Please use this identifier to cite or link to this item:
Studies on Pathogenesis of Rabbit hemorrhagic disease Virus
|關鍵字:||rabbit hemorrhagic disease;兔病毒性出血症;rabbit hemorrhagic disease virus;reverse transcription-polymerase chain reaction;field isolate;兔病毒性出血症病毒;反轉錄聚合鋂連鎖反應;野外分離株||出版社:||獸醫學系||摘要:||
由本省發病斃死兔隻肝臟中，以血球凝集試驗（HAT）證實分離到三株兔病毒性出血症病毒株。利用健康家兔行人工接種進行病毒的純化繼代及增殖。以連續氯化銫（CsCl）溶液行超高速離心，進行純化。三株病毒分別以負染色行電子顯微鏡觀察，可見無封套外形呈現圓形為正二十面體大小約40 nm之病毒顆粒。利用西方雜合反應證實每一分離株都存在一主要病毒結構蛋白其分子量為60 kDa，進一步利用反轉錄聚合鏈反應（RT-PCR），分別選取二組不同引子測試，結果顯示從三株病毒株均可增幅出不同的預期產物。RT-PCR產物不同分別以不同的限制進行切割，結果顯示三株病毒株經切割後有相同的切割圖譜。另以RHDV-R9株接種4月齡兔之後2-3天，以RT-PCR檢測兔隻各臟器樣品，除糞便外其餘樣品包括肝、膽、脾、血液、腸間淋巴、腎、胸腺、肺、肌肉及尿液均可檢測到病毒RNA。從肝臟乳劑樣品純化的RHDV-R9株病毒，其抽取RNA經10倍稀釋後供RT-PCR敏感性測定，結果顯示在ethidium bromide染色膠體中可測得最低量為300 fg，其產物經轉移後以南方吸漬雜合反應法測定，敏感度可提高10倍為30 fg。為進一步了解三株病毒株之間的關係，將病毒 capsid protein（VP60）基因定序，並進行親緣樹分析（phylogenetic analysis），顯示三株病毒株間基因核酸與氨基酸片段之序列幾乎沒有差異，與已知17株病毒核酸序列的差異為2.3-8.2﹪。
本試驗以兔病毒性出血症病毒經皮下接種，人工感染成兔及4-5週齡仔兔，並於接種後不同時間分別收集臟器，進行病毒核酸及抗原的檢測。利用血球凝集試驗（HAT）檢測病毒抗原則在感染後36小時於肝、膽汁和脾臟中檢測到。若以RT-PCR進行RHDV RNA的檢測，則早在接種後18小時於肝、膽汁和脾臟中即可檢出，而其他肺、腎、胸腺、腸間淋巴、白血球層等臟器亦於接種後26小時檢測得到。病毒核酸在尿及糞中在接種後36小時也出現，但檢出率則較不明顯。在離乳仔兔實驗中相同以RT-PCR檢測RHDV RNA，則在接種後一天在肝、膽汁和白血球層均可測得。2天後肺、腎、胸腺、腸間淋巴、糞便等陸續也被檢出。特別是在膽汁及脾臟則存有病毒核酸的時間持續整個實驗期。以帶毒家兔行注射合成葡萄醣類固醇（dexamethasone）及LPC兔化豬瘟疫苗誘發，均無法使兔病毒性出血症病毒再活化。利用血球凝集抑制試驗（HIT）及酵素接合吸附法（ELISA）測定其抗體之消長。於接種後一週其抗體即明顯上升，待接種後三週則達最高峰，並持續維持於整個試驗期間。
Liver tissues from animals that were suspected to have died of rabbit hemorrhagic disease (RHD) were used for isolation and characterization of the causative agent. Three strains of RHD virus were isolated as the supernatants of liver homogenate reacted positively in hemagglutination assays and were infective for rabbits after second passage in animals. Following extraction of liver homogenates from animals infected with each of three isolates, each virus strain was purified by CsCl density gradient ultracentrifugation for further characterization. In negative-stained preparations, the purified virions were icosahedral, measured approximately 40 nm in diameter, and nonenveloped. Morphologically, three isolates were similar. By immunoblotting, a protein with a molecular weight of 60,000 was identified as the major structural protein for each isolate. Furthermore, two sets of primer framed two different regions within RHD virus genome could amplify two fragments of the expected size, respectively, from each isolate, whereas, none were obtained from unifected control samples. The identity of the amplified products was further confirmed by using different restriction endonuclease digestion. Among three isolates of RHD virus, neither protein migration patterns of the virions nor cleavage patterns of the amplified product by restriction enzymes were found to differ. Amplified fragment with a length of 511 bp was detected in liver extracts of 4-month-old rabbits infected with each three strains of RHDV, whereas, none was obtained from mock-infected liver extracts. Furthermore, amplified fragment was also detected in tissue specimens prepared from liver, bile, spleen, blood, mesenteric lymph node, kidney, thymus, lung, muscle, and urine of rabbits experimentally infected with RHDV-R9, but not from fecal sample. Viral RNA extracted from purified virions of RHDV-R9 was used for testing the sensitivity of RT-PCR. The detection limit was 300 fg in ethidium bromide stained gel and could be further enhanced to 30 fg by hybridization after southern transfer. By using the RT-PCR test and sequencing, 99.9﹪homology was demonstrated in the capsid protein (VP60) of three isolates from Taiwan. Nucleotide sequence and phylogenetic analysis reveal these viruses a low rate (2.3-8.2)of change.
Adult and 4-to-5 week old rabbits were inoculated subcutaneously with rabbit hemorrhagic disease virus(RHDV). Samples were prepared from various tissues at intervals postinoculation (PI)for the detection of viral RNA and antigens. Using a hemagglutination test (HAT), viral antigens were detected in the liver, bile, and spleen of the adult rabbit at and after 36 h PI. The reverse transcription-polymerase chain reaction (RT-PCR) showed that RHDV RNA was present in the liver, bile, and spleen as early as 18 h PI, whereas lung, kidney, thymus, mesenteric lymph node, and buffy coat were found to be positive after more than 26 h PI. In addition, viral RNA in urine and faeces showed a variable positivity at and after 36 h PI. In the young rabbit, RT-PCR showed that RHDV RNA was present as early as 1 day PI in the liver, bile, spleen, and buffy coat; whereas lung, kidney, thymus, mesenteric lymph node, and faeces were found to be positive at and after 2 days PI. Bile and spleen were the only samples in which viral RNA was detected throughout the length of the experiment. Virus was not reactivated in six recovered virus inoculated rabbits treated with dexamethasome or a classical swine fever virus vaccine. Using a hemagglutination inhibition test and an ELISA, antibody titers increased rapidly from one week PI onwards, peaked at approximately three weeks of age, and were maintained throughout the length of the experiment.
The possibility of the transmission of rabbit hemorrhagic disease virus (RHDV) to swine through lapinized hog cholera virus (HCV) vaccine exists. To investigate the infectivity of RHDV in swine, 16 four- to six-week old piglets were inoculated subcutaneously with RHDV and samples of liver, lung, spleen, kidney, bile, adrenal gland, tonsil, mesenteric lymph node, thymus, urine, buffy coat, and feces were collected from each of 2 animals on days 0, 1, 2, 3, 5, 7, 14, and 28 postinfection. Using reverse transcription-polymerase chain reaction, viral RNA was detected in most tissues by day 3 and was absent after day 5, except in lung and liver tissues in which viral RNA was detected up to day 14. Viral RNA was not detected in kidney, urine, feces or bile. Antibody responses, as detected by hemagglutination inhibition, were of low titer and short duration, and were similar in animals inoculated with viable RHD and in those given formalin-inactivated RHDV (n=2). Neither viral RNA nor antibody were detected in the negative control or in the uninfected, in-contact animals.
|Appears in Collections:||獸醫學系所|
Show full item record
TAIR Related Article
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.