Six adenoviral vectored African swine fever virus genes protect against fatal disease caused by genotype I challenge.

African swine fever virus causes a lethal hemorrhagic disease in domestic swine and wild boar for which currently licensed commercial vaccines are only available in Vietnam. Development of subunit vaccines is complicated by the lack of information on protective antigens as well as suitable delivery systems. Our previous work showed that a pool of eight African swine fever virus genes vectored using an adenovirus prime and modified vaccinia virus boost could prevent fatal disease after challenge with a virulent genotype I isolate of the virus. Here, we identify antigens within this pool of eight that are essential for the observed protection and demonstrate that adenovirus-prime followed by adenovirus-boost can also induce protective immune responses against genotype I African swine fever virus. Immunization with a pool of adenoviruses expressing individual African swine fever virus genes partially tailored to genotype II virus did not protect against challenge with genotype II Georgia 2007/1 strain, suggesting that different antigens may be required to induce cross-protection for genetically distinct viruses. IMPORTANCE: African swine fever virus causes a lethal hemorrhagic disease in domestic pigs and has killed millions of animals across Europe and Asia since 2007. Development of safe and effective subunit vaccines against African swine fever has been problematic due to the complexity of the virus and a poor understanding of protective immunity. In a previous study, we demonstrated that a complex combination of eight different virus genes delivered using two different viral vector vaccine platforms protected domestic pigs from fatal disease. In this study, we show that three of the eight genes are required for protection and that one viral vector is sufficient, significantly reducing the complexity of the vaccine. Unfortunately, this combination did not protect against the current outbreak strain of African swine fever virus, suggesting that more work to identify immunogenic and protective viral proteins is required to develop a truly effective African swine fever vaccine.

Deletion of the gene for the African swine fever virus BCL-2 family member A179L increases virus uptake and apoptosis but decreases virus spread in macrophages and reduces virulence in pigs.

IMPORTANCE: African swine fever virus (ASFV) causes a lethal disease of pigs with high economic impact in affected countries in Africa, Europe, and Asia. The virus encodes proteins that inhibit host antiviral defenses, including the type I interferon response. Host cells also activate cell death through a process called apoptosis to limit virus replication. We showed that the ASFV A179L protein, a BCL-2 family apoptosis inhibitor, is important in reducing apoptosis in infected cells since deletion of this gene increased cell death and reduced virus replication in cells infected with the A179L gene-deleted virus. Pigs immunized with the BeninΔA179L virus showed no clinical signs and a weak immune response but were not protected from infection with the deadly parental virus. The results show an important role for the A179L protein in virus replication in macrophages and virulence in pigs and suggest manipulation of apoptosis as a possible route to control infection.

African Swine Fever Virus and Host Response: Transcriptome Profiling of the Georgia 2007/1 Strain and Porcine Macrophages.

African swine fever virus (ASFV) has a major global economic impact. With a case fatality in domestic pigs approaching 100%, it currently presents the largest threat to animal farming. Although genomic differences between attenuated and highly virulent ASFV strains have been identified, the molecular determinants for virulence at the level of gene expression have remained opaque. Here, we characterize the transcriptome of ASFV genotype II Georgia 2007/1 (GRG) during infection of the physiologically relevant host cells, porcine macrophages. In this study, we applied cap analysis gene expression sequencing (CAGE-seq) to map th0e 5' ends of viral mRNAs at 5 and 16 h postinfection. A bioinformatics analysis of the sequence context surrounding the transcription start sites (TSSs) enabled us to characterize the global early and late promoter landscape of GRG. We compared transcriptome maps of the GRG isolate and the lab-attenuated BA71V strain that highlighted GRG virulence-specific transcripts belonging to multigene families, including two predicted MGF 100 genes, I7L and I8L. In parallel, we monitored transcriptome changes in the infected host macrophage cells. Of the 9,384 macrophage genes studied, transcripts for 652 host genes were differentially regulated between 5 and 16 h postinfection compared with only 25 between uninfected cells and 5 h postinfection. NF-κB activated genes and lysosome components such as S100 were upregulated, and chemokines such as CCL24, CXCL2, CXCL5, and CXCL8 were downregulated. IMPORTANCE African swine fever virus (ASFV) causes hemorrhagic fever in domestic pigs, with case fatality rates approaching 100% and no approved vaccines or antivirals. The highly virulent ASFV Georgia 2007/1 strain (GRG) was the first isolated when ASFV spread from Africa to the Caucasus region in 2007, then spreading through Eastern Europe and, more recently, across Asia. We used an RNA-based next-generation sequencing technique called CAGE-seq to map the starts of viral genes across the GRG DNA genome. This has allowed us to investigate which viral genes are expressed during early or late stages of infection and how this is controlled, comparing their expression to the nonvirulent ASFV-BA71V strain to identify key genes that play a role in virulence. In parallel, we investigated how host cells respond to infection, which revealed how the ASFV suppresses components of the host immune response to ultimately win the arms race against its porcine host.

Differential Effect of Deleting Members of African Swine Fever Virus Multigene Families 360 and 505 from the Genotype II Georgia 2007/1 Isolate on Virus Replication, Virulence, and Induction of Protection.

African swine fever virus multigene family (MGF) 360 and 505 genes have roles in suppressing the type I interferon response and in virulence in pigs. The role of the individual genes is poorly understood. Different combinations of these genes were deleted from the virulent genotype II Georgia 2007/1 isolate. Deletion of five copies of MGF 360 genes, MGF360-10L, -11L, -12L, -13L, and -14L, and three copies of MGF505-1R, -2R, and -3R reduced virus replication in macrophages and attenuated virus in pigs. However, only 25% of the immunized pigs were protected against challenge. Deletion of MGF360-12L, -13L, and -14L and MGF505-1R in combination with a negative serology marker, K145R (GeorgiaΔK145RΔMGF(A)), reduced virus replication in macrophages and virulence in pigs, since no clinical signs or virus genome in blood were observed following immunization. Four of six pigs were protected after challenge. In contrast, deletion of MGF360-13L and -14L, MGF505-2R and -3R, and K145R (GeorgiaΔK145RΔMGF(B)) did not reduce virus replication in macrophages. Following immunization of pigs, clinical signs were delayed, but all pigs reached the humane endpoint. Deletion of genes MGF360-12L, MGF505-1R, and K145R reduced replication in macrophages and attenuated virulence in pigs since no clinical signs or virus genome in blood were observed following immunization. Thus, the deletion of MGF360-12L and MGF505-1R, in combination with K145R, was sufficient to dramatically attenuate virus infection in pigs. However, only two of six pigs were protected, suggesting that deletion of additional MGF genes is required to induce a protective immune response. Deletion of MGF360-12L, but not MGF505-1R, from the GeorgiaΔK145R virus reduced virus replication in macrophages, indicating that MGF360-12L was most critical for maintaining high levels of virus replication in macrophages. IMPORTANCE African swine fever has a high socioeconomic impact and no vaccines to aid control. The African swine fever virus (ASFV) has many genes that inhibit the host's interferon response. These include related genes that are grouped into multigene families, including MGF360 and 505. Here, we investigated which MGF360 and 505 genes were most important for viral attenuation and protection against genotype II strains circulating in Europe and Asia. We compared viruses with deletions of MGF genes. Deletion of just two MGF genes in combination with a third gene, K145R, a possible marker for vaccination, is sufficient for virus attenuation in pigs. Deletion of additional MGF360 genes was required to induce higher levels of protection. Furthermore, we showed that the deletion of MGF360-12L, combined with K145R, impairs virus replication in macrophages in culture. Our results have important implications for understanding the roles of the ASFV MGF genes and for vaccine development.

Role of African Swine Fever Virus Proteins EP153R and EP402R in Reducing Viral Persistence in Blood and Virulence in Pigs Infected with BeninΔDP148R.

The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress toward vaccine development. Previously, the DP148R gene was deleted from the genome of genotype I virulent Benin 97/1 isolate. This virus, BeninΔDP148R, induced transient moderate clinical signs after immunization and high levels of protection against challenge. However, the BeninΔDP148R virus and genome persisted in blood over a prolonged period. In the current study, deletion of either EP402R or EP153R genes individually or in combination from BeninΔDP148R genome was shown not to reduce virus replication in macrophages in vitro. However, deletion of EP402R dramatically reduced the period of infectious virus persistence in blood in immunized pigs from 28 to 14 days and virus genome from 59 to 14 days while maintaining high levels of protection against challenge. The additional deletion of EP153R (BeninΔDP148RΔEP153RΔEP402R) further attenuated the virus, and no viremia or clinical signs were observed postimmunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from BeninΔDP148R did not result in further virus attenuation and did not reduce the period of virus persistence in blood. These results show that EP402R and EP153R have a synergistic role in reducing clinical signs and levels of virus in blood. IMPORTANCE African swine fever virus (ASFV) causes a disease of domestic pigs and wild boar which results in death of almost all infected animals. The disease has a high economic impact, and no vaccine is available. We investigated the role of two ASFV proteins, called EP402R and EP153R, in determining the levels and length of time virus persists in blood from infected pigs. EP402R causes ASFV particles and infected cells to bind to red blood cells. Deletion of the EP402R gene dramatically reduced virus persistence in blood but did not reduce the level of virus. Deletion of the EP153R gene alone did not reduce the period or level of virus persistence in blood. However, deleting both EP153R and EP402R resulted in undetectable levels of virus in blood and no clinical signs showing that the proteins act synergistically. Importantly, the infected pigs were protected following infection with the wild-type virus that kills pigs.

Wild boar visits to commercial pig farms in southwest England: implications for disease transmission.

Contact between wild animals and farmed livestock may result in disease transmission with huge financial, welfare and ethical consequences. Conflicts between people and wildlife can also arise when species such as wild boar (Sus scrofa) consume crops or dig up pasture. This is a relatively recent problem in England where wild boar populations have become re-established in the last 20 years following a 500-year absence. The aim of this pilot study was to determine if and how often free-living wild boar visited two commercial pig farms near the Forest of Dean in southwest England. We placed 20 motion-sensitive camera traps at potential entry points to, and trails surrounding, the perimeter of two farmyards housing domestic pigs between August 2019 and February 2021, covering a total of 6030 trap nights. Forty wild boar detections were recorded on one farm spread across 27 nights, with a median (range) of 1 (0 to 7) night of wild boar activity per calendar month. Most of these wild boar detections occurred between ten and twenty metres of housed domestic pigs. No wild boar was detected at the other farm. These results confirm wild boar do visit commercial pig farms, and therefore, there is potential for contact and pathogen exchange between wild boar and domestic pigs. The visitation rates derived from this study could be used to parameterise disease transmission models of pathogens common to domestic pigs and wild boars, such as the African swine fever virus, and subsequently to develop mitigation strategies to reduce unwanted contacts.

Absence of Long-Term Protection in Domestic Pigs Immunized with Attenuated African Swine Fever Virus Isolate OURT88/3 or BeninΔMGF Correlates with Increased Levels of Regulatory T Cells and Interleukin-10.

Following short immunization protocols, naturally attenuated African swine fever virus (ASFV) isolate OURT88/3 and deletion mutant BeninΔMGF have previously been shown to induce high percentages of protection in domestic pigs against challenge with virulent virus. The results obtained in the present study show that a single intramuscular immunization of domestic pigs with OURT88/3 or BeninΔMGF followed by a challenge with the virulent Benin 97/1 isolate at day 130 postimmunization did not trigger the mechanisms necessary to generate immunological memory able to induce long-term protection against disease. All pigs developed acute forms of acute swine fever (ASF). Gamma interferon-producing cells peaked at day 24 postimmunization, declining thereafter. Surprisingly, the levels of regulatory T cells (Tregs) and interleukin-10 (IL-10) were elevated at the end of the experiment, suggesting that regulatory components of the immune system may inhibit effective protection.IMPORTANCE The duration of immunity for any vaccine candidate is crucial. In the case of African swine fever virus vaccine candidates, this issue has received little attention. Attenuated viruses have proven protective following short immunization protocols in which pigs were challenged a few weeks after the first immunization. Here, the duration of immunity and the immune responses induced over a duration of 130 days were studied during prechallenge and after challenge of pigs immunized with the naturally attenuated isolate OURT88/3 and an attenuated gene-deleted isolate, BeninΔMGF. After a single intramuscular immunization of domestic pigs with the OURT88/3 isolate or BeninΔMGF virus, animals were not protected against challenge with the virulent Benin 97/1 ASFV genotype I isolate at day 130 postimmunization. The levels of regulatory T cells and IL-10 were elevated at the end of the experiment, suggesting that regulatory components of the immune system may inhibit effective protection.

The African Swine Fever Virus Transcriptome.

African swine fever virus (ASFV) causes hemorrhagic fever in domestic pigs, presenting the biggest global threat to animal farming in recorded history. Despite the importance of ASFV, little is known about the mechanisms and regulation of ASFV transcription. Using RNA sequencing methods, we have determined total RNA abundance, transcription start sites, and transcription termination sites at single-nucleotide resolution. This allowed us to characterize DNA consensus motifs of early and late ASFV core promoters, as well as a polythymidylate sequence determinant for transcription termination. Our results demonstrate that ASFV utilizes alternative transcription start sites between early and late stages of infection and that ASFV RNA polymerase (RNAP) undergoes promoter-proximal transcript slippage at 5' ends of transcription units, adding quasitemplated AU- and AUAU-5' extensions to mRNAs. Here, we present the first much-needed genome-wide transcriptome study that provides unique insight into ASFV transcription and serves as a resource to aid future functional analyses of ASFV genes which are essential to combat this devastating disease.IMPORTANCE African swine fever virus (ASFV) causes incurable and often lethal hemorrhagic fever in domestic pigs. In 2020, ASF presents an acute and global animal health emergency that has the potential to devastate entire national economies as effective vaccines or antiviral drugs are not currently available (according to the Food and Agriculture Organization of the United Nations). With major outbreaks ongoing in Eastern Europe and Asia, urgent action is needed to advance our knowledge about the fundamental biology of ASFV, including the mechanisms and temporal control of gene expression. A thorough understanding of RNAP and transcription factor function, and of the sequence context of their promoter motifs, as well as accurate knowledge of which genes are expressed when and the amino acid sequence of the encoded proteins, is direly needed for the development of antiviral drugs and vaccines.

Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins.

African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs.IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates.

ICTV Virus Taxonomy Profile: Asfarviridae.

The family Asfarviridae includes the single species African swine fever virus, isolates of which have linear dsDNA genomes of 170-194 kbp. Virions have an internal core, an internal lipid membrane, an icosahedral capsid and an outer lipid envelope. Infection of domestic pigs and wild boar results in an acute haemorrhagic fever with transmission by contact or ingestion, or by ticks of the genus Ornithodoros. Indigenous pigs act as reservoirs in Africa, where infection is endemic, and from where introductions occur periodically to Europe. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Asfarviridae, which is available at www.ictv.global/report/asfarviridae.

Unraveling the Armor of a Killer: Evasion of Host Defenses by African Swine Fever Virus.

African swine fever is an acute hemorrhagic disease of pigs. Extensive recent spread in the Russian Federation and Eastern Europe has increased the risk to global pig production. The virus is a large DNA virus and is the only member of the Asfarviridae family. In pigs, the virus replicates predominantly in macrophages. We review how the virus overcomes the barriers to replication in the macrophage and the virus mechanism to inhibit key host defense pathways.

Structural Insight into African Swine Fever Virus A179L-Mediated Inhibition of Apoptosis.

Programmed cell death is a tightly controlled process critical for the removal of damaged or infected cells. Pro- and antiapoptotic proteins of the Bcl-2 family are pivotal mediators of this process. African swine fever virus (ASFV) is a large DNA virus, the only member of the Asfarviridae family, and harbors A179L, a putative Bcl-2 like protein. A179L has been shown to bind to several proapoptotic Bcl-2 proteins; however, the hierarchy of binding and the structural basis for apoptosis inhibition are currently not understood. We systematically evaluated the ability of A179L to bind proapoptotic Bcl-2 family members and show that A179L is the first antiapoptotic Bcl-2 protein to bind to all major death-inducing mammalian Bcl-2 proteins. We then defined the structural basis for apoptosis inhibition of A179L by determining the crystal structures of A179L bound to both Bid and Bax BH3 motifs. Our findings provide a mechanistic understanding for the potent antiapoptotic activity of A179L by identifying it as the first panprodeath Bcl-2 binder and serve as a platform for more-detailed investigations into the role of A179L during ASFV infection.IMPORTANCE Numerous viruses have acquired strategies to subvert apoptosis by encoding proteins capable of sequestering proapoptotic host proteins. African swine fever virus (ASFV), a large DNA virus and the only member of the Asfarviridae family, encodes the protein A179L, which functions to prevent apoptosis. We show that A179L is unusual among antiapoptotic Bcl-2 proteins in being able to physically bind to all core death-inducing mammalian Bcl-2 proteins. Currently, little is known regarding the molecular interactions between A179L and the proapoptotic Bcl-2 members. Using the crystal structures of A179L bound to two of the identified proapoptotic Bcl-2 proteins, Bid and Bax, we now provide a three-dimensional (3D) view of how A179L sequesters host proapoptotic proteins, which is crucial for subverting premature host cell apoptosis.

Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigs and Induces High Levels of Protection against Challenge.

Many of the approximately 165 proteins encoded by the African swine fever virus (ASFV) genome do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times postinfection. Deletion of this gene did not reduce virus replication in macrophages, showing that it is not essential for replication in these cells. However, deletion of this gene from a virulent isolate, Benin 97/1, producing the BeninΔDP148R virus, dramatically reduced the virulence of the virus in vivo All pigs infected with the BeninΔDP148R virus survived infection, showing only transient mild clinical signs soon after immunization. Following challenge with the parental virulent virus, all pigs immunized by the intramuscular route (11/11) and all except one immunized by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected, control nonimmune pigs developed signs of acute African swine fever (ASF). The virus genome and infectious virus were observed soon after immunization, coincident with the onset of clinical signs (∼106 genome copies or 50% tissue culture infective doses/ml). The levels of the virus genome declined over an extended period up to 60 days postimmunization. In contrast, infectious virus was no longer detectable by days 30 to 35. Gamma interferon (IFN-γ) was detected in serum between days 4 and 7 postimmunization, and IFN-γ-producing cells were detected in all pigs analyzed following stimulation of immune lymphocytes with whole virus. ASFV-specific antibodies were first detected from day 10 postimmunization.IMPORTANCE African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, the Russian Federation, and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterized. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines.

Dynamics of African swine fever virus shedding and excretion in domestic pigs infected by intramuscular inoculation and contact transmission.

African swine fever virus (ASFV) is a highly virulent swine pathogen that has spread across Eastern Europe since 2007 and for which there is no effective vaccine or treatment available. The dynamics of shedding and excretion is not well known for this currently circulating ASFV strain. Therefore, susceptible pigs were exposed to pigs intramuscularly infected with the Georgia 2007/1 ASFV strain to measure those dynamics through within- and between-pen transmission scenarios. Blood, oral, nasal and rectal fluid samples were tested for the presence of ASFV by virus titration (VT) and quantitative real-time polymerase chain reaction (qPCR). Serum was tested for the presence of ASFV-specific antibodies. Both intramuscular inoculation and contact transmission resulted in development of acute disease in all pigs although the experiments indicated that the pathogenesis of the disease might be different, depending on the route of infection. Infectious ASFV was first isolated in blood among the inoculated pigs by day 3, and then chronologically among the direct and indirect contact pigs, by day 10 and 13, respectively. Close to the onset of clinical signs, higher ASFV titres were found in blood compared with nasal and rectal fluid samples among all pigs. No infectious ASFV was isolated in oral fluid samples although ASFV genome copies were detected. Only one animal developed antibodies starting after 12 days post-inoculation. The results provide quantitative data on shedding and excretion of the Georgia 2007/1 ASFV strain among domestic pigs and suggest a limited potential of this isolate to cause persistent infection.

Transcription termination and readthrough in African swine fever virus.

Introduction: African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that encodes its own host-like RNA polymerase (RNAP) and factors required to produce mature mRNA. The formation of accurate mRNA 3' ends by ASFV RNAP depends on transcription termination, likely enabled by a combination of sequence motifs and transcription factors, although these are poorly understood. The termination of any RNAP is rarely 100% efficient, and the transcriptional "readthrough" at terminators can generate long mRNAs which may interfere with the expression of downstream genes. ASFV transcriptome analyses reveal a landscape of heterogeneous mRNA 3' termini, likely a combination of bona fide termination sites and the result of mRNA degradation and processing. While short-read sequencing (SRS) like 3' RNA-seq indicates an accumulation of mRNA 3' ends at specific sites, it cannot inform about which promoters and transcription start sites (TSSs) directed their synthesis, i.e., information about the complete and unprocessed mRNAs at nucleotide resolution. Methods: Here, we report a rigorous analysis of full-length ASFV transcripts using long-read sequencing (LRS). We systematically compared transcription termination sites predicted from SRS 3' RNA-seq with 3' ends mapped by LRS during early and late infection. Results: Using in-vitro transcription assays, we show that recombinant ASFV RNAP terminates transcription at polyT stretches in the non-template strand, similar to the archaeal RNAP or eukaryotic RNAPIII, unaided by secondary RNA structures or predicted viral termination factors. Our results cement this T-rich motif (U-rich in the RNA) as a universal transcription termination signal in ASFV. Many genes share the usage of the same terminators, while genes can also use a range of terminators to generate transcript isoforms varying enormously in length. A key factor in the latter phenomenon is the highly abundant terminator readthrough we observed, which is more prevalent during late compared with early infection. Discussion: This indicates that ASFV mRNAs under the control of late gene promoters utilize different termination mechanisms and factors to early promoters and/or that cellular factors influence the viral transcriptome landscape differently during the late stages of infection.

Full genome sequence analysis of African swine fever virus isolates from Cameroon.

African swine fever (ASF) is a devastating disease of domestic pigs that has spread across the globe since its introduction into Georgia in 2007. The etiological agent is a large double-stranded DNA virus with a genome of 170 to 180 kb in length depending on the isolate. Much of the differences in genome length between isolates are due to variations in the copy number of five different multigene families that are encoded in repetitive regions that are towards the termini of the covalently closed ends of the genome. Molecular epidemiology of African swine fever virus (ASFV) is primarily based on Sanger sequencing of a few conserved and variable regions, but due to the stability of the dsDNA genome changes in the variable regions occur relatively slowly. Observations in Europe and Asia have shown that changes in other genetic loci can occur and that this could be useful in molecular tracking. ASFV has been circulating in Western Africa for at least forty years. It is therefore reasonable to assume that changes may have accumulated in regions of the genome other than the standard targets over the years. At present only one full genome sequence is available for an isolate from Western Africa, that of a highly virulent isolate collected from Benin during an outbreak in 1997. In Cameroon, ASFV was first reported in 1981 and outbreaks have been reported to the present day and is considered endemic. Here we report three full genome sequences from Cameroon isolates of 1982, 1994 and 2018 outbreaks and identify novel single nucleotide polymorphisms and insertion-deletions that may prove useful for molecular epidemiology studies in Western Africa and beyond.

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

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

The Effect of Temperature on the Stability of African Swine Fever Virus BA71V Isolate in Environmental Water Samples.

African swine fever virus (ASFV) is known to be very stable and can remain infectious over long periods of time especially at low temperatures and within different matrices, particularly those containing animal-derived organic material. However, there are some gaps in our knowledge pertaining to the survivability and infectivity of ASFV in groundwater. This study aims to determine the stability and infectivity of the cell culture-adapted ASFV strain BA71V by plaque assay after incubation of the virus within river water samples at three different environmentally relevant temperatures (4 °C, 15 °C, and 21 °C) over the course of 42 days. The results from this study indicate that ASFV can remain stable and infectious when maintained at 4 °C in river water for more than 42 days, but as incubation temperatures are increased, the stability is reduced, and the virus is no longer able to form plaques after 28 days and 14 days, respectively, when stored at 15 °C and 21 °C. Characterizing the survivability of ASFV in groundwater can allow us to develop more appropriate inactivation and disinfection methods to support disease control and mitigate ASFV outbreaks.

Evaluation of the Efficacy of Commercial Disinfectants against African Swine Fever Virus.

African swine fever (ASF) is an economically important disease due to high morbidity and mortality rates and the ability to affect all ages and breeds of pigs. Biosecurity measures to prevent the spread of the causative agent, African swine fever virus (ASFV), include prescriptive cleaning and disinfection procedures. The aim of this study was to establish the biocidal effects of twenty-four commercially available disinfectants including oxidizing agents, acids, aldehydes, formic acids, phenol, and mixed-class chemistries against ASFV. The products were prepared according to the manufacturer's instructions and a suspension assay was performed with ASFV strain, BA71V using Vero cells (African green monkey cells) to test efficacy in reducing ASFV infection of cells. Generally, disinfectants containing formic acid and phenolic compounds, as well as oxidizing agents reduced viral titers of ASFV by over 4 log10 at temperatures ranging from 4 °C to 20 °C. Hydrogen peroxide, aldehyde, and quaternary ammonium compounds containing disinfectants were cytotoxic, limiting the detection of viral infectivity reductions to less than 4 log10. These preliminary results can be used to target research on disinfectants which contain active ingredients with known efficacy against ASFV under conditions recommended for the country where their use will be applied.

A protective multiple gene-deleted African swine fever virus genotype II, Georgia 2007/1, expressing a modified non-haemadsorbing CD2v protein.

African swine fever virus is a complex DNA virus that causes high fatality in pigs and wild boar and has a great socio-economic impact. An attenuated genotype II strain was constructed by replacing the gene for wildtype CD2v protein with versions in which single or double amino acid substitutions were introduced to reduce or abrogate the binding to red blood cells and reduce virus persistence in blood. The mutant CD2v proteins were expressed at similar levels to the wildtype protein on the surface of infected cells. Three recombinant viruses also had K145R, EP153R, and in one virus DP148R genes deleted. Following immunization of pigs, the virus with a single amino acid substitution in CD2v, Q96R, induced moderate levels of replication, and 100% protection against virulent ASFV. Two additional recombinant viruses had two amino acid substitutions in CD2v, Q96R, and K108D, and induced no binding to red blood cells in vitro. In immunized pigs, reduced levels of virus in blood and strong early ASFV-specific antibody and cellular responses were detected. After challenge low to moderate replication of challenge virus was observed. Reduced clinical signs post-challenge were observed in pigs immunized with the virus from which DP148R gene was deleted. Protection levels of 83-100% were maintained across a range of doses. Further experiments with virus GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D showed low levels of virus dissemination in tissue and transient clinical signs at high doses. The results support further evaluation of GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D as a vaccine candidate.