Nucleotide sequence, genome organization and phylogenetic analysis of pineapple mealybug wilt-associated virus-2

The genome of pineapple mealybug wilt-associated closterovirus-2 (PMWaV-2) was cloned from double-stranded RNA isolated from diseased pineapple and it s sequence determined. The 3'-terminal 14861 nt of the single-stranded RNA genome contains ten open reading frames (ORFs) which, from 5' to 3', potent ially encode a > 204 kDa polyprotein containing papain-like protease, methy ltransferase and helicase domains (ORF1a), a 65 kDa RNA-dependent RNA polym erase (ORF1b), a 5 kDa hydrophobic protein (ORF2), a 59 kDa heat shock prot ein 70 homologue (ORF3), a 46 kDa protein (ORF4), a 34 kDa coat protein (OR F5), a 56 kDa diverged coat protein (ORF6), a 20 kDa protein (ORF7), a 22 k Da protein (ORF8) and a 6 kDa protein (ORF9). A 132 nt untranslated region was present at the 3' terminus of the genome. This genome organization is t ypical of the monopartite closteroviruses, including the putative +1 riboso mal frameshift allowing expression of ORF1b. Phylogenetic analysis revealed that within the family Closteroviridae the mealybug-transmitted PMWaV-2 is more closely related to other mealybug-transmitted members than to those w hich are transmitted by aphids or whiteflies. Within this group, PMWaV-2 sh ares the greatest sequence identity with grapevine leafroll-associated viru s-3, another mealybug-transmitted closterovirus.