Raw reads were processed to remove adaptor and low-quality reads. The transcript sequences were de novo assembled into contigs using paired-end splicing method in the Trinity program. BLASTx analysis of assembled contigs revealed that one contig consisting of 4118 nt was most similar to the genome sequences of viruses of the family Tombusviridae. This result suggests that a Tombusviridae species could be responsible for the virus-disease-like symptoms in diseased Paris polyphylla var. yunnanensis plants. Sequencing of RT-PCR products of the total RNA isolated from virus-infected Paris polyphylla var. yunnanensis leaves produced a sequence of 4118 nt that includes the 5’ and 3’ RACE data for the isolated virus which was deposited in the GenBank database under the accession number MW423699.
The complete RNA genome of ParV2 (MW423699) consists of 4,118 nt, and contains six ORFs, 5’-UTR (19 nt) and 3’-UTRs (359 nt) (Fig. 1B). The first ORF of the ParV2 putatively encodes a P40 protein (nt:20-1096) of 40.14 kDa. If the amber codon UAG (nt:1094–1096) is readthrough, the ORF2 (nt:20-2683, 888 aa) predicted to encode a 100.26 kDa RNA-dependent RNA polymerase (RdRp, P100) protein is continuously translated. This ParV2 RdRp is similar to the highly conserved RdRp of members of the family Tombusviridae that contains a conserved 684GDD686 motif [7–9]. The ParV2 sequence 5’AAA UAG GGG 3’ (nt:1091–1099) surrounding the amber stop codon is consistent with the proposed relative efficiency of readthrough sequences (A/C/U) (A/U) AUAG (G/C) (G/A). This amber stop codon surrounded sequence of ParV2 is 100% identical to the corresponding sequence present in the members of Tombusviridae[10–12].ORF3 (nt:2641–2844,68aa,7.31kDa) encodes the putative movement protein 1 (MP1, P7), that may be involved in virus movement, contains the FNF motif (at the C-terminus) that is conserved within the putative movement proteins of Carmoviruses, Machlomoviruses and Panicoviruses[13, 14], an indication that ParV2 is a member of the Tombusviridae. As seen in some animal and plant viruses whose initiation codons for mRNA translation are non-AUG start codons CUG, ACG and GUG[13, 15, 16],the ORF4 (nt:2844–3062,72aa,7.85kDa,P8) encoding the putative movement protein 2 (MP2) of ParV2 has the non-canonical start codon 2844ACG2846. The last adenine base of ORF3 stop codon is the first base of ORF4 start codon. Similarly, the MP2 of panicoviruses such as Cocksfoot mild mosaic virus (CMMV), Panicum mosaic virus (PMV), Thin paspalum asymptomatic virus (TPAV) and Bermuda grass latent virus (BGLV) initiates with CUG, GUG, CUG and CUG start codons respectively[15, 17, 18]. ORF5 (nt:3031–3759, 243 aa) encodes the 26.09 kDa coat protein (CP, P26). In addition to pre-readthrough and readthrough proteins (RdRp), MP1, MP2, CP, ParV2 also contains ORF6 (nt:3207–3434,75aa) that is embedded within the CP gene, and encodes the P9 (accessory protein) of 8.77kDa. The existence of Tombusviridae motifs and other Tombusviridae-specific features in ParV2 genome suggest that ParV2 is evolutionally close to viruses in this family, and that ParV2 is a member of the family Tombusviridae.
Table 1
Nucleotide (nt) and amino acid (aa) sequence identities of ParV2 and other members of a few genus within Tombusviridae.
Genus of Tombusviridae
|
Virus Name
|
Accession number
|
Viral genome sizes(nt)
|
Sequence identity (%) complete genomes(nt)
|
Sequence identity (%)for encoded proteins
|
RdRP
|
MP1
|
MP2
|
CP
|
Unclassified
|
Ginger chlorotic fleck-associated tombusvirus
|
MN581046
|
4143
|
55.5
|
59.2
|
42.8
|
47.2
|
34.9
|
Panicovirus
|
Bermuda grass latent virus
|
NC-032405
|
4044
|
43.7
|
40.2
|
23.2
|
18.6
|
19.8
|
|
Thin paspalum asymptomatic virus
|
JX848616
|
4193
|
43.5
|
38.5
|
19.1
|
24.0
|
21.2
|
|
Thin paspalum asymptomatic virus
|
NC-021705
|
4195
|
41.9
|
38.5
|
20.5
|
24.0
|
18.1
|
|
Panicum mosaic virus
|
MH885652
|
4324
|
40.6
|
36.3
|
17.8
|
25.3
|
23.6
|
|
Cocksfoot mild mosaic virus
|
NC-011108
|
4198
|
42.4
|
38.6
|
24.3
|
16.0
|
24.2
|
Pelarspovirus
|
Jasmine mosaic-associated virus 2
|
MG958506
|
3885
|
31.7
|
31.1
|
12.8
|
13.0
|
7.7
|
|
Jasmine mosaic-associated virus 2
|
MF991300
|
3975
|
32.5
|
30.2
|
11.4
|
11.9
|
8.0
|
|
Jasmine virus H
|
MH231176
|
3867
|
33.4
|
30.4
|
11.4
|
10.8
|
8.0
|
|
Jasmine virus H
|
MH231180
|
3867
|
33.4
|
30.2
|
12.8
|
11.9
|
8.3
|
|
Jasmine mosaic-associated virus 1
|
MG958505
|
3867
|
32.5
|
29.8
|
11.4
|
10.8
|
8.6
|
Machlomovirus
|
Maize chlorotic mottle virus
|
MF510225
|
4439
|
38.9
|
38.9
|
25.7
|
14.8
|
17.0
|
|
Maize chlorotic mottle virus
|
MF510235
|
4439
|
39.1
|
38.9
|
25.7
|
14.8
|
17.0
|
Betacarmovirus
|
Turnip crinkle virus
|
MK301398
|
4061
|
33.8
|
30.6
|
15.0
|
7.6
|
10.1
|
|
Japanese iris necrotic ring virus
|
NC-002187
|
4041
|
37.8
|
26.3
|
10.5
|
13.6
|
10.1
|
Gammacarmovirus
|
Melon necrotic spot virus
|
AY122286
|
4271
|
32.2
|
29.3
|
15.7
|
23.6
|
9.3
|
|
Pea stem necrosis virus
|
NC-004995
|
4048
|
32.1
|
30.7
|
15.7
|
18.0
|
8.1
|
|
Cowpea mottle virus
|
NC-003535
|
4029
|
37.3
|
29.0
|
18.3
|
7.8
|
8.7
|
Alphacarmovirus
|
Angelonia flower break virus
|
NC-007733
|
3962
|
31.7
|
29.9
|
17.6
|
13.4
|
9.3
|
|
Pelargonium flower break virus
|
NC-005286
|
3923
|
32.2
|
29.4
|
18.0
|
11.1
|
8.5
|
Gallantivirus
|
Galinsoga mosaic virus
|
NC-001818
|
3803
|
34.4
|
29.3
|
16.9
|
14.8
|
9.7
|
Further pairwise comparison of the complete genome nucleotide sequence and ORFs of ParV2 indicated that ParV2 share 31.7–55.5% complete genome nt, 26.3–59.2% RdRp, 10.5–42.8% MP1, 7.6–47.2% MP2, and 7.7–34.9% CP aa identities with 21 virus isolates of the family Tombusviridae (Table 1). The highest identity (55.5%) was seen with GCFaV-1 (QKE30557) followed by members of the genus Panicovirus (40.6–43.7%).Again, the highest aa identities of the individual proteins of ParV2 were seen with GCFaV-1, followed by Machlomovirus (RdRp and MP1) or Panicovirus (MP2 and CP). These results indicate that ParV2 is a member of the Tombusviridae, although it may not have all the characteristics of specific genera within the family Tombusviridae.
To determine the phylogenetic status of ParV2 and the evolutionary relationship between ParV2 and other viruses of the family Tombusviridae, phylogenetic tree was constructed using the aa sequences of RdRp protein (a reliable indicator for virus taxonomy).It can be seen from the tree that ParV2 clustered with the unassigned/unclassified GCFaV-1, and machlomoviruses and panicoviruses of the subfamily Procedovirinae. GCFaV-1 is the closest known relative of ParV2 (Fig. 2). It can be inferred from the tree that ParV2 shares a recent common ancestor with viruses of the genera Machlomovirus and Panicovirus. ParV2 and GCFaV-1 diverged from the branch that gave rise to the genera Machlomovirus and Panicovirus (Fig. 2). It can be concluded that Parv2 is a member of the subfamily Procedovirinae of the family Tombusviridae, although it couldn’t be allocated to any Procedovirinae-specific genera. These results confirm our pairwise comparison, and genome analyses results which indicated that ParV2 is a novel member of the family Tombusviridae.