Characterization of a Novel RNA Virus Causing Massive Mortality in Yellow Catfish, Pelteobagrus fulvidraco, as an Emerging Genus in Caliciviridae (Picornavirales)

Abstract

An emerging disease in farmed yellow catfish (Pelteobagrus fulvidraco) causing massive mortality broke out in 2020 in Hubei, China. Histopathological examination indicated significant changes in kidneys and spleens of diseased fish. Electron microscopy revealed large numbers of viral particles in the kidneys and spleens. These particles were spherical with a diameter of approximately 35 nm. By using RNA sequencing and rapid identification of cDNA ends, the full nucleotide sequence of the virus was identified. The viral genome comprises 7,432 bp and contains three open reading frames sharing no nucleotide sequence similarity with other viruses; however, the amino acid sequence partially matched that of the nonstructural (NS) proteins from viruses in the order Picornavirales. Combined with the phylogenetic analysis, the conserved amino acid motifs and the domains of the viral genome predict a genome order typical of a calicivirus. Therefore, this virus was tentatively named yellow catfish calicivirus (YcCV). Cell culture showed that YcCV could cause a cytopathic effect in the channel catfish kidney cell line (CCK) at early passages. In artificial infection, this virus could infect healthy yellow catfish and led to clinical symptoms similar to those that occurred naturally. In situ hybridization analysis detected positive signals of the virus in kidney, spleen, liver, heart, and gill tissues of diseased fish. This study represents the first report of calicivirus infection in yellow catfish and provides a solid basis for future studies on the control of this viral disease. IMPORTANCE Caliciviruses are rapidly evolving viruses that cause pandemic outbreaks associated with significant morbidity and mortality globally. A novel calicivirus identified from yellow catfish also causes substantial mortality. Using an RNA sequencing (RNA-seq) and rapid amplification of cDNA ends (RACE) method, the full nucleotide sequence was identified and characterized, and this virus was tentatively named yellow catfish calicivirus (YcCV). A nucleotide sequence similarity search found no match with other viruses, and an amino acid sequence comparison indicated approximately 23.3% amino acid homology with the viruses in the order Picornavirales. These findings may represent a new avenue to explain virus evolution and suggest a need to further study the pathogenesis of calicivirus and characterize possible interactions among interspecific viruses in the aquaculture environment.

Keywords: Calicivirus; Pelteobagrus fulvidraco; Picornavirus; characterization; phylogenetic analysis; yellow catfish.

FIG 1

Clinical signs of diseased yellow catfish. (A) Diseased yellow catfish hanging head up in water and exhibiting disoriented behavior; (B) diseased yellow catfish showing hemorrhages on head, mouth, lower jaw, and fin base; (C) spleen of diseased fish, showing a dark color; (D) kidney showing severe hemorrhagic and necrosis; (E) diseased fish spleen showing various-sized vacuolation (red arrow) by H&E staining; (F) diseased fish kidney showing edema and a moderate to heavy infiltration of lymphocytes (blue arrows) and condensed and marginated nuclei of glomerulus cells (black arrow) by H&E staining.

FIG 2

Identification of the causative pathogen in diseased yellow catfish using RNA-seq, transmission electron microscopy, and virus isolation. (A) Kidney. Viral particles were observed in the cytoplasm. Nu, nucleus. Bar = 1 μm. (Aa) Higher magnification of the area bounded by the white rectangle in panel A showing magnified virus particles (white arrow). Bar = 200 nm. (B) Spleen. The mature viruses clustered in the cytoplasm near the cell nucleus. Bar = 1 μm. (Ba) Higher magnification of the area bounded by the white rectangle in panel B showing magnified virus particles (white arrow). Bar = 500 nm. (C) Workflow for RNA sequencing of kidney and spleen samples from diseased yellow catfish. (D and E) Viral mRNA sequence identification in kidney and spleen samples from diseased fish. Numbers and lengths of nonviral mRNA sequences (black solid dot) and predicted viral mRNA sequences (red solid dot) are shown. (F) Normal CCK cell. (G) CCK cell infected with the third passage of YcCV at 7 days postinfection. Typical CPE is visible. Bar = 100 μm. (H) Virus particles in CCK cells. Bar = 2.0 μm. (I) Higher magnification of the area bounded by the white rectangle in panel H showing magnified virus particles (white arrow). Bar = 500 nm.

FIG 3

Genome sequencing analysis of YcCV. (A) The assembled genome YcCV. The genome is composed of three ORFs (NS, VP1, and ORF3), a 5′ UTR at the 5′ terminus, and a 3′ poly(A) tail at the 3′ terminus. (B) Overlapping RT-PCR was carried out with six pairs of primers (YcCV-F1/YcCV-R1 to YcCV-F6/YcCV-R6) to obtain the main sequences of YcCV. (C) 5′ and 3′ RACE amplification of the full-length YcCV genomic sequence using the primers YcCV-rF1, -rF2, -rR1, and -rR2. (D) mRNA reads from the kidney tissue that mapped to the complete viral genome were confirmed by using RACE, RT-PCR from kidney tissue, and Sanger sequencing. Blue mapping indicates major splice donor and acceptor sites. The lower diagram shows the genomic structure of the YcCV sequence from the kidney of yellow catfish. Blue boxes indicate the nonstructural protein (NS), capsid protein (VP1), and ORF3 protein coding regions, and arrowheads show direction of YcCV transcription. (E) The location of conserved domains for an RNA helicase, RdRp, and capsid protein covering the regions found in YcCV. (F) Diagram comparing the genomic structures of members in the order of Picornavirales with that of the calicivirus isolated from yellow catfish. An asterisk indicates YcCV derived from the present study.

FIG 4

Detection, transmission, and distribution of YcCV in diseased yellow catfish. (A) Locations of RT-PCR primers on the YcCV genome. (B) Detection of YcCV in naturally infected yellow catfish using RT-PCR. Lane M, 1,000-bp DL1000 DNA ladder; lanes 1 to 3, results for kidney tissue samples taken from three naturally infected yellow catfish (samples 1 to 3); lane 4, negative control; lane 5, positive control. (C) RT-PCR detection of YcCV in the experimental group and the control group (i.p. injection of Dulbecco’s PBS). Lane M, DL1000 1,000-bp DNA ladder. Lanes 1 to 3, results for kidney tissues samples taken from three naturally infected yellow catfish; lanes 4 to 6, results for three mock-infected yellow catfish; lane 7, positive control (P); lane 8, negative control (N). (D) Mortality of yellow catfish in the experimental group and the control group. A homogenate of diseased fish tissues was prepared and filtered through a 0.22-μm filter (10 to 13 cm in length), and 0.5 mL was injected i.p. to challenge 30 fish in the experimental group. (E) Location of the in situ hybridization probe for YcCV. (F) Kidney samples from mock-infected yellow catfish. (G) Kidney samples with positive signals in infected yellow catfish (white arrow) using in situ hybridization. (H) Spleen samples from mock-infected yellow catfish. (I) Spleen samples with positive signals in infected yellow catfish (white arrow). (J) Distribution of YcCV in gill, brain, intestine, heart, kidney, spleen, and liver tissues of naturally infected yellow catfish. Lane M, 1,000-bp DL1000 DNA ladder; lane 1, liver; lane 2, spleen; lane 3, kidney; lane 4, heart; lane 5, gill; lane 6, brain; lane 7, intestine; lane 8, negative control. (K) Numbers of copies of YcCV in seven different tissues from naturally infected yellow catfish. Data are means and standard errors of the means for 3 independent replicates. (L) Locations of diseased yellow catfish samples collected in Hubei Province and Sichuan Province. Circles show sampling sites of yellow catfish; red indicates samples that were positive for YcCV, and black indicates negative samples.

FIG 5

FISH detection of YcCV in naturally infected yellow catfish. (A and C) FISH hybridization in YcCV-infected kidney and spleen cells, respectively; (Aa, Ca) detection of positive signals in kidney and spleen cells, respectively; (Ba, Da) absence of signal in healthy kidney and spleen cells. Arrows show positive signals.

FIG 6

Phylogenetic analysis based on amino acid sequences of the nonstructural protein from the order Picornavirales. Seven representative families in the order Picornavirales were used to construct the phylogenetic tree, using the maximum-likelihood method with 500 bootstrap replicates. The novel YcCV is indicated by pink shading, and the numbers under the branches indicate the bootstrap values (values lower than 50 are hidden). The genetic distance (as the number of substitutions per site) is represented by the scale bar.