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The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity
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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity
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a ˇ ˇ S. Cerni , J. Prpic´ b , L. Jemerˇsic´ b , D. Skori c´ a,∗ a b
University of Zagreb, Faculty of Science, Department of Biology, Maruli´cev trg 9A, Zagreb, Croatia Croatian Veterinary Institute, Department of Virology, Savska cesta 143, Zagreb, Croatia
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Article history: Received 5 February 2014 Received in revised form 14 April 2015 Accepted 16 April 2015 Available online xxx
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Keywords: Capsid protein Cloning HEV Quasispecies SSCP
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1. Introduction
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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, 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. 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. © 2015 Published by Elsevier B.V.
Intra-host RNA viral populations are composed of genetically related variants (Ojosnegros et al., 2011; Domingo et al., 2012) due to their high mutation and replication rates. These quasispecies are dynamic sources of viral adaptability enabling rapid evolution during different selective regimens (Schneider and Roossinck, 2001; Domingo et al., 2006). Evidently, virus pathogenicity depends on the structure of the quasispecies and the abundance of certain virus variants (Kumar et al., 2008; Ojosnegros et al., 2011). Quasispecies can hide components that in isolation would display dissimilar bioˇ et al., 2008; Domingo et al., 2012), while logical properties (Cerni reinfections may play a role in the accumulation of highly heterogeneous virus variants, some of which are distant phylogenetically. Hence, it is important to have an efficient laboratory screening tool for quick and accurate characterization of virus isolates. Although bulk cloning and sequencing is a good approach for detecting viral heterogeneity, it requires the preparation and sequencing of large numbers of clones to ensure that minor sequence variants are represented. Mainly due to the cost of too
∗ Corresponding author. Tel.: +385 1 4898079; fax: +385 1 4898081. E-mail addresses: [email protected], [email protected] ˇ ´ (D. Skori c).
many man-hours, it is not the most cost effective approach. Furthermore, the next generation sequencing approach, which enables quick detection of single nucleotide polymorphisms within different haplotypes, is still challenged with the reconstruction of virus quasispecies (Schirmer et al., 2012) and is not available widely for routine screenings. Single strand conformation polymorphism (SSCP) analysis is a good alternative that provides excellent insight into viral heterogeneity and reduces research costs (Gasser et al., 2006). This method enables the electrophoretic separation of single strand nucleic acids where a single nucleotide change could considerably affect strand electrophoretic mobility by altering intra-strand base paring and its resulting conformation (Orita et al., 1989). To date, it has been used widely for the rapid screening of selected genome fragments to identify most sequence variations between the mutants of many viruses, including hepatitis A, B and C (Hardie et al., 1996; Mackiewicz et al., 2005; Kumar et al., 2008). Although Hepatitis E virus (HEV) has only one serotype (Emerson and Purcell, 2003), its isolates display considerable genetic diversity (Lu et al., 2006; Smith et al., 2013). Increased testing and sequencing availability have resulted in an increased number of recorded HEV variants from wildlife and the environment (Smith et al., 2013). The recent Hepeviridae taxonomy proposal (Smith et al., 2014) recognizes four HEV human-infecting genotypes within the species Orthohepevirus A, though clear criteria are yet to be defined for the delineation of different HEV subgenotypes. To date, HEV
http://dx.doi.org/10.1016/j.jviromet.2015.04.020 0166-0934/© 2015 Published by Elsevier B.V.
ˇ Please cite this article in press as: Cerni, S., et al., The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.04.020
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genotypes 1 and 2 have been found to infect only humans. However, the zoonotic genotypes 3 and 4 have been detected in humans and in several animal species (Meng, 2010; Teshale et al., 2010). Domestic pigs are considered to be the main HEV reservoirs and have been studied the most extensively (Meng, 2011; Van der Poel, 2014). The quasispecies nature of HEV was first described by Grandadam et al. (2004) using restriction fragment length polymorphism (RFLP) analysis. Though two studies to date have addressed the direct sequencing of 20 randomly selected HEV clones (Zhang et al., 2010; Lhomme et al., 2012), RFLP remained the only method used in the characterization of intra-host heterogeneity of HEV. In this study, the suitability of SSCP, a method for the simultaneous detection of DNA polymorphisms at multiple positions (Orita et al., 1989; Gasser et al., 2006), was tested for the detection of HEV variants originating from swine and human samples. Clones containing a widely used conserved fragment of the HEV capsid protein (CP) gene (Meng et al., 1997; Huang et al., 2002; Lu et al., 2006), which is important phylogenetically and diagnostically, were subjected to SSCP analysis and sequencing under the assumption of sufficient SSCP reliability and sensitivity to characterize HEV intrahost populations. 2. Materials and methods 2.1. HEV isolates Five swine (CRO 2D, CRO 5D, CRO 3F, CRO 22F and CRO 5W) and three human serum samples (8, 16 and 22), all previously confirmed to be HEV positive, were analysed. Swine samples originating from four different Croatian counties were collected through the Classical Swine Fever National Monitoring Programme, in line with the standard international protocols for animal welfare. The samples CRO 2D and CRO 5D originated from fattening pigs bred on two small farms; CRO 3F and CRO 22F originated from sows bred on a large industrial farm; while sample CRO 5W originated from a wild boar. All five samples tested HEV positive by ELISA (MP Diagnostics HEV ELISA kit, Medical Technology Promedt, St. Ingbert, Germany). Human samples used in this analysis were deposited by Dr. A. R. Ciccaglione in the serum collection at the Istituto Superiore di Sanità (Rome, Italy) and purified 457 bp long PCR products of ORF2 region were kindly provided by Dr. G. La Rosa. Samples 8 and 22 originated from acutely infected Italian patients, while sample 16 was obtained from an acutely infected patient from Bangladesh. All human samples were previously confirmed to be HEV positive, both serologically and molecularly, as described by La Rosa et al. (2014). 2.2. cDNA synthesis and PCR amplification Swine sera were separated from cellular elements by centrifuging coagulated blood (blood clots were rimmed with a sterile glass stick to facilitate separation) for 15 min at 1000 g. For each serum sample, a 140 l aliquot was used for viral RNA purification using the QIAamp viral RNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA samples were stored at −80 ◦ C until analysis. RNA samples were reverse transcribed by SuperScript III reverse transcriptase using random hexamers (Invitrogen, Life Technologies) following the manufacturer’s instructions. The generated cDNA was used immediately for polymerase chain reaction (PCR) or stored at -20 ◦ C. Two sets of HEV primers (ORF2-3156/ORF2-3157 - direct and ORF2-3158/ORF2-3159 - nested) amplifying a 348 bp long fragment of the ORF2 conserved region (Huang et al., 2002) were used for the universal HEV detection by nested PCR assay.
The direct and nested step PCR parameters were identical (initial denaturation: 94 ◦ C, 3 min; 30 cycles: denaturation: 94 ◦ C, 1 min; annealing: 50 ◦ C, 1 min; extension: 72 ◦ C, 1 min; final extension: 72 ◦ C, 20 min). For human samples, the provided 457 bp PCR products were subjected to the nested PCR using a primer pair (ORF2-3158/ORF23159) and PCR parameters as described for swine samples. All PCR products were subjected to electrophoresis in 1.5% agarose gel and stained with ethidium bromide for UV light visualization. 2.3. Separation and identification of HEV variants To separate different genomic variants, nested PCR amplicons of each isolate were TA-cloned into the pTZ57R/T vector (Fermentas, Vilnius, Lithuania) as per manufacturer recommendations. Competent Escherichia coli XL-1Blue cells and their transformation were prepared using a commercial InsTAclone PCR cloning kit (Fermentas, Vilnius, Lithuania). Transformed colonies were selected by ␣ complementation, and the presence of the insert was confirmed by PCR using the same primer pair and nested PCR reaction conditions as described above. Ten transformed colonies per isolate were selected randomly and subjected to SSCP. Aliquots of the amplified products (1 l) were added to 9 l denaturing solution (95% formamide, 20 mM EDTA, pH 8.0, and 0.05% bromphenol blue), heated for 5 min at 90 ◦ C and immediately put on ice. Denatured products were separated by electrophoresis in native 8% polyacrylamide 0.75 mm thick gel in the standard TBE buffer under constant voltage (200 V) at 4 ◦ C for 3 h in the Mini PROTEAN Tetra Cell (Bio-Rad Laboratories, Hercules, CA, USA). The SSCP profiles were visualized by silver staining (Beidler et al., 1982) and PCR products displaying different SSCP patterns were considered different genomic variants (López-Labrador et al., 1999; Kong et al., 2000). Plasmids from all ten transformed colonies per samples (80 clones in total) were purified using a PureLinkTM Quick ˝ Plasmid Miniprep Kit (Invitrogen, Lohne, Germany) and sequenced in both directions (Macrogen, Seoul, Korea) using a pair of M13-pUC universal primers. 2.4. Nucleotide sequence and population structure analysis To determine the phylogenetic clustering of the obtained sequences, the reference ORF2 sequences and representatives of all Orthohepevirus A HEV genotypes (Smith et al., 2014) were retrieved from GenBank. Sequences were aligned using ClustalX 1.8 (Thompson et al., 1997) and analysed using MEGA 5 (Tamura et al., 2011). The phylogenetic tree was generated using the maximum likelihood method applying the Tamura Nei evolutionary model, selected as the most appropriate after performing Modeltest 3.7 analysis (Posada and Crandall, 1998). Tree topology was evaluated by bootstrap analysis based on 1000 repetitions. The overall mean genetic distance was calculated using MEGA 5 (Tamura et al., 2011) for the quasispecies of each sample. Population structure was considered at the haplotype level as determined by the SSCP analysis and compared to the structure obtained at the nucleotide sequence level. All sequences displaying nucleotide differences obtained in this work were submitted to GenBank under accession numbers KF366506-KF366524 and KP878281-KP878299. 3. Results The nested RT-PCR assay, using primers specific for HEV ORF2, gave strong amplification signals corresponding to the expected 348 bp long products in all tested samples. The comparison of SSCP patterns obtained after the analysis of 10 randomly selected genomic variants of each sample, separated by TA-cloning, suggested the coexistence of different genomic variants within all
ˇ Please cite this article in press as: Cerni, S., et al., The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.04.020
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tested samples. Generally, a higher number of variants was detected in human samples, with six different patterns observed in samples 16 and 22, and five different patterns observed in sample 8. In swine samples, the maximal number of different SSCP patterns obtained per sample was four (samples CRO 2D and CRO 22F). In two swine samples (CRO 5D and CRO 3F) three different patterns were detected, while one swine sample (CRO 5W) showed only two different SSCP patterns. In all five swine samples, the clear predominance of a single SSCP haplotype was identified, and ranged from 60 to 90%. In two human samples, the most dominant variants showed abundances of 40 and 50%, while one sample showed a co-domination of two variants, each represented by 30%. To verify the accuracy of the SSCP approach, the same 80 clones were sequenced in both directions and analysed. Supported by high bootstrap values, the phylogenetic relationship analysis of the obtained sequences (Fig. 1), including reference sequences, showed that the variants of swine samples clustered into phylogenetic lineages corresponding to genotype 3, while the variants of human HEV isolates clustered into phylogenetic lineages corresponding to genotype 1. Interestingly, variants clustering into two different subgroups (clusters CRO-B and CRO-C) within the same genotype were detected in two swine samples (CRO 3F and CRO 22F) (Fig. 1), while variants of all other samples clustered into single phylogenetic subgroups. The mean intra-host genetic distance calculated for individual samples was 0.005 for each human sample, while in swine samples it ranged from 0.001 (CRO 5W) to 0.002 (CRO 2D and CRO 5D). In the two swine samples in which variants clustered into two different subgroups, the mean genetic distances were 0.052 (CRO 22F) and 0.134 (CRO 3F). However, when calculated by subgroup, the mean values were 0.003 (CRO 3F - cluster CROB), 0.004 (CRO 22F - cluster CRO-C), and 0.010 (CRO 3F - cluster CRO-C). The comparison of the obtained SSCP patterns and nucleotide sequences and the nature of the detected mutations were presented for one representative sample (Fig. 2). In all analysed cases, the comparison of results confirmed that the sequenced clones displaying different SSCP patterns had different nucleotide compositions. The overall analysis showed that in comparison with predominant sample variants, the majority (68%) of minor variants had one point mutation, 25% had two point mutations, while 7% had an insertion. In 26% of variants with one point mutation, the SSCP analysis was not able to distinguish different genomic variants as the obtained patterns were identical. However, in the remaining 74% of variants with one point mutation, and in all cases of variants with two point mutations and variants with an insertion, SSCP was 100% effective in distinguishing the HEV variants.
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SSCP is a method previously proven to give reliable and rapid information on the relatedness of virus populations during outbreaks of hepatitis A and B viruses (Hardie et al., 1996; Mackiewicz et al., 2005). It has also been successfully applied in the characterization of highly heterogeneous hepatitis C virus quasispecies (Enomoto et al., 1994) with a range of nucleotide differences between subtypes comparable to those demonstrated for HEV genotypes 3 and 4 (Lu et al., 2006; Smith et al., 2014). Although not highly applicable in diagnostic screening, the knowledge of quasispecies structure and dynamics is crucial for understanding the emergence of new viral strains having altered virulence, infectivity, host range and other biological features. Quasispecies variations that enable a virus to adapt to a new ecological niche and that result in alterations to viral epidemiology and pathogenicity are also important in developing new diagnostic tools and disease management strategies, including vaccination
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Fig. 1. Phylogenetic tree obtained by maximum likelihood analysis of HEV ORF2 sequences of separated genomic variants from five swine (CRO 2D, CRO 5D, CRO 5W, CRO 3F, and CRO 22F) and three human samples (8, 16, and 22). Reference sequences are marked in bold. Bootstrap values are presented next to tree nodes. The bar represents 0.05 nucleotide substitutions per site. Sequence variants are named after the HEV isolate followed by the clone number and letter representing the characteristic SSCP pattern. Clusters are delineated in accordance with the proposed phylogenetic groups of Smith et al. (2014).
ˇ Please cite this article in press as: Cerni, S., et al., The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.04.020
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Fig. 2. Comparison of SSCP patterns, nucleotide sequences obtained and the nature of detected mutations for ten analysed variants of human HEV sample 22. The different SSCP haplotypes are presented with different letters. Identical nucleotides are marked with dots (•), while gaps are marked with dashes (-).
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programmes and innovative antiviral drugs. Many researchers have addressed the issue of underestimating HEV diversity (Grandadam et al., 2004; Lu et al., 2006; Reuter et al., 2009; Purdy and Khudyakov, 2011), though to date, no detailed studies have been performed on HEV quasispecies structure in human cases or from zoonotic reservoirs. One reason might be that in the majority of cases where human HEV is associated with self-limiting acute infections, high genomic variability was not expected (Grandadam et al., 2004). Contrary to this assumption, the present study, though performed on a small number of samples, suggests the presence of a notable number of genomic variants in all human samples. The diversity of human HEV quasispecies found in the present study was greater than the diversity in patients with acute and chronic infections observed by Lhomme and collaborators (2012). While those authors reported a mean genetic distance of 0.0016–0.0039, the mean genetic distance reported for all human samples in the present study was 0.005. The comparison with sequence identity analysis of four swine isolates of Zhang et al. (2010), where the lowest similarity reported was 96.8%, indicates a somewhat lower sequence diversity of the swine samples analysed in this study. In samples in which variants fell into a single cluster, the lowest sequence similarity observed was 99.3%. However, in samples in which variants clustered into two subgroups, the lowest sequence similarity reported was much lower (82.7%). Though these results may be case dependent, raising awareness of the existing variability of HEV quasispecies and its possible role in the outcome of disease, especially in chronic human infections, should prompt the use of an adequate laboratory tool for determining quasispecies structure (Dalton et al., 2009; Lhomme et al., 2012). In the present study, a rather conserved segment of the ORF2 gene (Huang et al., 2002) was selected for several reasons. Routine diagnostic protocols based on this HEV genome segment are
in place in many laboratories. Considering the method limitations regarding the investigated fragment length, the 348 bp long fragment is very convenient for the SSCP analysis (Orita et al., 1989; Gasser et al., 2006). Also, the current HEV classification scheme reflects the ORF2 phylogeny whilst its high degree of conservation (Lu et al., 2006; Smith et al., 2013) provides a discriminative system for assessing the SSCP resolution power in HEV quasispecies investigations. The results revealed that all analysed clones displaying different SSCP patterns had a different nucleotide composition. In 75 of 80 clones, the sequencing results were in complete agreement with the haplotype profiling obtained by the SSCP analysis. The inconsistencies observed in 5 variants, all with one point mutation, could be attributed to synonymous substitutions observed in all these cases that resulted in structurally similar changes. However, in the majority of single point mutation cases, both synonymous and nonsynonymous, differences between SSCP haplotypes could be observed (Fig. 2), though minor in some cases, despite very similar structural changes. It could be concluded that in the case of point mutations, the extent of the SSCP pattern differences was exclusively position related. Contrary to the slight pattern differences obtained in sequences differing in only one nucleotide, the patterns were drastically different in the cases of a single nucleotide insertion, as presented for pattern E of sample 22 (Fig. 2). Moreover, although comparison of SSCP patterns on different electrophoretic gels is not advisable due to the varying electrophoretic conditions on individual gels, a very high similarity was observed between SSCP haplotypes of the different swine HEV isolates with the same nucleotide sequences. This is in accordance with earlier observations for hepatitis A virus, which demonstrated that different SSCP electrophoresis of the same PCR product showed a similar banding pattern (Mackiewicz et al., 2005). This supports the predicted sensitivity and suitability of the SSCP approach in the characterization of HEV quasispecies. The phylogenetic analysis (Fig. 1) classified all analysed swine variants into different sequence clusters of the zoonotic genotype 3. In three swine samples (CRO 2D, CRO 5D, and CRO 5W), the sample variants clustered together, forming a single cluster for each sample, while in two samples (CRO 3F and CRO 22F) variants clustered into different groups. This may indicate multiple infection events, which should not be unexpected in large swine populations. Interestingly, only one clone of the sample CRO 22F clustered into a different sequence group, revealing that it could have been undetected in routine analysis. Consistent with the report of La Rosa et al. (2014), the variants of three human isolates clustered into three separate groups within the HEV genotype 1 (Fig. 1). Although the focus of the present study was not the analysis of HEV population structure, it provides the first assessment of the SSCP method in determining HEV isolate heterogeneity. The results are based on an intra-host population analysis of eight randomly selected samples. All analysed isolates showed a complex HEV population structure with the predominance of a single SSCP haplotype, confirming the quasispecies nature of the virus (Grandadam et al., 2004; Zhang et al., 2010; Lhomme et al., 2012). Individual samples showed a different population structure with a fraction of minor SSCP haplotypes ranging from 10 to 40% (average of 28% in swine and 53% in humans). This was much higher than the recorded average of 5% obtained by the RFLP method for human HEV samples (Grandadam et al., 2004). Unlike the poorly sensitive RFLP analysis that relies exclusively on the presence of a point mutation within a discrete restriction endonuclease site, SSCP provides accurate information on viral population structure completed by targeted sequencing of selected variants identified electrophoretically. In addition, it enables the precise quantitation of virus variants present in a sample simply by comparing SSCP haplotypes, thus diminishing the cost of random bulk sequencing.
ˇ Please cite this article in press as: Cerni, S., et al., The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.04.020
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From the evidence presented, SSCP is a reliable, cost effective and adequate method for assessing intra-host HEV diversity. Acknowledgements
We warmly thank Dr. Giuseppina La Rosa and Dr. Anna Rita 351 Ciccaglione, who kindly provided human HEV isolates. We also 352 thank Dr. Marin Jeˇzic´ and Ms. Ivana Prajdic´ for their technical assis353 Q2 tance. This study was supported by the Croatian Ministry of Science, 354 Education and Sports (grant no. 048-0481186-1183). 350
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Lhomme, S., Abravanel, F., Dubois, M., Sandres-Saune, K., Rostaing, L., Kamar, N., Izopet, J., 2012. Hepatitis E virus quasispecies and the outcome of acute hepatitis E in solid-organ transplant patients. J. Virol. 86, 10006–10014. López-Labrador, F.X., Ampurdanès, S., Giménez-Barcons, M., Guilera, M., Costa, J., Jiménez de Anta, M.T., Sánchez-Tapias, J.M., Rodés, J., Sáiz, J.C., 1999. Relationship of the genomic complexity of hepatitis C virus with liver disease severity and response to interferon in patients with chronic HCV genotype 1b infection [correction of interferon]. Hepatology 29, 897–903. Lu, L., Li, C., Hagedorn, C.H., 2006. Phylogenetic analysis of global hepatitis E virus sequences: genetic diversity, subtypes and zoonosis. Rev. Med. Virol. 16, 5–36. Mackiewicz, V., Roque-Afonso, A.-M., Marchadier, E., Nicand, E., Fki-Berrajah, L., Dussaix, E., 2005. Rapid investigation of hepatitis A virus outbreak by single strand conformation polymorphism analysis. J. Med. Virol. 76, 271–278. Meng, X.J., 2010. Hepatitis E virus: animal reservoirs and zoonotic risk. Vet. Microbiol. 140, 256–265. Meng, X.-J., 2011. From barnyard to food table: the omnipresence of hepatitis E virus and risk for zoonotic infection and food safety. Virus Res. 161, 23–30. Meng, X.J., Purcell, R.H., Halbur, P.G., Lehman, J.R., Webb, D.M., Tsareva, T.S., Haynes, J.S., Thacker, B.J., Emerson, S.U., 1997. A novel virus in swine is closely related to the human hepatitis E virus. Proc. Natl. Acad. Sci. USA 94, 9860–9865. Ojosnegros, S., Perales, C., Mas, A., Domingo, E., 2011. Quasispecies as a matter of fact: viruses and beyond. Virus Res. 162, 203–215. Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., Sekiya, T., 1989. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. USA 86, 2766–2770. Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818. Purdy, M.A., Khudyakov, Y.E., 2011. The molecular epidemiology of hepatitis E virus infection. Virus Res. 161, 31–39. Reuter, G., Fodor, D., Forgách, P., Kátai, A., Szucs, G., 2009. Characterization and zoonotic potential of endemic hepatitis E virus (HEV) strains in humans and animals in Hungary. J. Clin. Virol. 44, 277–281. Schirmer, M., Sloan, W.T., Quince, C., 2012. Benchmarking of viral haplotype reconstruction programmes: an overview of the capacities and limitations of currently available programmes. Brief. Bioinform. 15, 431–442. Schneider, W.L., Roossinck, M.J., 2001. Genetic diversity in RNA virus quasispecies is controlled by host-virus interactions. J. Virol. 75, 6566–6571. Smith, D.B., Purdy, M.A., Simmonds, P., 2013. Genetic variability and the classification of hepatitis E virus. J. Virol. 87, 4161–4169. Smith, D.B., Simmonds, P., Jameel, S., Emerson, S.U., Harrison, T.J., Meng, X.-J., Okamoto, H., Van der Poel, W.H.M., Purdy, M.A., 2014. Consensus proposals for classification of the family Hepeviridae. J. Gen. Virol. 95, 2223– 2232. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Teshale, E.H., Hu, D.J., Holmberg, S.D., 2010. The two faces of hepatitis E virus. Clin. Infect. Dis. 51, 328–334. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Van der Poel, W.H., 2014. Food and environmental routes of Hepatitis E virus transmission. Curr. Opin. Virol. 4C, 91–96. Zhang, X.-Y., Zhu, R.-L., Zhu, Y.-M., Dong, S.-J., Yu, R.-S., Shen, S.-Y., Wang, Z.-Y., Li, Z., 2010. Quasispecies investigation on swine hepatitis E viruses. Bing Du Xue Bao 26, 40–44.
ˇ Please cite this article in press as: Cerni, S., et al., The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.04.020
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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity
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a ˇ ˇ S. Cerni , J. Prpic´ b , L. Jemerˇsic´ b , D. Skori c´ a,∗ a b
University of Zagreb, Faculty of Science, Department of Biology, Maruli´cev trg 9A, Zagreb, Croatia Croatian Veterinary Institute, Department of Virology, Savska cesta 143, Zagreb, Croatia
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a b s t r a c t
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Article history: Received 5 February 2014 Received in revised form 14 April 2015 Accepted 16 April 2015 Available online xxx
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Keywords: Capsid protein Cloning HEV Quasispecies SSCP
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1. Introduction
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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, 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. 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. © 2015 Published by Elsevier B.V.
Intra-host RNA viral populations are composed of genetically related variants (Ojosnegros et al., 2011; Domingo et al., 2012) due to their high mutation and replication rates. These quasispecies are dynamic sources of viral adaptability enabling rapid evolution during different selective regimens (Schneider and Roossinck, 2001; Domingo et al., 2006). Evidently, virus pathogenicity depends on the structure of the quasispecies and the abundance of certain virus variants (Kumar et al., 2008; Ojosnegros et al., 2011). Quasispecies can hide components that in isolation would display dissimilar bioˇ et al., 2008; Domingo et al., 2012), while logical properties (Cerni reinfections may play a role in the accumulation of highly heterogeneous virus variants, some of which are distant phylogenetically. Hence, it is important to have an efficient laboratory screening tool for quick and accurate characterization of virus isolates. Although bulk cloning and sequencing is a good approach for detecting viral heterogeneity, it requires the preparation and sequencing of large numbers of clones to ensure that minor sequence variants are represented. Mainly due to the cost of too
∗ Corresponding author. Tel.: +385 1 4898079; fax: +385 1 4898081. E-mail addresses: [email protected], [email protected] ˇ ´ (D. Skori c).
many man-hours, it is not the most cost effective approach. Furthermore, the next generation sequencing approach, which enables quick detection of single nucleotide polymorphisms within different haplotypes, is still challenged with the reconstruction of virus quasispecies (Schirmer et al., 2012) and is not available widely for routine screenings. Single strand conformation polymorphism (SSCP) analysis is a good alternative that provides excellent insight into viral heterogeneity and reduces research costs (Gasser et al., 2006). This method enables the electrophoretic separation of single strand nucleic acids where a single nucleotide change could considerably affect strand electrophoretic mobility by altering intra-strand base paring and its resulting conformation (Orita et al., 1989). To date, it has been used widely for the rapid screening of selected genome fragments to identify most sequence variations between the mutants of many viruses, including hepatitis A, B and C (Hardie et al., 1996; Mackiewicz et al., 2005; Kumar et al., 2008). Although Hepatitis E virus (HEV) has only one serotype (Emerson and Purcell, 2003), its isolates display considerable genetic diversity (Lu et al., 2006; Smith et al., 2013). Increased testing and sequencing availability have resulted in an increased number of recorded HEV variants from wildlife and the environment (Smith et al., 2013). The recent Hepeviridae taxonomy proposal (Smith et al., 2014) recognizes four HEV human-infecting genotypes within the species Orthohepevirus A, though clear criteria are yet to be defined for the delineation of different HEV subgenotypes. To date, HEV
http://dx.doi.org/10.1016/j.jviromet.2015.04.020 0166-0934/© 2015 Published by Elsevier B.V.
ˇ Please cite this article in press as: Cerni, S., et al., The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.04.020
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genotypes 1 and 2 have been found to infect only humans. However, the zoonotic genotypes 3 and 4 have been detected in humans and in several animal species (Meng, 2010; Teshale et al., 2010). Domestic pigs are considered to be the main HEV reservoirs and have been studied the most extensively (Meng, 2011; Van der Poel, 2014). The quasispecies nature of HEV was first described by Grandadam et al. (2004) using restriction fragment length polymorphism (RFLP) analysis. Though two studies to date have addressed the direct sequencing of 20 randomly selected HEV clones (Zhang et al., 2010; Lhomme et al., 2012), RFLP remained the only method used in the characterization of intra-host heterogeneity of HEV. In this study, the suitability of SSCP, a method for the simultaneous detection of DNA polymorphisms at multiple positions (Orita et al., 1989; Gasser et al., 2006), was tested for the detection of HEV variants originating from swine and human samples. Clones containing a widely used conserved fragment of the HEV capsid protein (CP) gene (Meng et al., 1997; Huang et al., 2002; Lu et al., 2006), which is important phylogenetically and diagnostically, were subjected to SSCP analysis and sequencing under the assumption of sufficient SSCP reliability and sensitivity to characterize HEV intrahost populations. 2. Materials and methods 2.1. HEV isolates Five swine (CRO 2D, CRO 5D, CRO 3F, CRO 22F and CRO 5W) and three human serum samples (8, 16 and 22), all previously confirmed to be HEV positive, were analysed. Swine samples originating from four different Croatian counties were collected through the Classical Swine Fever National Monitoring Programme, in line with the standard international protocols for animal welfare. The samples CRO 2D and CRO 5D originated from fattening pigs bred on two small farms; CRO 3F and CRO 22F originated from sows bred on a large industrial farm; while sample CRO 5W originated from a wild boar. All five samples tested HEV positive by ELISA (MP Diagnostics HEV ELISA kit, Medical Technology Promedt, St. Ingbert, Germany). Human samples used in this analysis were deposited by Dr. A. R. Ciccaglione in the serum collection at the Istituto Superiore di Sanità (Rome, Italy) and purified 457 bp long PCR products of ORF2 region were kindly provided by Dr. G. La Rosa. Samples 8 and 22 originated from acutely infected Italian patients, while sample 16 was obtained from an acutely infected patient from Bangladesh. All human samples were previously confirmed to be HEV positive, both serologically and molecularly, as described by La Rosa et al. (2014). 2.2. cDNA synthesis and PCR amplification Swine sera were separated from cellular elements by centrifuging coagulated blood (blood clots were rimmed with a sterile glass stick to facilitate separation) for 15 min at 1000 g. For each serum sample, a 140 l aliquot was used for viral RNA purification using the QIAamp viral RNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA samples were stored at −80 ◦ C until analysis. RNA samples were reverse transcribed by SuperScript III reverse transcriptase using random hexamers (Invitrogen, Life Technologies) following the manufacturer’s instructions. The generated cDNA was used immediately for polymerase chain reaction (PCR) or stored at -20 ◦ C. Two sets of HEV primers (ORF2-3156/ORF2-3157 - direct and ORF2-3158/ORF2-3159 - nested) amplifying a 348 bp long fragment of the ORF2 conserved region (Huang et al., 2002) were used for the universal HEV detection by nested PCR assay.
The direct and nested step PCR parameters were identical (initial denaturation: 94 ◦ C, 3 min; 30 cycles: denaturation: 94 ◦ C, 1 min; annealing: 50 ◦ C, 1 min; extension: 72 ◦ C, 1 min; final extension: 72 ◦ C, 20 min). For human samples, the provided 457 bp PCR products were subjected to the nested PCR using a primer pair (ORF2-3158/ORF23159) and PCR parameters as described for swine samples. All PCR products were subjected to electrophoresis in 1.5% agarose gel and stained with ethidium bromide for UV light visualization. 2.3. Separation and identification of HEV variants To separate different genomic variants, nested PCR amplicons of each isolate were TA-cloned into the pTZ57R/T vector (Fermentas, Vilnius, Lithuania) as per manufacturer recommendations. Competent Escherichia coli XL-1Blue cells and their transformation were prepared using a commercial InsTAclone PCR cloning kit (Fermentas, Vilnius, Lithuania). Transformed colonies were selected by ␣ complementation, and the presence of the insert was confirmed by PCR using the same primer pair and nested PCR reaction conditions as described above. Ten transformed colonies per isolate were selected randomly and subjected to SSCP. Aliquots of the amplified products (1 l) were added to 9 l denaturing solution (95% formamide, 20 mM EDTA, pH 8.0, and 0.05% bromphenol blue), heated for 5 min at 90 ◦ C and immediately put on ice. Denatured products were separated by electrophoresis in native 8% polyacrylamide 0.75 mm thick gel in the standard TBE buffer under constant voltage (200 V) at 4 ◦ C for 3 h in the Mini PROTEAN Tetra Cell (Bio-Rad Laboratories, Hercules, CA, USA). The SSCP profiles were visualized by silver staining (Beidler et al., 1982) and PCR products displaying different SSCP patterns were considered different genomic variants (López-Labrador et al., 1999; Kong et al., 2000). Plasmids from all ten transformed colonies per samples (80 clones in total) were purified using a PureLinkTM Quick ˝ Plasmid Miniprep Kit (Invitrogen, Lohne, Germany) and sequenced in both directions (Macrogen, Seoul, Korea) using a pair of M13-pUC universal primers. 2.4. Nucleotide sequence and population structure analysis To determine the phylogenetic clustering of the obtained sequences, the reference ORF2 sequences and representatives of all Orthohepevirus A HEV genotypes (Smith et al., 2014) were retrieved from GenBank. Sequences were aligned using ClustalX 1.8 (Thompson et al., 1997) and analysed using MEGA 5 (Tamura et al., 2011). The phylogenetic tree was generated using the maximum likelihood method applying the Tamura Nei evolutionary model, selected as the most appropriate after performing Modeltest 3.7 analysis (Posada and Crandall, 1998). Tree topology was evaluated by bootstrap analysis based on 1000 repetitions. The overall mean genetic distance was calculated using MEGA 5 (Tamura et al., 2011) for the quasispecies of each sample. Population structure was considered at the haplotype level as determined by the SSCP analysis and compared to the structure obtained at the nucleotide sequence level. All sequences displaying nucleotide differences obtained in this work were submitted to GenBank under accession numbers KF366506-KF366524 and KP878281-KP878299. 3. Results The nested RT-PCR assay, using primers specific for HEV ORF2, gave strong amplification signals corresponding to the expected 348 bp long products in all tested samples. The comparison of SSCP patterns obtained after the analysis of 10 randomly selected genomic variants of each sample, separated by TA-cloning, suggested the coexistence of different genomic variants within all
ˇ Please cite this article in press as: Cerni, S., et al., The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.04.020
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tested samples. Generally, a higher number of variants was detected in human samples, with six different patterns observed in samples 16 and 22, and five different patterns observed in sample 8. In swine samples, the maximal number of different SSCP patterns obtained per sample was four (samples CRO 2D and CRO 22F). In two swine samples (CRO 5D and CRO 3F) three different patterns were detected, while one swine sample (CRO 5W) showed only two different SSCP patterns. In all five swine samples, the clear predominance of a single SSCP haplotype was identified, and ranged from 60 to 90%. In two human samples, the most dominant variants showed abundances of 40 and 50%, while one sample showed a co-domination of two variants, each represented by 30%. To verify the accuracy of the SSCP approach, the same 80 clones were sequenced in both directions and analysed. Supported by high bootstrap values, the phylogenetic relationship analysis of the obtained sequences (Fig. 1), including reference sequences, showed that the variants of swine samples clustered into phylogenetic lineages corresponding to genotype 3, while the variants of human HEV isolates clustered into phylogenetic lineages corresponding to genotype 1. Interestingly, variants clustering into two different subgroups (clusters CRO-B and CRO-C) within the same genotype were detected in two swine samples (CRO 3F and CRO 22F) (Fig. 1), while variants of all other samples clustered into single phylogenetic subgroups. The mean intra-host genetic distance calculated for individual samples was 0.005 for each human sample, while in swine samples it ranged from 0.001 (CRO 5W) to 0.002 (CRO 2D and CRO 5D). In the two swine samples in which variants clustered into two different subgroups, the mean genetic distances were 0.052 (CRO 22F) and 0.134 (CRO 3F). However, when calculated by subgroup, the mean values were 0.003 (CRO 3F - cluster CROB), 0.004 (CRO 22F - cluster CRO-C), and 0.010 (CRO 3F - cluster CRO-C). The comparison of the obtained SSCP patterns and nucleotide sequences and the nature of the detected mutations were presented for one representative sample (Fig. 2). In all analysed cases, the comparison of results confirmed that the sequenced clones displaying different SSCP patterns had different nucleotide compositions. The overall analysis showed that in comparison with predominant sample variants, the majority (68%) of minor variants had one point mutation, 25% had two point mutations, while 7% had an insertion. In 26% of variants with one point mutation, the SSCP analysis was not able to distinguish different genomic variants as the obtained patterns were identical. However, in the remaining 74% of variants with one point mutation, and in all cases of variants with two point mutations and variants with an insertion, SSCP was 100% effective in distinguishing the HEV variants.
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SSCP is a method previously proven to give reliable and rapid information on the relatedness of virus populations during outbreaks of hepatitis A and B viruses (Hardie et al., 1996; Mackiewicz et al., 2005). It has also been successfully applied in the characterization of highly heterogeneous hepatitis C virus quasispecies (Enomoto et al., 1994) with a range of nucleotide differences between subtypes comparable to those demonstrated for HEV genotypes 3 and 4 (Lu et al., 2006; Smith et al., 2014). Although not highly applicable in diagnostic screening, the knowledge of quasispecies structure and dynamics is crucial for understanding the emergence of new viral strains having altered virulence, infectivity, host range and other biological features. Quasispecies variations that enable a virus to adapt to a new ecological niche and that result in alterations to viral epidemiology and pathogenicity are also important in developing new diagnostic tools and disease management strategies, including vaccination
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Fig. 1. Phylogenetic tree obtained by maximum likelihood analysis of HEV ORF2 sequences of separated genomic variants from five swine (CRO 2D, CRO 5D, CRO 5W, CRO 3F, and CRO 22F) and three human samples (8, 16, and 22). Reference sequences are marked in bold. Bootstrap values are presented next to tree nodes. The bar represents 0.05 nucleotide substitutions per site. Sequence variants are named after the HEV isolate followed by the clone number and letter representing the characteristic SSCP pattern. Clusters are delineated in accordance with the proposed phylogenetic groups of Smith et al. (2014).
ˇ Please cite this article in press as: Cerni, S., et al., The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.04.020
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Fig. 2. Comparison of SSCP patterns, nucleotide sequences obtained and the nature of detected mutations for ten analysed variants of human HEV sample 22. The different SSCP haplotypes are presented with different letters. Identical nucleotides are marked with dots (•), while gaps are marked with dashes (-).
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programmes and innovative antiviral drugs. Many researchers have addressed the issue of underestimating HEV diversity (Grandadam et al., 2004; Lu et al., 2006; Reuter et al., 2009; Purdy and Khudyakov, 2011), though to date, no detailed studies have been performed on HEV quasispecies structure in human cases or from zoonotic reservoirs. One reason might be that in the majority of cases where human HEV is associated with self-limiting acute infections, high genomic variability was not expected (Grandadam et al., 2004). Contrary to this assumption, the present study, though performed on a small number of samples, suggests the presence of a notable number of genomic variants in all human samples. The diversity of human HEV quasispecies found in the present study was greater than the diversity in patients with acute and chronic infections observed by Lhomme and collaborators (2012). While those authors reported a mean genetic distance of 0.0016–0.0039, the mean genetic distance reported for all human samples in the present study was 0.005. The comparison with sequence identity analysis of four swine isolates of Zhang et al. (2010), where the lowest similarity reported was 96.8%, indicates a somewhat lower sequence diversity of the swine samples analysed in this study. In samples in which variants fell into a single cluster, the lowest sequence similarity observed was 99.3%. However, in samples in which variants clustered into two subgroups, the lowest sequence similarity reported was much lower (82.7%). Though these results may be case dependent, raising awareness of the existing variability of HEV quasispecies and its possible role in the outcome of disease, especially in chronic human infections, should prompt the use of an adequate laboratory tool for determining quasispecies structure (Dalton et al., 2009; Lhomme et al., 2012). In the present study, a rather conserved segment of the ORF2 gene (Huang et al., 2002) was selected for several reasons. Routine diagnostic protocols based on this HEV genome segment are
in place in many laboratories. Considering the method limitations regarding the investigated fragment length, the 348 bp long fragment is very convenient for the SSCP analysis (Orita et al., 1989; Gasser et al., 2006). Also, the current HEV classification scheme reflects the ORF2 phylogeny whilst its high degree of conservation (Lu et al., 2006; Smith et al., 2013) provides a discriminative system for assessing the SSCP resolution power in HEV quasispecies investigations. The results revealed that all analysed clones displaying different SSCP patterns had a different nucleotide composition. In 75 of 80 clones, the sequencing results were in complete agreement with the haplotype profiling obtained by the SSCP analysis. The inconsistencies observed in 5 variants, all with one point mutation, could be attributed to synonymous substitutions observed in all these cases that resulted in structurally similar changes. However, in the majority of single point mutation cases, both synonymous and nonsynonymous, differences between SSCP haplotypes could be observed (Fig. 2), though minor in some cases, despite very similar structural changes. It could be concluded that in the case of point mutations, the extent of the SSCP pattern differences was exclusively position related. Contrary to the slight pattern differences obtained in sequences differing in only one nucleotide, the patterns were drastically different in the cases of a single nucleotide insertion, as presented for pattern E of sample 22 (Fig. 2). Moreover, although comparison of SSCP patterns on different electrophoretic gels is not advisable due to the varying electrophoretic conditions on individual gels, a very high similarity was observed between SSCP haplotypes of the different swine HEV isolates with the same nucleotide sequences. This is in accordance with earlier observations for hepatitis A virus, which demonstrated that different SSCP electrophoresis of the same PCR product showed a similar banding pattern (Mackiewicz et al., 2005). This supports the predicted sensitivity and suitability of the SSCP approach in the characterization of HEV quasispecies. The phylogenetic analysis (Fig. 1) classified all analysed swine variants into different sequence clusters of the zoonotic genotype 3. In three swine samples (CRO 2D, CRO 5D, and CRO 5W), the sample variants clustered together, forming a single cluster for each sample, while in two samples (CRO 3F and CRO 22F) variants clustered into different groups. This may indicate multiple infection events, which should not be unexpected in large swine populations. Interestingly, only one clone of the sample CRO 22F clustered into a different sequence group, revealing that it could have been undetected in routine analysis. Consistent with the report of La Rosa et al. (2014), the variants of three human isolates clustered into three separate groups within the HEV genotype 1 (Fig. 1). Although the focus of the present study was not the analysis of HEV population structure, it provides the first assessment of the SSCP method in determining HEV isolate heterogeneity. The results are based on an intra-host population analysis of eight randomly selected samples. All analysed isolates showed a complex HEV population structure with the predominance of a single SSCP haplotype, confirming the quasispecies nature of the virus (Grandadam et al., 2004; Zhang et al., 2010; Lhomme et al., 2012). Individual samples showed a different population structure with a fraction of minor SSCP haplotypes ranging from 10 to 40% (average of 28% in swine and 53% in humans). This was much higher than the recorded average of 5% obtained by the RFLP method for human HEV samples (Grandadam et al., 2004). Unlike the poorly sensitive RFLP analysis that relies exclusively on the presence of a point mutation within a discrete restriction endonuclease site, SSCP provides accurate information on viral population structure completed by targeted sequencing of selected variants identified electrophoretically. In addition, it enables the precise quantitation of virus variants present in a sample simply by comparing SSCP haplotypes, thus diminishing the cost of random bulk sequencing.
ˇ Please cite this article in press as: Cerni, S., et al., The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.04.020
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From the evidence presented, SSCP is a reliable, cost effective and adequate method for assessing intra-host HEV diversity. Acknowledgements
We warmly thank Dr. Giuseppina La Rosa and Dr. Anna Rita 351 Ciccaglione, who kindly provided human HEV isolates. We also 352 thank Dr. Marin Jeˇzic´ and Ms. Ivana Prajdic´ for their technical assis353 Q2 tance. This study was supported by the Croatian Ministry of Science, 354 Education and Sports (grant no. 048-0481186-1183). 350
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ˇ Please cite this article in press as: Cerni, S., et al., The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.04.020
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