Treatment Of Influenza Virus Infection Research Articles

Global update on the susceptibilities of human influenza viruses to neuraminidase inhibitors and the cap-dependent endonuclease inhibitor baloxavir, 2017–2018

The global analysis of neuraminidase inhibitor (NAI) susceptibility of influenza viruses has been conducted since the 2012–13 period. In 2018 a novel cap-dependent endonuclease inhibitor, baloxavir, that targets polymerase acidic subunit (PA) was approved for the treatment of influenza virus infection in Japan and the United States. For this annual report, the susceptibilities of influenza viruses to NAIs and baloxavir were analyzed.A total of 15409 viruses, collected by World Health Organization (WHO) recognized National Influenza Centers and other laboratories between May 2017 and May 2018, were assessed for phenotypic NAI susceptibility by five WHO Collaborating Centers (CCs). The 50% inhibitory concentration (IC50) was determined for oseltamivir, zanamivir, peramivir and laninamivir. Reduced inhibition (RI) or highly reduced inhibition (HRI) by one or more NAIs was exhibited by 0.8% of viruses tested (n = 122). The frequency of viruses with RI or HRI has remained low since this global analysis began (2012–13: 0.6%; 2013–14: 1.9%; 2014–15: 0.5%; 2015–16: 0.8%; 2016–17: 0.2%). PA gene sequence data, available from public databases (n = 13523), were screened for amino acid substitutions associated with reduced susceptibility to baloxavir (PA E23G/K/R, PA A36V, PA A37T, PA I38F/M/T/L, PA E119D, PA E199G): 11 (0.08%) viruses possessed such substitutions. Five of them were included in phenotypic baloxavir susceptibility analysis by two WHO CCs and IC50 values were determined. The PA variant viruses showed 6–17-fold reduced susceptibility to baloxavir. Overall, in the 2017–18 period the frequency of circulating influenza viruses with reduced susceptibility to NAIs or baloxavir was low, but continued monitoring is important.

Effect of eleutheroside B1 on non‑coding RNAs and protein profiles of influenza A virus‑infected A549 cells

Influenza viruses often pose a serious threat to animals and human health. In an attempt to explore the potential of herbal medicine as a treatment for influenza virus infection, eleutheroside B1, a coumarin compound extracted from herba sarcandrae, was identified, which exhibited antiviral and anti-inflammatory activities against influenza A virus. In this study, high-throughput RNA sequencing and isobaric tags for relative and absolute quantification (iTRAQ) assays were performed to determine alterations in the non-coding RNA (ncRNA) transcriptome and proteomics. Bioinformatics and target prediction analyses were used to decipher the potential roles of altered ncRNAs in the function of eleutheroside B1. Furthermore, long ncRNA (lncRNA) and mRNA co-expressing networks were constructed to analyze the biological functions by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The analysis of RNA sequencing data revealed that 5 differentially expressed ncRNAs were upregulated and 3 ncRNAs were downregulated in the A549 cells infected with A/PR8/34/H1N1, with or without eleutheroside B1 treatment (PR8+eleu and PR8, respectively). Nuclear paraspeckle assembly transcript 1 (NEAT1) was differentially expressed between the PR8 and A549 cell groups. GO and KEGG pathway analyses indicated that eleutheroside B1 took advantage of the host cell biological processes and molecular function for its antiviral and anti-inflammatory activities, as well as for regulating cytokine-cytokine receptor interaction in the immune system, consistent with previous findings. The results of the iTRAQ assays indicated that L antigen family member 3 (LAGE3) protein, essential for tRNA processing, tRNA metabolic processes and ncRNA processing, was down-regulated in the PR8+eleu compared with the PR8 group. In the present study, these comprehensive, large-scale data analysis enhanced the understanding of multiple aspects of the transcriptome and proteomics that are involved in the antiviral and anti-inflammatory activities of eleutheroside B1. These findings demonstrate the potential of eleutheroside B1 for use in the prevention and treatment of influenza A virus-mediated infections.

Elucidating the Interactions between Influenza Virus Polymerase and Host Factor ANP32A.

The avian-origin influenza A virus polymerase is restricted in human cells. This restriction has been associated with species differences in host factor ANP32A. Avian ANP32A supports the activity of an avian-origin polymerase. However, the avian-origin polymerase is incompatible with human ANP32A. Avian ANP32A proteins harbor an additional 33 amino acids compared to human ANP32A proteins, which are crucial for their ability to support the avian-origin influenza virus polymerase. Here, we elucidate the interactions between ANP32A proteins and the influenza A virus polymerase using split luciferase complementation assays, coimmunoprecipitation, and in situ split Venus interaction assays. We show greater interaction of chicken ANP32A than human ANP32A with the viral polymerase and visualize these interactions in situ in the cell nucleus. We demonstrate that the 33 amino acids of chicken ANP32A and the PB2 627 domain of viral polymerase complex both contribute to this enhanced interaction. Finally, we show how these interactions are affected by the presence of viral RNA and the processivity of the polymerase, giving insights into the way that ANP32A might act during virus infection.IMPORTANCE Successful zoonotic transmission of influenza A virus into humans can lead to pandemics in an immunologically naive population. Host-encoded ANP32A proteins are required to support influenza A virus polymerase activity, and species differences in ANP32A can restrict the host range of influenza virus. Understanding how ANP32A proteins support the viral polymerase and how differences in ANP32A affect the ability of the polymerase to coopt these proteins will enhance our understanding of viral replication and species restriction as well as suggesting targeted antiviral approaches to treat influenza virus infection.

Gegen Qinlian Decoction Downregulates the TLR7 Signalling Pathway to Control Influenza A Virus Infection

Ethnopharmacological relevanceIn recent years, Gegen Qinlian decoction (GQD) has been applied to treat influenza virus infection, and its clinical effectiveness has been shown. However, the potential mechanism by which GQD acts on influenza A virus (IAV) has not been fully elucidated. Traditional Chinese medicine (TCM) formulas are well known to have multiple targets and effects. Our previous experiments examined the mechanism by which TCM can be used to treat influenza from the perspective of the influenza immune mechanism. Aim of the studyTo explore the possible mechanism by which GQD affects mice infected with the FM1 strain of influenza virus. Materials and MethodsForty-eight C57BL/6 mice were divided randomly into four groups: a normal control (NG) group, an IAV infection (VG) group, an IAV + oseltamivir (30.44 mg/kg) treatment (VO) group, and an IAV + GQD (9.74 g/kg) treatment (VQ) group. We also grouped forty-eight Toll-like receptor 7 knockout (TLR7−/−) mice in the same manner. The pulmonary mRNA expression of TLR7, myeloid differentiation factor 88 (MyD88), and nuclear factor (NF)-κB p65 was measured by RT-qPCR, and the pulmonary protein expression of TLR7, MyD88, and NF-κB p65 was measured by western blot. The proportions of T helper (Th) 1, Th2, Th17 and regulatory T (Treg) cells were measured by flow cytometry. ResultsIAV infection led to low body weights and high viral load. Compared with those in the NG group, the mRNA expression levels of TLR7, MyD88, and NF-κB p65 in the VG group were upregulated (P < 0.05). However, the mRNA and protein expression levels of TLR7, MyD88, and NF-κB p65 were lower in the VO and VQ groups than in the VG group (P < 0.05). IAV infection led to increased proportions of Th1/Th2 and Th17/Treg cells in the VG group. In the VO and VQ groups, both Th2 and Th1 cell numbers were increased, resulting in a lower Th1/Th2 proportion than that in the VG group. ConclusionsGQD downregulated the expression of some key TLR signalling pathway factors. GQD also affected the differentiation of CD4+ T cells, thereby exerting a protective systemic effect on influenza virus infection. In conclusion, GQD activated a balanced inflammatory response in the host to limit immune pathological injury and improve clinical and survival outcomes.

Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 feeding enhances humoral immune responses, which are suppressed by the antiviral neuraminidase inhibitor oseltamivir in influenza A virus–infected mice

Antiviral neuraminidase inhibitors, such as oseltamivir, zanamivir, and peramivir, are widely used for treatment of influenza virus infection. We reported previously that oseltamivir inhibits the viral growth cycle, ameliorates symptoms, and reduces viral antigen quantities. Suppressed viral antigen production, however, induces a reduction of acquired antiviral humoral immunity, and increases the incidence of re-infection rate in the following year. To achieve effective treatment of influenza virus infection, it is necessary to overcome these adverse effects of antiviral neuraminidase inhibitors. Feeding of yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) OLL1073R-1 is reported to have immune-stimulatory effects on influenza virus infection in mice and humans. In the present study, we assessed the effect of feeding L. bulgaricus OLL1073R-1 yogurt cultures (YC) on local and systemic humoral immune responses, which were suppressed by oseltamivir treatment, in mice infected with influenza A virus. Yogurt culture (1.14 × 108 cfu/0.4 mL per mouse per day) or sterile water (vehicle) was administered by intragastric gavage for 35 d. At d 22, influenza A virus/Puerto Rico/8/34 (H1N1) (PR8; 0.5 pfu/15 μL per mouse) was instilled intranasally, followed immediately by oral administration of oseltamivir (50 μg/100 μL per mouse, twice daily) or 5% methylcellulose (100 μL/mouse) as a vehicle for 13 d. Titers of anti-PR8-specific IgG and IgA in serum and mucosal secretory IgA (S-IgA) and IgG in bronchoalveolar lavage fluid (BALF) were analyzed by ELISA at 14 d after infection. Oseltamivir significantly suppressed the induction of anti-PR8-specific IgG and IgA in serum and S-IgA and IgG in BALF after infection. Feeding YC mildly but significantly stimulated production of PR8-specific IgA in serum, S-IgA in BALF, and IgG in serum without changing the IgG2a:IgG1 ratio. We analyzed the neutralizing activities against PR8 in serum and BALF and found that oseltamivir also reduced protective immunity, and YC feeding abrogated this effect. The immune-stimulatory tendency of YC on anti-PR8-specific IgA and IgG titers in serum and BALF was also detected in mice re-infected with PR8, but the effect was insignificant, unlike the effect of YC in the initial infection.

Diagnosis of Tamiflu-Resistant Influenza Virus in Human Nasal Fluid and Saliva Using Surface-Enhanced Raman Scattering.

Influenza viruses cause respiratory infection, spread through respiratory secretions, and are shed into the nasal secretion and saliva specimens. Therefore, nasal fluid and saliva are effective clinical samples for the diagnosis of influenza virus-infected patients. Although several methods have been developed to detect various types of influenza viruses, approaches for detecting mutant influenza viruses in clinical samples are rarely reported. Herein, we report for the first time a surface-enhanced Raman scattering (SERS)-based sensing platform for oseltamivir-resistant pandemic H1N1 (pH1N1) virus detection in human nasal fluid and saliva. By combining SERS-active urchin Au nanoparticles and oseltamivir hexylthiol, an excellent receptor for the pH1N1/H275Y mutant virus, we detected the pH1N1/H275Y virus specifically and sensitively in human saliva and nasal fluid samples. Considering that the current influenza virus infection testing methods do not provide information on the antiviral drug resistance of the virus, the proposed SERS-based diagnostic test for the oseltamivir-resistant virus will inform clinical decisions about the treatment of influenza virus infections, avoiding the unnecessary prescription of ineffective drugs and greatly improving therapy.

A physiologically-based pharmacokinetic model of oseltamivir phosphate and its carboxylate metabolite for rats and humans.

Oseltamivir phosphate (OP, Tamiflu®) is a widely used prodrug for the treatment of influenza viral infections. Orally administered OP is rapidly hydrolyzed by the carboxylesterases in animals to oseltamivir carboxylate (OC), a potent influenza virus neuraminidase inhibitor. The goals of this study were to develop and validate a physiologically-based pharmacokinetic (PBPK) model of OP/OC in rats and humans, and to predict the internal tissue doses for OP and OC in humans after receiving OP orally. To this end, a PBPK model of OP/OC was first developed in the rat, which was then scaled up to humans by replacing the physiological and biochemical parameters with human-specific values. The proposed PBPK model consisted of an OP and an OC sub-models each containing nine first-order, flow-limited tissue/organ compartments. OP metabolism to OC was assumed to carry out mainly by hepatic carboxylesterases although extra-hepatic metabolism also occurred especially in the plasma. The PBPK model was developed and validated by experimental data from our laboratories and from the literature. The proposed PBPK model accurately predicted the pharmacokinetic behavior of OP and OC in humans and rats after receiving a single or multiple doses of OP orally or an OC dose i.v. The PBPK model was used to predict the internal tissue doses of OP and OC in a hypothetical human after receiving the recommended dose of 75 mg/kg OP b.i.d. for 6 days. Steady-state OC concentrations in the plasma and major organs such as the lung and the brain were higher than the minimum in vitro IC50 reported for H1N1 influenza virus neuraminidase, confirming OP is an effective, anti-viral agent. OP side-effects in the gastrointestinal tract and brain of humans were explainable by the tissue doses found in these organs. The PBPK model provides a quantitative tool to evaluate the relationship between an externally applied dose of OP and the internal tissue doses in humans. As such the model can be used to adjust the dose regimens for adult patients in disease states e.g., renal failure and liver damage.

Detection of influenza A(H3N2) viruses exhibiting reduced susceptibility to the novel cap-dependent endonuclease inhibitor baloxavir in Japan, December 2018.

The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved for the treatment of influenza virus infection in Japan in February 2018. Two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA) were detected in baloxavir-treated children in December 2018. This mutation is known to confer reduced susceptibility to baloxavir, and the two mutant viruses exhibited 76- and 120-fold reduced susceptibility to baloxavir.

Consecutive influenza surveillance of neuraminidase mutations and neuraminidase inhibitor resistance in Japan.

BackgroundThe large consumption of neuraminidase inhibitors (NAIs) for the treatment of influenza virus infections places Japan at risk of becoming the epicenter of the global spread of NAI‐resistant viruses.ObjectiveTo clarify NA amino acid mutations of epidemic influenza viruses in Japan and their related NAI resistance.MethodsA total of 1791 samples, including 396 A/H1N1pdm09, 1117 A/H3N2, and 278 B isolates, were collected to determine of their 50% inhibitory concentration (IC 50) values by NAIs (oseltamivir, zanamivir, peramivir, and laninamivir) during the Japanese seasons from 2011‐2012 to 2016‐2017. Then, 380 samples including 49 A/H1N1pdm09, 251 A/H3N2, and 80 B isolates were sequenced for the entire NA genes.ResultsNeuraminidase inhibitor‐resistant A/H1N1pdm09 viruses were detected at a frequency of 1.3% (5/396 isolates) in the epidemic seasons. None of the A/H3N2 and B viruses developed resistance to any of the four NAIs during the six seasons. Only five and 13 AA mutations were detected in the NA catalytic sites of A/H1N1pdm09 and A/H3N2 viruses, respectively. No mutations were observed in the catalytic sites of B viruses. Four of the five mutations in the catalytic sites of A/H1N1pdm09 consisted of H275Y, which was related to high resistance to oseltamivir and peramivir. Most (10/13) of the catalytic site mutations in A/H3N2 were associated with MDCK‐passaged induction (D151G/N). Finally, no mutations related to substantial NAI resistance were detected in the A/H3N2 and B viruses examined.ConclusionThese findings suggest that the NA catalytic sites of influenza viruses are well preserved. Even in Japan, no spread of NAI‐resistant viruses has been observed, and A/H1N1pdm09 viruses carrying H275Y remain limited.

Combination treatment with the cap-dependent endonuclease inhibitor baloxavir marboxil and a neuraminidase inhibitor in a mouse model of influenza A virus infection.

ObjectivesBaloxavir marboxil (formerly S-033188) is a first-in-class, orally available, cap-dependent endonuclease inhibitor licensed in Japan and the USA for the treatment of influenza virus infection. We evaluated the efficacy of delayed oral treatment with baloxavir marboxil in combination with a neuraminidase inhibitor in a mouse model of lethal influenza virus infection.MethodsThe inhibitory potency of baloxavir acid (the active form of baloxavir marboxil) in combination with neuraminidase inhibitors was tested in vitro. The therapeutic effects of baloxavir marboxil and oseltamivir phosphate, or combinations thereof, were evaluated in mice lethally infected with influenza virus A/PR/8/34; treatments started 96 h post-infection.ResultsCombinations of baloxavir acid and neuraminidase inhibitor exhibited synergistic potency against viral replication by means of inhibition of cytopathic effects in vitro. In mice, baloxavir marboxil monotherapy (15 or 50 mg/kg twice daily) significantly and dose-dependently reduced virus titre 24 h after administration and completely prevented mortality, whereas oseltamivir phosphate treatments were not as effective. In this model, a suboptimal dose of baloxavir marboxil (0.5 mg/kg twice daily) in combination with oseltamivir phosphate provided additional efficacy compared with monotherapy in terms of virus-induced mortality, elevation of cytokine/chemokine levels and pathological changes in the lung.ConclusionsBaloxavir marboxil monotherapy with 96 h-delayed oral dosing achieved drastic reductions in virus titre, inflammatory response and mortality in a mouse model. Combination treatment with baloxavir acid and oseltamivir acid in vitro and baloxavir marboxil and oseltamivir phosphate in mice produced synergistic responses against influenza virus infections, suggesting that treating humans with the combination may be beneficial.

Structure-Based Optimization of N-Substituted Oseltamivir Derivatives as Potent Anti-Influenza A Virus Agents with Significantly Improved Potency against Oseltamivir-Resistant N1-H274Y Variant.

Due to the emergence of highly pathogenic and oseltamivir-resistant influenza viruses, there is an urgent need to develop new anti-influenza agents. Herein, five subseries of oseltamivir derivatives were designed and synthesized to improve their activity toward drug-resistant viral strains by further exploiting the 150-cavity in the neuraminidases (NAs). The bioassay results showed that compound 21h exhibited antiviral activities similar to or better than those of oseltamivir carboxylate (OSC) against H5N1, H5N2, H5N6, and H5N8. Besides, 21h was 5- to 86-fold more potent than OSC toward N1, N8, and N1-H274Y mutant NAs in the inhibitory assays. Computational studies provided a plausible rationale for the high potency of 21h against group-1 and N1-H274Y NAs. In addition, 21h demonstrated acceptable oral bioavailability, low acute toxicity, potent antiviral activity in vivo, and high metabolic stability. Overall, the above excellent profiles make 21h a promising drug candidate for the treatment of influenza virus infection.

The mechanism of resistance to favipiravir in influenza

Favipiravir is a broad-spectrum antiviral that has shown promise in treatment of influenza virus infections. While emergence of resistance has been observed for many antiinfluenza drugs, to date, clinical trials and laboratory studies of favipiravir have not yielded resistant viruses. Here we show evolution of resistance to favipiravir in the pandemic H1N1 influenza A virus in a laboratory setting. We found that two mutations were required for robust resistance to favipiravir. We demonstrate that a K229R mutation in motif F of the PB1 subunit of the influenza virus RNA-dependent RNA polymerase (RdRP) confers resistance to favipiravir in vitro and in cell culture. This mutation has a cost to viral fitness, but fitness can be restored by a P653L mutation in the PA subunit of the polymerase. K229R also conferred favipiravir resistance to RNA polymerases of other influenza A virus strains, and its location within a highly conserved structural feature of the RdRP suggests that other RNA viruses might also acquire resistance through mutations in motif F. The mutations identified here could be used to screen influenza virus-infected patients treated with favipiravir for the emergence of resistance.

Temperature Sensitive Mutations in Influenza A Viral Ribonucleoprotein Complex Responsible for the Attenuation of the Live Attenuated Influenza Vaccine.

Live attenuated influenza vaccines (LAIV) have prevented morbidity and mortality associated with influenza viral infections for many years and represent the best therapeutic option to protect against influenza viral infections in humans. However, the development of LAIV has traditionally relied on empirical methods, such as the adaptation of viruses to replicate at low temperatures. These approaches require an extensive investment of time and resources before identifying potential vaccine candidates that can be safely implemented as LAIV to protect humans. In addition, the mechanism of attenuation of these vaccines is poorly understood in some cases. Importantly, LAIV are more efficacious than inactivated vaccines because their ability to mount efficient innate and adaptive humoral and cellular immune responses. Therefore, the design of potential LAIV based on known properties of viral proteins appears to be a highly appropriate option for the treatment of influenza viral infections. For that, the viral RNA synthesis machinery has been a research focus to identify key amino acid substitutions that can lead to viral attenuation and their use in safe, immunogenic, and protective LAIV. In this review, we discuss the potential to manipulate the influenza viral RNA-dependent RNA polymerase (RdRp) complex to generate attenuated forms of the virus that can be used as LAIV for the treatment of influenza viral infections, one of the current and most effective prophylactic options for the control of influenza in humans.

PIN20 - A Cost-Effectiveness Analysis of Neuraminidase Inhibitors for Influenza Virus Infection in Japan

Influenza infections epidemics are responsible for medical and social concerns because they are associated with considerable morbidity and mortality in Japan. Neuraminidase inhibitors (NI) appear to be an effective treatment strategy. The study aimed to compare the relative costs and outcomes among four NI strategies for initiation of influenza treatment in adult-outpatient settings from a healthcare payer’s perspective. Using a CEA model, we compared four different NI drugs including oseltamivir, zanamivir, laninamivir, and peramivir. Costs included medical costs and pharmaceutical charges. Medical costs were obtained from the institutional medical fee scheduled table 2016. Authorized medication costs were applied to this study. Outcomes of effectiveness were measured using EQ-5D questionnaires that were applied to patients with influenza virus infections. The study time-horizon was defined as 14 days. Cost Effectiveness Ratios for oseltamivir, zanamivir, laninamivir, and peramivir were 341,076 (Yen/QALY), 351,818, 356,780, and 343,419, respectively. The CEA base-case analysis showed oseltamivir was the most cost-effective in NIs. Zanamivir and laninamivir were dominated. Incremental Cost Effectiveness Ratio (ICER) was 396,043 (Yen/QALY). One-way sensitivity analyses were performed in terms of “duration of treatment”, “QOL values”, “costs of pharmaceutical charges” (+-10%), “costs of physician visits” (+-10%), “costs of community pharmacy visits” (+-10%), “costs of hospital admission” (+-10%), and “effectiveness to high risk patients” (effective 92.9% and pneumonia 8.1%). The maximum ICER in terms of “effectiveness to high risk patients” was 1,352,104 (Yen/QALY) less than 5,000,000 (Yen/QALY). The study results show that oseltamivir was the most cost-effective NI drug for the treatment of influenza virus infection in adult-outpatient settings. Our findings can provide decision-makers with scientific evidence of clinical and economic evaluation for optimal therapeutic outcomes.

Effect of annexin II-mediated conversion of plasmin from plasminogen on airborne transmission of H9N2 avian influenza virus

Airborne transmission plays an important role in dissemination of H9N2 subtype avian influenza virus. Annexin II (A2)-mediated activation of plasminogen (PLG) promotes cleavage of the influenza virus HA protein and viral replication, resulting in enhanced pathogenesis. In this study, airborne transmission competent and defective strains of H9N2 influenza virus, SH7 and SH14, respectively, were used to investigate the effect of A2 on airborne spread. The results showed that A2 protein was increased in SH7 virions compared with SH14 particles, the binding ability of the SH7-infected MDCK cells to PLG was significantly higher than the SH14-infected cells, and influence efficiency of the PLG on replicated ability of SH7 virus was significantly stronger than that of SH14 virus, who spread without airborne route, indicating that the annexin 2 (A2) can bind PLG and contributes to SH7 with high replication ability. Furthermore, the copies of SH7 in the airborne infected chickens under inhibited by 6-AHA were significantly decreased, suggesting that the release of H9N2 avian influenza virus were reduced by inhibiting the conversion of PLG to PL, ultimately resulting in reduced airborne transmission of H9N2 avian influenza virus. In summary, A2-mediated conversion of PLG to PL plays a role in the airborne transmission capacity of H9N2 avian influenza viruses, and this interaction may represent potential targets for prevention and treatment of influenza virus infection.

Sheng Jiang San, a traditional multi-herb formulation, exerts anti-influenza effects in vitro and in vivo via neuraminidase inhibition and immune regulation

BackgroundSheng Jiang San (SJS), a multi-herb formulation, is used in treating high fever, thirsty and anxiety in ancient China and it is sometimes used to treat seasonal influenza nowadays. However, there is no evidence-based investigation and mechanism research to support the anti-influenza efficacy of SJS. This study aims at evaluating the anti-influenza effect of SJS and investigating its possible mechanism.MethodsThe inhibitory effect of SJS against different influenza virus strains on MDCK cells was examined. Influenza virus infected BALB/c mice were employed to evaluate the efficacy as in vivo model. Mice challenged with A/PR/8/34 (H1N1) were orally administrated 1 g/kg/day of SJS for seven days and monitored for 14 days. The survival rate, body weight changes, lung index, lung viral load, histopathologic changes and immune regulation of the mice were measured. The underlying anti-influenza virus mechanism of SJS was studied by a series of biological assays to determine if hemagglutinin, ribonucleoprotein complex or neuraminidase were targets of SJS.ResultsResults showed SJS exerted a broad-spectrum of inhibitory effects on multiple influenza strains in a dose-dependent manner. IC50 of SJS against A/WSN/33 (H1N1) was lower than 35 μg/ml. SJS also protected 50% of mice from A/PR/8/34 (H1N1) infection. The lung index and the lung viral load of SJS treated mice were significantly decreased compared with untreated mice. Meanwhile, SJS targeted on neuraminidase of influenza virus as SJS at 2 mg/ml inhibited 80% of neuraminidase enzymatic activity. SJS also significantly down-regulated TNF-α and up-regulated IL-2 of influenza virus induced mice.ConclusionsThus, SJS is a useful formulation for treating influenza virus infection.

Identification of the I38T PA Substitution as a Resistance Marker for Next-Generation Influenza Virus Endonuclease Inhibitors.

ABSTRACTThe clinical severity and annual occurrence of influenza virus epidemics, combined with the availability of just a single class of antivirals to treat infections, underscores the urgent need to develop new anti-influenza drugs. The endonuclease activity within the viral acidic polymerase (PA) protein is an attractive target for drug discovery due to the critical role it plays in viral gene transcription. RO-7 is a next-generation PA endonuclease inhibitor of influenza A and B viruses, but its drug resistance potential is unknown. Through serial passage of influenza A(H1N1) viruses in MDCK cells under selective pressure of RO-7, we identified an I38T substitution within the PA endonuclease domain that conferred in vitro resistance to RO-7 (up to a 287-fold change in 50% effective concentration [EC50]). I38T emerged between 5 and 10 passages, and when introduced into recombinant influenza A(H1N1) viruses, alone conferred RO-7 resistance (up to an 81-fold change in EC50). Cocrystal structures of mutant and wild-type endonuclease domains with RO-7 provided the structural basis of resistance, where a key hydrophobic interaction between RO-7 and the Ile38 side chain is compromised when mutated to the polar threonine. While Ile38 does not have a crucial role in coordinating the endonuclease active site, the switch to threonine does affect the polymerase activity of some viruses and influences RO-7 affinity for the PAN target (i.e., the ≈200-residue N-terminal domain of PA). However, the change does not lead to a complete loss of replication activity in vitro. Our results predict that RO-7-resistant influenza viruses carrying the I38T substitution may emerge under treatment. This should be taken into consideration for clinical surveillance and in refinement of these drugs.

Anti-influenza virus activity of a salcomine derivative mediated by inhibition of viral RNA synthesis.

Influenza virus infection is a major threat to global health. Although vaccines and anti-influenza virus drugs are available, annual influenza virus epidemics result in severe illness, and an influenza pandemic occurs every 20-30years. To identify candidate anti-influenza virus compounds, we screened approximately 5,000 compounds in an in-house library. We identified MZ7465, a salcomine derivative, as a potent inhibitor of influenza virus propagation. We analyzed the antiviral propagation mechanism of the hit compound by determining the amounts of viral proteins and RNA in infected cells treated with or without the hit compound. Treatment of infected cells with MZ7465 decreased both viral protein and RNA synthesis. In addition, an in vitro assay showed that viral RNA synthesis was directly inhibited by MZ7465. These results suggest that salcomine and its derivatives are potential candidates for the treatment of influenza virus infections.

Antiviral resistance due to deletion in the neuraminidase gene and defective interfering-like viral polymerase basic 2 RNA of influenza A virus subtype H3N2

Background and objectiveAntiviral treatment of influenza virus infections can lead to drug resistance of virus. This study investigates a selection of mutations in the full genome of H3N2 influenza A virus isolated from a patient in treatment with oseltamivir. Study designRespiratory samples from a patient were collected before, during, and after antiviral treatment. Whole genome sequencing of the influenza virus by next generation sequencing, and low-frequency-variant analysis was performed. Neuraminidase-inhibition tests were performed with oseltamivir and zanamivir, and viruses were propagated in sial-transferase gene transfected Madin-Darby Canine Kidney cells. ResultsA deletion at amino acid position 245–248 in the neuraminidase gene occurred after initiation of treatment with oseltamivir. The deleted virus had highly reduced inhibition against oseltamivir but was sensitive to zanamivir. Nine days after discontinuation of oseltamivir treatment the deleted H3N2 virus was still present in the patient. After three passages of the deleted virus in cell culture, the deletion was retained. Several variant mutations appeared in the other genes of the H3N2 virus, where most striking were two major out-of-frame deletions in the polymerase basic 2 (PB2) gene, indicating defective interfering-like viral RNA. ConclusionsThe viruses harboring the 245–248 deletion in the neuraminidase gene were still present after discontinuation of oseltamivir treatment and passages in cell cultures, indicating a potential risk for transmission of the deleted virus. Full genome deep sequencing was useful to reveal variant mutations that might be selected due to antiviral treatment, and defective interfering-like viral PB2 RNA in the respiratory samples was detected.

Antiviral Activity of the Sesquiterpene Lactones from Centipeda minima against Influenza a Virus in vitro

During the course of searching for antiviral agents from Chinese medicinal herbs, we found that the supercritical fluid extract (SFE) of Centipeda minima possessed good in vitro antiviral activity against influenza virus A/Puerto Rico/8/34 H1N1 (PR8). Bioassay-guided isolation and identification led to the isolation from this extract of seven pseudoguaianolides (1-7). These, as well as nine other sesquiterpene lactones (8-16) previously isolated from this herb were all tested for their anti-PR8 activity using both the cytopathogenic effect (CPE) reduction and cell counting kit 8 (CCK8) assays. As a result, eight pseudoguaianolides (1-8) possessing an α,β-unsaturated cyclopentenone moiety showed antiviral activity against PR8 to different extents. Of the active compounds, brevilin A (4) exhibited the strongest anti-PR8 activity, with an IC50 value much lower than that of the positive control ribavirin. Mechanistic study revealed that brevilin A affected the intracellular replication of PR8 via downregulating the expression of viral M2 protein. All these results suggest the potential application of the pseudoguaianolides containing an α,β-unsaturated cyclopentenone moiety (e.g. brevilin A) in the treatment of influenza virus infection.