H1N1 Influenza Virus Infection Research Articles

Guanylate-binding protein 1 inhibits inflammatory factors produced by H5N1 virus through Its GTPase activity

The combination of inflammatory factors resulting from an influenza A virus infection is one of the main causes of death in host animals. Studies have shown that guinea pig guanosine monophosphate binding protein 1 (guanylate-binding protein 1, gGBP1) can downregulate cytokine production induced by the influenza virus. Therefore, exploring the innate immune defense mechanism of GBP1 in the process of H5N1 influenza virus infection has important implications for understanding the pathogenic mechanism, disease prevention, and the control of influenza A virus infections. We found that, in addition to inhibiting the early replication of influenza virus, gGBP1 also inhibited the production of CCL2 and CXCL10 cytokines induced by the influenza virus as well as the proliferation of mononuclear macrophages induced by these cytokines. These findings further confirmed that gGBP1 inhibited the production of cytokines through its GTPase activity and cell proliferation through its C-terminal α-helix structure. This study revealed the effect of gGBP1 on the production of cellular inflammatory factors during influenza virus infection and determined the key amino acid residues that assist in the inhibitory processes mediated by gGBP1.

Highly pathogenic avian influenza H5N1 virus infections in pinnipeds and seabirds in Uruguay: Implications for bird-mammal transmission in South America.

The highly pathogenic avian influenza viruses of clade 2.3.4.4b have caused unprecedented deaths in South American wild birds, poultry, and marine mammals. In September 2023, pinnipeds and seabirds appeared dead on the Uruguayan Atlantic coast. Sixteen influenza virus strains were characterized by real-time reverse transcription PCR and genome sequencing in samples from sea lions (Otaria flavescens), fur seals (Arctocephalus australis), and terns (Sterna hirundinacea). Phylogenetic and ancestral reconstruction analysis showed that these strains have pinnipeds most likely as the ancestral host, representing a recent introduction of clade 2.3.4.4b in Uruguay. The Uruguayan and closely related strains from Peru (sea lions) and Chile (sea lions and a human case) carry mammalian adaptative residues 591K and 701N in the viral polymerase basic protein 2 (PB2). Our findings suggest that clade 2.3.4.4b strains in South America may have spread from mammals to mammals and seabirds, revealing a new transmission route.

Secreted phospholipase PLA2G2E contributes to regulation of T cell immune response against influenza virus infection.

The involvement of secreted phospholipase A2s in respiratory diseases, such as asthma and respiratory viral infections, is well-established. However, the specific role of secreted phospholipase A2 group IIE (PLA2G2E) during influenza virus infection remains unexplored. Here, we investigated the role of PLA2G2E during H1N1 influenza virus infection using a targeted mouse model lacking Pla2g2e gene (Pla2g2e-/-). Our findings demonstrated that Pla2g2e-/- mice had significantly lower survival rates and higher viral loads in lungs compared to wild-type mice following influenza virus infection. While Pla2g2e-/- mice displayed comparable innate and humoral immune responses to influenza virus challenge, the animals showed impaired influenza-specific cellular immunity and reduced T cell-mediated cytotoxicity. This indicates that PLA2G2E is involved in regulating specific T cell responses during influenza virus infection. Furthermore, transgenic mice expressing the human PLA2G2E gene exhibited resistance to influenza virus infection along with enhanced influenza-specific cellular immunity and T cell-mediated cytotoxicity. Pla2g2e deficiency resulted in perturbation of lipid mediators in the lung and T cells, potentially contributing to its impact on the anti-influenza immune response. Taken together, these findings suggest that targeting PLA2G2E could hold potential as a therapeutic strategy for managing influenza virus infections.IMPORTANCEThe influenza virus is a highly transmissible respiratory pathogen that continues to pose a significant public health concern. It effectively evades humoral immune protection conferred by vaccines and natural infection due to its continuous viral evolution through the genetic processes of antigenic drift and shift. Recognition of conserved non-mutable viral epitopes by T cells may provide broad immunity against influenza virus. In this study, we have demonstrated that phospholipase A2 group IIE (PLA2G2E) plays a crucial role in protecting against influenza virus infection through the regulation of T cell responses, while not affecting innate and humoral immune responses. Targeting PLA2G2E could therefore represent a potential therapeutic strategy for managing influenza virus infection.

Engineering of novel hemagglutinin biosensors for rapid detection and drug screening of Influenza A H7N9 virus

New pathogenic influenza virus strains are constantly emerging, posing a serious risk to both human health and economic growth. To effectively control the spread of this virus, there is an urgent need for early, rapid, sensitive, simple, and cost-effective detection technologies, as well as new and effective antiviral drugs. In this study, we have successfully achieved a significant milestone by successfully fusing the H7N9 influenza virus hemagglutinin (HA) protein with the nano-luciferase component, resulting in the development of a novel set of biosensors. This remarkable achievement marks the first instance of utilizing this biosensor technology for influenza antibody detection. Our biosensor technology also has the potential to facilitate the development of antiviral drugs targeting specific epitopes of the HA protein, providing a promising avenue for the treatment of H7N9 influenza virus infections. Furthermore, our biosensors have broad applications beyond H7N9 influenza virus detection, as they can be expanded for the detection of other pathogens and drug screening applications in the future. By providing a novel and effective solution to the detection and treatment of influenza viruses, our biosensors have the potential to revolutionize the field of infectious disease control.

Influenza virus immune imprinting dictates the clinical outcomes in ferrets challenged with highly pathogenic avian influenza virus H5N1.

Zoonotic transmission of H5N1 highly pathogenic avian influenza virus (HPAIV) into the human population is an increasing global threat. The recent 2022 HPAIV outbreak significantly highlighted this possibility, increasing concern in the general population. The clinical outcomes of H5N1 influenza virus exposure can be determined by an individual's primary influenza virus infection (imprinting) or vaccination status. Immunological imprinting with Group 1 - (H1N1, H2N2, and H2N3) increases survival rates following H5N1 viral infection compared to Group 2 - (H3N2) imprinted individuals. Vaccination against H5N1 influenza viruses can offer protection to at-risk populations; however, stockpiled inactivated H5N1 influenza vaccines are not readily available to the public. We hypothesize that the immunological response to vaccination and subsequent clinical outcome following H5N1 influenza virus infection is correlated with the immunological imprinting status of an individual. To test this hypothesis, our lab established a ferret pre-immune model of disease. Naïve ferrets were intranasally inoculated with seasonal influenza viruses and allowed to recover for 84 days prior to H5N1 virus infection. Ferrets imprinted following H1N1 and H2N3 virus infections were completely protected against lethal H5N1 influenza virus challenge (100% survival), with few to no clinical symptoms. In comparison, H3N2 influenza virus-imprinted ferrets had severe clinical symptoms, delayed disease progression, and a sublethal phenotype (40% mortality). Consecutive infections with H1N1 influenza viruses followed by an H3N2 influenza virus infection did not abrogate the immune protection induced by the original H1N1 influenza virus infection. In addition, ferrets consecutively infected with H1N1 and H2N3 viruses had no clinical symptoms or weight loss. H3N2 pre-immune ferrets were vaccinated with a broadly reactive H5 HA-based or H1 NA-based vaccine (Hu-CO 2). These ferrets were protected against H5N1 influenza virus challenge, whereas ferrets vaccinated with the H1N1 wild-type CA/09 rHA vaccine had similar phenotypes as non-vaccinated H3N2-imprinted ferrets with 40% survival. Overall, Group 2 imprinted ferrets, which were vaccinated with heterologous Group 1 HA vaccines, had redirected immune responses to Group 1 influenza viral antigens and rescued a sublethal phenotype to complete protection.

A Review on H3N2 Influenza Virus

The aim of this review is to discuss the influenza A i.e H3N2 influenza virus with their characteristics. Here we present the outline of the treatment regarding the H3N2 infection, precautions to be taken. The review focused on the symptoms and prevention of H3N2 influenza virus infection. It also highlites on the recent changes in new H3N2 influenza virus and focused on vaccinations against influenza, which researchers are currently developing to study these virus. Moreover this review states that the H3N2 influenza virus are rapidly altered themselves in different ways. The study also shows that the biology of influenza and adaptation acquired by new H3N2 influenza A virus create difficulties in future and current also to predicting the changes. Study of viral growth and their transmition. This review provides support and guide the researcher in the study and better understanding of influenza viruses.

Anti-influenza A (H1N1) virus effect of gallic acid through inhibition of virulent protein production and association with autophagy.

Influenza remains one of the most serious infectious diseases. Gallic acid is one of the most common and representative phenolic acids found in various plants. This is an interesting subject to explore how gallic acid could inhibit H1N1 influenza virus infection by reducing the production of virulent proteins and interrupting autophagy machinery for influenza virus replication on the host cell. Cellular viability was assessed by XTT assay. The inhibitory effects on the H1N1 influenza virus were assessed by hemagglutination assay, plaque assay, and qRT-PCR. Western blot analysis was used for detecting protein levels of M1, M2, NP, LC3B, and beclin-1. Autophagy activity was demonstrated by acridine orange staining assay. The result demonstrated that there was no cytotoxic effect of gallic acid on A549 cells, and gallic acid could restore the cellular viability of H1N1 influenza virus-infected A549 cells within the experimental concentration treatment. Moreover, gallic acid could effectively restrain viral activity of the H1N1 influenza virus. After the treatment of gallic acid, the production of virulent H1N1 influenza virus proteins, that is, M1, M2, and NP protein were reduced. As for autophagic mechanism, both of the LC3B II conversion and the level ratio of LC3B II to LC3B I were notably decreased. The acridine orange staining assay also revealed decreased accumulation of autophagosomes in H1N1 influenza virus-infected cells. In conclusion, gallic acid suppresses H1N1 influenza viral infectivity through restoration of autophagy pathway and inhibition of virulent M1, M2, and NP protein production.

Partially Hydrolyzed Guar Gum Intake Supports the Gut Microbiota and Attenuates Inflammation during Influenza H1N1 Virus Infection in Mice.

Partially hydrolyzed guar gum (PHGG) is a soluble dietary fiber that is effective for defecation control. It influences the gut microbiota, by which it is metabolized to yield short-chain fatty acids (SCFAs), and it was also recently shown to protect against influenza infection in humans. We here investigated the effects of PHGG in a mouse model of influenza H1N1 virus infection. Eight-week-old C57BL/6 mice were fed normal chow with or without PHGG (500 mg/kg per day) for 4 weeks, infected with H1N1 at 10 weeks of age, and analyzed at 12 weeks of age. Administration of PHGG attenuated the decline in body weight induced by H1N1 infection without affecting food intake. It also ameliorated intestinal atrophy and increased the production of SCFAs including acetic acid, propionic acid, and butyric acid in the cecum, thereby preventing the inhibitory effect of H1N1 infection on SCFA production. The H1N1-induced increases in the serum concentrations of inflammatory cytokines including interferon-γ and interleukin-6 and anti-inflammatory cytokine such as interleukin-10 were all inhibited by PHGG intake. In addition, PHGG administration attenuated inflammatory gene expression in the lung and promoted both natural killer cell activity and regulatory T-cell differentiation in the spleen. Our findings suggest that the consumption of PHGG may improve the gut environment and thereby limit the inflammatory response to H1N1 infection. They may thus provide the basis for novel dietary intervention strategies to suppress the excessive inflammation associated with virus infection.

Selenium Nanoparticles Control H1N1 Virus by Inhibiting Inflammatory Response and Cell Apoptosis.

The treatment of influenza caused by H1N1 has been the focus of much attention. Selenium nanoparticles (SeNPs) have been used in many aspects of research in the last two decades. They have shown excellent performance in antiviral, anti-inflammatory, and antioxidant functions. Previous anti-H1N1 cell experiments using SeNPs have shown that they have evident antiviral effects and low toxicities. This study focuses on the mechanism of selenium nanoparticles against an H1N1 influenza virus infection in vivo. The results showed that the selenium levels in the body decreased after an H1N1 virus infection, and inflammatory factors in the lung tissues increased abnormally, leading to the onset and aggravation of an inflammatory response. The H1N1 virus infection also led to the excessive activation of apoptotic pathways in the body and induced the apoptosis of tissue cells. In addition, this study found that SeNPs can alleviate this phenomenon. All results showed that SeNPs are promising inhibitors for controlling influenza H1N1 virus infections.

H1N1 influenza virus infection through NRF2-KEAP1-GCLC pathway induces ferroptosis in nasal mucosal epithelial cells.

Influenza A virus can induce nasal inflammation by stimulating the death of nasal mucosa epithelium, however, the mechanism is not clear. In this study, to study the causes and mechanisms of nasal mucosa epithelial cell death caused by Influenza A virus H1N1, we isolated and cultured human nasal epithelial progenitor cells (hNEPCs) and exposed them to H1N1 virus after leading differentiation. Then we performed high-resolution untargeted metabolomics and RNAseq analysis of human nasal epithelial cells (hNECs) infected with H1N1 virus. Surprisingly, H1N1 virus infection caused the differential expression of a large number of ferroptosis related genes and metabolites in hNECs. Furthermore, we have observed a significant reduction in Nrf2/KEAP1 expression, GCLC expression, and abnormal glutaminolysis. By constructing overexpression vector of GCLC and the shRNAs of GCLC and Keap1, we determined the role of NRF2-KEAP1-GCLC signaling pathway in H1N1 virus-induced ferroptosis. In addition, A glutaminase antagonist, JHU-083, also demonstrated that glutaminolysis can regulate the NRF2-KEAP1-GCLC signal pathway and ferroptosis. According to this study, H1N1 virus can induce the ferroptosis of hNECs via the NRF2-KEAP1-GCLC signal pathway and glutaminolysis, leading to nasal mucosal epithelial inflammation. This discovery is expected to provide an attractive therapeutic target for viral-induced nasal inflammation.

Potential mechanisms mediating PM2.5-induced alterations of H3N2 influenza virus infection and cytokine production in human bronchial epithelial cells

Exposure to particulate matter (PM) has been associated with increased hospital admissions for influenza. Airway epithelial cells are a primary target for inhaled environmental insults including fine PM (PM2.5) and influenza viruses. The potentiation of PM2.5 exposure on the effects of influenza virus on airway epithelial cells has not been adequately elucidated. In this study, the effects of PM2.5 exposure on influenza virus (H3N2) infection and downstream modulation of inflammation and antiviral immune response were investigated using a human bronchial epithelial cell line, BEAS-2B. The results showed that PM2.5 exposure alone increased the production of pro-inflammatory cytokines including interleukin-6 (IL-6) and IL-8 but decreased the production of the antiviral cytokine interferon-β (IFN-β) in BEAS-2B cells while H3N2 exposure alone increased the production of IL-6, IL-8, and IFN-β. Importantly, prior exposure to PM2.5 enhanced subsequent H3N2 infectivity, expression of viral hemagglutinin protein, as well as upregulation of IL-6 and IL-8, but reduced H3N2-induced IFN-β production. Pre-treatment with a pharmacological inhibitor of nuclear factor-κB (NF-κB) suppressed pro-inflammatory cytokine production induced by PM2.5, H3N2, as well as PM2.5-primed H3N2 infection. Moreover, antibody-mediated neutralization of Toll-like receptor 4 (TLR4) blocked cytokine production triggered by PM2.5 or PM2.5-primed H3N2 infection, but not H3N2 alone. Taken together, exposure to PM2.5 alters H3N2-induced cytokine production and markers of replication in BEAS-2B cells, which in turn are regulated by NF-κB and TLR4.

Identifying Hub Genes and miRNA-mRNA Regulatory Networks in Mice Infected with H1N1 Influenza Virus.

H1N1 influenza virus is a major factor in seasonal influenza outbreaks. After the body is infected with the influenza virus, the expression of certain mRNAs, including miRNAs, could be affected. However, the association between these mRNAs and miRNAs remains unclear. This study is aimed at identifying differentially expressed genes (DEGs) and miRNAs (DEmiRs) caused by H1N1 influenza virus infection and constructing a miRNA-mRNA regulatory network. Nine GSE datasets were downloaded from the Gene Expression Omnibus database, of which seven were mRNA data and two were miRNA data. The limma package in R language package was used to analyze array data, and edgeR package was used to analyze high-throughput sequencing data. At the same time, the genes related to H1N1 infection were further screened by WGCNA analysis. DEGs were subjected to Gene Ontology and KEGG pathway enrichment analyses by DAVID database, while the STRING database predicted the protein-protein interaction (PPI) network. The correspondence between miRNA and target mRNA was analyzed by the miRWalk database. Cytoscape software was used to output PPI results, identify hub genes, and construct a miRNA-mRNA regulatory network. 114 DEGs and 37 candidate DEmiRs were identified for subsequent analysis. These DEGs were significantly enriched in response to the virus, cytokine activity, and symbiont-containing vacuole membrane. According to KEGG analysis, DEGs were enriched in PD-L1 expression and PD-1 checkpoint pathway. The key point Cd274 (PD-L1) was highly expressed in the H1N1-infected group. Finally, a potential miRNA-mRNA regulatory network (containing 8 candidate DEmiRs and 69 candidate DEGs) and a PPI network were constructed. After that, three hub genes were identified: Ifit3, Stat2, and Irf7. These hub genes and Cd274 were validated by another independent high-throughput dataset and were highly expressed pattern. This study will help researchers gain insights into the intrinsic effects of H1N1 influenza virus infection on the host and suggest a novel association of H1N1 virus with the host immune system.

Evolution of highly pathogenic H5N1 influenza A virus in the central nervous system of ferrets.

Central nervous system (CNS) disease is the most common extra-respiratory tract complication of influenza A virus infections in humans. Remarkably, zoonotic highly pathogenic avian influenza (HPAI) H5N1 virus infections are more often associated with CNS disease than infections with seasonal influenza viruses. Evolution of avian influenza viruses has been extensively studied in the context of respiratory infections, but evolutionary processes in CNS infections remain poorly understood. We have previously observed that the ability of HPAI A/Indonesia/5/2005 (H5N1) virus to replicate in and spread throughout the CNS varies widely between individual ferrets. Based on these observations, we sought to understand the impact of entrance into and replication within the CNS on the evolutionary dynamics of virus populations. First, we identified and characterized three substitutions-PB1 E177G and A652T and NP I119M - detected in the CNS of a ferret infected with influenza A/Indonesia/5/2005 (H5N1) virus that developed a severe meningo-encephalitis. We found that some of these substitutions, individually or collectively, resulted in increased polymerase activity in vitro. Nevertheless, in vivo, the virus bearing the CNS-associated mutations retained its capacity to infect the CNS but showed reduced dispersion to other anatomical sites. Analyses of viral diversity in the nasal turbinate and olfactory bulb revealed the lack of a genetic bottleneck acting on virus populations accessing the CNS via this route. Furthermore, virus populations bearing the CNS-associated mutations showed signs of positive selection in the brainstem. These features of dispersion to the CNS are consistent with the action of selective processes, underlining the potential for H5N1 viruses to adapt to the CNS.

Nucleoside-Modified mRNA-Based Influenza Vaccines Circumvent Problems Associated with H3N2 Vaccine Strain Egg Adaptation.

Most human influenza vaccine antigens are produced in fertilized chicken eggs. Recent H3N2 egg-based vaccine antigens have limited effectiveness, partially due to egg-adaptive substitutions that alter the antigenicity of the hemagglutinin (HA) protein. The nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA-LNP) vaccine platform is a promising alternative for egg-based influenza vaccines because mRNA-LNP-derived antigens are not subject to adaptive pressures that arise during the production of antigens in chicken eggs. Here, we compared H3N2-specific antibody responses in mice vaccinated with either 3c.2A H3-encoding mRNA-LNP or a conventional egg-based Fluzone vaccine (which included an egg-adapted 3c.2A antigen) supplemented with an MF59-like adjuvant. We tested mRNA-LNP encoding wild-type and egg-adapted H3 antigens. We found that mRNA-LNP encoding wild-type H3 elicited antibodies that neutralized the wild-type 3c.2A H3N2 virus more effectively than antibodies elicited by mRNA-LNP encoding egg-adapted H3 or the egg-based Fluzone vaccine. mRNA-LNP expressing either wild-type or egg-adapted H3 protected mice against infection with the wild-type 3c2.A H3N2, whereas the egg-based Fluzone vaccine did not. We found that both mRNA-LNP vaccines elicited high levels of group 2 HA stalk-reactive antibodies, which likely contributed to protection in vivo. Our studies indicate that nucleoside-modified mRNA-LNP-based vaccines can circumvent problems associated with egg adaptations with recent 3c2.A H3N2 viruses. IMPORTANCE This study shows that the nucleoside-modified mRNA-LNP vaccine platform is a promising alternative for egg-based influenza vaccines. We show that mRNA-LNP vaccines expressing H3 antigens elicit high levels of antibodies in mice and protect against H3N2 influenza virus infection.

A replication-deficient H9N2 influenza virus carrying H5 hemagglutinin conferred protection against H9N2 and H5N1 influenza viruses in mice

H5N1 and H9N2 influenza viruses have been reported to cause human infections and are believed to have pandemic potential. The vaccine is an effective tool to prevent influenza virus infection. However, inactivated influenza vaccines sometimes result in low antigenicity as result leads to generating of incomplete immune protection in the form of low cellular and humoral immunity. While the low temperature adapted, traditional live attenuated influenza vaccine (LAIV) is associated with the potential risk to revert to a virulent phenotype, there appears an essential need for an alternative potent methodology to design and develop influenza vaccines with substantial safety and efficacy which may confer solid protection against H9N2 or H5N1 influenza virus infections. In the present study, a replication-deficient recombinant influenza virus, WM01ma-HA(H5), expressing hemagglutinin (HA) of both H9N2 and H5N1 subtypes was developed. The chimeric gene segment expressing HA(H5), was designed using the sequence of an open reading frame (ORF) of HA adopted from A/wild duck/Hunan/021/2005(H5N1)(HN021ma) which was flanked by the NA packaging signals of mouse-adapted strain A/Mink/Shandong/WM01/2014(H9N2)(WM01ma). Due to the absence of ORF of structural protein NA, the replication of this engineered H9N2 influenza viruses WM01ma-HA(H5) was hampered in vitro and in vivo but was well competent in MDCK cells stably expressing the NA protein of WM01ma. Intranasal vaccination of mice with WM01ma-HA(H5) stimulated robust immune response without any clinical signs and conferred complete protection from infection by H5N1 or H9N2 subtype influenza viruses.

Genome‑wide identification, organization, and expression profiles of the chicken fibroblast growth factor genes in public databases and Vietnamese indigenous Ri chickens against highly pathogenic avian influenza H5N1 virus infection.

Fibroblast growth factors (FGFs) play critical roles in embryo development, and immune responses to infectious diseases. In this study, to investigate the roles of FGFs, we performed genome-wide identification, expression, and functional analyses of FGF family members in chickens. Chicken FGFs genes were identified and analyzed by using bioinformatics approach. Expression profiles and Hierarchical cluster analysis of the FGFs genes in different chicken tissues were obtained from the genome-wide RNA-seq. A total of 20 FGF genes were identified in the chicken genome, which were classified into seven distinct groups (A-F) in the phylogenetic tree. Gene structure analysis revealed that members of the same clade had the same or similar exon-intron structure. Chromosome mapping suggested that FGF genes were widely dispersed across the chicken genome and were located on chromosomes 1, 4-6, 9-10, 13, 15, 28, and Z. In addition, the interactions among FGF proteins and between FGFs and mitogen‑activated protein kinase (MAPK) proteins are limited, indicating that the remaining functions of FGF proteins should be further investigated in chickens. Kyoto encyclopedia of genes and genomes pathway analysis showed that FGF gene interacts with MAPK genes and are involved in stimulating signaling pathway and regulating immune responses. Furthermore, this study identified 15 differentially expressed genes (DEG) in 21 different growth stages during early chicken embryo development. RNA-sequencing data identified the DEG of FGFs on 1- and 3-days post infection in two indigenous Ri chicken lines infected with the highly pathogenic avian influenza virus H5N1 (HPAIV). Finally, all the genes examined through quantitative real-time polymerase chain reaction and RNA-Seq analyses showed similar responses to HPAIV infection in indigenous Ri chicken lines (R2 = 0.92- 0.95, p<0.01). This study provides significant insights into the potential functions of FGFs in chickens, including the regulation of MAPK signaling pathways and the immune response of chickens to HPAIV infections.

Exploring the anticomplement components from Fagopyrum dibotrys for the treatment of H1N1-induced acute lung injury by UPLC-Triple-TOF-MS/MS

In the present study, a procyanidins-enriched fraction (PCE) from the rhizome of Fagopyrum dibotrys was obtained by anticomplement activity-guided fractionation. PCE could alleviate H1N1-induced ALI in mice by reducing weight loss, decreasing lung index, and regulating cytokine levels in lung tissue. PCE contained 76.5 ± 1.1% procyanidins with a mean degree of polymerization (mDP) of 5.24 ± 0.16. Meanwhile, thirty-three chemical constituents, including 27 procyanidins and 6 other compounds, were recognized by UPLC-Triple-TOF-MS/MS. Among them, twenty recognized procyanidins were composed of (epi)catechin with B-type link, while the rest consisted of (epi)catechin gallate. Furthermore, six compounds were obtained by preparative HPLC on a C18 column (250 × 10.0 mm, 5 µm), and their structures were confirmed by mass spectrum (MS), nuclear magnetic resonance (NMR), and specific rotation. The structure-activity relationship analysis indicated that DP and galloylation were closely related to the anticomplement activity of procyanidins. The obtained results revealed that anticomplement procyanidins were one kind of the potentially effective materials of F. dibotrys against H1N1 influenza virus infection, and the in vivo efficacy of these compounds was worthy of further investigation.

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

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. 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. 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. 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.

Refined polysaccharide from Dendrobium devonianum resists H1N1 influenza viral infection in mice by activating immunity through the TLR4/MyD88/NF-κB pathway.

Dendrobium polysaccharide exhibits multiple biological activities, such as immune regulation, antioxidation, and antitumor. However, its resistance to viral infection by stimulating immunity is rarely reported. In this study, we explored the effect and mechanism of DVP-1, a novel polysaccharide from Dendrobium devonianum, in the activation of immunity. After being activated by DVP-1, the ability of mice to prevent H1N1 influenza virus infection was investigated. Results of immune regulation showed that DVP-1 significantly improved the immune organ index, lymphocyte proliferation, and mRNA expression level of cytokines, such as IL-1β, IL-4, IL-6, and TNF-α in the spleen. Immunohistochemical results showed that DVP-1 obviously promoted the mucosal immunity in the jejunum tissue. In addition, the expression levels of TLR4, MyD88, and TRAF6 and the phosphorylation levels of TAK1, Erk, JNK, and NF-κB in the spleen were upregulated by DVP-1. The virus infection results showed that the weight loss of mice slowed down, the survival rate increased, the organ index of the lung reduced, and the virus content in the lung decreased after DVP-1 activated immunity. By activating immunity with DVP-1, the production of inflammatory cells and inflammatory factors in BALF, and alveolar as well as peribronchiolar inflammation could be prevented. The results manifested that DVP-1 could resist H1N1 influenza virus infection by activating immunity through the TLR4/MyD88/NF-κB pathway.

Insilico Identification of H5N1 Avian Influenza Neuraminidase Inhibitors

Background: H5N1 avian influenza virus is influenza-A virus subtype that is highly contagious and fatal among birds and humans. Although human-human transmission of H5N1 is rare, but once the virus has acquired this ability, a devastating pandemic may be inevitable. The virus is currently in WHO phase 3 pandemic alerts: A new influenza virus subtype causing disease in humans, but is yet to spread efficiently and sustainably among humans. Oseltamivir is a single most-win WHO recommended drug for the treatment and prophylaxis of H5N1 influenza virus infection. However, several oseltamivir resistant strains of H5N1 have been reported. Methods: In silico molecular docking algorithm and pharmacophore modelling simulations were used to identify a novel H5N1 avian influenza neuraminidase inhibitors. Results: Of the 518-neuraminidase analogue, five (eflornithine, eltrombopag, entecavir, aminosalicylic acid and pregabalin) demonstrated excellent binding affinities against viral neuraminidase similar to that of oseltamivir. Again, these drugs like oseltamivir interact mainly with arginine, tyrosine and glutamine at different positions of the neuraminidase. Similarly, oseltamivir and the five selected drugs superimpose to the active pocket of neuraminidase, further buttressing the previous findings. Furthermore, oseltamivir and the five drugs had common generated pharmacophores. Conclusion: This study was the first to demonstrate the anti-viral neuraminidase effect of eflornithine, eltrombopag, entecavir, aminosalicylic acid and pregabalin. Keywords: Neuraminidase, Oseltamivir, H5N1 avian influenza, Pharmacophore, molecular docking.