Characterization of a Fungal Virus Representing a Novel Genus in the Family Alphaflexiviridae

Abstract

Sclerotinia sclerotiorum is an ascomycetous fungus and hosts various mycoviruses. In this study, a novel fungal alphaflexivirus with a special genomic structure, named Sclerotinia sclerotiorum alphaflexivirus 1 (SsAFV1), was cloned from a hypovirulent strain, AHS31. Strain AHS31 was also co-infected with two botourmiaviruses and two mitoviruses. The complete genome of SsAFV1 comprised 6939 bases with four open reading frames (ORFs), a conserved 5'-untranslated region (UTR), and a poly(A) tail in the 3' terminal; the ORF1 and ORF3 encoded a replicase and a coat protein (CP), respectively, while the function of the proteins encoded by ORF2 and ORF4 was unknown. The virion of SsAFV1 was flexuous filamentous 480-510 nm in length and 9-10 nm in diameter. The results of the alignment and the phylogenetic analysis showed that SsAFV1 is related to allexivirus and botrexvirus, such as Garlic virus X of the genus Allexivirus and Botrytis virus X of the genus Botrevirus, both with 44% amino-acid (aa) identity of replicase. Thus, SsAFV1 is a novel virus and a new genus, Sclerotexvirus, is proposed to accommodate this novel alphaflexivirus.

Keywords: Alphaflexiviridae; Sclerotinia sclerotiorum; flexivirus; mycovirus.

Figure 1

Biological characteristics of strains Ep-1PNA367 and AHS31. (a) Colony morphology of strains Ep-1PNA367 and AHS31. All the strains were cultured on PDA plates at 20 °C for 7 days. (b) Average growth rates of Ep-1PNA367 and AHS31 strains. (c,d) The pathogenicity assay of strain AHS31 (36 hpi). Error bars indicate standard deviation (SD) from sample means. Different uppercase letters on the top of each column indicate significant differences (p < 0.01).

Figure 2

The viruses that infected the S. sclerotiorum strain, AHS31, and virions of SsAFV1. (a) Confirmation of the presence of five positive RNA viruses in strain AHS31 by RT–PCR. In control sets, ddH₂O was used instead of RT products. Primers specific to each virus are listed in Table 1. (b) Virions of SsAFV1 observed under a transmission-electron microscope (TEM) after negative staining. The bar is 200 nm. (c) A histogram of SsAFV1 particle length and diameter. Error bars indicate standard deviation (SD) from sample means.

Figure 3

Genomic characteristics of SsAFV1. (a) Alignments of the nucleotide sequences of 5′-untranslated regions (UTRs) of SsAFV1 and selected viruses. (b) Schematic diagram of three fungal alphaflexivirus, SsAFV1, BVX, and SsDRV. Four conserved domains are shown as rectangular boxes in different colors. MTR, methyl transferase; HEL, RNA helicase; RdRp, RNA-dependent RNA polymerase; CP, coat protein.

Figure 4

Multiple-alignment analysis of RdRp and CP of SsAFV1 and selected viruses. (a) Alignment of conserved RdRp of SsAFV1 and four selected alphaflexiviruses (Table S1). Eight conserved domains (I–VIII) among alphaflexiviruses are marked and GDD in the red rectangular box is the typical catalytic motif of positive single-RNA viral RdRp. Identical residues are indicated by asterisks and highlighted in blue. The conserved and semi-conserved amino-acid residues are indicated by colons and dots and highlighted in pink and green, respectively. (b) Alignment of conserved CP motifs of SsAFV1 and four selected alphaflexiviruses. Abbreviations of selected viruses are given in Table S1. (c) A percentage-identity matrix of viral replicase multiple-aligned via Clustal Omega 2.1 and generated by a custom R script. Abbreviations of viral names and GenBank accession numbers are listed in Table S1.

Figure 5

Phylogenetic analysis of SsAFV1 and representative members of the family Alphaflexiviridae based on the alignment of replicase (a) and CP (b). These ML trees were constructed as described with calculated best-fit model LG + I + G4 for replicase and LG + G4 for CP. The parameter of bootstrap was set as 1000 replicates and bootstrap values over 50% were indicated on branches. The virus studied in this paper was tagged with a red dot; the scale bar at the top left corresponds to the genetic distance. Details of these alphaflexiviruses are given in Table S1.