Viroporin-like activity of the hairpin transmembrane domain of African swine fever virus B169L protein.

African swine fever virus (ASFV) is the causative agent of a contagious disease affecting wild and domestic swine. The function of B169L protein, as a potential integral structural membrane protein, remains to be experimentally characterized. Using state-of-the-art bioinformatics tools, we confirm here earlier predictions indicating the presence of an integral membrane helical hairpin, and further suggest anchoring of this protein to the ER membrane, with both terminal ends facing the lumen of the organelle. Our evolutionary analysis confirmed the importance of purifying selection in the preservation of the identified domains during the evolution of B169L in nature. Also, we address the possible function of this hairpin transmembrane domain (HTMD) as a class IIA viroporin. Expression of GFP fusion proteins in the absence of a signal peptide supported B169L insertion into the ER as a Type III membrane protein and the formation of oligomers therein. Overlapping peptides that spanned the B169L HTMD were reconstituted into ER-like membranes and the adopted structures analyzed by infrared spectroscopy. Consistent with the predictions, B169L transmembrane sequences adopted α-helical conformations in lipid bilayers. Moreover, single vesicle permeability assays demonstrated the assembly of lytic pores in ER-like membranes by B169L transmembrane helices, a capacity confirmed by ion-channel activity measurements in planar bilayers. Emphasizing the relevance of these observations, pore-forming activities were not observed in the case of transmembrane helices derived from EP84R, another ASFV protein predicted to anchor to membranes through a α-helical HTMD. Overall, our results support predictions of viroporin-like function for the B169L HTMD.IMPORTANCEAfrican swine fever (ASF), a devastating disease affecting domestic swine, is widely spread in Eurasia, producing significant economic problems in the pork industry. Approaches to prevent/cure the disease are mainly restricted to the limited information concerning the role of most of the genes encoded by the large (160-170 kba) virus genome. In this report, we present the experimental data on the functional characterization of the African swine fever virus (ASFV) gene B169L. Data presented here indicates that the B169L gene encodes for an essential membrane-associated protein with a viroporin function.

African Swine Fever Virus Gene B117L Encodes a Small Protein Endowed with Low-pH-Dependent Membrane Permeabilizing Activity.

African swine fever virus (ASFV) is causing a devastating pandemic in domestic and wild swine in Central Europe to East Asia, resulting in economic losses for the swine industry. The virus contains a large double-stranded DNA genome that contains more than 150 genes, most with no experimentally characterized function. In this study, we evaluate the potential function of the product of ASFV gene B117L, a 115-amino-acid integral membrane protein transcribed at late times during the virus replication cycle and showing no homology to any previously published protein. Hydrophobicity distribution along B117L confirmed the presence of a single transmembrane helix, which, in combination with flanking amphipathic sequences, composes a potential membrane-associated C-terminal domain of ca. 50 amino acids. Ectopic transient cell expression of the B117L gene as a green fluorescent protein (GFP) fusion protein revealed the colocalization with markers of the endoplasmic reticulum (ER). Intracellular localization of various B117L constructs also displayed a pattern for the formation of organized smooth ER (OSER) structures compatible with the presence of a single transmembrane helix with a cytoplasmic carboxy terminus. Using partially overlapping peptides, we further demonstrated that the B117L transmembrane helix has the capacity to establish spores and ion channels in membranes at low pH. Furthermore, our evolutionary analysis showed the high conservation of the transmembrane domain during the evolution of the B117L gene, indicating that the integrity of this domain is preserved by the action of the purifying selection. Collectively our data support a viroporin-like assistant role for the B117L gene-encoded product in ASFV entry. IMPORTANCE ASFV is responsible for an extensively distributed pandemic causing important economic losses in the pork industry in Eurasia. The development of countermeasures is partially limited by the insufficient knowledge regarding the function of the majority of the more than 150 genes present on the virus genome. Here, we provide data regarding the functional experimental evaluation of a previously uncharacterized ASFV gene, B117L. Our data suggest that the B117L gene encodes a small membrane protein that assists in the permeabilization of the ER-derived envelope during ASFV infection.

Evaluation of the Deletion of MGF110-5L-6L on Swine Virulence from the Pandemic Strain of African Swine Fever Virus and Use as a DIVA Marker in Vaccine Candidate ASFV-G-ΔI177L.

African swine fever virus (ASFV) is responsible for an ongoing pandemic that is affecting central Europe, Asia, and recently the Dominican Republic, the first report of the disease in the Western Hemisphere in over 40 years. ASFV is a large, complex virus with a double-stranded DNA (dsDNA) genome that carries more than 150 genes, most of which have not been studied. Here, we assessed the role of the MGF110-5L-6L gene during virus replication in cell cultures and experimental infection in swine. A recombinant virus with MGF110-5L-6L deleted (ASFV-G-ΔMGF110-5L-6L) was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. ASFV-G-ΔMGF110-5L-6L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, indicating that the MGF110-5L-6L gene is nonessential for virus replication. Similarly, domestic pigs inoculated with ASFV-G-ΔMGF110-5L-6L presented with a clinical disease undistinguishable from that caused by the parental ASFV-G, confirming that the MGF110-5L-6L gene is not involved in producing disease in swine. Sera from animals inoculated with an efficacious vaccine candidate, ASFV-G-ΔMGF, strongly recognized the protein encoded by the MGF110-5L-6L gene as a potential target for the development of an antigenic marker differentiation of infected from vaccinated animals (DIVA) vaccine. To test this hypothesis, the MGF110-5L-6L gene was deleted from the highly efficacious ASFV vaccine candidate ASFV-G-ΔI177L, generating the recombinant ASFV-G-ΔI177L/ΔMGF110-5L-6L. Animals inoculated with ASFV-G-ΔI177L/ΔMGF110-5L-6L developed an ASFV-specific antibody response detected by enzyme-linked immunosorbent assay (ELISA). The sera strongly recognized ASFV p30 expressed in eukaryotic cells but did not recognize ASFV MGF110-5L-6L protein, demonstrating that deletion of the MGF110-5L-6L gene can enable DIVA capabilities in preexisting vaccine candidates. IMPORTANCE Currently, there are no African swine fever (ASF) commercial vaccines that can be used to prevent or control the spread of ASF. The only effective experimental vaccines against ASF are live-attenuated vaccines. However, these experimental vaccines, which rely on a deletion of a specific gene of the current circulating strain of ASF, make it hard to tell the difference between a vaccinated and an infected animal. In our search for a serological marker, we identified that the virus protein encoded by the MGF110-5L-6L gene induced an immune response, making a virus lacking this gene a vaccine candidate that allows the differentiation of infected from vaccinated animals (DIVA). Here, we show that deletion of MGF110-5L-6L does not affect virulence or virus replication. However, when the deletion of MGF110-5L-6L was added to vaccine candidate ASFV-G-ΔI177L, a reduction in the effectiveness of the vaccine occurred.

Deletion of the H108R Gene Reduces Virulence of the Pandemic Eurasia Strain of African Swine Fever Virus with Surviving Animals Being Protected against Virulent Challenge.

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a devastating disease affecting domestic and wild swine and currently causing a global pandemic, severely affecting swine production. Here, we demonstrate that the deletion of the previously uncharacterized ASFV gene, H108R from the highly virulent ASFV-Georgia2007 (ASFV-G) genome strain, reduces virulence in domestic swine. ASFV-G-ΔH108R, a recombinant virus with the H108R gene deleted, was used to evaluate the involvement of the H108R gene for ASFV replication and virulence in swine. ASFV-G-ΔH108R showed a delayed replication in swine macrophage cultures. A group of five pigs, intramuscularly inoculated with 102 HAD50 of ASFV-G-ΔH108R, was observed over a 28-day period and compared with a similar group of animals inoculated with similar doses of the parental virulent virus. While all animals inoculated with ASFV-G developed an acute fatal disease, ASFV-G-ΔH108R inoculated animals, with the exception of one animal showing a protracted but fatal form of the disease, all survived the infection, remaining clinically healthy during the observational period. The surviving animals presented protracted viremias with lower virus titers compared with those of animals inoculated with the parental virus, and all of them developed a strong virus-specific antibody response. Importantly, all animals surviving ASFV-G-ΔAH108R infection were protected when challenged with the virulent parental strain, ASFV-G. This report constitutes the first evidence that the H108R gene is involved in ASFV virulence in swine and that the deletion of this gene may be used as a tool to increase the attenuation of currently experimental vaccines to improve their safety profiles. IMPORTANCE Currently, there is no commercial vaccine available to prevent ASF. ASFV-Georgia2007 (ASFV-G) and its field isolate derivatives are producing a large pandemic which is drastically affecting pork production in Eurasia. We present here the discovery of a novel virus determinant of virulence, the H108R gene, which, when deleted from the ASFV-G genome, significantly reduces virus virulence in domestic swine. Additionally, animals that survive the inoculation with a recombinant virus harboring a deletion of the H108R gene, ASFV-G-ΔH108R, are protected against a challenge with the virulent parental virus. Although presenting residual virulence, ASFV-G-ΔH108R confers protection even at low doses (102 HAD50), demonstrating its potential to be used as an additional gene deletion to increase the safety profile of the preexisting vaccine candidate.

Deletion of E184L, a Putative DIVA Target from the Pandemic Strain of African Swine Fever Virus, Produces a Reduction in Virulence and Protection against Virulent Challenge.

African swine fever (ASF) is currently causing a major pandemic affecting the swine industry and protein availability from Central Europe to East and South Asia. No commercial vaccines are available, making disease control dependent on the elimination of affected animals. Here, we show that the deletion of the African swine fever virus (ASFV) E184L gene from the highly virulent ASFV Georgia 2010 (ASFV-G) isolate produces a reduction in virus virulence during the infection in swine. Of domestic pigs intramuscularly inoculated with a recombinant virus lacking the E184L gene (ASFV-G-ΔE184L), 40% experienced a significantly (5 days) delayed presentation of clinical disease and, overall, had a 60% rate of survival compared to animals inoculated with the virulent parental ASFV-G. Importantly, all animals surviving ASFV-G-ΔE184L infection developed a strong antibody response and were protected when challenged with ASFV-G. As expected, a pool of sera from ASFV-G-ΔE184L-inoculated animals lacked any detectable antibody response to peptides partially representing the E184L protein, while sera from animals inoculated with an efficacious vaccine candidate, ASFV-G-ΔMGF, strongly recognize the same set of peptides. These results support the potential use of the E184L deletion for the development of vaccines able to differentiate infected from vaccinated animals (DIVA). Therefore, it is shown here that the E184L gene is a novel ASFV determinant of virulence that can potentially be used to increase safety in preexisting vaccine candidates, as well as to provide them with DIVA capabilities. To our knowledge, E184L is the first ASFV gene product experimentally shown to be a functional DIVA antigenic marker. IMPORTANCE No commercial vaccines are available to prevent African swine fever (ASF). The ASF pandemic caused by the ASF virus Georgia 2010 (ASFV-G) strain is seriously affecting pork production in a contiguous geographical area from Central Europe to East Asia. The only effective experimental vaccines are viruses attenuated by deleting ASFV genes associated with virus virulence. Therefore, identification of such genes is of critical importance for vaccine development. Here, we report the discovery of a novel determinant of ASFV virulence, the E184L gene. Deletion of the E184L gene from the ASFV-G genome (ASFV-G-ΔE184L) produced a reduction in virus virulence, and importantly, animals surviving infection with ASFV-G-ΔE184L were protected from developing ASF after challenge with the virulent parental virus ASFV-G. Importantly, the virus protein encoded by E184L is highly immunogenic, making a virus lacking this gene a vaccine candidate that allows the differentiation of infected from vaccinated animals (DIVA). Here, we show that unlike what is observed in animals inoculated with the vaccine candidate ASFV-G-ΔMGF, ASFV-G-ΔE184L-inoculated animals do not mount a E184L-specific antibody response, indicating the feasibility of using the E184L deletion as the antigenic marker for the development of a DIVA vaccine in ASFV.

Confirming the absence of parental African swine fever virus as a potential contaminant of recombinant live attenuated ASF vaccines.

African swine fever (ASF) is a devastating disease that is currently producing a panzootic significantly impacting the swine industry worldwide. One of the major challenges for advancing the development of ASF vaccines has been the absence of international standards for ASF vaccine purity, potency, safety, and efficacy. To date, the most effective experimental vaccines have been live attenuated strains of viruses. Most of these promising vaccine candidates have been developed by deleting virus genes involved in the process of viral pathogenesis and disease production. This approach requires genomic modification of a parental virus field strain through a process of homologous recombination followed by purification of the recombinant attenuated virus. In this scenario, it is critical to confirm the absence of any parental virulent virus in the final virus stock used for vaccine production. We present here a protocol to establish the purity of virus stock using the live attenuated vaccine candidates ASFV-G-ΔMGF, ASFV-G-Δ9 GLΔUK and ASFV-G-ΔI177L. Procedures described here includes inoculation in susceptible pigs followed by the assessment of the obtained material by differential qPCRs that allows the identification of vaccine virus from ASFV field isolates. This protocol is proposed as a model to ensure that master seed virus stock used for vaccine production does not contain residual parental virulent virus. Procedures described here includes a passage in susceptible pigs followed by the assessment of the obtained material by differential qPCRs that allows the identification of vaccine virus from ASFV field isolates.

Deletion of the A137R Gene from the Pandemic Strain of African Swine Fever Virus Attenuates the Strain and Offers Protection against the Virulent Pandemic Virus.

African swine fever virus (ASFV) is causing a devastating pandemic in domestic and wild swine within an extended geographical area from Central Europe to East Asia, resulting in economic losses for the regional swine industry. There are no commercial vaccines; therefore, disease control relies on identification and culling of infected animals. We report here that the deletion of the ASFV gene A137R from the highly virulent ASFV-Georgia2010 (ASFV-G) isolate induces a significant attenuation of virus virulence in swine. A recombinant virus lacking the A137R gene, ASFV-G-ΔA137R, was developed to assess the role of this gene in ASFV virulence in domestic swine. Animals inoculated intramuscularly with 102 50% hemadsorption doses (HAD50) of ASFV-G-ΔA137R remained clinically healthy during the 28-day observational period. All animals inoculated with ASFV-G-ΔA137R had medium to high viremia titers and developed a strong virus-specific antibody response. Importantly, all ASFV-G-ΔA137R-inoculated animals were protected when challenged with the virulent parental strain ASFV-G. No evidence of replication of challenge virus was observed in the ASFV-G-ΔA137R-inoculated animals. Therefore, ASFV-G-ΔA137R is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce protection against the highly virulent ASFV Georgia virus that is the cause of the current Eurasian pandemic. IMPORTANCE No commercial vaccine is available to prevent African swine fever. The ASF pandemic caused by ASFV Georgia2007 strain (ASFV-G) is seriously affecting pork production in a contiguous area from Central Europe to East Asia. Here we report the rational development of a potential live attenuated vaccine strain by deleting a virus-specific gene, A137R, from the genome of ASFV-G. The resulting virus presented a completely attenuated phenotype and, importantly, animals infected with this genetically modified virus were protected from developing ASF after challenge with the virulent parental virus. ASFV-G-ΔA137R confers protection even at low doses (102 HAD50), demonstrating its potential as a vaccine candidate. Therefore, ASFV-G-ΔA137R is a novel experimental ASF vaccine protecting pigs from the epidemiologically relevant ASFV Georgia isolate.

Development of a Highly Effective African Swine Fever Virus Vaccine by Deletion of the I177L Gene Results in Sterile Immunity against the Current Epidemic Eurasia Strain.

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The disease is devastating the swine industry in Central Europe and East Asia, with current outbreaks caused by circulating strains of ASFV derived from the 2007 Georgia isolate (ASFV-G), a genotype II ASFV. In the absence of any available vaccines, African swine fever (ASF) outbreak containment relies on the control and culling of infected animals. Limited cross-protection studies suggest that in order to ensure a vaccine is effective, it must be derived from the current outbreak strain or at the very least from an isolate with the same genotype. Here, we report the discovery that the deletion of a previously uncharacterized gene, I177L, from the highly virulent ASFV-G produces complete virus attenuation in swine. Animals inoculated intramuscularly with the virus lacking the I177L gene, ASFV-G-ΔI177L, at a dose range of 102 to 106 50% hemadsorbing doses (HAD50), remained clinically normal during the 28-day observational period. All ASFV-G-ΔI177L-infected animals had low viremia titers, showed no virus shedding, and developed a strong virus-specific antibody response; importantly, they were protected when challenged with the virulent parental strain ASFV-G. ASFV-G-ΔI177L is one of the few experimental vaccine candidate virus strains reported to be able to induce protection against the ASFV Georgia isolate, and it is the first vaccine capable of inducing sterile immunity against the current ASFV strain responsible for recent outbreaks.IMPORTANCE Currently, there is no commercially available vaccine against African swine fever. Outbreaks of this disease are devastating the swine industry from Central Europe to East Asia, and they are being caused by circulating strains of African swine fever virus derived from the Georgia 2007 isolate. Here, we report the discovery of a previously uncharacterized virus gene, which when deleted completely attenuates the Georgia isolate. Importantly, animals infected with this genetically modified virus were protected from developing ASF after challenge with the virulent parental virus. Interestingly, ASFV-G-ΔI177L confers protection even at low doses (102 HAD50) and remains completely attenuated when inoculated at high doses (106 HAD50), demonstrating its potential as a safe vaccine candidate. At medium or higher doses (104 HAD50), sterile immunity is achieved. Therefore, ASFV-G-ΔI177L is a novel efficacious experimental ASF vaccine protecting pigs from the epidemiologically relevant ASFV Georgia isolate.

A neutralizing monoclonal antibody-based competitive ELISA for classical swine fever C-strain post-vaccination monitoring.

BACKGROUND: Virus neutralization test (VNT) is widely used for serological survey of classical swine fever (CSF) and efficacy evaluation of CSF vaccines. However, VNT is a time consuming procedure that requires cell culture and live virus manipulation. C-strain CSF vaccine is the most frequently used vaccine for CSF control and prevention. In this study, we presented a neutralizing monoclonal antibody (mAb) based competitive enzyme-linked immunosorbent assay (cELISA) with the emphasis on the replacement of VNT for C-strain post-vaccination monitoring. RESULTS: One monoclonal antibody (6B211) which has potent neutralizing activity against C-strain was generated. A novel cELISA was established and optimized based on the strategy that 6B211 can compete with C-strain induced neutralizing antibodies in pig serum to bind capture antigen C-strain E2. By testing C-strain VNT negative pig sera (n = 445) and C-strain VNT positive pig sera (n = 70), the 6B211 based cELISA showed 100% sensitivity (95% confidence interval: 94.87 to 100%) and 100% specificity (95% confidence interval: 100 to 100%). The C-strain antibody can be tested in pigs as early as 7 days post vaccination with the cELISA. By testing pig sera (n = 139) in parallel, the cELISA showed excellent agreement (Kappa = 0.957) with VNT. The inhibition rate of serum samples in the cELISA is highly correlated with their titers in VNT (r2 = 0.903, p < 0.001). In addition, intra- and inter-assays of the cELISA exhibited acceptable repeatability with low coefficient of variations (CVs). CONCLUSIONS: This novel cELISA demonstrated excellent agreement and high level correlation with VNT. It is a reliable tool for sero-monitoring of C-strain vaccination campaign because it is a rapid, simple, safe and cost effective assay that can be used to monitor vaccination-induced immune response at the population level.

Mutation-induced changes of transmembrane pore size revealed by combined ion-channel conductance and single vesicle permeabilization analyses.

Permeabilization of the Endoplasmic Reticulum (ER) is instrumental in the progression of host-cell infection by many viral pathogens. We have described that permeabilization of ER model membranes by the pore-forming domain of the Classical Swine Fever Virus (CSFV) p7 protein depends on two sequence determinants: the C-terminal transmembrane helix, and the preceding polar loop that regulates its activity. Here, by combining ion-channel activity measurements in planar lipid bilayers with imaging of single Giant Unilamellar Vesicles (GUVs), we demonstrate that point substitutions directed to conserved residues within these regions affect ER-like membrane permeabilization following distinct mechanisms. Whereas the polar loop appeared to be involved in protein insertion and oligomerization, substitution of residues predicted to face the lumen of the pore inhibited large conducting channels (>1 nS) over smaller ones (120 pS). Quantitative analyses of the ER-GUV distribution as a function of the solute size revealed a selective inhibition for the permeation of solutes with sizes larger than 4 kDa, further demonstrating that the mutation targeting the transmembrane helix prevented formation of the large pores. Collectively, our data support the idea that the pore-forming domain of p7 may assemble into finite pores with approximate diameters of 1 and 5 nm. Moreover, the observation that the mutation interfering with formation of the larger pores can hamper virus production without affecting ER localization or homo-oligomerization, suggests prospective strategies to block/attenuate pestiviruses.

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.

Simultaneous Deletion of the 9GL and UK Genes from the African Swine Fever Virus Georgia 2007 Isolate Offers Increased Safety and Protection against Homologous Challenge.

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Successful experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFV. Recombinant viruses harboring engineered deletions of specific virulence-associated genes induce solid protection against challenge with parental viruses. Deletion of the 9GL (B119L) gene in the highly virulent ASFV isolates Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (Δ9GL viruses) resulted in complete protection when challenged with parental isolates. When similar deletions were created within the ASFV Georgia 2007 (ASFV-G) genome, attenuation was achieved but the protective and lethal doses were too similar. To enhance attenuation of ASFV-G, we deleted another gene, UK (DP96R), which was previously shown to be involved in attenuation of the ASFV E70 isolate. Here, we report the construction of a double-gene-deletion recombinant virus, ASFV-G-Δ9GL/ΔUK. When administered intramuscularly (i.m.) to swine, there was no induction of disease, even at high doses (106 HAD50). Importantly, animals infected with 104 50% hemadsorbing doses (HAD50) of ASFV-G-Δ9GL/ΔUK were protected as early as 14 days postinoculation when challenged with ASFV-G. The presence of protection correlates with the appearance of serum anti-ASFV antibodies, but not with virus-specific circulating ASFV-specific gamma interferon (IFN-γ)-producing cells. ASFV-G-Δ9GL/ΔUK is the first rationally designed experimental ASFV vaccine that protects against the highly virulent ASFV Georgia 2007 isolate as early as 2 weeks postvaccination. IMPORTANCE: Currently, there is no commercially available vaccine against African swine fever. Outbreaks of the disease are devastating to the swine industry and are caused by circulating strains of African swine fever virus. Here, we report a putative vaccine derived from a currently circulating strain but containing two deletions in two separate areas of the virus, allowing increased safety. Using this genetically modified virus, we were able to vaccinate swine and protect them from developing ASF. We were able to achieve protection from disease as early as 2 weeks after vaccination, even when the pigs were exposed to a higher than normal concentration of ASFV.

The progressive adaptation of a georgian isolate of African swine fever virus to vero cells leads to a gradual attenuation of virulence in swine corresponding to major modifications of the viral genome.

UNLABELLED: African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified, or cell culture-adapted ASFV have been evaluated, but no commercial vaccine is available to control African swine fever (ASF). We report here the genotypic and phenotypic analysis of viruses obtained at different passages during the process of adaptation of a virulent ASFV field isolate from the Republic of Georgia (ASFV-G) to grow in cultured cell lines. ASFV-G was successively passaged 110 times in Vero cells. Viruses obtained at passages 30, 60, 80, and 110 were evaluated in vitro for the ability to replicate in Vero cells and primary swine macrophages cultures and in vivo for assessing virulence in swine. Replication of ASFV-G in Vero cells increased with successive passages, corresponding to a decreased replication in primary swine macrophages cultures. In vivo, progressive loss of virus virulence was observed with increased passages in Vero cells, and complete attenuation of ASFV-G was observed at passage 110. Infection of swine with the fully attenuated virus did not confer protection against challenge with virulent parental ASFV-G. Full-length sequence analysis of each of these viruses revealed significant deletions that gradually accumulated in specific areas at the right and left variable ends of the genome. Mutations that result in amino acid substitutions and frameshift mutations were also observed, though in a rather limited number of genes. The potential importance of these genetic changes in virus adaptation/attenuation is discussed. IMPORTANCE: The main problem in controlling ASF is the lack of vaccines. Attempts to produce vaccines by adaptation of ASFV to cultured cell lines have been made. These attempts led to the production of attenuated viruses that conferred only homologous protection. Specifics regarding adaptation of these isolates to cell cultures have been insufficiently described. Details like the numbers of passages required to obtain attenuated viruses, genetic modifications introduced into the virus genomes along passages, and the extent of attenuation and induced protective efficacy are not readily available. In this study, we assessed the changes that lead to decreased growth in swine macrophages and to attenuation in swine. Loss of virulence, probably associated with limited replication in vivo, may lead to the lack of protective immunity in swine observed after challenge. This report provides valuable information that can be used to further the understanding of ASFV gene function, virus attenuation, and protection against infection.

African Swine Fever Virus Georgia 2007 with a Deletion of Virulence-Associated Gene 9GL (B119L), when Administered at Low Doses, Leads to Virus Attenuation in Swine and Induces an Effective Protection against Homologous Challenge.

UNLABELLED: African swine fever virus (ASFV) is the etiological agent of an often lethal disease of domestic pigs. Disease control strategies have been hampered by the unavailability of vaccines against ASFV. Since its introduction in the Republic of Georgia, a highly virulent virus, ASFV Georgia 2007 (ASFV-G), has caused an epizootic that spread rapidly into Eastern European countries. Currently no vaccines are available or under development to control ASFV-G. In the past, genetically modified ASFVs harboring deletions of virulence-associated genes have proven attenuated in swine, inducing protective immunity against challenge with homologous parental viruses. Deletion of the gene 9GL (open reading frame [ORF] B119L) in highly virulent ASFV Malawi-Lil-20/1 produced an attenuated phenotype even when administered to pigs at 10(6) 50% hemadsorption doses (HAD50). Here we report the construction of a genetically modified ASFV-G strain (ASFV-G-Δ9GLv) harboring a deletion of the 9GL (B119L) gene. Like Malawi-Lil-20/1-Δ9GL, ASFV-G-Δ9GL showed limited replication in primary swine macrophages. However, intramuscular inoculation of swine with 10(4) HAD50 of ASFV-G-Δ9GL produced a virulent phenotype that, unlike Malawi-Lil-20/1-Δ9GL, induced a lethal disease in swine like parental ASFV-G. Interestingly, lower doses (10(2) to 10(3) HAD50) of ASFV-G-Δ9GL did not induce a virulent phenotype in swine and when challenged protected pigs against disease. A dose of 10(2) HAD50 of ASFV-G-Δ9GLv conferred partial protection when pigs were challenged at either 21 or 28 days postinfection (dpi). An ASFV-G-Δ9GL HAD50 of 10(3) conferred partial and complete protection at 21 and 28 dpi, respectively. The information provided here adds to our recent report on the first attempts toward experimental vaccines against ASFV-G. IMPORTANCE: The main problem for controlling ASF is the lack of vaccines. Studies on ASFV virulence lead to the production of genetically modified attenuated viruses that induce protection in pigs but only against homologous virus challenges. Here we produced a recombinant ASFV lacking virulence-associated gene 9GL in an attempt to produce a vaccine against virulent ASFV-G, a highly virulent virus isolate detected in the Caucasus region in 2007 and now spreading though the Caucasus region and Eastern Europe. Deletion of 9GL, unlike with other ASFV isolates, did not attenuate completely ASFV-G. However, when delivered once at low dosages, recombinant ASFV-G-Δ9GL induces protection in swine against parental ASFV-G. The protection against ASFV-G is highly effective after 28 days postvaccination, whereas at 21 days postvaccination, animals survived the lethal challenge but showed signs of ASF. Here we report the design and development of an experimental vaccine that induces protection against virulent ASFV-G.

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

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

Protection induced by a glycoprotein E-deleted bovine herpesvirus type 1 marker strain used either as an inactivated or live attenuated vaccine in cattle.

BACKGROUND: Bovine herpesvirus type 1 (BoHV-1) is the causative agent of respiratory and genital tract infections; causing a high economic loss in all continents. Use of marker vaccines in IBR eradication programs is widely accepted since it allows for protection of the animals against the disease while adding the possibility of differentiating vaccinated from infected animals.The aim of the present study was the development and evaluation of safety and efficacy of a glycoprotein E-deleted (gE-) BoHV-1 marker vaccine strain (BoHV-1ΔgEßgal) generated by homologous recombination, replacing the viral gE gene with the ß-galactosidase (ßgal) gene. RESULTS: In vitro growth kinetics of the BoHV-1ΔgEßgal virus was similar to BoHV-1 LA. The immune response triggered by the new recombinant strain in cattle was characterized both as live attenuated vaccine (LAV) and as an inactivated vaccine. BoHV-1ΔgEßgal was highly immunogenic in both formulations, inducing specific humoral and cellular immune responses. Antibody titers found in animals vaccinated with the inactivated vaccine based on BoHV-1ΔgEßgal was similar to the titers found for the control vaccine (BoHV-1 LA). In the same way, titers of inactivated vaccine groups were significantly higher than any of the LAV immunized groups, independently of the inoculation route (p < 0.001). Levels of IFN-γ were significantly higher (p < 0.001) in those animals that received the LAV compared to those that received the inactivated vaccine. BoHV-1ΔgEßgal exhibited an evident attenuation when administered as a LAV; no virus was detected in nasal secretions of vaccinated or sentinel animals during the post-vaccination period. BoHV-1ΔgEßgal, when used in either formulation, elicited an efficient immune response that protected animals against challenge with virulent wild-type BoHV-1. Also, the deletion of the gE gene served as an immunological marker to differentiate vaccinated animals from infected animals. All animals vaccinated with the BoHV-1ΔgE ßgal strain were protected against disease after challenge and shed significantly less virus than control calves, regardless of the route and formulation they were inoculated. CONCLUSIONS: Based on its attenuation, immunogenicity and protective effect after challenge, BoHV-1ΔgEßgal virus is an efficient and safe vaccine candidate when used either as inactivated or as live attenuated forms.

African Swine Fever Virus Protein-Protein Interaction Prediction.

The African swine fever virus (ASFV) is an often deadly disease in swine and poses a threat to swine livestock and swine producers. With its complex genome containing more than 150 coding regions, developing effective vaccines for this virus remains a challenge due to a lack of basic knowledge about viral protein function and protein-protein interactions between viral proteins and between viral and host proteins. In this work, we identified ASFV-ASFV protein-protein interactions (PPIs) using artificial intelligence-powered protein structure prediction tools. We benchmarked our PPI identification workflow on the Vaccinia virus, a widely studied nucleocytoplasmic large DNA virus, and found that it could identify gold-standard PPIs that have been validated in vitro in a genome-wide computational screening. We applied this workflow to more than 18,000 pairwise combinations of ASFV proteins and were able to identify seventeen novel PPIs, many of which have corroborating experimental or bioinformatic evidence for their protein-protein interactions, further validating their relevance. Two protein-protein interactions, I267L and I8L, I267L__I8L, and B175L and DP79L, B175L__DP79L, are novel PPIs involving viral proteins known to modulate host immune response.

Analysis of the Unique Historical Isolate of African Swine Fever Virus Isolate Spencer from Outbreaks in 1951.

African swine fever (ASF) is a deadly hemorrhagic disease of domestic and wild swine that was first described in the early 20th century after the introduction of European pigs to Kenya. The etiological agent, the African swine fever virus (ASFV), is a large DNA virus within the Asfarviridae family that is broadly categorized epidemiologically into genotypes based on the nucleotide sequence of B646L, the gene encoding the major capsid protein p72. ASF outbreaks in Africa have been linked historically to 25 genotypes by p72 nucleotide analysis and, recently, to 6 genotypes by amino acid comparison, whereas global outbreaks of ASF outside of Africa have only been linked to 2 genotypes: genotype I, which led to an outbreak in Europe during the 1960s that later spread to South America, and genotype II, responsible for the current pandemic that began in Georgia in 2007 and has since spread to Europe, Asia, and Hispaniola. Here, we present an analysis of the genome of ASFV Spencer, an isolate that was collected in 1951 near Johannesburg, South Africa. While nucleotide analysis of Spencer indicates the p72 coding sequence is unique, differentiating from the closest reference by five nucleotides, the predicted amino acid sequence indicates that it is 100% homologous to contemporary genotype 1. Full genome analysis reveals it is more similar to Mkuzi1979 and encodes genes that share similarity with either genotype 1 or genotype 2 outbreak strains.

African swine fever virus P72 genotyping tool.

Historically, genotyping of African swine fever virus was based on partial sequencing of B646L (p72). Until recently, the number of differences that defined genotypes was ambiguous. This tool allows a sequence to be uploaded and will report its closest matches along with its likely p72 genotype.

FlagT4G Vaccine Prevents Transplacental Transmission of Highly Virulent Classical Swine Fever Virus after Single Vaccination in Pregnant Sows.

The transplacental transmission of CSFV and the resulting persistent congenital infection in newborn piglets have been abundantly discussed in pregnant sows suffering from virus infection. Importantly, the availability of safe commercial vaccines with proven efficacy to prevent the generation of congenital and postnatal persistent infections in pregnant sows are critical tools for controlling the disease in CSF endemic areas. Here, we demonstrate the high efficacy of a single dose of the recombinant FlagT4G vaccine to provide solid protection in pregnant sows against transplacental transmission of a highly virulent CSFV. Pregnant sows vaccinated with FlagT4G at 44 days of gestation elicited a strong CSFV-specific antibody response, with neutralizing antibody levels above those required for protection against CSFV. Importantly, after the challenge with a highly virulent CSFV, all foetuses from FlagT4G-vaccinated sows lacked CSF macroscopic lesions and showed a complete absence of the challenge virus in their internal organs at day 79 of gestation. Therefore, pregnant sows safely vaccinated with FlagT4G without affecting reproductive efficacy are efficaciously protected, along with their foetuses, against the infection and disease caused by a CSFV virulent field strain.