engleski

The application of single strand conformation polymorphism (SSCP) in the molecular characterization of Hepatitis E virus intra-host populations

Intra-host populations of RNA viruses are genetically diverse and consist of a group of phylogenetically related variants or “quasispecies”. Moreover, due to the frequent reinfections, intra-host virus isolates can acquire phylogeneticaly distant variants having different biological properties. Since the genetic heterogeneity of viral populations influences their pathogenicity, epidemiology and evolution, accurate information regarding virus genetic structure is of great importance for both diagnostic and scientific purposes. Hepatitis E virus (HEV) is an RNA pathogen causing both acute and chronic hepatic disease in humans. Its’ zoonotic origin, with pigs as the main reservoirs, is well recognized and is a growing health concern in European countries. Also, in Croatia, where our previous study confirmed the prevalence of 30% in domestic pigs and 28.4% in wild boars, first autochthonous human cases have been reported recently. Although the quasispecies structure of HEV has been confirmed, its’ intra-host diversity has not been broadly investigated. For that reason, the objective of our study was to assess the suitability and the accuracy of single strand conformation polymorphism (SSCP) analysis, a well-established tool in the genetic variability research, for the characterization of HEV populations. SSCP is the electrophoretic separation of single-stranded DNA where a single nucleotide change can considerably affect the strand’s electrophoretic mobility by altering the intra-strand base paring and its resulting conformation. The analysis was conducted on 50 clones of five swine and 30 clones of three human HEV isolates. All swine isolates belonged to zoonotic genotype 3, while human isolates belonged to genotype 1, infecting humans only. To identify and quantify sequence variants present in each HEV isolate, 348 bp 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. In all tested samples, the coexistence of different genomic variants was reported, suggesting higher diversity of human HEV isolates. Moreover, the comparison of the SSCP haplotype profiling with the sequencing results showed a high correlation, confirming the sensitivity and reliability of this simple, rapid and low cost approach in the characterization of HEV quasispecies.

HEV; SSCP; quasispecies

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