Sequential deletion of genes from the African swine fever virus genome using the cre/loxP recombination system.
ABSTRACT: A method has been established to sequentially delete combinations of genes from the ASFV genome to test the effect on virus replication and host responses to infection. Initially the ASFV genes MGF505 2R and MGF505 3R and a truncated MGF360 9L gene were deleted from the genome of the tissue-culture adapted ASFV strain BA71V and replaced with bacteriophage loxP sequences flanking the beta-glucuronidase (GUS) marker gene to create recombinant virus V?MGF-GUS. Subsequently the GUS gene was removed by site-specific recombination between the two loxP sites involving expression of the bacteriophage Cre recombinase enzyme to create recombinant virus V?MGF?GUS. The EP402R and EP153R genes were subsequently deleted from the genome of V?MGF?GUS, using the same GUS marker gene, to construct virus V?MGF?CD2-Lectin-GUS. These sequential deletions of ASFV genes were shown not to alter virus replication significantly.
Project description:Pathogenic African swine fever virus (ASFV) isolates primarily target cells of the mononuclear-phagocytic system in infected swine and replicate efficiently in primary macrophage cell cultures in vitro. ASFVs can, however, be adapted to grow in monkey cell lines. Characterization of two cell culture-adapted viruses, MS16 and BA71V, revealed that neither virus replicated in macrophage cell cultures. Cell viability experiments and ultrastructural analysis showed that infection with these viruses resulted in early macrophage cell death, which occurred prior to viral progeny production. Genomic cosmid clones from pathogenic ASFV isolate E70 were used in marker rescue experiments to identify sequences capable of restoring MS16 and BA71V growth in macrophage cell cultures. A cosmid clone representing a 38-kbp region at the left terminus of the genome completely restored the growth of both viruses. In subsequent fine-mapping experiments, an 11-kbp subclone from this region was sufficient for complete rescue of BA71V growth. Sequence analysis indicated that both MS16 and BA71V had significant deletions in the region containing members of multigene family 360 (MGF 360) and MGF530. Deletion of this same region from highly pathogenic ASFV isolate Pr4 significantly reduced viral growth in macrophage cell cultures. These findings indicate that ASFV MGF360 and MGF530 genes perform an essential macrophage host range function(s) that involves promotion of infected-cell survival.
Project description: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.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.
Project description:The DP71L protein of African swine fever virus (ASFV) shares sequence similarity with the herpes simplex virus ICP34.5 protein over a C-terminal domain. We showed that the catalytic subunit of protein phosphatase 1 (PP1) interacts specifically with the ASFV DP71L protein in a yeast two-hybrid screen. The chimeric full-length DP71L protein, from ASFV strain Badajoz 71 (BA71V), fused to glutathione S-transferase (DP71L-GST) was expressed in Escherichia coli and shown to bind specifically to the PP1-alpha catalytic subunit expressed as a histidine fusion protein (6xHis-PP1alpha) in E. coli. The functional effects of this interaction were investigated by measuring the levels of PP1 and PP2A in ASFV-infected Vero cells. This showed that infection with wild-type ASFV strain BA71V activated PP1 between two- and threefold over that of mock-infected cells. This activation did not occur in cells infected with the BA71V isolate in which the DP71L gene had been deleted, suggesting that expression of DP71L leads to PP1 activation. In contrast, no effect was observed on the activity of PP2A following ASFV infection. We showed that infection of cells with wild-type BA71V virus resulted in decreased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha). ICP34.5 recruits PP1 to dephosphorylate the alpha subunit of eukaryotic translational initiation factor 2 (also known as eIF-2alpha); possibly the ASFV DP71L protein has a similar function.
Project description:African swine fever virus (ASFV) causes a lethal haemorrhagic disease of pigs. There are conflicting reports on the role of interferon in ASFV infection. We therefore analysed the interaction of ASFV with porcine interferon, in vivo and in vitro. Virulent ASFV induced biologically active IFN in the circulation of pigs from day 3-post infection, whereas low virulent OUR T88/3, which lacks genes from multigene family (MGF) 360 and MGF505, did not. Infection of porcine leucocytes enriched for dendritic cells, with ASFV, in vitro, induced high levels of interferon, suggesting a potential source of interferon in animals undergoing acute ASF. Replication of OUR T88/3, but not virulent viruses, was reduced in interferon pretreated macrophages and a recombinant virus lacking similar genes to those absent in OUR T88/3 was also inhibited. These findings suggest that as well as inhibiting the induction of interferon, MGF360 and MGF505 genes also enable ASFV to overcome the antiviral state.
Project description:The CD2-like African swine fever virus (ASFV) gene 8DR, (also known as EP402R) encodes for a structural transmembrane glycoprotein that has been shown to mediate hemadsorption and be involved in host immunomodulation as well as the induction of protective immune response. In addition, several natural ASFV isolates showing decreased virulence in swine has been shown to be non-hemadsorbing suggesting an association between altered or deleted forms of 8DR and virus attenuation. Here we demonstrate that deletion of 8DR gene from the genome of ASFV Georgia2010 isolate (ASFV-G-?8DR) does not significantly alter the virulence of the virus. ASFV-G-?8DR inoculated intramuscularly or intranasally (in a range of 102 to 104 TCID50) produced a clinical disease in domestic pigs indistinguishable from that induced by the same doses of the virulent parental ASFV Georgia2010 isolate. In addition, viremia values in ASFV-G-?8DR do not differ from those detected in animals infected with parental virus. Therefore, deletion of 8DR gene is not associated with a noticeable decrease in virulence of the ASFV Georgia isolate.
Project description:Introduction:African swine fever (ASF) is a pressing economic problem in a number of Eastern European countries. It has also depleted the Chinese sow population by 50%. Managing the disease relies on culling infected pigs or hunting wild boars as sanitary zone creation. The constraints on the development of an efficient vaccine are mainly the virus' mechanisms of host immune response evasion. The study aimed to adapt a field ASFV strain to established cell lines and to construct recombinant African swine fever virus (ASFV) strain. Material and Methods:The host immune response modulation genes A238L, EP402R, and 9GL were deleted using the clustered regularly interspaced short palindromic repeats/caspase 9 (CRISPR/Cas9) mutagenesis system. A representative virus isolate (Pol18/28298/Out111) from Poland was isolated in porcine primary pulmonary alveolar macrophage (PPAM) cells. Adaptation of the virus to a few established cell lines was attempted. The plasmids encoding CRISPR/Cas9 genes along with gRNA complementary to the target sequences were designed, synthesised, and transfected into ASFV-infected PPAM cells. Results:The reconstituted virus showed similar kinetics of replication in comparison to the parent virus isolate. Conclusion:Taking into account the usefulness of the developed CRISPR/Cas9 system it has been shown that modification of the A238L, EP402R, and 9GL genes might occur with low frequency, resulting in difficulties in separation of various virus populations.
Project description:African swine fever (ASF) is a devastating disease in pigs, with no vaccines for control. The genetic manipulation of African swine fever virus (ASFV) is often tedious and time consuming. Here, we describe a method to manipulate the virus genome to produce gene deletion viruses in a much-reduced time. This method combines the conventional homologous recombination with fluorescent-activated cells sorting (FACS), to isolate and purify viruses expressing fluorescent reporter genes. With three rounds of single cell isolation via FACS and two rounds of limiting dilution, we deleted two additional genes, EP153R and EP402R, from Benin 97/1 ASFV lacking the DP148R gene. By combining different fluorescent markers, this method has the potential to greatly facilitate studies on understanding ASFV gene functions and develop candidate live-attenuated vaccines.
Project description:African swine fever (ASF) is an infectious transboundary disease of domestic pigs and wild boar and spreading throughout Eurasia. There is no vaccine and treatment available. Complex immune escape strategies of African swine fever virus (ASFV) are crucial factors affecting immune prevention and vaccine development. MGF360 genes have been implicated in the modulation of the IFN-I response. The molecular mechanisms contributing to innate immunity are poorly understood. In this study, we demonstrated that ASFV MGF360-12L (MGF360 families 12L protein) significantly inhibited the mRNA transcription and promoter activity of IFN-? and NF-?B, accompanied by decreases of IRF3, STING, TBK1, ISG54, ISG56 and AP-1 mRNA transcription. Also, MGF360-12L might suppress the nuclear localization of p50 and p65 mediated by classical nuclear localization signal (NLS). Additionally, MGF360-12L could interact with KPNA2, KPNA3, and KPNA4, which interrupted the interaction between p65 and KPNA2, KPNA3, KPNA4. We further found that MGF360-12L could interfere with the NF-?B nuclear translocation by competitively inhibiting the interaction between NF-?B and nuclear transport proteins. These findings suggested that MGF360-12L could inhibit the IFN-I production by blocking the interaction of importin ? and NF-?B signaling pathway, which might reveal a novel strategy for ASFV to escape the host innate immune response.
Project description:African swine fever is a contagious and often lethal disease for domestic pigs with a significant economic impact for the swine industry. The etiological agent, African swine fever virus (ASFV), is a highly structurally complex double stranded DNA virus. No effective vaccines or antiviral treatment are currently commercially available. We present here the development of a strain of ASFV that has been shown to retain its ability to cause disease in swine, efficiently replicate in swine macrophage and that is fluorescently tagged. The insertion of an EGFP cassette replacing the reading frames for two neighboring genes, MGF360-13L and MGF360-14L, in highly virulent field isolate Georgia/2007, did not affect virus replication in cell cultures and did not affect disease progression in swine, the natural host for ASFV. A virulent fluorescently tagged ASFV is a suitable tool to conduct pathogenesis studies in swine, study on virus-macrophage interaction and to run large scale screens that require a sensitive high throughput output. Utilizing an EGFP reporter system for observing ASFV replication and infectivity can circumvent the time and labor-intensive steps associated with viral antigen-based assays such as the observation of hemadsorption or cytopathic effect.
Project description:The strain BA71V has played a key role in African swine fever virus (ASFV) research. It was the first genome sequenced, and remains the only genome completely determined. A large part of the studies on the function of ASFV genes, viral transcription, replication, DNA repair and morphogenesis, has been performed using this model. This avirulent strain was obtained by adaptation to grow in Vero cells of the highly virulent BA71 strain. We report here the analysis of the genome sequence of BA71 in comparison with that of BA71V. They possess the smallest genomes for a virulent or an attenuated ASFV, and are essentially identical except for a relatively small number of changes. We discuss the possible contribution of these changes to virulence. Analysis of the BA71 sequence allowed us to identify new similarities among ASFV proteins, and with database proteins including two ASFV proteins that could function as a two-component signaling network.