Enhanced Efficiency of the LLC-MK2 Cell Line with Stable Expression of TMPRSS2 in Isolating Human Parainfluenza Virus 3 from Respiratory Specimens.

OBJECTIVE: LLC-MK2/TMPRSS2 cells constitutively express TMPRSS2, eliminating the requirement for additional trypsin during HPIV3 culture. The efficiency of LLC-MK2/TMPRSS2 for isolating HPIV3 from respiratory specimens was evaluated in comparison with Madin-Darby Canine Kidney (MDCK). METHODS: HPIV3-positive respiratory specimens from children with acute respiratory infections (February-June 2025) were inoculated into LLC-MK2/TMPRSS2 and MDCK. The cytopathic effect (CPE) was monitored microscopically, and the proportion of positive cells was evaluated using direct immunofluorescence assay (DFA). Viral infection dynamics were assessed using the cycle threshold (Ct) values obtained by qPCR. RESULTS: Among 50 specimens, 35 strains (35/50, 70%) were successfully isolated using LLC-MK2/TMPRSS2, while 14 strains were isolated using MDCK (14/50, 28%). More pronounced CPE and a higher number of virus-infected positive cells were shown in LLC-MK2/TMPRSS2 compared to that in MDCK ( P < 0.001 and P = 0.001, respectively). Among specimens with an initial Ct < 27, the isolation rate of LLC-MK2/TMPRSS2 was higher and the Ct values were lower (< 27) (82.6%, 19/23). Among specimens with an initial Ct of 23 ≤ Ct < 27, the number of specimens with a supernatant Ct ≥ 27 (63.6%, 7/11) was significantly less than that in MDCK ( P = 0.003). CONCLUSION: LLC-MK2/TMPRSS2 exhibits superior adaptability and replication efficiency in the isolation of HPIV3 from respiratory specimens.