非洲猪瘟病毒的基因特征
2020-02-04谢春芳于瑞嵩董世娟陈冰清
谢春芳 于瑞嵩 董世娟 陈冰清
摘 要:由非洲猪瘟病毒(African Swine Fever Virus,ASFV)引起的非洲猪瘟(African Swine Fever,ASF)会给猪群的健康造成极其严重的危害。ASFV是大分子病毒,基因组成分复杂而且多变,这是研究ASFV的主要难点之一。ASFV以大分子DNA病毒的独特方式进行基因的转录、复制、翻译和表达。目前ASFV有超过150个开放阅读框编码的结构蛋白和各种酶等,能在宿主细胞中增殖并适应宿主细胞中的抗病毒应答环境。了解ASFV的基因特征可以为找到有效控制和治疗非洲猪瘟的方法提供基因组学方面的理论依据。
关键词:非洲猪瘟病毒;基因特征;复杂性;多样性
中图分类号:S852.6 文献标志码:C 文章編号:1001-0769(2020)12-0040-08
非洲猪瘟病毒(African Swine Fever Virus,ASFV)是非洲猪瘟病毒科(Asfarviridae)非洲猪瘟病毒属(Asfivirus)唯一的成员,是双链DNA大分子病毒,基因组长度介于170 kb~193 kb,编码150~167个蛋白,其基因组从DNA的两条链上读取,基因紧密间隔,病毒基因组末端由碱基不完全配对的发夹环共价闭合。发夹环以两种形式存在,互补并相互反转,与终端相邻的是多种串联的反向重复序列。大多数ASFV毒株的基因组GC含量*为38%,GC含量相对较低的区域位于基因组两端的左可变区(Left Variable Region,LVR)和右可变区(Right Variable Region,RVR),容易发生基因变异的多基因家族(Multigene Family,MGF)分布于LVR和RVR[1-2]。
1 与复制和转录相关的基因
细胞被ASFV感染后,病毒DNA开始在细胞核周围复制,F1055L蛋白在复制的起始时结合DNA,并与DNA起始酶融合。病毒基因组编码病毒基因转录和复制所需的酶、参与DNA碱基切除修复(Base Excision Repair,BER)途径的酶、逃逸免疫的酶、多基因家族蛋白(MGF360、MGF110、MGF100、MGF505)、功能性蛋白、结构蛋白和许多目前未知的蛋白。ASFV编码基因有150多个,ASFV基因编码早期表达的蛋白主要包括与基因组复制相关的蛋白、后期转录因子及与免疫逃逸相关的MGFs蛋白;ASFV结构蛋白主要在感染后期表达[3-6]。
ASFV的转录机制类似于真核RNA聚合酶Ⅱ(RNA polymerase Ⅱ,RNAPⅡ)系统,包括一个(8-亚基)ASFV RNAP和TATA结合蛋白(TATA Binding Protein,TBP)、转录起始因子Ⅱ B(Transcription Initiation Factor Ⅱ B,TFⅡB)、延伸因子(Transcription Elongation Factor Ⅱ,TFⅡ)以及DNA结合蛋白pA104R和拓扑异构酶Ⅱ pPⅡ92R[3,7-9]。
在感染ASFV的细胞中,病毒颗粒包含早期基因转录所需的所有酶和因子,病毒基因组转录不依赖宿主RNA聚合酶。在ASFV基因组中,与转录起始位点(Transcription Start Site,TSS)重叠的起始子(Initiator,Inr)元件是区分早期和晚期基因启动子的一个特征。早期基因Inr是一个TANA四核苷酸基序,其中N没有核苷酸偏好,ASFV基因的Inr与TSS重叠,早期表达基因启动子的特征序列是TA(TSS);而晚期基因Inr表现出对序列TATA的强烈偏好,晚期表达基因启动子的特征是序列TATA(TSS)。早期基因和晚期基因与5-非翻译区(5Untranslated Regions,5-UTRs)的长度有关,即mRNA N端与翻译起始密码子之间的距离,晚期基因的5-UTR比早期基因的5-UTR短,晚期基因的AT含量高于早期基因的;在TSS的上游有一个保守区,分别对应早期启动子基序(Early Promoter Motif,EPM)和晚期启动子基序(Late Promoter Motif,LPM),早期保守区的序列比晚期保守区的有更高的保守性。ASFV启动子没有一致的序列,但是编码链中7个或更多个连续的胸腺嘧啶残基(7 thymidylate residues,7T motif)是mRNA C端的形成信号[3,11-13]。
为了维持ASFV基因组的完整性,ASFV进化出了自己的碱基切除修复(Base Excision Repair,BER)系统。该系统包括abasic(AP)核酸内切酶(AP endonuclease)、修复聚合酶(repair Polymerase,PolX)和连接酶(Ligase,LIG)。这些酶的主要功能是维持病毒基因组的完整性,还在ASFV的基因突变和基因型形成中发挥重要作用,修复细胞内环境对ASFV产生的病毒DNA损伤。ASFV PolX包含5-磷酸基团(5-P)结合囊的结构域,ASFV LIG N端包含DNA结合域,其中ASFV AP是BER系统中的关键酶,ASFV pE296R基因编码病毒的AP。AP属于Ⅱ类AP内切酶家族,主要催化无碱基位点AP 5端的DNA裂解反应,生成3-羟基和5-脱氧核糖磷酸(deoxyRibose Phosphate,dRP)。ASFV AP具有3→5核酸外切酶、3-磷酸二酯酶和核苷酸切割修复(Nucleotide Incision Repair,NIR)活性,对ASFV在宿主细胞中的存活起着至关重要的作用,pE296R基因的缺失极大地降低了ASFV在细胞中的生长[14-19]。
2 与结构蛋白相关的基因
ASFV的形态为二十面体,病毒粒子分核心、内衣壳、内膜、外衣壳和外膜共五层,目前已知有19个基因编码结构蛋白。
ASFV A104R基因编码pA104R核蛋白,细胞被ASFV感染后2 h,转录病毒A104R基因,在感染后期细胞表达pA104R。pA104R是病毒的组蛋白样蛋白,与DNA高度亲和,与DNA结合位点长约11 nt~20 nt,pA104R上的精氨酸69残基和脯氨酸74残基对pA104R结合DNA的活性起关键作用[20-22]。
ASFV CP2475L基因编码病毒的多蛋白220(polyprotein 220,pp220),其水解产物是病毒核壳的主要结构蛋白p150、p37、p34和p14;CP530R基因编码病毒的另一个多蛋白pp62,其主要水解产物是蛋白p35和p15。p150、p37、p34和p14、p35和p15是形成核壳的主要蛋白[23]。
ASFV D117L基因编码p17蛋白,其既是内膜蛋白又是衣壳蛋白;二十面体衣壳蛋白(Capsid protein)包括p72、M1249L、p17、p49和H240R,其中p72是主要的衣壳结构蛋白,编码基因B646L是晚期表达的蛋白;B438L基因编码另一个衣壳蛋白p49,其位于二十面体衣壳的顶点,是一个顶点蛋白;M1249L基因编码的蛋白连接了二十面体中两个相邻的五边体,形成了三边体的边[24-26]。
p54蛋白是一个免疫原性衣壳结构蛋白,编码P54蛋白的基因在编码区有两组串联重复序列(tandem repeat sequence),第一组由15个核苷酸的六次重复组成,第二组由12个核苷酸的二次不完全重复组成[27]。
3 与酶相关的基因
ASFV基因编码转录并加工mRNAs需要的蛋白和酶,其中EP1242L、C147L、NP1450L、H359L、D205R、CP80R编码6个与RNAⅡ型多聚酶类似的亚基。NP868R基因编码mRNA帽酶的3个结构域:三磷酸酶、胍基转移酶和甲基转移酶。I226R基因和I243L基因在独立启动子的控制下,在ASFV感染的多个阶段能转錄多种mRNA[28-29]。
ASFV P1192R基因编码病毒的Ⅱ型拓扑异构酶(topoisomerase Ⅱ),pP1192R酶能够通过去乙酰化作用松弛DNA的超螺旋结构,P1192R基因在病毒感染后2 h开始转录,在感染后16 h转录达到高峰。ASFV B318L基因编码反式丙烯酰胺转移酶,反式丙烯酰胺转移酶在体外催化法尼基二磷酸和异戊烯基二磷酸缩合合成二磷酸或二磷酸长链,B318L基因包含一个氨基末端疏水序列和所有异戊二烯转移酶的特征区域[30-31]。
ASFV G1211R(G1207R)基因位于96 365 nt~ 99 997 nt,该基因阅读框内的起始密码子是第2个ATP,而不是第1个ATP,基因序列具有串联重复,对磷乙酸敏感。该基因在病毒感染的早期和晚期都表达蛋白,表达α样DNA螺旋酶,蛋白大小为139.8 KDa,等电点pI为8.2。ASFV RNA螺旋酶pQP509L和pQ706L在病毒感染的中晚期出现。ASFV C962R基因编码NTP酶,ASFV G1211R基因编码DNA多聚酶,E301R基因编码增殖细胞核抗原样蛋白(Proliferating Cell Nuclear Antigen,PCNA-like protein),该蛋白与DNA多聚酶直接作用于ASFV DNA,钳住DNA酶到DNA链[32-35]。
ASFV O174L基因编码DNA聚合酶β样蛋白,一个属于X族DNA聚合酶的修复性DNA聚合酶。聚合酶X在病毒感染时能有效通过碱基切除修复(Base Excision Repair,BER)修复单核苷酸DNA断裂,随着猪巨噬细胞突变频率的增加,ASFV聚合酶X基因的缺失导致DNA损伤的累积,这种特殊的基因对维持病毒的遗传信息至关重要。ASFV I1215L基因编码病毒的泛素结合酶,调节宿主细胞的泛素-蛋白酶体系统(ubiquitin-proteasome system, UPS)。pI1215L泛素结合酶能在pH 4~9、4 ℃~42 ℃环境中始终保持活性。I1215L基因在细胞感染ASFV的早期开始转录,在感染后2 h和16 h分别达到转录高峰,pI1215L泛素结合酶可能参与病毒生命周期的不同阶段。在病毒感染的中后期,即病毒DNA复制、转录和翻译的活跃时期,pI1215L泛素结合酶在细胞质内的“病毒工厂”内可以被检测到[36-39]。
4 与免疫相关的基因
ASFV的免疫抑制及免疫逃避主要与其多基因家族基因编码的蛋白相关。ASFV基因组内的多样性主要分布于基因组左右两端区域,左端35 kb和右端15 kb的两个区域包含了多个多基因家族,包括MGF360、MGF505、MGF110、MGF100、MGF530等。MGF505和MGF360是ASFV的毒力因子,可用于疫苗的设计;MGF360和MGF505/530能抑制诱导和影响Ⅰ型干扰素的作用;MGF360和MGF505拷贝数的减少不影响病毒的复制,但能减弱病毒的毒力;MGF505-7R(A 528R)基因编码的蛋白抑制Ⅰ型和Ⅱ型干扰素信号途径,抑制细胞内JAK激酶(一类信号分子)和信号转导与转录激活因子(Signal Transducer and Activator of Transcription,STAT)(一类信号分子)的通路途径(JAK-STAT passway)以及干扰素刺激基因(Interferon Stimulated Gene,ISG)的表达;MGF110基因包含一个信号肽,是一个多样性基因,位于基因组LVR,容易发生基因缺失;MGF基因受其自身启动子的控制[40-45]。
多基因家族具有相似的序列特征,在不同的基因组中,多家族基因的数量和序列是可变的。多基因家族成员MGF100、MGF110、MGF300、MGF360、MGF505/530和p22家族成员的得失是ASFV基因组变异最常见的原因,它们位于左端40 kb和右端20 kb内。MGF505和MGF360开放阅读框(Open Reading Frame, ORF)的突变与ASFV的毒力密切相关;ASFV MGF110基因在LVR中的缺失与衰减表型相关。MGF 110-7 L、MGF 505-5 R、K145R、I267L、DP60R第一个基因发生G→A突变,但突变是沉默的,对蛋白质氨基酸序列没有任何影响;在MGF505-5R基因中检测到其他G→A突变,导致缬氨酸突变为异亮氨酸,MGF-505-4R ORF的天冬氨酸突变为天冬酰胺;MGF 360-16 R ORF插入A后引起与下游编码DP63R的阅读框融合;单个碱基非同义突变导致MGF505-9R(赖氨酸突变为谷氨酸)单一氨基酸替换和NP419L(天冬酰胺突变为丝氨酸);MGF505-5R的缬氨酸突变为异亮氨酸[46-48]。
5 基因突变和变异
ASFV p72基因最早用于基因分型。p72基因中A、C、G和T所占的比例分别为: 0.273 3、0.240 8、0.187 4和0.298 6,根据C端p72基因分类法,ASFV的基因变异多达22种以上。感染基因Ⅱ型ASFV的猪会出现临床症状,感染基因Ⅸ型ASFV的猪则无临床症状。EP402R基因编码的CD2v是ASFV血红素吸附及抑制相关的蛋白,是ASFV血清学分型的依据[49-51]。
2014年至2018年,在欧洲和中国的ASFV分离株中发现了许多不同基因的累积突变。在不同基因和基因间区域的ASFV分离株中已经发现了多个单核苷酸的变化,包括一些预测翻译蛋白的移码和截短的变化。K145R基因内C→A突变导致另一种替代,即丝氨酸突变为酪氨酸。在I267L基因中发现了T→A突变,导致异亮氨酸突变为苯丙氨酸。在DP60R基因中检测到了其序列特有的最后一个变异,单个A的插入引起了阅读框移位,导致开放阅读框延长14 nt,编码O174L的ORF基因序列中有14个碱基插入;在QP383R、KP177R、CP204L和MGF 360-16 R ORFs之间的基因间区域(位置21 588 nt~24 589 nt)也容易有碱基的插入;QP383R ORF一个碱基的缺失或者一个A的插入使12个连续密码子移码;KP177R ORF中插入A导致了阅读框移位,从而导致假定的蛋白质截短(基因缺失);CP204L ORF中双重TT插入引起了阅读框移位,导致终止密码子的过早出现,并导致蛋白质C端最后几个残基的截短;E184L基因中还检测到其他独特的G→A突变;B602Ll基因中的小规模串联重复序列数量增加[52-58]。
6 小结
综上所述,ASFV基因组有150多个开放阅读框,编码150多個蛋白,其中50多个是结构蛋白;基因组两端主要编码与免疫相关的多基因家族,两端可变区LVR和RVR以及中央可变区的基因容易发生基因碱基突变和变异。ASFV基因组的数量庞大而且复杂、易变,导致对ASFV的研究充满了挑战。目前科技界对ASFV的认识还非常有限,还有许多未知基因等待去探索了解。
参考文献
[1] ALEXANDER SCHAFER,JANE HUHR,THERESA SCHWAIGER,et al. Porcine invariant natural killer T cells:functional profiling and dynamics in steady state and viral infections[J]. Frontiers in immunology,2019,10:1380.
[2] DIXON L K,SUN H,ROBERTS H. African swine fever[J]. Antiviral research,2019,165:34-41.
[3] DIXON L K,CHAPMAN D A G,NETHERTON C L,et al. African swine fever virus replication and genomics[J]. Virus Research,2013,173:3–14.
[4] NAN W,DONGMING Z,JIALING W,et al. Architecture of African swine fever virus and implications for viral assembly[J]. Science,2019,366:640–644.
[5] JIA N,OU Y,PEJSAK Z,et al. Roles of African swine fever virus structural proteins in viral infection[J]. Journal of veterinary research,2017,61(2):135–143.
[6] ALEJO A,MATAMOROS T,GUERRA M,et al. A proteomic atlas of the African swine fever virus particle[J]. Journal of virology,2018,92(23):e01293-18.
[7] KINYANYI D,OBIERO G,OBIERO G F O,et al. In silico structural and functional prediction of African swine fever virus protein-B263R reveals features of a TATA-binding protein[J]. PeerJ,2018,22:e4396.
[8] RODRIGUEZ J M,SALAS M L,VI?UELA E. Genes homologous to ubiquitin-conjugating proteins and eukaryotic transcription factor SII in African swine fever virus[J]. Virology,1992,186:40 –52.
[9] Y??EZ R J,RODR?GUEZ J M,NOGAL M L,et al. Analysis of the complete nucleotide sequence of African swine fever virus[J]. Virology,1995,208:249–278.
[10] FROUCO G,FREITAS F B,COELHO J,et al. DNA binding properties of African swine fever virus pA104R,a histone-like protein involved in viral replication and transcription[J]. Journal of virology,2017,91:e02498–16.
[11] CACKETT G,S?KORA M,WERNER F. Transcriptome view of a killer:African swine fever virus[J]. Biochemical society transactions,2020,48(4):1569–1581.
[12] ALMAZ?N F,RODR?GUEZ J M,ANDR?S G,et al. Transcriptional analysis of multigene family 110 of African swine fever virus[J]. Journal of virology,1992,66(11):6655-6667.
[13] ALMAZ?N F,RODR?GUEZ J M,ANGULO A,et al. Transcriptional mapping of a late gene coding for the p12 attachment protein of African swine fever virus[J]. Journal of virology,1993,67(1):553-556.
[14] WEN-JIN W,MEI-I S,JIAN-LI W,et al. How a low-fidelity DNA polymerase chooses non-Watson-Crick from Watson-Crick incorporation[J]. Journal of the American chemical society,2014,136(13):4927–4937.
[15] MACIEJEWSKI M W,SHIN R,PAN B,et al. Solution structure of a viral DNA repair polymerase[J]. Nature structural biology,2001,11(8):936–941.
[16] YIQING C,JING Z,HEHUA L,et al. Unique 5-P recognition and basis for dG:dGTP misincorporation of ASFV DNA polymerase X[J]. PLoS biology,2017,15(2):e1002599.
[17] REDREJO R M,GARCIA E R,YANEZ M R,et al. African swine fever virus protein pE296R is a DNA repair apurinic/apyrimidinic endonuclease required for virus growth in swine macrophages[J]. Journal of virology,2006,80(10):4847–4857.
[18] YIQING C,XI C,QI H,et al. A unique DNA-binding mode of African swine fever virus AP endonuclease[J]. Cell discovery,2020,17(6):13.
[19] REDREJO R M,ISHCHENKO A A,SAPARBAEV M K,et al. African swine fever virus AP endonuclease is a redox-sensitive enzyme that repairs alkylating and oxidative damage to DNA[J]. Virology,2009,390(1):102–109.
[20] BORCA M V,IRUSTA P M,KUTISH G F,et al. A structural DNA binding protein of African swine fever virus with similarity to bacterial histone-like proteins[J]. Archives of virology,1996,141(2):301-313.
[21] NEILAN J G,ZSAK Z L,KUTISH G F,et al. Novel swine virulence determinant in the left variable region of the African swine fever virus genome[J]. Journal of Virology,2002,76(7):3095–3104.
[22] FROUCO G,FREITAS F B,MARTINS C,et al. Sodium phenylbutyrate abrogates African swine fever virus replication by disrupting the virus-induced hypoacetylation status of histone H3K9/K14[J]. Virus research,2017,242:24-29.
[23] ANDRES G,ALEJO A,SALAS J,et al. African swine fever virus polyproteins pp220 and pp62 assemble into the core shell[J]. Journal of virology,2002,76(4):12473-12482.
[24] SALAS M L,ANDRES G. African swine fever virus morphogenesis[J]. Virus research,2013,173(1):29–41.
[25] SU?REZ C,GUTI?RREZ-BERZAL J,ANDR?S G,et al. African swine fever virus protein p17 is essential for the progression of viral membrane precursors toward icosahedral intermediates[J]. Journal of virology,2010,84(15):7484–7499.
[26] EPIFANO C,KRIJNSE-LOCKER J,SALAS M L,et al. Generation of filamentous instead of icosahedral particles by repression of African swine fever virus structural protein pB438L[J]. Journal of virology,2006,80(23):11456–11466.
[27] RODRIGUEZ R,ALCARAZ C,EIRAS A,et al. Characterization and molecular basis of African swine fever virus envelope protein p54[J]. Journal of virology,1994,68(11):7244-7252.
[28] PENA L,YANEZ R J,REVILLA Y. African swine fever guanylytransferase[J]. Virology,1993,193(1):319-328.
[29] RODR?GUEZ J M,SALAS M L,VI?UELA E,et al. Intermediate class of mRNAs in African swine fever virus[J]. Journal of virology,1996,70(12):8584–8589.
[30] FREITAS F B,FROUCO G,MARTINS C,et al. In vitro inhibition of African swine fever virus-topoisomerase Ⅱ disrupts viral replication[J]. Antiviral research,2016,134:34-41.
[31] ALEJO A,YANEZ R J,RODRIGUEZ J M,et al. African swine fever virus trans-prenyltransferase[J]. The journal of biological chemistry,1997,272(14):9417-9423.
[32] MORENO M A,CARRASCOSA A L,ORTIN L,et al. Inhibition of African swine fever(ASF) virus replication by phosphonoacetic acid[J]. Journal of genetic virology,1978,93:253-258.
[33] MARTINS A,RIBEIRO G,MARQUES M L,et al. Genetic identification and nucleotide sequence of the DNA polymerase gene of African swine fever virus[J]. Nucleic acids research,1994,22(2):208-213.
[34] YANEZ R J,RODRIGUEZ J M,NOGAL M L, et al. Analysis of the complete nucleotide sequence of African swine fever virus[J]. Virology,1995,208(1):249-278.
[35] SIMOES M,MARTINS C,FERREIRA F.Early intranuclear replication of African swine fever virus genome modifies the landscape of the host cell nucleus[J]. Virus research,2015,210:1-7.
[36] OLIVEROS M,Y??EZ R J,SALAS M L,et al. Characterization of an African Swine fever virus 20-kDa DNA polymerase involved in DNA repair[J]. The journal of biological chemistry,1997,272(49):30899-30910.
[37] COELHO J,MARTINS C,FERREIRA F,et al. African swine fever virus ORF P1192R codes for a functional type II DNA topoisomerase[J]. Virology,2015,474:82–93.
[38] FREITAS F B,FROUCO G,MARTINS C,et al. African swine fever virus encodes for an E2-ubiquitin conjugating enzyme that is mono- and di-ubiquitinated and required for viral replication cycle[J]. Scientific report. 2018,8(1):3471.
[39] RANDOW F,LEHNER P J. Viral avoidance and exploitation of the ubiquitin system[J]. Nature cell biology,2009,11(5):527–534.
[40] AFONSO C L,PICCONE M E,ZAFFUTO K M,et al. African swine fever virus multigene family 360 and 530 genes affect host interferon response[J]. Journal of virology,2004,78(4):1858–1864.
[41] REIS A L,ABRAMS C C,GOATLEY L C,et al. Deletion of African swine fever virus interferon inhibitors from the genome of a virulent isolate reduces virulence in domestic pigs and induces a protective response[J]. Vaccine,2016,39(34):4698-4705.
[42] ODONNELL V,HOLINKA L G,GIADUE D P,et al. 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[J]. Journal of virology,2015,89(11):6048-6056.
[43] CORREIA S,VENTURA S,PARKHOUSE R M. Identification and utility of innate immune system evasion mechanisms of ASFV[J]. Virus research,2013,173(1):87-100.
[44] ZANI L,FORTH J H,FORTH L,et al. Deletion at the 5-end of Estonian ASFV strains associated with an attenuated phenotype[J]. Scientific reports,2018,8:6510.
[45] NETHERTON C,ROUILLER I,WILEMAN T. The subcellular distribution of multigene family 110 proteins of African swine fever virus is determined by differences in C-terminal KDEL endoplasmic reticulum retention motifs[J]. Journal of virology,2004,78(7):3710–3721.
[46] BLASCO R,DE LA VEGA I,ALMAZAN F,et al. Genetic variation of African swine fever virus:variable regions near the ends of the viral DNA[J]. Virology,1989,173(1):251–257.
[47] KRUG P W,HOLINKA L G,ODONNELL V,et al. 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 modi?cations of the viral genome[J]. Journal of virology,2015,89(4):2324–2332.
[48] NATALIA M,GRZEGORZ W,KRZYSZTOF N. The first complete genomic sequences of African swine fever virus isolated in Poland[J]. Scientific reports,2019,9:4556.
[49] BAO J,WANG Q,LIN P,et al. Genome comparison of African swine fever virus China/2018/AnhuiXCGQ strain and related European p72 Genotype II strains[J]. Transboundary and emerging diseases,2019,66(3):1167–1176.
[50] JELLY S,CHARLES M,TIRUMALA B K S,et al. Symptomatic and asymptomatic cases of African swine fever in Tanzania[J]. Transboundary and emerging diseases,2019,66(6):2402-2410.
[51] CHAPMAN D A,TCHEREPANOV V,TCHEREPANOV V,et al. Comparison of the genome sequences of non-pathogenic and pathogenic African swine fever virus isolates[J]. Journal of general virology,2008,89:397–408.
[52] CISEK A A,DABROWSKA I,GREGORCZYK K P,et al. African swine fever virus:a new old enemy of Europe. Annals parasitology,2016,62(3):161–167.
[53] GALLARDO C,NURMOJA I,SOLER A,et al. Evolution in Europe of African swine fever genotype II viruses from highly to moderately virulent[J]. Veterinary microbiology,2018,219:70–79.
[54] GALLARDO C,SANCHEZ E G,PEREZ-NUNEZ D,et al. African swine fever virus (ASFV) protection mediated by NH/P68 and NH/P68 recombinant live-attenuated viruses[J]. Vaccine,2018,36(19):2694–2704.
[55] GARIGLIANY M,DESMECHT D,TIGNON M,et al. 2019. Phylogeographic Analysis of African Swine Fever Virus,Western Europe,2018[J]. Emerging infectious diseases,2019,25(1):184-186.
[56] ?MIETANKA K,WO?NIAKOWSKI G,KOZAK E,et al. African swine fever epidemic,Poland,2014-2015[J]. Emerging infectious diseases,2016,22(7):1201–1207.
[57] XINTAO Z,NAN L,YUZI L,et al. Emergence of african swine fever in China,2018[J]. Transboundary and emerging diseases,2018,65(6):1482–1484.
[58] MAZUR-PANASIUK N,WO?NIAKOWSKI G. The unique genetic variation within the O174L gene of Polish strains of African swine fever virus facilitates tracking virus origin[J]. Archives of Virology,2019,164:1667–1672.