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.

Human cytomegalovirus reactivation from latency: Validation of a "switch" model in vitro / Arcangeletti, Maria Cristina; Vasile Simone, Rosita; Rodighiero, Isabella; De Conto, Flora; Medici, Maria Cristina; Maccari, Clara; Chezzi, Carlo; Calderaro, Adriana. - In: VIROLOGY JOURNAL. - ISSN 1743-422X. - 13:1(2016), p. 179. [10.1186/s12985-016-0634-z]

Human cytomegalovirus reactivation from latency: Validation of a "switch" model in vitro

ARCANGELETTI, Maria Cristina;VASILE SIMONE, Rosita;RODIGHIERO, Isabella;DE CONTO, Flora;MEDICI, Maria Cristina;MACCARI, CLARA;CHEZZI, Carlo;CALDERARO, Adriana
2016

Abstract

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.
Human cytomegalovirus reactivation from latency: Validation of a "switch" model in vitro / Arcangeletti, Maria Cristina; Vasile Simone, Rosita; Rodighiero, Isabella; De Conto, Flora; Medici, Maria Cristina; Maccari, Clara; Chezzi, Carlo; Calderaro, Adriana. - In: VIROLOGY JOURNAL. - ISSN 1743-422X. - 13:1(2016), p. 179. [10.1186/s12985-016-0634-z]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11381/2818885
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