Characterization of the Complete Genome and P0 Protein for a Previously Unreported Genotype of Cotton Leafroll Dwarf Virus, an Introduced Polerovirus in the United States

Abstract

Virus-like disease symptoms consisting of leaf cupping, shortened internodes, and overall stunting were observed in commercial cotton fields in Alabama in 2017 to 2018. To determine the complete genome sequence of the suspected causal polerovirus, symptomatic leaf samples were collected in Macon County, Alabama, and subjected to Illumina RNA sequencing. Based on BLASTn analysis, the Illumina contig of 5,771 nt shared the highest nucleotide identity (approximately 95%) with members of the species Cotton leafroll dwarf virus (CLRDV) (genus Polerovirus; family Luteoviridae) from Argentina and Brazil. The full-length viral genome sequence was verified by reverse transcription (RT)-PCR amplification, cloning, and Sanger sequencing. The complete CLRDV genome of 5,865 nt in length shared 94.8 to 95.2% nucleotide identity with six previously reported CLRDV isolates. The genome of the CLRDV isolate amplified from Alabama samples (CLRDV-AL) has seven predicted open reading frames (ORFs). Viral proteins 1 to 5 (P1 to P5) shared 91.9 to 99.5% amino acid identity with the six CLRDV isolates from Argentina and Brazil. However, P0, the suppressor of host gene silencing, shared 82.4 to 88.5% pairwise amino acid identity with the latter CLRDV isolates. Phylogenetic analysis of the seven full-length CLRDV genomes resolved three sister clades: CLRDV-AL, CLRDV-typical, and CLRDV-atypical, respectively. Three recombination events were detected by the recombination detection program among the seven CLRDV isolates with breakpoints occurring along the genome. Pairwise nucleotide identity comparisons of ORF0 sequences for the three CLRDV-AL field isolates indicated that they were >99% identical, suggesting that this previously unknown CLRDV genotype represents a single introduction to Alabama.