Clinical Herpes Simplex Virus Type 1 Research Articles

CRISPR/Cas9-mediated genome editing of the thymidine kinase gene in a clinical HSV-1 isolate identifies F289S as novel acyclovir-resistant mutation

Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with intermittent reactivation of latent virus causing (a)symptomatic virus shedding. Whereas acyclovir (ACV) is a safe and highly effective antiviral to treat HSV-1 infections, long-term usage can lead to emergence of ACV resistant (ACVR) HSV-1 and subsequently ACV refractory disease. Here, we isolated an HSV-1 strain from a patient with reactivated herpetic eye disease that did not respond to ACV treatment. The isolate carried a novel non-synonymous F289S mutation in the viral UL23 gene encoding the thymidine kinase (TK) protein. Because ACV needs conversion by viral TK and subsequently cellular kinases to inhibit HSV-1 replication, the UL23 gene is commonly mutated in ACVR HSV-1 strains. The potential role of the F289S mutation causing ACVR was investigated using CRISPR/Cas9-mediated HSV-1 genome editing. Reverting the F289S mutation in the original clinical isolate to the wild-type sequence S289F resulted in an ACV-sensitive (ACVS) phenotype, and introduction of the F289S substitution in an ACVS HSV-1 reference strain led to an ACVR phenotype. In summary, we identified a new HSV-1 TK mutation in the eye of a patient with ACV refractory herpetic eye disease, which was identified as the causative ACVR mutation with the aid of CRISPR/Cas9-mediated genome engineering technology. Direct editing of clinical HSV-1 isolates by CRISPR/Cas9 is a powerful strategy to assess whether single residue substitutions are causative to a clinical ACVR phenotype.

Mechanism of resistance to acyclovir in thymidine kinase mutants from Herpes simplex virus type 1: a computational approach

Herpes simplex virus type 1 (HSV-1) infections affect about two-thirds of the world population, and the standard treatment consists of acyclovir (ACV) and its analogs, which interact with thymidine kinase (TK) blocking viral replication. Lately, the emergence of ACV-resistant strains has been reported, especially associated with TK mutations. In this context, ACV therapy fails against isolates encoding Y172C and Y53H/R163H TK mutants, but the molecular mechanism of drug resistance remains unclear. Thus, we examined the effects of these mutations on ACV and the cofactor ATP binding through molecular modeling approaches. We showed that Y172C prevents the anchoring of the aromatic ring of ACV through π–π stacking interactions, leading to an inversed binding mode and different interactions. On the other hand, Y53H/R163H remarkably affected the cofactor binding mode which shifted away from its binding site and also influenced the interaction network of ACV. This is likely due to the loss of polar interactions with R163 residue. Unlike what was observed in the wild-type complex, both drug and cofactor binding poses were not well positioned to allow the phosphorylation reaction which explains the resistance observed. Moreover, energy analysis corroborated the experimental data and showed lower theoretical affinity of ACV with mutant enzymes resulted from energetic loss in polar solvation in Y172C and electrostatic terms in Y53H/R163H mutant enzyme. Therefore, our study shed light on the resistance mechanism toward ACV of two mutant TKs identified in clinical HSV-1 strains and may further support the development of new anti-herpetic drugs to treat resistant infections. Communicated by Ramaswamy H. Sarma

Stepwise characterization of non-synonymous mutations in the HSV-1 thymidine kinase gene by different functional assays

Twenty amino acid substitutions in the thymidine kinase (TK) of clinical herpes simplex virus type 1 strains were assessed for conferring acyclovir (ACV) resistance. Site-directed mutagenesis, cell-free protein synthesis and protein expression in Escherichia coli were performed to obtain recombinant TK proteins, which were authenticated by Western blotting. A modified enzyme-linked immunosorbent assay (ELISA) was carried out to determine the phosphorylation activity of the mutants towards 5-bromo-2′-deoxyuridine (BrdU). The activity against ACV and deoxythymidine (dT) was analyzed by high performance liquid chromatography/ultraviolet spectroscopy (HPLC/UV) following incubation of recombinant TK with ACV and dT. Using ELISA, seven substitutions (G61E, A93V, M121K, R163G, P173del, V238F, G264V) showing negative activity could be classified likely as resistance-related, eleven (Q15K, R20C, R32H, E43A, E43D, R89H, A156V, P269S, G271V, S276N, I326V) with high activity as natural polymorphisms, and two (N244H and N376stop) with low phosphorylation activity. Since the N244H protein did not show any activity towards ACV, but activity towards dT using HPLC/UV, it was classified as TK with altered substrate specificity. In conclusion, the ELISA determining activity towards BrdU is suitable for the characterization of substitutions regarding their significance for resistance. Ambiguous results can be re-assessed by HPLC/UV, which classifies TK with altered substrate specificity.

Association between sensitivity of viral thymidine kinase-associated acyclovir-resistant herpes simplex virus type 1 and virulence

BackgroundAcyclovir (ACV)-resistant (ACVr) herpes simplex virus type 1 (HSV-1) infections are concern in immunocompromised patients. Most clinical ACVr HSV-1 isolates have mutations in the viral thymidine kinase (vTK) genes. The vTK-associated ACVr HSV-1 shows reduced virulence, but the association between the level of resistance and the virulence of the vTK-associated ACVr HSV-1 is still unclear.MethodsThe virulence in mice of 5 vTK-associated ACVr HSV-1 clones with a variety of ACV sensitivities, when inoculated through intracerebral and corneal routes, was evaluated in comparison with ACV-sensitive (ACVs) parent HSV-1 TAS.ResultsAlthough all the 5 ACVr HSV-1 clones and ACVs HSV-1 TAS showed a similar single-step growth capacity in vitro, the virulence of ACVr HSV-1 clones significantly decreased. A 50% lethal dose (LD50) of each clone was closely correlated with 50% inhibitory concentrations (IC50), demonstrating that the higher the ACV-sensitvity, the the higher the virulence among the ACVr clones. One of the ACVr HSV-1 clones with a relatively low IC50 value maintained similar virulence to that of the parent TAS. The infection in mice with ACVr HSV-1 due to a single amino acid substitution in vTK induced local diseases, keratitis and dermatitis, while vTK-deficient clone did not.ConclusionsA statistically significant correlation between the virulence and susceptibility to ACV among ACVr HSV-1 clones was demonstrated.

Genotyping of herpes simplex virus type 1 by whole-genome sequencing.

A previous phylogenetic analysis based on 32 full-length sequences of herpes simplex virus type 1 (HSV-1) suggested three major phylogenetic groups (phylogroups) with distinct geographic distribution: (1) western strains from Europe and North America, (2) isolates from Asia and one American strain and (3) isolates from Africa only. Here, we sequenced the genomes of additional 10 clinical HSV-1 isolates from Germany, and subsequently compared these sequences to 40 published HSV-1 genomes. The present data demonstrate that HSV-1 is the most diverse human alphaherpesvirus (mean pairwise p-distance of 0.756 %) and confirm the tripartite tree. However, as the German isolates cluster with strains of both phylogroups I and II, it is demonstrated that the latter is also present in Europe and thus is a Eurasian phylogroup. Tree-order scans indicate that HSV-1 evolution is massively influenced by recombination including all investigated strains regardless of the areal distribution of the phylogroups. Numerous recombination events in the evolution of HSV-1 may also influence genotyping as the present HSV-1 genotyping schemes do not yield results consistent with phylogroup classification. Genotyping of HSV-1 is currently based on analyses of intragenic sequence polymorphisms of US2, glycoprotein G (gG, US4) and gI (US7). Each of the 10 German HSV-1 isolates displayed a different US2/gG/gI-genotype combination, but clustered either in phylogroup I or II. In conclusion, the phylogroup concept provides a HSV-1 typing scheme that largely reflects human migration history, whereas the analysis of single-nucleotide polymorphisms fails to render significant biological properties, but allows description of individual genetic traits.

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

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.

Resistance testing of clinical herpes simplex virus type 2 isolates collected over 4 decades

There is only little information about the role of mutations of the thymidine kinase (TK) and DNA polymerase (pol) genes of herpes simplex virus type 2 (HSV-2) for the development of antiviral resistance. In this study, the polymorphism of TK and DNA pol genes was examined in 82 clinical isolates collected routinely between 1973 and 2013. If novel, presently unclear or resistance-related mutations were found, the resistance phenotype against acyclovir (ACV) and foscarnet (FOS) was analyzed. The four novel amino acid changes G150D, A157T, R248W, L342W and the hitherto phenotypically unclear substitution T131M within the TK gene were identified as natural polymorphisms. Within the DNA pol gene, 17 novel substitutions and the to-date unclear change R628C were characterized as part of natural gene polymorphism. Two novel DNA pol mutations were linked to resistance (M910T) and weak susceptibility to ACV (684 insertion ED), respectively. In one isolate, the genomic cause of ACV resistance could not be identified. Phylogenetic analysis including sequences of this study and of the GenBank revealed a hierarchy of mutation clusters in TK displaying G39E as first common mutation step, followed by N78D and L140F. In conclusion, the present findings allow a deeper insight in the variability of HSV-2 TK and DNA pol genes. The most common substitution G39E can be excluded as unique cause of HSV-2 resistance. This study supports once more the importance of phenotypic adjustment of genotypic results to enhance the clinical utility of genotypic findings.

Single nucleotide polymorphisms of thymidine kinase and DNA polymerase genes in clinical herpes simplex virus type 1 isolates associated with different resistance phenotypes

The role of mutations in the thymidine kinase (TK, UL23) and DNA polymerase (pol, UL30) genes of herpes simplex virus (HSV) for development of different resistance phenotypes has to be exactly determined before genotypic resistance testing can be implemented in patient’s care. Furthermore, the occurrence of cross-resistance is of utmost clinical importance. In this study, clinical HSV-1 isolates obtained between 2004 and 2011 from 26 patients after stem cell transplantation were examined in parallel by phenotypic and genotypic resistance testing. Thirteen isolates, which were phenotypically cross-resistant to acyclovir (ACV), penciclovir (PCV) and brivudin (BVDU), exhibited consistently frameshift or non-synonymous mutations in the TK gene known to confer resistance. One of these mutations (insertion of C at the nucleotide positions 1061–1065) has not been described before. Seven strains, phenotypically resistant to ACV and PCV and, except one each, sensitive to BVDU and resistant to foscarnet (FOS), carried uniformly resistance-related substitutions in the DNA pol gene. Finally, 3 isolates, resistant to ACV, PCV and 2 out of these also resistant to BVDU, had known but also unclear substitutions in the TK and DNA pol genes, and 3 isolates were completely sensitive. In conclusion, clinical ACV-resistant HSV-1 isolates, carrying resistance-associated mutations in the TK gene, can be regarded as cross-resistant to other nucleoside analogs such as BVDU. In contrast, clinical FOS-resistant HSV-1 strains which are cross-resistant to ACV may be sensitive to BVDU. This has to be considered for drug changes in antiviral treatment in case of ACV resistance.

Phylogenetic comparison of exonic US4, US7 and UL44 regions of clinical herpes simplex virus type 1 isolates showed lack of association between their anatomic sites of infection and genotypic/sub genotypic classification

BackgroundHSV-1 genome is a mosaic of recombinants. Clinical Herpes simplex virus -1 (HSV1) isolates were already genotyped as A, B and C types based on nucleotide variations at Unique Short (US) 4 (gG) and US 7 (gI) regions through phylogeny. Analysis of Glycoprotein C (gC) exon present on the Unique Long (UL) region had also revealed the existence of different genotypes. Glycoprotein C is mainly involved in initial viral attachment to heparan sulphate on host cell surface facilitating the virus's binding and penetration into cell. As the amount of heparan sulphate on the host cell surface varies according to the cell type, it is plausible that different genotypes bind differentially to cell types. Hence, this study was framed to determine the existence of novel genotypes/sub genotypes in the US or UL regions which could associate with clinical entities.ResultsAll the twenty five isolates analyzed in this study were of genotype A as per their gG gene sequences. In case of gI gene, 16 out of 25 were found to be type A and the remaining nine were type B putative intergenic recombinants. Intragenic recombinations were also encountered in both the US genes, with gG possessing novel subgenotypes, arbitrarily designated A1 and A2. The 9 type B isolates of gI genes also branched out into 2 clades due to genetic variations. Glycoprotein C of UL region had two distinct genotypic clades α and β, whose topological distribution was significantly different from that of the US region. Neither the US nor UL regions, however, showed any preference among the genotypes to a specific anatomic site of infection. Even the non synonymous variations identified in the functional domain of gC, were not confined to a particular genotype/clinical entity.ConclusionThe analyses of the US and UL regions of the HSV-1 genome showed the existence of variegated genotypes in these two regions. In contrary to the documented literature, in which Asian strains were concluded as more conserved than European ones, our study showed the existence of a higher degree of variability among Indian strains. However, the identified novel genotypes and subgenotypes were not found associated with clinical entities.

Prevalence of herpes simplex virus type 1 glycoprotein G (gG) and gI genotypes in patients with different herpetic diseases during the last four decades

The objective of this study was to genotype 375 clinical herpes simplex virus type 1 (HSV-1) isolates collected from the German Reference Laboratory of HSV and VZV between 1973 and 2010. The method is based on the amplification and the restriction fragment length polymorphism analysis of the glycoprotein G (gG) and gI. 45.1% of isolates were classified as genotype A, 28.5% as B, and 4.3% as C. 22.1% presented different cleavage patterns for gG and gI suggesting intergenic recombinants A/B in 7.7%, A/C in 0.5%, B/A in 9.3%, B/C in 1.9%, C/A in 1.6%, and C/B in 0.5% of isolates. Two isolates from 1982 and 2010 presented atypical gI cleavage pattern consistent with novel intragenic recombination between genotypes A and C. There were no significant differences of the prevalence of genotypes A, B as well as the recombinants A/B, B/A dependent on the age/gender of patients and the time period in which the strains were isolated. Likewise, there were no significant differences in the distribution of the genotypes A and B as etiological agents of eczema herpeticum, herpes labialis, herpes genitalis, and herpetic gingivostomatitis. The number of recombinants was not different significantly in the groups of the distinct herpetic diseases. In conclusion, the study confirms the high prevalence of recombinants in clinical HSV-1 strains. HSV-1 infections result in clinical manifestations which are independent of the gG/gI genotype and recombinants are not associated with special herpetic diseases.

A 12-year molecular survey of clinical herpes simplex virus type 2 isolates demonstrates the circulation of clade A and B strains in Germany

Background Recently two different herpes simplex virus type 2 (HSV-2) clades (A and B) were described on DNA sequence data of the glycoprotein E (gE), G (gG) and I (gI) genes. Objective To type the circulating HSV-2 wild-type strains in Germany by a novel approach and to monitor potential changes in the molecular epidemiology between 1997 and 2008. Study design A total of 64 clinical HSV-2 isolates were analyzed by a novel approach using the DNA sequences of the complete open reading frames of glycoprotein B (gB) and gG. Recombination analysis of the gB and gG gene sequences was performed to reveal intragenic recombinants. Results Based on the phylogenetic analysis of the gB coding DNA sequence 8 of 64 (12%) isolates were classified as clade A strains and 56 of 64 (88%) isolates were classified as clade B strains. Analysis of the gG coding DNA sequence classified 4 (6%) isolates as clade A strains and 60 (94%) isolates as clade B strains. In comparison, the 8 isolates classified as clade A strains using the gB sequence data were classified as clade B strains when using the gG coding DNA sequence, suggesting intergenic recombination events. Intragenic recombination events were not detected. Conclusion The first molecular survey of clinical HSV-2 isolates from Germany demonstrated the circulation of clade A and B strains and of intergenic recombinants over a period of 12 years.

Efficacy of anise oil, dwarf-pine oil and chamomile oil against thymidine-kinase-positive and thymidine-kinase-negative herpesviruses

Abstract The effect of anise oil, dwarf-pine oil and chamomile oil against different thymidine-kinase-positive (aciclovir-sensitive) and thymidine-kinase-negative (aciclovir-resistant) herpes simplex virus type 1 (HSV-1) strains was examined. Clinical HSV-1 isolates containing frameshift mutations in the thymidine kinase (TK) gene, an insertion or a deletion, yield a non-functional thymidine kinase enzyme resulting in phenotypical resistance against aciclovir. The inhibitory activity of three different essential oils against herpes simplex virus isolates was tested in-vitro using a plaque reduction assay. All essential oils exhibited high levels of antiviral activity against aciclovir-sensitive HSV strain KOS and aciclovir-resistant clinical HSV isolates as well as aciclovir-resistant strain Angelotti. At maximum noncytotoxic concentrations of the plant oils, plaque formation was significantly reduced by 96.6–99.9%, when herpesviruses were preincubated with drugs before attachment to host cells. No significant effect on viral infectivity could be achieved by adding these compounds during the replication phase. These results indicate that anise oil, dwarf-pine oil and chamomile oil affected the virus by interrupting adsorption of herpesviruses and in a different manner than aciclovir, which is effective after attachment inside the infected cells. Thus the investigated essential oils are capable of exerting a direct effect on HSV and might be useful in the treatment of drug-resistant viruses. Chamomile oil did not reveal any irritating potential on hen's egg chorioallantoic membrane, demonstrated the highest selectivity index among the oils tested and was highly active against clinically relevant aciclovir-resistant HSV-1 strains.

Asymptomatically shed recombinant herpes simplex virus type 1 strains detected in saliva

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen infecting most individuals worldwide. The majority of HSV-1-infected individuals have no clinical symptoms but shed HSV-1 asymptomatically in saliva. Recent phylogenetic analyses of HSV-1 have defined three genetic clades (A-C) and recombinants thereof. These data have all been based on clinical HSV-1 isolates and do not cover genetic variation of asymptomatically shed HSV-1. The primary goal of this study was to investigate such variation. A total of 648 consecutive saliva samples from five HSV-1-infected volunteers was collected. Asymptomatic shedding was detected on 7.6 % of the days from four subjects. The HSV-1 genome loads were quantified with real-time PCR and varied from 1x10(2) to 2.8x10(6) copies of virus DNA (ml saliva)(-1). Phylogenetic network analyses and bootscanning were performed on asymptomatically shed HSV-1. The analyses were based on DNA sequencing of the glycoprotein I gene, and also of the glycoprotein E gene for putative recombinants. For two individuals with clinical HSV-1 infection, the same HSV-1 strain was shed asymptomatically as induced clinical lesions, and sequence analyses revealed that these strains clustered distinctly to clades A and B, respectively. For one of the subjects with no clinical HSV-1 infection, a recombinant strain was identified. The other truly asymptomatic individual shed evolutionarily distinct HSV-1 strains on two occasions. The first strain was classified as a recombinant and the other strain clustered in clade A. High replication rates of different strains in the same person may facilitate the creation of recombinant clinical HSV-1 strains.

A 10-year molecular survey of herpes simplex virus type 1 in Germany demonstrates a stable and high prevalence of genotypes A and B

Background Recently three different herpes simplex virus type 1 (HSV-1) genotypes (A, B and C) were described on DNA sequence data of the glycoprotein E (gE), G (gG) and I (gI) genes. Objective To type the circulating HSV-1 wild-type strains in Germany and to monitor potential changes in the molecular epidemiology over the past 10 years. Study design A total of 569 clinical HSV-1 isolates from a 10-year survey in Germany were genotyped by a PCR-based restriction fragment length polymorphism analysis of gG and gI. Recombination analysis of the gE gene sequences was performed to reveal intragenic recombinants. Results Genotypes A and B strains represented 76% of all strains analyzed and showed a stable distribution within all age groups investigated, independently from the gender. Intergenic gG/gI and intragenic gE recombinants were demonstrated to be less prevalent. Interestingly, for one HIV infected patient a gG/gI genotype switch from A/A to C/A was observed within 3 years. Conclusion The first molecular survey of clinical HSV-1 isolates from Germany demonstrated a stable distribution of two different genotypes and recombinants within age groups and over a period of 10 years. Moreover, homologous recombination seems to be an important feature in the evolution of the HSV-1 genome.

Efficacy of anise oil, dwarf-pine oil and chamomile oil against thymidine-kinase-positive and thymidine-kinase-negative herpesviruses

The effect of anise oil, dwarf-pine oil and chamomile oil against different thymidine-kinase-positive (aciclovir-sensitive) and thymidine-kinase-negative (aciclovir-resistant) herpes simplex virus type 1 (HSV-1) strains was examined. Clinical HSV-1 isolates containing frameshift mutations in the thymidine kinase (TK) gene, an insertion or a deletion, yield a non-functional thymidine kinase enzyme resulting in phenotypical resistance against aciclovir. The inhibitory activity of three different essential oils against herpes simplex virus isolates was tested in-vitro using a plaque reduction assay. All essential oils exhibited high levels of antiviral activity against aciclovir-sensitive HSV strain KOS and aciclovir-resistant clinical HSV isolates as well as aciclovir-resistant strain Angelotti. At maximum noncytotoxic concentrations of the plant oils, plaque formation was significantly reduced by 96.6-99.9%, when herpesviruses were preincubated with drugs before attachment to host cells. No significant effect on viral infectivity could be achieved by adding these compounds during the replication phase. These results indicate that anise oil, dwarf-pine oil and chamomile oil affected the virus by interrupting adsorption of herpesviruses and in a different manner than aciclovir, which is effective after attachment inside the infected cells. Thus the investigated essential oils are capable of exerting a direct effect on HSV and might be useful in the treatment of drug-resistant viruses. Chamomile oil did not reveal any irritating potential on hen's egg chorioallantoic membrane, demonstrated the highest selectivity index among the oils tested and was highly active against clinically relevant aciclovir-resistant HSV-1 strains.

Prevalence of herpes simplex virus type 1 glycoprotein G (gG) and gI genotypes in patients with herpetic keratitis

Aim:Recent phylogenetic analyses on the herpes simplex virus type 1 (HSV-1) genes US4, encoding glycoprotein G (gG) and US7, encoding gI, of clinical HSV-1 isolates have led to the classification...

Divergence and recombination of clinical herpes simplex virus type 2 isolates

Divergence and recombination of clinical herpes simplex virus type 2 isolates

Glycoprotein I of herpes simplex virus type 1 contains a unique polymorphic tandem-repeated mucin region

Glycoprotein I (gI) of herpes simplex virus type 1 (HSV-1) contains a tandem repeat (TR) region including the amino acids serine and threonine, residues that can be utilized for O-glycosylation. The length of this TR region was determined for 82 clinical HSV-1 isolates and the results revealed a polymorphic distribution of two to six or eight repeated blocks with a majority harbouring between two and four repeats. Assessment of the O-glycosylation capacity of an acceptor peptide (STPSTTTSTPSTTT), representing two of the gI blocks, showed that the peptide was a universal substrate for O-glycosylation not only for the two most commonly expressed N-acetyl-d-galactosamine (GalNAc)-T1 and -T2 transferases, but also for the GalNAc-T3, -T4 and -T11 transferases. Immunoblotting of virus-infected cells showed that gI was exclusively O-glycosylated with GalNAc monosaccharides (Tn antigen). A polymorphic mucin region has not been described previously for HSV-1 and is a unique finding, as repeated blocks within gI homologues are lacking in other alphaherpesviruses.

Susceptibility of drug-resistant clinical herpes simplex virus type 1 strains to essential oils of ginger, thyme, hyssop, and sandalwood.

Acyclovir-resistant clinical isolates of herpes simplex virus type 1 (HSV-1) were analyzed in vitro for their susceptibilities to essential oils of ginger, thyme, hyssop, and sandalwood. All essential oils exhibited high levels of virucidal activity against acyclovir-sensitive strain KOS and acyclovir-resistant HSV-1 clinical isolates and reduced plaque formation significantly.

Genotyping of Clinical Herpes Simplex Virus Type 1 Isolates by Use of Restriction Enzymes

Recently, three distinct genotypes of clinical herpes simplex virus type 1 (HSV-1) isolates were identified based on DNA sequence information and phylogenetic analysis of clinical isolates and laboratory strains. We utilized single-nucleotide polymorphism within the genes coding for glycoproteins G and I for rapid genotype classification by PCR and restriction enzyme cleavage. The method is suitable for high-scale genotyping of clinical HSV-1 isolates and for the detection of recombinants.