Establishment of embryonic cell lines of Cydia pomonella (Lepidoptera: Tortricidae) and in vitro replication of Cydia pomonella granulovirus

Insect cell lines serve as essential in vitro models for biological, physiological, and virological research. The codling moth, Cydia pomonella, is a highly destructive invasive pest; no cell lines of C. pomonella has been reported in China currently.

Using eggs as starting material, embryonic cell lines of C. pomonella were established. From these, a cell line sensitive to Cydia pomonella granulovirus (CpGV) was screened, and its biological characteristics, including karyotype and growth were studied. The in vitroreplication of CpGV this cell line was analyzed by real-time fluorescence quantitative PCR (qPCR) and transmission electron microscopy (TEM).

The results showed that eight embryonic cell lines of C. pomonella were successfully established and could be stably passaged in TNM-FH medium containing 10% fetal bovine serum (FBS). One CpGV-sensitive cell line, designated QAU-Cp-E-11-L, was obtained. The cells grew adherently, the round, spindle-shaped, and rod-shaped cells accounted for 90.0%, 7.5%, and 2.5% of the total population, respectively. The amplified mitochondrial cytochrome oxidase Ⅰ subunit (COI) gene fragment was 576 bp in length. BLAST analysis revealed 100% nucleotide sequence identity with the COI gene of C. pomonella, confirming that the cell line derived from C. pomonella. The chromosome number of QAU-Cp-E-11-L cells varied from 23 ~ 440, indicating pronounced aneuploidy. The cell line QAU-Cp-E-11-L reached maximum density (2.27×10⁶ cells/mL) at 168 h with a population doubling time of 33.81 h. Following infection of QAU-Cp-E-11-L cells with CpGV, qPCR detected CpGV replication in the infected cells, with the viral copy number reaching a maximum of 2.48×10⁸ copies/μg DNA at 12 days post-infection (dpi). TEM observations results showed that the formation of CpGV occlusion bodies (OBs) in the cells, and the number of OBs in the cytoplasm increased with the inoculation time.

Hence, this study will enrich the resources of C. pomonella cell lines and provide critical tools for investigating granulovirus-host interactions.