Antiviral Activity of Some Compounds of Gasteroid Mushrooms from Western Siberia.

The antiviral activity of aqueous and ethanol extracts from the fruiting bodies of gasteroid Basidiomy-cetes of Western Siberia: Lycoperdon pyriforme, Lycoperdon perlatum, and Phallus impudicus, as well as an aqueous extract from cultivated mycelium of P. impudicus and total polysaccharides from it, on MDCK cell culture against influenza A virus, was studied. Aqueous and ethanol extracts from the fruiting bodies of all studied gasteroid fungi showed antiviral activity against human influenza virus A/Aichi/2/68 (H3N2) and bird A/chicken/Kurgan/05/2005 virus (H5N1). At the same time, extracts from P. impudicus and L. pyriforme showed more pronouncing antiviral activity compared to the activity of the reference drug Tamiflu against the A/H5N1 avian influenza virus. A high antiviral efficacy of an aqueous extract from cultivated mycelium of the P. impudicus and a sample of total polysaccharides from this extract against the A/H5N1 avian influenza virus was revealed.

Dietary lysozyme and avilamycin modulate gut health, immunity, and growth rate in broilers.

BACKGROUND: Attempts to use dietary lysozyme (LYZ) as an alternative to antibiotics in broilers have been successful, but further research is needed for effective use. Here, we compared the differences between LYZ and avilamycin (AVI) feed additives for growth performance, gut health and immunity of broilers. One-day old, one hundred and twenty broiler chicks (Ross 308) were randomly allocated into three groups consisting forty birds in each group. Standard diet without supplementation was applied as the control group (I), while the chicks of the other groups were supplemented with 100 mg of AVI per kg diet (AVI, group II), and 90 mg LYZ per kg diet (LYZ, group III) for five consecutive weeks. RESULTS: Body weight, feed conversion ratio, body weight gain, and European production efficiency factor were markedly (p < 0.05) increased in both AVI and LYZ groups in relation to CON group, but the feed intake and protein efficiency ratio were not affected. Both AVI and LYZ significantly (p < 0.001) upregulated the mRNA expression of ileal interleukin-18 (IL-18), interferon-gamma (IFN-γ), and interleukin-10 (IL-10), interleukin-2 (IL-2), and glutathione peroxidase (GSH-PX) genes compared to CON group. However, IL-2, IL-10, IL-18, and GSH-PX genes were markedly (p < 0.01) upregulated in LYZ compared to the AVI group. LYZ treated group had a significant increase (p < 0.05) in the serological haemagglutination inhibition titers of H5N1 vaccination and a significant decrease (p < 0.0001) in coliform counts compared to control and AVI groups, but all growth parameters were nearly similar between AVI and LYZ groups. The VH and VH/CD were markedly higher in LYZ than AVI and control groups. CONCLUSION: Exogenous dietary lysozyme supplementation by a dose of 90 mg/kg broilers' diet induced better effects on intestinal integrity, fecal bacterial counts, immune response, and growth performance which were comparable to avilamycin. Therefore, dietary lysozyme could safely replace avilamycin in the broiler chickens' diet. However, further experimental studies regarding the use of lysozyme in commercial broilers, both in vitro and in vivo, targeting more communities of intestinal microbiome and explaining more details about its beneficial effects need to be conducted.

Ambient atmospheric PM worsens mouse lung injury induced by influenza A virus through lysosomal dysfunction.

BACKGROUND: Particulate matter (PM) air pollution poses a significant risk to respiratory health and is especially linked with various infectious respiratory diseases such as influenza. Our previous studies have shown that H5N1 virus infection could induce alveolar epithelial A549 cell death by enhancing lysosomal dysfunction. This study aims to investigate the mechanisms underlying the effects of PM on influenza virus infections, with a particular focus on lysosomal dysfunction. RESULTS: Here, we showed that PM nanoparticles such as silica and alumina could induce A549 cell death and lysosomal dysfunction, and degradation of lysosomal-associated membrane proteins (LAMPs), which are the most abundant lysosomal membrane proteins. The knockdown of LAMPs with siRNA facilitated cellular entry of both H1N1 and H5N1 influenza viruses. Furthermore, we demonstrated that silica and alumina synergistically increased alveolar epithelial cell death induced by H1N1 and H5N1 influenza viruses by enhancing lysosomal dysfunction via LAMP degradation and promoting viral entry. In vivo, lung injury in the H5N1 virus infection-induced model was exacerbated by pre-exposure to silica, resulting in an increase in the wet/dry ratio and histopathological score. CONCLUSIONS: Our findings reveal the mechanism underlying the synergistic effect of nanoparticles in the early stage of the influenza virus life cycle and may explain the increased number of respiratory patients during periods of air pollution.

Inhibition of influenza a virus infection by natural stilbene piceatannol targeting virus hemagglutinin.

BACKGROUND: Given the magnitude of influenza pandemics as a threat to the global population, it is crucial to have as many prevention and treatment options as possible. Piceatannol (PIC) is a tetrahydroxylated stilbenoid (trans-3,4,3',5'-tetrahydroxystilbene), also known as 3'- hydroxy resveratrol, which has demonstrated many different biological activities such as anti-inflammatory and antiviral activities. PURPOSE: In this study, the anti-influenza A virus (IAV) activities and mechanisms of PIC in vitro and in vivo were investigated in order to provide reference for the development of novel plant-derived anti-IAV drugs. METHODS: The viral plaque assay, RT-PCR and western blot assay were used to evaluate the anti-IAV effects of PIC in vitro. The anti-IAV mechanism of PIC was determined by HA syncytium assay, DARTS assay and Surface Plasmon Resonance assay. The mouse pneumonia model combined with HE staining were used to study the anti-IAV effects of PIC in vivo. RESULTS: PIC shows inhibition on the multiplication of both H1N1 and H3N2 viruses, and blocks the infection of H5N1 pseudovirus with low toxicity. PIC may directly act on the envelope of IAV to induce the rupture and inactivation of IAV particles. PIC can also block membrane fusion via binding to HA2 rather than HA1 and cleavage site of HA0. PIC may interact with the two residues (HA2-T68 and HA2-I75) of HA2 to block the conformational change of HA so as to inhibit membrane fusion. Importantly, oral therapy of PIC also markedly improved survival and reduced viral titers in IAV-infected mice. CONCLUSION: PIC possesses significant anti-IAV effects both in vitro and in vivo and may block IAV infection mainly through interaction with HA to block membrane fusion. Thus, PIC has the potential to be developed into a new broad-spectrum anti-influenza drug for the prevention and treatment of influenza.

Effects of green tea powder on production performance, egg quality, and blood biochemical parameters in laying hens.

The paper aimed to evaluate the effects of dietary inclusion of green tea powder (GTP) on laying performance, egg quality, and blood biochemical parameters of laying hens. A total of 240 Jingfen No. 6 laying hens (age, 24 wk) were randomly allocated into 4 groups: control group (CON, basal diet), GTP0.5, GTP0.75, and GTP1.0 (basal diet included 0.5, 0.75, and 1.0% GTP, respectively). Each group has 5 replicates with 12 birds each. The feeding trial lasted 8 wk. The results showed that the hen-day egg production rate in GTP0.5 and GTP 0.75 group was higher than that of GTP1.0 group (P < 0.05), hen-day egg production rate in the GTP1.0 group was lower compared to the CON group (P > 0.05), the feed conversion ratio (FCR) in the GTP0.75 group was lower than that in CON and GTP 1.0 group (P < 0.05) during the entire experimental period. Albumen height and Haugh unit were higher in the GTP0.75 and GTP1.0 group compared to the CON group at d 56 (P < 0.05). At the end of experiment, plasma TG content in the GTP0.75 and GTP1.0 group was lower than that in the CON group (P < 0.05), the T-CH concentration in the GTP0.5 and GTP0.75 group was lower compared to the CON group (P < 0.05), plasma LDL-C and CORT concentrations were decreased by dietary GTP supplementation (P < 0.05), the HDL-C and BUN concentrations in the GTP0.75 and GTP1.0 group were higher than that in the CON group (P < 0.05). The antibody titers of H5N1 in the GTP0.75 and GTP1.0 group, and H7N9 in the GTP1.0 group were lower than that in the CON group (P < 0.05). In conclusion, dietary GTP inclusion could affect laying performance, regulate lipid metabolism, and have no favorable influence on antibody titers of H5N1 and H7N9, herein, dietary 0.5% GTP inclusion is suggested for Jingfen No. 6 laying hens during the peak laying period.

Anti-Influenza Activity of Cetraria islandica Lichen Extracts in In Vitro Experiments.

The toxicity and antiviral activity of extracts obtained by the methods of aqueous and ethanol extraction of bioactive substances from Cetraria islandica lichen as a raw material were studied. Aqueous and ethanol extracts of lichen were characterized by low toxicity with respect to the passaged MDCK cell culture and exhibited antiviral activity. The ethanol extract showed more potent in vitro antiviral activity against human A/H3N2 and avian A/H5N1 influenza viruses: in a concentration of 50 µg/ml, it suppressed replication of these viruses by 3.5 and 4 log10, respectively, while the aqueous extract inhibited replication of viruses by 2 and 6 log10, respectively, when taken in a concentration of 500 µg/ml that was 10-fold higher than the concentration of the ethanol extract.

Antiviral Effect of Ginsenosides rk1 against Influenza a Virus Infection by Targeting the Hemagglutinin 1-Mediated Virus Attachment.

Influenza A virus (IAV) infections have been a serious hazard to public health everywhere. With the growing concern of drug-resistant IAV strains, there is an urgent need for novel anti-IAV medications, especially those with alternative mechanisms of action. Hemagglutinin (HA), an IAV glycoprotein, plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a good target for developing anti-IAV drugs. Panax ginseng is a widely used herb in traditional medicine with extensive biological effects in various disease models, and its extract was reported to show protection in IAV-infected mice. However, the main effective anti-IAV constituents in panax ginseng remain unclear. Here, we report that ginsenoside rk1 (G-rk1) and G-rg5, out of the 23 screened ginsenosides, exhibit significant antiviral effects against 3 different IAV subtypes (H1N1, H5N1, and H3N2) in vitro. Mechanistically, G-rk1 blocked IAV binding to sialic acid in a hemagglutination inhibition (HAI) assay and an indirect ELISA assay; more importantly, we showed that G-rk1 interacted with HA1 in a dose-dependent manner in a surface plasmon resonance (SPR) analysis. Furthermore, G-rk1 treatment by intranasal inoculation effectively reduced the weight loss and mortality of mice challenged with a lethal dose of influenza virus A/Puerto Rico/8/34 (PR8). In conclusion, our findings reveal for the first time that G-rk1 possesses potent anti-IAV effects in vitro and in vivo. We have also identified and characterized with a direct binding assay a novel ginseng-derived IAV HA1 inhibitor for the first time, which could present potential approaches to prevent and treat IAV infections.

A Small Molecule RIG-I Agonist Serves as an Adjuvant to Induce Broad Multifaceted Influenza Virus Vaccine Immunity.

Retinoic acid-inducible gene I (RIG-I) is essential for activating host cell innate immunity to regulate the immune response against many RNA viruses. We previously identified that a small molecule compound, KIN1148, led to the activation of IFN regulatory factor 3 (IRF3) and served to enhance protection against influenza A virus (IAV) A/California/04/2009 infection. We have now determined direct binding of KIN1148 to RIG-I to drive expression of IFN regulatory factor 3 and NF-κB target genes, including specific immunomodulatory cytokines and chemokines. Intriguingly, KIN1148 does not lead to ATPase activity or compete with ATP for binding but activates RIG-I to induce antiviral gene expression programs distinct from type I IFN treatment. When administered in combination with a vaccine against IAV, KIN1148 induces both neutralizing Ab and IAV-specific T cell responses compared with vaccination alone, which induces comparatively poor responses. This robust KIN1148-adjuvanted immune response protects mice from lethal A/California/04/2009 and H5N1 IAV challenge. Importantly, KIN1148 also augments human CD8+ T cell activation. Thus, we have identified a small molecule RIG-I agonist that serves as an effective adjuvant in inducing noncanonical RIG-I activation for induction of innate immune programs that enhance adaptive immune protection of antiviral vaccination.

Orally administered BZL-sRNA-20 oligonucleotide targeting TLR4 effectively ameliorates acute lung injury in mice.

The global COVID-19 pandemic emerged at the end of December 2019. Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are common lethal outcomes of bacterial lipopolysaccharide (LPS), avian influenza virus, and SARS-CoV-2. Toll-like receptor 4 (TLR4) is a key target in the pathological pathway of ARDS and ALI. Previous studies have reported that herbal small RNAs (sRNAs) are a functional medical component. BZL-sRNA-20 (Accession number: B59471456; Family ID: F2201.Q001979.B11) is a potent inhibitor of Toll-like receptor 4 (TLR4) and pro-inflammatory cytokines. Furthermore, BZL-sRNA-20 reduces intracellular levels of cytokines induced by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (poly (I:C)). We found that BZL-sRNA-20 rescued the viability of cells infected with avian influenza H5N1, SARS-CoV-2, and several of its variants of concern (VOCs). Acute lung injury induced by LPS and SARS-CoV-2 in mice was significantly ameliorated by the oral medical decoctosome mimic (bencaosome; sphinganine (d22:0)+BZL-sRNA-20). Our findings suggest that BZL-sRNA-20 could be a pan-anti-ARDS ALI drug.

Evaluation of Efficacy of Oil Adjuvanted H5N6 Inactivated Vaccine against Highly Pathogenic H5N6 and H5N1 Influenza Viruses Infected Chickens.

BACKGROUND: Over the last 20 years, circulating highly pathogenic (HP) Asian H5 subtype avian influenza viruses have caused global pandemics in poultry and sporadic infections in humans. Vaccines are a desirable solution to prevent viral infections in poultry and reduce transmission to humans. Herein, we investigated the efficacy of an oil-adjuvanted inactivated H5N6 vaccine against highly pathogenic H5N6 and H5N1 influenza virus infections in chickens. METHODS: The polybasic amino acid cleavage site depleted HA gene and NA gene of A/Waterfowl/Korea/S57/2016 (clade 2.3.4.4) (H5N6) was assembled with the rest of the A/PR/8/34 (H1N1) genes to construct the vaccine virus. The vaccine virus was propagated in fertilized eggs, partially purified using a tangential flow filtration (TFF) system, and inactivated using formalin. The chickens were intramuscularly immunized with 384 HA, 192HA, and 96HA units of oil-adjuvanted inactivated H5N6 vaccine. Antibody titer, survival rate, and lung pathology were evaluated against the homologous H5N6: A/waterfowl/Korea/S57/2016 (clade 2.3.4.4) and heterologous H5N1: A/Hong Kong/213/2003 (clade 1) viruses 12 and 4 weeks post-vaccination (p.v.), respectively. Data were statistically analyzed using the Mann-Whitney U test. RESULTS: The 384HA (n = 10) and 192HA (n = 5) antigen-immunized chickens showed 100% survival after lethal infections with homologous H5N6, and no virus shedding was observed from tracheal and cloacal routes. All chickens that received the 384HA vaccine survived the challenge of heterologous H5N1 after 4 weeks of immunization. The chickens that received the 384HA vaccine showed mean HI titers of 60 and 240 after 12 and 4 weeks of vaccination, respectively, against HP H5N6, whereas a mean HI titer of 80 was observed in sera collected 4 weeks after vaccination against HP H5N1. CONCLUSIONS: Our findings indicate that one dose of 384HA oil-adjuvanted inactivated H5N6 vaccine can induce a long-lasting immune response against both homologous H5N6 and heterologous H5N1 infections in chickens.

Discovery and molecular elucidation of the anti-influenza material basis of Banlangen granules based on biological activities and ultra-high performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) has a wide range of applications, including human healthcare-associated treatments and bioactive compound discovery. However, complex chemical systems present a significant challenge for chemical-material-based research and quality control. For instance, Banlangen (BLG) granules is a well-acknowledged TCM preparation widely used in clinical treatment of virus infection. However, its chemical basis of anti-influenza efficacy remains unclear. AIM OF THE STUDY: In the present study, a systematic discovery strategy for identifying anti-influenza molecules based on biological activities and chemical analysis was established to contribute to the molecular elucidation of the anti-influenza material basis of Banlangen granules. MATERIALS AND METHODS: Hemagglutinase inhibition (HAI) and neuraminidase inhibition (NAI) assays were used to compare the anti-influenza activities of different fractions of BLG granules against H1N1, H5N1 and H7N9 viruses. A comparative qualitative analysis of the chemical constituents in BLG granules and their fractions was performed using ultra-high-performance liquid chromatography coupled with quadrupole orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap MS), in which a multiple mass spectrometry database platform and three compound identification strategies were used. The association between anti-influenza activities and chemical constituent characteristics was analyzed using multiple stoichiometries and data comparison strategies. RESULTS: The results showed that the chromatography fractions F3 and F4 of the BLG granules had the highest anti-influenza activity. A total of 88 compounds were identified in the BLG granules, including 31 alkaloids, 16 organic acids, 10 nucleosides, 8 phenylpropanoids, 6 sulfur-containing compounds, 5 amino acids, 4 aromatic compounds, 3 aldehydes and ketones, 2 flavonoids, 1 alcohol, 1 carbohydrate, and 1 aliphatic compound. Out of these, 31 characteristic compounds were identified in fractions F3-F4 as candidate compounds with anti-influenza activity. Additionally, 6-methoxyquinoline and 4-guanidinobutanal were identified in BLG granules and its raw material (Isatidis Radix) for the first time. CONCLUSION: In this study, we proposed a systematic discovery strategy to thoroughly investigate the anti-influenza activity, chemical identification, and constituents-activity relationship of BLG granules. These data not only provided a deeper understanding of the molecular mechanism of the activity of BLG granules, but also presented a basis for the discovery of potential novel drug candidates and quality evaluation and control of BLG granules.

Broad-spectrum antiviral activity of Spatholobus suberectus Dunn against SARS-CoV-2, SARS-CoV-1, H5N1, and other enveloped viruses.

The current COVID-19 pandemic caused by SARS-Cov-2 is responsible for more than 6 million deaths globally. The development of broad-spectrum and cost-effective antivirals is urgently needed. Medicinal plants are renowned as a complementary approach in which antiviral natural products have been established as safe and effective drugs. Here, we report that the percolation extract of Spatholobus suberectus Dunn (SSP) is a broad-spectrum viral entry inhibitor against SARS-CoV-1/2 and other enveloped viruses. The viral inhibitory activities of the SSP were evaluated by using pseudotyped SARS-CoV-1 and 2, HIV-1ADA and HXB2 , and H5N1. SSP effectively inhibited viral entry and with EC50 values ranging from 3.6 to 5.1 µg/ml. Pre-treatment of pseudovirus or target cells with SSP showed consistent inhibitory activities with the respective EC50 value of 2.3 or 2.1 µg/ml. SSP blocked both SARS-CoV-2 spike glycoprotein and the host ACE2 receptor. In vivo studies indicated that there was no abnormal toxicity and behavior in long-term SSP treatment. Based on these findings, we concluded that SSP has the potential to be developed as a drug candidate for preventing and treating COVID-19 and other emerging enveloped viruses.

Immunogenicity and cross-protective efficacy of recombinant H5HA1 protein of clade 2.3.2.1a highly pathogenic H5N1 avian influenza virus expressed in E.coli.

The global spread of H5N1 highly pathogenic avian influenza virus (HPAIV) in poultry has caused great economic loss to the poultry farmers and industry with significant pandemic threat. The current study involved production of recombinant HA1 protein of clade 2.3.2.1a H5N1 HPAIV (rH5HA1) in E.coli and evaluation of its protective efficacy in chickens. Purification under denaturing conditions and refolding by dialysis against buffers containing decreasing concentrations of urea was found to preserve the biological activity of the expressed recombinant protein as assessed by hemagglutination assay, Western blot and ELISA. The Montanide ISA 71 VGA adjuvanted rH5HA1 protein was used for immunization of chickens. Humoral response was maintained at a minimum of 4log2 hemagglutination inhibition (HI) titre till 154 days post 2nd booster. We evaluated the protective efficacy of rH5HA1 protein in immunized chickens by challenging them with homologous (2.3.2.1a) and heterologous (2.3.2.1c) clades of H5N1 HPAIV. In both the groups, the HI titre significantly increased (P < 0.05) after challenge and the virus shedding significantly (P < 0.05) reduced between 3rd and 14th day post challenge. The virus shedding ratio in oro-pharyngeal swabs did not differ significantly between both the groups except on 7 days post challenge and during the entire experimental period in cloacal swabs. These results indicate that rH5HA1 was able to induce homologous and cross protective immune response in chickens and could be a potential vaccine candidate used for combating the global spread of H5N1 HPAIV threat. To our knowledge, this is the first study to report immunogenicity and protective efficacy of prokaryotic recombinant H5HA1 protein in chicken.

Chitosan-based formulation of hemagglutinin antigens for oculo-nasal booster vaccination of chickens against influenza viruses.

Avian influenza viruses (AIVs) and especially highly pathogenic (HP) AIVs of the H5 and H7 subtypes are of both veterinary and public health concern worldwide. In response to the demand for effective vaccines against H5N1 HPAIVs, we produced recombinant protein based on hemagglutinin (HA), a protective viral antigen. A fragment of the HA ectodomain, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. Finally, the protein was formulated in Tris-HCl buffer of pH 8.0 or PBS of pH 7.4 to obtain antigens denoted rH5-1 and rH5-2, respectively. The systemic prime and boost immunizations proved that rH5-1 adsorbed to aluminum hydroxide induces anti-H5 HA neutralizing antibodies and protective immune responses against H5N1 HPAIVs in chickens. The present studies were aimed at stimulating immune responses via the mucosal routes using the systemic prime-mucosal boost strategy. Efficacy trials were performed in commercial layer chickens. For systemic and mucosal immunizations, H5 HA antigens were adjuvanted with aluminum hydroxide and chitosan glutamate, respectively. The first dose of rH5-2 was administered subcutaneously, while its second dose was administered subcutaneously, intraocularly, oculo-nasally, or intranasally. rH5-1 was delivered to the subcutaneously primed chickens by the intranasal route. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition tests. Intraocularly and oculo-nasally delivered rH5-2 mixed with chitosan glutamate was capable of stimulating anti-H5 HA IgY antibody responses in the subcutaneously primed chickens; however, it was ineffective when administered by the intranasal route. Efficient intranasal boosting was achieved using rH5-1. The enhanced production of antigen-specific antibodies was reflected in the development of H5-subtype specific and hemagglutination inhibiting antibodies. Conclusively, the subcutaneous prime and oculo-nasal boost vaccination is proposed as the target strategy for future optimization.

Development and application of a visual microarray for synchronously detecting H5N1, H7N9 and H9N2 avian influenza virus RNA.

The aim of this study was to develop a microarray assay for the simultaneous detection of the H5, H7, H9, N1, N9 and N2 genes of the avian influenza virus (AIV) using a Nanogold-streptavidin and silver-stain-enhanced nucleic acid dot-blot hybridisation system. The conserved sequences of H5 genes from H5N1, H7 genes from H7N9, H9 genes from H9N2, N9 genes from H7N9 and N2 genes from H9N2 AIV were cloned, together with that of N1 obtained commercially, and were used as templates for generating the probes using biotin-labeled primers, which targeted the conserved regions of H5, H7, H9, N1, N9 and N2 genes, respectively. The oligonucleotide probes were diluted using the spotting buffer and ddH2O, and each probe was then spotted to each specific position on the microarray. The PCR products including biotin-labeled lambda, NP, H5, H7, H9, N1, N9 and N2 were mixed, 200 µL of which was then added to the microarray chamber after denaturing. Following a hybridization incubation at 45℃ for 120 min, the microarray was then incubated with nanogold-streptavidin about 4 µg/mL for 30 min. After the supplementary of 200 µL of silver buffer A and silver buffer B in the chamber, the hybridization results were assessed by direct visualization in the dark at room temperature. The microarray assay was optimized and its specificity, sensitivity and stability were evaluated. The optimal conditions comprised a probe concentration of 50 µmol/L, a hybridization temperature of 45℃ and a hybridization time of 2 h. The optimal concentration of nanogold-streptavidin was 4 µg/mL and the optimal staining time was 7 min. The results of specificity evaluation showed that no cross-binding of the probes with each other and no cross-hybridization with Newcastle disease virus, infectious bronchitis virus and infectious laryngotracheitis virus was observed. The optimized microarray assay was significantly more sensitivity than the reverse-transcription PCR assay. The microarray was available after storing at less 90 d at 4 ℃. The optimized microarray assay was validated on clinical specimens and the results showed that it had over 95.6 % correlation with reverse-transcription PCR method. Therefore, the microarray assay could be used for the high throughput detection of AIV infections due to H5N1, H7N9 and H9N2.

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

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

Immunogenicity and Safety of AS03-adjuvanted H5N1 Influenza Vaccine in Children 6-35 Months of Age: Results From a Phase 2, Randomized, Observer-blind, Multicenter, Dose-ranging Study.

BACKGROUND: This phase 2 observer-blind, randomized, multicenter, dose-ranging study evaluated immunogenicity and safety of different formulations of an AS03-adjuvanted H5N1 influenza vaccine in children 6-35 months of age. METHODS: One hundred eighty-five children randomized into 5 groups [1.9 µg hemagglutinin (HA)/AS03B, 0.9 µg HA/AS03C, 1.9 µg HA/AS03C, 3.75 µg HA/AS03C or 3.75 µg HA/AS03D] were to receive 2 doses administered 21 days apart (primary vaccination). AS03 was classified by amount of DL-α-tocopherol, with AS03B the highest amount. One year later, all subjects were to receive unadjuvanted 3.75 µg HA as antigen challenge. Immunogenicity was assessed 21 days after primary vaccination (day 42) and 7 days after antigen challenge (day 392). Immunogenicity-fever index, based on hemagglutination inhibition and microneutralization antibody titers at day 42 and fever 7 days after each vaccination, was used to guide the selection of an acceptable formulation. RESULTS: After primary vaccination, formulations elicited strong homologous immune responses with all subjects' hemagglutination inhibition titers ≥1:40 post-vaccination. Immunogenicity-fever index based on hemagglutination inhibition and microneutralization assays showed that 1.9 µg HA/AS03B ranked the highest. Antibody levels persisted >4 times above baseline 12 months after primary vaccination with all formulations (day 385). Antibodies increased >4-fold after antigen challenge (day 392/day 385) with 1.9 µg HA/AS03B, 0.9 µg HA/AS03C and 1.9 µg HA/AS03C formulations. Overall per subject, the incidence of fever ranged from 28.6% (3.75 µg HA/AS03D) to 60.5% (1.9 µg HA/AS03B). CONCLUSIONS: All formulations were highly immunogenic and demonstrated acceptable safety profiles, with the 1.9 µg HA/AS03B providing the most favorable balance of immunogenicity versus reactogenicity for use in children 6-35 months of age.

The single-cell epigenomic and transcriptional landscape of immunity to influenza vaccination.

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.

Evodiamine Enhanced the Anti-Inflammation Effect of Clindamycin in the BEAS-2B Cells Infected with H5N1 and Pneumoniae D39 Through CREB-C/EBPß Signaling Pathway.

Pneumonia is a pulmonary disease among children. Evodiamine, a traditional Chinese medicine, is known for anti-inflammatory effect. This study aimed to investigate the impact of evodiamine on severe pneumonia-like cells and the underlying mechanism involved. H5N1 and pneumoniae D39 was used to induce severe pneumonia-like conditions in BEAS-2B cells. The cell viability in BEAS-2B cells after treatments with 0, 20, 40, 60, 80, and 100 µM evodiamine was examined using MTT assays. The protein concentrations of inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1ß, and Toll-like receptors (TLRs) were measured by enzyme-linked immunosorbent assay methods and the protein and mRNA changes in C/EBPß/CREB were measured using Real Time-quantitative polymerase chain reaction and Western blot methods. Our results revealed that Evodiamine significantly decreased TNF-α, IL-6, and IL-1ß in BEAS-2B cells. Moreover, evodiamine markedly reduced TLR2,3,4 protein expression and the phosphorylated protein of C/EBPß and CREB. Besides, evodiamine combined with clindamycin exerted more significant effects than clindamycin alone. Taken together, our results demonstrated that evodiamine enhanced the anti-inflammation effect of clindamycin in the BEAS-2B cells infected with H5N1 and pneumoniae D39 through CREB-C/EBPß signaling pathway.

Immune-adjuvant activity of lentinan-modified calcium carbonate microparticles on a H5N1 vaccine.

In recent years, the high prevalence of avian influenza viruses especially H5N1 subtype isolated from poultry and human has become a major public health concern. Vaccination is still a major strategy for preventing H5N1 infections. Lentinan (LNT), a ß-1,3-glucohexaose with ß-1,6-branches, is extracted from Lentinus edodes and has been extensively studied for its immunoenhancement effects. In this study, we synthesized and characterized calcium carbonate (CaCO3) microparticles which modified with LNT as an adjuvant for H5N1 vaccine and investigated their ability to enhance immune responses. We prepared spherical and uniform CaCO3-LNT microparticles with a mean hydrodynamic size was around 2 µm. The H5N1 antigen-loaded CaCO3-LNT particles were injected into mice to evaluate their effectiveness as an adjuvant for H5N1 vaccines. The results demonstrated that CaCO3-LNT/H5N1 significantly enhanced the expression of MHC-II and CD86 in lymph node dendritic cells, and increased the ratio of CD4+ to CD8+ T cells in lymphocytes. Moreover, CaCO3-LNT/H5N1 surprisingly increased the HI titers and induced the secretion of IgG subtypes (IgG1 and IgG2b) and Th-associated cytokines (TNF-α, IFN-γ and IL-4) in immunized mice. Therefore, by combining with the immunostimulatory activity of LNT and the drug/antigen delivery capabilities of CaCO3, the CaCO3-LNT/H5N1 could induce a stronger cellular and humoral immune response and could be a potential adjuvant for the H5N1 vaccine.