鳜传染性脾肾坏死病毒主衣壳蛋白单克隆抗体的制备及鉴定

为了建立鳜传染性脾肾坏死病毒(ISKNV)疫苗抗原含量的ELISA检测方法,制备了3株抗ISKNV主衣壳蛋白(MCP)的单克隆抗体,鉴定了其生物学特性。将大肠杆菌表达的重组MCP纯化复性后,连续3次免疫BALB/c小鼠,然后将免疫小鼠的脾细胞与SP2/0细胞融合,经过克隆、筛选,获得3株能稳定分泌抗ISKNV MCP蛋白的单克隆抗体阳性细胞株,分别命名为5F1、3D9和5B4,均为Ig G1亚型。间接ELISA实验表明,3株单抗可特异性识别ISKNV,与鳜弹状病毒、大鲵虹彩病毒等无交叉反应。将5F1株免疫小鼠后制备腹水,以重组MCP和ISKNV细胞培养物上清液为检测抗原,ELISA检测腹水效价分别为1∶51 200和1∶400。间接免疫荧光(IFA)和Western Blotting鉴定结果显示,5F1能够与ISKNV病毒发生特异性反应,并初步确定5F1单抗株制备的腹水用于IFA的使用浓度为1∶200、Western Blotting的使用浓度为1∶1000。结果证实,成功制备了抗ISKNV MCP的单克隆抗体,可特异性识别ISKNV病毒粒子和MCP蛋白,为建立ISKNV疫苗抗原含量检测方法奠定了基础。     

Competition of infectious hypodermal and haematopoietic necrosis virus (IHHNV) with white spot syndrome virus (WSSV) for binding to shrimp cellular membrane

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) are two widespread shrimp viruses. The interference of IHHNV on WSSV was the first reported case of viral interference that involved crustacean viruses and has been subsequently confirmed. However, the mechanisms underlying the induction of WSSV resistance through IHHNV infection are practically unknown. In this study, the interference mechanisms between IHHNV and WSSV were studied using a competitive ELISA. The binding of WSSV and IHHNV to cellular membrane of Litopenaeus vannamei was examined. The results suggested that there existed a mutual competition between IHHNV and WSSV for binding to receptors present on cellular membrane of L. vannamei and that the inhibitory effects of WSSV towards IHHNV were more distinct than those of IHHNV towards WSSV.     

传染性脾肾坏死病毒_ConA和L_省略_对体外鳜的头肾淋巴细胞转化的影响

采用MTT颜色反应法研究传染性脾肾坏死病毒（ISKNV）、伴刀豆球蛋白A（concanavallin A,ConA)和脂多糖(lipopolysaccharide,LPS）在离体状态下对健康和ISKNV感染后存活30d或60d的鳜的头肾淋巴细胞转化的影响。结果表明，ConA和LPS能促进健康鳜头肾淋巴细胞的转化，而对感染后存活30d或60d的鳜头肾淋巴细胞没有作用；纯化的ISKNV对健康鳜头肾淋巴细胞没有促进淋巴细胞转化的作用，但对ConA的作用有抑制，对感染后存活的30d或60d的鳜头肾淋巴细胞，纯化的ISKNV有一定的促进转化的作用，感染存活的鳜头肾中有对ISKNV的免疫记忆性T细胞。另外，用建立的ISKNV PCR检测方法对健康和ISKNV感染后存活的鲜组织进行了检测，结果显示，健康鳜为阴性，而ISKNV感染后存活的鱼，头肾、后肾、脾脏和部分鱼的心脏为阳性。ISKNV在鳜体内形成潜伏感染。     

Salmonid fish viruses and cell interactions at early steps of the infective cycle

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.     

鸡传染性支气管炎病毒肾致病株的致弱及免疫研究

用已鉴定的广西鸡传染性支气管炎病毒(IBV)分离株G,C,Z和Q分别通过鸡胚传代进行致弱,在一定代次时分别接种敏感鸡作毒力测定试验.然后用已致弱的毒株在实验室进行攻击保护试验,最后取已致弱而且抗原性良好的毒株在广西各地鸡场进行田间应用试验。结果表明,C株在传至40代时,G,Z和Q株分别传至20代时对鸡的毒性均已大大降低;免疫鸡用标准强毒株(M_(41))攻击,然后用病毒回收的技术来衡量其免疫力,结果各株经免疫两次后均有很好的保护率(60％～100％),均显著优于对照组(P<0.01);致弱株分别在南宁、柳州、容县、岑溪和博自5县市的11个鸡场进行田间试验,试验的鸡有30群共103761羽,试验时分别对疫苗免疫对鸡生产性能的影响、安全性和育成率进行观察和测算,结果表明,作者培育的IBV弱毒苗对鸡是安全的,使用C_(60)或C_(80)和G_(20)株先后两次免疫鸡,对IB有较好预防效果,这些弱毒株有希望成为供生产使用的疫苗。     

鸡传染性法氏囊病母源抗体对弱毒疫苗免疫效果的影响

雏鸡传染性法氏囊病 (IBD)母源抗体高低不一是造成免疫失败的主要原因 ,利用琼脂扩散试验对不同日龄的 2 76群鸡母源抗体以及免疫前后抗体的检测 ,了解鸡传染性法氏囊病母源抗体对IBD弱毒疫苗免疫效果的影响 ,从而找出最佳免疫时机     

一种非EDSV引起的鸭传染性减蛋症

自1999年5月以来，浙江省许多产蛋鸭发生了一种以产蛋率锐减为特征的新疾病，其中某地区67户养鸭专业户，共饲养蛋鸭15.7万羽，陆续进入产蛋高峰期后，有2375 羽鸭群的产蛋率突然下降，由原来的91.2%减到52.7%，同时伴有畸形蛋、沙壳蛋，蛋质低劣，蛋清稀薄，除少部分病鸭伴有烦躁不安和吵棚外，鸭群的精神体态基本正常，采食量略有下降，一般不引起死亡。起初养鸭户都误认为是当地饲料厂供应的饲料所致，后来发现该病具有传染性，疫病主要沿河流自上而下蔓延。在此后1个多月时间内，共有49户养鸭户12.3 万羽蛋鸭相继发病，产蛋率下降幅度为20%～80%，使用各种抗菌药物治疗无效，提高饲料营养也不起作用。该病发病急，传播迅速，通常在2～3天内产蛋率由80%～90%以上下降到30% ～40%，有的甚至更低，产蛋率越高，感染后下降幅度往往越大。病鸭从产蛋下降到康复需3 -7周时间，恢复后不能达到标准产蛋曲线，产蛋周期缩短。剖检病鸭主要表现为生殖器官病变，卵巢卵泡减少、萎缩，输卵管蛋白分泌部缩小，蛋白分泌腺减少，子宫萎缩，有的严重病例可见卵黄性腹膜炎，肝、心、肾和肺有斑点出血。该病的临床症状类似于减蛋综合征（ EDS）［1］，但是，用减蛋综合征灭活苗免疫预防不能有效保护。作者对病鸭体作无菌检查并分离了病毒（命名为YH99株），用0.2 μm滤器过滤后，经人工感染易感蛋鸭能成功诱导典型发病，再从病鸭中回收到同样病毒。将YH99株与鸡源减蛋综合征病毒（EDSV-AV127）［2～4］和鸭源减蛋综合征病毒［3］（EDSV-JE 1，由上海市畜牧兽医站周锦萍提供）进行比较，结果发现：     

家蚕生物反应器表达传染性法氏囊病病毒多聚蛋白的免疫原性研究

将传染性法氏囊病病毒(IBDV)浙江分离株JD1的多聚蛋白(VP2/4/3)基因克隆到家蚕杆状病毒转移载体pBacPAK8中,重组载体与杆状病毒Bm-BacPAK6的线性化基因组DNA共转染家蚕细胞后,将获得的重组病毒BacPAK-A感染家蚕5龄起幼虫进行虫体内表达.用感染后第5日的蚕血淋巴作抗原制备油佐剂苗免疫14日龄非免疫鸡,安全性试验表明,接种1倍和5倍免疫剂量的试验鸡在临床反应和剖检中均无异常变化;在免疫-攻毒试验中设杆状病毒表达的IBDV VP2蛋白和正常蚕血淋巴免疫对照组,并于28日龄加强免疫,25 d后用IBDV强毒BC6/85攻击.通过临床保护率、病理保护率及血清学试验表明,基于多聚蛋白制备的疫苗更成功地诱导了体液免疫应答,比VP2蛋白对IBDV强毒的攻击具有更高的保护力,更适于作为IBD基因工程亚单位疫苗的抗原成分.     

家养鲤科鱼暴发性传染病的病原研究

从不同地区、不同鱼种的濒死病鱼的实质性脏器中分离到一种革兰氏阴性、有运动力的短杆菌,具有如下特点:氧化酶和V-P反应均呈阳性,发酵葡萄糖产酸、产气,发酵甘露醇、水杨素和阿拉伯糖,不发酵肌醇,鸟氨酸脱羧酶阴性,对新生霉素不敏感。用API-20E系统试剂盒检测,证实其为嗜水气单胞菌。用该菌人工感染健康鱼,可引致与自然发病鱼类似的病症,其半数致死量(LD_(50))为5×10~3个菌。在该菌的培养上清中存在着一种具有溶血活性的毒素。研究证明,家养鲤科鱼暴发性传染病的病原为产毒素的嗜水气单胞菌,该病的准确名称应为嗜水气单胞菌败血症。     

传染性法氏囊病病毒dsRNA核酸提取

用免疫沉淀法从感染鸡法氏囊组织中分离传染性法氏囊病病毒（ＩＢＤＶ），用差速离心法从感染的鸡胚成纤维细胞（ＣＥＦ）中分离ＩＢＤＶ，再从分离的ＩＢＤＶ中抽提ｄｓＲＮＡ。在ｄｓＲＮＡ提取物受ＤＮＡ降解产物影响时，可用ＤＮａｓｅⅠ消化去除，核酸再经琼脂糖凝胶电泳纯化。