Abstract
Viral infection is a great challenge in healthcare and agriculture. The Singapore grouper iridovirus (SGIV) is highly infectious to numerous marine fishes and increasingly threatens mariculture and wildlife conservation. SGIV intervention is not available because little is known about key players and their precise roles in SGVI infection. Here we report the precise role of VP088 as a key player in SGIV infection. VP088 was verified as an envelope protein encoded by late gene orf088. We show that SGIV could be neutralized with an antibody against VP088. Depletion or deletion of VP088 significantly suppresses SGIV infection without altering viral gene expression and host responses. By precisely quantifying the genome copy numbers of host cells and virions, we reveal that VP088 deletion dramatically reduces SGIV infectivity through inhibiting virus entry without altering viral pathogenicity, genome stability and replication and progeny virus release. These results pinpoint that VP088 is a key player in SGIV entry and represents an ideal target for SGIV intervention.
Highlights
Quantitatively affects Singapore grouper iridovirus (SGIV) entry without altering viral pathogenicity, genome replication, and the release of progeny virus
On SDS-PAGE, rVP88 exhibited a molecular weight of 47 kDa as predicted for a protein of 428 amino acid residues including 248 amino acids of truncated protein at the C-terminus of VP088 and additional 180 amino acids of fused tags encoded by pET32a (Fig. 1b)
With Western blot analysis, αVP088 clearly detected the purified rVP88, and it detected a protein with a molecule weight of approximately 54 kDa in the cell lysate from SGIV-infected HX1 cells, which was absent in mock-infected HX1 cells (Fig. 1c)
Summary
The medaka (Oryzias latipes) haploid ES cell line HX1 was maintained at 28 °C in medium. ESM4 as previously described[33,34]. The grouper spleen cell line GS was maintained at 25 °C in medium Leibovitz L-15 as described[31]. SGIV (strain A3/12/98) was propagated in GS cells as described[5]. SGIV was inoculated onto confluent GS cells at a multiplicity of infection (MOI) of ∼0.1. Upon the appearance of apparent cytopathic effect (CPE), cells and medium were collected, followed by three cycles of rapid freezing/thawing. Medium containing virus was centrifuged at 12,000 g (Eppendorf) for 30 min at 4 °C, the supernatant was centrifuged at 200,000 g (Beckman) for 1 h at 4 °C to concentrate virus, and the pellet was resuspended in L-15 and stored at −8 0 °C until use
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