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.     

Susceptibility to acyclovir of herpes simplex virus isolates obtained between 1977 and 1996 in Japan

The susceptibility of genital herpes to acyclovir (ACV) in immunocompetent women was examined, as was the frequency of ACV-resistant viruses by analyzing 56 clinical isolates in Japan between 1977 and 1996. The mean susceptibilities of herpes simplex virus (HSV) type 1 and type 2 were 0.13+/-0.74 and 0.42+/-0.14 microg/ml, respectively, assessed by the 50% inhibitory concentration of plaque formation. The susceptibility to ACV of clinical isolates did not changed since 1977, and also that of nine pairs of HSV-1 and HSV-2 isolates was not affected by ACV treatment. In order to characterize the change in the virus population, the quantitation of the ACV-resistant virus in 10(4) plaque forming units (PFU) of clinical isolates was adopted. The mean frequencies of ACV-resistant viruses per 10(4) PFU for all strains of HSV-1 and HSV-2 were 0.31+/-0.41 and 9.74+/-14.83, respectively, and were not influenced by ACV treatment. Additionally, the phenotypes of ACV-resistance were not influenced by ACV treatment, and more than 90% of ACV-resistant viruses were found to be thymidine kinase-deficient. This study characterized clinical isolates with respect to ACV susceptibility as a population and the quantitative and qualitative characterization of ACV-resistant virus in the virus population of clinical isolates was also studied. The susceptibility of isolates from genital lesions, the frequency of ACV-resistant viruses, and also the phenotypic characterization of ACV-resistant viruses was maintained between 1977 and 1996, even after the introduction of ACV treatment for genital herpes in Japan.     

Herpes simplex virus antiviral drug resistance--current trends and future prospects.

The various manifestations of herpes simplex virus (HSV) have been widely treated using antiviral agents for more than 40 years. Acyclovir (ACV) is the drug that has been most commonly used to date. When tested in cell culture, the majority of isolates of HSV are sensitive to ACV with ED50 values of approximately 0.1 microg/ml. ACV-resistant strains (defined as having ED50>2 microg/ml) are rarely encountered in clinical practice among normal patients (<1% isolates) and there is no firm evidence, to date, that this incidence is increasing. Resistant HSV occurs much more frequently, however, among immunocompromised patients during treatment (approximately 5% isolates) where this is recognised to be an important clinical problem leading to ineffective therapy. In this review it is argued that the rapid establishment of neuronal latency in the normal pathogenesis of HSV is the key to the low incidence of resistance development and leads to some optimism concerning future trends.     

Surveillance network for herpes simplex virus resistance to antiviral drugs: 3-year follow-up

Herpes simplex virus (HSV) infections are very common in the general population and among immunocompromised patients. Acyclovir (ACV) is an effective treatment which is widely used. We deemed it essential to conduct a wide and coordinated survey of the emergence of ACV-resistant HSV strains. We have formed a network of 15 virology laboratories which have isolated and identified, between May 1999 and April 2002, HSV type 1 (HSV-1) and HSV-2 strains among hospitalized subjects. The sensitivity of each isolate to ACV was evaluated by a colorimetric test (C. Danve, F. Morfin, D. Thouvenot, and M. Aymard, J. Virol. Methods 105:207-217, 2002). During this study, 3900 isolated strains among 3357 patients were collected; 55% of the patients were immunocompetent. Only six immunocompetent patients excreted ACV-resistant HSV strains (0.32%), including one female patient not treated with ACV who was infected primary by an ACV-resistant strain. Among the 54 immunocompromised patients from whom ACV-resistant HSV strains were isolated (3.5%), the bone marrow transplantation patients showed the highest prevalence of resistance (10.9%), whereas among patients infected by human immunodeficiency virus, the prevalence was 4.2%. In 38% of the cases, the patients who excreted the ACV-resistant strains were treated with foscarnet (PFA), and 61% of them developed resistance to PFA. The collection of a large number of isolates enabled an evaluation of the prevalence of resistance of HSV strains to antiviral drugs to be made. This prevalence has remained stable over the last 10 years, as much among immunocompetent patients as among immunocompromised patients.     

Survey of acyclovir-resistant herpes simplex virus in the Netherlands: prevalence and characterization.

BACKGROUND: Widespread and frequent use of acyclovir (ACV) for treatment, suppressive therapy and prophylaxis of herpes simplex virus (HSV) infections and its over the counter availability may be associated with emergence of HSV resistance. OBJECTIVES: To determine the prevalence of ACV-resistant HSV isolates in different patient groups between 1999 and 2002 in the Netherlands. STUDY DESIGN: A total of 542 isolates, 410 HSV-1 and 132 HSV-2, from 496 patients were screened for reduced susceptibility to ACV. A newly developed ELVIRA HSV screening assay was used that allowed a high throughput screening. The genotypic analysis of the HSV thymidine kinase gene was performed to identify resistance-associated mutations. RESULTS: Thirteen isolates, 8 HSV-1 and 5 HSV-2, from 10 patients (2%) were found resistant to ACV. A single ACV-resistant strain was identified among isolates from 368 immunocompetent patients (0.27%; 95% confidence interval [CI], 0.007%-1.5%), whereas in nine isolates from 128 immunocompromised patients resistant HSV was identified (7%; 95% CI, 3.26%-12.93%). The highest frequency of ACV-resistant HSV was associated with bone marrow transplantation: four patients out of 28 (14.3%) shed resistant virus. In addition, resistant virus was obtained from two HIV-positive patients, one patient with a hematological malignancy and two patients on immunosuppressive drugs. Further testing showed that none of the isolates was resistant to foscarnet. Several new mutations were identified in the thymidine kinase gene of these resistant isolates, and their effect on ACV-resistance is discussed. CONCLUSIONS: Our study shows that the prevalence of ACV resistance is low in immunocompetent patients (0.27%), whereas ACV-resistant HSV infections occur relatively frequently in immunocompromised patients (7%; P < 0.0001). This emphasizes the need for drug susceptibility monitoring of HSV infections in immunocompromised patients with persisting infections despite antiviral therapy.     

Comparison of methods for identifying resistant herpes simplex virus and measuring antiviral susceptibility.

BACKGROUND: A number of in vitro assays are used to determine susceptibility of HSV to antiviral agents, but results from these in vitro assays do not necessarily correlate with treatment outcome. OBJECTIVES: A method with improved capability for identifying an isolate as acyclovir (ACV) or penciclovir (PCV) resistant when resistance is borderline could greatly improve the management of HSV disease. STUDY DESIGN: A comparative evaluation of four in vitro assays, plaque reduction (PRA), DNA hybridization, plating efficiency (PEA) and plaque autoradiography (PAR) was performed to accurately identify and measure resistance of a TK-altered clinical HSV isolate (HSV-1 N4) from a patient who was non-responsive to ACV treatment. Two established criteria for the prediction of antiviral resistance, IC(50)> or =2.0 microg/ml or an IC(50) greater than 10x above a sensitive virus IC(50), as well as testing in human (MRC-5) and nonhuman (Vero and CV-1 monkey kidney) cell lines were evaluated. RESULTS: The PRA and DNA hybridization assays accurately identified HSV-1 N4 as ACV(r) in human cells when using the 10x above sensitive virus IC(50) resistance criterion. Moreover, the PEA and PAR assays failed to classify HSV-1 N4 as drug resistant and indicate that these technologies alone are inadequate for identifying resistant virus. CONCLUSIONS: The data presented herein indicate that the PRA and DNA hybridization assays most accurately identified an otherwise borderline-resistant isolate as drug resistant: (i) when a sensitive virus is used within each individual assay as a control, (ii) when ACV and PCV susceptibility is evaluated in human cells, and (iii) when the 10x above sensitive IC(50) criterion is used to classify a virus as drug-resistant. Testing of additional clinical samples is warranted to further confirm these findings.     

Frequency of acyclovir-resistant herpes simplex virus in clinical specimens and laboratory isolates

The proportion of acyclovir (ACV)-resistant herpes simplex virus (HSV) isolates in clinical specimens and laboratory isolates was determined. HSV isolates in clinical specimens and laboratory isolates were cultured in the absence or presence of 5 microg of ACV per ml. The frequency of ACV-resistant HSV was calculated as (average virus titer in the wells with ACV)/(average virus titer in the wells without ACV). The mutation frequency of HSV type 1 isolates in clinical samples (directly from patient lesions) was 7.5 x 10(-4) +/- 2.5 x 10(-4) (mean +/- standard error), and that of HSV type 2 isolates was 15.0 x 10(-4) +/- 4.6 x 10(-4). The mutation frequencies of isolates derived in the laboratory from these clinical samples were very similar. Similarly, the 50% inhibitory concentrations for isolates in clinical samples and laboratory isolates were identical. The frequencies of ACV-resistant HSV types 1 and 2 were in a narrow range of 7.5 x 10(-4) to 15.0 x 10(-4) among isolates in clinical specimens and did not change for laboratory-derived pools of viral isolates.     

Occurrence and characterization of acyclovir-resistant herpes simplex virus isolates: report on a two-year sensitivity screening survey.

For the past 2 years, a survey network was established for the screening of acyclovir (ACV)-resistant clinical isolates of herpes simplex virus (HSV). Among 889 strains tested for in vitro ACV sensitivity, 14 HSV-1 and 6 HSV-2 were resistant to ACV concentrations exceeding 3 micrograms/ml. These resistant isolates were most often obtained after prolonged ACV treatment of severely immunocompromised patients. For five patients, the emergence of ACV-resistant virus correlated with treatment failure. In particular, a decrease in the in vitro sensitivity to ACV was observed for eight successive HSV-1 isolates from one immunodeficient patient undergoing therapy. All ACV-resistant isolates were studied for their sensitivity to different antiherpetic compounds and showed various cross-sensitive and -resistant patterns. The examination of viral populations by plaque autoradiography procedures frequently revealed their heterogeneity in terms of thymidine kinase (TK) phenotype and allowed the detection of various proportions of TK-positive (TK+), TK-deficient (TKD), or TK-altered (TKA) viruses. Our data underline the importance of monitoring the emergence of drug-resistant virus during the course of antiviral therapy, and the need for the detection and characterization of TK mutants in clinical specimens. The routine examination of drug sensitivity of HSV isolates provides useful information to clinicians for the management of ACV treatment in the hope of preventing ACV-resistant mutants from becoming predominant in mixed viral populations.     

The effect of gramicidin, a topical contraceptive and antimicrobial agent with anti-HIV activity,against herpes simplex viruses type 1 and 2 in vitro

Summary.  The effect of an anti-HIV compound, gramicidin, previously used as a topical antibiotic and vaginal contraceptive, on the replication of herpes simplex viruses (HSV) type 1 and 2 has been examined. Human WI-38 fibroblasts were inoculated with either HSV type in the presence of serial dilutions of gramicidin and reduction in viral yield was measured by ELISA. The 50% inhibitory dose (IC₅₀) of gramicidin against 3 HSV-1 and 4 HSV-2 isolates was equal to 0.3 μg/ml and was comparable to the efficacy of the anti-HSV agent acyclovir (ACV). The IC₅₀ of gramicidin required to protect WI-38 from cytolytic effect of HSV was 10 μg/ml at day 5 postinfection, indicating that at this time point the activity of gramicidin was inferior than that of ACV. Nevertheless, gramicidin suppressed the replication of ACV-resistant thymidine kinase and DNA polymerase HSV mutants at doses effective against ACV-sensitive strains. The results suggest that the antimicrobial and spermostatic agent, gramicidin, has potential against sexually transmitted diseases (STDs) and for prophylaxis of sex-borne HIV and HSV infections. Received November 8, 1996 Accepted June 26, 1997     

Serotyping of herpes simplex virus isolates: A comparison of BVDU sensitivities,indirect immunofluorescence with monoclonal antibodies,and indirect immunofluorescence with cross-adsorbed rabbit antibodies

Four methods for typing of herpes simplex virus (HSV) isolates were compared for 43 recent clinical isolates and 3 reference strains of HSV, These isolates were subjected to indirect immunofluorescence using both monoclonal antibodies and cross-adsorbed rabbit antisera. Sensitivity to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was also examined.All isolates which fluoresced with HSV-1 monoclonals were found to be sensitive to BVDU with ID₅₀'s ranging from 0.001 to 0.006 μg/ml. All isolates labelled as HSV-2 using monoclonal antibodies had ID₅₀'s to BVDU ranging from 0.4 to 3.5 μg/ml. Results of typing with rabbit cross-adsorbed antisera were less accurate, however. When dilutions were predetermined according to manufacturer's instructions, only 3 of 22 isolates (14%) of HSV-1 were correctly typed. HSV-2 isolates were correctly labelled in 24 of 26 situations (92%).When fluorescence dilution endpoints were compared, however, 21 of 22(95%) HSV-1 isolates had fluorescence endpoints at a greater dilution with HSV-1 antiserum. Twenty-three of 24 HSV-2 isolates were also correctly typed (96%).     

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 microM) 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.     

Infection due to acyclovir resistant herpes simplex virus in patients undergoing allogeneic hematopoietic stem cell transplantation

Over an eight-month period from October 1997 to May 1998, four patients who had received bone marrow transplant (BMT) from unrelated donor presented with severe mucosal cutaneous infections involving acyclovir resistant herpes simplex virus 1 (HSV-1). The four isolates were acyclovir (ACV) resistant, three of which were also foscarnet resistant as determined by the dye uptake method. The sequencing of the thymidine kinase (TK) gene did not permit to establish a relation between mutations and resistance to ACV. Three patients were considered as clinically cured of their HSV infection by replacement of ACV or foscarnet with either valacyclovir (one case) or cidofovir (two cases) but eventually two of them died of graft vs host disease. One patient died of extensive HSV infection despite administration of cidofovir. This study emphasizes the importance of monitoring the herpes virus resistance to antiviral drugs in bone marrow transplant recipients and the usefulness of the evaluation of novel antiviral drug for treatment of infections due to strains of HSV resistant to ACV and foscarnet that occur in about 5% of immunocompromised patients.     

Phenotypic and genotypic methods for the detection of herpes simplex virus serotypes

Typing of herpes simplex virus (HSV) into its serotypes plays a major role in epidemiology and management of reactivation. To develop and evaluate a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) was employed using Hae III and Taq I against neutralization test, allele-specific PCR and DNA sequencing for the detection of HSV serotypes. Neutralization test, allele-specific PCR, DNA sequencing and PCR-based RFLP were applied simultaneously to 2 standard strains (HSV-1 and HSV-2) and 23 clinical isolates. PCR-based RFLP was applied further to 20 culture negative PCR positive clinical specimens. The 179 bp product of the clinical isolates and specimens amplified using the type-common primers of HSV was subjected to DNA sequencing and PCR-based RFLP. Allele-specific PCR was absolutely specific and highly sensitive. All the typing methods differentiated concordantly 23 clinical isolates into 12 HSV-1 and 11 HSV-2. DNA sequencing did not reveal any nucleotide variations within the serotypes among the isolates sequenced. PCR-based RFLP typed a further 20 culture negative clinical specimens into 15 HSV-1 and 5 HSV-2. PCR-based RFLP was a reliable, less laborious and cost-effective molecular biological tool for the determination of HSV serotypes both for the clinical isolates and culture negative specimens.     

Demonstration of either endogenous recurrence or exogenous reinfection by resiriction endonuclease cleavage analysis of herpes simplex virus from patients with recrudescent genital herpes

By using restriction endonuclease (RE) cleavage analysis, either endogenous recurrence or exogenous reinfection of herpes simplex virus (HSV) was clarified in 45 male and 20 female subjects with recrudescent genital herpes. All of the plural (two to ten) isolates from 63 (205 isolates) out of 65 subjects (97%) were HSV-2. Two isolates from only one of 65 subjects (1.5%) were HSV-1, and they showed the same RE profile. In addition, an HSV-1 and five HSV-2 isolates were obtained from the remaining one female patient (1.5%), indicating that an exogenous HSV-1 strain has been reinfected during HSV-2 recrudescences. HSV-2 isolates were furthermore classified into genotypes of HSV-2 using 16 different RE markers with five REs. Two hundred and ten HSV-2 isolates from 64 subjects were classified into 27 distinct genotypes, in which some predominant genotypes and seven new genotypes were found. Plural HSV-2 isolates obtained from 63 out of 64 subjects, including one subject from whom an HSV-1 and five HSV-2 strains were isolated, were classified into the same genotypes, indicating that they may be regarded as recrudescent genital herpes by the reactivation of the same endogenous strain. However, the RE profiles of two HSV-2 strains from the remaining one subject were different. Thus, it was finally found that only two out of 65 subjects (3%) were reinfected with exogenous strains. These results lead to the conclusion that almost all recrudescent genital herpes are due to the reactivation of an initially infected HSV-2 strain, and are occasionally due to reinfection with distinct HSV strains of either the same or a different type. © 1995 Wiley-Liss, Inc.     

Effect of metal complexes of acyclovir and its acetylated derivative on Herpes simplex virus 1 and Herpes simplex virus 2 replication

The effect of zinc, nickel, cobalt and cadmium complexes of acyclovir (ACV) and its omicron-acetylated derivative (Ac-ACV) on the replication of wild type (wt) and ACV-resistant (ACV(R)) strains of Herpes simplex virus 1 (HSV-1) and Herpes simplex virus 2 (HSV-2) was examined. According to cytotoxicity, these compounds followed the order Ni-ACV chloride > Cd-ACV 3 Ni-ACV nitrate > ACV = Zn-ACV nitrate = Ac-ACV = Zn-Ac-ACV > Zn-ACV chloride > Co-ACV. Besides Ac-ACV, the only active complexes in inhibiting virus replication were Zn-ACV nitrate and Zn-Ac-ACV, which effectively suppressed the growth of both wt and ACVR strains of HSV-1 and HSV-2. The most active and most selective inhibitor of the growth of ACVR strains of HSV-1 and HSV-2 was Ac-ACV; its EC50 and SI were 100 and 10 times higher than those of ACV, respectively. Zn-Ac-ACV was less active than Ac-ACV, obviously due to the stability of the complex. Zn-ACV nitrate was active against both wt and ACVR strains of HSV-1; its activity and selectivity were 100 and 75 times higher than those of ACV, respectively. Ac-ACV and Zn-Ac-ACV suppressed the pre-mitotic arrest caused by HSV-1 infection during the first 2 hrs of infection and later on restored the cell division.     

Zinc salts inactivate clinical isolates of herpes simplex virus in vitro

Using a standard plaque assay and clinical isolates of herpes simplex virus (HSV), we have tested the ability of zinc salts to inactivate HSV. Virus was treated by incubation at 37 degrees C with zinc salts in morpholinepropanesulfonic acid-buffered culture medium and was then diluted and plated onto CV-1 cells for detection and quantitation of remaining infectious virus. Of 10 randomly chosen clinical isolates (five HSV type 1 [HSV-1] isolates and five HSV-2 isolates), seven were inactivated >98% by treatment in vitro with 50 mM zinc gluconate for 2 h and nine were inactivated >97% by treatment with zinc lactate. The effect was concentration dependent. With an HSV-1 isolate, 50 mM zinc gluconate or zinc lactate caused 100% inactivation, 15 mM caused 98 to 99% inactivation, and 5 mM caused 63 to 86% inactivation. With an HSV-2 isolate, 50 and 15 mM zinc gluconate caused 30% inactivation and 5 and 1 mM caused less than 9% inactivation, whereas 50 and 15 mM zinc lactate caused greater than 92% inactivation and 5 and 1 mM caused 37 and 26% inactivation, respectively. The ability of the zinc salts to inactivate HSV was not related to pH in the pH range of 6.1 to 7.6 since inactivation by zinc gluconate or zinc lactate in that pH range was 99.7 to 100% with a 2-h treatment with 50 mM zinc salt. Short (5-min) treatments of selected isolates with zinc gluconate, zinc lactate, zinc acetate, or zinc sulfate yielded inactivation rates of 0 to 55%.     

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 μg 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.     

Vangueria infausta root bark: in vivo and in vitro antiplasmodial activity

Vangueria infausta burch subsp. infausta (Rubiaceae) produces fruits eaten by humans and animals. The leaf, fruit, stem bark and root bark are used as a remedy for many ailments and the roots are used to treat malaria. In this study, concentrations of fractions of the V. infausta root bark extract that produce 50% inhibition (IC50) are determined using the ability of the extract to inhibit the uptake of [G3H]-hypoxanthine by P. falciparum cultured in vitro. The root bark extract showed antimalarial activity against Plasmodium berghei in mice. It gave a parasite suppression of 73.5% in early infection and a repository effect of 88.7%. One fraction obtained from a chloroform extract gave an IC50 value of 3.8 +/- 1.5 microg/mL and 4.5 +/- 2.3 microg/mL against D6 and W2 strains of P. falciparum, respectively, and another from the butanol extract gave an IC50 value of 3.9 +/- 0.3 microg/mL against the D6 strain. Chloroquine had an IC50 value of 0.016 microg/mL and 0.029 microg/mL against D6 and W2 strains, respectively. The plant showed the presence of flavonoids, coumarins, tannins, terpenoids, anthraquinones and saponins.     

Typing of herpes simplex virus types 1 and 2 by immunoblotting analysis using polyclonal antisera to herpes simplex virus glycoproteins

Herpes simplex virus (HSV) isolates were differentiated by immunoblotting analysis using a mixture of polyclonal antisera directed against HSV type 1 (HSV-1) and HSV type 2 (HSV-2) glycoprotein fractions (gB/gC of HSV-1 and gC/gE/84-kDa protein of HSV-2), since the mixed antisera recognized viral polypeptides with different molecular weights in HSV-1- and HSV-2-infected cells. Results of typing by immunoblotting analysis were consistent with those obtained by restriction endonuclease analysis of DNAs extracted from 10 HSV isolates. These results suggest that the immunoblotting technique will be applicable to reliable typing of HSV isolates using polyclonal antisera showing the difference in reaction patterns between HSV-1- and HSV-2-infected cells.