Abstract:

Since the autumn and winter of 2018, a newly emerged infectious disease characterized by decreased egg production and diarrhea in laying hens has been observed in China's poultry farming regions. To investigate the etiology of this condition. Intestinal tissue samples from affected chickens were collected and pathogenic agents were isolated through 10 consecutive blind passages in specific pathogen-free (SPF) chicken embryos. The isolated viral strain was characterized for physicochemical and hemagglutination properties. Suspected virus-specific primers were designed to perform PCR detegtion, gene sequencing and nucleotide homology analysis on the extracted viral nucleic acid. Based on the alignment result, primers were designed to obtain the whole genome nucleotide sequence of the virus. The novel avian coronavirus strain AvCoV/CH/SD/24 was used to challenge SPF chickens, AA+ white-feathered broilers, and Hy-Line laying hens. Intestinal tissues from infected SPF chickens were processed for histopathological sections and serum antibody titers against infectious bronchitis virus (IBV) in different chicken groups post-challenge were measured using enzyme-linked immunosorbent assay (ELISA). Four novel avian coronaviruses (AvCoV/CH/BZ/24, AvCoV/CH/JS/24, AvCoV/CH/SD/24 and AvCoV/CH/HB/24) were successfully isolated, these viruses exhibited no hemagglutinating activity.The results of nucleic acid detection showed that four samples tested positive for both the N and S1 genes of avian infectious bronchitis virus (IBV). The N gene sequences showed the highest homology with avian infectious bronchitis virus strains in the GenBank database, while the S1 gene sequences exhibited maximum identity with the avian coronavirus AvCoV/TZ/CA127/19. Whole genome amplification and sequencing revealed that the four novel avian coronaviruses demonstrated 97.1%-98.6% nucleotide homology with two previously reported avian coronavirus strains: one isolated from Anhui Province, China in 2016 and another from Arusha, Tanzania in 2019. Phylogenetic analysis revealed that the S gene of the novel avian coronavirus is closely related to a turkey coronavirus-like, forming distinct subclades within the same evolutionary branch, while its other genes clustered within the evolutionary branch of the GⅠ-19 lineage infectious bronchitis virus. Animal experiments demonstrated that the AvCoV/CH/SD/24 strain induced diarrhea in SPF chickens, accompanied by duodenal villus detachment, congestion and hemorrhage at the villus tips and lamina propria and granular degeneration of renal tubular epithelial cells with interstitial congestion. The virus was primarily excreted through the cloaca with the highest viral loads detected in the intestines and bursa of Fabricus among tested organs. Infection did not affect the weight gain rate of chickens. ELISA results indicated serological cross-reactivity between the recombinant novel avian coronavirus and infectious bronchitis virus. In laying hens, infection led to reduced egg production and diarrhea, which was consistent with the disease in the field. Four novel avian coronaviruses were isolated from the intestines of laying hens exhibiting egg production decline and diarrhea, these newly isolated avian coronaviruses are recombinant viruses capable of inducing egg production decline and diarrhea in chickens.

Key words: enteropathogenic, novel recombinant avian coronavirus, diarrhea, decline in egg production