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-⌬MGF) 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-⌬MGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-⌬MGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 10 2 or 10 4 50% hemadsorbing doses (HAD 50 ) 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-⌬MGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G. A frican swine fever (ASF) is a contagious viral disease of swine caused by ASF virus (ASFV), a large enveloped virus containing a double-stranded DNA genome of approximately 180 to 190 kbp (1). ASF causes a spectrum of disease manifestations, from highly lethal to subclinical, depending on host characteristics and the virus strain (2). Virulent ASFV infections in domestic pigs are fatal and characterized by fever, hemorrhages, ataxia, and severe depression.Currently, ASF is endemic in several sub-Saharan African countries. In Europe, the disease is endemic in Sardinia (Italy), and outbreaks have been recorded in the Caucasus region since 2007, affecting Georgia, Armenia, Azerbaijan, and Russia, and, more recently, in Ukraine, Belarus, Lithuania, Latvia, and Poland, threatening to disseminate into neighboring western European countries (3).There is no vaccine available for ASF, and outbreaks are usually controlled by animal quarantine and elimination of the affected animals. Experimental vaccines based on the use of different inactivated virus preparations have failed to induce protective immunity (4-6). Protective immunity against reinfection with homologous viruses and (rarely) against reinfection with heterologous viruses does develop in pigs surviving viral infection (7,8). Pigs im...

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.

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. IMPORTANCEThe 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.A frican swine fever (ASF) is a contagious disease of swine caused by ASF virus (ASFV), an enveloped virus containing a large (190-kbp) double-stranded DNA (dsDNA) genome. ASFV infection of domestic pigs can induce a spectrum of disease severity, from highly lethal to subclinical, depending on host characteristics and the particular circulating virus strain (1).A...

African swine fever virus (ASFV) is IMPORTANCEThe 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-⌬9GL 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.A frican swine fever (ASF) is a contagious viral disease of swine. The causative agent, ASF virus (ASFV), is a large enveloped virus containing a double-stranded DNA (dsDNA) genome of approximately 190 kbp. ASFV shares aspects of genome structure and replication strategy with other large dsDNA viruses, including the Poxviridae, Iridoviridae, and Phycodnaviridae (1). ASF causes a spectrum of disease that ranges from highly lethal to subclinical, depending on host characteristics and the virulence of circulating virus strains (2). ASFV infections in domestic pigs are often fatal and are characterized by high fever, hemorrhages, ataxia, and severe depression.Currently, the disease is endemic in more than 20 sub-Saharan African countries. In Europe, ASF is endemic on the island of Sardinia (Italy), and outbreaks of ASF have been recorded in the Caucasus region since 2007, affecting Georgia, Armenia, Azerbaijan, and Russia. Isolated outbreaks have been recently reported in Ukraine, Belarus, Lithuania, Latvia, and Poland, posing the risk of further dissemination into neighboring countries. The epidemic

Foot-and-mouth disease virus (FMDV) is the type species of the Aphthovirus genus within the Picornaviridae family. Infection of cells with positive-strand RNA viruses results in a rearrangement of intracellular membranes into viral replication complexes. The origin of these membranes remains unknown; however induction of the cellular process of autophagy is beneficial for the replication of poliovirus, suggesting that it might be advantageous for other picornaviruses. By using confocal microscopy we showed in FMDV-infected cells co-localization of non-structural viral proteins 2B, 2C and 3A with LC3 (an autophagosome marker) and viral structural protein VP1 with Atg5 (autophagy-related protein), and LC3 with LAMP-1. Importantly, treatment of FMDV-infected cell with autophagy inducer rapamycin, increased viral yield, and inhibition of autophagosomal pathway by 3-methyladenine or small-interfering RNAs, decreased viral replication. Altogether, these studies strongly suggest that autophagy may play an important role during the replication of FMDV.

The genome of foot-and-mouth disease virus (FMDV) differs from that of other picornaviruses in that it encodes a larger 3A protein (>50% longer than poliovirus 3A), as well as three copies of protein 3B (also known as VPg). Previous studies have shown that a deletion of amino acids 93 to 102 of the 153-codon 3A protein is associated with an inability of a Taiwanese Foot-and-mouth disease (FMD) is an extremely contagious viral disease of cattle, pigs, sheep, goats, and many wild animals. The disease is characterized by fever and vesicular lesions of the epithelium of the mouth, tongue, feet, and teats. The causal agent, FMD virus (FMDV), is a positive-stranded RNA virus that is the type species of the Aphthovirus genus of the Picornaviridae. The FMDV genome is over 8 kb in length and contains a protein cap (3B, also known as VPg) (27). During replication, the genome is expressed as a single open reading frame (ORF) that is processed into mature polypeptide products. Translation of the ORF begins with a proteinase (L pro ), which is followed in the ORF by the structural proteins (1A, 1B, 1C, and 1D), a short autoproteinase (2A), and the remaining nonstructural proteins (2B, 2C, 3A, 3B, 3C pro , and 3D pol ). 3C pro is responsible for proteolytic cleavage of the majority of the cleavage sites in the FMDV polyprotein (33), and 3D pol is the core subunit of the picornavirus RNA-dependent RNA polymerase (7). Protein 3B, which is represented in three nonidentical copies in FMDV (8), is covalently bound to the 5Ј end of the genome and antigenome, and functions in priming picornavirus RNA synthesis (see reference 34 for review). The functions of the nonstructural proteins 2B, 2C, and 3A are less well understood, although all three have hydrophobic domains (9, 26), and all have been found physically associated with intracellular membranes that proliferate in picornavirus-infected cells (2,3,29,30,32).We have previously shown that a deletion in the 3A protein of FMDV is a characteristic of a virus (O/TAW/97) that devastated the Taiwanese pork industry in 1997 and that the deleted 3A is responsible for the virus' inability to cause disease in cattle (1). However, this deleted 3A does not interfere with production of an acute and highly and readily transmissible disease in pigs (5). The deletion in O/TAW/97 occurs at positions 93 to 102 of the 153-amino-acid 3A protein and is similar in size and position to deletions that were found in eggadapted derivatives of FMDV that were developed for use as vaccines in South America (12). Interestingly, an investigation of the 3A coding regions of a number of Asian serotype O FMDVs revealed that viruses harboring this deletion have been circulating in Asia for more than 30 years and that viruses with 3As harboring a deletion spanning residues 133 to 143 have been circulating in Southeast Asia since the mid 1990s (18). Recently, Sobrino and coworkers have shown that adaptation of an FMDV isolate to cause disease in guinea pigs is dependent on a point mutation in 3A, adding further supp...

In 1997, an epizootic in Taiwan, Province of China, was caused by a type O foot-and-mouth disease virus which infected pigs but not cattle. The virus had an altered 3A protein, which harbored a 10-amino-acid deletion and a series of substitutions. Here we show that this deletion is present in the earliest type O virus examined from the region (from 1970), whereas substitutions surrounding the deletion accumulated over the last 29 years. Analyses of the growth of these viruses in bovine cells suggest that changes in the genome in addition to the deletion, per se, are responsible for the porcinophilic properties of current Asian viruses in this lineage.

It has been demonstrated that foot-and-mouth disease virus (FMDV) can utilize at least four members of the ␣ V subgroup of the integrin family of receptors in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the ␤G-␤H loop of VP1. While there have been extensive studies of virus-receptor interactions at the cell surface, our understanding of the events during viral entry into the infected cell is still not clear. We have utilized confocal microscopy to analyze the entry of two FMDV serotypes (types A and O) after interaction with integrin receptors at the cell surface. In cell cultures expressing both the ␣ V ␤ 3 and ␣ V ␤ 6 integrins, virus adsorbed to the cells at 4°C appears to colocalize almost exclusively with the ␣ V ␤ 6 integrin. Upon shifting the infected cells to 37°C, FMDV capsid proteins were detected within 15 min after the temperature shift, in association with the integrin in vesicular structures that were positive for a marker of clathrin-mediated endocytosis. In contrast, virus did not colocalize with a marker for caveola-mediated endocytosis. Virus remained associated with the integrin until about 1 h after the temperature shift, when viral proteins appeared around the perinuclear region of the cell. By 15 min after the temperature shift, viral proteins were seen colocalizing with a marker for early endosomes, while no colocalization with late endosomal markers was observed. In the presence of monensin, which raises the pH of endocytic vesicles and has been shown to inhibit FMDV replication, viral proteins were not released from the recycling endosome structures. Viral proteins were not observed associated with the endoplasmic reticulum or the Golgi. These data indicate that FMDV utilizes the clathrin-mediated endocytosis pathway to infect the cells and that viral replication begins due to acidification of endocytic vesicles, causing the breakdown of the viral capsid structure and release of the genome by an as-yet-unidentified mechanism.Foot-and-mouth disease is a highly contagious viral disease of cloven-hoofed livestock caused by Foot-and-mouth disease virus (FMDV), the type species of the genus Aphthovirus of the family Picornaviridae. The virus is a positive-strand RNA virus containing a genome of approximately 8,400 nucleotides (reference 33 and references therein).FMDV initiates infection in cultured cells by binding to any of four members of the ␣ V subgroup of the integrin family of cellular receptors (␣ V ␤ 1 , ␣ V ␤ 3 , ␣ V ␤ 6 , and ␣ V ␤ 8 ) (16,27,36,39,40,60) via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the ␤G-␤H loop of VP1 (6, 30, 46, 52). We have recently shown that the ␣ V ␤ 6 integrin acts as a high-affinity receptor for the virus while ␣ V ␤ 3 interacts with virus with a much lower affinity (28). In addition, viruses of the type A serotype utilize both the ␣ V ␤ 3 and ␣ V ␤ 6 integrins as receptors in cultured cells, while serotype O...