鸡传染性喉气管炎病毒致病性评价及2株分离株基因组序列分析

发布时间：2019-05-27 04:31

【摘要】：鸡传染性喉气管炎病毒(Infectious laryngotracheitis virus,ILTV)能引起鸡的一种呼吸道的传染病,具有高度接触性和急性感染的特征,该病被称为鸡传染性喉气管炎(Infectious laryngotracheitis,ILT)。急性感染的鸡群可出现高的发病率和死亡率,慢性感染可引起结膜炎、产蛋和蛋品质下降,从1925年首次报道本病以来,已在全球各地流行,近些年来,在我国多地也呈流行趋势,给养鸡业造成的损失不容忽视。为研究ILTV LJS09株鸡胚源毒经在LMH上适应后致病性的变化,本研究以ILTV LJS09株鸡胚源毒和LMH细胞适应第四代的毒分别人工感染SPF鸡,通过评价两感染组的临床症状、临床得分、临床指数(1.4和0.7)、发病率(80%和60%)和死亡率(10%和0)、剖检变化以及鸡胚源毒感染组病理组织学变化,血清抗体水平变化规律,ILTV在鸡体内的嗜性器官以及各个器官中病毒的载量这些指标,继而评价鸡胚源毒和LMH细胞适应毒的致病效果,并比较其致病力的强弱,结果表明两感染组有相似的临床症状和剖检变化,血清抗体水平变化趋势相同,ILTV嗜性器官相同,但鸡胚源毒感染组的临床得分、临床指数、发病率和死亡率、血清抗体水平较LMH细胞适应毒感染组高,表明ILTV LJS09株鸡胚源毒比LMH细胞适应毒的致病力强。这些评价指标及结果为了解我国传染性喉气管炎病毒的致病性提供了参考。本实验室于2014年从黑龙江省和河北省分离得到两株ILTV,分别命名为LHLJ1403和LHB1409株。经SPF鸡胚分离,并经PCR和测序鉴定后确定其为ILTV。将这2株分离株在LMH细胞上适应,经噬斑纯化后得到了纯化的病毒,通过电镜观察和间接免疫荧光检测确定了纯化的病毒为ILTV。为进一步了解我国ILTV毒株的流行病学特性及分子生物学特点,用PCR的方法测定了这两株分离株的全基因组序列。通过将全基因组片段化,形成106个短片段,利用106对引物,分别进行克隆测序,由于内部重复区和末端重复区具有倒置重复的特点,因此共利用了17对引物,通过锚定PCR和嵌套PCR相结合的方法准确确定其位置和序列。将得到的两株分离株的所有克隆片段的序列拼接在一起,获得两株分离株的基因组全长序列。LHLJ1403株的基因组全长为153222 bp,具有79个ORF;LHB1409株的基因组全长为153213 bp,同样具有79个ORF。利用DNAStar、MEGA、DNAMAN和ORF Finder等生物学软件对序列进行分析,并将其与已公布的参考序列进行比对(用于比对的参考序列:SA2、A20、LT Blen、1874C5、USDA、81658、63140、Laryngo、Serva、LJS09)。通过同源性比对,LHLJ1403和LHB1409株相似性为99.02%,LHLJ1403和LT Blen株的相似性最高达99.49%,LHB1409和63140株的相似性最高达99.19%。与SA2和A20株相比,LHLJ1403和LHB1409株UL区693~706位缺失14 bp,3297~3485位缺失189 bp,3672~3690位缺失19 bp,4662~4693位缺失了32 bp的序列。将参考序列的重复区相互比较,发现LHLJ1403和LHB1409株的内部(IRS)和末端重复区(TRS)都有857 bp片段的大段缺失,而在USDA株的重复序列中并不缺失这段片段,但与Serva株相比,LHLJ1403和LHB1409株的IRS和TRS均存在217 bp的缺失。此外,LHLJ1403和LHB1409株重复区的DNA复制起点Ori S只含有结合蛋白(OBP)的结合位点,而AT富集区及周围序列存在缺失。LHLJ1403和LHB1409株的糖蛋白g B、g C、g D和g H/g L与UASD株相比,部分氨基酸位点发生了突变。将分离毒株的全基因组序列与参考毒株进行比对并进行遗传进化分析,结果表明LHLJ1403与美国疫苗株LT Blen和Laryngo的亲缘关系较近,并位于同一分支;LHB1409与美国疫苗株Serva和美国强毒株63140株缘关系较近,且它们位于同一分支。本研究测定了我国两株ILTV分离毒株的全基因组序列,并将其与已发表的其他强毒株、弱毒株或疫苗株的基因组序列进行比对和分析,其结果为ILTV分子遗传特性和分子生物学的研究奠定了基础,为了解我国ILT的流行情况、该病的防控及新型疫苗的研发提供了信息。
[Abstract]:The infectious laryngotracheitis virus (ILTV) can cause an infectious disease of the respiratory tract of the chicken, which is characterized by high contact and acute infection, which is called infectious laryngotracheitis (ILT). Acute infection can cause high morbidity and mortality, and chronic infection can cause a decline in conjunctivitis, egg-laying and egg quality. Since the first report of this disease in 1925, it has been popular around the world. In recent years, it has also become a popular trend in China. The loss to the chicken industry cannot be ignored. In order to study the changes of the pathogenicity of ILTV LJS09 chicken embryo source in LMH, this study uses ILTV LJS09 strain of chicken embryo source and LMH cell to adapt to the fourth generation of toxicity of SPF chicken respectively, and the clinical symptoms, clinical scores and clinical index (1.4 and 0.7) of the two infected groups are evaluated. The incidence rate (80% and 60%) and the death rate (10% and 0), the change of the profile and the pathological changes of the chicken embryo source virus infection group, the change of the serum antibody level, the eosinophilic organ of the ILTV in the chicken and the load of the virus in the various organs, The pathogenic effects of chicken embryo source and LMH cells were evaluated, and the pathogenic force was compared. The results showed that there were similar clinical symptoms and cross-section changes in the two infected groups, the changes of serum antibody level were the same, and the level of ILTV was the same, but the clinical score of the chicken embryo source virus infection group was the same. The clinical index, morbidity and mortality, serum antibody level were higher than that of LMH cells, indicating that ILTV LJS09 chicken embryo-source toxicity was stronger than that of LMH cells. These evaluation indexes and results provide a reference for understanding the pathogenicity of the infectious laryngotracheitis virus in China. The laboratory was isolated from Heilongjiang and Hebei in 2014 to obtain two ILTV, named LHLJ1403 and LHB1409, respectively. It was isolated by SPF chicken embryo and identified as ILTV by PCR and sequencing. The two isolates were adapted to LMH cells and purified by plaque purification. The purified virus was determined by electron microscope and indirect immunofluorescence. In order to further understand the epidemiological characteristics and molecular biological characteristics of the ILTV strain in China, the whole genome sequence of the two isolates was determined by PCR. By fragmenting the whole genome to form 106 short segments, the primers are respectively cloned and sequenced by using 106 pairs of primers, and since the internal repeat region and the terminal repeat region have the characteristics of inverted repeat,17 pairs of primers are used, The position and sequence of the method can be accurately determined by the combination of the anchor PCR and the nested PCR. And the sequence of all the cloned fragments of the two isolated strains is spliced together to obtain the genome full-length sequence of the two isolates. The total length of the LHLJ1403 strain is 153222bp, and has 79 ORFs; the total length of the LHJ1403 strain is 153213bp, and the total length of the LHJ1403 strain is 15322bp, and the total length of the LHJ1403 strain is 15322bp, and the total length of the LH@@ The sequence is analyzed using biological software such as DNA Star, MEGA, DNMAN, and ORFs and is compared with the published reference sequence (for the specific reference sequence: SA2, A20, LT Blen,1874 C5, USDA,81658,63140, Laryngo, Serva, LJS09). The similarity of the LHLJ1403 and the LH1409 strains was 99.02%, the similarity of the LHLJ1403 and the LT Blen strains was 99.49%, and the similarity of the LHJ1403 and the LH1409 strains was up to 99.19%. Compared with SA2 and A20, the deletion of 14bp,3297-3485, 189bp,3672-3690 and the deletion of 19bp,4662-4693 in the UL region of LHLJ1403 and LHB1409 deleted the sequence of 32bp. The regions of the LHLJ1403 and LHB1409 strains (both the IRS) and the terminal repeat region (TRS) were found to have a large segment deletion of the 857 bp segment compared to the repeat region of the reference sequence, while the IRS and TRS of the LHLJ1403 and the LHB1409 strains all had a deletion of 217 bp in comparison to the Sera strain. In addition, the DNA replication origin, Ori S, of the repeat region of the LHLJ1403 and the LHB1409 strain, contains only the binding site of the binding protein (OBP), while the AT-rich region and the surrounding sequence are absent. The glycoprotein g B, g C, g D and g H/ g L of the LHLJ1403 and LHB1409 strains were mutated in part of the amino acid site as compared to the UASD strains. The whole genome sequence of the isolated strain is compared with the reference strain and the genetic evolution analysis is carried out, the result shows that the relationship between the LHLJ1403 and the American vaccine strain LT Blen and Laryngo is relatively close, and is located in the same branch; and the LHB1409 is close to the American vaccine strain Serva and the American strong strain 63140, And they are located in the same branch. The whole genome sequence of two ILTV isolates in China was determined and compared with the published other virulent strains, weak strains or vaccine strains, and the results provided a basis for the study of the molecular genetic characteristics and molecular biology of ILTV. In order to understand the prevalence of ILT in China, the prevention and control of the disease and the development of new vaccine have provided information.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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