鸡马立克病疫苗株CVI988/Rispens meq基因编辑及缺失毒株的构建与鉴定

meq是鸡马立克病病毒(MDV)最重要的致瘤基因,在马立克病(MD)肿瘤发生中发挥关键作用。同时,它在疫苗株和强毒株之间具有明显的序列差异性。本文利用CRISPR/Cas9基因编辑技术,以MDV疫苗株CVI988/Rispens meq基因为靶点,设计合成gRNA,克隆构建pX459-gRNA质粒,转染CEF并感染CVI988/Rispens,然后对meq基因编辑的病毒噬斑进行克隆纯化,经过PCR扩增、测序分析及IFA鉴定,成功构建1株meq基因编辑的缺失毒株CVI988Δmeq-C7,为后续筛选和鉴定抗MD疫苗株MEQ单抗及鉴别诊断研究奠定了基础。     

鸡马立克病疫苗株CVI988/Rispens meq基因编辑及缺失毒株的构建与鉴定

meq是鸡马立克病病毒（MDV）最重要的致瘤基因，在马立克病（MD）肿瘤发生中发挥关键作用。同时，它在疫苗株和强毒株之间具有明显的序列差异性。本文利用CRISPR/Cas9基因编辑技术，以MDV疫苗株CVI988/Rispens meq基因为靶点，设计合成gRNA，克隆构建pX459-gRNA质粒，转染CEF并感染CVI988/Rispens，然后对meq基因编辑的病毒噬斑进行克隆纯化，经过PCR扩增、测序分析及IFA鉴定，成功构建1株meq基因编辑的缺失毒株CVI988Δmeq-C7，为后续筛选和鉴定抗MD疫苗株MEQ单抗及鉴别诊断研究奠定了基础。     

整合REV-LTR基因的MDV活疫苗与亲本CVI988/Rispens株的免疫效力比较

利用同源重组技术将禽网状内皮组织增生症病毒（REV）的长末端重复序列（LTR）整合进CVI988/Rispens疫苗株基因组中所构建成的重组马立克氏病病毒株（r MDV-LTR株）,具有较高的增殖效率。为比较r MDV-LTR株与亲本CVI988/Rispens株的免疫效力差异,开展了对这两种疫苗的比较试验。试验将这两种疫苗分别按照三个不同剂量经颈背部皮下接种1日龄SPF鸡。接种后第7 d,攻毒马立克氏病强毒株rMd5,连续观察60 d后进行剖检。临床病理观察结果发现,以不低于250 PFU/羽剂量的rMDV-LTR株或CVI988/Rispens株免疫SPF雏鸡都可以提供大于80%的免疫保护效率;以125 PFU/羽剂量的两种疫苗虽均不能提供大于80%的免疫保护效力,但rMDV-LTR株免疫组试验鸡未出现死亡,仅有2只鸡出现消瘦,剖解脏器无肿瘤,而CVI988/Rispens株免疫组鸡出现2只死亡,其中1只剖解后检出肿瘤。rMDV-LTR株和CVI988/Rispens株免疫组的免疫保护指数分别为77.78%和66.67%。由实验结果可见,在较小免疫剂量下,rMDV-LTR株免疫保护...     

整合进禽反转录病毒长末端重复序列的马立克氏病病毒的分离鉴定

应用鸭胚成纤维细胞(DEF)从曾免疫过CVI988/Rispens株疫苗的患马立克氏病(MD)肿瘤的三黄鸡中分离到一株马立克氏病病毒(MDV,命名为GXY2株。用禽肿瘤病聚合酶链式反应(PCR)鉴别诊断技术对患鸡的肿瘤组织病料及克隆纯化毒株的DEF培养物进行检测,结果均扩增到MDV-1强毒株的132-bpr特异性带和网状内皮组织增殖病病毒(REV)的长末端重复序列(LTR)。用基于抗MDV-1的gB蛋白单克隆抗体BA4、MEQ蛋白单克隆抗体3G12E6和抗REV的单克隆抗体11B118分别对毒株的培养物进行间接免疫荧光试验(IFA),结果样品只与抗MDV-1的单克隆抗体呈现阳性反应,而与抗REV的单克隆抗体呈现阴性反应。应用PCR技术扩增并测定了毒株的致瘤相关基因meq的核苷酸序列,并与其他MDV-1参考毒株的序列进行比较分析,结果发现其序列与我们之前分离鉴定的MDV-1野强毒株G2和YL040920高度同源。研究的结果表明,分离株GXY2为整合有REVLTR片段的重组MDV强毒株。     

表达鸡新城疫病毒F蛋白的重组马立克氏病毒的构建及其鉴定

为构建表达鸡新城疫病毒(NDV)融合蛋白(F)的重组马立克氏病毒(MDV),本研究采用RT-PCR方法从NDV强毒株F48E9基因组中扩增出病毒的融合蛋白F基因,构建由CMV启动子和BGH polyA组成的2.7 kb F基因表达盒。将其插入带有黄嘌呤-鸟嘌呤磷酸核糖基转移酶基因(gpt)和MDV US2同源臂的中间转移载体pUAB-gpt中获得重组MDV转移载体pUAB-gpt-wF。将该转移载体与MDV-814疫苗株感染的鸡胚成纤维细胞(CEF)总DNA共转染CEF,经同源重组及gpt选择系统筛选,获得带有F基因表达盒的重组MDV(rMDV814-wF)。其体外增殖与亲本病毒没有差异。经间接免疫荧光试验、PCR、Southern-blot及western blot等试验证明,重组病毒在CEF中传至13代以上仍稳定表达NDV的F蛋白。该重组病毒的构建为MDV活载体疫苗的筛选及应用奠定了基础。     

马立克氏病毒CVI988的TK及gB基因启动子的扩增

根据GenBank公布的MDV GA株全基因组序列设计引物,以CVI 988/R ispens感染鸡胚成纤维细胞的基因组总DNA为模板,利用PCR的方法分别扩增了CVI 988/R ispens的TK及gB基因启动子基因,克隆入pGEM-T-easy载体并进行测序,结果表明CVI 988/R ispens的TK及gB基因启动子序列均存在TATA盒和CAAT盒等真核启动子所具有的保守序列,且与MDV GA株的相应基因序列完全一致,为进一步研究该启动子在MDV病毒载体中的表达调控提供了物质条件。     

表达H5亚型禽流感病毒HA基因重组火鸡疱疹病毒的构建

为构建表达禽流感病毒(AIV)血凝素(HA)基因的重组疱疹病毒(HVT),本研究将AIV A/Goose/Guangdong/3/96(H5N1)的HA基因克隆于真核表达质粒中,构建了转移载体pUAB-gpt-HA。通过同源重组及霉酚酸筛选技术,将HA基因表达盒插入到HVT US10基因区,构建了一株表达H5亚型AIV HA基因的重组病毒HVT(rHVT-US10-HA)。经PCR、间接免疫荧光、western blot及红细胞凝集试验鉴定,重组病毒能稳定表达具有生物学活性的HA蛋白。rHVT-US10-HA的构建为禽流感基因工程病毒活载体疫苗的研究奠定了基础。     

鸡马立克病病毒强毒与疫苗毒PCR鉴别检测方法的建立

在Ⅰ型马立克病病毒(MDV)基因组中针对132 bp串联重复序列的两侧合成一对引物,应用PCR技术对临床病例采集的疑似马立克病肿瘤病变鸡肝组织和1日龄接种CVI988弱毒疫苗的健康雏鸡羽髓样本进行检测。结果表明,从临床病例采集的10份肝脏组织,7份扩增出一条314 bp的条带,相当于2个拷贝数的132 bp串联重复序列;在接种疫苗健康雏鸡的羽髓样本中,扩增出与CVI988弱毒一致的PCR图谱,相当于6个~8个或更多拷贝数的132 bp串联重复序列。根据PCR图谱的差异即可鉴别MDV强毒株与CVI988疫苗弱毒株。     

脂质体介导马立克病病毒DNA转染鸡胚成纤维细胞

利用粗提的马立克病病毒(MDV)CVI988感染的鸡胚成纤维细胞(CEF)总DNA与脂质体包裹转染CEF,获得具有感染性的MDV。结果表明,使用3μg MDV感染的CEF总DNA转染CEF,6 d~7 d后可形成0~16个MDV特有空斑。     

3株马立克氏病病毒的分离鉴定及其主要致病基因分析

为了解国内鸡群马立克病(MD)毒株目前的流行情况,在我国3个已免疫MD疫苗的白羽肉种鸡场暴发MD的鸡群中,通过临床剖检,组织病理学变化,病毒分离,IFA检测从发病鸡的淋巴细胞中分离到3株马立克氏病毒(MDV),并分别命名为SDAU1501、SDAU1502和SDAU1503。采用PCR方法扩增3株MDV的vIL8、pp38、meq致病基因,所得氨基酸序列与已发表的参考毒株序列进行比较分析,结果显示SDAU1501和SDAU1502的致病基因与强毒株的同源性达到97%以上,具有强毒特征;而SDAU1503的meq基因在第194位具有与CVI988同样的缺失(P),但其没有CVI988中177个核苷酸的插入突变,其具有与弱毒疫苗株的特征。MDV新的变异在不断发生,出现了不同类型的变异株,因此,对MD的流行情况与基因监测应当继续下去;同时,在MDV疫苗的研制更新上还有待于进一步研究。     

重组马立克病毒通用转移载体的构建及初步应用

本实验以独立启动子控制的增强型绿色荧光基因(GFP)作为报告基因,同时将CMV启动子及其多克隆位点与之连接,构成外源基因表达盒,插入到马立克病毒(MDV)复制非必需区基因(短独特区US2等)构成的同源臂中,构建成重组马立克病毒的通用载体。鉴定正确后,将转移载体与提取的MDV基因组共转染鸡胚成纤维细胞(CEF),同源重组获得具有感染性的重组病毒,待病毒蚀斑出现后,荧光显微镜下观察,可见到明显的绿色荧光病毒蚀斑,经三次筛选,初步分离到重组病毒。结果表明,转移载体与MDV基因组共转染可获得感染性病毒,US2基因可作为重组病毒构建中的外源基因插入位点,证实通用转移载体的构建是可行的,为重组马立克病毒新型疫苗的研究奠定物质基础。     

A comparative evaluation of the protective efficacy of rMd5deltaMeq and CVI988/ Rispens against a vv+ strain of Marek's disease virus infection in a series of recombinant congenic strains of White Leghorn chickens

Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a highly infectious, oncogenic alpha-herpesvirus known as Marek's disease virus (MDV). MD is presently controlled by vaccination. Current MD vaccines include attenuated serotype 1 strains (e.g., CVI988/Rispens), avirulent serotype 2 (SB-1), and serotype 3 (HVT) MDV strains. In addition, recombinant MDV strains have been developed as potential new and more efficient vaccines to sustain the success of MD control in poultry. One of the candidate recombinant MDV strains, named rMd5deltaMeq, was derived from Md5, a very virulent strain of MDV lacking the MDV oncogene Meq. Our earlier reports suggest that rMd5deltaMeq provided protection equally well or better than commonly used MD vaccines in experimental and commercial lines of chickens challenged with very virulent plus (vv+) strains of MDV. In this study, maternal antibody-positive (trial 1) and negative (trial 2) chickens from a series of relatively MD resistant lines were either vaccinated with the rMd5deltaMeq or CVI988/Rispens followed by infection of a vv+ strain of MDV, 648A, passage 10. This report presents experimental evidence that the rMd5deltaMeq protected significantly better than the CVI988/Rispens (P < 0.01) in the relatively resistant experimental lines of chickens challenged with the vv+ strain of MDV. Together with early reports, the rMd5deltaMeq appeared to provide better protection, comparing with the most efficacious commercially available vaccine, CVI988/Rispens, for control of MD in lines of chickens regardless of their genetic background.     

Differential quantification of cloned CVI988 vaccine strain and virulent RB-1B strain of Marek’s disease viruses in chicken tissues, using real-time PCR

The ‘gold standard’ vaccine against Marek’s disease in poultry is the CVI988/Rispens virus, which is not easily distinguishable, antigenically or genetically, from virulent Marek’s disease herpesvirus. Accurate differential measurement of the CVI988 vaccine and virulent viruses is important to investigate mechanisms of vaccinal protection. Minimal sequence differences between CVI988 and virulent MDV strains restrict the application of molecular diagnostic methods such as real-time PCR to distinguish between these viruses. The use of bacterial-artificial-chromosome (BAC) cloned CVI988 virus, which carries the BAC vector sequences in place of the U_s2 gene, allows its differential quantification from virulent strains using real-time PCR assays that target the BAC vector sequence and the U_S2 gene respectively. These novel assays allowed investigation of replication of both serotype-1 vaccine virus (cloned CVI988) and challenge virus (RB-1B strain) in tissues of individual chickens in an experimental vaccination-challenge model of Marek’s disease.     

The efficacy of recombinant fowlpox vaccine protection against Marek's disease: its dependence on chicken line and B haplotype

Earlier studies have shown that the B haplotype has a significant influence on the protective efficacy of vaccines against Marek's disease (MD) and that the level of protection varies dependent on the serotype of MD virus (MDV) used in the vaccine. To determine if the protective glycoprotein gene gB is a basis for this association, we compared recombinant fowlpox virus (rFPV) containing a single gB gene from three serotypes of MDV. The rFPV were used to vaccinate 15.B congenic lines. Nonvaccinated chickens from all three haplotypes had 84%-97% MD after challenge. The rFPV containing gB1 provides better protection than rFPV containing gB2 or gB3 in all three B genotypes. Moreover, the gB proteins were critical, since the B*21/*21 chickens had better protection than chickens with B*13/*13 or B*5/*5 using rFPV with gB1, gB2, or gB3. A newly described combined rFPV/gB1gEgIUL32 + HVT vaccine was analyzed in chickens of lines 15 x 7 (B*2/*15) and N (B*21/*21) challenged with two vv+ strains of MDV. There were line differences in protection by the vaccines and line N had better protection with the rFPV/gB1gEgIUL32 + HVT vaccines (92%-100%) following either MDV challenge, but protection was significantly lower in 15 X 7 chickens (35%) when compared with the vaccine CVI988/Rispens (94%) and 301B1 + HVT (65%). Another experiment used four lines of chickens receiving the new rFPV + HVT vaccine or CVI988/Rispens and challenge with 648A MDV. The CVI 988/Rispens generally provided better protection in lines P and 15 X 7 and in one replicate with line TK. The combined rFPV/gB1gEgIUL32 + HVT vaccines protected line N chickens (90%) better than did CVI988/Rispens (73%). These data indicate that rFPV + HVT vaccines may provide protection against MD that is equivalent to or superior to CVI988/ Rispens in some chicken strains. It is not clear whether the rFPV/gB1gEgIUL32 + HVT vaccine will offer high levels of protection to commercial strains, but this vaccine, when used in line N chickens, may be a useful model to study interactions between vaccines and chicken genotypes and may thereby improve future MD vaccines.     

马立克氏病病毒肿瘤Meq基因缺失株的保护性免疫作用

马立克氏病病毒（MDV）的致病性一直在不断增强中,甚至已出现了能抵抗CV1988/Rispens疫苗的特超强株。本实验室用BAC克隆技术构建了MDV中国野毒株的Meq基因缺失株,显示出在抗马立克氏病方面具有与美国Meq基因缺失株同样有效的保护性免疫效果,而且其自身没有明显的免疫抑制作用。对美国和中国的两个MDV的Meg基因缺失株的优缺点做了比较。     

鸡马立克氏病CVI988／Rispens疫苗免疫效力试验

应用荷兰农业部提供的鸡马立克氏病（ＭＤ）ＣＶＩ９８８／Ｒｉｓｐｅｎｓ Ⅰ型致弱种毒, 在农业部批准的符合ＧＭＰ 要求的生产车间研制出鸡马立克氏病ＣＶＩ９８８／Ｒｉｓｐｅｎｓ 疫苗。将按国际标准检验合格的三批疫苗及进口商品ＣＶＩ９８８／Ｒｉｓｐｅｎｓ 疫苗接种１ 日龄ＳＰＦ 雏鸡, 于７ 日龄经腹腔攻击鸡马立克氏病强毒（北京－ １ 株） 血毒, 全部鸡只隔离饲养观察至６０ 日龄并作全群剖检。经测定： 非免疫攻毒组１００％ 发病,健康对照组全部阴性, 三批国产ＣＶＩ９８８／Ｒｉｓｐｅｎｓ 疫苗保护指数分别为９０－０, ９０－０, ９３－３ , 进口商品苗保护率为９３－３ 。结果表明国产和进口ＣＶＩ９８８／Ｒｉｓｐｅｎｓ疫苗均能提供对ＭＤ 较高的免疫保护力, 国产疫苗的保护效果达到了国际同类产品的先进水平。     

鸡马立克氏病CVI988/Rispens疫苗免疫效力试验

本试验用北京市农林科学院畜牧兽医研究所制备的ＣＶＩ９８８／Ｒｉｓｐｅｎｓ疫苗和进口的ＣＶＩ９８８／Ｒｉｓｐｅｎｓ疫苗免疫１日龄来航鸡,７日龄以ＭＤＶ北京－１株血毒进行攻击,６０日龄全群剖检。经免疫效力试验两次测定,３批北京所制备的ＣＶＩ９８Ｒｉｓｐｅｎｓ疫苗产品保护指数分别为试验的９０．０、９０．０、９３．３和试验（２）的１００．０、１００．０、９４．５与进口商品ＣＶＩ／９８８Ｒｉｓｐｅｎｓ苗的保