In vivo secondary structural analysis of Influenza A virus genomic RNA.

Influenza A virus (IAV) is a respiratory virus that causes epidemics and pandemics. Knowledge of IAV RNA secondary structure in vivo is crucial for a better understanding of virus biology. Moreover, it is a fundament for the development of new RNA-targeting antivirals. Chemical RNA mapping using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) coupled with Mutational Profiling (MaP) allows for the thorough examination of secondary structures in low-abundance RNAs in their biological context. So far, the method has been used for analyzing the RNA secondary structures of several viruses including SARS-CoV-2 in virio and in cellulo. Here, we used SHAPE-MaP and dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) for genome-wide secondary structure analysis of viral RNA (vRNA) of the pandemic influenza A/California/04/2009 (H1N1) strain in both in virio and in cellulo environments. Experimental data allowed the prediction of the secondary structures of all eight vRNA segments in virio and, for the first time, the structures of vRNA5, 7, and 8 in cellulo. We conducted a comprehensive structural analysis of the proposed vRNA structures to reveal the motifs predicted with the highest accuracy. We also performed a base-pairs conservation analysis of the predicted vRNA structures and revealed many highly conserved vRNA motifs among the IAVs. The structural motifs presented herein are potential candidates for new IAV antiviral strategies.

Antiviral Effect of Isoquercitrin against Influenza A Viral Infection via Modulating Hemagglutinin and Neuraminidase.

Isoquercitrin (IQC) is a component abundantly present in many plants and is known to have an anti-viral effect against various viruses. In this study, we demonstrate that IQC exhibits strong anti-influenza A virus infection, and its effect is closely related to the suppression of hemagglutinin (HA) and neuraminidase (NA) activities. We used green fluorescent protein-tagged Influenza A/PR/8/34 (H1N1), A/PR/8/34 (H1N1), and HBPV-VR-32 (H3N2) to evaluate the anti-IAV effect of IQC. The fluorescence microscopy and fluorescence-activated cell sorting analysis showed that IQC significantly decreases the levels of GFP expressed by IAV infection, dose-dependently. Consistent with that, IQC inhibited cytopathic effects by H1N1 or H3N2 IAV infection. Immunofluorescence analysis confirmed that IQC represses the IAV protein expression. Time-of-addition assay showed that IQC inhibits viral attachment and entry and exerts a strong virucidal effect during IAV infection. Hemagglutination assay confirmed that IQC affects IAV HA. Further, IQC potently reduced the NA activities of H1N1 and H3N2 IAV. Collectively, IQC prevents IAV infection at multi-stages via virucidal effects, inhibiting attachment, entry and viral release. Our results indicate that IQC could be developed as a potent antiviral drug to protect against influenza viral infection.

The traditional herbal formulation, Jianpiyifei II, reduces pulmonary inflammation induced by influenza A virus and cigarette smoke in mice.

Chronic obstructive pulmonary disease (COPD) is a worldwide chronic inflammatory lung disease, and influenza A virus (IAV) infection is a common cause of acute exacerbations of COPD (AECOPD). Therefore, targeting viral infections represents a promising strategy to prevent the occurrence and development of inflammatory flare ups in AECOPD. Jianpiyifei II (JPYFII) is a traditional herbal medicine used in China to treat patients with COPD, and its clinical indications are not well understood. However, investigation of the anti-inflammatory effects and underlying mechanism using an animal model of smoking have been reported in a previous study by our group. In addition, some included herbs, such as Radix astragali and Radix aupleuri, were reported to exhibit antiviral effects. Therefore, the aim of the present study was to investigate whether JPYFII formulation relieved acute inflammation by clearing the IAV in a mouse model that was exposed to cigarette smoke experimentally. JPYFII formulation treatment during smoke exposure and IAV infection significantly reduced the number of cells observed in bronchoalveolar lavage fluid (BALF), expression of proinflammatory cytokines, chemokines, superoxide production, and viral load in IAV-infected and smoke-exposed mice. However, JPYFII formulation treatment during smoke exposure alone did not reduce the number of cells in BALF or the expression of Il-6, Tnf-a, and Il-1ß. The results demonstrated that JPYFII formulation exerted an antiviral effect and reduced the exacerbation of lung inflammation in cigarette smoke (CS)-exposed mice infected with IAV. Our results suggested that JPYFII formulation could potentially be used to treat patients with AECOPD associated with IAV infection.

Cellular Protein Phosphatase 2A Regulates Cell Survival Mechanisms in Influenza A Virus Infection.

Influenza A viruses (IAVs) are respiratory pathogens that are able to hijack multiple cellular mechanisms to drive their replication. Consequently, several viral and cellular proteins undergo posttranslational modifications such as dynamic phosphorylation/dephosphorylation. In eukaryotic cells, dephosphorylation is mainly catalyzed by protein phosphatase 2A (PP2A). While the function of kinases in IAV infection is quite well studied, only little is known about the role of PP2A in IAV replication. Here, we show, by using knockdown and inhibition approaches of the catalytic subunit PP2Ac, that this phosphatase is important for efficient replication of several IAV subtypes. This could neither be attributed to alterations in the antiviral immune response nor to changes in transcription or translation of viral genes. Interestingly, decreased PP2Ac levels resulted in a significantly reduced cell viability after IAV infection. Comprehensive kinase activity profiling identified an enrichment of process networks related to apoptosis and indicated a synergistic action of hyper-activated PI3K/Akt, MAPK/JAK-STAT and NF-kB signaling pathways, collectively resulting in increased cell death. Taken together, while IAV seems to effectively tap leftover PP2A activity to ensure efficient viral replication, reduced PP2Ac levels fail to orchestrate cell survival mechanisms to protect infected cells from early cell death.

Development of an RNA Strand-Specific Hybridization Assay To Differentiate Replicating versus Nonreplicating Influenza A Viruses.

Replication of influenza A virus (IAV) from negative-sense viral RNA (vRNA) requires the generation of positive-sense RNA (+RNA). Most molecular assays, such as conventional real-time reverse transcriptase PCR (rRT-PCR), detect total RNA in a sample without differentiating vRNA from +RNA. These assays are not designed to distinguish IAV infection versus exposure of an individual to an environment enriched with IAVs but wherein no viral replication occurs. We therefore developed a strand-specific hybridization (SSH) assay that differentiates between vRNA and +RNA and quantifies relative levels of each RNA species. The SSH assay exhibited a linearity of 7 logs with a lower limit of detection of 6.0 × 102 copies of molecules per reaction. No signal was detected in samples with a high load of nontarget template or influenza B virus, demonstrating assay specificity. IAV +RNA was detected 2 to 4 h postinoculation of MDCK cells, whereas synthesis of cold-adapted IAV +RNA was significantly impaired at 37°C. The SSH assay was then used to test IAV rRT-PCR positive nasopharyngeal specimens collected from individuals exposed to IAV at swine exhibitions (n = 7) or while working at live bird markets (n = 2). The SSH assay was able to differentiate vRNA and +RNA in samples collected from infected, symptomatic individuals versus individuals who were exposed to IAV in the environment but had no active viral replication. Data generated with this technique, especially when coupled with clinical data and assessment of seroconversion, will facilitate differentiation of actual IAV infection with replicating virus versus individuals exposed to high levels of environmental contamination but without virus infection.

Anti-Influenza Virus Activity and Chemical Components from the Parasitic Plant Cuscuta japonica Choisy on Dimocarpus longans Lour.

Dodder (Cuscuta spp.) is a parasitic weed damaging many plants and agricultural production. The native obligate parasite Cuscuta japonica Choisy (Japanese dodder) parasitizes Dimocarpus longans Lour., Ficus septica Burm. F., Ficus microcarpa L.f., Mikania micrantha H.B.K. and Melia azedarach Linn, respectively. Five Japanese dodders growing on different plants exhibit slightly different metabolites and amounts which present different pharmacological effects. Among these plants, a significant antiviral activity against influenza A virus (IAV) was found in Japanese dodder parasitizing on D. longans Lour. (CL). To further explore methanol extract components in Japanese dodder (CL), four undescribed aromatic glycosides, cuscutasides A-D (compounds 1-4) were isolated, together with twenty-six known compounds 5-30. The chemical structures of 1-4 were elucidated using a combination of spectroscopic techniques. The eighteen isolated compounds were evaluated for antiviral activity against IAV activity. Among them, 1-monopalmitin (29) displayed potent activity against influenza A virus (A/WSN/1933(H1N1)) with EC50 2.28 ± 0.04 µM and without noteworthy cytotoxicity in MDCK cells. The interrupt step of 29 on the IAV life cycle was determined. These data provide invaluable information for new applications for this otherwise harmful weed.

Guava flavonoid glycosides prevent influenza A virus infection via rescue of P53 activity.

Influenza is a highly infectious disease caused by three types of viruses, including influenza A virus (IAV), influenza B virus, and, rarely, influenza C virus. IAV is a major, global public health threat, causing approximately 500 000 deaths per year worldwide. The new strains of IAV have emerged due to a mutation called antigenic shift, which results in a new subtype of the virus that shows resistance to common antiviral drugs. Here, guava and lemon extracts, including green leaves and flowers, were investigated for their activity against IAV replication in human A549 cells. Concomitantly, the cytotoxicity of a potent extract on host-cell multiplication was assessed. Our results reveal that guava extracts inhibit IAV replication, indicated by viral nucleoprotein expression profile and traditional plaque assay. Interestingly, treatment with guava extract inactivates Akt protein kinase and stimulates the pro-apoptotic protein P53, at early stages of infection. Furthermore, purified guava flavonoid glycosides (GFGs) show competitive inhibition of IAV-virus replication via early regulation of IL-1ß and IL-8 in association with P53 gene expression. The docking analysis of GFGs and the protein structure of upstream targets for the Akt signaling pathway indicates a sufficient interaction and stabilization with Gbr2 protein. These data indicate that treatment with GFGs disturbs IAV replication via activation of P53 and its apoptotic related factors after infection. Collectively, these data show that targeting of essential host kinases that are involved in the replication cycle of IAV and rescue of P53 activity by GFGs could represent a new strategy to eradicate IAV.

RNA epitranscriptomics: Regulation of infection of RNA and DNA viruses by N6 -methyladenosine (m6 A).

N6 -methyladenosine (m6 A) was discovered 4 decades ago. However, the functions of m6 A and the cellular machinery that regulates its changes have just been revealed in the last few years. m6 A is an abundant internal mRNA modification on cellular RNA and is implicated in diverse cellular functions. Recent works have demonstrated the presence of m6 A in the genomes of RNA viruses and transcripts of a DNA virus with either a proviral or antiviral role. Here, we first summarize what is known about the m6 A "writers," "erasers," "readers," and "antireaders" as well as the role of m6 A in mRNA metabolism. We then review how the replications of numerous viruses are enhanced and restricted by m6 A with emphasis on the oncogenic DNA virus, Kaposi sarcoma-associated herpesvirus (KSHV), whose m6 A epitranscriptome was recently mapped. In the context of KSHV, m6 A and the reader protein YTHDF2 acts as an antiviral mechanism during viral lytic replication. During viral latency, KSHV alters m6 A on genes that are implicated in cellular transformation and viral latency. Lastly, we discuss future studies that are important to further delineate the functions of m6 A in KSHV latent and lytic replication and KSHV-induced oncogenesis.

Lung Aerosol Dynamics of Airborne Influenza A Virus-Laden Droplets and the Resultant Immune System Responses: An In Silico Study.

Influenza A Virus (IAV) replications start from the deposition of inhaled virus-laden droplets on the epithelial cells in the pulmonary tracts. In order to understand the local deposition patterns and within-host dynamics of infectious aerosols, accurate information of high-resolution imaging capabilities, as well as real-time flow cytometry analysis, are required for tracking infected cells, virus agents, and immune system responses. However, clinical and animal studies are in deficit to meet the above-mentioned demands, due to their limited operational flexibility and imaging resolution. Therefore, this study developed an experimentally validated multiscale epidemiological computational model, i.e., the Computational Fluid-Particle Dynamics (CFPD) plus Host Cell Dynamics (HCD) model, to predict the transport and deposition of the low-strain IAV-laden droplets, as well as the resultant regional immune system responses. The hygroscopic growth and shrinkage of IAV-laden droplets were accurately modeled. The subject-specific respiratory system was discretized by generating the new polyhedral-core mesh. By simulating both mouth and nasal breathing scenarios, the inhalations of isotonic IAV-laden droplets with three different compositions were achieved. It is the first time that parametric analysis was performed using the multiscale model on how different exposure conditions can influence the virus aerodynamics in the lung and the subsequent immune system responses. Numerical results show a higher viral accretion followed by a faster immune system response in the supraglottic region when droplets with the higher salt concentration were inhaled. Consequently, more severe symptoms and longer recovery are expected at the pharynx. Furthermore, local deposition maps of IAV-laden droplets and post-deposition infection dynamics provide informative and direct evidence which significantly enhance the fundamental understanding of the underlying mechanisms for upper airway and lower airway infections.

CD151, a novel host factor of nuclear export signaling in influenza virus infection.

BACKGROUND: Despite advances in our understanding of the mechanisms of influenza A virus (IAV) infection, the crucial virus-host interactions during the viral replication cycle still remain incomplete. Tetraspanin CD151 is highly expressed in the human respiratory tract, but its pathological role in IAV infection is unknown. OBJECTIVES: We sought to characterize the functional role and mechanisms of action of CD151 in IAV infection of the upper and lower respiratory tracts with H1N1 and H3N2 strains. METHODS: We used CD151-null mice in an in vivo model of IAV infection and clinical donor samples of in vitro-differentiated human nasal epithelial cells cultured at air-liquid interface. RESULTS: As compared with wild-type infected mice, CD151-null infected mice exhibited a significant reduction in virus titer and improvement in survival that is associated with pronounced host antiviral response and inflammasome activation together with accelerated lung repair. Interestingly, we show that CD151 complexes newly synthesized viral proteins with host nuclear export proteins and stabilizes microtubule complexes, which are key processes necessary for the polarized trafficking of viral progeny to the host plasma membrane for assembly. CONCLUSIONS: Our results provide new mechanistic insights into our understanding of IAV infection. We show that CD151 is a critical novel host factor of nuclear export signaling whereby the IAV nuclear export uses it to complement its own nuclear export proteins (a site not targeted by current therapy), making this regulation unique, and holds promise for the development of novel alternative/complementary strategies to reduce IAV severity.

Monoclonal antibody against the universal M2 epitope of influenza A virus.

M2 protein, a highly conserved protein of influenza A virus (IAV), plays an important role in virus particle uncoating, assembly, and budding. In the present study, eight monoclonal antibodies (mAbs) against the M2 protein of the H3N2 IAV strain were generated with recombinant truncated M2 protein or BSA-coupled M2 peptides as immunogens. The linear epitopes recognized by the mAbs were defined by IFA and peptide ELISA. The results showed that mAb 10F4 recognized an epitope located in the N-terminal 6-12 amino acids of the M2 peptide, and the mAbs 10D9, 1E2, 4B5, and 5G10 recognized the epitopes located in the C-terminal 62-77 amino acids of the M2 peptide. Importantly, mAb 10D9 recognized the M2 protein of H1-H13 IAV subtypes, which stained M2 protein located on the membrane of host cells and could be applied in immunoprecipitation and immunohistochemistry assays. The mAb 10D9 which recognizes the universal M2 epitope of IAVs will be a useful tool for studies on the function of IAV M2 protein and for the development of vaccines or detection methods for IAV infection.

FcγRIIB modulates splenic germinal center response against immune subversion during acute influenza A virus infection.

BACKGROUND: B cells are essential for providing humoral protection against acute influenza A virus (IAV) infection. FcγRIIB, a regulator of antibody (Ab) production, influences immune responses during pathogen infections, but its specific impact on humoral protection and B cell-mediated responses against IAV remains unclear. METHODS: To investigate FcγRIIB's role in host defense and B cell function during acute IAV infection, we generated mice with systemic FcγRIIB deficiency, functional impairment, and B cell-specific FcγRIIB deletion. We infected these mice with PR8 (H1N1) or Hkx31 (H3N2) IAVs and evaluated body weight preservation, survival rates, Ab production, viral neutralization, Ab affinity maturation, and germinal center B cell development. RESULTS: Mice lacking FcγRIIB or with impaired function showed improved protection, preserved body weight, and increased survival rates during IAV infection. Notably, mice with haploinsufficient FcγRIIB function displayed protective effects. Selective deficiency of FcγRIIB in B cells led to enhanced Ab production, resulting in elevated IAV-specific Abs in the serum with superior viral neutralizing potency. However, the impact on the affinity maturation index of virus-specific Abs was modest. Accordingly, FcγRIIB-deficient B cells maintained normal germinal center B cell development during IAV infection, whereas wild-type mice exhibited delayed differentiation. CONCLUSION: Our research underscores the pivotal role of FcγRIIB in host defense and B cell-mediated immunity during acute IAV infection. Additionally, our discoveries hold implications for antiviral treatments, particularly during the initial stages of IAV infection, aimed at enhancing the host's humoral immune response.

Mutual antagonism of mouse-adaptation mutations in HA and PA proteins on H9N2 virus replication.

Avian H9N2 viruses have wide host range among the influenza A viruses. However, knowledge of H9N2 mammalian adaptation is limited. To explore the molecular basis of the adaptation to mammals, we performed serial lung passaging of the H9N2 strain A/chicken/Hunan/8.27 YYGK3W3-OC/2018 (3W3) in mice and identified six mutations in the hemagglutinin (HA) and polymerase acidic (PA) proteins. Mutations L226Q, T511I, and A528V of HA were responsible for enhanced pathogenicity and viral replication in mice; notably, HA-L226Q was the key determinant. Mutations T97I, I545V, and S594G of PA contributed to enhanced polymerase activity in mammalian cells and increased viral replication levels in vitro and in vivo. PA-T97I increased viral polymerase activity by accelerating the viral polymerase complex assembly. Our findings revealed that the viral replication was affected by the presence of PA-97I and/or PA-545V in combination with a triple-point HA mutation. Furthermore, the double- and triple-point PA mutations demonstrated antagonistic effect on viral replication when combined with HA-226Q. Notably, any combination of PA mutations, along with double-point HA mutations, resulted in antagonistic effect on viral replication. We also observed antagonism in viral replication between PA-545V and PA-97I, as well as between HA-528V and PA-545V. Our findings demonstrated that several antagonistic mutations in HA and PA proteins affect viral replication, which may contribute to the H9N2 virus adaptation to mice and mammalian cells. These findings can potentially contribute to the monitoring of H9N2 field strains for assessing their potential risk in mammals.

Location, Age, and Antibodies Predict Avian Influenza Virus Shedding in Ring-Billed and Franklin's Gulls in Minnesota.

Influenza A virus (IAV) is a multi-host pathogen maintained in water birds and capable of spillover into humans, wildlife, and livestock. Prior research has focused on dabbling ducks as a known IAV reservoir species, yet our understanding of influenza dynamics in other water birds, including gulls, is lacking. Here, we quantify morphological and environmental drivers of serological (antibody detection by ELISA) and virological (viral RNA detection by PCR) prevalence in two gull species: ring-billed (Larus delawarensis) and Franklin's (Leucophaeus pipixcan) gulls. Across 12 months and 10 locations, we tested over 1500 gulls for influenza viral RNA, and additionally tested antibody levels in nearly 1000 of these. We find substantial virus prevalence and a large, nonoverlapping seroprevalence, with significant differences across age and species classifications. The body condition index had minimal explanatory power to predict (sero)positivity, and the effect of the surrounding environment was idiosyncratic. Our results hint at a nontrivial relationship between virus and seropositivity, highlighting serological surveillance as a valuable counterpoint to PCR. By providing indication of both past infections and susceptibility to future infections, serosurveillance can help inform the distribution of limited resources to maximize surveillance effectiveness for a disease of high human, wildlife, and livestock concern.

Filling two needs with one deed: a combinatory mucosal vaccine against influenza A virus and respiratory syncytial virus.

Influenza A Virus (IAV) and Respiratory Syncytial Virus (RSV) are both responsible for millions of severe respiratory tract infections every year worldwide. Effective vaccines able to prevent transmission and severe disease, are important measures to reduce the burden for the global health system. Despite the strong systemic immune responses induced upon current parental immunizations, this vaccination strategy fails to promote a robust mucosal immune response. Here, we investigated the immunogenicity and efficacy of a mucosal adenoviral vector vaccine to tackle both pathogens simultaneously at their entry site. For this purpose, BALB/c mice were immunized intranasally with adenoviral vectors (Ad) encoding the influenza-derived proteins, hemagglutinin (HA) and nucleoprotein (NP), in combination with an Ad encoding for the RSV fusion (F) protein. The mucosal combinatory vaccine induced neutralizing antibodies as well as local IgA responses against both viruses. Moreover, the vaccine elicited pulmonary CD8+ and CD4+ tissue resident memory T cells (TRM) against the immunodominant epitopes of RSV-F and IAV-NP. Furthermore, the addition of Ad-TGFß or Ad-CCL17 as mucosal adjuvant enhanced the formation of functional CD8+ TRM responses against the conserved IAV-NP. Consequently, the combinatory vaccine not only provided protection against subsequent infections with RSV, but also against heterosubtypic challenges with pH1N1 or H3N2 strains. In conclusion, we present here a potent combinatory vaccine for mucosal applications, which provides protection against two of the most relevant respiratory viruses.

Integrated serum pharmacochemistry and investigation of the anti-influenza A virus pneumonia effect of Qingjin Huatan decoction.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingjin Huatan Decoction (QJHTT) consists of 11 herbal medicines: Scutellaria baicalensis Georgi, Gardenia jasminoides J. Ellis, Platycodon grandiflorus (Jacq.) A. DC., Ophiopogon japonicus (Thunb.) Ker Gawl., Morus alba L., Fritillaria thunbergii Miq., Anemarrhena asphodeloides Bunge, Trichosanthes kirilowii Maxim., Citrus reticulata Blanco, Poria cocos (Schw.) Wolf, and Glycyrrhiza uralensis Fisch. As a traditional Chinese medicinal formula, QJHTT has been used for more than 400 years in China. It has shown promising results in treating influenza A virus (IAV) pneumonia. AIM OF THE STUDY: To elusive the specific pharmacological constituents and mechanisms underlying its anti-IAV pneumonia effects. MATERIALS AND METHODS: The components in QJHTT were analyzed through the use of a serum pharmacology-based ultra high-performance liquid chromatography Q- Exactive Orbitrap mass spectrometry (UHPLC-Q Exactive Orbitrap-MS) method. Simultaneously, the dynamic changes in IAV-infected mouse lung viral load, lung index, and expression of lung inflammation factors were monitored by qRT-PCR. RESULTS: We successfully identified 152 chemical components within QJHTT, along with 59 absorbed chemical prototype constituents found in the serum of mice treated with QJHTT. 43.45% of these chemical components and 43.10% of the prototype constituents were derived from the monarch drugs, namely Huangqin and Zhizi, aligning perfectly with traditional Chinese medicine theory. Notably, our analysis led to the discovery of 14 compounds within QJHTT for the first time, three of which were absorbed into the bloodstream. Simultaneously, we observed that QJHTT not only reduced the viral load but also modulated the expression of inflammation factors in the lung tissue including TNF-α, IL-1ß, IL-4, IL-6, IFN-γ, and IL17A. A time-effect analysis further revealed that QJHTT intervention effectively suppressed the peak of inflammatory responses, demonstrating a robust anti-IAV pneumonia effect. CONCLUSIONS: We comprehensively analyzed the pharmacological material basis of QJHTT by a highly sensitive and high-resolution UHPLC-Q Exactive Orbitrap-MS method, and demonstrated its efficacy in combating IAV pneumonia by reducing lung viral load and inflammatory factors. This study has significant importance for elucidating the pharmacological basis and pharmacological mechanism of QJHTT in combating IAV pneumonia.

Sangju Cold Granule exerts anti-viral and anti-inflammatory activities against influenza A virus in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Sangju Cold Granule (SJCG) is a classical traditional Chinese medicine (TCM) prescription described in "Item Differentiation of Warm Febrile Diseases". Historically, SJCG was employed to treat respiratory illnesses. Despite its popular usage, the alleviating effect of SJCG on influenza A virus infection and its mechanisms have not been fully elucidated. AIM OF THE STUDY: Influenza is a severe respiratory disease that threatens human health. This study aims to assess the therapeutic potential of SJCG and the possible molecular mechanism underlying its activity against influenza A virus in vitro and in vivo. MATERIALS AND METHODS: Ultrahigh-performance liquid chromatography (UPLC)-Q-Exactive was used to identify the components of SJCG. The 50% cytotoxic concentration of SJCG in MDCK and A549 cells were determined using the CCK-8 assay. The activity of SJCG against influenza A virus H1N1 was evaluated in vitro using plaque reduction and progeny virus titer reduction assays. RT-qPCR was performed to obtain the expression levels of inflammatory mediators and the transcriptional regulation of RIG-I and MDA5 in H1N1-infected A549 cells. Then, the mechanism of SJCG effect on viral replication and inflammation was further explored by measuring the expressions of proteins of the RIG-I/NF-kB/IFN(I/III) signaling pathway by Western blot. The impact of SJCG was explored in vivo in an intranasally H1N1-infected BALB/c mouse pneumonia model treated with varying doses of SJCG. The protective role of SJCG in this model was evaluated by survival, body weight monitoring, lung viral titers, lung index, lung histological changes, lung inflammatory mediators, and peripheral blood leukocyte count. RESULTS: The main SJCG chemical constituents were flavonoids, carbohydrates and glycosides, amino acids, peptides, and derivatives, organic acids and derivatives, alkaloids, fatty acyls, and terpenes. The CC50 of SJCG were 24.43 mg/mL on MDCK cells and 20.54 mg/mL on A549 cells, respectively. In vitro, SJCG significantly inhibited H1N1 replication and reduced the production of TNF-α, IFN-ß, IL-6, IL-8, IL-13, IP-10, RANTES, TRAIL, and SOCS1 in infected A549 cells. Intracellularly, SJCG reduced the expression of RIG-I, MDA5, P-NF-κB P65 (P-P65), P-IκBα, P-STAT1, P-STAT2, and IRF9. In vivo, SJCG enhanced the survival rate and decreased body weight loss in H1N1-infected mice. Mice with H1N1-induced pneumonia treated with SJCG showed a lower lung viral load and lung index than untreated mice. SJCG effectively alleviated lung damage and reduced the levels of TNF-α, IFN-ß, IL-6, IP-10, RANTES, and SOCS1 in lung tissue. Moreover, SJCG significantly ameliorated H1N1-induced leukocyte changes in peripheral blood. CONCLUSIONS: SJCG significantly reduced influenza A virus and virus-mediated inflammation through inhibiting the RIG-I/NF-kB/IFN(I/III) signaling pathway. Thus, SJCG could provide an effective TCM for influenza treatment.

Allopregnanolone targets nucleoprotein as a novel influenza virus inhibitor.

Influenza A virus (IAV) poses a global public health concern and remains an imminent threat to human health. Emerging antiviral resistance to the currently approved influenza drugs emphasizes the urgent need for new therapeutic entities against IAV. Allopregnanolone (ALLO) is a natural product that has been approved as an antidepressant drug. In the present study, we repurposed ALLO as a novel inhibitor against IAVs. Mechanistic studies demonstrated that ALLO inhibited virus replication by interfering with the nucleus translocation of viral nucleoprotein (NP). In addition, ALLO showed significant synergistic activity with compound 16, a hemagglutinin inhibitor of IAVs. In summary, we have identified ALLO as a novel influenza virus inhibitor targeting NP, providing a promising candidate that deserves further investigation as a useful anti-influenza strategy in the future.

Revisiting influenza A virus life cycle from a perspective of genome balance.

Influenza A virus (IAV) genome comprises eight negative-sense RNA segments, of which the replication is well orchestrated and the delicate balance of multiple segments are dynamically regulated throughout IAV life cycle. However, previous studies seldom discuss these balances except for functional hemagglutinin-neuraminidase balance that is pivotal for both virus entry and release. Therefore, we attempt to revisit IAV life cycle by highlighting the critical role of "genome balance". Moreover, we raise a "balance regression" model of IAV evolution that the virus evolves to rebalance its genome after reassortment or interspecies transmission, and direct a "balance compensation" strategy to rectify the "genome imbalance" as a result of artificial modifications during creation of recombinant IAVs. This review not only improves our understanding of IAV life cycle, but also facilitates both basic and applied research of IAV in future.

The antiviral activity of Thuja orientalis folium against Influenza A virus.

Thuja orientalis Folium (TOF) has been prescribed traditionally as an expectorant for inflammatory airway disease. In this study, we evaluated the anti-influenza A virus (IAV) activity of TOF by detecting GFP expressed by influenza A virus (A/PR/8/34-GFP) infection. The fluorescence microscopy and fluorescence-activated cell sorting analysis showed that TOF potently inhibited IAV infection, dose-dependently. Consistently, immunofluorescence and Q-PCR analysis results confirmed TOF significantly represses IAV protein and RNA expression. TOF inhibited IAV infection at the binding and entry step upon viral infection and interferes with HA protein. Further, TOF exhibited a virucidal effect and inhibited the neuraminidase activity of IAV. Additionally, TOF prevented the cytopathic effect caused by H1N1 and H3N2 IAV infection. Amentoflavone among the constituents in TOF exerted the strongest anti-IAV effect. Myricetin, quercetin, and quercitrin also inhibited IAV infection. However, the potent anti-IAV effect of TOF may be related to the synergistic effect of constituents, not by a single specific compound. Our results suggest TOF exhibits a significant inhibitory effect against IAV infection at multi-stages via the blockage of viral attachment and entry, inhibition of neuraminidase, and induction of virucidal effects.