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.     

极速实时荧光聚合酶链反应与常规方法对新型布尼亚病毒检测的评价

目的探讨极速实时荧光聚合酶链反应(polymerase chain reaction,PCR)、实时荧光PCR、酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)和胶体金免疫层析法(gold immunochromatography assay,GICA)4种方法检测新型布尼亚病毒的特异度和灵敏度,为发热伴血小板减少综合征的早期诊断提供依据。方法采集2017年6月1日至9月30日山东大学附属济南市传染病医院86例临床诊断为发热伴血小板减少综合征患者的血清样本,分别应用极速实时荧光PCR、实时荧光PCR、ELISA和GICA 4种方法进行检测。统计学分析采用χ^2检验。结果86份患者血清标本中,极速实时荧光PCR、实时荧光PCR、IgM-ELISA、IgG-ELISA、IgM-GICA、IgG-GICA的新型布尼亚病毒阳性分别为82份(95.34%)、79份(91.86%)、41份(47.67%)、8份(9.3%)、19份(22.09%)和3份(3.49%)。极速实时荧光PCR特异度为100%,灵敏度达到1×103拷贝/mL,3次重复扩增试验显示其Ct值变异系数均<2%。在发热伴血小板减少综合征进展的1期、2期、3期病程中,极速实时荧光PCR的阳性检出率为41份(97.62%)、34份(94.44%)、7份(87.50%),实时荧光PCR的阳性检出率为39份(92.86%)、33份(91.67%)、7份(87.50%),在1期和2期两个病程,极速实时荧光PCR阳性检出率略高;IgM-ELISA阳性检出率从1期(28.57%)到3期(87.50%)显著增高,2期、3期与1期相比,差异均有统计学意义(χ^2=8.347、7.561,均P<0.01);IgM-GICA的阳性检出率从1期(14.29%)到2期(33.33%)也有增高,差异有统计学意义(χ^2=3.962,P<0.05),但与其他方法相比,其检出率偏低。1期,实时荧光PCR阳性检出率显著高于ELISA(IgM和IgG)和GICA(IgM和IgG),差异均有统计学意义(χ^2=33.740、55.080、49.010、64.340,均P<0.01)。2期,实时荧光PCR的阳性检出率高于ELISA(IgM和IgG)和GICA(IgM和IgG),差异均有统计学意义(χ^2=7.700、46.720、23.700、50.630,均P<0.01)。3期,极速实时荧光PCR、实时荧光PCR和IgM-ELISA表现出同样高的阳性检出率,远高于IgG-ELISA和GICA(IgM和IgG)。实时荧光PCR阳性检出率和IgG-ELISA、IgM-GICA、IgG-GICA之间差异均有统计学意义(均χ^2=6.250,P<0.05)。结论极速实时荧光PCR在新型布尼亚病毒的早期检测中有更高的灵敏度和特异度,且重复性好、稳定度高,与传统实时荧光PCR相比大大缩短了扩增时间,对发热伴血小板减少综合征的早期快速诊断具有重要价值。     

一腓骨肌萎缩症家系临床及基因分析

目的分析一腓骨肌萎缩症家系的临床表现及不同基因检测方法的特点。方法收集一CMT家系8名成员临床资料,并应用等位基因特异性PCR-双酶切方法及多重连接依赖的探针扩增技术(MLPA)检测PMP22基因突变情况,同时选择60名性别、年龄无明显差异的健康人做为对照组。结果该家系中患病者以行走不稳、跨阈步态,伴有弓形足为主要临床表现。该家系中5名成员经等位基因特异性PCR-双酶切及MLPA方法均检测出PMP22基因重复序列,其中出现临床症状的有4名(Ⅱ3、Ⅱ9、Ⅱ11、Ⅲ7),未出现临床症状但基因检测结果示PMP22基因重复序列的为携带者有1名(Ⅲ5),家系中余3名成员及对照组60名均未见重复序列。结论基因检测在明确CMT诊断中起重要作用,且MLPA法筛查基因时操作更简便、灵敏度更高、特异性更好。     

间期细胞原位PCR检测B细胞系恶性淋巴瘤

目的：应用原位PCR技术检测B细胞系恶性淋巴瘤。方法：采用免疫球蛋白重链（IgH)基因第三互补决定区（CDR－Ⅲ）的引物，dig-11-dUTP标记物，应用直接原位PCR技术检测Ⅲ-Ⅳ级恶性淋巴瘤的间期淋巴细胞，结果：在初诊的15例恶性淋巴瘤患中，12例检测到IgH基因重排；4例临床表现呈现淋巴瘤性白血病患，治疗后骨髓完全缓解，2例间期细胞原位PCR仍显示有IgH基因重排，6个月复发，结论：间期细胞原位PCR技术能敏感、准确地检测Ⅲ级以上的恶性淋巴瘤，并帮助预测复发，为一有效的基因诊断方法。     

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。