To Phylogenetic trees were constructed by MEGA7 [11]

To construct the phylogenetic tree, nucleotide and amino acid
sequences of all the species were retrieved from public databases. Sequence
alignment was done by Clustal omega with default parameters. Phylogenetic trees
were constructed by MEGA7 11 using Maximum likelihood method based on JTT
matrix model 12. Numbers at each node represent percentage bootstrap values
with 1000 replicates.

Recombination
analysis

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Recombination
violates the branching pattern and thus challenges arise in performing
straightforward phylogenetic analysis 13. In order to understand the
evolution of Wuhan poty-like virus 1,
genome-wide screening of recombination signals was screened. Available 10
macluravirus whole genome sequences (4 CYNMV, 3 ArLV, YCMV, BDVA and Wuhan poty-like virus 1) were retrieved
from NCBI and aligned using Clustal omega with default parameters. Pairwise
Homoplasy Index (PHI test) for the aligned sequence calculated by SPLITSTREE 14.
The alignment file was then analysed using RDP4 package version 4.82 by default
settings and a Bonferroni corrected P-value cut-off of 0.05 and 0.01.
Recombination signals of a given alignment file were analysed by RDP, GENECONV,
BOOTSCAN, MAXCHI, CHIMAERA, SISCAN and 3SEQ as implemented in RDP4 package 15.
Recombination events predicted by three or more programs were considered as
likely recombinants.

Results

The
Wuhan poty like virus 1 genome
consists 8197 nts, excluding poly A tail and relatively similar to other
macluraviruses 8224 nts in CYNMV, 8287 nts in ArLV and 8208 nts in YCMV. Length
of 5′ UTR is 164 nts and 3′ UTR is 221 nts with polyprotein coding region of
7812 nts. The genome does not maintain plant consensus start codon UUGCAAUG
like CYNMV and YCMV 3, 7 with UAA stop codon. The genomic RNA of Wuhan poty-like virus 1 lacks the
complete P1 gene and has short HC-Pro gene as like other macluraviruses 3, 4,
7, 8. Similarly, the deduced amino acid sequences of Wuhan poty-like virus 1 polyprotein is about ~290kDa. It consists
of nine proteins HC-Pro, P3, 7K, CI, 9K, VPg, NIa, NIb, and CP. The second ORF,
P3N-PIPO starting motif GAAAAAA is positioned at 1370-1376 nts and encodes 56
amino acids. In CI protein, the helicase motifs GSGKSX3P and DEXH are found at
amino acids of 713-721 and 806-809 respectively 16. The highly-conserved
tyrosine residue in VPg responsible for genome linked is observed at 1424th
position of polyprotein 17. Motif of NIa protease (H-X31-D-X61-T-X4-C-X15-H)
is also found 18. Predicted NIb/CP cleavage sites of this polyprotein
consists LQ/M which is similar to all macluraviruses except Zingiberaceae infecting viruses those
has FQ/M. Mostly, the cleavage restricted residue methionine has been found at
P1′ position in macluraviruses 19. The RNA dependent RNA polymerase (RdRP)
specific domain GDD is present at 2146-2148. As like other macluravirus
members, N- terminal of CP does not have DAG triplet peptide for aphid
transmission 20.

Phylogenetic
tree constructed using available full-length polyprotein sequences of Macluravirus and of representatives of
each genera of Potyviridae revealed
that Wuhan poty-like virus 1 shared
the same clade with BDVA under the genus Macluravirus
(Fig.1a). Phylogenetic analysis of coat proteins of Macluravirus formed two distinct major groups (Fig.1b).
Mediterranean isolates NLV, MacMV and ArLV are clustered in one branch whereas
Asian isolates CdMV, LCCV, CYNMV, YCMV, AlpMV and Wuhan poty-like virus 1 are clustered in another branch. BDVA is
reported in New Zealand which is very close to Wuhan poty-like virus 1. The entire branching pattern is well supported
by the bootstrap values.

Recombination
of genetic material, between host and viruses or between viruses during
co-infection is a common phenomenon 21. In monopartite RNA viruses,
recombination may occur in two ways. First is occurred by switching over of
RNA-dependant RNA polymerase (RdRP) from one RNA genome to another and thereby
generating chimeric RNA molecule. In other case, pieces of RNA molecules are
ligated randomly to form recombinant RNA molecule which is known as
non-replicative recombination 22. In either way, recombination results in an
evolutionary breakthrough and thus decreasing the confident about phylogenetic
positioning of candidates. After aligning the whole genome sequences of
Macluraviruses, Pairwise Homoplasy Index (PHI
test) results showed statistically significant evidence for recombination with
a P-value of 2.32E-8 by SPLITSTREE. Among
all macluraviral isolates, identified events with significant cut off values
suggest that YCMV and ArLV are recombinants. Absence of recombination signals
in Wuhan poty-like virus 1 genome gives
additional supports to its phylogenetic classification. The details about
parental isolates, recombinants, breakpoint positions, detected events and the
corresponding P values are listed in
Table 1. The most significant interspecific recombination in YCMV is found at
nucleotide position of 7181-7225 between the major parent CYNMV and minor
parent Wuhan poty-like virus 1
(Fig.1d).  The major and minor parent
nucleotide sequences shared 72% and 56% similarity respectively with
recombinant isolate. This may reflect the predicted event might have occurred
due to template switching of RdRP.  The
corresponding nucleotide region spans the beginning of coat protein. This sharing
of genetic element is consistently supported by five out of seven programs
including RDP and maximum chi-square, which are robust in finding recombination
sites in viral sequences 23, 24, 25. The second intraspecific event observed
within the different isolates of ArLV. Recombination occurred at the position
of the P3 protein of ArLV (KF155694). Isolates FR37 (NC_026759) and FR50
(KP405233) are major and minor parents respectively (Fig.1e).