title: 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.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, New York, USA.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Krug
authors.affiliation: Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, New York, USA.
authors.forename: Lauren G
authors.initials: LG
authors.lastname: Holinka
authors.affiliation: Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, New York, USA Department of Pathobiology and Veterinary Science, CANHR, University of Connecticut, Storrs, Connecticut, USA.
authors.forename: Vivian
authors.initials: V
authors.lastname: O'Donnell
authors.affiliation: Center for Applied Genetics and Technology, University of Connecticut, Storrs, Connecticut, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Reese
authors.affiliation: Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, New York, USA.
authors.forename: Brenton
authors.initials: B
authors.lastname: Sanford
authors.affiliation: Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, New York, USA Department of Pathobiology and Veterinary Science, CANHR, University of Connecticut, Storrs, Connecticut, USA.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Fernandez-Sainz
authors.affiliation: Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, New York, USA Department of Pathobiology and Veterinary Science, CANHR, University of Connecticut, Storrs, Connecticut, USA.
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Gladue
authors.affiliation: Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, New York, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Arzt
authors.affiliation: Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, New York, USA.
authors.forename: Luis
authors.initials: L
authors.lastname: Rodriguez
authors.affiliation: Department of Pathobiology and Veterinary Science, CANHR, University of Connecticut, Storrs, Connecticut, USA.
authors.forename: Guillermo R
authors.initials: GR
authors.lastname: Risatti
authors.affiliation: Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, New York, USA manuel.borca@ars.usda.gov.
authors.forename: Manuel V
authors.initials: MV
authors.lastname: Borca
mesh_terms: D000220:Adaptation, Biological; D000358:African Swine Fever Virus / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification / Q000502:physiology; D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D016679:Genome, Viral; D005838:Genotype; D005846:Georgia (Republic); D009154:Mutation; D010641:Phenotype; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D012692:Serial Passage; D013552:Swine; D014774:Virulence
chemical_list: D004279:DNA, Viral