A Novel Antiviral Target Structure Involved in the RNA Binding,Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein

Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP.     

The NF‐κB inhibitor SC75741 efficiently blocks influenza virus propagation and confers a high barrier for development of viral resistance

Ongoing human infections with highly pathogenic avian H5N1 viruses and the emergence of the pandemic swine‐origin influenza viruses (IV) highlight the permanent threat elicited by these pathogens. Occurrence of resistant seasonal and pandemic strains against the currently licensed antiviral medications points to the urgent need for new and amply available anti‐influenza drugs. The recently identified virus‐supportive function of the cellular IKK/NF‐κB signalling pathway suggests this signalling module as a potential target for antiviral intervention. We characterized the NF‐κB inhibitor SC75741 as a broad and efficient blocker of IV replication in non‐toxic concentrations. The underlying molecular mechanism of SC75741 action involves impaired DNA binding of the NF‐κB subunit p65, resulting in reduced expression of cytokines, chemokines, and pro‐apoptotic factors, subsequent inhibition of caspase activation and block of caspase‐mediated nuclear export of viralribonucleoproteins. SC75741 reduces viral replication and H5N1‐induced IL‐6 and IP‐10 expression in the lung of infected mice. Besides its virustatic effect the drug suppresses virus‐induced overproduction of cytokines and chemokines, suggesting that it might prevent hypercytokinemia that is discussed to be an important pathogenicity determinant of highly pathogenic IV. Importantly the drug exhibits a high barrier for development of resistant virus variants. Thus, SC75741‐derived drugs may serve as broadly non‐toxic anti‐influenza agents.     

Assessment of the antiviral properties of recombinant porcine SP-D against various influenza A viruses in vitro

The emergence of influenza viruses resistant to existing classes of antiviral drugs raises concern and there is a need for novel antiviral agents that could be used therapeutically or prophylacticaly. Surfactant protein D (SP-D) belongs to the family of C-type lectins which are important effector molecules of the innate immune system with activity against bacteria and viruses, including influenza viruses. In the present study we evaluated the potential of recombinant porcine SP-D as an antiviral agent against influenza A viruses (IAVs) in vitro. To determine the range of antiviral activity, thirty IAVs of the subtypes H1N1, H3N2 and H5N1 that originated from birds, pigs and humans were selected and tested for their sensitivity to recombinant SP-D. Using these viruses it was shown by hemagglutination inhibition assay, that recombinant porcine SP-D was more potent than recombinant human SP-D and that especially higher order oligomeric forms of SP-D had the strongest antiviral activity. Porcine SP-D was active against a broad range of IAV strains and neutralized a variety of H1N1 and H3N2 IAVs, including 2009 pandemic H1N1 viruses. Using tissue sections of ferret and human trachea, we demonstrated that recombinant porcine SP-D prevented attachment of human seasonal H1N1 and H3N2 virus to receptors on epithelial cells of the upper respiratory tract. It was concluded that recombinant porcine SP-D holds promise as a novel antiviral agent against influenza and further development and evaluation in vivo seems warranted.     

The viral nucleoprotein determines Mx sensitivity of influenza A viruses

Host restriction factors play a crucial role in preventing trans-species transmission of viral pathogens. In mammals, the interferon-induced Mx GTPases are powerful antiviral proteins restricting orthomyxoviruses. Hence, the human MxA GTPase may function as an efficient barrier against zoonotic introduction of influenza A viruses into the human population. Successful viruses are likely to acquire adaptive mutations allowing them to evade MxA restriction. We compared the 2009 pandemic influenza A virus [strain A/Hamburg/4/09 (pH1N1)] with a highly pathogenic avian H5N1 isolate [strain A/Thailand/1(KAN-1)/04] for their relative sensitivities to human MxA and murine Mx1. The H5N1 virus was highly sensitive to both Mx GTPases, whereas the pandemic H1N1 virus was almost insensitive. Substitutions of the viral polymerase subunits or the nucleoprotein (NP) in a polymerase reconstitution assay demonstrated that NP was the main determinant of Mx sensitivity. The NP of H5N1 conferred Mx sensitivity to the pandemic H1N1 polymerase, whereas the NP of pandemic H1N1 rendered the H5N1 polymerase insensitive. Reassortant viruses which expressed the NP of H5N1 in a pH1N1 genetic background and vice versa were generated. Congenic Mx1-positive mice survived intranasal infection with these reassortants if the challenge virus contained the avian NP. In contrast, they succumbed to infection if the NP of pH1N1 origin was present. These findings clearly indicate that the origin of NP determines Mx sensitivity and that human influenza viruses acquired adaptive mutations to evade MxA restriction. This also explains our previous observations that human and avian influenza A viruses differ in their sensitivities to Mx.     

Differential nucleocytoplasmic shuttling of the nucleoprotein of influenza a viruses and association with host tropism

The nucleoprotein (NP) of influenza A virus plays a crucial role in virus replication, infectivity, and host adaptation. As a major component of the viral ribonucleoprotein complexes (vRNP), NP initiates vRNP shuttling between the nucleus and cytoplasm in the host cell. However, the characteristics of the nucleocytoplasmic shuttling of NP from H1N1 influenza A virus still remain unclear. In the present study, the subcellular localization and the related key residues of the H1N1 influenza virus NP were identified and evaluated. The NP of influenza virus A/WSN/33 (H1N1; WSN) displayed a more obvious nuclear accumulation than A/Anhui/1/2013 (H7N9; AH) and A/chicken/Shandong/lx1023/2007 (H9N2; SD). NP residue K4, located in NLS1, and residue F253, located in NES3, from WSN NP are not conserved in H7N9 and H9N2, which instead encode Q4 and I253, respectively. Crucially, these residues are involved in the regulation of NP nucleocytoplasmic shuttling through interactions with CRM1 and importin‐α. Moreover, residues at position 253 also play important roles in the replication of the virus, resulting in an increase in vRNP polymerase activity and an alteration of the cell tropism and pathogenicity in mice. The present data revealed a pivotal role of the Q4 and I253 residues of NP from H7N9 in enhancing the cytoplasmic accumulation of NP and vRNP activity compared to the K4 and F253 residues in WSN‐NP. In addition, an F253I substitution in the NP of WSN altered the survival ratio of infected mice and the growth curve in infected avian‐origin cells (DF‐1). The current data indicate that the F253I mutation results in attenuated pathogenicity of the virus in mice and altered cell tropism. The present study demonstrated the dissimilarity in subcellular NP transport processes between H1N1 virus WSN and other influenza A virus strains, as well as uncovered the mechanism responsible for this difference.     

The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus

By nature of their segmented RNA genome, influenza A viruses (IAVs) have the potential to generate variants through a reassortment process. The influenza nonstructural (NS) gene is critical for a virus to counteract the antiviral responses of the host. Therefore, a newly acquired NS segment potentially determines the replication efficiency of the reassortant virus in a range of different hosts. In addition, the C-terminal PDZ-binding motif (PBM) has been suggested as a pathogenic determinant of IAVs. To gauge the pandemic potential from human and avian IAV reassortment, we assessed the replication properties of NS-reassorted viruses in cultured cells and in the lungs of mice and determined their transmissibility in guinea pigs. Compared with the recombinant A/Korea/01/2009 virus (rK09; 2009 pandemic H1N1 strain), the rK09/VN:NS virus, in which the NS gene was adopted from the A/Vietnam/1203/2004 virus (a human isolate of the highly pathogenic avian influenza H5N1 virus strains), exhibited attenuated virulence and reduced transmissibility. However, the rK09/VN:NS-PBM virus, harboring the PBM in the C-terminus of the NS1 protein, recovered the attenuated virulence of the rK09/VN:NS virus. In a guinea pig model, the rK09/VN:NS-PBM virus showed even greater transmission efficiency than the rK/09 virus. These results suggest that the PBM in the NS1 protein may determine viral persistence in the human and avian IAV interface.     

The highly pathogenic H5N1 influenza A virus down‐regulated several cellular MicroRNAs which target viral genome

Higher and prolonged viral replication is critical for the increased pathogenesis of the highly pathogenic avian influenza (HPAI) subtype of H5N1 influenza A virus (IAV) over the lowly pathogenic H1N1 IAV strain. Recent studies highlighted the considerable roles of cellular miRNAs in host defence against viral infection. In this report, using a 3′UTR reporter system, we identified several putative miRNA target sites buried in the H5N1 virus genome. We found two miRNAs, miR‐584‐5p and miR‐1249, that matched with the PB2 binding sequence. Moreover, we showed that these miRNAs dramatically down‐regulated PB2 expression, and inhibited replication of H5N1 and H1N1 IAVs in A549 cells. Intriguingly, these miRNAs expression was differently regulated in A549 cells infected with the H5N1 and H1N1 viruses. Furthermore, transfection of miR‐1249 inhibitor enhanced the PB2 expression and promoted the replication of H5N1 and H1N1 IAVs. These results suggest that H5N1 virus may have evolved a mechanism to escape host‐mediated inhibition of viral replication through down‐regulation of cellular miRNAs, which target its viral genome.     

Incorporation of privileged structures into 3-O-β-chacotriosyl ursolic acid can enhance inhibiting the entry of the H5N1 virus

The glycoprotein hemagglutinin of influenza virus plays a key role in the initial stage of virus infection, making it a potential target for novel influenza viruses entry inhibitors. Two “privileged fragments”, 2-(piperidin-1-yl)ethan-1-amine and 2-(1,3-oxazinan-3-yl)ethan-1-amine were integrated into 3-O-β-chacotriosyl ursolic acid producing new derivatives 5 and 6 with improved activity against IAVs in vitro. Mechanistically, compound 6 was effective in inhibiting infection of H1-, H3-, and H5-typed influenza A viruses by interfering with the viral hemagglutinin. Furthermore, the docking studies were in agreement with the antiviral data. These results showed that the title compound 6 as a new lead compound was meriting further optimization and development.     

A Novel Small Molecule Inhibitor of Influenza A Viruses that Targets Polymerase Function and Indirectly Induces Interferon

Influenza viruses continue to pose a major public health threat worldwide and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The antiviral cytokine, interferon (IFN) is an essential mediator of the innate immune response and influenza viruses, like many viruses, have evolved strategies to evade this response, resulting in increased replication and enhanced pathogenicity. A cell-based assay that monitors IFN production was developed and applied in a high-throughput compound screen to identify molecules that restore the IFN response to influenza virus infected cells. We report the identification of compound ASN2, which induces IFN only in the presence of influenza virus infection. ASN2 preferentially inhibits the growth of influenza A viruses, including the 1918 H1N1, 1968 H3N2 and 2009 H1N1 pandemic strains and avian H5N1 virus. In vivo, ASN2 partially protects mice challenged with a lethal dose of influenza A virus. Surprisingly, we found that the antiviral activity of ASN2 is not dependent on IFN production and signaling. Rather, its IFN-inducing property appears to be an indirect effect resulting from ASN2-mediated inhibition of viral polymerase function, and subsequent loss of the expression of the viral IFN antagonist, NS1. Moreover, we identified a single amino acid mutation at position 499 of the influenza virus PB1 protein that confers resistance to ASN2, suggesting that PB1 is the direct target. This two-pronged antiviral mechanism, consisting of direct inhibition of virus replication and simultaneous activation of the host innate immune response, is a unique property not previously described for any single antiviral molecule.     

Efficacy of the New Neuraminidase Inhibitor CS-8958 against H5N1 Influenza Viruses

Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 µg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants.     

Design of deoxyribozymes for inhibition of influenza a virus reproduction

Influenza A viruses play a significant role in human and animal pathologies that cause epidemics and epizootics. Therefore, the development of new anti-flu drugs has become increasingly urgent. Deoxyribozymes can be considered as promising antiviral agents due to their ability to efficiently cleave RNA molecules with high specificity. In this study, a number of genomic sequences of the most relevant influenza A virus subtypes, i.e., H5N1, H3N2, and H1N1, were analyzed. Conserved regions were revealed in the five least variable segments of the fragmented viral RNA genome, and potential sites of their cleavage with 10–23 deoxyribozymes were determined. We designed and synthesized 46 virus-specific 33-mer deoxyribozymes with the general structure of 5′N₈AGGCTAGCTACAACGAN₉. Screening of the antiviral activity of these agents in combination with lipofectin on the Madin-Darby Canine Kidney cells infected with highly pathogenic avian influenza virus A/chicken/Kurgan/05/2005(H5N1) revealed 17 deoxyribozymes that suppressed the titer of virus cytopathicity by more than 2.5 logTCID₅₀/mL (i.e. the neutralization index of the virus was more than 300), five of which suppressed the virus titer by a factor of 1000 or more. The most active deoxyribozymes appeared to be specific to segment 5 of the influenza A virus genome, which encoded NP nucleoprotein.     

Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.     

DBA/2 mouse as an animal model for anti-influenza drug efficacy evaluation

Influenza viruses are seasonally recurring human pathogens. Vaccines and antiviral drugs are available for influenza. However, the viruses, which often change themselves via antigenic drift and shift, demand constant efforts to update vaccine antigens every year and develop new agents with broad-spectrum antiviral efficacy. An animal model is critical for such efforts. While most human influenza viruses are unable to kill BALB/c mice, some strains have been shown to kill DBA/2 mice without prior adaptation. Therefore, in this study, we explored the feasibility of employing DBA/2 mice as a model in the development of anti-influenza drugs. Unlike the BALB/c strain, DBA/2 mice were highly susceptible and could be killed with a relatively low titer (50% DBA/2 lethal dose = 10^2.83 plaque-forming units) of the A/Korea/01/2009 virus (2009 pandemic H1N1 virus). When treated with a neuraminidase inhibitor, oseltamivir phosphate, infected DBA/2 mice survived until 14 days post-infection. The reduced morbidity of the infected DBA/2 mice was also consistent with the oseltamivir treatment. Taking these data into consideration, we propose that the DBA/2 mouse is an excellent animal model to evaluate antiviral efficacy against influenza infection and can be further utilized for combination therapies or bioactivity models of existing and newly developed anti-influenza drugs.     

Inhibitory effect of 2,4‐dichlorophenoxyacetic acid on ROS,autophagy formation,and mRNA replication for influenza virus infection

Influenza virus has had a high rate of antigenic shift and drift that causes significant morbidity and mortality in humans and animals. The lack of excellent pharmacological treatment underlines the importance of the development of the novel antiviral drugs. We investigated the anti‐influenza A and B viruses of 2,4‐dichlorophenoxyacetic acid (2,4‐D), which is the synthetic analog to auxin and is used as a popular herbicide in the agricultural practices. 2,4‐D was evaluated using a cytopathic effect reduction method; assay results showed that 2,4‐D possessed strong anti‐influenza A and B viruses inhibiting the formation of a visible cytopathic effect. Influenza viral RNA expression was performed by quantitative real‐time polymerase chain reaction. 2,4‐D also inhibited virus replication in the early stage of influenza virus infection without direct interaction with virus particles. Additionally, 2,4‐D significantly inhibited various factors occur during influenza virus infection as the acidic vesicular formation and reactive oxygen species production. Moreover, 2,4‐D represented no cytotoxicity in normal kidney cell. Therefore, these findings provide an understanding of the mechanism and efficient use of 2,4‐D in pharmacological applications against influenza virus infection.     

Oseltamivir Resistance in Influenza A(H6N2) Caused by an R292K Substitution in Neuraminidase Is Not Maintained in Mallards without Drug Pressure

Background

Wild waterfowl is the natural reservoir of influenza A virus (IAV); hosted viruses are very variable and provide a source for genetic segments which can reassort with poultry or mammalian adapted IAVs to generate novel species crossing viruses. Additionally, wild waterfowl act as a reservoir for highly pathogenic IAVs. Exposure of wild birds to the antiviral drug oseltamivir may occur in the environment as its active metabolite can be released from sewage treatment plants to river water. Resistance to oseltamivir, or to other neuraminidase inhibitors (NAIs), in IAVs of wild waterfowl has not been extensively studied.

Aim and Methods

In a previous in vivo Mallard experiment, an influenza A(H6N2) virus developed oseltamivir resistance by the R292K substitution in the neuraminidase (NA), when the birds were exposed to oseltamivir. In this study we tested if the resistance could be maintained in Mallards without drug exposure. Three variants of resistant H6N2/R292K virus were each propagated during 17 days in five successive pairs of naïve Mallards, while oseltamivir exposure was decreased and removed. Daily fecal samples were analyzed for viral presence, genotype and phenotype.

Results and Conclusion

Within three days without drug exposure no resistant viruses could be detected by NA sequencing, which was confirmed by functional NAI sensitivity testing. We conclude that this resistant N2 virus could not compete in fitness with wild type subpopulations without oseltamivir drug pressure, and thus has no potential to circulate among wild birds. The results of this study contrast to previous observations of drug induced resistance in an avian H1N1 virus, which was maintained also without drug exposure in Mallards. Experimental observations on persistence of NAI resistance in avian IAVs resemble NAI resistance seen in human IAVs, in which resistant N2 subtypes do not circulate, while N1 subtypes with permissive mutations can circulate without drug pressure. We speculate that the phylogenetic group N1 NAs may easier compensate for NAI resistance than group N2 NAs, though further studies are needed to confirm such conclusions.     

Novel Pandemic Influenza A(H1N1) Viruses Are Potently Inhibited by DAS181, a Sialidase Fusion Protein

Background

The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase™) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses.

Methods and Findings

The activity of DAS181 against several pandemic influenza A(H1N1) virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice. DAS181 efficiently inhibited viral replication in each of these models and against all tested pandemic influenza A(H1N1) strains. DAS181 treatment also protected mice from pandemic influenza A(H1N1)-induced pathogenesis. Furthermore, DAS181 antiviral activity against pandemic influenza A(H1N1) strains was comparable to that observed against seasonal influenza virus including the H274Y oseltamivir-resistant influenza virus.

Conclusions

The sialidase fusion protein DAS181 exhibits potent inhibitory activity against pandemic influenza A(H1N1) viruses. As inhibition was also observed with oseltamivir-resistant IFV (H274Y), DAS181 may be active against the antigenically novel pandemic influenza A(H1N1) virus should it acquire the H274Y mutation. Based on these and previous results demonstrating DAS181 broad-spectrum anti-IFV activity, DAS181 represents a potential therapeutic agent for prevention and treatment of infections by both emerging and seasonal strains of IFV.     

Small Interfering RNA Targeting M2 Gene Induces Effective and Long Term Inhibition of Influenza A Virus Replication

RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection.     

Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China

As pigs are susceptible to both human and avian influenza viruses, they have been proposed to be intermediate hosts or mixing vessels for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we reported avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Homology and phylogenetic analyses showed that the H1N1 virus (A/swine/Zhejiang/1/07) was closely to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses, which was for the first time reported in China; and the two H1N2 viruses (A/swine/Shanghai/1/07 and A/swine/Guangxi/13/06) were novel ressortant H1N2 influenza viruses containing genes from the classical swine (HA, NP, M and NS), human (NA and PB1) and avian (PB2 and PA) lineages, which indicted that the reassortment among human, avian, and swine influenza viruses had taken place in pigs in China and resulted in the generation of new viruses. The isolation of avian-like H1N1 influenza virus originated from the European swine H1N1 viruses, especially the emergence of two novel ressortant H1N2 influenza viruses provides further evidence that pigs serve as intermediate hosts or “mixing vessels”, and swine influenza virus surveillance in China should be given a high priority.