Graphene oxide nanoparticles inhibit H9N2 influenza A virus infectivity by destroying viral coat proteins.

Nanoparticles have gained attention as potential antiviral agents, but the effects of graphene oxide nanoparticles (GONPs) on influenza virus remain unclear. In this study, we evaluated the antiviral activity of GONPs against influenza virus strain A/Hunan-Lengshuitan/11197/2013(H9N2). Our results show that GONPs with a diameter of 4 nm exerted an antiviral effect, whereas those with a diameter of 400 nm had no effect. Treatment with 4-nm GONPs reduced viral titers by more than 99% and inhibited viral nucleoprotein expression in a dose-dependent manner. We also confirmed that 4-nm GONPs inhibited the infectivity of H9N2 in MDCK cells. A transmission electron microscopic analysis revealed morphological abnormalities in the GONP-treated virus, including the destruction of the envelope glycoprotein spikes and an irregular shape, suggesting that GONPs cause the destruction of the viral coat proteins. Our results highlight the potential utility of GONPs in the prevention and treatment of viral infections, especially those of emerging and re-emerging viruses.

The therapeutic efficacy of neem (Azadirecta indica) leaf extract against coinfection with Chlamydophila psittaci and low pathogenic avian influenza virus H9N2 in broiler chickens.

Avian chlamydiosis is a serious avian infection that carries a significant zoonotic danger to the poultry industry. The respiratory co-infections caused by the low pathogenic avian influenza virus H9N2 (LPAIV H9N2) also cause significant financial losses in the poultry industry. The purpose of this study was to examine the pathogenicity of Chlamydophila psittaci, and LPAIV H9N2 individually and in combination in broiler chickens, as well as to determine whether or not aqueous neem (Azadirachta indica) leaf extract is effective against infections caused by these pathogens. Therefore, 120 broiler cobb chicks were equally divided into 4 groups (30 birds each) with triplicates with 10 birds. Broilers in group 1 (G1) were infected with only C. psittaci, broilers in group 2 (G2) were infected with only LPAIV H9N2, broilers in group 3 (G3) were infected with C. psittaci and LPAIV H9N2, and broilers in group 4 (G4) remained not challenged and non-treated with any therapeutic or preventive treatment (negative control). At 21 d postinfection (dpi), birds in G1, G2, and G3 were divided into 3 subgroups of 10 birds each: subgroup (A) remained infected and untreated (positive control), subgroup (B) infected and received oxytetracycline for 5 consecutive d, and subgroup (C) infected and received 8% aqueous neem leaf extract for 5 consecutive d. The multiplication of C. psittaci in birds in G1, in various tissues was evaluated using Giemsa staining and the data showed that multiplication was much higher in the lung, spleen, and liver from 6 h to 21 dpi, but low in the heart from 8 to 21 dpi. During simultaneous co-infection in G3, the birds developed significant clinical symptoms and postmortem lesions (PM). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect viral shedding from oropharyngeal and cloacal swabs between 2 dpi and 8 dpi, with cycle threshold (CT) values ranging from 22 to 24. In contrast, bacterial shedding began 6 h after infection and continued until 21 dpi, with CT values ranging from 23 to 26. Administration of an aqueous neem leaf extract at an 8% concentration (Group C) resulted in a numerical rise in average body weight across all treatment groups in the third and fourth week, as well as a reduction in LPAIV H9N2 and C. psittaci replication in the respiratory and gut of treated birds compared to those treated with oxytetracycline (Group B). Overall, respiratory co-infections pose a considerable risk to the poultry business, which is a big threat. To control C. psittaci and LPAIV H9N2 in broiler chickens, oral supplementation of 8% aqueous neem leaf extract is recommended. This treatment improves the birds' performance, as evidenced by an increase in their average body weight. In addition, the application of 8% aqueous neem leaf extract lowers C. psittaci replication within tissues and diminishes LPAIV H9N2 shedding.

Extraction and immunomodulatory effects of acid Lagenaria siceraria (Molina) Standl. Polysaccharide on chickens.

Herbal polysaccharides are extensively studied as vaccine adjuvants due to their safety and potent immunoenhancing activity. This study aimed to analyze the structure of Lagenaria siceraria (Molina) Standl polysaccharide (LSP50) and investigate its adjuvant activity for the H9N2 vaccine in broiler chickens. Structural analysis revealed that LSP50 primarily consisted of rhamnose, arabinose, xylose, mannose, glucose, and galactose with molar ratios of 23.12: 12.28: 10.87: 8.26: 2.64: 22.82 respectively. The adjuvant activity of LSP50 was evaluated, which showing significant enhancements compared to the H9N2 group. Parameters including the immune organ index, H9N2 specific IgG level, cytokines contents (IFN-γ, IL-2, IL-4, and IL-5), and the proportion of CD3e+CD8aT+cells were significantly increased in the LSP50 group (P < 0.05). Additionally, sequencing results showed that LSP50 modulates the immune response by regulating PLA2G12B and PTGDS genes involved in the arachidonic acid pathway. These findings were further validated through qPCR analysis to affirm the reliability of the sequencing data. In conclusion, our results demonstrate that LSP50 exhibits potent adjuvant activity, enhancing both cellular and humoral immunity.

Genotypic and phenotypic susceptibility of emerging avian influenza A viruses to neuraminidase and cap-dependent endonuclease inhibitors.

Avian influenza outbreaks, including ones caused by highly pathogenic A(H5N1) clade 2.3.4.4b viruses, have devastated animal populations and remain a threat to humans. Risk elements assessed for emerging influenza viruses include their susceptibility to approved antivirals. Here, we screened >20,000 neuraminidase (NA) or polymerase acidic (PA) protein sequences of potentially pandemic A(H5Nx), A(H7Nx), and A(H9N2) viruses that circulated globally in 2010-2023. The frequencies of NA or PA substitutions associated with reduced inhibition (RI) or highly reduced inhibition (HRI) by NA inhibitors (NAIs) (oseltamivir, zanamivir) or a cap-dependent endonuclease inhibitor (baloxavir) were low: 0.60% (137/22,713) and 0.62% (126/20,347), respectively. All tested subtypes were susceptible to NAIs and baloxavir at sub-nanomolar concentrations. A(H9N2) viruses were the most susceptible to oseltamivir, with IC50s 3- to 4-fold lower than for other subtypes (median IC50: 0.18 nM; n = 22). NA-I222M conferred RI of A(H5N1) viruses by oseltamivir (with a 26-fold IC50 increase), but NA-S246N did not reduce inhibition. PA-E23G, PA-K34R, PA-I38M/T, and the previously unreported PA-A36T caused RI by baloxavir in all subtypes tested. Avian A(H9N2) viruses endemic in Egyptian poultry predominantly acquired PA-I38V, which causes only a <3-fold decrease in the baloxavir EC50 and fails to meet the RI criteria. PA-E199A/D in A(H7Nx) and A(H9N2) viruses caused a 2- to 4-fold decrease in EC50 (close to the borderline for RI) and should be closely monitored. Our data indicate antiviral susceptibility is high among avian influenza A viruses with pandemic potential and present novel markers of resistance to existing antiviral interventions.

Cistanche deserticola polysaccharide- functionalized dendritic fibrous nano-silica -based adjuvant for H9N2 oral vaccine enhance systemic and mucosal immunity in chickens.

Avian influenza virus subtype H9N2 has the ability to infect birds and humans, further causing significant losses to the poultry industry and even posing a great threat to human health. Oral vaccine received particular interest for preventing majority infection due to its ability to elicit both mucosal and systemic immune responses, but their development is limited by the bad gastrointestinal (GI) environment, compact epithelium and mucus barrier, and the lack of effective mucosal adjuvants. Herein, we developed the dendritic fibrous nano-silica (DFNS) grafted with Cistanche deserticola polysaccharide (CDP) nanoparticles (CDP-DFNS) as an adjuvant for H9N2 vaccine. Encouragingly, CDP-DFNS facilitated the proliferation of T and B cells, and further induced the activation of T lymphocytes in vitro. Moreover, CDP-DFNS/H9N2 significantly promoted the antigen-specific antibodies levels in serum and intestinal mucosal of chickens, indicating the good ability to elicit both systemic and mucosal immunity. Additional, CDP-DFNS facilitate the activation of CD4 + and CD8 + T cells both in spleen and intestinal mucosal, and the indexes of immune organs. This study suggested that CDP-DFNS may be a new avenue for development of oral vaccine against pathogens that are transmitted via mucosal route.

Inhibitory effects of Belamcanda extract on inflammatory response and antiviral mechanism in H9N2 Avian influenza virus: insights from in vitro and in vivo studies.

Avian influenza, particularly the H9N2 subtype, presents significant challenges to poultry health, underscoring the need for effective antiviral interventions. This study explores the antiviral capabilities of Belamcanda extract, a traditional Chinese medicinal herb, against H9N2 Avian influenza virus (AIV) in specific pathogen-free (SPF) chicks. Through a comprehensive approach, we evaluated the impact of the extract on cytokine modulation and crucial immunological signaling pathways, essential for understanding the host-virus interaction. Our findings demonstrate that Belamcanda extract significantly modulates the expression of key inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), interleukin-2 (IL-2), and interleukin-6 (IL-6), which are pivotal to the host's response to H9N2 AIV infection. Western blot analysis further revealed that the extract markedly reduces the expression of critical immune signaling molecules such as toll-like receptor 3 (TLR3), TIR-domain-containing adapter-inducing interferon-ß (TRIF), and nuclear factor kappa B (NF-κB). These insights into the mechanisms by which Belamcanda extract influences host immune responses and hinders viral replication highlight its potential as an innovative antiviral agent for poultry health management. The study advances our comprehension of natural compounds' antiviral mechanisms and lays the groundwork for developing strategies to manage viral infections in poultry. The demonstrated ability of Belamcanda extract to modulate immune responses and inhibit viral replication establishes it as a promising candidate for future antiviral therapy development, especially in light of the need for effective treatments against evolving influenza virus strains and the critical demand for enhanced poultry health management strategies.

Oxymatrine Modulation of TLR3 Signaling: A Dual-Action Mechanism for H9N2 Avian Influenza Virus Defense and Immune Regulation.

In our research, we explored a natural substance called Oxymatrine, found in a traditional Chinese medicinal plant, to fight against a common bird flu virus known as H9N2. This virus not only affects birds but can also pose a threat to human health. We focused on how this natural compound can help in stopping the virus from spreading in cells that line the lungs of birds and potentially humans. Our findings show that Oxymatrine can both directly block the virus and boost the body's immune response against it. This dual-action mechanism is particularly interesting because it indicates that Oxymatrine might be a useful tool in developing new ways to prevent and treat this type of bird flu. Understanding how Oxymatrine works against the H9N2 virus could lead to safer and more natural ways to combat viral infections in animals and humans, contributing to the health and well-being of society. The H9N2 Avian Influenza Virus (AIV) is a persistent health threat because of its rapid mutation rate and the limited efficacy of vaccines, underscoring the urgent need for innovative therapies. This study investigated the H9N2 AIV antiviral properties of Oxymatrine (OMT), a compound derived from traditional Chinese medicine, particularly focusing on its interaction with pulmonary microvascular endothelial cells (PMVECs). Employing an array of in vitro assays, including 50% tissue culture infectious dose, Cell Counting Kit-8, reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot, we systematically elucidated the multifaceted effects of OMT. OMT dose-dependently inhibited critical antiviral proteins (PKR and Mx1) and modulated the expression of type I interferons and key cytokines (IFN-α, IFN-ß, IL-6, and TNF-α), thereby affecting TLR3 signaling and its downstream elements (NF-κB and IRF-3). OMT's antiviral efficacy extended beyond TLR3-mediated responses, suggesting its potential as a versatile antiviral agent. This study not only contributes to the growing body of research on the use of natural compounds as antiviral agents but also underscores the importance of further investigating the broader application of OMT for combating viral infections.

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

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

Arctiin suppresses H9N2 avian influenza virus-mediated inflammation via activation of Nrf2/HO-1 signaling.

BACKGROUND: H9N2 avian influenza viruses (AIVs) infect avian and mammalian hosts and provide internal genes for new emerging highly pathogenic avian viruses that cause severe pneumonia with high mortality, for which few medications are available. Arctiin, a bioactive lignan glycoside, has been reported to possess multiple pharmacological properties. However, the effect of arctiin on H9N2 virus infection is unclear. In the current study, we analyzed the effect of arctiin on H9N2 virus infection and the underlying molecular mechanism in vitro. METHODS: The antiviral effect against H9N2 virus was determined by plaque reduction assay (PRA) and progeny virus reduction assay. We employed MTT assay, qRT-PCR, ELISA, immunofluorescence and Western blotting to better understand the anti-inflammatory effect and corresponding mechanism of arctiin on H9N2 virus-infected cells. RESULTS: The results showed that arctiin had antiviral activity against H9N2 virus. Arctiin treatment reduced H9N2 virus-triggered proinflammatory cytokines, such as IL-6, and TNF-α. Moreover, arctiin significantly suppressed H9N2 virus-mediated expression of COX-2 and PGE2. Furthermore, we found that arctiin inhibited H9N2 virus-mediated activation of RIG-I/JNK MAPK signaling. Interestingly, arctiin treatment obviously reversed H9N2 virus-induced reduction of Nrf2, increased the nuclear translocation of Nrf2, and upregulated Nrf2 signaling target genes (HO-1 and SOD2). Zinc protoporphyrin (Znpp)-an HO-1 inhibitor-weakened the inhibitory effect of arctiin on H9N2 virus-induced RIG-I/JNK MAPK and proinflammatory mediators. CONCLUSION: Taken together, these results suggested that the anti-inflammatory effects of arctiin on H9N2 virus infection may be due to the activation of Nrf2/HO-1 and blocked RIG-I/JNK MAPK signaling; thus, arctiin may be a promising agent for prevention and treatment of H9N2 virus infections.

Pre-treatment with chicken IL-17A secreted by bioengineered LAB vector protects chicken embryo fibroblasts against Influenza Type A Virus (IAV) infection.

The recent advances in our understanding of the host factors in orchestrating qualitatively different immune responses against influenza Type A virus (IAV) have changed the perception of conventional approaches for controlling avian influenza virus (AIV) infection in chickens. Given that infection-induced pathogenicity and replication of influenza virus largely rely on regulating host immune responses, immunoregulatory cytokine profiles often determine the disease outcomes. However, in contrast to the function of other inflammatory cytokines, interleukin-17A (IL-17A) has been described as a 'double-edged sword', indicating that in addition to antiviral host responses, IL-17A has a distinct role in promoting viral infection. Therefore, in the present study, we investigated the chicken IL-17A mediated antiviral immune effects on IAVs infection in primary chicken embryo fibroblasts cells (CEFs). To this end, we first bioengineered a food-grade Lactic Acid Producing Bacteria (LAB), Lactococcus lactis (L. lactis), secreting bioactive recombinant chicken IL-17A (sChIL-17A). Next, the functionality of sChIL-17A was confirmed by transcriptional upregulation of several genes associated with antiviral host responses, including granulocyte-monocyte colony-stimulating factor (GM-CSF) (CSF3 in the chickens), interleukin-6 (IL-6), interferon-α (IFN-α), -ß and -γ genes in primary CEFs cells. Consistent with our hypothesis that such a pro-inflammatory state may translate to immunoprotection against IAVs infection, we observed that sChIL-17A pre-treatment could significantly limit the viral replication and protect the primary CEFs cells against two heterotypic IAVs such as A/turkey/Wisconsin/1/1966(H9N2) and A/PR/8/1934(H1N1). Together, the data presented in this work suggest that exogenous application of sChIL-17A secreted by modified LAB vector may represent an alternative strategy for improving antiviral immunity against avian influenza virus infection in chickens.

Elucidating antiviral activity of Curcuma longa against H9 N2 influenza virus using embryonated chicken egg model.

Curcumin is a potent antimicrobial herb used traditionally as a spice in culinary. This study was designed to evaluate the antiviral effects of curcuma longa extract against H9 influenza virus. A total of 60 embryonated eggs were equally divided into 5 groups with 12 eggs in each group. Group 1 (G1) served as uninfected negative control. Whereas Group 2 (G2) was kept as positive control infected with known virus @ 0.2 ml with 10-9.3 EID50. Group 3 (G3) was offered Curcuma longa @ 0.2 mg/0.2 ml and H9N2 virus (@ 0.2 ml with 10-9.3 EID50. Similarly, Group 4 (G4) was inoculated with extract of Curcuma longa @ 0.2 mg/0.2 ml per egg, whereas Group 5 (G5) was given Ribazole @ 0.2 ml/ egg. The crude extract and virus were administered on the 15th day of incubation and were checked after every 24 hours up to 96th hour post inoculation by random selection of 3 eggs. Death and survival rate were noted in all groups. Gross and histopathological lesions were also observed. Results revealed that Curcuma longa extract had significantly (p<0.05) reduced the mortality rate of embryos infected with H9N2 virus. In G3, increased lymphocytes and mild fatty changes were seen in liver. Whereas, mature RBCs, plasma cells and some lymphoblast's were observed in Spleen. Similarly, the bursa follicles were with lymphocytic aggregation. The G4 (Curcuma longa) and G5 (Ribazole) were with maximum embryo survival after 48 and 72 h post inoculation. This study revealed potential antiviral activity of Curcuma longa against H9N2 influenza viruses and can be opted as alternative to antibiotics and antiviral drugs to minimize the antimicrobial resistance in human and animal population.

Antiviral activity of diallyl trisulfide against H9N2 avian influenza virus infection in vitro and in vivo.

BACKGROUND: Diallyl trisulfide (DATS) is a garlic-derived organosulfur compound. As it has been shown to have anti-viral activity, we hypothesized that it may alleviate infections caused by H9N2 avian influenza virus (AIV), which is prevalent in poultry with pandemic potential. METHODS: Human lung A549 epithelial cells were treated with three different concentrations of DATS 24 h before (pre-treatment) or one hour after (post-treatment) H9N2 AIV infection. Culture supernatants were collected 24 h and 48 h post-infection and analyzed for viral titers and levels of inflammatory and anti-viral immune responses. For in vivo experiments, BABL/c mice were administered daily by intraperitoneal injection with DATS (30 mg/kg) for 2 weeks starting 1 day after H9N2 AIV infection. Clinical signs, lung pathology, and inflammatory and anti-viral immune responses were assessed 2, 4, and 6 days after infection. RESULTS: Both pre-treatment and post-treatment of A549 cells with DATS resulted in reduced viral loads, increased expression of anti-viral genes (RIG-I, IRF-3, and interferon-ß), and decreased expression of inflammatory cytokines (TNF-α and IL-6). These effects were also observed in H9N2 AIV-infected mice treated with DATS. Such treatment also reduced lung edema and inflammation in mice. CONCLUSIONS: Results suggest that DATS has anti-viral activity against H9N2 AIV and may be used as an alternative treatment for influenza virus infection.

Inhibitory effects of aprotinin on influenza A and B viruses in vitro and in vivo.

Influenza viruses cause significant morbidity and mortality worldwide. Long-term or frequent use of approved anti-influenza agents has resulted in drug-resistant strains, thereby necessitating the discovery of new drugs. In this study, we found aprotinin, a serine protease inhibitor, as an anti-influenza candidate through screening of compound libraries. Aprotinin has been previously reported to show inhibitory effects on a few influenza A virus (IAV) subtypes (e.g., seasonal H1N1 and H3N2). However, because there were no reports of its inhibitory effects on the other types of influenza viruses, we investigated the inhibitory effects of aprotinin in vitro on a wide range of influenza viruses, including avian and oseltamivir-resistant influenza virus strains. Our cell-based assay showed that aprotinin had inhibitory effects on seasonal human IAVs (H1N1 and H3N2 subtypes), avian IAVs (H5N2, H6N5, and H9N2 subtypes), an oseltamivir-resistant IAV, and a currently circulating influenza B virus. We have also confirmed its activity in mice infected with a lethal dose of influenza virus, showing a significant increase in survival rate. Our findings suggest that aprotinin has the capacity to inhibit a wide range of influenza virus subtypes and should be considered for development as a therapeutic agent against influenza.

In Vitro and In Vivo Antiviral Activity of Gingerenone A on Influenza A Virus Is Mediated by Targeting Janus Kinase 2.

Janus kinase (JAK) inhibitors have been developed as novel immunomodulatory drugs and primarily used for treating rheumatoid arthritis and other inflammatory diseases. Recent studies have suggested that this category of anti-inflammatory drugs could be potentially useful for the control of inflammation "storms" in respiratory virus infections. In addition to their role in regulating immune cell functions, JAK1 and JAK2 have been recently identified as crucial cellular factors involved in influenza A virus (IAV) replication and could be potentially targeted for antiviral therapy. Gingerenone A (Gin A) is a compound derived from ginger roots and a dual inhibitor of JAK2 and p70 S6 kinase (S6K1). Our present study aimed to determine the antiviral activity of Gin A on influenza A virus (IAV) and to understand its mechanisms of action. Here, we reported that Gin A suppressed the replication of three IAV subtypes (H1N1, H5N1, H9N2) in four cell lines. IAV replication was also inhibited by Ruxolitinib (Rux), a JAK inhibitor, but not by PF-4708671, an S6K1 inhibitor. JAK2 overexpression enhanced H5N1 virus replication and attenuated Gin A-mediated antiviral activity. In vivo experiments revealed that Gin A treatment suppressed IAV replication in the lungs of H5N1 virus-infected mice, alleviated their body weight loss, and prolonged their survival. Our study suggests that Gin A restricts IAV replication by inhibiting JAK2 activity; Gin A could be potentially useful for the control of influenza virus infections.

Antiviral activity of a novel mixture of natural antimicrobials, in vitro, and in a chicken infection model in vivo.

The aim of this study was to test in vitro the ability of a mixture of citrus extract, maltodextrin, sodium chloride, lactic acid and citric acid (AuraShield L) to inhibit the virulence of infectious bronchitis, Newcastle disease, avian influenza, porcine reproductive and respiratory syndrome (PRRS) and bovine coronavirus viruses. Secondly, in vivo, we have investigated its efficacy against infectious bronchitis using a broiler infection model. In vitro, these antimicrobials had expressed antiviral activity against all five viruses through all phases of the infection process of the host cells. In vivo, the antimicrobial mixture reduced the virus load in the tracheal and lung tissue and significantly reduced the clinical signs of infection and the mortality rate in the experimental group E2 receiving AuraShield L. All these effects were accompanied by a significant reduction in the levels of pro-inflammatory cytokines and an increase in IgA levels and short chain fatty acids (SCFAs) in both trachea and lungs. Our study demonstrated that mixtures of natural antimicrobials, such AuraShield L, can prevent in vitro viral infection of cell cultures. Secondly, in vivo, the efficiency of vaccination was improved by preventing secondary viral infections through a mechanism involving significant increases in SCFA production and increased IgA levels. As a consequence the clinical signs of secondary infections were significantly reduced resulting in recovered production performance and lower mortality rates in the experimental group E2.

Cross protection by inactivated recombinant influenza viruses containing chimeric hemagglutinin conjugates with a conserved neuraminidase or M2 ectodomain epitope.

Influenza virus neuraminidase (NA) contains a universally conserved epitope (NAe, NA222-230). However, no studies have reported vaccines targeting this NA conserved epitope and inducing antibodies recognizing NAe. The extracellular domain of M2 (M2e) is considered as an attractive target for a universal influenza vaccine. We generated recombinant influenza H1N1 viruses expressing conserved epitopes in hemagglutinin (HA) molecules: NAe (NAe-HA) or M2e (M2e-HA) within the HA head domain. Inactivated recombinant NAe-HA and M2e-HA viruses were more effective in inducing IgG antibodies specific for an inserted conserved epitope than live recombinant virus. Recombinant inactivated M2e-HA virus vaccination induced cross protection against H3N2 virus with less weight loss compared to NAe-HA and was more effective in inducing humoral and cellular M2e immune responses. This study provides insight into developing recombinant influenza virus vaccines compatible with current platforms to induce antibody responses to conserved poorly immunogenic epitopes.

Taishan Pinus massoniana pollen polysaccharide inhibits H9N2 subtype influenza virus infection both in vitro and in vivo.

The H9N2 subtype avian influenza virus (AIV) is one of the most prevalent AIV subtypes that can be found throughout most countries. Currently, due to the neglect of low pathogenic avian influenza virus (LPAIV) and monotonous control technique, an expanding H9N2 virus epizootic have been arisen and causes great economic losses in the poultry industry. Therefore, novel anti-influenza drugs are necessary for the prevention and control of H9N2 AIV. Our previous studies have found that Taishan Pinus massoniana pollen polysaccharides (TPPPS) have antiviral effects, but whether they can inhibit the H9N2 AIV remains unclear. Here, we further investigated the effects of TPPPS on the H9N2 virus and its underlying mechanisms of action. We found that TPPPS significantly inhibited the replication of the H9N2 virus in a dose-dependent manner, especially during the period of virus adsorption in vitro. Transmission electron microscopy demonstrated that TPPPS reduce infection by interfering with virus entry into host cells rather than by interacting with the H9N2 virus particles. A fluorescence quantitative PCR (qPCR) assay and an animal experiment were performed to evaluate the anti-viral effect of TPPPS in vivo. As expected, the lungs of chickens treated with TPPPS had fewer lesions and lower virus contents compared with the PBS group. In addition, pre-treatment with TPPPS clearly enhanced host disease resistance and delayed infection by the H9N2 virus. Taken together, our results reveal that TPPPS suppress H9N2 virus replication both in vitro and in vivo and therefore shows promising as an anti-AIV agent.

Antiviral activity of furanocoumarins isolated from Angelica dahurica against influenza a viruses H1N1 and H9N2.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. (Umbelliferae family) is an herbaceous, perennial plant native to northern and eastern Asia. The root of A. dahurica has traditionally been used under the name "Bai Zhi" as a medicinal plant for colds, dizziness, ulcers, and rheumatism. Moreover, it is also an important ingredient of various prescriptions, such as Gumiganghwal-Tang, for the common cold and influenza. AIM OF THE STUDY: Even though various biological activities of the root of A. dahurica have been reported along with its chemical components, the detailed mechanism of how it exerts anti-influenza activity at the compound level has not been studied. Therefore, we investigated the anti-influenza properties of furanocoumarins purified by bioactivity-guided isolation. MATERIALS AND METHODS: Bioactivity-guided isolation from a 70% EtOH extract of the root of A. dahurica was performed to produce four active furanocoumarins. The inhibition of cytopathic effects (CPEs) was evaluated to ascertain the antiviral activity of these compounds against influenza A (H1N1 and H9N2) viruses. The most potent compound was subjected to detailed mechanistic studies such as the inhibition of viral protein synthesis, CPE inhibition in different phases of the viral replication cycle, neuraminidase (NA) inhibition, antiapoptotic activity using flow cytometry, and immunofluorescence. RESULTS: The bioactivity-guided isolation produced four active furanocoumarins, isoimperatorin (1), oxypeucedanin (2), oxypeucedanin hydrate (3) and imperatorin (4) from the n-BuOH fraction. Among them, compound 2 (followed by compounds 1, 4 and 3) showed a significant CPE inhibition effect, which was stronger than that of the positive control ribavirin, against both H1N1 and H9N2 with an EC50 (µM) of 5.98 ± 0.71 and 4.52 ± 0.39, respectively. Compound 2 inhibited the synthesis of NA and nucleoprotein (NP) in a dose-dependent manner. In the time course assays, the cytopathic effects of influenza A-infected MDCK cells were reduced by 80-90% when treated with compound 2 for 1 and 2 h after infection and declined drastically 3 h after infection. The level of viral NA and NP production was markedly reduced to less than 20% for both proteins in compound 2 (20 µM)-treated cells compared to untreated cells at 2 h after infection. In the molecular docking analysis, compound 2 showed a stronger binding affinity for the C-terminus of polymerase acidic protein (PAC; -36.28 kcal/mol) than the other two polymerase subunits. Compound 2 also exerted an antiapoptotic effect on virus infected cells and significantly inhibited the mRNA expression of caspase-3 and Bax. CONCLUSION: Our results suggest that compound 2 might exert anti-influenza A activity via the inhibition of the early phase of the viral replication cycle, not direct neutralization of surface proteins, such as hemagglutinin and NA, and abnormal apoptosis induced by virus infection. Taken together, these findings suggest that furanocoumarins predominant in A. dahurica play a pivotal role in its antiviral activity. These findings can also explain the reasons for the ethnopharmacological uses of this plant as an important ingredient in many antiviral prescriptions in traditional Chinese medicine (TCM).

Bacillus subtilis spores as adjuvants against avian influenza H9N2 induce antigen-specific antibody and T cell responses in White Leghorn chickens.

Low-pathogenicity avian influenza H9N2 remains an endemic disease worldwide despite continuous vaccination, indicating the need for an improved vaccine strategy. Bacillus subtilis (B. subtilis), a gram-positive and endospore-forming bacterium, is a non-pathogenic species that has been used in probiotic formulations for both animals and humans. The objective of the present study was to elucidate the effect of B. subtilis spores as adjuvants in chickens administered inactivated avian influenza virus H9N2. Herein, the adjuvanticity of B. subtilis spores in chickens was demonstrated by enhancement of H9N2 virus-specific IgG responses. B. subtilis spores enhanced the proportion of B cells and the innate cell population in splenocytes from chickens administered both inactivated H9N2 and B. subtilis spores (Spore + H9N2). Furthermore, the H9N2 and spore administration induced significantly increased expression of the pro-inflammatory cytokines IL-1ß and IL-6 compared to that in the H9N2 only group. Additionally, total splenocytes from chickens immunized with inactivated H9N2 in the presence or absence of B. subtilis spores were re-stimulated with inactivated H9N2. The subsequent results showed that the extent of antigen-specific CD4+ and CD8+ T cell proliferation was higher in the Spore + H9N2 group than in the group administered only H9N2. Taken together, these data demonstrate that B. subtilis spores, as adjuvants, enhance not only H9N2 virus-specific IgG but also CD4+ and CD8+ T cell responses, with an increase in pro-inflammatory cytokine production. This approach to vaccination with inactivated H9N2 together with a B. subtilis spore adjuvant in chickens produces a significant effect on antigen-specific antibody and T cell responses against avian influenza virus.

Dendrigraft poly-L-lysines delivery of DNA vaccine effectively enhances the immunogenic responses against H9N2 avian influenza virus infection in chickens.

Biodegradable nanomaterials can protect antigens from degradation, promote cellular absorption, and enhance immune responses. We constructed a eukaryotic plasmid [pCAGGS-opti441-hemagglutinin (HA)] by inserting the optimized HA gene fragment of H9N2 AIV into the pCAGGS vector. The pCAGGS-opti441-HA/DGL was developed through packaging the pCAGGS-opti441-HA with dendrigraft poly-l-lysines (DGLs). DGL not only protected the pCAGGS-opti441-HA from degradation, but also exhibited high transfection efficiency. Strong cellular immune responses were induced in chickens immunized with the pCAGGS-opti441-HA/DGL. The levels of IFN-γ and IL-2, and lymphocyte transformation rate of the vaccinated chickens increased at the third week post the immunization. For the vaccinated chickens, T lymphocytes were activated and proliferated, the numbers of CD3+CD4+ and CD4+/CD8+ increased, and the chickens were protected completely against H9N2 AIV challenge. This study provides a method for the development of novel AIV vaccines, and a theoretical basis for the development of safe and efficient gene delivery carriers.