Recombinant herpes simplex virus type 1 strains with targeted mutations relevant for aciclovir susceptibility

Abstract

Here, we describe a novel reliable method to assess the significance of individual mutations within the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) to nucleoside analogue resistance. Eleven defined single nucleotide polymorphisms that occur in the TK gene of clinical HSV-1 isolates and a fluorescence reporter were introduced into the HSV-1 strain 17 + that had been cloned into a bacterial artificial chromosome. The susceptibility of these different strains to aciclovir, penciclovir, brivudin, and foscarnet was determined with a modified cytopathic effect reduction assay. The strains were also tested for their aciclovir susceptibility by measuring the relative fluorescence intensity as an indicator for HSV-1 replication and by quantifying the virus yield. Our data indicate that the amino acid substitutions R41H, R106H, A118V, L139V, K219T, S276R, L298R, S345P, and V348I represent natural polymorphisms of the TK protein, whereas G61A and P84L mediate broad cross-resistance against aciclovir, penciclovir, brivudin, and susceptibility to foscarnet. This method allows the definition of the resistance genotype of otherwise unclear mutations in the TK gene of HSV-1. Thus, it provides a scientific basis for antiviral testing in clinical isolates of patients suffering from serious diseases and will facilitate testing of new antivirals against HSV-1.Herpes simplex virus type 1 (HSV-1) belongs to the genus Simplexvirus of the Alphaherpesvirinae, a subfamily within the Herpesviridae 1 . With its high seroprevalence ranging in Europe from 50% to 90%, this human pathogen is of great medical importance 2 . After onset of primary infection, HSV-1 establishes a lifelong latency followed by endogenous reactivations 2 .Severe HSV-1 infections are mainly observed in immunosuppressed patients 3 who suffer from extensive, disseminated, or persistent herpetic lesions but also from more serious complications such as pneumonia 4,5 , encephalitis 6,7 , or hepatitis 8,9 . Thus, abundant and prolonged antiviral treatment is often inevitable but promotes the selection of resistant HSV-1 variants 3 . Approximately 2% to 10% of the immunosuppressed patients develop an infection or reactivation with HSV-1 variants resistant against antivirals in clinical use 10,11 . The highest rates of up to 45% have been described in patients who have received stem-cell transplantation 12 . So far, aciclovir (ACV) remains the drug of choice for prophylaxis and treatment of HSV infections 3 . Resistance-associated substitutions are predominantly observed within the viral thymidine kinase (TK) that otherwise converts the guanosine analogue to its active monophosphate form 13 . The incorporation of ACV results in DNA chain termination due to the missing hydroxyl group on the acyclic molecule. Accordingly, alterations of the TK-encoding gene UL23 are reported in 95% of clinical isolates resistant to ACV 14,15 . The high degree of UL23 sequence variability includes nucleotide deletions or insertions, which usually result in frame shifts or s...

Identification and Pathogenic Potential of Orofacial Herpetic Clinical Isolates in Northeast Mexico

“…As we know these variations can also affect the TK gene, and are related to its resistance to acyclovir [16,17]. The identification of new HSV-1strains and the obtaining of the sequences of their genomes will be useful for the development of effective molecular diagnostic tools that allow to solve the obtaining of erroneous results [9,16].…”

Relative Contributions of the cGAS-STING and TLR3 Signaling Pathways to Attenuation of Herpes Simplex Virus 1 Replication

“…Herpes simplex virus 1 (HSV-1) is a DNA virus that belongs to the family Herpesviridae (16,17). HSV-1 causes a range of diseases, including herpes keratitis, herpes labialis, and herpes encephalitis, which can be fatal (18).…”

“…HSV-1 causes a range of diseases, including herpes keratitis, herpes labialis, and herpes encephalitis, which can be fatal (18). HSV-1 establishes latency in the peripheral nervous system, hence causing lifelong infections, and is highly prevalent in both North America and worldwide (16). Like other viral infections, HSV-1 triggers more than one signaling pathway to induce type I IFNs and other cytokines (4).…”

Input of recombinant phenotyping for the characterization of a novel acyclovir-resistance mutation identified in a patient with recurrent herpetic keratitis

“…However, the roles of individual mutations in HSV drug resistance have to be formally assessed by recombinant phenotyping [7]. The use of BACcloned genomes of cytomegalovirus (CMV), varicella-zoster virus (VZV), and HSV constitutes the gold-standard strategy to assess the formal role of unknown mutations potentially implicated in drug resistance (Boutolleau D et al, 2017;Brunnemann et al, 2016Brunnemann et al, , 2015Lurain and Chou, 2010). Classically, each mutation of interest is generated by site-directed mutagenesis into a wild-type gene, previously cloned into a plasmid, from a susceptible viral reference strain.…”