Toll-like receptor 3 (TLR3) is an important membrane-bound receptor for recognizing double-stranded RNA in innate immunity. In this study, we described the cloning and characterization of the Muscovy duck TLR3 (MdTLR3) gene. The full-length MdTLR3 cDNA (2,836 bp) encoded a polypeptide of 895 amino acids. The deduced amino acid sequence contained 4 main structural domains: a signal peptide, an extracellular leucine rich repeats domain, a transmembrane domain, and a Toll/IL-1 receptor domain. Quantitative real-time PCR analysis indicated that MdTLR3 mRNA was constitutively expressed in all sampled tissues of uninfected Muscovy duck except muscle. Expression of MdTLR3 in brain was significantly upregulated at 24 h (1.94-fold, P < 0.05), reached a peak at 48 h (4.64-fold, P < 0.05), and recovered to normal levels at 72 h postinfection with the H5N1 highly pathogenic avian influenza virus. In contrast, MdTLR3 expression was downregulated during the test period in spleen and lung. These results implicated MdTLR3 was a novel member of the TLR family, which is involved in the early stage of antiviral innate immunity.

Newcastle disease virus (NDV) is distributed worldwide and has caused significant losses to the poultry industry. Almost all virulent NDV strains belong to class II, among which genotype VII is the predominant genotype in China. However, the molecular evolution and phylodynamics of class II genotype VII NDV strains in China remained largely unknown. In this study, we identified 13 virulent NDV including 11 genotype VII strains and 2 genotype IX strains, from clinical samples during 1997 to 2019. Combined NDV sequences submitted to GenBank, we investigate evolution, and transmission dynamics of class II NDVs in China, especially genotype VII strains. Our results revealed that East and South China have the most genotypic diversity of class II NDV, and East China might be the origin of genotype VII NDVs in China. In addition, genotype VII NDVs in China are presumably transmitted by chickens, as the virus was most prevalent in chickens. Furthermore, codon usage analysis revealed that the F genes of genotype VII NDVs have stronger adaptation in chickens, and six amino acids in this gene are found under positive selection via selection model analysis. Collectively, our results revealed the genetic diversity and evolutionary dynamics of genotype VII NDVs in China, providing important insights into the epidemiology of these viruses in China.

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.