Ticks, being vectors for a variety of pathogens such as tick-borne encephalitis virus (TBEV), have developed defense mechanisms and pathways against infections, allowing them to control the virus at a level that does not hinder their fitness and development. At the present moment, only a few studies focused on interactions between ticks and TBEV on a molecular level have been published. Here, a possible application of MALDI-TOF MS as a research tool for the investigation of tick-virus interactions was shown. Mass spectrometry (MS) profiles of TBEV-infected and non-infected IRE/CTVM19 tick cell line were compared using principal component analysis. MS spectra were clustered based on the cultivation time of cells, but not their infection status. Nevertheless, the analysis of loading plots revealed different factors (peaks) being involved in the clustering of infected and non-infected cells. Out of them, nine were assigned with proteins: five and four for non-infected and infected cells, respectively. Peak with m/z 8565 was found to be of interest because it was suppressed upon TBEV infection and assigned to proteasome subunit alpha type (B7QE67).
- Klíčová slova
- Biotyping, MALDI-TOF MS, TBEV, Tick cell line, Tick-virus interaction,
- MeSH
- buněčné linie virologie MeSH
- klíště virologie MeSH
- viry klíšťové encefalitidy fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) latency represents the major barrier to virus eradication in infected individuals because cells harboring latent HIV-1 provirus are not affected by current antiretroviral therapy (ART). We previously demonstrated that DNA methylation of HIV-1 long terminal repeat (5' LTR) restricts HIV-1 reactivation and, together with chromatin conformation, represents an important mechanism of HIV-1 latency maintenance. Here, we explored the new issue of temporal development of DNA methylation in latent HIV-1 5' LTR. RESULTS: In the Jurkat CD4(+) T cell model of latency, we showed that the stimulation of host cells contributed to de novo DNA methylation of the latent HIV-1 5' LTR sequences. Consecutive stimulations of model CD4(+) T cell line with TNF-α and PMA or with SAHA contributed to the progressive accumulation of 5' LTR DNA methylation. Further, we showed that once established, the high DNA methylation level of the latent 5' LTR in the cell line model was a stable epigenetic mark. Finally, we explored the development of 5' LTR DNA methylation in the latent reservoir of HIV-1-infected individuals who were treated with ART. We detected low levels of 5' LTR DNA methylation in the resting CD4(+) T cells of the group of patients who were treated for up to 3 years. However, after long-term ART, we observed an accumulation of 5' LTR DNA methylation in the latent reservoir. Importantly, within the latent reservoir of some long-term-treated individuals, we uncovered populations of proviral molecules with a high density of 5' LTR CpG methylation. CONCLUSIONS: Our data showed the presence of 5' LTR DNA methylation in the long-term reservoir of HIV-1-infected individuals and implied that the transient stimulation of cells harboring latent proviruses may contribute, at least in part, to the methylation of the HIV-1 promoter.
- Klíčová slova
- Chromatin conformation, DNA methylation, HIV-1, HIV-1-infected individuals, Latent HIV-1 provirus reactivation, Latent reservoir,
- MeSH
- buněčné linie virologie MeSH
- časové faktory MeSH
- HIV - dlouhá koncová repetice genetika MeSH
- HIV infekce farmakoterapie genetika virologie MeSH
- HIV-1 genetika MeSH
- Jurkat buňky virologie MeSH
- latence viru genetika fyziologie MeSH
- látky proti HIV terapeutické užití MeSH
- lidé MeSH
- metylace DNA * MeSH
- proviry genetika fyziologie MeSH
- vysoce aktivní antiretrovirová terapie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- látky proti HIV MeSH
A survey was performed on ornamental fish imported into the EU to detect viral agents belonging to the genus Ranavirus. The objective was to gain knowledge of the potential for these systemic iridoviruses to gain entry into the EU via international trade in ornamental fish. A total of 208 pooled samples, representing 753 individual fish, were tested. The samples included 13 orders and 37 families, originating from different countries and continents. Tissues from fish that died during or just after transport were collected and examined by standard virological techniques in epithelioma papulosum cyprini cells, by transmission electron microscopy and by PCR for the detection of the major capsid protein and DNA polymerase gene sequences of ranaviruses. Virus was isolated from nine fish species but ranavirus was not identified in those samples. The results suggest that ranaviruses are not highly prevalent in ornamental fish imported into the EU.
- MeSH
- buněčné linie virologie MeSH
- DNA-dependentní DNA-polymerasy analýza genetika MeSH
- Evropská unie MeSH
- fylogeneze MeSH
- infekce DNA virem genetika veterinární MeSH
- karcinom virologie MeSH
- nemoci ryb virologie MeSH
- polymerázová řetězová reakce MeSH
- Ranavirus klasifikace enzymologie genetika ultrastruktura MeSH
- ryby virologie MeSH
- transmisní elektronová mikroskopie MeSH
- virové plášťové proteiny analýza genetika MeSH
- virové proteiny analýza genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA-dependentní DNA-polymerasy MeSH
- virové plášťové proteiny MeSH
- virové proteiny MeSH
CpG islands are important in the protection of adjacent housekeeping genes from de novo DNA methylation and for keeping them in a transcriptionally active state. However, little is known about their capacity to protect heterologous genes and assure position-independent transcription of adjacent transgenes or retroviral vectors. To tackle this question, we have used the mouse aprt CpG island to flank a Rous sarcoma virus (RSV)-derived reporter vector and followed the transcriptional activity of integrated vectors. RSV is an avian retrovirus which does not replicate in mammalian cells because of several blocks at all levels of the replication cycle. Here we show that our RSV-derived reporter proviruses linked to the mouse aprt gene CpG island remain undermethylated and keep their transcriptional activity after stable transfection into both avian and nonpermissive mammalian cells. This effect is most likely caused by the protection from de novo methylation provided by the CpG island and not by enhancement of the promoter strength. Our results are consistent with previous finding of CpG islands in proximity to active but not inactive proviruses and support further investigation of the protection of the gene transfer vectors from DNA methylation.
- MeSH
- adeninfosforibosyltransferasa genetika MeSH
- buněčné linie virologie MeSH
- CpG ostrůvky * MeSH
- defektní viry genetika MeSH
- DNA virů chemie genetika MeSH
- DNA-(cytosin-5-)methyltransferasa metabolismus MeSH
- experimentální sarkom genetika virologie MeSH
- fibroblasty virologie MeSH
- genetická transkripce * MeSH
- genetické vektory genetika fyziologie MeSH
- integrace viru MeSH
- koncové repetice MeSH
- křečci praví MeSH
- křeček rodu Mesocricetus MeSH
- kuřecí embryo MeSH
- metylace DNA MeSH
- myši MeSH
- proviry genetika MeSH
- regulace exprese virových genů * MeSH
- replikace viru MeSH
- reportérové geny MeSH
- umlčování genů * MeSH
- viry ptačího sarkomu genetika fyziologie MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- kuřecí embryo MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- adeninfosforibosyltransferasa MeSH
- DNA virů MeSH
- DNA-(cytosin-5-)methyltransferasa MeSH
Continuous cell lines offer a level of reproducibility, and thus standardization, which cannot normally be achieved using primary cells. However, even with continuous cell lines adoption of correct cell banking and appropriate quality control procedures are critical to the provision of reliable, reproducible and safe cell stocks. These procedures enable establishment of cryopreserved stocks of pure cultures of correct identity and phenotype which are free from adventitious agents. In addition to quality control techniques, culture conditions and growth medium used often require standardization. In particular different sources of serum, growth factors and cell attachment substrates may lead to significant variation in the 'performance' of cell lines. To ensure a high degree of reliability it is essential to obtain cells from authenticated and quality controlled sources. In culture collections, the principles of correct cell banking should be applied with appropriate quality control for which the minimum standard should be confirmation of viability and mycoplasma testing. Such approaches will afford increased confidence in research data and avoid the waste of time and resources which result from the use of cross-contaminated or infected cells.
- MeSH
- banky biologického materiálu normy MeSH
- buněčné kultury normy MeSH
- buněčné linie mikrobiologie virologie MeSH
- kryoprezervace MeSH
- řízení kvality MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The effect of influenza strains A (H3N2) and B, isolated during the seasons of 1994 and 1995 in the Czech Republic, on MDCK cells was studied. Various concentrations of virus and conditions of nutrition were used during the cell culture. The virus replication and consequently fragmentation of genomic DNA together with cytotoxicity were investigated in the absence and presence of 10 per cent calf serum. Virus replication, regardless of type A or B, caused earlier DNA fragmentation in comparison to non-infected cells in tissue culture. The results showed that the influenza B strain had a greater cytotoxic effect on MDCK cells than influenza A. A higher infection dose of influenza A virus accelerated the onset of apoptosis; conversely, a higher infection dose of influenza B virus delayed the onset of apoptosis. The absence of serum enhanced the progress of influenza-induced apoptosis in conditions in vitro.
- MeSH
- buněčná smrt fyziologie MeSH
- buněčné linie virologie MeSH
- fragmentace DNA účinky léků fyziologie MeSH
- hemaglutininy farmakologie MeSH
- infekce viry z čeledi Orthomyxoviridae patofyziologie MeSH
- kultivační média bez séra farmakologie MeSH
- kultivační média farmakologie MeSH
- Orthomyxoviridae fyziologie MeSH
- virus chřipky A fyziologie MeSH
- virus chřipky B fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- hemaglutininy MeSH
- kultivační média bez séra MeSH
- kultivační média MeSH
