The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity.
Genetic heterogeneity of RNA populations influences virus pathogenesis, epidemiology and evolution. Therefore, accurate information regarding virus genetic structure is highly important for both diagnostic and scientific purposes. For the Hepatitis E virus (HEV), the causal agent of hepatitis in humans, the intra-host population structure has been poorly investigated, mainly using the less sensitive RFLP-based approach. The objective of this study was to assess the suitability and the accuracy of single strand conformation polymorphism (SSCP) analysis, a well-established tool in genetic variation research, for the characterization of HEV quasispecies. The analysis was conducted on 50 clones of five swine isolates and 30 clones of three human HEV isolates. To identify and quantify the sequence variants present in each HEV isolate, 348bp long fragments of the amplified conserved ORF2 region were separated by cloning. Ten clones per isolate were subjected to SSCP and sequenced in a parallel experiment. The results show a high correlation of SSCP haplotype profiling with the sequencing results, confirming the sensitivity and reliability of this simple, rapid and low cost approach in the characterization of HEV quasispecies.
University of Zagreb, Faculty of Science, Department of Biology, Marulićev trg 9A, Zagreb, Croatia.
S
S
Černi
Croatian Veterinary Institute, Department of Virology, Savska cesta 143, Zagreb, Croatia.
J
J
Prpić
Croatian Veterinary Institute, Department of Virology, Savska cesta 143, Zagreb, Croatia.
L
L
Jemeršić
University of Zagreb, Faculty of Science, Department of Biology, Marulićev trg 9A, Zagreb, Croatia. Electronic address: dijana.skoric@biol.pmf.hr.
D
D
Škorić
D000818:Animals; D014644:Genetic Variation; D005838:Genotype; D006239:Haplotypes; D016752:Hepatitis E virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity; D013552:Swine
Capsid protein; Cloning; HEV; Quasispecies; SSCP