Transcriptional activity of the long control region in human papillomavirus type 33 intratype variants.

BACKGROUND: High-risk human papillomaviruses (HPVs) are responsible for the development of cervical and other anogenital cancers. Intratype sequence variants of certain high-risk HPV types (e.g. 16, 18 and 31) are thought to have different oncogenic potential, partly due to nucleotide sequence variation in the viral long control region (LCR). The LCR has an important role in the regulation of viral replication and transcription. The purpose of this study was to explore sequence variation in the LCR of HPV 33 intratype variants in Hungary and to see whether there are differences in the transcriptional activities of the variants. METHODS: The complete HPV 33 LCR was amplified from HPV 33 positive cervical samples. After sequencing the LCR variants, multiple sequence alignment and phylogenetic analyses were carried out. Representative HPV 33 LCR sequence variants were selected for cloning and functional analysis. After transient transfection of HeLa cells, luciferase reporter assays were used to analyse the transcriptional activities of different LCR variants. RESULTS: Altogether 10 different variants were identified by sequence analysis of the HPV 33 LCR. The results of phylogenetic analysis showed that 3 variants belonged to sublineage A1, while the other 7 variants clustered with sublineage A2. Variants belonging to sublineage A2 had significantly lower transcriptional activities than variants belonging to sublineage A1. Within sublineage A2, the two variants analysed had significantly different transcriptional activities, which was shown to be caused by the A7879G variation. CONCLUSIONS: Nucleotide variation in the HPV 33 LCR can result in altered transcriptional activity of the intratype variants. Our results can help to understand the correlation between LCR polymorphism and the oncogenic potential of HPV 33 variants.

Coordinated action of human papillomavirus type 16 E6 and E7 oncoproteins on competitive endogenous RNA (ceRNA) network members in primary human keratinocytes.

BACKGROUND: miRNAs and lncRNAs can regulate cellular biological processes both under physiological and pathological conditions including tumour initiation and progression. Interactions between differentially expressed diverse RNA species, as a part of a complex intracellular regulatory network (ceRNA network), may contribute also to the pathogenesis of HPV-associated cancer. The purpose of this study was to investigate the global expression changes of miRNAs, lncRNAs and mRNAs driven by the E6 and E7 oncoproteins of HPV16, and construct a corresponding ceRNA regulatory network of coding and non-coding genes to suggest a regulatory network associated with high-risk HPV16 infections. Furthermore, additional GO and KEGG analyses were performed to understand the consequences of mRNA expression alterations on biological processes. METHODS: Small and large RNA deep sequencing were performed to detect expression changes of miRNAs, lncRNAs and mRNAs in primary human keratinocytes expressing HPV16 E6, E7 or both oncoproteins. The relationships between lncRNAs, miRNAs and mRNAs were predicted by using StarBase v2.0, DianaTools-LncBase v.2 and miRTarBase. The lncRNA-miRNA-mRNA regulatory network was visualized with Cytoscape v3.4.0. GO and KEEG pathway enrichment analysis was performed using DAVID v6.8. RESULTS: We revealed that 85 miRNAs in 21 genomic clusters and 41 lncRNAs were abnormally expressed in HPV E6/E7 expressing cells compared with controls. We constructed a ceRNA network with members of 15 lncRNAs - 43 miRNAs - 358 mRNAs with significantly altered expressions. GO and KEGG functional enrichment analyses identified numerous cancer related genes, furthermore we recognized common miRNAs as key regulatory elements in biological pathways associated with tumorigenesis driven by HPV16. CONCLUSIONS: The multiple molecular changes driven by E6 and E7 oncoproteins resulting in the malignant transformation of HPV16 host cells occur, at least in part, due to the abnormal alteration in expression and function of non-coding RNA molecules through their intracellular competing network.

Nutraceuticals Induced Changes in the Broiler Gastrointestinal Tract Microbiota.

Effects of nutraceuticals on the intestinal microbiota are receiving increased attention; however, there are few studies investigating their effects on broiler meat production. The aim of this study was to implement feeding strategies and carry out a comprehensive trial examining the interplay between natural biologically active compounds such as carotenoids, anthocyanins, fermentable oligosaccharides, and synbiotics and the gastrointestinal tract microbiota. Our feeding program was applied to an intensive production system with a flock of 1,080 Ross 308 broilers. Aging induced significant changes through the feeding experiment. Nutraceuticals were shown to modulate broiler intestinal diversity and differentially enriched Lactobacillus, Enterococcus, Campylobacter, and Streptococcus in the core microbiome during the different stages of broiler rearing. Additionally, they did not remarkably affect animal growth performance; nevertheless, a positive correlation was found between body weight and Corynebacteriales and Pseudomonadales Furthermore, a diet high in carotenoid, fermentable oligosaccharide, and anthocyanin contents affected the number of beneficial genera such as Faecalibacterium, Lactobacillus, Blautia, and Ruminococcus With this comprehensive trial, we revealed that nutraceuticals induced modulations in broiler gastrointestinal tract microbiota. We believe that plant-derived immunostimulants, recycled from plant food waste products, can supplement antibiotic-free broiler meat production.IMPORTANCE In this trial, nutraceuticals were manufactured from waste products of food industry processing of Hungarian red sweet pepper and sour cherry and incorporated into the diet of poultry to investigate their effects on broilers' growth and the broiler gastrointestinal tract microbiota. To avoid the generation of food waste products, we believe that this approach can be developed into a sustainable, green approach that can be implemented in commercial antibiotic-free poultry to provide safe and high-quality meat.

Reconsidering the technical aspects and quality management background of faecal microbiota transplantation due to the novel coronavirus pandemic / A székletmikrobiota-transzplantáció technológiájának és minoségirányítási hátterének újragondolása a SARS-CoV-2 víruspandémia kapcsán.

Összefoglaló. A székletmikrobiota-transzplantáció (faecalismikrobiota-transzplantáció - FMT) a Clostridioides difficile fertozés (CDI) kezelésében nemzetközileg széles körben elfogadott, megfelelo szakmai háttér mellett végezve biztonságos, potenciálisan életmento, költséghatékony, valamint a hospitalizációs ido és az orvos-beteg találkozások jelentos redukálására képes eljárás. Az FMT elvégzésére egyes országokban magas szintu minoségirányítási háttérrel muködo, célfeladatra szervezodött donor- és székletbankok rendezkedtek be. Máshol, így például hazánkban, az eljáráshoz az egyértelmu jogi szabályozási környezet, a standardizált technológiai háttér és a finanszírozás hiánya miatt nem egységes a hozzáférés. Régóta idoszeru továbbá, hogy a heterogén, nemegyszer háztartási eszközökkel elokészített beavatkozások helyett a nemzetközi és legújabban már a hazai ajánlásokban is megfogalmazott, a betegbiztonságot legjobban garantáló elvárások mellett történjen a széklettranszplantáció. Az új koronavírus (SARS-CoV-2) okozta pandémia megjelenése eroteljes szakmai érv országos szinten az FMT minoségirányítási környezetének és technológiai hátterének újragondolására, mert a SARS-CoV-2 egyszerre jelent kockázatot a CDI miatt kórházban kezelt sérülékeny betegpopulációnak, és egyben veszélyezteti az FMT biztonságosságát mind a recipiens, mind pedig az eljárást végzo egészségügyi személyzet tekintetében. Ezekre a szakmai és társadalmi kihívásokra reagálva, a széles köru beteghozzáférés és a legmagasabb szintu betegbiztonság garantálására, a Debreceni Egyetemen új eljárásrendet dolgoztunk ki az FMT végzésére. Ezen eljárásrendnek a COVID-19-pandémia miatt módosított, a fagyasztottgraftbank üzemeltetése és a rendszerszemlélet tekintetében releváns elemeit ismertetjük. Javasolt, hogy országos szinten hasonló, megfelelo minoségirányítási és technológiai környezettel, a SARS-CoV-2-fertozés kizárását is integráló donorszurési rendszerrel, továbbá fagyasztottgraft-banki háttérrel muködo laboratóriumok vegyenek részt a széklettranszplantációk végzésében. Felmerül továbbá, hogy az eljárást a számos analógia és a donor-recipiens koncepció alapján a sejt- és szövettranszplantációkra vonatkozó szabályozórendszer keretei közé ajánlott beágyazni. Orv Hetil. 2020; 161(44): 1858-1871. Summary. Stool transplantation (faecal microbiota transplantation - FMT) is a widely accepted, potentially life-saving, cost-effective medical intervention for the treatment of Clostridioides difficile infection (CDI), which has an acceptable safety profile if performed with an appropriate professional background. FMT can significantly reduce hospitalization time and the number of patient visits. National donor and stool banks with high-standard quality management systems were established in certain countries for supporting the procedures. In other regions, including Hungary, patient access is not uniform due to the lack of clear legal regulations, standardized technology or financial reimbursement. It has been expected for a long time to replace the heterogenous techniques, occasionally utilizing household equipment with a technology providing improved patient safety and fulfilling international and recently published local FMT guidelines. The emergence of the novel coronavirus (SARS-CoV-2) pandemic is a very powerful argument in favour of urgently reconsidering the quality management and technological background of FMT procedures. SARS-CoV-2 is a major threat to the vulnerable patients suffering from CDI and also impose risks for the recipient and healthcare personnel involved in carrying out the transplantation. New FMT guidelines were implemented at the University of Debrecen to address these professional and public challenges, to provide wide patient access and to guarantee the highest achievable patient safety. Relevant elements of this new protocol are presented, focusing on a systemic quality management approach, on the operation of a frozen stool bank and on a modified donor screening algorithm taking the risks of COVID-19 into consideration. We suggest that laboratories with proper quality assurance and technological conditions, implementing SARS-CoV-2 donor screening and operating a frozen graft bank should participate in faecal microbiota transplantations. It is also recommended that, based on the analogies and the similar donor-recipient concept, FMT should be embedded under the organ tissue and cell transplantation polices in Hungary. Orv Hetil. 2020; 161(44): 1858-1871.

Orientation-dependent toxic effect of human papillomavirus type 33 long control region DNA in Escherichia coli cells.

The functional analysis of human papillomavirus (HPV) sequence variation requires the molecular cloning of different genomic regions of virus variants. In this study, we report an unexpected difficulty experienced when trying to clone HPV33 long control region (LCR) variants in Escherichia coli. Standard cloning strategies proved to be inappropriate to clone HPV33 LCR variants in the forward orientation into a eukaryotic reporter vector (pGL2-Basic). However, by slight modification of culture conditions (incubation at 25 °C instead of 37 °C), constructs containing the HPV33 LCR variants in the forward orientation were obtained. Transformation experiments performed with different HPV33 LCR constructs indicated that there is a sequence element in the 5' LCR of HPV33 causing temperature-dependent toxic effect in E. coli. Sequence analysis revealed the presence of an open reading frame (ORF) in the 5' part of HPV33 LCR potentially encoding a 116-amino acid polypeptide. Protein structure prediction suggested that this putative protein might have a structural similarity to transmembrane proteins. Even a low-level expression of this protein may cause significant toxicity in the host bacteria. In silico analysis of the LCR of HPV33 and some other HPV types belonging to the species Alphapapillomavirus 9 (HPV31, 35 and 58) seemed to support the assumption that the ORFs found in the 5' LCR of these HPVs are protein-coding sequences. Further studies should be performed to prove that these putative proteins are really expressed in the infected host cells and to identify their function.

Patient-related factors, antibiotic prescribing and antimicrobial resistance of the commensal Staphylococcus aureus and Streptococcus pneumoniae in a healthy population - Hungarian results of the APRES study.

BACKGROUND: Antimicrobial resistance (AMR) is an increasing public health problem worldwide. We studied some patient-related factors that might influence the antimicrobial resistance. and whether the volume of antibiotic prescribing of the primary care physicians correlate with the antibiotic resistance rates of commensal nasal Staphylococcus aureus and Streptococcus pneumoniae. METHODS: The socio-demographic questionnaires, the antibiotic prescription and resistance data of commensal nasal S. aureus and S. pneumoniae were collected in the 20 participating Hungarian practices of the APRES study. Multivariate logistic regression analyses were performed on the patient-related data and the antimicrobial resistance of the S. aureus and S. pneumoniae on individual, patient level. Ecological analyses were performed with Spearman's rank correlations at practice level, the analyses were performed in the whole sample (all practices) and in the cohorts of primary care practices taking care of adults (adult practices) or children (paediatric practices). RESULTS: According to the multivariate model, age of the patients significantly influenced the antimicrobial resistance of the S. aureus (OR = 0.42, p = 0.004) and S. pneumoniae (OR = 0.89, p < 0.001). Living with children significantly increased the AMR of the S. pneumoniae (OR = 1.23, p = 0.019). In the cohorts of adult or paediatric practices, neither the age nor other variables influenced the AMR of the S. aureus and S. pneumoniae. At practice level, the prescribed volume of penicillins significantly correlated with the resistance rates of the S. aureus isolates to penicillin (rho = 0.57, p = 0.008). The volume of prescribed macrolides, lincosamides showed positive significant correlations with the S. pneumoniae resistance rates to clarithromycin and/or clindamycin in all practices (rho = 0.76, p = 0.001) and in the adult practices (rho = 0.63, p = 0.021). CONCLUSIONS: The age is an important influencing factor of antimicrobial resistance. The results also suggest that there may be an association between the antibiotic prescribing of the primary care providers and the antibiotic resistance of the commensal S. aureus and S. pneumoniae. The role of the primary care physicians in the appropriate antibiotic prescribing is very important to avoid the antibiotic resistance.

Study of fast and slow consecutive processes by heterogeneous isotope exchange using P-32 radiotracer.

The sorption of phosphate on soils is studied by radioisotopic tracer method. Two consecutive processes with rather different rates were differentiated: namely the heterogeneous isotope exchange between the phosphate in the soil solution and the weakly sorbed phosphate (fast reaction), and the transformation of weakly sorbed phosphate to tightly sorbed phosphate (slow reaction). In this paper, it is shown how the rate constants of these two processes can be determined by a radiotracer with a relatively short half-life.

Down-regulation of increased TRAF6 expression in the peripheral mononuclear cells of patients with primary Sjögren's syndrome by an EBV-EBER1-specific synthetic single-stranded complementary DNA molecule.

AIM: We described earlier a simultaneously increased that the increased expression of miRNA-146a/b was accompanied by an increase in the expression of and TRAF6 and a decrease in the expression of IRAK1 genes in the peripheral mononuclear cells (PBMCs) of patients with primary Sjogren's syndrome (pSS) patients. Recently, the expression of EBV encoded. RNA (EBER) was published in the B cells of salivary glands of in pSS. In the present study, we applied an EBV-EBER1 specific synthetic single stranded complementary DNA molecule (EBV-EBER1-cDNA) to test whether any EBER1 related effect exists also in PBMCs of pSS patients. METHODS: In the PBMCs of pSS patients and healthy controls, we investigated in vitro the effects of a synthetic single stranded EBV-EBER1-cDNA molecule, synthetic double-stranded (ds)RNA polyinosinic-polycytidylic acid [poly (I:C)] and polyadenylic acid potassium salt poly-adenylic acid [poly-(A)] on the expression of TRAF6 gene tested by qRTPCR. The release of interferon -α was detected by ELISA. RESULTS: EBV-EBER1-cDNA resulted in a significant reduction in the expression of TRAF6 in the cells of patients, but in the healthy controls not, whereas the treatments with poly (I:C) and poly-(A) could not reduce the TRAF6 over-expression. No release of EBER1 could be observed in the culture supernatants of patients with pSS. Only the treatment with poly (I:C) resulted in a significant increase of interferon -α release, and only in the heathy controls. No release of EBER1 molecules took place during the culturing of cells. EBV-EBER- cDNA acted functionally on the cells of patients only. CONCLUSION: These findings give a further evidence of the linkage between EBV and pSS, furthermore, they show the possible role of EBV-EBER1 in the induction of increased TRAF6 expression in the peripheral B cells of Sjögren's patients.

The PTPN14 Tumor Suppressor Is a Degradation Target of Human Papillomavirus E7.

Activation of signaling pathways ensuring cell growth is essential for the proliferative competence of human papillomavirus (HPV)-infected cells. Tyrosine kinases and phosphatases are key regulators of cellular growth control pathways. A recently identified potential cellular target of HPV E7 is the cytoplasmic protein tyrosine phosphatase PTPN14, which is a potential tumor suppressor and is linked to the control of the Hippo and Wnt/beta-catenin signaling pathways. In this study, we show that the E7 proteins of both high-risk and low-risk mucosal HPV types can interact with PTPN14. This interaction is independent of retinoblastoma protein (pRb) and involves residues in the carboxy-terminal region of E7. We also show that high-risk E7 induces proteasome-mediated degradation of PTPN14 in cells derived from cervical tumors. This degradation appears to be independent of cullin-1 or cullin-2 but most likely involves the UBR4/p600 ubiquitin ligase. The degree to which E7 downregulates PTPN14 would suggest that this interaction is important for the viral life cycle and potentially also for the development of malignancy. In support of this we find that overexpression of PTPN14 decreases the ability of HPV-16 E7 to cooperate with activated EJ-ras in primary cell transformation assays.IMPORTANCE This study links HPV E7 to the deregulation of protein tyrosine phosphatase signaling pathways. PTPN14 is classified as a potential tumor suppressor protein, and here we show that it is very susceptible to HPV E7-induced proteasome-mediated degradation. Intriguingly, this appears to use a mechanism that is different from that employed by E7 to target pRb. Therefore, this study has important implications for our understanding of the molecular basis for E7 function and also sheds important light on the potential role of PTPN14 as a tumor suppressor.

Phylogenetic and functional analysis of sequence variation of human papillomavirus type 31 E6 and E7 oncoproteins.

High-risk human papillomaviruses (HPV) are the causative agents of cervical and other anogenital cancers as well as a subset of head and neck cancers. The E6 and E7 oncoproteins of HPV contribute to oncogenesis by associating with the tumour suppressor protein p53 and pRb, respectively. For HPV types 16 and 18, intratypic sequence variation was shown to have biological and clinical significance. The functional significance of sequence variation among HPV 31 variants was studied less intensively. HPV 31 variants belonging to different variant lineages were found to have differences in persistence and in the ability to cause high grade cervical intraepithelial neoplasia. In the present study, we started to explore the functional effects of natural sequence variation of HPV 31 E6 and E7 oncoproteins. The E6 variants were tested for their effects on p53 protein stability and transcriptional activity, while the E7 variants were tested for their effects on pRb protein level and also on the transcriptional activity of E2F transcription factors. HPV 31 E7 variants displayed uniform effects on pRb stability and also on the activity of E2F transcription factors. HPV 31 E6 variants had remarkable differences in the ability to inhibit the trans-activation function of p53 but not in the ability to induce the in vivo degradation of p53. Our results indicate that natural sequence variation of the HPV 31 E6 protein may be involved in the observed differences in the oncogenic potential between HPV 31 variants.

CpG methylation in human papillomavirus (HPV) type 31 long control region (LCR) in cervical infections associated with cytological abnormalities.

The mechanisms that regulate papillomavirus gene expression include DNA methylation. The transcription of papillomavirus oncogenes E6 and E7 is controlled by certain regulatory elements in the LCR, which include binding sites for the E2 protein, a viral regulator of oncogene expression. In HPV-31-infected exfoliated cervical cells, the CpG methylation of the entire LCR was determined by next-generation sequencing after bisulfite modification. Six of the 22 cases had methylated CpG sites in the HPV-31 LCR, including position 7479 and/or 7485, at the promoter distal E2 binding site, thus suggesting a potential regulatory mechanism for papillomavirus transcription.

Combining standard clinical methods with PCR showed improved diagnosis of invasive pulmonary aspergillosis in patients with hematological malignancies and prolonged neutropenia.

BACKGROUND: We assessed the diagnostic value of standard clinical methods and combined biomarker testing (galactomannan assay and polymerase chain reaction screening) in a prospective case-control study to detect invasive pulmonary aspergillosis in patients with hematological malignancies and prolonged neutropenia. METHODS: In this observational study 162 biomarker analyses were performed on samples from 27 febrile neutropenic episodes. Sera were successively screened for galactomannan antigen and for Aspergillus fumigatus specific nucleic acid targets. Furthermore thoracic computed tomography scanning was performed along with bronchoscopy with lavage when clinically indicated. Patients were retrospectively stratified to define a case-group with "proven" or "probable" invasive pulmonary aspergillosis (25.93 %) and a control-group of patients with no evidence for of invasive pulmonary aspergillosis (74.07 %). In 44.44 % of episodes fever ceased in response to antibiotic treatment (group II). Empirical antifungal therapy was administered for episodes with persistent or relapsing fever (group I). 48.15 % of patients died during the study period. Postmortem histology was pursued in 53.85 % of fatalities. RESULTS: Concordant negative galactomannan and computed tomography supported by a polymerase chain reaction assay were shown to have the highest discriminatory power to exclude invasive pulmonary aspergillosis. Bronchoalveolar lavage was performed in 6 cases of invasive pulmonary aspergillosis and in 15 controls. Although bronchoalveolar lavage proved negative in 93 % of controls it did not detect IPA in 86 % of the cases. Remarkably post mortem histology convincingly supported the presence of Aspergillus hyphae in lung tissue from a single case which had consecutive positive polymerase chain reaction assay results but was misdiagnosed by both computed tomography and consistently negative galactomannan assay results. For the galactomannan enzyme-immunoassay the diagnostic odds ratio was 15.33 and for the polymerase chain reaction assay it was 28.67. According to Cohen's kappa our in-house polymerase chain reaction method showed a fair agreement with the galactomannan immunoassay. Combined analysis of the results from the Aspergillus galactomannan enzyme immunoassay together with those generated by our polymerase chain reaction assay led to no misdiagnoses in the control group. CONCLUSION: The data from this pilot-study demonstrate that the consideration of standard clinical methods combined with biomarker testing improves the capacity to make early and more accurate diagnostic decisions.

Transcriptional regulation of genes involved in keratinocyte differentiation by human papillomavirus 16 oncoproteins.

The life cycle of human papillomaviruses (HPVs) is strictly linked to the differentiation of their natural host cells. The HPV E6 and E7 oncoproteins can delay the normal differentiation program of keratinocytes; however, the exact mechanisms responsible for this have not yet been identified. The goal of this study was to investigate the effects of HPV16 oncoproteins on the expression of genes involved in keratinocyte differentiation. Primary human keratinocytes transduced by LXSN (control) retroviruses or virus vectors expressing HPV16 E6, E7 or E6/E7 genes were subjected to gene expression profiling. The results of microarray analysis showed that HPV 16 E6 and E7 have the capacity to downregulate the expression of several genes involved in keratinocyte differentiation. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed to confirm the microarray data. To investigate the effects of the HPV oncoproteins on the promoters of selected keratinocyte differentiation genes, luciferase reporter assays were performed. Our results suggest that the HPV 16 E6 and/or E7 oncogenes are able to downregulate the expression of several genes involved in keratinocyte differentiation (such as desmocollin 1, keratin 4, S100 calcium-binding protein A8 and small proline-rich protein 1A), at least partially by downregulating their promoter activity. This activity of the HPV oncoproteins may have a role in the productive virus life cycle, and also in virus-induced carcinogenesis.

Serum interleukin-6 levels in murine models of Candida albicans infection.

Two Balb/C mouse models of Candida infection were used to detect serum interleukin-6 (IL-6) responses. The first model used systemic infection by Candida albicans ATCC 10231 strain infected through the lateral tail vein of mice without any specific pretreatment. The median Candida burdens of the kidneys were 1.5 × 106 CFU/ml 24 h postinoculation (p.i.) and 1.2 × 107 CFU/ml 72 h p.i., while median serum IL-6 levels were 479.3 pg/ml and 934.5 pg/ml, respectively. The Candida burden showed significant correlation with serum IL-6 24 h p.i. (R2 = 0.6358; P = 0.0082) but not 72 h p.i.The second model was a mouse vaginitis model applying intravaginal inoculation of mice pretreated with subcutaneous estradiol-valerate (10 mg/ml) 3 days before infection. Candida cell count in vaginal lavage fluid was 2.8 × 106 CFU/ml 24 h p.i. and 1.4 × 108 CFU/ml 72 h p.i. Serum IL-6 response was detected in 4 of 15 mice 24 h p.i. and 9 of 15 mice 72 h p.i. Even the responders had low IL-6 serum levels (mean values 29.9 pg/ml and 60.1 pg/ml, respectively) not correlating with Candida cell count in vaginal lavage fluid.In conclusion, serum IL-6 had strong relationship with systemic C. albicans infection while the local C. albicans infection of the vagina led to partial, prolonged and limited serum IL-6 response.

Molecular typing of human herpesvirus 8 isolates from patients with Kaposi's sarcoma in Hungary.

BACKGROUND: Kaposi's sarcoma (KS) shows a distinct geographical and ethnic distribution. Genes at both ends of the human herpesvirus 8 (HHV-8) genome have been shown to vary considerably. Seven major molecular subtypes of HHV-8 were defined based on the amino acid sequence of the open reading frame K1 (orf-K1). The aim of the present study was to characterize HHV8 isolates from hospitalized patients in Hungary. MATERIALS AND METHODS: A total of 36 archival paraffin-embedded Kaposi's sarcoma tissue samples were collected. Polymerase chain reaction (PCR) was carried out on the extracted DNA, using specific primers for HHV-8. After identifying the presence of HHV-8 by amplification of its orf26 region, the orf-K1 region was amplified, sequenced and used for phylogenetic analysis. RESULTS: From the 36 orf26-positive cases, orf-K1 was amplified and was analyzed successfully in 12 cases. Phylogenetic studies, based on the complete K1 gene/protein sequences, indicate that all strains belong to the A subtype. Specifically, six of them were related to the A1 subgroup, six to the A2 subgroup and three previously reported to the A3 subgroup. Nucleotide sequence data are reported and are available in the Genbank database under accession numbers KF829938-KF829947.

Microbiological profile of adenoid hypertrophy correlates to clinical diagnosis in children.

OBJECTIVE: Adenoid hypertrophy is a common condition in childhood, which may be associated with recurring acute otitis media (RAOM), otitis media with effusion (OME), and obstructive sleep apnea syndrome (OSAS). These different clinical characteristics have some clinical overlap; however, they might be explained by distinct immunologic and infectious profiles and result in various histopathologic findings of adenoid specimens. METHODS: A total of 59 children with adenoid hypertrophy undergoing adenoidectomy were studied. Three series of identical adenoid specimens were processed to hematoxylin-eosin (H.E.) and Gram staining and to respiratory virus specific real-time PCR, respectively. RESULTS: According to the clinical characteristics, patients were recruited into three groups: RAOM (n = 25), OME (n = 19), and OSAS (n = 15). Bacterial biofilms were detected in 21 cases, while at least one of the studied respiratory viruses was detected in 52 specimens. RAOM cases were significantly associated with biofilm existence (n = 20, P < 0.001). In contrast, OME group was characterized by the absence of bacterial biofilm and by normal mucosa. Showing a statistically significant correlation, all OME cases were positive for human bocavirus (HBoV, P < 0.001). CONCLUSIONS: Bacterial biofilms might contribute to the damage of respiratory epithelium and recurring acute infections resulting in RAOM. In OME cases persisting respiratory viruses, mainly HBoV, can cause subsequent lymphoid hyperplasia leading to ventilation disorders and impaired immunoreactivity of the middle ear cleft.

Sequence variation of human papillomavirus type 31 long control region: phylogenetic and functional implications.

About one-third of human papillomavirus (HPV) types infect the anogenital tract. High-risk genital HPV types (such as HPV 16, 18, 31, 33, and 35) are linked causally to the development of cervical cancer. The long control region (LCR) of the HPV genome regulates the replication and transcription of the viral genome. In this study, the functional significance of nucleotide sequence variation within the LCR of HPV 31 was investigated. The LCR was amplified by polymerase chain reaction (PCR) from 41 HPV 31 positive cervical samples of Hungarian women. A phylogenetic tree constructed from the nucleotide sequences of the LCR variants revealed the presence of three intratypic variant lineages of HPV 31, in accordance with previous results. In order to explore the functional consequences of sequence variation in the LCR of HPV 31, selected LCR variants were cloned into a luciferase reporter vector, transfected into C33-A cells and tested in luciferase reporter assays. Significant differences were found between the transcriptional activities of HPV 31 LCR variants belonging to different variant lineages. As the LCR is governing the transcription of the E6 and E7 oncogenes, the differences in the transcriptional activities of LCR variants may be associated with differences in their oncogenic potential.

Elevated tumor necrosis factor-alpha expression in periapical lesions infected by Epstein-Barr virus.

INTRODUCTION: In apical periodontitis, there is an intense inflammatory response to endodontopathogenic bacteria, an essential component of the pathogenic microbiota. The inflammation can be aggravated by herpesviruses acting as nonessential pathogens in periapical lesions. This study aimed to determine the levels of tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-ß) in periapical lesions in relation to local occurrence of Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6), and human herpesvirus 8 (HHV-8). METHODS: Fifty-eight samples with apical periodontitis and 20 clinically healthy gingival control tissues were collected. Viral DNA was determined with nested polymerase chain reaction, and cytokine mRNA expression was detected with real-time polymerase chain reaction assays. RESULTS: Periapical lesions harbored EBV (75.9%) and HHV-6 (22.4%) at significantly higher frequencies compared with controls (P < .000001 and P < .05, respectively), whereas HCMV (12%) and HHV-8 (0%) occurred rarely. The median TNF-α expression was 13 times higher (P < .001) and TGF-ß expression was 5 times higher in periapical lesions than in controls (P < .001). TNF-α expression was significantly higher in EBV-positive lesions than in EBV-negative lesions (P = .032). Presence of symptoms, lesion size, and infection by HCMV or HHV-6 had no significant association with either TNF-α or TGF-ß expression. CONCLUSIONS: The herpesviral component of the endodontic microbiota did not correlate with TGF-ß expression, whereas EBV infection was associated with a median 1.5 times further elevation of the high TNF-α expression characteristic for periapical lesions.

Activation of Src, Fyn and Yes non-receptor tyrosine kinases in keratinocytes expressing human papillomavirus (HPV) type 16 E7 oncoprotein.

BACKGROUND: The Src family tyrosine kinases (SFK) are cellular regulatory proteins that influence cell adhesion, proliferation, invasion and survival during tumor development. Elevated activity of Src was associated with increased cell proliferation and invasivity in human papillomavirus (HPV)-associated malignancies; therefore, transduced human foreskin keratinocytes (HFK) were used to investigate whether SFK activation is a downstream effect of papillomaviral oncoproteins. Activation of ubiquitously expressed SFKs, namely Src, Yes and Fyn, was investigated in both proliferating and differentiating keratinocytes. RESULTS: In proliferating keratinocytes, Src, Yes and Fyn mRNA levels were not affected by HPV 16 E6 or E7 oncoproteins, while at the protein level as detected by western blot, the presence of both E6 and E7 resulted in substantial increase in Src and Yes expression, but did not alter the high constitutive level of Fyn. Phospo-kinase array revealed that all ubiquitously expressed SFKs are activated by phosphorylation in the presence of HPV 16 E7 oncoprotein. Keratinocyte differentiation led to increased Yes mRNA and protein levels in all transduced cell lines, while it did not influence the Src transcription but resulted in elevated Src protein level in HPV16 E7 expressing lines. CONCLUSIONS: This study revealed that HPV 16 oncoproteins upregulate Src family kinases Src and Yes via posttranscriptional mechanisms. A further effect of HPV 16 E7 oncoprotein is to enhance the activating phosphorylation of SFKs expressed in keratinocytes.

Surveillance of human rotaviruses in 2007-2011, Hungary: exploring the genetic relatedness between vaccine and field strains.

BACKGROUND: The availability of rotavirus vaccines has resulted in an intensification of post vaccine strain surveillance efforts worldwide to gain information on the impact of vaccines on prevalence of circulating rotavirus strains. OBJECTIVES: In this study, the distribution of human rotavirus G and P types in Hungary is reported. In addition, the VP4 and VP7 genes of G1P[8] strains were sequenced to monitor if vaccine-derived strains were introduced and/or some strains/lineages were selected against. STUDY DESIGN: The study was conducted in 8 geographic areas of Hungary between 2007 and 2011. Rotavirus positive stool samples were collected from diarrheic patients mostly <5 years of age. Viral RNA was amplified by multiplex genotyping RT-PCR assay, targeting the medically most important G and P types. When needed, sequencing of the VP7 and VP4 genes was performed. RESULTS: In total, 2380 strains were genotyped. During the 5-year surveillance we observed the dominating prevalence of genotype G1P[8] (44.87%) strains, followed by G4P[8] (23.4%), G2P[4] (14.75%) and G9P[8] (6.81%) genotypes. Uncommon strains were identified in a low percentage of samples (4.12%). Phylogenetic analysis of 318 G1P[8] strains identified 55 strains similar to the Rotarix strain (nt sequence identities; VP7, up to 97.9%; VP4, up to 98.5%) although their vaccine origin was unlikely. CONCLUSIONS: Current vaccines would have protected against the majority of identified rotavirus genotypes. A better understanding of the potential long-term effect of vaccine use on epidemiology and evolutionary dynamics of co-circulating wild type strains requires continuous strain surveillance.