Clinical Herpes Simplex Virus Type 1 Research Articles

A Viral Model for Corneal Scarring and Neovascularization Following Ocular Infection of Rabbits With a Herpes Simplex Virus Type 1 (HSV-1) Mutant

Herpes simplex virus type 1 (HSV-1) remains a major cause of corneal scarring and visual loss. Although efforts have been made, no reproducible animal model is available to examine recurrent corneal disease. Here we propose a rabbit ocular model to study recurrent corneal disease using an HSV-1 mutant that reactivates with high efficiency. Rabbits were ocularly infected with 2 x 10 PFU/eye of the parental McKrae, dLAT2903 (a LAT-null virus with a low-reactivation phenotype), or CJLAT (a high-reactivation virus). Acute ocular disease [days 2, 4, 7, and 10 postinfection (pi)], recurrent ocular disease, and neovascularization (days 30 to 58 pi) were monitored. All acute ocular disease symptoms, including conjunctivitis and corneal disease, were similar with all 3 viruses. No corneal scarring was detected in any eyes up to day 30 pi. Between days 35 and 58 pi, corneal scarring was observed in 11/14 (experiment 1) and 18/22 (experiment 2) eyes of CJLAT-infected rabbits. Significantly less corneal scarring was seen in eyes of rabbits infected with McKrae (0/18 and 0/16) or dLAT2903 (0/16 and 3/24) (P < 0.0001). Many of the eyes with corneal scarring developed obvious, measurable neovascularization. Rabbits infected with CJLAT developed corneal scarring and neovascularization similar to that of clinical ocular HSV-1 recurrent disease. Because this occurred well after the acute infection had resolved, the corneal scarring and neovascularization appeared to be recurrent disease. Thus, CJLAT ocular infection of rabbits may provide a good and reproducible animal model to study factors involved in corneal scarring and neovascularization from recurrent ocular HSV-1.

Phylogenetic analysis of clinical herpes simplex virus type 1 isolates identified three genetic groups and recombinant viruses.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen which establishes lifelong infections. In the present study, we determined the sequence diversity of the complete genes coding for glycoproteins G (gG), I (gI), and E (gE), comprising 2.3% of the HSV-1 genome and located within the unique short (US) region, for 28 clinical HSV-1 isolates inducing oral lesions, genital lesions, or encephalitis. Laboratory strains F and KOS321 were sequenced in parallel. Phylogenetic analysis, including analysis of laboratory strain 17 (GenBank), revealed that the sequences were separated into three genetic groups. The identification of different genogroups facilitated the detection of recombinant viruses by using specific nucleotide substitutions as recombination markers. Seven of the isolates and strain 17 displayed sequences consistent with intergenic recombination, and at least four isolates were intragenic recombinants. The observed frequency of recombination based on an analysis of a short stretch of the US region suggests that most full-length HSV-1 genomes consist of a mosaic of segments from different genetic groups. Polymorphic tandem repeat regions, consisting of two to eight blocks of 21 nucleotides in the gI gene and seven to eight repeats of 3 nucleotides in the gG gene, were also detected. Laboratory strain KOS321 displayed a frameshift mutation in the gI gene with a subsequent alteration of the deduced intracellular portion of the protein. The presence of polymorphic tandem repeat regions and the different genogroup identities can be used for molecular epidemiology studies and for further detection of recombination in the HSV-1 genome.

Oral bioavailability and in vivo efficacy of the helicase-primase inhibitor BILS 45 BS against acyclovir-resistant herpes simplex virus type 1.

This study investigated the oral bioavailability and efficacy of BILS 45 BS, a selective herpes simplex virus (HSV) helicase-primase inhibitor, against acyclovir (ACV)-resistant (ACV(r)) infections mediated by the HSV type 1 (HSV-1) dlsptk and PAA(r)5 mutant strains. In vitro, the compound was more potent than ACV against wild-type clinical and laboratory HSV-1 strains and ACV(r) HSV isolates, as determined by a standard plaque reduction assay, with a mean 50% effective concentration of about 0.15 microM. The oral bioavailability of BILS 45 BS in hairless mice was 49%, with a peak concentration in plasma of 31.5 microM after administration of a single dose of 25 mg/kg. Following cutaneous infection of nude mice, both the HSV-1 dlsptk and PAA(r)5 mutant strains induced significant, reproducible, and persistent cutaneous lesions that lasted for more than 2 weeks. Oral treatment with ACV (100 or 125 mg/kg/day, three times a day by gavage) did not affect either mutant-induced infection. In contrast, BILS 45 BS at an oral dose of 100 mg/kg/day almost completely abolished cutaneous lesions mediated by both ACV(r) HSV-1 mutants. The 50% effective doses of BILS 45 BS were 56.7 and 61 mg/kg/day against dlsptk- and PAA(r)5-induced infections, respectively. Taken together, our results demonstrate very effective oral therapy of experimental ACV(r) HSV-1 infections in nude mice and support the potential use of HSV helicase-primase inhibitors for the treatment of nucleoside-resistant HSV disease in humans.

Protective immunity against lethal HSV-1 challenge in mice by nucleic acid-based immunisation with herpes simplex virus type-1 genes specifying glycoproteins gB and gD.

DNA-based vaccines were employed to assess protective immunity against herpes simplex virus in experimental infections of hairless (strain SKH1) and BALB/c mice. Mice were vaccinated with plasmids containing the herpes simplex virus type-1 (HSV-1) glycoprotein B (gB) or D (gD) genes under the human cytomegalovirus immediate-early promoter control. Vaccines were injected intramuscularly (i.m.) or intraperitoneally (i.p.) as purified DNA alone or as formulations supplemented with different non-ionic block copolymers. Antibody responses were assessed by immunofluorescence and radio-immunoprecipitation assays. Mice inoculated with either gB or gD plasmid, alone or with non-ionic block copolymers CRL 1029 and CRL 1190, produced high levels of antibodies specific for gB or gD. Three weeks after the last vaccination, mice were challenged with a clinical HSV-1 isolate (ABGK-1) by inoculation of a shaved and subsequently scarified area between the third and fourth lumbar vertebrae. Mice immunised with either gD or gB plasmid alone or mixed with copolymers were protected against lethal HSV-1 challenge when immunisation was performed via the i.m. route. Immunisations given via the i.p. route induced humoral responses in some mice and protected the animals against lethal HSV-1 challenge only when the formulations contained copolymers. The BALB/c mouse model was shown to be as good a model as the hairless mouse model.

Inhibition of herpes simplex virus type 1 by the experimental immunosuppressive agent leflunomide.

Despite advances in antiviral chemotherapy, herpes simplex virus type 1 (HSV-1), continues to complicate the clinical course of many allograft recipients. We have previously demonstrated that the experimental immunosuppressive agent leflunomide inhibits production of cytomegalovirus by interference with virion assembly. We test the hypothesis that this agent exerts similar antiviral activity against HSV-1. Plaque assay of virus yield from endothelial or Vero cells after inoculation with each of four clinical HSV-1 isolates demonstrated a dose-dependent reduction of virus production in the presence of pharmacologic concentrations of A77 1726, the active metabolite of leflunomide. DNA dot blot and biochemical assay of viral DNA polymerase activity indicated that A77 does not inhibit viral DNA synthesis. Rather, as visualized by transmission electron microscopic method, this agent seems to disrupt virion assembly by preventing nucleocapsid tegumentation. These findings, in demonstrating that leflunomide exerts antiviral activity against HSV-1 by mechanisms similar to those we have previously shown with cytomegalovirus, imply that this agent may possess broad spectrum activity against other herpesviruses.

Typing of Clinical Herpes Simplex Virus Type 1 and Type 2 Isolates with Monoclonal Antibodies

Typing of Clinical Herpes Simplex Virus Type 1 and Type 2 Isolates with Monoclonal Antibodies

Variability of the glycoprotein G gene in clinical isolates of herpes simplex virus type 1.

Glycoprotein G (gG) of herpes simplex virus type 1 (HSV-1) has been used as a prototype antigen for HSV-1 type-specific serodiagnosis, but data on the sequence variability of the gene coding for this protein in wild-type strains are lacking. In this study, direct DNA sequencing of the gG-1 genes from PCR products was performed with clinical HSV-1 isolates from 11 subjects as well as with strains Syn 17(+), F, and KOS 321. The reference strains Syn 17(+) and F showed a high degree of conservation, while KOS 321 carried 13 missense mutations and, in addition, 12 silent mutations. Three clinical isolates showed mutations leading to amino acid alterations: one had a mutation of K(122) to N, which is a gG-1-to-gG-2 alteration; another contained all mutations which were observed in KOS 321 except two silent mutations; and the third isolate carried five missense mutations. Two clinical isolates as well as strain KOS 321 showed a mutation (F(111)-->V) within the epitope of a gG-1-reactive monoclonal antibody (MAb). When all viruses were tested for reactivity with the anti-gG-1 MAb, the three strains with the F(111)-->V mutation were found to be unreactive. Furthermore, gG-1 antibodies purified from sera from the two patients carrying strains mutated in this epitope were less reactive when they were tested by an HSV-1-infected-cell assay. Therefore, our finding that the sequence variability of the gG-1 gene also affects B-cell epitope regions of this protein in clinical isolates may have consequences for the use of this protein as a type-specific antigen for serodiagnosis.

Typing of clinical herpes simplex virus type 1 and type 2 isolates with monoclonal antibodies.

The purpose of this study was to evaluate the performance of a herpes simplex virus (HSV) type 1-specific anti-glycoprotein C-1 monoclonal antibody (MAb) and a type 2-specific anti-glycoprotein G-2 MAb for typing of 2,400 clinical HSV-1 isolates and 2,400 clinical HSV-2 isolates, respectively, using an enzyme immunoassay. The anti-HSV-1 MAb showed sensitivity and specificity of 100%, and the anti-HSV-2 MAb showed a sensitivity of 99.46% and 100% specificity, indicating that these MAbs are suitable for typing of clinical HSV isolates.

Amplification of reiterated sequences of herpes simplex virus type 1 (HSV-1) genome to discriminate between clinical HSV-1 isolates.

Herpes simplex virus type 1 (HSV-1)-related disease ranges from a localized, self-limiting illness to fatal disease in immunocompromised individuals. The corneal disease herpetic keratitis may develop after reactivation of a latent virus or reinfection with an exogenous herpesvirus. Molecular analysis of the virus involved may allow distinction between these two options. The HSV-1 genome contains several hypervariable regions that vary in numbers of reiterating regions (reiterations I to VIII [ReI to ReVIII]) between individual strains. Twenty-four HSV-1 clones, derived by subcloning of HSV-1 (strain F) twice in limiting dilutions, were tested in a PCR-based assay to analyze the stabilities of ReI, ReIII, ReIV, and ReVII. ReI and ReIII proved to vary in size upon subcloning, whereas ReIV and ReVII were stable. Subsequently, 37 unrelated isolates and 10 sequential isolates from five patients, all with HSV-1-induced keratitis, were genotyped for ReIV and ReVII. Of the 37 unrelated samples, 34 (92%) could be discriminated, while the genotypes of the viruses in sequential samples were identical for each individual. Conclusively, the data show that the approach presented allows the rapid and accurate discrimination of HSV-1 strains in studies that address the transmission and pathogenesis of HSV-1 infections.

Herpes simplex virus-1 and varicella virus infections in familial dysautonomia patients

Familial dystautonomia (FD) patients are deficient in type C fibers, suggesting that there may be a different pattern of infection and clinical presentation when infected by Herpes simplex virus type 1 (HSV-1) or Varicella-Zoster virus (VZV). These viruses infect and are reactivated in the periphery of the body through type C sensory nerve fibers. HSV-1 infects epithelial cells, penetrates into type C fibers, and migrates to the ganglia to generate latent infection. In reactivation, the viral DNA migrates through type C fibers, infecting the epidermis at the entry site. VZV infects through the respiratory tract, causing systemic viral infection and latency in the ganglia, from which it is reactivated and reaches the skin. The study was carried by clinical questionnaire and by HSV and VZV IgG antibodies on fifty-one FD patients and eighty matched controls. The questionnaire revealed that no FD patient had a history of clinical HSV-1 infection, compared to 15% in the control group (P < 0.05), while 50% FD patients had been infected by varicella, compared to 66% in the VZV control group. However in FD, VZV clinical manifestations were mild in comparison to controls. There was no difference in infection rates for some other viral diseases. HSV-1 antibodies were detected in 24% of the FD patients, compared to 38% in the control group (P < 0.1). VZV antibodies were similar in FD and controls (66%, 63%). We concluded that the rate of HSV infection in FD is low and clinical reactivation is rare. The rate of varicella infection appears to be the same for patients and controls, but in FD the clinical presentation is mild. We suggest that these differences are due to the lack of type C fibers in FD patients.

Herpes simplex virus‐1 and varicella virus infections in familial dysautonomia patients

Familial dystautonomia (FD) patients are deficient in type C fibers, suggesting that there may be a different pattern of infection and clinical presentation when infected by Herpes simplex virus type 1 (HSV-1) or Varicella-Zoster virus (VZV). These viruses infect and are reactivated in the periphery of the body through type C sensory nerve fibers. HSV-1 infects epithelial cells, penetrates into type C fibers, and migrates to the ganglia to generate latent infection. In reactivation, the viral DNA migrates through type C fibers, infecting the epidermis at the entry site. VZV infects through the respiratory tract, causing systemic viral infection and latency in the ganglia, from which it is reactivated and reaches the skin. The study was carried by clinical questionnaire and by HSV and VZV IgG antibodies on fifty-one FD patients and eighty matched controls. The questionnaire revealed that no FD patient had a history of clinical HSV-1 infection, compared to 15% in the control group (P < 0.05), while 50% FD patients had been infected by varicella, compared to 66% in the VZV control group. However in FD, VZV clinical manifestations were mild in comparison to controls. There was no difference in infection rates for some other viral diseases. HSV-1 antibodies were detected in 24% of the FD patients, compared to 38% in the control group (P < 0.1). VZV antibodies were similar in FD and controls (66%, 63%). We concluded that the rate of HSV infection in FD is low and clinical reactivation is rare. The rate of varicella infection appears to be the same for patients and controls, but in FD the clinical presentation is mild. We suggest that these differences are due to the lack of type C fibers in FD patients. J. Med. Virol. 54:158–161, 1998. © 1998 Wiley-Liss, Inc.

Typing and strain differentiation of clinical Herpes simplex virus type 1 and 2 isolates by Polymerase Chain reaction and subsequent restriction fragment length polymorphism analysis

In the present study, different combinations of primers were investigated for PCR amplification and typing of clinical herpes simplex virus type 1 (HSV-1) (n = 22) and herpes simplex virus type 2 (HSV-2) (n = 12) isolates. Intratypic strain differentiation was performed by restriction fragment length polymorphism (RFLP) analysis of different PCR amplified HSV genome regions. Enzymatic amplification of HSV DNA from all the clinical isolates could be achieved using primer combinations DNAP5/DNAp3-1 (HSV-1), DNAP5/DNAP3-2 (HSV-2). With the primer pair corresponding to the thymidine kinase (TK) genome region, amplification of all the HSV-1 isolates was only possible by nested PCR. With primers HSV UP/DOWN, 10 of 12 HSV-2 isolates could be detected. HSV typing by type-specific primers DNAP5/DNAP3-1 (HSV-1), DNAP5/DNAP3-2 (HSV-2) or restriction enzyme analysis (Ava II) of amplified DNA with HSV-specific UP/DOWN primers showed results which concorded with serotyping by monoclonal antibodies. RFLP analysis of the PCR products using selected restriction enzymes showed sufficient diversity of profiles among strains to differentiate all the HSV-1 isolates and to distinguish four groups of HSV-2 isolates. Amplification of HSV DNA from clinical isolates with subsequent typing and strain differentiation represents a valuable alternative to conventional methods (virus isolation, serotyping and restriction fragment analysis of the entire genomic DNA) and may be suitable for the study of HSV transmission and pathogenesis, especially in labour-intensive clinical samples with low levels of virus replication (i.e. cerebrospinal fluid, vitreous fluid and corneal transplants).

Analysis of the thymidine kinase of a herpes simplex virus type 1 isolate that exhibits resistance to (E)-5-(2-bromovinyl)-2'-deoxyuridine.

The mechanism responsible for the decreased sensitivity of a clinical herpes simplex virus type 1 (HSV-1) isolate, HSV-145, to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was examined. Measurements of 50% inhibitory doses of several drugs demonstrated that although HSV-145 was sensitive to phosphonoacetic acid, adenine arabinoside and acyclovir, its sensitivity to BVDU and 5-(2-chloroethyl)-2'-deoxyuridine was significantly less than that normally observed for HSV-1. Analysis of the thymidylate kinase (TMP-K) activity of HSV-145 thymidine kinase (TK) demonstrated a decreased level of TMP-K activity when compared to HSV-1 TK. The TMP-K activity of HSV-145 resembled that observed for HSV-2 and the TK-deficient strain HSV-1 TK-7. When the nucleotide sequence of the HSV-145 TK gene was compared to that of the HSV-1 strains C1(101) and SC16 a single nucleotide substitution (G changed to A at base position 502) was detected which would result in the substitution of threonine at amino acid position 168 for alanine. The substitution is the same as that for the laboratory-derived BVDU-resistant virus HSV-1 SC16B3. Collectively, these studies highlight the importance of amino acid conservation at position 168 of the HSV-1 TK in conferring efficient TMP-K activity and BVDU sensitivity.

Treatment of experimental herpes simplex virus type 1 encephalitis in mice with ( E)-5-(2-bromovinyl)- and 5-vinyl-1-β- d-arabinofuranosyluracil: Comparison with bromovinyldeoxyuridine and acyclovir

The efficiency of ( E)-5-(2-bromovinyl)- and 5-vinyl-1-β- d-arabinofuranosyluracil (BrVaraU, VaraU) as inhibitors of three herpes simplex virus type 1 (HSV-1) strains was assessed in comparison to ( E)-5-(2-bromovinyl)-2′-deoxyuridine (BrVUdR), 9-(2-hydroxyethoxymethyl)guanine (ACV), and trisodium phosphonoformate (Na 3PFA) using a plaque assay in human embryonic lung fibroblast (HELF) cell cultures. The following order of decreasing activity was found: BrVaraUt>VaraU>BrVUdR>ACV>>Na 3PFA. In HELF cell cultures, the selectivity indexes of VaraU and BrVaraU were 10 times higher than those of BrVUdR and ACV. Protection of mice from encephalitis and death due to intracerebral (i.c.) infection with a clinical HSV-1 isolate was nearly complete if mice were treated intraperitoneally (i.p.) with two daily doses of VaraU and BrVaraU (100 or 200 mg/kg per day) over a period of 5 or 10 days. The efficacy was similar to ACV, but, using a treatment schedule of three daily i.p. doses over 10 days, with equimolar amounts of the nucleoside analogs, VaraU and BrVaraU (140 and 180 mg/kg per day) were superior to ACV (130 mg/kg per day) ( P<0.05).