RESULTS: A PCR of the gtrIC cluster showed that serotype 1c isolates from different geographical origins were genetically conserved. An analysis of sequences flanking the gtrIC cluster revealed remnants of a prophage genome, in particular integrase and tRNA(Pro) genes. Meanwhile, Southern blot analyses on serotype 1c, 1a and 1b strains indicated that all the tested serotype 1c strains may have had a common origin that has since remained distinct from the closely related 1a and 1b serotypes. The identification of prophage genes upstream of the gtrIC cluster is consistent with the notion of bacteriophage-mediated integration of the gtrIC cluster into a pre-existing serotype.
CONCLUSIONS: This is the first study to show that serotype 1c isolates from different geographical origins share an identical pattern of genetic arrangement, suggesting that serotype 1c strains may have originated from a single parental strain. Analysis of the sequence around the gtrIC cluster revealed a new site for the integration of the serotype converting phages of S. flexneri. Understanding the origin of new pathogenic serotypes and the molecular basis of serotype conversion in S. flexneri would provide information for developing cross-reactive Shigella vaccines.
IMPORTANCE: EV-A71 is one of many viruses that cause HFMD, a common syndrome that largely affects infants and children. HFMD usually causes only mild illness with no long-term consequences. Occasionally, however, severe infection may arise, especially in very young children, causing neurological complications and even death. EV-A71 is highly contagious and is associated with the most severe HFMD cases, with large and frequent epidemics of the virus recorded worldwide. Although major advances have been made in the development of a potential EV-A71 vaccine, there is no current prevention and little is known about the patterns and dynamics of EV-A71 spread. In this study, we utilize full-length genome sequence data obtained from HFMD patients in Viet Nam, a geographical region where the disease has been endemic since 2003, to characterize the phylodynamics of this important emerging virus.
METHODS AND RESULTS: Total nucleic acids were extracted from leaf samples harvested from frond 20 of seven Dura × Pisifera (D × P) African oil palm (Elaeis guineensis Jacq.) aged between 13 and 21 years old collected from local plantations. The nucleic acids were fractionated using 5% non-denaturing polyacrylamide gel electrophoresis (PAGE) before being subjected to detection by reverse transcribed polymerase chain reaction (RT-PCR). The PCR products were cloned into a plasmid vector and the sequence of the clones was analyzed. CCCVd variants were quantified using real-time qPCR assay with CCCVd specific primers. Sixteen randomly selected clones of (OP246) had an arbitrary 100% identity with CCCVdOP246 (GeneBank Accession No: HQ608513). Meanwhile, four clones had >93% similarity with several minor sequence variations forming variants of OP234, OP235, OP251 and OP279.
CONCLUSION: The OS symptoms observed in the field were characterized into three categories based on the size and morphology of the orange spots on the affected fronds. In addition, there was no direct correlation between disease severity and the accumulation of CCCVd variants in oil palm. This finding is the first report describing the sequence variation of the CCCVd RNA and symptom variation in OS oil palm field samples.