Discussion
Ranaviruses have been recognized as agents of emerging infectious
disease that are now globally distributed (Duffus et al., 2015).
Establishing a rapid and effective taxonomic method plays an important
role in virus identification and epidemiological investigation. Multiple
molecular biology methods, such as
RFLP (Restriction endonuclease
fragment length polymorphism) profiles and cross-hybridization, are used
for virus classification (King et al., 2011). But, with the development
of DNA sequencing technology, genomic DNA sequencing and analysis as
demarcation criteria are becoming more common. An increasing number of
ranavirus genome sequences have been published in the public databases,
which can contribute to the investigation of the evolutionary history
and taxonomy study. In this study, we employed genomic phylogenic
analysis and dot plot comparison to gain a better understanding of
classification within ranavirus subspecies. Dot plot analysis can
provide insight into genome architecture changes (containing deletions,
inversions and duplications) among ranaviruses (Jancovich, Steckler, et
al., 2015). These genome architecture changes can be visualized in a
picture using JDotter software (Brodie et al., 2004). In previous
research, dot plot analysis was widely used in the investigation of the
evolutionary history by comparison of genome architecture changes (Chen
et al., 2013; Jancovich et al., 2003). In this study, we found that the
same group of ranavirus subspecies approximately showed similar
linearity pattern (Figure 4) according to dot plot analysis maps of the
FV3-AY548484 genome versus other ranavirues genomes. Neither NJ-Tree nor
CV-tree can clearly determine taxonomic position of ToRV (Figure 2 and
Figure 3), while ToRV should belong to CMTV-like group by Dot plot
analysis (Figure 4 and Figure S1). Therefore, dot plot analysis as an
important supplementary method can assist phylogenic analysis to confirm
taxonomic position of ranaviruses (Table S4).
Presently, the most commonly used classification of newly isolated
ranaviruses is phylogenetic analysis based on a single gene (e.g, MCP
gene, NF-H1 gene and DNA polymerase gene (Allender et al., 2013; George
et al., 2015; Jancovich, Steckler, et al., 2015; Zhou et al., 2013).
While phylogenic analysis of a single gene is convenient, it is unlikely
to be as robust as whole genome analysis and even causes mistakes. For
example, MCP gene is a highly conserved gene and is most widely applied
in phylogenic analysis (Jancovich, Steckler, et al., 2015). The
Neighbor-Joining phylogenic tree using MCP gene showed that
ToRV and CMTV-E was closely related
to the FV3-like group, which is not consistent with taxonomic
identification based on genome analysis (Figure S5, Cluster10). As a
structural protein, MCP gene is too highly conserved sequence to
distinguish among various virus strains (Duffus et al., 2013). In
addition, Anke C. Stöhr et (Stöhr et al., 2015) constructed a
phylogenic tree using concatenated sequences (3223 bp) of MCP, DNApol,
RNR-α and RNR-β genes. The tree showed that ToRV was most closely to the
FV3-like group, but otherwise shows CMTV-like characteristics in regard
to the global arrangement of its genome (Figure S1) (Stöhr et al.,
2015). The reason for the difference observed is that the genes used in
phylogenic analysis experienced substantial substitution saturation or
contained recombinant fragments. For example, the RNR-β gene (Cluster
28) contained recombinant fragments (Table S5). In this study,
recombination analysis and substitution saturation analysis were
performed to select qualified genes, and the phylogenetic positions of
the concatenated 4 selected sequences were also consistent with
phylogenetic analysis based on core genes. These results can further
improve the accuracy of single-gene or multiple-genes phylogenetic trees
in ranavirus taxonomy.
In recent years, there has been a suggestion that GIV/SGIV should be
considered
as a new genus (Jancovich, Steckler, et al., 2015). We tend to agree
with the view based on our study. Firstly, GIV/SGIV share little
collinearity with FV3 (Figure 4) and other ranaviruses, which indicated
that GIV/SGIV genome architecture were significant different with those
of ranaviruses. Secondly, branch length of GIV/SGIV in phylogenetic tree
based on ranavirus core genes are 0.27 (Figure 3A). In contrast, branch
length among FV3- like, CMTV-like and EHNV-like group are about
0.01~0.03. The branch lengths represent the evolutionary
distances. The phylogenetic tree revealed that the evolutionary
distances between GIV/SGIV and other ranaviruses (FV3- like, CMTV-like
and EHNV-like gourp) are far greater than the evolutionary distances
among FV3- like, CMTV-like and EHNV-like group. In addition, in the
process of core pan analysis, it is interesting that only 2 genes within
GIV and SGIV genomes can be strictly classified into the same cluster
when compared without “-nsl” parameter. Meanwhile, 30 ranaviruses
(remove GIV and SGIV) share 50 strictly core genes (Figure S6). The
default program of PanX is without “-nsl” parameter, which means that
long branches gene will be removed from cluster. The branch lengths
reflect evolutionary distances among genes within one cluster (Ding et
al., 2017). Therefore, the results of core pan analysis indicated that
significant portions of strictly core genes within GIV/SGIV were far
from the others of 30 ranaviruses in evolutionary distances. In
conclusion, genomic characterizations of GIV/SGIV are significantly
different from other ranaviruses, and need to be considered as a new
genus.