Human cytomegalovirus reactivation from latency: validation of a 'switch' model in vitro
| Autor: | Carlo Chezzi, Rosita Vasile Simone, Flora De Conto, Clara Maccari, Maria Cristina Arcangeletti, Adriana Calderaro, M.C. Medici, Isabella Rodighiero |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Human cytomegalovirus viruses Cytomegalovirus Biology Real-Time Polymerase Chain Reaction Models Biological Virus Monocytes Cell Line 03 medical and health sciences 0302 clinical medicine Latent Virus Virology medicine Humans THP-1 monocytes Latency (engineering) HCMV Reverse Transcriptase Polymerase Chain Reaction Research Virus Activation medicine.disease Reactivation 030104 developmental biology Infectious Diseases Real-time polymerase chain reaction Lytic cycle Cell culture 030220 oncology & carcinogenesis DNA Viral Latency RNA Viral THP-1 differentiation |
| Zdroj: | Virology Journal |
| ISSN: | 1743-422X |
| Popis: | Background Human cytomegalovirus (HCMV) is an opportunistic pathogen leading to severe and even fatal diseases in ‘at-risk’ categories of individuals upon primary infection or the symptomatic reactivation of the endogenous virus. The mechanisms which make the virus able to reactivate from latency are still matter of intense study. However, the very low number of peripheral blood monocytes (an important latent virus reservoir) harbouring HCMV DNA makes it very difficult to obtain adequate viral quantities to use in such studies. Thus, the aim of the present study was to demonstrate the usefulness of human THP-1 monocytes, mostly employed as HCMV latent or lytic infection system, as a reactivation model. Methods THP-1 monocytes were infected with HCMV TB40E strain (latency model) at multiplicities of infection (MOI) of 0.5, 0.25 or 0.125. After infection, THP-1 aliquots were differentiated into macrophages (reactivation model). Infections were carried out for 30 h, 4, 6 and 7 days. Viral DNA evaluation was performed with viable and UV-inactivated virus by q-Real-Time PCR. RNA extracted from latency and reactivation models at 7 days post-infection (p.i.) was subjected to RT-PCR to analyse viral latency and lytic transcripts. To perform viral progeny analysis and titration, the culture medium from infected THP-1 latency and reactivation models (7 days p.i.) was used to infect human fibroblasts; it was also checked for the presence of exosomes. For viral progeny analysis experiments, the Towne strain was also used. Results Our results showed that, while comparable TB40E DNA amounts were present in both latent and reactivation models at 30 h p.i., gradually increased quantities of viral DNA were only evident in the latter model at 4, 6, 7 days p.i.. The completion of the lytic cycle upon reactivation was also proved by the presence of HCMV lytic transcripts and an infectious viral yield at 7 days p.i. Conclusions Our data demonstrate the effectiveness of THP-1 cells as a “switch” model for studying the mechanisms that regulate HCMV reactivation from latency. This system is able to provide adequate quantities of cells harbouring latent/reactivated virus, thereby overcoming the intrinsic difficulties connected to the ex vivo system. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0634-z) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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