發(fā)布時間：2019-05-27 04:31

【摘要】：雞傳染性喉氣管炎病毒(Infectious laryngotracheitis virus,ILTV)能引起雞的一種呼吸道的傳染病,具有高度接觸性和急性感染的特征,該病被稱為雞傳染性喉氣管炎(Infectious laryngotracheitis,ILT)。急性感染的雞群可出現(xiàn)高的發(fā)病率和死亡率,慢性感染可引起結(jié)膜炎、產(chǎn)蛋和蛋品質(zhì)下降,從1925年首次報道本病以來,已在全球各地流行,近些年來,在我國多地也呈流行趨勢,給養(yǎng)雞業(yè)造成的損失不容忽視。為研究ILTV LJS09株雞胚源毒經(jīng)在LMH上適應(yīng)后致病性的變化,本研究以ILTV LJS09株雞胚源毒和LMH細胞適應(yīng)第四代的毒分別人工感染SPF雞,通過評價兩感染組的臨床癥狀、臨床得分、臨床指數(shù)(1.4和0.7)、發(fā)病率(80%和60%)和死亡率(10%和0)、剖檢變化以及雞胚源毒感染組病理組織學變化,血清抗體水平變化規(guī)律,ILTV在雞體內(nèi)的嗜性器官以及各個器官中病毒的載量這些指標,繼而評價雞胚源毒和LMH細胞適應(yīng)毒的致病效果,并比較其致病力的強弱,結(jié)果表明兩感染組有相似的臨床癥狀和剖檢變化,血清抗體水平變化趨勢相同,ILTV嗜性器官相同,但雞胚源毒感染組的臨床得分、臨床指數(shù)、發(fā)病率和死亡率、血清抗體水平較LMH細胞適應(yīng)毒感染組高,表明ILTV LJS09株雞胚源毒比LMH細胞適應(yīng)毒的致病力強。這些評價指標及結(jié)果為了解我國傳染性喉氣管炎病毒的致病性提供了參考。本實驗室于2014年從黑龍江省和河北省分離得到兩株ILTV,分別命名為LHLJ1403和LHB1409株。經(jīng)SPF雞胚分離,并經(jīng)PCR和測序鑒定后確定其為ILTV。將這2株分離株在LMH細胞上適應(yīng),經(jīng)噬斑純化后得到了純化的病毒,通過電鏡觀察和間接免疫熒光檢測確定了純化的病毒為ILTV。為進一步了解我國ILTV毒株的流行病學特性及分子生物學特點,用PCR的方法測定了這兩株分離株的全基因組序列。通過將全基因組片段化,形成106個短片段,利用106對引物,分別進行克隆測序,由于內(nèi)部重復(fù)區(qū)和末端重復(fù)區(qū)具有倒置重復(fù)的特點,因此共利用了17對引物,通過錨定PCR和嵌套PCR相結(jié)合的方法準確確定其位置和序列。將得到的兩株分離株的所有克隆片段的序列拼接在一起,獲得兩株分離株的基因組全長序列。LHLJ1403株的基因組全長為153222 bp,具有79個ORF;LHB1409株的基因組全長為153213 bp,同樣具有79個ORF。利用DNAStar、MEGA、DNAMAN和ORF Finder等生物學軟件對序列進行分析,并將其與已公布的參考序列進行比對(用于比對的參考序列:SA2、A20、LT Blen、1874C5、USDA、81658、63140、Laryngo、Serva、LJS09)。通過同源性比對,LHLJ1403和LHB1409株相似性為99.02%,LHLJ1403和LT Blen株的相似性最高達99.49%,LHB1409和63140株的相似性最高達99.19%。與SA2和A20株相比,LHLJ1403和LHB1409株UL區(qū)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。將參考序列的重復(fù)區(qū)相互比較,發(fā)現(xiàn)LHLJ1403和LHB1409株的內(nèi)部(IRS)和末端重復(fù)區(qū)(TRS)都有857 bp片段的大段缺失,而在USDA株的重復(fù)序列中并不缺失這段片段,但與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株重復(fù)區(qū)的DNA復(fù)制起點Ori S只含有結(jié)合蛋白(OBP)的結(jié)合位點,而AT富集區(qū)及周圍序列存在缺失。LHLJ1403和LHB1409株的糖蛋白g B、g C、g D和g H/g L與UASD株相比,部分氨基酸位點發(fā)生了突變。將分離毒株的全基因組序列與參考毒株進行比對并進行遺傳進化分析,結(jié)果表明LHLJ1403與美國疫苗株LT Blen和Laryngo的親緣關(guān)系較近,并位于同一分支;LHB1409與美國疫苗株Serva和美國強毒株63140株緣關(guān)系較近,且它們位于同一分支。本研究測定了我國兩株ILTV分離毒株的全基因組序列,并將其與已發(fā)表的其他強毒株、弱毒株或疫苗株的基因組序列進行比對和分析,其結(jié)果為ILTV分子遺傳特性和分子生物學的研究奠定了基礎(chǔ),為了解我國ILT的流行情況、該病的防控及新型疫苗的研發(fā)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.
【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.31

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