Adriana A. Giri

Differential expression of the human homologue of drosophila discs large oncosuppressor in histologic samples from human papillomavirus–associated lesions as a marker for progression to malignancy

High‐risk HPVs play a causal role in the development of cervical cancer, and their E6 oncoproteins target h‐Dlg for ubiquitin‐mediated proteolysis. The h‐Dlg oncosuppressor is associated with cell–cell interactions, and deregulation of these structures leads to defective cell adhesion, loss of cell polarity and unregulated proliferation. We evaluated the contribution of this E6 activity in the progression to malignancy in HPV infections by analyzing h‐Dlg expression in HPV‐associated lesions. We analyzed h‐Dlg in cervical, laryngeal, vulvar, colon and kidney histologic samples by Dlg immunohistochemistry. HPV association was ascertained by a PCR‐colorimetric method. Although Dlg was certainly expressed in intraepithelial cervical, vulvar and laryngeal HPV‐associated lesions, its cellular and tissue distribution patterns were altered compared to normal tissue. However, marked reduction in Dlg levels was observed in HPV‐positive invasive cervical carcinomas. To elucidate whether the loss of Dlg was significant for carcinogenesis in general, we investigated Dlg expression in tumors not associated with HPV. In colon and kidney carcinomas, Dlg was expressed, albeit with a different pattern of distribution with respect to the normal tissue. The loss of Dlg may be considered a late‐stage marker in cervical carcinogenesis, but alterations in its expression and localization take place during the different dysplastic stages. Dlg downregulation and/or alterations in its localization may contribute to transformation and may explain some of the characteristics of the malignant cells, such as loss of polarity and high migration ability.

New generic primer system targeting mucosal/genital and cutaneous human papillomaviruses leads to the characterization of HPV 115, a novel Beta-papillomavirus species 3

We explored the cutaneotropic HPV genetic diversity in 71 subjects from Argentina. New generic primers (CUT) targeting 88 mucosal/cutaneous HPV were designed and compared to FAP primers. Overall, 69 different HPV types/putative types were identified, being 17 of them novel putative types. Phylogenetic analysis of partial L1 sequences grouped 2 novel putative types in the Beta-PV, 14 in the Gamma-PV and 1 in the Mu-PV genera. CUT primers showed broader capacity than FAP primers in detecting different genera/species and novel putative types (p<0.01). Using overlapping PCR, the full-length genome of a Beta-PV putative type was amplified and cloned. The new virus, designated HPV 115, encodes five early genes and two late genes. Phylogenetic analysis indicated HPV 115 as the most divergent type within the genus Beta-PV species 3. This report is the first providing data on cutaneous HPVs circulating in South America and expands our knowledge of the Papillomaviridae family.

Altered Cortisol/DHEA Ratio in Tuberculosis Patients and its Relationship with Abnormalities in the Mycobacterial-driven Cytokine Production by Peripheral Blood Mononuclear Cells

We have investigated the relationship between cortisol and dehydroepiandrosterone (DHEA) levels and the immune response to mycobacterial antigens in peripheral venous blood, from a male population of active tuberculosis patients and age‐matched healthy controls of the same sex (HCo). Peripheral blood mononuclear cells were cultured for 36 or 96 h with whole sonicated Mycobacterium tuberculosis (WSA) for measurement of proliferation, interferon gamma (IFN‐γ) and interleukin‐10 (IL‐10) in culture supernatants. Comparisons on the in vitro mycobacterial‐driven immune responses demonstrated that TB patients had a higher IL‐10 production, a decreased lymphoproliferation and a trend to reduced IFN‐γ synthesis, in relation to HCo. Active disease was also characterized by increases in the plasma levels of glucocorticoids (GC) and reduced concentrations of DHEA which resulted in a higher cortisol/DHEA ratio respect the HCo group. Plasma DHEA levels were positively correlated with IFN‐γ values. An inverse correlation was found between the cortisol/DHEA ratio and IFN‐γ levels. Novel evidence is provided showing that the balance between cortisol and DHEA is partly responsible for the immune perturbations seen in TB patients.

Analysis of the genetic diversity and phylogenetic relationships of putative human papillomavirus types.

More than 170 human papillomavirus (HPV) types have been completely sequenced, curated and divided into five genera: Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus and Nupapillomavirus. With the application of PCR methods, hundreds of putative novel HPV types have been identified as PCR amplicons in mucosa and skin. However, at present there are no studies reporting a systematic search of the currently known L1 amplicons and their phylogenetic relationships. This survey revealed the existence of at least 202 different putative HPV types that are pending for full-genome characterization: five alphapapillomaviruses, 37 betapapillomaviruses, 159 gammapapillomaviruses and one mupapillomavirus. All potential viruses of the genera Alphapapillomavirus and Betapapillomavirus were grouped in the defined species, while 59 putative gammapapillomaviruses types were segregated in 21 unidentified putative species. These data highlight the need for progress in the identification of additional taxa of the family Papillomaviridae in order to elucidate the diversity, evolution and medical implications of these viruses.

Identification of human papillomavirus type 156, the prototype of a new human gammapapillomavirus species, by a generic and highly sensitive PCR strategy for long DNA fragments.

This study developed a hanging-droplet long PCR, a generic and highly sensitive strategy to facilitate the identification of new human papillomavirus (HPV) genomes. This novel procedure used for the first time the hanging-droplet PCR technique for the amplification of long DNA fragments with generic primers targeting the L1 and E1 regions. It was first applied to the amplification of types belonging to the highly divergent genus Gammapapillovirus (γ-PV). The hanging-droplet long PCR was 100-fold more sensitive than a simple long PCR procedure, detecting as few as ten copies of HPV-4. Nineteen skin samples, potentially containing putative HPV types from the γ-PV genus, were also screened. The method identified four γ-PV genomic halves from new and previously described putative types, and made the full characterization of HPV-156 possible. This novel virus meets the criteria for a new species within the γ-PV genus, with nucleotide identities in the L1 ORF ranging from 58.3 to 67.3 % compared with representative types of the current γ-PV species. HPV-156 showed the highest identity to HPV-60 (67.3 %) from species γ-4, and was consistently closely related to it in both late- and early-gene-derived phylogenies. In conclusion, this report provides a versatile and highly sensitive approach that allowed identification of the prototype of a new species within the γ-PV genus. Its application with primers targeting the different genera in which both human and non-human PVs are distributed may facilitate characterization of the missing members of the family Papillomaviridae.

Global Genomic Diversity of Human Papillomavirus 11 Based on 433 Isolates and 78 Complete Genome Sequences

ABSTRACT Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution. IMPORTANCE This study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage- and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.

Regulation of translational efficiency by different splice variants of the Disc large 1 oncosuppressor 5'-UTR.

Human Disc large (DLG1) has been demonstrated to be involved in the control of cell polarity and maintenance of tissue architecture, and is frequently lost in human tumours. However, the mechanisms controlling DLG1 expression are poorly understood. To further examine the regulation of DLG1 expression, we analysed the 5′ ends of DLG1 transcripts by rapid amplification of cDNA ends polymerase chain reaction. We identified an alternative splicing event in the 5′ region of DLG1 mRNA that generates transcripts with two different 5′ untranslated regions (5′‐UTRs). We show by reporter assays that the DLG1 5′‐UTR containing an alternatively spliced exon interferes with the translation of a downstream open reading frame (ORF). However, no significant differences in mRNA stability among the DLG1 5′‐UTR variants were observed. Sequence analysis of the additional exon present in the larger DLG1 5′‐UTR showed the presence of an upstream short ORF which is lost in the short version of the 5′‐UTR DLG1. By mutagenesis and luciferase assays, we analysed the contribution of this upstream short ORF in reducing translation efficiency, and showed that its disruption can revert, to some extent, the negative regulation of large 5′‐UTR. Using computational modelling we also show that the large DLG1 5′‐UTR isoform forms a more stable structure than the short version, and this may contribute to its ability to repress translation. This represents the first analysis of the 5′ region of the DLG1 transcripts and shows that differential expression of alternatively spliced 5′‐UTRs with different translational properties could result in changes in DLG1 abundance.

Strategic approach to produce low-cost, efficient, and stable competitive internal controls for detection of RNA viruses by use of reverse transcription-PCR.

ABSTRACT Molecular diagnostics based on reverse transcription (RT)-PCR are routinely complicated by the lack of stable internal controls, leading to falsely negative results. We describe a strategy to produce a stable competitive internal control (CIC) based on a Qβ phage derivative (recombinant Qβ [rQβ]) bearing primers KY78 and KY80, which are widely used in the detection of hepatitis C virus (HCV). rQβ was RNase resistant and stable at 4°C for 452 days in SM medium (0.1 M NaCl, 8 mM MgSO4·7H2O, 50 mM Tris HCl [pH 7.5], 2% gelatin) and for 125 days after lyophilization and reconstitution. rQβ performance as a CIC was evaluated. rQβ was added to HCV-positive samples, followed by RNA extraction and a CIC-HCV RT-PCR assay. This method combines RT-PCR, liquid hybridization with nonradioactive probes, and enzyme immunoanalysis. No influence of the CIC on qualitative HCV detection was observed independently of viral load, and results had high concordance with those of commercial kits. In conclusion, we describe a versatile, low-cost alternative strategy to armored RNA technology that can be adapted for detection or real-time applications of any RNA target. Moreover, the CIC reported here is an essential reagent for HCV screening in blood banks in resource-limited settings.

Prevalence of human papillomavirus infection in Argentinean women attending two different hospitals prior to the implementation of the National Vaccination Program

Cervarix vaccine was included in the National Immunization Program of Argentina in 2011 but data about the local distribution of human papillomavirus (HPV) infection in women exposed to the virus are scarce. This cross‐sectional study determined the prevalence and type distribution of HPV infection in unvaccinated women attending routine gynecological screening in two public hospitals located in Buenos Aires and Santa Fe, Argentina. Socio‐demographic, sexual behavior, and co‐factors information was obtained from all participants (Buenos Aires, n = 429; Santa Fe, n = 433). Cervicovaginal swabs were tested with an MY11/09 primer‐based assay and with the CUT primer system targeting mucosal/cutaneous HPVs. Participants from Buenos Aires showed significantly higher rates of HPV infection (52.4% vs. 40.6%), of multiple infections (24.2% vs. 16.4%), and of low‐risk (20.3% vs. 13.9%) and high‐risk types (44.1% vs. 33.3%) than those from Santa Fe. HPV‐66 (Buenos Aires: 17%) and HPV‐16 (Santa Fe: 8.5%) were the most prevalent types. Novel HPV‐66 putative subtype and variants were identified. Vaccine types 16 and 18 were frequent (Buenos Aires: 13.5%; Santa Fe: 10.2%) but few participants had co‐infections with both (Buenos Aires: 1.4%; Santa Fe: 0.2%). A common risk factor for HPV infection was having a new sexual partner in the last year (Buenos Aires: OR 2.53, P < 0.001; Santa Fe: OR 1.85, P = 0.04). This study provides valuable baseline data for future assessment of the impact of massive vaccination in Argentina and it underlines the use of additional HPV testing strategies, such as the CUT system, for surveillance and vaccinology. J. Med. Virol. 85:655–666, 2013.

mRNA expression of alpha and beta isoforms of glucocorticoid receptor in peripheral blood mononuclear cells of patients with tuberculosis and its relation with components of the immunoendocrine response.

We have analyzed the expression of glucocorticoid receptor (GR) isoforms by real time RT-qPCR in PBMCs from 19 controls (HCo) and 28 TB patients (8 mild; 12 moderate; 8 severe), HIV(-) and similar sex and age distribution. mRNA hGRα/β ratios were found higher in TB patients respect to those in HCo. However, when analyzing for disease severity such overall trend was at the expense of mild and moderate patients, with severe cases showing a lower mRNA hGRα/β ratio with respect to the other patient groups. This suggested some degree of resistance to endogenous glucocorticoids in patients with severe TB, since hGRαα dimer mediates the biological functions of GC, with the GRβ isoform acting as an inhibitor of GC activity. Levels of IL-6, IL-18, IFN-γ and Cortisol were significantly increased in severe and moderate cases, whereas DHEA values were found decreased in them (p<0.05 respect to HCo). Analysis on the relationship between plasma levels of these immuno-endocrine mediators with the mRNA expression of hGRα and hGRβ showed that IL-6 was positively associated with hGRα in mild TB patients (p<0.01), whereas a negative correlation between IFN-γ and hGRβ was observed in severe cases (p<0.01). As regard to hormones, DHEA was positively associated with hGRα in moderate and severe cases (p<0.01). This group also showed a negative correlation between hGRα and Cortisol/DHEA ratios (p<0.05). Changes in the systemic levels of cytokine and adrenal hormones are likely to affect GR expression in a differential fashion and according to the amount of pulmonary involvement.