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Molecular studies on HSV: replication rate, infection capacity and progeny

2016-09Conference object Restricted access
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dc.contributor.authorAzevedo, Aleksandra
dc.contributor.authorNunes, Alexandra
dc.contributor.authorRoque, Carla
dc.contributor.authorCosta, Inês
dc.contributor.authorGomes, João Paulo
dc.contributor.authorLopo, Sílvia
dc.date.accessioned2017-03-15T11:51:48Z
dc.date.available2017-03-15T11:51:48Z
dc.date.issued2016-09
dc.description.abstractIntroduction: In the last years genital herpes has emerged as one of the most prevalent sexually transmitted infections. Herpes simplex virus (HSV) is the most common cause of genital ulcer disease, with infections caused by both sub-types HSV-1 and HSV-2. A better understanding of the virus replication cycle is relevant to the pathogenesis of human diseases and is essential for the development of antiviral chemotherapy. Objectives: We aimed to shed some light on the HSV-1 and HSV-2 infectious cycle, namely their capacity of infection, replication rate and progeny, in three distinct cell lines (Vero, Vero E6 and HeLa229). We also aimed to evaluate whether the concentration of virus has any influence on the degree of the infection. Methodology: Preliminary assays were performed in order to understand which cellular concentration, viral load, nutrients’ availability and inoculation modus operandi (centrifugation versus agitation) best mimic the HSV infection. Confluent cell monolayers were infected with two HSV-2 and two HSV-1 at MOIs of 1:10, 1:1, 10:1 and 100:1. Inoculations were performed in parallel in two 24-well plates, one for quantitative real-time PCR (kPCR) and one for immunofluorescence assays, which were incubated for 30 hours at 37ºC and 5% CO2. At different times-points of infection (6, 12, 18, 24 and 30 hours p.i.), the wells were scratched for kPCR and the slides were stained with monoclonal antibodies. For kPCR assays, appropriate standard curves were generated by serial diluting plasmids cloned with HSV-1 and HSV-2 single copy genes. Results and Conclusions: Preliminary assays showed that, regardless of the viral load, it takes approximately 23 hours for the virus to complete the infectious cycle taking into account that no replication is observed after this time point. Considering the comparison between the two inoculation procedures (centrifugation versus agitation), we only observed relevant differences for lower viral loads, with centrifugation yielding more viral progeny. More specific data regarding both the HSV-1 and HSV-2 replication capacity for different MOIs are currently under evaluation.pt_PT
dc.description.versionN/Apt_PT
dc.identifier.urihttp://hdl.handle.net/10400.18/4661
dc.language.isoengpt_PT
dc.peerreviewednopt_PT
dc.publisherInstituto Nacional de Saúde Doutor Ricardo Jorge, IPpt_PT
dc.subjectHSV1pt_PT
dc.subjectHSV2pt_PT
dc.subjectHerpes Ssimplex Viruspt_PT
dc.subjectSexually Transmitted Diseasespt_PT
dc.subjectReplication Ratept_PT
dc.subjectInfection Capacitypt_PT
dc.subjectProgenypt_PT
dc.subjectInfecções Sexualmente Transmissíveispt_PT
dc.titleMolecular studies on HSV: replication rate, infection capacity and progenypt_PT
dc.typeconference object
dspace.entity.typePublication
oaire.citation.conferencePlaceLisboa, Portugalpt_PT
oaire.citation.title19th Annual Meeting of the European Society for Clinical Virology (ESCV), 14-17 September 2016pt_PT
rcaap.rightsclosedAccesspt_PT
rcaap.typeconferenceObjectpt_PT

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