Development of primary cell cultures from mud crab, Scylla serrata, and their potential as an in vitro model for the replication of white spot syndrome virus

Abstract

Primary cell cultures were developed from haemocytes and testis of Scylla serrata. Haemocytes were collected from live animals and cultured in double-strength L-15 medium (2× L-15) prepared in crab saline, supplemented with 5% foetal bovine serum and antibiotic-antimycotic solution (penicillin 100U/mL, streptomycin 100μg/mL and amphotericin B 0.25μg/mL) with osmolality adjusted to 894mOsm/kg. The haemocytes adhered within 2h after seeding and showed proliferation up to 72h. The disaggregated testis tissue fragments were seeded in 3× L-15 supplemented with non-essential amino acid mixture, lipid concentrate and antibiotic-antimycotic solution, with osmolality adjusted to 1,035mOsm/kg with crab saline. Cells from the testis could be subcultured and maintained up to 21d as suspension culture. Different dilutions of white spot syndrome virus (WSSV) inoculum (known virus copy number) prepared from infected Penaeus monodon were inoculated in the cultured cells, and the cytopathic effects like detachment, rounding of cells and clear areas of depleted cells were observed after 48h in haemocyte cultures. However, WSSV-exposed testis cells did not show any obvious change until 72h post-infection. WSSV was detected in both haemocyte and testis cultures at different time-points of infection by conventional and real-time PCR using WSSV-specific primers. The transcripts of WSSV were found to be much higher in haemocytes than in testis culture. The virus harvested from the cultured haemocytes after three passages could infect healthy P. monodon. The present study showed that mud crab haemocyte culture can support WSSV replication, and it can be used as an in vitro tool for WSSV replication.