【摘要】：鴨肝炎病毒(Duck hepatitis virus,DHV)導致的鴨病毒性肝炎(Duck viral hepatitis,DVH)以侵害三周齡以內(nèi)的雛鴨為主,是一種急性高度致死性傳染病。近年來,由3型鴨甲肝病毒(Duck hepatitis A virus,DHAV-3)引起的DVH廣泛流行于中國和韓國,致死率高達80%,嚴重危害養(yǎng)鴨業(yè)的發(fā)展。為進一步了解山東省DHAV-3的流行近況、遺傳變異規(guī)律和分子生物學特征,提高對該病的診斷速度和質(zhì)量,加強對其的科學防控,本研究進行了以下兩部分的研究:第一部分山東地區(qū)DHAV-3的病毒分離及分離株P1基因的序列分析2012年7月至2014年7月,對山東省四個地區(qū)(濟南市、濰坊市、煙臺市、臨沂市)DHAV-3的流行情況進行了流行病學調(diào)查。共采集了18批167份疑似鴨病毒性肝炎致死的鴨肝組織等病料,經(jīng)病料處理、鴨胚接種和雛鴨人工感染實驗對病毒進行分離,用DHAV-3特異性檢測引物對分離病毒進行RT-PCR擴增檢測,共分離鑒定出70株DHAV-3野毒株。結果表明18批病料中均檢出DHAV-3,批次檢出率為100%;167份病料中有70份樣品為DHAV-3陽性,個體陽性率為41.9%。從此次山東分離株中選取18株DHAV-3代表株,對其P1基因進行克隆測序和序列分析。同源性分析顯示,18株DHAV-3分離株P1基因高度保守,其核苷酸和氨基酸序列同源性高達95.9%~99.8%和97.4%~100%。與27株DHAV-3參考株的核苷酸和氨基酸序列同源性分別為91.9%~99.0%和95.2%~99.6%,屬同一血清型。進化樹分析顯示,中國分離株(除B63株)均屬于GⅠ型。本研究分離的18株DHAV-3代表株均屬于GⅠ型的S1亞型,其中濟南、臨沂、濰坊三地分離株遺傳距離較近,位于同一小分支。越南分離株與中國B63株屬于GⅡ型中的S3亞型,而所有韓國分離株組成GⅡ型中的S4亞型。DHAV-3的基因分型呈現(xiàn)明顯的地域特征。氨基酸比對結果顯示不同分支彼此之間存在多個氨基酸變異位點,其中共有22個氨基酸穩(wěn)定變異位點。P1基因第671~712位氨基酸處于高變區(qū)(HVR),是病毒抗原變異的關鍵所在。在HVR內(nèi),所有的RGD序列變異為QSD序列。第二部分SD1101株單克隆抗體的制備為研制DHAV-3單克隆抗體,用已完成全基因組測序的SD1101株DHAV-3的全病毒免疫適齡BALB/c小鼠,取免疫效果最佳的小鼠的脾細胞和骨髓瘤細胞SP2/0進行融合,通過間接ELISA法對雜交瘤細胞進行篩選,經(jīng)三次亞克隆后獲得2株穩(wěn)定分泌針對DHAV-3的單克隆抗體4A2和4C3。對這2株單抗的腹水效價、亞類、染色體數(shù)、特異性和穩(wěn)定性進行了鑒定,結果如下:用間接ELISA檢測連續(xù)傳代和凍存復蘇的細胞上清,發(fā)現(xiàn)其分泌抗體的能力無顯著差異,證明其穩(wěn)定性好;染色體數(shù)目在100左右,呈現(xiàn)典型的雜交瘤細胞染色體特征;經(jīng)亞類鑒定,2株單抗分泌的抗體類型均為Ig G1亞類、κ亞型;單抗腹水效價為1:10240;IFA結果顯示,2株單抗與DHAV-3和DHAV-1均發(fā)生特異性反應,但與DHAV-3的反應更強烈。
[Abstract]:Duck hepatitis virus (Duck hepatitis virus,DHV) caused by duck hepatitis (Duck viral hepatitis,DVH is an acute and highly fatal infectious disease, which mainly infects ducklings within three weeks of age. In recent years, DVH caused by duck hepatitis A virus type 3 (Duck hepatitis A virus,DHAV-3) is widely prevalent in China and South Korea. The fatality rate is as high as 80%, which seriously harms the development of duck breeding. In order to further understand the epidemic status, genetic variation and molecular biological characteristics of DHAV-3 in Shandong Province, improve the speed and quality of diagnosis of the disease, and strengthen the scientific prevention and control of the disease. The results of this study are as follows: the first part is the isolation of DHAV-3 virus and the sequence analysis of P1 gene in Shandong province from July 2012 to July 2014. Four regions of Shandong Province (Jinan, Weifang, Yantai) were studied. Epidemiological investigation on the prevalence of DHAV-3 was carried out in Linyi City. A total of 18 batches of 167 samples of duck liver tissue were collected. The virus was isolated by inoculation of duck embryo and artificial infection of ducklings. The virus was amplified by RT-PCR with DHAV-3 specific detection primers. A total of 70 DHAV-3 wild strains were isolated and identified. The results showed that the detection rate of DHAV-3, batches in 18 batches was 100 and 167. 70 samples were positive for DHAV-3, and the positive rate was 41.9%. Eighteen representative strains of DHAV-3 were selected from this Shandong isolate and their P1 genes were cloned and sequenced. Homology analysis showed that P1 gene of 18 DHAV-3 isolates was highly conserved, and the nucleotide and amino acid sequence homology was up to 99.8% and 97.4100% respectively. The homology of nucleotide and amino acid sequences with 27 DHAV-3 reference strains were 99.0% and 95.2%, respectively, belonging to the same serotype. Phylogenetic tree analysis showed that all Chinese isolates (except B63) belonged to G 鈪，

本文編號：2228085