Inhibitors of the Hepatitis C Virus Polymerase; Mode of Action and Resistance

The hepatitis C virus (HCV) is a pandemic human pathogen posing a substantial health and economic burden in both developing and developed countries. Controlling the spread of HCV through behavioural prevention strategies has met with limited success and vaccine development remains slow. The development of antiviral therapeutic agents has also been challenging, primarily due to the lack of efficient cell culture and animal models for all HCV genotypes, as well as the large genetic diversity between HCV strains. On the other hand, the use of interferon-α-based treatments in combination with the guanosine analogue, ribavirin, achieved limited success, and widespread use of these therapies has been hampered by prevalent side effects. For more than a decade, the HCV RNA-dependent RNA polymerase (RdRp) has been targeted for antiviral development. Direct acting antivirals (DAA) have been identified which bind to one of at least six RdRp inhibitor-binding sites, and are now becoming a mainstay of highly effective and well tolerated antiviral treatment for HCV infection. Here we review the different classes of RdRp inhibitors and their mode of action against HCV. Furthermore, the mechanism of antiviral resistance to each class is described, including naturally occurring resistance-associated variants (RAVs) in different viral strains and genotypes. Finally, we review the impact of these RAVs on treatment outcomes with the newly developed regimens.     

PRNCR1在前列腺癌诊断及预后的价值

目的 探讨前列腺癌非编码RNA1（PRNCR1）在前列腺癌早期诊断及预后的价值。方法 选取2012年1月至2015年10月在本院诊治前列腺癌患者45例、良性前列腺增生患者88例，进行PRNCR1检验。结果 PRNCR1 检验的灵敏度84.4%（38/45）、特异度90.9%（80/88）、阳性预测值82.6%（38/46）、阴性预测值92.0%（80/87)、符合率88.7%（118/133），PRNCR1在前列腺癌组织中含量高于癌旁组织和良性前列腺增生组织，差异有统计学意义（P<0.05）。两年内转移、复发、肿瘤死亡患者30例，其PRNCRA表达量高于两年内无转移、复发、死亡的患者（15例），差异有统计学意义（P<0.05）。结论 PRNCR1表达在诊断前列腺癌有价值，PRNCR1高表达与患者预后不良有关。     

Inactivated influenza vaccine formulated with single-stranded RNA-based adjuvant confers mucosal immunity and cross-protection against influenza virus infection

Influenza vaccination is considered the most valuable means to prevent and control seasonal influenza infections, which causes various clinical symptoms, ranging from mild cough and fever to even death. Among various influenza vaccine types, the inactivated subunit type is known to provide improved safety with reduced reactogenicity. However, there are some drawbacks associated with inactivated subunit type vaccines, with the main ones being its low immunogenicity and the induction of Th2-biased immune responses. In this study, we investigated the role of a single-stranded RNA (ssRNA) derived from the intergenic region in the internal ribosome entry site of the Cricket paralysis virus as an adjuvant rather than the universal vaccine for a seasonal inactivated subunit influenza vaccine. The ssRNA adjuvant stimulated not only well-balanced cellular (indicated by IgG2a, IFN-γ, IL-2, and TNF-α) and humoral (indicated by IgG1 and IL-4) immune responses but also a mucosal immune response (indicated by IgA), a key protector against respiratory virus infections. It also increases the HI titer, the surrogate marker of influenza vaccine efficacy. Furthermore, ssRNA adjuvant confers cross-protective immune responses against heterologous influenza virus infection while promoting enhanced viral clearance. Moreover, ssRNA adjuvant increases the number of memory CD4⁺ and CD8⁺ T cells, which can be expected to induce long-term immune responses. Therefore, this ssRNA-adjuvanted seasonal inactivated subunit influenza vaccine might be the best influenza vaccine generating robust humoral and cellular immune responses and conferring cross-protective and long-term immunity.     

静脉毒瘾者84例HGV感染状况

目的调查庚型肝炎病毒（ＨＧＶ）在静脉毒瘾者中的感染状况。方法采用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）检测８４例静脉毒瘾者血浆标本。ＨＧＶＲＮＡ经热变性法提取后逆转录为ｃＤＮＡ，在ＨＧＶ５′非编码区（５′ＮＣＲ）设计两对引物进行巢式扩增，产物为２３８ｂｐ，并经限制性内切酶ＨｐａⅡ鉴定扩增产物来自ＨＧＶ。结果８４例中有１５例为ＨＧＶＲＮＡ阳性，阳性率为１７．９％。ＨＧＶＲＮＡ阳性病例中１１例合并丙型肝炎病毒感染（１１／１５）。结论静脉毒瘾者是ＨＧＶ感染的高危人群；不洁注射是获得ＨＧＶ感染的重要途径。     

犬MC1R基因多态性的研究

目的 利用PCR—RFLP和PCR—SSCP技术分别检测犬MC1R基因中两个序列T105A和R306Ter的基因多态性，为遗传育种、培育出更加优良的实验用犬奠定基础。方法 以224只犬为实验材料，对犬MC1R基因T105A片段和R306Ter片段分别进行PCR-RFLP和PCR—SSCP分析，并且分别对具有RFLP和SSCP多态性的片段进行克隆测序，找出等位基因的差异位点。结果 经对犬MClR基因的PCR-RFLP分析，发现犬MC1R基因的T105A序列具有多态性，表现为三种基因型：AA、AB和BB。通过对具有RFLP多态性的片段进行克隆测序发现MC1R基因在编码第105位氨基酸的密码子处存在由G到A的单碱基突变，该突变导致第105位氨基酸发生由精氨酸向苏氨酸的改变，此外在第207位氨基酸的密码子处还发现由A到G的单碱基突变，并产生了一个HhαI限制性内切酶位点。同时，经过犬MC1R基因的PCR—SSCP分析，发现犬MC1R基因的R306Ter序列具有多态性，表现为三种基因型：CC、CD、DD.通过对具有SSCP多态性的片段进行了克隆测序发现，MC1R基因在编码第306位氨基酸的密码子处存在一个由CGA到TGA的终止突变，而使翻译终止。结论 利用PCR—RFLP和PCR—SSCP分析技术证实犬MC1R基因具有明显的多态性。     

人端粒酶RNA的cDNA探针制备及其对胃粘膜细胞端粒酶RNA表达的检测

目的 建立人端粒酶RNA表达的检测方法。方法 制备人端粒酶RNA，(human telomeraseRNA，hTR)的cDNA探针，分别应用RNA斑点杂交与端粒重复序列扩增法(TRAP)分析检测不同胃粘膜的端粒酶RNA的表达与端粒酶活性。结果 人端粒酶RNA的cDNA探针制备成功。18例活检胃癌组织及45例手术胃癌组织RNA斑点杂交检测的阳性率均为l00％，相应TRAP分析的阳性率分别为88．89％、86．67％，低于RNA斑点杂交(P＜0．05)。同时RNA斑点杂交结果提示在非癌胃组织中随着肠化程度增高人端粒酶RNA表达也增强。结论 RNA斑点杂交检测人端粒酶RNA，具有高度的敏感性和特异性，弥补了TRAP分析敏感性不足的缺点。     

外周血细胞DNA、RNA及血红蛋白同时提取法

采用淋巴细胞分离液分离外周血中的有核细胞,经细胞裂解液处理,上清提取RNA,沉淀提取DNA,用分离液分离所得红细胞沉淀制备血红蛋白溶液。经对抽提物质量进行评价,发现3种提取物质量均高。本法能从少量外周血中同时分离DNA、RNA及血红蛋白,高效易操作,值得推广。     

肿瘤坏死因子β基因多态性在中国汉族SLE病人中的分布特点及与不同人种的比较研究

目的 :探讨中国江苏地区汉族人群TNFβ基因多态性在SLE病人中分布特点 ,并与不同人种进行比较研究。 方法 :收集江苏地区 16 8名无血缘关系健康个体及 6 6例SLE病人的静脉血提取DNA ,应用聚合酶链反应限制性片段长度多态性(PCR RFLP) ,分析TNFβ基因的多态性。 结果 :中国汉族SLE病人与白种SLE病人TNFβ基因频率差异无统计学意义 ;中国汉族健康个体TNFβ 1等位基因频率明显高于白种健康个体 ;SLE病人TNFβ 2基因频率较正常人明显升高 (SLE病人 6 7 4% ,正常人 5 5 1% ,P <0 0 5 ,R =1 6 8) ;中国汉族SLE病人与白种SLE病人TNFβ基因频率差异无统计学意义。 结论 :TNFβ等位基因频率在正常人分布具有种族差异 ,但在SLE病人分布无种族差异。     

慢性低氧大鼠肺动脉内皮素mRNA的表达及定位

目的：探讨慢性低氧大鼠肺动脉内皮素－1基因表达及分布。方法：采用生物素标记cRNA探针，对Wistar大鼠肺动脉进行原位杂交。结果：低氧1周组及低氧2周组大鼠大部分肺动脉内皮细胞ET－1mRNA表达呈阳性信号，而对照组大鼠仅极少数阳性信号（P＜0．01）；低氧1周组大鼠部分显示强阳性信号。低氧1周组及低氧2周组大鼠部分肺动脉平滑肌细胞呈现ET-1mRNA表达阳性信号，而对照组大鼠则无阳性信号表达（P＜0．01及0．05）。结论：慢性低氧对大鼠肺动脉ET－1分泌的影响是在基因转录水平进行，且主要细胞定位在肺动脉内皮细胞，此外还包括肺动脉平滑肌细胞。