Mycoviruses identified in fungi and oomycetes, have genomic types of double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), and DNA [1]. DsRNA mycoviruses belong to seven families including Birnaviridae, Chrysoviridae, Cystoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, and Quadriviridae, and a new established genus Botybirnavirus [2, 3]. The family Chrysoviridae contains two genera, namely Alphachrysovirus (17 species) and Betachrysovirus (8 species). Members of Chrysoviridae possess 3–7 dsRNA segments with each segment being separately encapsulated. Among these dsRNA segments, the dsRNA1 and dsRNA2 encodes RNA-dependent RNA polymerase (RdRp) and capsid protein (CP), respectively. Virions of chrysoviruses, whose hosts include fungi, plants, and insects, are isometric and non-enveloped, with the size of about 40 nm in diameter [4, 5].
In 1988, viral dsRNAs were detected in Alternaria alternata isolated from Japanese pear trees, which is the first reported mycovirus in Alternaria [6]. Later, many mycoviruses had been identified in A. alternata, such as Alternaria alternata virus-1 (AAV-1) [7], Alternaria alternata chrysovirus 1 (AaCV1) [8], Alternaria alternata partitivirus 1 (AtPV1) [9], Alternaria alternata botybirnavirus 1 (AaBRV1) [3], and Alternaria alternata hypovirus 1 (AaHV1) [10]. In addition, several mycoviruses including Alternaria tenuissima partitivirus 1 (AttPV1) [11], Alternaria brassicicola endornavirus 1 (AbEV1) [12], Alternaria longipes dsRNA virus 1 (AlRV1) [13], Alternaria arborescens victorivirus 1 (AaVV1) [14], and Alternaria dianthicola dsRNA virus 1 (AdRV1) [15] had been reported in five other species (A. tenuissima, A. brassicicola, A. longipes, A. arborescens, and A. dianthicola) of Alternaria. Recently, an ssRNA mycovirus named Alternaria solani fusarivirus 1 (AsFV1) had been isolated from A. solani [16].
In our previous study, A. solani had been documented to cause potato early blight in China [17, 18]. In this study, A. solani strain DT-10, which was recovered from diseased leaves of potato with the symptoms of early blight in Datong city, Shanxi province of China, was identified according to the methods described previously [17, 18]. A new alphachrysovirus was found to be associated with the A. solani strain DT-10 and designated as Alternaria solani chrysovirus 1 (AsCV1).
Provenance of the virus material
Alternaria solani strain DT-10 was cultured on potato dextrose agar (PDA) plates overlaid with cellophane membranes for 7 days in the dark at 25°C. Multiple dsRNAs were extracted from the mycelia by binding to the CF-11 cellulose powder in 15% ethanol as described by Morris and Dodds [19]. After treatment with S1 nuclease and DNase I (TaKaRa, Dalian, China), the dsRNAs were analyzed by 1.0% agarose gel electrophoresis (Fig. 1A) and purified with a gel extraction kit (Aidlab Biotechnologies, Beijing, China). The partial complementary DNA (cDNA) sequences of the dsRNA segments were obtained and assembled as described previously [20]. Using specific primers designed based on the obtained sequences of cDNA (Supplementary Table 1), the terminal sequences were determined by RNA-ligase-mediated rapid amplification of cDNA ends (RLM-RACE) method as described previously [21, 22]. The full genome sequences of AsCV1 were obtained through assembling all the partial cDNA and terminal sequences with DNAMAN 7.0 (Lynnon Biosoft, USA), and deposited into the GenBank database with the accession numbers of MW656210, MW656211, MW656212, and MW656213 for dsRNA1, dsRNA2, dsRNA3, and dsRNA4, respectively.
Sequence properties
The full sequence of the four dsRNA fragments are 3,600 bp, 3,128 bp, 2,996 bp, and 2,714 bp, respectively. The open reading frame (ORF) of each dsRNA sequence was analyzed using ORFfinder online tool (https://www.ncbi.nlm.nih.gov/orffinder/). Each dsRNA contains a single ORF, which was named ORF1, ORF2, ORF3, and ORF4 for dsRNA1, dsRNA2, dsRNA3, and dsRNA4, respectively (Fig. 1B). BLASTp search for the four ORFs resulted in the highest similarity to the counterparts of Helminthosporium victoriae virus 145S (HvV145S) in the genus Alphachrysovirus of the family Chrysoviridae. ORF1 encoded a protein of 1084 amino acids (aa) (124.56 kDa) with 72.38% sequence similarity to the RdRp of HvV145S-A9 (GenBank No. NC_005978). The predicted RdRp domain at 351–803 aa contained eight conserved motifs (Supplementary Fig. 1A). The protein encoded by ORF2 contained 905 aa (102.29 kDa), which showed 61.40% sequence identity to the putative CP of Bipolaris maydis chrysovirus 1 (BmCV1) (GenBank No. KY489955), an isolate of HvV145S [23]. BLASTp also presented that ORF3-encoded protein (835 aa, 92.84 kDa) and ORF4-encoded protein (729 aa, 82.31 kDa) had 69.84% identity to Alphachryso-P3 of HvV145S-A9 and 41.77% identity to Alphachryso-P4 of BmCV1, respectively.
The 5′-untranslated regions (UTRs) and 3′-UTRs of AsCV1 were 242 bp and 103 bp long in dsRNA1, 312 bp and 98 bp long in dsRNA2, 317 bp and 171 bp long in dsRNA3, and 315 bp and 209 bp long in dsRNA4, respectively (Supplementary Table 2). Multiple alignments of the 5′-UTR and 3′-UTR sequences of the four dsRNAs were performed using DNAMAN 7.0 (Lynnon Biosoft). The 5′-UTRs presented strong sequence similarities in their internal parts, sharing strictly conserved terminal sequence of CGATAAAAACAAAAA. Meanwhile, the 3′-UTRs among the four dsRNA segments shared conserved GCTTTAAGTGT termini (Supplementary Fig. 1B). The 5′-UTRs and 3′-UTRs of AsCV1 dsRNA1-4 had 51.65%-68.01% and 30.04%-70.34% identities to those of HvV145S-A9 and BmCV1. Intriguingly, several unique inserts (3–37 bp) and deletions (5–64 bp) were found in 5′-UTRs of AsCV1, which were different from those of HvV145S-A9 and BmCV1 (Fig. 2).
The RdRp sequences of AsCV1 and 24 members of the family Chrysoviridae were analyzed by ClustalX 2.0 and MEGA 6.0 [22]. The phylogenetic tree constructed by the neighbor-joining algorithm with 1000 replicates based on the RdRp sequences showed that AsCV1 formed a well-supported taxonomic cluster with the members of the genus Alphachrysovirus and was closely related to HvV145S-A9 and BmCV1 (Fig. 1C).
Virion particles were extracted from the mycelium of A. solani strain DT-10 and stained according to the methods previously described [24, 3]. Viewed under transmission electron microscope (TEM) (JEM-1230, JEOL, Tokyo, Japan), the particles of AsCV1 were isometric with a diameter of approximately 40 nm (Supplementary Fig. 1C).
According to ICTV, the host of isolation, nucleotide and deduced amino acid sequence data, the length of genome segments, and 5′-UTRs are useful species demarcation criteria for the genus Alphachrysovirus [4]. Based on host fungus A. solani, low sequence identities of proteins encoded, unusual characteristics of 5′-UTRs, and phylogenetic relationships to known members of Alphachrysovirus, AsCV1 is a novel member in the genus Alphachrysovirus. This is the first report of the complete genome sequence of a new chrysovirus infecting A. solani.