Intranasally administered protein coated chitosan nanoparticles encapsulating influenza H9N2 HA2 and M2e mRNA molecules elicit protective immunity against avian influenza viruses in chickens.

Chitosan nanoparticles (CNPs) represent an efficient vaccination tool to deliver immunogenic antigens to the antigen-presenting cells (APCs), which subsequently stimulate protective immune responses against infectious diseases. Herein, we prepared CNPs encapsulating mRNA molecules followed by surface coating with conserved H9N2 HA2 and M2e influenza proteins. We demonstrated that CNPs efficiently delivered mRNA molecules into APCs and had effectively penetrated the mucosal barrier to reach to the immune initiation sites. To investigate the potential of CNPs delivering influenza antigens to stimulate protective immunity, we intranasally vaccinated chickens with empty CNPs, CNPs delivering HA2 and M2e in both mRNA and protein formats (CNPs + RNA + Pr) or CNPs delivering antigens in protein format only (CNPs + Pr). Our results demonstrated that chickens vaccinated with CNPs + RNA + Pr elicited significantly (p < 0.05) higher systemic IgG, mucosal IgA antibody responses and cellular immune responses compared to the CNPs + Pr vaccinated group. Consequently, upon challenge with either H7N9 or H9N2 avian influenza viruses (AIVs), efficient protection, in the context of viral load and lung pathology, was observed in chickens vaccinated with CNPs + RNA + Pr than CNPs + Pr vaccinated group. In conclusion, we show that HA2 and M2e antigens elicited a broad spectrum of protection against AIVs and incorporation of mRNAs in vaccine formulation is an effective strategy to induce superior immune responses.

The Microbiota Contributes to the Control of Highly Pathogenic H5N9 Influenza Virus Replication in Ducks.

Ducks usually show little or no clinical signs following highly pathogenic avian influenza virus infection. In order to analyze whether the microbiota could contribute to the control of influenza virus replication in ducks, we used a broad-spectrum oral antibiotic treatment to deplete the microbiota before infection with a highly pathogenic H5N9 avian influenza virus. Antibiotic-treated ducks and nontreated control ducks did not show any clinical signs following H5N9 virus infection. We did not detect any significant difference in virus titers neither in the respiratory tract nor in the brain nor spleen. However, we found that antibiotic-treated H5N9 virus-infected ducks had significantly increased intestinal virus excretion at days 3 and 5 postinfection. This was associated with a significantly decreased antiviral immune response in the intestine of antibiotic-treated ducks. Our findings highlight the importance of an intact microbiota for an efficient control of avian influenza virus replication in ducks.IMPORTANCE Ducks are frequently infected with avian influenza viruses belonging to multiple subtypes. They represent an important reservoir species of avian influenza viruses, which can occasionally be transmitted to other bird species or mammals, including humans. Ducks thus have a central role in the epidemiology of influenza virus infection. Importantly, ducks usually show little or no clinical signs even following infection with a highly pathogenic avian influenza virus. We provide evidence that the microbiota contributes to the control of influenza virus replication in ducks by modulating the antiviral immune response. Ducks are able to control influenza virus replication more efficiently when they have an intact intestinal microbiota. Therefore, maintaining a healthy microbiota by limiting perturbations to its composition should contribute to the prevention of avian influenza virus spread from the duck reservoir.

Avian influenza viruses in humans: lessons from past outbreaks.

BACKGROUND: Human infections with avian influenza viruses (AIV) represent a persistent public health threat. The principal risk factor governing human infection with AIV is from direct contact with infected poultry and is primarily observed in Asia and Egypt where live-bird markets are common. AREAS OF AGREEMENT: Changing patterns of virus transmission and a lack of obvious disease manifestations in avian species hampers early detection and efficient control of potentially zoonotic AIV. AREAS OF CONTROVERSY: Despite extensive studies on biological and environmental risk factors, the exact conditions required for cross-species transmission from avian species to humans remain largely unknown. GROWING POINTS: The development of a universal ('across-subtype') influenza vaccine and effective antiviral therapeutics are a priority. AREAS TIMELY FOR DEVELOPING RESEARCH: Sustained virus surveillance and collection of ecological and physiological parameters from birds in different environments is required to better understand influenza virus ecology and identify risk factors for human infection.

Immunological and pathological effects of vitamin E with Fetomune Plus® on chickens experimentally infected with avian influenza virus H9N2.

Avian influenza virus (AIV) H9N2 infection causes economic losses on poultry farms, and immunostimulants are essential for improving chicken immunity. This study evaluated the immunological and pathological effects of vitamin E with Fetomune Plus® (a commercial product based on a yeast extract and vitamins) on chickens experimentally infected with AIV H9N2. Three groups of white Hy-Line chicks were included. The G1 group was kept as an uninfected untreated control, the G2 group was intranasally infected with the AIV H9N2 strain (0.5 ml of 106 50% egg infectious dose (EID50)), and the G3 group was infected and treated with vitamin E (200 mg/kg of diet) and Fetomune Plus® (1 ml/liter of drinking water) for four weeks. The gene expression of interferon-gamma (IFN-γ), interleukin (IL)-6, and IL-2 was determined at 3, 5 and 7 days post-infection (PI). Virus shedding titers and rates and haemagglutination inhibition (HI) antibody titers were detected. Clinical signs, mortalities and post-mortem lesions were recorded. The birds were weighed, and relative organ weights were calculated. Tissue specimens were taken for histopathological examination and immunohistochemistry (IHC). The expression of IFN-γ in the duodenum revealed a significant increase in G2 compared to G3 at 3 days PI, while the duodenal and splenic expression of IL-6 was significantly increased in G2 compared to G3 at 5 days PI. IL-2 was overexpressed in the duodenum in G3 compared to G2 at 3 and 5 days PI. A significant decrease (P ≤ 0.05) in the virus shedding titer and an increase in the HI titers were detected in G3 compared to G2. The clinical signs and the mortality rate were clearly appeared in G2 than in G3. By IHC, lower H9N2 staining intensity was observed in the examined organs from G3 than in those from G2. In conclusion, as a first report, vitamin E with Fetomune Plus® supplementation for four weeks could improve the immunological and pathological effects of H9N2 infection on chickens.

Characterization of immunogenicity of avian influenza antigens encapsulated in PLGA nanoparticles following mucosal and subcutaneous delivery in chickens.

Mucosal vaccine delivery systems have paramount importance for the induction of mucosal antibody responses. Two studies were conducted to evaluate immunogenicity of inactivated AIV antigens encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). In the first study, seven groups of specific pathogen free (SPF) layer-type chickens were immunized subcutaneously at 7-days of age with different vaccine formulations followed by booster vaccinations two weeks later. Immune responses were profiled by measuring antibody (Ab) responses in sera and lachrymal secretions of vaccinated chickens. The results indicated that inactivated AIV and CpG ODN co-encapsulated in PLGA NPs (2x NanoAI+CpG) produced higher amounts of hemagglutination inhibiting antibodies compared to a group vaccinated with non-adjuvanted AIV encapsulated in PLGA NPs (NanoAI). The tested adjuvanted NPs-based vaccine (2x NanoAI+CpG) resulted in higher IgG responses in the sera and lachrymal secretions at weeks 3, 4 and 5 post-vaccination when immunized subcutaneously. The incorporation of CpG ODN led to an increase in Ab-mediated responses and was found useful to be included both in the prime and booster vaccinations. In the second study, the ability of chitosan and mannan coated PLGA NPs that encapsulated AIV and CpG ODN was evaluated for inducing antibody responses when delivered via nasal and ocular routes in one-week-old SPF layer-type chickens. These PLGA NPs-based and surface modified formulations induced robust AIV-specific antibody responses in sera and lachrymal secretions. Chitosan coated PLGA NPs resulted in the production of large quantities of lachrymal IgA and IgG compared to mannan coated NPs, which also induced detectable amounts of IgA in addition to the induction of IgG in lachrymal secretions. In both mucosal and subcutaneous vaccination approaches, although NPs delivery enhanced Ab-mediated immunity, one booster vaccination was required to generate significant amount of Abs. These results highlight the potential of NPs-based AIV antigens for promoting the induction of both systemic and mucosal immune responses against respiratory pathogens.

Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens.

Several types of avian influenza virus (AIV) vaccines exist, including live-attenuated, vectored, and whole inactivated virus (WIV) vaccines. Inactivated vaccines offer some advantages compared to other types of vaccines, including ease of production and lack of ability to revert to a virulent state. However, WIV are poorly immunogenic, especially when these vaccines are delivered to mucosal surfaces. There are several factors that contribute to the immunogenicity of vaccines, one of which is the method used to inactivate viruses. Several methods exist for producing influenza WIVs, including formaldehyde, a chemical that affects protein structures leading to virus inactivation. Other methods include treatment with beta-propiolactone (BPL) and the application of gamma radiation, both of which have less effects on protein structures compared to formaldehyde, and instead alter nucleic acids in the virion. Here, we sought to determine the effect of the above inactivation methods on immunogenicity of AIV vaccines. To this end, chickens were vaccinated with three different H9N2 WIVs using formaldehyde, BPL, and gamma radiation for inactivation. In addition to administering these three WIVs alone as vaccines, we also included CpG ODN 2007, a synthetic ligand recognized by Toll-like receptor (TLR)21 in chickens, as an adjuvant for each WIV. Subsequently, antibody- and cell-mediated immune responses were measured following vaccination. Antibody-mediated immune responses were increased in chickens that received the BPL and Gamma WIVs compared to the formaldehyde WIV. CpG ODN 2007 was found to significantly increase antibody responses for each WIV compared to WIV alone. Furthermore, we observed the presence of cell-mediated immune responses in chickens that received the BPL WIV combined with CpG ODN 2007. Based on these results, the BPL WIV + CpG ODN 2007 combination was the most effective vaccine at inducing adaptive immune responses against H9N2 AIV. Future studies should characterize mucosal adaptive immune responses to these vaccines.

Immunogenicity of Concentrated and Purified Inactivated Avian Influenza Vaccine Formulation.

Avian influenza (AI) H9N2 is a low pathogenic virus subtype belonging to Orthomyxoviridae family. Given the prevalence of this subtype as an infectious agent in poultry industry, special attention has been always directed toward the development of vaccine production against this infection. The vaccine of this infection is produced by killing the virus and using a mixture of inactivated antigen and oil phase. Egg-based viral antigens have high levels of unwanted proteins that may adversely affect the vaccine formulation. In addition, it is required to raise the antigen concentration for the production of combination vaccines, especially in low doses. This underscores the need to the improvement of the downstream purification process and concentration of antigens. The optimization of downstream processing would decrease the cost of vaccine procurement and maintenance. Regarding this, the present study was conducted to evaluate a downstream procedure for the concentration and purification of avian influenza virus (H9N2) and investigate the immunogenicity of the vaccine containing these antigens. To this end, after harvesting and clarifying virus-containing allantoic fluid, it was concentrated and purified using ultrafiltration and chromatography, respectively. The concentrated and purified samples were checked for their ovalbumin level and emulsified with oil adjuvant to access their immunogenicity. The results showed that one dose of both formulated antigens (i.e., concentrated and purified) was effective in raising the immune response in the vaccinated chicks for a long time. The applied formulation had a one-year stability in the refrigerator. Furthermore, the concentrated antigen showed a high hemagglutination activity through a year when storing in the refrigerator. Based on the findings, the optimization of downstream process of influenza (H9N2) vaccine production and use of new technologies could be considered in the large-scale preparation of a sustainable vaccine without any unwanted risk factors.

Effects of isolation and slaughter strategies in different species on emerging zoonoses.

Zoonosis is the kind of infectious disease transmitting among different species by zoonotic pathogens. Different species play different roles in zoonoses. In this paper, we established a basic model to describe the zoonotic pathogen transmission from wildlife, to domestic animals, to humans. Then we put three strategies into the basic model to control the emerging zoonoses. Three strategies are corresponding to control measures of isolation, slaughter or similar in wildlife, domestic animals and humans respectively. We analyzed the effects of these three strategies on control reproductive numbers and equilibriums and we took avian influenza epidemic in China as an example to show the impacts of the strategies on emerging zoonoses in different areas at beginning.

Highly Pathogenic Avian Influenza A(H7N9) Virus, Tennessee, USA, March 2017.

In March 2017, highly pathogenic avian influenza A(H7N9) was detected at 2 poultry farms in Tennessee, USA. Surveillance data and genetic analyses indicated multiple introductions of low pathogenicity avian influenza virus before mutation to high pathogenicity and interfarm transmission. Poultry surveillance should continue because low pathogenicity viruses circulate and spill over into commercial poultry.

Influenza A H5N1 and H7N9 in China: A spatial risk analysis.

BACKGROUND: Zoonotic avian influenza poses a major risk to China, and other parts of the world. H5N1 has remained endemic in China and globally for nearly two decades, and in 2013, a novel zoonotic influenza A subtype H7N9 emerged in China. This study aimed to improve upon our current understanding of the spreading mechanisms of H7N9 and H5N1 by generating spatial risk profiles for each of the two virus subtypes across mainland China. METHODS AND FINDINGS: In this study, we (i) developed a refined data set of H5N1 and H7N9 locations with consideration of animal/animal environment case data, as well as spatial accuracy and precision; (ii) used this data set along with environmental variables to build species distribution models (SDMs) for each virus subtype in high resolution spatial units of 1km2 cells using Maxent; (iii) developed a risk modelling framework which integrated the results from the SDMs with human and chicken population variables, which was done to quantify the risk of zoonotic transmission; and (iv) identified areas at high risk of H5N1 and H7N9 transmission. We produced high performing SDMs (6 of 8 models with AUC > 0.9) for both H5N1 and H7N9. In all our SDMs, H7N9 consistently showed higher AUC results compared to H5N1, suggesting H7N9 suitability could be better explained by environmental variables. For both subtypes, high risk areas were primarily located in south-eastern China, with H5N1 distributions found to be more diffuse and extending more inland compared to H7N9. CONCLUSIONS: We provide projections of our risk models to public health policy makers so that specific high risk areas can be targeted for control measures. We recommend comparing H5N1 and H7N9 prevalence rates and survivability in the natural environment to better understand the role of animal and environmental transmission in human infections.

Vaccine Efficacy Against a New Avian Influenza (H9N2) Field Isolate from the Middle East (Serology and Challenge Studies).

Avian influenza subtype H9N2 is endemic in many countries in the Middle East. The reported prevalence of infection was variable between countries and ranged from 28.7% in Tunisia to 71% in Jordan. Several commercial killed whole-virus vaccine products are used as monovalent or bivalent mixed with Newcastle disease virus. Recently, we have noticed that many of the vaccinated broiler flocks did not show a production advantage over nonvaccinated flocks in the field. A new avian influenza field virus (H9N2) was isolated from these vaccinated and infected broiler flocks in 2013. This virus had 89.1% similarity of its hemagglutinin (HA) gene to the classical virus used for manufacturing the classical vaccine. Inactivated autogenous vaccine was manufactured from this new field isolate to investigate its serological response and protection in specific-pathogen-free (SPF) and breeder-male chickens compared to the classical vaccine. Oropharyngeal virus shedding of vaccinated breeder-male chickens was evaluated at 3, 9, 10, and 14 days postchallenge (DPC). Percentage of chickens shedding the virus at 3 DPC was 64%, 50%, and 64% in the classical vaccine group, autogenous vaccine group, and the control challenged group, respectively. At 7 DPC percentage of virus shedding was 42%, 7%, and 64% in the classical vaccine group, autogenous vaccine group, and the control challenged group, respectively. At 10 DPC only 9% of classical vaccine group was shedding the virus and there was no virus shedding in any of the groups at 14 DPC. There was statistical significance difference (P < 0.05) in shedding only at 7 DPC between the autogenous vaccine group and the other two groups. At 42 days of age (14 DPC), average body weight was 2.720, 2.745, 2.290, and 2.760 kg for the classical vaccine group, autogenous vaccine group, control challenged group, and control unchallenged group, respectively. Only the control challenged group had significantly (P < 0.05) lower average body weight. In another experiment, vaccinated SPF chicks had hemagglutination inhibition (HI) geometric mean titers (GMTs), with classical antigen, of 8.7 and 3.1 log 2 for classical and autogenous vaccine groups, respectively. When the autogenous antigen was used for HI, GMTs were 6.0 and 8.1 log 2, respectively. Both vaccines protected against body weight suppression after challenge. However, autogenous vaccine elicited significantly higher HI titer and reduced viral shedding at 7 DPC. In conclusion, it is important to revise the vaccine virus strains used in each region to protect against and control infection from new field strains. Further field experiments are needed to demonstrate the efficacy of new vaccines under field conditions.

Identifying innate immune pathways of the chicken may lead to new antiviral therapies.

Zoonotic viruses, such as highly pathogenic avian influenza (HPAI), present a significant threat to both the poultry industry and public health. The present method of controlling avian influenza (AI) relies on good farming practice with limited use of vaccination in some countries. However, new ways to control disease outbreaks might be possible with additional knowledge of the natural host response to virus. Moreover, manipulation of the innate immune system in mammals improves the outcomes following viral infection. A similar approach might be applied to the chicken, nevertheless, a greater knowledge of the chicken innate immune system is required. This review outlines important mammalian antiviral mechanisms that have been modulated to strengthen viral immunity and highlights the potential application of these strategies in the chicken, especially in regards, to AI.

Pathogenesis of influenza: virus-host interactions.

Since their inception in March 1972, Keystone Symposia on Molecular and Cellular Biology have brought together scientists from across the globe to discuss key biological topics. Now in its 40th year, it is a completely independent, nonprofit organization devoted solely to providing outstanding scientific conferences in all areas of the biological and biomedical sciences. Towards the end of May 2011, over 200 virologists and immunologists came to Hong Kong, an appropriate setting given the emergence of H5N1, to discuss influenza virus and host interactions. The meeting, expertly organized by Siamon Gordon (University of Oxford, Oxofrd, UK), Malik Peiris (University of Hong Kong, Hong Kong, China) and Kanta Subbarao (NIAID, NIH, MD, USA), took place in the aftermath of the first pandemic in 40 years and provided great insight into both pandemic H1N1 and H5N1. This article focuses on some of the recurring themes that were discussed during the week.

[MiR26a and miR939 regulate the replication of H1N1 influenza virus in MDCK cell].

OBJECTIVE: MicroRNAs (miRNAs) play an important role in the process of infection and replication of virus in host cells. In this study, we cloned two miRNAs expression vectors and examined their effects on the replication of H1N1 type influenza virus in MDCK (Madin dardy canine kidney) cells. METHODS: We constructed the plasmids expressing miR26a and miR939 and performed the transfection study in MDCK cells. Subsequently, the cells were infected with H1N1 type influenza virus 24 h after transfection. Then we tested the hemagglutination titer at 72 h time point to investigate the effect of miR26a and miR939 on the replication H1N1 type influenza virus in MDCK cells. RESULTS: The transfection study showed that miR26a and miR939 can achieve efficiently expression in MDCK cells. miR26a and miR939 can influence the replication of H1N1 influenza virus differently in two different ways. miR26a inhibits the replication, whereas miR939 stimulates the process. CONCLUSION: Cellular miRNAs can regulate the replication of H1N1 influenza virus in host cells, and our paper should report the role of miR26a and miR939 in this regulation for the first time.

Retrocyclin 2: a new therapy against avian influenza H5N1 virus in vivo and vitro.

We tested the ability of retrocyclin 2, a type of theta defensin, to protect cells and chicken embryos from infection by H5N1 highly pathogenic avian influenza virus. A gene fragment of retrocyclin 2 was designed based on the protein sequence of retrocyclin 2 and cloned into the eukaryotic expression vector pcDNA4.01 (HismaxA), named pcDNA4-RC2. The expression vector pcDNA4-RC2 protected MDCK cells and chicken embryos from infection by the H5N1 virus through inhibition of virus replication and viral mRNA transcription. Retrocyclin 2 is therefore effective in preventing H5N1 virus infection in vivo and in vitro and could be considered as a new therapy for H5N1 influenza and other diseases.

El virus de la gripe aviar: nuevos aspectos relativos a su patogenicidad y a estrategias para combatirlo / Avian influenza virus: new features related to its pathogenicity and strategies for fighting against it

Reconstruido por ingeniería genética el virus que causó la terriblepandemia de gripe de 1918-19 y comprobada su capacidad deinfección, se ha deducido que la gran patogenicidad del mismo puededeberse a una desregulación de los mecanismos inmunitarios queejercen normalmente citocinas de las células hospedadoras.Asimismo, el subtipo H5N1 del virus de la gripe aviar, por unadesregulación análoga, posee una patogenicidad que es aún mayorque la del subtipo de virus de la gripe de 1918-19.Intentos para prevenir los riesgos (leves, moderados o muy graves)de la esperada pandemia que produciría en humanos el subtipoH5N1 —u otros subtipos peligrosos que puedan surgir— se estánefectuando con la preparación de nuevas vacunas, así como mediantela interrupción del ciclo biológico del virus con inhibidores de laenzima vírica neuraminidasa (= sialidasa), tales como el oseltamiviry el zanamivir, o con diversos agentes que bloquean el funcionamientode otros componentes de dicho virus.Últimamente, se está analizando si es o no conveniente tratar dereforzar estas medidas con el uso de moduladores de ciertas citocinas, tales como algunas estatinas u otros compuestos de acción eíndole varias, cuyo reducido coste —por pertenecer al grupo de los«genéricos»— constituye un aliciente adicional (AU)

Avian influenza virus: new features related to its pathogenicity and strategies for fighting against itSince Taubenberger’s team recreated each of the genes of influenzavirus that caused the terrible pandemic of 1918-19 using thetechnique called reverse genetics and checked the infectious capacityof the newborn virus, it seems that its high pathogenicity may bedue to disregulation of the innate immune mechanisms normallyperformed by the cytokines of host cell.Furthermore, the H5N1 subtype of avian influenza, which alsocauses a similar dysfunction, has higher pathogenicity than that of1918-19 influenza virus.Assays to prevent the mild, moderate or severe risks of an eventualfuture pandemic affecting humans caused by the H5N1 subtypeare now being carried out by preparing new vaccines and neuraminidase(= sialidase) inhibitors such as oseltamivir and zanamivir, aswell as other agents that block the function of several componentsof the virus.Recently, has been debated whether it would be convenient to usemodulators of the immune system, such as statins, against the secondaryeffects of the viral infection caused by the disregulation mechanismsof cytokines, to reinforce the above therapeutic measures. Thelow cost of statins offers additional justification for their use (AU)

Avian influenza vaccines and therapies for poultry.

Vaccines have been used in avian influenza (AI) control programs to prevent, manage or eradicate AI from poultry and other birds. The best protection is produced from the humoral response against the hemagglutinin (HA) protein. A variety of vaccines have been developed and tested under experimental conditions with a few receiving licensure and field use following demonstration of purity, safety, efficacy and potency. Current licensed vaccines are predominately inactivated whole AI vaccines, typically produced from low pathogenicity (LP) AI virus strains, or occasionally from high pathogenicity AI virus strains. Recently, reverse genetic procedures have been developed that allow construction of vaccine strains using a genetically altered HA gene (changing HP HA proteolytic cleavage site to LP) and a backbone of internal gene segments for safe, high growth production. Other licensed AI vaccines include recombinant fowl poxvirus vector with an AI H5 insert and a recombinant Newcastle disease virus vector with an AI H5 gene insert. The latter vaccine can be mass administered via aerosol application.