Low Pathogenic Avian Influenza H9N2 Research Articles

Infection dynamics of subtype H9N2 low pathogenic avian influenza A virus in turkeys

While mammals can be infected by influenza A virus either sporadically or with well adapted lineages, aquatic birds are the natural reservoir of the pathogen. So far most of the knowledge on influenza virus dynamics was however gained on mammalian models. In this study, we infected turkeys using a low pathogenic avian influenza virus and determined the infection dynamics with a target-cell limited model. Results showed that turkeys had a different set of infection characteristics, compared with humans and ponies. The viral clearance rates were similar between turkeys and ponies but higher than that in humans. The cell death rates and cell to cell transmission rates were similar between turkeys and humans but higher than those in ponies. Overall, this study indicated the variations of within-host dynamics of influenza infection in avian, humans, and other mammalian systems.

Structural and functional characterization of avian influenza H9N2 virus neuraminidase with a combination of five novel mutations

The influenza virus neuraminidase (NA) protein is responsible for actively cleaving the sialic acid (SA) bound to the viral hemagglutinin. In the present study we identified a combination of five novel amino acid substitutions in the NA, conferring increased substrate binding and altered surface characteristics to a low pathogenic avian influenza (LPAI) H9N2 strain. The H9N2 virus strain reported from India, namely, A/Environmental/India/1726265/2017 (H9N2-1726265) showed the combination of amino acid substitutions T149I, R249W, G346A, W403R and G435R, which were in the vicinity of the enzyme active site cavity. The strain A/chicken/India/99321/2009 (H9N2-99321) did not show these substitutions and was used for comparison. Virus elution was studied using turkey red blood cells (tRBCs). NA enzyme kinetics assays were carried out using the MUNANA substrate, which is a SA analogue. Homology modelling and molecular docking were performed to determine alterations in the surface characteristics and substrate binding. H9N2-1726265 showed enhanced elution from turkey red blood cells. Enzyme kinetics revealed a lower KM of H9N2-1726265 (111.5 μM) as compared to H9N2-99321 (135.2 μM), indicating higher substrate binding affinity of H9N2-1726265 due to which the NA enzyme cleaved the SA more efficiently, leading to faster elution. Molecular docking revealed a greater number of binding interactions of H9N2-1726265 to SA as compared to H9N2-99321 which explained the greater substrate binding affinity. Changes in the surface charge, hydrophobicity, and contour, were observed in H9N2-1726265 NA due to the substitutions. Thus, the novel combination of five amino acids near the sialic acid binding site of NA, resulted in altered surface characteristics, higher substrate binding affinity and virus elution.

Pathogenicity Evaluation of Low Pathogenic Avian Influenza (H9N2) Virus Isolated from Layer Flocks in Malaysia in Specific-pathogen-free Chickens

Infection with the low pathogenic avian influenza (LPAI) H9N2 virus has been reported worldwide and poses a health risk to poultry as well as to global health due to its ability to re-assort with other avian influenza viruses. Besides, the silent spread of the H9N2 infection causes significant economic damage to the poultry industry. Recently, Malaysia reported major outbreaks of LPAI H9N2 in commercial layer chicken flocks. Genome sequence analysis indicated that the predominant LPAI H9N2 viruses are of the Y280/BJ94-like lineages. However, the pathogenicity of the virus has not been evaluated. This study determines the pathogenicity of LPAI H9N2 strain UPM994/2018, previously isolated from commercial layer chickens, in one-week-old specific-pathogen-free (SPF) chickens. Clinical signs such as ruffled feathers, mild tracheal rales, facial edema, sero-nasal discharge, and diarrhea were observed from days 6 to 10 post-inoculation (PI). However, no mortality was recorded. Based on a real-time polymerase chain reaction assay, the viruses can be detected in the lungs, trachea, and kidneys of the inoculated chickens on the second day, increased until day 10, then declined at day 16 PI. However, swab samples collected from the oropharyngeal and cloacal regions remain positive from day 2 to day 14 PI, with the highest viral load detected at day 10 PI. In conclusion, although the virus is an LPAI, it is pathogenic in SPF chickens, causing respiratory, gastrointestinal, and renal-associated illnesses.

First Report of Low Pathogenic Avian Influenza Subtype H9N2 in African Houbara Bustards (Chlamydotis undulata undulata) and Gamebirds in Morocco: Clinico-Pathological Findings, Molecular Characterization, and Associated Coinfections.

We report in this paper the first detection of low pathogenic avian influenza (LPAI) subtype H9N2 in houbara bustards and in gamebirds in Morocco. Starting in 2019, an increase in mortality rates related to respiratory distress was recorded in these species. Necropsy of the specimens revealed fibrinous sinusitis and tracheitis with intra-bronchial fibrin casts, which are consistent with H9N2 infection in chickens; therefore, implication of the virus in these outbreaks was strongly suspected. Consequently, between January 2020 and June 2023, birds with respiratory signs were necropsied for pathological lesions, tissue samples were examined by histopathology, and samples of trachea, lungs, and cecal tonsils were analyzed using quantitative real-time PCR for the detection of H9N2 virus. In addition, the sequencing of isolates was performed and lastly differential diagnosis with other respiratory pathogens was carried out. During the study period, 93 samples were collected from suspected H9N2 outbreaks, of which 30 tested positive for H9N2 virus: 23 Houbara bustards, 4 partridges, 2 quails, and 1 pheasant. Moreover, sequencing of the HA gene of the virus showed 97.33% nucleotide identity with strains reported previously in broilers in Morocco in 2017 and in 2022. Phylogenetic analysis grouped the Moroccan partridge isolates in the same cluster as viruses isolated in Morocco between 2016 and 2022, Algeria (2017), Burkina Faso (2017), Nigeria (2019), and Togo (2020). Additionally, 10 house sparrows from the premises of these birds were examined for the presence of H9N2 virus, revealing a 30% positivity rate. In conclusion, LPAIV H9N2 is circulating in houbara bustards and gamebirds in Morocco, and house sparrows might be a possible source of the infection. To our knowledge, this is the first report of LPAI H9N2 in the African species of houbara bustards worldwide and in gamebirds in Morocco.

Low Pathogenic Avian Influenza H9N2 Viruses in Morocco: Antigenic and Molecular Evolution from 2021 to 2023.

Avian influenza viruses pose significant threats to both the poultry industry and public health worldwide. Among them, the H9N2 subtype has gained substantial attention due to its high prevalence, especially in Asia, the Middle East, and Africa; its ability to reassort with other influenza viruses; and its potential to infect humans. This study presents a comprehensive phylogenetic and molecular analysis of H9N2 avian influenza viruses circulating in Morocco from 2021 to 2023. Through an active epidemiological survey, a total of 1140 samples (trachea and lungs) and oropharyngeal swabs pooled into 283 pools, collected from 205 farms located in 7 regions of Morocco known for having a high density of poultry farms, were analyzed. Various poultry farms were investigated (159 broiler farms, 24 layer farms, 10 breeder farms, and 12 turkey breeder farms). A total of 21 AI H9N2 strains were isolated, and in order to understand the molecular evolution of the H9N2 avian influenza virus, their genetic sequences were determined using the Sanger sequencing technique. Phylogenetic analysis was performed using a dataset comprising global H9N2 sequences to determine the genetic relatedness and evolutionary dynamics of the Moroccan strains. The results revealed the continued circulation and diversification of H9N2 avian influenza viruses in Morocco during the study period. Real-time RT-PCR showed a positivity rate of 35.6% (73/205), with cycle threshold values ranging from 19.2 to 34.9. The phylogenetic analysis indicated that all Moroccan strains belonged to a G1-like lineage and regrouped into two distinct clusters. Our newly detected isolates aggregated distinctly from the genotypes previously isolated in Morocco, North and West Africa, and the Middle East. This indicats the potential of virus evolution resulting from both national circulation and cross-border transmission. A high genetic diversity at both nucleotide and amino-acid levels was observed among all the strains isolated in this study, as compared to H9N2 strains isolated in Morocco since 2016, which suggests the co-circulation of genetically diverse H9N2 variants. Newly discovered mutations were detected in hemagglutinin positions 226, 227, and 193 (H3 numbering), which highlights the genetic evolution of the H9N2 AIVs. These findings contribute to our understanding of the evolution and epidemiology of H9N2 in the region and provide valuable insights for the development of effective prevention and control strategies against this emerging avian influenza subtype.

The influence of Spirulina extract on pathogenicity, immune response, and vaccine efficacy against H9N2 avian influenza virus in specific pathogen free chickens

Avian influenza (AI) viruses pose a risk to the worldwide poultry industry. Ultimately, improving the efficiency of the H9N2 vaccine is necessary to better control low-pathogenic avian influenza-H9N2 by using natural immunostimulant. Therefore, the goal of the present study was to examine varying doses of the cyanobacterium Spirulina extract on the effectiveness of H9N2 vaccine. Thus, a total of 150 specific pathogen-free (SPF) chickens were allocated into 6 groups, 25 birds each, as follow: G1, G2, and G6 were supplemented with 200, 400, and 400 mg Spirulina extract/kg feed, respectively, whilst the feed in G3, G4, and G5 were not supplemented with Spirulina extract. At 21-days-old, only the chickens in G1, G2, and G3 were vaccinated with the H9N2 AI vaccine. After 4 wk postvaccination, the chickens in G1, G2, G3, G4, and G6 were challenged with H9N2 AI Egyptian strain. The challenged virus was selected from a recent circulating Egyptian strain during 2022, and it was related to A/quail/Hong Kong/G1/97-like virus lineage and clustered with G1-B sub-lineage EGY-2 group. It had a high amino acids identity percentage of 92.6% with the A/chicken/Iran/av1221/1998 (Boehringer Ingelheim) vaccine. The results of real-time reverse-transcriptase polymerase-chain-reaction (rRT-PCR) revealed that no shedding of the virus was reported in G1, G2, G3, and G5. The supplementation of Spirulina extract in low (200 mg/kg of feed) and high (400 mg/kg of feed) concentration with the birds vaccinated with H9N2 AI vaccine (G1 and G2) induced prominent immuno-stimulatory effect in a dose dependent manner where it strongly enhanced the phagocytic activities of broilers' peripheral blood monocytes, and lysozyme at all days postvaccination (dpv) and days postchallenge (dpc) compared to other groups with significant differences at all day of experiment and 21st dpv, 28th dpv, 7th dpc, and 14th dpc, respectively. The supplementation with Spirulina extract in G1 and G2 induced the highest hemagglutination inhibition antibody titer in a dose-dependent manner at all-time intervals. The antibody titer postvaccination was significantly increased in G1 and G2 at 14th, and 21st dpv, in comparison with G3. Furthermore, G1 and G2 showed higher significant antibody titers at 7th and 14th dpc, compared to other groups. Furthermore, Spirulina extract (200 and 400 mg/kg feed) in G1 and G2 showed anti-inflammatory effect in a dose dependant manner by downregulating nitric oxide levels at all times postchallenge with a significant difference at 3 to 7 dpc compared to G3, G4, and G6, with improved histopathological alterations in the trachea, lung, kidney, spleen, and bursa of Fabricius. G6 supplied with 400 mg/kg Spirulina extract feed only without vaccination had a similar effect as vaccinated groups on innate immunity. However, it delayed the production of antibodies and did not prevent viral shedding as in vaccinated groups. In conclusion, vaccination in conjunction with either dose of Spirulina extract (G1, and G2) prevents viral shedding, increases the immune response, and reduces inflammation and histopathological change caused by H9N2 AI infection in a dose dependent manner. We recommend the use of 400 mg Spirulina extract/kg feed as a natural immunostimulant in conjunction with the H9N2 vaccine to achieve the highest possible level of protection against H9N2 AI infection.

Dissection of key factors correlating with H5N1 avian influenza virus driven inflammatory lung injury of chicken identified by single-cell analysis.

Chicken lung is an important target organ of avian influenza virus (AIV) infection, and different pathogenic virus strains lead to opposite prognosis. Using a single-cell RNA sequencing (scRNA-seq) assay, we systematically and sequentially analyzed the transcriptome of 16 cell types (19 clusters) in the lung tissue of chickens infected with H5N1 highly pathogenic avian influenza virus (HPAIV) and H9N2 low pathogenic avian influenza virus (LPAIV), respectively. Notably, we developed a valuable catalog of marker genes for these cell types. Compared to H9N2 AIV infection, H5N1 AIV infection induced extensive virus replication and the immune reaction across most cell types simultaneously. More importantly, we propose that infiltrating inflammatory macrophages (clusters 0, 1, and 14) with massive viral replication, pro-inflammatory cytokines (IFN-β, IL1β, IL6 and IL8), and emerging interaction of various cell populations through CCL4, CCL19 and CXCL13, potentially contributed to the H5N1 AIV driven inflammatory lung injury. Our data revealed complex but distinct immune response landscapes in the lung tissue of chickens after H5N1 and H9N2 AIV infection, and deciphered the potential mechanisms underlying AIV-driven inflammatory reactions in chicken. Furthermore, this article provides a rich database for the molecular basis of different cell-type responses to AIV infection.

Predominance of low pathogenic avian influenza virus H9N2 in the respiratory co-infections in broilers in Tunisia: a longitudinal field study, 2018–2020

Respiratory diseases are a health and economic concern for poultry production worldwide. Given global economic exchanges and migratory bird flyways, respiratory viruses are likely to emerge continuously in new territories. The primary aim of this study was to investigate the major pathogens involved in respiratory disease in Tunisian broiler poultry and their epidemiology. Between 2018 and 2020, broilers farms in northeastern Tunisia were monitored, and 39 clinically diseased flocks were sampled. Samples were screened for five viral and three bacterial respiratory pathogens using a panel of real-time PCR assays. The reemergence of H9N2 low pathogenic avian influenza virus (LPAIV) in commercial poultry was reported, and the Northern and Western African GI lineage strain was typed. The infectious bronchitis virus (IBV) GI-23 lineage and the avian metapneumovirus (aMPV) subtype B also were detected for the first time in broilers in Tunisia. H9N2 LPAIV was the most detected pathogen in the flocks tested, but rarely alone, as 15 of the 16 H9N2 positive flocks were co-infected. Except for infectious laryngotracheitis virus (ILTV), all of the targeted pathogens were detected, and in 61% of the respiratory disease cases, a combination of pathogens was identified. The major combinations were H9N2 + aMPV (8/39) and H9N2 + IBV (6/39), showing the high contribution of H9N2 LPAIV to the multifactorial respiratory diseases. This field survey provided evidence of the emergence of new respiratory viruses and the complexity of respiratory disease in Tunisia. A comprehensive and continuous surveillance strategy therefore is needed to better control respiratory pathogens in Tunisia.

The emergence of poultry-adapted H9 subtype avian influenza viruses. Proceeding of The First International Avian Influenza Summit, University of Arkansas- October 16-17, 2023"

Avian influenza is a highly contagious disease that severely affects poultry production and food security. Several countries have developed vaccination programs to prevent and control avian influenza. Unlike currently available vaccines, modified live virus (MLV) vaccines have the potential to be easily mass-applied. Concerns of reversion, recombination, and unintended transmission have slowed the development of MLVs against avian influenza for use in poultry. To address these concerns, we generated reassortment-impaired, non-transmissible, safe, immunogenic, and protective MLVs based on the rearrangement of internal gene segments and additional modifications of the surface gene segments HA and NA. The HA was produced to carry the unique DRPAVIAN peptide marker, whereas NA was modified as a chimera encoding the chicken interleukin-18 (ckIL18) gene (MLV-IL). As a proof of principle and given its economic importance and widespread geographic distribution, we tested the MLV and MLV-IL approaches in chickens against a prototypic H9N2 low pathogenic avian influenza virus (MLV-H9N2 and MLV-H9N2-IL, respectively). \textit{In-vitro}, the MLV-H9N2 and MLV-H9N2-IL candidates were stable and achieved yields similar to the wild-type H9N2 strain. Co-infection studies with a wild-type H9N2 virus showed that the altered HA segment in the MLV-H9N2 and MLV-H9N2-IL candidates has a fitness disadvantage and did not reassort. Likewise, the modified NA segment in the MLV-H9N2-IL candidate was impaired in reassortment. \textit{In-vivo}, the MLV-H9N2 and MLV-H9N2-IL candidates did not transmit chicken-to-chicken via direct contact. MLV-H9N2- and MLV-H9N2-IL-vaccinated chickens showed no clinical signs upon intranasal vaccination, and all were seroconverted. More importantly, including ckIL18 in the MLV-H9N2-IL vaccine enhanced the generation of neutralizing antibodies, suggesting a role in the host immune response. A significant decrease in viral loads post-challenge demonstrated the protective effect of the MLVs. The MLV-H9N2-IL vaccine administered via drinking water was immunogenic in chickens in a dose-dependent manner and generated levels of neutralizing antibodies after prime and boost predictive of protection, which was later confirmed upon aggressive homologous virus challenge. In summary, this work provides the basis for novel and safe MLVs against avian influenza amenable for mass vaccination.

Prevalence of major infectious diseases in backyard chickens from rural markets in Morocco.

Raising backyard chickens is a common practice in Morocco, mainly in rural or periurban areas. Constraints due to devastating avian diseases have been recognized as a major limiting factor in backyard poultry production. Consequently, these flocks could potentially be implicated as reservoirs for poultry diseases. However, there is a considerable lack of information on disease prevalence in this production system, and the risk represented by these small flocks remains under debate. This study aimed to estimate the seroprevalence and identify related risk factors of a range of bacterial and viral pathogens of outstanding importance for the economy and public health in backyard poultry in Morocco. A total of 712 sera samples and 258 cloacal swabs were collected from 712 backyard chickens from 15 rural markets in the Khemisset and Skhirat-Temara provinces. None of the sampled chickens received any vaccination. Sera samples were screened for antibodies against Newcastle disease virus (NDV) and low pathogenic avian influenza H9N2 subtype (LPAI H9N2) using a hemagglutination-inhibition test, against bursal infectious disease virus (IBDV) and infectious bronchitis virus (IBV) using enzyme-linked immunosorbent assay, and against Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) using a rapid serum agglutination test. Swab samples were compiled into 86 pools and submitted for molecular detection using real-time reverse-transcription-polymerase chain reaction (RT-PCR). The seroprevalences in backyard chickens for NDV, LPAI H9N2, IBDV, IBV, MG, and MS were 52.1% (371/712), 63.5% (452/712), 84.7% (603/712), 82.2% (585/712), 58% (413/712), and 74.8% (533/712), respectively. Based on the RT-PCR results, 2.3% (2/86), 62.8% (54/86), 2.3% (2/86), 63.9% (55/86), 40.7% (35/86), and 29.1% (25/86) of the pools were positive for NDV, H9N2 LPAI, IBDV, IBV, MG, and MS, respectively. Multiple coinfections (H9N2-IBV-MG), (H9N2-IBV-MS), or (IBV-MG-MS) were observed in 15.1%, 8.5%, and 8.5% of the tested samples, respectively. The results show that backyard chicken flocks and rural markets have the potential to serve as reservoirs or amplifiers for poultry pathogens and could pose a risk to the commercial poultry sector. This highlights the need for a comprehensive and adapted vaccination plan for backyard chickens, and extension of efforts to increase flock owners' awareness of avian diseases and incite the implementation of biosecurity measures at the farm level.

Emergence of a new genotype of clade 2.3.4.4b H5N1 highly pathogenic avian influenza A viruses in Bangladesh

ABSTRACT Influenza virological surveillance was conducted in Bangladesh from January to December 2021 in live poultry markets (LPMs) and in Tanguar Haor, a wetland region where domestic ducks have frequent contact with migratory birds. The predominant viruses circulating in LPMs were low pathogenic avian influenza (LPAI) H9N2 and clade 2.3.2.1a highly pathogenic avian influenza (HPAI) H5N1 viruses. Additional LPAIs were found in both LPM (H4N6) and Tanguar Haor wetlands (H7N7). Genetic analyses of these LPAIs strongly suggested long-distance movement of viruses along the Central Asian migratory bird flyway. We also detected a novel clade 2.3.4.4b H5N1 virus from ducks in free-range farms in Tanguar Haor that was similar to viruses first detected in October 2020 in The Netherlands but with a different PB2. Identification of clade 2.3.4.4b HPAI H5N1 viruses in Tanguar Haor provides continued support of the role of migratory birds in transboundary movement of influenza A viruses (IAV), including HPAI viruses. Domestic ducks in free range farm in wetland areas, like Tangua Haor, serve as a conduit for the introduction of LPAI and HPAI viruses into Bangladesh. Clade 2.3.4.4b viruses have dominated in many regions of the world since mid-2021, and it remains to be seen if these viruses will replace the endemic clade 2.3.2.1a H5N1 viruses in Bangladesh.

Tissue Tropism of H9N2 Low-Pathogenic Avian Influenza Virus in Broiler Chickens by Immunohistochemistry

The H9N2 subtype of low-pathogenic avian influenza viruses (LPAIV) is a widespread pathogen of poultry that can also infect humans. The characterization of viral infections is a complex process, involving clinical, pathological, and virological investigations. The aim of this study was to adapt and optimize an immunohistochemical (IHC) technique developed for LPAIVs specifically for the detection of H9N2 virus antigens in infected tissues. Twenty-one-day-old broiler chickens were inoculated with three different strains of H9N2 virus by different infection routes (i.e., intranasal-intratracheal and intravenous) or co-infected with infectious bronchitis virus (IBV) and observed for 11 days post infection. The suggested IHC protocol was modified: (i) DAB (diamino-benzidine) was substituted with AEC (3-amino-9-ethyl carbazole) as chromogen; and (ii) indirect two-step immune reactions of monoclonal primary and peroxidase-labeled anti-mouse secondary antibodies were used instead of avidin-biotin complexes. Avian influenza virus antigen appears as a red precipitate in the nuclei of affected cells but can also be identified in the cytoplasm. Mild hyperemia and congestion were observed in the trachea, air sac, and lungs of the challenged birds, and fibrinous exudate was found at the bifurcation in a few cases. Neither gross pathological nor IHC lesions were found in the control group. Using the optimized protocol and an associated scoring scheme, it was demonstrated that the H9N2 strains tested exhibited respiratory and urinary tract tropism irrespective of the route of inoculation. On day 5, viral antigen was detected in the respiratory tract and kidney in 30-50% of the samples. On day 11, no IHC signal was observed, indicating the lack of viral replication. Slight differences in viral antigen expression were found between the different H9N2 virus strains, but, in contrast to highly pathogenic avian influenza (HPAI), no viral antigen was detected in the brain and pancreas. Thus, IHC can be considered as an informative, visual addition to the toolkit for the characterization of H9N2 LPAIV infections.

Protection of SPF Chickens by H9N2 Y439 and G1 Lineage Vaccine against Homologous and Heterologous Viruses.

Prior to the identification of low pathogenic avian influenza H9N2 viruses belonging to the Y280 lineage in 2020, Y439 lineage viruses had been circulating in the Republic of Korea since 1996. Here, we developed a whole inactivated vaccine (vac564) by multiple passage of Y439 lineage viruses and then evaluated immunogenicity and protective efficacy in specific-pathogen-free chickens. We found that LBM564 could be produced at high yield in eggs (108.4EID50/0.1 mL; 1024 hemagglutinin units) and was immunogenic (8.0 ± 1.2 log2) in chickens. The vaccine showed 100% inhibition of virus in the cecal tonsil with no viral shedding detected in either oropharyngeal or cloacal swabs after challenge with homologous virus. However, it did not induce effective protection against challenge with heterologous virus. An imported commercial G1 lineage vaccine inhibited viral replication against Y280 and Y439 lineage viruses in major tissues, although viral shedding in oropharyngeal and cloacal swabs was observed up until 5 dpi after exposure to both challenge viruses. These results suggest that a single vaccination with vac564 could elicit immune responses, showing it to be capable of protecting chickens against the Y439 lineage virus. Thus, our results suggest the need to prepare suitable vaccines for use against newly emerging and re-emerging H9N2 viruses.

Experimental Pathogenicity of H9N2 Avian Influenza Viruses Harboring a Tri-Basic Hemagglutinin Cleavage Site in Sonali and Broiler Chickens.

Low-pathogenic avian influenza (LPAI) H9N2 virus is endemic in Bangladesh, causing huge economic losses in the poultry industry. Although a considerable number of Bangladeshi LPAI H9N2 viruses have been molecularly characterized, there is inadequate information on the pathogenicity of H9N2 viruses in commercial poultry. In this study, circulating LPAI H9N2 viruses from recent field outbreaks were characterized, and their pathogenicity in commercial Sonali (crossbred) and broiler chickens was assessed. Phylogenetic analysis of currently circulating field viruses based on the hemagglutinin (HA) and neuraminidase (NA) gene sequences revealed continuous circulation of G1 lineages containing the tri-basic hemagglutinin cleavage site (HACS) motif (PAKSKR*GLF) at the HA protein. Both the LPAI susceptible Sonali and broiler chickens were infected with selected H9N2 isolates A/chicken/Bangladesh/2458-LT2/2020 or A/chicken/Bangladesh/2465-LT56/2021 using intranasal (100 µL) and intraocular (100 µL) routes with a dose of 106 EID50/mL. Infected groups (LT_2-So1 and LT_56-So2; LT_2-Br1 and LT_56-Br2) revealed no mortality or clinical signs. However, at gross and histopathological investigation, the trachea, lungs, and intestine of the LT_2-So1 and LT_56-So2 groups displayed mild to moderate hemorrhages, congestion, and inflammation at different dpi. The LT 2-Br1 and LT 56-Br2 broiler groups showed nearly identical changes in the trachea, lungs, and intestine at various dpi, indicating no influence on pathogenicity in the two commercial bird species under study. Overall, the prominent lesions were observed up to 7 dpi and started to disappear at 10 dpi. The H9N2 viruses predominantly replicated in the respiratory tract, and higher titers of virus were shed through the oropharyngeal route than the cloacal route. Finally, this study demonstrated the continuous evolution of tri-basic HACS containing H9N2 viruses in Bangladesh with a low-pathogenic phenotype causing mild to moderate tracheitis, pneumonia, and enteritis in Sonali and commercial broiler chickens.

Comparative assessment of lyophilized and wet reagents for the molecular detection of H5N1 high pathogenic avian influenza virus and H9N2 low pathogenic avian influenza virus

Global surveillance for Avian Influenza Virus (AIV) in birds is essential for assessing public and animal health risks and real-time polymerase chain reaction (RT-qPCR) is among the official methods recommended by the World Organisation for Animal Health (WOAH) to confirm the presence of the virus in laboratory specimens. Yet, in low-resource setting laboratories, the detection of AIV can be hampered by the need to maintain a cold chain for wet reagents. In such cases, alternatives should be ready to maximize surveillance capacities and mining of AIV. Therefore, we compared two lyophilized RT-qPCR reagents (1st - 5 × CAPITAL™ 1-Step qRT-PCR Probe Reagent, lyophilized kit, and 2nd - Qscript lyo 1-step-kit) to the WOAH recommended protocol by Nagy et al., 2020 using QuantiTect Probe RT-PCR-kit as wet reagent. The comparative study panel comprised 102 RNA samples from two AIV subtypes, i.e. H5 and H9 subtypes. Despite that the wet reagent exhibited the lowest limit of detection (LOD) compared to the two lyophilized reagents, the inter-assay agreement was substantial between the 1st lyophilized reagent and the comparator with 95.1% of shared positive results. Cohen’s-kappa was fair between the 2nd lyophilized reagent and the comparator with 75.5% of shared positive results. Agreement using the statistical test Bland-Altman was good for samples with Cq-values < 25 for all reagents, revealing discrepancies when the viral load is low. This trend was especially evident while using the 2nd lyophilized reagent. Similar trends were obtained using the same lyophilized reagents but following the protocol by Heine et al., 2015 with AgPath-ID™ One-Step RT-PCR as a comparator, showing that Cq-values increase using lyophilized reagents but correlate strongly with the wet reagent. Further, inter-assay agreement between reagents improved when the protocol from Heine et al., 2015 was applied, suggesting a higher resilience to chemistry changes allowing easier reagents interchangeability.

The evolution, characterization and phylogeography of avian influenza H9N2 viruses from India

The low pathogenic avian influenza H9N2 virus is a significant zoonotic agent and contributes genes to highly pathogenic avian influenza (HPAI) viruses. H9N2 viruses are prevalent in India with a reported human case. We elucidate the spatio-temporal origins of the H9N2 viruses from India. A total of 30H9N2 viruses were isolated from poultry and environmental specimens (years 2015–2020). Genome sequences of H9N2 viruses (2003-2020) from India were analyzed, revealing several substitutions. We found five reassortant genotypes. The HA, NA and PB2 genes belonged to the Middle-Eastern B sublineage; NP and M to the classical G1 lineage; PB1, PA and NS showed resemblance to genes from either HPAI-H7N3/H5N1 viruses. Molecular clock and phylogeography revealed that the introduction of all the genes to India took place around the year 2000. This is the first report of the genesis and evolution of the H9N2 viruses from India, and highlights the need for surveillance.

Protective Efficacy of Inactivated H9N2 Vaccine in Turkey Poults under Both Experimental and Field Conditions.

Low pathogenic avian influenza (LPAI) H9N2 virus is one of the major poultry pathogens associated with severe economic losses in the poultry industry (broiler, layers, breeders, and grandparents' flocks), especially in endemic regions including the Middle East, North Africa, and Asian countries. This work is an attempt to evaluate the efficacy of whole inactivated H9N2 vaccine (MEFLUVACTM H9) in turkey poults kept under laboratory and commercial farm conditions. Here, 10,000 white turkey poults (1-day old) free from maternally derived immunity against H9N2 virus were divided into four groups; G1 involved 10 vaccinated birds kept under biosafety level-3 (BLS-3) as a laboratory vaccinated and challenged group, while G2 had 9970 vaccinated turkeys raised on a commercial farm. Ten of those birds were moved to BLS-3 for daily cloacal and tracheal swabbing to check for the absence of any life-threating disease, before conducting analyses. G3 (10 birds) served as a non-vaccinated challenged control under BSL-3 conditions, while G4 (10 birds) was used as a non-vaccinated and non-challenged control under BSL-3 conditions. Sera were collected on days 7-, 14-, 21-, and 28-post-vaccinations to monitor the humoral immune response using a hemagglutination-inhibition (HI) test. At these same intervals, cloacal and tracheal swabs were also checked for any viral infection. The challenge was conducted 28 days post-vaccination (PV) using AI-H9N2 in BSL-3 by intranasal inoculation of 6-log10 embryo infective dose50 (EID50). At 3-, 6-, and 10-days post-challenge, oropharyngeal swabs were taken from challenged birds to quantify viral shedding by quantitative polymerase chain reaction (qRT-PCR). The results of this study showed that vaccinated groups (G1/2) developed HI titers of 1.38, 4.38, 5.88, and 7.25 log2 in G1 vs. 1.2, 3.8, 4.9 and 6.2 log2 in G2 when measured at 7-, 14-, 21- and 28-days PV, respectively, while undetectable levels were recorded in non-vaccinated groups (G3/4). Birds in G3 showed 90% clinical sickness vs. 10% and 20% in G1/2, respectively, over a 10-day monitoring period following challenge. Vaccinated birds showed a significant reduction in virus shedding in terms of the number of shedders, amount of shed virus and shedding interval over the non-vaccinated challenged birds. Regarding mortality, all groups did not show any mortality, which confirms that the circulating H9N2 virus still has low pathogenicity and cannot cause mortality. However, the virus may cause up to 90% clinical sickness in non-vaccinated birds vs. 10% and 20% in laboratory- and farm-vaccinated birds, respectively, highlighting the role of the vaccine in limiting clinical sickness cases. In conclusion, under the current trial circumstances, MEFLUVACTM-H9 provided protective seroconversion titers, significant clinical sickness protection and significant reduction in virus shedding either in laboratory- or farm-vaccinated groups after a single vaccine dose.

Live recombinant Newcastle disease virus vectored vaccine expressing the haemagglutinin of H9N2 avian influenza virus suppresses viral replication in chickens

ABSTRACT In 2020, the Y280-lineage H9N2 low-pathogenic avian influenza virus (LPAIV) was introduced into South Korea for the first time. Current vaccines are focused on the control of Y439-like viruses; however, there are continuous reports of decrease in egg production and secondary infections caused by Y280-lineage H9N2 LPAI infection in chickens. Therefore, there is an urgent need to develop effective novel vaccines against Y280-lineage H9N2 LPAI. Most commercialized avian influenza vaccines are oil-adjuvanted inactivated vaccines, which are labour-intensive to administer and require higher dosage. In this study, rK148/Y280-HA, a novel recombinant Newcastle disease virus (NDV) vectored vaccine against Y280-lineage H9N2 LPAI, was developed and evaluated using two mass-applicable administration methods, spray vaccination and drinking water vaccination. Regardless of low serum antibody haemagglutination inhibition titres against NDV and Y280-lineage H9N2 LPAI after applying the rK148/Y280-HA vaccine, vaccination with either administration method protected chickens against virulent NDV and Y280-lineage H9N2 LPAIV after the challenge. Taken together, these results indicate that the rK148/Y280 vaccine can be administered using facile mass-application methods to provide protection against the Y280-lineage LPAI. RESEARCH HIGHLIGHTS NDV vectored vaccine harbouring Y280-lineage H9N2 HA protein was successfully generated. NDV vectored vaccine provides protection against NDV. NDV vectored vaccine with H9N2 HA protects against homologous H9N2 LPAIV.

Effect of Co-infection of Low Pathogenic Avian Influenza H9N2 Virus and Avian Pathogenic E. coli on H9N2-Vaccinated Commercial Broiler Chickens.

In the last 40 years, low pathogenic avian influenza virus (LPAIV) subtype H9N2 has been endemic in most Middle Eastern countries and of course Egypt which is one of the biggest poultry producers in the middle east region. The major losses with the H9N2 virus infections come from complicated infections in commercial broiler chickens, especially E. coli infection. In this work, 2,36,345 Arbor acres broiler chickens from the same breeder flock were placed equally in four pens, where two pens were vaccinated against LPAIV of subtype H9N2 virus, and the other two pens served as non-vaccinated controls. All were placed on the same farm under the same management conditions. A total of twenty birds from each pen were moved to biosafety level−3 chicken isolators (BSL-3) on days 21 and 28 of life and challenged with LPAIV-H9N2 or E. coli. Seroconversion for H9N2 was evaluated before and after the challenge. The recorded results revealed a significant decrease in clinical manifestations and virus shedding in terms of titers of shedding virus and number of shedders in vaccinated compared to non-vaccinated chickens. In groups early infected with LPAIV-H9N2 virus either vaccinated or not vaccinated, there was no significant difference in clinical sickness or mortalities in both groups, but in late infection groups with H9N2 alone, non-vaccinated infected group showed significantly higher clinical sickness in comparison with infected vaccinated group but also without mortality. In groups co-infected with E. coli (I/M) and H9N2, it showed 100% mortalities either in vaccinated or non-vaccinated H9N2 groups and thus reflect the high pathogenicity of used E. coli isolates, whereas in groups co-infected with E. coli (per os to mimic the natural route of infection) and LPAIV-H9N2, mortality rates were significantly higher in non-vaccinated groups than those vaccinated with H9N2 vaccine (15 vs. 5%). In conclusion, the use of the LPAIV H9N2 vaccine has significantly impacted the health status, amount of virus shed, and mortality of challenged commercial broilers, as it can minimize the losses and risks after co-infection with E. coli (orally) and LPAIV-H9N2 virus under similar natural route of infection in commercial broilers.

Genetic Variations among Different Variants of G1-like Avian Influenza H9N2 Viruses and Their Pathogenicity in Chickens.

Since it was first discovered, the low pathogenic avian influenza (LPAI) H9N2 subtype has established linages infecting the poultry population globally and has become one of the most prevalent influenza subtypes in domestic poultry. Several different variants and genotypes of LPAI H9N2 viruses have been reported in Egypt, but little is known about their pathogenicity and how they have evolved. In this study, four different Egyptian LPAI H9N2 viruses were genetically and antigenically characterized and compared to representative H9N2 viruses from G1 lineage. Furthermore, the pathogenicity of three genetically distinct Egyptian LPAI H9N2 viruses was assessed by experimental infection in chickens. Whole-genome sequencing revealed that the H9N2 virus of the Egy-2 G1-B lineage (pigeon-like) has become the dominant circulating H9N2 genotype in Egypt since 2016. Considerable variation in virus shedding at day 7 post-infections was detected in infected chickens, but no significant difference in pathogenicity was found between the infected groups. The rapid spread and emergence of new genotypes of the influenza viruses pinpoint the importance of continuous surveillance for the detection of novel reassortant viruses, as well as monitoring the viral evolution.