Krug Peter W, Holinka Lauren G, O'Donnell Vivian, Reese Bo, Sanford Brenton, Fernandez-Sainz Ignacio, Gladue Douglas P, Arzt Jonathan, Rodriguez Luis, Risatti Guillermo R, Borca Manuel V
Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, New York, USA.
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.
J Virol. 2015 Feb;89(4):2324-32. doi: 10.1128/JVI.03250-14. Epub 2014 Dec 10.
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.
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.
非洲猪瘟病毒（ASFV）可引发野生和家养猪的一种具有传染性且往往致命的疾病。源自自然发生、基因改造或适应细胞培养的ASFV的实验性疫苗已得到评估，但尚无用于控制非洲猪瘟（ASF）的商业疫苗。我们在此报告了在将一株来自格鲁吉亚共和国的强毒ASFV野外分离株（ASFV-G）适应在培养细胞系中生长的过程中，不同传代时获得的病毒的基因型和表型分析。ASFV-G在Vero细胞中连续传代110次。对传代30、60、80和110时获得的病毒进行了体外评估，以检测其在Vero细胞和原代猪巨噬细胞培养物中的复制能力，并进行了体内评估以测定其对猪的毒力。ASFV-G在Vero细胞中的复制随着连续传代而增加，这与在原代猪巨噬细胞培养物中的复制减少相对应。在体内，随着在Vero细胞中传代次数增加，观察到病毒毒力逐渐丧失，并且在传代110时观察到ASFV-G完全减毒。用完全减毒的病毒感染猪并不能使其免受强毒亲本ASFV-G攻击的保护。对这些病毒中的每一种进行的全长序列分析揭示了在基因组左右可变末端的特定区域逐渐积累的显著缺失。还观察到导致氨基酸替换和移码突变的突变，不过仅在相当有限数量的基因中出现。讨论了这些基因变化在病毒适应/减毒中的潜在重要性。
控制ASF的主要问题是缺乏疫苗。已尝试通过使ASFV适应培养细胞系来生产疫苗。这些尝试导致产生了仅提供同源保护的减毒病毒。关于这些分离株适应细胞培养的具体情况描述不足。诸如获得减毒病毒所需的传代次数、沿传代过程引入病毒基因组的基因修饰以及减毒程度和诱导的保护效力等细节并不容易获得。在本研究中，我们评估了导致在猪巨噬细胞中生长减少和在猪体内减毒的变化。毒力丧失可能与体内有限的复制有关，这可能导致在攻击后观察到猪缺乏保护性免疫。本报告提供了有价值的信息，可用于进一步了解ASFV基因功能、病毒减毒以及抗感染保护。
