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
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
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DOI：
10.1007/s11626-013-9718-x
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发表时间：
2014-05-01
影响因子：
2.1
通讯作者：
Rajendran, K. V.
中科院分区：
文献类型：
--
作者：
Deepika, A.;Makesh, M.;Rajendran, K. V.
采用锯缘青蟹（Scylla serrata）的血细胞和精巢进行原代细胞培养。从活动物中采集血细胞，并在蟹盐水中制备的双倍浓度L-15培养基（2 x L-15）中培养，该培养基补充有5%胎牛血清和抗真菌药-抗霉菌剂溶液（青霉素100 U/mL、链霉素100 μ g/mL和链霉素B 0.25 μ g/mL），渗透压摩尔浓度调节至894 mOsm/kg。血细胞在接种后2 h内粘附，并显示增殖至72 h。将分解的睾丸组织碎片接种在补充有非必需氨基酸混合物、脂质浓缩物和抗增殖剂-抗霉菌剂溶液的3x L-15中，用蟹盐水将渗透压摩尔浓度调节至1，035 mOsm/kg。睾丸细胞可进行传代培养，并以悬浮培养的方式维持21 d。将感染斑节对虾的白色斑点综合征病毒（WSSV）接种液（已知病毒拷贝数）按不同稀释度接种于血细胞培养物中，48 h后观察到细胞脱落、变圆和细胞耗竭区清晰等细胞病变。然而，暴露于WSSV的睾丸细胞没有表现出任何明显的变化，直到72小时后感染。采用WSSV特异性引物，通过常规和实时荧光PCR方法，在不同感染时间点的血细胞和睾丸培养物中检测到WSSV。WSSV在血细胞中的表达量明显高于在睾丸中的表达量。从血细胞中分离的病毒经3次传代后可感染健康斑节对虾。本研究表明，青蟹血细胞培养物可以支持WSSV的复制，可以作为WSSV体外复制的工具。
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 (2x L-15) prepared in crab saline, supplemented with 5% foetal bovine serum and antibiotic-antimycotic solution (penicillin 100 U/mL, streptomycin 100 mu g/mL and amphotericin B 0.25 mu g/mL) with osmolality adjusted to 894 mOsm/kg. The haemocytes adhered within 2 h after seeding and showed proliferation up to 72 h. The disaggregated testis tissue fragments were seeded in 3x L-15 supplemented with non-essential amino acid mixture, lipid concentrate and antibiotic-antimycotic solution, with osmolality adjusted to 1,035 mOsm/kg with crab saline. Cells from the testis could be subcultured and maintained up to 21 d 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 48 h in haemocyte cultures. However, WSSV-exposed testis cells did not show any obvious change until 72 h 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.