Genetic variability of the HPV16 early genes and LCR. Present and future perspectives.

Human papillomavirus 16 (HPV16) infection is the aetiologic factor for the development of cervical dysplasia and is regarded as highly carcinogen, because it is implicated in more than 50% of cervical cancer cases, worldwide. The tumourigenic potential of HPV16 has triggered the extensive sequence analysis of viral genome in order to identify nucleotide variations and amino acid substitutions that influence viral oncogenicity and subsequently the initiation and progression of cervical cancer. Nowadays, specific mutations of HPV16 DNA have been associated with an increased risk of high-grade squamous intraepithelial lesions and invasive cervical cancer (ICC) development, including E6: Q14H, H78Y, L83V, Ε7: N29S, S63F, E2: H35Q, P219S, T310K, E5: I65V, whereas highly conserved regions of viral DNA have been extensively characterised. In addition, numerous novel HPV16 mutations are observed among the studied populations from various geographic regions, hence advocating that different HPV16 strains seem to emerge with different tumourigenic capacities. The present review focuses on the variability of the early genes and the long control region, emphasising on the association of specific mutations with the development of severe dysplasia. Finally, it evaluates whether specific regions of HPV16 DNA are able to serve as valuable biomarkers for cervical cancer risk.

A colorimetric IsoPCR for the rapid and sensitive visual detection of high-risk HPV16 in clinical samples with hydroxynaphthol blue.

HPV16 infection is found in more than 50 % of cervical cancer cases worldwide, triggering the development of numerous molecular techniques for viral diagnosis. The present study focuses on the development of a colorimetric IsoPCR for HPV16 DNA detection. The methodology combines the advantages of PCR and LAMP, while the most significant aspect of the new established methodology is the visual detection of amplification products through hydroxynapthol blue dye, thus minimizing the time and labor needed. An experimental cut-off value was tested through reconstitution experiments, while the specificity was evaluated by assessing clinical samples. The analytical sensitivity of the new colorimetric IsoPCR was found to be 0.1 viral DNA copy per reaction, while the specificity was 100 % for the detection of HPV16 DNA. The assay enabled the amplification of viral DNA in cases with viral load lower than 1 copy. In conclusion, the new established colorimetric IsoPCR can be regarded as an attractive molecular tool that facilitates the specific, rapid and highly sensitive visual detection of HPV16 DNA even at the very early stages of viral infection.

WarmStart colorimetric RT-LAMP for the rapid, sensitive and specific detection of Enteroviruses A-D targeting the 5'UTR region.

AIMS: The aim of the present study was to develop a colorimetric LAMP assay for the detection of enteroviruses belonging to species A-D targeting the 5' untranslated region (5' UTR) of enteroviruses genome. METHODS AND RESULTS: The RNA was converted to cDNA by the reverse transcriptase and then amplified via LAMP by the WarmStart®Bst DNA polymerase, simultaneously in a single reaction tube, so we shortened the reaction time to 50 min. The sensitivity of the assay regarding Enterovirus B, C and D was determined to be 0·30 CCID50 assay-1 while the sensitivity for Enterovirus A was 3·00 CCID50 assay-1 . The assay demonstrated high specificity and sensitivity for the detection of 45 reference strains of Enteroviruses A-D and validated on 20 clinical isolates. CONCLUSIONS: This assay can be used as a diagnostic tool for the rapid, sensitive and specific detection of enteroviruses, easily implemented in small clinical and research laboratories since LAMP amplicons were visualized by colour changes eliminating the requirement for post-amplification processing steps. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a colorimetric assay ideal for field situations for the detection of enteroviruses, by targeting the 5' UTR. This assay demonstrated high specificity and sensitivity, based on its performance on 45 EV A-D reference strains, on 20 EV B clinical isolates and on three non-enteroviral RNA viruses.

Development of a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) that detects enteroviruses by targeting the highly conserved 5'-UTR region.

Enteroviruses are positive sense single-stranded RNA viruses. Most infections from enteroviruses are asymptomatic and can circulate "silently", increasing the risk of an outbreak. For preventing such outbreaks, a rapid, cost-effective, and simple assay for the detection of enteroviruses is of great importance. In this study, we developed a rapid, simple, sensitive, and specific isothermal reverse transcription assay (RT-Loop-Mediated Amplification, RT-LAMP) for the detection of EV-A, B, C, and D species of enteroviruses, by targeting the highly conserved 5'UTR region. The assay was designed and validated based on reference sequences of the four species and on clinical and environmental isolates. The limit of detection of the assay is 0.75 CCID50/assay for Enterovirus A, B, and D and 0.075 CCID50/assay for Enterovirus C. LAMP allows immediate diagnosis in just 30-50 min, instead of a minimum of 120 min needed for PCR with an equal or better sensitivity. Moreover, due to its isothermal nature, there is no need for expensive equipment, thus decreasing the cost of each reaction. Therefore, this assay is ideal for use in resource-limited settings such as primary care facilities and environmental and clinical laboratories in developing countries.

WarmStart colorimetric LAMP for the specific and rapid detection of HPV16 and HPV18 DNA.

BACKGROUND AND OBJECTIVES: Persistent infection with High-Risk HPV genotypes is the principal cause for the development of cervical cancer with HPV16 and HPV18 to be the most frequently identified HPV genotypes observed in approximately 70% of cervical cancer cases worldwide. The present study focused on the development of a simple molecular methodology based on WarmStart colorimetric LAMP for the specific identification of HPV16 and HPV18. METHODS: The method was developed by designing LAMP type-specific primer sets that target the E6 gene. The assay was applied using HPV-positive clinical samples along with control cases in order to evaluate the specificity of the newly designed isothermal protocol. In addition, an experimental cutoff value was estimated through reconstitution experiments with HPV-DNA plasmids. LAMP amplicons were visualized by color changes, thus eliminating the requirement for post-amplification processing steps. RESULTS: The WarmStart colorimetric LAMP facilitates the isothermal amplification of 10 copies per reaction of both HPV16 and HPV18 DNA, while it exhibits 100% specificity for the detection of the corresponding genotypes in LSIL and HSIL cases. Moreover, the assay demonstrates 100% PPV and 100% NPV. Finally, the sensitivity of conventional PCR with the type-specific LAMP primer sets (B3/F3) for the HPV16, HPV18 DNA detection was 100 copies/reaction and 10 copies/reaction, respectively. CONCLUSIONS: The newly established WarmStart colorimetric LAMP can be considered as a powerful molecular tool that it can be easily implemented in small clinical and research laboratories for a rapid and efficient identification of the most tumorigenic HPV genotypes.

Α 2-stage, nested-like nucleic acid amplification method (IsoPCR) for the highly sensitive detection of HPV16 and HPV18 DNA.

Molecular detection of HPV DNA is considered as the gold standard for the diagnosis of cervical disease. Although the molecular assays for the identification of HPV16 and HPV18 have helped identify cervical cancer incidents, they are restricted to specialized laboratories. Thus, we developed a novel 2-stage, nested-like nucleic acid amplification method, named IsoPCR, to amplify the E6 gene of HPV16 and HPV18 with high analytical sensitivity and specificity. The performance of IsoPCR was compared to that of conventional PCR and LAMP. The analytical sensitivity of IsoPCR (1 copy/test) was 10-fold higher than conventional PCR and 25-fold higher than conventional LAMP. IsoPCR displayed significant amplification specificity (100%) and efficiency, as well. In conclusion, IsoPCR is a highly sensitive and specific diagnostic tool and it is suitable for the detection of low copy number of viral DNA in clinical specimens, providing critical information to healthcare providers.

NAT/NCS2-hound: a webserver for the detection and evolutionary classification of prokaryotic and eukaryotic nucleobase-cation symporters of the NAT/NCS2 family.

Nucleobase transporters are important for supplying the cell with purines and/or pyrimidines, for controlling the intracellular pool of nucleotides, and for obtaining exogenous nitrogen/carbon sources for metabolism. Nucleobase transporters are also evaluated as potential targets for antimicrobial therapies, since several pathogenic microorganisms rely on purine/pyrimidine salvage from their hosts. The majority of known nucleobase transporters belong to the evolutionarily conserved and ubiquitous nucleobase-ascorbate transporter/nucleobase-cation symporter-2 (NAT/NCS2) protein family. Based on a large-scale phylogenetic analysis that we performed on thousands of prokaryotic proteomes, we developed a webserver that can detect and distinguish this family of transporters from other homologous families that recognize different substrates. We can further categorize these transporters to certain evolutionary groups with distinct substrate preferences. The webserver scans whole proteomes and graphically displays which proteins are identified as NAT/NCS2, to which evolutionary groups and subgroups they belong to, and which conserved motifs they have. For key subgroups and motifs, the server displays annotated information from published crystal-structures and mutational studies pointing to key functional amino acids that may help experts assess the transport capability of the target sequences. The server is 100% accurate in detecting NAT/NCS2 family members. We also used the server to analyze 9,109 prokaryotic proteomes and identified Clostridia, Bacilli, ß- and γ-Proteobacteria, Actinobacteria, and Fusobacteria as the taxa with the largest number of NAT/NCS2 transporters per proteome. An analysis of 120 representative eukaryotic proteomes also demonstrates the server's capability of correctly analyzing this major lineage, with plants emerging as the group with the highest number of NAT/NCS2 members per proteome.

Detection of negative and positive RNA strand of poliovirus Sabin 1 and echovirus E19 by a stem-loop reverse transcription PCR.

In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the assay was the use of a specific reverse transcription primer targeting the 5'-UTR of enteroviruses that consisted of a stem-loop structure at the 5'-end and an enteroviral-specific sequence at the 3'-end. The stem loop RT-PCR was found to be an accurate and sensitive method, detecting even 10-2 CCID50 of poliovirus sabin 1 (Sabin1) and E19 strains 6 h postinfection (p.i.), while CPE appeared 3 days later. This assay was also validated in SiHa and Caski cell lines that are not used for the detection of enteroviruses. The negative RNA strand was detected 6 h and 12 h p.i. in SiHa and Caski cells, when these cell lines were inoculated with 105 and 1 CCID50 respectively, whereas CPE was observed 5 days p.i for SiHa cells and 8 days p.i for Caski cells and that only at 105 CCID50 . The results show that this approach may be used for replacing the time-consuming cell cultures in order to detect the active replication of enteroviruses. SIGNIFICANCE AND IMPACT OF THE STUDY: Enteroviruses are positive stranded RNA viruses that may cause severe diseases. The conventional method for detection of active viral replication involves virus isolation in sensitive cell cultures followed by titration and seroneutralization. In this report, we describe the use of a stem-loop secondary structured oligonucleotide in RT-PCR assay for the detection of the replicative negative strand of the positive-stranded RNA of poliovirus sabin 1 and E19 strains. This approach proved to be a useful tool that may be used for replacing the time-consuming cell culture assays in order to detect the active replication of enteroviruses.

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.

One billion years of bZIP transcription factor evolution: conservation and change in dimerization and DNA-binding site specificity.

The genomic era has revealed that the large repertoire of observed animal phenotypes is dependent on changes in the expression patterns of a finite number of genes, which are mediated by a plethora of transcription factors (TFs) with distinct specificities. The dimerization of TFs can also increase the complexity of a genetic regulatory network manifold, by combining a small number of monomers into dimers with distinct functions. Therefore, studying the evolution of these dimerizing TFs is vital for understanding how complexity increased during animal evolution. We focus on the second largest family of dimerizing TFs, the basic-region leucine zipper (bZIP), and infer when it expanded and how bZIP DNA-binding and dimerization functions evolved during the major phases of animal evolution. Specifically, we classify the metazoan bZIPs into 19 families and confirm the ancient nature of at least 13 of these families, predating the split of the cnidaria. We observe fixation of a core dimerization network in the last common ancestor of protostomes-deuterostomes. This was followed by an expansion of the number of proteins in the network, but no major dimerization changes in interaction partners, during the emergence of vertebrates. In conclusion, the bZIPs are an excellent model with which to understand how DNA binding and protein interactions of TFs evolved during animal evolution.