Infectious bronchitis virus (IBV) and H9N2 avian influenza virus (AIV) are frequently identified in chickens with respiratory disease. However, the role and mechanism of IBV and H9N2 AIV co-infection remain largely unknown. Specific-pathogen-free (SPF) chickens were inoculated with IBV 2 days before H9N2 virus inoculation (IBV/H9N2); with IBV and H9N2 virus simultaneously (IBV+H9N2); with H9N2 virus 2 days before IBV inoculation (H9N2/IBV); or with either IBV or H9N2 virus alone. Severe respiratory signs, pathological damage, and higher morbidity and mortality were observed in the co-infection groups compared with the IBV and H9N2 groups. In general, a higher virus load and a more intense inflammatory response were observed in the three co-infection groups, especially in the IBV/H9N2 group. The same results were observed in the transcriptome analysis of the trachea of the SPF chickens. Therefore, IBV might play a major role in the development of respiratory disease in chickens, and secondary infection with H9N2 virus further enhances the pathogenicity by inducing a severe inflammatory response. These findings may provide a reference for the prevention and control of IBV and H9N2 AIV in the poultry industry and provide insight into the molecular mechanisms of IBV and H9N2 AIV co-infection in chickens.

Newcastle disease virus (NDV) belongs to the Paramyxoviridae family and has had a great impact on the poultry industry. In the past, NDV strains were generally pathogenic only to fowls, but goose paramyxovirus (goose-origin APVM-1) is highly infectious to waterfowl, and, thus, there have been frequent outbreaks in China since 1997. In this study three pairs of specific primers were designed to detect the virulence of different NDV and goose-origin APVM-1 isolates and to differentiate between NDV and goose-origin APVM-1 using multiplex reverse transcription-polymerase chain reaction (RT-PCR). Pathogenicity tests were performed to confirm multiplex RT-PCR results. Data from our study indicate that multiplex RT-PCR is a convenient, low-cost, and effective technique for rapid identification. Twenty-six viral isolates of NDV and four goose-origin APVM-1 were analyzed, and we found that the VII genotype of NDV is the most prevalent type in South China and that it is not closely related to viral strains common to Europe.