Isolation, Identification and Genomic Analysis of Orange-Spotted Grouper Iridovirus Hainan Strain in China

Abstract

The orange-spotted grouper (Epinephelus coioides) is an important mariculture fish in China. However, in recent years, with the rapid development of aquaculture activities, outbreaks of viral diseases have affected the grouper aquaculture industry, causing severe economic losses. In the present study, we isolated and identified a virus from diseased, orange-spotted groupers from an aquaculture farm in Hainan Province, China. The isolated virus was identified as orange-spotted grouper iridovirus, hence named the orange-spotted grouper iridovirus Hainan strain (OSGIV-HN-2018-001). OSGIV-HN-2018-001 induces a cytopathic effect after the infection of mandarin fish (Siniperca chuatsi) brain clonal passage (SBC) cells. In addition, the cytoplasm of the OSGIV-HN-2018-001-infected SBC cells was found to contain a large number of hexagonal virus particles with a diameter of approximately 134 nm. Using the Illumina NovaSeq system, we assembled the sequence data and annotated the complete genome of OSGIV-HN-2018-001 (GenBank accession number: PP974677), which consisted of 110,699 bp and contained 122 open reading frames (ORFs). Phylogenetic tree analysis showed that OSGIV-HN-2018-001 was most closely related to ISKNV-ASB-23. The cumulative mortality rate of groupers infected with OSGIV-HN-2018-001 reached 100% on day 8. The spleens were enlarged and blackened after the dissection of the dying groupers. These results contribute to the understanding of the molecular regulatory mechanism of the iridovirus infection and provide a basis for iridovirus prevention.

Keywords: Epinephelus coioides; OSGIV-HN-2018-001; characterization; isolation; megalocytivirus.

Figure 1

OSGIV-HN-2018-001 infection in SBC cells. CPE induced by OSGIV-HN-018-001 in SBC cells. SBC cells were infected with OSGIV-HN-018-001 at indicated time points (2 d p.i. and 4 d p.i.). Cell morphologies were observed by light microscopy.

Figure 2

Viral particles were examined by electron microscopy.

Figure 3

OSGIV-HN-2018-001 genome circle mapping using the online software Proksee. The orange portion represents the predicted open reading frame (ORF), with the arrow direction indicating the approximate size and transcription direction of the ORFs. The blue part is the information on CDs, which were subjected to a homology search using the NCBI BLASTp (https://blast.ncbi.nlm.nih.gov/Blastp (accessed on 20 September 2024)) program. This circle diagram also shows the CG content and CG Skew distribution of the genome.

Figure 4

Phylogenetic relationship of OSGIV-HN-2018-001 and other fish iridoviruses. Phylogenetic trees based on the amino acid sequences of DNA polymerase (A), MCP (B), ATPase (C), and ribonuclease III (D) were constructed using MEGA software. All virus sequences were taken from the GenBank database (accession number: EHNV, FJ433873; FV3, NC_005946; ISKNV, AF371960; LCDV-C, AY300826; OSGIV, AY894343; PIV, MK098185; SDDV, MN562489; SGIV, AY521625; TRBIV, GQ273492; ISKNV-ASB-23, PP151097; LBUSV-GZ, OP009387; BCIV-2015, MW883595; SKIV-SD, MT986830).

Figure 5

Genome collinearity between OSGIV-HN-2018-001 and other megalocytiviruses. Data were obtained from the Mauve 2.3.1 software that analyzes population evolutionary events through multiple sequence comparisons. Accession numbers of viral genomes are as follows: LBUSV-GZ, OP009387; BCIV/WVL17393/2012, MN432490; BCIV, MN432490; BCIV-2015, MW883595; RSIV-Ku, KT781098; SKIV-SD, MT986830; TSGIV, MG570132; TTIV-2015, MW883599; OSGIV, AY894343; RBIV-C1, KC244182; TRBIV, GQ279492; ISKNV, AF371960; ISKNV-ASB-23, PP151097.

Figure 6

Pathogenicity of OSGIV-HN-2018-001 in groupers. Cumulative mortality of groupers after infection with OSGIV-HN-2018-001 was recorded up to 10 d p.i.

Figure 7

Healthy and clinical signs of groupers infected with OSGIV-HN-2018-001. (A,B) Healthy groupers; (C,D) groupers infected with OSGIV-HN-2018-001.

Figure 8

The results of PCR. The spleens of normal and dying groupers were taken for PCR. Agarose (1%) gel showed the PCR product (1362 bp) (lanes 1–5: normal fish in the control group; lanes 6–10: dead fish in the infected group), “+” lane is the positive control and “-” lane is the negative control.