Proliferation of Trionyx sinensis hemorrhagic syndrome virus using primary alveolar macrophages in vitro

Trionyx Sinensis (Chinese soft-shelled turtle) parotitis manifests as bacterial or viral infections. Viral parotitis often leads to rapid mortality, posing a significant threat to the T. sinensis aquaculture industry. The causative agent of viral parotitis is T. sinensis Hemorrhagic Syndrome Virus (TSHSV). However, due to the lack of sensitive cells, conducting comprehensive studies on this virus is challenging. In this study, primary macrophages were isolated from the lung tissue of juvenile T. sinensis to establish a method for TSHSV proliferation in vitro. Macrophages were isolated using irrigation method. These cells were cultured in M199 medium supplemented with 25 % fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin, and incubated at 30 °C in a 5 % CO₂ incubator. Macrophages were initially distinguished through morphological observation and an acidic phosphatase assay. In this study, the changes were observed in macrophages infected with TSHSV, and then RT-qPCR, immunofluorescence, and immunohistochemistry techniques were used to evaluate the sensitivity of macrophages for TSHSV. The results demonstrated that the cells washed out from the lung were predominantly identified as alveolar macrophages. After infection with TSHSV, cytopathic effects were observed. Viral nucleic acid segments could be detected in the infected cells, and the expression of antiviral immune-related genes Mx1, OASL, and RSAD2 was significantly upregulated. Moreover, a large amount of viral protein was expressed within the infected macrophages, indicating viral replication. Our results suggested that the developed approach for isolating and culturing primary alveolar macrophages of turtles can be employed for in vitro cultivation of TSHSV.