The Isolation and Full-Length Transcriptome Sequencing of a Novel Nidovirus and Response of Its Infection in Japanese Flounder (Paralichthys olivaceus)

Abstract

A novel nidovirus, CSBV Bces-Po19, was isolated from the marine fish, Japanese flounder (Paralichthys olivaceus). The viral genome was 26,597 nucleotides long and shared 98.62% nucleotide identity with CSBV WHQSR4345. PacBio Sequel and Illumina sequencing were used to perform full-length transcriptome sequencing on CSBV Bces-Po19-sensitive (S) and -resistant (R) Japanese flounder. The results of negative staining revealed bacilliform and spherical virions. There were in total 1444 different genes between CSBV Bces-Po19 S and R groups, with 935 being up-regulated and 513 being down-regulated. Metabolism-, immune-, and RNA-related pathways were significantly enriched. Furthermore, CSBV Bces-Po19 infection induced alternative splicing (AS) events in Japanese flounder; the S group had a higher numbers of AS events (12,352) than the R group (11,452). The number of long non-coding RNA (lncRNA) in the S group, on the other hand, was significantly lower than in the R group. In addition to providing valuable information that sheds more light on CSBV Bces-Po19 infection, these research findings provide further clues for CSBV Bces-Po19 prevention and treatment.

Keywords: alternative splicing; isoform sequencing; marine fish; metabolism; viral genome.

Figure 1

Clinical signs of CSBV Bces-Po19 infected Japanese flounder (Paralichthys olivaceus). The clinical signs observed in the moribund fish were partial body surface reddening, especially hemorrhages in fins and abdominal muscles. (A) The blind side of fish with CSBV Bces-Po19 disease; (B) visceral organs of the abdomen. Arrows indicate the hemorrhage positions.

Figure 2

Histopathology of CSBV Bces-Po19 infected Japanese flounder (Paralichthys olivaceus). (A) Gill, with black arrow indicating the gill lamella with slight hyperplasia; red arrow indicates the gill lamella in stick shape with obvious hyperplasia. (B) Liver, with black arrow indicating the vesicular degeneration of hepatocytes; red arrow indicates sinus dilatation and blood cells extravasation. (C) Spleen, with black arrows indicating the hemolytic plaque; asterisk indicates vacuolated splenocytes. (D) Kidney, with black arrows indicating tubular epithelial cells necrosis; red arrow indicates renal stroma hemolysis. (E) Heart, with black arrows indicating the congestive tissue with erythrocytes. (F) Stomach, with black arrows indicating infiltrated hemocytes in the submucosa; asterisk indicates necrotic loose connective tissue of submucosa. Scale bars: 40 µm.

Figure 3

The genetic characteristics of CSBV Bces-Po19. (A) Structural organization of the CSBV Bces-Po19 genome. (B) Phylogenetic analysis of CSBV Bces-Po19 and other aquatic nidoviruses.

Figure 4

RT-PCR detection of CSBV Bces-Po19 in JFB cells. M, DL2000 DNA marker; Lanes 1–5, control JFB cells cultured for 4 days; Lanes 6–10, control JFB cells cultured for 7 days; Lanes 11–15, CSBV Bces-Po19-infected JFB cells for 4 days; Lanes 16–20, CSBV Bces-Po19-infected JFB cells for 7 days.

Figure 5

Transmission electron micrographs of liver, kidney, ovary, and spleen from CSBV Bces-Po19-infected Japanese flounder (Paralichthys olivaceus). (A) Liver, with arrows indicating the mature virions; asterisk indicates the mitochondrion with virion-like, granular osmiophilic substances. (B) Kidney, with arrows indicating the mature virions. (C) Ovary, with asterisk indicating viral budding from the infected cell membrane. (D) Spleen, with arrow indicating the mature virion; asterisk indicates the mitochondrion with virion-like, granular osmiophilic substances. Scale bars: 400 nm.

Figure 5

Transmission electron micrographs of liver, kidney, ovary, and spleen from CSBV Bces-Po19-infected Japanese flounder (Paralichthys olivaceus). (A) Liver, with arrows indicating the mature virions; asterisk indicates the mitochondrion with virion-like, granular osmiophilic substances. (B) Kidney, with arrows indicating the mature virions. (C) Ovary, with asterisk indicating viral budding from the infected cell membrane. (D) Spleen, with arrow indicating the mature virion; asterisk indicates the mitochondrion with virion-like, granular osmiophilic substances. Scale bars: 400 nm.

Figure 6

Transmission electron micrographs of JFB cells inoculated with CSBV Bces-Po19. (A) Arrows indicate the spherical virions; asterisk indicates viral-like tubular structure. (B) Asterisk indicates bacilliform virion. (C) Arrow indicates the spherical virion budding from the infected cell membrane; asterisk indicates bacilliform virion. (D) Transmission electron microscopy (negative staining) of purified virions from the JFB cell supernatant. Orange asterisks indicate bacilliform virions; white asterisks indicate spherical virions. Scale bars: 200 nm.

Figure 6

Transmission electron micrographs of JFB cells inoculated with CSBV Bces-Po19. (A) Arrows indicate the spherical virions; asterisk indicates viral-like tubular structure. (B) Asterisk indicates bacilliform virion. (C) Arrow indicates the spherical virion budding from the infected cell membrane; asterisk indicates bacilliform virion. (D) Transmission electron microscopy (negative staining) of purified virions from the JFB cell supernatant. Orange asterisks indicate bacilliform virions; white asterisks indicate spherical virions. Scale bars: 200 nm.

Figure 7

The differentially expressed genes (DEG) between CSBV Bces-Po19-sensitive and -resistant Japanese flounder (Paralichthys olivaceus). (A) Volcano map of DEGs. Each dot represents one gene, red dots represent the significantly up-regulated genes, and green dots represent the significantly down-regulated genes. Blue dots represent genes that are not significantly differentially expressed. (B) Heatmap analysis of the hierarchical clustering of DEGs. Different colors indicate differences in the expression level.

Figure 8

GO and KEGG enrichment of differentially expressed genes (DEG) between CSBV Bces-Po19-sensitive and -resistant Japanese flounder (Paralichthys olivaceus). (A) GO enrichment of DEGs. The names of the GO categories are listed along the y-axis; the degree of GO enrichment is represented by the −log₁₀ (p-value). (B) KEGG enrichment of DEGs. The x-axis represents the gene ratio, which is a proportion of the DEGs out of the total genes in a KEGG term. y-axis is the gene function classification of KEGG. Different plot colors indicate different q values. Plot diameter represents the DEG numbers in a KEGG term.

Figure 9

Alternative splicing (AS) analysis of the full-length transcriptome of CSBV Bces-Po19-sensitive and -resistant Japanese flounder (Paralichthys olivaceus). (A) AS events statistics of CSBV Bces-Po19-sensitive (S) group. (B) AS events statistics of CSBV Bces-Po19-resistant (R) group. (C) Comparison of the number of different AS in genes between CSBV Bces-Po19 S and R group.

Figure 10

The differentially expressed isoforms between CSBV Bces-Po19-sensitive and -resistant Japanese flounder (Paralichthys olivaceus). (A) Volcano map of differentially expressed isoforms. Each dot represents one isoform, blue dots represent the significantly up-regulated isoforms, and red dots represent the significantly down-regulated isoforms. Green dots represent isoforms that are not significantly differentially expressed. (B) Heatmap analysis of the hierarchical clustering of differentially expressed isoforms with their genes. Different colors indicate differences in the expression level.

Figure 11

Identification of lncRNAs in CSBV Bces-Po19-sensitive (S) and -resistant (R) Japanese flounder (Paralichthys olivaceus). (A) Venn diagram of the number of lncRNAs predicted by CNCI, PLEK, CPC, and Pfam in the CSBV Bces-Po19-resistant group. (B) Distribution of lncRNAs identified in HCSBV Bces-Po19-resistant group. (C) Venn diagram of the number of lncRNAs predicted by CNCI, PLEK, CPC, and Pfam in the CSBV Bces-Po19-sensitive group. (D) Distribution of lncRNAs identified in CSBV Bces-Po19-sensitive group.