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.

Which Is More Toxic? Evaluation of the Short-Term Toxic Effects of Chlorpyrifos and Cypermethrin on Selected Biomarkers in Common Carp (Cyprinus carpio, Linnaeus 1758).

The general aim of this study was to investigate the negative short-term effects of different concentrations of chlorpyrifos (CPF) and cypermethrin (CYP), based on the EU legislation (MAC-EQS) in common carp (Cyprinus carpio Linnaeus, 1758) under laboratory conditions and to compare their toxicity. The fish were exposed to the pesticides for 96 h and then different histological and biochemical biomarkers were investigated in the gills and liver, and bioaccumulation analyses were conducted. The chemical studies showed increased pesticide concentrations in the gills as the first site for pollutants compared to the liver at the 96th hour. In addition, the histological analyses showed severe alterations in the gills and liver after exposure to both tested pesticides. In the gills, we found mainly intense proliferative and, to a lesser extent, degenerative changes and alterations in the circulatory system, such as necrosis and vasodilation. In the liver, regressive and progressive lesions, as well as circulatory disturbances and inflammation, were observed. The regressive lesions showed a higher degree of expression compared to the other changes. Furthermore, we found altered enzymatic activities-catalase, glutathione reductase, and glutathione peroxidase-in the liver, compared to the control. Overall, both tested pesticides impacted the studied biomarkers in common carp, even at concentrations lower than those permitted by law. However, the results of the comparative analysis showed a relatively higher toxicity of CYP compared to CPF in the fish. Still, questions persist as to whether the observed changes are adaptive or entirely destructive. To avoid any danger or risk, these pesticides must be applied cautiously, especially near water bodies.

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.

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.

Phylogenetic evidence for a new species of Barbus in the Danube River basin.

Three species of small-sized rheophilic Barbus fishes are endemic to and widely distributed throughout the mountain regions in the Danube River basin. In Hungary, barbels referred to as B. petenyi occur in streams in the foothills of the Carpathians near the borders with Slovakia, Ukraine and Romania. However, up to now, no genetic investigations were carried out on rheophilic barbels in this region. This study aims to clarify the taxonomic identity and distribution of the rheophilic barbels in the Hungarian plain based on molecular and morphological analyses. Two mitochondrial genes (cytochrome b, ATPase 6/8) and one nuclear gene (beta-actin intron 2) were sequenced and several morphometric and meristic characters were recorded. Phylogenetic and morphological analyses revealed that there are four genetically distinct lineages among the rheophilic barbels in the Carpathian Basin. The results demonstrated that North-Hungarian Barbus populations belong to B. carpathicus and that B. petenyi presumably does not occur in Hungary. As expected, B. balcanicus was only recorded in samples from the Balkans analyzed for reference. A distinct species, new to science, was discovered to be present in Sebes-Körös River (Crisul Repede) in eastern Hungary and western Romania and is formally described here as B. biharicus Antal, László, Kotlík - sp. nov.

Large-scale whole genome sequencing identifies country-wide spread of an emerging G9P[8] rotavirus strain in Hungary, 2012.

With the availability of rotavirus vaccines routine strain surveillance has been launched or continued in many countries worldwide. In this study relevant information is provided from Hungary in order to extend knowledge about circulating rotavirus strains. Direct sequencing of the RT-PCR products obtained by VP7 and VP4 genes specific primer sets was utilized as routine laboratory method. In addition we explored the advantage of random primed RT-PCR and semiconductor sequencing of the whole genome of selected strains. During the study year, 2012, we identified an increase in the prevalence of G9P[8] strains across the country. This genotype combination predominated in seven out of nine study sites (detection rates, 45-83%). In addition to G9P[8]s, epidemiologically major strains included genotypes G1P[8] (34.2%), G2P[4] (13.5%), and G4P[8] (7.4%), whereas unusual and rare strains were G3P[8] (1%), G2P[8] (0.5%), G1P[4] (0.2%), G3P[4] (0.2%), and G3P[9] (0.2%). Whole genome analysis of 125 Hungarian human rotaviruses identified nine major genotype constellations and uncovered both intra- and intergenogroup reassortment events in circulating strains. Intergenogroup reassortment resulted in several unusual genotype constellations, including mono-reassortant G1P[8] and G9P[8] strains whose genotype 1 (Wa-like) backbone gene constellations contained DS1-like NSP2 and VP3 genes, respectively, as well as, a putative bovine-feline G3P[9] reassortant strain. The conserved genomic constellations of epidemiologically major genotypes suggested the clonal spread of the re-emerging G9P[8] genotype and several co-circulating strains (e.g., G1P[8] and G2P[4]) in many study sites during 2012. Of interest, medically important G2P[4] strains carried bovine-like VP1 and VP6 genes in their genotype constellation. No evidence for vaccine associated selection, or, interaction between wild-type and vaccine strains was obtained. In conclusion, this study reports the reemergence of G9P[8] strains across the country and indicates the robustness of whole genome sequencing in routine rotavirus strain surveillance.

Review of global rotavirus strain prevalence data from six years post vaccine licensure surveillance: is there evidence of strain selection from vaccine pressure?

Comprehensive reviews of pre licensure rotavirus strain prevalence data indicated the global importance of six rotavirus genotypes, G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] and G12P[8]. Since 2006, two vaccines, the monovalent Rotarix (RV1) and the pentavalent RotaTeq (RV5) have been available in over 100 countries worldwide. Of these, 60 countries have already introduced either RV1 or RV5 in their national immunization programs. Post licensure vaccine effectiveness is closely monitored worldwide. This review aimed at describing the global changes in rotavirus strain prevalence over time. The genotype distribution of the nearly 47,000 strains that were characterized during 2007-2012 showed similar picture to that seen in the preceding period. An intriguing finding was the transient predominance of heterotypic strains, mainly in countries using RV1. Unusual and novel antigen combinations continue to emerge, including some causing local outbreaks, even in vaccinated populations. In addition, vaccine strains have been found in both vaccinated infants and their contacts and there is evidence for genetic interaction between vaccine and wild-type strains. In conclusion, the post-vaccine introduction strain prevalence data do not show any consistent pattern indicative of selection pressure resulting from vaccine use, although the increased detection rate of heterotypic G2P[4] strains in some countries following RV1 vaccination is unusual and this issue requires further monitoring.

One year survey of human rotavirus strains suggests the emergence of genotype G12 in Cameroon.

In this study the emergence of rotavirus A genotype G12 in children <5 years of age is reported from Cameroon during 2010/2011. A total of 135 human stool samples were P and G genotyped by reverse transcriptase PCR. Six different rotavirus VP7 genotypes were detected, including G1, G2, G3, G8, G9, and G12 in combinations with P[4], P[6] and P[8] VP4 genotypes. Genotype G12 predominated in combination with P[8] (54.1%) and P[6] (10.4%) genotypes followed by G1P[6] (8.2%), G3P[6] (6.7%), G2P[4] (5.9%), G8P[6] (3.7%), G2P[6] (0.7%), G3P[8] (0.7%), and G9P[8] (0.7%). Genotype P[6] strains in combination with various G-types represented a substantial proportion (N=44, 32.6%) of the genotyped strains. Partially typed strains included G12P[NT] (2.2%); G3P[NT] (0.7%); G(NT)P[6] (1.5%); and G(NT)P[8] (0.7%). Mixed infections were found in five specimens (3.7%) in several combinations including G1+ G12P[6], G2+ G3P[6] + P[8], G3+ G8P[6], G3 + G12P[6] + P[8], and G12P[6] +P[8]. The approximately 10% relative frequency of G12P[6] strains detected in this study suggests that this strain is emerging in Cameroon and should be monitored carefully as rotavirus vaccine is implemented in this country, as it shares neither G- nor P-type specificity with strains in the RotaTeq® and Rotarix® vaccines. These findings are consistent with other recent reports of the global spread and increasing epidemiologic importance of G12 and P[6] strains.

Review of group A rotavirus strains reported in swine and cattle.

Group A rotavirus (RVA) infections cause severe economic losses in intensively reared livestock animals, particularly in herds of swine and cattle. RVA strains are antigenically heterogeneous, and are classified in multiple G and P types defined by the two outer capsid proteins, VP7 and VP4, respectively. This study summarizes published literature on the genetic and antigenic diversity of porcine and bovine RVA strains published over the last 3 decades. The single most prevalent genotype combination among porcine RVA strains was G5P[7], whereas the predominant genotype combination among bovine RVA strains was G6P[5], although spatiotemporal differences in RVA strain distribution were observed. These data provide important baseline data on epidemiologically important RVA strains in swine and cattle and may guide the development of more effective vaccines for veterinary use.

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.

Systematic review of regional and temporal trends in global rotavirus strain diversity in the pre rotavirus vaccine era: insights for understanding the impact of rotavirus vaccination programs.

Recently, two rotavirus vaccines have been recommended for routine immunization of infants worldwide. These vaccines proved efficacious during clinical trials and field use in both developing and developed countries, and appear to provide good protection against a range of rotavirus genotypes, including some that are not included in the vaccines. However, since conclusive data that the vaccines will protect against a wide variety of rotavirus strains are still lacking and since vaccines may exert some selection pressure, a detailed picture of global strain prevalence from the pre-rotavirus vaccine era is important to evaluate any potential changes in circulating strains observed after widespread introduction of rotavirus vaccines. Thus, we systematically reviewed rotavirus genotyping studies spanning a 12-year period from 1996 to 2007. In total, ~110,000 strains were genotyped from 100 reporting countries. Five genotypes (G1-G4, and G9) accounted for 88% of all strains, although extensive geographic and temporal differences were observed. For example, the prevalence of G1 strains declined from 2000 onward, while G3 strains re-emerged, and G9 and G12 strains emerged during the same period. When crude strain prevalence data were weighted by region based on the region's contribution to global rotavirus mortality, the importance of genotypes G1 and G9 strains that were more prevalent in regions with low mortality was reduced and conversely the importance of G8 strains that were more prevalent in African settings with greater contribution to global rotavirus mortality was increased. This study provides the most comprehensive, up-to-date information on rotavirus strain surveillance in the pre-rotavirus vaccine era and will provide useful background to examine the impact of rotavirus vaccine introduction on future strain prevalence.

[Post vaccination rotavirus surveillance in Hungary, in 2007]. / Posztvakcinációs rotavírus-surveillance Magyarországon, 2007.

Vaccination is the main strategy to control severe dehydrating gastroenteritis caused by rotaviruses in early childhood. The availability of new generation rotavirus vaccines has led to an intensification of strain surveillance worldwide, in part, to gauge the impact of the possible vaccine-driven immune selection of wild-type rotavirus strains. In the present study, authors describe the strain prevalence data obtained in 2007, with the involvement of different regions of Hungary. Genomic RNA was extracted from rotavirus-positive stool samples collected mainly from children and then subjected to genotyping using multiplex RT-PCR assay. Type-specific primers targeted G1 to G4, G6, G8 to G10, and G12 VP7 specificities, and P[4], P[6], and P[8] to P[11] VP4 specificities were used. Out of 489 rotavirus-positive specimens, collected from 482 patients, 466 and 474 were successfully G and P typed, respectively, and both G and P type specificities could be assigned for 457 strains. Prevalence data showed the predominance of G4P[8] (31.5%) strains, followed by G1P[8] (28.3%), G2P[4] (19.3%), and G9P[8] (10.2%). Minority strains were G1P[4] (0.4%), G2P[8] (1.3%), G3P[9] (0.2%), G4P[6] (0.7%), G6P[9] (0.4%), G8P[8] (0.2%), G9P[4] (0.2%), G9P[6] (0.8%), and G12P[8] (0.4%). Mixed infections were found in 1.2% of the samples, while 4.9% remained partially or fully non-typified. Our data indicate that the antigen specificities of medically important rotavirus strains identified in this 1-year study are well represented in the vaccines available in the pharmaceutical private market in Hungary. Depending on the vaccination coverage achievable in the forthcoming years, the post-vaccination rotavirus strain surveillance may allow us to gain comprehensive information on the impact of rotavirus vaccines on the prevalence of circulating rotavirus strains.

First detection of P[6],G9 rotaviruses in Hungary--an imported strain from India?

EuroRotaNet was launched to monitor rotavirus strain prevalence during and after introduction of rotavirus vaccines in Europe. In early 2007, we detected P[6],G9 rotaviruses to appear in Hungary, representing the first documented occurrence of this strain in our surveillance area. Epidemiologic data suggested that this strain was introduced from India.

Lineage-specific silencing of human IL-10 gene expression by promoter methylation in cervical cancer cells.

Epigenetic analysis was performed to demonstrate that the normal and neoplastic epithelial cells do not serve as the source of the locally elevated IL-10 production during cervical carcinogenesis. Bisulfite sequencing was used to correlate promoter CpG methylation with the transcription of the gene. Lack of IL-10 transcription in HeLa, SiHa, Caski, HT-3, C33-A, HaCaT cell lines and in primary human keratinocytes correlated consistently with the methylated state of the proximal CpG residues, particularly with the two most proximal CpGs at positions -185 and -110. These two sites were also highly methylated in normal and malignant cervical cells directly isolated from patient material. On the other hand, IL-10 producing peripheral blood mononuclear cells had unmethylated CpG residues in the proximal promoter associated with acetylated H3 and H4 histones as determined by chromatin immunoprecipitation. In HeLa carrying epigenetically silenced endogeneous IL-10 promoter, the transfected non-CpG methylated 1 kb and 0.6 kb proximal promoter fragments could drive reporter gene expression, which was reversed by cassette methylation of these promoter fragments. In conclusion, the CpG methylation pattern of the proximal promoter is implicated as a major determinant of transcriptional silencing of human IL-10 expression in cells of cervical epithelial origin.

[EuroRotaNet--European rotavirus strain surveillance network established with Hungarian participation]. / Hazai részvétellel megalakult az Európai Rotavírus-törzs-figyelô Hálózat (EuroRotaNet).

Group A rotaviruses are the most common cause of severe gastroenteritis worldwide. The incidence and distribution of group A rotavirus sero/genotypes varies between geographical areas during a rotavirus season, and from one season to the next. In addition, cocirculation of genetically diverse multitypic rotaviruses and of intratypic variants in any one place and time is common. Assuming widespread use of rotavirus vaccine in the near future, comprehensive surveillance of natural rotavirus infections is vital. EuroRotaNet has been established in order to gather comprehensive information on the rotavirus types co-circulating throughout Europe. The main objectives of the network are to (i) develop methods and algorithms for effective rotavirus strain typing and characterisation, (ii) describe in detail the molecular epidemiology of rotavirus infections in Europe, (iii) monitor the effectiveness of current genotyping methods and respond to changes associated with genetic drift and shift, and (iv) monitor the emergence and spread of novel rotavirus strains within Europe. This infrastructure may serve as a platform for future surveillance activities and nested studies for evaluating the effectiveness of a rotavirus vaccine in the general population. Studies to monitor the reduction in disease associated with common rotavirus types, the possible vaccine-induced emergence of antibody escape mutants of genotypes other than those included in the vaccine and of reassortment between vaccine and naturally circulating wildtype strains are required.