專利名稱：：豬繁殖與呼吸綜合征病毒強(qiáng)毒株、其弱毒疫苗株及其應(yīng)用的制作方法
技術(shù)領(lǐng)域：
：本發(fā)明涉及一株病毒強(qiáng)毒株以及由其傳代致弱的弱毒疫苗株，尤其涉及一株豬繁殖與呼吸綜合征病毒強(qiáng)毒株HuN4、由該強(qiáng)毒株HuN4傳代致弱的弱毒疫苗株HuN4-F112，本發(fā)明還涉及弱毒疫苗株在預(yù)防或治療高致病性豬藍(lán)耳病中的應(yīng)用，該屬于生物醫(yī)藥領(lǐng)域。
背景技術(shù)：
：2006年春夏之際，我國(guó)南方部分地區(qū)發(fā)生以發(fā)熱、皮膚發(fā)紅、高發(fā)病率和高死亡率為主要特征的豬"高熱病"，隨后逐漸蔓延至全國(guó)大部分省份。經(jīng)大量的病原分離鑒定、動(dòng)物試驗(yàn)證實(shí)，豬"高熱病"的主要病原為高致病性豬藍(lán)耳病病毒變異株，該病也被稱為"高致病性豬藍(lán)耳病"。直至今日，疫病形勢(shì)依然嚴(yán)峻，生豬飼養(yǎng)是我國(guó)廣大農(nóng)民賴以增收的主要產(chǎn)業(yè)，如何防控該病已經(jīng)成為目前最為關(guān)心的問題。高致病性豬藍(lán)耳病(高致病性豬繁殖與呼吸綜合征)有以下特點(diǎn)高熱，體溫可達(dá)4rc以上，并伴有高發(fā)病率和死亡率，通常情況下發(fā)病率高達(dá)50%以上，死亡率約40%90%。其中，感染豬、康復(fù)豬及其污染物是本病的主要傳染源。不同日齡、不同品種的豬均可發(fā)生。本病主要通過呼吸道或通過公豬的精液經(jīng)生殖道在同豬群間進(jìn)行水平傳播，也可以進(jìn)行母子間的垂直傳播。病毒可通過鼻、眼分泌物、胎兒及子宮甚至公豬的精液排出，感染健康豬只。此外，風(fēng)媒傳播在本病流行中具有重要的意義，通過氣源性感染可以使本病在3km以內(nèi)的豬群中傳播，傳播速度極快。高致病性豬藍(lán)耳病有以下癥狀病豬精神沉郁，采食量下降，發(fā)病嚴(yán)重者食欲廢絕，嗜睡，嘔吐，拉稀，流鼻涕，呼吸困難。病死豬耳尖多因發(fā)紺而呈藍(lán)紫色，體表常有出血尤其皮表、肢體遠(yuǎn)端出血；有的臀部、腹部及后肢皮膚出現(xiàn)紐扣狀潰瘍或結(jié)痂。起初個(gè)別豬只發(fā)燒，隨后迅速傳播至大部分豬，體溫升高至4041.5'C，出現(xiàn)個(gè)別豬突然死亡。病豬呼吸困難，喜伏臥，部分豬出現(xiàn)嚴(yán)重的腹式呼吸，有的表現(xiàn)喘氣或呈不規(guī)則呼吸。部分患豬眼分泌物增多，出現(xiàn)結(jié)膜炎癥狀。部分豬伴有腹瀉。發(fā)病豬群死亡率很高，有的豬場(chǎng)高達(dá)50%以上，其中以保育肥豬最為嚴(yán)重。部分母豬在懷孕后期(100110天)出現(xiàn)流產(chǎn)、死胎。病程稍長(zhǎng)的病豬全身蒼白，出現(xiàn)貧血現(xiàn)象，被毛粗亂，部分病豬后肢無力，個(gè)別病豬瀕死前不能立，最后全身抽搐而死。剖檢病理變化病豬的淋巴結(jié)一般均有腫大、出血，其中以腹股溝淋巴結(jié)、頜下淋巴結(jié)和腸系膜淋巴結(jié)出血嚴(yán)重，有的腹股溝淋巴結(jié)只是腫大，無出血現(xiàn)象；肺有不同程度的實(shí)變；喉頭氣管基本正常，個(gè)別喉頭有黏液；大部分豬心內(nèi)膜和心外膜有出血點(diǎn)；脾臟基本正常，個(gè)別邊緣有壞死；肝臟基本正常；腎臟多數(shù)有條狀出血斑；胃基本正常，個(gè)別有胃底潰瘍和出血；小腸多數(shù)管壁透明，個(gè)別豬腸道卡他出血。高致病性豬藍(lán)耳病的病原為豬繁殖與呼吸綜合征病毒(PorcineReproductiveandRespiratorySyndromeVirus,PRRSV)變異株。通過對(duì)全國(guó)各地分離的PRRSV變異株進(jìn)行完整的0RF5和部分Nsp2基因序列比較分析發(fā)現(xiàn)，來自不同豬場(chǎng)的PRRSV高度同源，而且在Nsp2基因上都有兩個(gè)相同位點(diǎn)的缺失，Nsp2基因的第483位和533-561位氨基酸發(fā)生缺失。而與2005年以前的PRRSV的Nsp2同源性僅為69.8-86.2%。新分離毒株的GP5基因相互之間也是高度同源(98.5%_100.0%)，與以前的PRRSV毒株相比變異也比較大。同其它病毒性疾病一樣，目前尚無有效的治療性藥物用于PRRS的治療，免疫接種是防制該病的最有效方法。目前商品化的PRRS疫苗主要是由經(jīng)典株制備的弱毒苗和由變異株制備的滅活苗。經(jīng)由經(jīng)典株制備的弱毒苗免疫后，在變異株的攻擊下多數(shù)豬會(huì)發(fā)病、增重減慢，部分豬發(fā)生死亡；經(jīng)由變異株制備的滅活苗免疫豬后，在變異株的攻擊下絕大多數(shù)豬會(huì)發(fā)病、死亡。由此可見目前的PRRS疫苗雖然種類繁多，但沒有任何一種疫苗可對(duì)豬體產(chǎn)生足夠的保護(hù)作用。面對(duì)依然十分嚴(yán)峻的疫情，開發(fā)新型高效的PRRS疫苗已是迫在眉睫。
發(fā)明內(nèi)容本發(fā)明目的之一是提供一株豬繁殖與呼吸綜合征病毒強(qiáng)毒株。本發(fā)明目的之二是提供一株由該豬繁殖與呼吸綜合征病毒強(qiáng)毒株傳代致弱的弱毒疫苗株，用該弱毒疫苗株所制備的疫苗對(duì)于高致病性豬藍(lán)耳病具有良好的保護(hù)效力。本發(fā)明目的之三是提供一種預(yù)防或治療高致病性豬藍(lán)耳病的疫苗組合物。本發(fā)明的上述目的分別是通過以下技術(shù)方案來實(shí)現(xiàn)的豬繁殖與呼吸綜合征病毒強(qiáng)毒株的分離從我國(guó)湖南省暴發(fā)"高熱綜合癥"豬場(chǎng)的病豬的臟器為病料，將所采集的病料研磨，離心、過濾取濾液凍存待用，Marc-145細(xì)胞在37。C下培養(yǎng)4872h形成單層后，每瓶接種lml濾過的病料懸液上清，吸附24h后，換含4%胎牛血清的MEM培養(yǎng)液繼續(xù)在37°C下培養(yǎng)，觀察45天有無細(xì)胞病變產(chǎn)生。當(dāng)607(F。細(xì)胞出現(xiàn)CPE變化時(shí)收毒，將有病變的毒株傳23代后用PRRSV的N、GP5弓I物進(jìn)行PCR擴(kuò)增；用PRRSVCH-la株的N、GP5特異單抗通過間接免疫熒光方法進(jìn)行鑒定。試驗(yàn)證明，該分離毒株為PRRS病毒，命名為PRRSVHuN4株；通過對(duì)HuN4株的毒力的測(cè)定發(fā)現(xiàn)該株病毒對(duì)仔豬具有較強(qiáng)的致死性。對(duì)HuN4株進(jìn)行全序列測(cè)定，其序列結(jié)果為SEQIDN0:1所示。PRRSVHuN4株與PRRSV-N、GP5蛋白特異性單克隆抗體呈陽(yáng)性反應(yīng)，而與HCV、PRV、PPV、TGEV、PEDV和HEV無交叉反應(yīng)。采用RT-PCR技術(shù)對(duì)我國(guó)分離的HuN4株的0RF7和0RF5基因進(jìn)行進(jìn)行了擴(kuò)增，分別擴(kuò)增出2個(gè)目的基因片段，在此基礎(chǔ)上對(duì)分離到的PRRSVHuN4株做了PRRSVNsp2和GP5的序列分析，證明HuN4株屬于北美洲型，與已分離鑒定的JXA-1具有較高的同源性。本發(fā)明PRRSV弱毒株HuN4-F112的培育將所分離的豬繁殖與呼吸綜合征病毒強(qiáng)毒株HUN4在Marc-145細(xì)胞上連續(xù)傳代，經(jīng)3次蝕斑克隆，對(duì)傳代過程中不同代次病毒的培養(yǎng)特性、純粹性和生物學(xué)特性進(jìn)行了檢測(cè)，結(jié)果表明各代次病毒無外源病毒、細(xì)菌和霉菌污染，且能被高致病性豬藍(lán)耳病病毒特異性的陽(yáng)性血清中和。根據(jù)以上結(jié)果并結(jié)合不同代次病毒的序列分析結(jié)果比較，本發(fā)明選擇豬繁殖與呼吸綜合征病毒HUN4株第112代細(xì)胞毒(命名為HuN4-F112)作為高致病性豬藍(lán)耳病活疫苗(HUN4株-F112)的基礎(chǔ)種子代次。本發(fā)明PRRSV弱毒株HuN4-F112的分類命名為豬繁殖與呼吸綜合征病毒，其保藏號(hào)是CGMCCN0.2484;保藏時(shí)間是2008年5月8日；保藏地址是北京市朝陽(yáng)區(qū)大屯路，中國(guó)科學(xué)院微生物研究所；保藏單位中國(guó)微生物菌種保藏管理委員會(huì)普通微生物中心。本發(fā)明HUN4-F112株的安全性和免疫原性試驗(yàn)結(jié)果表明，本發(fā)明弱毒株的安全性好，本發(fā)明弱毒株對(duì)妊娠母豬、仔豬等各年齡豬只均安全、無副反應(yīng)。對(duì)本發(fā)明HuN4-F112株進(jìn)行全序列測(cè)定，其序列結(jié)果為SEQIDN0:2所示。本發(fā)明弱毒株HuN4-F112的形態(tài)學(xué)觀察傳至80代以后其病毒滴度達(dá)到最高并保持穩(wěn)定，電鏡觀察發(fā)現(xiàn)低代次病毒與高代次的病毒粒子形態(tài)有所改變，蝕斑形態(tài)比較發(fā)現(xiàn)低代次病毒的蝕斑大小不一，而高代次病毒形成的蝕斑大小均一。電鏡負(fù)染觀察發(fā)現(xiàn)HuN4-F112病毒粒子以圓形為主，直徑為40-100nm。將本發(fā)明弱毒株HuN4-F112與藥學(xué)上可接受的佐劑相配伍，可得到一種預(yù)防或治療高致病性豬藍(lán)耳病的疫苗組合物。具體的，本發(fā)明疫苗組合物可參考以下方法制備得到選用生長(zhǎng)良好的Marc-145細(xì)胞，倒掉培養(yǎng)液后，換以含1%病毒種子液的新鮮培養(yǎng)液(DMEM培養(yǎng)液)，置于37。C繼續(xù)培養(yǎng)；接本發(fā)明弱毒株HuN4-F112后，每日觀察；發(fā)現(xiàn)生長(zhǎng)異常、污染的細(xì)胞應(yīng)廢棄；培養(yǎng)2-3天后，約有80%的細(xì)胞出現(xiàn)病變，并于對(duì)照細(xì)胞差異顯著時(shí)，即可收獲，經(jīng)3次凍融后，置-2(TC保存；將收獲的病毒取樣進(jìn)行無菌檢驗(yàn)，應(yīng)無細(xì)菌生長(zhǎng)；效價(jià)均應(yīng)>106'5TCID5。/ml;將檢驗(yàn)合格的病毒液與蔗糖明膠保護(hù)劑(蔗糖280g，明膠49g,加去離子水700ml加熱溶解后，分裝，110-116'C滅菌30-60分鐘)按照7:1混合(病毒液保持在10-20°C，保護(hù)劑40-5CTC),充分搖勻后分裝，邊分裝邊搖動(dòng)，每只疫苗瓶分裝量為2ml，迅速進(jìn)行冷凍真空干燥，得到本發(fā)明疫苗組合物。本發(fā)明疫苗組合物的使用方法(1)、接種途徑頸部肌肉注射。，(2)、接種劑量哺乳仔豬0.5頭份，斷奶仔豬及架子豬l頭份。圖l用單克隆抗體通過間接免疫熒光試驗(yàn)檢測(cè)新分離的PRRSV:A為PRRSVCH-laN蛋白單抗；B為PRRSVCH-laGP5蛋白單抗。PRRSV聚集在感染細(xì)胞的胞漿區(qū)(400X)。圖2不同代次病毒蝕斑大小；高代次病毒蝕斑大小均一，低代次病毒蝕斑大小不一。圖3豬繁殖與呼吸綜合征病毒HuN4株F5和F112在Marc-145細(xì)胞上形成的病變結(jié)果。圖4豬繁殖與呼吸綜合征病毒HuN4株不同代次的病毒滴度。圖5豬繁殖與呼吸綜合征病毒HuN4株F5和F112代電鏡負(fù)染照片。F5代病毒粒子以橢圓形為主，F(xiàn)112病毒粒子以圓形為主，直徑40-100nm。具體實(shí)施方式下面結(jié)合具體實(shí)施例來進(jìn)一步描述本發(fā)明，本發(fā)明的優(yōu)點(diǎn)和特點(diǎn)將會(huì)隨著描述而更為清楚。但這些實(shí)施例僅是范例性的，并不對(duì)本發(fā)明的范圍構(gòu)成任何限制。本領(lǐng)域技術(shù)人員應(yīng)該理解的是，在不偏離本發(fā)明的精神和范圍下可以對(duì)本發(fā)明技術(shù)方案的細(xì)節(jié)和形式進(jìn)行修改或替換，但這些修改和替換均落入本發(fā)明的保護(hù)范圍內(nèi)。實(shí)施例1豬繁殖與呼吸綜合征病毒強(qiáng)毒株HuN4的分離與鑒定1.材料與方法1.1Marc-145細(xì)胞、抗PRRSV-N、GP5蛋白的單克隆抗體由哈爾濱獸醫(yī)研究所保存。1.2病料采集和處理采集南方某市豬場(chǎng)發(fā)生高熱病豬的抗凝血、肺、腎臟和淋巴結(jié)。1.3Marc-145細(xì)胞培養(yǎng)從液氮中取出凍存的Marc-145細(xì)胞23個(gè)安瓶，在37。C水浴中迅速解凍。將每個(gè)安瓶用1000r/min離心510min，棄去上清液，用23ml含有100IU/ml青霉素、80Pg/ml鏈霉素、6%犢牛血清和0.5%水解乳蛋白的DMEM重新懸浮細(xì)胞，然后吸入裝有10ml培養(yǎng)液的培養(yǎng)瓶中搖勻，置37'C、5%0)2的培養(yǎng)箱中靜止培養(yǎng)，72h后待其形成良好單層細(xì)胞后擴(kuò)大培養(yǎng)，每隔7296h傳代l次。1.4病毒分離將采集的病料研磨，離心、過濾取濾液凍存待用，Marc-145細(xì)胞在37。C下培養(yǎng)4872h形成單層后，每瓶接種lml濾過的病料懸液上清，吸附24h后，換含4%胎牛血清的MEM培養(yǎng)液繼續(xù)在37'C下培養(yǎng)，觀察45天有無細(xì)胞病變產(chǎn)生。當(dāng)607(F。細(xì)胞出現(xiàn)CPE變化時(shí)收毒，將有病變的毒株傳23代后用PRRSV的N、GP5引物進(jìn)行PCR擴(kuò)增；用PRRSVCH-la株的N、GP5特異單抗通過間接免疫熒光方法進(jìn)行鑒定。1.5病毒鑒定1.5.1間接熒光試驗(yàn)將出現(xiàn)CPE變化的細(xì)胞培養(yǎng)物接種Marc-145細(xì)胞，培養(yǎng)48h后刮取涂片，冷丙酮固定10min，用PBS沖洗后，分別滴加抗PRRSV-N、GP5蛋白的單克隆抗體，設(shè)立的PRRSVVR-2332毒株感染的Marc-145細(xì)胞作陽(yáng)性對(duì)照，未接毒的Marc-145細(xì)胞作陰性對(duì)照也作同樣處理，37'C作用45min，用PBS沖洗后，分別滴加熒光二抗于37"C作用45min，PBS沖洗后熒光顯微鏡下觀察。1.5.2致病性試驗(yàn)1.5.2.1對(duì)仔豬的毒力將經(jīng)蝕斑克隆純化過的PRRSVHuN4株強(qiáng)毒稀釋為每ml病毒含量為105°TCID5。，用60日齡健康易感PRRSV陰性(IFA抗體效價(jià)《1:4)仔豬5頭，每頭滴鼻接種2ml，連續(xù)觀察21日。1.5.2.2理化特性試驗(yàn)分離毒株的3代細(xì)胞毒懸液(lX105。TCID5。/ml)5ml，平均分裝于5試管中，試管l于56'C水浴作用45min;試管2加20%的乙醚震蕩10min后，4。C過夜，無菌揮去乙醚；試管3先用0.1MHC1調(diào)pH至3.0，于4'C作用2h后用0.1NaOH調(diào)pH至7.2;試管4加5-碘脫氧尿核苷(5-IUDR)50iil，4"C作用12h;試管5作未處理的正常對(duì)照，分別測(cè)定TCIDso。1.5.3紅細(xì)胞凝集試驗(yàn)采用微量板法，分別用0.5%的雞、豚鼠、大鼠、兔和綿羊的紅細(xì)胞懸液測(cè)定PRRSV的HA滴度，以50%紅細(xì)胞凝集的最高稀釋度作為判定終點(diǎn)。1.5.4形態(tài)學(xué)觀察分離毒株的3代細(xì)胞毒懸液60ml反復(fù)凍融3次，5000r/min離心20min取上清(Sl)待用；將沉淀用0.01M的PBS(pH7.2)5ml懸浮，用超聲波反復(fù)裂解沉淀3次，IOOW，30S/次，再用6000r/min離心20min取上清(S2),將Sl和S2混合，27000r/min(SW28轉(zhuǎn)子)離心3h，沉淀用2ml的TNC緩沖液懸浮，取lml懸浮液用5g/L的磷鉤酸復(fù)染；另lml懸浮液與等量的1:80的膠體金標(biāo)記的PRRSV抗體在37'C作用30min，分別取小樣作電鏡觀察。1.5.5交叉反應(yīng)試驗(yàn)用豬瘟病毒(HCV)、豬偽狂犬病毒(PRV)、豬傳染性胃腸炎病毒(TGEV)、豬流行性腹瀉病毒(PEDV)、豬血凝性腦脊髓炎病毒(HEV)的抗血清分別作l:40稀釋，然后將上述血清分別與出現(xiàn)CPE變化的細(xì)胞培養(yǎng)物(HuN4株)和未感染Marc-145細(xì)胞反應(yīng)。1.6分離毒株基因擴(kuò)增及序列分析對(duì)檢測(cè)到PRRSV(HuN4株)擴(kuò)增其Nsp2(擴(kuò)增區(qū)域是2535nt-3307nt)、0RF7和GP5全基因(引物序列見表1)，克隆并測(cè)序。并與GenBank中的PRRSV對(duì)應(yīng)序列進(jìn)行比較并繪制系統(tǒng)進(jìn)化樹。表l分離毒株基因擴(kuò)增使用的引物引物名稱引物序列擴(kuò)增基因的位置擴(kuò)增片段的長(zhǎng)度PRRSVORF75'CTGTGCCCTGGCTGCGTTGA3'5，TAGAGTCTGCCCTTGGTGT3'14129-14228117bpcompleteORF55'TGGCAATTTGAATGTTCAAGTATG3'5'CTGTGCTATCATTGCAGAAGTCGT3'13677-14301640bppartialNsp25，CAACACCCAGGCGACTTCAGAAATG3'5'CCAAGTCAGCATGTCAACCCTATC3，2535-3307796bp2、試驗(yàn)結(jié)果2.l病毒分離將處理好的病料接種于生長(zhǎng)良好的Marc-145細(xì)胞，34天左右，可見病變細(xì)胞發(fā)生聚叢成堆現(xiàn)象，并且越來越嚴(yán)重。將該毒傳23代，經(jīng)RT-PCR可擴(kuò)增出PRRSV的N、GP5基因；用PRRSVCH-la株的N、GP5單抗進(jìn)行間接免疫熒光試驗(yàn)，可見感染細(xì)胞的胞漿有特異性的熒光(圖1)。細(xì)胞分離物用PCR或RT-PCR檢測(cè)豬瘟病毒、豬圓環(huán)病毒、豬偽狂犬病毒均為陰性。該分離株在Marc-145細(xì)胞上傳12代后，產(chǎn)生的CPE變化規(guī)律、典型，一般在接種48h產(chǎn)生明顯CPE變化，細(xì)胞呈灶狀變化、突起；72h后，細(xì)胞皺縮、呈灶狀脫落，接種后45天細(xì)胞病變發(fā)展迅速，80%100%細(xì)胞產(chǎn)生CPE變化。2.2病毒鑒定2.2.1間接免疫熒光染色方法用抗PRRSV-N、GP5蛋白的單克隆抗體對(duì)PRRSVHuN4毒株感染的Marc-145細(xì)胞進(jìn)行間接免疫熒光染色，未接種的Marc-145細(xì)胞作為陰性對(duì)照。結(jié)果感染該分離株和Marc-145細(xì)胞胞漿均呈現(xiàn)強(qiáng)陽(yáng)性熒光，而對(duì)照細(xì)胞則呈陰性(見表2)。因此證明該分離毒株為PRRS病毒，命名為PRRSVHuN4株。PRRSVHuN4株的細(xì)胞培養(yǎng)物用PCR或RT-PCR檢測(cè)豬瘟病毒、豬圓環(huán)病毒、豬偽狂犬病毒均為陰性。表2PRRSV-N、GP5蛋白的單抗對(duì)PRRSVHuN4株感染細(xì)胞的間接熒光染色結(jié)果CH-laHuN4Marc-145N-單抗##-GP5-單抗#-2.2.2對(duì)仔豬的毒力試驗(yàn)5頭仔豬攻毒后第4天體溫開始升高，并持續(xù)到死亡前1-2天體溫才下降，體溫最高均超過40.5。C，其中兩頭超過41。C，攻毒豬還表現(xiàn)出精神沉郁、厭食、消瘦、眼圈發(fā)黑、眼瞼水腫、體表有大量脂溢性皮炎狀紅斑。從攻毒后第7天開始有豬死亡，至攻毒后21天全部死亡，死亡豬的體表皮膚由于末梢血管中的血液循環(huán)發(fā)生障礙而表現(xiàn)出皮膚變紅變紫，耳朵發(fā)紺。病理剖檢顯示，死亡最主要的原因是由于肺部感染引發(fā)炎癥反應(yīng)，肺實(shí)變導(dǎo)致呼吸衰竭。體表淋巴結(jié)腫大，部分豬胃部有出血和潰瘍，心、肝、腦、脾臟、腎臟眼觀病變不明顯。病理組織變化結(jié)果顯示1)全身性的免疫系統(tǒng)損傷；2)全身性的血管炎、血栓和淤血；3)呼吸系統(tǒng)的肺臟以淋巴細(xì)胞浸潤(rùn)為主造成肺泡隔增寬而形成的間質(zhì)性肺炎；而腎臟在死亡早期的豬表現(xiàn)為部分腎小球富核、核濃縮進(jìn)而形成腎小球壞死，隨著死亡時(shí)間的延長(zhǎng)表現(xiàn)為腎間質(zhì)有大量淋巴細(xì)胞浸潤(rùn)形成嚴(yán)重的間質(zhì)性腎炎。腦組織血管由于大量的淋巴細(xì)胞浸潤(rùn)形成嚴(yán)重的血管套并有嚴(yán)重的血栓和淤血，部分神經(jīng)細(xì)胞液化壞死。肝臟的匯管區(qū)內(nèi)有淋巴細(xì)胞和嗜酸性粒細(xì)胞浸潤(rùn)，肝小葉內(nèi)有淋巴細(xì)胞和紅細(xì)胞浸潤(rùn)并有小灶狀壞死。2.2.3理化特性試驗(yàn)分別測(cè)定經(jīng)過處理和未經(jīng)處理的細(xì)胞毒懸液的TCID5。，結(jié)果見表3。表3HuN4株理化特性試驗(yàn)結(jié)果處理方法56°C20%的乙醚pH3.05-IUDR未感染處理時(shí)間45min4'C過夜4°C、2h4。C12h-TCID50/ml0010111046105.12.2.4紅細(xì)胞凝集試驗(yàn)經(jīng)微量板法檢測(cè)細(xì)胞毒懸液對(duì)0.5%的雞、豚鼠、大鼠、兔和綿羊的紅細(xì)胞均無凝集作用。2.2.5形態(tài)學(xué)觀察純化后的PRRSV，經(jīng)磷鎢酸復(fù)染的電鏡觀察結(jié)果為，在鏡下可見大量直徑4278nm呈球型的病毒粒子，經(jīng)膠體金標(biāo)記的PRRSV抗體作用后的電鏡觀察結(jié)果為，在鏡下可見有大量的金顆粒吸附在病毒粒子周圍。2.2.6交叉反應(yīng)試驗(yàn)用HCV、PRV、PPV、TGEV、PEDV、HEV抗血清對(duì)HuN4株感染的Marc-145細(xì)胞間接免疫熒光染色結(jié)果(見表4)表明該分離毒株與HCV、PPV、TGEV、PEDV、HEV之間無交叉抗原。表4各種病毒的陽(yáng)性血清對(duì)HuN4株感染Marc-145細(xì)胞間接免疫熒光染色結(jié)果抗血清HuNlHuN2HuN3HuN4未感染HCV-—-—PrV———_PPV—_———TGEV-———-PEDV-----HEV■-—--—PRRSVVR2332####-2.2.7序列分析本發(fā)明強(qiáng)毒株的基因組全長(zhǎng)15297個(gè)堿基，Nsp2基因的第483位和535-563位氨基酸發(fā)生缺失。對(duì)其它省市發(fā)病豬場(chǎng)PRRSV陽(yáng)性樣品中擴(kuò)增的Nsp2序列分析發(fā)現(xiàn)，它們都有和HuN4株完全一致缺失特征。這48株P(guān)RRSV的Nsp2序列相互之間高度同源，同源性為98.2%100%，而與2005年以前的PRRSV的Nsp2同源性僅為69.886.2%。PRRSVHuN4株與其他新分離毒株間的GP5基因相互之間也是高度同源(98.5%100.0%)，與以前的PRRSV毒株相比變異也比較大，尤其是第151位氨基酸的改變被認(rèn)為是與毒力增強(qiáng)有關(guān)。HuN4株的Nsp2基因、GP5基因和全基因組核苷酸與JXA1株相比同源性分別為99.7%、99.8%和99.6%，兩個(gè)病毒全基因組氨基酸同源為99.4%。試驗(yàn)結(jié)論(1)本發(fā)明PRRSVHuN4株的細(xì)胞適應(yīng)能力與2005年前的毒株相比顯著提高，先前毒株很難直接適應(yīng)Marc-145細(xì)胞，往往需適應(yīng)原代肺泡巨噬細(xì)胞后才能適應(yīng)Marc-145細(xì)胞，且細(xì)胞病變不顯著。而新分離的毒株第1代即可適應(yīng)Marc-145細(xì)胞，并且產(chǎn)生明顯的細(xì)胞病變，正是因?yàn)檫@個(gè)特點(diǎn)，本發(fā)明人短時(shí)間內(nèi)在10個(gè)省份分離到15株病毒，這種現(xiàn)象在以前PRRSV分離中是很罕見的。(2)臨床癥狀和病理變化比以往毒株嚴(yán)重，例如本發(fā)明以PRRSVCH-la毒株攻擊仔豬臨床癥狀只表現(xiàn)為中等程度的發(fā)熱，體溫一般不超過40.5'C，其他臨床癥狀并不明顯，同樣的飼養(yǎng)環(huán)境也沒發(fā)生繼發(fā)細(xì)菌感染的癥狀，攻毒豬幾乎沒有致死情況的發(fā)生。而PRRSVHuN4株攻豬后臨床癥狀明顯，并具有顯著的致死能力，病理變化中的腦部海馬區(qū)血管細(xì)胞腫大增厚明顯，這是否與現(xiàn)地許多發(fā)病豬出現(xiàn)神經(jīng)癥狀有關(guān)，這也說明新分離的毒株具有感染腦組織的能力，這可能也是毒力增強(qiáng)的一種表現(xiàn)，另外胃部有出血和潰瘍這也是以往PRRSV攻毒試驗(yàn)所未見到的。(3)PRRSVHuN4株與其他新分離毒株間分子特征高度一致，而與先前毒株又有顯著差異，尤其是Nsp2基因的缺失和結(jié)構(gòu)蛋白基因序列的突變，這些變化中哪些對(duì)毒力的改變起決定作用還有待進(jìn)一步的研究。綜上所述，2006年從爆發(fā)所謂"高熱綜合癥"的豬場(chǎng)分離到的PRRSV分離株有一個(gè)顯著特征就是在Nsp2基因上有兩處缺失，分子流行病學(xué)分析發(fā)現(xiàn)在不同省市豬場(chǎng)流行的PRRSV相互之間高度同源，與以往PRRSV有很大的區(qū)別，尤其是致病能力大大增強(qiáng)，因此，可以初步認(rèn)定這種新的PRRSV變異株是南方豬"高熱綜合癥"流行的主要病因(可能有部分豬場(chǎng)發(fā)病是由其它原因引起的)。實(shí)施例2豬繁殖與呼吸綜合征病毒弱毒疫苗株HuN4-F112的培育1.材料方法1.1病毒細(xì)胞培養(yǎng)及克隆化進(jìn)行病毒培育的Marc-145細(xì)胞用DMEM細(xì)胞培養(yǎng)液培養(yǎng)，其中含8%滅活犢牛血清、100U/ml的青霉素、80Pg/ml的鏈霉素，并用NaHC03調(diào)整pH值至7.2左右，過濾除菌。用此細(xì)胞培養(yǎng)液復(fù)蘇Marc-145細(xì)胞或使之傳代至容量為100ml的滅菌的細(xì)胞培養(yǎng)瓶里，待長(zhǎng)成良好細(xì)胞單層后用于接種病毒。1.2種毒的克隆化將實(shí)施例1所分離的高致病性豬藍(lán)耳病病毒HuN4株在Marc-145細(xì)胞上傳代，分別在傳至3、15、20代時(shí)進(jìn)行病毒蝕斑克隆，并選取直徑lmm的蝕斑繼續(xù)傳代培養(yǎng)。1.3種毒的毒價(jià)測(cè)定，選取不同代次的種毒按常規(guī)方法測(cè)定TCID5Q。1.4病毒粒子形態(tài)觀察取HuN4株F5和F112代毒的細(xì)胞培養(yǎng)物，3000轉(zhuǎn)離心15分鐘，與PRRSVCH-laN蛋白單抗作用l小時(shí)，12000轉(zhuǎn)離心20分鐘，取沉淀懸浮后，電鏡負(fù)染觀察。1.5理化特性試驗(yàn)取分離毒株的5、20、40、65、84、112代細(xì)胞毒懸液(105Q108°TCID5。/ml)5ml，平均分裝于5支試管中，試管1于56。C水浴作用45min;試管2加20%的乙醚震蕩10min后，4'C過夜，無菌揮去乙醚；試管3先用0.1MHC1調(diào)pH至3.0,于4。C作用2h后用0.l麗aOH調(diào)pH至7.2;試管4加5-碘脫氧尿核苷(5-IUDR)50ul，4"C作用12h;同時(shí)設(shè)未做任何處理的正常對(duì)照，分別測(cè)定TCID5。。1.6無菌檢驗(yàn)取分離毒株的84、90、112代細(xì)胞毒懸液(107°108°TCID5。/ml)按照按《中國(guó)獸藥典》進(jìn)行。1.7支原體檢驗(yàn)取分離毒株的84、90、112代細(xì)胞毒懸液(107°108°TCID5Q/ml)按《中國(guó)獸藥典》進(jìn)行。1.8外源病毒檢測(cè)按《中國(guó)獸藥典》進(jìn)行。2試驗(yàn)結(jié)果2.1傳代毒的克隆純化在致弱毒種的過程中進(jìn)行了3次克隆純化，每次克隆均選擇lmm蝕斑。高致病性豬藍(lán)耳病病毒HuN4株低代次病毒蝕斑大小不一，而高代次病毒蝕斑大小均一(圖2)，高、低代次病毒的病變形態(tài)也不同低代次點(diǎn)狀、聚叢，高代次均勻、整體病變(圖3)。2.2理化特性試驗(yàn)測(cè)定經(jīng)處理和未經(jīng)處理的細(xì)胞毒懸液的TCID5Q，結(jié)果見表5。表5理化特性試驗(yàn)結(jié)果<table>tableseeoriginaldocumentpage13</column></row><table>2.3紅細(xì)胞凝集試驗(yàn)經(jīng)微量板法檢測(cè)80、90、100、112代細(xì)胞毒懸液對(duì)0.5%的雞、豚鼠、大鼠、兔和綿羊的紅細(xì)胞均無凝集作用。2.4種毒的培育及其滴度測(cè)定當(dāng)HuN4株在Marc-145細(xì)胞系傳代時(shí)，其病毒滴度在低代次時(shí)病變速度較快，48小時(shí)即可收毒毒價(jià)在105°—6°TCID5。/ml左右，然后病毒毒價(jià)下降、病變速度變慢，30代以后病變速度加快，毒價(jià)也升高，70代以后病變速度和毒價(jià)保持穩(wěn)定，毒價(jià)達(dá)到1075—85TCID5。/ml(圖4)。2.6病毒粒子形態(tài)高致病性豬藍(lán)耳病病毒HuN4株F5和F112代電鏡負(fù)染觀察發(fā)現(xiàn)，F(xiàn)5代病毒粒子以橢圓形為主，F(xiàn)112病毒粒子以圓形為主，直徑40-100nm(圖5)。2.7無菌檢驗(yàn)通過對(duì)分離毒株的80、90、100、112代細(xì)胞毒懸液進(jìn)行無菌檢驗(yàn)，結(jié)果無雜菌污染。2.8支原體檢驗(yàn)通過對(duì)分離毒株的80、90、100、112代細(xì)胞毒懸液進(jìn)行支原體檢驗(yàn)，結(jié)果無支原體污染。2.9外源病毒檢測(cè)利用豬三價(jià)(HCV、PPV、BVDV)熒光抗體、豬偽狂犬熒光抗體檢測(cè)，均未檢測(cè)到80、90、100、112代細(xì)胞毒感染細(xì)胞有特異性熒光；采用細(xì)胞病變(CPE)方法檢測(cè)，未見Vero細(xì)胞出現(xiàn)CPE;采用紅細(xì)胞吸附法檢測(cè)，未見Vero細(xì)胞出現(xiàn)血細(xì)胞吸附現(xiàn)象出現(xiàn)。試驗(yàn)證明，高致病性豬藍(lán)耳病病毒HuN4株種毒，未檢測(cè)到外源病毒污染。高致病性豬藍(lán)耳病病毒HuN4株在Marc-145細(xì)胞上經(jīng)蝕斑克隆和連續(xù)傳代后，其病變速度和病毒滴度有很大的提高，病毒粒子形態(tài)也有所改變，傳至80代以后病毒的生物學(xué)特性保持穩(wěn)定，結(jié)合病毒序列分析，我們選擇高致病性豬藍(lán)耳病病毒HuN4株第110120代細(xì)胞毒作為高致病性豬藍(lán)耳病活疫苗(HuN4-F112)的基礎(chǔ)種子代次，并以此為基礎(chǔ)研究其安全性和免疫原性。試驗(yàn)例1高致病性豬藍(lán)耳病病毒HuN4株致弱過程中不同代次病毒的分子特征1、材料與方法1.1毒株及細(xì)胞高致病性豬藍(lán)耳病病毒HuN4株(實(shí)施例1所分離)、傳代細(xì)胞Marc-145由本研究室分離和保存。1.2親本毒HuN4株的傳代致弱將HuN4株種毒100倍稀釋后接種其傳代細(xì)胞Marc-145，于37°C0)2培養(yǎng)箱中培養(yǎng)，觀察細(xì)胞病變，當(dāng)病變達(dá)到80%時(shí)，收獲病毒，以傳代獲取的病毒作為種毒繼續(xù)在Marc-145細(xì)胞上傳代培養(yǎng)，一直傳至100代以上。1.3HuN4株全長(zhǎng)基因擴(kuò)增引物設(shè)計(jì)根據(jù)本研究室分離的高致病性PRRSVHuN4株(GenBankno.EF635006)的全長(zhǎng)基因序列，將HuN4株的全長(zhǎng)基因分為八個(gè)相互重疊的基因片段，設(shè)計(jì)合成了八對(duì)引物(見表6)。表6高致病性PRRSV親本毒HuN4株及其不同代次毒株全某因RT-PCR擴(kuò)增引物、位置及片段大小<table>tableseeoriginaldocumentpage15</column></row><table>利用RNA提取試劑盒(購(gòu)自Qiagen公司)，分別提取親本毒HuN4株及其傳代毒HuN4-F20、HuN4-F40、HuN4-F65、HuN4-F84、HuN4-FIOO、HuN4-F112的病毒RNA，-8(TC保存?zhèn)溆谩?.5親本毒HuN4株及其傳代毒株RT-PCR擴(kuò)增及全基因克隆以提取的HuN4株及其不同代次病毒的RNA反轉(zhuǎn)錄產(chǎn)物為模板，利用合成的8個(gè)片段引物分別對(duì)不同代次的病毒進(jìn)行RT-PCR擴(kuò)增，通過PCR產(chǎn)物膠回收試劑盒分別回收8個(gè)片段的PCR產(chǎn)物，克隆于pMD18-T載體上，各基因片段連接產(chǎn)物經(jīng)PCR鑒定后，分別隨機(jī)選取8-10個(gè)陽(yáng)性克隆，送測(cè)序。1.6親本毒HuN4株及其傳代毒株全基因序列分析將8個(gè)基因片段測(cè)序結(jié)果經(jīng)序列分析軟件比對(duì)后進(jìn)行全長(zhǎng)基因拼接，以獲得各代次病毒的全基因組序列，然后利用分子生物學(xué)軟件對(duì)各個(gè)代次病毒結(jié)構(gòu)蛋白和非結(jié)構(gòu)蛋白基因的核苷酸和氨基酸序列進(jìn)行序列比較分析。2、試驗(yàn)結(jié)果2.1親本毒HuN4株及不同代次毒株全基因RT-PCR擴(kuò)增選取親本毒HuN4株及其Marc-145細(xì)胞上傳代致弱的不同代次的病毒HuN4-F20、HuN4-F40、HuN4-F65、HuN4-F84、HuN4-F100、HuN4-F112，提取病毒RNA，反轉(zhuǎn)錄后，利用8片段PCR擴(kuò)增引物，對(duì)親本毒HuN4株及選取的不同代次病毒進(jìn)行RT-PCR擴(kuò)增，均可以獲得特異性的PCR產(chǎn)物，與預(yù)期大小相一致(見圖1)。2.2不同代次毒株與親本毒的基因序列分析利用DNAStar軟件對(duì)親本毒HuN4株及其不同代次病毒各個(gè)結(jié)構(gòu)基因及其非結(jié)構(gòu)基因的氨基酸進(jìn)行序列比較，結(jié)果發(fā)現(xiàn)親本毒HuN4經(jīng)傳代后不同代次的病毒在非結(jié)構(gòu)蛋白區(qū)和結(jié)構(gòu)蛋白區(qū)都有不周數(shù)量的氮基酸發(fā)生突變，其數(shù)量統(tǒng)計(jì)結(jié)果見表8和表3。表7親本毒HuN4株及其不同代次毒株在不同開放閱讀框中氨基酸突變的數(shù)量<table>tableseeoriginaldocumentpage16</column></row><table>表8親本毒HuN4及其不同代次病毒氨基酸突變數(shù)量統(tǒng)計(jì)不同毒株H畫F20HUN4F40HUN4F65HUN4F84■HUNF100H眼F"2突變數(shù)量142025353537通過對(duì)高致病性豬藍(lán)耳病病毒HuN4株及其不同代次病毒全基因進(jìn)行克隆測(cè)序及遺傳演化分析，結(jié)果發(fā)現(xiàn)親本毒HuN4株在Marc-145細(xì)胞中進(jìn)行連續(xù)傳代過程中，在非結(jié)構(gòu)蛋白編碼區(qū)和結(jié)構(gòu)蛋白編碼區(qū)均有氨基酸的突變，但是發(fā)生突變的氨基酸比較分散，其中突變最多的主要集中在0RFla和0RFlb復(fù)制酶基因區(qū)域，其中0RFla基因中發(fā)生突變的氨基酸主要在NSP2蛋白，占ORFla基因突變氨基酸的61.5%，ORFlb基因發(fā)生突變的氨基酸主要在NSP9、NSP11和NSP12蛋白。在結(jié)構(gòu)蛋白區(qū)域氨基酸突變較少，0RF2基因在F65代以前僅有1個(gè)氨基酸發(fā)生突變，到了F84代以后卻出現(xiàn)4個(gè)或5個(gè)氨基酸突變；0RF3基因在F65代以前都未發(fā)生突變，但是到了F84代以后開始出現(xiàn)突變；0RF4-6也都出現(xiàn)了l-4個(gè)氨基酸的突變。其中氨基酸穩(wěn)定遺傳的是0RF7編碼的核衣殼蛋白，即使傳至100代以上，也未發(fā)生氨基酸的突變或缺失、插入等現(xiàn)象。原始種毒HuN4株與F20至F112代的傳代病毒在以前報(bào)道的與毒力相關(guān)的氨基酸處，都保持了和野毒株相一致的氨基酸特性，未發(fā)生突變，暗示以前所報(bào)道和毒力相關(guān)的氨基酸與高致病性分離株致弱似乎并不相關(guān)。從不同代次病毒氨基酸變化結(jié)果來看，親本毒HuN4株傳至F20時(shí)有14個(gè)氨基酸發(fā)生突變，F(xiàn)40代有20個(gè)氨基酸發(fā)生突變，F(xiàn)65代氨基酸發(fā)生突變達(dá)到25個(gè)，突變率達(dá)到0.49%，F(xiàn)84代發(fā)生突變的氨基酸達(dá)到了35個(gè)，突變率達(dá)到0.68%，在整個(gè)過程中突變率達(dá)到了最高，從F85代以后氨基酸遺傳性能基本趨于穩(wěn)定，當(dāng)親本毒HuN4株傳至FIOO、F112代以后，氨基酸遺傳性能基本穩(wěn)定，從F100代到F112代僅發(fā)生2個(gè)氨基酸的突變，其氨基酸突變率僅為0.03%。從以上研究中可以看出，高致病性豬藍(lán)耳病病毒HuN4株在傳代致弱過程中，F(xiàn)84代以前中間代次的各個(gè)毒株氨基酸仍然可以繼續(xù)發(fā)生突變，具有遺傳性能的不穩(wěn)定性，F(xiàn)84代以后其氨基酸突變率降低，基本趨于平衡，而經(jīng)過傳代致弱F100代以后的毒株其生物學(xué)遺傳性能更加趨于穩(wěn)定，適合作為弱毒疫苗的種毒。試驗(yàn)結(jié)論1、高致病性豬藍(lán)耳病病毒HuN4株在連續(xù)傳代過程中，整個(gè)基因組未發(fā)生缺失和插入現(xiàn)象，但存在氨基酸的點(diǎn)突變，且發(fā)生突變的氨基酸比較分散，其中突變最多的主要集中在ORFla和ORFlb復(fù)制酶基因區(qū)域。2、親本毒HuN4株在細(xì)胞傳代致弱過程中，F(xiàn)84代以前中間代次的各個(gè)毒株氨基酸仍然可以繼續(xù)發(fā)生突變，具有遺傳的不穩(wěn)定性。F84代以后其氨基酸突變率降低，基本趨于平衡。經(jīng)過傳代致弱F100代以后的毒株其生物學(xué)遺傳性能更加趨于穩(wěn)定，適合作為弱毒疫苗的種毒。試驗(yàn)例2高致病性豬藍(lán)耳病活疫苗(HuN4-F112)的保護(hù)效力試驗(yàn)1材料和方法1.1高致病性豬藍(lán)耳病活疫苗(HuN4-F112)為HuN4株第112代細(xì)胞毒，病毒滴度10775TCID5Q/ml。1.2動(dòng)物試驗(yàn)30頭40日齡PRRS病毒和抗體均為陰性，PRV、PCV2和CSFV陰性的健康仔豬，隨機(jī)分成6組每組5頭，1-5組，分別接種高致病性豬藍(lán)耳病活疫苗(HuN4-F112)接種劑量為102°TCID5。、103°TCID5。、104°TCID5。、105.。TCID50、106°TCID5。，第6組為對(duì)照組，免疫后21天，所有試驗(yàn)豬每頭攻擊HuN4F5強(qiáng)毒3X104。TCIDso，攻毒后每天測(cè)溫，7、10、14、21采血，分離病毒和檢測(cè)抗體。2試驗(yàn)結(jié)果2.1臨床癥狀免疫后各組豬均正常，無發(fā)燒、厭食等臨床癥狀，攻毒后24小時(shí)1和6組的豬體溫就達(dá)到4rC以上最高達(dá)42.2'C，41。C以上最多累計(jì)達(dá)10天，而第2組l頭豬體溫4rC以上累計(jì)2天，并且消瘦，試驗(yàn)結(jié)束時(shí)病理剖檢符合臨床發(fā)病標(biāo)準(zhǔn)，另外2組還有3頭豬體溫達(dá)到41°C以上1天，其它各組豬體溫都沒有超過41°C，病理剖檢結(jié)果全部保護(hù)。1和6組臨床表現(xiàn)有厭食、咳嗽、呼吸困難、腹瀉和后肢麻痹等癥狀，大部分豬體重表現(xiàn)為負(fù)增長(zhǎng)，攻毒后10天開始死亡，至試驗(yàn)結(jié)束時(shí)1組死亡3頭，6組死亡2頭，剩余豬病理剖檢全部符合臨床發(fā)病標(biāo)準(zhǔn)。2.2抗體變化免疫后10天3-5組ELISA抗體開始陽(yáng)轉(zhuǎn)，免疫后21天2-5組所有豬LISA抗體陽(yáng)轉(zhuǎn)，1和6組均為陰性。2.3病毒分離免疫后21天除4和5組各有一頭豬可以分離到疫苗毒外，其余各組均為陰性。攻毒后7天3、4、5組全部產(chǎn)生中和抗體并分離不到病毒，而1和6組剩余豬到攻毒后14天仍能分離到病毒。表9高致病性豬藍(lán)耳病活疫苗(HuN4-F112)不同接種劑量對(duì)仔豬免疫保護(hù)試驗(yàn)的結(jié)果<table>tableseeoriginaldocumentpage18</column></row><table>試驗(yàn)結(jié)果表明，高致病性豬藍(lán)耳病活疫苗(HuN4-F112)對(duì)仔豬的最小免疫劑量是103flTCID5。。試驗(yàn)例3高致病性豬藍(lán)耳病活疫苗(HuN4—F112)對(duì)仔豬的安全性試驗(yàn)1、材料與方法l.l試驗(yàn)用疫苗高致病性豬藍(lán)耳病活疫苗(HuN4—F112)為HuN4株第112代細(xì)胞毒(實(shí)施例2所分離)，病毒滴度l(f75TCID5。/ml。1.2實(shí)驗(yàn)動(dòng)物選取14頭40日齡左右PRRS病毒和抗體均為陰性的健康仔豬。PRRS病毒檢測(cè)方式為RT-PCR，分別使用針對(duì)0RF5和0RF7基因的2對(duì)引物進(jìn)行檢測(cè)?？贵w檢測(cè)采用IDEXX公司生產(chǎn)的PRRSVELISA試劑盒。14頭仔豬隨機(jī)分成3組，第一組6頭為安全性試驗(yàn)組，第二組4頭為空白對(duì)照。空白對(duì)照組與安全性試驗(yàn)組分圈飼養(yǎng)，自由采食。第三組4頭不接種，與安全試驗(yàn)組飼養(yǎng)于同一圈中，作為同居感染組。1.3動(dòng)物試驗(yàn)安全性試驗(yàn)組免疫劑量為2X107。TCID5。/頭，接種方式為頸部肌肉注射。臨床監(jiān)測(cè)方式與指標(biāo)接種疫苗后觀察疫苗注射部位有無紅腫、潰爛等炎癥反應(yīng)，并每日觀察豬只的臨床表現(xiàn)，如采食量、精神狀態(tài)、生長(zhǎng)狀況、耳部眼瞼以及體表有無異常。于每日清晨伺喂后進(jìn)行一次直腸測(cè)溫，直至剖殺。1.4組織病理變化于接種后第7、14和21天各剖殺疫苗組2頭和對(duì)照組1頭，取剖殺動(dòng)物的心、肝、肺、腎、腦、胃、腸、脾、扁桃體、腹股溝淋巴結(jié)、頜下淋巴結(jié)、腸系膜淋巴結(jié)做病理組織學(xué)檢査。2.試驗(yàn)結(jié)果2.1試驗(yàn)豬的臨床表現(xiàn)試驗(yàn)豬接種高致病性豬藍(lán)耳病活疫苗(HuN4—F112)后采食量以及精神狀態(tài)正常，與空白對(duì)照組無明顯差別。接種部位無炎癥反應(yīng)。耳部及體表無淤血或潰瘍斑、眼瞼無腫脹，眼分泌物無明顯增多。空白對(duì)照組無臨床癥狀、無體溫升高。試驗(yàn)豬無發(fā)熱，僅在接種后第2天、第3天有一過性的體溫升高，最高時(shí)達(dá)到40.3。C(體溫超過41。C即為發(fā)熱)，第4天全部回落，直至剖殺體溫始終在40.3°C以下。2.2組織病理變化試驗(yàn)豬分別于接種后第7、14、21天各剖殺2頭，解剖學(xué)檢查結(jié)果顯示高劑量高致病性豬藍(lán)耳病活疫苗(HuN4-F112)接種后仔豬的各實(shí)質(zhì)臟器與淋巴結(jié)均沒有肉眼可見的病變。進(jìn)一步的組織病理學(xué)檢査顯示各種實(shí)質(zhì)臟器(心、肝、脾、肺、腎、小腸、大腦、胃)均沒有明顯的病理?yè)p傷，對(duì)淋巴系統(tǒng)(腹股溝淋巴結(jié)、頜下淋巴結(jié)以及腸系膜淋巴結(jié))也沒有發(fā)現(xiàn)明顯的病理?yè)p傷，僅在腹股溝淋巴結(jié)有少量的嗜酸性粒細(xì)胞浸潤(rùn)，而腸系膜、頜下淋巴結(jié)無病理?yè)p傷。2.3同居感染組病毒含量與抗體的檢測(cè)4頭健康仔豬與肌注高致病性豬藍(lán)耳病活疫苗(HuN4—F112)107°TCID5。組混養(yǎng)，采集3、5、7、10、14、21天的血清，經(jīng)Real-TimePCR方法檢測(cè)血清中的病毒，結(jié)果未發(fā)現(xiàn)有病毒。同時(shí)利用IDEXXELISA試劑盒也未檢測(cè)到抗體。另外，本發(fā)明在其它試驗(yàn)中，累計(jì)以10"TCID5。接種7日齡仔豬10頭，斷奶仔豬75頭，所有被接種動(dòng)物在一個(gè)月內(nèi)的觀察期內(nèi)無體溫升高、無咳嗽和厭食等臨床癥狀，接種部位無炎癥反應(yīng)，以上結(jié)果表明本發(fā)明高致病性豬藍(lán)耳病活疫苗(HuN4—F112)對(duì)各年齡段的豬都是安全的。試驗(yàn)結(jié)果表明，高劑量接種高致病性豬藍(lán)耳病活疫苗(HuN4—F112)后對(duì)仔豬的各種臟器均沒有明顯的損傷；同居感染結(jié)果顯示高致病性豬藍(lán)耳病活疫苗(HuN4—F112)水平傳播能力極低。序列表<110>中國(guó)農(nóng)業(yè)科學(xué)院哈爾濱獸醫(yī)研究所<120〉豬繁殖與呼吸綜合征病毒強(qiáng)毒株、其弱毒疫苗株及其應(yīng)用<130>KLPI0789<160〉2<170>Patentlnversion3.1<210>1<211>15352<212>RNA<213>PorcineReproductiveandRespiratorySyndromeVirus,HuN4strain<400>1augacgU3uagguguuggcucuaugccacggcauuugueiuugucaggagcugugaccauu60ggcacagcccaaaacuugcugcacgggaacacccuccugugacagcccucuucaggggga120uuaggggucugucccu肪caccuugcuuccggaguugcacugcuuuacggucucuccacc180ccuuuaaxxaugucugggauacuugaucggugcacguguaccccc肌ugccagggug醒240guggcggagggccaggucuacugcacacgaugucucagugcacggucucuccuuccucug300肪ucucc肌guuccugagcuuggggugcugggUCUElUUUUauaggcccgaagagccacuc360cgguggacguugccacgugcmiuccccacugucgagugcucccccgccggggccugcugg420cuuucugcgauuuuuccgauugcacgaaugacuaguggaaaccug肌cuuucaaca犯ga480肌ggugcgggucgcagcugaaBUcuacagagccggccaacucaccccuacaguucuaaag540acucuscaaguuumjgaacgggguugucguugguaccccauugucgggcccgucccuggg600gugggcguuuacgccaacucccugcaugugagugacaaaccuuucccggg3gC肌CUC3U660guguuaacca3cmmccgcucccgcagaggccc肌eiccugaggacuuuugcccuuuugag720UgUgCUELUggCUgElCgUCUEtugacauuggucguggcgccgucmigUEiuguggccggagga780aaggucucuugggccccucguggugggaaugaagugaaauuugaaccuguccccaaggag840uugaaguugguugcgaaccgacuccscaccuccuucccgccccaucacgu3guggacaug900uccagguuuaCCUUC3Ug3CcccugggaguggugucuccaugcggguugagU3CC3aC3C960ggcugccuccccgcugacacugucccugaaggaaacugcugguggcgcuuguuugacucg1020cucccaccggaaguucaguaca犯gaaaxmcgccaugcuaacc幼u(yù)uuggCU3UC3犯CC讓aagcauggugucccuggcaaguaccuacagcggaggcugcaaguu犯uggucuucgagca1140gugaccgacacacauggaccimucgucauacaguauuucucuguuaaggagaguuggauc1200cgccaccugaaguugguggaagaacccagccuccccggguuugaggaucuCCUC3g肪UC1260agggu卿gccc肪uacgucaccacuggcuggaa^ggaugagaagauuuuccgguuuggc1320agucauaagugguacggugccggaa柳gagc卿gaaaacacgcucuggugcgacuacu1380miggucgcucaucacgcuucguccgcucauga犯cccggcaggccacgaagcacgsgggu1440gccggcgcuaacaaggcugagcaucucaagcgcuacucuccgccugccgaagggsacugu1500ggUUggC3CUgcauuuccgcc肌cgcc肌ccggaugguga3u11ccaacuuUg3gaCCElCC1560cuuccuga犯gaguaaggccuucagaugacugggccscugacgaggaucuugugaacacc16203ucca肪uccucaggcucccugcggccuuggacagg肌cggcgcuugcgguagcgccaag1680uacgugcuu3aacuggagggugagcmiuggacugucucugugaiicccugggauguccccu1740acuuugcucccccuugaauguguucaggguuguug卿gcauaagggcggucuuguuucc1800ccggmjgcggucg3aanuuccggauuugauccugccugccuugaccgacuggcu肌ggua1860augcacuugccuagcaguaccaucccagccgc隨ggccg犯uuguccgacgacuccimc1920cguccgguuuccccggccgcuacuacguggacuguuucgcaauucuaugcucguuauaga1980ggagg哪ucaucaugacc3ggugugcuuggggaaaaucaucagccuuugUC犯gUU3UU2040gaggauugcugcugccaucaaaccgggcuacuccggaagaggucgcggca2100aagauugaucaguaccuccguggcgcaacaagucuugaggaaugcuuggccaaacuugag2160agaguuuccccgccgagcgcugcggacaccuccuuugauugg咖guugugcuuccuggg2220guugaggcggcgaaucagacaaccg犯caaccucacgucaacucaugcugcacccugguc2280ccucccgugacucaagagccuuugggcaaggscucggucccucugaccgccuucuceicug2340uccaauugcuauuacccugcacaaggugacgagguucaucaccgugagagguu犯aiuucc2400gU3CUCUCUEL3guuggEiagagguuguccuggaagaauaugggcucauguccacuggacuu2460ggcccgcgacccgugcugccgagcgggcucgacgagcuua肪gacc3gauggaggaggeiu2520cugcuaaascuagcc幼cacccaggcgacuucagaaaugauggccugggcggcugagcag2580gucgauuuaaaagcuuggguc肪aagcu3cccgcgguggacaccaccacccccuccacca2640agaguucaaccucgaagaac肪agucugucaaaagcuugccagagggc幼gccugucccu2700gcuccgcgcaggaaggucagauccgauugcggcagcccgguuuugaugggcgacaauguc2760ccu犯cgguucgg肪gaaacugucggugguccccucaauuuuccgacaccauccgagccg2820augacaccuaugagugagcccguacuuaugcccgcgucgcgacguguccccaagcugaug2880acaccuuugagugggucggc8ccaguuccugcaccgcguag犯cugugacaacaacgcug2940acgcaccaggaugagccucuggauuugccugcguccucacagacggaauaugaggcuuuc3000ccccuagcaccaucgcagaacaugggcauccuggaggcgggggggcaagaaguugaggaa3060guccugagug肪aucucggaumiacuaaaugacaccaacccugcaccugugucaucaagc3120agcucccuguca3guguuaagaucacacgcccaaaauacucagcucaagcc肌caucgac3180ucuggcgggccuugcagugggcaucuccsaaaggaaaeiagaagcaugccucagcaucmig3240cgugaggcuugugaugcguccaagcuuggugauccugcuacgcaggaguggcucucucgc3300augugggauagggu卿caugcugacuuggcgcaacacgucugcuuaccaggcg圓cgc3360aucuuaaauggcagguuugaguuucucccaaagaugauucucgagacaccgccgccccac3420ccgugcggguuugugauguuaccucgcacgccugcaccuuccgugagugcagagagugac3480cuc3ccauugguucaguggccaccgaggauguuccacgcauccucgggaa犯u3ggagac3540acugacgagcugcuugaccggggucccucggC3CCCUCC3agggagaaccggucugugac3600C肪CCUgCC3aagauccccggaugucgccgcgggagucuggauagcuccg3660cccgcagauacagguggugucggcucauucacugauuugccgucuuc兆augguguggau3720guggacgggggggggccguuaagaacgguaaaaac肪肪gC3gggaggcucuuagaccaa3780cugagcugccagguuuuuagccucguuucccmjcucccuauuuucuucucac3ccucuuc3840a犯ucugacagugguuauucuccgggugauugggguuuugcagcuuuuacUCU3UUUUgC3900CUCUUUCU3UguuacaguuaCCC3UUCUUCgguuuugcuccccucuuggguguauuuucu3960gggucuucucggcgugugcg肌uggggguuuuuggcugcugguuggcuuuugcuguuggu4020cuguucaagccuguguccgacccagucggc3CUgCUUgUgaguuugacucgcc柳gugu4080aggaacguccuucauucuuuugagcuucucaaaccuugggacccuguccgcagccuuguu4140gugggccccgucggucucggccuugcc叫ucuuggcagguuacugggcggggcacgcuac4200aucuggcacuuuuugcuuaggcuuggcauuguuacagacuguaucuuggcUggagCUU2lU4260gugcuuucucaagguagguguaaaaagugcuggggaucuuguguaag幼cugcuccuaau4320gagaucgccuucaacguguucccuuuuacacgugcgaccaggucgucacucaucgaccug4380ugcgaucgguuuugcgcaccaaaaggcauggaccccmiuuUUCUCgCC3Cuggguggcgu4440gggugcuggaccggccggagucccauugagc犯ccuucugaaaaficcc3ucgcguucgcc4500cagcuggaugagaagaggauuacggcuagaacuguggucacuceigccuuaugaucccaac4560caggccguaaagugcuugcggguauuacaggcggguggggcgaugguggccgaggcaguc4620cc88aagugguca肪guuuccgcuauuccauuccgagcuccuuucuuucccgcuggagug4680aaaguugauccugagugcagaaucgugguugaucccgauacuuuuacuacELgCCCUCCgg4740ucuggcuauuccaccgcgaaccucguccuugguacggggg21CUUUgCCC3gcugaaugga4800ggcaaauuuccaagccuucagggggaggcccac3ccucmiugcugccuug4860cauguugccugcucgauggcguuacacaugcuugcugguguuuaugu犯cugcagugggg4920uccugcgguaccgguaccaacgauccguggugcacuaacccguuugccgucccuggcuac4980ggaccuggcucucuuugcacgucimgauugugcaucucccaacacggccucaccuugccc5040uugacetgcacuuguggcgggauucggccuucaagagauugccuuggucguuuugauuuuu5100gucuccaucggaggcauggcucauagguugaguuguaaggcugacauguugugcaucuua5160cucgcaaucgcuaguuauguuuggguaccucuuaccugguugcuuuguguguuuccuugu5220ugguugcgcugguucucuuugcacccccucELCCgUJCCUgUgguugguguuuuucuugauu5280ucugu犯aimuacccucgggaaucuuggccgugguguuauugguuucucucuggcuuuua5340ggucguimuBcu肪cmiugcuggucucgucacccccuaugacauuc3ucauuacaccagu5400ggcccccgcggugucgccgccuuggccaccgcaccagauggsaccuacuuggcugccguc5460cgccgugcugcgcugacuggucguaccaugcuguucaccccgucucagcucgggucccuc5520cuug鄉(xiāng)gcgCUUUC8g犯Cuc犯aagcccucacugascaccgucaauguggucgggucc5580uccaugggcucuggcggaguguucacuauugacgggaaaaucaagugcgugacugccgca5640cmiguccuuacggguaacucagcuaggguuuccggggucggcuucaauca肪ugcuugac5700uuugauguaacgccauagcugauugcccgaauuggceiELgggguugcuccc5760aaggcccaguucugcgaggaUgggUgg3CUggucgcgccuauuggcugacauccucuggc5820guugaacccgguguuauugggaauggguucgccuucugcuucaccgcguguggcgauucu5880ggauccccagugauuaccgaagccggugagcuugucggcguucacacaggEiuc犯acaaa5940caaggaggaggcauugucacgCgCCCCUCElggucaguuuuguaaugugaagcccaucaag6000cugagcgaguugagugaauucuucgcuggaccuaaggucccgcucggugaugugaaaauu6060ggcagucacauaauuaaagacacaugcgaggugccuucagaucuuugugcccugcuugcu6120gccaaacccgaacuggaaggaggccuuuccEicaguucaacuucuguguguguuuuuccuc6180cuguggagaa卿uggggcaugccuggacgcccuugguugcugugggguuUUUC3UCCUg6240aaugag3uucucccagcuguccugguccggaguguuuucuccuuugggauguuugugcua6300ucuuggcucacaccauggucugcgcaaguccug肌gaucaggcuucugacagcagcccuu6360aacagaaacagauggucucuUggUUUUU3Cagccuuggugcagu犯ccaguuuugucgca6420gaucuugcgguaacucaagggcauccguimcaggugguaaugaacuu幼gcaccuaugcc6480uuccugccccggaugaugguugugaccucgccagucccagugaucgcgugugguguugug6540caccuccuugccau肪uuuuguacuuguuuaaguaccgcugccuucacaauguccuuguu6600ggcgaugggguguucucuucggcuuucuucuugcgauacuuugccg鄉(xiāng)g6660g卿gggugucgcaauccugcgggaugsgucaugagucgc卿cuggugcccucgccaug6720agacucacugacgaggacuuggauuuccuuacgaaauggacugauuuuaagugcuuuguu6780ucugcguccaacaugaggaaugcagcgggccaauuuaucgaggcugcuuaugcaaaagca6840cuaag犯uugaacuugcucaguugguacagguugaua鄉(xiāng)uccgaggcaccauggccaaa6900cucgaggcuuuugccgauaccguggcaccccaacucucgcccggugacauuguuguugcc6960cuuggccacacgccuguuggcagcmicuucgaccuaaagguugguagcaccaagcauacu7020cuccaagccauugagacuagaguccuugccgggucc犯肪ugacuguggcgcgugucguu7080gacccaacccccgcacccccacccguaccugugcccaucccucucccaccgaaaguucug7140g柳acggucccaaugccuggggggaugaggaccguuugaacaagaagaagsggcgcagg7200gtuggaagccgucggcauuuuugucauggacggg肪aaaguEiccagaaauuuugggacaag7260aauuccggugauguguuuuaugaggagguccauauuagcacagacgagugggagugccuu7320柳acuggcg3CCCUgUCg3CU叫g(shù)3UCCUuucagugugggC3U3UC3CC7380auugaagaua3gguuuacaeiugucuucaccUCCCC2LUCUgguaggagauucuuggucccc7440gccaaccccgagaauagaagagcucagugggaagccgccaagcuuuccguggagcaagcc7500cuugguaugaugaacgucgacggcgaacugacugccaaagaacuggagaaacugaagaga7560auaauugac3幼cuccagggccugacuaaggagcaguguuuaaacugcuagccgccagcg7620gcuugacccgcuguggucgcggcggcuuaguuguimcugagacagcggua肌aauaguca7680aauuucacaaccggaccuucacccuaggaccugugaacuuaaaaguggccagugagguug7740cgcgguugagmiccgguugccaggccgguugaugguggug7800uugugcuccugcgcucugcaguuccuucgcuuauagaugucuugaucuccggcgcugaug7860caucuccu幼guuacucgccCgCCElCgggCcgggaaacacugggauugauggcacgcuuu7920gggauuuugaggccgaggcuacuaaagaggaaguugcacucagugcgcaaauammcagg7980cuugugauauuaggcgcggcgacgcgccugaaauuggucucccuuau幼guuguacccug8040uuaggggcaacccugagcggguaaaaggaguuuuacag幼u(yù)acaagguuuggagacauac8100cuuscaaaaccccuagugacacuggaagcccggugcacgcggcugccugccucacgccua8160augcu3cuccggugac卿ugggcgcuccgucuuggcuac幼ccaugcccucuggcuuug8220aguuguaugugccgaccauuccagcguccguccuugauimucuugauucuaggccugacu8280guu幼cagagcacgguugugaggaugcugcauu卿3gaccucucc肪gu8340augauuuguccacccaaggcuuuguuuugccuggaguucuucgccucgugcggaaguacc8400uguucgcccacguggguaagugcccgcccguucaucggccuuccacuuscccugcu肪ga8460auucuauggcuggaauaaauggg33C3ggUuuccaaccaaggacauucagagcgucccug8520aasucgacguucugugcgcacaggcugugcg3g幼aacuggcaaacuguuaccccuugim8580cccuceiagaaacaguacugugggaagaagaagacuaggacaauacuuggcaccaauaacu8640uuauugcguuggccceiucgggcagcguugagugguguuacccagggcuucaugasaaaag8700cguucaacucgcccaucgcccucgggaa犯acaamiuu犯ggagcuacaagccccggucc8760U3ggC3ggUgccuugaagcugaucuugcguccugcgaucg3UCC3C3CCUgcaauugucc8820gcugguuugccgccaaucuucuuumig犯cucgccugugcugaggagcaucuaccgucgu8880acgugcugaacugcugccacgacuuacuggucacgcaguccggcgcggugacuaagagag8940guggccugucgucuggcgacccg3uuaccucugugucaaaC3CC3UUU3C站cuuaguga9000uauaugceic3gcacauggugcuceiguuscuuca犯aguggucacccuc3uggccuucugu9060UUCUgCg3g3ccagcua幼guuugaggacaugcucaagguuc幼ccccugaucgucuauu9120cggacgaccuugugcuguaugccgagucucCCUCC3UgCCaaacuaccacugguggguug9180aacaucugaaucuumigcuggguuuccagacggacccaaagaagac肪cc3UC3C6lgaCU9240caccaucauuccuagguugcaggauaauaaaugggcgccagcuagucccuaaccgugaca9300ggauccucgcggcccucgccimcc3uaugaaggc肪gu犯uguuucug犯imcuacgccu9360cggcggcugcgacetgcugugCUUgUUU3g3gU3iigmiccugaaugguuug9420aagagcucgugguugggauagcgcagugcgcccgcaaggacggcuacagcuuuccuggcc9480caccggucuucuuguccauguggg犯犯acucagguccaaucaugaggggaagaagucca9540gaaugugcggguacugcggggccccggcuccguacgccacugccuguggucucgaugucu9600guguuuaccacacccacuucC3CC3gC3UUguccuguuauaaucugguguggccacccgg9660cggguucugguucuiiguagug3gUgCg咖CCCCCCU3ggaaaaggcacaagcccucuag9720augagguguuag犯caaguuccguacaagccuccgcggacugugaucmigcauguggagc9780agggucucacCCCUCUUg3Cccagguagauaccagacucgccgcggauuagucuccguua9840ggcguggcaucagggga肪ugaagucgaccuaccagacggugmiuacgcuaguaccgccu9900ugcucccimcuuguaaagagaucaacmiggucgcugucgccucu犯cguguugcgcagca9960ggUUU3UC3Ucggcccacccggugcugggaa朋cac加uggcuucuuca3c肪guccagg10020auggugaugucauuusic3cgcc幼cucaccagaccaugcucgacaugmjuagggcuuugg10080ggacgugccgguucaacguuccagcagguac肪cgcugcaauucccugcccccucccgua10140ccggccceiuggguucgcaucuuggccggcgguugguguccuggcaagaacuccuuccugg10200aug卿cggcguauugcaaucaccuugaugucuugaggcuucucaguaaa翻Ocuugccuaggggacuucaaac肌cuccacccuguggguuuugacucccauugcuaugimu10320UUg3C3UC3Ugccucagaccccaucuggagguucgggcagaauaucugug10380augcc￡iuucaaccagauuac3gggacaaacuuauguccauggucaacacgacccguguga10440ccuacguggaaaaaccugucagguaugggcagguccucacccccuaccacagggaccgag10500aggacggcgcceiuu8cuaucgacuccagucaaggcgccacauuugaugugguuacacugc10560過uuu3cccacu犯agauucacucaacaggcei犯gagcucuuguugcuaucaccagggcaa10620gacaugcuaucuucguguaugacccacacaggcaauugcagagcauguuugaucuccccg10680cgaaaggcacacccgucaaccucgcagugcaccgugacgaacagcugaucguauu3ggica10740gaaacaacagagaaeiucacgguugcucaggcucuaggcaauggag肌a犯uucagggcca10800cagauaagcgcguuguagauucucuccgcgcuauuugcgcagaccuggaagggucgagcu10860ccccgcuc.cccaaggucgcgcauaacuugggauucuauuucucaccugauuugacucagu10920UUgCU3犯CUcccggcagaacuugcaccccacuggcccguggugacaacccagaacaaug109808卿gliggCC3gmicggcugguagccagccuucgcccuauccaua犯uauagccgcgcgu11040gc簡(jiǎn)ggugccggcuauauggugggccccucgguguuuuuaggcaccccugggguugugu11100cmiacuaucucaca肌auuuguu柳ggcgaggcucaamigcuuccggagaceigucuuca11160gcacuggccgaauugagguagauugccgagaguaucuugaug肌cgggagcgagaaguug11220cugagucccucccacaugccuucaucggcg3UgUC3犯gguaccaccguugggggauguc11280aucacguu3cCUCC3犯U3Ccuuccgcgcuuccuuccc幼ggaaucaguugcgguggucg11340ggguuucgeigccccgggaaagccgcgaaagcaguuugcacauugacggauguguaccucc11400cag3ccuugaagcguaccuccacccagagacccaguccaggugcuggaaagugmiguugg11460acuuu貼ggagguucgacugaugguauggaaagacaagacggccuauuuucaacuugaag11520gccgucaiiuuuaccugguauc幼cuugc肪gcuacgccuc3U3C3UCCgaguucctiguu過115803UUCU3CUgUguacuuggaccccugcaugggcccugcucuuugcaacagaaggguugucg11640gguccacccauuggggagcugaccucgcagucaccccuuaugauuacggugccaa幼u(yù)u311700uucugucimgugcauaccmiggugaaaugccuccagguimc肪aauucuggcgugcgcgg11760aguucucgcuugsugauccaguaagguacaaacacaccuggggauuugaaucggauacag11820cguaucugimCg3gUUU3CUggaaauggugaggacugggaggauuacaaugaugcguuuc11880gggcgcgccag犯agggaaaauuuauaaagcuaaugccacCEigcaugagguuucauuuuc11940ccccgggcccugucauugaacc幼cuuuaggccugaauugaamigsiaauggggucuaugc12000犯agccucuuugacaaaauuggccaacuuuuuguggaugcuuucacggaauuucuggugu12060ccauuguugauaucmjcauauuuuuggccauuuuguuuggcuucac肌ucgccgguuggc12120ugguggucuuaugcaucagacugguuugcuccgcgguacuccgugcgcgcucuaccguuc12180acccugagcaauuacagaagaucuuaugaggccuuucuuuCUCELgUgUCELgguggacauu12240cccaccuggggcgucaaacacccuuugggggugcuuuggcaccauaaggugucaacccug12300acugaugaaauggugucgcgucgaauguaccgcaucauggaaaaagcagggcaggcugcc12360uggaaacagguggugagcgaggcuacauugucucgcauuagugguuuggaugugguggcu12420cacuuucaacaucuugccgcuauugaagccgagacuuguaaauauuuggcuucccggcua12480cccaugcugcacaaccugcgcuugacagggucaaaugu幼ccauagugua12540uuggaucagguguuugccauuuucccaeiccccugguucccggccaaagcuucacgauuuu12600cagcaauggcu肌usgcuguacauuccucc3USIUUUUCCUccguugcagcuucuuguacu12660cuuuuuguugugcugugguugcgaauuccaaugcuacguucuguuuuugguuuccgcugg12720uuaggggcaacuuuucuuuugaacucauggugamjuacacgguaugcccgcuuugcccaa12780cccggcaggcagccgcugagauccuugagcccggcaagucuuuuuggugcaggauagggc12840augaccgauguagugagaacgaucaugacgaacuaggguucaugguuccgccuggccucu12900ccagcgaaggccacuugaccaguguuuacgccugguuggcguuccuguccuucagcimca12960cggcccaguuccaucccgagauauuugggauagggaaugugagucaaguuuauguugaca匿Oucaagcaccaauucaucugcgcuguucacgacggggauaacgccaccuugccucgcceiug13080ac肪uauuucagccguauuucagaccuacuaccaac3cc3ggucgacggcggcaauuggu13140uucaccuggaauggcugcguccuuucuuuuccucuugguugguuuu犯auguuucguggu13200uucucaggcguucgccugcaagccauguuucaguucgagucuuucggacauc肌aaccaEi13260caccaccgcagcaucagacuucguuguccuccaggacaucagcugccuuaggcauggcga13320cucguccucuccgacgauucgc幼aauuucucagugccgcacggcgauagggacgcccgu13380guacauc3cc3ucacugccaaugucacagauga貼auuaucuacauucuucugaucuccu13440caugcuuucuucuugccuuuucimugcuuccgagaugagugaa卿ggauucaaaguggu13500guuuggc犯ugugucaggcaucguggcugugugcgucaacuuua>ccagcuacguccaaca13560cguc幼ggaguuuacccaacgcuccuuaguggucgaucaugugcgacugcuuc肌uucau13620g3C3CCUg3g3CC肌g3ggUgggcaaccguuuuagccuguCUUUUUgCC3uccimcuggc13680aauuugaauguucaaguauguugggg卿ugcuugaccgcgugcuguugcucgcgauugc13740uuii叫uuguggugimucgugccguucimucuugcugugcucguc犯cgcc13800acagcucucauauucaguugauuuauaacuuaacgcuaugugagcugaauggcacagauu13860ggcuggcaca犯aauuugacugggcaguggagscuuuuguceiucuuccccguguugacuc139203cauuguuuccuauggggcacucaccaccagCC3UUUCCUugacacaguuggucuggcca13980cuguguccaccgccggauauuaucacgggcgguaugucuugaguagcauuuacgcagucu14040gugcucuggcugcgcugauuugcuuugucauuaggcuugcgaagaacugcauguccuggc14100gcuacucuuguaccagEiuauaccaacuuccuucuggacacuaagggc柳cucuaucguu14160ggcggucgcccgucauuguggagaaagggggua郎guugaggucgaaggucaccugaucg14220accuc助gsgaguugugcuugaugguuccgcggcaaccccuuuaaccagaguuucagcgg14280aac幼u(yù)ggggucgucucuagacgacuucugcaaugauagcacagcuccacagaaggugcu14340uuuggcguuuuccauuaccuacacgccEigugaugeiuauaugcucu肪agguaagucgcgg14400ccgacugcuagggcuucugcaccuuuugaucuuucugaauugugcuuuuaccuucgggua14460caugacauucgugcacuuugagagpacaaaU3gggUCgCgCUCELCU肌gggagcaguagu14520ugcacuucuuuggggaguguacucagcc3Uagaaaccugg犯auucaucaccuccagaug14580ccguuugugcuugcuaggccgcaag咖auucuggccccugcccaccacgucgaaagugc14640cgcgggcuuucauccgauugcggcaaaugauaacc3cgceiuuugucguccggcgucccgg14700cuccacuacggucaacggcacauuggugcccggguuga幼agccucguguuggguggcag14760aaaagcuguu卿c鄉(xiāng)gaguggusaaccuuguu幼auaugccaaau肌caacggcaagc14820agcaa幼gaaaaagaaggggaauggcc3gccagucamicagcugugccaaaugcugggua14880agaucaucgcccaacaaB3Ccaguccagaggc肪gggaccgggg幼ga幼aauaggaaga14940aaaacccggagaagccccauUUCCCUCU3gcgacugaagaugacgucaggcauceicuuua15000ccccuagugagcggc貼uugugucugucgucgauccagacugccuuc肪uc鄉(xiāng)gcgcug15060g肪cuugugcccugucagauucagggaggauaaguuacacuguggaguuuaguuugccga15120cgc肪ceiuacugugcgucugauccgcgccacagcaucacccucagcaugeiugggcuggca15180UUCUUUggC3CCUC3gUgUUagaauugggag幼u(yù)guguggug肪uggcacugauugacac15240ugugccucuaagucaccuauucaauuagggcgaccgugugggggua卿uuuaauuggcg15300agaaccaugcggccguaami肪犯犯aa肪幼15352<210〉2<211>15323<212>腿<213>PorcineReproductiveandRespiratorySyndromeVirus,HuN4-Fl12strain<400>2augacguauagguguuggcucuaugccacggcauimguauugucaggagcugugaccmau60ggcacagcccaa助cuugcugcacgggaacacccuccugugacagcccucuucaggggga120uuaggggucugucccuaacaccuugcuuccggaguugceicugcuuuacggUCUCUCC3CC180CCUUU3aCC3ugucugggmiacuugaucggugcacguguacccccaaugccaggguguuu240guggcggagggccaggucuacugcacacgaugucucagugcacggucucuccuuccucug300aaucucc肪guuccugagcuuggggugcugggucumiuuuauaggcccgaagagccacuc360cgguggacguugccacgugcauuccccEicugucgagugcucccccgccggggccugcugg420cuuucugcgauuuuuccgauugceicg貼ugacuaguggaasccugaacuu480auggugcgggucgcagcugaaaucuacagagccggccaacucaccccuacaguucua肪g540acucueicaaguuuaugaacgggguugucguuggiiaccccauugucgggcccgucccuggg600gugggcguuu3CgCCa3CUCccugcaugugagugacaaaccuuucccgggagc肌cucau660guguu幼ccaacuugccgcucccgcagaggcccaaaccugaggacuuuugcccuuuugag720ugugcuauggcug3cgucueiugac3uuggucguggcgccgucauguauguggccggaggg780aBggucuuuugggccccucgugguggg幼u(yù)gaagugaaauuugaaccuguccccaaggag840u卿aguugguugcgaaccgacuccaceiccuccuucccgccccmicacguaguggacaug900ucc明guuuaccuucauggiccccugggaguggugucuccaugcggguugaguaccaacac960ggcugccuccccgcugacacugucccugaagga犯cugcugguggcgcuuguu卿cucg1020cucccaccggaaguucaguaC3肪gasauucgccaugcuaaccaauuuggcuaucaaacc1080aagcauggugucccuggc肪gUaCCUELCElgcggaggcugcaaguuaauggucuucgagca1140gugaccgacacacauggacccaguauuucuCUgUU幼gg3gaguuggauc1200cgccaccugaaguuggcggaagaacccagccuccccggguuugaggaucuccucaggauc1260aggguugagcccfiauacgucaccacuggcuggaaaggaugagaagauuuuccgguuuggc1320agucaimagugguacggugccggaaagagagcgiaggaaaacacgcucuggugcgacuacu1380auggucgcucgucacgcuucguccgcucaugaaacccggcaggccacgaagcacgsgggu1440gccggcgcuaacaaggcugagcaucucaagcgcuacucuccgccugccgaagggaacugu1500gguuggcacugcauuuccgcC3UCgCCgl3Ccggauggugaauucc幼cuuug卿ccacc1560cuuccug犯agagu犯ggccUUC3g3Ug3Cugggccacugacgaggmicuugugaacauc1620aucca肪uccucaggcucccugcggccuuggeiceLgg￡iacggcgcuugcggimgcgccaag1680uacgugcuuaaacuggagggugagcauuggacugucucugugaucccugggauguccccu17403CUUUgCUCCcccuugaauguguucaggguuguugugagcmiaagggcggucuuguuucc1800ccggaugcggucga犯uuuccggauuug肌ccugccugccuugaccgacuggcu幼ggua1860augcacuugccuagcaguaccaucccagccgcucuggccgaauuguccgacgacuccuac1920cguccgguuuccccggccgcuacuacguggacuguuucgc幼u(yù)ucueiugcucguuauaga1980ggaggagaucaucaugacc3ggugugcuuggggaaaaucaucagccuuugucasguuauu2040gaggauugcugcugccaucagaau犯幼ccaaccgggcuacuccggaagaggucgcggca2100犯gauugaucaguaccuccguggcgc幼caagucuugaggaaugcuuggccaaacuugag2160agaguuuccccgccgagcgcugcggacaccuccuuugauuggaauguugugcuuccuggg2220guugaggcggcgaaucagacaaccgeiacaaccucgicgucaacucaugcugcacccugguc2280ccucccgugacucaagagcc畫gggcaaggacucggucccucugaccgccuucucacug2340ucc幼u(yù)ugcuauuEicccugcacaaggugacgagguucaucaccgugagagguu幼auucc2400guacucucuaaguugg卿agguuguccuggaagaauaugggcucauguccacuggacuu2460ggcccgcgacccgugcugccgagcgggcucgacgagcuuaaagaccagauggaggaggau2520cugcua肌acuagccaacacccaggcgacuucagaaaugauggccugggcggcugagcag2580gucaeiuuima卿cuugggucaei犯gcuacccgcgguggacaccaccacccccuccacca2640agaguucaaccucgaagaacaaagucuguc幼肪gcuugccagagggcaagccugucccu2700gcuccgcgcagg幼ggucagauccgauugcggcagcccgguuuugaugggcgacaauguc2760ccu肪cgguucgg犯gaaacugucggugguccccuc犯uuuuccgacaccauccgagccg28203ugacaccua卿gugagcccguacuuaugcccgcgucgcgacgugcccccaagcugaug2880acaccuuugagugggucggcaccaguuccugcaccgcguagaacugugacaacaacgcug2940acgcaccaggaugagccucuggauuugccugcguccucacagacggaauaugaggcuuuc3000ccccimgcacC3ucgceigaacaugggcauccuggaggcggaguugagg幼3060guccugagugaeiaucucggauauacimaaugacaccaacccugcaccugugucaucaagc3120agcccccuguc犯guguuaagaucacacgcccEL犯mmcucagcucaagccaucaucgac3180ucuggcgggccuugcagugggcaucuccaaaagg肪aaagaagcaugccucagcaucaug3240cg卿ggcuugugaugcguccaagcuuggugauccugcuacgcaggaguggcucucucgc3300augugggauaggguugacaugcugacuuggcgcaacacgucugcuuaccsiggcguuucgc3360aucuuaaguggcagguuugaguuucucccaaag3ugauucucgagacaccgccgccccac3420ccgugcggguuugugauguuaccucgcacgccugcaccuuccgugagugcagagagugac3480CUC3CC3UUgguucaguggcC3CCgElgg3Uguuccacgcauccucgggaaaauaggagac3540acugacgagcugcuugaccggggucccucggcacccuccaaggg柳accggucagugac3600C肪CCUgCC3aagauccccggaugucgccgcgggag固gacgagagcaugauagcuccg3660cccgcagauacagguggugucggcucauucacugauuugccgucuucagaugguguggau3720guggacgggggggggccguuaagaacgguacggggaggcuCUU3g3CC肪3780cugagcugccagguuuuuagccucguuucccaucucccuauuuucuucucacaccucuuc3840肌aucugacgigugguuauucuccgggugauugggg薩ugcagcuuuuacucuauuuugc3900CUCUUUCLI3UCCC3UUCUUCgguuuugcuccccucuugggugumiuuucu3960gggucuucucggcgugugcg幼u(yù)ggggguuuuuggcugcugguuggcuuuugcuguuggu4020cuguuc幼gccuguguccgacccagucggcacugcuugugaguuugacucgccagagugu4080aggaacgimcUUC3UUCUUUugagcuucucaaaccuugggacccuguccgcagccuuguu4140gugggccccgucggucucggccuugccauucuuggcagguimcugggcggggcacgcuau4200aucuggcacuuuuugcuuaggcuuggcauuguuacagacuguaucuuggcuggagcuimu4260gugcuuucuc肪ggimggugU犯3犯gUgCuggggaucuuguguaag犯cugcuccimau4320gagaucgccuucaacguguucccuuuuacacgugcgaccaggucgucacucaucgaccug4380ugcgaucgguuuugcgcaccaaaaggcauggaccccauuuuucucgccscuggguggcgu4440gggugcuggaccggccggagucccauugagc肪ccuucugcgcguucgcc4500cagcuggmigagaagaggmjuacggcuagaacuguggucgcucagccuuaugaucccaac4560caggccguaaagugcuugcggguauuacaggcggguggggcgaugguggccgaggcaguc4620cc肪膽guggucaaaguuuccgcuauuccauuccgagcuccuuucuuucccgcuggagug4680aaaguugauccugagugcagaaucgugguugaucccgauacuuuuacuacagcccuccgg4740ucuggcuauuccaccgcgaaccucguccuugguacgggggacuuugcccagcugaaugga4800cuei朋gaucaggca幼u(yù)uucc膽gccuucagggggaggcccacaccuc3uugcugccuug4860cauguugccugcucgauggcgUU3C3C3Ugcuugcugguguuuauguaacugcagugggg4920uccugcgguaccggcaccaacgauccguggugcacuaacccguuugccgucccuggcuac4980ggaccuggcucucuuugcacgucuagauugugcaucucccaacacggccucaccuugccc5040uugacagcacuuguggcgggauucggccuucaagagauugccuuggucguuuugauuuuu5100gucuccaucggaggcaugguucau明guugaguuguaaggcugacauguugugcaucuua5160cucgcaaucgcuaguuauguuuggguaccucuimccugguugcuuuguguguuuccuugu5220ugguugcgcugguucucuuugcacccccucaccauccugugguugguguuuuucuugauu5280ucugu犯auauacccucgggaaucuuggccgugguguuauugguuucucucuggcuuuua5340ggucguuauacuaacauugcuggucucgucacccccimug3CaUUC￡lUC3uimcgiccagu5400gguccccgcggugucgccgccuuggccaccgcaccagaugg肌ccuacuuggcugccguc5460cgccgugcugcgcugacuggucguaccaugCUgUUCELCCCcgucucagcucgggucccuc5520cuugagggcgcuuuceigaacUC肪36lgCCCucacugaeicaccgucaauguggucgggucc5580uccaugggcucuggcggaguguucacumiugacgggaa肪ucaagugcgugacugccgca5640C3UgUCCUU3cggguaacucagcuaggguuucuggggucggcuucaauc3肪ugcuugac5700uuugaugu膽犯ggggacuucgccauagcugauugcccgaauuggc卿ggguugcuccc5760aaggcccaguucugcgaggauggguggacuggucgcgccuauuggcugacauccucuggc5820guugaacccgguguuauugggaauggguucgccuucugcuucaccgcguguggcgauucu5880ggauccccagugauuaccgaagccggugagcuugucggcguucacacagg5940caaggaggaggcauugucacgcgccccucaggccaguuuuguetaugugaagcccaucaag6000cugagcgaguugagugaauucuucgcuggaccuaaggucccgcucggugaugugaa幼u(yù)u6060ggcagucacau肪uu肪3gacacaugcgaggugccuucagaucuuugugcccugcuugcu6120guca犯cccg犯cugg肪ggaggccuuuccuucuguguguguuuuuccuc6180cuguggcg肪ugauggggcaugccuggacgcccuugguugcugugggguuuuucauccug6240aaugsgauucucccagcuguccugguccgg3gUgUUUUCUccuuugggauguuugugcua6300ucuuggcucacaccauggucugcacaaguccugaugaucaggcuucugacagcagcccuu6360肪caga幼cag￡LUggUCUCUugguuuuuacagccuuggugca_guaaccaguuuugucgca6420gaucuugcgguaacucaagggcmiccguuacaggugguaaug肌cuuaagcaccumigcc6480uuccugccccggaugaugguugugaccucgccagucccag卿ucgcgugugguguugug6540caccuccuugccau犯uuuuguacuuguuuaaguaccgcugccuucac幼u(yù)guccuuguu6600ggcgaugggguguucucuucggcuuucuucuugcgauacuuugccg鄉(xiāng)gaaaguug鄉(xiāng)6660gaagggg卿cgcaauccugcgggaugagucaugagucgcugacuggugcccucgccaug6720agacucacug3Cgaggacuugg3UUUCCUUacga幼u(yù)ggacugauuuua3gugcuuuguu6780ucugcguccaatcsLUgaggeLEicaaimuaucgaggcugcmiaugcaaaagca6840cua卿guug助CUUgCUC3guuggimcagguugacaaggUCCg3ggC3Cc加ggccaaa6900cucgaggcuuuugccgauaccguggcacccc幼cucucgcccggugacauugmiguugcc6960cuuggccacacgccuguuggceigcmicuuc印ccu站agguugguagc犯csagcauacu7020cuccaagccauugagacuagaguccuugccggguccaaaaugacuguggcgcgugucguu7080gaccc犯cccccgcacccccacccguaccugugcccaucccucucccaccgaaaguucug7140gag犯cggucccaaugccuggggggaugaggaccguuugaacaagaagsagaggcgcagg7200augg肪gccgucggcauuuuugucauggacgggaaa卿uacc站a肪uuuugggacaag7260aauuccggugaugugmnmaugaggagguccauauuagcsc卿cgagugggagugccuu7320卿acuggcgacccugucgacuuugmjccug柳cagggauucagugugggc3U叫cacc7380auugaagauaagguuuacaaugucuucaccuccccaiicuggCELgg3gaUUcuuggucccc7440gccaaccccgag肪uag犯g縛cucaguggg犯gccgccaagcuuuccguggagc幼gcc7500cuugguaugaug犯cgucgacggcgaacugacugcc犯agaacuggagaaacug幼aaga7560auaauugacaaacuccaaggccugacuaaggagcag卿uua肪cugcuagccgccagcg7620gcuugacccgcuguggucgcggcggcuuaguuguuacugaggcagcggua朋aauaguca7680犯uuucac幼ccggaccuucacccuaggaccugugaacuu幼犯guggccagugagguug7740agcu幼aagacgcggu卿gcacaacc犯cauccgguugccagaccgguugaugguggug7800uugugcuccugcgcucugcaguuccuucgcuumiagaugucuugaucuccggcgcugaug7860ceiucuccuaaguuacucgcc.cgccacgggccggg犯ac3cugggsu卿uggcacgcuuu7920gggauuuugaggccgsggcuacuaaagagg肪guugcacucagugugcaaauaauacagg7980cuugugauauuaggcgcggcgacgcgccixgeiELauuggucucccuuauaaguuguacccug8040gggcaacccugagcggguaaaagguuuuacaguacaagguuuggagac3imc8100CUUEICCcccuagugacacuggaagcccggugcacgcggcugccugccuc8cgccim8160gCU3CUCCggcugaugggcgcuccgucuuggcuacccaugcccucuggcuuug8220aguugumigugccgaccauuccagcgcccguccuug3uuaucuugucuaggccugacu8280gcccuasacaguuaacagagC3CggUUgUgaggaugcugcgiuuaagagaccucuccgu8340giugauuuguccacccaaggcuuuguuuugccuggaguucuucgccucgugcggaaguacc8400uguucgcccacguggguaagugcccgcccguucmicggccuuccacuuacccugcuga8460auucuauggcuggaauaaaugggaacagguuuccaaccaaggacauucagagcgucccug8520aaaucgacguucugugcgcacaggcugugcgagaaaacuggcacuguuaccccuugua8580cccucgaaacagimcugugggaaga3gEicuaggacaauacuuggc8640UU3UUgCgUUggcccaucgggcagcgugugguguuacccagggcuucaugaaaaaag,cguucaacucgcccaucgcccucgggaaaaacauuuaaggagcuacaagccccggucc8760uaggcaggugccuugaagcugaucuugcguccugcgaucgauccacaccugcaauugucc8820gcugguuugccgccamicuucuuumigcucgccugugcugaggagcmicuaccgucgu8880acgugcugaacugcugccacgacuuacuggucacgcaguccggcgcggugacuaagsgag8940guggccugucgucuggcgacccgauuacccuguguceLcaccavuuiacagcuuaguga9000gcacauggugcucaguuacuucaaguggucacccucauggccuucugu9060uucugcaagaCCCU3guuugaggacaugcucaagguucccccugcucgucuauu9120cggacgaccuuguguuguaugccgagucucccuccaugccacuacc3cugguggguug9180aacmicugaaUCUU3UgCUgggUUUCC3g3cggacccaaagaagacaacc3UCElC8g3CU9240caccaucauuccuagguugeaggauaauaa&ugggcgcca_gcuagucccuaaccgugaca9300ggauccucgcggcccuugccU2tccemaug3aggcaaguaauguuucugaaU8cuacgccu9360cggcggcugcuacucauggacagcugugcuuguuuagaguaugauccugaaugguuug'9420aagagcucgugguugggaimgcgcagugcgcccgcaaggacggcueicagcuuuccuggcc9480ceiccguucuucuuguccaugugggaaaaacucagguccaaucaugaggggaagaagucca9540gugugcggguacugcggggccccggcuccguacgccacugccuguggucucgaugucu9600guguuuaccaC3CCC3CUUCC3cccauuguccuguuau幼u(yù)cugguguggccacccgg9660cggguucugguucuuguagugagugcgaacccccccuaggaagaggcacaagcccucuag9720augagguguuaggacaaguuccguacaagccuccgcggacugugeiucaugcmiguggagc9780agggucucaccccucuugacccagguagauaccagacucgccgcggauuagucuccguua9840ggcguggcaucagggg犯augaagucgaccuaccagscggugauuacgcuaguaccgccu9900UgCUCCCUELCuuguaaagagaucaacauggucgcugucgccucuaacguguugcgcagca9960gguuuaucaucggcccacccggugcugggaaaac3cgicuggcuucuuc犯caaguccagg0auggugauguC3uuueicacgcc幼cucaccagaccaugcucgacaugauuagggcgg10080ggacgugccgguucaacguuccagcagguacaacgcugcaELUUCCCUgCCcccucccgua10140ccggcccauggguucgcmicuuggccggcgguugguguccuggcaagaacuccuuccugg10200augaagcggcguauugcaaucaccuugaugucuugaggcuucucagu幼a3C33CUCUC310260cuugccuaggggacuuca肌caacuccacccuguggguuuugacucccmjugcuauguau10320uugacaucaugccucagaccc犯uua犯gaccaucuggagguucgggcagaauaucugug10380augccauuca3ccagauuacagggacaaacuumiguccauggucsacacgacccguguga10440ccuacguggaaaaaccugucagguaugggcaaguccucacccccuEiccacagggaccgag10500aggacggcgccauuacu3ucgacuccagucaaggcgccacauuug肌gugguuacacugc10560miuimcccacuaa叫suucacucaacaggcaaagagcucuuguugcuaucaccagggcaa10620gacaugcuaucuucgugumigacccacacaggcaauugcagagcauguuugaucuccccg10680cgaaaggcac3CCCgUC犯Ccuugcagugcaccgugacgaacagcugaucguauuagaca10740gaaacaacagagaaaucacgguugcucaggcucuaggcaauggagauaaauucagggcca10800cagau肪gcgcguuguagmjucucuccgcgcuauuugcgcagaccuggaagggucgagcu10860ccccgcuccccaaggucgcgcauaacuugggauucuauuucucaccugauuugacucagu10920uugcuaaEicucccggcagaacuugcacccc3CUggCCCgUggugacaacccagaacaaug10980aaagguggccagaucggcugguagccagccuucgcccuauccaua肪umiagccgcgcgul畫gcauuggugccggcu肌auggugggccccucgguguuuuuaggcaccccugggguugugu11100cauEicuaucucacaaaauiiuguuagaggcgaggcuc幼augcuuccggagacagucuuca11160gcacuggccg貼uugagguagauugccgagagumicuugaugmicggg￡igcgagaaguug11220cugagucccucccacaugccuucaucggcgmiguca肪gguaccaccguugggggauguc112803UC3CgUU3CcuuccgcgcuUCCUUCCCEIEIggaaucaguugcgguggucg11340ggguuucgagccccgggaaagccgcgaaagcaguuugcacauugacggauguguaccucc11400cagaccuugaagcguaccucuacccagagacccaguccaggugcuggaaagugauguuggl函acuuu肪ggagguucgacugmigguaugga肌gacaagacggCCU2lUUUUcaacuug肌g11520gccgucauuuuaccugguaucaacuugcaagcuacgccuca_uacauccgaguuccuguua11580auucimcuguguacuuggaccccugcaugggcccugcucuuugcaacagaaggguugucg11640gguccacccauuggggagcugaccucgcaguceiccccuimugauuacggugccaaaauua11700uucugucuagugcEiuaccaugguga幼u(yù)gccuccagguuacaaaauucuggcgugcgcgg11760aguucucgcuugaugacccagu犯ggimcaaacacaccuggggauuugaaucggauacag11820cguaucuguacgaguuuacuggaaauggugaggacugggaggauuacaaugaugeguuuc11880gggcgcgccaga肪gg卿aauuuaua犯gcu犯ugccaucagcaugeigguuucauuuuc11940ccccgggcccugucauug肪ccaacuuuaggccugamjugaaaugaaauggggucuaugc12000犯ElgCCUCUUu幼caaaauuggccaacuuuuuguggaugcuuucacggaauuucuggugu12060ccauuguugauaucaucanauuuuuggccauuuuguuuggcuucBcaaucgccgguuggc12120ugguggucuucugcaucagacugguuugcuccgcgguacuccgugcgcgcucuaccguuc12180acccugagcaauuacagaagaucuuaugaggccuuucuuucucagugucagguggacauu12240cccaccuggggcgucaaacacccuuugggggugcuuuggcaccmiaagguguceiacccug12300auugaugaaauggugucgcgucgaauguaccgcgucauggauaaagcagggcaggcugcc12360uggaaacagguggugagcgaggcuac3imgucucgcauimgugguuuggaugugguggcu12420cacuuucaacaucuugccgcuauugaagccgagacuugUEtaauEiuuuggcuucccggcua12480cccaugcugcacaaccugcgcuugacaggggcaaauguaaccauaguguau幼u(yù)aguacu12540uuggaucagguguuugccauuuucccaaccCCUggUUCCGggccaaagcuucacgmjuuu12600cagcaauggcuaauagcuguacauuccuccausuuuuccuccguugcagcUUCUUgU3CU12660cuuuuuguugugcugugguugcg犯uuccagiugcuacguucuguuuuugguuuccgcugg12720uuaggggc肌uuuuucuuuugaacucguggug犯uimcacggumjgcccgcuuugcccaa12780cccggcaggcagccgcugagauccuugagcccggcaagucuuuuuggugcaggauagggc12840augaccgauguagugagaacgaucaugacgaacuaggguucaugguuccgccuggccucu12900ccagcgaaggccacuugsccaguguuimcgccugguuggcguuccuguccuucagcimca12960cggcccaguuccaucccgagaumiuugggauagggaaugugagucaaguuuauguugaca13020ucaagc3ccaauuc3ucugcgcuguucacgcgccaccuugccucgccaug13080acaaimuuucagccguauuucagsiccuacu8ccaacacc3ggucgacggcggca謹(jǐn)ggu13140uucaccuggaauggcugcguccuuucuuuuccucuugguugguuuuaaauguuucguggu13200uucucaggcguucgccugcaagccauguuucaguucg3gucuuucggacauca犯acc犯13260caccaccgcagcaucaaacuucguuguccuccaggacaucagcugccmmggcauggcga13320cucguccucuccgacgauucgcaaamiuccucagugccgcacggcgauagggacgcccgu13380gimcmic3ccaucscugccaaugucacagaug幼aauuaucuacmiucuucugaucuccu13440caugcuuucuucuugccuuuucumigcuuccgagaugagugaaaagggauucaaaguggu13500guuuggcaaugugucaggcgucguggcugugugcaucaacuuuaccagcuacguccaaca13560cgucaaggagUUU3CCC犯Cgcuccuuaguggucgaucaugugcgacugcuucauuucan13620gacaccugggacaaugaggugggceiaccguuuuagccugucuuuuugccauccuacuggc13680犯uuugaauguucaaguauguuggggaagugcuugaccgcgugcuguugcucgcgauugc13740uuuuuuugugguguaucgugccguucuaucuugcugugcucgucaacgccagcaacgaca13800acagcucucauauucaguugauuuauaacuuaacguuaugugagcugaauggcacagauu13860ggcuggcacaaagiauuugacugggcaguggagacuuuugucaucuuccccguguugacuc13920acauuguuuccuauggggcacucacceiccagccauuuccuugacacaguuggucuggcca13980cuguguccaccgccggauauuaucacgggcgguaugucuugaguagcauuuacgcagucu14040gugcucuggcugcgcugauuugcuuugucauuaggcuugcgaagaacugcauguccuggc14100gcuacucuugU3cc3gauauacc肪cuuccuucuggacacuaagggc卿cucu叫cguu14160ggcggucgcccgucauuguggagaaaggggguaagguugaggucgaaggucaccugaucg14220accucaagagaguugugcuugaugguuccgcggcaaccccuuuaaccagagu腦agcgg14280犯cgauggggucgucucuagacgacuucugcaaugauagcacagcuccacagaaggugcu14340uuuggcguuuuccauuacGUacacgccagugaugauauaugcucuaaagguaagucgcgg14400ccgacugcuagggcuucugcaccuuuugaucuuucug肪uugugcuuuimccuucgggua14460caugacauucgcgcacuuugagagcacaaauagggucgcgcucacimugggagcaguagu14520ugcacuucuuuggggaguguacucagccauagaaaccuggaaauucaucaccuccagaug14580ccguuugugcuugcuaggccgcaaguacauucuggccccugcccaccacgucgaaagugc14640cgcgggcuuucauccgauugcggcaaaugau肌ccacgcauuugucguccggcgucccgg14700cuccacuacggucaacggcacauuggugcccggguugaaaagccucguguuggguggcag14760ugguaaaccuuguuaaauaugccaaauaacaacggcaagc14820agcaaaagaaaaagaaggggaauggccagccagucaaucagcugugccaaaugcugggua14880agaucaucgcccaacaaaaccaguccagaggcaagggaccggggaag犯aaauaggaaga14940aaaacccggagaagccccauuucccucuagcgacugaagaugacgucaggcaucacuuua15000ccccuagugagcggcaauugugucugucgucgauccagacugcauucaaucagggcgcug15060gaacuugugcccugucagauucagggaggauaaguuacacuguggaguuuaguuugccga15120cgcaacauacugugcgucugauccgcgccacagcaucacccucagcaugaugggcuggca15180uucuuuggcaccucaguguuagaauugggagaauguguggugaauggcacugauugacac15240ugugccucuaagucaccuauucaauuagggcgaccguguggggguaaaguuuaauuggcg15300agaaccaugcggccguaauuaaa1532權(quán)利要求1.一株豬繁殖與呼吸綜合征病毒(PorcineReproductiveandRespiratorySyndromeVirus)強(qiáng)毒株HuN4，其特征在于其核苷酸序列為SEQIDNO1所示。2、由權(quán)利要求1所述豬繁殖與呼吸綜合征病毒強(qiáng)毒株HuN4傳代致弱所得到的弱毒疫苗株HuN4-F112，其特征在于其核苷酸序列為SEQIDN0:2所示。3、按照權(quán)利要求2所述豬繁殖與呼吸綜合征病毒弱毒疫苗株HuN4-F112，其特征在于，其微生物保藏號(hào)是CGMCCN0.2484。4、一種治療或預(yù)防高致病性豬藍(lán)耳病的疫苗組合物，其特征在于由權(quán)利要求2或3所述的弱毒疫苗株HuN4-F112和藥學(xué)上可接受的佐劑組成。5、權(quán)利要求2或3所述的弱毒疫苗株HuN4-F112在制備預(yù)防或治療高致病性豬藍(lán)耳病藥物中的用途。6、權(quán)利要求2或3所述的弱毒疫苗株HuN4-F112在制備診斷或檢測(cè)高致病性豬藍(lán)耳病藥物中的用途。全文摘要本發(fā)明公開了一株豬繁殖與呼吸綜合征病毒強(qiáng)毒株HuN4以及由該強(qiáng)毒株HuN4傳代致弱的弱毒疫苗株HuN4-F112(微生物保藏號(hào)是CGMCCNo.2484)，本發(fā)明還公開了該弱毒疫苗株在預(yù)防或治療高致病性豬藍(lán)耳病中的應(yīng)用，屬于生物醫(yī)藥領(lǐng)域。本發(fā)明弱毒疫苗株HuN4-F112可制備成單苗或聯(lián)苗(活疫苗或滅活疫苗)等，可有效預(yù)防或治療高致病性豬藍(lán)耳病，也可將其制備成診斷高致病性豬藍(lán)耳病的診斷試劑。本發(fā)明弱毒疫苗株HuN4-F112具有安全性好、保護(hù)效率高等優(yōu)點(diǎn)。文檔編號(hào)A61P31/14GK101280292SQ20081009754公開日2008年10月8日申請(qǐng)日期2008年5月13日優(yōu)先權(quán)日2008年5月13日發(fā)明者周艷君,安同慶,彭金美,田志軍,童光志申請(qǐng)人:中國(guó)農(nóng)業(yè)科學(xué)院哈爾濱獸醫(yī)研究所