黄芩苷抗呼吸道合胞病毒感染作用的体外研究

目的探讨黄芩苷（baicalin）的体外抗呼吸道合胞病毒（RSV）感染作用及最佳浓度。方法应用微量细胞培养技术，观察RSV感染人喉癌（Hep-2）细胞病变效应（CPE）。观察不同浓度黄芩苷对人肺癌腺上皮（A549）细胞的CPE及RSV感染后CPE的抑制作用。应用四甲基偶氮唑蓝（MTT）比色法检测黄芩苷对A549细胞的毒性作用和RSV感染后细胞存活率的影响。结果黄芩苷浓度≤0．156mg／ml时，A549细胞未显示明显CPE，MTT比色法结果与CPE观察结果相符；浓度在0．078～1．25mg／ml时，RSV感染的A549细胞仅出现轻微CPE，0．625mg／ml时对CPE有最好的保护作用；治疗指数（Ⅱ）为19．23；MTT比色法也显示以上浓度范围黄芩苷组的细胞存活率明显高于RSV感染组，差异有统计学意义（P均〈0．05），其中以0．3125mg/ml组的细胞存活率最高，达82．09％。结论黄芩苷具有体外抗RSV感染作用，有效浓度为0．078～1．25mg／ml，最佳作用浓度为0．156mg／ml。     

病毒唑体外抑制RSV和ADV3作用强度的研究

目的：了角病毒唑在体外抑制呼吸道合胞病毒（RSV）和腺病毒3型（ADV3）的作用强度，对临床应用该药的理论依据进行验证，方法：采用观察Hep-2和Hela细胞病理变化方法，判断该药抗病毒作用的强度。结果：病毒唑在Hep-2和Hela细胞上完全抑制RSV和ADV3的剂量为7．8μg/ml。结论：在体外，小剂量的病毒唑有很强的抗RSV和ADV3的作用。     

咳喘平与病毒唑联合应用体外对呼吸道合胞病毒抑制作用的研究

目的了解咳喘平、病毒唑及二者联合应用时对呼吸道合胞病毒(RSV)的体外抑制作用。方法观察不同药物浓度下RSV对Hep-2细胞的致病作用。结果病毒唑完全抑制RSV复制的最小浓度为7·8μg/ml,咳喘平为13·75mg/ml,两药联合应用时,病毒唑和咳喘平完全抑制RSV的最小浓度分别减少到1·95μg/ml和3·44mg/ml,比单独用药浓度降低了4倍。结论两种药物对RSV都有体外抑制作用,联合应用时效果最强。     

五种中药制剂体外抗呼吸道病毒效果及细胞毒性的研究

目的了解5种中药制剂体外抗流感病毒、柯萨奇病毒和呼吸道合胞病毒的效果,为抗病毒性感冒新药研发提供依据。方法建立流感病毒感染MDCK细胞、柯萨奇病毒和呼吸道合胞病毒感染VeroE6细胞的模型,以细胞病变效应（CPE）为观察指标,了解不同浓度5种中药制剂体外抗病毒效果及细胞毒性。实验中以病毒唑作为对照药。结果1～5号中药制剂对MDCK细胞未出现细胞毒性的最小稀释度分别为1：20、1：40、1：20、1：80和1：400,对VeroE6细胞则为1：40、1：80、1：20、1：20和1：100。1～5号中药制剂出现对流感病毒的IC50值分别为1：200、1：100、1：400、1：200和1：400,对柯萨奇病毒的IC50值分别为1：50、1：50、1：100、1：200和1：200．对呼吸道舍胞病毒的IC50值分别为〈1：25、〈1：50、1：200、〈1：25和1：100。结论综合药效和细胞毒性实验结果,3号中药制剂可以作为抗病毒性感冒新药的首选制剂．其次为4号中药制剂。     

黄菊液抗单纯疱疹病毒I型的实验研究

目的 :测定黄菊液在细胞培养条件下抗单纯疱疹病毒Ⅰ型 (HSV -I)的作用。方法 :将不同浓度的黄菊液以 4种方法与不同感染剂量的HSV -Ⅰ作用后 ,加入细胞培养板内观察病毒致细胞病变作用 (CPE) ,以能抑制最大病毒感染量的药物浓度为判断结果的标准。结果 :管外法和治疗法在病毒感染剂量为 2 5TCID5 0 时 ,可抑制CPE的出现。     

商陆提取蛋白体外抗柯萨奇病毒的实验研究

目的:探讨商陆提取蛋白(PAP)在体外抗柯萨奇病毒(CVB3)的效应。方法:(1)用MTT法检测PAP对Hela细胞的毒性作用;(2)在Hela细胞上采用微量细胞病变抑制法观察PAP直接灭活CBV3的作用。结果:(1)连续作用6dPAP对Hela细胞的50%毒性浓度依次降低;(2)实验的第1、2、3d,0.1至25mg/L-3浓度的PAP能抑制100TCID50和1000TCID50柯萨奇病毒在胞内复制;(3)实验的第4、5、6d,0.1至25mg/L-3浓度的PAP作用于感染了CVB3的Hela细胞发生了病变。结论:在体外细胞培养上,商陆提取蛋白能起到阻断或减缓柯萨奇病毒吸附细胞的作用。     

呼吸道合胞病毒多克隆抗体的制备及临床意义

目的 制备呼吸道合胞病毒（RSV）多克隆抗体，为RSV的检测提供条件。方法 用RSV颗粒抗原免疫新西兰家兔3只，采用细胞病变中和实验检测RSV多克隆抗体。结果 细胞病变中和实验结果显示，本研究制备的RSV多克隆抗体在1：1102的稀释度能保护50％Hela细胞免受RSV的攻击，而阴性对照血清不能保护细胞对病毒的感染。结论 RSV颗粒抗原免疫新西兰家兔可成功制备RSV多克隆抗体，1：1102稀释的血清可保护50％的细胞不产生病变。     

金丝桃素和盐酸赖氨酸抗疱疹病毒作用体外试验研究

目的：通过评价金丝桃素、盐酸赖氨酸体外抗疱疹病毒活性，为进一步研究疱疹后遗神经痛(PHN)以及继发抑郁的治疗提供更多理论支持。方法：采用对病毒昕致细胞病变(CPE)抑制法，观察金丝桃素和盐酸赖氨酸对Vero细胞和2BS细胞的细胞毒性，以及对单纯疱疹病毒1型(HSV-1)和水痘-带状疱疹病毒(VZV)的抗病毒活性。结果：金丝桃素对HSV-1和VZV抑制的最小有效浓度和治疗指数相同，分别为0．39mg／L和32；盐酸赖氨酸对HSV-1和VZV抑制的最小有效浓度都是19．5mg／L，治疗指数分别为64和129。结论：金丝桃素和盐酸赖氨酸在体外具有抗HSV-1和VZV作用。     

黄菊液抗单纯疱疹病毒Ⅰ型的实验研究

目的：测定黄菊液在细胞培养条件下抗单纯疱疹病毒Ⅰ型（ＨＳＶ－Ⅰ）的作用。方法：将不同浓度的黄菊液以４种方法与不同感染剂量的ＨＳＶ－Ⅰ作用后，加入细胞培养板内观察病毒致细胞病变作用（ＣＰＥ），以能抑制最大病毒感染量的药物浓度为判断结果的标准。结果：管外法和治疗法在病毒感染剂量为２５ＴＣＩＤ５０时，可抑制ＣＰＥ的出现。     

人巨细胞病毒临床分离株对更昔洛韦耐药的表型分析

目的检测分析人巨细胞病毒（HCMV）临床分离株的更昔洛韦（GCV）耐药表型。方法收集临床接受GCV治疗过的各类移植受体的血液或尿液标本以及婴幼儿HCMV感染症患儿的尿液标本,分离HCMV。然后通过噬斑减少试验测试其对GCV药物的耐受性。病毒株对GCV的耐受性程度表示以不加药物孔中病毒致细胞病变效应（CPE）为参照,能够抑制50％CPE的药物浓度（IC50）为GCV的耐受性。GCV敏感型毒株的IC50为≤5．000μmol／L。结果以标本接种人胚肺成纤维细胞（MRC-5）,最终获得病毒株共33株。其中6株来自移植受体,27株来自婴幼儿HCMV感染症患儿。以MRC-5为敏感细胞,调整所测病毒浓度为100×50％组织培养感染量（TCID50）,并加入不同浓度的GCV药物,检测发现32株GCV的IC50均≤5．000μmol／L（1．500～5．000μmol／L）,只有1株GCVIC50为12．500μmoL／L。同步平行检测的标准毒株HCMVAD169的GCVIC50为1．500μmol／L。结论药物耐受表型测定能够了解并评估病毒对GCV药物的敏感与否,但全过程周期较长,技术要求比较高,尤其必须以获得活病毒为前提,尚难以直接推向临床实验室。     

RSV感染气液相界面培养人支气管上皮细胞模型的建立及其对HMGB1和pMLKL表达的影响

目的 构建呼吸道合胞病毒（RSV）感染气液相界面（ALI）培养的人支气管上皮细胞（hBEC）模型,为深入研究呼吸道病毒致病机制提供更接近体内环境的细胞模型;通过分析RSV感染对高迁移率族蛋白1(HMGB1)和磷酸化混合系列蛋白激酶样结构域（pMLKL）表达的影响,探讨RSV感染损伤支气管上皮的致病机制。方法 将hBEC接种到Transwell膜上,进行液体浸没式培养,细胞汇合度达100%后转ALI培养,倒置显微镜观察细胞生长状态。细胞分化成熟后按照以下分组分别感染RSV病毒：6 h对照组,6 h感染复数（MOI）1.0组,6 h MOI 3.0组,24 h对照组,24 h MOI 1.0组,24 h MOI 3.0组。每组3个复孔。通过免疫荧光染色确认RSV感染效果和RSV感染对细胞核蛋白HMGB1和pMLKL表达的影响。结果 经细胞扩展期液体浸没式培养4～10 d后,细胞汇合度达100%。转ALI培养约4周后,细胞条索状分布越来越清晰,且分泌肉眼可见的黏液,形成黏液层,将Transwell膜石蜡包埋切片,得到典型的假复层上皮HE染色结果。经抗RSV抗体免疫荧光染色确认,MOI为3.0...     

Double-Blind Evaluation of Oral Ribavirin (Virazole) in Experimental Influenza A Virus Infection in Volunteers

The prophylactic effectiveness of oral administration of ribavirin (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) against experimentally induced influenza A infection was evaluated in a double-blind clinical trial in normal volunteers. Fourteen men received ribavirin capsules (1,000 mg/day in four divided doses) and 15 other men received identical-appearing placebo capsules beginning 6 h after the intranasal inoculation of 3.4 log₁₀ 50% tissue culture infectious doses of influenza virus A/Victoria/3/75 H3N2 and continuing for 5 days after challenge. The total number of moderate-to-severe symptom scores and the total number of temperatures ≥100°F (37.8°C) were significantly lower in the ribavirin group compared with the placebo group. The mean quantity of virus shed in nasal wash specimens and the total number of days that there were viral titers greater than 1.0 log₁₀ 50% tissue culture infectious doses per ml were significantly greater in the placebo group. There was no difference between the frequencies of virus isolated or the antibody responses in the two groups. Therefore, prophylactic ribavirin ameliorated symptoms and fever indicative of moderate-to-severe illness, but had no effect on the manifestations of mild illness in response to influenza A challenge. A transient rise in total serum bilirubin occurred in 29% of the ribavirin-treated volunteers and in none of the placebo-treated volunteers.     

利巴韦林雾化联合免疫球蛋白治疗年龄小于3个月婴儿呼吸道合胞病毒感染疗效观察

【目的】评价利巴韦林雾化联合免疫球蛋白治疗年龄小于3个月婴儿呼吸道合胞病毒（RSV）感染疗效。【方法】将入住本院治疗的年龄小于3个月婴儿呼吸道合胞病毒感染患儿100例按不同治疗方法分为联合治疗组和常规治疗组两组各50例,所有患儿均给予保持呼吸道通畅、止咳化痰、预防感染、维持内环境稳定等对症支持治疗,在此基础上联合治疗组给予利巴韦林雾化及免疫球蛋白（IVIG）静脉注射,常规组单用利巴韦林雾化,观察比较两组患儿的临床疗效。【结果】联合治疗组喘憋、呼吸急促持续时间显著短于常规组（P〈0．05）;肺部哆音消失时间明显早于常规组（P〈0．05）;总有效率（96．0％）显著高于常规组（82％）。【结论】利巴韦林雾化联合免疫球蛋白治疗年龄小于3个月婴儿婴儿呼吸道合胞病毒感染可明显提高临床疗效。     

Significance of serum ribavirin concentration in combination therapy of interferon and ribavirin for chronic hepatitis C

OBJECTIVE: The purpose of this clinical retrospective cohort study was to determine the most suitable ribavirin concentration on combination therapy of interferon (IFN)-ribavirin. METHODS: Entry criteria were serum HCV-RNA level >/=100 KIU/ml, HCV-genotype 1b, chronic hepatitis, and initial combination treatment of IFN-alpha-2b (6 million units daily for 2 weeks and then 3 times weekly for 6 weeks) and ribavirin (600-800 mg/day) for 8 weeks without stopping or decreasing the dosage of IFN and/or ribavirin. Sixty-eight consecutive patients who satisfied the above criteria were given maintenance therapy for another 16 weeks. RESULTS: A sustained virological response (SVR) rate of 25.0% (17/68) was seen in all subjects. The SVR rate was 44.0% (11/25) in the high ribavirin group with a serum ribavirin concentration of >/=3,000 ng/ml at 8 weeks after initiation of combination therapy. SVR was significantly dependent at a serum ribavirin level of >/=3,000 ng/ml (p = 0.005). The incidence of discontinuations and dose modifications for combination therapy in patients having a serum ribavirin concentration of >/=3,500 ng/ml 8 weeks after initiation of therapy was 57.1% (4/7). This value was statistically higher than that in patents with <3,500 ng/ml (p = 0.033). CONCLUSION: Our results showed that the most suitable serum ribavirin concentrations are from 3,000 to 3,500 ng/ml 8 weeks after initiation of combination therapy.     

GS-5806 Inhibits a Broad Range of Respiratory Syncytial Virus Clinical Isolates by Blocking the Virus-Cell Fusion Process

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants and young children. In addition, RSV causes significant morbidity and mortality in hospitalized elderly and immunocompromised patients. Currently, only palivizumab, a monoclonal antibody against the RSV fusion (F) protein, and inhaled ribavirin are approved for the prophylactic and therapeutic treatment of RSV, respectively. Therefore, there is a clinical need for safe and effective therapeutic agents for RSV infections. GS-5806, discovered via chemical optimization of a hit from a high-throughput antiviral-screening campaign, selectively inhibits a diverse set of 75 RSV subtype A and B clinical isolates (mean 50% effective concentration [EC₅₀] = 0.43 nM). The compound maintained potency in primary human airway epithelial cells and exhibited low cytotoxicity in human cell lines and primary cell cultures (selectivity > 23,000-fold). Time-of-addition and temperature shift studies demonstrated that GS-5806 does not block RSV attachment to cells but interferes with virus entry. Follow-up experiments showed potent inhibition of RSV F-mediated cell-to-cell fusion. RSV A and B variants resistant to GS-5806, due to mutations in F protein (RSV A, L138F or F140L/N517I, and RSV B, F488L or F488S), were isolated and showed cross-resistance to other RSV fusion inhibitors, such as VP-14637, but remained fully sensitive to palivizumab and ribavirin. In summary, GS-5806 is a potent and selective RSV fusion inhibitor with antiviral activity against a diverse set of RSV clinical isolates. The compound is currently under clinical investigation for the treatment of RSV infection in pediatric, immunocompromised, and elderly patients.     

A randomized,placebo‐controlled study to evaluate safety and pharmacokinetics of inhaled ribavirin

Ribavirin is an inosine monophosphate dehydrogenase inhibitor. Studies suggest ribavirin aerosol could be a safe and efficacious treatment option in the fight against coronaviruses. However, current treatment is long (12–18 h per day, 3–7 days), limiting clinical utility. A reduction in treatment time would reduce treatment burden. We aimed to evaluate safety and pharmacokinetics (PK) of four, single‐dose regimens of ribavirin aerosol in healthy volunteers. Thirty‐two subjects were randomized, to four cohorts of aerosolized ribavirin (active) or placebo. Cohort 1 received 50 mg/ml ribavirin/placebo (10 ml total volume); cohort 2, 50 mg/ml ribavirin/placebo (20 ml total volume); cohort 3, 100 mg/ml ribavirin/placebo (10 ml total volume); and cohort 4, 100 mg/ml ribavirin/placebo (20 ml total volume). Intense safety monitoring and PK sampling took place on days 1, 2, 3, and 40. Subjects were (mean ± SD, active vs. placebo) aged 57 ± 4.5 vs. 60 ± 2.5 years; 83% vs. 88% were female; and 75% vs. 50% were Caucasian. Some 12.5% (3/24) and 25% (2/8) experienced at least one treatment‐emergent adverse event (TEAE) (two moderate; five mild) in the active and placebo groups, respectively. No clinically significant safety concerns were reported. Mean maximum observed concentration (C _max) and area under the curve (AUC) values were higher in cohort 4, whereas cohorts 2 and 3 showed similar PK values. Ribavirin absorption reached C _max within 2 h across cohorts. Four single‐dose regimens of ribavirin aerosol demonstrated systemic exposure with minimal systemic effects. Results support continued clinical development of ribavirin aerosol as a treatment option in patients with coronaviruses.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

Ribavirin is an inosine monophosphate dehydrogenase inhibitor, an enzyme in the synthesis of purine nucleotides, and a broad‐spectrum antiviral agent. It is approved in the USA and Canada for the treatment of lower respiratory tract infections in hospitalized infants and children due to the respiratory syncytial virus (RSV). Early data suggest that ribavirin is safe and effective in the treatment of COVID‐19. However, RSV treatment procedures are lengthy (12–18 h per day for 3−7 days), limiting wider clinical utility. A shorter treatment time, while maintaining safety and efficacy, is required for ribavirin to become a practical treatment option for coronaviruses.

• WHAT QUESTION DID THIS STUDY ADDRESS?

We performed this study to evaluate the safety and pharmacokinetics (PK) of four, single‐dose, clinically relevant regimens of ribavirin aerosol in healthy volunteers. Doses ranged from 50 to 100 mg/ml, delivered in a single inhalation of either 20 or 40 min duration.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

Our results showed that four single‐dose regimens of ribavirin aerosol were safe and well‐tolerated, without dose‐limiting toxicities, and a comparable safety profile to placebo. The PK were linear and well‐tolerated across the four single‐dose regimens, demonstrating systemic exposure with minimal systemic effects.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

In the context of coronaviruses, delivery of drug directly to the site of infection (the respiratory tract) is key. As such, these results support the continued clinical development of ribavirin aerosol as a new treatment option in patients with coronaviruses.     

Phase I study of intravenous ribavirin treatment of respiratory syncytial virus pneumonia after marrow transplantation.

Respiratory syncytial virus (RSV) pneumonia in marrow transplant recipients is associated with significant mortality. Ribavirin is a nucleoside analog with activity against RSV and in its aerosolized formulation is the only drug approved for treatment of RSV pneumonia in the United States. The clinical use of aerosolized ribavirin has been limited by caregivers' concerns about drug exposure and potential teratogenic effects. Since there is lack of proven efficacy and safety of the aerosolized ribavirin in this setting, we performed a phase I study of intravenous ribavirin treatment. Between November 1993 and May 1994, 10 patients with clinically significant RSV pneumonia at the Fred Hutchinson Cancer Research Center were enrolled. Only 2 of the 10 survived (20%; 95% CI, 3-56). Two of the 10 patients developed acute hemolysis that necessitated discontinuation of the medication. In conclusion, treatment of marrow transplant recipients with RSV pneumonia with intravenous ribavirin did not improve mortality compared with historical controls treated with the aerosolized drug.     

间充质干细胞对脐血CIK／NK细胞CD69表达及培养体系中T调节细胞量比的影响

本研究通过Transwell细胞隔离培养体系探讨骨髓源间充质干细胞（MSC）对脐血（CB）来源细胞因子诱导杀伤细胞（CIK）／自然杀伤细胞（NK）CD69的表达以及对培养体系中CD4^＋CD25^＋T调节（Treg）细胞量比的影响。利用聚碳酯膜孔径0．4μm的Transwell隔离细胞培养系统将实验组分为隔离培养（Transwell）组和直接混合（mixture）培养组;将MSC和CIK／NK细胞按1：20、1：50和1：100的比例分别在Transwell组和mixture组中进行隔离和直接混合培养,每组6个重复孔;培养48小时后通过流式细胞术检测各纽CIK／NK细胞上CD69的表达以及培养体系中CD4^＋CD25^＋细胞量比的变化。结果显示：加入MSC的实验各组CB-CIK细胞和NK细胞上表达的CD69比例均显著低于对照组（P值均〈0．001）．Transwell组中的CIK和NK细胞上表达的CD69,在MSC的高浓度组（1：20组）均显著低于低浓度组（1：50组和1：100组）,其中CIK细胞组间的P值分别为0．046、0．020;NK细胞组间的P值均为0．000。mixture组中不同比例组问CIK细胞上表达的CD69无显著性差异,而NK细胞上CD69的表达在MSC的高浓度组（1：20组）均显著低于低浓度组（1：50组和1：100组）。加入MSC的CB—CIK／NK细胞体系中的CD4^＋CD25^＋细胞的量比不论在Transwell还是mixture组中均显著高于对照组（P值均〈0．01）;在Transwell组中．CIK／NK细胞体系中的CD4^＋CD25^＋细胞的量比在1：20组、1：50组均显著高于1：100组;在mixture各组中,CIK／NK细胞体系中的CD4^＋CD25^＋T调节细胞的量比均有显著性差异。MSC的高浓度组（1：20组）,CIK／NK细胞体系中的CD4^＋CD25^＋细胞量比在mixture组显著高于Transwell组;而在MSC的低浓度组（1：50纽和1：100组）,2纽体系中的C04^＋CD25^＋细胞量比无显著性差异。结论：MSC能以直接接触或间接作用的方式抑制异基因CIK／NK细胞的活化,这可能与MSC能上调培养体系中的CD4^＋CD25^＋Treg细胞量比有关,且这种作用与MSC的数量呈浓度依赖关系。     

Efficacy of Oral Ribavirin in Hematologic Disease Patients with Paramyxovirus Infection: Analytic Strategy Using Propensity Scores

Few antiviral agents are available for treating paramyxovirus infections, such as those involving respiratory syncytial virus (RSV), parainfluenza virus (PIV), and human metapneumovirus (hMPV). We evaluated the effect of oral ribavirin on clinical outcomes of paramyxovirus infections in patients with hematological diseases. All adult patients with paramyxovirus were retrospectively reviewed over a 2-year period. Patients who received oral ribavirin were compared to those who received supportive care without ribavirin therapy. A propensity-matched case-control study and a logistic regression model with inverse probability of treatment weighting (IPTW) were performed to reduce the effect of selection bias in assignment for oral ribavirin therapy. A total of 145 patients, including 64 (44%) with PIV, 60 (41%) with RSV, and 21 (15%) with hMPV, were analyzed. Of these 145 patients, 114 (78%) received oral ribavirin and the remaining 31 (21%) constituted the nonribavirin group. Thirty-day mortality and underlying respiratory death rates were 31% (35/114) and 12% (14/114), respectively, for the oral ribavirin group versus 19% (6/31) and 16% (5/31), respectively, for the nonribavirin group (P = 0.21 and P = 0.56). In the case-control study, the 30-day mortality rate in the ribavirin group was 24% (5/21) versus 19% (4/21) in the nonribavirin group (P = 0.71). In addition, the logistic regression model with IPTW revealed no significant difference in 30-day mortality (adjusted hazard ratio of 1.3; 95% confidence interval [95% CI] of 0.3 to 5.8) between the two groups. Steroid use (adjusted odds ratio, 5.67; P = 0.01) and upper respiratory tract infection (adjusted odds ratio, 0.07; P = 0.001) was independently associated with mortality. Our data suggest that oral ribavirin therapy may not improve clinical outcomes in hematologic disease patients infected with paramyxovirus.