Intramuscularly administered neuraminidase inhibitor peramivir is effective against lethal H5N1 influenza virus in mice

The replication efficiency and multi-organ dissemination of some influenza A (H5N1) viruses requires a rapid re-evaluation of the available antiviral strategies. We assessed five regimens of the neuraminidase (NA) inhibitor peramivir in mice inoculated with H5N1 virus. The regimens differed by: (1) frequency of administration on first day (once vs twice); (2) duration of administration (1 day vs 8 days); (3) route of administration (intramuscular [IM] injection alone or followed by oral administration). In all regimens, BALB/c mice were administered 30 mg/kg peramivir IM 1 h after lethal challenge with 5 MLD(50) of A/Vietnam/1203/04 (H5N1) influenza virus. When given only on the day of inoculation, a single IM injection produced a 33% survival rate, which increased to 55% with two injections. Eight-day regimens significantly increased survival; two IM injections followed by seven daily IM injections was the most effective regimen (100% survival; inhibition of replication in lungs and brain). When this 8-day regimen began at 24h after inoculation, 78% of mice survived; 56% survived when treatment began at 48 hours. Anti-HA antibody titer differed with the peramivir regimen and corresponded to the severity of disease. Overall, our results demonstrate that IM administration of peramivir is effective in promoting the survival of mice infected with systemically replicating H5N1 virus.     

Activity of ultra-low doses of antibodies to gamma-interferon against lethal influenza A(H1N1)2009 virus infection in mice

Background

The influenza A virus is a highly infective agent that causes acute pulmonary diseases. In serious cases, it causes pneumonia which is particularly fatal in patients with cardiopulmonary diseases, obesity, young children and elderly people. The present study shows a protective effect of ultra-low doses of purified antibodies to gamma-interferon (Anaferon for children®, AC®) against lethal influenza virus infection caused by pandemic influenza virus A(H1N1) in mice.

Methods

Balb/c mice were infected with mouse-adapted pandemic influenza virus A/California/07/09 (H1N1)v. Mortality, weight loss, infectious titer of the virus in lungs and lung morphology were monitored in the groups of AC®-, oseltamivir- and placebo-treated animals.

Results

The protective action of AC® was demonstrated by prolongation of life of the infected animals, reduction of infectious titer of the virus in the lung tissue, normalization of weight dynamics in the course of disease, decrease in mortality of treated animals compared to a placebo control and normalization of lung tissue structure. The protective activity of AC® was similar to that of the reference compound oseltamivir. Combination of AC® with oseltamivir resulted in a higher protective effect comparing to oseltamivir alone.

Conclusion

Based on the results obtained, AC® should be considered as an important part of anti-influenza prophylaxis and therapy, in particular in severe cases of the disease.     

Evaluation of intravenous zanamivir against experimental influenza A (H5N1) virus infection in cynomolgus macaques

We investigated the prophylactic and therapeutic efficacy of an intravenous (IV) formulation of zanamivir in a macaque infection model for highly pathogenic influenza A (H5N1) virus. Antiviral efficacy was dose-dependent, with no reduction in viral load observed at 2 mg/kg, but a significant reduction observed at 10 mg/kg (p=0.039) and at 20 mg/kg in the combined prophylactic and therapeutic groups (p=0.049) with both prophylaxis (commencing 12 h before infection) and therapy (commencing 4 h after infection) showing similar reductions in viral load. Combined gross pathology and microscopic pneumonia scores in the treated animals relative to untreated controls were significantly reduced at 10 mg/kg (p=0.02) and at 20 mg/kg in the prophylaxis group (p=0.02), but were not significant in the treatment group (p=0.145). In this new animal model for evaluation of influenza antivirals, despite variability observed between individual animals, IV zanamivir showed evidence of efficacy against highly pathogenic H5N1 virus.     

甲型H1N1流感的抗病毒治疗

2009年4月初,甲型H1N1流感开始在墨西哥和美国出现,2009年6月11日WHO将甲型H1N1流感大流行警告级别提升至6级,表明此次流感疫情已经进入大流行阶段,目前,甲型H1N1流感正在令球流行.     

CL-385319 inhibits H5N1 avian influenza A virus infection by blocking viral entry

CL-385319, an N-substituted piperidine, is effective in inhibiting infection of H1-, H2-, and to a lesser extent, H3-typed influenza A viruses by interfering with the fusogenic function of the viral hemagglutinin. Here we show that CL-385319 is effective in inhibiting infection of highly pathogenic H5N1 influenza A virus in Madin-Darby Canine Kidney (MDCK) cells with an IC50 of 27.03±2.54 μM. This compound with low cytotoxicity (CC50=1.48±0.01 mM) could also inhibit entry of pseudoviruses carrying hemagglutinins from H5N1 strains that were isolated from different places at different times, while it had no inhibitory activity on the entry of VSV-G pseudotyped particles. CL385319 could not inhibit N1-typed neuraminidase activity and the adsorption of H5-typed HA to chicken erythrocytes at the concentration as high as 1 mg/ml (2.8 mM). Computer-aid molecular docking analysis suggested that CL-385319 might bind to the cavity of HA2 stem region which was known to undergo significant rearrangement during membrane fusion. Pseudoviruses with M24A mutation in HA1 or F110S mutation in HA2 were resistant to CL-385319, indicating that these two residues in the cavity region may be critical for CL-385319 bindings. These findings suggest that CL-385319 can serve as a lead for development of novel virus entry inhibitors for preventing and treating H5N1 influenza A virus infection.     

湘西地区甲型H1N1流感危重患儿4例临床特点分析

目的探讨湘西自治州少数民族地区甲型H1N1流感危重患儿临床特点及诊疗体会。方法回顾性分析资料完整的本院儿科住院4例甲型H1N1流感危重患儿临床表现、实验室检查及影像学资料。结果 4例患儿早期无特异性表现,均为流感样症状。3例发热,1例病初体温未测,4例均咳嗽,少量痰液,病情加重可出现咳嗽加重,均有呼吸困难,无腹泻呕吐,2例3d未排大便,伴腹胀;随着病情进展都有多脏器功能不全,其中以肺脏受累最为突出。实验室检查：白细胞计数正常、降低或升高,多有肝肾功能异常。X线胸片提示双肺均有广泛受损害,表现为大片状高密度影。结论甲型H1N1流感危重患儿病情凶险,进展迅速,病死率高。因此,为了降低发病率,特别是降低危重症发病率,必须加强小于5岁的农村儿童甲型H1N1流感疫苗接种及防控工作,同时注重针对合并营养性缺铁性贫血儿童的早期治疗。早发现,早诊断,早期综合治疗,是降低甲型H1N1流感危重症病死率的关键。     

CpG oligodeoxynucleotides protect against the 2009 H1N1 pandemic influenza virus infection in a murine model

The 2009 H1N1 influenza virus pandemic poses a global public health threat, and there is a critical need for antiviral drugs for pandemic control. CpG oligodeoxynucleotides have strong immunostimulatory properties and are expected to be used as prophylactic agents to protect against microbial infections. The present study evaluated the efficacy of synthetic CpG oligodeoxynucleotide (ODN) 1826 against pandemic H1N1 virus infection in a murine model. A single injection of 15 μg ODN 1826 intraperitoneally prior to virus challenge inhibits virus replication in lungs, reduces lung lesions and prevents mortality in mice, indicating CpG ODNs as a possible strategy for future influenza pandemics control.     

热毒宁注射液体外抑制甲型H1N1流感病毒的研究

目的 研究热毒宁注射液体外抑制甲型H1N1流感病毒的作用。方法 以奥司他韦为阳性对照,采用CPE和MTT法观察热毒宁注射液对甲型H1N1流感病毒的抑制作用。结果 CPE法结果表明热毒宁注射液最大无毒浓度（TC₀）为16.2 mg/mL,半数中毒浓度为（TC₅₀）为（24.5±8.1）mg/mL;MTT法测定结果表明热毒宁注射液TC₀为16.2 mg/mL,TC₅₀为（21.7±9.4）mg/mL。热毒宁注射液体外抑制甲型H1N1流感病毒结果显示,CPE法和MTT法热毒宁注射液作用感染细胞1次组半数有效浓度（IC₅₀）为（900.0±173.2）、（933.3±57.7）μg/mL,治疗指数（TI）为27.2、23.2;热毒宁注射液作用感染细胞3次组IC₅₀为（666.7±115.5）、（866.7±208.1）μg/mL,TI为36.7、25.0。结论 热毒宁注射液具有明显体外抗甲型H1N1流感病毒的作用。     

Drug discovery against H1N1 virus (influenza A virus) via computational virtual screening approach

The H1N1 virus is the causative agent of the recent outbreak of Swine flu pandemic. Neuraminidase is an enzyme that cleave glycosidic linkage of neuraminic acid on viral cell surface and is known to occur as antigen determinant to evoke immune response in host cell. It plays an important role in life cycle of influenza virus. Inhibitors of neuraminidase are, therefore, believed to have a potential in development of new drugs against swine flu. Using a recently published model structure of neuraminidase, we have carried out virtual screening of 70 compounds obtained from Ligand databases. The ligands library also included 57 natural plant metabolites from medicinal plants. The virtual screening was performed via PatchDock & GemDock softwares. Two of the plant metabolites, Hesperidin & Narirutin showed significantly higher docking score than the currently marketed anti-influenza drug Oseltamivir (Tamiflu).     

Lactobacillus fermentum CJL-112 protects mice against influenza virus infection by activating T-helper 1 and eliciting a protective immune response

We have previously reported that nasally administered Lactobacillus fermentum CJL-112 (CJL-112) efficiently improves resistance against lethal influenza infection in both mice and chicken. The aim of the present study was to understand the underlying mechanisms of the significant anti-influenza activity of this lactobacilli strain. In vitro, co-culturing of the chicken macrophage cell line HD-11 with CJL-112 significantly increased nitric oxide (NO) production. In vivo, CJL-112 was nasally administered to BALB/c mice for 21 days prior to influenza A/NWS/33 (H1N1) virus (IFV) infection. Significant up-regulation of T-helper 1 (Th1) cytokines (IL-2, IFN-γ) was observed, while the levels of T-helper 2 (Th2) cytokines (IL-4, IL-5, IL-10) was either reduced or unchanged than that in control mice were. Furthermore, IgA and specific anti-influenza IgA levels increased significantly in the treated mice than those in untreated mice. Therefore, CJL-112 likely protects the mice against lethal IFV infection via stimulation of macrophages, activation of Th1 and augmentation of IgA production, when directly delivered into the respiratory tract.     

A case of pneumonia following human infection with avian influenza a (H5N1)

The H5N1 infection was diagnosed in 12 patients in Turkey and confirmed by the WHO. Of these 12 patients so far, 8 have been published. In this case, we are presenting a case of pneumonia that developed following avian influenza infection in Eskisehir. Our case is one of the 4 patients who were not reported previously.     

Computational investigation of oseltamivir resistance in influenza A (H5N1) virus

Oseltamivir is the most effective antiviral drug used for the treatment and prevention of influenza A infections. Neuraminidase is the principal target for treating patients with H5N1 infection. Until recently, only a low prevalence of neuraminidase inhibitors (NAIs) resistance (<1 %) had been detected in circulating viruses. However, there have been reports of significant numbers of influenza A (H5N1) strains with a H274Y neuraminidase mutation that was highly resistant to the NAI, oseltamivir. In this study, we used molecular docking and molecular dynamics (MD) approach to characterize the effect of H274Y mutation in drug–target interactions. Docking results suggest that oseltamivir was found to adopt the most promising conformations to the wild type NA (WT) by identifying the guanidinium side chain of R-156 and R-152 as a prospective partner for making polar contacts as compared to the mutant type NA. The MD results showed that the average atom, especially atoms of the wild type NA–oseltamivir complex, movements were small, displayed fast convergence of energy and charges in geometry. This highlights the stable binding of the oseltamivir with wild type NA as compared to mutant type NA. Overall, our study may be helpful for the rational design of more powerful, selective, and more robust NAI against drug-resistant H274Y mutation.     

新型甲型H1N1流感危重症23例临床分析

目的探讨新型甲型H1N1流感危重症的临床特征、诊断及治疗方法。方法对已经确诊的23例新型甲型H1N1流感危重症患者的临床特点、治疗过程、治疗效果进行综合分析。结果新型甲型H1N1流感危重症孕妇患病率达到47.9%,60%患者体温高于38.5℃。症状以咳嗽、咳痰、胸闷为主,且80%患者有湿啰音。影像为片絮状影,弥散实变影。血常规检查提示白细胞计数偏低,中性粒细胞、淋巴细胞、血小板与正常相比无统计学意义。生化检查中,谷草转氨酶、碱性磷酸酶明显升高,总胆红素及直接胆红素升高。乳酸脱氢酶及α-羟丁酸脱氢酶明显升高。患者的血肌酐、尿素氮正常,尿酸升高。血浆中钠、钾、氯、钙均降低。结论新型甲型H1N1流感危重症的体征、影像、生化均有别于其他病毒性肺炎,预后与就诊时间、妊娠密切相关,尽早终止妊娠与机械通气,糖皮质激素的早期大量应用可改善预后。     

Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1

Effective diagnostic and therapeutic strategies are needed to control and combat the highly pathogenic avian influenza virus (AIV) subtype H5N1. To this end, we developed human monoclonal antibodies (mAbs) in single chain fragment variable (scFv) format towards the H5N1 avian influenza virus to gain new insights for the development of immunotherapy against human cases of H5N1. Using a biopanning based approach a large array of scFvs against H5N1 virus were isolated from the human semi-synthetic ETH-2 phage antibody library. H5N1 ELISA-positive scFvs with unique variable heavy (VH) and light (VL) chain gene sequences showed different biochemical properties and neutralization activity across H5N1 viral strains. In particular, the scFv clones AV.D1 and AV.C4 exerted a significant inhibition of the H5N1 A/Vietnam/1194/2004 virus infection in a pseudotype-based neutralization assay. Interestingly, these two scFvs displayed a cross-clade neutralizing activity versus A/whooping swan/Mongolia/244/2005 and A/Indonesia/5/2005 strains. These studies provide proof of the concept that human mAbs in scFv format with well-defined H5N1 recognition patterns and in vitro neutralizing activity can be easily and rapidly isolated by biopanning selection of an entirely artificial antibody repertoire using inactivated H5N1 virus as a bait.     

H1N1 influenza A virus neuraminidase modulates infectivity in mice

In the 2years since the onset of the H1N1 2009 pandemic virus (H1N1pdm09), sporadic cases of oseltamivir-resistant viruses have been reported. We investigated the impact of oseltamivir-resistant neuraminidase from H1N1 Brisbane-like (seasonal) and H1N1pdm09 viruses on viral pathogenicity in mice. Reassortant viruses with the neuraminidase from seasonal H1N1 virus were obtained by co-infection of a H1N1pdm09 virus and an oseltamivir-resistant H1N1 Brisbane-like virus. Oseltamivir-resistant H1N1pdm09 viruses were also isolated from patients. After biochemical characterization, the pathogenicity of these viruses was assessed in a murine model. We confirmed a higher infectivity, in mice, of the H1N1pdm09 virus compared to seasonal viruses. Surprisingly, the oseltamivir-resistant H1N1pdm09 virus was more infectious than its sensitive counterpart. Moreover, the association of H1N1pdm09 hemagglutinin and an oseltamivir-resistant neuraminidase improved the infectivity of reassortant viruses in mice, regardless of the NA origin: seasonal (Brisbane-like) or pandemic strain. This study highlights the need to closely monitor the emergence of oseltamivir-resistant viruses.     

众志成城，抗击“甲流”

2009年，一个名词震惊了世界——“甲流”。2009年4月，自在墨西哥出现第1例“甲流”患者以后，短短几个月内甲型流感病毒感染就迅速蔓延至200多个国家。同年6月11日，世界卫生组织（WHO）正式宣布把甲型H1N1流感警戒级别提升至6级，这意味着WHO认为疫情已经发展为全球性的“流感大流行”。由于疫情肆虐，“甲流”疫苗作为对抗流感最有效的手段在第一时间内被提上了国家相关部门的议事日程。上海生物制品研究所是国内疫苗生产的龙头企业。在这国家危难之际，     

人用H5N1禽流感病毒疫苗生产工艺的优化

目的 建立优化的人用H5N1禽流感病毒疫苗生产的工艺。方法 在不同的稀释倍数、收获时间及灭活剂添加量下,通过测量收获液的病毒滴度和血凝效价,来确定病毒的最佳生产条件,并对离心法和凝胶过滤层析法的纯化效果进行对比。结果 103~104半数鸡胚感染量(50% egg infective dose,EID50)病毒接种鸡胚,收获的鸡胚尿囊液的病毒滴度和血凝效价最高,分别为10-8.3EID50和1∶480;在56~72 h血凝效价最高。甲醛浓度1∶10 000灭活144 h为灭活最佳条件。两种纯化方法得到的样品纯度和卵清蛋白的去除率相近,但离心纯化法和凝胶过滤层析纯化法病毒回收率有较大的差异,分别为19%和70%。结论 成功建立了高产毒的鸡胚基质H5N1禽流感病毒培养、灭活及纯化工艺。