1300807 九、發明說明： 【發明所屬之技術領域】 本發明係有關於一種鳥類病毒的疫苗，尤其係有關於 一種鳥傳染性支氣管炎病毒之減毒疫苗。 【先前技術】 傳染性支氣管炎病毒(Infectious bronchitis virus)屬於 冠狀病毒科(Coronaviridae)、冠狀病毒屬的病毒。該病毒具 • 有多形性，但多數呈圓形，大小約80〜120nm。該病毒具有 囊膜，表面有杆狀纖突，長約20nm。該病毒傳染雞隻時會 表現出急性呼吸道症狀，如呼吸時的異常呼吸音、咳漱、 喷嚏、開口呼吸等，及食慾不振、無精神、下痢，並會造 成慢性或急性的腎炎，有時亦會造成蛋雞產蛋的障礙。 堂灣地區自1958年首度有傳染性支氣管炎(infecti〇us bronchitis，IB)的病例報告以來，該病一直為我國養雞產業 上的重要疾病，對我國家禽產業危害甚大。傳染我國雞隻 _ 之傳染性支氣管炎主要係以腎毒型的傳染性支氣管炎為 主。已知傳染性支氣管炎具有許多種血清型，且不同血清 型間缺乏交叉保護力。 目前台灣養雞業者為防治傳染性支氣管炎，已廣泛使 用疫苗進行防治，其主要是以進口的麻州血清型病毒疫苗 為主’例如 H120 (Merial，Cevac，Intervet，and others)、H52 (Abie)、Ma5 (Intervet)、M41 (Merial，Charoen Pokphand)、 M48 (Cevac)、Mass (Merial)、Mass II (Tungying)與 Ava-Bron 5 1300807 (mass type Ava-Bron stain，Schering-Plough)等。亦有人使用 康州的 Conn (Merial)、荷蘭株的 Broilerbron-H (Schering-Plough)、英國變異株的 4_91 (Intervet)，以及曰 本的ON株(Nisseiken)與TM86 (Dafung)。然而雖然養雞業 ， 者已大量施用前述之疫苗，但此疾病仍不斷發生，甚至有 不斷擴散漫延之趨勢。這是由於傳染台灣雞隻之病毒的血 清型與所施用之疫苗的血清型不同，致使前述習知的傳染 ^ 性支氣管炎疫苗無法充分保護雞隻免於台灣田間傳染性支 氣管炎病毒之攻擊所致。另外，根據先前的研究結果顯示， 台灣田間分離之傳染性支氣管炎病毒株大致上可區分為二 種不同血清型（台灣一型與台灣二型），但皆與麻州型或其 • 他已知的血清型不同。 ， 傳染性支氣管炎病毒疫苗目前一般以油質死毒疫苗及 活毒減毒疫苗為主。但是由於死毒疫苗免疫效果不佳，A 法弓丨發足夠的抗體反應，達到免疫保護效果，故一般僅用 • 於補強免疫。活毒減毒疫苗是採用經雞胚胎多次繼代剧丨化 培養的病毒株，因此可以減少病毒的致病性，其通常用作 基礎免疫。活毒減毒疫苗接種方式一般是以喷霧、點眼、 點鼻、飲水或蛋内接種等方式給予，藉此可刺激淋巴細胞 ，哈氏腺產生特異性抗體IgA抵抗病毒攻擊。前述幾種給 某方式中’以點眼、點鼻及飲水給予的方式較佳，其中和 抗體力價上升較快。 如前所述’麻州型疫苗業已無法防治我國傳染性支氣 盲戈之病毒傳染，因此開發具本土性傳染性支氣管炎灰清 6 1300807 型之不活化與活毒減毒疫苗便成為當務之急。 【發明内容】 為解决Sil述習知技術之問題，本發明之目的即在於提 n轉染性支氣管炎病毒之減毒病毒株，以及包含此 減毒病毒株之活毒減毒疫苗。 為達成本發明之目的，根據本發明所指出之一種鳥傳 染性支氣官炎病毒之減毒病毒株，其係選自經減毒馴化後 參 之傳染性支氣管炎病毒株25乃（寄存編號970039)與傳染 性支氣官炎病毒株2296 (寄存編號97〇〇4〇)所組成之族 群。前述傳染性支氣管炎病毒株攻5與傳染性找管炎病 毒株2296係為發明人所賦予相對應病毒株之編號，其血清 型分別為台灣-型與台灣二型。該減毒病毒株可誘發鳥類 的免疫系統產生免疫反應，以抵抗台灣本土之傳染性支氣 管炎病毒的入侵，且其於足以誘發免疫反應的免疫劑量下1300807 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a vaccine for avian viruses, and more particularly to an attenuated vaccine for avian infectious bronchitis virus. [Prior Art] Infectious bronchitis virus belongs to the genus Coronaviridae and the virus of the genus Coronavirus. The virus has pleomorphism, but most of them are round and about 80~120nm in size. The virus has a capsule with a rod-like fiber on the surface and is about 20 nm long. When the virus infects chickens, it will show acute respiratory symptoms, such as abnormal breathing sounds during breathing, coughing, sneezing, open breathing, etc., and loss of appetite, lack of spirit, squatting, and can cause chronic or acute nephritis, sometimes It also causes obstacles in laying eggs for laying hens. Since the first case report of infectious bronchitis (IB) in the Tangwan area in 1958, the disease has been an important disease in the chicken industry in China, and it is very harmful to the poultry industry in China. Infectious bronchitis, which is transmitted to Chinese chickens, is mainly caused by nephrotoxic infectious bronchitis. Infectious bronchitis is known to have many serotypes and lacks cross-protection between different serotypes. At present, Taiwanese chicken manufacturers have widely used vaccines for the prevention and treatment of infectious bronchitis, mainly based on imported Maize serotype vaccines such as H120 (Merial, Cevac, Intervet, and others), H52 (Abie ), Ma5 (Intervet), M41 (Merial, Charoen Pokphand), M48 (Cevac), Mass (Merial), Mass II (Tungying), and Ava-Bron 5 1300807 (mass type Ava-Bron stain, Schering-Plough). Some people also use Conn (Merial) from Connecticut, Broilerbron-H (Schering-Plough) from the Netherlands strain, 4_91 (Intervet) from the British mutant, and ON strain (Nisseiken) and TM86 (Dafung) from Sakamoto. However, although the chicken industry has already applied a large amount of the aforementioned vaccine, the disease continues to occur and even has a tendency to spread continuously. This is because the serotype of the virus infecting Taiwanese chickens is different from the serotype of the vaccine administered, so that the aforementioned infectious bronchitis vaccine cannot adequately protect chickens from the attack of Taiwan infectious bronchitis virus. To. In addition, according to previous research results, the infectious bronchitis virus strain isolated from Taiwan field can be roughly divided into two different serotypes (Taiwan type 1 and Taiwan type 2), but all with the type of Ma or its The serotypes are known to be different. Infectious bronchitis virus vaccines are currently mainly based on oily dead vaccines and live attenuated vaccines. However, due to the poor immune effect of the dead vaccination vaccine, the A method has sufficient antibody response to achieve an immunoprotective effect, so it is generally only used for tonifying immunity. The live attenuated vaccine is a virus strain cultured by multiple successive passages of chicken embryos, thereby reducing the pathogenicity of the virus, which is usually used as a basic immunity. The live attenuated vaccination method is generally administered by spraying, eye-catching, nose-nosed, drinking water or intra-egg inoculation, thereby stimulating lymphocytes, and the Hasan gland produces a specific antibody IgA against virus attack. The above-mentioned several methods are preferred in the manner of giving eye, nose and drinking water, and the antibody price increases rapidly. As mentioned above, the “Massachus-type vaccine industry” has been unable to control the infection of the infectious stagnation and blindness of the country. Therefore, it is imperative to develop a non-activated and live attenuated vaccine with local infectious bronchitis. SUMMARY OF THE INVENTION In order to solve the problem of the prior art of Sil, the object of the present invention is to provide an attenuated virus strain of transfective bronchitis virus, and a live attenuated vaccine comprising the attenuated virus strain. For the purpose of the present invention, an attenuated virus strain of avian infectious bronchitis virus according to the present invention is selected from attenuated domesticated bronchitis virus strain 25 (a registered number) 970039) A group consisting of the infectious bronchitis virus strain 2296 (accession number 97〇〇4〇). The infectious bronchitis virus strain 5 and the infectious tract tuberculosis strain 2296 are the numbers assigned to the corresponding virus strains by the inventors, and the serotypes are Taiwan-type and Taiwanese type II, respectively. The attenuated virus strain can induce an immune response in the bird's immune system to resist the invasion of infectious bronchitis virus in Taiwan, and it is sufficient to induce an immune response.
對鳥類不具致病性。前述之鳥類尤其係指家禽類動物 • 如雞。 J 根據本發明所指出之活毒減毒疫苗，其係包含一鳥傳 染性支氣官炎病毒之減毒病毒株及一藥理上可接受之载 體，其中该減毒病毒株係選自經減毒馴化之傳染性支氣管 炎病毋株2575 (寄存編號wo，)與傳染性支氣管炎病毒 株2296 (寄存編號97〇〇4〇)所組成之族群。本發明活毒滅毒 疫苗於適當之免疫劑量下，能誘發雞隻產生免疫反應，並 具有良好的中和指數值，且對接種之雞隻不具致病性。 1300807 本發明將藉由參考下列的實施方式做進一步的說明， 在此所述之實施方式並不限制本發明前面所揭示之内容。 熟習本發明之技藝者，可做些許之改良與修飾，但仍不脫 離本發明之範疇。 【實施方式】 根據本發明所指出經減毒馴化之鳥傳染性支氣管炎病 毒株2575與2296，係篩選自具有強毒性之鳥傳染性支氣 官炎病毒的野生株，並藉由習知雞胚胎連續繼代培養方 式，經高度繼代後(繼代70次以上），馴化所得之減毒病毒 株。 本發明減毒病毒株2575與2296業已分別寄存於食品 工業發展研究所，其寄存編號分別為970039與970040。 本發明減毒病毒株2575與2296分別具有如SEQ ID NO : 1與SEQIDNO : 2所示之基因序列。 本發明減毒病毒株可藉由習知方法製得活毒減毒疫 苗。例如，將本發明減毒病毒株以雞胚胎培養後，收取雞 胚胎的尿囊液(allantoic fluid)，再將此具有大量增殖後之病 毒的尿囊液加入一藥理上可接受之載體中。最後，經冷凍 乾秌即可製得本發明活毒減毒疫苗。前述方法僅為一實施 例，並不用以限制本發明之範圍，熟習本發明領域之技藝 者，可藉由閱讀本發明說明書之内容後，輕易地利用本發 明減:病毒株以其他習知活毒減毒疫苗製備方法製得所需 之活毋減t疫田。可應用於本發明中之藥理上可接受之載 1300807 體，例如佐劑(adjuvants)，但並不僅限於此。可應用於本發 明中作為佐劑的例子，包含由3%乳糖、1%麩胺酸鈉、與 1%脫脂奶粉所組成之佐劑，或5%乳糖、0.15%聚乙烯吡咯 烧酮(PVP-40)與0.1 %麩胺酸納所組成之佐劑，但並不僅限 於此。 本發明減毒病毒株或以該減毒病毒株所製備的活毒減 毒疫苗，均具有良好的安全性，其在i 〇2·3〜丨04·3 EID5Q (5〇% egg-infective doses)/隻的劑量下均不會對接種之鳥類引發 致病性。此外，其尚具有大於4·4的中和指數，且經攻毒 試驗證實，本發明活毒減毒疫苗確實可誘發鳥的免疫反 應’以抵抗傳染性支氣管炎病毒的侵襲(9〇%以上）。另外， 本發明活毒減毒疫苗於雞隻中經五次逆繼代(back passage) 後，並無毒力回復(virulence reversi〇n)的情形。這些結果均 顯示’本發明活毒減毒疫苗符合我國動物用藥品檢驗標準。 另外，以本發明減毒病毒株所製得之活毒減毒疫苗， 不論在特性試驗、無菌試驗、真空試驗及含濕度試驗上， 亦均符合我國動物用藥品檢驗標準。 實施例一 病毒分離、繼代及增殖 選用9〜11日齡、無特定病原（speciflc_path〇gen_free， 以I簡稱SPF)之雞胚胎(取自農委會家畜衛生試驗所動物 用藥品檢定分所，崎頂，台灣）’經照蛋後，晝出氣室位置， 並以75 %酒精喷灑蛋设表面。於酒精揮發後，以蛾液 1300807 (:〇Vi=e4Gdlne)塗抹氣室範圍的蛋殼表面，再以馨子於所 !之氣至界線上方鑿出一個小洞。 以具有26-gaUge、13 mm針頭的】址針筒吸取病毒 液，劑，由小洞將整個針頭插入，每一顆蛋接種〇 ι虹 - &誠_至尿囊腔⑽編ie咖），再财性填封膠將 小洞封住，並置於3π：孵蛋器中培養。 每天知蛋觀察，接種後24小時内死亡之雞胚胎，判為 細菌污料以丟棄。接種48小時後，將雜胎置於代冷 藏4 J守以上以75%酒精喷灑蛋殼表面。於酒精揮發後 再用普威隆溶液(P0vid0ne soluti〇n)塗抹氣室範圍的蛋殼表 面，以無菌恭械將氣室的蛋殼移去，並以具有24_职以弘、 25 mm 針頭的 3 mL 針筒（Terum〇 Medical c〇rp〇rati〇n， Elkton，MD)收取尿囊液。 冷凍乾燥疫苗的做法是將大量增殖之病毒尿囊液加入 等量佐劑，經冷凍乾燥後備用。 Φ 實施例二 安全性測試 取3日齡SPF小雞分成4組，每組1〇隻，飼養於已 消毒的實驗動物舍内。食物及飲水不予限制，每日清洗消 毒飼料槽及飲水器，並且更換足量乾淨之飼料及飲水。控 制組以點鼻（intranasal)滴入〇.〇1 mL的磷酸鹽緩衝溶液 (phosphate-buffered saline，以下簡稱 PBS)，而試驗組則點 鼻滴入0.01 mL的減毒毒株(2575與2296)病毒液，其接種 之病毒力價為 1〇2·3、103 3、104·3 EID50 (50% egg-infective 1300807 doses)/隻。接種後每日觀察是否有呼吸道症狀，或是其他 不良反應發生。另外，於接種21天後，翼靜脈抽血，所得 血液放置於37°C培養箱中數小時，之後將所得血清存在_2〇 °C冰箱内備用。另外，於不同時期使用1〇日齡Hy_VacSpF 小雞（Hy_Vac，Adel，I〇wa，USA)，分成兩組，每組1〇隻， 控制組同樣點鼻滴入0·01 mL PBS，試驗組則點鼻接種1〇3·3 EID%的減毒病毒液，同樣觀察21天。並於接種前及接種 後21天進行翼靜脈抽血，將所得血清亦存在_2〇它冰箱内 備用。 經測試後所有雞隻在觀察期中皆無任何臨床症狀與不 良反應。顯示本發明減毒毒株在102·3、103·3、104·3 eid%/ 隻的劑量之下，對於小雞並沒有致病性，此即表示本發明 活毒減毒疫苗的安全範圍大，且合乎我國動物用藥品檢驗 標準之規定。 實施例三 攻毒測試(challenge test) 將已依實施例二中之方法免疫過減毒病毒株的試驗組 小雞及接種過PBS的控制組小雞，以攻毒毒株TW97-4 (取 自中興大學張伯俊教授1997年1月間在雲林縣所分離之病 毒株，其係屬台灣一型）進行點鼻攻毒，其攻毒病毒力價為 10 EID%/隻。經攻毒1〇天後，將所有小雞以二氧化碳安 樂死並解剖，以觀察病變情形，及採集腎和氣管，混合後 製成乳劑，以反轉錄酶聚合鏈鎖反應(RT_PCR)檢測病毒核 11 1300807 酸的存在。並使用SPF胚胎蛋進行f目病毒分離兩代後， 再以RT_PCR檢測病毒核酸。 攻毋之後的觀察期間中，試驗組(免疫減毒毒株)的雞 隻均無呼吸迢症狀，剖檢後亦無氣管及腎臟的病變；控制 組雞隻則1G隻中有3隻(3/1G)出現呼吸症狀，剖檢後雖無 氣管病變，但10隻中有5隻(5/1〇)出現腎臟病變。 另外，臟器乳劑以RT-PCR進行分析及病毒分離分析 的結果，分示於表一。從表一中可以看出，大部分控制組 雞隻經攻毒後，可於其檢體中驗出病毒，顯示此攻毒測試 疋適當的。而經本發明減毒病毒株免疫過之實驗組的雞 隻，其檢體中幾乎檢測不出病毒的存在。此一結果顯示， 以本發明減毒病毒株進行免疫，確實可保護雞隻減少被病 毒侵襲的機會。 實施例四 抗體力價試驗（中和指數） 採取依實施例二中方法免疫3週後之血清及對照組血 清，於56艺水浴槽内非動化30分鐘。將病毒株TW97-4 以磷酸緩衝液連續10倍稀釋，將各階段稀釋液分為三群： 第一群加入試驗組之混合血清、第二群加入對照組混合血 清及第三群加入磷酸緩衝液做為病毒對照，等量混合於4 °C感作18_24小時後，各取〇·ι mL接種於5個8曰齡SPF 雞胚胎尿囊腔内，於37。(：培養箱培養8日。除注射後24 小時内死亡者不計外，檢查胚胎之變化，計算中和指數 12 1300807 (neutralization index，NI)。 以兩次安全試驗之後鬼集的血清進行中和指數的計 异，結果洋如表一。從表一中可得知，以減毒毒株2575 免疫後的中和指數大於4.4，以減毒毒株2296免疫後的中 和指數大於6.7,均高於對照組的中和指數。即接種此減毒 毒株的小雞能夠產生出抗體來中和中和試驗用病毒，此點 也合乎我國動物用藥品檢驗標準的規定。Not pathogenic to birds. The aforementioned birds are especially referred to as poultry animals such as chickens. J is a live attenuated vaccine according to the present invention, which comprises an attenuated virus strain of avian infectious bronchitis virus and a pharmaceutically acceptable carrier, wherein the attenuated virus strain is selected from the group consisting of Attenuated domesticated infectious bronchitis disease strain 2575 (registered number wo,) and infectious bronchitis virus strain 2296 (accession number 97〇〇4〇). The live poisoning and anti-virus vaccine of the present invention can induce an immune response in chickens under appropriate immunization doses, has a good neutralization index value, and is not pathogenic to the inoculated chicken. The invention will be further illustrated by reference to the following embodiments, which are not intended to limit the invention. A person skilled in the art can make some modifications and modifications without departing from the scope of the invention. [Embodiment] According to the present invention, the attenuated domesticated avian infectious bronchitis virus strains 2575 and 2296 are selected from wild strains of the highly infectious avian infectious bronchitis virus, and by conventional chickens. The embryos were successively subcultured, and after a high degree of subculture (more than 70 times), the attenuated virus strains were domesticated. The attenuated virus strains 2575 and 2296 of the present invention have been separately deposited in the Food Industry Development Research Institute, and their registration numbers are 970039 and 970040, respectively. The attenuated virus strains 2575 and 2296 of the present invention have the gene sequences shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The attenuated virus strain of the present invention can be obtained by a conventional method to obtain a live attenuated vaccine. For example, after the attenuated virus strain of the present invention is cultured in chicken embryos, the allantoic fluid of the chicken embryo is collected, and the allantoic fluid having a large amount of the proliferated virus is added to a pharmaceutically acceptable carrier. Finally, the live attenuated vaccine of the present invention can be prepared by freeze drying. The foregoing method is only an embodiment and is not intended to limit the scope of the present invention. Those skilled in the art can easily use the present invention to reduce the virus strain to other conventional activities by reading the contents of the present specification. The attenuated vaccine preparation method produces the desired live mites and reduces the epidemic field. The pharmacologically acceptable carrier 1300807, such as adjuvants, can be applied to the present invention, but is not limited thereto. An example of an adjuvant which can be used in the present invention comprises an adjuvant consisting of 3% lactose, 1% sodium glutamate, and 1% skim milk powder, or 5% lactose, 0.15% polyvinylpyrrolidone (PVP). -40) An adjuvant composed of 0.1% sodium glutamate, but is not limited thereto. The attenuated virus strain of the invention or the live attenuated vaccine prepared by the attenuated virus strain has good safety, and the i 〇2·3~丨04·3 EID5Q (5〇% egg-infective doses) / / only doses will not cause pathogenicity to the inoculated birds. In addition, it still has a neutralization index of more than 4.4, and it has been confirmed by the challenge test that the live attenuated vaccine of the present invention can induce the bird's immune response to resist the invasion of infectious bronchitis virus (more than 9%) ). In addition, the live attenuated vaccine of the present invention has no virulence reversi〇n after five times of back passage in the chicken. These results all show that the live attenuated vaccine of the present invention meets the Chinese animal drug testing standards. In addition, the live attenuated vaccine prepared by the attenuated virus strain of the invention conforms to the test standard for animal drugs in China, regardless of the characteristic test, the sterility test, the vacuum test and the humidity test. Example 1 Virus isolation, subculture and proliferation Select chicken embryos of 9 to 11 days old and without specific pathogens (speciflc_path〇gen_free, I referred to as SPF) (taken from the Animal Drug Testing Branch of the Animal Health Laboratory of the Council of Agriculture, Sakid, Taiwan) 'After the egg, take out the position of the air chamber and spray the surface with 75% alcohol. After the alcohol is volatilized, apply the moth liquid 1300807 (: 〇Vi=e4Gdlne) to the surface of the eggshell in the air chamber range, and then cut a small hole above the boundary line with the sesame seed. The virus solution is taken by a syringe with a 26-gaUge, 13 mm needle, and the entire needle is inserted by a small hole. Each egg is inoculated with 〇ι虹- & Cheng _ to the allantoic cavity (10) The small hole is sealed by the refilling sealant and placed in a 3π: incubator. The eggs were observed every day, and the chicken embryos that died within 24 hours after inoculation were classified as bacterial contamination to be discarded. After 48 hours of inoculation, the miscellaneous tires were placed on the surface of the eggshell with 75% alcohol in the sub-cold storage. After the alcohol is volatilized, the surface of the eggshell on the air chamber is applied with the Pweedon solution (P0vid0ne soluti〇n), and the eggshell of the air chamber is removed by a sterile knives, and has a needle of 24_ occupation, 25 mm needle The 3 mL syringe (Terum〇 Medical c〇rp〇rati〇n, Elkton, MD) received the allantoic fluid. The freeze-dried vaccine is prepared by adding a large amount of the proliferating virus allantoic fluid to an equal amount of adjuvant, and lyophilizing for use. Φ Example 2 Safety test Three-day-old SPF chicks were divided into 4 groups, each group was placed in a sterile laboratory animal house. Food and drinking water are not restricted. Clean the disinfection feed tank and drinker daily, and replace enough clean feed and drinking water. The control group was intranasally instilled with 〇.〇1 mL of phosphate-buffered saline (hereinafter referred to as PBS), while the experimental group was intranasally instilled with 0.01 mL of attenuated strains (2575 and 2296). Viral fluid, the viral price of the inoculation is 1〇2·3, 103 3, 104·3 EID50 (50% egg-infective 1300807 doses)/only. Observe daily respiratory symptoms or other adverse reactions after vaccination. Further, 21 days after the inoculation, blood was drawn from the wing vein, and the obtained blood was placed in a 37 ° C incubator for several hours, after which the resulting serum was stored in a refrigerator at _2 ° C for use. In addition, 1 day old Hy_VacSpF chicks (Hy_Vac, Adel, I〇wa, USA) were used in different periods, and divided into two groups, each group was only 1 ,, and the control group was also intranasally instilled with 0·01 mL PBS. Then, the nose was inoculated with 1〇3·3 EID% of the attenuated virus solution, and the same observation was made for 21 days. The blood was taken from the wing vein before the inoculation and 21 days after the inoculation. The obtained serum was also stored in the refrigerator for use. After testing, all chickens did not have any clinical symptoms or adverse reactions during the observation period. It shows that the attenuated strain of the present invention has no pathogenicity to chickens at doses of 102·3, 103·3, 104·3 eid%/only, which means the safe range of the live attenuated vaccine of the present invention. Large, and in line with China's animal drug testing standards. Example 3 challenge test (challenge test) The chickens in the test group that had been immunized with the attenuated virus strain according to the method of Example 2 and the control group chicks inoculated with PBS were used to challenge the strain TW97-4 (taken Professor Zhang Bojun from Zhongxing University, a virus strain isolated in Yunlin County in January 1997, which is a Taiwanese type, was attacked by a nose. The price of the virus was 10 EID%/only. One day after the attack, all the chickens were euthanized with carbon dioxide and dissected to observe the lesions, and the kidneys and trachea were collected, mixed and made into an emulsion, and the virus core was detected by reverse transcriptase polymerase chain reaction (RT_PCR). 1300807 The presence of acid. After SPF embryonic eggs were used to separate the F-eye virus for two generations, the viral nucleic acid was detected by RT-PCR. During the observation period after the attack, the chickens in the test group (immunized attenuated strains) had no symptoms of respiratory sputum, and there were no lesions of the trachea and kidney after the necropsy; 3 of the 1G in the control group (3) /1G) Respiratory symptoms occurred, although there was no tracheal lesion after necropsy, but 5 of 5 (5/1 〇) had renal lesions. In addition, the results of analysis of the organ emulsion by RT-PCR and virus separation analysis are shown in Table 1. As can be seen from Table 1, most of the control group chickens can be tested for viruses in their samples after being challenged, indicating that this attack test is appropriate. On the other hand, in the experimental group immunized with the attenuated virus strain of the present invention, the presence of the virus was hardly detected in the specimen. This result shows that immunization with the attenuated virus strain of the present invention does protect the chicken from the chance of being attacked by the virus. Example 4 Antibody valence test (neutralization index) The serum of the immunized body for 3 weeks according to the method of Example 2 and the control group were subjected to immobilization for 30 minutes in a bath of 56 art water. The virus strain TW97-4 was serially diluted 10-fold with phosphate buffer, and the dilutions of each stage were divided into three groups: the first group was added to the mixed serum of the test group, the second group was added to the control group, and the third group was added with phosphate buffer. The solution was used as a virus control, and the same amount was mixed at 4 °C for 18-24 hours, and each 〇·ι mL was inoculated into 5 8-inch-old SPF chicken embryos in the allantoic cavity, at 37. (: Incubator culture for 8 days. Except for the death within 24 hours after the injection, check the embryo change and calculate the neutralization index 12 1300807 (neutralization index, NI). Neutralize the serum of the ghost after the two safety tests. The results of the index are as follows: As shown in Table 1, the neutralization index after immunization with the attenuated strain 2575 is greater than 4.4, and the neutralizing index after immunization with the attenuated strain 2296 is greater than 6.7. It is higher than the neutralization index of the control group. That is, the chicken inoculated with the attenuated strain can produce antibodies to neutralize the virus for testing, which is also in line with the Chinese animal drug testing standards.
實施例五 毒性回復(virulence reversi〇n)測試 將1〜10日齡的SPF小雞分成兩組，每組至少3隻。 試，組雞隻以點鼻滴入mL的減毒毒株2575病毒液 (10 EID50 / 〇·ι mL)，每兩天觀察一次臨床症狀，觀察5 天:控制組雞隻則點鼻滴入〇1 mLpBS。於接種後5天， ，難隻犧牲亚進行剖檢。於剖檢雜錄和氣管，之後以 病毒。將前述腎和氣管混合後製成乳劑接種至 下-個實驗組_4，重複此步驟五次。 檢測===檢測中，實驗組的内臟中均可 中，實驗％雞隹、'：人;丙毋’但控制組則無。於觀察期間 二=:;二=察到有臨床症狀或死亡。此結 回復。 減，“株贿有在這些雞隻中發生毒力 貫施例六 1300807 特性試驗、無菌試驗、真空試驗、含濕度試驗 冷凍乾燥疫苗的特性、無菌、真空、含溼度試驗依照 動物用藥品檢驗標準規定進行。 冷凍乾燥疫苗的特性試驗結果為正常，無異物及異 味，溶解後均一；無菌試驗結果為無任何活菌；真空試驗 結果為正常；含濕度試驗結果為2.8%，低於規定的4% ; 並且在其中並無測到新城雞病及家禽流行性感冒的病毒核 酸。 因此，依據上述研究結果，加上毒力回歸試驗中雞體 内繼代五代後並無發現毒力恢復，顯示本發明減毒病毒株 極適合作為一良好的減毒疫苗。Example 5 Virulence reversi〇n test SPF chicks of 1 to 10 days old were divided into two groups of at least three. The rats in the group were instilled with mL of attenuated strain 2575 virus solution (10 EID50 / 〇·ι mL), and the clinical symptoms were observed every two days for 5 days: the control group was given nose drops. 〇 1 mLpBS. Five days after the inoculation, it is difficult to sacrifice the subtotal for necropsy. After the necropsy and trachea, the virus was used. The aforementioned kidney and trachea were mixed and made into an emulsion to be inoculated to the next experimental group _4, and this step was repeated five times. In the test === in the test, the viscera of the experimental group was available, and the experiment was % chicken, ': human; Bing' but the control group was absent. During the observation period, two =:; two = observed clinical symptoms or death. This is a reply. Less, "the bribes have toxic effects in these chickens. Example 6 1300807 Characteristic test, sterility test, vacuum test, humidity test, freeze-dried vaccine characteristics, sterility, vacuum, humidity test according to animal drug test standards The test results of the freeze-dried vaccine are normal, no foreign matter and odor, uniform after dissolution; the results of the sterility test are no live bacteria; the vacuum test results are normal; the humidity test result is 2.8%, lower than the prescribed 4 %; and there is no virus nucleic acid detected in Newcastle disease and poultry influenza. Therefore, based on the above findings, no virulence recovery was found after five generations of chicken in the virulence regression test. The attenuated virus strain of the present invention is highly suitable as a good attenuated vaccine.
14 1300807 表一藉由RT-PCR及病毒分離檢測傳染性支氣管炎病毒 組另1j RT-PCR 病毒分離 2575 0/10 1/10 控制組 9/10 8/10 2296 0/13 1/13 控制組 14/14 8/1414 1300807 Table 1 Detection of infectious bronchitis virus by RT-PCR and virus isolation Another 1j RT-PCR virus separation 2575 0/10 1/10 Control group 9/10 8/10 2296 0/13 1/13 Control group 14/14 8/14
表二抗體力價測試的中和指數 組另U Logio EID50 中和指數(NI) 2575 <3 >4.4 控制組 >7 <0.4 2296 <3 >6.7 控制組 7 2.7 m 15 1300807 【圖式簡單說明】 無 【主要元件符號說明】 無Table 2 Neutralization index group of antibody strength test U Logio EID50 Neutralization Index (NI) 2575 <3 >4.4 Control Group >7 <0.4 2296 <3 >6.7 Control Group 7 2.7 m 15 1300807 [Simple description of the diagram] No [Main component symbol description] None
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