Susceptibility of primary cultured cells from the lymphoid organ of Penaeus monodon to Monodon Baculovirus (MBV) and White Spot Syndrome Virus (WSSV)

Issue Date

12-2008

Abstract

A primary cell culture system, susceptible to monodon baculovirus (MBV) and white spot syndrome virus (WSSV), was developed from the lymphoid organ of Penaeus monodon. Lymphoid organs from shrimps (15–20 g) were dissected out and then placed in 2X Leibovitz-15 (L-15) medium with antibiotics (1 h) and subsequently minced. Four to five fragments were seeded in each well of a 24-well culture plate and incubated in supplemented 2X L-15. The plates were incubated at 28 ºC in normal atmospheric condition. The basal culture medium was not supplemented with growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF) or interleukin-2 (IL-2), but results showed excellent cell proliferation and migration. Cells were 80% confluent after 4–6 d of culture, with predominantly fibroblast-like type of cells, which were eventually overgrown with epithelioid type of cells after 7 d of culture. The cell monolayers lasted for 3–4 wk with regular replacement of the growth medium. Subsequent experiments showed that MBV and WSSV replicated in the primary lymphoid cell cultures.Seven-day-old cell monolayers inoculated with two-fold dilutions of viral suspensions of either MBV orWSSV exhibited localized cytopathic effects (CPE) after 1–2 d of incubation at 28 ºC. Early CPE showed the presence of highly refractile bodies and cell shrinkage; advanced cellular degeneration was observed after 3-4 d incubation, especially in monolayers which were inoculated with the higher viral dilutions. Significant cellular degeneration took a longer time to develop in cells inoculated with the lowest dilutions of WSSV. Polymerase chain reaction (PCR) assays were done to confirm MBV and WSSV infection of the cells. Positive MBV and WSSV infections were detected in all samples, except in those inoculated with the lowest viral dilutions, at the nested PCR step only. Subsequent passages (2nd and 3rd) of harvested cell suspension on cultured lymphoid cells likewise produced CPE from day-3 post-inoculation. This further confirms viral susceptibility and replication in the primary cultured lymphoid cells.

Source or Periodical Title

Philippine Agricultural Scientist, The (Formerly: The Philippine Agriculturist)

ISSN

0031-7454

Volume

91

Issue

4

Page

450-458

Document Type

Article

College

College of Agriculture and Food Science (CAFS)

Frequency

quarterly

Physical Description

illustrations; references

Language

English

Subject

Penaeus monodon, monodon baculovirus, white spot syndrome virus, primary shrimp cell cultures, lymphoidorgan, cytopathic effects

En – AGROVOC descriptors

MONODON; BACULOVIRUS; WHITE SPOT SYNDROME VIRUS; CELL CULTURE; PCR; PENAEUS MONODON; SHRIMP CULTURE

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