Suspension Adaptation of Immortalized Lamb Testis (iLT) Cells in Serum-Reduced Media and Evaluation of Goatpox Virus (GTPV-AV41) Replication.

BACKGROUND

Goatpox is a severe viral disease in goats, and vaccination with live attenuated strains remains the most effective strategy for control. Conventional vaccine production relies on primary or Vero cells, but these substrates are limited by unstable supply, high cost, and suboptimal virus yields.

OBJECTIVE

This study aimed to establish a scalable suspension culture system using immortalized lamb testis (iLT) cells to support efficient replication of the goatpox virus (GTPV) AV41 strain for vaccine production.

METHODS

Adherent iLT cells were adapted to suspension by stepwise passaging under reduced serum. Eight commercial suspension media were screened, and four were selected for growth evaluation in 125 mL shaker flasks (20 mL working volume; 1 × 106 cells/mL), with stability assessed up to the 40th passage. For virus production, suspension cultures at 2-3 × 106 cells/mL were inoculated with GTPV-AV41 at 5-10% (v/v), samples were collected at 72-144 h post-inoculation, and viral replication was quantified by real-time quantitative PCR (RT-qPCR) using a cycle threshold (Ct) -50% tissue culture infectious dose (TCID50) calibration curve, with infection confirmed by indirect immunofluorescence.

RESULTS

Suspension-adapted iLT cells maintained robust growth for over 40 continuous passages, reaching densities exceeding 1.2 × 107 cells/mL with >95% viability. After GTPV-AV41 inoculation, the suspension culture system supported efficient viral replication, yielding a maximum viral titer of 108.00 TCID50 equivalents/mL in cell lysates at 72 h post-inoculation. Efficient viral entry and replication were further confirmed by indirect immunofluorescence assays.

CONCLUSIONS

Suspension-cultured iLT cells represent a promising platform for scalable goatpox vaccine production, combining high growth performance with efficient viral replication.

HIGHLIGHTS: This study presents a scalable suspension-culture platform by adapting iLT cells under serum-reduced conditions, enabling stable high-density growth and efficient replication of goatpox virus for cost-effective vaccine manufacturing.