Influenza Virus H5N1 Research Articles

Molecular docking analysis of N-substituted oseltamivir derivatives with the SARS-Cov-2 main protease

The identification of chemotherapeutic drugs against Novel Coronavirus (2019-nCoV) is a significant requirement due to the rapid rise in deaths due to Corona Viral Infection all around the world. Therefore, it is of interest to document the molecular docking analysis data of 32 N-substituted Oseltamivir derivatives inhibitors of influenza virus H5N1 with the Novel Coronavirus main protease (2019-nCoV). We describe the optimal binding features of Oseltamivir derivatives with the SARS-Cov-2 main protease (Code PDB: 6LU7) for further consideration.

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-1β 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.

Transmission of highly pathogenic avian influenza in the nomadic free-grazing duck production system in Viet Nam

The presence of free-grazing ducks (FGD) has consistently been shown to be associated with highly pathogenic avian influenza virus (HPAIV) H5N1 outbreaks in South-East Asia. However, the lack of knowledge about the transmission pathways limits the effectiveness of control efforts. To address this gap, we developed a probabilistic transmission model of HPAIV H5N1 in the nomadic FGD production system in Viet Nam, assuming different scenarios to address parameter uncertainty. Results suggested that HPAIV H5N1 could spread within the nomadic FGD production system, with an estimated flock-level effective reproduction number (re) ranging from 2.16 (95% confidence interval (CI): 1.39-3.49) to 6.10 (95%CI: 3.93-9.85) depending on the scenario. Indirect transmission via boats and trucks was shown to be the main transmission route in all scenarios. Results suggest that re could be reduced below one with 95% confidence if 86% of FGD flocks were vaccinated in the best-case scenario or 95% in the worst-case scenario. If vaccination was combined with cleaning and disinfection of transport vehicles twice a week, vaccination coverage could be lowered to 60% in the best-case scenario. These findings are of particular relevance for prioritising interventions for effective control of HPAIV in nomadic free-grazing duck production systems.

Experimental PCEP-Adjuvanted Swine Influenza H1N1 Vaccine Induced Strong Immune Responses but Did Not Protect Piglets against Heterologous H3N2 Virus Challenge.

Vaccination is the most efficient method of protection against influenza infections. However, the rapidly mutating viruses and development of new strains make it necessary to develop new influenza vaccines annually. Hence, vaccines that stimulate cross-protection against multiple influenza subtypes are highly sought. Recent evidence suggests that adjuvants such as PCEP that promote Th1-type T cell and Th2-type T cell immune responses and broad-spectrum immune responses may confer cross-protection against heterologous influenza strains. In this study, we evaluated whether the immunogenic and protective potential of PCEP-adjuvanted inactivated swine influenza virus H1N1 vaccine can protect pigs immunized against live H3N2 virus. Piglets were vaccinated via the intradermal route with PCEP-adjuvanted inactivated swine influenza virus (SIV) H1N1 vaccine, boosted at day 21 with the same vaccines then challenged with infectious SIV H3N2 virus at day 35 via the tracheobronchial route. The pigs showed significant anti-H1N1 SIV specific antibody titres and H1N1 SIV neutralizing antibody titres, and these serum titres remained after the challenge with the H3N2 virus. In contrast, vaccination with anti-H1N1 SIV did not trigger anti-H3N2 SIV antibody titres or neutralizing antibody titres and these titres remained low until pigs were challenged with H3N2 SIV. At necropsy (six days after challenge), we collected prescapular lymph nodes and tracheobronchial draining the vaccination sites and challenge site, respectively. ELISPOTs from lymph node cells restimulated ex vivo with inactivated SIV H1N1 showed significant production of IFN-γ in the tracheobronchial cells, but not the prescapular lymph nodes. In contrast, lymph node cells restimulated ex vivo with inactivated SIV H1N1 showed significantly higher IL-13 and IL-17A in the prescapular lymph nodes draining the vaccination sites relative to unchallenged animals. Lung lesion scores show that intradermal vaccination with H1N1 SIV plus PCEP did not prevent lesions when the animals were challenged with H3N2. These results confirm previous findings that PCEP is effective as a vaccine adjuvant in that it induces strong immune responses and protects against homologous swine influenza H1N1 virus, but the experimental H1N1 vaccine failed to cross-protect against heterologous H3N2 virus.

Protection of Chickens with Maternal Immunity Against Avian Influenza Virus (AIV) by Vaccination with a Novel Recombinant Newcastle Disease Virus Vector.

Newcastle disease virus (NDV) vectors expressing avian influenza virus (AIV) hemagglutinin of subtype H5 protect specific pathogen-free chickens from Newcastle disease and avian influenza. However, maternal AIV antibodies (AIV-MDA+) are known to interfere with active immunization by influencing vaccine virus replication and gene expression, resulting in inefficient protection. To overcome this disadvantage, we inserted a transgene encoding a truncated soluble hemagglutinin (HA) in addition to the gene encoding membrane-bound HA from highly pathogenic avian influenza virus (HPAIV) H5N1 into lentogenic NDV Clone 30 genome (rNDVsolH5_H5) to overexpress H5 antigen. Vaccination of 3-wk-old AIV-MDA+ chickens with rNDVsolH5_H5 and subsequent challenge infection with HPAIV H5N1 3 wk later resulted in 100% protection. Vaccination of younger chickens with higher AIV-MDA levels 1 and 2 wk after hatch resulted in protection rates of 40% and 85%, respectively. However, all vaccinated chickens showed strongly reduced shedding of challenge virus compared with age-matched, nonvaccinated control chickens. All control chickens succumbed to the HPAIV infection with a grading in disease progression between the three groups, indicating the influence of AIV-MDAs even at a low level. Furthermore, the shedding and serologic data gathered after immunization indicate sufficient replication of the vaccine virus, which leads to the assumption that lower protection rates in younger AIV-MDA+ chickens are caused by an H5 antigen-specific block and not by the interference of the AIV-MDA and the vaccine virus itself. In summary, solid protective efficacy and reduced virus transmission were achieved in 3-wk-old AIV-MDA+ chickens, which is relevant especially in regions endemically infected with HPAIV H5N1.

A Systematic Review Analyzing the Prevalence and Circulation of Influenza Viruses in Swine Population Worldwide.

The global anxiety and a significant threat to public health due to the current COVID-19 pandemic reiterate the need for active surveillance for the zoonotic virus diseases of pandemic potential. Influenza virus due to its wide host range and zoonotic potential poses such a significant threat to public health. Swine serve as a “mixing vessel” for influenza virus reassortment and evolution which as a result may facilitate the emergence of new strains or subtypes of zoonotic potential. In this context, the currently available scientific data hold a high significance to unravel influenza virus epidemiology and evolution. With this objective, the current systematic review summarizes the original research articles and case reports of all the four types of influenza viruses reported in swine populations worldwide. A total of 281 articles were found eligible through screening of PubMed and Google Scholar databases and hence were included in this systematic review. The highest number of research articles (n = 107) were reported from Asia, followed by Americas (n = 97), Europe (n = 55), Africa (n = 18), and Australia (n = 4). The H1N1, H1N2, H3N2, and A(H1N1)pdm09 viruses were the most common influenza A virus subtypes reported in swine in most countries across the globe, however, few strains of influenza B, C, and D viruses were also reported in certain countries. Multiple reports of the avian influenza virus strains documented in the last two decades in swine in China, the United States, Canada, South Korea, Nigeria, and Egypt provided the evidence of interspecies transmission of influenza viruses from birds to swine. Inter-species transmission of equine influenza virus H3N8 from horse to swine in China expanded the genetic diversity of swine influenza viruses. Additionally, numerous reports of the double and triple-reassortant strains which emerged due to reassortments among avian, human, and swine strains within swine further increased the genetic diversity of swine influenza viruses. These findings are alarming hence active surveillance should be in place to prevent future influenza pandemics.

Historical origins and zoonotic potential of avian influenza virus H9N2 in Tunisia revealed by Bayesian analysis and molecular characterization.

During 2009-2012, several outbreaks of avian influenza virus H9N2 were reported in Tunisian poultry. The circulating strains carried in their hemagglutinins the human-like marker 226L, which is known to be important for avian-to-human viral transmission. To investigate the origins and zoonotic potential of the Tunisian H9N2 viruses, five new isolates were identified during 2012-2016 and their whole genomes were sequenced. Bayesian-based phylogeny showed that the HA, NA, M and NP segments belong to the G1-like lineage. The PB1, PB2, PA and NS segments appeared to have undergone multiple intersubtype reassortments and to be only distantly related to all of the Eurasian lineages (G1-like, Y280-like and Korean-like). The spatiotemporal dynamic of virus spread revealed that the H9N2 virus was transferred to Tunisia from the UAE through Asian and European pathways. As indicated by Bayesian analysis of host traits, ducks and terrestrial birds played an important role in virus transmission to Tunisia. The subtype phylodynamics showed that the history of the PB1 and PB2 segments was marked by intersubtype reassortments with H4N6, H10N4 and H2N2 subtypes. Most of these transitions between locations, hosts and subtypes were statistically supported (BF > 3) and not influenced by sampling bias. Evidence of genetic evolution was observed in the predicted amino acid sequences of the viral proteins of recent Tunisian H9N2 viruses, which were characterized by the acquisition of new mutations involved in virus adaptation to avian and mammalian hosts and amantadine resistance. This study is the first comprehensive analysis of the evolutionary history of Tunisian H9N2 viruses and highlights the zoonotic risk associated with their circulation in poultry, indicating the need for continuous surveillance of their molecular evolution.

Structures of the MHC-I molecule BF2*1501 disclose the preferred presentation of an H5N1 virus-derived epitope

Lethal infections by strains of the highly-pathogenic avian influenza virus (HPAIV) H5N1 pose serious threats to both the poultry industry and public health worldwide. A lack of confirmed HPAIV epitopes recognized by cytotoxic T lymphocytes (CTLs) has hindered the utilization of CD8+ T-cell-mediated immunity and has precluded the development of effectively diversified epitope-based vaccination approaches. In particular, an HPAIV H5N1 CTL-recognized epitope based on the peptide MHC-I-β2m (pMHC-I) complex has not yet been designed. Here, screening a collection of selected peptides of several HPAIV strains against a specific pathogen-free pMHC-I (pBF2*1501), we identified a highly-conserved HPAIV H5N1 CTL epitope, named HPAIV-PA123-130 We determined the structure of the BF2*1501-PA123-130 complex at 2.1 Å resolution to elucidate the molecular mechanisms of a preferential presentation of the highly-conserved PA123-130 epitope in the chicken B15 lineage. Conformational characteristics of the PA123-130 epitope with a protruding Tyr-7 residue indicated that this epitope has great potential to be recognized by specific TCRs. Moreover, significantly increased numbers of CD8+ T cells specific for the HPAIV-PA123-130 epitope in peptide-immunized chickens indicated that a repertoire of CD8+ T cells can specifically respond to this epitope. We anticipate that the identification and structural characterization of the PA123-130 epitope reported here could enable further studies of CTL immunity against HPAIV H5N1. Such studies may aid in the development of vaccine development strategies using well-conserved internal viral antigens in chickens.

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Evaluation of Viral-Induced Stress by Quantitating Corticosterone in Feathers of Virus-Infected Specific Pathogen-Free Chicks

Physiological stress is known to promote economic losses by causing immunosuppression. Various factors induce stress, including non-optimal management and infection with various pathogens. The concentration of the stress hormone, corticosterone, is indicative of stress in birds. However, its measurement is difficult due to its elevation caused by catching, handling, bleeding, short half-life in the blood, and short secretion period in feces. We describe a novel method to assess corticosterone concentration by sampling feathers. The viruses studied were 2 immunosuppressive viruses, Marek's disease virus and chicken anemia virus, the respiratory viruses, influenza virus H9N2, Newcastle disease virus, and Infectious bronchitis virus. The study contributes novelty as virus-induced stress by feather examination was described sporadically in chickens previously, and feathers were used to detect corticosterone mainly in dead wild and captive birds. Also, the development of extraction method from feathers is novel by using PBS instead of methanol, and by preparing the feather homogenate with steroid displacement reagent. The study findings revealed that virus infections increased the corticosterone concentrations in feather tips to various extents, from 2× to 10× fold, demonstrating that birds endure stress in addition to the clinical and pathological effects induced by the virus infections.

Trend of Changes in the Titer of Antibody against Avian Influenza Virus H9n2 during Raising Period in Vaccinated and Unvaccinated Broiler Farms in Qazvin Province, Iran: A Cohort Study.

Avian influenza virus (AIV) H9N2 is endemic in Iran and its large-scale circulation in the poultry industry of the country is devastating. This virus was first reported in the industrial poultry populations of Iran in July 1998. Some of the published studies showed that inactivated avian influenza (AI) vaccines are capable of inducing an immune response and providing protection against morbidity and mortality in different countries (Vasfi et al., 2002; Tavakkoli et al., 2011). Low pathogenicity avian influenza subtype H9N2 virus has been reported to have a zoonotic potential and widespread distribution in Iran. Therefore, water-in-oil emulsion vaccines are employed to control the disease in chickens (Nili and Asasi, 2003). This cohort study was conducted during July 2016-November 2017 in broiler chicken farms of Qazvin province, Iran to investigate the serological change trends in broiler chickens in this region. Level of immunity against the H9N2 virus was evaluated by hemagglutination inhibition assay. Fifteen farms out of thirty enrolled units used AI H9N2 killed vaccines. The minimum of mean antibody titers (MATs) was 4.54-2.42 and the maximum of MATs was 4.54+2.42 on day 3. In addition, the minimum and maximum MATs on day 50 were 0.4-0.64 and 0.4+0.064, respectively. The transfer rate of H9N2 AIV antibodies from the serum of breeders to the serum of chickens was calculated as 60.35% in our study. A significant difference was revealed between the maternal mean antibody titers (MMATs) and the MATs on day 3 (P<0.001). In addition, the difference between the MATs on day 3 and the MATs on day 10 was found to be significant (P<0.01). Moreover, MATs were significantly different between the vaccinated and unvaccinated herds on day 40 (P<0.05), while no significant difference was observed on days 3, 10, 20, and 30 (P>0.05). According to the results of this study, antibody titers in the vaccinated farms did not reach the protective level until the end of the rearing period. Most of the unvaccinated herds experienced a spurt in antibody titers due to exposure to the virus. Consequently, biosecurity measures must be implemented more seriously and strictly in broiler farms.

Assessment of experimental pathogenicity of avian influenza virus H9N2 isolates by intravenous pathogenicity index (IVPI) test and histopathology

A total of 1,423 number of tissue samples received from various parts of India were processed for virus isolation in chicken embryos of 9-11 d old as a part of programme to monitor Avian Influenza Virus (AIV) infection in Indian poultry population and a total number of three samples were positive by HA test and the titre of the virus isolates ranged from 1:8 to 1:64. The virus isolates had been subjected to HI test with the reference positive serum imported from NVSL, USA for various subtypes. The virus isolates gave a HI titre of 1:32 with H9 subtype specific reference positive serum indicating that the virus isolates were influenza A H9 subtype. One of the isolates (accession no. 3722/04) was subjected to neuraminidase assay and the virus was found by N2 subtype and also virus sub typing was done by RT-PCR for further confirmation. The influenza H9N2 virus isolates (accession no. 2543/04, 2544/04, 2317/04, 2424/04, and 3722/04) were used in the intravenous pathogenicity index test. In the present study no, clinical signs and/or gross lesions in experimentally inoculated chickens were observed. All the five isolates studied were found to be non-pathogenic according to the European Union definition. The organs collected from the experimental chickens did not show significant lesions, on histopathological examination, except for some minor changes in some of the organs. Immunofluorescence test was used to study the tissue tropism of avian influenza viruses’ isolates experimentally infected in chickens. The antigen was detected only in the brain and trachea of the chickens experimentally infected with the isolates having accession no. 2543/04 and intestine of chickens experimentally infected with the isolate having accession number 2544/04. Avian influenza virus re-isolation from experimentally infected chickens was attempted in 9-11 d old embryonated chicken eggs. Virus could be re-isolated from some organs, three groups infected with the isolates with accession numbers 2543/04, 2544/04 and 2317/04. Thus, the present work was envisaged to fill in the gap formed by this lack of information on avian influenza in India.

Optimization of 4-Aminopiperidines as Inhibitors of Influenza A Viral Entry That Are Synergistic with Oseltamivir.

Vaccination is the most prevalent prophylactic means for controlling seasonal influenza infections. However, an effective vaccine usually takes at least 6 months to develop for the circulating strains. Therefore, new therapeutic options are needed for the acute treatment of influenza infections to control this virus and prevent epidemics/pandemics from developing. We have discovered fast-acting, orally bioavailable acylated 4-aminopiperidines with an effective mechanism of action targeting viral hemagglutinin (HA). Our data show that these compounds are potent entry inhibitors of influenza A viruses. We present docking studies that suggest an HA binding site for these inhibitors on H5N1. Compound 16 displayed a significant decrease of viral titer when evaluated in the infectious assays with influenza virus H1N1 (A/Puerto Rico/8/1934) or H5N1 (A/Vietnam/1203/2004) strains and the oseltamivir-resistant strain with the most common H274Y mutation. In addition, compound 16 showed significant synergistic activity with oseltamivir in vitro.

Small Animal Review

Summary: Canine influenza virus (CIV) H3N2 is a highly transmissible virus with the potential to cause significant respiratory disease in dogs, and occasionally death. Sources of the virus are predominantly rescue dogs imported from endemic regions e.g. Asia. Outbreaks have major economic implications for kennels and veterinary practices.

Characterization of thermostable Newcastle disease virus recombinants expressing the hemagglutinin of H5N1 avian influenza virus as bivalent vaccine candidates

Newcastle disease virus (NDV) has been used as a vector in the development of vaccines and gene delivery. In the present study, we generated the thermostable recombinant NDV (rNDV) expressing the different forms of hemagglutinin (HA) of highly pathogenic avian influenza virus (HPAIV) H5N1 based on the full-length cDNA clone of thermostable TS09-C strain. The recombinant thermostable Newcastle disease viruses, rTS-HA, rTS-HA1 and rTS-tPAs/HA1, expressed the HA, HA1 or modified HA1 protein with the tissue plasminogen activator signal sequence (tPAs), respectively. The rNDVs displayed similar thermostability, growth kinetics and pathogenicity compared with the parental TS09-C virus. The tPAs facilitated the expression and secretion of HA1 protein in cells infected with rNDV. Animal studies demonstrated that immunization with rNDVs elicited effective H5N1- and NDV-specific antibody responses and conferred immune protection against lethal H5N1 and NDV challenges in chickens and mice. Importantly, vaccination of rTS-tPAs/HA1 resulted in enhanced protective immunity in chickens and mice. Our study thus provides a novel thermostable NDV-vectored vaccine candidate expressing a soluble form of a heterologous viral protein, which will greatly aid the poultry industry in developing countries.

QSAR study of N-substituted oseltamivir derivatives as potent avian influenza virus H5N1 inhibitors using quantum chemical descriptors and statistical methods

In silico modelling studies were executed on thirty two N-substituted oseltamivir derivatives as inhibitors of influenza virus H5N1.

Novel pathogenic characteristics of highly pathogenic avian influenza virus H7N9: viraemia and extrapulmonary infection

ABSTRACT The H7N9 virus mutated in 2017, resulting in new cases of highly pathogenic avian influenza (HPAI) H7N9 virus infection. H7N9 was found in a viraemic patient in Guangdong province, China. The present study aimed to clarify the pathogenic characteristics of HPAI H7N9. Virus was isolated from the plasma and sputum of the patient with HPAI H7N9. Liquid phase chip technology was used to detect the plasma cytokines from the infected patient and healthy controls. Mice were infected with strains A/Guangdong/GZ8H002/2017(H7N9) and A/Zhejiang/DTID-ZJU01/2013(H7N9) to observe the virus’s pathogenic characteristics. Serum and brain tissue were collected at 2, 4, and 6 days after infection. The viruses in serum and brain tissue were detected and isolated. The two strains were infected into A549 cells, exosomes were extracted, and virus genes in the exosomes were assessed. Live virus was isolated from the patient’s plasma. An acute cytokine storm was detected during the whole course of the disease. In animal experiments, A/Guangdong/GZ8H002/2017(H7N9) was more pathogenic than A/Zhejiang /DTID-ZJU01/2013(H7N9) and resulted in the death of mice. Live virus was isolated from infected mouse serum. Virus infection was also detected in the brain of mice. Under viral stress, A549 cells secreted exosomes containing the entire viral genome. The viraemic patient was confirmed to have an HPAI H7N9 infection. A/Guangdong/GZ8H002/2017(H7N9) showed significantly enhanced toxicity. Patient deaths might result from cytokine storms and brain infections. Extrapulmonary tissue infection might occur via the exosome pathway. The determined pathogenic characteristics of HPAI H7N9 will contribute to its future treatment.

Purification method for cell-cultured influenza virus H5N1

Objective To reduce the residual proteins and DNA of host cells in the preparation of H5N1 influenza A virus. Methods Core 700 was firstly used to remove residual host cell proteins, and then Capto Q was used to remove host cell DNA. Several batches of H5N1 influenza A virus cultured in Madin-Darby canine kidney (MDCK) cells were purified using this method. The efficiency of purification was evaluated using many methods including quantitative real-time PCR, hemagglutination (HA) test and single radial immunodiffusion assay. Moreover, Benzonase nuclease was used for comparison. Results Without the use of Benzonase nuclease, the overall removal rates of host cell DNA and residual proteins were 99.62% and 98.1%, and the HA antigen recovery rate was 66.96%. Conclusions This study established a purification strategy with good effect for cell-based influenza vaccines. It can efficiently remove host cell DNA and proteins and achieve a high HA recovery rate. The purification result is no worse than that of adding Benzonase nuclease, suggesting the potential of its application in actual vaccine production. Key words: Influenza virus; MDCK cells; Host cell protein; Host cell DNA; Purification

Flavonoids from Houttuynia cordata attenuate H1N1-induced acute lung injury in mice via inhibition of influenza virus and Toll-like receptor signalling

BackgroundInfluenza virus is one of the most important human pathogens, causing substantial seasonal and pandemic morbidity and mortality. Houttuynia cordata is a traditionally used medicinal plant for the treatment of pneumonia. Flavonoids are one of the major bioactive constituents of Houttuynia cordata. PurposeThis study was designed to investigate the therapeutic effect and mechanism of flavonoid glycosides from H. cordata on influenza A virus (IAV)-induced acute lung injury (ALI) in mice. MethodsFlavonoids from H. cordata (HCF) were extracted from H. cordata and identified by high-performance liquid chromatography. Mice were infected intranasally with influenza virus H1N1 (A/FM/1/47). HCF (50, 100, or 200 mg/kg) or Ribavirin (100 mg/kg, the positive control) were administered intragastrically. Survival rates, life spans, weight losses, lung indexes, histological changes, inflammatory infiltration, and inflammatory markers in the lungs were measured. Lung virus titers and neuraminidase (NA) activities were detected. The expression of Toll-like receptors (TLRs) and levels of NF-κB p65 phosphorylation (NF-κB p65(p)) in the lungs were analysed. The effects of HCF on viral replication and TLR signalling were further evaluated in cells. ResultsHCF contained 78.5% flavonoid glycosides. The contents of rutin, hyperin, isoquercitrin, and quercitrin in HCF were 8.8%, 26.7%, 9.9% and 31.7%. HCF (50, 100 and 200 mg/kg) increased the survival rate and life span of mice infected with the lethal H1N1 virus. In H1N1-induced ALI, mice treated with HCF (50, 100 and 200 mg/kg) showed lesser weight loss and lower lung index than the model group. The lungs of HCF-treated ALI mice presented more intact lung microstructural morphology, milder inflammatory infiltration, and lower levels of monocyte chemotactic protein 1 (MCP-1), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α) and malondialdehyde (MDA) than in the model group. Further investigation revealed that HCF exerted antiviral and TLR-inhibitory effects in vivo and in vitro. HCF (50, 100 and 200 mg/kg) reduced lung H1N1 virus titers and inhibited viral NA activity in mice. HCF (100 and 200 mg/kg) elevated the levels of interferon-β in lungs. HCF also decreased the expression of TLR3/4/7 and level of NF-κB p65(p) in lung tissues. In vitro experiments showed that HCF (50, 100 and 200 μg/ml) significantly inhibited viral proliferation and suppressed NA activity. In RAW 264.7 cells, TLR3, TLR4, and TLR7 agonist-stimulated cytokine secretion, NF-κB p65 phosphorylation, and nuclear translocation were constrained by HCF treatment. Furthermore, among the four major flavonoid glycosides in HCF, hyperin and quercitrin inhibited both viral replication and TLR signalling in cells. ConclusionHCF significantly alleviated H1N1-induced ALI in mice, which were associated with its dual antiviral and anti-inflammatory effects via inhibiting influenzal NA activity and TLR signalling. among the four major flavonoid glycosides in HCF, hyperin and quercitrin played key roles in the therapeutic effect of HCF.

Analysis of antibody response to an epitope in the haemagglutinin subunit 2 of avian influenza virus H5N1 for differentiation of infected and vaccinated chickens

ABSTRACT The H5N1 subtype of highly pathogenic avian influenza virus has been circulating in poultry in Indonesia since 2003 and vaccination has been used as a strategy to eradicate the disease. However, monitoring of vaccinated poultry flocks for H5N1 infection by serological means has been difficult, as vaccine antibodies are not readily distinguishable from those induced by field viruses. Therefore, a test that differentiates infected and vaccinated animals (DIVA) would be essential. Currently, no simple and specific DIVA test is available for screening of a large number of vaccinated chickens. Several epitopes on E29 domain of the haemagglutinin H5N1 subunit 2 (HA2) have recently been examined for their antigenicity and potential as possible markers for DIVA in chicken. In this study, the potential of E29 as an antigen for DIVA was evaluated in detail. Three different forms of full-length E29 peptide, a truncated E29 peptide (E15), and a recombinant E29 were compared for their ability to detect anti-E29 antibodies. Preliminary ELISA experiments using mono-specific chicken and rabbit E29 sera, and a mouse monoclonal antibody revealed that the linear E29 peptide was the most antigenic. Further examination of the E29 antigenicity in ELISA, using several sera from experimentally infected or vaccinated chickens, revealed that the full-length E29 peptide had the greatest discrimination power between infected and vaccinated chicken sera while providing the least non-specific reaction. This study demonstrates the usefulness of the HPAI H5N1 HA2 E29 epitope as a DIVA antigen in HPAI H5N1-vaccinated and -infected chickens. RESEARCH HIGHLIGHTS E29 (HA2 positions 488-516) epitope is antigenic in chickens. Antibodies to E29 are elicited following live H5N1 virus infection in chickens. E29 epitope is a potential DIVA antigen for use in ELISA.