A novel bioluminescent herpes simplex virus 1 for in vivo monitoring of herpes simplex encephalitis

Olus Uyar,Jacques Corbeil,Pier-Luc Plante,Guy Boivin,Marie-Christine Venable,Jocelyne Piret,Julie Carbonneau

doi:10.1038/s41598-021-98047-z

Abstract

Herpes simplex virus 1 (HSV-1) is responsible for herpes simplex virus encephalitis (HSE), associated with a 70% mortality rate in the absence of treatment. Despite intravenous treatment with acyclovir, mortality remains significant, highlighting the need for new anti-herpetic agents. Herein, we describe a novel neurovirulent recombinant HSV-1 (rHSV-1), expressing the fluorescent tdTomato and Gaussia luciferase (Gluc) enzyme, generated by the Clustered regularly interspaced short palindromic repeats (CRISPR)—CRISPR-associated protein 9 (Cas9) (CRISPR-Cas9) system. The Gluc activity measured in the cell culture supernatant was correlated (P = 0.0001) with infectious particles, allowing in vitro monitoring of viral replication kinetics. A significant correlation was also found between brain viral titers and Gluc activity in plasma (R2 = 0.8510, P < 0.0001) collected from BALB/c mice infected intranasally with rHSV-1. Furthermore, evaluation of valacyclovir (VACV) treatment of HSE could also be performed by analyzing Gluc activity in mouse plasma samples. Finally, it was also possible to study rHSV-1 dissemination and additionally to estimate brain viral titers by in vivo imaging system (IVIS). The new rHSV-1 with reporter proteins is not only as a powerful tool for in vitro and in vivo antiviral screening, but can also be used for studying different aspects of HSE pathogenesis.

Highlights

Introduction of reporter genes intoUL26‐UL27 intergenic region did not alter in vitro viral replicative capacity.The replicative capacity of the WT and recombinant Herpes simplex virus 1 (HSV-1) (rHSV-1) strains was evaluated in Vero cells by real-time cell analysis (RTCA) and confirmed by plaque assay
The transfection/infection approach was performed to generate rHSV-1 expressing Gaussia luciferase (Gluc) and tdTomato reporter proteins, using the Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas[9] s ystem[13]. This method consists of transfecting host cells with a mixture of CRISPR-CRISPR-associated protein 9 (Cas9)/guide RNA (gRNA) and the donor plasmid prior to their infection with the virus to modify (Fig. 1, two workflows: (i) gRNA selection and (ii) rHSV-1 production and validation)
We found that the viral replicative capacity of rHSV-1 remained unchanged compared to WT HSV-1 for both MOIs (Fig. 4a,b)

Summary

Introduction

UL26‐UL27 intergenic region did not alter in vitro viral replicative capacity. The replicative capacity of the WT and rHSV-1 strains was evaluated in Vero cells by real-time cell analysis (RTCA) and confirmed by plaque assay. Two different multiplicity of infection (MOIs) of 0.01 and 0.001 were used to infect cells, and the cell index (CI) was recorded for 4 days. We found that the viral replicative capacity of rHSV-1 remained unchanged compared to WT HSV-1 for both MOIs (Fig. 4a,b). We confirmed our data by comparing the replication capabilities of WT and rHSV-1 by plaque assay. We observed similar trends in replication kinetics (Fig. 4c) of both viruses, suggesting that neither the large foreign genes in the intergenic region of UL26-UL27 nor previously described SNPs in the rHSV-1 genome seem to have a significant impact on in vitro replication properties

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