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.

Our study aimed to investigate the virulence of three recent H9N2 LPAIV strains belonging to the G1 lineage, isolated from field infections in North Africa and the Middle East. Three-week-old commercial broiler chickens (in total 62) were included and randomly allocated into three infected test groups and one control group. Each test group was inoculated intranasally/intratracheally with one of the three H9N2 isolates at a dose of 10⁸ EID₅₀ virus. The control group received phosphate-buffered saline (PBS) via the same route of application. The pathogenicity was evaluated based on clinical signs and gross pathological and histopathological lesions, the viral antigen load was assessed through immunohistochemistry staining (IHC), and a semi-quantitative detection of the genetic material was conducted via a real-time PCR. Our findings confirmed the obvious respiratory tract tropism of the virus strains with variable renal tropism. In contrast to the highly pathogenic AIVs, the tested H9N2 strains did not show replication in the central nervous system. The virus presence and lesions, mainly in the respiratory tract, were predominant on dpi 5 and significantly reduced or disappeared by dpi 11. A clear difference was demonstrated among the three isolates: the A/chicken/Morocco/2021/2016 strain proved to be significantly more virulent than the Egyptian and Saudi Arabian ones, which showed no remarkable difference.