Selection pressure and codon usage bias analysis
To analyze the selection pressure on the ORF of TMUV, 4 different methods were used: Single Likelihood Ancestor Counting (SLAC), Fixed Effects Likelihood (FEL), Fast Unconstrained Bayesian AppRoximation for inferring selection (FUBAR), and mixed effects model of evolution (MEME) on DATAMONKEY (http://www.datamonkey.org/) (Katoh & Standley, 2013; Murrell et al., 2013; Murrell et al., 2012; Smith et al., 2015). The basic nucleotide composition (A%, T%, C%, and G%), nucleotide at the third position of synonymous codons (A3s%, T3s%, C3s%, and G3s%), GC content on the third synonymous codon position (GC3s), and the effective number of codons (ENC) were calculated for the VP2 gene sequence using CodonW (1.4.2).
The ENC value ranged from 20 to 61. The higher the ENC value, the smaller is the codon usage bias. Therefore, when the evaluation value is 20, the deviation is stronger. When the ENC value is less than 35, codon usage bias is considered very strong (Comeron & Aguade, 1998; He et al., 2019). Subsequently, an ENC-plot (ENC plotted against GC3s) was drawn to reflect the factors influencing codon usage bias (Fuglsang, 2004; Wright, 1990). The expected ENC was calculated as follows (‘s ’ is the frequency of G + C at the third position of synonymous codons):
\begin{equation} ENC\ expect\ =\ 2+s+\frac{29}{s^{2}+{(1-s)}^{2}}\nonumber \\ \end{equation}
At the same time, the neutrality plot analysis was also performed, which can reflect the degree of the influence of mutation pressure and natural selection on codon use patterns. We can judge whether natural selection or mutation is dominant based on the slope of the curve. All data were plotted using Origin 2018 software.