Meq基因簇microRNAs调控马立克氏病病毒致病表型的研究
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摘要
马立克氏病（Marek’s disease，MD）是由马立克氏病病毒（Marek’s diseasevirus，MDV）感染鸡而引发的一种高传染性、免疫抑制性和肿瘤性疾病。虽然MD肿瘤的发生可以用疫苗来进行免疫预防，但由于MDV毒力的不断增强，近年来在疫苗免疫鸡群中仍不断有MD的发生，给养鸡业造成了严重的经济损失。
MicroRNA(miRNA)在多种生物学过程中发挥重要的转录后基因调控功能，研究发现MDV也编码数十种miRNA基因，它们在病毒的复制、潜伏感染、诱导肿瘤发生等方面可能具有重要的调控作用。MDV-1编码的14个miRNA基因全部位于病毒基因组的反转重复序列区中，且形成3个miRNA基因簇，其中Meq基因簇miRNAs可能在MDV致病和致瘤中发挥关键作用。为了进一步探究MDV致病和致瘤的分子机制，本研究在vv MDV GX0101BAC克隆的基础上，采用RecE/T重组技术构建MDV-1Meq基因簇miRNAs及Meq基因簇内各miRNA单基因缺失的MDV毒株，经动物实验研究该基因簇内miRNAs基因缺失对MDV-1致病表型的影响，最终探明具有调控MDV-1致病表型作用的miRNA，为进一步阐明其分子调控机制奠定基础。
首先，在超强毒MDV分离株GX0101BAC克隆的基础上，分别采用2轮RecE/T重组突变对MDV meq基因簇内miR-M9、miR-M5、miR-M12、miR-M3、miR-M2、miR-M4及整个Meq基因簇miRNAs分别进行缺失，并对各缺失后的BAC克隆进行多基因的PCR鉴定及DNA序列分析。结果表明，经过2轮RecE/T重组突变，成功获得了缺失整个Meq基因簇miRNAs和Meq基因簇内各miRNA单基因的BAC克隆，分别命名为GX0101ΔmiR-M2-BAC、GX0101ΔmiR-M3-BAC、 GX0101ΔmiR-M4-BAC、 GX0101ΔmiR-M5-BAC、GX0101ΔmiR-M9-BAC、GX0101ΔmiR-M12-BAC和GX0101△Meq-miRs-BAC。
其次，将GX0101ΔmiR-M2、 GX0101ΔmiR-M3、 GX0101ΔmiR-M4、GX0101ΔmiR-M5、GX0101ΔmiR-M9、GX0101ΔmiR-M12和GX0101ΔMeq-miRs的BAC DNA分别转染单层CEF进行病毒的拯救，并对拯救的病毒进行鉴定。结果表明，成功获得了MDV miRNA基因缺失株GX0101ΔmiR-M2、GX0101ΔmiR-M3、GX0101ΔmiR-M4、GX0101ΔmiR-M5、GX0101ΔmiR-M9、GX0101ΔmiR-M12和GX0101ΔMeq-miRs。采用SYBR Green I qRT-PCR对感染CEF后不同时间各GX0101△miRNA毒株的Meq基因及gB基因进行定量，检测各GX0101ΔmiRNA毒株在CEF上的复制动力学。结果在感染CEF后0-144h内各MDV GX0101ΔmiRNA毒株均显示与亲本株GX0101BAC相似的增殖曲线，表明Meq基因簇内各miRNA不是MDV复制的必需基因。
最后，将MDV GX0101ΔmiR-M2、GX0101ΔmiR-M3、GX0101ΔmiR-M4、GX0101ΔmiR-M5、GX0101ΔmiR-M9、GX0101ΔmiR-M12和GX0101ΔMeq-miRs毒株分别感染1日龄SPF白来航鸡，进行各病毒株致死率、致肿瘤率、在攻毒鸡体内复制力、鸡生长性能和免疫器官发育及显微肿瘤发生情况等方面的检测、分析。结果表明，亲本株GX0101BAC在75dpi时的致死率已为100%，而各GX0101ΔmiRNA毒株在90dpi的累计致死率均仍低于亲本株GX0101BAC，且GX0101△miR-M5的致死率最高，其次分别为GX0101△miR-M3、GX0101△miR-M9、GX0101△miR-M2、GX0101△miR-M4、GX0101△miR-M12和GX0101△Meq-miRs；在75dpi时，而各GX0101ΔmiRNA毒株累计肉眼肿瘤发生率均低于GX010BAC，在90dpi时GX0101Meq-miRs的累计肿瘤发生率最低，其次分别为GX0101△miR-M12、 GX0101△miR-M3、 GX0101△miR-M4、GX0101△miR-M5、GX0101△miR-M2、GX0101△miR-M9；Meq基因簇内miRNAs并不是MDV的体内复制所必需，但Meq基因簇内miRNAs的缺失影响病毒的体内复制。除GX0101△miR-M3在30dpi时、GX0101△miR-M5在21dpi-30dpi时抑制感染鸡的生长外， GX0101△miR-M2、 GX0101△miR-M4、GX0101△miR-M9、GX0101△miR-M12及GX0101△Meq-miRs对感染鸡的体重增长没有影响；各miRNA缺失株在14-21dpi期间的法氏囊指数与胸腺指数均显著低于CEF对照组，但各miRNA缺失株和GX0101BAC毒株之间没有持续性的显著差异。各GX0101ΔmiRNA感染鸡后肝脏显微肿瘤发生率也低于亲本株GX0101BAC，且GX0101Meq-miRs组的肿瘤形成率最低，其次分别为GX0101ΔmiR-M12、GX0101ΔmiR-M2、GX0101ΔmiR-M4、GX0101ΔmiR-M3、GX0101ΔmiR-M9、GX0101ΔmiR-M5。
综上所述，Meq基因簇内miRNAs的缺失虽不能完全废除MDV的致瘤性，但却能阻止肿瘤的发生进程，Meq基因簇miRNAs对MDV的致病表型具有重要的调控作用，但Meq基因簇内的各miRNA在MDV致瘤过程中可能具有不同的调控作用，其中MDV1-miR-M12的潜在调控作用可能最为重要，其次为MDV1-miR-M2、 MDV1-miR-M3、 MDV1-miR-M4和MDV1-miR-M9，而MDV1-miR-M5的调控作用可能最弱。本研究的结果将为进一步研究Meq基因簇miRNAs调控MD致病及致瘤的的分子机制奠定了重要基础。
Marek’s disease(MD) is a highly contagious, neoplastic, and immunosuppressivedisease of chicken caused by Marek’s disease virus (MDV). The tumorigenesis can beprevented effectively by immunization with attenuated or nonpathogenic forms ofMDV viruses. However, due to high-density poultry production and possible selectionpressure from vaccination, the virulence of MDV field strains has been seen to haveincreased. MD outbreaks happen sporadically every year in worldwide, and it hascaused huge losses in the poultry industry.
MicroRNAs play important post-transcriptional regulatory roles in variousimportant biological processes. In recent years, a large number of viral miRNAs havebeen found to be encoded in the genomes of MDV, and these miRNAs may playcritical regulatory roles in infection, latency, and oncogenesis. Three distinct serotypesof MDV have been characterized, and the viral miRNAs are all located in the invertedrepeat sequences of MDV-1genomes and focused in three gene clusters. TheMeq-clustered miRNAs are sequentially located upstream from the Meq oncogene,and these miRNAs were hypothesized to play more important roles in MDVpathogenesis and oncogenesis.
In order to further explore the molecular mechanism of MDV pathogenesis andtumorigenesis, we intend to construct a series of single-cluster-deleted orsingle-miRNA-deleted MDV strains using RecE/T homologous recombination basedon the BAC clones of vv MDV GX0101. Then the pathogenic or tumorigenicphenotypes would be evaluated by challenge SPF chicken with the miRNA-deletedmutants. The study will provide a means for further investigating the regulatoryfunctions of miRNAs in MDV pathogenic phenotypes and a basis for furtherelucidating the molecular regulation mechanism, and the research will also providereference for clarifying the regulation roles of miRNAs on biology, genetics, andimmunology of tumorigenesis.
Firstly, the single Meq-clustered miRNAs and MDV1-miR-M2, MDV1-miR-M3, MDV1-miR-M4, MDV1-miR-M5, MDV1-miR-M9, MDV1-miR-M12were deletedrespectively based on the BAC clones of vv MDV GX0101by two rounds RecE/Tmutagenesis，then the existence of the GX0101genome and deletion of the individualMeq-clustered miRNAs were confirmed by PCR analysis using the different primersthat amplify different genes and by DNA sequence analysis. The results showed that aseries of BAC clones with the corresponding deletions of single Meq-clusteredmiRNAs and individual miRNA in Meq cluster were constructed successfully, namedGX0101Δ miR-M2-BAC, GX0101Δ miR-M3-BAC, GX0101Δ miR-M4-BAC,GX0101Δ miR-M5-BAC, GX0101Δ miR-M9-BAC, GX0101Δ miR-M12-BACand GX0101Δ Meq-miRs respectively.
Secondly, the BAC DNA of GX0101ΔmiR-M2, GX0101ΔmiR-M3,GX0101ΔmiR-M4, GX0101ΔmiR-M5, GX0101ΔmiR-M9, GX0101ΔmiR-M12andGX0101ΔMeq-miRs were transfected into CEF respectively for the rescue of MDVvirus. Then the rescued viruses were confirmed by PCR analysis and indirectimmunofluorescence assay (IFA). The results indicated that GX0101ΔmiRNA viruseswere reconstituted successfully. To investigate the in vitro virus proliferation rates,real-time qPCR were performed on Meq and gB genes with the CEFs infectedGX0101BAC or GX0101ΔmiRNA viruses. The results showed that both the parentand the miRNAs-deleted mutant viruses have very similar replication kinetics,indicating that the six Meq-clustered miRNAs are not essential for MDV replicationin vitro.
Finally, one-day-old white Leghorn SPF chickens were separately challengedwith CEFs containing MDV GX0101BAC, GX0101ΔmiR-M2, GX0101ΔmiR-M3,GX0101ΔmiR-M4, GX0101ΔmiR-M5, GX0101ΔmiR-M9，GX0101ΔmiR-M12orGX0101ΔMeq-miRs viruses by abdominal cavity inoculation, then the mortality,gross tumor incidences and in vivo replication of GX0101ΔmiRNA viruses, thegrowth rates, immune organs and microscopic tumorigenesis of birds wereexamined and analyzed. The results showed that the rates of cumulative mortality ofMDV GX0101ΔmiRNA viruses were all lower than the parent GX0101BAC, andMDV GX0101ΔmiR-M5was higher than that of GX0101ΔmiR-M3,GX0101ΔmiR-M9, GX0101ΔmiR-M2, GX0101ΔmiR-M4, GX0101ΔmiR-M12andGX0101ΔMeq-miRs. By75dpi, the rates of cumulative gross tumor generation ofMDV GX0101ΔmiRNA viruses were all lower than that of the parent GX0101BAC. By90dpi, the rates of cumulative gross tumor generation of GX0101ΔMeq-miRswere lower than that of GX0101ΔmiR-M12, GX0101ΔmiR-M3, GX0101ΔmiR-M4,GX0101ΔmiR-M5, GX0101ΔmiR-M2and GX0101ΔmiR-M9. These results showedthat the pathogenicity and oncogenicity of the serial mutant viruses were stronglysuppressed when compared to the parental GX0101BAC virus; The Meq-clusteredmiRNAs were not essential for replication of MDV in vivo, and some effect of thedeletions in the Meq-cluster locus affecting the viral replication in vivo; There was nodifference in body weight of birds except GX0101ΔmiR-M3orGXΔmiR-M5-challenged birds whose body weights were lower at30dpi or at21to30dpi than that of the CEF controls. The ratios of bursa/body weight andthymus/body weight of birds inoculated with the mutant viruses were all significantlylower than that of the control birds at14and/or21dpi. However, there was noconsistent significant difference between the parental GX0101BAC and themiRNA-deleted mutants viruses.The rates of microscopic tumorigenesis in liver ofbirds showing no gross organic tumors showed that GX0101ΔmiRNA viruses were alllower that of the parental GX0101BAC, and the microscopic tumorigenesis ofGX0101Meq-miRs was the lowest, then followed by GX0101ΔmiR-M12,GX0101ΔmiR-M2, GX0101ΔmiR-M4, GX0101ΔmiR-M3, GX0101ΔmiR-M9andGX0101Δ miR-M5.
In conclusion, these results suggest that the oncogeneicity was not completelyabolished in the mutant viruses the Meq-clustered miRNAs deleted but tumorprogression was hindered compared to that of the parental GX0101virus. Theseresults further indicate that the Meq-clustered miRNAs have important reglatory rolesin the pathogenic phenotype of Marek’s disease virus. The data suggests that in MDVoncogenesis, the Meq-clustered miRNAs possibly play different roles, soMDV1-miR-M12may be a more important regulator, followed by MDV1-miR-M2,MDV1-miR-M3, MDV1-miR-M4and MDV1-miR-M9，while MDV1-miR-M5maybe less significant. Further work will be needed to elucidate which other fundamentalmolecular mechanisms trigger the development of MD lymphoma.
MicroRNA(miRNA)在多种生物学过程中发挥重要的转录后基因调控功能，研究发现MDV也编码数十种miRNA基因，它们在病毒的复制、潜伏感染、诱导肿瘤发生等方面可能具有重要的调控作用。MDV-1编码的14个miRNA基因全部位于病毒基因组的反转重复序列区中，且形成3个miRNA基因簇，其中Meq基因簇miRNAs可能在MDV致病和致瘤中发挥关键作用。为了进一步探究MDV致病和致瘤的分子机制，本研究在vv MDV GX0101BAC克隆的基础上，采用RecE/T重组技术构建MDV-1Meq基因簇miRNAs及Meq基因簇内各miRNA单基因缺失的MDV毒株，经动物实验研究该基因簇内miRNAs基因缺失对MDV-1致病表型的影响，最终探明具有调控MDV-1致病表型作用的miRNA，为进一步阐明其分子调控机制奠定基础。
首先，在超强毒MDV分离株GX0101BAC克隆的基础上，分别采用2轮RecE/T重组突变对MDV meq基因簇内miR-M9、miR-M5、miR-M12、miR-M3、miR-M2、miR-M4及整个Meq基因簇miRNAs分别进行缺失，并对各缺失后的BAC克隆进行多基因的PCR鉴定及DNA序列分析。结果表明，经过2轮RecE/T重组突变，成功获得了缺失整个Meq基因簇miRNAs和Meq基因簇内各miRNA单基因的BAC克隆，分别命名为GX0101ΔmiR-M2-BAC、GX0101ΔmiR-M3-BAC、 GX0101ΔmiR-M4-BAC、 GX0101ΔmiR-M5-BAC、GX0101ΔmiR-M9-BAC、GX0101ΔmiR-M12-BAC和GX0101△Meq-miRs-BAC。
其次，将GX0101ΔmiR-M2、 GX0101ΔmiR-M3、 GX0101ΔmiR-M4、GX0101ΔmiR-M5、GX0101ΔmiR-M9、GX0101ΔmiR-M12和GX0101ΔMeq-miRs的BAC DNA分别转染单层CEF进行病毒的拯救，并对拯救的病毒进行鉴定。结果表明，成功获得了MDV miRNA基因缺失株GX0101ΔmiR-M2、GX0101ΔmiR-M3、GX0101ΔmiR-M4、GX0101ΔmiR-M5、GX0101ΔmiR-M9、GX0101ΔmiR-M12和GX0101ΔMeq-miRs。采用SYBR Green I qRT-PCR对感染CEF后不同时间各GX0101△miRNA毒株的Meq基因及gB基因进行定量，检测各GX0101ΔmiRNA毒株在CEF上的复制动力学。结果在感染CEF后0-144h内各MDV GX0101ΔmiRNA毒株均显示与亲本株GX0101BAC相似的增殖曲线，表明Meq基因簇内各miRNA不是MDV复制的必需基因。
最后，将MDV GX0101ΔmiR-M2、GX0101ΔmiR-M3、GX0101ΔmiR-M4、GX0101ΔmiR-M5、GX0101ΔmiR-M9、GX0101ΔmiR-M12和GX0101ΔMeq-miRs毒株分别感染1日龄SPF白来航鸡，进行各病毒株致死率、致肿瘤率、在攻毒鸡体内复制力、鸡生长性能和免疫器官发育及显微肿瘤发生情况等方面的检测、分析。结果表明，亲本株GX0101BAC在75dpi时的致死率已为100%，而各GX0101ΔmiRNA毒株在90dpi的累计致死率均仍低于亲本株GX0101BAC，且GX0101△miR-M5的致死率最高，其次分别为GX0101△miR-M3、GX0101△miR-M9、GX0101△miR-M2、GX0101△miR-M4、GX0101△miR-M12和GX0101△Meq-miRs；在75dpi时，而各GX0101ΔmiRNA毒株累计肉眼肿瘤发生率均低于GX010BAC，在90dpi时GX0101Meq-miRs的累计肿瘤发生率最低，其次分别为GX0101△miR-M12、 GX0101△miR-M3、 GX0101△miR-M4、GX0101△miR-M5、GX0101△miR-M2、GX0101△miR-M9；Meq基因簇内miRNAs并不是MDV的体内复制所必需，但Meq基因簇内miRNAs的缺失影响病毒的体内复制。除GX0101△miR-M3在30dpi时、GX0101△miR-M5在21dpi-30dpi时抑制感染鸡的生长外， GX0101△miR-M2、 GX0101△miR-M4、GX0101△miR-M9、GX0101△miR-M12及GX0101△Meq-miRs对感染鸡的体重增长没有影响；各miRNA缺失株在14-21dpi期间的法氏囊指数与胸腺指数均显著低于CEF对照组，但各miRNA缺失株和GX0101BAC毒株之间没有持续性的显著差异。各GX0101ΔmiRNA感染鸡后肝脏显微肿瘤发生率也低于亲本株GX0101BAC，且GX0101Meq-miRs组的肿瘤形成率最低，其次分别为GX0101ΔmiR-M12、GX0101ΔmiR-M2、GX0101ΔmiR-M4、GX0101ΔmiR-M3、GX0101ΔmiR-M9、GX0101ΔmiR-M5。
综上所述，Meq基因簇内miRNAs的缺失虽不能完全废除MDV的致瘤性，但却能阻止肿瘤的发生进程，Meq基因簇miRNAs对MDV的致病表型具有重要的调控作用，但Meq基因簇内的各miRNA在MDV致瘤过程中可能具有不同的调控作用，其中MDV1-miR-M12的潜在调控作用可能最为重要，其次为MDV1-miR-M2、 MDV1-miR-M3、 MDV1-miR-M4和MDV1-miR-M9，而MDV1-miR-M5的调控作用可能最弱。本研究的结果将为进一步研究Meq基因簇miRNAs调控MD致病及致瘤的的分子机制奠定了重要基础。
Marek’s disease(MD) is a highly contagious, neoplastic, and immunosuppressivedisease of chicken caused by Marek’s disease virus (MDV). The tumorigenesis can beprevented effectively by immunization with attenuated or nonpathogenic forms ofMDV viruses. However, due to high-density poultry production and possible selectionpressure from vaccination, the virulence of MDV field strains has been seen to haveincreased. MD outbreaks happen sporadically every year in worldwide, and it hascaused huge losses in the poultry industry.
MicroRNAs play important post-transcriptional regulatory roles in variousimportant biological processes. In recent years, a large number of viral miRNAs havebeen found to be encoded in the genomes of MDV, and these miRNAs may playcritical regulatory roles in infection, latency, and oncogenesis. Three distinct serotypesof MDV have been characterized, and the viral miRNAs are all located in the invertedrepeat sequences of MDV-1genomes and focused in three gene clusters. TheMeq-clustered miRNAs are sequentially located upstream from the Meq oncogene,and these miRNAs were hypothesized to play more important roles in MDVpathogenesis and oncogenesis.
In order to further explore the molecular mechanism of MDV pathogenesis andtumorigenesis, we intend to construct a series of single-cluster-deleted orsingle-miRNA-deleted MDV strains using RecE/T homologous recombination basedon the BAC clones of vv MDV GX0101. Then the pathogenic or tumorigenicphenotypes would be evaluated by challenge SPF chicken with the miRNA-deletedmutants. The study will provide a means for further investigating the regulatoryfunctions of miRNAs in MDV pathogenic phenotypes and a basis for furtherelucidating the molecular regulation mechanism, and the research will also providereference for clarifying the regulation roles of miRNAs on biology, genetics, andimmunology of tumorigenesis.
Firstly, the single Meq-clustered miRNAs and MDV1-miR-M2, MDV1-miR-M3, MDV1-miR-M4, MDV1-miR-M5, MDV1-miR-M9, MDV1-miR-M12were deletedrespectively based on the BAC clones of vv MDV GX0101by two rounds RecE/Tmutagenesis，then the existence of the GX0101genome and deletion of the individualMeq-clustered miRNAs were confirmed by PCR analysis using the different primersthat amplify different genes and by DNA sequence analysis. The results showed that aseries of BAC clones with the corresponding deletions of single Meq-clusteredmiRNAs and individual miRNA in Meq cluster were constructed successfully, namedGX0101Δ miR-M2-BAC, GX0101Δ miR-M3-BAC, GX0101Δ miR-M4-BAC,GX0101Δ miR-M5-BAC, GX0101Δ miR-M9-BAC, GX0101Δ miR-M12-BACand GX0101Δ Meq-miRs respectively.
Secondly, the BAC DNA of GX0101ΔmiR-M2, GX0101ΔmiR-M3,GX0101ΔmiR-M4, GX0101ΔmiR-M5, GX0101ΔmiR-M9, GX0101ΔmiR-M12andGX0101ΔMeq-miRs were transfected into CEF respectively for the rescue of MDVvirus. Then the rescued viruses were confirmed by PCR analysis and indirectimmunofluorescence assay (IFA). The results indicated that GX0101ΔmiRNA viruseswere reconstituted successfully. To investigate the in vitro virus proliferation rates,real-time qPCR were performed on Meq and gB genes with the CEFs infectedGX0101BAC or GX0101ΔmiRNA viruses. The results showed that both the parentand the miRNAs-deleted mutant viruses have very similar replication kinetics,indicating that the six Meq-clustered miRNAs are not essential for MDV replicationin vitro.
Finally, one-day-old white Leghorn SPF chickens were separately challengedwith CEFs containing MDV GX0101BAC, GX0101ΔmiR-M2, GX0101ΔmiR-M3,GX0101ΔmiR-M4, GX0101ΔmiR-M5, GX0101ΔmiR-M9，GX0101ΔmiR-M12orGX0101ΔMeq-miRs viruses by abdominal cavity inoculation, then the mortality,gross tumor incidences and in vivo replication of GX0101ΔmiRNA viruses, thegrowth rates, immune organs and microscopic tumorigenesis of birds wereexamined and analyzed. The results showed that the rates of cumulative mortality ofMDV GX0101ΔmiRNA viruses were all lower than the parent GX0101BAC, andMDV GX0101ΔmiR-M5was higher than that of GX0101ΔmiR-M3,GX0101ΔmiR-M9, GX0101ΔmiR-M2, GX0101ΔmiR-M4, GX0101ΔmiR-M12andGX0101ΔMeq-miRs. By75dpi, the rates of cumulative gross tumor generation ofMDV GX0101ΔmiRNA viruses were all lower than that of the parent GX0101BAC. By90dpi, the rates of cumulative gross tumor generation of GX0101ΔMeq-miRswere lower than that of GX0101ΔmiR-M12, GX0101ΔmiR-M3, GX0101ΔmiR-M4,GX0101ΔmiR-M5, GX0101ΔmiR-M2and GX0101ΔmiR-M9. These results showedthat the pathogenicity and oncogenicity of the serial mutant viruses were stronglysuppressed when compared to the parental GX0101BAC virus; The Meq-clusteredmiRNAs were not essential for replication of MDV in vivo, and some effect of thedeletions in the Meq-cluster locus affecting the viral replication in vivo; There was nodifference in body weight of birds except GX0101ΔmiR-M3orGXΔmiR-M5-challenged birds whose body weights were lower at30dpi or at21to30dpi than that of the CEF controls. The ratios of bursa/body weight andthymus/body weight of birds inoculated with the mutant viruses were all significantlylower than that of the control birds at14and/or21dpi. However, there was noconsistent significant difference between the parental GX0101BAC and themiRNA-deleted mutants viruses.The rates of microscopic tumorigenesis in liver ofbirds showing no gross organic tumors showed that GX0101ΔmiRNA viruses were alllower that of the parental GX0101BAC, and the microscopic tumorigenesis ofGX0101Meq-miRs was the lowest, then followed by GX0101ΔmiR-M12,GX0101ΔmiR-M2, GX0101ΔmiR-M4, GX0101ΔmiR-M3, GX0101ΔmiR-M9andGX0101Δ miR-M5.
In conclusion, these results suggest that the oncogeneicity was not completelyabolished in the mutant viruses the Meq-clustered miRNAs deleted but tumorprogression was hindered compared to that of the parental GX0101virus. Theseresults further indicate that the Meq-clustered miRNAs have important reglatory rolesin the pathogenic phenotype of Marek’s disease virus. The data suggests that in MDVoncogenesis, the Meq-clustered miRNAs possibly play different roles, soMDV1-miR-M12may be a more important regulator, followed by MDV1-miR-M2,MDV1-miR-M3, MDV1-miR-M4and MDV1-miR-M9，while MDV1-miR-M5maybe less significant. Further work will be needed to elucidate which other fundamentalmolecular mechanisms trigger the development of MD lymphoma.
引文
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