Insights from Avian Influenza: A Review of Its Multifaceted Nature and Future Pandemic Preparedness.

Avian influenza viruses (AIVs) have posed a significant pandemic threat since their discovery. This review mainly focuses on the epidemiology, virology, pathogenesis, and treatments of avian influenza viruses. We delve into the global spread, past pandemics, clinical symptoms, severity, and immune response related to AIVs. The review also discusses various control measures, including antiviral drugs, vaccines, and potential future directions in influenza treatment and prevention. Lastly, by summarizing the insights from previous pandemic control, this review aims to direct effective strategies for managing future influenza pandemics.

Recombinant parainfluenza virus 5 expressing clade 2.3.4.4b H5 hemagglutinin protein confers broad protection against H5Ny influenza viruses.

The global circulation of clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) in poultry and wild birds, increasing mammal infections, continues to pose a public health threat and may even form a pandemic. An efficacious vaccine against H5Ny HPAIVs is crucial for emergency use and pandemic preparedness. In this study, we developed a parainfluenza virus 5 (PIV5)-based vaccine candidate expressing hemagglutinin (HA) protein of clade 2.3.4.4b H5 HPAIV, termed rPIV5-H5, and evaluated its safety and efficacy in mice and ferrets. Our results demonstrated that intranasal immunization with a single dose of rPIV5-H5 could stimulate H5-specific antibody responses, moreover, a prime-boost regimen using rPIV5-H5 stimulated robust humoral, cellular, and mucosal immune responses in mice. Challenge study showed that rPIV5-H5 prime-boost regimen provided sterile immunity against lethal clade 2.3.4.4b H5N1 virus infection in mice and ferrets. Notably, rPIV5-H5 prime-boost regimen provided protection in mice against challenge with lethal doses of heterologous clades 2.2, 2.3.2, and 2.3.4 H5N1, and clade 2.3.4.4h H5N6 viruses. These results revealed that rPIV5-H5 can elicit protective immunity against a diverse clade of highly pathogenic H5Ny virus infection in mammals, highlighting the potential of rPIV5-H5 as a pan-H5 influenza vaccine candidate for emergency use.IMPORTANCEClade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) have been widely circulating in wild birds and domestic poultry all over the world, leading to infections in mammals, including humans. Here, we developed a recombinant PIV5-vectored vaccine candidate expressing the HA protein of clade 2.3.4.4b H5 virus. Intranasal immunization with rPIV5-H5 in mice induced airway mucosal IgA responses, high levels of antibodies, and robust T-cell responses. Importantly, rPIV5-H5 conferred complete protection in mice and ferrets against clade 2.3.4.4b H5N1 virus challenge, the protective immunity was extended against heterologous H5Ny viruses. Taken together, our data demonstrate that rPIV5-H5 is a promising vaccine candidate against diverse H5Ny influenza viruses in mammals.

Mucosal immune responses and protective efficacy elicited by oral administration AMP-ZnONPs-adjuvanted inactivated H9N2 virus in chickens.

The avian influenza virus is infected through the mucosal route, thus mucosal barrier defense is very important. While the inactivated H9N2 vaccine cannot achieve sufficient mucosal immunity, adjuvants are needed to induce mucosal and systemic immunity to prevent poultry from H9N2 influenza virus infection. Our previous study found that polysaccharide from Atractylodes macrocephala Koidz binding with zinc oxide nanoparticles (AMP-ZnONPs) had immune-enhancing effects in vitro. This study aimed to evaluate the mucosal immune responses of oral whole-inactivated H9N2 virus (WIV)+AMP-ZnONPs and its impact on the animal challenge protection, and the corresponding changes of pulmonary metabolomics after the second immunization. The results showed that compared to the WIV, the combined treatment of WIV and AMP-ZnONPs significantly enhanced the HI titer, IgG and specific sIgA levels, the number of goblet cells and intestinal epithelial lymphocytes (iIELs) as well as the expression of J-chain, polymeric immunoglobulin receptor (pIgR), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and transforming growth factor-ß (TGF-ß). In viral attack experiments, WIV combing with AMP-ZnONPs effectively reduced lung damage and viral titers in throat swabs. Interestingly, significant changes of both the IgA intestinal immune network and PPAR pathway could also be found in the WIV+AMP-ZnONPs group compared to the non-infected group. Taken together, these findings suggest that AMP-ZnONPs can serve as a potential mucosal vaccine adjuvant, thereby avoiding adverse stress and corresponding costs caused by vaccine injection.

Multifaceted analysis of temporal and spatial distribution and risk factors of global poultry HPAI-H5N1, 2005-2023.

The purpose of this study was to analyze the characteristics of occurrence and spread of highly pathogenic avian influenza H5N1 (HPAI-H5N1) globally, understand its spatiotemporal characteristics, investigate the risk factors influencing outbreaks, and identify high-risk areas for disease occurrence. We collected the data on global poultry HPAI-H5N1 outbreaks from January 2005 to April 2023, and conducted a thorough analysis of the spatial and temporal characteristics of the disease through time series decomposition and directional distribution analysis. Additionally, an ecological niche model was established to explore the major factors influencing the occurrence of HPAI-H5N1 and to pinpoint high-risk areas. Our findings revealed that HPAI-H5N1 outbreaks were cyclical, and seasonal, exhibiting a rising trend, with a predominant northwest-southeast transmission direction. The ecological niche model highlighted that species factors and economic trade factors are critical in influencing the outbreak of HPAI-H5N1. Variables such as chicken and duck density, population density, isothermality, and road density, contributed to importantly risk of outbreaks. High-risk areas for HPAI-H5N1 occurrence were primarily identified in Europe, West Africa, Southeast Asia, and Southeast China. This study provided valuable insights into the spatial and temporal distribution characteristics and risk factors of global poultry HPAI-H5N1 outbreaks. The identification of high-risk areas provides essential information that can be used to develop more effective prevention and control policies.

Unraveling frontiers in poultry health (part 1) - Mitigating economically important viral and bacterial diseases in commercial Chicken and Turkey production.

This symposium offered up-to-date perspectives on field experiences and the latest research on significant viral and bacterial diseases affecting poultry. A highlight was the discussion on the use of enteroids as advanced in vitro models for exploring disease pathogenesis. Outcomes of this symposium included identifying the urgent need to improve the prevention and control of avian influenza by focusing research on vaccine effectiveness. In this regard, efforts should focus on enhancing the relatedness of vaccine antigen to the field (challenge) virus strain and improving immunogenicity. It was also revealed that gangrenous dermatitis could be controlled through withholding or restricting the administration of ionophores during broiler life cycle, and that administration of microscopic polymer beads (gel) based-live coccidia vaccines to chicks could be used to reduce necrotic enteritis-induced mortality. It was emphasized that effective diagnosis of re-emerging Turkey diseases (such as blackhead, fowl cholera, and coccidiosis) and emerging Turkey diseases such as reoviral hepatitis, reoviral arthritis, Ornithobacterium rhinotracheale infection, and strepticemia require complementarity between investigative research approaches and production Veterinarian field approaches. Lastly, it was determined that the development of a variety of functionally-specific enteroids would expedite the delineation of enteric pathogen mechanisms and the identification of novel vaccine adjuvants.

Vaccination of Poultry Against Influenza.

The complexity of influenza A virus (IAV) infections in avian hosts leads to equally complex scenarios for the vaccination of poultry. Vaccination against avian influenza strains can be used to prevent infections from sources with a single strain of IAV. It has been used as a part of outbreak control strategies as well as a way to maintain production for both low and high pathogenicity outbreaks. Unlike other viral pathogens of birds, avian influenza vaccination when used against highly pathogenic avian influenza virus, is tied to international trade and thus is not freely available for use without specific permission.

Vacunación de aves comerciales contra la influenza aviar. La complejidad de las infecciones por el virus de la influenza A en las aves hospedadoras conduce a escenarios igualmente complejos para la vacunación en la avicultura. La vacunación contra cepas de influenza aviar se puede utilizar para prevenir infecciones provenientes de fuentes con una sola cepa del virus de influenza. Se ha utilizado como parte de las estrategias de control de brotes, así como como una forma de mantener la producción tanto en brotes de baja como de alta patogenicidad. A diferencia de otros patógenos virales de las aves, la vacunación contra la influenza aviar, cuando se usa contra el virus de la influenza aviar altamente patógeno, está vinculada al comercio internacional y por lo tanto, no está disponible para su uso sin un permiso específico.

A case-control study of the infection risk of H5N8 highly pathogenic avian influenza in Japan during the winter of 2020-2021.

In Japan, outbreaks of H5N8 highly pathogenic avian influenza (HPAI) were reported between November 2020 and March 2021 in 52 poultry farms. Understanding HPAI epidemiology would help poultry industries improve their awareness of the disease and enhance the immediate implementation of biosecurity measures. This study was a simulation-based matched case-control study to elucidate the risk factors associated with HPAI outbreaks in chicken farms in Japan. Data were collected from 42 HPAI-affected farms and 463 control farms that were within a 5-km radius of each case farm but remained uninfected. When infected farms were detected as clusters, one farm was randomly selected from each cluster, considering the possibility that the cluster was formed by farm-to-farm transmission within an epidemic area. For each case farm, up to three control farms were selected within a 5-km radius. Overall, 26 case farms (16 layer and 10 broiler farms) and 75 control farms (45 layer and 30 broiler farms) were resampled 1000 times for the conditional logistic regression model with explanatory variables comprising geographical factors and farm flock size. A larger flock size and shorter distance to water bodies from the farm were found to increase infection risk in layer farms. Similarly, in broiler farms, a shorter distance to water bodies increased infection risk. On larger farms, frequent access of farm staff and instrument carriages to premises could lead to increased infection risk. Waterfowl visiting water bodies around farms may also be associated with infection risk.

UK flockdown: A survey of smallscale poultry keepers and their understanding of governmental guidance on highly pathogenic avian influenza (HPAI).

The scale of the current outbreak of highly pathogenic avian influenza (HPAI) due to the A/H5N1 virus in the United Kingdom is unprecedented. In addition to its economic impact on the commercial poultry sector, the disease has devastated wild bird colonies and represents a potential public health concern on account of its zoonotic potential. Although the implementation of biosecurity measures is paramount to reducing the spread of HPAI in domestic and commercial settings, little is known about the attitudes and perspectives of backyard poultry keepers, who often keep their flocks in close proximity to the public. A large nationwide survey of backyard poultry keepers was undertaken in December 2021-March 2022, contemporaneous with the enforcement of an Avian Influenza Prevention Zone (AIPZ) and additional housing measures in England, Scotland and Wales. The survey explored keepers' understanding of the clinical manifestations of HPAI, compliance with housing and biosecurity measures, attitudes towards obligatory culling on confirmation of HPAI in their flocks, and the potential use of vaccination to control HPAI. Summary statistical analysis of the closed question responses was supplemented with qualitative data analysis and corpus linguistic approaches to draw out key themes and salient patterns in responses to open text questions. Survey responses were received from 1559 small-scale poultry keepers across the United Kingdom. Awareness of the HPAI outbreak was very high (99.0%). The majority of respondents learned of it via social media (53%), with Defra (49.7%), British Hen Welfare Trust (33.8%) and the APHA (22.0%) identified as the principal sources of information. Analysis revealed that backyard keepers lacked knowledge of the clinical signs of avian influenza and legal requirements relating to compliance with biosecurity measures. Some respondents dismissed the seriousness of HPAI and were unwilling to comply with the measures in force. The issue of obligatory culling proved highly emotive, and some expressed a lack of trust in authorities. Most respondents (93.1%) indicated a willingness to pay for vaccination if the option was available. Communications on biosecurity measures that are relevant to large-scale industrial setups are inappropriate for backyard contexts. Understanding the barriers that backyard keepers face is essential if official agencies are to communicate biosecurity information effectively to such groups. Lack of trust in authorities is likely to make elimination of the virus in the UK difficult. We make recommendations for tailoring HPAI-related information for backyard contexts, to aid future HPAI control measures in the UK.

Differential Protection of Chickens against Highly Pathogenic H5 Avian Influenza Virus Using Polybasic Amino Acids with H5 Cleavage Peptide.

BACKGROUND: Highly pathogenic H5Nx viruses cause avian influenza, a zoonotic disease that can infect humans. The vaccine can facilitate the prevention of human infections from infected poultry. Our previous study showed that an H5 cleavage-site peptide vaccine containing the polybasic amino acid RRRK could protect chickens from lethal infections of the highly pathogenic H5N6 avian influenza virus. METHODS: Chickens immunized with the various polybasic amino combinations (RRRK, RRR, RR, R, RK, and K) of H5 cleavage-site peptides were challenged with highly pathogenic H5N6 avian influenza viruses. The challenged chickens were monitored for survival rate, and viral titers in swabs and tissue samples were measured in Madin-Darby canine kidney (MDCK) cells using the median tissue culture infectious dose 50 (log10 TCID50/mL). RESULTS: Most H5 cleavage-site vaccines containing various combinations of polybasic amino acids protected chickens from lethal infection. Chickens immunized with the RK-containing peptide combination of the H5 cleavage site were not protected. CONCLUSIONS: The polybasic amino acids (RRRK) of H5 cleavage cleavage-site peptide vaccines are important for protecting chickens against HP H5N6 avian influenza virus. The H5 cleavage cleavage-site peptide containing RK did not protect chickens against the virus.

New Patterns for Highly Pathogenic Avian Influenza and Adjustment of Prevention, Control and Surveillance Strategies: The Example of France.

From 2020 up to summer 2023, there was a substantial change in the situation concerning the high pathogenic avian influenza (HPAI) virus in Europe. This change concerned mainly virus circulation within wildlife, both in wild birds and wild mammals. It involved the seasonality of HPAI detections, the species affected, excess mortality events, and the apparent increased level of contamination in wild birds. The knock-on effect concerned new impacts and challenges for the poultry sector, which is affected by repeated annual waves of HPAI arriving with wild migratory birds and by risks due to viral circulation within resident wild birds across the year. Indeed, exceeding expectations, new poultry sectors and production areas have been affected during the recent HPAI seasons in France. The HPAI virus strains involved also generate considerable concern about human health because of enhanced risks of species barrier crossing. In this article, we present these changes in detail, along with the required adjustment of prevention, control, and surveillance strategies, focusing specifically on the situation in France.

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.

Efficiency of natural oils as alternative adjuvants to mineral oils in inactivated avian influenza vaccine formulation.

1. The effectiveness of inactivated vaccines depends on selecting the suitable adjuvant for vaccine formulation. The potency of vaccines with low antigen content can be improved with the appropriate adjuvant. This could allow production of more doses and lower the production cost.2. This study evaluated the efficiency of vaccines prepared using oil extracted from natural sources including argan oil, almond oil, sesame seed oil, pumpkin oil, cactus oil and black seed oil as alternative adjuvants for improving the protection capacity of inactivated influenza virus vaccine as compared to commonly used mineral oils.3. Each vaccine formulation was evaluated for stability, safety and immunogenicity in chickens, as well as for reducing the viral shedding after challenge infection.4. The cactus, sesame and pumpkin seed oil-based vaccines were found to be potent and successfully induced the production of humoral immunity in vaccinated chickens.

Research Note: Factors influencing highly pathogenic avian influenza preventive behavior among live poultry market vendors.

In China, fresh food has always been a top priority and live poultry has been a staple in the diet for thousands of years. As a result, the live poultry market (LPM) remains a popular and important source for purchasing live poultry among the public. However, LPMs also play a crucial role in spreading and retaining highly pathogenic avian influenza (HPAI) due to the high poultry movement and trade volume. Therefore, the preventive behavior of LPM vendors is essential in blocking the transmission of HPAI and reducing occupational exposure. Based on the health belief model, this study utilized structural equation modeling to examine the effect of risk perceptions on preventive behavior among vendors in the live poultry wholesale market (wLPM) and the live poultry retail market (rLPM) in Guangdong Province. The results indicated that perceived severity and perceived benefits positively influenced the wLPM vendors' ability to adopt preventive behavior (i.e., self-efficacy) while perceived barriers negatively affected self-efficacy in both wLPM and rLPM. Moreover, cues to action positively mediated the relationship between perceived severity, perceived benefits, and self-efficacy of wLPM and rLPM vendors. Cues to action also positively mediated the effect of perceived susceptibility among wLPM vendors. To promote preventive behavior among vendors, the market management companies and the government must provide timely and effective HPAI information through various channels and develop differentiated health campaigns according to the market types to raise vendors' awareness about HPAI.

Vaccination of African penguins (Spheniscus demersus) against high-pathogenicity avian influenza.

BACKGROUND: High-pathogenicity avian influenza (HPAI) has become a conservation threat to wild birds. Therefore, suitable vaccine technology and practical application methods require investigation. METHODS: Twenty-four African penguins (Spheniscus demersus) were vaccinated with either a conventional inactivated clade 2.3.4.4b H5N8 HPAI whole virus or a tobacco leaf-produced H5 haemagglutinin-based virus-like particle (VLP). Six birds received a second dose of the inactivated vaccine. Antibody responses were assessed and compared by employing haemagglutination inhibition tests. RESULTS: A second dose of inactivated vaccine was required to induce antibody titres above the level required to suppress virus shedding, while a single dose of VLP vaccine produced these levels by day 14, and one bird still had antibodies on day 430. LIMITATIONS: Bacterial contamination of the VLP vaccine limited the monitoring period and sample size in that treatment group, and it was not possible to perform a challenge study with field virus. CONCLUSION: VLP vaccines offer a more practical option than inactivated whole viruses, especially in logistically challenging situations involving wild birds.

Avian sarcoma/leukosis virus (RCAS)-mediated over-expression of IFITM3 protects chicks from highly pathogenic avian influenza virus subtype H5N1.

Broad-spectrum antiviral activities of interferon-induced transmembrane proteins (IFITMs) are primarily attributed to in vitro inhibition of viral entry. Here, we used an avian sarcoma-leukosis virus (RCAS)-based gene transfer system and successfully generated chicks that constitutively express chicken IFITM3 (chIFITM3). The chIFITM3-overexpressing chicks showed significant protection and disease tolerance against highly pathogenic avian influenza virus (HPAIV) H5N1 (Clade 2.2.1.2). The chicks, overexpressing chIFITM3, also showed delayed onset of clinical symptoms, reduced viral shedding, and alleviated histopathologic alterations compared to control and challenged chicks. These findings highlight that overexpression of chIFITM3 provide a substantial defense against zoonotic H5N1 in vivo.

The application of biosecurity practices for preventing avian influenza in North-Eastern Italy turkey farms: An analysis of the point of view and perception of farmers.

INTRODUCTION: Italian and international outbreaks of highly pathogenic avian influenza (HPAI), particularly in densely populated poultry areas (DPPAs), have increased over the past few decades. These emerging risks, which endanger both human and animal health and the entire poultry industry, can be effectively limited by biosecurity measures implemented at human-animal food chain interfaces. Some problems, however, persist in the application of these measures on the part of poultry farmers, prompting the need to explore those aspects and causes that limit their implementation. MATERIAL AND METHODS: A qualitative approach was selected for the study and a semi-structured interview technique was applied to collect data among turkey farmers (n = 29) working in the north-east of Italy. The aim of this technique was to gather data on farms in order to understand the biosecurity practices adopted and the reasons for and impediments to farmer implementation, or lack thereof. This article presents and discusses the main data collected. RESULTS: The study revealed that farmers were familiar with the biosecurity measures necessary to contain avian influenza (AI) and other poultry diseases; personal disinfection and animal isolation practices were particularly prominent. Based on the reported procedures, managerial, economic, and psychosocial factors were among the barriers behind the failure to implement biosecurity measures. These obstacles were variously intertwined and associated with the different action settings. In particular management factors, such as lack of time to apply the rules and difficulties contingent on the farm's structural characteristics, mediate the application of biosecurity measures. In terms of communication channels, the company, particularly its technicians, proved to be the primary source of information for farmers in case of emergencies, as well as the primary source of information on the application of biosecurity measures. However, other sources of information were indicated, such as word of mouth among farmers or other non-institutional figures (relatives and acquaintances). CONCLUSIONS: What emerged, was the need to improve not only the biosecurity management skills, but also to implement forms of cooperation among the various key stakeholders in the poultry sector. The information presented in this pilot study needs to be discussed among competent authorities, public and company veterinarians, company technicians, and farmers. Furthermore, this information will help in participatory co-planning of risk prevention and communication strategies to implement a long-term, sustainable, effective approach to address future epidemic emergencies.

Construction and Immunogenicity Evaluation of Recombinant Bacillus subtilis Expressing HA1 Protein of H9N2 Avian Influenza Virus.

The H9N2 subtype of the avian influenza virus (AIV) is one of the main subtypes of low pathogenic AIV, and it seriously affects the poultry breeding industry. Currently, vaccination is still one of China's main strategies for controlling H9N2 avian influenza. In this study, we selected MW548848.1 on the current popular main branch h9.4.2.5 as the reference strain, and we optimized the amino acid sequence of HA1 to make it suitable for expression in Bacillus subtilis. The B. subtilis expression vector showed good safety and stress resistance; therefore, this study constructed a recombinant B. subtilis expressing H9N2 HA1 protein and evaluated its immunogenicity in mice. The following results were obtained: the sIgA level of HA1 protein in small intestine fluid and the IgG level of PHT43-HA1/B. subtilis in serum were significantly improved (P < 0.01); PHT43-HA1/B. subtilis can cause a special immune response in mice; and cytokine detection interferon-gamma (IFN-γ) (P < 0.05) and Interleukin 2 (IL-2) (P < 0.01) expressions significantly increased. Additionally, the study found that PHT43-HA1/B. subtilis can alleviate the attack of H9N2 AIV in the spleen, lungs, and small intestine of mice. This study was the first to use an oral recombinant B. subtilis-HA1 vaccine candidate, and it provides theoretical data and technical reference for the creation of a new live vector vaccine against H9N2 AIV.

In ovo administration of retinoic acid enhances cell-mediated immune responses against an inactivated H9N2 avian influenza virus vaccine.

The H9N2 subtype avian influenza virus (AIV) is a low pathogenic AIV that infects avian species and lead to huge economical losses in the poultry industry. The unique immunomodulatory properties of Retinoic acid (RA), an active component of vitamin A, highlights its potential to enhance chicken's resistance to infectious diseases and perhaps vaccine-induced immunity. Therefore, the present study evaluated the effects of in ovo supplementation of RA on the immunogenicity and protective efficacy of an inactivated avian influenza virus vaccine. On embryonic day 18, eggs were inoculated with either 90 µmol RA/200 µL/egg or diluent into the amniotic sac. On days 7 and 21 post-hatch, birds were vaccinated with 15 µg of ß-propiolactone (BPL) inactivated H9N2 virus via the intramuscular route. One group received BPL in combination with an adjuvant, while the other group received saline solution and served as a non-vaccinated control group. Serum samples were collected on days 7, 14, 21, 28, 35, and 42 post-primary vaccination (ppv) for antibody analysis. On day 24 ppv, spleens were collected, and splenocytes were isolated to analyze cytokine expression, interferon gamma (IFN-γ) production, and cell population. On day 28 ppv, birds in all groups were infected with H9N2 virus and oral and cloacal swabs were collected for TCID50 (50 % Tissue Culture Infectious Dose) assay up to day 7 post-infection. The results demonstrated that in ovo administration of RA did not significantly enhance the AIV vaccine-induced antibody response against H9N2 virus compared to the group that received the vaccine alone. However, RA supplementation enhanced the frequency of macrophages (KUL01+), expression of inflammatory cytokines and production of IFN-γ by splenocytes. In addition, RA administration reduced oral shedding of AIV on day 5 post-infection. In conclusion, these findings suggest that RA can be supplemented in ovo to enhance AIV vaccine efficacy against LPAIV.