| Summary: | A viral chimera in which the <i>P1-HCPro</i> bi-cistron of a plum pox virus construct (PPV-GFP) was replaced by that of potato virus Y (PVY) spread slowly systemically in <i>Nicotiana benthamiana</i> plants and accumulated to levels that were 5−10% those of parental PPV-GFP. We tested whether consecutive mechanical passages could increase its virulence, and found that after several passages, chimera titers rose and symptoms increased. We sequenced over half the genome of passaged chimera lineages infecting two plants. The regions sequenced were <i>5′NCR-P1-HCPro-P3</i>; <i>Vpg/NIa</i>; <i>GFP-CP</i>, because of being potential sites for mutations/deletions leading to adaptation. We found few substitutions, all non-synonymous: two in one chimera (nt 2053 <i>HCPro</i>, and 5733 <i>Vpg/NIa</i>), and three in the other (2359 <i>HCPro</i>, 5729 <i>Vpg/NIa</i>, 9466 <i>CP</i>). <i>HCPro</i> substitutions 2053 A<b>U</b>U(Ile)→A<b>C</b>U(Thr), and 2359 C<b>U</b>G(Leu)→C<b>G</b>G(Arg) occurred at positions where single nucleotide polymorphisms were observed in NGS libraries of sRNA reads from agroinfiltrated plants (generation 1). Remarkably, position 2053 was the only one in the sequenced protein-encoding genome in which polymorphisms were common to the four libraries, suggesting that selective pressure existed to alter that specific nucleotide, previous to any passage. Mutations 5729 and 5733 in the <i>Vpg</i> by contrast did not correlate with polymorphisms in generation 1 libraries. Reverse genetics showed that substitution 2053 alone increased several-fold viral local accumulation, speed of systemic spread, and systemic titers.
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