T.G. Dimitriou

Sites of disruption within E1 and E2 genes of HPV16 and association with cervical dysplasia

Integration of HPV16 DNA into the host chromosome usually disrupts the E1 and/or E2 genes. The present study investigated the disruption of E1, E2 genes in a total of eighty four HPV16‐positive precancerous and cervical cancer specimens derived from Greek women (seventeen paraffin‐embedded cervical biopsies and sixty seven Thin Prep samples). Complete E2 and E1 genes were amplified using three and nine overlapping primer sets respectively, in order to define the sites of disruption. Extensive mapping analysis revealed that disruption/deletion events within E2 gene occurred in high grade and cervical cancer samples (x2 test, P < 0.01), while no evidence of E2 gene disruption was documented among low grade cervical intraepithelial neoplasias. In addition, disruptions within the E1 gene occur both in high and low grade cervical intraepithelial neoplasia. This leads to the assumption that in low grade cervical intraepithelial neoplasias only E1 gene disruption was involved (Fishers exact test, P < 0.05), while in high grade malignancies and cervical cancer cases deletions in both E1 and E2 genes occurred. Furthermore, the most prevalent site of disruption of E1 gene was located between nucleotides 1059 and 1323, while the most prevalent deleted region of the E2 gene was located between nucleotides 3172 and 3649 (E2 hinge region). Therefore, it is proposed that each population has its own profile of frequencies and sites of disruptions and extensive mapping analysis of E1 and E2 genes is mandatory in order to determine suitable markers for HPV16 DNA integration analysis in distinct populations. J. Med. Virol. 87:1973–1980, 2015.

Characterization of novel intergenogroup and intergenotype recombinant noroviruses from central Greece.

Noroviruses (NoVs) are a major causative agent of acute gastroenteritis in humans. They are members of the Caliciviridae family and based on the genetic analysis of the RdRp and capsid regions, human NoVs are divided into three genogroups (Gs), GI, GII, and GIV. The three genogroups further segregate into distinct lineages called genotypes. The NoV genus is genetically diverse and recombination of viral RNA is known to depend upon various immunological and intracellular constraints that may allow the emergence of viable recombinants. In this study, three Noroviral strains detected in clinical samples revealed two hitherto unobserved recombination events between GII.9/GII.4 and GII.9/GI.7 genogroups. To our knowledge, these intergenotype and intergenogroup recombination events of GII.9/GII.4 and GII.9/GI.7, in ORF1 and ORF2 genes respectively are reported for the first time and highlight the ongoing evolution of noroviruses.

Molecular epidemiology and evolutionary dynamics of Echovirus 3 serotype

Echovirus 3 (E3) serotype has been related with several neurologic diseases, although it constitutes one of the rarely isolated serotypes, with no report of epidemics in Europe. The aim of the present study was to provide insights into the molecular epidemiology and evolution of this enterovirus serotype, while an E3 strain was isolated from sewage in Greece, four years after the initial isolation of the only reported E3 strain in the same geographical region. Phylogenetic analysis of the complete VP1 genomic region of that E3 strain and of those available in GenBank suggested three main genogroups that were further subdivided into seven subgenogroups. Further evolutionary analysis suggested that VP1 genomic region of E3 was dominated by purifying selection, as the vast majority of genetic diversity presumably occurred through synonymous nucleotide substitutions and the substitution rate for complete and partial VP1 sequences was calculated to be 8.13×10(-3) and 7.72×10(-3) substitutions/site/year respectively. The partial VP1 sequence analysis revealed the composite epidemiology of this serotype, as the strains of the three genogroups presented different epidemiological characteristics.

Molecular and evolutionary analysis of HPV16 E6 and E7 genes in Greek women

Human papillomavirus type 16 (HPV16) non-European variants have been associated with persistent infection and cervical cancer development, while the L83V variant of the E6 gene has been correlated with the progression of cervical malignancy. The present study investigated the presence of the HPV16 L83V variant in Greek women. Molecular evolutionary analysis of the HPV16 E6 and E7 oncogenes was conducted in order to estimate the evolution of the HPV16 genome in the Greek population. The E6 L83V variant was found in 78.2 % of high- and 64.28 % of low-grade specimens. Moreover, the prototype and E6 L83V variants were both prevalent in high- and low-grade malignancies in Greek women. Selective pressure analysis of the individual amino acid residues of HPV16 sequences from the Greek population indicates that codon 83 of the E6 protein, as well as codon 85 of the E7 protein, are undergoing positive selection. Novel sequence variations were recorded within the E6 and E7 genes in cervical samples, characterized as (T350G) European variants. However, no signal of intratypic recombination event was identified within the E6-E7 region. Molecular and evolutionary analyses of HPV16 genomes from distinct geographical locations might provide valuable information about viral evolution and oncogenecity.

Determination of human papillomavirus 16 physical status through E1/E6 and E2/E6 ratio analysis.

Human papillomavirus (HPV) 16 genome integration into the host chromosome is a crucial event during the life cycle of the virus and a major step towards carcinogenesis. The integration of HPV16 DNA promotes a constitutive high expression level of E6 and E7 oncoproteins, resulting in the extensive proliferation of the infected epithelial cells. In the present report the physical status of the HPV16 genome was studied, through determination of E1/E6 and E2/E6 DNA copy number ratios in 61 cervical samples of low- and high-grade malignancy and 8 cervical cancer samples, all of them associated with HPV16 infection. The selection of E1, E2 and E6 amplification target regions was performed according to the most prevalent deleted/disrupted sites of E1 and E2 genes. For this target selection we also considered the most conserved regions of E1, E2 and E6 genes among the same HPV16 isolates that were recently reported by our group. The analysis of HPV16 DNA form revealed a significant association among the mixed DNA forms in low-grade and high-grade malignancies, (χ(2), P<0.01). The comparative analysis of E1/E6 and E2/E6 in the same cervical samples provides an accurate picture of HPV16 DNA form and may reveal whether different HPV16 DNA integrants coexist in the same cervical sample or not. This study proposes that E1/E6 and E2/E6 ratios determine with accuracy the HPV16 DNA integration pattern and may predict multiple integration events in the examined sample, thus providing significant information about the progression of cervical dysplasia.

Duplex Real-time PCR assay and SYBR green I melting curve analysis for molecular identification of HPV genotypes 16, 18, 31, 35, 51 and 66.

Long-term infection with high-risk HPV genotypes is the leading cause of cervical cancer. In the present study a Duplex Real-time PCR assay was developed in order to identify HPV types 16, 18, 31, 35, 51 and 66 in three reactions, through SYBR green I melting curve analysis. The method utilizes type-specific primer sets that allowed the amplification of highly conserved regions of L1 gene. Reconstitution experiments were conducted by using HPV DNA plasmids in order to determine the sensitivity of the assay. The newly designed assay has a limit of detection of 10 copies per reaction. The most prevalent HPV genotype in single and in multiple HPV infections was HPV16 followed by HPV18, HPV51, HPV31, HPV35 and HPV66. The proposed method is a simple, specific, sensitive and cost-effective assay that can be easily incorporated in small and medium size laboratories for the rapid identification of the most clinically important HPV genotypes.

Identification of rearranged sequences of HPV16 DNA in precancerous and cervical cancer cases.

Integration of HPV16 DNA into the host chromosome is considered to be a crucial step towards genomic instability and cervical cancer development. Aim of the present study was to investigate the presence of HPV16 rearranged intra-viral sequences in HPV16-positive normal, precancerous and cervical cancer samples using the method of Restriction Site-PCR (RS-PCR). Sequence analysis of HPV16 integrants revealed for the first time in clinical samples two distinct rearranged intra-viral sequences, concerning the conjunction of E2 and L1 genes and the conjunction of E1 and L1 genes with inverted orientation. Furthermore mapping analysis of the E1 and E2 genes in cervical samples with rearranged intra-viral sequences of HPV16 genome was conducted in order to determine the integrity of viral genes. The identification of intra-viral rearrangements provides valuable information regarding the HPV16 DNA integration, and may be a significant biomarker for the presence of chromosomal instability and DNA damages in clinical samples.

PCR assays for the identification of rare recombination types from VP1 to 3D genomic region of vaccine derived poliovirus strains

Poliomyelitis has been effectively controlled by the use of inactivated poliovirus vaccine (IPV) or trivalent live attenuated oral poliovirus vaccine (OPV). Since 1964, the use of OPV in mass vaccinations has resulted in drastic reductions of the number of poliomyelitis cases caused by wild-type polioviruses. However, the characterization of OPV derivatives with increased neurovirulence, constituted a real problem with respect to OPV safety. Mutations at attenuating sites of the genome and recombination events between Sabin strains of the trivalent OPV vaccine have been correlated with the loss of the attenuated phenotype of OPV strains and the acquisition of traits characteristic of wild polioviruses. In consequence, early detection and characterization of recombinant evolved derivatives of vaccine strains is highly important. In this report, ten PCR assays are described which allow for the identification of rare recombination events located in VP1, 2A, 2C, 3A, 3C and 3D genomic regions and predominant recombination events located in 2C and 3D genomic regions of OPV derivatives. These assays could be readily implemented in diagnostics laboratories lacking sequencing facilities as a first approach for the early detection and characterization of recombinant OPV derivatives.

Nucleotide polymorphisms of the human papillomavirus 16 E1 gene.

The E1 ORF is one of the most conserved regions in the human papillomavirus (HPV) genome. The complete E1 gene of the HPV16 genome was amplified with four overlapping primer sets in 16 high-grade (CIN II, III) and 13 low-grade cervical (CIN I) intraepithelial neoplasias as well as in one cervical cancer case. Sequence analysis of the E6 and E7 genes was also carried out in the same cervical samples in order to confirm the association between nucleotide sequence variations in the HPV16 E1 ORF and HPV16 variant lineages. Analysis of the E1 ORF revealed 27 nucleotide changes, and these changes were correlated with those found in HPV16 Asian American and African type II variants. Of these nucleotide variations, A1668G, G2073A, T2169C, T2189C, A2453T, C2454T, A2587T and G2650A were identified only in high-grade dysplasia cases. A phylogenetic tree of the E1 ORF and nucleotide sequence analysis of the E1, E6 and E7 genes revealed that intratypic nucleotide sequence polymorphisms located in the E1 ORF can be used to identify the major phylogenetic branch to which a HPV16 genome belongs. Moreover, amplification of the E1 ORF revealed a disruption between nucleotides 878 and 1523 in five high- and two low-grade cervical cases, indicating that integration of HPV DNA occurs at an early stage of viral infection.

Prevalence of HPV16 E1-1374^63nt variants in Greek women.

Recent studies have focused on sequence variation of the HPV16 E1 gene. The present study investigates the prevalence of E1‐1374^63nt duplication in the Greek population, and the sequence variation at the 5′ end of the E1 and E6 genes from samples that harbored this genetic alteration. Fifty HPV16 positive cervical samples, derived from Greek patients were investigated. The 5′ end of the E1 gene was amplified through PCR and the variant amplicons were cloned, sequenced, and bioinformatically analyzed for selective pressure. The E1‐1374^63nt duplication was identified in 24% of the examined samples, with the same prevalence in both high and low‐grade cervical malignancies. The E1‐1374^63nt duplication was linked to the European variant lineage (x2 = 5.076, P < 0.024) and it was significantly associated with the nucleotide variation A1053C (x2 = 23.102, P < 0.0001). Molecular evolution analyses anticipate that the E1‐1374^63nt duplication induces functional constraints on the 5′ end of E1 gene, and it is proposed that this duplication might not affect negatively the function or structure of the E1 protein. The E1‐1374^63nt duplication is prevalent in the Greek population, whereas the A1053C variation might constitute a significant marker for the characterization of the E1‐1374^63nt variant in the Greek population, thus providing significant information about viral pathogenicity. J. Med. Virol. 86:778–784, 2014.