Quantitation of viruses is practised widely in both basic and applied virology. Infectious titration in cell cultures, the most common approach to it, is quite labour-intensive and alternative protocols are therefore sought. One of the alternatives is transmission electron microscope (TEM) quantitation using latex particles at a known concentration as a reference for counting virus particles. If virus TCID₅₀ is determined in parallel, the ratio of infectious to non-infectious virus particles may be established. This study employs such an approach to compute the number of virus particles and TCID₅₀, and establish their correlation for three viruses: Canine adenovirus 1 (CAdV-1), Feline calicivirus (FCV) and Bovine herpesvirus 1 (BoHV-1). Each of the viruses was grown in five replicates until complete cytopathology was recorded (time 0), then frozen. They were thawed, filter-sterilised and left for additional periods of 16, 32 and 48 h at 37°C. At each time point, the infectious ability of the virus was characterised by TCID50 and the number of virions quantified by TEM, in order to evaluate the influence of timing on virus harvest. The virus particle count determined by TEM did not change for any of the viruses throughout the experiment. The relationship between virus particle counts with TCID₅₀ at time 0 showed good linearity response; their ratio was almost constant. The virus particle-to-TCID₅₀ ratio varied between 146 and 426 (mean±SD: 282±103) for CAdV-1, between 36 and 79 (57±18) for FCV and between 110 and 249 (167±53) for BoHV-1. The proportion of non-infectious particles did not change throughout the experiment for either CAdV-1 or BoHV-1. However, a decrease in virus infectious ability disclosed by TCID₅₀ indicated that the fraction of non-infectious particles in FCV increased 300,000 times when time 0 and 48 h were compared. The quantitation of viruses with TEM is a simple and rapid protocol for virus quantitation but account must be taken of the type of virus and harvesting time as virus counts need not necessarily correlate with virus infectious ability.
- MeSH
- bovinní herpesvirus 1 izolace a purifikace fyziologie MeSH
- buněčné linie MeSH
- časové faktory MeSH
- kočičí kalicivirus izolace a purifikace fyziologie MeSH
- kultivace virů MeSH
- mikrobiální viabilita * MeSH
- psí adenoviry izolace a purifikace fyziologie MeSH
- transmisní elektronová mikroskopie metody MeSH
- virová nálož metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We designed and synthesised a series of new cationic lipids based on spermine linked to various hydrophobic anchors. These lipids could be potentially useful for the preparation of stable cationic liposomes intended for the construction of drug targeting systems applicable in the field of anticancer/antiviral therapy, vaccine carriers, and vectors for the gene therapy. Low in vitro toxicity was found for these compounds, especially for LD1, in several cell lines. The delivery of both a fluorescence marker (calcein) and antiviral drugs into cells has been achieved owing to a large extent of internalization of cationic liposomes (labelled by Lyssamine-Rhodamine PE or fluorescein-PE) as demonstrated by fluorescent microscopy and quantified by flow cytometry. The bovine herpes virus type 1 (BHV-1) virus infection in vitro model using MDBK cells was employed to study the effect of the established antiviral drug HPMPC (Cidofovir®) developed by Prof. A. Holý. Inhibition of BHV-1 virus replication was studied by quantitative RT-PCR and confirmed by both Hoffman modulation contrast microscopy and transmission electron microscopy. We found that in vitro antiviral activity of HPMPC was significantly improved by formulation in cationic liposomes, which decreased the viral replication by about 2 orders of magnitude.
- MeSH
- antivirové látky aplikace a dávkování farmakologie MeSH
- bovinní herpesvirus 1 účinky léků fyziologie MeSH
- buněčné kultury MeSH
- buněčné linie MeSH
- cytopatogenní efekt virový MeSH
- cytosin aplikace a dávkování analogy a deriváty farmakologie MeSH
- fluorescenční mikroskopie MeSH
- kationty MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- ledviny cytologie virologie MeSH
- lipidy chemie MeSH
- liposomy MeSH
- nosiče léků chemie MeSH
- organofosfonáty aplikace a dávkování farmakologie MeSH
- replikace viru účinky léků MeSH
- skot MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- bovinní herpesvirus 1 imunologie MeSH
- protilátky virové analýza MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Geografické názvy
- Česká republika MeSH
