Generation and characterization of a nanobody against the avian influenza virus H7 subtype.

Avian influenza virus (AIV) H7N9 diseases have been recently reported, raising concerns about a potential pandemic. Thus, there is an urgent need for effective therapeutics for AIV H7N9 infections. Herein, camelid immunization and yeast two-hybrid techniques were used to identify potent neutralizing nanobodies (Nbs) targeting the H7 subtype hemagglutinin. First, we evaluated the binding specificity and hemagglutination inhibition activity of the screened Nbs against the H7 subtype hemagglutinin. Nb-Z77, with high hemagglutination inhibition activity was selected from the screened Nbs to optimize the yeast expression conditions and construct oligomeric forms of Nb-Z77 using various ligation methods. The oligomers Nb-Z77-DiGS, Nb-Z77-TriGS, Nb-Z77-Fc and Nb-Z77-Foldon were successfully constructed and expressed. Nb-Z77-DiGS and Nb-Z77-Foldon exhibited considerably greater activity than did Nb-Z77 against H7 subtype hemagglutinin, with median effective concentrations of 384.7 and 27.33 pM and binding affinity values of 213 and 5.21 pM, respectively. Nb-Z77-DiGS and Nb-Z77-Foldon completely inhibited the hemagglutination activity of the inactivated virus H7-Re1 at the lowest concentration of 0.938 µg/mL. This study screened a strain of Nb with high hemagglutination inhibition activity and enhanced its antiviral activity through oligomerization, which may have great potential for developing effective agents for the prevention, diagnosis, and treatment of AIV H7 subtype infection.

Factors affecting highly pathogenic avian influenza vaccination practices at poultry farms in Tra Vinh, Vietnam.

Background: The increased number of cases of highly pathogenic avian influenza (HPAI) as a zoonosis has raised concerns in terms of poultry and human health. Farmers' preventive practices are an effective way of reducing zoonosis. However, this practice may have been affected by many factors, including production behaviors, awareness, and farmers' perceptions of farmers toward zoonosis. Aim: This study was conducted on 166 poultry farms in Tra Vinh Province with 14,894 poultry heads to determine the socioeconomic profiles and production characteristics of poultry farms and analyze the effect of these factors on HPAI vaccination practices. Methods: Respondents were selected from lists provided by government officers. Descriptive statistics were used to describe all variables, and factors affecting HPAI vaccination practices were analyzed using binary regression analysis. Results: The results showed that most farmers raised poultry with other livestock using the free-range method, which is a semi-intensive system. The primary objectives of poultry farming are meat sales and augmenting household consumption, with farmers primarily raising chicks produced on their farms. The implementation of the vaccine was less than 50% on the surveyed farms, with a small number of farmers administering an HPAI booster dose. However, only 6% of the farmers confirmed that their livestock had been exposed to HPAI. In addition, HPAI vaccination and booster dose practices significantly increased when farmers had 4-6 family members and received HPAI prevention training. Moreover, increased poultry numbers have led to increased vaccination rates and the implementation of booster doses for poultry. The study also reported that the vaccination rate decreased when poultry was used for household consumption. Conclusion: Sociodemographic characteristics and production behaviors can affect the implementation of HPAI vaccination on small poultry farms.

Evolution of H7N9 highly pathogenic avian influenza virus in the context of vaccination.

Human infections with the H7N9 influenza virus have been eliminated in China through vaccination of poultry; however, the H7N9 virus has not yet been eradicated from poultry. Carefully analysis of H7N9 viruses in poultry that have sub-optimal immunity may provide a unique opportunity to witness the evolution of highly pathogenic avian influenza virus in the context of vaccination. Between January 2020 and June 2023, we isolated 16 H7N9 viruses from samples we collected during surveillance and samples that were sent to us for disease diagnosis. Genetic analysis indicated that these viruses belonged to a single genotype previously detected in poultry. Antigenic analysis indicated that 12 of the 16 viruses were antigenically close to the H7-Re4 vaccine virus that has been used since January 2022, and the other four viruses showed reduced reactivity with the vaccine. Animal studies indicated that all 16 viruses were nonlethal in mice, and four of six viruses showed reduced virulence in chickens upon intranasally inoculation. Importantly, the H7N9 viruses detected in this study exclusively bound to the avian-type receptors, having lost the capacity to bind to human-type receptors. Our study shows that vaccination slows the evolution of H7N9 virus by preventing its reassortment with other viruses and eliminates a harmful characteristic of H7N9 virus, namely its ability to bind to human-type receptors.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Immunoinformatic based designing of potential immunogenic novel mRNA and peptide-based prophylactic vaccines against H5N1 and H7N9 avian influenza viruses.

Influenza viruses are the most common cause of serious respiratory illnesses worldwide and are responsible for a significant number of annual fatalities. Therefore, it is crucial to look for new immunogenic sites that might trigger an effective immune response. In the present study, bioinformatics tools were used to design mRNA and multiepitope-based vaccines against H5N1 and H7N9 subtypes of avian influenza viruses. Several Immunoinformatic tools were employed to extrapolate T and B lymphocyte epitopes of HA and NA proteins of both subtypes. The molecular docking approach was used to dock the selected HTL and CTL epitopes with the corresponding MHC molecules. Eight (8) CTL, four (4) HTL, and Six (6) linear B cell epitopes were chosen for the structural arrangement of mRNA and of peptide-based prophylactic vaccine designs. Different physicochemical characteristics of the selected epitopes fitted with suitable linkers were analyzed. High antigenic, non-toxic, and non-allergenic features of the designed vaccines were noted at a neutral physiological pH. Codon optimization tool was used to check the GC content and CAI value of constructed MEVC-Flu vaccine, which were recorded to be 50.42% and 0.97 respectively. the GC content and CAI value verify the stable expression of vaccine in pET28a + vector. In-silico immunological simulation the MEVC-Flu vaccine construct revealed a high level of immune responses. The molecular dynamics simulation and docking results confirmed the stable interaction of TLR-8 and MEVC-Flu vaccine. Based on these parameters, vaccine constructs can be regarded as an optimistic choice against H5N1 and H7N9 strains of the influenza virus. Further experimental testing of these prophylactic vaccine designs against pathogenic avian influenza strains may clarify their safety and efficacy.Communicated by Ramaswamy H. Sarma.

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.