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A novel Asfarvirus-like virus identified as a potential cause of mass mortality of abalone - PubMed

  • ️Wed Jan 01 2020

A novel Asfarvirus-like virus identified as a potential cause of mass mortality of abalone

Tomomasa Matsuyama et al. Sci Rep. 2020.

Erratum in

Abstract

A novel Asfarvirus-like virus is proposed as the etiological agent responsible for mass mortality in abalone. The disease, called abalone amyotrophia, originally was recognized in the 1980s, but efforts to identify a causative agent were unsuccessful. We prepared a semi-purified fraction by nuclease treatment and ultracentrifugation of diseased abalone homogenate, and the existence of the etiological agent in the fraction was confirmed by a challenge test. Using next-generation sequencing and PCR-based epidemiological surveys, we obtained a partial sequence with similarity to a member of the family Asfarviridae. BLASTP analysis of the predicted proteins against a virus database resulted in 48 proteins encoded by the novel virus with top hits against proteins encoded by African swine fever virus (ASFV). Phylogenetic analyses of predicted proteins of the novel virus confirmed that ASFV represents the closest relative. Comparative genomic analysis revealed gene-order conservation between the novel virus and ASFV. In situ hybridization targeting the gene encoding the major capsid protein of the novel virus detected positive signals only in tissue from diseased abalone. The results of this study suggest that the putative causative agent should be considered a tentative new member of the family Asfarviridae, which we provisionally designate abalone asfa-like virus (AbALV).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

Results of the artificial infection tests. (A) Cumulative mortalities of black abalone (Haliotis discus discus) in experimental infections. Two groups of abalone were challenged with semi-purified fractions by intramuscular injection (dotted line). The positive control group (bold line) and negative control group (fine line) were treated in the same way with homogenates of diseased and healthy abalone, respectively. (B) Histological observation of a survivor from one of the semi-purified fraction-injected groups. Note abnormal cell masses (*). Black bar indicates 50 µm.

Figure 2
Figure 2

Electrophoresis images of PCR products obtained from individual DNA samples. PCR products from healthy abalone in 2015 and 2016, diseased black abalone (Haliotis discus discus), and giant abalone (Haliotis madaka) (N = 6 per group) were electrophoresed, stained with GelGreen, and visualized using an LED transilluminator. M indicates the DNA molecular-weight size marker (2,000, 1,000, 500, and 100 bp). Full-length gels are presented in Supplemental Fig. 3.

Figure 3
Figure 3

Phylogenetic tree based on the deduced amino acid sequences of abalone asfa-like virus (AbALV)-encoded DNA polymerase (A), topoisomerase (B), major capsid protein (MCP) (C), DNA-directed RNA polymerase 1 (D), and DNA-directed RNA polymerase 2 (E) sequences compared to homologous proteins in other nucleocytoplasmic DNA viruses. The tree was constructed by the maximum-likelihood method using MEGA7; the numbers at nodes indicate percentages of bootstrap support from 1,000 replicates each. Bar indicates expected amino acid substitutions per site. Two MCP proteins predicted from the AbALV genome were analyzed separately, but full-length MCP protein after splicing was analyzed from faustovirus and kaumoebavirus.

Figure 4
Figure 4

Syntenic analysis of abalone asfa-like virus (AbALV) and African swine fever virus (ASFV). Comparative genomics with AbALV (as obtained in the present study; incomplete genome) is shown at the top and reference genome of ASFV Georgia 2007/1 is shown at the bottom; the figure was generated using GenomeMatcher. Genomic regions with >30% nucleotide identity are joined (top vs. bottom). The color scale on the right-hand side indicates percent protein identity. Black areas with no joining represent regions that lack synteny. Proteins used for phylogenetic analysis (top) and inversion and translocation sites (bottom) are indicated by arrows, and the corresponding ASFV genes are indicated by locus tag, locus, and protein name.

Figure 5
Figure 5

In situ hybridization showing the presence of the viral genome in diseased black abalone (Haliotis discus discus). Arrows indicate cells positive for staining. (A) Cross-section of a diseased abalone. The foot muscle is located on the upper side and the visceral mass is located on the lower side. Squares indicate the magnified fields shown in Panels C and D. The letters d, i, m, r, and s indicate digestive gland, intestine, mantle, stomach, and radula, respectively. (B) Higher-magnification image of infected cells. Positive signals were observed in entire cells, including the cytoplasm. Arrowheads indicate the cell nuclei of cells negative for staining. (C) Enlarged view around the nerve cord. Note that nerve cells are negative for staining (arrowheads). (D) Enlarged view around the digestive tract.

Figure 6
Figure 6

External appearance and histopathological characteristics of abalone with amyotrophia. (A) Photograph of diseased black abalone shells with symptomatic incisions on the front margin of the shells (arrowheads) and brown pigmentation inside the shell (arrows). Histological observation of healthy (B) and diseased (C) black abalone (Haliotis discus discus) and healthy (D) and diseased (E) giant abalone (Haliotis madaka). Abnormal cell masses (*) are observed around the ganglia in both species. Black bars indicate 200 µm.

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