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 (x(2) 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 (Fisher's 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.

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.

Multiplex PCR assay for the rapid identification of human papillomavirus genotypes 16, 18, 45, 35, 66, 33, 51, 58, and 31 in clinical samples.

The causal association between persistent human papillomavirus (HPV) infection and cervical cancer has lead to the development of a variety of molecular assays for HPV detection. The present study focused on the development of a simple, sensitive and cost-effective HPV genotyping method based on multiplex PCR methodology that could be easily performed in small laboratories. Three multiplex PCR assays were developed to identify the HPV genotypes 16, 18, 45, 35, 66, 33, 51, 58, and 31 together with an internal control. The method was established by designing nine type-specific primer sets that target conserved regions of the L1 gene. The assay was applied using HPV-positive cervical specimens, and cloning and sequencing of all of the amplicons that were generated were performed to examine the specificity of the newly designed primers. Moreover, an experimental cutoff value was determined through reconstitution experiments using HPV DNA plasmids. Amplicons of expected size were obtained, while cloning and sequencing of PCR products confirmed the genomic specificity of the amplicons. The sensitivity of this method was determined to be 10 copies of each individual HPV genotype per test. Multiple and single HPV infections were documented in 42.2 % and 57.8 % of cervical specimens, respectively. The most prevalent HPV genotype was HPV16, followed by HPV18, HPV66 and HPV51. The present multiplex PCR assay is a simple method with high specificity and sensitivity that can be applied in clinical or epidemiological analyses for rapid identification of the most clinically important HPV genotypes present in cervical intraepithelial neoplasias.

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.

Prevalence of HPV16 E1-137463nt 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.

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.

Circulation of intergenotype recombinant noroviruses GII.9/GII.6 from 2006 to 2011 in central Greece.

Noroviruses (NoVs) are members of the Caliciviridae family and are recognized as a worldwide cause of acute nonbacterial gastroenteritis. Based on the genetic analysis of the RdRp and capsid regions, human NoVs are divided into three genogroups (Gs), GI, GII, and GIV, which further segregate into distinct lineages called genotypes. In this study, in an attempt to discern the circulation of an intergenotypic recombinant GII.9/GII.6, which was previously reported by our group in central Greece, we investigated NoVs in raw sewages from 2006 to 2011 and compared the results with the viruses detected from clinical samples in the same area and in the same time period. Two specific primer pairs for NoVs were designed which amplified in a single PCR fragment from polymerase to capsid gene covering the widespread recombination point in ORF1/ORF2 junction. Based on the genetic analysis, recombinant NoV strains GII.9/GII.6 were identified. Fourteen out of 15 environmental and eight out of ten clinical samples that were used in the present study were positive, with both primer pairs, confirming that the intergenotypic recombinant GII.9/GII.6 was circulating in the population of central Greece from 2006 to 2011. The crossover point was identified to be within the overlapping region of ORF1/ORF2 (GII.9/GII.6, respectively) and was determined by Simplot at nucleotide position 5,032 bp.

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.

Identification of novel E6-E7 sequence variants of human papillomavirus 16.

The rate of evolution of the human papillomavirus 16 (HPV16) genome is low. However, the ability of the E6 oncoprotein to interact with distinct p53 variants causes selective pressure on the E6 gene. In addition, intratypic recombination events in the HPV16 E6 and E7 genes have been characterized as extraordinary phenomena during the evolutionary history of virus. In the present study, we identified two new sequence variants through nucleotide analysis of the E6-E7 region of the HPV16 genome. Maximum-likelihood and empirical Bayesian methods were used in order to identify positive selection at particular residues of the E6 and E7 genes. Using the single recombination breakpoint (SBP) method, we found evidence of recombination events in the E6 ORF. Nucleotide sequence analysis showed that the new sequence variants are phylogenetically distant from the other members of the population. Our results indicate that new evolutionary intermediates of HPV16 might be formed either though positive selective pressure or through recombination events by multiple infections with distinct HPV16 variants.

Molecular and phylogenetic analysis of the HPV 16 E4 gene in cervical lesions from women in Greece.

The HPV16 E1(∧)E4 protein is thought to contribute to the release of newly formed viral particles from infected epithelia. In order to investigate amino acid mutations in the HPV16 E1(∧)E4 protein, the complete E4 ORF was amplified by PCR in 27 HPV16-positive cervical samples, and the amplicons were cloned. Fifteen nucleic acid variations were identified in the E4 ORF, including seven silent nucleic acid mutations. In addition, nine amino acid mutations (A7V, A7P, L16I, D45E, L59I, L59T, Q66P, S72F, H75Q) were detected in the E1(∧)E4 protein, and these were associated with the severity of cervical malignancy. A maximum-likelihood phylogenetic tree was constructed based on the E4 ORF, and nucleotide sequence analysis of the E4, E6 and E7 genes from the same samples was conducted in order to determine the phylogenetic origin of the cloned sequences from the amplified HPV16 E4. Based on the nucleotide sequence and phylogenetic analysis it was revealed that even though E4 ORF constitutes a small polymorphic portion of the viral genome (288 bp), it could provide valuable information about the origins of the HPV16 genome. In addition, molecular evolutionary analysis of the E4 coding region revealed that neutral selection is dominant in the overlapping region of the E4 and E2 ORFs.

Sequence variation analysis of the E2 gene of human papilloma virus type 16 in cervical lesions from women in Greece.

The E2 gene of human papilloma virus is expressed at the early stage of the viral life cycle, encoding the E2 transcription factor, and regulates the expression of E6 and E7 oncogenes. Disruption of E2 gene due to viral integration inhibits the transcriptional suppression of the HPV oncogenes, inducing cell proliferation. In the present study, a total of 22 HPV16-positive cytological specimens derived from high- and low-grade cervical intraepithelial lesions were investigated in order to identify sequence variations in the HPV16 E2 ORF. The E2 gene was amplified by PCR using external and internal overlapping sets of primers. Amplicons were cloned and sequenced. Disruption sites were detected in cervical samples diagnosed as high-grade cervical intraepithelial lesions. Moreover, sequence variations were identified in the E2 ORF and specific variations were associated with non-European variants such as African type I, African type II and Asian American. A total of three new sequence variations were identified at positions 2791, 2823 (transactivation domain) and 3361 (hinge region). Distinct phylogenetic branches were formed according to E2 analysis that characterized the different HPV16 variants. It was ascertained that non-European variants are circulating in the Greek population.

Molecular characterization of a new intergenotype Norovirus GII recombinant.

Human noroviruses (NoVs) of the Caliciviridae family are a major cause of epidemic gastroenteritis. 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 the present study, we report the development of a broadly reactive RT-PCR assay, which allowed the characterization of strain A6 at molecular level, established its genetic relationship at the sub-genogroup level and classified A6 strain at the sub-genotype level. The detection was carried out initially by enzyme-linked immunosorbent assay (ELISA) and the subsequent detection and molecular characterization of NoV strain was achieved by reverse transcription-PCR and sequencing. Based on the sequence analysis, A6 strain was revealed to belong to the GII genogroup of NoVs. Partial ORF1 gene sequencing analysis and complete ORF2 gene sequencing revealed that ORF1 and ORF2 belonged to two distinct genotypes GII/9 and GII/6, respectively, making obvious that A6 strain is a rare intergenotypic recombinant within the genogroup GII between GII.9 and GII.6 genotypes. A6 strain represents the first human NoV from Greece, whose genome has been partially (ORF1&ORF3) and completed (ORF2) sequenced. To our knowledge the recombination event GII.9/GII.6 in RdRp and capsid gene, respectively, that was revealed in the present study is reported for the first time.

Complete nucleotide sequence analysis of the VP1 genomic region of Echoviruses 6 isolated from sewage in Greece revealed 98% similarity with Echoviruses 6 that were characterized from an aseptic meningitis outbreak 1 year later.

The molecular characterization of two enterovirus strains (LR51A5 and LR61G3) isolated from the sewage treatment plant unit in Larissa, Greece, in May and June 2006 and the investigation of their relationship with enteroviruses of the same serotype isolated in Greece in 2001 and 2007 were performed by complete VP1 sequence analysis of the isolates. The close phylogenetic relationship and the high nucleotide similarity (98%) led to the conclusion that the virus isolated from sewage in 2006 was associated with that isolated from an aseptic meningitis outbreak 1 year later. Bootscan analysis of the VP1 genomic region revealed that intraserotypic multi-recombination events might have been involved in the evolutionary past history of the LR51A5 and LR61G3 isolates.

Retrospective molecular and phenotypic analysis of poliovirus vaccine strains isolated in Greece.

The live oral poliovirus vaccine (OPV) strains are genetically unstable, causing, in rare cases, vaccine-associated paralytic poliomyelitis. Reversions of the known attenuating mutations in OPV strains and intertypic recombination have been identified as the underlying causes of the increased neurovirulence of poliovirus isolates. In this study, three OPV isolates (one non-recombinant and two recombinants) were tested in order to correlate phenotypic traits such as temperature sensitivity (Rct test) and growth kinetics (one-step growth curve test) with mutations and recombination events of the viral genome. Moreover, the immunity level of the western Greek population aged 1-40 years was evaluated against OPV isolates and Sabin vaccine strains, with a microneutralization assay. Members of the 1-40-year age group (both pooled and individual sera) showed no significant differences in neutralization test (NT) titres against OPV isolates in comparison with the Sabin vaccine strains. However, all three OPV isolates showed reverted phenotypic traits in Rct or one-step growth curve assays. The results of our study revealed a significant decrease in immunity level from the 1-10-year age group to the 21-30-year age group (pooled sera) for both poliovirus types 1 and 3. For both poliovirus types, the highest NT titres were observed in the 1-10-year age group, and the lowest NT titre was observed in the 21-30-year age group, towards poliovirus type 3. Our study underlines the need for immunological studies in all age groups, in order to allow reconsideration of the current vaccination policies and to avoid epidemics caused by the circulation of highly evolved OPV derivatives.

Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains.

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.

A seroprevalence study of poliovirus antibody against a collection of recombinant and non-recombinant poliovirus vaccine strains in the population of southern Greece.

In this study, the serological status of the southern Greek population in the 1­10-year, 11­20-year, 21­30-year and 31­40-year age groups with regard to Sabin vaccine strains and a collection of 15 recombinant and four non-recombinant poliovirus vaccine strains was determined. For all three poliovirus types, the highest neutralization test (NT) titres were observed in the 1­10- year age group, indicating a good response to vaccination. In general, the serological status of the population of southern Greece with regard to poliovirus is better for types 1 and 2 than for type 3. The presence of the lowest NT titre in the 21­ 30-year age group against poliovirus type 3 suggests the need for a booster dose of monovalent Sabin3 vaccine to ensure personal and herd immunity.