A Novel Virus Causes Scale Drop Disease in Lates calcarifer

Abstract

From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch's postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.

Fig 1. Phylogenetic clustering of SDDV within the Iridoviridae family.

Deduced amino acid sequences of 26 conserved genes (see S2 Table) were aligned with the corresponding genes of Iridoviridae of which the full length genome sequence is available. The genes of each virus are placed in the same order and the total was used for phylogenetic analysis, similar as described by Eaton et al. [9]. The neighbor-joining method with pairwise deletion within the MEGA-5 package was used, bootstrap values (for 500 replications) are provided at the root of the clusters and the scale bar is a measure of the proportion of divergence. SDDV is indicated with • in the tree.

Fig 2. Replication of SDDV on SK21 cells.

SK21 cells were inoculated with an MOI of 0.01 TCID₅₀/cell SDDV (passage 3). (A/C) Negative control confluent monolayer of SK21 cells on day 3 (A) and 4 (C); (B/D) SDDV-infected SK21 cells on day 3 (B) and 4 (D) after inoculation; (E) SDDV genome copy number/mL (red line and left y-axis) and infectious titer (blue diamonds and right y-axis) of the infected SK21 cells in time. Error bars represent the standard deviation.

Fig 3. Electron microscopy of SDDV.

Hexagon particles with (upper left) or without (lower right) outer membrane as visualized by by negative staining (A) followed by tomography (B). In (C) the outer membrane (1), capsid (2) and inner core (3) of a different enveloped particle are indicated by arrows. In (B) and (C) (ortho)slices through virus particles are shown.

Fig 4. Inoculation of SDDV in Lates calcarifer.

(A) Kaplan-Meier survival curve of infected fish with 5x10⁶ TCID₅₀/fish (IP—black line) or 5.5 x10⁵ TCID₅₀/fish (IM—blue line) or both (IP + IM—yellow line). IP (1:10; green line) dose equaled 5.5 x10⁵ TCID₅₀/fish. (B) Control fish at day 10; (C) SDDV-infected fish (IP-high dose) at day 10 after infection. Note the fin erosion, tail erosion, body color variations, white or less mucus (inset), and changes in eye color. (D) SDDV genome copy number in serum of the fish on day 1, 7, 10, and 14 after infection. Diamond—black line: IP (mortality 60%); Triangle—blue line: IM (mortality 13%); Single cross—yellow line: IP + IM (mortality 47%); Square—green line: IP 1:10 (mortality 20%).

Fig 5. Protection of scale drop syndrome by vaccination of Lates calcarifer.

Kaplan-Meier survival curve of fish vaccinated with formalin-inactivated virus (FK-SDDV), binary ethyleneimine-inactivated virus (BEI-SDDV), recombinant MCP protein (recMCP), or diluent control vaccine (placebo). Twenty-eight days after vaccination the fish were challenged with 2 x 10⁷ TCID₅₀ SDDV per fish (intraperitoneal), mortality was scored until day 28 after challenge. All three vaccines provided >70% RPS against disease (p<0.001, Tarone-Ware test).