Lettuce necrotic yellows virus (LNYV) is the type species of the genus Cytorhabdovirus, family Rhabdoviridae. The virus is restricted to Australia and New Zealand and causes an economically important disease of lettuce. The complete genome sequence of only one cytorhabdovirus, Northern cereal mosaic virus (NCMV) has been reported. This study describes the completion and analysis of the LNYV genome sequence, and analysis of the sequence variability of its nucleocapsid (N) gene.
The variability of the nucleocapsid gene sequence of nine isolates, from five states of Australia, was analysed. Only the 3' untranslated region of these isolates varied in length. The majority of nucleotide substitutions in the N gene ORF were third codon position changes. Two distinct subgroups were evident from phylogenetic analysis and were supported by pairwise sequence alignments. Sequence identity between the subgroups was -20% and -4% at the nucleotide and amino acid levels, respectively. The sequence of the N gene of these isolates was examined for potential phosphorylation sites, translation and transcription start and stop signals, intergenic sequence variation, eukaryotic polyadenylation signals and hydropathy. A putative RNA-binding motif was identified by comparison to analogous sequences of other rhabdovirus N proteins. A LNYV N gene RT-PCR and restriction enzyme typing assay has also been devised for future validation.
The glycoprotein (G) gene of 1836 nt was cloned and sequenced and analysed with respect to potential eukaryotic polyadenylation and transcription start/stop signals. Three potential glycosylation sites were identified in the LNYV G protein sequence, as well as heptad repeat sequences and putative signal and transmembrane domains. Sequence identity of the LNYV G protein to other rhabdoviruses was ̴20% and eleven cysteine residues were identified that appear to be specific to G proteins of plant-infecting rhabdoviruses. Three possible non-viral nucleotides including a potential m7G5'ppp5' cap were identified at the 5' end of LNYV G gene mRNA.
The large (L) polymerase gene of 6335 nt was cloned and sequenced to complete the genome of 12,807 nucleotides. While limited sequence identity was found between the L protein of LNYV and that of other rhabdoviruses, its "polymerase module" sequence displayed higher sequence conservation. Phylogenetic analysis of both of these sequences for a selection of rhabdoviruses supported current taxanomic groupings. These phylogenetic and sequence identity analyses suggest that LNYV may be more closely related to Strawberry crinkle virus, than NCMV. Previously described motifs within the polymerase module sequences of negative-sense, non-segmented RNA viruses were also identified in the LNYV L gene and that of representatives of all six rhabdovirus genera.
An error was identified and corrected in the previously published LNYV 5' trailer sequence. Analysis of at least one complete genome sequence from each of the rhabdovirus genera found only plant-infecting rhabdoviruses contained additional genes between the phosphoprotein and matrix protein genes. An extended stretch of sequence could be found in all rhabdoviruses between the glycoprotein gene translation stop codon and the polymerase gene transcription start sequence and some genera-specific trends were evident with respect to genome complexity, coding capacity and gene and intergenic sequence sizes. The complete LNYV genome sequence and cloned probes developed during this research will be useful molecular tools for future study of the biology, cytopathology and transcription and replication processes of LNYV.