Susceptibility Of Herpes Simplex Virus Research Articles

Rare Genetic Variants in Immune Genes and Neonatal Herpes Simplex Viral Infections.

Neonatal herpes simplex virus (HSV) infection is a devastating disease with high mortality, particularly when disseminated. Studies in adults and children suggest that susceptibility to herpes simplex encephalitis (HSE) may represent phenotypes for inborn errors in toll-like receptor 3 (TLR3) signaling. However, the genetic basis of susceptibility to neonatal HSV including disseminated disease remains unknown. To test the hypothesis that variants in known HSE-susceptible genes as well as genes mediating HSV immunity will be identified in neonatal HSV, we performed an unbiased exome sequencing study in 10 newborns with disseminated, HSE, and skin, eyes, and mouth disease. Determination of potential impact on function was determined by following American College of Medical Genetics and Genomics guidelines. We identified deleterious and potentially deleterious, rare variants in known HSE-related genes including a stop IRF3 variant (disseminated), nonsynonymous variants in TLR3 and TRAF3 (HSE), STAT1 (skin, eyes, and mouth), and DBR1 (disseminated) in our cohort. Novel and rare variants in other immunodeficiency genes or HSV-related immune genes GRB2, RAG2, PRF1, C6, C7, and MSR1 were found in 4 infants. The variant in GRB2, essential for T-lymphocyte cell responses to HSV, is a novel stop variant not found in public databases. In this pilot study, we identified deleterious or potentially deleterious variants in TLR3 pathway and genes that regulate anti-HSV immunity in neonates with HSV including disseminated disease. Larger, definitive studies incorporating functional analysis of genetic variants are required to validate these data and determine the role of immune genetic variants in neonatal HSV susceptibility.

Identification and characterization of acyclovir-resistant clinical HSV-1 isolates from children

Background The occurrence of herpes simplex virus (HSV) with acyclovir (ACV) resistance is a cause for concern due to the frequent use of ACV for treatment, suppressive therapy, and prophylaxis of HSV infection. Although HSV infection is prevalent among children, very little is known about the drug susceptibility of HSV circulating in this patient population. Objective To determine the status of ACV resistant HSV-1 among children. Study design A reporter cell-based HSV infection assay (mVILA) was developed to conveniently evaluate the ACV susceptibility of HSV-1 clinical strains and used to analyze 68 HSV-1 primary isolates from oral lesions in children. Results Compared with PRA, mVILA is easier to perform. Using mVILA, HSV-1 isolates C106, C153, and C174 were found completely resistant to ACV, with a greater than 100-fold increase in IC50s. Sequence analysis of thymidine kinase (TK) and DNA polymerase (DNA POL) genes identified 11 new mutations. Structural modeling of the TK and DNA POL proteins suggested structural changes that might alter their interactions with ACV and ACV triphosphate, respectively. The insertion of a single G in a seven-guanine homopolymeric repeat sequence generated a truncated TK protein in C106. Conclusion This study provides preliminary data on the ACV susceptibility status of HSV-1 in children. The prevalence rate of ACV-resistant HSV-1 in children was higher than predicted. Moreover, multiple mechanisms leading to the resistance were identified. These results suggest that new anti-herpetics with different working mechanisms should be valuable.

A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus

A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities.

Bispecific Adapter-Mediated Retargeting of a Receptor-Restricted HSV-1 Vector to CEA-Bearing Tumor Cells

The safety and efficacy of viral therapies for solid tumors can be enhanced by redirecting the virus infection to tumor-specific cell-surface markers. Successful retargeting of herpes simplex virus type 1 (HSV-1) has been achieved using vectors that carry a modified envelope glycoprotein D (gD) engineered to interact directly with novel receptors. In addition, soluble bridging molecules (adapters) have been used to link gD indirectly to cell-specific receptors. Here, we describe the development of an adapter connecting gD to the common tumor antigen carcinoembryonic antigen (CEA). The adapter consisted of a CEA-specific single-chain antibody fused to the gD-binding region of the gD receptor, herpes virus entry mediator (HVEM). We used this adapter in combination with a vector that is detargeted for recognition of the widely expressed gD receptor nectin-1, but retains an intact binding region for the less common HVEM. We show that the adapter enabled infection of HSV-resistant Chinese hamster ovary (CHO) cells expressing ectopic CEA and nectin-1/CEA-bearing human gastric carcinoma cells that are resistant to the vector alone. We observed cell-to-cell spread following adapter-mediated infection in vitro and reduced tumor growth in vivo, indicating that this method of vector retargeting may provide a novel strategy for tumor-specific delivery of tumoricidal HSV.

Development of Herpes Simplex Virus Stomatitis during Receipt of Cidofovir Therapy

We report 3 children who, after undergoing hematopoietic stem cell transplant, developed herpes simplex virus (HSV) stomatitis while receiving weekly cidofovir as preemptive treatment for cytomegalovirus infection. All patients responded well to treatment with either acyclovir or ganciclovir. Despite the in vitro susceptibility of HSV to cidofovir, once-weekly treatment with this agent may not be adequate prophylaxis in pediatric patients.

Frequency of acyclovir‐resistant herpes simplex viruses isolated from the general immunocompetent population and patients with acquired immunodeficiency syndrome

Herpes simplex virus (HSV) infections are usually chronically recurrent in the normal population and represent a significant cause of morbidity in immunocompromised patients. Acyclovir (ACV) is widely used for the treatment and prophylaxis of HSV infections. The emergence of ACV-resistant strains has been frequently reported as a result of long-term ACV therapy. Despite the widespread use of ACV, there are no data available in our area on the frequency of ACV-resistant HSVs. The purpose of this study was to evaluate the susceptibility of HSV isolated from normal subjects and patients with acquired immunodeficiency syndrome (AIDS) to ACV. HSVs were isolated from the orofacial region of normal individuals and patients with AIDS. The susceptibility of isolated HSV strains to various concentrations of ACV was determined by plaque reduction assay. The sensitivity of the viral strains was expressed as IC(50) (the concentration of drug reducing the viral plaque by 50%). One hundred and thirty-three isolates from 102 normal subjects and 31 patients with AIDS were tested. One HSV-1 isolate from normal individuals had intermediate susceptibility. Two ACV-resistant isolates (one HSV-1 and one HSV-2), with IC(50) > or = 2 to < or = 3 microg/mL, and one highly resistant HSV-2 isolate, with IC(50) > or = 5 microg/mL, were detected in patients with AIDS. Our data show that the prevalence of ACV-resistant strains is very low in the general immunocompetent population; however, in patients with AIDS, the prevalence of ACV-resistant strains is remarkable (P = 0.001). Alternative antiherpetic agents should be employed to control and reduce the emergence of ACV-resistant strains in patients with AIDS.

ACICLOVIR RESISTANCE AMONG INDIAN STRAINS OF HERPES SIMPLEX VIRUS AS DETERMINED USING A DYE UPTAKE ASSAY

Resistance to aciclovir (ACV) among Herpes simplex virus (HSV) isolates is increasingly being reported in the literature particularly in immunocompromised patients. However, there is only limited data available from India despite widespread use of ACV in our hospital. A cross-sectional study was hence conducted to determine the aciclovir (ACV) susceptibility of HSV 1 and 2 isolates using a dye uptake (DU) assay. This study showed a 3.0% prevalence of ACV resistance among HSV-1 strains (2/66, median IC500.098 µg/mL) while in HSV-2 strains, it was 7.8% (5/64, median IC500.195 µg/mL). The IC50 for the HSV-1 and HSV-2 strains resistant to ACV was greater than or equal to 6.25 µg/mL.

Aciclovir resistance among indian strains of &lt;i&gt;Herpes simplex&lt;/i&gt; virus as determined using a dye uptake assay

Resistance to aciclovir (ACV) among Herpes simplex virus (HSV) isolates is increasingly being reported in the literature particularly in immunocompromised patients. However, there is only limited data available from India despite widespread use of ACV in our hospital. A cross-sectional study was hence conducted to determine the aciclovir (ACV) susceptibility of HSV 1 and 2 isolates using a dye uptake (DU) assay. This study showed a 3.0% prevalence of ACV resistance among HSV-1 strains (2/66, median IC 50 0.098 microg/mL) while in HSV-2 strains, it was 7.8% (5/64, median IC 50 0.195 microg/mL). The IC 50 for the HSV-1 and HSV-2 strains resistant to ACV was greater than or equal to 6.25 microg/mL.

Factors that affect in vitro measurement of the susceptibility of herpes simplex virus to nucleoside analogues

Objectives To identify factors that contribute to variability of HSV antiviral susceptibility breakpoints. Methods Acyclovir and penciclovir IC 50's for 12 HSV clinical isolates were measured in two laboratories using plaque reduction assay (PRA), an enzyme immunoassay (EIA)-based antigen reduction, and DNA hybridization on Vero, A549, MRC-5, HEL299 and HELG monolayers. Pair-wise comparisons were performed to evaluate variables including testing laboratory, technique, monolayer, and antiviral. The proportion of false results was analyzed using a conventional susceptibility IC 50 breakpoint of 2 μg/ml. Results Acyclovir-resistant HSV isolates were correctly identified by all methods. In contrast, there were 6–67% of susceptible isolates incorrectly characterized as drug-resistant. Variables associated with these errors included testing site, assay method, cell line and antiviral. A549, DNA hybridization, and penciclovir were associated with the highest IC 50's, whereas the PRA, EIA, and human fibroblast-monolayers provided the best differentiation between susceptible and resistant HSV isolates. Conclusions The current recommendations to use a single discriminating value to define HSV resistance to nucleoside analogues can be problematic. False results are influenced in various degrees by the laboratory method, tissue culture and antivirals.

Rapid determination of antiviral drug susceptibility of herpes simplex virus types 1 and 2 by real-time PCR

An antiviral drug susceptibility assay of herpes simplex virus (HSV) was developed using real-time PCR quantification of intracellular viral DNA load. The number of HSV DNA copies within Vero cells after 24 h infection was strongly correlated with the number of plaques obtained after 72 h infection. Antiviral drug susceptibility of HSV was determined after virus growth for 24 h by measuring the reduction of intracellular HSV DNA in the presence of increasing concentrations of either acyclovir (ACV) or foscarnet (PFA). This assay required neither preliminary titration of infectious stock nor follow-up of cytopathic effect. The 50% inhibitory concentrations (IC 50s) obtained with 27 isolates of HSV types 1 and 2 by using this test were significantly correlated with those obtained in parallel with plaque reduction assay taken as the reference method ( r = 0.91, p < 0.0001 and r = 0.51, p = 0.009 for ACV and PFA, respectively). The threshold real-time PCR IC 50s for ACV and PFA resistance did not differ according to HSV type and were determined to be 1.0 and 100 μM, respectively. The real-time PCR susceptibility assay reported here is rapid, reproducible, applicable for HSV-1 as well as HSV-2, and suitable for automation.

Risk factors for herpes simplex virus transmission to pregnant women: A couples study

This study was undertaken to determine risk factors for herpes simplex virus (HSV) acquisition among at risk pregnant women. Women in a prospective study of HSV acquisition in pregnancy invited their sexual partners for HSV type-specific serologic testing. Risk factors for HSV susceptibility, exposure, and acquisition were examined. A total of 3192 couples enrolled; 22% included women at risk for HSV-1 or HSV-2. Among 582 HSV-1 seronegative women with HSV-1 seropositive partners, 14 (3.5% adjusted for gestation length) acquired HSV-1. Having a partner with a history of oral herpes was associated with HSV-1 acquisition (odds ratio [OR] 8.1, 95% CI: 1.8-36.0) and accounted for 75% of incident infections. Among 125 HSV-2 seronegative women with HSV-2 seropositive partners, 17 (20% adjusted for gestation length) acquired HSV-2. Duration of partnership of 1 year or less was associated with HSV-2 acquisition (OR 7.8, 95% CI: 2.3-25.7) and accounted for 63% of incident infections. No combination of clinical characteristics could identify the majority of susceptible women with serologically discordant partners. HSV acquisition rates in pregnancy are high in discordant couples, especially for HSV-2. Interventions that address risk factors for HSV acquisition should be studied in pregnancy. Clinical profiles cannot replace serologic screening to identify susceptible women with serologically discordant partners.

Comparative usage of herpesvirus entry mediator A and nectin-1 by laboratory strains and clinical isolates of herpes simplex virus

The herpesvirus entry mediator A (HVEM/HveA) and nectin-1 (HveC/CD111) are two major receptors for herpes simplex virus (HSV). Although structurally unrelated, both receptors can independently mediate entry of wild-type (wt) HSV-1 and HSV-2 by interacting with the viral envelope glycoprotein D (gD). Laboratory strains with defined mutations in gD (e.g. rid1) do not use HVEM but use nectin-2 (HveB/CD112) for entry. The relative usage of HVEM and nectin-1 during HSV infection in vivo is not known. In the absence of a defined in vivo model, we used in vitro approaches to address this question. First, we screened HSV clinical isolates from various origins for receptor tropism and found that all used both HVEM and nectin-1. Second, we determined the numbers of surface receptors on various susceptible and resistant cell lines as well as on primary fibroblasts derived from an individual with cleft lip/palate ectodermal dysplasia (CLPED1). Although CLPED1 cells can only express a defective form of nectin-1, they allowed entry of wild type and mutant HSV strains by usage of either HVEM or nectin-2. Finally, we compared the ability of HVEM and nectin-1 to mediate entry when expressed at varying cell surface densities. Both receptors showed a direct relationship between the number of receptors and HSV susceptibility. Direct comparison of receptors suggests that nectin-1 is more efficient at promoting entry than HVEM. Overall, our data suggest that both receptors play a role during HSV infection in vivo and that both are highly efficient even at low levels of expression.

Application of real-time PCR for determination of antiviral drug susceptibility of herpes simplex virus.

A quantitative real-time PCR (TaqMan) assay was developed for determination of antiviral drug susceptibility of herpes simplex virus (HSV). After short-time culture of the virus, the antiviral drug susceptibility of HSV isolates for acyclovir (ACV) was determined by measuring the reduction of the HSV type 1 (HSV-1) DNA levels in culture supernatants using real-time PCR. The 50% inhibitory concentration was reported as the concentration of antiviral drug that reduced the number of HSV-1 DNA copies by 50%. A total of 15 well-characterized ACV-sensitive or -resistant strains and clinical isolates were used for assay evaluation. The new assay with real-time PCR readout permitted rapid (3 days), objective, and reproducible determination of HSV-1 drug susceptibilities with no need for stringent control of initial multiplicity of infection. Furthermore, the real-time PCR assay results showed good correlation (r = 0.86) with those for the plaque reduction assay. In conclusion, the real-time PCR assay described here is a suitable quantitative method for determination of antiviral susceptibility of HSV-1, amenable for use in the routine diagnostic virology laboratory.

A screening dye-uptake assay to evaluate in vitro susceptibility of herpes simplex virus isolates to acyclovir

The widespread use of acyclovir (ACV) could increase the prevalence of herpes simplex virus (HSV) ACV-resistant isolates, and a screening assay are thus important for routine surveillance of the ACV susceptibility of HSV. A screening dye-uptake assay was developed, based on the conventional dye-uptake assay [J. Biol. Stand. 14 (1986) 201]. The susceptibility of HSV was measured by testing two virus dilutions (10 −1 and 10 −2) against two ACV concentrations (5 and 10 μM) on Vero cells and expressed as a reduced percentage of viral replication. The reproducibility was evaluated with HSV1 and HSV2 ACV-sensitive and ACV-resistant reference strains introduced as controls in successive series. The dye-uptake by Vero cells, the growth capacity of the HSV strains and the reduction of the viral replication in the presence of acyclovir varied by less than 14, 20 and 30%, respectively. This assay allowed the detection of a heterogenous population containing as few as 20% of ACV-resistant strain. The screening test was applied to 500 HSV isolates in a prospective study, and over 95% of the HSV isolates tested were characterised using a single test. This test appeared to be half the cost and much easier to carry out than the conventional dye-uptake assay, and consequently is well suited for large scale surveillance.

Highly reliable heterologous system for evaluating resistance of clinical herpes simplex virus isolates to nucleoside analogues.

Clinical resistance of herpes simplex virus (HSV) types 1 and 2 to acyclovir (ACV) is usually caused by the presence of point mutations within the coding region of the viral thymidine kinase (TK) gene. The distinction between viral TK mutations involved in ACV resistance or part of viral polymorphism can be difficult to evaluate with current methodologies based on transfection and homologous recombination. We have developed and validated a new heterologous system based on the expression of the viral TK gene by the protozoan parasite Leishmania, normally devoid of TK activity. The viral TK genes from 5 ACV-susceptible and 13 ACV-resistant clinical HSV isolates and from the reference strains MS2 (type 2) and KOS (type 1) were transfected as part of an episomal expression vector in Leishmania. The susceptibility of TK-recombinant parasites to ganciclovir (GCV), a closely related nucleoside analogue, was evaluated by a simple measurement of the absorbance of Leishmania cultures grown in the presence of the drug. Expression of the TK gene from ACV-susceptible clinical isolates resulted in Leishmania susceptibility to GCV, whereas expression of a TK gene with frameshift mutations or nucleotide substitutions from ACV-resistant isolates gave rise to parasites with high levels of GCV resistance. The expression of the HSV TK gene in Leishmania provides an easy, reliable, and sensitive assay for evaluating HSV susceptibility to nucleoside analogues and for assessing the role of specific viral TK mutations.

Type I interferons and herpes simplex virus infection: a naked DNA approach as a therapeutic option?

Herpes simplex virus (HSV) types 1 and 2 are highly successful human pathogens that can elicit blinding herpetic keratoconjunctivitis, fatal sporadic encephalitis, aseptic meningitis, and increase the risk of acquiring additional sexually transmitted diseases. Type I interferons (IFN) play a significant role in controlling HSV pathogenesis by antagonizing viral replication and spread. Taking advantage of the susceptibility of HSV to IFNs, a novel approach of employing plasmid DNA cassettes expressing type 1 IFNs to antagonize viral pathogenesis has been undertaken. This review will describe recent work in our lab and those of others using naked DNA encoding cytokines to antagonize HSV replication and virus trafficking or immune-mediated pathogenesis as a result of viral assault to ocular tissue.

Cytopathic effect inhibition assay for determining the in-vitro susceptibility of herpes simplex virus to antiviral agents.

We compare a rapid dilution method for the determination of antiviral susceptibility of herpes simplex virus (HSV) with the plaque reduction assay. A total of 84 HSV clinical isolates were studied by both methods to detect in-vitro resistance to acyclovir and foscarnet. The rapid method showed for the detection of HSV isolates resistant to acyclovir and foscarnet, a sensitivity of 96. 8% and 100% and specificity of 100% and 100%, respectively. This method provides an easy and accurate screening procedure for the susceptibility testing of HSV to antiviral agents.

Rapid screening tests for determining in vitro susceptibility of herpes simplex virus clinical isolates.

The susceptibility of human herpes simplex virus (HSV) to acyclovir (ACV) was determined with the use of a single dose of the drug (1 and 2 micrograms of ACV per ml for HSV-1 and HSV-2, respectively) in two rapid assays: a rapid cytopathic effect inhibitory assay (Rapid CIA) and a rapid dye uptake assay (Rapid DUA). These tests allow the simultaneous determination of virus titer and susceptibility to ACV at a determined viral concentration (100 50% tissue culture infective doses and 100 50% dye uptake units). These tests were compared with a conventional susceptibility assay (dye uptake assay) and showed similar results. Indeterminate results with the Rapid CIA appeared in 3 of 30 samples. With the use of both Rapid CIA and Rapid DUA, we were able to determine the susceptibility of 100% of the isolates. The rapid tests, unlike conventional assays, are able to provide susceptibility results within 3 days after the virus has been isolated from a clinical specimen and could thus play a direct role in therapeutic decisions.

Variable region usage in B cell deficient mice in a model of experimental herpes simplex virus retinitis.

By using PCR, we have found previously that differences in T cell receptor alpha/beta chain variable region (TCR alpha/beta) gene expression in lymph nodes (LN) existed between susceptible and resistant mice following anterior chamber (AC) inoculation with herpes simplex virus (HSV). We now report and compare the immunoglobulin gene variable region (Ig VH) and the TCR V beta mRNA expression in normal congenic mice (BALB/c and C.B-17) and also in B cell modulated C.B-17 mice (B-). RNA prepared from spleen and LN of these mice before and after HSV-AC inoculation was analysed by PCR using oligoprimers specific for 11 VH gene families and 19 V beta gene families. Densitometry analysis revealed that VH family mRNA expression levels in spleen and lymph nodes did not correlate with HSV susceptibility or resistance patterns in B-, BALB/c and C.B-17 mice. Analysis of TCR V beta chain genes showed that spleen of resistant C.B-17 and B-Ab transferred mice showed a preference for V beta 11 gene expression not seen in susceptible mice. In contrast, LN of BALB/c and B-, both susceptible mice, made TCR V beta transcripts that were indistinguishable from those generated by resistant C.B-17 mice, following HSV-AC inoculation. Finally, TCR V beta gene family repertoire appears to be severely diminished in uninfected B- mice (50% in LN and 45% in spleen) by anti-mu treatment.

A comparative evaluation of three methods of antiviral susceptibility testing of clinical herpes simplex virus isolates

Background: Current methods of antiviral susceptibility testing for herpes simplex virus (HSV) are poorly standardized and have rarely been compared critically. Objectives: To compare the three most commonly utilized HSV susceptibility assays for accuracy of result, method of implementation, and time required. Study design: We compared susceptibility results for acyclovir and foscarnet using the plaque reduction, dye uptake and DNA hybridization assays in 30 patient isolates of HSV, of varying susceptibility pattern. Compared parameters included: values for ID 50 (the concentration of drug required to inhibit virus growth by 50% or more), ratio of ID 90 to ID 50, and correlation of susceptibility result with clinical response to antiviral therapy, when available. In addition, we compared ease of the assay, presence of objective endpoint, time required to generate the susceptibility result, and necessary equipment for implementation. Results: The dye uptake yielded ID 50 results that were approximately two-fold greater than those from the plaque reduction assay, while ID 50 values from the DNA hybridization assay were one-half those from the plaque reduction assay. Comparison of the correlation of susceptibility result with clinical response to acyclovir therapy in 17 instances and to foscarnet therapy in 10 instances suggested the possibility of a somewhat greater discriminative ability of the dye uptake assay, and a somewhat lesser discriminative ability of the DNA hybridization assay, when compared with results from the plaque reduction assay in isolates with borderline acyclovir susceptibility. Conclusions: Larger comparative studies are necessary to further differences in discriminative ability of the three assays for HSV. All three assays were deemed suboptimal due to an overly long turnaround time, associated expense, and/or level of equipment required for their performance. Continued evaluation of alternative, more rapid assays is therefore warranted.