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jwk3029金蟲(chóng) (小有名氣)
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雞新城疫(Newcastle Disease,ND)是由新城疫病毒(Newcastle Disease Virus,NDV)引起禽的一種高度接觸性、急性敗血性傳染病。新城疫是威脅世界養(yǎng)禽業(yè)的主要疾病之一，被世界動(dòng)物衛(wèi)生組織(OIE)歸為A類(lèi)傳染病。新城疫病毒幾乎可感染全部鳥(niǎo)類(lèi)，并可隨侯鳥(niǎo)的遷徙而廣泛傳播，NDV屬于副粘病毒科、副粘病毒亞科的腮腺炎病毒屬，單股負(fù)鏈RNA病毒。我國(guó)長(zhǎng)期以來(lái)普遍開(kāi)展了以接種疫苗為主的新城疫綜合防制措施，很好控制了ND的暴發(fā)，但近年來(lái)ND的流行又出現(xiàn)了新的特點(diǎn)：非典型新城疫日益增多，甚至高抗體雞群也發(fā)生該病，這些流行特點(diǎn)很可能是由變異或超強(qiáng)毒株引起。近年來(lái)鵝源病毒新城疫變異株的出現(xiàn)使人們對(duì)于非典型新城疫發(fā)病原因的尤為感興趣。本研究在2008年從新鄉(xiāng)某雞場(chǎng)不表現(xiàn)典型ND癥狀且高抗體蛋雞中分離到的一株新城疫野毒（NDV ND-xx08株），在對(duì)其進(jìn)行生物學(xué)特性研究的基礎(chǔ)上，擴(kuò)增出其F和HN基因，并做了免疫保護(hù)試驗(yàn)，為探討新城疫是否發(fā)生變異和其預(yù)防提供理論依據(jù)，為河南省NDV分子流行病學(xué)研究提供有益參考，豐富了我國(guó)ND分子流行病學(xué)特征，為制定控制ND方法提供重要依據(jù)。首先對(duì)NDV ND-xx08株進(jìn)行了一系列生物學(xué)特性研究。SPF雞胚接種培養(yǎng)后，經(jīng)過(guò)試驗(yàn)鑒定，該分離株具有血凝性，且可被ND標(biāo)準(zhǔn)陽(yáng)性血清所抑制和中和，不能被禽流感（H5 、H9亞型）標(biāo)準(zhǔn)陽(yáng)性血清和EDS-76標(biāo)準(zhǔn)陽(yáng)性血清所抑制，因此該分離株為新城疫病毒。該病毒的最小致死量病毒致死雞胚的平均時(shí)間（MDT）為50h；1日齡SPF雞腦內(nèi)接種分離病毒致病指數(shù)(ICPI)為1.71；6周齡SPF雞靜脈接種致病指數(shù)（IVPI）為2.38；由此NDV ND-xx08株確定屬于NDV強(qiáng)毒型。其次分別設(shè)計(jì)了一對(duì)引物，Trizol法提取了其RNA，RT-PCR擴(kuò)增出NDV ND-xx08株F基因和HN基因的全基因序列，并分別兩條基因的核苷酸序列進(jìn)行測(cè)定和分析，并對(duì)氨基酸序列進(jìn)行了分析。用DNAStar軟件對(duì)本研究毒株與國(guó)內(nèi)外發(fā)表的一些有代表性的46株參考毒株的NDV F基因47nt-420nt區(qū)域進(jìn)行同源性比較，繪制了基因進(jìn)化樹(shù)，分析其遺傳變異關(guān)系。經(jīng)測(cè)定NDV ND-xx08 毒株的F基因開(kāi)放性閱讀框架全長(zhǎng)1662bp, 共編碼553個(gè)氨基酸，F(xiàn)蛋白的裂解位點(diǎn)為112R-R-Q-K-R-F117。系統(tǒng)發(fā)育分析得出該分離株屬于基因Ⅶe型，將其與國(guó)內(nèi)外發(fā)表的部分新城疫病毒毒株之間F基因核苷酸和氨基酸序列進(jìn)行比較，其核苷酸序列的同源性在82.7％-97.8％之間，氨基酸同源性在87.5％-97.7％之間，與F48E9株的氨基酸同源性為91.5%，與 Lasota，Clone30和V4株的同源性分別為88.1%，88.6%和88.3%。從分子水平上說(shuō)明NDV ND-xx08 株為發(fā)生了一定程度變異的新城疫強(qiáng)毒株。NDV ND-xx08 毒株的HN基因開(kāi)放性閱讀框架全長(zhǎng)1 716 bp，編碼571個(gè)氨基酸，屬于C群。它與其他24株的 HN 基因核苷酸序列同源性在80％－98.4％。它與北京毒株（SRZ03）和江蘇毒株（zj1）同源性較高，分別經(jīng)測(cè)定為98.1%和98.4%。從基因進(jìn)化樹(shù)來(lái)看，該毒株與北京和江蘇所分離的毒株同源性較近，基本上屬于同一基因群；與 Lasota 和Clone30等疫苗株的同源性較遠(yuǎn)，屬不同的基因群。ND-xx08株 HN 蛋白氨基酸序列與其他24株同源性在 87.2％－98.2％，與 Lasota 和Clone30 的同源性分別為88.2 %和89.3 %，同源性較低。最后用該毒株做了免疫保護(hù)性試驗(yàn)，分別將NDV ND-xx08株尿囊液稀釋10、100、1000倍，將原尿囊液和3種稀釋倍數(shù)的尿囊液按照常規(guī)方法做成油苗。將四種疫苗對(duì)雞群免疫了2次，研究其抗體增長(zhǎng)規(guī)律。組后對(duì)免疫雞群進(jìn)行攻毒，看油苗的保護(hù)率，篩選出效果最好的油苗和免疫劑量。 [ Last edited by jwk3029 on 2010-5-19 at 17:29 ] |
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2樓2010-05-20 03:59:21
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jwk3029(金幣+90, 翻譯EPI+1): 2010-05-20 17:16:48
sirljz(金幣+3):謝謝交流 2010-05-20 17:33:41
jwk3029(金幣+90, 翻譯EPI+1): 2010-05-20 17:16:48
sirljz(金幣+3):謝謝交流 2010-05-20 17:33:41
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僅供LZ參考。 Chicken Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious, acute septic infectious disease. ND is among the most serious disease threats to the poultry industry worldwide. It’s included in the list A infectious disease by OIE. NDV, is a negative-sense single-stranded RNA virus, a member of rubulavirus, paramyxoviridae family, affects most avian species, and spread as the immigration of birds. For a long time, ND had been well prevented by a comprehensive measure mainly by vaccination in China. But, ND has new features in recent years, such as more and more non-typical ND, even in chicken flocks have high antibodies concentration, which might induced by variant or highly virulent strains. Newly identified geese-derived NDV variant attracted researchers’ attention on the pathogenesis of inapparent ND. In this study, we isolated a wild NDV strain (strain NDV ND-xx08) from layers with no apparent ND symptoms, in a hennery of Xinxiang. After basic assay on its biological characteristic, the F and HN gene were amplified, immune protection efficiency was evaluated, which would provide theoretical foundation for investigation on ND variation and prevention, provide important basis for formulation of ND prevention methods, and also be a useful reference for NDV molecular epidemiology in Henan, enrichment for ND molecular epidemiology features in China. Firstly, biological characteristics of NDV ND-xx08 were examined. Cultured by inoculating SPF chicken embryo, this new strain was able of inhibiting blood coagulation, and was neutralized by NDV standard positive serum, can’t be inhibited by bird flu (subtype H5, H9) and EDS-76 standard positive serum. So we identified this isolation as NDV. It took average 50h (MDT) for minimum lethal dose of this virus to kill chick embryo. Pathogenicity index for 1-day SPF chicken inoculated the isolation in brain (ICPI) was 1.71, Pathogenicity index for 6-week chicken inoculated the virus through vein (IVPI) was 2.38. These data revealed NDV ND-xx08 was a highly virulent strain. Secondly, the F and HN gene were amplified by RT-PCR from RNA (extracted by Trizol method) using primers we designed. After sequencing, we analyzed the nucleotide sequences and the protein sequences. DNAstar software was used to compare the NDVF gene 47nt-420nt region of this strain with other 46 typical strains published in China and other countries, draw the gene phylogenetic tree and analyze the genetic variation. The open reading frame of NDV ND-xx08 strain F gene is 1662 bp, encodes 553 amino acids. The lysis site locates at 112R-R-Q-K-R-F117. Phylogenetic analysis shows this strain belongs to VIIe genotype. Comparison of the nucleotide sequence for this strain and other published strains, revealed 82.7%-97.8% homology, 87.5%-97.7% homology in amino acid sequence. A 91.5% amino acids sequence homology was found when compared to F48E9, 88.1%, 88.6% and 88.3% to Lasota, Clone30 and V4 respectively. This means NDV ND-xx08 is a highly virulent NDV strain has some variation in the molecular level. The open reading frame of NDV ND-xx08 strain HN gene is 1716 bp, encodes 571 amino acids, belonging to C group. The nucleotide sequence of this strain has 80-98.4% homology to other 24 strains. High homology was found to Beijing strain (SRZ03) and Jiangsu strain (zj1), 98.1% and 98.4% respectively. On the gene phylogenetic tree, ND-xx08 strain is near to Beijing strain (SRZ03) and Jiangsu strain (zj1), they are basically in the same gene group; but far away from Lasota and Clone30, belonging to different groups. The amino acid sequence of ND-xx08 has 87.2%-98.2% homology with other 24 strains, a low homology of 88.2% and 89.3% to Lasota and Clone30 respectively. At last, immune protection experiments were carried out using this strain. Inactivated oil emulsified vaccine were prepared by original amniotic fluid and amniotic fluid diluted 10, 100 and 1000 times according to traditional method. Chicken flocks were inoculated twice with these four oil-emulsified vaccines, following with examination of antibody concentrations. Subsequently, we evaluated the protection efficiency of chicken flocks to the challenge of the virus strain, found the vaccine with high efficiency and best dose. 順便說(shuō)一句，一個(gè)摘要，這么多車(chē)轱轆話。 |
3樓2010-05-20 10:30:37
jwk3029
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