cdna sbs Archives - Gen9 Genetics
Infectious cDNA clones are a powerful tool for RNA virus studies using reverse genetics. Potato virus S (PVS) is a carlavirus with worldwide distribution. Although the complete genomic sequences of many isolates of PVS have been reported, the construction of an infectious cDNA clone of PVS has not yet been reported. The objective of this study is the development and molecular characterization of an infectious cDNA clone of PVS.
A full length cDNA clone pPVS-H-FL-AB was constructed by connecting eight isolated PVS H95 cDNA clones. Blocked RNA transcripts of pPVS-H-FL-AB and a modified clone pPVS-H-FL-H, containing the consensus genome sequence of PVS-H95, were shown to be non-infectious. Therefore, a full-length cDNA clone pPVS-H-FL-β was reconstructed from PVS-H00, isolated from PVS-H95 populations by repeating the isolation of a single local lesion in Chenopodium quinoa three times; PVS-H00 appeared to be a selected variant that survived genetic bottlenecks. The sequence of the cDNA clone pPVS-H-FL-β was determined as the sequence of the PVS-H00 genome and compared with the consensus sequence of the PVS-H95 genome.
All Nicotiana occidentalis plants inoculated with capped RNA transcripts ≥0.2 μg of pPVS-H-FL-β developed symptoms in the upper leaves, as observed with inoculation of PVS-H00. Similar levels of viral genomic and subgenomic RNA and coat protein were detected in systemically infected leaves. Sequence comparison of PVS-H95 and PVS-H00 revealed polymorphisms of 370 nucleotides (4.4% of the complete genome sequence), resulting in 91 amino acid substitutions in six open reading frames (ORFs). The infectivity of the recombinant-derived chimeric RNAs between the two cDNA clones revealed that the lack of infectivity of the pPVS-H-FL-H transcripts was due to ORF1, which encodes replicase and harbors 80 amino acid substitutions compared to pPVS-H-FL- β. Approximately 71.3% of the amino acid substitutions in the replicase were located within the variable region of unknown function between the putative methyltransferase and ovarian tumor-like protease domains.
This is the first report of the development of an infectious PVS cDNA clone. Our analyzes suggest that the PVS population within a plant exists as a quasispecies and the diversity of PVS replicase sequences obstructs the construction of a full-length infectious cDNA clone.
The potato virus S (PVS) is one of the most common viruses of the potato (Solanum tuberosum L.) with worldwide distribution. PVS alone generally causes little or no symptoms in most potato varieties; however, potato yield losses of up to 20% have been reported in case of secondary infection [1, 2]. Two important biologically distinct strains of PVS, PVSO (ordinary) and PVSA (Andean), have been identified based on the ability to systemically infect plants of Chenopodium quinoa and C. amaranticolor. PVSO induces local chlorotic lesions in the inoculated leaves of C. quinoa and C. amaranticolor without systemic infection, whereas PVSA induces systemic chlorotic mottling in plants of C. quinoa and C. amaranticolor after inducing chlorotic lesions in the inoculated leaves. Furthermore, PVSA causes more severe symptoms on potato leaves and is more easily transmitted by aphids and contact with infected plants than PVSO [3, 4]. Recently, however, some isolates exhibiting different biological properties than PVSO and PVSA strains have been reported from Tasmania, Australia . Unlike PVSO, 13 out of 44 Tasmanian isolates cause local but asymptomatic infection only in inoculated leaves of C. quinoa. In contrast, nine isolates, unlike PVSA, cause systemic infection without the typical symptoms of C. quinoa. Of these nine isolates, five induce symptoms only in inoculated leaves and four cause asymptomatic systemic infection in inoculated and non-inoculated upper leaves .
Matoušek et al.  suggest that PVS-Chenopodium-systemic (CS) isolates from Central Europe are genetically distinct from PVSA isolates, and are closely related to European PVSO isolates. Cox and Jones  proposed the term PVSO-CS for isolates that systemically infect Chenopodium spp. but they do not cluster within the PVSA clade, based on phylogenetic analysis based on coat protein (CP) gene sequences. However, there is no evidence that CP is involved in systemic infection in Chenopodium spp. For example, the Vltava CP gene sequence isolated from Czech Republic clusters within the PVSA clade , although Vltava does not infect C. quinoa  systemically. The analysis