FOUR-WEEK ORAL ADMINISTRATION OF BALOXAVIR MARBOXIL AS AN ANTI-INFLUENZA VIRUS DRUG SHOWS NO TOXICITY IN CHICKENS.

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.

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

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

Effect of enrofloxacin on clinical parameters and mucociliary system of broilers challenged with H9N2 avian influenza/infectious bronchitis viruses.

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.

Probenecid Inhibits Influenza A(H5N1) and A(H7N9) Viruses In Vitro and in Mice.

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.

Baloxavir marboxil use for critical human infection of avian influenza A H5N6 virus.

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).

Evaluation of chlorine dioxide in liquid state and in gaseous state as virucidal agent against avian influenza virus and infectious bronchitis virus.

The antiviral activity of chlorine dioxide (ClO2) in liquid (ClO2 gas dissolved liquid) and gaseous state against avian influenza virus (AIV) and infectious bronchitis virus (IBV) was evaluated. To evaluate the effect of ClO2 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 ClO2 gas against IBV in aerosols was evaluated. After the exposure of sprayed IBV to ClO2 gas for a few seconds, 94.2% reduction of the virus titer was observed, as compared to the pre-treatment control. Altogether, hence, ClO2 has an evident potential to be an effective disinfectant for the prevention and control of AIV and IBV infections on poultry farms.

ß-Glucans from Trametes versicolor (L.) Lloyd Is Effective for Prevention of Influenza Virus Infection.

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 ß-glucans from C. versicolor have various physiological activities, including activating macrophages to protect against Salmonella infection. However, whether ß-glucans have antiviral effects has not been reported. Hence, the objective of this study was to confirm whether ß-glucans could boost the immune response to combat influenza virus in mouse and chick models. The results show that ß-glucans induced the expression of Dectin-1, costimulatory molecules (CD80/86) and cytokines IL-6, IL-1ß, IFN-ß and IL-10 in murine bone marrow dendritic cells (BMDCs). In addition, orally administered ß-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 ß-glucans improved survival and reduced lung viral titers in chickens infected with H9N2 avian influenza virus. These results suggest that ß-glucans have a significant antiviral effect. Therefore, ß-glucans could become a potential immunomodulator against influenza virus.

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

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

Substitution of I222L-E119V in neuraminidase from highly pathogenic avian influenza H7N9 virus exhibited synergistic resistance effect to oseltamivir in mice.

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 IC50 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 6log10 TCID50/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.

Single dose of multi-clade virus-like particle vaccine protects chickens against clade 2.3.2.1 and clade 2.3.4.4 highly pathogenic avian influenza viruses.

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 log2). 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 PD50 (protective dose 50%) of the multi-clade VLP against clades 2.3.2.1c and 2.3.4.4c was < 50 PD50 (28 and 42 PD50, 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.

Virucidal efficacy of acidic electrolyzed water (AEW) against African swine fever virus and avian influenza virus.

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.

Evaluation of Baloxavir Marboxil and Peramivir for the Treatment of High Pathogenicity Avian Influenza in Chickens.

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.

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

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

Development of ssDNA Aptamers for Diagnosis and Inhibition of the Highly Pathogenic Avian Influenza Virus Subtype H5N1.

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.

Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development.

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.

Immunostimulant effect of a mixed herbal extract on infectious bursal disease virus (IBDV) vaccinated chickens in the context of a co-infection model of avian influenza virus H9N2 and IBDV.

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.

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

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

Antiviral responses against chicken respiratory infections: Focus on avian influenza virus and infectious bronchitis virus.

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.

Influenza A/H4N2 mallard infection experiments further indicate zanamivir as less prone to induce environmental resistance development than oseltamivir.

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 IC50 increased 5.5-fold) and E119G (ZA IC50 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 virus transmission is suppressed in chickens fed Lactobacillus paracasei expressing the 3D8 single-chain variable fragment protein.

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 109 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 107.5 EID50/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.