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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., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In search of novel therapeutic options to treat influenza virus (IV) infections, we previously identified a series of inhibitors that act by disrupting the interactions between the PA and PB1 subunits of the viral RNA polymerase. These compounds showed broad-spectrum antiviral activity against human influenza A and B viruses and a high barrier to the induction of drug resistance in vitro. In this short communication, we investigated the effects of combinations of the PA-PB1 interaction inhibitor 54 with oseltamivir carboxylate (OSC), zanamivir (ZA), favipiravir (FPV), and baloxavir marboxil (BXM) on the inhibition of influenza A and B virus replication in vitro. We observed a synergistic effect of the 54/OSC and 54/ZA combinations and an antagonistic effect when 54 was combined with either FPV or BXM. Moreover, we demonstrated the efficacy of 54 against highly pathogenic avian influenza viruses (HPAIVs) both in cell culture and in the embryonated chicken eggs model. Finally, we observed that 54 enhances OSC protective effect against HPAIV replication in the embryonated eggs model. Our findings represent an advance in the development of alternative therapeutic strategies against both human and avian IV infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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-β (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., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

High pathogenicity avian influenza is an acute zoonotic disease with high mortality in birds caused by a high pathogenicity avian influenza virus (HPAIV). Recently, HPAIV has rapidly spread worldwide and has killed many wild birds, including endangered species. Baloxavir marboxil (BXM), an anti-influenza agent used for humans, was reported to reduce mortality and virus secretion from HPAIV-infected chickens ( Gallus domesticus , order Galliformes) at a dosage of ≥2.5 mg/kg when administered simultaneously with viral challenge. Application of this treatment to endangered birds requires further information on potential avian-specific toxicity caused by repeated exposure to BXM over the long term. To obtain information of potential avian-specific toxicity, a 4-wk oral repeated-dose study of BXM was conducted in chickens ( n = 6 or 7 per group), which are commonly used as laboratory avian species. The study was conducted in reference to the human pharmaceutical guidelines for nonclinical repeated-dose drug toxicity studies to evaluate systemic toxicity and exposure. No adverse changes were observed in any organs examined, and dose proportional increases in systemic exposure to active pharmaceutical ingredients were noted from 12.5 to 62.5 mg/kg per day. BXM showed no toxicity to chickens at doses of up to 62.5 mg/kg per day, at which systemic exposure was approximately 71 times higher than systemic exposure at 2.5 mg/kg, the reported efficacious dosage amount, in HPAIV-infected chickens. These results also suggest that BXM could be considered safe for treating HPAIV-infected endangered birds due to its high safety margin compared with the efficacy dose. The data in this study could contribute to the preservation of endangered birds by using BXM as a means of protecting biodiversity.

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-β, 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.

Background: Effect of antibacterials on mucociliary system and clinical outcome of chickens with mixed viral respiratory conditions is not properly addressed., Objective: We evaluated enrofloxacin effects on clinical parameters and mucociliary system of broilers challenged with H9N2/IB viruses., Methods: Broilers (105), at the age of 25 days, were randomly allocated into three groups: Group 1 (negative control), no treatment; Group 2 (positive control [PC]) challenged by intranasal and intraocular route. Group 3 (antibiotic [AB]-treated) challenged and also received enrofloxacin started after manifestation of clinical signs (day 2 post-challenge [pc]) and continued for 5 days., Results: Administration of AB was not associated with appreciable changes in body weight, feed conversion ratio (FCR) or the severity of clinical signs although it slightly reduced mortality rate as compared to PC group (p > 0.05). Virus shedding period and number of virus positive tracheal and caecal tonsil samples were also statistically similar between PC and AB groups. In necropsy, the most profound effect of AB was decreased pleuropneumonia severity score on day 12 pc. Histopathological lesion scores were statistically the same between PC and AB groups. However, the administration of AB increased the number of tracheal goblet cells, with no effect on ciliostasis., Conclusions: We found a weak positive effect of enrofloxacin administration in H9N2/IB-infected chickens. Considering the risks of AB treatment in broiler chickens, the results of this small-scale study do not encourage the benefit of enrofloxacin use in these viral diseases., (© 2024 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Avian influenza (AI) viruses cause infection in birds and humans. Several H5N1 and H7N9 variants are highly pathogenic avian influenza (HPAI) viruses. H5N1 is a highly infectious bird virus infecting primarily poultry, but unlike other AIs, H5N1 also infects mammals and transmits to humans with a case fatality rate above 40%. Similarly, H7N9 can infect humans, with a case fatality rate of over 40%. Since 1996, there have been several HPAI outbreaks affecting humans, emphasizing the need for safe and effective antivirals. We show that probenecid potently inhibits H5N1 and H7N9 replication in prophylactically or therapeutically treated A549 cells and normal human broncho-epithelial (NHBE) cells, and H5N1 replication in VeroE6 cells and mice.

Background: Recent outbreaks of avian influenza and ongoing virus reassortment have drawn focus on spill-over infections. The increase in human infections with highly pathogenic avian influenza H5N6 virus and its high fatality rate posed a potential threat, necessitating the search for a more effective treatment., Methods: Longitudinal clinical data and specimens were collected from five H5N6 patients after admission. All patients received antiviral treatment of either sequential monotherapy of oseltamivir and baloxavir or the two drugs in combination. Severity of illness; viral load in sputum, urine, and blood; and cytokine levels in serum and sputum were serially analyzed., Findings: All patients developed acute respiratory distress syndrome (ARDS) and viral sepsis within 1 week after disease onset. When delayed oseltamivir showed poor effects, baloxavir was administered and rapidly decreased viral load. In addition, levels of IL-18, M-CSF, IL-6, and HGF in sputum and Mig and IL-18 in serum that reflected ARDS and sepsis deterioration, respectively, were also reduced with baloxavir usage. However, three patients eventually died from exacerbation of underlying disease and secondary bacterial infection. Nonsurvivors had more severe extrapulmonary organ dysfunction and insufficient H5N6 virus-specific antibody response., Conclusions: For critical human cases of H5N6 infection, baloxavir demonstrated effects on viral load and pulmonary/extrapulmonary cytokines, even though treatment was delayed. Baloxavir could be regarded as a first-line treatment to limit continued viral propagation, with potential future application in avian influenza human infections and poultry workers exhibiting influenza-like illness., Funding: This work was funded by the National Natural Science Foundation of China (81761128014)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The antiviral activity of chlorine dioxide (ClO 2 ) in liquid (ClO 2 gas dissolved liquid) and gaseous state against avian influenza virus (AIV) and infectious bronchitis virus (IBV) was evaluated. To evaluate the effect of ClO 2 in liquid state, suspension tests (10 ppm) and carrier tests in dropping / wiping techniques (100 ppm) were performed. In the suspension test, virus titers were reduced below the detection limit within 15 sec after treatment, in spite of the presence of an accompanying organic matter. In the carrier test by dropping technique, AIV and IBV were reduced to below the detection limit in 1 and 3 min, respectively. Following wiping technique, no virus was detected in the wiping sheets after 30 sec of reaction. Both viruses adhering to the carriers were also reduced by 3 logs, thereby indicating that they were effectively inactivated. In addition, the effect of ClO 2 gas against IBV in aerosols was evaluated. After the exposure of sprayed IBV to ClO 2 gas for a few seconds, 94.2% reduction of the virus titer was observed, as compared to the pre-treatment control. Altogether, hence, ClO 2 has an evident potential to be an effective disinfectant for the prevention and control of AIV and IBV infections on poultry farms.

Coriolus versicolor ( C. versicolor ) is a higher fungi or mushroom which is now known by its accepted scientific names as Trametes versicolor (L.) Lloyd. Many studies have shown that β-glucans from C. versicolor have various physiological activities, including activating macrophages to protect against Salmonella infection. However, whether β-glucans have antiviral effects has not been reported. Hence, the objective of this study was to confirm whether β-glucans could boost the immune response to combat influenza virus in mouse and chick models. The results show that β-glucans induced the expression of Dectin-1 , costimulatory molecules ( CD80 / 86 ) and cytokines IL-6 , IL-1β , IFN-β and IL-10 in murine bone marrow dendritic cells (BMDCs). In addition, orally administered β-glucans reduced weight loss, mortality and viral titers in the lungs of mice infected with influenza virus and attenuated pathological lung damage caused by the virus in the mice. Orally administered β-glucans improved survival and reduced lung viral titers in chickens infected with H9N2 avian influenza virus. These results suggest that β-glucans have a significant antiviral effect. Therefore, β-glucans could become a potential immunomodulator against influenza virus.

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-β), 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., (© 2021. The Author(s).)

That the high frequency and good replication capacity of strains with reduced susceptibility to neuraminidase inhibitors (NAIs) in highly pathogenic avian influenza H7N9 (HPAI H7N9) virus made it a significance to further study its drug resistance. HPAI H7N9 viruses bearing NA I222L or E119V substitution and two mutations of I222L-E119V as well as their NAIs-sensitive counterpart were generated by reverse genetics for NA inhibition test and replication capability evaluation in vitro. The attenuated H7N9/PR8 recombinant viruses were developed to study the pathogenicity and drug resistance brought by the above substitutions to mice. The IC 50 fold change of oseltamivir to HPAI H7N9 with NA222L-119V is 306.34 times than that of its susceptible strain, and 3.5 times than the E119V mutant virus. HPAI H7N9 bearing NA222L-119V had good replication ability with peak value of more than 6log 10 TCID 50 /ml in MDCK cells. H7N9/PR8 virus bearing NA222L-119V substitutions leaded to diffuse pneumonia, significant weight loss and fatality in mice. NA E119V made H7N9/PR8 virus resistant to oseltamivir, and I222L-E119V had synergistic resistance to oseltamivir in mice. Due to the good fitness of drug resistant strains of HPAI H7N9 virus, it is necessary to strengthen drug resistance surveillance and new drug research., (© 2021. The Author(s).)

Virus-like particles (VLPs) are recognized as an alternative vaccine platform that provide effective protection against various highly pathogenic avian influenza viruses (HPAIVs). Here, we developed multi-clade VLPs expressing two HAs (a chimera of clade 2.3.2.1c and clade 2.3.4.4c HA) within a single vector. We then compared its protective efficacy with that of a monovalent VLP and evaluated its potency against each homologous strain. Chickens vaccinated with the multi-clade VLP shed less virus and were better protected against challenge than birds receiving monovalent vaccines. Single vaccination with a multi-clade VLP resulted in 100% survival, with no clinical symptoms and high levels of pre-challenge protective immunity (7.6-8.5 log 2 ). Moreover, the multi-clade VLP showed high productivity (128-256 HAU) both in the laboratory and on a large scale, making it cheaper than whole inactivated vaccines produced in eggs. However, the PD 50 (protective dose 50%) of the multi-clade VLP against clades 2.3.2.1c and 2.3.4.4c was < 50 PD 50 (28 and 42 PD 50 , respectively), and effective antibody response was maintained for 2-3 months. This multi-clade VLP protects against both clades of HPAI viruses and can be produced in high amounts at low cost. Thus, the vaccine has potential as a pandemic preparedness vaccine.

This study evaluated the virucidal efficacy of acidic electrolyzed water (AEW) against African swine fever virus (ASFV) and avian influenza virus (AIV), according to the Animal and Plant Quarantine Agency (APQA) guidelines for efficacy testing of veterinary disinfectants. AEW (pH 5.0-6.5) was prepared using a commercially available "Electrolyzed Water Generator" with a free chlorine concentration (FCC) of 5-140 ppm, and its efficiency in reducing the titer of ASFV and AIV was tested in a suspension under low- and high-level organic soiling. Under low-level organic soiling conditions, AEW with FCC ≥40 ppm was effective against ASFV; under high-level organic soiling conditions, AEW with FCC ≥80 ppm was effective against ASFV. Under low-level organic soiling conditions, AEW with FCC ≥60 ppm was effective against AIV; under high-level organic soiling conditions, AEW with FCC ≥100 ppm was effective against AIV. The virucidal effect of AEW seemed dependent on the FCC and the presence of organic soiling. Based on these data, we recommend the following minimum FCCs in AEW treatment for routine disinfection in veterinary field under low- and high-level organic soiling conditions: for ASFV, 50 ppm and 100 ppm; and for AIV, 75 ppm and 125 ppm, respectively. In conclusion, the virucidal effects of AEW against ASFV and AIV emphasize its potential utility as a disinfectant, and we suggest considering organic soiling conditions while using AEW for implementing effective control measures for field applications.

Control measures in the case of high pathogenicity avian influenza (HPAI) outbreaks in poultry include culling, surveillance, and biosecurity; wild birds in captivity may also be culled, although some rare bird species should be rescued for conservation. In this study, two anti-influenza drugs, baloxavir marboxil (BXM) and peramivir (PR), used in humans, were examined in treating HPAI in birds, using chickens as a model. Chickens were infected with H5N6 HPAI virus and were treated immediately or 24 h from challenge with 20 mg/kg BXM or PR twice a day for five days. As per our findings, BXM significantly reduced virus replication in organs and provided full protection to chickens compared with that induced by PR. In the 24-h-delayed treatment, neither drug completely inhibited virus replication nor ensured the survival of infected chickens. A single administration of 2.5 mg/kg of BXM was determined as the minimum dose required to fully protect chickens from HPAI virus; the concentration of baloxavir acid, the active form of BXM, in chicken blood at this dose was sufficient for a 48 h antiviral effect post-administration. Thus, these data can be a starting point for the use of BXM and PR in treating captive wild birds infected with HPAI virus.

This study was conducted to assess the comparative effects of a mixed herbal extract (MHE) containing Ocimum sanctum, Withania somnifera, Emblica officinalis, Tinospora cordifolia, Mangifera indica, and Asphaltum (shilajit) on infectious bursal disease virus (IBDV)-vaccinated (VAC) chickens infected with IBDV and avian influenza virus (AIV) H9N2. The experiment included three groups (G1-G3): G1, the negative control group; G2, the VAC + challenged (Ch) group; and G3, the VAC + Ch + MHE group. MHE was orally administered continuously for 5 weeks post-vaccination (PV) with IBDV at 12 days of age, and the chicks were simultaneously challenged with virulent IBDV (intraocularly) and AIV H9N2 (intranasally) at 21 days PV. Blood and tissue samples as well as tracheal and cloacal swabs were gathered at different times PV and post-challenge. Immunological and haematological parameters, histopathological lesions, relative organ weights and final live weights revealed significant differences (P ≤ 0.05) between G2 and G3 groups. Furthermore, in the G3 group, the protection rates, ELISA and HI titers and CD4+/CD8+ ratio were significantly increased, whereas viral shedding titers and the heterophil/lymphocyte ratio were decreased. In conclusion, the oral administration of the mixed herbal extract for 5 weeks can stimulate the immune response to IBDV vaccination and relieves the pathogenicity of an AIV H9N2 and IBDV co-infection in chickens., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Highly pathogenic emerging and re-emerging viruses continuously threaten lives worldwide. In order to provide prophylactic prevention from the emerging and re-emerging viruses, vaccine is suggested as the most efficient way to prevent individuals from the threat of viral infection. Nonetheless, the highly pathogenic viruses need to be handled in a high level of biosafety containment, which hinders vaccine development. To shorten the timeframe of vaccine development, the pseudovirus system has been widely applied to examine vaccine efficacy or immunogenicity in the emerging and re-emerging viruses., Methods: We developed pseudovirus systems for emerging SARS coronavirus 2 (SARS-CoV-2) and re-emerging avian influenza virus H5 subtypes which can be handled in the biosafety level 2 facility. Through the generated pseudovirus of SARS-CoV-2 and avian influenza virus H5 subtypes, we successfully established a neutralization assay to quantify the neutralizing activity of antisera against the viruses., Results: The result of re-emerging avian influenza virus H5Nx pseudoviruses provided valuable information for antigenic evolution and immunogenicity analysis in vaccine candidate selection. Together, our study assessed the potency of pseudovirus systems in vaccine efficacy, antigenic analysis, and immunogenicity in the vaccine development of emerging and re-emerging viruses., Conclusion: Instead of handling live highly pathogenic viruses in a high biosafety level facility, using pseudovirus systems would speed up the process of vaccine development to provide community protection against emerging and re-emerging viral diseases with high pathogenicity., Competing Interests: Conflicts of Interest The authors have declared that there is no conflict of interest., (Copyright © 2020 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Neuraminidase inhibitors (NAIs) are the gold standard treatment for influenza A virus (IAV). Oseltamivir is mostly used, followed by zanamivir (ZA). NAIs are not readily degraded in conventional wastewater treatment plants and can be detected in aquatic environments. Waterfowl are natural IAV hosts and replicating IAVs could thus be exposed to NAIs in the environment and develop resistance. Avian IAVs form the genetic basis for new human IAVs, and a resistant IAV with pandemic potential poses a serious public health threat, as NAIs constitute a pandemic preparedness cornerstone. Resistance development in waterfowl IAVs exposed to NAIs in the water environment has previously been investigated in an in vivo mallard model and resistance development was demonstrated in several avian IAVs after the exposure of infected ducks to oseltamivir, and in an H1N1 IAV after exposure to ZA. The N1 and N2 types of IAVs have different characteristics and resistance mutations, and so the present study investigated the exposure of an N2-type IAV (H4N2) in infected mallards to 1, 10 and 100 µg l -1 of ZA in the water environment. Two neuraminidase substitutions emerged, H274N (ZA IC 50 increased 5.5-fold) and E119G (ZA IC 50 increased 110-fold) at 10 and 100 µg l -1 of ZA, respectively. Reversion towards wild-type was observed for both substitutions in experiments with removed drug pressure, indicating reduced fitness of both resistant viruses. These results corroborate previous findings that the development of resistance to ZA in the environment seems less likely to occur than the development of resistance to oseltamivir, adding information that is useful in planning for prudent drug use and pandemic preparedness.

Avian influenza (AI) has severely affected the poultry industry worldwide and has caused the deaths of millions of birds. Highly pathogenic avian influenza virus is characterized by high mortality and the ability to transmit from birds to humans. Early diagnosis is difficult because of the variation in pathogenicity and the genetic diversity between virus subtypes. Therefore, development of a sensitive and accurate diagnostic system is an urgent priority. We developed ssDNA aptamer probes to detect AI viruses. Through seven rounds of SELEX to search for a probe specific to the highly pathogenic AI virus subtype H5N1, we identified 16 binding aptamers and selected two with the highest binding frequency. These two aptamers had strong binding affinities and low detection limits. We found that they could bind more specifically to H5N1, as compared to other subtypes. Furthermore, these aptamers inhibited hemagglutination, which is caused by the virus surface protein hemagglutinin. Our results indicate that our screened aptamers are effective molecular probes for diagnosing H5N1 and can be used as therapeutic agents to inhibit viral surface proteins. Sensitive diagnosis and suppression of avian influenza will help maintain a stable and healthy livestock industry, as well as protect human health.

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., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Some of the respiratory viral infections in chickens pose a significant threat to the poultry industry and public health. In response to viral infections, host innate responses provide the first line of defense against viruses, which often act even before the establishment of the infection. Host cells sense the presence of viral components through germinal encoded pattern recognition receptors (PRRs). The engagement of PRRs with pathogen-associated molecular patterns leads to the induction of pro-inflammatory and interferon productions. Induced antiviral responses play a critical role in the outcome of the infections. In order to improve current strategies for control of viral infections or to advance new strategies aimed against viral infections, a deep understanding of host-virus interaction and induction of antiviral responses is required. In this review, we summarized recent progress in understanding innate antiviral responses in chickens with a focus on the avian influenza virus and infectious bronchitis virus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The 3D8 single-chain variable fragment (scFv) is a mini-antibody sequence with independent nuclease activity that shows antiviral effects against all types of viruses in chickens and mice. In this study, chickens were treated daily with an oral dose of 10 9 CFU Lactobacillus paracasei (L. paracasei) expressing either a secreted or anchored 3D8 scFv for three weeks. After L. paracasei administration, the chickens were challenged with avian influenza virus (AIV). From each experimental group, three chickens were directly infected with 100 µL of 10 7.5 EID 50 /mL H9N2 AIV and seven chickens were indirectly challenged through contact transmission. oropharyngeal and cloacal swab samples were collected at 3, 5, 7, and 9 days post-inoculation (dpi) from AIV-challenged chickens, AIV Shedding titres were measured by quantitative real-time PCR. Contact transmission in the chickens that were fed 3D8 scFv-secreting L. paracasei showed a significant reduction in viral shedding when compared with other groups. These results suggest that L. paracasei secreting 3D8 provides a basis for the development of ingestible antiviral probiotics with activity against AIV.

Avian influenza or bird flu is a common problem of domestic and wild birds. Some of its strains are able to cross the species barrier and cause infection in various members of class Mammalia. In view of relatively lesser efficacy of vaccines, antiviral therapies remain the only choice for the sustenance of mammals acquiring this highly devastating infection. This study is based on the evaluation of antiviral potential of methanol extracts of eleven selected Cholistani plants. The methanol extracts were prepared by using dried plants material followed by concentrating in a rotary evaporator and finally air dried before dissolving in nanopure water. The suspension was filter sterilized and subjected to in ovo antiviral assays. The allantoic fluids were harvested and haemagglutinin (HA) titers were determined. Among the eleven plants evaluated all methanol extracts were found effective against AIV H9N2 except S. baryosma extract. The medicinal plants O. compressa, N. procumbens, and S. surattense were found to be more effective than others and they retained HA titers at 0 after challenge. The next in order were extracts of O. esculentum, H. salicornicum and S. fruticosa which kept HA titers at 4, 8 and 16 respectively. The extracts of H. recurvum, P. antidotale, S. icolados and A. aspera were found less effective than above mentioned plant extracts and they kept the HA titers at 32, 64, 128 and 256 respectively. These results led us to conclude that the medicinal plants of Cholistan region are a rich source of antiviral agent(s) against AIV H9N2 and could be a source of cost effective alternate therapeutics.

Influenza A virus (IAV) is one of the most common infectious pathogen and associated with significant morbidity and mortality. Although processing the IAV hemagglutinin (HA) envelope glycoprotein precursor is a pre-requisite for viral membrane fusion activity, viral entry and transmission, HA-processing protease is not encoded in the IAV genome and thus the cellular trypsin-type serine HA-processing proteases determine viral infectious tropism and viral pathogenicity. The initial process of IAV infection of the airway is followed by marked upregulation of ectopic trypsin in various organs and endothelial cells through the induction of various proinflammatory cytokines, and this process has been termed the "influenza virus-cytokine-trypsin" cycle. In the advanced stage of IAV infection, the cytokine storm induces disorders of glucose and lipid metabolism and the "metabolic disorders-cytokine" cycle is then linked with the "influenza virus-cytokine-trypsin" cycle, to advance the pathogenic process into energy crisis and multiple organ failure. Application of protease inhibitors and treatment of metabolic disorders that break these cycles and their interconnection is therefore a promising therapeutic approach against influenza. This review discusses IAV pathogenicity on trypsin type serine HA-processing proteases, cytokines, metabolites and therapeutic options., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background & Objectives: The susceptibility of influenza viruses to neuraminidase inhibitors (NAIs) is studied using enzyme-based assays, sequence analysis and in vitro and in vivo studies. Oseltamivir carboxylate (OC) is the active prodrug of the NAI oseltamivir. There is lack of information on the use of embryonated chicken eggs for studying susceptibility of highly pathogenic avian influenza (HPAI) H5N1 viruses to antiviral drugs. The aim of the present study was to assess the use of 10 day old embryonated chicken eggs for studying antiviral susceptibility of HPAI H5N1 viruses., Methods: Two HPAI H5N1 viruses isolated from India were used in the study. Fluorescence-based NAI assay was performed to determine antiviral susceptibility of these viruses. In ovo antiviral assays were carried out using 10 day old embryonated chicken eggs. The virus dilutions were incubated with 14 μg/ml of OC and inoculated in the allantoic cavity. In the eggs, 50 per cent egg infectious dose (EID 50 ) titres as well as mortality were quantitated., Results: The two viruses used were susceptible to OC in the NAI assay. It was found that there was a significant drop in EID 50 titres; however, no significant protection from mortality after OC treatment was observed., Interpretation & Conclusions: By measuring viral titres, the egg model was suitable to study the susceptibility of HPAI viruses to antiviral drugs along with NAI assay. The present study highlights the use of eggs as a model to study susceptibility of HPAI viruses to OC., Competing Interests: None

Baloxavir showed broad-spectrum in vitro replication inhibition of 4 types of influenza viruses (90% effective concentration range 1.2-98.3 nmol/L); susceptibility pattern was influenza A ˃ B ˃ C ˃ D. This drug also inhibited influenza A viruses of avian and swine origin, including viruses that have pandemic potential and those resistant to neuraminidase inhibitors.

Human infections with avian-origin influenza A(H7N9) virus represent a serious threat to global health; however, treatment options are limited. Here, we show the inhibitory effects of baloxavir acid (BXA) and its prodrug baloxavir marboxil (BXM), a first-in-class cap-dependent endonuclease inhibitor, against A(H7N9), in vitro and in vivo. In cell culture, BXA at four nanomolar concentration achieved a 1.5-2.8 log reduction in virus titers of A(H7N9), including the NA-R292K mutant virus and highly pathogenic avian influenza viruses, whereas NA inhibitors or favipiravir required approximately 20-fold or higher concentrations to achieve the same levels of reduction. A(H7N9)-specific amino acid polymorphism at position 37, implicated in BXA binding to the PA endonuclease domain, did not impact on BXA susceptibility. In mice, oral administration of BXM at 5 and 50 mg/kg twice a day for 5 days completely protected from a lethal A/Anhui/1/2013 (H7N9) challenge, and reduced virus titers more than 2-3 log in the lungs. Furthermore, the potent therapeutic effects of BXM in mice were still observed when a higher virus dose was administered or treatment was delayed up to 48 hours post infection. These findings support further investigation of BXM for A(H7N9) treatment in humans.

In this paper, we report a soluble expression based on Escherichia coli and two-step purification of a novel thioredoxin-tagged chicken interferon-α fusion protein (Trx-rChIFN-α) by using pET32a(+) expression system. The mature ChIFN-α gene was amplified by Reverse transcriptase-polymerase chain reaction (RT-PCR) and subcloned into pET-32a (+) vector prior to transformation into Rosetta (DE3) competent cells. After IPTG induction, the recombinant fusion protein was expressed efficiently in the soluble fraction. The protein purification was performed by nickel affinity chromatography and DEAE anion exchange chromatography. The purified product has a purity of 95% with a yield of 47.3 mg/L of culture. The specific activity of the fusion protein reaches to 2.0 × 10 7  IU/mg as determined in the CEF/VSV titration system. After excision of the Trx tag by enterokinase, the remaining solo protein was confirmed as rChIFN-α protein by SDS-PAGE, N-terminal sequencing and mass spectrometry. The effects of this Trx-rChIFN-α fusion protein against H9N2 influenza virus infection were also evaluated in ovo. The results showed that the Trx-rChIFN-α protein could significantly reduce the hemagglutination titer of H9N2 virus, and the H9N2 viruses HA gene copy numbers. These findings will enable us to produce large amount and bio-active rChIFN-α protein for future applications.

Introduction: The emergence of influenza virus strains with drug resistance to antiviral drugs requires finding new compounds, potential direct-acting inhibitors. Аdamantane compounds drugs used since the 1960s have lost their activity the resulting due to resistance. Only neuraminidase inhibitors such as zanamivir and oseltamivir have been approved by WHO for influenza treatment. The Russian pharmaceutical drug Arbidol (Umifenovirum) is actively used in Russia. This drug is used to treat influenza in Russia, China and most post-Soviet republics. This work presents a new derivative of aminoadamantane - dichlorohydrate L-histidyl-1-adamantayl ethylamine (2HCl*H-His-Rim), which showed a high level of inhibition of strains of influenza virus A in vitro., Objectives: Comparison of antiviral properties of the new synthetic low-molecular inhibitor of influenza A virus replication and Arbidol drug pharmacy., Methods: The compound 2HCl*H-His-Rim was obtained by classical peptide synthesis methods. It was identified by methods of mass spectrometry, infrared spectroscopy (IR) and nuclear magnetic resonance spectroscopy (NMR). Its antiviral properties have been studied in vitro for monolayer of cells Vero-E6 infected with a high-virulent strain of A/duck/Novosibirsk/56/06 (H5N1) influenza virus at various injection schemes of the investigated compounds., The Results: The antiviral activity of the 2HCl*H-His-Rim compound against the highly pathogenic strain of the influenza A/H5N1 virus was slightly higher than for the known pharmacy drug arbidol., Discussion: The difference in antiviral activity of these two compounds is explained by different mechanisms of action on the viral particle., Conclusion: The 2HCl*H-His-Rim compound can be recommended as a candidate for preclinical and clinical trials in order to obtain an etiotropic antiviral drug based on it, due to its high efficacy and economic and synthetic availability. The synthetic compound 2HCl*H-His-Rim acts on influenza A virus variants resistant to Rimantadine and Amantadine., Competing Interests: The authors declare no conflict of interest.

A sensitive competitive immunoassay with simple operation was developed for the detection of the anti-virus drug amantadine (AMD). The single-chain variable fragment (scFv) antibody against AMD was site-specific biotinylated and overexpressed as a secreted body in Escherichia coli AVB101. Horseradish peroxidase-labeled streptavidin-biotinylated scFv antibody (HRP-SA-BIO-scFv) could specifically bind to AMD-functionalized magnetic beads (MBs) and then the immune complexes were separated from the matrix solution by magnet. The concentration of the AMD could be known by the measurement of the signal produced by the horseradish peroxidase. The newly established assay provides a significant improvement in comparison to the conventional ELISA without SA-BIO signal amplification and MBs separation. The limit of detection and assay time was 0.64 vs. 8.4 ng/mL and 50 vs. 150 min, respectively. The recoveries ranged from 77.8 to 112% with the coefficient of variation less than 13%. The immunoassay exhibited an obvious cross-reactivity to rimantadine (84%), 1-(1-adamantyl)ethylamine (72%), and somantadine (63%). These results demonstrated that the developed immunoassay provided a sensitive, rapid, and accurate approach for the detection of AMD in chicken by employing MBs as solid phase and SA-BIO as signal amplification. When applied in natural chicken samples, the newly established method provided results consistent with those from UPLC-MS/MS, suggesting that the proposed method could be used for rapid screening of the target of interest; the new immunoassay could also be extended to other small molecular contaminants and thus represents a universal strategy for food safety analysis. Graphical abstract ᅟ.

1. The effect of Zataria multiflora essential oil on replication rate of the H9N2 virus in target organs was determined by real-time PCR. One-day-old broiler chicks were randomly divided into six groups and were challenged with H9N2 influenza. Two groups received either 20 or 40 µl/kg body weight/day Zataria multiflora essential oils (ZM) seven days before the challenge while two other groups received the essential oil at the same dosage but after H9N2 challenge. One group received 4 mg/kg body weight/day of the anti-viral compound amantadine after challenge and the last group received no treatment and served as the control. 2. Groups that received the ZM, before or after H9N2 challenge, and the amantadine treated group showed reduced viral replication in the respiratory and gastrointestinal tracts compared to the control. Supplementation with ZM improved weight gain and FCR in broilers in comparison with the control. 3. The results showed that ZM had a positive effect on reducing viral replication in both the intestine and trachea of H9N2 influenza infected broiler chickens, that led to milder clinical symptoms and better performance.

We evaluated the anti-influenza-virus effects of Melia components and discuss the utility of these components. The effects of leaf components of Melia azedarach L. on viruses were examined, and plaque inhibition tests were performed. The in vivo efficacy of M. azedarach L. was tested in a mouse model. Leaf components of Melia azedarach L. markedly inhibited the growth of various influenza viruses. In an initial screening, multiplication and haemagglutination (HA) activities of H1N1, H3N2, H5, and B influenza viruses were inactivated by the liquid extract of leaves of M. azedarach L. (MLE). Furthermore, plaque inhibition titres of H1N1, H3N2, and B influenza viruses treated with MLE ranged from 10 3.7 to 10 4.2 . MLE possessed high plaque-inhibitory activity against pandemic avian H5N1, H7N9, and H9N2 vaccine candidate strains, with a plaque inhibition titre of more than 10 4.2 . Notably, the buoyant density decreased from 1.175 to 1.137 g/cm 3 , and spikeless particles appeared. We identified four anti-influenza virus substances: pheophorbide b, pheophorbide a, pyropheophorbide a, and pheophytin a. Photomorphogenesis inside the envelope may lead to removal of HA and neuraminidase spikes from viruses. Thus, MLE could efficiently remove floating influenza virus in the air space without toxicity. Consistent with this finding, intranasal administration of MLE in mice significantly decreased the occurrence of pneumonia. Additionally, leaf powder of Melia (MLP) inactivated influenza viruses and viruses in the intestines of chickens. MLE and MLP may have applications as novel, safe biological disinfectants for use in humans and poultry.

Toll-like receptor (TLR)3 signaling pathway is known to induce type 1 interferons (IFNs) and proinflammatory mediators leading to antiviral response against many viral infections. Double-stranded ribonucleic acid (dsRNA) has been shown to act as a ligand for TLR3 and, as such, has been a focus as a potential antiviral agent in many host-viral infection models. Yet, its effectiveness and involved mechanisms as a mediator against low pathogenic avian influenza virus (LPAIV) have not been investigated adequately. In this study, we used avian fibroblasts to verify whether dsRNA induces antiviral response against H 4 N 6 LPAIV and clarify whether type 1 IFNs and proinflammatory mediators such as interleukin (IL)-1β are contributing to the dsRNA-mediated antiviral response against H 4 N 6 LPAIV. We found that dsRNA induces antiviral response in avian fibroblasts against H 4 N 6 LPAIV infection. The treatment of avian fibroblasts with dsRNA increases the expressions of TLR3, IFN-α, IFN-β, and IL-1β. We also confirmed that this antiviral response elicited against H 4 N 6 LPAIV infection correlates, but is not attributable to type 1 IFNs or IL-1β. Our findings imply that the TLR3 ligand, dsRNA, can elicit antiviral response in avian fibroblasts against LPAIV infection, highlighting potential value of dsRNA as an antiviral agent against LPAIV infections. However, further investigations are required to determine the potential role of other innate immune mediators or combination of the tested cytokines in the dsRNA-mediated antiviral response against H 4 N 6 LPAIV infection.

The antiviral cytokine interferon-alpha (IFN-α) plays a critical role in the innate immune system. Previous studies have shown that recombinant chicken IFN-α inhibits avian influenza virus (AIV) replication in vivo; however, the antiviral effect of recombinant duck IFN-α (rDuIFN-α) on highly pathogenic AIV remains unknown. In this study, the duck IFN-α gene was cloned, expressed, and purified. The antiviral effects of the resulting rDuIFN-α were further evaluated in vitro and in vivo. Our results showed that rDuIFN-α inhibited the replication of vesicular stomatitis virus (VSV) and AIV in duck embryo fibroblasts in vitro, with antiviral activities against VSV and AIV of 2.1 × 10 5 and 4.1 × 10 5 U/mg, respectively. We next investigated the anti-H5N1 AIV effect of intramuscular injection of rDuIFN-α in vivo. rDuIFN-α reduced viral titers in the brains, lungs, and spleens of 2-day-old (2D) ducks compared with that in the virus-challenged control group, and pretreatment with rDuIFN-α reduced mortality from 60% to 10% in 2D ducks. Moreover, rDuIFN-α increased the expression of IFN-stimulated genes in the brains and spleens of 2D ducks. Our results demonstrate that rDuIFN-α blocks VSV and H5N1 influenza virus infection in vitro and exhibits antiviral effects against H5N1 influenza virus infection in 2D ducks.

Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs. IMPORTANCE The frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs., (Copyright © 2017 American Society for Microbiology.)

Human infections with novel influenza A viruses are of global public health concern, and antiviral medications have a potentially important role in treatment and prevention of human illness. Initial guidance was developed by the U.S. Centers for Disease Control and Prevention after the emergence of human infections with avian influenza A(H5N1) and has evolved over time, with identification of influenza A(H7N9) virus infections in humans, as well as detection of avian influenza viruses in birds in the United States. This commentary describes the historical context and current guidance for the use of influenza antiviral medications for treatment and post-exposure chemoprophylaxis of human infections with novel influenza A viruses associated with severe human illness, or with the potential to cause severe human disease, and provides the scientific rationale behind current recommendations., (Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Mutation and reassortment of highly pathogenic avian influenza A(H5N1) viruses at the animal-human interface remain a major concern for emergence of viruses with pandemic potential. To understand the relationship of H5N1 viruses circulating in poultry and those isolated from humans, comprehensive phylogenetic and molecular analyses of viruses collected from both hosts in Vietnam between 2003 and 2010 were performed. We examined the temporal and spatial distribution of human cases relative to H5N1 poultry outbreaks and characterized the genetic lineages and amino acid substitutions in each gene segment identified in humans relative to closely related viruses from avian hosts. Six hemagglutinin clades and 8 genotypes were identified in humans, all of which were initially identified in poultry. Several amino acid mutations throughout the genomes of viruses isolated from humans were identified, indicating the potential for poultry viruses infecting humans to rapidly acquire molecular markers associated with mammalian adaptation and antiviral resistance., (Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Inhibition of influenza A virus infection by multivalent sialic acid inhibitors preventing viral hemagglutinin binding to host cells of the respiratory tract is a promising strategy. However, optimal geometry and optimal ligand presentation on multivalent scaffolds for efficient inhibition both in vitro and in vivo application are still unclear. Here, by comparing linear and dendritic polyglycerol sialosides (LPGSA and dPGSA) we identified architectural requirements and optimal ligand densities for an efficient multivalent inhibitor of influenza virus A/X31/1 (H3N2). Due to its large volume, the LPGSA at optimal ligand density sterically shielded the virus significantly better than the dendritic analog. A statistical mechanics model rationalizes the relevance of ligand density, morphology, and the size of multivalent scaffolds for the potential to inhibit virus-cell binding. Optimized LPGSA inhibited virus infection at IC 50 in the low nanomolar nanoparticle concentration range and also showed potent antiviral activity against two avian influenza strains A/Mallard/439/2004 (H3N2) and A/turkey/Italy/472/1999 (H7N1) post infection. In vivo application of inhibitors clearly confirmed the higher inhibition potential of linear multivalent scaffolds to prevent infection. The optimized LPGSA did not show any acute toxicity, and was much more potent than the neuraminidase inhibitor oseltamivir carboxylate in vivo. Combined application of the LPGSA and oseltamivir carboxylate revealed a synergistic inhibitory effect and successfully prevented influenza virus infection in mice., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Successful immunization against avian influenza virus (AIV) requires eliciting an adequate polyclonal response to AIV hemagglutinin (HA) subunit 1 (HA1) epitopes. Outbreaks of highly-pathogenic (HP) AIV subtype H5N1 can occur in vaccinated flocks in many endemic areas. Protection against emerging AIV is partly hindered by the limitations of vaccine production and transport, the use of leaky vaccines, and the use of multiple, and often antigenically-diverse, vaccines. It was hypothesized that the majority of alternative functional configurations (AFC) within the AIV HA1 can be represented by the pool of vaccine seed viruses currently in production because only a finite number of AFC are possible within each substructure of the molecule. Therefore, combinations of commercial vaccines containing complementing structural units (CSU) to each HA1 substructure can elicit responses to the totality of a given emerging AIV HA1 substructure isoforms. Analysis of homology-based 3D models of vaccine seed and emerging viruses facilitated the definition of HA1 AFC isoforms. CSU-based plots were used to predict which commercial vaccine combinations could have been used to cover nine selected AFC isoforms on recent Egyptian HP AIV H5N1 outbreak viruses. It is projected that expansion of the vaccine HA1 3D model database will improve international emergency responses to AIV.

Background: Understanding the regulatory functions of the biological peptide from the humoral central immune organ bursa of Fabricius on vaccine immune responses and antibody production is of vital importance., Objectives: Here we thoroughly verified the immunomodulatory functions of the new tetrapeptide BP4 from the bursa of Fabricius on vaccine immune responses in mice and chicken immunizaiton model, and on potential intracellular signaling during antibody production., Method: BP4 was isolated and identified by Reverse Phase High Performance Liquid Chromatography and matrix-assisted laser desorption ionization time of flight mass spectrometry. immunomodulatory functions of BP4 was verified by AIV vaccine immunization on mice and chickens regarding roles in vivo, by monitoring the impact of signalling inhibitors in hybridoma cells on antibody production in vitro., Results: Our investigation revealed the strong inducing roles of new isolated BP4 on immune responses in mice immunization, the immunomodulatory effects in the immunized chicken, four potential key intracellular signaling during antibody production in hybrdoma cells., Conclusion: The new bursal-derived peptide BP4 was isolated and identified, and the immunomodulatory effects on antigen-specific immune responses in vivo and in vitro were verified, suggesting BP4 might be highly relevant to the humoral immune responses, and PI3K/Akt, p38 MAPK, NF-κB and tyrosine phosphorylation signaling might be the key activated intracellular signaling during antibody production during BP4 stimulation, which provided a novel potential adjuvant candidate for vaccine immunization improvement and precaution on animal epidemic disease., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The authors retract the above paper due to: 1) conflict of interest among the authors; and 2) addition of coauthor Dr. Muhammad Younus without his knowledge or permission. The authors apologize for these two grave mistakes., (© 2016 Poultry Science Association Inc.)

The present study has two aims: to optimize the antiviral activity of oseltamivir in chicken embryos against an avian influenza-H9N2 strain (P0) and to apply the optimized protocol for studying the drug susceptibility of 4 H9N2 mutants (M1, M2, M3, and M4). As for the first aim, oseltamivir antiviral activity was monitored upon its delivery into 9-day-old chicken embryo at a concentration of 0.27 mg/100 μl, against 7 doses of the P0 strain, ranging between 1.2 x 10(-5) and 2.0 Hemagglutination (HA) units. Oseltamivir showed its highest efficacy in reduction of viral propagation (95% reduction in HA titer) (P 〈0.05), when the inoculum level contained a minimum HA units of 1.2 x 10(-5). For the second aim of this study, the application of the 1.2 x 10-(5) HA units of the virus in inocula for the evaluation of oseltamivir-antiviral effect against the 4 H9N2 mutants revealed an emergence of a resistant mutant (M1), associated with 2 adjacent point mutations in its neuraminidase (N) amino acid (aa) sequence at positions 46 and 47. The other 3 mutants maintained a variable sensitivity to oseltamivir, resulting in the following reduction in HA titers: M2 (82.9%), M3 (61.5%), and M4 (100.0%). How the point mutations of the neuraminidase sequences affected the susceptibility of H9N2 virus to oseltamivir is still to be determined and deserve further investigations.

Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is a threat to animal and public health worldwide. Till date, the H5N1 virus has claimed 402 human lives, with a mortality rate of 58 percent and has caused the death or culling of millions of poultry since 2003. In this study, we have designed three siRNAs (PB2-2235, PB2-479 and NP-865) targeting PB2 and NP genes of avian influenza virus and evaluated their potential, measured by hemagglutination (HA), plaque reduction and Real time RT-PCR assay, in inhibiting H5N1 virus (A/chicken/Navapur/7972/2006) replication in MDCK cells. The siRNAs caused 8- to 16-fold reduction in virus HA titers at 24 h after challenged with 100TCID50 of virus. Among these siRNAs, PB2-2235 offered the highest inhibition of virus replication with 16-fold reduction in virus HA titer, 80 percent reduction in viral plaque counts and 94 percent inhibition in expression of specific RNA at 24 h. The other two siRNAs had 68-73 percent and 87-88 percent reduction in viral plaque counts and RNA copy number, respectively. The effect of siRNA on H5N1 virus replication continued till 48h (maximum observation period). These findings suggest that PB2-2235 could efficiently inhibit HPAI H5N1 virus replication.

Mannose-binding lectin (MBL), a pattern-recognition molecule in serum, recognizes specific hexose sugars rich in mannose and N-acetylglucosamine on bacterium, yeasts, viruses as well as apoptotic cells. It has been well-identified that MBL has antiviral effects via binding to seasonal influenza H1 and H3 subtype viruses. Influenza A (H7N9) virus, a novel reassortant virus to human population, possesses the surface hemagglutinin (HA) and neuraminidase (NA) genes from duck and wild-bird influenza viruses and internal genes from poultry H9N2 viruses. As of Dec 7th, 2014, a total of 467 human infections and 183 fatal cases have been identified. Here, recombinant human (rh) MBL was tested for its binding and effects on hemagglutination inhibition (HI) and NA activity inhibition (NAI) of avian H7N9, H9N2 and human H3N2 viruses. We discovered that rhMBL exhibited a strong binding to H7N9 virus as human H3N2 did at high virus titers. However, it performed a significantly weaker HI activity effect on H7N9 comparing to those of H3N2 and H9N2, even at a much higher concentration (3.67 ± 0.33 vs. 0.026 ± 0.001 and 0.083 ± 0.02 μg/mL, respectively). Similarly, minor NAI effect of rhMBL, even at up to 10 μg/mL, was found on H7N9 virus while it displayed significant effects on both H3N2 and H9N2 at a lowest concentration of 0.0807 ± 0.009 and 0.0625 μg/mL, respectively. The HI and NAI effects of rhMBL were calcium-dependent and mediated by lectin domain. Our findings suggest that MBL, the host innate molecule, has differential interference effects with human and avian influenza virus and limited antiviral effect against H7N9 virus., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Fucoidan from the sporophyll (Mekabu) of brown seaweed Undaria pinnatifida (wakame) is interesting due to its various biological activities. Mekabu fucoidan (Mw ∼ 9 kDa) of this study (MF) was previously isolated and characterized by chemical and separation methods including GPC and methylation analysis (Lee, Hayashi, Hashimoto, Nakano, & Hayashi, 2004). It was found that this fucoidan composed of partially sulphated (DS ∼ 0.72) fucose and galactose at approximately equal amounts. Methylation analyses revealed complex structure of MF. However, it has been still unclear about the linkages between units and substitution patterns. To solve these structural tasks, spectroscopic methods (FTIR, FT Raman and NMR) were used in the analysis of native MF and its deesterified derivatives. According to obtained results, this polysaccharide was defined as O-acetylated sulphated fucogalactan. The defensive effects of MF were evaluated on mice infected with avian influenza A viruses (H5N3 and H7N2 subtypes); its efficacy was determined in reducing viral replication and increasing antibody production. Oral administration of MF resulted in suppressing virus yields. In addition, the production of neutralizing antibodies and mucosal IgA in the animals inoculated with the avian influenza A viruses was significantly increased. These results suggested that MF could be used for the prevention of viral infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Ethnopharmacological Relevance: Peperomia sui Lin and Lu (Peperomia sui), a well-known Taiwanese folk medicine, has a broad range of biological effects, especially in treatment of upper respiratory tract diseases. However, no previous study has explored the activity of Peperomia sui against influenza virus infections. This study was carried out to evaluate the anti-influenza virus activity and the potential virucidal effect of the ethanolic extract of Peperomia sui (PSE)., Methods: The anti-H6N1 avian influenza viral activity of PSE against the influenza virus A/Chicken/TW/0518/2011 (H6N1) in chicken fibroblast DF-1 cells was evaluated by cell viability assay, hemagglutination assay, neuraminidase activity assay, indirect immunofluorescence assay and quantitative RT-PCR assay., Results: PSE significantly increased the viability of cells that were infected by the H6N1 virus. PSE also suppressed the synthesis of viral nucleoprotein (NP), and inhibited the growth of the virus in DF-1 cells. Further, PSE inhibited the neuraminidase activity of H6N1 virus., Conclusions: The findings of this study provide important information for the exploitation and utilization of Peperomia sui in treatment of influenza infection., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: The herb formulation Deva-5 is used in traditional medicine to treat acute infectious diseases. Deva-5 is composed of five herbs: Gentiana decumbens L., Momordica cochinchinensis L., Hypecoum erectum L., Polygonum bistorta L., and Terminalia chebula Retz. Deva-5 and its five components were investigated for in vitro antiviral activity against avian influenza A virus subtype H3N8., Methods: The water extracts of the herbal parts of G. decumbens, H. erectum and P. bistorta, the seeds of T. chebula and M. cochinchinensis and Deva-5 were prepared by boiling and clarified by low-speed centrifugation and filtration. To assess the antiviral properties, avian influenza virus isolate A/Teal/Tunka/7/2010(H3N8) was incubated at 37°C for 30 min in the presence and absence of the extracts of five plants and DEVA-5 in various concentrations. Subsequently, the concentration of infectious virus in each sample was determined by plaque assays. Neutralisation indexes and 90% plaque reduction concentrations were estimated for each extract, and the significance of the data was evaluated using statistical methods., Results: The extracts of G. decumbens, H. erectum, P. bistorta and Deva-5 demonstrated no significant toxicity at concentrations up to 2%, whereas extracts of T. chebula and M. cochinchinensis were well-tolerated by Madin-Darby canine kidney cells at concentrations up to 1%. The extracts of H. erectum, M. cochinchinensis and T. chebula reduced the titre of A/Teal/Tunka/7/2010 (H3N8) by approximately five-fold (p ≤ 0.05). The other three extracts did not significantly reduce the infectivity of the virus. The plaque reduction neutralisation tests revealed that none of the extracts tested were able to inhibit formation of plaques by 90%. However, three extracts, H. erectum, T. chebula and M. cochinchinensis, were able to inhibit formation of plaques by more than 50% at low dilutions from 1:3 to 1:14. The T. chebula extract had a concentration-dependent inhibitory effect., Conclusions: For the first time, the consistent direct antiviral action of the extracts of H. erectum, T. chebula and M. cochinchinensis was detected. These extracts significantly reduced the infectivity of influenza A virus H3N8 in vitro when used at high concentrations (0.5-1%). However, Deva-5 itself and the remainder of its components did not exhibit significant antiviral action. The results suggest that H. erectum, T. chebula and M. cochinchinensis plants contain substances with direct antiviral activity and could be promising sources of new antiviral drugs.

Purpose of Review: In the last decade, sporadic and lethal human disease caused by zoonotic avian influenza viruses, and the seasonal activity of human H1N1 2009 pandemic type have driven intense epidemiological and laboratory studies into the virus life cycle. This article highlights major developments from mid-2012 to early 2014., Recent Findings: Advances in molecular techniques and efficient rollout of diagnostic tests have enabled the rapid identification of clinical cases and detailed genetic sequencing of viral genomes. Studies have contributed widely to the understanding of how and when influenza viruses circulate, what determines their innate pathogenicity in particular hosts and whether host cofactors influence disease severity. Other imperatives include investigations into how influenza can be better prevented by vaccination, or treated with antiviral drugs., Summary: Avian influenza viruses present a continuous threat to human populations. There is a need for sustained surveillance and downstream research to evaluate the potential for future pandemics.