Antiviral Susceptibilities of Avian Influenza A(H5), A(H7), and A(H9) Viruses Isolated in Japan.

The circulation of avian influenza A viruses in poultry is a public health concern due to the potential transmissibility and severity of these viral infections. Monitoring the susceptibility of these viruses to antivirals is important for developing measures to strengthen the level of preparedness against influenza pandemics. However, drug susceptibility information on these viruses is limited. Here, we determined the susceptibilities of avian influenza A(H5N1), A(H5N2), A(H5N8), A(H7N7), A(H7N9), A(H9N1), and A(H9N2) viruses isolated in Japan to the antivirals approved for use there: an M2 inhibitor (amantadine), neuraminidase inhibitors (oseltamivir, peramivir, zanamivir, and laninamivir) and RNA polymerase inhibitors (baloxavir and favipiravir). Genotypic methods that detect amino acid substitutions associated with antiviral resistance and phenotypic methods that assess phenotypic viral susceptibility to drugs have revealed that these avian influenza A viruses are susceptible to neuraminidase and RNA polymerase inhibitors. These results suggest that neuraminidase and RNA polymerase inhibitors currently approved in Japan could be a treatment option against influenza A virus infections in humans.

Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza.

Limited efficacy of current antivirals and antiviral-resistant mutations impairs anti-influenza treatment. Here, we evaluate the in vitro and in vivo antiviral effect of three defective interfering genes (DIG-3) of influenza virus. Viral replication is significantly reduced in cell lines transfected with DIG-3. Mice treated with DIG-3 encoded by jetPEI-vector, as prophylaxis and therapeutics against A(H7N7) virus, respectively, have significantly better survivals (80% and 50%) than control mice (0%). We further develop a dual-functional peptide TAT-P1, which delivers DIG-3 with high efficiency and concomitantly exerts antiviral activity by preventing endosomal acidification. TAT-P1/DIG-3 is more effective than jetPEI/DIG-3 in treating A(H7N7) or A(H1N1)pdm09-infected mice and shows potent prophylactic protection on A(H7N7) or A(H1N1)pdm09-infected mice. The addition of P1 peptide, which prevents endosomal acidification, can enhance the protection of TAT-P1/DIG-3 on A(H1N1)pdm09-infected mice. Dual-functional TAT-P1 with DIG-3 can effectively protect or treat mice infected by avian and seasonal influenza virus.

SMARCA2-regulated host cell factors are required for MxA restriction of influenza A viruses.

The human interferon (IFN)-induced MxA protein is a key antiviral host restriction factor exhibiting broad antiviral activity against many RNA viruses, including highly pathogenic avian influenza A viruses (IAV) of the H5N1 and H7N7 subtype. To date the mechanism for how MxA exerts its antiviral activity is unclear, however, additional cellular factors are believed to be essential for this activity. To identify MxA cofactors we performed a genome-wide siRNA-based screen in human airway epithelial cells (A549) constitutively expressing MxA using an H5N1 reporter virus. These data were complemented with a proteomic screen to identify MxA-interacting proteins. The combined data identified SMARCA2, the ATPase subunit of the BAF chromatin remodeling complex, as a crucial factor required for the antiviral activity of MxA against IAV. Intriguingly, our data demonstrate that although SMARCA2 is essential for expression of some IFN-stimulated genes (ISGs), and the establishment of an antiviral state, it is not required for expression of MxA, suggesting an indirect effect on MxA activity. Transcriptome analysis of SMARCA2-depleted A549-MxA cells identified a small set of SMARCA2-regulated factors required for activity of MxA, in particular IFITM2 and IGFBP3. These findings reveal that several virus-inducible factors work in concert to enable MxA restriction of IAV.

Therapeutic efficacy of peramivir against H5N1 highly pathogenic avian influenza viruses harboring the neuraminidase H275Y mutation.

High morbidity and mortality associated with human cases of highly pathogenic avian influenza (HPAI) viruses, including H5N1 influenza virus, have been reported. The purpose of the present study was to evaluate the antiviral effects of peramivir against HPAI viruses. In neuraminidase (NA) inhibition and virus replication inhibition assays, peramivir showed strong inhibitory activity against H5N1, H7N1 and H7N7 HPAI viruses with sub-nanomolar activity in enzyme assays. In H5N1 viruses containing the NA H275Y mutation, the antiviral activity of peramivir against the variant was lower than that against the wild-type. Evaluation of the in vivo antiviral activity showed that a single intravenous treatment of peramivir (10 mg/kg) prevented lethality in mice infected with wild-type H5N1 virus and also following infection with H5N1 virus with the H275Y mutation after a 5 day administration of peramivir (30 mg/kg). Furthermore, mice injected with peramivir showed low viral titers and low levels of proinflammatory cytokines in the lungs. These results suggest that peramivir has therapeutic activity against HPAI viruses even if the virus harbors the NA H275Y mutation.

Identification of a novel small-molecule compound targeting the influenza A virus polymerase PB1-PB2 interface.

The PB1 C-terminal domain and PB2 N-terminal domain interaction of the influenza A polymerase, which modulates the assembly of PB1 and PB2 subunits, may serve as a valuable target for the development of novel anti-influenza therapeutics. In this study, we performed a systematic screening of a chemical library, followed by the antiviral evaluation of primary hits and their analogues. Eventually, a novel small-molecule compound PP7 that abrogated the PB1-PB2 association and impaired viral polymerase activity was identified. PP7 exhibited antiviral activities against influenza virus subtypes A (H1N1)pdm09, A(H7N9) and A(H9N2) in cell cultures and partially protected mice against lethal challenge of mouse-adapted influenza A (H1N1)pdm09 virus. Surprisingly, a panel of other subtypes of influenza virus, including A(H5N1) and A(H7N7), showed various degrees of resistance to the compound. Biochemical studies revealed a similar pattern of resistance on the impairment of polymerase activity. Molecular docking analyses suggested a PP7-binding site that appeared to be completely conserved among the subtypes of the virus mentioned above. Thus, we propose that alternative/additional binding site (s) may exist for the regulation of PB1-PB2 subunits assembly of influenza A virus.

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model.

BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.

A new class of synthetic anti-lipopolysaccharide peptides inhibits influenza A virus replication by blocking cellular attachment.

Influenza A viruses are a continuous threat to human health as illustrated by the 2009 H1N1 pandemic. Since circulating influenza virus strains become increasingly resistant against currently available drugs, the development of novel antivirals is urgently needed. Here, we have evaluated a recently described new class of broad-spectrum antiviral peptides (synthetic anti-lipopolysaccharide peptides; SALPs) for their potential to inhibit influenza virus replication in vitro and in vivo. We found that particularly SALP PEP 19-2.5 shows high binding affinities for the influenza virus receptor molecule, N-Acetylneuraminic acid, leading to impaired viral attachment and cellular entry. As a result, replication of several influenza virus subtypes (H7N7, H3N2 and 2009 pandemic H1N1) was strongly reduced. Furthermore, mice co-treated with PEP 19-2.5 were protected against an otherwise 100% lethal H7N7 influenza virus infection. These findings show that SALPs exhibit antiviral activity against influenza viruses by blocking virus attachment and entry into host cells. Thus, SALPs present a new class of broad-spectrum antiviral peptides for further development for influenza virus therapy.

The NF-kappaB inhibitor SC75741 protects mice against highly pathogenic avian influenza A virus.

The appearance of pandemic H1N1 and highly pathogenic avian H5N1 viruses in humans as well as the emergence of seasonal H1N1 variants resistant against neuraminidase inhibitors highlight the urgent need for new and amply available antiviral drugs. We and others have demonstrated that influenza virus misuses the cellular IKK/NF-kappaB signaling pathway for efficient replication suggesting that this module may be a suitable target for antiviral intervention. Here, we show that the novel NF-kappaB inhibitor SC75741 significantly protects mice against infection with highly pathogenic avian influenza A viruses of the H5N1 and H7N7 subtypes. Treatment was efficient when SC75741 was given intravenously in a concentration of 5mg/kg/day. In addition, application of SC75741 via the intraperitoneal route resulted in a high bioavailability and was also efficient against influenza when given 15 mg/kg/day or 7.5 mg/kg/twice a day. Protection was achieved when SC75741 was given for seven consecutive days either prior to infection or as late as four days after infection. SC75741 treatment showed no adverse effects in the concentrations required to protect mice against influenza virus infection. Although more pre-clinical studies are needed SC75741 might be a promising candidate for a novel antiviral drug against influenza viruses that targets the host cell rather than the virus itself.

A plant extract of Ribes nigrum folium possesses anti-influenza virus activity in vitro and in vivo by preventing virus entry to host cells.

Infections with influenza A viruses (IAV) are still amongst the major causes of highly contagious severe respiratory diseases not only bearing a devastating effect to human health, but also significantly impact the economy. Besides vaccination that represents the best option to protect from IAV infections, only two classes of anti-influenza drugs, inhibitors of the M2 ion channel and the neuraminidase, often causing resistant IAV variants have been approved. That is why the need for effective and amply available antivirals against IAV is of high priority. Here we introduce LADANIA067 from the leaves of the wild black currant (Ribes nigrum folium) as a potent compound against IAV infections in vitro and in vivo. LADANIA067 treatment resulted in a reduction of progeny virus titers in cell cultures infected with prototype avian and human influenza virus strains of different subtypes. At the effective dose of 100 µg/ml the extract did not exhibit apparent harming effects on cell viability, metabolism or proliferation. Further, viruses showed no tendency to develop resistance to LADANIA067 when compared to amantadine that resulted in the generation of resistant variants after only a few passages. On a molecular basis the protective effect of LADANIA067 appears to be mainly due to interference with virus internalisation. In the mouse infection model LADANIA067 treatment reduces progeny virus titers in the lung upon intranasal application. In conclusion, an extract from the leaves of the wild black currant might be a promising source for the development of new antiviral compounds to fight IAV infections.

Small molecule inhibitors of the c-Jun N-terminal kinase (JNK) possess antiviral activity against highly pathogenic avian and human pandemic influenza A viruses.

C-Jun N-terminal kinases (JNK) are activated in course of many viral infections. Here we analyzed the activity of JNK inhibitors on influenza A virus (IAV) amplification. Human lung epithelial cells were infected with either the highly pathogenic avian virus strain A/FPV/Bratislava/79 (H7N7) or the pandemic swine-origin influenza virus A/Hamburg/4/09 (H1N1v). The application of the JNK inhibitors SP600125 and AS601245 reduced IAV amplification by suppressing viral protein and RNA synthesis. Although AS601245 appeared to generally block the transcription of newly introduced genes, SP600125 specifically affected viral RNA synthesis. Overexpression of a dominant negative mutant of SEK/MKK4 and siRNA-mediated suppression of JNK2 expression confirmed that specific manipulation of the JNK pathway attenuates virus propagation. An IAV minigenome replication assay revealed that SP600125 did not directly affect the activity of the viral RNA polymerase complex but seems to suppress an anti-influenza nonstructural protein 1-mediated virus supportive function. Finally, when H7N7- or H1N1v-infected mice were treated with SP600125, the viral load is reduced in lungs of treated compared with untreated mice. Our data suggest that this class of ATP competitive inhibitors once optimized for antiviral action potentially represent novel drugs for antiviral intervention.

Oseltamivir inhibits H7 influenza virus replication in mice inoculated by the ocular route.

The majority of human infections associated with H7 influenza viruses have resulted in ocular and not respiratory disease. While oseltamivir has been prescribed to individuals presenting with conjunctivitis following H7 virus exposure, it is unknown if oseltamivir inhibits virus replication in ocular tissue. We demonstrate that H7 viruses possess sensitivity to neuraminidase inhibitors and that administration of oseltamivir before ocular virus challenge in mice inhibits H7N7 and H7N3 virus replication in ocular and respiratory tissues.

[Investigation of antiviral activity of adamantan boron derivaties on pandemic influenza virus models].

Comparative investigation of the virus-inhibiting activity of some boron-containing compounds showed that products BG 12 and BG 4 had the highest inhibitory effect on pandemic viruses. The minimum inhibitory concentration (MIC) of the products was 0.1 mcg/ml. The use of liposomes loaded with BG 12 molecules in the optimal concentration (0.1 mcg/ml) resulted in inhibition of the avian plague virus growth in the MDCK cells. Possible design of efficient drugs for antiviral protection based on the complexes liposomes--boron-containing compounds is discussed.

Inhibition of lung serine proteases in mice: a potentially new approach to control influenza infection.

BACKGROUND: Host serine proteases are essential for the influenza virus life cycle because the viral haemagglutinin is synthesized as a precursor which requires proteolytic maturation. Therefore, we studied the activity and expression of serine proteases in lungs from mice infected with influenza and evaluated the effect of serine protease inhibitors on virus replication both in cell culture and in infected mice. RESULTS: Two different inbred mouse strains were investigated: DBA/2J as a highly susceptible and C57Bl/6J as a more resistant strain to influenza virus infection. The serine proteases from lung homogenates of mice exhibited pH optima of 10.00. Using the substrate Bz-Val-Gly-Arg-p-nitroanilide or in zymograms, the intensities of proteolysis increased in homogenates from both mouse strains with time post infection (p.i.) with the mouse-adapted influenza virus A/Puerto Rico/8/34 (H1N1; PR8). In zymograms at day 7 p.i., proteolytic bands were stronger and numerous in lung homogenates from DBA/2J than C57Bl/6J mice. Real-time PCR results confirmed differential expression of several lung proteases before and after infecting mice with the H1N1 virus. The most strongly up-regulated proteases were Gzma, Tmprss4, Elane, Ctrl, Gzmc and Gzmb. Pretreatment of mouse and human lung cell lines with the serine protease inhibitors AEBSF or pAB or a cocktail of both prior to infection with the H1N1 or the A/Seal/Massachusetts/1/80 (H7N7; SC35M) virus resulted in a decrease in virus replication. Pretreatment of C57Bl/6J mice with either AEBSF or a cocktail of AEBSF and pAB prior to infection with the H1N1 virus significantly reduced weight loss and led to a faster recovery of treated versus untreated mice while pAB alone exerted a very poor effect. After infection with the H7N7 virus, the most significant reduction of weight loss was obtained upon pretreatment with either the protease inhibitor cocktail or pAB. Furthermore, pretreatment of C57BL/6J mice with AEBSF prior to infection resulted in a significant reduction in the levels of both the H1N1 and H7N7 nucleoproteins in mice lungs and also a significant reduction in the levels of the HA transcript in the lungs of the H1N1--but not the H7N7-infected mice. CONCLUSION: Multiple serine protease activities might be implicated in mediating influenza infection. Blocking influenza A virus infection in cultured lung epithelia and in mice by the used serine protease inhibitors may provide an alternative approach for treatment of influenza infection.

Anti-viral properties and mode of action of standardized Echinacea purpurea extract against highly pathogenic avian influenza virus (H5N1, H7N7) and swine-origin H1N1 (S-OIV).

BACKGROUND: Influenza virus (IV) infections are a major threat to human welfare and animal health worldwide. Anti-viral therapy includes vaccines and a few anti-viral drugs. However vaccines are not always available in time, as demonstrated by the emergence of the new 2009 H1N1-type pandemic strain of swine origin (S-OIV) in April 2009, and the acquisition of resistance to neuraminidase inhibitors such as Tamiflu (oseltamivir) is a potential problem. Therefore the prospects for the control of IV by existing anti-viral drugs are limited. As an alternative approach to the common anti-virals we studied in more detail a commercial standardized extract of the widely used herb Echinacea purpurea (Echinaforce, EF) in order to elucidate the nature of its anti-IV activity. RESULTS: Human H1N1-type IV, highly pathogenic avian IV (HPAIV) of the H5- and H7-types, as well as swine origin IV (S-OIV, H1N1), were all inactivated in cell culture assays by the EF preparation at concentrations ranging from the recommended dose for oral consumption to several orders of magnitude lower. Detailed studies with the H5N1 HPAIV strain indicated that direct contact between EF and virus was required, prior to infection, in order to obtain maximum inhibition in virus replication. Hemagglutination assays showed that the extract inhibited the receptor binding activity of the virus, suggesting that the extract interferes with the viral entry into cells. In sequential passage studies under treatment in cell culture with the H5N1 virus no EF-resistant variants emerged, in contrast to Tamiflu, which produced resistant viruses upon passaging. Furthermore, the Tamiflu-resistant virus was just as susceptible to EF as the wild type virus. CONCLUSION: As a result of these investigations, we believe that this standard Echinacea preparation, used at the recommended dose for oral consumption, could be a useful, readily available and affordable addition to existing control options for IV replication and dissemination.

[Synthesis of dihydroquinopymaric acid conjugates with amino acids].

Synthesis of dihydroquinopymaric acid amides and their 2beta-succinyl and 2beta-phthalyl derivatives containing residues of amino acids was carried out for the first time. Antiviral properties of the compounds synthesized were investigated.

Characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol.

The antiviral drug arbidol (ARB), which is licensed in Russia for use against influenza, is known to inhibit early membrane fusion events in influenza A and B virus replication. To investigate in more detail the target and mechanism of ARB action we generated and studied the characteristics of ARB-resistant influenza virus mutants. Observations of the ARB susceptibility of reassortants between A/Singapore/1/57(H2N2) and A/chicken/Germany/27(H7N7, "Weybridge" strain) and of mutants of the latter virus identified the virus haemagglutinin (HA) as the major determinant of ARB sensitivity. ARB-resistant mutants, selected from the most sensitive reassortant, possessed single amino acid substitutions in the HA2 subunit which caused an increase in the pH of fusion and the associated conformational change in HA. ARB was shown to stabilize the HA by causing a 0.2 pH unit reduction in the pH of the transition to the low pH form, which was specifically abrogated by the resistance mutations. Some of the resistance mutations, which reduce acid stability and would disrupt ARB-HA interactions, are located in the vicinity of a potential ARB binding site identified using the docking programme Gold. Together, the results of these investigations indicate that ARB falls within a class of inhibitor which interacts with HA to stabilize it against the low pH transition to its fusogenic state and consequently inhibit HA-mediated membrane fusion during influenza virus infection.

A polyphenol rich plant extract, CYSTUS052, exerts anti influenza virus activity in cell culture without toxic side effects or the tendency to induce viral resistance.

Infections with influenza A viruses still pose a major threat to humans and several animal species. The occurrence of highly pathogenic avian influenza viruses of the H5N1 subtype capable to infect and kill humans highlights the urgent need for new and efficient countermeasures against this viral disease. Here we demonstrate that a polyphenol rich extract (CYSTUS052) from the Mediterranean plant Cistus incanus exerts a potent anti-influenza virus activity in A549 or MDCK cell cultures infected with prototype avian and human influenza strains of different subtypes. CYSTUS052 treatment resulted in a reduction of progeny virus titers of up to two logs. At the effective dose of 50 microg/ml the extract did not exhibit apparent harming effects on cell viability, metabolism or proliferation, which is consistent with the fact that these plant extracts are already used in traditional medicine in southern Europe for centuries without any reported complications. Viruses did not develop resistance to CYSTUS052 when compared to amantadine that resulted in the generation of resistant variants after only a few passages. On a molecular basis the protective effect of CYSTUS052 appears to be mainly due to binding of the polymeric polyphenol components of the extract to the virus surface, thereby inhibiting binding of the hemagglutinin to cellular receptors. Thus, a local application of CYSTUS052 at the viral entry routes may be a promising approach that may help to protect from influenza virus infections.

Contribution of H7 haemagglutinin to amantadine resistance and infectivity of influenza virus.

In the present study we determined the antiviral effect of amantadine against influenza A/Netherlands/219/03 (H7N7) virus in cell culture and in a mouse model. Amantadine at concentrations <100 muM failed to inhibit virus replication in Madin-Darby canine kidney (MDCK) cells. When orally administered to mice for 5 days, amantadine at 15 mg kg(-1) day(-1) did not protect animals against lethal challenge with H7N7 infection, and virus titres in mouse organs were not reduced. However, sequence analysis of the M2 protein revealed none of the mutations previously described as being associated with amantadine resistance. We used reverse genetics to generate viruses containing the haemagglutinin (HA) or M gene of A/Netherlands/219/03 virus to investigate the role of these genes in amantadine sensitivity. All recombinant viruses carrying the HA segment of A/Netherlands/219/03 (H7N7) virus were amantadine-resistant, regardless of the origin of their other genes. To study the role of fusion activity in the mechanism of drug resistance, we introduced the Gly(23)-->Cys mutation in the H7 fusion peptide. This substitution resulted in a decrease of the pH of fusion and was also associated with reduced virus replication in both MDCK cells and mice, as compared to that of the wild-type virus. We suggest that H7 HA protein plays a role in amantadine resistance, although all HA amino acids that participate in drug resistance still remain to be characterized. Our finding reveals that sequence analysis of the transmembrane domain of M2 protein may not adequately identify all drug-resistant variants.

Acetylsalicylic acid (ASA) blocks influenza virus propagation via its NF-kappaB-inhibiting activity.

Influenza is still one of the major plagues worldwide. The statistical likeliness of a new pandemic outbreak highlights the urgent need for new and amply available antiviral drugs. We and others have shown that influenza virus misuses the cellular IKK/NF-kappaB signalling pathway for efficient replication suggesting that this module may be a suitable target for antiviral intervention. Here we examined acetylsalicylic acid (ASA), also known as aspirin, a widely used drug with a well-known capacity to inhibit NF-kappaB. We show that the drug efficiently blocks influenza virus replication in vitro and in vivo in a mechanism involving impaired expression of proapoptotic factors, subsequent inhibition of caspase activation as well as block of caspase-mediated nuclear export of viral ribonucleoproteins. As ASA showed no toxic side-effects or the tendency to induce resistant virus variants, existing salicylate-based aerosolic drugs may be suitable as anti-influenza agents. This is the first demonstration that specific targeting of a cellular factor is a suitable approach for anti-influenza virus intervention.

Purification and characterisation of a protease inhibitor from Streptomyces chromofuscus 34-1 with an antiviral activity.

The aim of this study was to purify and characterise a novel protease inhibitor (PISC-2002) isolated from culture supernatants of Streptomyces chromofuscus. PISC-2002 was purified by anion-exchange chromatography, and RP-HPLC analysis. PISC-2002 had a molecular mass of 11.2 kDa and a high content of hydrophobic amino acids and proline. N-terminal sequence gave two sequences differing by one residue. The main sequence is ASLPAVSALVLTV and the shorter sequence is SLPAVSALVLTV. This shows its homology to Streptomyces subtilisin inhibitor family. Besides its large spectrum of powerful inhibitory activities against various serine proteases, PISC-2002 displayed significant antiviral effect against influenza virus A/Rostock/34 (H7N7).