Modelling the transmission dynamics of H9N2 avian influenza viruses in a live bird market.

H9N2 avian influenza viruses (AIVs) are a major concern for the poultry sector and human health in countries where this subtype is endemic. By fitting a model simulating H9N2 AIV transmission to data from a field experiment, we characterise the epidemiology of the virus in a live bird market in Bangladesh. Many supplied birds arrive already exposed to H9N2 AIVs, resulting in many broiler chickens entering the market as infected, and many indigenous backyard chickens entering with pre-existing immunity. Most susceptible chickens become infected within one day spent at the market, owing to high levels of viral transmission within market and short latent periods, as brief as 5.3 hours. Although H9N2 AIV transmission can be substantially reduced under moderate levels of cleaning and disinfection, effective risk mitigation also requires a range of additional interventions targeting markets and other nodes along the poultry production and distribution network.

Mapping the interaction sites of human and avian influenza A viruses and complement factor H.

The complement system is an innate immune mechanism against microbial infections. It involves a cascade of effector molecules that is activated via classical, lectin and alternative pathways. Consequently, many pathogens bind to or incorporate in their structures host negative regulators of the complement pathways as an evasion mechanism. Factor H (FH) is a negative regulator of the complement alternative pathway that protects "self" cells of the host from non-specific complement attack. FH has been shown to bind viruses including human influenza A viruses (IAVs). In addition to its involvement in the regulation of complement activation, FH has also been shown to perform a range of functions on its own including its direct interaction with pathogens. Here, we show that human FH can bind directly to IAVs of both human and avian origin, and the interaction is mediated via the IAV surface glycoprotein haemagglutinin (HA). HA bound to common pathogen binding footprints on the FH structure, complement control protein modules, CCP 5-7 and CCP 15-20. The FH binding to H1 and H3 showed that the interaction overlapped with the receptor binding site of both HAs, but the footprint was more extensive for the H3 HA than the H1 HA. The HA - FH interaction impeded the initial entry of H1N1 and H3N2 IAV strains but its impact on viral multicycle replication in human lung cells was strain-specific. The H3N2 virus binding to cells was significantly inhibited by preincubation with FH, whereas there was no alteration in replicative rate and progeny virus release for human H1N1, or avian H9N2 and H5N3 IAV strains. We have mapped the interaction between FH and IAV, the in vivo significance of which for the virus or host is yet to be elucidated.

Pandemic preparedness through vaccine development for avian influenza viruses.

Influenza A viruses pose a significant threat to global health, impacting both humans and animals. Zoonotic transmission, particularly from swine and avian species, is the primary source of human influenza outbreaks. Notably, avian influenza viruses of the H5N1, H7N9, and H9N2 subtypes are of pandemic concern through their global spread and sporadic human infections. Preventing and controlling these viruses is critical due to their high threat level. Vaccination remains the most effective strategy for influenza prevention and control in humans, despite varying vaccine efficacy across strains. This review focuses specifically on pandemic preparedness for avian influenza viruses. We delve into vaccines tested in animal models and summarize clinical trials conducted on H5N1, H7N9, and H9N2 vaccines in humans.

Mapping Genetic Markers Associated with Antigenicity and Host Range in H9N2 Influenza A Viruses Infecting Poultry in Pakistan.

The aim of the current study was to map the genetic diversity in the haemagglutinin (HA) glycoprotein of influenza A viruses (IAVs) of the H9N2 subtype. Twenty-five H9N2 IAVs were isolated from broiler chickens from March to July 2019. The HA gene was amplified, and phylogenetic analysis was performed to determine the evolutionary relationship. Important antigenic amino acid residues of HA attributed to immune escape and zoonotic potential were compared among H9N2 IAVs. Phylogenetic analysis revealed that sublineage B2 under the G1 lineage in Pakistan was found to be diversified, and newly sequenced H9N2 isolates were nested into two clades (A and B). Mutations linked to the antigenic variation and potential immune escape were observed as G72E (1/25, 4%), A180T (3/25, 12%), and A180V (1/25, 4%). A twofold significant reduction (P < 0.01) in log2 hemagglutination inhibition titers was observed with H9N2 IAV naturally harboring amino acid V180 instead of A180 in HA protein. Moreover, in the last 20 years, complete substitution at residues (T127D, D135N, and L150N) and partial substitution at residues (72, 74, 131, 148, 180, 183, 188, 216, 217, and 249, mature H9 HA numbering) associated with changes in antigenicity were observed. The presence of L216 in all H9N2 IAV isolates and T/V180 in four isolates in the receptor-binding site reveals the potential of these viruses to cross the species barrier to infect human or mammals. The current study observed the circulation of antigenically diverse H9N2 IAV variants that possess potential mutations that can escape the host immune system.

Nota de investigación- Mapeo de marcadores genéticos asociados con la antigenicidad y el rango de huéspedes en los virus de la influenza tipo A subtipo H9N2 que infectan a la avicultura en Pakistán. El objetivo del presente estudio fue mapear la diversidad genética en la glicoproteína hemaglutinina (HA) de los virus de la influenza A (IAV) del subtipo H9N2. Se aislaron veinticinco virus de influenza H9N2 de pollos de engorde de marzo a julio del 2019. Se amplificó el gene HA y se realizó un análisis filogenético para determinar la relación evolutiva. Se compararon importantes residuos de aminoácidos antigénicos de la hemaglutinina atribuidos al escape inmunológico y al potencial zoonótico entre los virus de la influenza aviar H9N2. El análisis filogenético reveló que el sublinaje B2 bajo el linaje G1 en Pakistán estaba diversificado, y los aislados de H9N2 recién secuenciados se agruparon en dos clados (A y B). Se observaron mutaciones relacionadas con la variación antigénica y el posible escape inmunológico como los residuos de aminoácidos G72E (1/25, 4%), A180T (3/25, 12%) y A180V (1/25, 4%). Se observó una reducción significativa al doble (P < 0.01) en los títulos de inhibición de la hemaglutinación log2 cuando el virus de la influenza aviar H9N2 albergaba naturalmente el aminoácido V180 en lugar del A180 en la proteína HA. Además, en los últimos 20 años, sustitución completa en los residuos (T127D, D135N y L150N) y sustitución parcial en los residuos (72, 74, 131, 148, 180, 183, 188, 216, 217 y 249, de acuerdo con la numeración de la HA subtipo madura) asociados con cambios en la antigenicidad. La presencia del residuo L216 en todos los aislados de influenza aviar H9N2 y T/V180 en cuatro aislados en el sitio de unión al receptor revela el potencial de estos virus para cruzar la barrera de las especies para infectar a humanos o mamíferos. El estudio actual observó la circulación de variantes antigénicamente diversas del virus de influenza aviar H9N2 que poseen mutaciones potenciales que pueden escapar del sistema inmunológico del huésped.

Immunogenicity and protective efficacy of a multivalent herpesvirus vectored vaccine against H9N2 low pathogenic avian influenza in chicken.

The application of recombinant herpesvirus of turkey, expressing the H9 hemagglutinin gene from low pathogenic avian influenza virus (LPAIV) H9N2 and the avian orthoavulavirus-1 (AOAV-1) (commonly known as Newcastle Disease virus (NDV)) fusion protein (F) as an rHVT-H9-F vaccine, is an alternative to currently used classical vaccines. This study investigated H9- and ND-specific humoral and mucosal responses, H9-specific cell-mediated immunity, and protection conferred by the rHVT-H9-F vaccine in specific pathogen-free (SPF) chickens. Vaccination elicited systemic NDV F- and AIV H9-specific antibody response but also local antibodies in eye wash fluid and oropharyngeal swabs. The ex vivo H9-specific stimulation of splenic and pulmonary T cells in the vaccinated group demonstrated the ability of vaccination to induce systemic and local cellular responses. The clinical protection against a challenge using a LPAIV H9N2 strain of the G1 lineage isolated in Morocco in 2016 was associated with a shorter duration of shedding along with reduced viral genome load in the upper respiratory tract and reduced cloacal shedding compared to unvaccinated controls.

Recent H9N2 avian influenza virus lost hemagglutination activity due to a K141N substitution in hemagglutinin.

The H9N2 avian influenza virus (AIV) represents a significant risk to both the poultry industry and public health. Our surveillance efforts in China have revealed a growing trend of recent H9N2 AIV strains exhibiting a loss of hemagglutination activity at 37°C, posing challenges to detection and monitoring protocols. This study identified a single K141N substitution in the hemagglutinin (HA) glycoprotein as the culprit behind this diminished hemagglutination activity. The study evaluated the evolutionary dynamics of residue HA141 and studied the impact of the N141K substitution on aspects such as virus growth, thermostability, receptor-binding properties, and antigenic properties. Our findings indicate a polymorphism at residue 141, with the N variant becoming increasingly prevalent in recent Chinese H9N2 isolates. Although both wild-type and N141K mutant strains exclusively target α,2-6 sialic acid receptors, the N141K mutation notably impedes the virus's ability to bind to these receptors. Despite the mutation exerting minimal influence on viral titers, antigenicity, and pathogenicity in chicken embryos, it significantly enhances viral thermostability and reduces plaque size on Madin-Darby canine kidney (MDCK) cells. Additionally, the N141K mutation leads to decreased expression levels of HA protein in both MDCK cells and eggs. These findings highlight the critical role of the K141N substitution in altering the hemagglutination characteristics of recent H9N2 AIV strains under elevated temperatures. This emphasizes the need for ongoing surveillance and genetic analysis of circulating H9N2 AIV strains to develop effective control and prevention measures.IMPORTANCEThe H9N2 subtype of avian influenza virus (AIV) is currently the most prevalent low-pathogenicity AIV circulating in domestic poultry globally. Recently, there has been an emerging trend of H9N2 AIV strains acquiring increased affinity for human-type receptors and even losing their ability to bind to avian-type receptors, which raises concerns about their pandemic potential. In China, there has been a growing number of H9N2 AIV strains that have lost their ability to agglutinate chicken red blood cells, leading to false-negative results during surveillance efforts. In this study, we identified a K141N mutation in the HA protein of H9N2 AIV to be responsible for the loss of hemagglutination activity. This finding provides insight into the development of effective surveillance, prevention, and control strategies to mitigate the threat posed by H9N2 AIV to both animal and human health.

Effect of Vaccination on Distribution and Immune Response of Avian Influenza Virus H9N2 in Coturnix coturnix.

Influenza viruses can multiply in quails and be transmitted to other animal species. As vaccination reduces virus shedding in chickens, the effect of the killed H9N2 avian influenza virus (AIV) on tissue distribution and virus shedding was evaluated in quails. One hundred 20-day-old quails were divided into six equal groups, kept in separate pens, and fed ad libitum. Before vaccination, blood samples were randomly collected from the wing veins. Four groups were vaccinated with the inactivated H9N2 Razi Institute vaccine at 21 days subcutaneously at the back of neck. Three weeks later, two groups were re-vaccinated. Two weeks later, at the age of 56 days, three groups were challenged with 100 µL of allantoic fluid containing 105 EID50 H9N2 through the oculonasal route. Blood samples were collected from quails at 42, 56, 63, and 70 days from each group to determine AIV antibodies by the hemagglutination inhibition test. Three quails were randomly selected and euthanized from each group on days 1, 3, and 6 post-inoculation (PI). Tissue samples were collected, and the RT-PCR test was performed. No clinical signs or gross lesions existed in any of the groups during the experiment. However, the virus was detected in different tissues on the first, third, and sixth days after the challenge in unvaccinated challenged birds. Virus detection was significantly more frequent in the quails vaccinated once and challenged than in the twice-vaccinated challenged group (P≤0.05). On the third day of PI, the virus was detected in some organs of the challenged groups. On the sixth day of PI, the virus was detected only in the lungs of two unvaccinated and once-vaccinated challenged birds. It was concluded that the vaccination of quails against AIV H9 is necessary to protect them from clinical signs, as well as respiratory tract and intestine replication. Two-time vaccination significantly protects the respiratory and intestine tracts, compared to one-time vaccination (P≤0.05).

Paper-based electrochemical immunosensor for label-free detection of multiple avian influenza virus antigens using flexible screen-printed carbon nanotube-polydimethylsiloxane electrodes.

Many studies have been conducted on measuring avian influenza viruses and their hemagglutinin (HA) antigens via electrochemical principles; most of these studies have used gold electrodes on ceramic, glass, or silicon substrates, and/or labeling for signal enhancement. Herein, we present a paper-based immunosensor for label-free measurement of multiple avian influenza virus (H5N1, H7N9, and H9N2) antigens using flexible screen-printed carbon nanotube-polydimethylsiloxane electrodes. These flexible electrodes on a paper substrate can complement the physical weakness of the paper-based sensors when wetted, without affecting flexibility. The relative standard deviation of the peak currents was 1.88% when the electrodes were repeatedly bent and unfolded twenty times with deionized water provided each cycle, showing the stability of the electrodes. For the detection of HA antigens, approximately 10-µl samples (concentration: 100 pg/ml-100 ng/ml) were needed to form the antigen-antibody complexes during 20-30 min incubation, and the immune responses were measured via differential pulse voltammetry. The limits of detections were 55.7 pg/ml (0.95 pM) for H5N1 HA, 99.6 pg/ml (1.69 pM) for H7N9 HA, and 54.0 pg/ml (0.72 pM) for H9N2 HA antigens in phosphate buffered saline, and the sensors showed good selectivity and reproducibility. Such paper-based sensors are economical, flexible, robust, and easy-to-manufacture, with the ability to detect several avian influenza viruses.

Molecular and Antigenic Characterization of Avian H9N2 Viruses in Southern China.

The H9N2 subtype avian influenza virus (AIV) has become endemic in poultry globally; however due to its low pathogenicity, it is not under primary surveillance and control in many countries. Recent reports of human infection caused by H9N2 AIV has increased public concern. This study investigated the genetic and antigenic characteristics of H9N2 AIV isolated from local markets in nine provinces in Southern China from 2013 to 2018. We detected an increasing annual isolation rate of H9N2 AIV. Phylogenetic analyses of hemagglutinin (HA) genes suggests that isolated strains were rooted in BJ94 lineage but have evolved into new subgroups (II and III), which derived from subgroup I. The estimated substitution rate of the subgroup III strains was 6.23 × 10-3 substitutions/site/year, which was 1.5-fold faster than that of the average H9N2 HA rate (3.95 × 10-3 substitutions/site/year). Based on the antigenic distances, subgroup II and III strains resulted in two clear antigenic clusters 2 and 3, separated from the vaccine strain F98, cluster 1. New antigenic properties of subgroup III viruses were associated with 11 amino acid changes in the HA protein, suggesting antigenic drift in H9N2 viruses. Our phylogenetic and antigenic analyses of the H9N2 strains circulating in local markets in Southern China provide new insights on the antigenic diversification of H9N2 viruses. IMPORTANCE The H9N2 low pathogenicity avian influenza (LPAI) virus has become endemic in poultry globally. In several Asian countries, vaccination against H9N2 avian influenza virus (AIV) was approved to reduce economic losses in the poultry industry. However, surveillance programs initiated after the introduction of vaccination identified the persistence of H9N2 AIV in poultry (especially in chicken in South Korea and China). Recent reports of human infection caused by H9N2 AIV has increased public concern. Surveillance of H9N2 circulating in poultry in the fields or markets was essential to update the vaccination strategies. This study investigated the genetic and antigenic characteristics of H9N2 AIVs isolated from local markets in nine provinces in Southern China from 2013 to 2018. The discovery of mutations in the hemagglutinin (HA) gene that result in antigenic changes provides a baseline reference for evolutionary studies of H9N2 viruses and vaccination strategies in poultry.

Transcriptomics of chicken cecal tonsils and intestine after infection with low pathogenic avian influenza virus H9N2.

Influenza viruses cause severe respiratory infections in humans and birds, triggering global health concerns and economic burden. Influenza infection is a dynamic process involving complex biological host responses. The objective of this study was to illustrate global biological processes in ileum and cecal tonsils at early time points after chickens were infected with low pathogenic avian influenza virus (LPAIV) H9N2 through transcriptome analysis. Total RNA isolated from ileum and cecal tonsils of non-infected and infected layers at 12-, 24- and 72-h post-infection (hpi) was used for mRNA sequencing analyses to characterize differentially expressed genes and overrepresented pathways. Statistical analysis highlighted transcriptomic signatures significantly occurring 24 and 72 hpi, but not earlier at 12 hpi. Interferon (IFN)-inducible and IFN-stimulated gene (ISG) expression was increased, followed by continued expression of various heat-shock proteins (HSP), including HSP60, HSP70, HSP90 and HSP110. Some upregulated genes involved in innate antiviral responses included DDX60, MX1, RSAD2 and CMPK2. The ISG15 antiviral mechanism pathway was highly enriched in ileum and cecal tonsils at 24 hpi. Overall, most affected pathways were related to interferon production and the heat-shock response. Research on these candidate genes and pathways is warranted to decipher underlying mechanisms of immunity against LPAIV in chickens.

Recombinant HA1-ΔfliC enhances adherence to respiratory epithelial cells and promotes the superiorly protective immune responses against H9N2 influenza virus in chickens.

H9N2 subtype avian influenza virus (AIV) is an ongoing threat causing substantial loss to the poultry industry and thus necessitating the development of safe and effective vaccines against AIV. Given that inactivated vaccines are less effective in activating the mucosal immune system, we aimed to generate a vaccine that can actively engage the mucosal immunity which is the front line of the immune system. We generated a group of flagellin-based hemagglutinin globular head (HA1) fusion proteins and characterized their immunogenicity and efficacy. We found that Salmonella typhimurium flagellin (fliC) lacking the hypervariable domain (called herein as HA1-ΔfliC) was recognized by TLR5 and induced a moderate innate immune response compared to N-terminus of fliC (HA1-fliC) and C-terminus of fliC (fliC-HA1). The HA1-ΔfliC protein had increased adherence to the nasal cavity and trachea than HA1-fliC and fliC-HA1 and significantly increased the HA-specific sIgA titers. Our in vivo results revealed that chickens treated with HA1-ΔfliC had a significantly reduced level of viral loads in the cloaca and throat compared with chickens treated with inactivated vaccine. Overall, these results revealed that HA1-ΔfliC can protect chickens against H9N2 AIV by eliciting the efficient mucosal immune responses.

Bacillus subtilis Spore-Trained Dendritic Cells Enhance the Generation of Memory T Cells via ICAM1.

Immunological memory is a cardinal feature of the immune system. The intestinal mucosa is the primary exposure and entry site of infectious organisms. For an effective and long-lasting safeguard, a robust immune memory system is required, especially by the mucosal immunity. It is well known that tissue-resident memory T cells (Trms) provide a first response against infections reencountered at mucosal tissues surfaces, where they accelerate pathogen clearance. However, their function in intestinal immunization remains to be investigated. Here, we report enhanced local mucosal and systemic immune responses through oral administration of H9N2 influenza whole inactivated virus (H9N2 WIV) plus Bacillus subtilis spores. Subsequently, H9N2 WIV plus spores led to the generation of CD103+ CD69+ Trms, which were independent of circulating T cells during the immune period. Meanwhile, we also found that Bacillus subtilis spores could stimulate Acrp30 expression in 3T3-L1 adipocytes. Moreover, spore-stimulated adipocyte supernatant also upregulated the expression of intercellular adhesion molecule-1 (ICAM1) in dendritic cells (DCs). Furthermore, the proportion of HA-tetramer+ cells was severely curtailed upon suppressed ICAM1 expression, which also depended on HA-loaded DCs. Taken together, our data demonstrated that spore-promoted H9N2 WIV induced an immune response by enhancing Trms populations, which were associated with the activation of ICAM1 in DCs.

Pathogenesis of Avian Influenza Virus Subtype H9N2 in Turkeys and Evaluation of Inactivated Vaccine Efficacy.

Avian influenza H9N2 viruses circulate in all types of poultry species, including turkeys, and cause significant losses for the poultry industry in many parts of the word. The aim of this study was to assess the pathogenesis of the Moroccan avian influenza virus (AIV) H9N2 under experimental conditions in turkeys and the protection efficacy of an inactivated commercial vaccine against AIV H9N2. Unvaccinated turkeys showed marked depression sinusitis, respiratory distress characterized by bronchiolar and tracheal rales of moderate severity, and a mortality rate of 50%. Postmortem examinations of dead and euthanatized birds revealed the presence of fibrinous tracheitis and airsacculitis lesions. Vaccination reduced the mortality rate to 20%. Vaccinated birds recovered at day 10 postchallenge, and only 12.5% (1/8) and 37.5% of birds still displayed fibrinous and nonfibrinous airsacculitis lesions, respectively, at day 15 postinoculation. Viral shedding in cloacal and tracheal swabs was lower in vaccinated than in control birds. Although viral RNA was detected in the cloacal swabs of all unvaccinated turkeys at day 3 postinoculation, only 50% of the vaccinated turkeys were positive for virus detection. At day 11 postinoculation, no viral RNA was detected in oropharyngeal swabs of vaccinated turkeys, whereas 40% of the unvaccinated turkeys were still shedding virus.

Artículo regular­Patogenia del subtipo H9N2 del virus de la influenza aviar en pavos y evaluación de la eficacia de una vacuna inactivada. Los virus de la influenza aviar H9N2 circulan en todo tipo de especies de aves comerciales, incluidos los pavos, y causan pérdidas significativas para la industria avícola en muchas partes del mundo. El objetivo de este estudio fue evaluar la patogenia del virus de la influenza aviar de Marruecos (AIV) H9N2 bajo condiciones experimentales en pavos y la eficacia de protección de una vacuna comercial inactivada contra el virus de la influenza aviar H9N2. Los pavos no vacunados mostraron una marcada sinusitis, depresión, dificultad respiratoria caracterizada por estertores bronquiolares y traqueales de severidad moderada y una tasa de mortalidad del 50%. Los exámenes post mortem de aves muertas y sacrificadas revelaron la presencia de traqueítis fibrinosa y aerosaculitis. La vacunación redujo la tasa de mortalidad al 30%. Las aves vacunadas se recuperaron en el día 10 después del desafío, y solo el 12.5% (1/8) y el 37.5% de las aves todavía mostraban aerosaculitis fibrinosa y no fibrinosa, respectivamente, el día 15 después de la inoculación. La diseminación viral en los hisopos cloacales y traqueales fue menor en las aves vacunadas que en las aves control. Aunque se detectó ARN viral en los hisopados cloacales de todos los pavos no vacunados en el día tres después de la inoculación, solo el 50% de los pavos vacunados dieron positivo para la detección del virus. En el día 11 después de la inoculación, no se detectó ARN viral en hisopados orofaríngeos de pavos vacunados, mientras que el 40% de los pavos no vacunados todavía estaban diseminando virus.

Antibody Response of an H9 Subtype Avian Influenza Poultry Vaccine on Three Kinds of Wild Birds in Shanghai Zoo.

A semiannual immunization with a commercial inactivated H9 subtype avian influenza virus (AIV) vaccine developed for poultry has been used to prevent and control the avian influenza (AI) infections among captured wild birds in Shanghai Zoo. However, the overall safety and effectiveness of the poultry vaccine for housed birds in the zoo remain unclear. To verify the safety and efficacy of the commercial inactivated H9 AI vaccine on zoo birds and to explore a more reasonable and effective immunization procedure, 48 zoo birds, including 11 Oriental white storks, 25 peafowl, and 12 silver pheasants, were administered the AI vaccine developed for poultry use. Then, the clinical signs of the immunized birds were observed for 2 weeks, and the antibodies against H9 AI were determined via the hemagglutination inhibition test. Results showed that no harmful effects related to the vaccination were observed, and the antibody titers of the Oriental white stork, peafowl, and silver pheasants were all higher than 7 log 2 at 21 days, 30 days, 60 days, 120 days, and 180 days postimmunization. For further study, the H9 AIV titers of 11 peafowls and 6 Oriental storks, which were raised in the nursing ground, were continuously monitored for 15 months. All of their antibody titers were above the national standards of China (5 log 2; GB/T18936-2003), even at 12 months and 15 months postimmunization. We concluded that the commercial inactivated H9 AI vaccine used at the present time in Shanghai Zoo can induce high and prolonged immune responses in vaccinated birds.

Artículo regular­Respuesta de anticuerpos de una vacuna contra influenza aviar subtipo H9 en tres tipos de aves silvestres en el zoológico de Shanghai. Se ha utilizado una inmunización semestral con una vacuna comercial inactivada contra el virus de la influenza aviar del subtipo H9 (AIV) desarrollada para la avicultura comercial para prevenir y controlar las infecciones por influenza aviar (IA) entre aves silvestres cautivas en el zoológico de Shanghai. Sin embargo, la seguridad y eficacia generales de la vacuna avícola para las aves alojadas en el zoológico siguen sin estar completamente determinadas. Para verificar la seguridad y eficacia de la vacuna comercial contra la influenza aviar H9 inactivada en las aves de zoológico y para explorar un procedimiento de inmunización más razonable y eficaz, una vacuna contra la influenza desarrollada para su uso en avicultura comercial se administró a 48 aves de zoológico, incluyendo 11 cigüeñas blancas orientales, 25 pavos reales y 12 faisanes plateados. Posteriormente, se observaron los signos clínicos de las aves inmunizadas durante 2 semanas y se determinaron los anticuerpos contra el subtipo H9 del virus de influenza aviar mediante la prueba de inhibición de la hemaglutinación. Los resultados mostraron que no se desarrollaron efectos nocivos relacionados con la vacunación, y los títulos de anticuerpos de la cigüeña blanca oriental, el pavo real y los faisanes plateados fueron todos superiores a 7 log2 a los 21 días, 30 días, 60 días, 120 días y 180 días posinmunización. Para un estudio adicional, los títulos contra el subtipo H9 de 11 pavos reales y 6 cigüeñas orientales que se criaron en el área de recría, se evaluaron continuamente durante 15 meses. Todos sus títulos de anticuerpos estaban por encima de los estándares nacionales de China (5 log2; GB/T18936-2003), incluso a los 12 y 15 meses posteriores a la inmunización. Se concluye que la vacuna comercial de influenza aviar inactivada con el subtipo H9 que se utiliza actualmente en el zoológico de Shanghai puede inducir respuestas inmunitarias elevadas y prolongadas en las aves vacunadas.

Development of PDA Nanoparticles for H9N2 Avian Influenza BPP-V/BP-IV Epitope Peptide Vaccines: Immunogenicity and Delivery Efficiency Improvement.

The protection of current influenza vaccines is limited due to the viral antigenic shifts and antigenic drifts. The universal influenza vaccine is a new hotspot in vaccine research that aims to overcome these problems. Polydopamine (PDA), a versatile biomaterial, has the advantages of an excellent biocompatibility, controllable particle size, and distinctive drug loading approach in drug delivery systems. To enhance the immunogenicities and delivery efficiencies of H9N2 avian influenza virus (AIV) epitope peptide vaccines, PDA nanoparticles conjugated with the BPP-V and BP-IV epitope peptides were used to prepare the nano BPP-V and BP-IV epitope peptide vaccines, respectively. The characteristics of the newly developed epitope peptide vaccines were then evaluated, revealing particle sizes ranging from approximately 240 to 290 nm (PDI<0.3), indicating that the synthesized nanoparticles were stable. Simultaneously, the immunoprotective effects of nano BPP-V and BP-IV epitope peptide vaccines were assessed. The nano BPP-V and BP-IV epitope vaccines, especially nano BP-IV epitope vaccine, quickly induced anti-hemagglutinin (HA) antibody production and a sustained immune response, significantly promoted humoral and cellular immune responses, reduced viral lung damage and provided effective protection against AIV viral infection. Together, these results reveal that PDA, as a delivery carrier, can improve the immunogenicities and delivery efficiencies of H9N2 AIV nano epitope vaccines, thereby providing a theoretical basis for the design and development of PDA as a carrier of new universal influenza vaccines.

Generation and Evaluation of Recombinant Thermostable Newcastle Disease Virus Expressing the HA of H9N2 Avian Influenza Virus.

H9N2 avian influenza virus (AIV) has become endemic in many countries, causing great economic losses when co-infected with other pathogens. So far, several live vaccines based on Newcastle disease virus (NDV) vectors expressing influenza hemagglutinin (HA) have been developed. However, the thermostable recombinant NDV is rarely reported. In this study, using a thermostable NDV rAHR09 strain as the vector, three recombinant NDVs expressing native HA, chimeric HA ectodomain with transmembrane domain/C-terminal cytoplasmic tail domain from fusion protein of NDV, and HA ectodomain were generated, designated rAHR09-HA, rAHR09-HAF, and rAHR09-HAE. The MDT value of three recombinant NDVs was above 120 h, their ICPI value was about 0.03, and the recombinant NDVs were still infectious when treated for 100 min under 56 °C, which demonstrated that the recombinant NDVs kept the lentogenic and thermostable nature of rAHR09. The immunization data showed that rAHR09-HA and rAHR09-HAF induced a higher HI antibody titer against H9N2 AIV and NDV. After being challenged with H9N2 AIV, the rAHR09-HA and rAHR09-HAF could significantly reduce the virus shedding in cloacal and tracheal swab samples. Our results suggest that rAHR09-HA and rAHR09-HAF might be vaccine candidates against H9N2 AIV.

Development of a Rapid Fluorescent Diagnostic System to Detect Subtype H9 Influenza A Virus in Chicken Feces.

The circulation of the H9N2 virus results in significant economic losses in the poultry industry, and its zoonotic transmission highlights the need for a highly sensitive and rapid diagnostic and detection system for this virus. In this study, the performance of lateral flow test strips for a fluorescent immunochromatographic test (FICT) was optimized for the diagnosis of H9N2 virus-infected animal samples. The novel monoclonal antibodies (McAbs) against influenza A H9 viruses were developed, and two categories of McAbs with linear and conformational epitopes were compared for the performance of rapid diagnostic performance in the presence of feces sample at different time points (2, 4, and 6 days) post-infection (dpi). The limit of detection (LOD) of FICT and Kd values were comparable between linear and conformational epitope McAbs. However, superior performance of linear epitope McAbs pairs were confirmed by two animal studies, showing the better diagnostic performance showing 100% relative sensitivity in fecal samples at 6 dpi although it showed less than 80% sensitivity in early infection. Our results imply that the comparable performance of the linear epitope McAbs can potentially improve the diagnostic performance of FICT for H9N2 detection in feces samples. This highly sensitive rapid diagnostic method can be utilized in field studies of broiler poultry and wild birds.

Plant-based, adjuvant-free, potent multivalent vaccines for avian influenza virus via Lactococcus surface display.

Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHAH5N6 + H9N2 BLP or a combination of tHAH5N6 BLP and tHAH9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.

Egyptian Fruit Bats (Rousettus aegyptiacus) Were Resistant to Experimental Inoculation with Avian-Origin Influenza A Virus of Subtype H9N2, But Are Susceptible to Experimental Infection with Bat-Borne H9N2 Virus.

Influenza A viruses (IAV) of subtype H9N2, endemic in world-wide poultry holdings, are reported to cause spill-over infections to pigs and humans and have also contributed substantially to recent reassortment-derived pre-pandemic zoonotic viruses of concern, such as the Asian H7N9 viruses. Recently, a H9N2 bat influenza A virus was found in Egyptian fruit bats (Rousettus aegyptiacus), raising the question of whether this bat species is a suitable host for IAV. Here, we studied the susceptibility, pathogenesis and transmission of avian and bat-related H9N2 viruses in this new host. In a first experiment, we oronasally inoculated six Egyptian fruit bats with an avian-related H9N2 virus (A/layer chicken/Bangladesh/VP02-plaque/2016 (H9N2)). In a second experiment, six Egyptian fruit bats were inoculated with the newly discovered bat-related H9N2 virus (A/bat/Egypt/381OP/2017 (H9N2)). While R. aegyptiacus turned out to be refractory to an infection with H9N2 avian-type, inoculation with the bat H9N2 subtype established a productive infection in all inoculated animals with a detectable seroconversion at day 21 post-infection. In conclusion, Egyptian fruit bats are most likely not susceptible to the avian H9N2 subtype, but can be infected with fruit bat-derived H9N2. H9-specific sero-reactivities in fruit bats in the field are therefore more likely the result of contact with a bat-adapted H9N2 strain.

Phenotypic and Functional Characteristics of a Novel Influenza Virus Hemagglutinin-Specific Memory NK Cell.

Immune memory represents the most efficient defense against invasion and transmission of infectious pathogens. In contrast to memory T and B cells, the roles of innate immunity in recall responses remain inconclusive. In this study, we identified a novel mouse spleen NK cell subset expressing NKp46 and NKG2A induced by intranasal influenza virus infection. These memory NK cells specifically recognize N-linked glycosylation sites on influenza hemagglutinin (HA) protein. Different from memory-like NK cells reported previously, these NKp46+ NKG2A+ memory NK cells exhibited HA-specific silence of cytotoxicity but increase of gamma interferon (IFN-γ) response against influenza virus-infected cells, which could be reversed by pifithrin-µ, a p53-heat shock protein 70 (HSP70) signaling inhibitor. During recall responses, splenic NKp46+ NKG2A+ NK cells were recruited to infected lung and modulated viral clearance of virus and CD8+ T cell distribution, resulting in improved clinical outcomes. This long-lived NK memory bridges innate and adaptive immune memory response and promotes the homeostasis of local environment during recall response.IMPORTANCE In this study, we demonstrate a novel hemagglutinin (HA)-specific NKp46+ NKG2A+ NK cell subset induced by influenza A virus infection. These memory NK cells show virus-specific decreased cytotoxicity and increased gamma interferon (IFN-γ) on reencountering the same influenza virus antigen. In addition, they modulate host recall responses and CD8 T cell distribution, thus bridging the innate immune and adaptive immune responses during influenza virus infection.