上海地区在校中学生接种甲型H1N1流感疫苗前后抗体水平监测分析

 目的  了解上海地区在校中学生接种甲型H1N1流感疫苗前后的抗体水平;观察甲型H1N1流感疫苗对该人群的免疫保护作用。 方法    应用常规微量血凝抑制试验（micro-hemagglutination inhibition test, HIT）对上海地区在校中学生分3个时间段进行甲型H1N1流感病毒抗体的血清学监测,3个时间段的抗体阳性率比较采用Pearson χ2 检验进行分析。 结果  2009年甲型H1N1流感流行前上海地区在校中学生的血清抗体阳性率仅为1.3%。经过一段时间流行后,血清抗体阳性率升至8.5%。接种甲型H1N1流感疫苗后,血清抗体阳性率升至87.3%。经Pearson χ² 检验分析,3个时间段在校中学生的血清甲型H1N1流感抗体阳性率差异有统计学意义（χ²=243.7,P<0.05）。 结论  在甲型H1N1流感流行前,上海地区在校中学生对其几乎没有免疫力,接种甲型H1N1流感疫苗能为该人群提供较好的免疫保护作用。     

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.     

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.     

A single intramuscular injection of neuraminidase inhibitor peramivir demonstrates antiviral activity against novel pandemic A/California/04/2009 (H1N1) influenza virus infection in mice

New and emerging influenza virus strains, such as the pandemic influenza A (H1N1) virus require constant vigilance for antiviral drug sensitivity and resistance. Efficacy of intramuscularly (IM) administered neuraminidase (NA) inhibitor, peramivir, was evaluated in mice infected with recently isolated pandemic A/California/04/2009 (H1N1, swine origin, mouse adapted) influenza virus. A single IM injection of peramivir (four dose groups), given 1h prior to inoculation, significantly reduced weight loss (p < 0.001) and mortality (p < 0.05) in mice infected with LD90 dose of pandemic A/California/04/2009 (H1N1) influenza virus compared to vehicle group. There was 20% survival in the vehicle-treated group, whereas in the peramivir-treated groups, survival increased in a dose-dependent manner with 60, 60, 90 and 100% survivors for the 1, 3, 10, and 30 mg/kg doses, respectively. Weight loss on day 4 in the vehicle-treated group was 3.4 gm, and in the peramivir-treated groups was 2.1, 1.5, 1.8 and 1.8 g for the 1, 3, 10 and 30 mg/kg dose groups, respectively. In the treatment model, peramivir given 24h after infection as a single IM injection at 50mg/kg dose, showed significant protection against lethality and weight loss. There was 13% survival in the vehicle-treated group while in the peramivir-treated group at 24, 48, and 72 h post infection, survival was 100, 40, and 50%, respectively. Survival in the oseltamivir groups (10 mg/kg/d twice a day, orally for 5 days) was 90, 30 and 20% at 24, 48 and 72 h, respectively. These data demonstrate efficacy of parenterally administered peramivir against the recently isolated pandemic influenza virus in murine infection models.     

In vitro and in vivo effects of a long-acting anti-influenza agent CS-8958 (laninamivir octanoate, Inavir) against pandemic (H1N1) 2009 influenza viruses

Laninamivir is a novel neuraminidase inhibitor of influenza viruses and it has been reported that its prodrug, CS-8958 shows a long-lasting characteristics. Using viruses isolated in Nagasaki of pandemic (H1N1) 2009 influenza virus which cause pandemic in 2009, it was shown that laninamivir has a strong inhibitory activities against their neuraminidases and virus replication in cultured cells, and strong binding stability to the virus NA. Furthermore, a single intranasal administration of CS-8958 showed a superior reduction of virus load in lungs in mouse infection model. These suggest that CS-8958 will work as a long-acting neuraminidase inhibitor to an infection with pandemic (H1N1) 2009 influenza viruses as well.     

对禽流感H5N1灭活疫苗不同免疫方式诱导的免疫应答及异亚型保护研究

目的 观察禽流感H5N1灭活疫苗以不同方式免疫后诱导的免疫应答及抗异亚型病毒攻击的保护作用.方法 将H5N1灭活疫苗分别通过腹腔和滴鼻方式免疫BALB/c小鼠,同时以PBS作为对照;免疫后分别以PR8和H9N2病毒攻击,观察小鼠的体重变化和生存情况.采用ELISA对各组小鼠攻毒后不同时间的血清IgG及其亚类水平进行动态检测;流式细胞仪检测脾淋巴细胞亚群情况.采用t检验对各组数据进行比较.结果 PR8和H9N2病毒攻击后,各组小鼠体重均下降,但疫苗组小鼠于后期体重恢复正常,存活率分别为100%和70%-80%,而PBS组小鼠则全部死亡.无论以何种方式免疫,疫苗组的特异性IgG及其亚类水平均明显升高,其中以IgG2a水平升高更为明显.攻毒后疫苗组小鼠脾CD4+与CD8+T淋巴细胞比值出现下降(t=6.8017,P<0.01);滴鼻免疫组与腹腔免疫组相比降低更明显(t=3.9701,P<0.05).结论 H5N1疫苗免疫原性良好,可诱导较高水平的特异性抗体产生,诱导的抗体亚类以IgG2a为主.两种免疫方式均可对小鼠提供很好的异亚型保护,而滴鼻免疫能够诱导更强的CD8+T细胞应答.

Abstract：

Objective To observe immune responses and heterosubtypic protection elicited by an inactivated influenza H5N1 vaccine with different immunization routes in mice. Methods BALB/c mice were intraperitoneally injected or intranasally immunized with the inactivated H5N1 vaccine, the mice administered with PBS were used as control. Weight loss and survival in mice were observed after PR8 or a H9N2 virus challenge. Serum specific IgG antibody and its subclasses were detected by ELISA kits. Ratios of CD4+ /CD8+ lymphocytes in spleens of mice were assayed. The t-test was used in the comparison of different groups.Results After challenge, weight loss was found in all groups, but the weight of mice in vaccination groups returned to normal later. The mice in PBS groups all died. The vaccinated mice were completely protected against PR8 and partly protected against H9N2 virus. The level of IgG antibody increased significantly after vaccination, and the increase magnitude of IgG2a was higher than that of IgG1. The ratios of CD4+ /CD8+ lymphocytes in spleens of vaccinated mice decreased after challenge (t=6. 8017,P<0. 01) , especially in the intranasal group (t = 3. 9701, P < 0. 05 ). Conclusions Both intraperitoneal injection and intranasal immunization can induce a high level of specific IgG, especially the level of IgG2a, and provide protection against heterosubtypic viruses. Intranasal immunization seems to induce a higher level of CD8+ T cell response.     

对禽流感H5N1灭活疫苗与不同免疫方式诱导的免疫应答及异亚型保护研究

目的  观察禽流感H5N1灭活疫苗以不同方式免疫后诱导的免疫应答及抗异亚型病毒攻击的保护作用.  方法 将H5N1灭活疫苗分别通过腹腔和滴鼻方式免疫BALB/c小鼠,同时以PBS作为对照;免疫后分别以PR8和H9N2病毒攻击,观察小鼠的体重变化和生存情况.采用ELISA对各组小鼠攻毒后不同时间的血清IgG及其亚类水平进行动态检测;流式细胞仪检测脾淋巴细胞亚群情况.采用t检验对各组数据进行比较.   结果 PR8和H9N2病毒攻击后,各组小鼠体重均下降,但疫苗组小鼠于后期体重恢复正常,存活率分别为100%和70%-80%,而PBS组小鼠则全部死亡.无论以何种方式免疫,疫苗组的特异性IgG及其亚类水平均明显升高,其中以IgG2a水平升高更为明显.攻毒后疫苗组小鼠脾CD4+与CD8+T淋巴细胞比值出现下降(t=6.8017,P<0.01);滴鼻免疫组与腹腔免疫组相比降低更明显(t=3.9701,P<0.05).    结论   H5N1疫苗免疫原性良好,可诱导较高水平的特异性抗体产生,诱导的抗体亚类以IgG2a为主.两种免疫方式均可对小鼠提供很好的异亚型保护,而滴鼻免疫能够诱导更强的CD8+T细胞应答.     

Antiviral effects of Psidium guajava Linn. (guava) tea on the growth of clinical isolated H1N1 viruses: its role in viral hemagglutination and neuraminidase inhibition

Rapid evolution of influenza RNA virus has resulted in limitation of vaccine effectiveness, increased emergence of drug-resistant viruses and occurrence of pandemics. A new effective antiviral is therefore needed for control of the highly mutative influenza virus. Teas prepared by the infusion method were tested for their anti-influenza activity against clinical influenza A (H1N1) isolates by a 19-h influenza growth inhibition assay with ST6Gal I-expressing MDCK cells (AX4 cells) using fluorogenic quantification and chromogenic visualization. Guava tea markedly inhibited the growth of A/Narita/1/2009 (amantadine-resistant pandemic 2009 strain) at an IC(50) of 0.05% and the growth of A/Yamaguchi/20/06 (sensitive strain) and A/Kitakyushu/10/06 (oseltamivir-resistant strain) at similar IC(50) values ranging from 0.24% to 0.42% in AX4 cells, being 3.4- to 5.4-fold more potent than green tea (IC(50) values: 0.27% for the 2009 pandemic strain and 0.91% to 1.44% for the seasonal strains). In contrast to both teas, oseltamivir carboxylate (OC) demonstrated high potency against the growth of A/Narita/1/09 (IC(50) of 3.83nM) and A/Yamaguchi/20/06 (IC(50) of 11.57nM) but not against that of A/Kitakyushu/10/06 bearing a His274-to-Tyr substitution (IC(50) of 15.97μM). Immunofluorescence analysis under a confocal microscope indicated that both teas inhibited the most susceptible A/Narita/1/2009 virus at the initial stage of virus infection. This is consistent with results of direct inhibition assays showing that both teas inhibited viral hemagglutination at concentrations comparable to their growth inhibition concentrations but inhibited sialidase activity at about 8-times higher concentrations. Guava tea shows promise to be efficacious for control of epidemic and pandemic influenza viruses including oseltamivir-resistant strains, and its broad target blockage makes it less likely to lead to emergence of viral resistance.     

Oseltamivir-resistant pandemic A(H1N1) 2009 influenza viruses detected through enhanced surveillance in the Netherlands, 2009–2010

Enhanced surveillance of infections due to the pandemic A(H1N1) influenza virus, which included monitoring for antiviral resistance, was carried out in the Netherlands from late April 2009 through late May 2010. More than 1100 instances of infection with the pandemic A(H1N1) influenza virus from 2009 and 2010 [A(H1N1) 2009] distributed across this period were analyzed. Of these, 19 cases of oseltamivir-resistant virus harboring the H275Y mutation in the neuraminidase (NA) were detected. The mean 50% inhibitory concentration (IC₅₀) levels for oseltamivir- and zanamivir-susceptible A(H1N1) 2009 viruses were 1.4-fold and 2-fold, respectively, lower than for the seasonal A(H1N1) influenza viruses from 2007/2008; for oseltamivir-resistant A(H1N1) 2009 virus the IC₅₀ was 2.9-fold lower. Eighteen of the 19 patients with oseltamivir-resistant virus showed prolonged shedding of the virus and developed resistance while on oseltamivir therapy. Sixteen of these 18 patients had an immunodeficiency, of whom 11 had a hematologic disorder. The two other patients had another underlying disease. Six of the patients who had an underlying disease died; of these, five had received cytostatic or immunosuppressive therapy. No indications for onward transmission of resistant viruses were found. This study showed that the main association for the emergence of cases of oseltamivir-resistant A(H1N1) 2009 virus was receiving antiviral therapy and having drug-induced immunosuppression or an hematologic disorder. Except for a single case of a resistant virus not linked to oseltamivir therapy, the absence of detection of resistant variants in community specimens and in specimens from contacts of cases with resistant virus suggested that the spread of resistant A(H1N1) 2009 virus was limited. Containment may have been the cumulative result of impaired NA function, successful isolation of the patients, and prophylactic measures to limit exposure.     

Grippe A (H1N1) 2009. Revue générale

Influenza A(H1N1) 2009 is an acute contagious respiratory infection caused by influenza A virus subtype H1N1 appeared in 2009 and responsible for a pandemic. The new virus, different from the avian virus H5N1, is a variant containing genes from several known viruses from porcine, avian and human origin. Appeared originally in the northern hemisphere, the epidemic wave reached early most countries, and up to 24% of the population in metropolitan France. It is characterized by a low mortality estimated at 0.04 to 0.2%, and by a benign, or even asymptomatic presentation. However, more severe clinical expression has been observed in some subgroups, carrying or not risk factors (young, pregnant women). The preexisting immunity in a significant proportion of the population, the remarkable stability of the virus, determination of early antigenic characteristics of the virus, the development and rapid availability of suitable vaccines, the efficacy of antiviral drugs, and health care system contributed to the control the morbidity and mortality of the first pandemic wave. Other virological, clinical and epidemiological investigations, are needed to identify all potential risk factors for severity and determine the role of mutation and the diffusion of pandemic and seasonal viruses, which may alter the virulence and transmissibility of influenza A(H1N1)v 2009.     

Treatment of influenza A (H1N1) virus infections in mice and ferrets with cyanovirin-N

Cyanovirin-N (CV-N), a protein derived from Nostoc ellipsosporum, neutralizes influenza virus infectivity by binding to specific high-mannose oligosaccharides (oligomannose-8 and -9) at glycosylation sites on the viral hemagglutinin HA1 subunit. Mouse-adapted viruses lose sensitivity to CV-N due to HA1 mutations that eliminate these glycosylation sites. Recently we created a hybrid (reassortant) influenza A/WSN/33 (H1N1) virus containing the HA gene of A/New Caledonia/20/99 (H1N1) with an Asp225Gly mutation in the HA1, that was lethal to mice yet retained sensitivity to CV-N. We then utilized this model system to test the efficacy of CV-N against influenza. CV-N efficacy was dose-responsive from 0.0625 to 1 mg/kg/day when administered intranasally (i.n.) twice daily for 4 days starting 4 h prior to virus exposure. In a second study, survival benefit was seen with CV-N treatments (0.5 mg/kg/day for 4 days) beginning at −4 or +6 h, but was significantly reduced at +12 h. The early treatment resulted in up to 100% survival and 1000-fold reduction in lung virus titer on day 3 of the infection. In contrast, ribavirin (a positive control—75 mg/kg/day) treatment resulted in 30% survival and 30-fold decrease in lung virus titers. Lung consolidation scores and lung weights were significantly reduced by CV-N and ribavirin treatment on day 6 of the infection. Ferrets infected with a non-animal adapted influenza A/Charlottesville/31/95 (H1N1) virus were treated intranasally with CV-N (50 μg twice daily for 5 days starting 24 h before virus challenge). They exhibited 100-fold lower viral titers in nasal washes than placebos 1 day after treatment, but virus titers were equivalent on days 2–7. CV-N has the potential for prophylaxis and early initiation of treatment of influenza virus infections.     

Antiviral activity of the MEK-inhibitor U0126 against pandemic H1N1v and highly pathogenic avian influenza virus in vitro and in vivo

The emergence of the 2009 H1N1 pandemic swine influenza A virus is a good example of how this viral infection can impact health systems around the world in a very short time. The continuous zoonotic circulation and reassortment potential of influenza A viruses (IAV) in nature represents an enormous public health threat to humans. Beside vaccination antivirals are needed to efficiently control spreading of the disease. In the present work we investigated whether the MEK inhibitor U0126, targeting the intracellular Raf/MEK/ERK signaling pathway, is able to suppress propagation of the 2009 pandemic IV H1N1v (v = variant) as well as highly pathogenic avian influenza viruses (HPAIV) in cell culture and also in vivo in the mouse lung. U0126 showed antiviral activity in cell culture against all tested IAV strains including oseltamivir resistant variants. Furthermore, we were able to demonstrate that treatment of mice with U0126 via the aerosol route led to (i) inhibition of MEK activation in the lung (ii) reduction of progeny IAV titers compared to untreated controls (iii) protection of IAV infected mice against a 100× lethal viral challenge. Moreover, no adverse effects of U0126 were found in cell culture or in the mouse. Thus, we conclude that U0126, by inhibiting the cellular target MEK, has an antiviral potential not only in vitro in cell culture, but also in vivo in the mouse model.     

黄芩苷联合帕拉米韦体内外抗甲型H1N1流感病毒作用

目的评价黄芩苷与帕拉米韦联合用药体内外抗甲型H1N1流感病毒作用。方法体外试验中,以甲型H1N1流感病毒感染MDCK细胞,黄芩苷与帕拉米韦联合用药,终点稀释法检测细胞上清液病毒滴度;体内试验中,以甲型H1N1流感病毒感染BALB/c小鼠,黄芩苷灌胃,帕拉米韦肌肉注射,两者联合给药,观察试验小鼠存活情况及体重变化。试验结果以MacSynergyⅡ软件分析两种药物体内外联合作用结果。结果细胞试验中,黄芩苷与帕拉米韦联用抗甲型H1N1流感病毒在95%置信区间内的协同值为3.2,表现为相加作用;小鼠试验中,黄芩苷与帕拉米韦联用,对提高感染流感病毒小鼠的存活率和抑制其体重下降表现为显著协同作用,协同值分别为69.0和105.2。结论黄芩苷与帕拉米韦联合抗流感作用比单独使用效果好,在临床上具有重要的应用价值。     

Protective immunity elicited by a pseudotyped baculovirus-mediated bivalent H5N1 influenza vaccine

The development of novel H5N1 influenza vaccines to elicit a broad immune response is a priority in veterinary and human public health. In this report, a baculovirus vector was used to construct bivalent recombinant baculovirus vaccine encoding H5N1 influenza virus hemagglutinin proteins (BV-HAs) from clade 2.3.4 and clade 9 influenza viruses. Mice immunized with 5 × 10⁷ IFU BV-HAs developed significantly high levels of H5-specific neutralizing antibodies and cellular immunity that conferred 100% protection against infection with H5N1 influenza viruses. This study suggests that baculovirus-delivered multi-hemagglutinin proteins might serve as a candidate vaccine for the prevention of pre-pandemic and pandemic H5N1 influenza viruses.     

Treatment of influenza A (H1N1) virus infections in mice and ferrets with cyanovirin-N

Cyanovirin-N (CV-N), a protein derived from Nostoc ellipsosporum, neutralizes influenza virus infectivity by binding to specific high-mannose oligosaccharides (oligomannose-8 and -9) at glycosylation sites on the viral hemagglutinin HA1 subunit. Mouse-adapted viruses lose sensitivity to CV-N due to HA1 mutations that eliminate these glycosylation sites. Recently we created a hybrid (reassortant) influenza A/WSN/33 (H1N1) virus containing the HA gene of A/New Caledonia/20/99 (H1N1) with an Asp225Gly mutation in the HA1, that was lethal to mice yet retained sensitivity to CV-N. We then utilized this model system to test the efficacy of CV-N against influenza. CV-N efficacy was dose-responsive from 0.0625 to 1 mg/kg/day when administered intranasally (i.n.) twice daily for 4 days starting 4 h prior to virus exposure. In a second study, survival benefit was seen with CV-N treatments (0.5 mg/kg/day for 4 days) beginning at −4 or +6 h, but was significantly reduced at +12 h. The early treatment resulted in up to 100% survival and 1000-fold reduction in lung virus titer on day 3 of the infection. In contrast, ribavirin (a positive control—75 mg/kg/day) treatment resulted in 30% survival and 30-fold decrease in lung virus titers. Lung consolidation scores and lung weights were significantly reduced by CV-N and ribavirin treatment on day 6 of the infection. Ferrets infected with a non-animal adapted influenza A/Charlottesville/31/95 (H1N1) virus were treated intranasally with CV-N (50 μg twice daily for 5 days starting 24 h before virus challenge). They exhibited 100-fold lower viral titers in nasal washes than placebos 1 day after treatment, but virus titers were equivalent on days 2–7. CV-N has the potential for prophylaxis and early initiation of treatment of influenza virus infections.     

Progress in identifying virulence determinants of the 1918 H1N1 and the Southeast Asian H5N1 influenza A viruses

The 1918 pandemic H1N1 influenza virus and the recently emerged Southeast Asian H5N1 avian influenza virus are unique among influenza A virus isolates in their high virulence for humans and their lethality for a variety of animal species without prior adaptation. Reverse genetic studies have implicated several viral genes as virulence determinants. For both the 1918 and H5N1 viruses, the hemagglutinin and the polymerase complex contribute to high virulence. Non-structural proteins NS1 and PB1-F2, which block host antiviral responses, also influence pathogenesis. Additionally, recent studies correlate high levels of viral replication and induction of strong proinflammatory responses with the high virulence of these viruses. Defining how individual viral proteins promote enhanced replication, inflammation and severe disease will provide insight into the pathogenesis of severe influenza virus infections and suggest novel therapeutic approaches.     

Long-lasting protective immunity against H7N9 infection is induced by intramuscular or CpG-adjuvanted intranasal immunization with the split H7N9 vaccine

There is an urgent need for efficient vaccines against the highly pathogenic avian influenza A viral strain H7N9. The duration and intensity of the immune response to H7N9 critically impacts the epidemiology of influenza viral infection at the population level. However, the insufficient immunogenicity of H7N9 raises concerns about vaccine efficacy. In this study, we evaluated the impact of immunization routes and the adjuvant CpG on the immune response to a split H7N9 vaccine in mice. Determination of humoral and cellular responses to the vaccine revealed that after four vaccine doses, high titers of H7N9-specific serum IgG, determined by the influenza hemagglutination inhibition (HI) assay, were induced through the intramuscular (i.m.) route and lasted for at least 40 weeks. CpG-adjuvanted immunization increased the levels of long-lived IFN-γ⁺ T cells and raised the Th1-biased IgG2a/IgG1 response ratio. In addition, aside from mucosal IgA, CpG-adjuvanted intranasal (i.n.) immunization elicited serum IgG and cellular responses of a similar duration and intensity to CpG-adjuvanted i.m. immunization. Mouse challenge assays demonstrated that 24 weeks following i.m. immunization without CpG or CpG-adjuvanted immunization through the i.m. or i.n. routes, both offered a high level of protection against H7N9 infection. These results indicate that efficient long-term protection against H7N9 can be achieved via the optimization of vaccination strategies, such as immunization doses, routes, and adjuvants.     

In vitro and in vivo efficacy of fluorodeoxycytidine analogs against highly pathogenic avian influenza H5N1, seasonal, and pandemic H1N1 virus infections

Various fluorodeoxyribonucleosides were evaluated for their antiviral activities against influenza virus infections in vitro and in vivo. Among the most potent inhibitors was 2′-deoxy-2′-fluorocytidine (2′-FdC). It inhibited various strains of low and highly pathogenic avian influenza H5N1 viruses, pandemic H1N1 viruses, an oseltamivir-resistant pandemic H1N1 virus, and seasonal influenza viruses (H3N2, H1N1, influenza B) in MDCK cells, with the 90% inhibitory concentrations ranging from 0.13 to 4.6 μM, as determined by a virus yield reduction assay. 2′-FdC was then tested for efficacy in BALB/c mice infected with a lethal dose of highly pathogenic influenza A/Vietnam/1203/2004 H5N1 virus. 2′FdC (60 mg/kg/d) administered intraperitoneally (i.p.) twice a day beginning 24 h after virus exposure significantly promoted survival (80% survival) of infected mice (p = 0.0001). Equally efficacious were the treatment regimens in which mice were treated with 2′-FdC at 30 or 60 mg/kg/day (bid X 8) beginning 24 h before virus exposure. At these doses, 70-80% of the mice were protected from death due to virus infection (p = 0.0005, p = 0.0001; respectively). The lungs harvested from treated mice at day four of the infection displayed little surface pathology or histopathology, lung weights were lower, and the 60 mg/kg dose reduced lung virus titers, although not significantly compared to the placebo controls. All doses were well tolerated in uninfected mice. 2′-FdC could also be administered as late as 72 h post virus exposure and still significantly protect 60% mice from the lethal effects of the H5N1 virus infection (p = 0.019). Other fluorodeoxyribonucleosides tested in the H5N1 mouse model, 2′-deoxy-5-fluorocytidine and 2′-deoxy-2′,2′-difluorocytidine, were very toxic at higher doses and not inhibitory at lower doses. Finally, 2′-FdC, which was active in the H5N1 mouse model, was also active in a pandemic H1N1 influenza A infection model in mice. When given at 30 mg/kg/d (bid X 5) beginning 24 h before virus exposure), 2′-FdC also significantly enhanced survival of H1N1-infected mice (50%, p = 0.038) similar to the results obtained in the H5N1 infection model using a similar dosing regimen (50%, p < 0.05). Given the demonstrated in vitro and in vivo inhibition of avian influenza virus replication, 2′FdC may qualify as a lead compound for the development of agents treating influenza virus infections.     

Genotypic and phenotypic resistance of pandemic A/H1N1 influenza viruses circulating in Germany

In response to the rapid global spread of an antigenically novel A/H1N1 influenza virus in 2009, the World Heath Organization (WHO) recommended surveillance and monitoring for antiviral resistance of influenza viruses. We designed and evaluated pyrosequencing (PSQ)-based genotypic assays for high-throughput analysis of the susceptibility of pandemic A/H1N1 influenza viruses to neuraminidase (NA) inhibitors. A total of 1570 samples circulating in Germany between April 2009 and April 2010 were tested for determination of molecular markers of resistance to the NA inhibitors oseltamivir and zanamivir, and 635 of them were evaluated by phenotypic fluorescence-based assay with MUNANA substrate. Eight (0.5%) viruses were resistant to oseltamivir due to the H274Y NA substitution (N2 numbering). Six of these oseltamivir-resistant cases were treatment-related; four of them were selected in immunocompromised patients, two in patients suffered from chronic diseases. The two remaining oseltamivir-resistant viruses seem to have evolved in the absence of drug treatment and were isolated from immunocompetent healthy patients. All tested A/H1N1 pandemic viruses were sensitive to zanamivir. In addition, analysis of 1011 pandemic A/H1N1 virus samples by a PSQ-based assay according to the WHO protocol revealed the presence of mutation S31N in the M2 protein that conferred resistance to M2 ion channel inhibitors. Our data demonstrate a low incidence of oseltamivir-resistant pandemic A/H1N1 influenza variants isolated under drug selection pressure as well as community-acquired or naturally evolving viruses.     

Oseltamivir-resistant influenza A(H1N1) 2009 virus in Greece during the post-pandemic 2010-2011 season

Information on the drug susceptibility of influenza epidemic strains is important for antiviral resistance monitoring. In Greece, the 2009-2010 pandemic waves were very mild and seroprevalence rates remained low after this influenza season, resulting in exclusive detection of the pandemic strain during the 2010-2011 influenza season. In the present study during the post-pandemic 2010-2011 season, 50 consecutive influenza A(H1N1) 2009 virus-positive samples from patients hospitalised in Greek hospitals were analysed for resistance to the neuraminidase inhibitor oseltamivir. All patients were hospitalised with severe influenza complications and had previously received oseltamivir. Influenza A(H1N1) 2009 virus detection and testing for oseltamivir resistance were performed with real-time PCR amplification assays. The H275Y substitution associated with resistance to oseltamivir was identified in two immunocompetent patients who received oseltamivir treatment for 3 days and 5 days, respectively. In both cases, patients were discharged in good condition despite development of resistance to antiviral treatment.