Attenuated African swine fever virus vaccine

FIELD OF THE INVENTION

The present invention relates to attenuated African Swine Fever Viruses. The engineered viruses protect pigs against subsequent challenge with virulent virus. The present invention also relates to the use of such attenuated viruses to treat and/or prevent African Swine Fever. The invention further relates to intranasal administration of attenuated African Swine Fever Viruses.

BACKGROUND TO THE INVENTION

African Swine Fever (ASF)

African swine fever is a devastating haemorrhagic disease of domestic pigs caused by a double-stranded DNA virus, African swine fever virus (ASFV). ASFV is the only member of the Asfarviridae family and replicates predominantly in the cytoplasm of cells. Virulent strains of ASFV can kill domestic pigs within about 5-14 days of infection with a mortality rate approaching 100%.

ASFV can infect and replicate in warthogs (Phacochoerus sp.), bushpigs (Potamocherus sp.) and soft ticks of the Ornithodoros species, but in these species few if any clinical signs are observed and long term persistent infections can be established. The disease is currently endemic in many sub-Saharan countries and in Europe in Sardinia. Following its introduction to Georgia in the Trans Caucasus region in 2007, ASFV has spread extensively through neighbouring countries including the Russian Federation. In 2012 the first outbreak was reported in Ukraine and in 2013 the first outbreaks in Belarus. In 2014 further outbreaks were reported in pigs in Ukraine and detection in wild boar in Lithuania and Poland.

There is currently no treatment for ASF. Prevention in countries outside Africa has been attempted on a national basis by restrictions on incoming pigs and pork products, compulsory boiling of waste animal products under license before feeding to pigs and the application of a slaughter policy when the disease is diagnosed. Prevention in Africa is based on measures to keep warthogs and materials contaminated by warthogs away from the herd.

To date, no effective attenuated or inactivated vaccines have been developed.

There is thus a need for improved measures to control ASFV infection and prevent spread of the disease.

African Swine Fever Virus (ASFV)

The ASFV genome encodes five multigene families (MGF 100, MGF 110, MGF 360 and MGF 505/530) located within the left hand 35 kb or right hand 15 kb terminal variable regions. The MGFs constitute between 17 and 25% of the total coding capacity of the ASFV genome. They lack similarity to other known genes. Although the function of individual MGF genes is unknown, it has been shown the MGF 360 and 505 families encode genes essential for host range function that involves promotion of infected-cell survival and suppression of type I interferon response.

The OURT88/3 Isolate

OURT88/3 is a non-pathogenic isolate of ASFV from Portugal. Previous infection with ASFV OURT88/3 has been shown to confer protection against challenge with related virulent viruses (Boinas et al (2004) J Gen Virol 85:2177-2187; Oura et al (2005) J. Gen. Virol. 86:2445-2450).

It has been demonstrated that CD8+ T cells are required for the protection induced by the OURT88/3 strain, since antibody mediated depletion of CD8+ T cells abrogates protection (Oura et al., 2005, as above).

Studies were carried out using the NIH inbred pig lines cc and dd. In these studies, a control group of 3 dd pigs and a control group of 6 dd pigs were immunised with OURT88/3. In the first of these experiments, following OURT88/3 immunisation one dd pig developed a transient low viremia of log 10 2-3 TCID 50/ml, but no fever or clinical signs of disease. It was observed that 3 of 6 of the cc inbred pigs immunised with OURT88/3 were not protected following lethal challenge with OURT88/3 isolate, whereas protective responses were induced in all dd pigs. These results indicate that the genetic background of the pig influences the response to OURT88/3 inoculation.

In subsequent experiments 5 dd and 5 cc pigs were immunised with OURT88/3 and challenged with OURT88/1 (Takamatsu et al., 2003 unpublished results). This confirmed that protective responses were induced in all dd pigs, but not induced in all cc pigs following immunisation with OURT88/3 and that adverse reactions including transient pyrexia, joint swelling and lameness were induced in some of the cc pigs.

Subsequent experiments were carried out in France using the Anses herd of SPF pigs. In these pigs, similar to observations with cc pigs, some pigs developed adverse reactions including transient fever, joint swelling and lameness following immunisation with OURT88/3 (unpublished results).

Although OURT88/3 has been shown to induce a protective immune response in certain animals, this effect does not appear to be universal. Immunisation with OURT88/3 appears to be ineffective in protecting some pigs from subsequent challenge. It is also associated with the induction of adverse immune responses, such as joint swelling, in some pigs.

There is therefore a need for alternative ASFV vaccine candidates with improved efficacy and safety profiles.

DESCRIPTION OF THE FIGURES

FIG. 1--Schematic diagram showing the generation of recombinant virus Benin.DELTA.MGF with the deletion of five MGF 360 genes 10L, 11L, 12L, 13L, 14L and the deletion of three MGF 505 genes 1R, 2R, 3R. Recombinant virus Benin.DELTA.MGF was created by the homologous recombination between the MGF 360 gene 9L and the MGF 505 4R gene on the wild type Benin 97/1 genome and the transfer vector plasmid p.DELTA.MGFGUS resulting in the deletion of the eight MGF genes and the insertion of the GUS marker gene.

FIG. 2--Analysis of genomic viral DNA gene deletions and insertions by PCR. Viral DNA was extracted from wild type Benin 97/1 and the recombinant virus Benin .DELTA.MGF. Specific fragments were amplified by PCR and the products were analysed on a 1% agarose TAE gel. The following primer sets were used in lane 1 (BeninD8F and BeninD8R), lanes 2 and 3 (BeninD8F and RGUS), lanes 4 and 5 (BeninD8INTF and BeninD8INT). Lane 0 contains a DNA ladder.

FIG. 3--DNA sequence analysis of recombinant virus Benin.DELTA.MGF at the deletion/insertion site compared to the published sequence of wild type Benin 97/1. Viral gDNA was isolated from Benin.DELTA.MGF infected cells and the left flank of the deletion/insertion was sequenced with primer 9LF and the right flank sequenced with primer 4RR. The left flank sequence of Benin.DELTA.MGF shows the insertion of sequences vp72 promoter, loxP and 5' GUS gene and the deletion of eight MGF genes and deletion of the first five nucleotides of the MGF 360 9L gene including the ATG start codon. The right flank sequence of Benin.DELTA.MGF shows the insertion of the 3' GUS gene and the deletion of the first seven nucleotides of the MGF 505 4R gene, including the ATG start codon.

FIG. 4--Replication kinetics of Benin 97/1 and recombinant Benin.DELTA.MGF viruses. Pig bone marrow macrophages were infected at high multiplicity of infection with parental Benin.DELTA.97/1 or recombinant Benin.DELTA.MGF viruses. At various hours post-infection, as indicated on the x-axis, total virus was harvested and infectious virus titrated on 96 well plates by analysis of haemadsorption on cultures of pig bone marrow macrophages. The virus titre (HAD.sub.50/ml) is the mean of three individual observations.

FIG. 5--Clinical scores post first inoculation. Clinical scores (y-axis) of individual pigs at different days (x-axis) post inoculation. Pigs 16, 18, 19 and 20 inoculated with 10.sup.4 TCID.sub.50 OURT88/3 virus. Pigs 21, 22, 23 inoculated with 10.sup.2 HAD.sub.50 Benin.DELTA.MGFA2 virus. Pigs 24 and 25 inoculated with 10.sup.2 HAD.sub.50 Benin.DELTA.MGFA1 virus. Clinical scoring system as designed by King et al 2011.

FIG. 6--Temperatures post first inoculation. Temperatures (y-axis) of individual pigs at different days (x-axis) post inoculation. Pigs 16, 18, 19 and 20 inoculated with OURT88/3 virus. Pigs 21, 22, 23 inoculated with Benin.DELTA.MGFA2 virus. Pigs 24 and 25 inoculated with Benin.DELTA.MGFA1 virus.

FIG. 7--IFN-.gamma. ELISPOT assays. Peripheral blood mononuclear cells collected day 20 post first inoculation were stimulated ex vivo with either medium alone, OURT88/3 or Benin.DELTA.MGF. Results are shown as IFN-.gamma. production per 10.sup.6 lymphocytes (y-axis) and pig number (x-axis). Spots per 10.sup.6 cells produced by PBMC purified on day 20 post inoculation with OUR T88/3 (pigs 16 to 20) or Benin.DELTA.MGF (pigs 21 to 25) in a porcine IFN.gamma. ELIspot. Error bars represent standard deviation from the mean of duplicate wells. Dilution 1 contained twice as many cells as dilution 2.

FIG. 8--Clinical scores post boost inoculation. Clinical scores (y-axis) of individual pigs at different days (x-axis) post boost inoculation. Pigs 16, 18, 19 and 20 boost inoculated with 10.sup.4 TCID.sub.50 OURT88/3 virus. Pigs 21, 22, 23 boost inoculated with 10.sup.4 HAD.sub.50 Benin.DELTA.MGFA2 virus. Pigs 24 and 25 boost inoculated with 10.sup.4 TCID.sub.50 Benin.DELTA.MGFA1 virus.

FIG. 9--Temperatures post boost inoculation. Temperatures (y-axis) of individual pigs at different days (x-axis) post inoculation. Pigs 16, 18, 19 and 20 inoculated with 10.sup.4 TCID.sub.50 OURT8813 virus. Pigs 21, 22, 23 inoculated with 10.sup.4 HAD.sub.50 Benin.DELTA.MGFA2 virus. Pigs 24 and 25 inoculated with 10.sup.4 HAD.sub.50 Benin.DELTA.MGFA1 virus.

FIG. 10--Clinical scores of pigs after challenge with Benin 97/1. Pigs from Groups 1 (pigs 21, 22, 23), Group 2 (pigs 24, 25), Group 3 (pigs 16, 18, 19, 20) and an unvaccinated Group 4 (pigs 26, 27, 28) were challenged with 10.sup.4 HAD.sub.50 Benin 97/1 virus and the clinical scores (y-axis) recorded at different days (x-axis) post challenge.

FIG. 11--Temperatures of pigs after challenge with Benin 97/1. Pigs from Groups 1 (pigs 21, 22, 23), Group 2 (pigs 24, 25), Group 3 (pigs 16, 18, 19, 20) and an unvaccinated Group 4 (pigs 26, 27, 28) were challenged with 10.sup.4 HAD.sub.50 Benin 97/1 virus and the temperatures (y-axis) recorded at different days (x-axis) post challenge.

FIG. 12--Results of survival rates for all four groups of pigs. The y-axis shows the percentage of pigs which survived following initial inoculation (Day 0), boost inoculation (Day 25) and challenge (Day 46) with virulent virus Benin 97/1. Groups 1 and 2 .star-solid., Group 3 .circle-solid., Group 4 .quadrature..

FIG. 13--IFN-.gamma. ELISPOT assays. Peripheral blood mononuclear cells or spleen homogenate cells isolated on day 63 post first inoculation were stimulated ex vivo with either medium alone or Benin 97/1 virus. Results are shown as IFN-.gamma. production per 10.sup.6 lymphocytes (y-axis) and pig number (x-axis).

FIG. 14--Percentage of circulating total lymphocytes in peripheral blood samples (as compared with day 0) collected at different days post infection (x-axis) from pigs infected with OURT88/3 (pigs 16 to 20) or Benin.DELTA.MGF (pigs 21 to 25).

FIG. 15--Percentage of circulating CD4+ cells in peripheral blood samples (as compared with day 0) collected at different days post infection (x-axis) from pigs infected with OURT88/3 (pigs 16 to 20) or Benin.DELTA.MGF (pigs 21 to 25).

FIG. 16--Percentage of circulating CD8+ total cells in peripheral blood samples (as compared with day 0) collected at different days post infection (x-axis) from pigs infected with OURT88/3 (pigs 16 to 20) or Benin.DELTA.MGF (pigs 21 to 25).

FIG. 17--Percentage of circulating CD8+CD4-.gamma..delta.TCR- (CD8 only) cells in peripheral blood samples (as compared with day 0) collected at different days post infection (x-axis) from pigs infected with OURT88/3 (pigs 16 to 20) or Benin.DELTA.MGF (pigs 21 to 25).

FIG. 18--Percentage of circulating CD8+/CD3- cells in peripheral blood samples (as compared with day 0) collected at different days post infection (x-axis) from pigs infected with OURT88/3 (pigs 16 to 20) or Benin.DELTA.MGF (pigs 21 to 25).

FIG. 19--Percentage of circulating gamma delta cells in peripheral blood samples (as compared with day 0) collected at different days post infection (x-axis) from pigs infected with OURT88/3 (pigs 16 to 20) or Benin.DELTA.MGF (pigs 21 to 25).

FIG. 20--Percentage of circulating gamma delta/CD8+ cells in peripheral blood samples (as compared with day 0) collected at different days post infection (x-axis) from pigs infected with OURT88/3 (pigs 16 to 20) or Benin.DELTA.MGF (pigs 21 to 25).

FIG. 21--Temperatures of pigs in Example 6 Groups A, B, C, D, E, F on different days post-immunisation and challenge and of control group G after challenge.

FIG. 22--Mean temperatures of all pigs in groups A to F of Example 6 after immunisation and challenge are shown in panel A. Panel B shows mean temperatures of surviving pigs in groups A to F and panel C temperatures of non-survivors.

FIG. 23--Clinical scores of pigs in Example 6 Groups A, B, C, D, E, F on different days post-immunisation and challenge and of control group G after challenge.

FIG. 24--Mean clinical scores of all pigs in Example 6 groups A to F after immunisation and challenge are shown in panel A. Panel B shows mean temperatures of surviving pigs in groups A to F and panel C temperatures of non-survivors.

FIG. 25--Scoring of lesions observed at necroscopy for different groups of pigs in Example 6. Mean scores for numbers of lesions observed in different groups from A to F are shown. Panel A shows scores of macroscopic lesions, Panel B of pulmonary lesions and Panel C of skin and musculoskeletal lesions.

FIG. 26--IFN-.beta. mRNA induction by different ASFV isolates in primary porcine alveolar macrophages

FIG. 27--Shaded in grey is the sequence of the DP148R gene which was deleted from the ASFV genome. The start and stop codons are shown in bold. Sequences used to amplify the left and right flanking regions are underlined. The sequences amplified are between these primers.

FIG. 28--Growth curves of Benin97/1, Benin.DELTA.DP148R and Benin.DELTA.MGF in porcine alveolar macrophages.

FIG. 29--Temperatures of pigs immunised with Benin.DELTA.MGF at HAD.sub.50 doses of 10.sup.2 (A), 10.sup.3 (B) and 10.sup.4 (C) using the IM route, 10.sup.3 HAD.sub.50 using the intranasal route (D) and Benin.DELTA.DP148R at 10.sup.3 HAD.sub.50 using IM route (E), on different days post-immunisation and challenge and of control group (F) after challenge.

FIG. 30--Clinical scores of pigs in groups A to E as described for FIG. 29 on different days post-immunisation and challenge and of control group F after challenge.

FIG. 31--Mean temperatures of all pigs in groups A to E as described for FIG. 29 after immunisation and challenge.

FIG. 32--Mean clinical scores of all pigs in groups A to E as described for FIG. 29 after immunisation and challenge

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have surprisingly found that deletion of five multi-gene family (MGF) 360 genes 10L, 11L, 12L, 13L, 14L and three MGF 505 genes 1R, 2R, 3R from the left hand end of the ASF virus genome and interruption of two additional genes (MGF360 9L and MGF 505 4R) resulted in attenuation of a virulent virus and induction of 100% protection against challenge with parental ASFV virulent virus.

Thus, in a first aspect; the present invention provides an attenuated African Swine Fever (ASF) virus which lacks a functional version of the following genes: multigene-family 360 genes 9L, 10L, 11L, 12L, 13L and 14L; and multigene-family 505 genes 1R, 2R, 3R and 4R.

The following genes may be at least partially (i.e. partially or completely) deleted: multigene-family 360 genes 10L, 11L, 12L, 13L and 14L; and multigene-family 505 genes 1R, 2R and 3R.

The following genes may be interrupted: multigene-family 360 gene 9L; and multigene-family 505 gene 4R.

The inventors have also surprisingly found that deletion of only one gene, namely the DP148R gene, from a region close to the right end of the virulent Benin97/1 genome, resulted in attenuation of a virulent virus and does not reduce virus replication in macrophages. A group of 5 pigs immunised with Benin.DELTA.DP148R intramuscularly with 10.sup.3 HAD.sub.50 units developed a transient fever and loss of appetite for 1 or 2 days at day 5 post-immunisation. No further clinical signs were observed following boost 21 days later or at challenge 42 days after the first immunisation. All 5 pigs survived challenge.

Accordingly, the invention further provides an attenuated African Swine Fever (ASF) virus which lacks a functional version of the DP148R gene. The gene may be partially or completely deleted, or interrupted.

The DP148R mutation may also be made in combination with mutation of the multigene-family 360 genes 9L, 10L, 11L, 12L, 13L and 14L and multigene-family 505 genes 1R, 2R, 3R and 4R as described herein. Therefore in one embodiment the invention provides an attenuated African Swine Fever (ASF) virus which lacks a functional version of the following genes: multigene-family 360 genes 9L, 10L, 11L, 12L, 13L and 14L; multigene-family 505 genes 1R, 2R, 3R and 4R; and DP148R.

The virus may be derivable from a virulent ASFV virus isolate. In other words, the genome of the attenuated virus of the invention (other than the genes MGF 360 genes 9L, 10L, 11L, 12L, 13L and 14L, and MGF 505 genes 1R, 2R, 3R and 4R; and/or DP148R) may correspond to the genome of a virulent ASFV virus isolate. The attenuated virus of the invention may be made by deleting/interrupting the genes: MGF 360 genes 9L, 10L, 11L, 12L, 13L and 14L and MGF 505 genes 1R, 2R, 3R and 4R; and/or DP148R; from a virulent ASFV virus isolate.

The virus may be derivable from one of the following virulent ASFV virus isolates: Georgia 2007/1, Benin 97/1, Kenyan, Malawi Lil20/1, Pretorisuskop/96/4 and Tengani 62.

The virus may be derivable from Benin 97/1.

The attenuated ASF virus, when administered to a subject, may induce an immune response which is protective against subsequent challenge with virulent ASF virus.

The attenuated ASF virus, when administered to a subject, may induce a reduced T cell mediated immune response compared to the immune response induced by attenuated virus OURT88/3. The immune response may involve a lower number of CD8+ positive .gamma..delta.T cells

In a second aspect, the present invention provides a vaccine comprising an attenuated ASF virus according to the first aspect of the invention.

The vaccine may comprise a plurality of attenuated ASF viruses of different genotypes.

In a third aspect, the present invention provides a vaccine according to the second aspect of the invention for use in treating and/or preventing African Swine Fever.

The vaccine may induce a cross-protective immune response against a plurality of ASF virus genotypes.

In a fourth aspect, the present invention provides a method of attenuating an African Swine Fever (ASF) virus, which comprises the step of partially or completely deleting, or interrupting the expression of, the following genes: multigene-family 360 genes 9L, 10L, 11L, 12L, 13L and 14L; and multigene-family 505 genes 1R, 2R, 3R and 4R.

The following genes may be partially or completely deleted: multigene-family 360 genes 10L, 11L, 12L, 13L and 14L; and multigene-family 505 genes 1R, 2R and 3R.

The following genes may be interrupted: multigene-family 360 gene 9L; and multigene-family 505 gene 4R.

The invention further provides a method of attenuating an African Swine Fever (ASF) virus, which comprises the step of partially or completely deleting, or interrupting the expression of, the DP148R gene.

In a fifth aspect the present invention provides a method for treating and/or preventing African Swine Fever in a subject which comprises the step of administering to the subject an effective amount of a vaccine according to the second aspect of the invention.

The subject may, for example, be a domestic pig.

The vaccine may be administered following a prime-boost regime.

The engineered virus Benin.DELTA.MGF of the present invention has several advantages over OURT88/3, for example: It is effective in inducing a protective immune response in a greater proportion of pigs than OURT88/3 It has an improved safety profile compared with OURT88/3 and does not induce adverse immunological reactions such as joint swelling. It is more efficient and is effective at a lower dose than OURT88/3 It induces a "better" type of immune response than OURT8813, as OURT88/3 induces a much higher IFN.gamma. response. Benin.DELTA.MGF has a reduced T cell mediated response than OURT88/3.

The engineered virus Benin.DELTA.DP148R of the present invention also has several advantages over OURT88/3, for example: It has an improved safety profile compared with OURT88/3 since no pulmonary, musculoskeletal or gross lesions were observed at post-mortem examination. It has higher efficacy than OURT88/3 since 100% of pigs tested were protected against Benin 97/1 challenge.

The present inventors have furthermore surprisingly found that intranasal administration of an attenuated African Swine Fever (ASF) virus results in improved protection and fewer side effects compared to administration via the intramuscular route, and may require a lower dose to obtain protection.

Accordingly, in a sixth aspect the present invention provides an attenuated ASF virus for use in the treatment and/or prevention of African Swine Fever, wherein the attenuated virus is administered intranasally.

The invention further provides a method for treating and/or preventing African Swine Fever in a subject which comprises the step of administering an effective amount of an attenuated ASF virus to the subject by the intranasal route.

The invention also relates to a vaccine comprising an attenuated ASF virus, wherein the vaccine is formulated for intranasal administration.

Further provided is a kit for delivery of an intranasal vaccine formulation comprising: a) an attenuated ASF virus vaccine; and b) an intranasal delivery device.

The invention also provides an intranasal delivery device comprising an attenuated ASF virus vaccine.

The attenuated ASF virus may be any attenuated ASF virus, such as OURT88/13, Benin.DELTA.MGF or Benin.DELTA.DP148R.

DETAILED DESCRIPTION

African Swine Fever Virus (ASFV)

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF). The virus causes a haemorrhagic fever with high mortality rates in pigs, but persistently infects its natural hosts, warthogs, bushpigs with no disease signs. It also infects soft ticks of the Ornithodoros genus, which are thought to be used as a vector.

ASFV replicates in the cytoplasm of infected cells, and is the only member of the Asfarviridae family. ASFV is endemic to sub-Saharan Africa and exists in the wild through a cycle of infection between ticks and wild pigs, bushpigs and warthogs. ASFV was first described after European settlers brought pigs into areas endemic with ASFV and, as such, is an example of an `emerging infection`.

ASFV is a large, icosahedral, double-stranded DNA virus with a linear genome containing at least 150 genes. The number of genes differs slightly between different isolates of the virus. ASFV has similarities to the other large DNA viruses, e.g., poxvirus, iridovirus and mimivirus. In common with other viral haemorrhagic fevers, the main target cells for replication are those of monocyte, macrophage lineage.

Based on sequence variation in the C-terminal region of the B646L gene encoding the major capsid protein p72, 22 ASFV genotypes (I-XXII) have been identified. All ASFV p72 genotypes have been circulating in eastern and southern Africa. Genotype I has been circulating in Europe, South America, the Caribbean and western Africa, Genotype VIII is confined to four East African countries.

Examples of strains from some of the genotypes are given below:

Genotype I: OURT88/3; Brazil/79; Lisbon/60; BA715; Pret; Benin 97/1; IC/1/96; IC/576; CAM/82; Madrid/62; Malta/78; ZAR85; Katange63; Togo; Dakar59; Ourt88/1; BEN/1/97; Dom_Rep; VAL/76; IC/2/96; Awoshie/99; NIG/1/99; NIG/1/98; ANG/70; BEL/85; SPEC120; Lisbon/57; ASFV-Warm; GHA/1/00; GAM/1/00; Ghana; HOL/86; NAM/1/80; NUR/90/1; CAM/4/85; ASFV-Teng; Tegani; ASFV-E75. Genotype II: Georgia 2007/1 Genotype III: BOT 1/99 Genotype IV: ASFV-War; RSA/1/99NV Genotype VI: MOZ 94/1 Genotype VII: VICT/90/1; ASFV-Mku; RSA/1/98 Genotype VIII: NDA/1/90; KAL88/1; ZAM/2/84; JON89/13; KAV89/1; DEZda; AFSV-Mal; Malawi LIL 20/1 Genotype IX. UGA/1/95 Genotype X: BUR/1/84; BUR/2/84; BUR/90/1; UGA/3/95; TAN/Kwh12; Hindell; ASFV-Ken; Virulent Uganda 65. Multigene Families

ASFV contains five multi-gene families which are present in the left and right variable regions of the genome. The MGFs are named after the average number of codons present in each gene: MGF100, 110, 300, 360 and 505/530. The N-terminal regions of members of MGFs 300, 360 and 505/530 share significant similarity with each other. Several genes in MGF360 and 505/530 determine host range and virulence.

The five multigene families of the ASFV genome (MGF 100, MGF 110, MGF 360 and MGF 505/530) are located within the left hand 35 kb or right hand 15 kb terminal variable regions. The MGFs constititute between 17 and 25% of the total coding capacity of the ASFV genome. They lack similarity to other known genes.

The complete genome sequences of isolate Benin 97/1 (a highly pathogenic virus from West Africa, Group 1), isolate OURT88/3 (non-pathogenic, attenuated virus from Portugal, Group 1) and isolate BA71V (Vero cell tissue culture adapted non-pathogenic virus, Group 1) have been compared (Chapman et al (2008) J. Gen Virol. 89:397-408). The isolate OURT88/3 has a deletion of 10007 bp compared to Benin 97/1 extending from nucleotide number (nt no.) 19448 to nt no. 29525 in the Benin 97/1 genome. In all, five MGF 360 genes (10L, 11L, 12L, 13L and 14L) are deleted from the OURT88/3 genome. Two MGF 505 genes (1R, 2R) are deleted and the MGF 505R 3R gene is truncated in the OURT88/3 genome. These genes are present in the genomes of all eight other pathogenic isolates of ASFV that have been sequenced.

In the attenuated tissue-culture adapted BA71V isolate, the genome contains the MGF 505 3R gene but lacks the other seven MGF genes and in addition also has the MGF 360 9L gene truncated (total deletion of 8250 bp).

Previously six MGF 360 genes (9L, 10L, 11L, 12L, 13L and 14L) and two MGF 505 genes (1R and 2R) were deleted from the highly pathogenic South African isolate Pr4. This deletion markedly reduced viral growth in primary macrophage cell cultures by 100- to 1000-fold (Zsak et al 2001, as above) and led to attenuation of the virus (cited as unpublished results, Afonso et al 2004 J. Virol 78:1858-1864). However no experiments were carried out to challenge the recovered pigs to determine if they were protected. In fact, at an African swine fever virus workshop at the Biosecurity Research Institute, Manhattan Kans. in May 2011 it was mentioned that the Pr4 deletion mutant was not protective and induced a chronic form of the disease.

It has also been shown that deletion of three MGF 360 genes (12L, 13L and 14L) and four MGF 505 genes (1R, 2R, 3R and 4R truncation) from the pathogenic virus Malawi.DELTA.DP71 reduces virus replication in pigs and in addition attenuated the virus (Neilan et al (2002) J. Virol. 76:3095-3104). However again, no experiments were reported to determine if the recovered pigs were protected against challenge.

The attenuated ASFV according to one embodiment of the present invention lacks a functional version of the following genes: multigene-family 360 genes 9L, 10L, 11L, 12L, 13L and 14L; and multigene-family 505 genes 1R, 2R, 3R and 4R.

The location of these genes in the genomes of a variety of ASFV strains is provided below (Table 1). The sequence identity of each gene to the corresponding Benin 97/1 gene is also provided.

TABLE-US-00001 TABLE 1 Start Nucleotide Stop Nucleotide % Nucleotide Strain Number number Identity a - MGF 360-9L Benin 97/1 19487 18435 100 Georgia 2007/1 25215 24163 98 BA71V 17495 17057 100 OURT88/3 20355 19937 99 Kenya 1950 29478 28424 86 Malawi-Lil/20 23685 22630 86 Mkuzi 1979 26939 25887 99 Pretorisuskop/96/4 26016 24958 85 Tengani 62 20398 19346 98 Warmbaths 25244 24186 86 Warthog 22020 20962 85 E75 18802 17750 99 b - MGF 360-10L Benin 97/1 20677 19607 100 Georgia 2007/1 26438 25368 93 BA71V Deleted OURT88/3 Deleted Kenya 1950 30628 29563 82 Malawi-Lil/20 24802 23763 91 Mkuzi 1979 28139 27069 99 Pretorisuskop/96/4 27186 26116 96 Tengani 62 21600 20530 92 Warmbaths 26361 25322 94 Warthog 23130 22091 95 E75 19981 18911 100 c - MGF 360-11L Benin 97/1 21764 20703 100 Georgia 2007/1 27526 26465 97 BA71V Deleted OURT88/3 Deleted Kenya 1950 31716 30655 84 Malawi-Lil/20 25892 24831 86 Mkuzi 1979 29225 28164 99 Pretorisuskop/96/4 28275 27214 97 Tengani 62 22688 21627 97 Warmbaths 27449 26388 97 Warthog 24218 23157 97 E75 21068 20007 100 d - MGF 360-12L Benin 97/1 24668 23616 100 Georgia 2007/1 30434 29382 96 BA71V Deleted OURT88/3 Deleted Kenya 1950 34566 33549 90 Malawi-Lil/20 28731 27682 92 Mkuzi 1979 32125 31073 97 Pretorisuskop/96/4 31150 30098 95 Tengani 62 25592 24540 96 Warmbaths 30346 29294 96 Warthog 27088 26036 96 E75 23971 22920 99 e - MGF 360-13L Benin 97/1 25901 24840 100 Georgia 2007/1 31656 30595 97 BA71V Deleted OURT88/3 Deleted Kenya 1950 35812 34757 90 Malawi-Lil/20 29980 28925 91 Mkuzi 1979 33347 32286 99 Pretorisuskop/96/4 32368 31307 95 Tengani 62 26814 25753 95 Warmbaths 31559 30498 95 Warthog 28338 27277 95 E75 25204 24143 99 f - MGF 360-14L Benin 97/1 27146 26073 100 Georgia 2007/1 32913 31840 99 BA71V Deleted OURT88/3 Deleted Kenya 1950 37194 36121 92 Malawi-Lil/20 31266 30193 92 Mkuzi 1979 34620 33547 99 Pretorisuskop/96/4 33598 32525 97 Tengani 62 28056 26983 97 Warmbaths 32820 31747 96 Warthog 29568 28495 97 E75 26449 25376 100 g - MGF 505-1R Benin 97/1 21971 23566 100 Georgia 2007/1 27734 29329 95 BA71V Deleted OURT88/3 Deleted Kenya 1950 31904 33496 91 Malawi-Lil/20 26041 27627 93 Mkuzi 1979 29425 31020 96 Pretorisuskop/96/4 28449 30044 94 Tengani 62 22891 24486 95 Warmbaths 27651 29246 95 Warthog 24387 25982 95 E75 21275 22870 100 h - MGF 505-2R Benin 97/1 27352 28932 100 Georgia 2007/1 33119 34699 99 BA71V 17725 19304 99 OURT88/3 29532 29981 76 Kenya 1950 37419 38985 90 Malawi-Lil/20 31541 33121 93 Mkuzi 1979 34826 36406 99 Pretorisuskop/96/4 33795 35374 97 Tengani 62 28261 29830 98 Warmbaths 33029 34597 96 Warthog 29773 31352 97 E75 26655 28236 99 i - MGF 505-3R Benin 97/1 29019 29861 100 Georgia 2007/1 34786 35625 96 BA71V 20398 21915 100 OURT88/3 20850 22367 100 Kenya 1950 40295 41815 89 Malawi-Lil/20 34322 35842 89 Mkuzi 1979 37500 39017 99 Pretorisuskop/96/4 36480 37985 89 Tengani 62 30926 32443 90 Warmbaths 35712 37232 93 Warthog 32449 33954 90 E75 29330 30847 100 j - MGF 505-4R Benin 97/1 30026 31543 100 Georgia 2007/1 35796 37316 94 BA71V 20394 21915 100 OURT88/3 20850 22367 100 Kenya 1950 40295 41815 89 Malawi-Lil/20 34322 35842 89 Mkuzi 1979 37500 39017 99 Pretorisuskop/96/4 36480 37985 89 Tengani 62 30926 32443 90 Warmbaths 35712 37232 93 Warthog 32449 33954 90 E75 29330 30847 100

The translation products of these genes are given below:

TABLE-US-00002 SEQ ID No. 1-translation of 360-91_ MVLSLQILTKKVLASQYPAKCHPHFLKCCGLWWHNGPIMYHQKK IWTPYFKNGTNLNAALVKAVEENNHDLIELFTEWGANINYGLLSVNTEHT RDLCRQLGAKEQLNDQEILRFFYTLKRDLTSSNIIFCHEVFSNNPILDTI NRFEVKGMIYEQLEGLMVETDILSEMFTKYWYAMAIEFNLKEAICYFYQR YAHLHRWRLMCALFYNNVFDLHELYAKEKVRMDMDEMLRWACRKNYNYLT IYYCCVALGADINQAMFHSIQFYNIGNIFFCIDLGANAFEEGKILAHQKD NSFIASMLSLNCYSMNDSLSLKETDPEVIKRMLKDYHSKNLSIAHKHYIN DGFNDI SEQ ID No. 2-translation of 360-10L MVPSLQSFAKKVLASQHVSIDYHVILERCGLWWYKAPISLDCKH MLIKLPNFADGLDLNTALMLATKENNYQLIKMFTDWGADINYGLICANTP PIREFCWELGAKYQVDKKKIMHIFFKLIHPNTTSNNIILCLKFFNDNPFS AYVIIREIKSCHWKLKNLAEDTNVLSNISDGDMLTIYCFIVALQDNLREA ISYVYQHFKYLNTWWLICALCYNKLFDLHNLYEKEKIRMDMDEMMRIACT KDNNFLTIYYCFILGANINLAMIASIRFYNMDNLFFCIDLGADAFEEAKA LAEQQNYYLISHRLSLDIYSPDSSLLTLKEADPNKIYRLLKNYKSKSMLA YLNYDINDTSL SEQ ID No. 3-translation of 360-11L MLPSLQSLTKKVLAGQCVSVDHYHILKCCGLWWHNGPIMLHIRR NKLFIRSTCFSQGIELNIGLMKAVKENNHDLIKLFTEVVGADINYGMICA LTENTRDLCKELGAKEYLEREYILKIFFDTTRDKTSSNIIFCHEVFSNNP NLRIIDNLDLRGEIMVVELRGLMEITFMLDHDDSFSTVLTKYWYAIAVDY DLKDAIRYFYQKYPRLHRWRLMCALFYNNVFDLHELYEIERVRMDIDEMM HIACIQDYSYSAIYYCFIMGANINQAMLVSIQNYNLGNLFFCIDLGANAF EEGKALAEQKENYLIAHALSLKHYNPVISLLSNVMDPEKINYMLKNYHSI NMGIFLDYEQR SEQ ID No. 4-translation of 360-12L MLPSLQSLTKKVLAGQCVPTNQHYLLKYYDLWWYNAPITFDHNL RLIKSSGIKEGLDLNTALVKAVRENNYSLIKLFTEWGADINYGLVSVNTE HTRDLCQELGAKEILNEEEILQIFIDLKFHKTSSNIILCHEVFSNNPILQ KVNNLKLRIEIFWELRELIEKTDLLNNEFLLSTLLLKYWYAIAVRYSLKE AIQYFYQKYTHMNTWRLTCALCFNNVFDLHEAYEKDKIHMDIEEMMRIAC IKDHNLSTMYYCYMLGANINQAMLTSIQYYNIENMFFCMDLGADVFEEGT TALGEGYELIKNILSLKIYSPTTIPLPKSTDPEIIDHALKNYFSKNMMIF LSYDLR SEQ ID No. 5-translation of 360-131_ MSLPLSLQTLVKKTVASQCLSIDEHCILKYCGLWWHDAPLKLCM DRGRIQIKSGFLGEDIDLRVALIIAVKENNYSLIKLFTEWGANINYSLLS INTKHIRELCRQLGAKETLEDNDIFRIFTRIMHNKTSGSIILCHEIFMNN PMLENKFVIQLRGLIYKRLWGLIEIKETDELNDLLVKYWYAKAVQYVCKN AICFLDEKYTDLNEWRLKCLLYYNKIYELHEMYHKKKVQIDVHDMICLAC AKDNNLLTIYYCYALGGNINQAMLTSVQYYNVGNIFFCIDLGGNAFEEGR AIAEQKGYNFLSHSLILDIYSSDASLPLNLKDPEKISSLLKDYKSKNLSI IWEYSHNIL SEQ ID No. 6-translation of 360-14E_ MLSLQTLAKKVVACNYLSSDYDYTLQRFGLWWDLGPIHLCNNCK QVFSYKHLQCFSEDDLCLEAALVKAVKSDNLELIRLFVDWGANPEYGLIR VPAVYLKRLCAELGGLTPVSEPRLLEILKEVANLKSCAGVLLGYDMFCHN PLLETVTRTTLDTVTYTCSNIPLTGDTAHLLLTKFWFALALRHNFTKAIH YFYKRHKNQLYVVRVACSLYFNNIFDIHELCREKEICISPNLMMKFACLR EKNYAAIYYCHRLGASLDYGMNLSIYNNNTLNMFFCIDLGAADFDRAQLI AHKAYMYNLSNIFLVKQLFSRDVTLVLDVTEPQEIYDMLKTYTSKNMKRA EEYLTAHPEIIVID SEQ ID No. 7-translation of 505-1R MFSLQNLCRKTLPDCKLPEFFDDYILQLLGLYWENHGTIQRAGN NCVLIQQHTLIPVNEALRIAASEENYEIVGLLLAWEGNLYYAIIGALEGN RYNLIRKYDDQIKDHHDILPFIDDPIIFHKCHIMRRCFFDCILYQAVKYS KFRVLLYFKYTLEDDLPLVHLLIEKACEDHNYEVIKWIYENLHVCHIIDT FDCAIAHKDLRLYCLGYTFIYNRIVPYKYHHLDILILSSLQLLHKVAAKG YLDFILETLKYDHNIDNLDVILTQAATYNHRKILTYFIPQSTYAQIEQCL FVAIKTKSSKKTLNLLLSHLNLSIKLIQKISQYVATFNSTNIIGILSMKR KKKIYLDIILTKFVKNAIFNKFVVRCMERFSINPERIVKMAARINKMMLV KKISEHVWKNHAARLKHLKHAVHTMKHKDGKNRLMNFIYEHCYYHMQGEE IFSLARFYAIHHAPKLFDVFYNCCILDTIRFKSLLLDCSHIIGKNAHDAT NINIVNKYIGNLFAMGVLSKKEILQDYPSIYSKHYMP SEQ ID No. 8-translation of 505-2R MFSLQDLCRKHLFILPDVFGEHVLQRLGLYWRCHGSLQRIGDDH ILIRRDLILSTNEALRMAGEEGNNEVVKLLLLWKGNLHYAVIGALQGDQY DLIHKYENQIGDFHFILPLIQDANTFEKCHALERFCGVSCLLKHATKYNM LPILQKYQEELSMRAYLHETLFELACLWQRYDVLKWIEQTMHVYDLKIMF NIAISKRDLTMYSLGYIFLFDRGNTEATLLTQFILEKTAAKGLLFIFVLE TLKYGGNIDTVLTQAVKYNHRKLLDYFLRQLPRKHIEKLILLAVQEKASK KTLNLLLSFILNYSVKRIKKLLRYVIEYESTLVIKILLKKRVNLIDAMLE KMVRYFSATKVRTIMDELSISPERVIKMAIQKMRTDIVIHTSYVWEDDLE RLTRLKNMVYTIKYEHGKKMLIKVMHGIYKNLLYGEREKVMFHLAKLYVA QNAATQFRDICKDCYKLDVARFKPRFKQLILDCLEIVTKKSCYSILEILE KHIISLFTMKVMTEEEKNLCLEILYKVIHYKTIQC SEQ ID No. 9-translation of 505-3R MSSSLQELCRKKLPDCILPEFFDDYVLQLLGLHWQDHGSLQRIE KNQILVQQEPIHINEALKVAASEGNYEIVELLLSWEADPRYAWGALESKY YDLVYKYYDLVKDCHDILPLIQNPETFEKCHELNNPCSLKCLFKHAVIHD MLPILQKYTYFLDGWEYCNQMLFELACSKKKYEMWWIEGVLGIGKVTSL FTIAISNRDLHLYSLGHLIILERMQSCGQDPTFLLNHFLRDVSIKGLLPF VLKTIEYGGSKEIAITLAKKYQHKHILKYFETGKC SEQ ID No. 10-translation of 505-4R MFSLQDICRKYLFQLPDSFDEYTLQVLGLYWEKHGSLQRIRKDA VFVQRNLIISINEALRIAASEGNGRVVKLLLSWEGNFHYVIIGALEGDHY DLIHKYGSQIEDYHMILSSIHNANTFEKCHELSNCDMWCLIQNAIKYNML PILQKHRNILTHEGENQELFEMACEEQKYDIVLWIGQILMLNEPEFIFDI AFERIDFSLLTMGYSLLFNNKMSSIDIHDEEDLISLLTEHLEKAATKGCF FFMLETLKHGGNVNMAVLSKAVEYNHRKILDYFIRQKCLSRKDIEKLLLV AISNSASKKTLNLLLSYLNHSVKNIIGKIVQSVLKNGDFTIIIFLKKKKI NLVEPALIGFINYYYSYCFLEQFIHEFDIRPEKMIKMAARKGKLNMIIEF LNEKYVHKDDLGAIFKFLKNLVCTMKHKKGKETLIVLIHKIYQVIQLETK EKFKLLRFYVMHDATIQFISMYKDCFNLAGFKPFLLECLDIAIKKNYPDM IRNIETLLKCE

The complete genome for the African swine fever virus Benin 97/1 pathogenic isolate is given in Genbank Locus: AM712239.1

From the study described by Chapman et al (2008--as above) it was determined that the complete BA71 isolate genome encodes 151 open reading frames (ORFs), the Benin 97/1 isolate encodes 157 ORFs and the OURT88/3 isolate encodes 151 ORFs.

According to another embodiment of the invention, there is provided an attenuated ASFV which lacks a functional version of gene DP148R.

The DP148R mutation may be made on its own, or in combination with mutations of the multigene-family 360 genes 9L, 10L, 11L, 12L, 13L and 14L and multigene-family 505 genes 1R, 2R, 3R and 4R as described herein. This combination of gene mutations may result in a better safety profile for the attenuated virus.

DP148R is located at genome position 177915 to 178679 on the Benin 97/1 genome. The protein sequence is:

TABLE-US-00003 Benin 97/1 177915-178679- SEQ ID No. 11 MQNKIPNFNLFFFFLYRMLEIVLATLLGDLQRLRVLTPQQRAVAFFRANT KELEDFLRSDGQSEEILSGPLLNRLLEPSCPLDILTGYHLFRQNPKAGQL RGLEVKMLERLYDANIYNILSRLRPKKVRNKAIELYWVFRAIHICHAPLV LDIVRYEEPDFAELAFICAAYFGEPQVMYLLYKYMPLTRAVLTDAIQISL ESNNQVGICYAYLMGGSLKGLVSAPLRKRLRAKLRSQRKKKDVLSPHDFL LLLQ.

Orthologous DP148R sequences from other genomes share between 74 and 99% amino acid identity. The orthologous DP148R genes from other ASFV isolates are located in positions:

TABLE-US-00004 Warthog: 181103 to 181549 SEQ ID No. 12 MLERLYDANIYNILSRLRPEKVRNKAVELYWVFRAINMCHAPLVLDIVRY EEPDFAELAFICAAYFGEPQVMYLLYKYMPLTRAVLTDAIQISLESNSQV GICYAYLMGGSLKGLVRAPLRKRLRAKLRSQRKKKDVLPPHDFLLLLQ Kenya: 189417 to 189872 SEQ ID No. 13 MLERLYDANIYNMLARLRPELVRDKAIELYWLFRAILMCHSPLVLEIVRH ETMDFAETAFICAAYFSEPQVMYALYKFIPISRAVLADAIQMCLESNSEA GICYAYLMGGSLKGKVPGSLRKRLRASPLRQERKKKNVLPPHEFLLMLHG I Malawi LIL20/1: 183687 to 184346 SEQ ID No. 14 MQRAVAFFRVNTKELEDFLYPDGQSEELLPGLLLNRLLEPSGPIDILTGY HLFRENPKAGRLRGLEVKLLERLYDANIYNMLAQIRPELVRIKAIELYWL FRAILMCHSPLVLEIVRHETMDFAELAFICAAYFSEPQVMYALYKFIPIS RAVLADAIEMSLESNSETGICYAYLMGGSLKGKVPGPLRKRLRASPLRQE RKKKNVLPPHEFLLMLHGI Mkuzi: 185751 to 186515 SEQ ID No. 15 MQNKIPNFNLFFFFLYRMLEIVLATLLGDLQRLRVLTPQQRAVAFFRANT KELEDFLCSDGQSEEILSGPLLNRLLEPSGPLDILTGYHLFRQNPKAGQL RGLEVKMLERLYDANIYNILSRLRPEKVRNKAIELYWVFRAIHICHAPLV LDIVRYEEPDFAELAFICAAYFGEPQVMYLLYKYMPLTRAVLTDAIRISL ESNNQVGICYAYLMGGSLKGLVSAPLRKRLCAKLRSQRKKKDVLSPHDFL LLLQ Pretoriskup: 185416 to 185862 SEQ ID No, 16 MLERLYDANIYNILSRLRPEKVRNKAVELYWVFRAINMCHAPLVLDIVRY EEPDFAELAFICAAYFGEPQVMYLLYKYMPLSRAVLTDAIQISLESNSQV GICYAYLMGGSLKGLVRAPLRKRLRAKLRSQRKKKDVLPPHDFLLLLQ Tengani: 180112 to 180558 SEQ ID No. 17 MLERLYDANIYNILSRLRPEKVRNKAVELYWVFRAINMCHAPLVLDIVRN EELDFAELAFICAAYFGEPQVMYLLYKYMPLTRAVLTDAIQISLESNSQV GICYAYLMGGSLKGLVRAPLRKRLRAKLRSQRKKKDVLPPHDFLLLLQ Warmbaths: 184606 to 185052 SEQ ID No. 18 MLERLYDANIYNILSRLRPEKVRNKAIELYWVFRAIHICHAPLVLDIVRY EEPDFAELAFICAAYFGEPQVMYLLYKYMPLTRAVLTDAIRISLESNNQV GICYAYLMGGSLKGLVSAPLRKRLRAKLRSQRKKKDVLSPHDFLLLLQ OURT88/3: 169146 to 169592 SEQ ID No. 19 MLERLYDANIYNILSRLRPEKVRNKAIELYWVFRAIHICHAPLVLDIVRY EEPDFAELAFICAAYFGEPQVMYLLYKYMPLTRAVLTDAIQISLESNNQV GICYAYLMGGSLKGLVSAPLRKRLRAKLRSQRKKKDVLSPHDFLLLLQ

ASF Virus Isolates

The attenuated ASF virus of the present invention may be derivable from a wild-type ASF virus isolate, but includes mutations in its genome such that the following genes are completely or partially deleted or otherwise made non-functional: MGF 360 genes 9L, 10L, 11L, 12L, 13L and 14L and MGF 505 genes 1R, 2R, 3R and 4R; and/or DP148R gene.

The term "wild-type" indicates that the virus existed (at some point) in the field, and was isolated from a natural host, such as a domestic pig, tick or wart hog. Table 2 below lists known ASF virus isolates.

The genome structure of ASFVs is known in the art, as detailed in Chapman et al (2008) J. Gen. Virol, 89:397-408.

The term "corresponds to" means that the remainder of the genome is the same, or substantially the same, as the wild type strain. Thus the genome of the attenuated virus of the present invention may include the genes of the wild type strain, other than MGF 360 genes 9L, 10L, 11L, 12L, 13L and 14L and MGF 505 genes 1R, 2R, 3R and 4R; and/or DP148R gene.

The genome of the attenuated virus may comprise the ORFs conserved in all 10 genome sequences available and completely or partially deleted or otherwise made non-functional: MGF 360 genes 9L, 10L, 11L, 12L, 13L and 14L and MGF 505 genes 1R, 2R, 3R and 4R; and/or DP148R gene.

The genome of the attenuated recombinant ASF virus of the invention may correspond to that of a virulent ASF virus strain (with the exception of the MGF 360 genes 9L, 10L, 11L, 12L, 13L and 14L and MGF 505 genes 1R, 2R, 3R and 4R; and/or DP148R gene).

African swine fever virus isolates described to date are summarised in Table 2, together with their Genbank Accession numbers.

Known virulent ASF virus strains include; Georgia 2007/1, Benin 97/1, Kenyan, Malawi Lil20/1, Pretorisuskop/96/4 and Tengani 62.

TABLE-US-00005 TABLE 2 GenBank accession Isolate Country Host Year Virulence No Georgia 2007/1 Georgia Domestic 2007 High FR862468 pig BA71qqV Spain Domestic 1971 Tissue U18466 pig culture adapted Benin 97/1 Spain Domestic 1997 High AM712239 pig OURT88/3 Portugal Tick 1988 Low AM712240 Kenya Kenya Domestic 1950 High AY261360 pig Malawi Lil20/1 Malawi Tick 1983 High AY261361 Mkuzi Zululand Tick 1978 Unknown AY261362 Pretorisuskop/ South Tick 1996 High AY261363 96/4 Africa Tangani 62 Malawi Domestic 1962 High AY261364 pig Warmbaths South Tick 1987 Unknown AY261365 Africa Warthog Namibia Warthog 1980 Unknown AY261366

The genome of the attenuated recombinant ASF virus of the invention (with the exception of the MGF 360 genes 9L, 10L, 11L, 12L, 13L and 14L and MGF 505 genes 1R, 2R, 3R and 4R; and/or DP148R gene) may correspond to that of the Benin 97/1 isolate.

The genome of the attenuated recombinant ASF virus of the invention (with the exception of the MGF 360 genes 9L, 10L, 11L, 12L, 13L and 14L and MGF 505 genes 1R, 2R, 3R and 4R, and/or DP148R) may correspond to that of an ASF virus strain whose virulence is currently unknown, for example: Mkuzi, Warmbaths and Warthog.

The present invention also provides a vaccine composition which comprises a plurality of attenuated ASF viruses. The plurality of attenuated ASF viruses may correspond to a plurality of different isolates, for example, different isolates of high or unknown virulence.

Such a vaccine composition may elicit a cross-protective immune response to several or substantially all ASF viruses.

Deletions

The attenuated African Swine Fever (ASF) virus of one embodiment of the present invention lacks a functional version of the following genes: multigene-family 360 genes 9L, 10L, 11L, 12L, 13L and 14L; and multigene-family 505 genes 1R, 2R, 3R and 4R.

The genes may, for example, be wholly or partially deleted.

In particular, the following genes may be at least partially deleted: multigene-family 360 genes 10L, 11L, 12L, 13L and 14L; and multigene-family 505 genes 1R, 2R and 3R.

In another embodiment, the present invention provides an attenuated ASFV which lacks a functional version of DP148R. The gene may be wholly or partially deleted.

The deletion may be continuous, or may comprise a plurality of sections of sequence. The deletion should remove a sufficient amount of nucleotide sequence such that the gene no longer encodes a functional protein. The deletion may, for example, remove at least 50, 60, 70, 80 or 90% of the coding portion of the gene.

The deletion may be total, in which case 100% of the coding portion of the gene is absent, when compared to the corresponding genome of the wild-type isolate.

Interruptions

In the attenuated African Swine Fever (ASF) virus of one embodiment of the present invention one or more of the following genes may be interrupted: multigene-family 360 genes 9L, 10L, 11L, 12L, 13L and 14L; and multigene-family 505 genes 1R, 2R, 3R and 4R

In particular, the following genes may be interrupted: multigene-family 360 gene 9L; and multigene-family 505 gene 4R

In another embodiment, the present invention provides an attenuated ASF virus wherein the DP148R gene is interrupted.

The gene may be interrupted, for example, by deleting or otherwise modifying the ATG start codon of the gene.

The genome may comprise one or more nucleotide change(s) that ablate expression of the gene. For example, expression of the gene may be ablated by a frame shift or introduction of one or more stop codons in the open reading frame of the gene or a modification of a translational start site.

The interruption may cause the gene to not be transcribed and/or translated.

Functional Genes

As mentioned above, the complete genome sequence of the attenuated ASFV strain OURT88/3 has been determined and compared with that of virulent viruses (Chapman et al., 2008 as above). In addition to the deletions mentioned above, the OURT88/3 strain also has interruptions in 3 other genes: EP402R, EP153R and DP148R (previously referred to as MGF360 18R).

EP402R codes for CD2v protein which is incorporated in the external layer of the virus and may have a role in virus entry or spread. It may also be a target for antibodies that inhibit infection. EP153R is a C-type lectin. DP148R inhibits type I interferon.

The genome of the attenuated virus of the present invention may comprise complete, uninterrupted and functional versions of one or more of the genes EP402R and EP 153R. In the attenuated virus of the present invention, both of these genes may be complete, uninterrupted and functional.

Vaccine/Pharmaceutical Composition

The present invention also provides a vaccine comprising an attenuated ASF virus of the invention.

The term `vaccine` as used herein refers to a preparation which, when administered to a subject, induces or stimulates a protective immune response. A vaccine can render an organism immune to a particular disease, in the present case ASF. The vaccine of the present invention thus induces an immune response in a subject which is protective against subsequent ASF virus challenge.

The vaccine may comprise a plurality of attenuated ASF viruses of different genotypes. Such a vaccine may be capable of inducing a cross-protective immune response against a plurality of ASF virus genotypes.

The vaccine may be useful in preventing African Swine Fever.

The present invention also provides a pharmaceutical composition which comprises one or more attenuated ASF virus(es) of the invention. The pharmaceutical composition may be used for treating African Swine Fever.

The vaccine or pharmaceutical composition may comprise one or more attenuated ASF virus(es) of the invention and optionally one or more adjuvants, excipients, carriers and diluents.

Immunisation with OURT88/3 induces a high IFN.gamma. response, suggesting that it is mediated by T cells. The attenuated virus of the present invention appears to induce a reduced T cell mediated response (see FIGS. 7 and 13). The attenuated virus of the present invention therefore appears to induce a more "useful" (i.e. more protective) cellular immune response than OURT88/3.

The attenuated virus of the present invention may induce a reduced T-cell mediated response compared to OURT88/3. Methods for analysing and comparing T-cell mediated responses are known in the art, for example by assaying T-cell proliferation in response to antigen (e.g, cell counts, thymidine incorporation or BrdU incorporation), assaying cytokine secretion and/or expression in response to antigen (e.g. ELISA, ELISPOT, flow cytometry) or determination of lymphocyte subpopulations present following inoculation of a subject with the virus (e.g. using flow cytometry).

The attenuated virus of the present invention may induce an immune response comprising a reduced percentage of circulating gamma delta/CD8+ cells and/or a reduced percentage of CD8+/CD4-/.gamma..delta.TCR- (CD8 only cells) and or a reduced percentage of CD8+/CD3- (NK) cells compared to OURT88/3.

Methods of Prevention/Treatment

The present invention also provides a method of preventing and/or treating ASF in a subject by administration of an effective amount of an attenuated virus, vaccine, or pharmaceutical composition of the invention.

The term `preventing` is intended to refer to averting, delaying, impeding or hindering the contraction of ASF. The vaccine may, for example, prevent or reduce the likelihood of an infectious ASFV entering a cell.

The term "treating" is intended to refer to reducing or alleviating at least one symptom of an existing ASF infection.

The subject may be any animal which is susceptible to ASF infection. ASF susceptible animals include domestic pigs, warthogs, bush pigs and ticks.

The subject vaccinated according to the present invention may be a domestic pig.

Administration

The vaccine of the invention may be administered by any convenient route, such as by intramuscular injection. Other suitable routes of administration include intranasal, oral, subcutaneous, transdermal and vaginal (e.g, during artificial insemination). In one embodiment, oral administration comprises adding the vaccine to animal feed or drinking water. In another embodiment, the vaccine may be added to a bait for a wild animal, for example a bait suitable for wild boar, wild pigs, bushpigs or warthogs.

The present inventors have found the attenuated virus Benin.DELTA.MGF of the invention is effective at a lower dose compared to OURT88/3. For example, in Example 3, pigs received 10.sup.2 HAD of Benin.DELTA.MGF which was compared with a dose of 10.sup.4 TCID.sub.50 of the virus OURT8813.

The dose for pig immunisation may therefore be less than 10.sup.4 HAD.sub.50 or TCID.sub.50 per pig. For example the dose may be between 10.sup.2-10.sup.3 HAD.sub.50 or TCID.sub.50 The dose may be about 10.sup.2 HAD.sub.50 or TCID.sub.50 per pig.

The vaccine may be administered following a prime-boost regime. For example, after the first inoculation, the subjects may receive a second boosting administration some time (such as about 7, 14, 21 or 28 days) later. Typically the boosting administration is at a higher dose than the priming administration. The boosting dose may be about 10.sup.2, 10.sup.3 or 10.sup.4 HAD.sub.50 or TCID.sub.50 of the recombinant attenuated virus per pig.

Method for Preparing Attenuated Virus

The present invention also provides a method of attenuating an African Swine Fever (ASF) virus, which comprises the step of at least partially deleting, or interrupting, the expression of the following genes: multigene-family 360 genes 9L, 10L, 11L, 12L, 13L and 14L; and multigene-family 505 genes 1R, 2R, 3R and 4R.

The following genes may be partially or completely deleted: multigene-family 360 genes 10L, 11L, 12L, 13L and 14L; and multigene-family 505 genes 1R, 2R and 3R.

The following genes may be interrupted: multigene-family 360 gene 9L; and multigene-family 505 gene 4R.

The present invention further provides a method of attenuating an African Swine Fever (ASF) virus, which comprises the step of partially or completely deleting, or interrupting, the expression of the DP148R gene.

The DP148R mutation may also be made in combination with mutations of the multigene-family 360 genes 9L, 10L, 11L, 12L, 13L and 14L and multigene-family 505 genes 1R, 2R, 3R and 4R as described herein.

Methods for deletion of viral genes are known in the art. For example, homologous recombination may be used, in which a transfer vector is created in which the relevant gene(s) are missing and used to transfect virus-infected cells. Recombinant viruses expressing the new portion of sequence may then be selected. Similar procedures may be used in order to interrupt gene expression, for example by deletion of the ATG start codon.

Intranasal Administration

The present inventors have surprisingly found that intranasal administration of an attenuated African Swine Fever (ASF) virus results in improved protection and fewer side effects compared to administration via the intramuscular route, and may require a lower dose to obtain protection. In particular, intranasal administration of attenuated strain OURT88/3 demonstrated complete protection (100%) against challenge with parental ASFV virulent virus, which represents a significant improvement over survival rates using the same attenuated virus via the intramuscular route.

Accordingly, in one embodiment the invention provides an attenuated ASF virus for use in the treatment and/or prevention of African Swine Fever, wherein the attenuated virus is administered intranasally.

The invention further provides a method for treating and/or preventing African Swine Fever in a subject which comprises the step of administering an effective amount of an attenuated ASF virus to the subject by the intranasal route.

The invention also relates to a vaccine comprising an attenuated ASF virus, wherein the vaccine is formulated for intranasal administration.

Further provided is a kit for delivery of an intranasal vaccine formulation comprising: a) an attenuated ASF virus vaccine; and b) an intranasal delivery device.

The invention also provides an intranasal delivery device comprising an attenuated ASF virus vaccine. Suitable devices for intranasal administration of vaccine are well known in the art, for example, a syringe or dropper, an aerosol device (e.g. Omron, Philips respironics InnoSpire Deluxe, Devilbiss mask/nebuliser and compressor BreathEazy), a mucosal atomisation device (e.g. LMA MAD Nasal.TM., Teleflex VaxlNator.TM.), a single or multidose spray pump, a unit dose powder dispenser or a bidose powder dispenser.

The attenuated ASF virus may be any suitable attenuated ASF virus. In one embodiment, the attenuated ASF virus may be one which is not considered suitable for use in the prevention or treatment of African Swine Fever when administered by a different route of administration, such as the intramuscular route, for example due to an unacceptable safety profile or low efficacy. In particular, the attenuated ASF virus may be OURT88/3. The attenuated ASF virus may alternatively be an attenuated ASF virus which lacks a functional version of the multigene-family 360 genes 9L, 10L, 11L, 12L, 13L and 14L and multigene-family 505 genes 1R, 2R, 3R and 4R, and/or lacks a functional version of DP148R. The attenuated ASF virus may be any suitable attenuated ASF virus as described herein, such as Benin.DELTA.MGF or Benin.DELTA.DP148R.

Intranasal administration according to the invention may be in a droplet, spray or dry powder form, or may be a nebulised or aerosolised vaccine formulation. The dose is typically 2 ml, administered as 1 ml per nostril.

The present inventors have found that intranasal administration of attenuated ASF virus is effective at a lower dose compared to intramuscular administration. For example, in Example 6, pigs immunised intranasally with 10.sup.3 or 10.sup.4 of the OURT883 had complete (100%) protection against lethal challenge, compared to much lower survival rates in pigs immunised with the same doses via the intramuscular route.

The dose for intranasal immunisation may therefore be 10.sup.4 HAD.sub.50 or TCID.sub.50 or less per pig. For example the dose may be between 10.sup.2-10.sup.3 HAD.sub.50 or TCID.sub.50. The dose may be about 10.sup.2 HAD.sub.50 or TCID.sub.50 per pig.

The vaccine may be administered following a prime-boost regime. For example, after the first inoculation, the subjects may receive a second boosting administration some time (such as about 7, 14, 21 or 28 days) later. Typically the boosting administration is at a higher dose than the priming administration. The boosting dose may be about 10.sup.2, 10.sup.3 or 10.sup.4 HAD.sub.50 or TCID.sub.50 of the recombinant attenuated virus per pig.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

Example 1--Isolation of Recombinant Virus Benin.DELTA.MGF

The MGF 360 genes 10L, 11L, 12L, 13L, 14L and MGF 505 genes 1R, 2R, 3R were deleted from the ASFV Benin 97/1 isolate. In addition the ATG codons of MGF 360 9L and of MGF 505 4R were deleted to interrupt expression of these genes. The genes deleted were replaced with the GUS gene under control of the ASFV p72 promoter.

This was achieved by homologous recombination between plasmid p.DELTA.MGFGUS and the virus genome (see Materials and Methods and FIG. 1). Recombinant viruses were identified by expression of the GUS gene and purified by infection at limiting dilution. Two independent viruses Benin.DELTA.MGFA2 and Benin.DELTA.MGFA1 were isolated using this method to reduce the possibility that the phenotype of the gene deletion was associated with mutations which may have occurred elsewhere on the genome.

Recombinant virus Benin.DELTA.MGFA2, was further characterised. Genomic DNA was isolated from wild type Benin 97/1 and Benin.DELTA.MGFA2 and analysed by PCR to test for the insertion of the GUS marker gene and deletion of the eight MGF genes (FIG. 2). Primers BeninD8F and BeninD8R were designed to anneal within the MGF 360 9L gene, and the MGF360 4R gene, that flank the insertion site. PCR using these primers amplified a 2046 bp fragment using recombinant virus Benin.DELTA.MGFA2 gDNA as template (FIG. 2 lane 1).

The size of this band is consistent with that of a PCR product in which the eight MGF genes have been deleted and have been replaced by the GUS marker gene under the control of the ASFV vp72 promoter. To confirm that recombinant virus Benin.DELTA.MGFA2 contains the GUS gene, a PCR was carried out using an internal GUS gene primer, RGUS, and primer Benin.DELTA.8F. This PCR amplified a fragment consistent with the expected 1552 bp size (FIG. 2 lane 2) when DNA from Benin.DELTA.MGFA2 was used as template. As expected, no PCR fragment was detected using these primers with wild type Benin 97/1 gDNA as template (FIG. 2 lane 3). To confirm that the recombinant virus Benin.DELTA.MGFA2 did not contain the MGF 360 10L gene, PCR reactions were carried out using primers Benin.DELTA.8INTF and Benin.DELTA.8INTR located at positions 19613 and 20110 within the MGF 360 10L gene. No PCR fragment was isolated using the recombinant virus Benin.DELTA.MGFA2 gDNA as template (FIG. 2 lane 3), but as expected a 498 bp fragment was isolated using wild type Benin 97/1 gDNA as template (FIG. 2 lane 4). Taken together, the PCR data showed that recombinant virus Benin.DELTA.MGFA2 contains the GUS gene in place of the eight MGF genes. To additionally confirm that the eight MGF genes had been deleted, gDNA from virus Benin.DELTA.MGFA2 was isolated and the junction at the site of deletion/insertion was sequenced using the primers X and Y. Analysis of the sequence revealed that the eight MGF genes had been deleted and the GUS marker gene had been inserted (FIG. 3). The first five bp of the flanking gene MGF 360 9L and the first seven bp of the MGF 505 4R flanking gene had also been deleted. Because the additional deleted sequences each contained the ATG start codon, it can be concluded that the MGF 360 9L and MGF 505 4R genes are not expressed by the recombinant virus Benin.DELTA.MGFA2.

Example 2--Growth Characteristics of Recombinant Virus Benin.DELTA.MGFA2

To investigate whether deletion of the eight MGF genes affected virus replication, the growth of Benin.DELTA.MGFA2 was compared to parental Benin 97/1 virus in primary porcine bone marrow macrophages. Cells were infected at high (10 HAD50/cell) multiplicity of infection (m.o.i.) and total virus was harvested from supernatants at different times post-infection. FIG. 4 shows that there were no significant differences between the titres of wild type Benin 97/1 and those of Benin.DELTA.MGFA2 viruses recovered at any of the time points measured. This shows that deletion of the eight MGF genes did not significantly affect the replication of Benin.DELTA.MGFA2 in primary porcine bone marrow macrophages.

Example 3--Inoculation of Pigs with Recombinant Virus Benin.DELTA.MGF and Attenuated Virus OURT88/3

One group of three pigs (Group 1) were immunised intramuscularly with 10.sup.2 HAD of deletion virus Benin.DELTA.MGFA2, a second group of two pigs (Group 2) were inoculated with 10.sup.2 HAD of deletion virus Benin.DELTA.MGFA1 and a third group of four pigs (Group 3) were inoculated with 10.sup.4 TCID.sub.50 of attenuated virus OURT88/3. Clinical scores and temperatures for each pig were recorded every day and blood samples were taken every seven days. The results of these recordings are shown in FIG. 5 and FIG. 6 and show that none of the pigs had a clinical score above 3 on any day post-inoculation. Four pigs (Group 1 pigs 21 and 22, Group 2 pigs 23 and 25) had a temperature above 40.degree. C. but this was for one or two days only.

On day 20 post-inoculation, 20 ml of blood was taken from each pig, PBMCs were purified and Group 1 and 2 pig cells were stimulated with Benin.DELTA.MGFA2 and Benin.DELTA.MGFA1 viruses respectively. PBMCs isolated from Group 3 pigs were stimulated with OURT88/3 virus isolate and the numbers of IFNg producing cells from all three Groups were measured by ELIspot. The results show that three out of four pigs from Group 3 had a high IFNg response to OURT88/3, whereas none of the pigs from Groups 1 and 2 had a high IFNg response to Benin.DELTA.MGF or OURT88/3 (FIG. 7). At 21 days after the first inoculation, pigs in Groups 1 and 2 were boosted intramuscularly with 10.sup.4 HAD of viruses Benin.DELTA.MGFA2 and Benin.DELTA.MGFA1 respectively and pigs in Group 3 with 10.sup.4 TCID.sub.50 of OURT88/3. Clinical observations showed that only one pig (Group 3, pig 19) had a clinical score above score 2 (FIG. 8) and that only one pig (Group 3 pig 19) had a temperature above 40.degree. C. for 1 day (FIG. 9).

Example 4--Challenge of all Pigs with Virulent Benin 97/1

On day 46 post initial inoculation (21 days post-boost) all three groups of pigs (Groups 1, 2 and 3) were challenged intramuscularly with 10.sup.4 HAD of virulent virus Benin 97/1. In addition, a control group of three un-inoculated pigs (Group 4) were also challenged with 10.sup.4 HAD of virulent virus Benin 97/1. All three pigs from Group 4 and one pig (pig 19) from Group 3 developed high temperatures and high clinical scores (above score 4) and were terminated at five days post-challenge because the humane end-point on the animal license had been reached (FIGS. 10 and 11). All five pigs from Groups 1 and 2 and the remaining three pigs from Group 3 were protected against challenge with virulent virus Benin 97/1 and continued to be healthy up to day 63 when the experiment was terminated (FIG. 12).

At day 63 post initial inoculation, PBMCs were isolated from blood and cells isolated from spleen and these were stimulated with virus strains and numbers of IFN.gamma. producing cells detected by ELIspot assay. Results showed that all three pigs from Group 3 showed high IFNg responses in cells from both blood and spleen, whereas only one pig (pig 24, Group 2) from Groups 1 and 2 showed high IFN.gamma. responses in blood and spleen (FIG. 13).

Example 5--Characterisation of the Immune Response Generated by Benin.DELTA.MGF

Infection with Benin.DELTA.MGF was shown to decrease the number of circulating gamma delta CD8+ T cells as early as day 10 post infection. Higher numbers of circulating CD8+CD4-.gamma..delta.TCR- (CD8 only) and CD8+CD3- (NK cells) cells were observed in pigs infected with OURT88/3 at late days post infection (days 53 and 63). No other significant differences between OURT88/3 and Benin.DELTA.MGF infected animals were observed for the other cell populations studied (total CD4+, CD4+CD8+ and total gamma delta T cells) (see FIGS. 14 to 20).

Conclusions

Inoculation of Pigs with Benin.DELTA.MGF Demonstrates the Viruses are Attenuated

Group 1 and 2 pigs were inoculated with 10.sup.2 HAD of the independently isolated recombinant viruses Benin.DELTA.MGFA2 and Benin.DELTA.MGFA1. None of the pigs showed a clinical score above score 3 and no pigs showed a temperature above 40.5.degree. C. This showed that deletion viruses Benin.DELTA.MGFA2 and Benin.DELTA.MGFA1 were attenuated and short transient fever (1 or 2 days) was detected in 4 of the 5 pigs but no other clinical signs associated with ASFV infection.

Pigs in Groups 1 and 2 were boosted intramuscularly with 10.sup.4 HAD of viruses Benin.DELTA.MGFA2 and Benin.DELTA.MGFA1. None of the pigs showed a clinical score above score 2 and no pigs showed a temperature above 40.1.degree. C. Similar results were observed following boost of the pigs in Group 3 with the attenuated strain OURT88/3.

Challenge of Pigs Immunised with Benin.DELTA.MGF with a Lethal Dose of Benin 97/1 Demonstrates 100% are Protected

Pigs from Groups 1, 2 and 3 and three un-inoculated control pigs were challenged with 10.sup.4 HAD of virulent virus isolate Benin 97/1. All five pigs (100%) from Groups 1 and 2 (Benin.DELTA.MGFA2 and Benin.DELTA.MGFA1) were protected against virulent virus challenge. 75% of pigs in Group 3 and 0% of pigs in Group 4 were protected against virulent Benin 97/1 (FIG. 12). After challenge with Benin 97/1 no pigs in Groups 1 and 2 showed a temperature above 39.6.degree. C. and no pigs had a clinical score above 2. In contrast all pigs in Group 4 showed temperatures above 40.8.degree. C. and had clinical scores above score 8.

The deletion viruses Benin.DELTA.MGFA2 and Benin.DELTA.MGFA1 showed a better level of protection (100%) compared to the attenuated strain OURT88/3 (75%) against challenge with virulent Benin 97/1.

Materials and Methods

Cells and Viruses

Non-virulent, non-heamabsorbing ASFV isolate OUR T88/3 was obtained from Ourique in Portgual. Virulent, haemabsorbing isolate Benin 97/1 has been previously described. Both OUR T88/3 and Benin 97/1 are p72 genotype I viruses were grown in primary macrophage cultures derived from bone marrow. Titres of virus were determined as the amount of virus causing haemadsorption (for HAD isolates) or cytopathic effects (for non-HAD isolates) in 50% of infected cultures (HAD.sub.50/ml or TCID.sub.50/ml).

Construction of Plasmid Transfer Vector pDMGFGUS

The plasmid vector p.DELTA.MGFGUS was constructed to facilitate deletion of the eight MGF genes (MGF 360 10L, 11L, 12L, 13L, 14L and MGF 505 1R, 2R, 3R) from the genome of Benin 97/1. Using Benin 97/1 genomic DNA as template a 479 bp fragment (Flank L) located at the 3' terminus of the MGF 360 9L gene at position 19004 19482 immediately upstream of the MGF 360 10L gene was amplified using the primers FlankLF (ACGTTGCAAAGCTTCCATTAATCCCTCCAGTTGTTC) (SEQ ID NO: 20) and FlankLR (ACGTTGCAGGTACCCCTCTCTCTGCAGACTCTCACC) SEQ ID NO: 21). Using Benin 97/1 genomic DNA as template, a 501 bp fragment (Flank R) located at the 5' terminus of the MGF 505 4R gene at position 30033 30533 immediately downstream of the MGF 505 3R gene was amplified using the primer FlankRF (ACGTTGCAGCGGCCGCCTCTCCAAGACATCTGTCGG) (SEQ ID NO: 22) and primer FlankRR (ACGTACGTCTCGAGCCTCACATGCCATCTCAAACAATTCC) (SEQ ID NO: 23). The FlankL fragment was digested with HindIII and KpnI and ligated into vector pP72loxPGUS which was also digested with HindIII and KpnI to create plasmid pFlankL-GUS. The FlankR fragment was digested with NotI and XhoI enzymes and ligated into into the vector pFlankL-GUS digested with the same enzymes to create the transfer vector pDMGFGUS. The plasmid pDMGFGUS contains a GUS marker gene flanked on the left hand side by the 3' terminal section of the MGF 360 9L gene, an ASFV vp72 promoter sequence and a loxP sequence. To the right side of the GUS gene is located the 5' terminal sequence of the MGF 505 4R gene. (FIG. 1).

Construction and Isolation of Recombinant Virus Benin.DELTA.MGF

Primary porcine alveolar macrophages (35 mm dish, 10.sup.6 cells) were infected with Benin 97/3 at a multiplicity of infection (m.o.i). of 10 and incubated at 37.degree. C. for 5 hours, and then washed with Earle's saline (10% porcine serum, penicillin/streptomycin 10,000 u mg-1 ml-1). A transfection mixture containing 250 .mu.l Optimem (Gibco-Life Technologies), 5 .mu.g p.DELTA.MGFGUS and 7.5 .mu.l TRANS-IT LT-1 (Mirus) transfection reagent was incubated at 20.degree. C. for 20 minutes before adding it to the infected cells. Incubation was continued at 37.degree. C. for 4 hours before the addition of 1 ml Earle's saline and continued incubation at 37.degree. C. Virus was harvested from the infected and transfected cells 72 hours post-infection and cell debris removed by centrifugation. Aliquots of virus containing supernatant were used to infect bone marrow macrophages on 96 well plates. At 90 hours post-infection Earle's saline containing 100 .mu.g/ml 5-Bromo-4-chloro-1H-indol-3-yl .beta.-D-glucopyranosiduronic acid (X-Gluc) was added and wells appearing `blue` containing recombinant GUS expressing viruses were harvested. Infections at limiting dilution were further carried out on pig bone marrow macrophages containing X-Gluc until only one blue well per 96 well plate was observed to indicate infection with the recombinant deletion virus. No evidence of virus infection (cytopathic effect (cpe)) was observed in the other 95 wells. Two independent recombinant viruses (Benin.DELTA.MGFA2 and Benin.DELTA.MGFA1) were isolated using this method. High titre stocks of recombinant viruses Benin.DELTA.MGFA2 and Benin.DELTA.MGFA1 were grown up on porcine bone marrow macrophages.

Growth Characteristics of Recombinant Virus Benin.DELTA.MGFA2

The growth of Benin.DELTA.MGFA2 was compared to parental Benin 97/1 virus in primary porcine bone marrow macrophages. Cells were infected at high (10 HAD.sub.50/cell) multiplicity of infection (m.o.i.) and total virus was harvested from supernatants at different times post-infection.

Purification of Viral Genomic DNA and PCR

Viral genomic DNA from Benin.DELTA.MGF and Benin 97/1 virus harvests were purified from 300 ul of supernatant from infected pig bone marrow macrophage infected cells using a GE Healthcare Illustra Genomic Prep Mini Spin kit. Analysis of viral genomic DNA was carried out by PCR using the specific DNA primers BeninD8F (GGTGAGAGTCTGCAGAGAGAGG (SEQ ID NO: 24)), BeninD8R (GCCCTAGCACTTGTAACG) (SEQ ID NO: 25), RGUS (CCTTCTCTGCCGTTTCCAAATCGCCGC) (SEQ ID NO: 26), BeninD8INTF (CGATGTATCATTGATGTC) (SEQ ID NO: 27), BeninD8INTR (GGATAATCTTAGGGAGGCC) (SEQ ID NO: 28).

Sequencing of Recombinant Virus Benin.DELTA.MGFA2

Viral gDNA was isolated from the harvest of BeninDMGF infected cells and sequenced using the following primers 9LF (ATGACGCATTAAACCGGCG) (SEQ ID NO: 29) and 4RR (CAGTATAGCCCTAGCACTTG) (SEQ ID NO: 30)

ELIspot

IFN-g ELIspot was carried out as previously described (Gerner et al 2006 Virus Res>121:223-228).

Pig Inoculation and Challenge

Pigs used were cross-bred, large white.times.Landrace, of average weight 15 kg at the first inoculation. All pigs were maintained in high security SAPO4 facilities throughout and the experiment performed under Home Office licence PPL 70/7198-. One group of three pigs (Group 1, pigs 21, 22 and 23) were inoculated intramuscularly with 10.sup.2HAD.sub.50 of recombinant virus BeninDMGFA2. A second group of two pigs (Group 2, pigs 24 and 25) were inoculated intramuscularly with 10.sup.2HAD.sub.50 of recombinant virus Benin.DELTA.MGFA1. A third group of four pigs (Group 3, pigs 16, 18, 19 and 20) were inoculated with 10.sup.4 TCID.sub.50 of attenuated strain OURT88/3. The inventors had previously determined that a dose of 10.sup.4 TCID.sub.50 of OURT88/3 was needed to induce protection and that 10.sup.3 was less effective.

Three weeks later to boost the ASFV specific adaptive immune responses, pigs in Groups 1 and 2 were inoculated with 10.sup.4HAD.sub.50 of recombinant viruses BeninDMGFA2 and BeninDMGFA1 respectively and Group 3 pigs were immunised with 10.sup.4TCID.sub.50 of OURT88/3. Three weeks post-boost Groups 1, 2, 3 and a fourth group of three pigs (Group 4, pigs 26, 27 and 28) containing three non-immunised pigs, were challenged intramuscularly with 10.sup.4HAD.sub.50 of virulent ASFV isolate Benin 97/1. ASFV-inoculated and challenged pigs were monitored daily for body temperature and clinical signs and these were scored as reported by King et al., 2011. All pigs were examined by post-mortem at termination and spleen and lymph tissues were collected.

Characterisation of T Cell Subpopulations

Blood samples were collected from the infected pigs at different times post infection and the different T cell populations were identified by using the following Zenon (Invitrogen) labelled antibodies: mouse IgG1 anti-porcine CD3 (Alexa Fluor 405), mouse IgG2b anti-porcine CD4 (Alexa Fluor 647), mouse IgG2a anti-porcine CD8.alpha..alpha. (R-PE) and mouse IgG2b anti-porcine gamma delta TCR (Alexa Fluor 488). Briefly, 100 .mu.l of whole blood was incubated with 5 .mu.l of each conjugated antibody for 20 minutes at room temperature. Red blood cells were lysed by adding 4 ml of 1.times.BD FACS lysing solution (BD Biosciences) and vortexing. Cells were pelleted by centrifugation at 1200 rpm for 7 minutes and the supernatant was discarded. The cells were then fixed with 4% paraformaldehyde for 30 minutes, washed twice with PBS and finally analysed by flow cytometry (MACSQuant, Miltenyi Biotec). Lymphocyte subsets were gated based on cell surface markers staining using FCS express software.

Example 6--Immunisation with OUR T88/3 Via Different Routes

The experiments were carried out in level-3 biocontainment facilities (BSL-3) of Centre de Recerca en Sanitat Animal (CReSA, Barcelona, Spain). All animal experiments were carried out under UK Home Office Licence number 70/7198 with the approval of the Ethics Committees for Animal Experiments of the Autonomy University of Barcelona (No. 1189R5) and the Regional Government of Catalonia, Spain (No. 5796), and complied fully with the regulated procedures from the Animals (Scientific Procedures) Act 1986.

Materials and Methods

7-week-old Large White and Pietrain crossbred male piglets in good health, vaccinated against Porcine Circovirus type 2 and Mycoplasma hyopneumoniae, average weight 15 kg, from a high health herd tested negative for Porcine Respiratory and Reproductive syndrome (PRRS) and Aujeszky's disease were used. After a 5-day acclimatisation period, three groups of six pigs each were immunised intramuscularly (IM) with 1 ml containing 10.sup.3 (group A), 10.sup.4 (group C) and 10.sup.5 (group E) TCID.sub.50/ml of low virulent ASFV isolate OUR T88/3 respectively. An additional three groups of six pigs each were immunised intranasally (IN), using a mucosal atomization device, with 1 ml per nostril containing 10.sup.3 (group B), 10.sup.4 (group D) and 10.sup.5 (group F) TCID.sub.50/ml of low virulent isolate OUR T88/3. Three weeks later all immunised groups, together with a control group (group G) containing three non-immunised pigs, were challenged intramuscularly with 1 ml containing 10.sup.4 TCID.sub.50/ml of the closely related virulent ASFV isolate OUR T88/1. This control group was housed separately (room 4), while pigs immunised with the same ASFV titre, but by different inoculation routes (IN or IM), were allocated in the same isolation room separated by 1 m high partition as follows: experiment 1 (groups A and B), experiment 2 (groups C and D), experiment 3 (groups E and F).

Sampling, Clinical and Post-Mortem Examination

Immunisation day was defined as day 0 (0 dpi). Rectal temperatures and clinical signs were monitored daily prior to immunisation and throughout the study, following a clinical score previously reported (King et al., 2011). EDTA blood and serum samples were collected from all pigs prior to virus immunisation (0 dpi), after immunisation (at 3, 5, 7, 14 and 21 dpi) and after challenge (at 3, 5, 7, 14 and 19 dpc).

A post-mortem examination also was carried out in order to evaluate gross lesions of dead pigs or pigs euthanized during the experiment upon reaching a predetermined humane endpoint, as well as at the end of the experiment at 19 days post-challenge (dpc). Macroscopic lesions were evaluated in accordance with the standardized pathological framework of ASFV infections (Galindo-Cardiel et al., 2013). The humane end point was determined in accordance with the welfare regulations specified in the UK Home Office Licence, so that pigs with a rectal temperature over 40.5.degree. C. for three consecutive days, or showing three or more clinical signs of disease combined on a single day, were euthanized. Euthanasia was conducted by intravenous injection of pentobarbital sodium.

ASFV Detection and Immune Response Evaluation.

After collection, EDTA blood samples were frozen at -80.degree. C. until ASFV detection by quantitative PCR (qPCR) as described previously (King et al., 2003). Frozen tissues samples (spleen, tonsil, lung, submandibular, retropharyngeal and gastrohepatic lymph nodes) were also analysed for the presence of ASFV by qPCR. Serum samples were also frozen at -80.degree. C. until assayed by commercially available ELISA kits for detection of antibodies against Vp72 structural protein of ASFV (INGEZIM PPA Compac, Ingenasa Madrid, Spain). In addition, serum samples were used to evaluate, by commercially available ELISA kits (R&D Systems, Abingdon, UK), porcine cytokines with inflammatory and immunological functions during humoral and cell mediated immunity in pigs (IL-1.beta., TNF.alpha., IFN.gamma., IL-4 and IL-10). Cytokine concentrations were presented as pg/ml.

Statistical Analyses

Data were analysed using the statistical analysis program GraphPad Prism Version 6.0 (GraphPad Software). The values of rectal temperature, clinical signs, macroscopic lesions, viraemia, ASFV antibodies and cytokine levels were assessed to calculate means.+-.standard deviations (SD). For analysis of temperature and clinical signs, differences between baseline values (day 0) and the values obtained at each time-point in the un-inoculated control group and inoculated groups were analysed using One-way ANOVA with Bonferroni post-test. Furthermore, differences between the un-inoculated control group and infected groups as well as differences between both infected groups at the same time-point were analysed using a two-way ANOVA with Bonferroni post-test. For all comparisons, differences were considered significant at P<0.05.

Results from Control Non-Immune Pigs

From 3 dpc, pigs in control Group G (non-immune) displayed non-specific symptoms such as fever (40.8-41.7.degree. C.) and apathy (see FIG. 21 G). These clinical signs increased progressively until 5 dpc, with rectal temperatures between 41.4-41.6.degree. C., recumbence and the presence of skin erythema and cyanotic areas on tip of ears, being euthanized for ethical reasons. After euthanasia of pigs at 5 dpc, necropsies revealed the presence of gross lesions characteristic of acute forms of ASF such as hemorrhagic lymphadenitis (gastrohepatic and renal lymph nodes being the most severely affected), hyperemic splenomegaly, non-collapsed lungs with interstitial and alveolar edema as well as foam in trachea, petechiae in kidneys (cortex and medulla) and lungs, retroperitoneal edema and moderate hepatic congestion.

Results from Groups Immunised by Intranasal Route

Of those pigs immunised with OURT88/3 by the intranasal route 100% (n=6) of those in groups B (10.sup.3 TCID.sub.50) and D (10.sup.4 TCID.sub.50) survived challenge. Some of the pigs that survived displayed a transient moderate joint swelling before and after challenge. Two pigs from group B (B3 and B4) and 3 from group D (D2, D4 and D5) showed other short transient clinical signs post-challenge (temperature, inappetence, apathy). In group F (10.sup.5 TCID.sub.50) 66% survived challenge. The two pigs which did not survive (F3 and F5) were euthanized at day 5 post-challenge showing gross lesions characteristic of ASF and other some other clinical signs (elbow swelling and erosion of skin on the nose). Three surviving pigs (F1, F2 and F4) also showed clinical signs and lesions including severe joint swelling, laboured breathing, erythema on ears, conjunctivitis as well as skin erosions/ulcers in nose, flanks and limbs that lasted until the end of the experiment.

Results from Groups Immunised by the Intramuscular Route

Of the pigs immunised by the intramuscular route, 3 (E2, E3 and E6) from group E (10.sup.5 TCID.sub.50) were euthanized or found dead between 13 and 14 dpi and before challenge showing signs typical of ASF. Of those pigs challenged 50% (3/6) from Group A (10.sup.3 TCID.sub.50), 66% (4/6) from group C (10.sup.4 TCID.sub.50) and 33% (2/6) from Group E (10.sup.5 TCID.sub.50) survived challenge.

Statistical Analysis of Results

Comparative statistical analysis of clinical scores (5 days before death) developed by pigs that died or were euthanized inside each experimental group (including pigs dead before challenge in group E and non-immunised control pigs), revealed similar kinetics as well as non-significant differences in reached temperatures among groups (FIG. 22). In addition, kinetics of clinical scores was similar, significant differences appeared just between pigs dead in group F (IN, 10.sup.5) and group A (IM, 10.sup.3) (FIG. 24). On the other hand, statistical analysis of clinical scores in pigs that survived revealed that after challenge, surviving pigs in group F (IN, 10.sup.5) showed significant differences in clinical scores with respect to surviving pigs including in other groups until the end of the study (FIG. 24).

During macroscopic evaluation of surviving pigs, lesions were mainly observed in cardiorespiratory system, skin and musculoskeletal system. In general, surviving pigs immunised intramuscularly (9 pigs in all) displayed no lesions or minimal pulmonary lesions (6/9 pigs), while surviving pigs immunised intranasally (16 pigs in all) showed cardiorespiratory lesions (12/16 pigs) as well as injuries in skin and joints (7/16 pigs). In this sense, while lesions were moderate in surviving pigs immunised intranasally with 10.sup.3 and 10.sup.4 (groups B and D), surviving pigs inoculated intranasally with 10.sup.5 (group F) displayed the most intense cardiorespiratory and musculoskeletal injuries (fibrinous pleuritis, fibrinonecrotic pleuropneumonia, fibrinous pericarditis and serofibrinous/purulent periarthritis), cardiorespiratory lesions being compatible with the presence of secondary bacterial infections. Statistical analysis of macroscopic lesion scores confirmed the significant differences among surviving pigs in group F (IN, 10.sup.5) and the other groups of surviving animals (FIG. 25).

Discussion

These results have demonstrated a complete protection (100%) in pigs immunised intranasally with 10.sup.3 and 10.sup.4 TCID.sub.50/ml of low virulent ASFV isolate OUR T88/3. Pigs displayed minimal and transient adverse clinical reactions before and after challenge with the virulent ASFV isolate OUR T88/1 as well as mild lesions in lungs, associated to secondary bacterial infections, and joints mainly. We predict from our results that a lower dose (eg 10.sup.2 TCID.sub.50) may also induce high levels of protection against lethal challenge.

However, in both the group of pigs immunised intranasally with 10.sup.5 TCID.sub.50/ml and in all groups immunised intramuscularly, the rate of protection conferred was lower. The lowest survival rates were observed in pigs immunised intramuscularly with 10.sup.5 TCID.sub.50/ml, where 3 of the 6 immunised pigs (E2, E3 and E6) died before challenge. The results show that intranasal immunisation of pigs with live attenuated ASFV vaccines is an alternative to the previously reported intramuscular route. Intranasal immunisation induced fewer clinical signs before challenge compared to the intramuscular route and a higher percentage of pigs were protected.

Example 7--Immunisation with Benin.DELTA.MGF by Different Routes

This involved testing delivery of the gene deleted attenuated ASFV Benin.DELTA.MGF at three different doses using the IM route (10.sup.2, 10.sup.3, 10.sup.4 HAD.sub.50) and by one dose (10.sup.3 HAD.sub.50) using the intranasal route and challenge with parental virulent virus.

Materials and Methods

The experiments were carried out in SAPO-4 biocontainment facilities at The Pirbright Institute. All animal experiments were carried out under UK Home Office Licence number 70/7198 and complied fully with the regulated procedures from the Animals (Scientific Procedures) Act 1986.

Large White Landrace crossbred female piglets in good health, average weight 15-20 kg, from a high health herd were used. After a 5-day acclimatisation period, three groups of six pigs each were immunised intramuscularly (IM) with 1 ml containing 10.sup.2 (group A), 10.sup.3 (group B) and 10.sup.4 (group C) HAD.sub.50 of attenuated ASFV gene deleted Benin.DELTA.MGF strain respectively. An additional group of six pigs were immunised intranasal (IN), using a mucosal atomization device, with 1 ml per nostril containing 10.sup.3 Benin.DELTA.MGF (group D). Three weeks later all immunised groups were boosted with the same dose of virus by the same route. After a further 19 days pigs in groups A to D, together with a control group (group F) containing six non-immunised pigs, were challenged intramuscularly with 1 ml containing 10.sup.4 TCID.sub.50/ml of the parental virulent ASFV isolate Benin 97/1. This control group was housed separately, while the other pigs were allocated in the same isolation room separated by 1 m high partition as follows: room 1 (groups A and B), room 2 (groups C and D).

Sampling, Clinical and Post-Mortem Examination.

Immunisation day was defined as day 0 (0 dpi). Rectal temperatures and clinical signs were monitored daily prior to immunisation and throughout the study, following a clinical score previously reported (King et al., 2011). EDTA blood and serum samples were collected from all pigs prior to virus immunisation (0 dpi), after immunisation (at 2, 4, 7, 10, 14 and 21 dpi) boost and after challenge (at 3, 5, 7, 14 and 19 dpc).

A post-mortem examination also was carried out in order to evaluate gross lesions of dead pigs or euthanized during the experiment upon reaching a predetermined humane endpoint, as well as at the end of the experiment. Macroscopic lesions were evaluated in accordance with the standardized pathological framework of ASFV infections (Galindo-Cardiel et al., 2013). The humane end point was determined in accordance with the welfare regulations specified in the UK Home Office Licence, so that pigs with a rectal temperature over 40.5.degree. C. for three consecutive days, or showing three or more clinical signs of disease combined on a single day, were euthanized. Euthanasia was conducted by intravenous injection of pentobarbital sodium.

Results Pigs were immunised and observed daily for clinical signs including temperatures. Results are shown in FIG. 29. Of the pigs in groups A (10.sup.2) and B (10.sup.3) 4/6 developed a transient low fever for 1 or 2 days from day 4 or 5 post-immunisation. In group C (IM 10.sup.4) 3/6 pigs developed a transient low fever for 1 or 2 days. In group D (IN 10.sup.3) 1/6 pigs developed a low transient fever at day 5 post-immunisation. No other clinical signs were observed.

The results confirm that increasing the dose of Benin.DELTA.MGF does not increase clinical signs post-immunisation by the IM route. FIG. 29 shows that minimal clinical signs were observed in these and remaining pigs following the boost and until after challenge. Following challenge several pigs were euthanized because they reached moderate severity end-point (3 days fever above 40.5 or 2 days without eating). These included 3 of the 6 pigs from the group immunised with 10.sup.2 IM, 2 pigs immunised with 10.sup.3 IM, 1 pig immunised with 10.sup.4 IM and 2 pigs immunised with 10.sup.3 IN. At post-mortem, lesions were not observed in any group except for 1 pig from group Benin.DELTA.MGF 10.sup.2 IM which had a single lung lesion.

A comparison of mean temperatures of the different groups (FIG. 31) showed statistically significant differences between the group immunised with Benin.DELTA.MGF 10.sup.2 IM and other groups post-immunisation at day 4 (higher compared to all other groups), day 15 (higher compared to Benin.DELTA.MGF 10.sup.3 IM, 10.sup.3 IN and 10.sup.4 IM). Post-boost statistically significant differences were observed between Benin.DELTA.MGF IM (higher compared to Benin.DELTA.MGF 10.sup.3 IN and Benin.DELTA.DP148R). At day 12 post-challenge pigs immunised with Benin.DELTA.MGF 10.sup.2 IM had a significantly higher temperature compared to pigs immunised with Benin.DELTA.MGF 10.sup.4 IM.

Pigs immunised with Benin.DELTA.MGF 10.sup.3 IN had significantly different temperatures at days post-immunisation 4 (lower compared to Benin.DELTA.MGF 10.sup.3 IM, 10.sup.4 IM and Benin.DELTA.DP148R), post-immunisation day 6 (higher compared to Benin.DELTA.DP148R), day 11 post-challenge (higher compared to Benin.DELTA.MGF 10.sup.4 IM). Pigs immunised with Benin.DELTA.MGF 10.sup.4 IM had statistically significant temperatures at day 8 post-immunisation (higher compared to Benin.DELTA.MGF 10.sup.3 IM, 10.sup.3 IN) and day 9 post-immunisation (higher compared to Benin.DELTA.DP148R). Pigs immunised with Benin.DELTA.MGF 10.sup.3 IM had significantly different temperatures at day 12 post-immunisation (lower compared to Benin.DELTA.MGF 10.sup.4 IM and 10.sup.3 IN).

A comparison of mean clinical scores of the different groups (FIG. 32) showed statistically significant differences between the group immunised with Benin.DELTA.MGF10.sup.2 IM and other groups post-immunisation at day 2 (higher than Benin.DELTA.MGF 10.sup.3 IM, 10.sup.3 IN, Benin.DELTA.DP148R), day 4 (higher than Benin.DELTA.MGF 10.sup.3 IM, 10.sup.4 IM, 10.sup.3 IN) and day 5 (lower than Benin.DELTA.DP148R, higher than Benin.DELTA.MGF 10.sup.3 IM, 10.sup.3 IN).

In conclusion, Benin.DELTA.MGF 10.sup.2 IM showed significantly higher clinical scores and lower temperatures than other groups post-immunisation.

Example 8--IFN-.beta. mRNA Induction by Different ASFV Isolates

Porcine alveolar macrophages were infected with ASFV isolates indicated at multiplicity of infection 3 (Benin 97/1, Benin.DELTA.MGF, OURT88/3) or were mock-infected. At different times post-infection (2, 4, 6, 8, 10, 12, 16, 20 hours) RNA was harvested from infected cells and levels of mRNA for IFN-pa RNA, GAPDH and ASFV B646L gene (VP72) were measured by quantitative reverse transcriptase PCR. FIG. 26 panel A shows levels of IFN-.beta. compared to control housekeeping gene GAPDH. Panel B shows levels of mRNA for ASFV B646L (VP72) gene.

The deletion mutant Benin.DELTA.MGF has a deletion or interruption of 6 members of MGF360 and 4 members of MGF 505 from a region close to the left end of the Benin 97/1 genome. Deletion of these genes increases the induction of IFN-.beta. mRNA in macrophages infected with Benin.DELTA.MGF360 compared to parental virulent virus in which IFN-.beta. mRNA was barely detected (see FIG. 26). Type I IFNs induce expression of IFN-stimulated genes which are involved in activation of host innate and adaptive immune response pathways and in creating an antiviral state. Our hypothesis is that induction of type I IFN is important for attenuation of virulent ASFV and induction of a protective immune response.

Example 9--Deletion Mutant Benin.DELTA.DP148R

A single gene, DP148R, was deleted from a region close to the right end of the virulent Benin97/1 genome. To achieve this a 529 bp fragment from the left and 740 bp fragment from the right regions flanking the MGF36018R gene were amplified by PCR. FIG. 27 shows the primers used to amplify the left flanking region (360-18RFlankL) and those to amplify the right flanking region (360-18RFlankR). The sequences amplified are between these primers. Shaded in grey is the sequence of the MGF360-18R gene which was deleted from the ASFV genome. The start and stop codons are shown in bold. The flanking regions were cloned either side of a reporter gene consisting of the .beta.-Glucorinidase (.beta.-GUS) gene downstream from the ASFV VP72 promoter. This plasmid was transfected into pig macrophages infected with the Benin 97/1 isolate. Progeny virus was tested for expression of the .beta.-GUS gene and recombinant viruses in which the .beta.-GUS gene replaced the MGF360-18R gene was isolated by limiting dilution. The deletion of the gene and location of the insertion was confirmed by PCR analysis using primers that were from the ASFV genome outside of the regions cloned in the transfer plasmid (see FIG. 27).

Deletion of the DP148R Gene does not Reduce Virus Replication in Macrophages

Replication of the Benin.DELTA.MGF36018R virus strain in porcine macrophages was compared with parental Benin 97/1 virus. Porcine alveolar macrophages were infected at a multiplicity of 3 and at different times post-infection (0, 24, 48, 72, 96 hours) virus from cells and supernatants was harvested and titrated using porcine alveolar macrophages. The results (FIG. 28) showed that deletion of the MGF360 18R gene did not reduce virus replication in macrophages.

Deletion of the DP148R Gene Attenuates ASFV in Pigs

A group of 5 male large white/landrace crossbred pigs (15-20 kg) were immunised intramuscularly with 10.sup.3 HAD.sub.50 Benin.DELTA.DP148R and observed for clinical signs. All 5 pigs displayed transient clinical signs for 1 or 2 days at days 4 or 5 post-immunisation. These signs included transient fever, loss of appetite and lethargy.

Pigs were boosted with the same dose of virus by the same route at day 21 post-immunisation and were challenged with virulent Benin97/1 in parallel with control non-immunised pigs at day 39 post-immunisation. No further clinical signs were observed following the boost and all pigs survived challenge (terminated at 19-21 days post-challenge). No lesions were observed at post-mortem.

A comparison of mean temperatures of the different groups (FIG. 31) showed statistically significant differences between the group immunised with Benin.DELTA.DP148R and other groups post-immunisation at day 2 (lower compared to groups Benin.DELTA.MGF 10.sup.2 IM and 10.sup.4 IM) and day 5 (higher compared to groups Benin.DELTA.MGF 10.sup.3 IM, 10.sup.4 IM and 10.sup.3 IN). At days post-boost statistically lower temperatures were observed between Benin.DELTA.DP148R and other groups: 5, (Benin.DELTA.MGF10.sup.2 IM, 10.sup.3 IM) 6 (Benin.DELTA.MGF 10.sup.2 IM), 12 (Benin.DELTA.MGF 10.sup.2 IM, 10.sup.3 IN), 13 (Benin.DELTA.MGF 10.sup.4 IM, 10.sup.3 IN), 14 (Benin.DELTA.MGF 10.sup.2 IM, 10.sup.3 IM, 10.sup.3 IN), 15 (Benin.DELTA.MGF 10.sup.2 IM, 10.sup.3 IM), 17-19 (Benin.DELTA.MGF all groups). Post-challenge significantly lower temperatures were observed between pigs immunised with Benin.DELTA.DP148R and other immunised groups at day 3 (Benin.DELTA.MGF 10.sup.3 IN), 5 (10.sup.2 IM), 6 (10.sup.3 IM).

A comparison of mean clinical scores of the different groups (FIG. 32) showed statistically significant differences between the group immunised with Benin.DELTA.DP148R and other groups post-immunisation at day 4 (higher than Benin.DELTA.MGF 10.sup.2 IM, 10.sup.3 IM, 10.sup.3 IN), day 5 (Benin.DELTA.MGF 10.sup.2 IM, 10.sup.3 IM, 10.sup.3IN, 10.sup.4 IM), day 6 (Benin.DELTA.MGF 10.sup.3IM; 10.sup.3 IN, 10.sup.4 IM) and day 6 (Benin.DELTA.MGF 10.sup.3 IM; 10.sup.3 IN, 10.sup.4 IM).

In conclusion; Benin.DELTA.DP148R demonstrated significantly higher clinical scores and lower temperatures than the other groups post-mortem, and no lesions at post-mortem.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in virology, molecular biology or related fields are intended to be within the scope of the following claims.

SEQUENCE LISTINGS

1

451350PRTAfrican swine fever virus 1Met Val Leu Ser Leu Gln Thr Leu Thr Lys Lys Val Leu Ala Ser Gln1 5 10 15Tyr Pro Ala Lys Cys His Pro His Phe Leu Lys Cys Cys Gly Leu Trp 20 25 30Trp His Asn Gly Pro Ile Met Tyr His Gln Lys Lys Ile Trp Thr Pro 35 40 45Tyr Phe Lys Asn Gly Thr Asn Leu Asn Ala Ala Leu Val Lys Ala Val 50 55 60Glu Glu Asn Asn His Asp Leu Ile Glu Leu Phe Thr Glu Trp Gly Ala65 70 75 80Asn Ile Asn Tyr Gly Leu Leu Ser Val Asn Thr Glu His Thr Arg Asp 85 90 95Leu Cys Arg Gln Leu Gly Ala Lys Glu Gln Leu Asn Asp Gln Glu Ile 100 105 110Leu Arg Phe Phe Tyr Thr Leu Lys Arg Asp Leu Thr Ser Ser Asn Ile 115 120 125Ile Phe Cys His Glu Val Phe Ser Asn Asn Pro Ile Leu Asp Thr Ile 130 135 140Asn Arg Phe Glu Val Lys Gly Met Ile Tyr Glu Gln Leu Glu Gly Leu145 150 155 160Met Val Glu Thr Asp Ile Leu Ser Glu Met Phe Thr Lys Tyr Trp Tyr 165 170 175Ala Met Ala Ile Glu Phe Asn Leu Lys Glu Ala Ile Cys Tyr Phe Tyr 180 185 190Gln Arg Tyr Ala His Leu His Arg Trp Arg Leu Met Cys Ala Leu Phe 195 200 205Tyr Asn Asn Val Phe Asp Leu His Glu Leu Tyr Ala Lys Glu Lys Val 210 215 220Arg Met Asp Met Asp Glu Met Leu Arg Trp Ala Cys Arg Lys Asn Tyr225 230 235 240Asn Tyr Leu Thr Ile Tyr Tyr Cys Cys Val Ala Leu Gly Ala Asp Ile 245 250 255Asn Gln Ala Met Phe His Ser Ile Gln Phe Tyr Asn Ile Gly Asn Ile 260 265 270Phe Phe Cys Ile Asp Leu Gly Ala Asn Ala Phe Glu Glu Gly Lys Thr 275 280 285Leu Ala His Gln Lys Asp Asn Ser Phe Ile Ala Ser Met Leu Ser Leu 290 295 300Asn Cys Tyr Ser Met Asn Asp Ser Leu Ser Leu Lys Glu Thr Asp Pro305 310 315 320Glu Val Ile Lys Arg Met Leu Lys Asp Tyr His Ser Lys Asn Leu Ser 325 330 335Ile Ala His Lys His Tyr Ile Asn Asp Gly Phe Asn Asp Ile 340 345 3502356PRTAfrican swine fever virus 2Met Val Pro Ser Leu Gln Ser Phe Ala Lys Lys Val Leu Ala Ser Gln1 5 10 15His Val Ser Ile Asp Tyr His Val Ile Leu Glu Arg Cys Gly Leu Trp 20 25 30Trp Tyr Lys Ala Pro Ile Ser Leu Asp Cys Lys His Met Leu Ile Lys 35 40 45Leu Pro Asn Phe Ala Asp Gly Leu Asp Leu Asn Thr Ala Leu Met Leu 50 55 60Ala Thr Lys Glu Asn Asn Tyr Gln Leu Ile Lys Met Phe Thr Asp Trp65 70 75 80Gly Ala Asp Ile Asn Tyr Gly Leu Ile Cys Ala Asn Thr Pro Pro Ile 85 90 95Arg Glu Phe Cys Trp Glu Leu Gly Ala Lys Tyr Gln Val Asp Lys Lys 100 105 110Lys Ile Met His Ile Phe Phe Lys Leu Ile His Pro Asn Thr Thr Ser 115 120 125Asn Asn Ile Ile Leu Cys Leu Lys Phe Phe Asn Asp Asn Pro Phe Ser 130 135 140Ala Tyr Val Ile Ile Arg Glu Ile Lys Ser Cys Ile His Trp Lys Leu145 150 155 160Lys Asn Leu Ala Glu Asp Thr Asn Val Leu Ser Asn Ile Ser Asp Gly 165 170 175Asp Met Leu Thr Ile Tyr Cys Phe Ile Val Ala Leu Gln Asp Asn Leu 180 185 190Arg Glu Ala Ile Ser Tyr Val Tyr Gln His Phe Lys Tyr Leu Asn Thr 195 200 205Trp Trp Leu Thr Cys Ala Leu Cys Tyr Asn Lys Leu Phe Asp Leu His 210 215 220Asn Leu Tyr Glu Lys Glu Lys Ile Arg Met Asp Met Asp Glu Met Met225 230 235 240Arg Ile Ala Cys Thr Lys Asp Asn Asn Phe Leu Thr Ile Tyr Tyr Cys 245 250 255Phe Ile Leu Gly Ala Asn Ile Asn Leu Ala Met Ile Ala Ser Ile Arg 260 265 270Phe Tyr Asn Met Asp Asn Leu Phe Phe Cys Ile Asp Leu Gly Ala Asp 275 280 285Ala Phe Glu Glu Ala Lys Ala Leu Ala Glu Gln Gln Asn Tyr Tyr Leu 290 295 300Ile Ser His Arg Leu Ser Leu Asp Ile Tyr Ser Pro Asp Ser Ser Leu305 310 315 320Leu Thr Leu Lys Glu Ala Asp Pro Asn Lys Ile Tyr Arg Leu Leu Lys 325 330 335Asn Tyr Lys Ser Lys Ser Met Leu Ala Tyr Leu Asn Tyr Asp Ile Asn 340 345 350Asp Thr Ser Leu 3553353PRTAfrican swine fever virus 3Met Leu Pro Ser Leu Gln Ser Leu Thr Lys Lys Val Leu Ala Gly Gln1 5 10 15Cys Val Ser Val Asp His Tyr His Ile Leu Lys Cys Cys Gly Leu Trp 20 25 30Trp His Asn Gly Pro Ile Met Leu His Ile Arg Arg Asn Lys Leu Phe 35 40 45Ile Arg Ser Thr Cys Phe Ser Gln Gly Ile Glu Leu Asn Ile Gly Leu 50 55 60Met Lys Ala Val Lys Glu Asn Asn His Asp Leu Ile Lys Leu Phe Thr65 70 75 80Glu Trp Gly Ala Asp Ile Asn Tyr Gly Met Ile Cys Ala Leu Thr Glu 85 90 95Asn Thr Arg Asp Leu Cys Lys Glu Leu Gly Ala Lys Glu Tyr Leu Glu 100 105 110Arg Glu Tyr Ile Leu Lys Ile Phe Phe Asp Thr Thr Arg Asp Lys Thr 115 120 125Ser Ser Asn Ile Ile Phe Cys His Glu Val Phe Ser Asn Asn Pro Asn 130 135 140Leu Arg Ile Ile Asp Asn Leu Asp Leu Arg Gly Glu Ile Met Trp Glu145 150 155 160Leu Arg Gly Leu Met Glu Ile Thr Phe Met Leu Asp His Asp Asp Ser 165 170 175Phe Ser Thr Val Leu Thr Lys Tyr Trp Tyr Ala Ile Ala Val Asp Tyr 180 185 190Asp Leu Lys Asp Ala Ile Arg Tyr Phe Tyr Gln Lys Tyr Pro Arg Leu 195 200 205His Arg Trp Arg Leu Met Cys Ala Leu Phe Tyr Asn Asn Val Phe Asp 210 215 220Leu His Glu Leu Tyr Glu Ile Glu Arg Val Arg Met Asp Ile Asp Glu225 230 235 240Met Met His Ile Ala Cys Ile Gln Asp Tyr Ser Tyr Ser Ala Ile Tyr 245 250 255Tyr Cys Phe Ile Met Gly Ala Asn Ile Asn Gln Ala Met Leu Val Ser 260 265 270Ile Gln Asn Tyr Asn Leu Gly Asn Leu Phe Phe Cys Ile Asp Leu Gly 275 280 285Ala Asn Ala Phe Glu Glu Gly Lys Ala Leu Ala Glu Gln Lys Glu Asn 290 295 300Tyr Leu Ile Ala His Ala Leu Ser Leu Lys His Tyr Asn Pro Val Ile305 310 315 320Ser Leu Leu Ser Asn Val Met Asp Pro Glu Lys Ile Asn Tyr Met Leu 325 330 335Lys Asn Tyr His Ser Ile Asn Met Gly Ile Phe Leu Asp Tyr Glu Gln 340 345 350Arg4350PRTAfrican swine fever virus 4Met Leu Pro Ser Leu Gln Ser Leu Thr Lys Lys Val Leu Ala Gly Gln1 5 10 15Cys Val Pro Thr Asn Gln His Tyr Leu Leu Lys Tyr Tyr Asp Leu Trp 20 25 30Trp Tyr Asn Ala Pro Ile Thr Phe Asp His Asn Leu Arg Leu Ile Lys 35 40 45Ser Ser Gly Ile Lys Glu Gly Leu Asp Leu Asn Thr Ala Leu Val Lys 50 55 60Ala Val Arg Glu Asn Asn Tyr Ser Leu Ile Lys Leu Phe Thr Glu Trp65 70 75 80Gly Ala Asp Ile Asn Tyr Gly Leu Val Ser Val Asn Thr Glu His Thr 85 90 95Arg Asp Leu Cys Gln Glu Leu Gly Ala Lys Glu Ile Leu Asn Glu Glu 100 105 110Glu Ile Leu Gln Ile Phe Ile Asp Leu Lys Phe His Lys Thr Ser Ser 115 120 125Asn Ile Ile Leu Cys His Glu Val Phe Ser Asn Asn Pro Ile Leu Gln 130 135 140Lys Val Asn Asn Leu Lys Leu Arg Ile Glu Ile Phe Trp Glu Leu Arg145 150 155 160Glu Leu Ile Glu Lys Thr Asp Leu Leu Asn Asn Glu Phe Leu Leu Ser 165 170 175Thr Leu Leu Leu Lys Tyr Trp Tyr Ala Ile Ala Val Arg Tyr Ser Leu 180 185 190Lys Glu Ala Ile Gln Tyr Phe Tyr Gln Lys Tyr Thr His Met Asn Thr 195 200 205Trp Arg Leu Thr Cys Ala Leu Cys Phe Asn Asn Val Phe Asp Leu His 210 215 220Glu Ala Tyr Glu Lys Asp Lys Ile His Met Asp Ile Glu Glu Met Met225 230 235 240Arg Ile Ala Cys Ile Lys Asp His Asn Leu Ser Thr Met Tyr Tyr Cys 245 250 255Tyr Met Leu Gly Ala Asn Ile Asn Gln Ala Met Leu Thr Ser Ile Gln 260 265 270Tyr Tyr Asn Ile Glu Asn Met Phe Phe Cys Met Asp Leu Gly Ala Asp 275 280 285Val Phe Glu Glu Gly Thr Thr Ala Leu Gly Glu Gly Tyr Glu Leu Ile 290 295 300Lys Asn Ile Leu Ser Leu Lys Ile Tyr Ser Pro Thr Thr Ile Pro Leu305 310 315 320Pro Lys Ser Thr Asp Pro Glu Ile Ile Asp His Ala Leu Lys Asn Tyr 325 330 335Phe Ser Lys Asn Met Met Ile Phe Leu Ser Tyr Asp Leu Arg 340 345 3505353PRTAfrican swine fever virus 5Met Ser Leu Pro Leu Ser Leu Gln Thr Leu Val Lys Lys Thr Val Ala1 5 10 15Ser Gln Cys Leu Ser Ile Asp Glu His Cys Ile Leu Lys Tyr Cys Gly 20 25 30Leu Trp Trp His Asp Ala Pro Leu Lys Leu Cys Met Asp Arg Gly Arg 35 40 45Ile Gln Ile Lys Ser Gly Phe Leu Gly Glu Asp Ile Asp Leu Arg Val 50 55 60Ala Leu Ile Ile Ala Val Lys Glu Asn Asn Tyr Ser Leu Ile Lys Leu65 70 75 80Phe Thr Glu Trp Gly Ala Asn Ile Asn Tyr Ser Leu Leu Ser Ile Asn 85 90 95Thr Lys His Ile Arg Glu Leu Cys Arg Gln Leu Gly Ala Lys Glu Thr 100 105 110Leu Glu Asp Asn Asp Ile Phe Arg Ile Phe Thr Arg Ile Met His Asn 115 120 125Lys Thr Ser Gly Ser Ile Ile Leu Cys His Glu Ile Phe Met Asn Asn 130 135 140Pro Met Leu Glu Asn Lys Phe Val Ile Gln Leu Arg Gly Leu Ile Tyr145 150 155 160Lys Arg Leu Trp Gly Leu Ile Glu Ile Lys Glu Thr Asp Glu Leu Asn 165 170 175Asp Leu Leu Val Lys Tyr Trp Tyr Ala Lys Ala Val Gln Tyr Val Cys 180 185 190Lys Asn Ala Ile Cys Phe Leu Asp Glu Lys Tyr Thr Asp Leu Asn Glu 195 200 205Trp Arg Leu Lys Cys Leu Leu Tyr Tyr Asn Lys Ile Tyr Glu Leu His 210 215 220Glu Met Tyr His Lys Lys Lys Val Gln Ile Asp Val His Asp Met Ile225 230 235 240Cys Leu Ala Cys Ala Lys Asp Asn Asn Leu Leu Thr Ile Tyr Tyr Cys 245 250 255Tyr Ala Leu Gly Gly Asn Ile Asn Gln Ala Met Leu Thr Ser Val Gln 260 265 270Tyr Tyr Asn Val Gly Asn Ile Phe Phe Cys Ile Asp Leu Gly Gly Asn 275 280 285Ala Phe Glu Glu Gly Arg Ala Ile Ala Glu Gln Lys Gly Tyr Asn Phe 290 295 300Leu Ser His Ser Leu Thr Leu Asp Ile Tyr Ser Ser Asp Ala Ser Leu305 310 315 320Pro Leu Asn Leu Lys Asp Pro Glu Lys Ile Ser Ser Leu Leu Lys Asp 325 330 335Tyr Lys Ser Lys Asn Leu Ser Ile Ile Trp Glu Tyr Ser His Asn Ile 340 345 350Leu6357PRTAfrican swine fever virus 6Met Leu Ser Leu Gln Thr Leu Ala Lys Lys Val Val Ala Cys Asn Tyr1 5 10 15Leu Ser Ser Asp Tyr Asp Tyr Thr Leu Gln Arg Phe Gly Leu Trp Trp 20 25 30Asp Leu Gly Pro Ile His Leu Cys Asn Asn Cys Lys Gln Val Phe Ser 35 40 45Tyr Lys His Leu Gln Cys Phe Ser Glu Asp Asp Leu Cys Leu Glu Ala 50 55 60Ala Leu Val Lys Ala Val Lys Ser Asp Asn Leu Glu Leu Ile Arg Leu65 70 75 80Phe Val Asp Trp Gly Ala Asn Pro Glu Tyr Gly Leu Ile Arg Val Pro 85 90 95Ala Val Tyr Leu Lys Arg Leu Cys Ala Glu Leu Gly Gly Leu Thr Pro 100 105 110Val Ser Glu Pro Arg Leu Leu Glu Ile Leu Lys Glu Val Ala Asn Leu 115 120 125Lys Ser Cys Ala Gly Val Leu Leu Gly Tyr Asp Met Phe Cys His Asn 130 135 140Pro Leu Leu Glu Thr Val Thr Arg Thr Thr Leu Asp Thr Val Thr Tyr145 150 155 160Thr Cys Ser Asn Ile Pro Leu Thr Gly Asp Thr Ala His Leu Leu Leu 165 170 175Thr Lys Phe Trp Phe Ala Leu Ala Leu Arg His Asn Phe Thr Lys Ala 180 185 190Ile His Tyr Phe Tyr Lys Arg His Lys Asn Gln Leu Tyr Trp Arg Val 195 200 205Ala Cys Ser Leu Tyr Phe Asn Asn Ile Phe Asp Ile His Glu Leu Cys 210 215 220Arg Glu Lys Glu Ile Cys Ile Ser Pro Asn Leu Met Met Lys Phe Ala225 230 235 240Cys Leu Arg Glu Lys Asn Tyr Ala Ala Ile Tyr Tyr Cys His Arg Leu 245 250 255Gly Ala Ser Leu Asp Tyr Gly Met Asn Leu Ser Ile Tyr Asn Asn Asn 260 265 270Thr Leu Asn Met Phe Phe Cys Ile Asp Leu Gly Ala Ala Asp Phe Asp 275 280 285Arg Ala Gln Leu Ile Ala His Lys Ala Tyr Met Tyr Asn Leu Ser Asn 290 295 300Ile Phe Leu Val Lys Gln Leu Phe Ser Arg Asp Val Thr Leu Val Leu305 310 315 320Asp Val Thr Glu Pro Gln Glu Ile Tyr Asp Met Leu Lys Thr Tyr Thr 325 330 335Ser Lys Asn Met Lys Arg Ala Glu Glu Tyr Leu Thr Ala His Pro Glu 340 345 350Ile Ile Val Ile Asp 3557531PRTAfrican swine fever virus 7Met Phe Ser Leu Gln Asn Leu Cys Arg Lys Thr Leu Pro Asp Cys Lys1 5 10 15Leu Pro Glu Phe Phe Asp Asp Tyr Ile Leu Gln Leu Leu Gly Leu Tyr 20 25 30Trp Glu Asn His Gly Thr Ile Gln Arg Ala Gly Asn Asn Cys Val Leu 35 40 45Ile Gln Gln His Thr Leu Ile Pro Val Asn Glu Ala Leu Arg Ile Ala 50 55 60Ala Ser Glu Glu Asn Tyr Glu Ile Val Gly Leu Leu Leu Ala Trp Glu65 70 75 80Gly Asn Leu Tyr Tyr Ala Ile Ile Gly Ala Leu Glu Gly Asn Arg Tyr 85 90 95Asn Leu Ile Arg Lys Tyr Asp Asp Gln Ile Lys Asp His His Asp Ile 100 105 110Leu Pro Phe Ile Asp Asp Pro Ile Ile Phe His Lys Cys His Ile Met 115 120 125Arg Arg Cys Phe Phe Asp Cys Ile Leu Tyr Gln Ala Val Lys Tyr Ser 130 135 140Lys Phe Arg Val Leu Leu Tyr Phe Lys Tyr Thr Leu Glu Asp Asp Leu145 150 155 160Pro Leu Val His Leu Leu Ile Glu Lys Ala Cys Glu Asp His Asn Tyr 165 170 175Glu Val Ile Lys Trp Ile Tyr Glu Asn Leu His Val Cys His Ile Ile 180 185 190Asp Thr Phe Asp Cys Ala Ile Ala His Lys Asp Leu Arg Leu Tyr Cys 195 200 205Leu Gly Tyr Thr Phe Ile Tyr Asn Arg Ile Val Pro Tyr Lys Tyr His 210 215 220His Leu Asp Ile Leu Ile Leu Ser Ser Leu Gln Leu Leu His Lys Val225 230 235 240Ala Ala Lys Gly Tyr Leu Asp Phe Ile Leu Glu Thr Leu Lys Tyr Asp 245 250 255His Asn Ile Asp Asn Leu Asp Val Ile Leu Thr Gln Ala Ala Thr Tyr 260 265 270Asn His Arg Lys Ile Leu Thr Tyr Phe Ile Pro Gln Ser Thr Tyr Ala 275 280 285Gln Ile Glu Gln Cys Leu Phe Val Ala Ile Lys Thr Lys Ser Ser Lys 290 295 300Lys Thr Leu Asn Leu Leu Leu Ser His Leu Asn Leu Ser Ile Lys Leu305 310 315 320Ile Gln Lys Ile Ser Gln Tyr

Val Ala Thr Phe Asn Ser Thr Asn Ile 325 330 335Ile Gly Ile Leu Ser Met Lys Arg Lys Lys Lys Ile Tyr Leu Asp Ile 340 345 350Ile Leu Thr Lys Phe Val Lys Asn Ala Ile Phe Asn Lys Phe Val Val 355 360 365Arg Cys Met Glu Arg Phe Ser Ile Asn Pro Glu Arg Ile Val Lys Met 370 375 380Ala Ala Arg Ile Asn Lys Met Met Leu Val Lys Lys Ile Ser Glu His385 390 395 400Val Trp Lys Asn His Ala Ala Arg Leu Lys His Leu Lys His Ala Val 405 410 415His Thr Met Lys His Lys Asp Gly Lys Asn Arg Leu Met Asn Phe Ile 420 425 430Tyr Glu His Cys Tyr Tyr His Met Gln Gly Glu Glu Ile Phe Ser Leu 435 440 445Ala Arg Phe Tyr Ala Ile His His Ala Pro Lys Leu Phe Asp Val Phe 450 455 460Tyr Asn Cys Cys Ile Leu Asp Thr Ile Arg Phe Lys Ser Leu Leu Leu465 470 475 480Asp Cys Ser His Ile Ile Gly Lys Asn Ala His Asp Ala Thr Asn Ile 485 490 495Asn Ile Val Asn Lys Tyr Ile Gly Asn Leu Phe Ala Met Gly Val Leu 500 505 510Ser Lys Lys Glu Ile Leu Gln Asp Tyr Pro Ser Ile Tyr Ser Lys His 515 520 525Tyr Met Pro 5308526PRTAfrican swine fever virus 8Met Phe Ser Leu Gln Asp Leu Cys Arg Lys His Leu Phe Ile Leu Pro1 5 10 15Asp Val Phe Gly Glu His Val Leu Gln Arg Leu Gly Leu Tyr Trp Arg 20 25 30Cys His Gly Ser Leu Gln Arg Ile Gly Asp Asp His Ile Leu Ile Arg 35 40 45Arg Asp Leu Ile Leu Ser Thr Asn Glu Ala Leu Arg Met Ala Gly Glu 50 55 60Glu Gly Asn Asn Glu Val Val Lys Leu Leu Leu Leu Trp Lys Gly Asn65 70 75 80Leu His Tyr Ala Val Ile Gly Ala Leu Gln Gly Asp Gln Tyr Asp Leu 85 90 95Ile His Lys Tyr Glu Asn Gln Ile Gly Asp Phe His Phe Ile Leu Pro 100 105 110Leu Ile Gln Asp Ala Asn Thr Phe Glu Lys Cys His Ala Leu Glu Arg 115 120 125Phe Cys Gly Val Ser Cys Leu Leu Lys His Ala Thr Lys Tyr Asn Met 130 135 140Leu Pro Ile Leu Gln Lys Tyr Gln Glu Glu Leu Ser Met Arg Ala Tyr145 150 155 160Leu His Glu Thr Leu Phe Glu Leu Ala Cys Leu Trp Gln Arg Tyr Asp 165 170 175Val Leu Lys Trp Ile Glu Gln Thr Met His Val Tyr Asp Leu Lys Ile 180 185 190Met Phe Asn Ile Ala Ile Ser Lys Arg Asp Leu Thr Met Tyr Ser Leu 195 200 205Gly Tyr Ile Phe Leu Phe Asp Arg Gly Asn Thr Glu Ala Thr Leu Leu 210 215 220Thr Gln His Leu Glu Lys Thr Ala Ala Lys Gly Leu Leu His Phe Val225 230 235 240Leu Glu Thr Leu Lys Tyr Gly Gly Asn Ile Asp Thr Val Leu Thr Gln 245 250 255Ala Val Lys Tyr Asn His Arg Lys Leu Leu Asp Tyr Phe Leu Arg Gln 260 265 270Leu Pro Arg Lys His Ile Glu Lys Leu Leu Leu Leu Ala Val Gln Glu 275 280 285Lys Ala Ser Lys Lys Thr Leu Asn Leu Leu Leu Ser His Leu Asn Tyr 290 295 300Ser Val Lys Arg Ile Lys Lys Leu Leu Arg Tyr Val Ile Glu Tyr Glu305 310 315 320Ser Thr Leu Val Ile Lys Ile Leu Leu Lys Lys Arg Val Asn Leu Ile 325 330 335Asp Ala Met Leu Glu Lys Met Val Arg Tyr Phe Ser Ala Thr Lys Val 340 345 350Arg Thr Ile Met Asp Glu Leu Ser Ile Ser Pro Glu Arg Val Ile Lys 355 360 365Met Ala Ile Gln Lys Met Arg Thr Asp Ile Val Ile His Thr Ser Tyr 370 375 380Val Trp Glu Asp Asp Leu Glu Arg Leu Thr Arg Leu Lys Asn Met Val385 390 395 400Tyr Thr Ile Lys Tyr Glu His Gly Lys Lys Met Leu Ile Lys Val Met 405 410 415His Gly Ile Tyr Lys Asn Leu Leu Tyr Gly Glu Arg Glu Lys Val Met 420 425 430Phe His Leu Ala Lys Leu Tyr Val Ala Gln Asn Ala Ala Thr Gln Phe 435 440 445Arg Asp Ile Cys Lys Asp Cys Tyr Lys Leu Asp Val Ala Arg Phe Lys 450 455 460Pro Arg Phe Lys Gln Leu Ile Leu Asp Cys Leu Glu Ile Val Thr Lys465 470 475 480Lys Ser Cys Tyr Ser Ile Leu Glu Ile Leu Glu Lys His Ile Ile Ser 485 490 495Leu Phe Thr Met Lys Val Met Thr Glu Glu Glu Lys Asn Leu Cys Leu 500 505 510Glu Ile Leu Tyr Lys Val Ile His Tyr Lys Thr Ile Gln Cys 515 520 5259280PRTAfrican swine fever virus 9Met Ser Ser Ser Leu Gln Glu Leu Cys Arg Lys Lys Leu Pro Asp Cys1 5 10 15Ile Leu Pro Glu Phe Phe Asp Asp Tyr Val Leu Gln Leu Leu Gly Leu 20 25 30His Trp Gln Asp His Gly Ser Leu Gln Arg Ile Glu Lys Asn Gln Ile 35 40 45Leu Val Gln Gln Glu Pro Ile His Ile Asn Glu Ala Leu Lys Val Ala 50 55 60Ala Ser Glu Gly Asn Tyr Glu Ile Val Glu Leu Leu Leu Ser Trp Glu65 70 75 80Ala Asp Pro Arg Tyr Ala Val Val Gly Ala Leu Glu Ser Lys Tyr Tyr 85 90 95Asp Leu Val Tyr Lys Tyr Tyr Asp Leu Val Lys Asp Cys His Asp Ile 100 105 110Leu Pro Leu Ile Gln Asn Pro Glu Thr Phe Glu Lys Cys His Glu Leu 115 120 125Asn Asn Pro Cys Ser Leu Lys Cys Leu Phe Lys His Ala Val Ile His 130 135 140Asp Met Leu Pro Ile Leu Gln Lys Tyr Thr Tyr Phe Leu Asp Gly Trp145 150 155 160Glu Tyr Cys Asn Gln Met Leu Phe Glu Leu Ala Cys Ser Lys Lys Lys 165 170 175Tyr Glu Met Val Val Trp Ile Glu Gly Val Leu Gly Ile Gly Lys Val 180 185 190Thr Ser Leu Phe Thr Ile Ala Ile Ser Asn Arg Asp Leu His Leu Tyr 195 200 205Ser Leu Gly His Leu Ile Ile Leu Glu Arg Met Gln Ser Cys Gly Gln 210 215 220Asp Pro Thr Phe Leu Leu Asn His Phe Leu Arg Asp Val Ser Ile Lys225 230 235 240Gly Leu Leu Pro Phe Val Leu Lys Thr Ile Glu Tyr Gly Gly Ser Lys 245 250 255Glu Ile Ala Ile Thr Leu Ala Lys Lys Tyr Gln His Lys His Ile Leu 260 265 270Lys Tyr Phe Glu Thr Gly Lys Cys 275 28010505PRTAfrican swine fever virus 10Met Phe Ser Leu Gln Asp Ile Cys Arg Lys Tyr Leu Phe Gln Leu Pro1 5 10 15Asp Ser Phe Asp Glu Tyr Thr Leu Gln Val Leu Gly Leu Tyr Trp Glu 20 25 30Lys His Gly Ser Leu Gln Arg Ile Arg Lys Asp Ala Val Phe Val Gln 35 40 45Arg Asn Leu Ile Ile Ser Ile Asn Glu Ala Leu Arg Ile Ala Ala Ser 50 55 60Glu Gly Asn Gly Arg Val Val Lys Leu Leu Leu Ser Trp Glu Gly Asn65 70 75 80Phe His Tyr Val Ile Ile Gly Ala Leu Glu Gly Asp His Tyr Asp Leu 85 90 95Ile His Lys Tyr Gly Ser Gln Ile Glu Asp Tyr His Met Ile Leu Ser 100 105 110Ser Ile His Asn Ala Asn Thr Phe Glu Lys Cys His Glu Leu Ser Asn 115 120 125Cys Asp Met Trp Cys Leu Ile Gln Asn Ala Ile Lys Tyr Asn Met Leu 130 135 140Pro Ile Leu Gln Lys His Arg Asn Ile Leu Thr His Glu Gly Glu Asn145 150 155 160Gln Glu Leu Phe Glu Met Ala Cys Glu Glu Gln Lys Tyr Asp Ile Val 165 170 175Leu Trp Ile Gly Gln Thr Leu Met Leu Asn Glu Pro Glu Phe Ile Phe 180 185 190Asp Ile Ala Phe Glu Arg Ile Asp Phe Ser Leu Leu Thr Met Gly Tyr 195 200 205Ser Leu Leu Phe Asn Asn Lys Met Ser Ser Ile Asp Ile His Asp Glu 210 215 220Glu Asp Leu Ile Ser Leu Leu Thr Glu His Leu Glu Lys Ala Ala Thr225 230 235 240Lys Gly Cys Phe Phe Phe Met Leu Glu Thr Leu Lys His Gly Gly Asn 245 250 255Val Asn Met Ala Val Leu Ser Lys Ala Val Glu Tyr Asn His Arg Lys 260 265 270Ile Leu Asp Tyr Phe Ile Arg Gln Lys Cys Leu Ser Arg Lys Asp Ile 275 280 285Glu Lys Leu Leu Leu Val Ala Ile Ser Asn Ser Ala Ser Lys Lys Thr 290 295 300Leu Asn Leu Leu Leu Ser Tyr Leu Asn His Ser Val Lys Asn Ile Ile305 310 315 320Gly Lys Ile Val Gln Ser Val Leu Lys Asn Gly Asp Phe Thr Ile Ile 325 330 335Ile Phe Leu Lys Lys Lys Lys Ile Asn Leu Val Glu Pro Ala Leu Ile 340 345 350Gly Phe Ile Asn Tyr Tyr Tyr Ser Tyr Cys Phe Leu Glu Gln Phe Ile 355 360 365His Glu Phe Asp Ile Arg Pro Glu Lys Met Ile Lys Met Ala Ala Arg 370 375 380Lys Gly Lys Leu Asn Met Ile Ile Glu Phe Leu Asn Glu Lys Tyr Val385 390 395 400His Lys Asp Asp Leu Gly Ala Ile Phe Lys Phe Leu Lys Asn Leu Val 405 410 415Cys Thr Met Lys His Lys Lys Gly Lys Glu Thr Leu Ile Val Leu Ile 420 425 430His Lys Ile Tyr Gln Val Ile Gln Leu Glu Thr Lys Glu Lys Phe Lys 435 440 445Leu Leu Arg Phe Tyr Val Met His Asp Ala Thr Ile Gln Phe Ile Ser 450 455 460Met Tyr Lys Asp Cys Phe Asn Leu Ala Gly Phe Lys Pro Phe Leu Leu465 470 475 480Glu Cys Leu Asp Ile Ala Ile Lys Lys Asn Tyr Pro Asp Met Ile Arg 485 490 495Asn Ile Glu Thr Leu Leu Lys Cys Glu 500 50511254PRTAfrican swine fever virus 11Met Gln Asn Lys Ile Pro Asn Phe Asn Leu Phe Phe Phe Phe Leu Tyr1 5 10 15Arg Met Leu Glu Ile Val Leu Ala Thr Leu Leu Gly Asp Leu Gln Arg 20 25 30Leu Arg Val Leu Thr Pro Gln Gln Arg Ala Val Ala Phe Phe Arg Ala 35 40 45Asn Thr Lys Glu Leu Glu Asp Phe Leu Arg Ser Asp Gly Gln Ser Glu 50 55 60Glu Ile Leu Ser Gly Pro Leu Leu Asn Arg Leu Leu Glu Pro Ser Cys65 70 75 80Pro Leu Asp Ile Leu Thr Gly Tyr His Leu Phe Arg Gln Asn Pro Lys 85 90 95Ala Gly Gln Leu Arg Gly Leu Glu Val Lys Met Leu Glu Arg Leu Tyr 100 105 110Asp Ala Asn Ile Tyr Asn Ile Leu Ser Arg Leu Arg Pro Lys Lys Val 115 120 125Arg Asn Lys Ala Ile Glu Leu Tyr Trp Val Phe Arg Ala Ile His Ile 130 135 140Cys His Ala Pro Leu Val Leu Asp Ile Val Arg Tyr Glu Glu Pro Asp145 150 155 160Phe Ala Glu Leu Ala Phe Ile Cys Ala Ala Tyr Phe Gly Glu Pro Gln 165 170 175Val Met Tyr Leu Leu Tyr Lys Tyr Met Pro Leu Thr Arg Ala Val Leu 180 185 190Thr Asp Ala Ile Gln Ile Ser Leu Glu Ser Asn Asn Gln Val Gly Ile 195 200 205Cys Tyr Ala Tyr Leu Met Gly Gly Ser Leu Lys Gly Leu Val Ser Ala 210 215 220Pro Leu Arg Lys Arg Leu Arg Ala Lys Leu Arg Ser Gln Arg Lys Lys225 230 235 240Lys Asp Val Leu Ser Pro His Asp Phe Leu Leu Leu Leu Gln 245 25012148PRTAfrican swine fever virus 12Met Leu Glu Arg Leu Tyr Asp Ala Asn Ile Tyr Asn Ile Leu Ser Arg1 5 10 15Leu Arg Pro Glu Lys Val Arg Asn Lys Ala Val Glu Leu Tyr Trp Val 20 25 30Phe Arg Ala Ile Asn Met Cys His Ala Pro Leu Val Leu Asp Ile Val 35 40 45Arg Tyr Glu Glu Pro Asp Phe Ala Glu Leu Ala Phe Ile Cys Ala Ala 50 55 60Tyr Phe Gly Glu Pro Gln Val Met Tyr Leu Leu Tyr Lys Tyr Met Pro65 70 75 80Leu Thr Arg Ala Val Leu Thr Asp Ala Ile Gln Ile Ser Leu Glu Ser 85 90 95Asn Ser Gln Val Gly Ile Cys Tyr Ala Tyr Leu Met Gly Gly Ser Leu 100 105 110Lys Gly Leu Val Arg Ala Pro Leu Arg Lys Arg Leu Arg Ala Lys Leu 115 120 125Arg Ser Gln Arg Lys Lys Lys Asp Val Leu Pro Pro His Asp Phe Leu 130 135 140Leu Leu Leu Gln14513151PRTAfrican swine fever virus 13Met Leu Glu Arg Leu Tyr Asp Ala Asn Ile Tyr Asn Met Leu Ala Arg1 5 10 15Leu Arg Pro Glu Leu Val Arg Asp Lys Ala Ile Glu Leu Tyr Trp Leu 20 25 30Phe Arg Ala Ile Leu Met Cys His Ser Pro Leu Val Leu Glu Ile Val 35 40 45Arg His Glu Thr Met Asp Phe Ala Glu Thr Ala Phe Ile Cys Ala Ala 50 55 60Tyr Phe Ser Glu Pro Gln Val Met Tyr Ala Leu Tyr Lys Phe Ile Pro65 70 75 80Ile Ser Arg Ala Val Leu Ala Asp Ala Ile Gln Met Cys Leu Glu Ser 85 90 95Asn Ser Glu Ala Gly Ile Cys Tyr Ala Tyr Leu Met Gly Gly Ser Leu 100 105 110Lys Gly Lys Val Pro Gly Ser Leu Arg Lys Arg Leu Arg Ala Ser Pro 115 120 125Leu Arg Gln Glu Arg Lys Lys Lys Asn Val Leu Pro Pro His Glu Phe 130 135 140Leu Leu Met Leu His Gly Ile145 15014219PRTAfrican swine fever virus 14Met Gln Arg Ala Val Ala Phe Phe Arg Val Asn Thr Lys Glu Leu Glu1 5 10 15Asp Phe Leu Tyr Pro Asp Gly Gln Ser Glu Glu Leu Leu Pro Gly Leu 20 25 30Leu Leu Asn Arg Leu Leu Glu Pro Ser Gly Pro Ile Asp Ile Leu Thr 35 40 45Gly Tyr His Leu Phe Arg Glu Asn Pro Lys Ala Gly Arg Leu Arg Gly 50 55 60Leu Glu Val Lys Leu Leu Glu Arg Leu Tyr Asp Ala Asn Ile Tyr Asn65 70 75 80Met Leu Ala Gln Ile Arg Pro Glu Leu Val Arg Ile Lys Ala Ile Glu 85 90 95Leu Tyr Trp Leu Phe Arg Ala Ile Leu Met Cys His Ser Pro Leu Val 100 105 110Leu Glu Ile Val Arg His Glu Thr Met Asp Phe Ala Glu Leu Ala Phe 115 120 125Ile Cys Ala Ala Tyr Phe Ser Glu Pro Gln Val Met Tyr Ala Leu Tyr 130 135 140Lys Phe Ile Pro Ile Ser Arg Ala Val Leu Ala Asp Ala Ile Glu Met145 150 155 160Ser Leu Glu Ser Asn Ser Glu Thr Gly Ile Cys Tyr Ala Tyr Leu Met 165 170 175Gly Gly Ser Leu Lys Gly Lys Val Pro Gly Pro Leu Arg Lys Arg Leu 180 185 190Arg Ala Ser Pro Leu Arg Gln Glu Arg Lys Lys Lys Asn Val Leu Pro 195 200 205Pro His Glu Phe Leu Leu Met Leu His Gly Ile 210 21515254PRTAfrican swine fever virus 15Met Gln Asn Lys Ile Pro Asn Phe Asn Leu Phe Phe Phe Phe Leu Tyr1 5 10 15Arg Met Leu Glu Ile Val Leu Ala Thr Leu Leu Gly Asp Leu Gln Arg 20 25 30Leu Arg Val Leu Thr Pro Gln Gln Arg Ala Val Ala Phe Phe Arg Ala 35 40 45Asn Thr Lys Glu Leu Glu Asp Phe Leu Cys Ser Asp Gly Gln Ser Glu 50 55 60Glu Ile Leu Ser Gly Pro Leu Leu Asn Arg Leu Leu Glu Pro Ser Gly65 70 75 80Pro Leu Asp Ile Leu Thr Gly Tyr His Leu Phe Arg Gln Asn Pro Lys 85 90 95Ala Gly Gln Leu Arg Gly Leu Glu Val Lys Met Leu Glu Arg Leu Tyr 100 105 110Asp Ala Asn Ile Tyr Asn Ile Leu Ser Arg Leu Arg Pro Glu Lys Val 115 120 125Arg Asn Lys Ala Ile Glu Leu Tyr Trp Val Phe Arg Ala Ile His Ile 130 135 140Cys His Ala Pro Leu Val Leu Asp Ile Val Arg Tyr Glu Glu Pro

Asp145 150 155 160Phe Ala Glu Leu Ala Phe Ile Cys Ala Ala Tyr Phe Gly Glu Pro Gln 165 170 175Val Met Tyr Leu Leu Tyr Lys Tyr Met Pro Leu Thr Arg Ala Val Leu 180 185 190Thr Asp Ala Ile Arg Ile Ser Leu Glu Ser Asn Asn Gln Val Gly Ile 195 200 205Cys Tyr Ala Tyr Leu Met Gly Gly Ser Leu Lys Gly Leu Val Ser Ala 210 215 220Pro Leu Arg Lys Arg Leu Cys Ala Lys Leu Arg Ser Gln Arg Lys Lys225 230 235 240Lys Asp Val Leu Ser Pro His Asp Phe Leu Leu Leu Leu Gln 245 25016148PRTAfrican swine fever virus 16Met Leu Glu Arg Leu Tyr Asp Ala Asn Ile Tyr Asn Ile Leu Ser Arg1 5 10 15Leu Arg Pro Glu Lys Val Arg Asn Lys Ala Val Glu Leu Tyr Trp Val 20 25 30Phe Arg Ala Ile Asn Met Cys His Ala Pro Leu Val Leu Asp Ile Val 35 40 45Arg Tyr Glu Glu Pro Asp Phe Ala Glu Leu Ala Phe Ile Cys Ala Ala 50 55 60Tyr Phe Gly Glu Pro Gln Val Met Tyr Leu Leu Tyr Lys Tyr Met Pro65 70 75 80Leu Ser Arg Ala Val Leu Thr Asp Ala Ile Gln Ile Ser Leu Glu Ser 85 90 95Asn Ser Gln Val Gly Ile Cys Tyr Ala Tyr Leu Met Gly Gly Ser Leu 100 105 110Lys Gly Leu Val Arg Ala Pro Leu Arg Lys Arg Leu Arg Ala Lys Leu 115 120 125Arg Ser Gln Arg Lys Lys Lys Asp Val Leu Pro Pro His Asp Phe Leu 130 135 140Leu Leu Leu Gln14517148PRTAfrican swine fever virus 17Met Leu Glu Arg Leu Tyr Asp Ala Asn Ile Tyr Asn Ile Leu Ser Arg1 5 10 15Leu Arg Pro Glu Lys Val Arg Asn Lys Ala Val Glu Leu Tyr Trp Val 20 25 30Phe Arg Ala Ile Asn Met Cys His Ala Pro Leu Val Leu Asp Ile Val 35 40 45Arg Asn Glu Glu Leu Asp Phe Ala Glu Leu Ala Phe Ile Cys Ala Ala 50 55 60Tyr Phe Gly Glu Pro Gln Val Met Tyr Leu Leu Tyr Lys Tyr Met Pro65 70 75 80Leu Thr Arg Ala Val Leu Thr Asp Ala Ile Gln Ile Ser Leu Glu Ser 85 90 95Asn Ser Gln Val Gly Ile Cys Tyr Ala Tyr Leu Met Gly Gly Ser Leu 100 105 110Lys Gly Leu Val Arg Ala Pro Leu Arg Lys Arg Leu Arg Ala Lys Leu 115 120 125Arg Ser Gln Arg Lys Lys Lys Asp Val Leu Pro Pro His Asp Phe Leu 130 135 140Leu Leu Leu Gln14518148PRTAfrican swine fever virus 18Met Leu Glu Arg Leu Tyr Asp Ala Asn Ile Tyr Asn Ile Leu Ser Arg1 5 10 15Leu Arg Pro Glu Lys Val Arg Asn Lys Ala Ile Glu Leu Tyr Trp Val 20 25 30Phe Arg Ala Ile His Ile Cys His Ala Pro Leu Val Leu Asp Ile Val 35 40 45Arg Tyr Glu Glu Pro Asp Phe Ala Glu Leu Ala Phe Ile Cys Ala Ala 50 55 60Tyr Phe Gly Glu Pro Gln Val Met Tyr Leu Leu Tyr Lys Tyr Met Pro65 70 75 80Leu Thr Arg Ala Val Leu Thr Asp Ala Ile Arg Ile Ser Leu Glu Ser 85 90 95Asn Asn Gln Val Gly Ile Cys Tyr Ala Tyr Leu Met Gly Gly Ser Leu 100 105 110Lys Gly Leu Val Ser Ala Pro Leu Arg Lys Arg Leu Arg Ala Lys Leu 115 120 125Arg Ser Gln Arg Lys Lys Lys Asp Val Leu Ser Pro His Asp Phe Leu 130 135 140Leu Leu Leu Gln14519148PRTAfrican swine fever virus 19Met Leu Glu Arg Leu Tyr Asp Ala Asn Ile Tyr Asn Ile Leu Ser Arg1 5 10 15Leu Arg Pro Glu Lys Val Arg Asn Lys Ala Ile Glu Leu Tyr Trp Val 20 25 30Phe Arg Ala Ile His Ile Cys His Ala Pro Leu Val Leu Asp Ile Val 35 40 45Arg Tyr Glu Glu Pro Asp Phe Ala Glu Leu Ala Phe Ile Cys Ala Ala 50 55 60Tyr Phe Gly Glu Pro Gln Val Met Tyr Leu Leu Tyr Lys Tyr Met Pro65 70 75 80Leu Thr Arg Ala Val Leu Thr Asp Ala Ile Gln Ile Ser Leu Glu Ser 85 90 95Asn Asn Gln Val Gly Ile Cys Tyr Ala Tyr Leu Met Gly Gly Ser Leu 100 105 110Lys Gly Leu Val Ser Ala Pro Leu Arg Lys Arg Leu Arg Ala Lys Leu 115 120 125Arg Ser Gln Arg Lys Lys Lys Asp Val Leu Ser Pro His Asp Phe Leu 130 135 140Leu Leu Leu Gln1452036DNAArtificial SequencePrimer FlankLF 20acgttgcaaa gcttccatta atccctccag ttgttc 362136DNAArtificial SequencePrimer Flank LR 21acgttgcagg tacccctctc tctgcagact ctcacc 362236DNAArtificial SequencePrimer Flank RF 22acgttgcagc ggccgcctct ccaagacatc tgtcgg 362340DNAArtificial SequencePrimer Flank RR 23acgtacgtct cgagcctcac atgccatctc aaacaattcc 402422DNAArtificial SequencePrimer BeninD8F 24ggtgagagtc tgcagagaga gg 222518DNAArtificial SequencePrimer BeninD8R 25gccctagcac ttgtaacg 182627DNAArtificial SequencePrimer RGUS 26ccttctctgc cgtttccaaa tcgccgc 272718DNAArtificial SequencePrimer BeninD8INTF 27cgatgtatca ttgatgtc 182819DNAArtificial SequencePrimer BeninD8INTR 28ggataatctt agggaggcc 192919DNAArtificial SequencePrimer 9LF 29atgacgcatt aaaccggcg 193020DNAArtificial SequencePrimer 4RR 30cagtatagcc ctagcacttg 203196DNAArtificial SequenceRecombinant virus sequence (left flank of deletion/insertion) 31agagaggggt accatttaat aaaaacaata aattattttt tataacatta tatataactt 60tgtatagcat acattatacg aagttatatg ttacgt 963296DNAAfrican swine fever virus 32agagaggacc atatttcttt ttttgaaaaa atcaaattaa aaaaaaacat gcttgtttgg 60catacatgta actatgttat aaccatgtta taacca 963373DNAArtificial SequenceRecombinant virus sequence (right flank of deletion/insertion) 33cagccaggga ggcaaacaat gaggaattct gcagatatcc atcactggcg gccgcctctc 60caagacatct tgt 733477DNAAfrican swine fever virus 34ttttatcata cattaaaatt ccagtaaaat ttatattttt tttggtaaac aaatgttttc 60tctccaagac atcttgt 77352160DNAAfrican swine fever virus 35aaatgaatag atttaatcca agtagtatta aaatttttta gaaatagtgt tctacaaata 60atgaaatgaa tagcccaaaa aaaataaggt gtacattaat gtaatatatt gttaggctaa 120gtaaatttaa tatttaaagt acttggaaaa atatttttta acatatgctg tctaggaatt 180tttttagaca tttaaaacca tatagttact ttatttatta cacttaactt gaaaagaatt 240attgcctaaa atattaatag atgaagtaat attgtgtaat tgagtccata acatgggtgg 300gaaacaaaaa tctcgtaata tgaaaaataa acatcctaaa aagagtgcaa ttgttataag 360tttatgtaac tttattttaa agtaagaata taaaaatatg agtacaagag gaataggggc 420cattactaat attggctcca acatcctgtt gtctacaaaa aaaaatattt ttttggcaaa 480aaaaaatcca tggaaggata ttaatacaca taattgtttg acatcatatt ggtatactta 540ccaaatagta atatacaacc atcctaatat tcactttatg aaatgacccc cccccccacc 600tatacagtaa aatagtatag gttttaataa agaaaaaaga tattatgtag tttttatttt 660tgtatagtgc gtgaatgcaa aataaaatcc caaattttaa ccttttcttt ttttttctat 720acaggatgtt agaaatagta ttggcaacgc tgctaggcga cctgcagcgg ctccgggttc 780ttacccctca gcagcgggcg gttgccttct ttcgagccaa tactaaggag ctagaggact 840tcttacgctc agatgggcaa tctgaggaga tactgtctgg ccccctcctt aaccgtctac 900tagaaccctc atgccctctt gatattttaa ccggatatca cctatttcgt cagaatccca 960aggcaggtca gttgcgcggc cttgaggtca agatgcttga acggttatac gatgctaata 1020tttacaatat attgtctcgg ctgcgaccta aaaaagtccg caacaaggct attgagctat 1080actgggtttt ccgagctatc catatttgtc atgctccttt agttttagat attgtacgat 1140atgaggaacc ggactttgct gaactggcct ttatttgtgc tgcttacttt ggtgaacctc 1200aggtaatgta tttgctctac aaatatatgc ctctgacccg cgcagttctt acggatgcca 1260tccagataag tcttgagagc aacaaccagg tagggatttg ctatgcttac ttgatgggag 1320gcagcctcaa gggactagtc tccgccccac tgcgtaaacg tctgcgcgcc aaactacgct 1380cgcagcgcaa aaagaaggac gttctttcac cccacgactt cttactgctg ctccagtagc 1440tttttttgcc gcaggagcac cgtggatagg agctcctcca cgctcgcgat ccggcgctgg 1500aagcggaacc gatcgaccgc cacctgctcc cagggaccct tgcgctcgat gtcgtcggct 1560tcccatacct cgacggctgc agcaaagcgg acgtgcttcg tgtcattcgt ccgttttttg 1620cgccgccgcc ccccccccat tattcctgta agattagtgt ttaataccta taataacata 1680attttaagat ttaatatacc aaaacttaaa ctatttttgt atagtaacta ttagcatgtc 1740tacacatgat tgtttttcaa aagagaaacc agttgatatg aacgatatat ctgagaaatc 1800atctgtcgtg gataatgcac ccgagaaacc agctggagcg aatcatatac ctgagaagtc 1860ggcccgcgaa atgacatcat cagaatggat tgctgaatat tggaaaggta taaaacgtgg 1920aaatgacgtg ccatgttgtt gtccaagaaa aatgaccagt gcagacaaaa agttttcagt 1980atttggtaag ggatccctaa tacgctccat ccagaagaat aattaaaaaa aatatttttt 2040ttggtaagtt tataaactat atagttaaat atggtaaaaa aaatcacata ataattaaac 2100tgaacgtgtt agaattaata tttttttata atcggatata atatccatta aatcaataaa 21603638DNAArtificial SequencePrimer 360-18RFlankL-for-Benin 36gctatccaag cttctacaaa taatgaaatg aatagccc 383738DNAArtificial SequencePrimer 360-18RFlankL-rev-Benin2 37gctatccggt acccataaag tgaatattag gatggttg 383837DNAArtificial SequencePrimer 360-18RFlankR-for 38gctatccgcg gccgccgact tcttactgct gctccag 373936DNAArtificial SequencePrimer 360-18RFlankR-rev 39gctatcctct agatcttctg gatggagcgt attagg 364022DNAArtificial SequencePrimer 18R-Benin-Pair1-For 40gagtacaaga ggaatagggg cc 224123DNAArtificial SequencePrimer 18R-OURT-Pair1-Rev 41gcatgacaaa tatggatagc tcg 234221DNAArtificial SequencePrimer 18R-OURT-Pair2-For 42gtccgcaaca aggctattga g 214319DNAArtificial SequencePrimer 18R-OURT-Pair2-Rev 43gacgtttacg cagtggggc 194424DNAArtificial SequencePrimer 18R-OURT-Pair3-For 44caaccaggta gggatttgct atgc 244516DNAArtificial SequencePrimer 18R-OURT-Pair3-Rev 45gctgcagccg tcgagg 16