Influenza Virus In Chickens Research Articles

Hemagglutinin and neuraminidase of a non-pathogenic H7N7 avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens.

Polybasic amino acid residues at the hemagglutinin (HA) cleavage site are insufficient to induce the highly pathogenic phenotype of avian influenza viruses in chickens. In our previous study, an H7N7 avian influenza virus named "Vac2sub-P0", which is nonpathogenic despite carrying polybasic amino acids at the HA cleavage site, was passaged in chick air sacs, and a virus with high intravenous pathogenicity, Vac2sub-P3, was obtained. Intranasal infection with Vac2sub-P3 resulted in limited lethality in chickens; therefore, in this study, this virus was further passaged in chicken lungs, and the resultant virus, Vac2sub-P3L4, acquired high intranasal pathogenicity. Experimental infection of chickens with recombinant viruses demonstrated that mutations in HA and neuraminidase (NA) found in consecutive passages were responsible for the increased pathogenicity. The HA and NA functions of Vac2sub-P3L4 were compared with those of the parental virus in vitro; the virus growth at 40 °C was faster, the binding affinity to a sialic acid receptor was lower, and the rate of release by NA from the cell surface was lower, suggesting that these changes enabled the virus to replicate efficiently in chickens with high intranasal pathogenicity. This study demonstrates that viruses that are highly pathogenic when administered intranasally require additional adaptations for increased pathogenicity to be highly lethal to intranasally infected chickens.

A Rationally Designed H5 Hemagglutinin Subunit Vaccine Provides Broad-Spectrum Protection against Various H5Nx Highly Pathogenic Avian Influenza Viruses in Chickens.

The evolution of the H5 highly pathogenic avian influenza (HPAI) viruses has led to the emergence of distinct groups with genetically similar clusters of hemagglutinin (HA) sequences. In this study, a consensus H5 HA sequence was cloned into the baculovirus expression system. The HA protein was expressed in baculovirus-infected insect cells and utilized as the antigen for the production of an oil emulsion-based H5 avian influenza vaccine (rBacH5Con5Mut). Twenty-one-day-old SPF chickens were immunized with this vaccine and then challenged at 21 days post-vaccination with clade 2.3.2.1, clade 2.3.4.4, and clade 7.2 of H5 HPAI viruses. The sera of vaccinated chickens exhibited high hemagglutination inhibition (HI) titers against the rBacH5 vaccine antigen, while lower HI titers were observed against the different challenge virus H5 hemagglutinins. Furthermore, the rBacH5Con5Mut vaccine provided 100% protection from mortality and clinical signs. Virus isolation results showed that oropharyngeal and cloacal shedding was prevented in 100% of the vaccinated chickens when challenged with clade 2.3.2.1 and clade 2.3.4.4 H5 viruses. When the rBacH5Con5Mut vaccine candidate was administrated at one day of age, 100% protection was demonstrated against the challenge of clade 2.3.4.4 virus at three weeks of age, indicating the potential of this vaccine for hatchery vaccination. Overall, A single immunization of rBacH5Con5Mut vaccine candidate with a consensus HA antigen can protect chickens against different clades of H5 HPAI viruses throughout the rearing period of broiler chickens without a boost, thus fulfilling the criteria for an efficacious broad-spectrum H5 avian influenza vaccine.

Efficacy of commercial recombinant HVT vaccines against a North American clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus in chickens.

The outbreak of clade 2.3.4.4b H5 highly pathogenic avian influenza (HPAI) in North America that started in 2021 has increased interest in applying vaccination as a strategy to help control and prevent the disease in poultry. Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade 2.3.4.4b H5 HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both chicken types COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken types. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both types of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.

Pathogenicity and transmission of novel highly pathogenic H7N2 variants originating from H7N9 avian influenza viruses in chickens

The H7 subtype avian influenza viruses are circulating widely worldwide, causing significant economic losses to the poultry industry and posing a serious threat to human health. In 2019, H7N2 and H7N9 co-circulated in Chinese poultry, yet the risk of H7N2 remained unclear. We isolated and sequenced four H7N2 viruses from chickens, revealing them as novel reassortants with H7N9-derived HA, M, NS genes and H9N2-derived PB2, PB1, PA,NP, NA genes. To further explore the key segment of pathogenicity, H7N2-H7N9NA and H7N2-H9N2HA single-substitution were constructed. Pathogenicity study showed H7N2 isolates to be highly pathogenic in chickens, with H7N2-H7N9NA slightly weaker than H7N2-Wild type. Transcriptomic analysis suggested that H7N9-derived HA genes primarily drove the high pathogenicity of H7N2 isolates, eliciting a strong inflammatory response. These findings underscored the increased threat posed by reassorted H7N2 viruses to chickens, emphasizing the necessity of long-term monitoring of H7 subtype avian influenza viruses.

Cecropin AD reduces viral load and inflammatory response against H9N2 avian influenza virus in chickens.

This study focuses on evaluating the therapeutic efficacy of cecropin AD, an antimicrobial peptide, against H9N2 avian influenza virus (AIV) in chickens. Given the global impact of H9N2 AIV on poultry health, identifying effective treatments is crucial. To assess the impact of cecropin AD, we conducted in vivo experiments involving 108 5-week-old chickens divided into control, infected, and various treatment groups based on cecropin AD dosage levels (high, medium, and low). The methodologies included hemagglutination (HA) tests for viral titers, histopathological examination and toluidine blue (TB) staining for lung pathology, real-time PCR for viral detection, and enzyme-linked immunosorbent assays for measuring serum levels of inflammatory markers. The findings revealed that cecropin AD substantially reduced lung pathology and viral load, especially at higher dosages, comparing favorably with the effects seen from conventional treatments. Moreover, cecropin AD effectively modulated mast cell activity and the levels of inflammatory markers such as IL-6, TNF-α, IFN-γ, and 5-HT, indicating its potential to diminish inflammation and viral spread. Cecropin AD presents a significant potential as an alternative treatment for H9N2 AIV in chickens, as evidenced by its ability to lessen lung damage, decrease viral presence, and adjust immune responses. This positions cecropin AD as a promising candidate for further exploration in the management of H9N2 AIV infections in poultry.

The Chinese Hamster Ovary Cell-Based H9 HA Subunit Avian Influenza Vaccine Provides Complete Protection against the H9N2 Virus Challenge in Chickens.

(1) Background: Avian influenza has attracted widespread attention because of its severe effect on the poultry industry and potential threat to human health. The H9N2 subtype of avian influenza viruses was the most prevalent in chickens, and there are several commercial vaccines available for the prevention of the H9N2 subtype of avian influenza viruses. However, due to the prompt antigenic drift and antigenic shift of influenza viruses, outbreaks of H9N2 viruses still continuously occur, so surveillance and vaccine updates for H9N2 subtype avian influenza viruses are particularly important. (2) Methods: In this study, we constructed a stable Chinese hamster ovary cell line (CHO) to express the H9 hemagglutinin (HA) protein of the major prevalent H9N2 strain A/chicken/Daye/DY0602/2017 with genetic engineering technology, and then a subunit H9 avian influenza vaccine was prepared using the purified HA protein with a water-in-oil adjuvant. (3) Results: The results showed that the HI antibodies significantly increased after vaccination with the H9 subunit vaccine in specific-pathogen-free (SPF) chickens with a dose-dependent potency of the immunized HA protein, and the 50 μg or more per dose HA protein could provide complete protection against the H9N2 virus challenge. (4) Conclusions: These results indicate that the CHO expression system could be a platform used to develop the subunit vaccine against H9 influenza viruses in chickens.

Efficacy of an inactivated influenza vaccine adjuvanted with Toll-like receptor ligands against transmission of H9N2 avian influenza virus in chickens

Avian influenza viruses (AIV), including the H9N2 subtype, pose a major threat to the poultry industry as well as to human health. Although vaccination provides a protective control measure, its effect on transmission remains uncertain in chickens. The objective of the present study was to investigate the efficacy of beta-propiolactone (BPL) whole inactivated H9N2 virus (WIV) vaccine either alone or in combination with CpG ODN 2007 (CpG), poly(I:C) or AddaVax™ (ADD) to prevent H9N2 AIV transmission in chickens. The seeder chickens (trial 1) and recipient chickens (trial 2) were vaccinated twice with different vaccine formulations. Ten days after secondary vaccination, seeder chickens were infected with H9N2 AIV (trial 1) and co-housed with healthy recipient chickens. In trial 2, the recipient chickens were vaccinated and then exposed to H9N2 AIV-infected seeder chickens. Our results demonstrated that BPL+ CpG and BPL+ poly(I:C) treated chickens exhibited reduced oral and cloacal shedding in both trials post-exposure (PE). The number of H9N2 AIV+ recipient chickens in the BPL+ CpG group (trial 1) was lower than in other vaccinated groups, and the reduction was higher in BPL+ CpG recipient chickens in trial 2. BPL+ CpG vaccinated chickens demonstrated enhanced systemic antibody responses with high IgM and IgY titers with higher rates of seroprotection by day 21 post-primary vaccination (ppv). Additionally, the induction of IFN-γ expression and production was higher in the BPL+ CpG treated chickens. Interleukin (IL)− 2 expression was upregulated in both BPL+ CpG and BPL+ poly(I:C) groups at 12 and 24 hr post-stimulation.

Mutations in HA and PA affect the transmissibility of H7N9 avian influenza virus in chickens

Low pathogenic (LP) H7N9 avian influenza virus (AIV) emerged in 2013 and had spread widely over several months in China, experienced a noteworthy reduction in isolation rate in poultry and human since 2017. Here, we examined the transmission of H7N9 viruses to better understand viral spread and dissemination mechanisms. Three out of four viruses (2013–2016) could transmit in chickens through direct contact, and airborne transmission was confirmed in the JT157 (2016) virus. However, we did not detect the transmission of the two 2017 viruses, WF69 and AH395, through either direct or airborne exposure. Molecular analysis of genome sequence of two viruses identified eleven mutations located in viral proteins (except for matrix protein), such as PA (K362R and S364N) and HA (D167N, H7 numbering), etc. We explored the genetic determinants that contributed to the difference in transmissibility of the viruses in chickens by generating a series of reassortants in the JT157 background. We found that the replacement of HA gene in JT157 by that of WF69 abrogated the airborne transmission in recipient chickens, whereas the combination of HA and PA replacement led to the loss of airborne and direct contact transmission. Failure with contact transmission of the viruses has been associated with the emergence of the mutations D167N in HA and K362R and S364N in PA. Furthermore, the HA D167N mutation significantly reduced viral attachment to chicken lung and trachea tissues, while mutations K362R and S364N in PA reduced the nuclear transport efficiency and the PA protein expression levels in both cytoplasm and nucleus of CEF cells. The D167N substitution in HA reduced the H7N9 viral acid stability and avian-like receptor binding, while enhanced human-like receptor binding. Further analysis revealed these mutants grew poorly in vitro and in vivo. To conclude, H7N9 AIVs that contain mutations in the HA and PA protein reduced the viral transmissibility in chicken, and may pose a reduced threat for poultry but remain a heightened public health risk.

Efficacy of inactivated and RNA particle vaccines against a North American Clade 2.3.4.4b H5 highly pathogenic avian influenza virus in chickens

Highly pathogenic avian influenza virus (HPAIV) has caused widespread outbreaks in poultry in the Americas. Because of the duration and extent of these outbreaks, vaccine use may be an additional tool to limit virus spread. Three vaccines were evaluated for efficacy in chickens against a current North American clade 2.3.4.4b H5 HPAIV isolate, A/turkey/Indiana/3703-003/2022 H5N1. The vaccines included: 1) a commercial inactivated reverse genetics (rg) generated H5N1 product with a clade 2.3.4.4c H5 hemagglutinin (HA) (rgH5N1); 2) a commercial alphavirus RNA particle (RP) vaccine with the TK/IN/22 HA; and 3) an in-house inactivated rg produced vaccine with the TK/IN/22 HA and a North American lineage N9 neuraminidase (NA) (SEP-22-N9). Both inactivated vaccines were produced with HA genes that were modified to be low pathogenic and with the remaining genes from the PR8 influenza strain. All vaccines provided 100% protection against mortality and morbidity and all vaccines reduced virus shed by the oropharyngeal and cloacal routes significantly compared to sham vaccinates. However, differences were observed among the vaccines in quantities of virus shed at two- and four-days post challenge (DPC). To determine if infected birds could be identified after vaccination to aid surveillance programs, serum was collected from the RP and SEP-22-N9 vaccine groups at 7, 10, and 14 DPC to detect antibody to the NA and nucleoprotein (NP) of the challenge virus by enzyme linked lectin assay (ELLA) and ELISA. As early as 7DPC ELLA detected antibody in sera from 100% of the chickens in the RP vaccinated group and 70% of the chickens in the SEP-22-N9 vaccinated group. Antibody to the NP was detected by commercial ELISA in more than 50% of the birds in the RP vaccinated group at each time point.

Pathogenicity of H5N8 avian influenza virus in chickens and in duck breeds and the role of MX1 and IFN-α in infection outcome and transmission to contact birds

This study examined the pathogenicity, immunogenicity, and transmission potential of the H5N8 HPAI clade 2.3.4.4b virus in three breeds of ducks and in broiler chickens. Chickens, Muscovy, Pekin, and Mallard ducks (n = 10) received a dose of 6 log10 EID50 of HPAIV H5N8 directly. Nine contact chickens were introduced to each group on the day of infection. All infected chickens died, with MDT of 7.6 days. Muscovy and Pekin ducks died by 11.1% and 10%, respectively, with MDTs of 7 and 6 days. No Mallards died but showed more severe clinical disease than Pekin ducks. Mallards had the highest MX1 gene expression in the lung and spleen and IFN-α in the spleen. MX1 expression levels were lower in the spleen and lung of Pekin ducks, in the spleen of chickens and in the lung of Muscovy ducks than in noninfected controls. However, viral shedding was higher in ducks than in chickens and was highest in Mallards. 66.7% of chickens placed in contact with infected chickens died and 77.8% of in-contact chickens to infected three duck breeds died. In conclusion, there was a diversity in sensitivity and immunogenicity for HPAIV H5N8 among duck breeds, resulting in diverse infection outcomes and transmissibility to contacts. This study provides duck/chicken interface models for HPAIV transmission to poultry.

Loss of amino acids 67-76 in the neuraminidase protein under antibody selection pressure alters the tropism, transmissibility and innate immune response of H9N2 avian influenza virus in chickens

H9N2 virus has become the most widespread subtype of avian influenza in Chinese poultry. Although many studies have been published on this disease, the pathogenesis of the H9N2 virus remains to be fully understood. In our previous work, we identified 44 viral strains with 67–76 amino acid deletions in the neuraminidase protein (NA∆67–76) from trachea and lung tissues after 20 successive generations in vaccinated chickens. Interestingly, these 10 amino acid deletions are located in the stalk of the NA protein, and all mutations were unique to the viruses under the selection pressure of vaccine antibodies. To investigate the effect of NA∆67–76 on the H9N2 virus, the NA∆67–76 deletion mutant (rF/NAΔ67–76) was constructed in the H9N2 virus A/Chicken/Shanghai/F/98 (F/98) to assess the phenotypic changes between the parental and mutant strains. The results showed that the recombinant virus rF/NAΔ67–76 had no significantly effect on the antigenicity of the virus or on the infectivity of the host cells, but it significantly inhibited the release of virions from host cells. In addition, rF/NAΔ67–76 efficiently enhanced the neuraminidase activity and improved the receptor binding ability of the virus, indicating that the influence of receptor binding ability on the rF/NAΔ67–76 virus is much greater than that of neuraminidase activity. Furthermore, this study revealed that rF/NAΔ67–76 reduced the viral replication ability at 6 and 12 h post-infection, but improved it at 24, 48, and 72 h post-infection. Chicken experiments showed that rF/NAΔ67–76 exhibits a much higher tissue tropism for the trachea rather than lung tissue. rF/NAΔ67–76 still had the ability to infect the upper respiratory tract through aerosol, but its cloaca replication capacity was significantly reduced. Both in vivo and in vitro experiments confirmed that rF/NAΔ67–76 could produce a stronger innate immune response after infecting cells and chickens, especially significantly enhancing the transcription levels of TLR3, TLR4, TLR7, TLR21, MDA5, and NLRP3. Altogether, the results of this study propose that antibody selection pressure plays an important role in the evolution of H9N2 avian influenza virus.

Comparative Investigation of Coincident Single Nucleotide Polymorphisms Underlying Avian Influenza Viruses in Chickens and Ducks.

Avian influenza is a severe viral infection that has the potential to cause human pandemics. In particular, chickens are susceptible to many highly pathogenic strains of the virus, resulting in significant losses. In contrast, ducks have been reported to exhibit rapid and effective innate immune responses to most avian influenza virus (AIV) infections. To explore the distinct genetic programs that potentially distinguish the susceptibility/resistance of both species to AIV, the investigation of coincident SNPs (coSNPs) and their differing causal effects on gene functions in both species is important to gain novel insight into the varying immune-related responses of chickens and ducks. By conducting a pairwise genome alignment between these species, we identified coSNPs and their respective effect on AIV-related differentially expressed genes (DEGs) in this study. The examination of these genes (e.g., CD74, RUBCN, and SHTN1 for chickens and ABCA3, MAP2K6, and VIPR2 for ducks) reveals their high relevance to AIV. Further analysis of these genes provides promising effector molecules (such as IκBα, STAT1/STAT3, GSK-3β, or p53) and related key signaling pathways (such as NF-κB, JAK/STAT, or Wnt) to elucidate the complex mechanisms of immune responses to AIV infections in both chickens and ducks.

A Comparison of Host Responses to Infection with Wild-Type Avian Influenza Viruses in Chickens and Tufted Ducks.

Cross-species transmission of influenza A virus (IAV) from wild waterfowl to poultry is the first step in a chain of events that can ultimately lead to exposure and infection of humans. Herein, we study the outcome of infection with eight different mallard-origin IAV subtypes in two different avian hosts: tufted ducks and chickens. We found that infection and shedding patterns as well as innate immune responses were highly dependent on viral subtypes, host species, and inoculation routes. For example, intraoesophageal inoculation, commonly used in mallard infection experiments, resulted in no infections in contrast to oculonasal inoculation, suggesting a difference in transmission routes. Despite H9N2 being endemic in chickens, inoculation of mallard-origin H9N2 failed to cause viable infection beyond 1 day postinfection in our study design. The innate immune responses were markedly different in chickens and tufted ducks, and despite the presence of retinoic acid-inducible gene-I (RIG-I) in tufted duck transcriptomes, it was neither up nor downregulated in response to infection. Overall, we have revealed the heterogeneity of infection patterns and responses in two markedly different avian hosts following a challenge with mallard-origin IAV. These virus-host interactions provide new insights into important aspects of interspecies transmission of IAV. IMPORTANCE Our current findings highlight important aspects of IAV infection in birds that have implications for our understanding of its zoonotic ecology. In contrast to mallards where the intestinal tract is the main site of IAV replication, chickens and tufted ducks show limited or no signs of intestinal infection suggesting that the fecal-oral transmission route might not apply to all bird IAV host species. Our results indicate that mallard-origin IAVs undergo genetic changes upon introduction into new hosts, suggesting rapid adaptation to a new environment. However, similar to the mallard, chickens and tufted ducks show a limited immune response to infection with low pathogenic avian influenza viruses. These findings and future studies in different IAV hosts are important for our understanding of barriers to IAV transmission between species and ultimately from the wild reservoir to humans.

Diverse infectivity, transmissibility, and pathobiology of clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses in chickens

ABSTRACT Clade 2.3.4.4 Eurasian lineage H5Nx highly pathogenic avian influenza virus (HPAIV) has become the globally dominant clade and caused global outbreaks since 2014. The clade 2.3.4.4 viruses have evolved into eight hemagglutinin subgroups (2.3.4.4a-h). In this study, we evaluated the infectivity, pathobiology, and transmissibility of seven clade 2.3.4.4 viruses (two 2.3.4.4a, two 2.3.4.4b, one 2.3.4.4c and two 2.3.4.4e) in chickens. The two clade 2.3.4.4e viruses caused 100% mortality and transmissibility in chickens. However, clade 2.3.4.4a and c viruses showed 80–90% mortality and 67% transmissibility. Clade 2.3.4.4b viruses showed 100% mortality, but no transmission to co-housed chickens was observed based on lack of seroconversion. All the infected chickens died showing systemic infection, irrespective of subgroup. The results highlight that all the clade 2.3.4.4 HPAIVs used in this study caused high mortality in infected chickens, but the transmissibility of the viruses in chickens was variable in contrast to that of previous Eurasian-lineage H5N1 HPAIVs. Changes in the pathogenicity and transmissibility of clade 2.3.4.4 HPAIVs warrant careful monitoring of the viruses to establish effective control strategies.

Vaccination with recombinant Lactococcus lactis expressing HA1-IgY Fc fusion protein provides protective mucosal immunity against H9N2 avian influenza virus in chickens

BackgroundH9N2 virus is mainly transmitted through the respiratory mucosal pathway, so mucosal immunity is considered to play a good role in controlling avian influenza infection. It is commonly accepted that no adequate mucosal immunity is achieved by inactivated vaccines, which was widely used to prevent and control avian influenza virus infection. Thus, an improved vaccine to induce both mucosal immunity and systemic immunity is urgently required to control H9N2 avian influenza outbreaks in poultry farms.MethodsIn this study, we constructed a novel Lactococcus lactis (L. lactis) strain expressing a recombinant fusion protein consisting of the HA1 proteins derived from an endemic H9N2 virus strain and chicken IgY Fc fragment. We evaluated the immunogenicity and protective efficacy of this recombinant L. lactis HA1-Fc strain.ResultsOur data demonstrated that chickens immunized with L. lactis HA1-Fc strain showed significantly increased levels of serum antibodies, mucosal secretory IgA, T cell-mediated immune responses, and lymphocyte proliferation. Furthermore, following challenge with H9N2 avian influenza virus, chickens immunized with L. lactis HA1-Fc strain showed reduced the weight loss, relieved clinical symptoms, and decreased the viral titers and the pathological damage in the lung. Moreover, oropharyngeal and cloacal shedding of the H9N2 influenza virus was detected in chicken immunized with L. lactis HA1-Fc after infection, the results showed the titer was low and reduced quickly to reach undetectable levels at 7 days after infection.ConclusionOur data showed that the recombinant L. lactis HA1-Fc strain could induce protective mucosal and systemic immunity, and this study provides a theoretical basis for improving immune responses to prevent and control H9N2 virus infection.

The role of transforming growth factor beta-1 protein in Escherichia coli secondary infection induced by H9N2 avian influenza virus in chickens

The H9N2 subtype of avian influenza virus (AIV) is common in poultry production. It causes mild clinical signs but rarely leads to poultry mortalities. However, higher mortality can occur in chickens with co-infections, especially avian pathogenic Escherichia coli (APEC), which results in huge economic losses for the poultry industry. Unfortunately, the mechanism of co-infection remains unknown. Our previous studies screened several proteins associated with bacterial adhesion, including transforming growth factor beta-1 (TGF-β1), integrins, cortactin, E-cadherin, vinculin, and fibromodulin. Herein, we investigated the contribution of TGF-β1 to APEC adhesion after H9N2 infection. We first infected H9N2 and APEC in chicken, chicken embryo and DF-1cells, and demonstrated that H9N2 infection promotes APEC adhesion to hosts in vitro and in vivo by plate count method. Through real-time fluorescence quantification and enzyme-linked immunosorbent assay, it was demonstrated that H9N2 infection not only increases TGF-β1 expression but also its activity in a time-dependent manner. Then, through exogenous addition of TGF-β1 and overexpression, we further demonstrated that TGF-β1 can increase the adhesion of endothelial cells to DF-1cells. Furthermore, the capacity of APEC adhesion to DF-1cells was significantly decreased either by adding a TGF-β1 receptor inhibitor or using small interfering RNAs to interfere with the expression of TGF-β1. To sum up, H9N2 infection can promote the upregulation of TGF-β1 and then increase the adhesion ability of APEC. Targeting TGF-β1 and its associated pathway will provide valuable insights into the clinical treatment of E. coli secondary infection induced by H9N2 infection.

Efficacy of a recombinant turkey herpesvirus (H9) vaccine against H9N2 avian influenza virus in chickens with maternal-derived antibodies.

Although vaccines have been widely used for many years, they have failed to control H9N2 avian influenza virus (AIV) in the field in China. The high level of maternal-derived antibodies (MDAs) against H9N2 virus contributes to the H9N2 influenza vaccine failure in poultry. The study aimed to generate a new vaccine to overcome MDAs interference in H9N2 vaccination in chickens. We used turkey herpesvirus (HVT) as a vaccine vector to express H9 hemagglutinin (HA) proteins. The recombinant HVT expressing H9 HA proteins (rHVT-H9) was successfully generated and characterized in primary chicken embryonic fibroblasts (CEFs). Western blot and indirect immunofluorescence assay (IFA) showed that the rHVT-H9 consistently expressed HA proteins. In addition, the rHVT-H9 had similar growth kinetics to the parent HVT. Preliminary animal experiments showed that compared to the conventional inactivated whole virus (IWV) vaccine, the rHVT-H9 stimulated robust humoral immunity in chickens with passively transferred antibodies (PTAs) that were used to mimic MDAs. Transmission experiments showed that the rHVT-H9 induced both humoral and cellular immunity in chickens with PTAs. Furthermore, we used mathematical models to quantify the vaccine's efficacy in preventing the transmission of H9N2 AIV. The results showed that the rHVT-H9 reduced the virus shedding period and decreased the reproduction ratio (R) value in chickens with PTAs after homologous challenge. However, the vaccination in this trial did not yet bring R < 1. In summary, we generated a new rHVT-H9 vaccine, which stimulated strong humoral and cellular immunity, reducing virus shedding and transmission of H9N2 AIV even in the presence of PTAs in chickens.

Structural analysis of N-glycans in chicken trachea and lung reveals potential receptors of chicken influenza viruses

Although avian influenza A viruses (avian IAVs) bind preferentially to terminal sialic acids (Sia) on glycans that possess Siaα2-3Gal, the actual glycan structures found in chicken respiratory tracts have not been reported. Herein, we analyzed N-glycan structures in chicken trachea and lung, the main target tissues of low pathogenic avian IAVs. 2-Aminopyridine (PA)-labeled N-glycans from chicken tissues were analyzed by combined methods using reversed-phase liquid chromatography (LC), electrospray ionization (ESI)-mass spectrometry (MS), MS/MS, and multistage MS (MSn), with or without modifications using exoglycosidases, sialic acid linkage-specific alkylamidation (SALSA), and/or permethylation. The results of SALSA indicated that PA-N-glycans in both chicken trachea and lung harbored slightly more α2,6-Sia than α2,3-Sia. Most α2,3-Sia on N-glycans in chicken trachea was a fucosylated form (sialyl Lewis X, sLex), whereas no sLex was detected in lung. By contrast, small amounts of N-glycans with 6-sulfo sialyl LacNAc were detected in lung but not in trachea. Considering previous reports that hemagglutinins (HAs) of avian IAVs originally isolated from chicken bind preferentially to α2,3-Sia with or without fucosylation and/or 6-sulfation but not to α2,6-Sia, our results imply that avian IAVs do not evolve to possess HAs that bind preferentially to α2,6-Sia, regardless of the abundance of α2,6-Sia.

Pathogenicity of three genetically distinct and highly pathogenic Egyptian H5N8 avian influenza viruses in chickens

In late 2016, Egypt encountered multiple cases of the highly pathogenic avian influenza (HPAI) virus of the H5N8 subtype. In a previous study, three distinct genotypes, including A/common-coot/Egypt/CA285/2016 (H5N8) (CA285), A/duck/Egypt/SS19/2017 (H5N8) (SS19), and A/duck/Egypt/F446/2017 (H5N8) (F446), were isolated from wild birds, a backyard, and a commercial farm, respectively, during the first wave of infection. In this current study, we investigated the differences in the pathogenicity, replication and transmissibility of the three genotypes and A/chicken/Egypt/15S75/2015 (H5N1) (S75) was used as the control. The intravenous pathogenicity index was between 2.68 and 2.9. The chicken lethal dose 50 values of F446, SS19 and CA285 were 103.7, 103.7, an 104 with a natural route of infection, respectively. These strains took longer than S75 to cause death when infection was carried out through the natural route (HPAI H5N1). After inoculation with the original concentration of 105 and 106 egg infective dose 50 (EID50), F446 had a higher mortality rate with short mean death times of 4, and 7 days, respectively compared with the other H5N8 viruses. Chickens inoculated with F446 and contacted exposed chickens infected with F446 showed the highest viral titer with remarkable differences in all H5N8 tested swabs at 2-4 days postinfection (dpi) compared to S75 at 2 dpi. This indicates that F446 had a more efficient transmission and spread from contact exposed birds to other birds. All H5N8 viruses were able to replicate systematically in all organs (trachea, brain, lung, and spleen) of the chicken with high viral titer with significantly different and more pathological changes observed in F446 than in other H5N8 viruses at 2 and 4 dpi. Compared with H5N1, we recorded a significantly high viral titer in the samples obtained from the lung, brain and both cloacal and tracheal swabs at 2 and 4 dpi, respectively and in the samples obtained from the spleen at 2 and 4 dpi among the experimental chicken. The comparative pathogenesis study revealed that in comparison with the other HPAI H5N8 viruses, the genotype F446 was more pathogenic, and showed more efficient viral replication and transmissibility in chickens in Egypt. The genotype F446 also showed a high viral titer than HPAI H5N1 and short mean death time at the third day after inoculation with 106 and 105 EID50, which revealed a conservation of certain H5N8 genotypes and a decrease in the incidence of H5N1.

Opposite Outcomes of the Within-Host Competition between High- and Low-Pathogenic H5N8 Avian Influenza Viruses in Chickens Compared to Ducks.

Highly pathogenic avian influenza viruses (HPAIV) emerge from low-pathogenic avian influenza viruses (LPAIV) through the introduction of basic amino acids at the hemagglutinin (HA) cleavage site. Following viral evolution, the newly formed HPAIV likely represents a minority variant within the index host, predominantly infected with the LPAIV precursor. Using reverse genetics-engineered H5N8 viruses differing solely at the HA cleavage, we tested the hypothesis that the interaction between the minority HPAIV and the majority LPAIV could modulate the risk of HPAIV emergence and that the nature of the interaction could depend on the host species. In chickens, we observed that the H5N8LP increased H5N8HP replication and pathogenesis. In contrast, the H5N8LP antagonized H5N8HP replication and pathogenesis in ducks. Ducks mounted a more potent antiviral innate immune response than chickens against the H5N8LP, which correlated with H5N8HP inhibition. These data provide experimental evidence that HPAIV may be more likely to emerge in chickens than in ducks and underscore the importance of within-host viral variant interactions in viral evolution. IMPORTANCE Highly pathogenic avian influenza viruses represent a threat to poultry production systems and to human health because of their impact on food security and because of their zoonotic potential. It is therefore crucial to better understand how these viruses emerge. Using a within-host competition model between high- and low-pathogenic avian influenza viruses, we provide evidence that highly pathogenic avian influenza viruses could be more likely to emerge in chickens than in ducks. These results have important implications for highly pathogenic avian influenza virus emergence prevention, and they underscore the importance of within-host viral variant interactions in virus evolution.