Ⅰ型鸭肝炎病毒内部核糖体进入位点的结构与功能研究

为验证Ⅰ型鸭肝炎病毒(DHV-Ⅰ)基因组5'非编码区(UTR)是否含有内部核糖体进入位点元件,本研究利用体外翻译系统,结合细胞转染试验对DHV-Ⅰ5'UTR的结构和功能进行分析。结果表明:DHV-Ⅰ的5'UTR具有典型的内部核糖体结合元件(IRES)功能,能够内部起始下游蛋白的翻译,IRES元件位于360 nt～620 nt,共260个碱基。对IRES的二级结构进行预测和分析显示,DHV-ⅠIRES的结构与丙型肝炎病毒相似。利用定点突变方法,破坏IRES的颈环结构(SL)1、2以及IIIe区的关键性核苷酸,检测该突变对IRES功能的影响。研究结果显示,SL1、SL2和IIIe结构的破坏使得DHV-ⅠIRES的功能消失,表明SL1、SL2和IIIe区是维持IRES启动内部翻译起始功能的关键性结构域。     

Seroprevalence of neutralizing antibodies to human adenovirus type 5, human adenovirus type 26 and chimpanzee adenovirus type 68 in patients with chronic hepatitis B and patients with primary liver cancer

AIM: To investigate the seroprevalence of neutralizing antibodies to human adenovirus type 5 (AdHu5), human adenovirus type 26 (AdHu26) and chimpanzee adenovirus type 68 (AdC68) in the patients with chronic hepatitis B (CHB) and the patients with primary liver cancer (PLC), and to provide guidance for developing safe and effective biotherapy vectors against CHB and PLC. METHODS: The blood samples from 196 patients with CHB and 193 patients with PLC were examined to assess the presence of neutralizing antibodies against AdHu5, AdHu26 and AdC68 by adenovirus neutralization assays. RESULTS: The seroprevalence rates of neutralizing antibodies to AdHu5, AdHu26 and AdC68 in the CHB patients were 84.7%, 58.2% and 39.8%, respectively. Among the patients with PLC, the prevalence rates of neutralizing antibodies were as follows: AdHu5, 75.1%; AdHu26, 66.8%; AdC68, 32.1%. CONCLUSION: The prevalence rates and titers of neutralizing antibodies against AdC68 were the lowest among the 3 adenoviruses. Therefore, AdC68 serves as more suitable biological therapy vectors for CHB and PLC than AdHu5 and AdHu26.     

应用干扰素治疗肝炎研究进展

干扰素联合用药是治疗慢性乙型肝炎和丙型肝炎中最常用的治疗方案,联合用药不但可以提高干扰素的治疗效果,还可减轻干扰素的不良反应.但是,干扰素联合用药也存在着一定问题,如何改善现有的治疗方案和利用基因工程手段寻找新的治疗肝炎药物是此后一段时期该研究领域学者将要面临的新的挑战.     

一株鸭肝炎病毒的分离与鉴定

从山东某养殖场送检的疑似鸭病毒性肝炎病料中分离到一株病毒（暂命名为SD株）,分别用鸭胚、雏鸭进行传代和病毒含量测定,E1-E8代鸭胚适应毒的ELD50在1015^-5.0-10^-6.0／0．1mL之间,E1、E2代鸭肝组织毒的LD50分别为10^-5.5／0．1mL、10^-6.7／0．1mL。动物回归试验结果表明,SD株鸭胚适应毒E2对4日龄雏鸭的致死率为100％;雏鸭毒力测定试验结果表明SD株雏鸭肝组织毒E,代对12日龄内雏鸭具有较高致死率,12日龄后随着日龄的增大,雏鸭的死亡率逐渐降低;理化特性鉴定结果表明,SD株为无囊膜病毒,对脂溶剂（乙醚和氯仿）、胰酶、酸、热均不敏感,且无血凝性。血清交叉中和试验和交叉保护试验结果表明,在血清型上,SD株与DHV-1无关。通过对SD株与韩国N-DHVVP1基因的序列比对和进化树分析,发现二者同源性在99％以上。结果证实,SD株是新型鸭病毒性肝炎病毒,而不是DHV-1。     

血清3型鸭甲型肝炎病毒阳性血清的制备

通过使用血清3型鸭甲型肝炎疫苗免疫SPF鸡,制备了一批血清3型鸭甲型肝炎诊断用阳性血清,并对其进行了无菌、支原体和特异性检验以及中和效价测定。结果表明,该血清无菌检验合格,无支原体污染,特异性良好,无禽类常见13种外源病毒抗体,血清中和效价为10-2.8。本研究为鸭甲型肝炎病毒血清学鉴定及相关研究提供了重要的物质基础。     

鸭肝炎病毒的分离与鉴定

1999-2008年从山东、河南、浙江、江苏、广西、山西、江西、广东、福建和辽宁等地有典型鸭病毒性肝炎症状的病死雏鸭中共分离得到22株病毒。用鸭肝炎病毒（DHV）Ⅰ型（DHV-1）抗血清和新型DHV （N-DHV）抗血清对分离的22株病毒进行血清中和试验;对22株病毒的抗原相关VP1基因进行RT-PCR扩增和测序,然后对VP1基因的核苷酸与推导的氨基酸序列进行分析。结果表明,目前我国存在DHV-1和N-DHV两种血清型。其中分离到的12株为DHV-1,10株为N-DHV。依据VP1基因序列同源性进行的血清型分型结果与根据抗原性鉴定进行的血清型分型结果一致。12株DHV-1的氨基酸序列同源性为95%左右,10株N-DHV的氨基酸同源性为98%左右;DHV-1分离株与N-DHV分离株间氨基酸同源性为72%-77%。同血清型病毒株间的VP1基因变异很小,表明DHV的抗原性稳定。     

鸭胚肝细胞培养的研究

取１５日龄左右的鸭胚肝脏，经清洗、剪碎后，加入最终浓度为０．１２５％的胰酶，３７℃消化１５～２０分钟，以含犊牛血清的１９９培养基为营养液，置含５％ＣＯ＿２培养箱内培养７２小时，可制成鸭胚肝细胞单层培养物，若向培养基中加入１０％的鸭胚尿囊液，能促进细胞的生长，接种鸭肝炎病毒后，感染细胞可在４８小时出现ＣＰＥ，９６小时形成空斑。     

C1qa deficiency in mice increases susceptibility to mouse hepatitis virus A59 infection

BackgroundMouse hepatitis virus (MHV) A59 is a highly infectious pathogen and starts in the respiratory tract and progresses to systemic infection in laboratory mice. The complement system is an important part of the host immune response to viral infection. It is not clear the role of the classical complement pathway in MHV infection.ObjectivesThe purpose of this study was to determine the importance of the classical pathway in coronavirus pathogenesis by comparing C1qa KO mice and wild-type mice.MethodsWe generated a C1qa KO mouse using CRISPR/Cas9 technology and compared the susceptibility to MHV A59 infection between C1qa KO and wild-type mice. Histopathological and immunohistochemical changes, viral loads, and chemokine expressions in both mice were measured.ResultsMHV A59-infected C1qa KO mice showed severe histopathological changes, such as hepatocellular necrosis and interstitial pneumonia, compared to MHV A59-infected wild-type mice. Virus copy numbers in the olfactory bulb, liver, and lungs of C1qa KO mice were significantly higher than those of wild-type mice. The increase in viral copy numbers in C1qa KO mice was consistent with the histopathologic changes in organs. These results indicate that C1qa deficiency enhances susceptibility to MHV A59 systemic infection in mice. In addition, this enhanced susceptibility effect is associated with dramatic elevations in spleen IFN-γ, MIP-1 α, and MCP-1 in C1qa KO mice.ConclusionsThese data suggest that C1qa deficiency enhances susceptibility to MHV A59 systemic infection, and activation of the classical complement pathway may be important for protecting the host against MHV A59 infection.