Scale Drop Disease Virus Associated Yellowfin Seabream (Acanthopagrus latus) Ascites Diseases, Zhuhai, Guangdong, Southern China: The First Description

Abstract

Scale drop disease virus (SDDV), an emerging piscine iridovirus prevalent in farmed Asian seabass Lates calcarifer in Southeast Asia, was firstly scientifically descripted in Singapore in 2015. Here, an SDDV isolate ZH-06/20 was isolated by inoculating filtered ascites from diseased juvenile yellowfin seabream into MFF-1 cell. Advanced cytopathic effects were observed 6 days post-inoculation. A transmission electron microscopy examination confirmed that numerous virion particles, about 140 nm in diameter, were observed in infected MFF-1 cell. ZH-06/20 was further purified and both whole genome and virion proteome were determined. The results showed that ZH-06/20 was composed of 131,122 bp with 135 putative viral proteins and 113 of them were further detected by virion proteome. Western blot analysis showed that no (or weak) cross-reaction was observed among several major viral proteins between ZH-06/20 and ISKNV-like megalocytivirus. An artificial challenge showed that ZH-06/20 could cause 100% death to juvenile yellowfin seabream. A typical sign was characterized by severe ascites, but not scale drop, which was considerably different from SDD syndrome in Asian seabass. Collectively, SDDV was confirmed, for the first time, as the causative agent of ascites diseases in farmed yellowfin seabream. Our study offers useful information to better understanding SDDV-associated diseases in farmed fish.

Keywords: genome; pathogenicity; proteome; scale drop disease virus; yellowfin seabream ascites diseases.

Figure 1

A recent mortality event of YFSBAD and the clinical symptoms of naturally infected large-sized yellowfin seabream (80~140 g per fish). (A) A recent outbreak of YFSBAD in a local farm in Zhuhai. (B) The diseased fish were characterized by swollen abdomens, ascites, splenomegaly, and petechial to ecchymotic hemorrhage in the liver. Over 2 mL of ascites were extracted from each fish. The red arrows indicate enlarged spleens. The black arrows indicate bloodless liver.

Figure 2

Replication of ZH-06/20 in MFF-1 cells. (A) Normal MFF-1 cells; (B) ascites-inoculated MFF-1 cells on day 6 post-inoculation; (C/D) ZH-06/20 at passage 2 (C) and passage 7 (D) in MFF-1 cells at 2 days post-infection.

Figure 3

Transmission electron photomicrograph of ZH-06/20-infected MFF-1 cells and purified viral particles from infected MFF-1 cells. (A,B) Numerous hexagonal viral particles with a diameter of about 140 nm were observed in infected MFF-1 cells with low and large magnifications. (C,D) Spherical viral particles purified from infected MFF-1 cells with low and large magnifications.

Figure 4

Liner schematic organization of ZH-06/20 genome. The 26 common core genes in iridovirus, 27 ZH-06/20 specific genes and ZH-06/20 tandem repeat areas are indicated in red, blue, and yellow, respectively. Generally, ZH-06/20 is composed of 131,122 nucleotides with 135 ORFs and 27 unique genes are never found in other iridoviruses, except for SDDV.

Figure 5

Phylogenetic relationship of ZH-06/20 and 32 other members in the genus Megalocytivirus based on the mcp gene. A red solid triangle indicated the ZH-06/20. The FV3 in genus Ranavirus was used as an outgroup. Generally, megalocytivirus could be divided into two subgroups. The ISKNV-like subgroup includes RSIV, ISKNV, and TRBIV and the SDDV-like subgroup includes SDDV and ECIV. Genomic sizes of viral members in ISKNV group are about 111,000 bp. By contrast, genomic sizes of members in the SDDV-like group are 128,000~132,000 bp. FV3 in genus Ranavirus was used as an outgroup.

Figure 6

(A,B) Viral protein of the purified ZH-06/20. (A) Protein profile of the crude purification of ZH-06/20 by SDS-PAGE. (B) Western blot analysis of ZH-06/20 proteins recognized by anti-recombinant MCP pAb of ZH-06/20. (C,D) Cross-reaction between ZH-06/20 and ISKNV using different anti-ISKNV and anti-ZH-06/20 antibodies by western blot analysis. (C) The purified ZH-06/20 and ISKNV virions were recognized by anti-ISKNV-MCP, -VP007, -VP101, and anti-ZH-06/20 MCP pAbs. (D) ZH-06/20 and ISKNV-infected MFF-1 cells were recognized by anti-ISKNV VP101 and VP023 pAbs and anti-ISKNV 2D8 and VP023 mAbs. M: marker; Lane 1–2: Purified ZH-06/20 and ISKNV; Lane 3–5: ZH-06/20, ISKNV, and mock infected-MFF-1 cells, respectively. pAb, poly-antibody; mAb, monoclonal antibody.

Figure 7

Infectivity of ZH-06/20 to juvenile yellowfin seabream under artificial conditions. (A) The infected fish under artificial conditions showed ocular proptosis and swollen abdomens. (B) The mortality of artificial-infected yellowfin seabream was observed on the 6th day post-challenge.

Figure 8

Histopathological observation of the spleen and kidney of ZH-06/20-infected yellowfin seabream. (A,B) were the normal spleen and kidney, respectively. (C) Infected spleen tissue showed the most severe lesions with abundant vacuolated cells (green arrows) and diffuse karyolysis (red arrows). (D) Infected kidney tissue exhibited some karyolysis (red arrows) and distinct pyknosis (black arrows).

Figure 9

Immunofluorescence observation of infected tissues of spleen (Sp), kidney (Ki) and liver (Li). The infected cells are labeled by green fluorescence, which are associated with anti-ZH-06/20 MCP. Strongest fluorescence signals are observed in the spleen, then in the kidney and liver.