virus (ASFV) isolates obtained mainly from the field, but also engineered in the laboratory, is a critical step for diagnosis, titration, or virus infection studies. This unit describes a set of methods and protocols to produce and titrate any ASFV strain in cell cultures. The procedures include (1) basic techniques to prepare virus-sensitive target cells; (2) strategies for growth, concentration, and purification of virus stocks; and (3) the semi-quantitative (end dilution) and quantitative (plaque) assays for the determination of viral titers, and the use of different ASFV-sensitive cells as targets for virus production and titration.

Intranuclear detection of African swine fever virus DNA in several cell types from formalin-fixed and paraffin-embedded tissues using a new in situ hybridisation protocol.

In this study, a new in situ hybridisation (ISH) protocol has been developed to identify African swine fever virus (ASFV) genome in formalin-fixed, paraffin-embedded tissues. Different digoxigenin labelled ASFV-probes were tested, including single ASFV-specific oligonucleotides, an 18.5kb restriction fragment from the viral genome and the entire ASFV genome. The latter showed the highest sensitivity in all tissues tested, independently of the virus used for challenge: E75L or Ba71L. Although a similar ASFV genome distribution was observed, the number of ISH-positive cells was higher for Ba71L compared to E75L infected tissues. As expected, the monocyte-macrophage cell lineage was the main target cell for ASFV infection. Corresponding with the last stages of infection, ISH-positive signals were also found in other cell types, including endothelial cells, hepatocytes and neutrophils. Furthermore, two unexpected findings were also noticed: the detection of a specific ISH-signal in lymphocytes and a tendency to find the signal in the nucleus of infected cells. In summary, the present findings demonstrate the utility of this new ISH protocol to study ASFV pathogenesis and its potential use as a diagnostic tool.

Enhanced discrimination of African swine fever virus isolates through nucleotide sequencing of the p54, p72, and pB602L (CVR) genes.

Complete sequencing of p54-gene from 67 European, American, and West and East African Swine Fever virus (ASFV) isolates revealed that West African and European ASFV isolates classified within the predominant Genotype I according to partial sequencing of p72 were discriminated into four major sub-types on the basis of their p54 sequences. This highlighted the value of p54 gene sequencing as an additional, intermediate-resolution, molecular epidemiological tool for typing of ASFV viruses. We further evaluated p54-based genotyping, in combination with partial sequences of two other genes, for determining the genetic relationships and origin of viruses responsible for disease outbreaks in Kenya. Animals from Western and central Kenya were confirmed as being infected with ASFV using a p72 gene-based PCR assay, following outbreaks of severe hemorrhagic disease in domestic pigs in 2006 and 2007. Eleven hemadsorbing viruses were isolated in macrophage culture and genotyped using a combination of full-length p54-gene sequencing, partial p72-gene sequencing, and analysis of tetrameric amino acid repeat regions within the variable region of the B602L gene (CVR). The data revealed that these isolates were identical in their p72 and p54 sequence to viruses responsible for ASF outbreaks in Uganda in 2003. There was a minor difference in the number of tetrameric repeats within the B602L sequence of the Kenyan isolates that caused the second Kenyan outbreak in 2007. A practical implication of the genetic similarity of the Kenyan and Ugandan viral isolates is that ASF control requires a regional approach.

African swine fever virus isolate, Georgia, 2007.

African swine fever (ASF) is widespread in Africa but is rarely introduced to other continents. In June 2007, ASF was confirmed in the Caucasus region of Georgia, and it has since spread to neighboring countries. DNA fragments amplified from the genome of the isolates from domestic pigs in Georgia in 2007 were sequenced and compared with other ASF virus (ASFV) isolates to establish the genotype of the virus. Sequences were obtained from 4 genome regions, including part of the gene B646L that encodes the p72 capsid protein, the complete E183L and CP204L genes, which encode the p54 and p30 proteins and the variable region of the B602L gene. Analysis of these sequences indicated that the Georgia 2007 isolate is closely related to isolates belonging to genotype II, which is circulating in Mozambique, Madagascar, and Zambia. One possibility for the spread of disease to Georgia is that pigs were fed ASFV-contaminated pork brought in on ships and, subsequently, the disease was disseminated throughout the region.

Macrophage transcriptional responses following in vitro infection with a highly virulent African swine fever virus isolate.

We used a porcine microarray containing 2,880 cDNAs to investigate the response of macrophages to infection by a virulent African swine fever virus (ASFV) isolate, Malawi LIL20/1. One hundred twenty-five targets were found to be significantly altered at either or both 4 h and 16 h postinfection compared with targets after mock infection. These targets were assigned into three groups according to their temporal expression profiles. Eighty-six targets showed increased expression levels at 4 h postinfection but returned to expression levels similar to those in mock-infected cells at 16 h postinfection. These encoded several proinflammatory cytokines and chemokines, surface proteins, and proteins involved in cell signaling and trafficking pathways. Thirty-four targets showed increased expression levels at 16 h postinfection compared to levels at 4 h postinfection and in mock-infected cells. One host gene showed increased expression levels at both 4 and 16 h postinfection compared to levels in mock-infected cells. The microarray results were validated for 12 selected genes by quantitative real-time PCR. Levels of protein expression and secretion were measured for two proinflammatory cytokines, interleukin 1beta and tumor necrosis factor alpha, during a time course of infection with either the virulent Malawi LIL20/1 isolate or the OUR T88/3 nonpathogenic isolate. The results revealed differences between these two ASFV isolates in the amounts of these cytokines secreted from infected cells.