A Therapeutically Active Minibody Exhibits an Antiviral Activity in Oseltamivir-Resistant Influenza-Infected Mice via Direct Hydrolysis of Viral RNAs

Emerging Oseltamivir-resistant influenza strains pose a critical public health threat due to antigenic shifts and drifts. We report an innovative strategy for controlling influenza A infections by use of a novel minibody of the 3D8 single chain variable fragment (scFv) showing intrinsic viral RNA hydrolyzing activity, cell penetration activity, and epidermal cell penetration ability. In this study, we examined 3D8 scFv’s antiviral activity in vitro on three different H1N1 influenza strains, one Oseltamivir-resistant (A/Korea/2785/2009pdm) strain, and two Oseltamivir-sensitive (A/PuertoRico/8/1934 and A/X-31) strains. Interestingly, the 3D8 scFv directly digested viral RNAs in the ribonucleoprotein complex. scFv’s reduction of influenza viral RNA including viral genomic RNA, complementary RNA, and messenger RNA during influenza A infection cycles indicated that this minibody targets all types of viral RNAs during the early, intermediate, and late stages of the virus’s life cycle. Moreover, we further addressed the antiviral effects of 3D8 scFv to investigate in vivo clinical outcomes of influenza-infected mice. Using both prophylactic and therapeutic treatments of intranasal administered 3D8 scFv, we found that Oseltamivir-resistant H1N1-infected mice showed 90% (prophylactic effects) and 40% (therapeutic effects) increased survival rates, respectively, compared to the control group. The pathological signs of influenza A in the lung tissues, and quantitative analyses of the virus proliferations supported the antiviral activity of the 3D8 single chain variable fragment. Taken together, these results demonstrate that 3D8 scFv has antiviral therapeutic potentials against a wide range of influenza A viruses via the direct viral RNA hydrolyzing activity.     

Antiviral Activity of 3D,a Butene Lactone Derivative Against Influenza A Virus In Vitro and In Vivo

Influenza A virus is a highly variable and contagious respiratory pathogen that can cause annual epidemics and it poses an enormous threat to public health. Therefore, there is an urgent need for a new generation of antiviral drugs to combat the emergence of drug-resistant strains of the influenza virus. A novel series of butene lactone derivatives were screened and the compound 3D was selected, as it exhibited in vitro potential antiviral activity against A/Weiss/43 H1N1 virus with low toxicity. In addition, 3D dose-dependently inhibited the viral replication, expression of viral mRNA and viral proteins. 3D exerted a suppressive effect on A/Virginia/ATCC2/2009 H1N1 and A/California/2/2014 H3N2 in vitro. The time-of-addition analysis indicated that 3D suppressed H1N1 in the early stage of its life cycle. A/Weiss/43 H1N1-induced apoptosis in A549 cells was reduced by 3D via the mitochondrial apoptosis pathway. 3D could decrease the production of H1N1-induced pro-inflammatory cytokines that are induced by H1N1 in vitro and in vivo. The administration of 3D reduced lung lesions and virus load in vivo. These results suggest that 3D, which is a butene lactone derivative, is a promising agent for the treatment of influenza A virus infection.     

Mucosal antibody responses are directed by viral burden in children with acute influenza infection

Please cite this paper as: He et al. (2012) Mucosal antibody responses are directed by viral burden in children with acute influenza infection. Influenza and Other Respiratory Viruses DOI: 10.1111/j.1750‐2659.2012.00346.x. Background Influenza infection causes excess hospitalizations and deaths in younger patients, but susceptibility to severe disease is poorly understood. While mucosal antibodies can limit influenza‐associated infection and disease, little is known about acute mucosal antibody responses to influenza infection. Objectives These studies characterize mucosal antiviral antibody production in children during lower respiratory infection (LRI) with H1N1 influenza versus other viral LRI and examine the relationship between mucosal antiviral antibodies and protection against severe disease. Methods B lymphocytes were assessed by immunohistochemistry in lung tissue from infants with fatal acute seasonal influenza infection. Nasopharyngeal secretions (NPS) were obtained at presentation from children with acute respiratory illness, including H1N1 (2009) influenza infection. Total and antiviral antibodies, and inflammatory and immune mediators, were quantified by ELISA. Neutralizing activity in NPS was detected using a pseudotyped virus assay. Viral burden was assessed by qPCR. Results and conclusions B lymphocytes were abundant in lung tissue of infants with fatal acute influenza LRI. Among surviving children with H1N1 infection, only a small subset (11%) demonstrated H1N1 neutralizing activity in NPS. H1N1 neutralizing activity coincided with high local levels of antiviral IgM, IgG and IgA, greater detection of inflammatory mediators, and higher viral burden (P = 0·016). Patients with mucosal antiviral antibody responses demonstrated more severe respiratory symptoms including greater hypoxia (P = 0·0018) and pneumonia (P = 0·038). These patients also trended toward younger age, longer duration of illness and longer hospital stays. Prophylaxis strategies that heighten neutralizing antibody production in the mucosa are likely to benefit both older and younger children.     

DAS181, a novel sialidase fusion protein, protects mice from lethal avian influenza H5N1 virus infection

Increasing resistance to currently available influenza antivirals highlights the need to develop alternate approaches for the prevention and/or treatment of influenza. DAS181 (Fludase), a novel sialidase fusion protein that enzymatically removes sialic acids on respiratory epithelium, exhibits potent antiviral activity against influenza A and B viruses. Here, we use a mouse model to evaluate the efficacy of DAS181 treatment against a highly pathogenic avian influenza H5N1 virus. When used to treat mice daily beginning 1 day before infection with A/Vietnam/1203/2004(H5N1) virus, DAS181 treatment at 1 mg/kg/day protected 100% of mice from fatal disease, prevented viral dissemination to the brain, and effectively blocked infection in 70% of mice. DAS181 at 1 mg/kg/day was also effective therapeutically, conferring enhanced survival of H5N1 virus-challenged mice when treatment was begun 72 h after infection. This notable antiviral activity underscores the potential utility of DAS181 as a new class of drug that is effective against influenza viruses with pandemic potential.     

Sublingual vaccination with influenza virus protects mice against lethal viral infection

We assessed whether the sublingual (s.l.) route would be an effective means of delivering vaccines against influenza virus in mice by using either formalin-inactivated or live influenza A/PR/8 virus (H1N1). Sublingual administration of inactivated influenza virus given on two occasions induced both systemic and mucosal antibody responses and conferred protection against a lethal intranasal (i.n.) challenge with influenza virus. Coadministration of a mucosal adjuvant (mCTA-LTB) enhanced these responses and resulted in complete protection against respiratory viral challenge. In addition, s.l. administration of formalin-inactivated A/PR/8 plus mCTA-LTB induced systemic expansion of IFN-gamma-secreting T cells and virus-specific cytotoxic T lymphocyte responses. Importantly, a single s.l. administration of live A/PR/8 virus was not pathogenic and induced protection mediated by both acquired and innate immunity. Moreover, s.l. administration of live A/PR/8 virus conferred heterosubtypic protection against respiratory challenge with H3N2 virus. Unlike the i.n. route, the A/PR/8 virus, whether live or inactivated, did not migrate to or replicate in the CNS after s.l. administration. Based on these promising findings, we propose that the s.l. mucosal route offers an attractive alternative to mucosal routes for administering influenza vaccines.     

Differences in clinical outcomes after 2009 influenza A/H1N1 and seasonal influenza among hematopoietic cell transplant recipients

It is not known whether pandemic 2009 influenza A/H1N1 (2009 H1N1) leads to more serious disease than seasonal influenza in hematopoietic cell transplant (HCT) recipients. In a retrospective study in HCT recipients with virologically proven influenza virus infection, a total of 161 HCT recipients (18 2009 H1N1, 103 seasonal influenza A, and 40 seasonal influenza B) were analyzed. In multivariable analyses, more patients with 2009 H1N1 had lower respiratory tract disease (LRD), hypoxemia, and prolonged viral shedding compared with seasonal influenza A. Seasonal influenza A and B outcomes were similar. There was no difference in overall and influenza-associated mortality among influenza virus types. Both early and delayed administration of antiviral therapy was shown to be beneficial in terms of decreased rates of development of LRD, although earlier intervention appeared to be more effective. Profound lymphopenia and lack of early antiviral therapy were associated significantly with LRD, hypoxemia, and death. High-dose corticosteroid treatment (≥ 1 mg/kg) given at the time of influenza diagnosis was associated with a reduced risk for mechanical ventilation. Thus, our data suggest that infection with 2009 influenza A/H1N1 resulted in more severe respiratory disease in HCT recipients compared with seasonal influenza.     

福建省首例人感染H7N9禽流感病例实验室诊断和病毒序列分析

摘要 目的 应用real-time RT-PCR和病毒序列测定等方法对福建省首例人感染H7N9禽流感病例开展实验室诊断。方法 采集人感染H7N9禽流感病例呼吸道标本并提取RNA,分别采用甲乙型流感通用引物和探针、季节性流感（包括H3N2、H1N1、H1N1pdm）特异性引物和探针以及H7N9特异性引物和探针进行荧光PCR检测。利用自行设计的引物扩增病毒基因组节段,测定并分析病毒基因组序列。结果 real-time RT-PCR结果表明,应用甲型通用引物扩增结果阳性,乙型及季节性流感（包括H3N2、H1N1以及H1N1pdm）扩增结果均为阴性,特异性H7N9亚型流感病毒扩增结果阳性。序列测定获得的病毒4个节段的序列与已公布的人感染H7N9禽流感病毒序列高度一致。结论 病例呼吸道标本中存在人感染H7N9禽流感病毒,病毒的基因与近期国内流行的人感染H7N9禽流感病毒高度类似。     

Characterization of the Anti-Influenza Activity of the Chinese Herbal Plant Paeonia lactiflora

Bai Shao (BS, the root of Paeonia lactiflora Pall.), a common Chinese herb in many recipes used to treat viral infection and liver diseases, is recognized for its ability to nourish menstruation, its Yin convergence, and as an antiperspirant. However, the mechanism and components for its antiviral function remain to be elucidated. In this study, an ethanolic extract of BS was further partitioned into aqueous and organic parts (EAex) for in vitro functional study and in vivo efficacy testing. EAex exhibited an IC₅₀ of 0.016 ± 0.005 mg/mL against influenza virus A/WSN/33 (H1N1), with broad-spectrum inhibitory activity against different strains of human influenza A viruses, including clinical oseltamivir-resistant isolates and an H1N1pdm strain. The synthesis of both viral RNA and protein was profoundly inhibited when the cells were treated with EAex. A time-of-addition assay demonstrated that EAex exerted its antiviral activity at various stages of the virus replication cycle. We addressed its antiviral activity at virus entry and demonstrated that EAex inhibits viral hemagglutination and viral binding to and penetration into host cells. In vivo animal testing showed that 200 mg/kg/d of EAex offered significant protection against viral infection. We conclude that BS possesses antiviral activity and has the potential for development as an anti-influenza agent.     

Cross-protection against H5N1 influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine

BACKGROUND: Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice. METHODS: BALB/c mice were inoculated 3 times, either intranasally or subcutaneously, with the trivalent inactivated influenza vaccine licensed in Japan for the 2005-2006 season. The vaccine included A/NewCaledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2), and B/Shanghai/361/2002 viral strains and was administered together with poly(I):poly(C(12)U) (Ampligen) as an adjuvant. At 14 days after the final inoculation, the inoculated mice were challenged with either the A/HongKong/483/97, the A/Vietnam/1194/04, or the A/Indonesia/6/05 strain of H5N1 influenza virus. RESULTS: Compared with noninoculated mice, those inoculated intranasally manifested cross-reactivity of mucosal IgA and serum IgG with H5N1 virus, as well as both a reduced H5N1 virus titer in nasal-wash samples and increased survival, after challenge with H5N1 virus. Subcutaneous inoculation did not induce a cross-reactive IgA response and did not afford protection against H5N1 viral infection. CONCLUSIONS: Intranasal inoculation with annual influenza vaccine plus the Toll-like receptor-3 agonist, poly(I):poly(C(12)U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.     

Severity of pandemic H1N1 2009 influenza virus infection may not be directly correlated with initial viral load in upper respiratory tract

Please cite this paper as: Wu et al. (2012) Severity of pandemic H1N1 2009 influenza virus infection may not be directly correlated with initial viral load in upper respiratory tract. Influenza and Other Respiratory Viruses 6(5), 367–373. Background Recent studies have demonstrated that rapid influenza diagnostic tests (RIDTs) have a relatively low sensitivity in detecting severe cases of pandemic H1N1 2009 influenza virus (pH1N1) infection. We hypothesized that viral load in upper respiratory specimens obtained on presentation may not be correlated with disease severity. Methods We conducted a prospective study to compare patterns of viral shedding using nasopharyngeal swab specimens, according to the number of days of post‐symptom onset and post‐antiviral therapy, between patients with and without complications. Results From July 15, 2009 through July 23, 2010, we collected and processed a total of 141 nasopharyngeal swab specimens from 64 inpatients and outpatients with laboratory‐confirmed pH1N1 infection. These included 46 patients without any complications (uncomplicated group) and 18 patients who required hospital admission (complicated group). The mean initial viral load was higher in the uncomplicated group than in the complicated group (3·4 ± 1·6 log₁₀ copies/μl versus 1·9 ± 1·7, P = 0·02). However, prolonged viral shedding was only detected in the complicated group (44% by day 7 of antiviral therapy). By multivariate analysis, we found that age (OR, 1·1; 95% CI, 1·0–1·1) and initial nasopharyngeal viral load (OR, 0·5; 95% CI, 0·3–0·8) were significant factors associated with complications. Conclusion Given that patients with severe pH1N1 infection may have relatively lower initial viral load in the upper respiratory tract, cautious interpretation of negative RIDT results is particularly warranted in this patient population.     

Protection against lethal influenza virus challenge by RNA interference in vivo

Influenza virus infection is responsible for hundreds of thousands of deaths annually. Current vaccination strategies and antiviral drugs provide limited protection; therefore, new strategies are needed. RNA interference is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with small interfering RNAs (siRNAs) specific for highly conserved regions of the nucleoprotein or acidic polymerase inhibits influenza A virus replication in vivo. Delivery of these siRNAs significantly reduced lung virus titers in infected mice and protected animals from lethal challenge. This protection was specific and not mediated by an antiviral IFN response. Moreover, influenza-specific siRNA treatment was broadly effective and protected animals against lethal challenge with highly pathogenic avian influenza A viruses of the H5 and H7 subtypes. These results indicate that RNA interference is promising for control of influenza virus infection, as well as other viral infections.     

H1N1 infection in a cohort of hematopoietic stem cell transplant recipients: prompt antiviral therapy might be life saving

E. Suyani, Z. Aki, Ö. Güzel, ?. Altindal, E. ?enol, G. Sucak. H1N1 infection in a cohort of hematopoietic stem cell transplant recipients: prompt antiviral therapy might be life saving.
Transpl Infect Dis 2011: 13: 208–212. All rights reserved Abstract: Influenza A H1N1 virus, causing a pandemic since spring 2009, has been an important cause of morbidity and mortality worldwide. Patients with hematological malignancies and hematopoietic stem cell transplant (HCT) recipients are in a high‐risk group and might require hospitalization more commonly because of H1N1 infection. Early demonstration of H1N1 influenza virus and commencing antiviral therapy promptly can be life saving particularly in immunosuppressed patients. We retrospectively reviewed the data of 10 HCT recipients who were diagnosed with influenza H1N1 infection at the Stem Cell Transplantation Unit of Gazi University Hospital in Turkey, from October through December 2009. All patients, except 1, were started empirically on oseltamivir on admission, after nasopharyngeal and oropharyngeal sampling for H1N1 virus. Four of the patients, 2 of whom developed pneumonia, required hospitalization. One of the patients with pneumonia died of respiratory failure caused by bacterial co‐infection. The course of the remaining patients was uneventful. In conclusion, HCT recipients infected with H1N1 during the influenza H1N1 pandemic did not necessarily have an adverse prognosis, particularly with prompt administration of the appropriate antiviral therapy.     

3-Indoleacetonitrile Is Highly Effective in Treating Influenza A Virus Infection In Vitro and In Vivo

Influenza A viruses are serious zoonotic pathogens that continuously cause pandemics in several animal hosts, including birds, pigs, and humans. Indole derivatives containing an indole core framework have been extensively studied and developed to prevent and/or treat viral infection. This study evaluated the anti-influenza activity of several indole derivatives, including 3-indoleacetonitrile, indole-3-carboxaldehyde, 3-carboxyindole, and gramine, in A549 and MDCK cells. Among these compounds, 3-indoleacetonitrile exerts profound antiviral activity against a broad spectrum of influenza A viruses, as tested in A549 cells. Importantly, in a mouse model, 3-indoleacetonitrile with a non-toxic concentration of 20 mg/kg effectively reduced the mortality and weight loss, diminished lung virus titers, and alleviated lung lesions of mice lethally challenged with A/duck/Hubei/WH18/2015 H5N6 and A/Puerto Rico/8/1934 H1N1 influenza A viruses. The antiviral properties enable the potential use of 3-indoleacetonitrile for the treatment of IAV infection.     

人甲型H1N1流行性感冒病毒全基因组分析与反向遗传载体的克隆

目的 明确一株人甲型H1N1流行性感冒(流感)病毒的遗传背景,建立流感病毒反向遗传的平台.方法 对分离自汕头市儿童的一株人甲型H1N1流感病毒进行空斑纯化;利用甲型流感病毒通用引物,对该病毒全基因组的8条片段(PB2、PB1、PA、HA、NP、NA、M和NS)进行RT-PCR全长克隆、DNA测序及初步生物信息学分析;将其8条全长基因组片段分别插入反向遗传通用载体,构建人甲型H1N1流感病毒的反向遗传系统.结果 RT-PCR克隆得到该H1N1毒株的8条全长片段,经测序分析确认该毒株的基因序列,该毒株与2006年至2008年分离的人甲型H1N1流感病毒具有很高的同源性,未出现奥司他韦或金刚乙胺抗药性的遗传突变.PCR与测序证实,插入该菌株8个全长基因组片段的载体序列完全正确.结论 成功构建了该毒株的感染性克隆.获得了该毒株全基因组序列,为明确病毒遗传信息、提供流行病学监测数据、建立流感病毒反向遗传平台奠定了研究基础.     

Infection by rhinovirus: Similarity of clinical signs included in the case definition of influenza IAn/H1N1

Introduction

Although new influenza virus (IAn/H1N1) infections are mild and indistinguishable from any other seasonal influenza virus infections, there are few data on comparisons of the clinical features of infection with (IAn/H1N1) and with other respiratory viruses. The incidence, clinical aspects and temporal distribution of those respiratory viruses circulating during flu pandemic period were studied.

Methods

Respiratory samples from patients with acute influenza-like symptoms were collected from May 2009 to December 2009. Respiratory viruses were detected by conventional culture methods and genome amplification techniques.

Results

Although IAn/H1N1 was the virus most frequently detected, several other respiratory viruses co-circulated with IAn/H1N1 during the pandemic period, especially rhinovirus. The similarity between clinical signs included in the clinical case definition for influenza and those caused by other respiratory viruses, particularly rhinovirus, suggest that a high percentage of viral infections were clinically diagnosed as case of influenza.

Conclusions

Our study offers useful information to face future pandemics caused by influenza virus, indicating that differential diagnoses are required in order to not overestimate the importance of the pandemic.     

IFN-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and Sindbis viruses

Type I interferons (IFNs) play an essential role in the host response to viral infection through the induction of numerous IFN-stimulated genes (ISGs), including important antiviral molecules such as PKR, RNase L, Mx, and iNOS. Yet, additional antiviral ISGs likely exist. IFN-stimulated gene 15 (ISG15) is a ubiquitin homolog that is rapidly up-regulated after viral infection, and it conjugates to a wide array of host proteins. Although it has been hypothesized that ISG15 functions as an antiviral molecule, the initial evaluation of ISG15-deficient mice revealed no defects in their responses to vesicular stomatitis virus or lymphocytic choriomeningitis virus, leaving open the important question of whether ISG15 is an antiviral molecule in vivo. Here we demonstrate that ISG15 is critical for the host response to viral infection. ISG15-/- mice are more susceptible to influenza A/WSN/33 and influenza B/Lee/40 virus infections. ISG15-/- mice also exhibited increased susceptibility to both herpes simplex virus type 1 and murine gammaherpesvirus 68 infection and to Sindbis virus infection. The increased susceptibility of ISG15-/- mice to Sindbis virus infection was rescued by expressing wild-type ISG15, but not a mutant form of ISG15 that cannot form conjugates, from the Sindbis virus genome. The demonstration of ISG15 as a novel antiviral molecule with activity against both RNA and DNA viruses provides a target for the development of therapies against important human pathogens.     

Cross-Reactive Fc-Fused Single-Domain Antibodies to Hemagglutinin Stem Region Protect Mice from Group 1 Influenza a Virus Infection

The continued evolution of influenza viruses reduces the effectiveness of vaccination and antiviral drugs. The identification of novel and universal agents for influenza prophylaxis and treatment is an urgent need. We have previously described two potent single-domain antibodies (VHH), G2.3 and H1.2, which bind to the stem domain of hemagglutinin and efficiently neutralize H1N1 and H5N2 influenza viruses in vivo. In this study, we modified these VHHs with Fc-fragment to enhance their antiviral activity. Reformatting of G2.3 into bivalent Fc-fusion molecule increased its in vitro neutralizing activity against H1N1 and H2N3 viruses up to 80-fold and, moreover, resulted in obtaining the ability to neutralize H5N2 and H9N2 subtypes. We demonstrated that a dose as low as 0.6 mg/kg of G2.3-Fc or H1.2-Fc administered systemically or locally before infection could protect mice from lethal challenges with both H1N1 and H5N2 viruses. Furthermore, G2.3-Fc reduced the lung viral load to an undetectable level. Both VHH-Fc antibodies showed in vivo therapeutic efficacy when delivered via systemic or local route. The findings support G2.3-Fc as a potential therapeutic agent for both prophylaxis and therapy of Group 1 influenza A infection.     

Tropism and Infectivity of Pandemic Influenza A H1N1/09 Virus in the Human Placenta

Influenza virus infection in pregnant women may put the fetus at higher risk; however, to date, there has been no detailed research about the expression of influenza virus receptors in the human placenta. We employed the lectin staining technique, which is a classic influenza virus receptor research method for studying the distribution of viral receptors in the human placenta. In addition, we examined the susceptibility of the human placenta to H1N1/09, by detecting viral proteins and RNA at different time points post-infection. We found that the human placenta expressed both avian and human influenza A virus receptors (α-2, 3-linked sialic acid and α-2, 6-linked sialic acid). In addition, H1N1/09 did not only infect the human placenta, but also replicated and was released into the culture media. We concluded that the human placenta is susceptible to the 2009 influenza A virus (H1N1/09) infection, and that particular attention should be paid to shielding pregnant women from infection during influenza season.