Enhanced antiglioma activity of chimeric HCMV/HSV-1 oncolytic viruses.

Oncolytic herpes simplex virus (HSV)-1 gamma(1)34.5-deletion mutants (Deltagamma(1)34.5 HSV) are promising agents for tumor therapy. The attenuating mutation renders the virus aneurovirulent but also limits late viral protein synthesis and efficient replication in many tumors. We tested whether one function of gamma(1)34.5 gene, which mediates late viral protein synthesis through host protein kinase R (PKR) antiviral response evasion, could be restored, without restoring the neurovirulence. We have previously reported the construction of two chimeric Deltagamma(1)34.5 HSV vectors (chimeric HSV), C130 and C134, which express the human cytomegalovirus (HCMV) PKR-evasion genes TRS1 and IRS1, respectively. We now demonstrate the following. The HCMV/HSV-1 chimeric viruses (i) maintain late viral protein synthesis in the human malignant glioma cells tested (D54-MG, U87-MG and U251-MG); (ii) replicate to higher titers than Deltagamma(1)34.5 HSV in malignant glioma cells in vitro and in vivo; (iii) are aneurovirulent; and (iv) are superior to other Deltagamma(1)34.5 HSV with both improved reduction of tumor volumes in vivo, and improved survival in two experimental murine brain tumor models. These findings demonstrate that transfer of HCMV IRS1 or TRS1 gene into Deltagamma(1)34.5 HSV significantly improves replication in malignant gliomas without restoring wild-type neurovirulence, resulting in enhanced tumor reduction and prolonged survival.

Historical correlates of genital herpes simplex virus type 2 infection in men attending an STD clinic.

BACKGROUND/OBJECTIVES: While nearly one in four Americans has antibodies to HSV-2, only one of 40 reports a history of genital herpes (GH). The goal of this study was to correlate questions designed to elicit a GH history with serological evidence of HSV-2 in male STD clinic attendees. METHODS: Consecutive males were enrolled in a study of the epidemiology of GH. Consenting men answered questionnaires regarding their histories of possible GH and underwent serological testing for HSV-1 and HSV-2. Association statistics between response to each question and HSV-2 serological status were calculated. RESULTS: Of 328 men enrolled, 148 (46%) had HSV-2 antibodies. 14 (4.3%) reported a history of GH when queried as part of a list of other STD (sensitivity (S) 0.08). 17 (5.2%) reported a history of GH when asked directly "Do you have genital herpes?" (S 0.09). 75 (21.1%) participants reported a history of a recurring genital sore, ulcer, or zipper cut (S 0.32). Overall, 64.2% of HSV-2 seropositive men answered "no" to all three questions. A "yes" response to any of the questions was only 36% sensitive for predicting HSV-2 infection. CONCLUSION: Few HSV-2 infected men report either a history of GH or are aware that they are infected. Asking about a history of recurrent genital sores was a more sensitive historical marker of HSV-2 infection than asking about a history of "genital herpes."

Natural history of neonatal herpes simplex virus infections in the acyclovir era.

OBJECTIVE: During the 2 decades in which effective antiviral therapies have been available for neonatal herpes simplex virus (HSV) disease, changes have been documented not only in the outcomes of infected infants, but also in the natural history of the disease itself. Numerous studies previously have reported that early institution of antiviral therapy is beneficial to the outcome of the disease. The objective of this study was to provide an update of neonatal HSV disease to identify means by which future improvements in the management of HSV-infected neonates can be made. DESIGN/METHODS: Neonates enrolled in 2 studies of parenteral acyclovir for the treatment of neonatal HSV disease provided the data source. The National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group conducted the studies between 1981 and 1997. A total of 186 patients are summarized, all of whom were treated with acyclovir. Demographic and clinical characteristics of these patients are reported. RESULTS: Comparisons between patients treated in the periods between 1981-1988 and 1989-1997 according to extent of disease revealed that the mean time between the onset of disease symptoms and initiation of therapy has not changed significantly from the early 1980s to the late 1990s. Of all patients evaluated, 40% had fetal scalp monitors during the delivery process. A significant minority of patients did not have skin vesicles at the time of their presentation and did not develop them during the acute HSV disease (39% of patients with disseminated disease; 32% of patients with central nervous system [CNS] disease; and 17% of patients with skin, eye, and/or mouth disease). Among patients with CNS disease, mortality was associated with prematurity. Among patients with disseminated HSV disease treated with acyclovir at 30 mg/kg/d, mortality was associated with aspartate transaminase elevations of >/=10 times the upper limit of normal at the time of initiation of acyclovir therapy. Mortality was also associated with lethargy at initiation of antiviral therapy for patients with disseminated disease. Patients' morbidity status was associated with the extent of disease (skin, eye, and/or mouth disease vs CNS vs disseminated). For those patients with CNS disease, morbidity was also associated with seizures at initiation of antiviral therapy. CONCLUSION: Data presented in the current comparison of neonatal HSV disease over the 2 periods (1981-1988 vs 1989-1997) demonstrate that no progress has been made in decreasing the interval between onset of HSV symptoms and initiation of antiviral therapy. Additional strides in the improvement of disease outcome may occur only if the interval between onset of symptoms and initiation of therapy is shortened. The means by which this will be accomplished lie in increased consideration of neonatal HSV infections in acutely ill infants. Specific data and recommendations to facilitate this goal are contained within.

Safety and efficacy of high-dose intravenous acyclovir in the management of neonatal herpes simplex virus infections.

OBJECTIVE: The objective of this investigation was to establish the safety of high-dose (HD) acyclovir for the treatment of neonatal herpes simplex virus (HSV) disease. In addition, an estimate of therapeutic efficacy was sought, both with respect to mortality and to morbidity. Virologic efficacy of HD acyclovir was also assessed. PARTICIPANTS: Infants who were

Viral shedding and biodistribution of G207, a multimutated, conditionally replicating herpes simplex virus type 1, after intracerebral inoculation in aotus.

G207 is a multimutated, conditionally replicating herpes simplex virus type 1 (HSV-1) that is currently in clinical trial for patients with malignant glioma. G207 exhibits an efficient oncolytic activity in tumor cells, yet minimal toxicity in normal tissue when injected into the brains of HSV-susceptible mice or nonhuman primates. In this study, we evaluated the shedding and biodistribution of clinical-grade G207 after intracerebral inoculation (3 x 10(7) pfu) in four New World owl monkeys (Aotus nancymae). Using PCR analyses and viral cultures, neither infectious virus nor viral DNA was detected from tear, saliva, or vaginal secretion samples at any time point up to 1 month postinoculation. Analyses of tissues obtained at necropsy at 1 month from two of the four monkeys, plus one monkey inoculated with laboratory-grade G207 (10(9) pfu) 2 years earlier, showed the distribution of G207 DNA restricted to the brain, although infectious virus was not isolated. Histopathology revealed normal brain tissues including the sites of inoculation. A measurable increase of serum anti-HSV antibody titer was observed in all monkeys, as early as 21 days postinoculation. The results ascertain the safety of G207 in the brain and indicate that strict biohazard management may not be required for G207-treated patients.

Comparison of antiviral compounds against human herpesvirus 6 and 7.

Four classes of antiviral compounds were evaluated for inhibitory activity against two variants of human herpesvirus 6 (HHV-6A and -6B) and human herpesvirus 7 (HHV-7). These included: (1) a pyrophosphate analog, phosphonoformic acid (PFA); (2) beta-guanine analogs, 9-(2-hydroxyethoxymethyl)guanine (acyclovir or ACV), 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir or GCV) and 9-(4-hydroxy-3-hydroxy-3-hydroxymethylbutylyl)guanine (penciclovir or PCV); (3) acyclic nucleoside phosphonates, (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine [cidofovir or (S)-HPMPC] and its cyclic derivative (S)-cyclic-HPMPC (cHPMPC), 9-[[2-hydroxy-1-phosphonomethoxy)ethoxy]methyl]guanine (HPMEMG) and 9-[(2-phosphonylmethoxy)ethyl]-2,6-diaminopurine (PMEDAP), and the seven other related compounds; and (4) a series of benzimidazole ribonucleosides, including 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB). End-point inhibitory concentration (EPC) and 50% effective inhibitory concentration (EC50) values were determined by a dot-blot antigen detection method in cord blood mononuclear cells infected with HHV-6A, HHV-6B or HHV-7 at a multiplicity of infection of 0.004 CCID50/cell. (S)-HPMPC and cHPMPC had an EC50 value of approximately 0.3 microg/ml for HHV-6A, 1.2 microg/ml for HHV-6B and 3.0 microg/ml for HHV-7. These compounds were the most active of those tested against each virus. The EC50 value of GCV for HHV-6A was 0.65 microg/ml, 1.33 microg/ml for HHV-6B, and >7 microg/ml for HHV-7. The EC50 values of ACV and PCV were approximately 6-8 microg/ml for HHV-6A, 16-24 microg/ml for HHV-6B and 121-128 microg/ml for HHV-7. These drugs were the least active. The sensitivity of HHV-7 to the guanine analogs was different from HHV-6, suggesting a difference in selectivity of specific viral enzymes.

Pharmacokinetics of oral valacyclovir and acyclovir in late pregnancy.

OBJECTIVE: The objective was to obtain preliminary pharmacokinetic data for acyclovir from gravid women receiving herpes simplex virus suppressive therapy with the acyclovir prodrug valacyclovir. STUDY DESIGN: In a prospective, double-blind trial, 20 women with a history of recurrent genital herpes simplex virus infection and positive herpes simplex virus 2 serologic results were randomly assigned at 36 weeks' gestation to receive oral valacyclovir (500 mg twice daily) or acyclovir (400 mg 3 times daily). Acyclovir pharmacokinetic profiles were obtained after the initial dose (36 weeks) and at steady state (38 weeks). Amniotic fluid samples were obtained during labor and simultaneous umbilical cord and maternal plasma samples were collected at delivery. Laboratory studies were performed to screen for laboratory evidence of toxicity in mothers and infants. RESULTS: Peak acyclovir plasma concentrations (mean +/- standard deviation) were higher in valacyclovir recipients than in acyclovir recipients after the initial dose (3.14 +/- 0.7 microg/mL vs 0.74 +/- 0.6 microg/mL, P < .0001) and at steady state (3.03 +/- 1.0 microg/mL vs 0.94 +/- 0.7 microg/mL, P < .001). The daily area under the curve values were higher in valacyclovir recipients than acyclovir recipients after the initial dose (17.8 +/- 3.6 h x microg/mL vs 7.71 +/- 2.5 h x microg/mL, P < .001) and at steady state (19.65 +/- 6.4 h x microg/mL versus 11.0 +/- 4.5 h x microg/mL, P = .009). There was no significant difference in drug elimination half-life or in time to peak concentration between valacyclovir and acyclovir recipients at either sampling interval. Acyclovir was concentrated in the amniotic fluid; however, there was no evidence of preferential fetal drug accumulation (mean maternal/umbilical vein plasma ratios at delivery were 1.7 for valacyclovir and 1.3 for acyclovir). Valacyclovir was well tolerated, and no significant laboratory or clinical evidence of toxicity was detected. CONCLUSION: In this phase I trial maternal valacyclovir therapy resulted in higher plasma acyclovir levels, with significantly higher peak concentrations and daily area under the curve values, than did acyclovir therapy. Additional trials are needed to further evaluate the safety and efficacy of suppressive valacyclovir therapy during late pregnancy.

Application of competitive PCR to cerebrospinal fluid samples from patients with herpes simplex encephalitis.

The purpose of the present study was to determine if the quantity of herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) of patients with herpes encephalitis would be useful in establishing the prognosis of the disease and to determine the effect of antiviral therapy on the clearance of viral DNA from the CSF. Quantitation of HSV DNA was done by constructing an internal standard (IS) from the glycoprotein B amplicon which had a 25-bp deletion between primer annealing sites. Each CSF specimen was coamplified with the IS and the ratio of the amount of HSV/amount of IS was compared to the ratios on a standard curve constructed with the same IS plus known amounts of HSV DNA. CSF specimens were available from 16 patients who were treated with intravenous acyclovir, and the amount of HSV DNA ranged from < 25 to 18,000 copies per microliter in CSF obtained before or within 4 days of the initiation of acyclovir therapy. Patients with > 100 copies of HSV DNA per microliter were older, were found by computed tomography to have lesions, and had poorer outcomes than patients with < 100 copies. Follow-up CSF specimens were available from seven patients. In six of these seven patients, the HSV DNA levels decreased during therapy. One patient had a twofold increase in HSV DNA levels after 1 week of therapy and died on day 8. The application of this assay may be helpful in establishing the prognosis and in the monitoring of patients with herpes simplex encephalitis.

Diagnosis of herpes simplex encephalitis by magnetic resonance imaging and polymerase chain reaction assay of cerebrospinal fluid.

The early diagnosis of herpes simplex encephalitis (HSE) is essential because early introduction of antiviral therapy can significantly reduce the mortality of this disease. Herpes simplex virus (HSV) DNA detection in cerebrospinal fluid (CSF) samples is a rapid, noninvasive, specific, and highly sensitive method for HSE diagnosis. Neurodiagnostic methods have also been studied for noninvasive diagnosis of HSE. Magnetic resonance imaging (MRI) seems to be the most sensitive of them but it has not been compared to PCR in terms of efficacy for HSE diagnosis. In this study, 17 patients with focal encephalitis were prospectively evaluated by PCR analysis of CSF samples and MRI examination. MRI lesions involving the inferomedial region of one or both temporal lobes were observed in all PCR-positive patients but one. No PCR-negative patient presented with the same pattern of MRI lesions. MRI was also important for the establishment of an alternative diagnosis in three of eight PCR-negative patients. Both methods should be routinely applied in the evaluation of presumed HSE cases.

Evaluation of the range of clinical presentations of herpes simplex encephalitis by using polymerase chain reaction assay of cerebrospinal fluid samples.

Detection of DNA from herpes simplex virus in cerebrospinal fluid (CSF) samples by polymerase chain reaction (PCR) analysis has been shown to be more sensitive and specific for the diagnosis of herpes simplex encephalitis than isolation of herpes simplex virus from biopsy specimens of brain tissue. Because of the invasiveness of brain biopsy, it has been suggested that PCR analysis of CSF may reveal a wider spectrum of the disease than has been previously recognized by brain biopsy studies. In this study, PCR assay of CSF samples obtained from 29, 12, and 8 patients with focal, mild, and diffuse encephalitis, respectively, was performed. PCR assay was positive for 15 (51.7%) of 29 patients with focal encephalitis and three (25%) of 12 patients with mild encephalitis. The correlation between temporal abnormalities shown by electroencephalography, computed tomography of the brain, or cranial magnetic resonance imaging and a positive PCR assay was high. PCR analysis has revealed that atypical and less severe forms of encephalitis are caused by herpes simplex virus.

Advantage of polymerase chain reaction in the diagnosis of herpes simplex encephalitis: presentation of 5 atypical cases.

Four case of herpes encephalitis (HSVE) are described. The diagnosis was established by polymerase chain reaction (PCR) assay of cerebrospinal fluid (CSF). These reports illustrate different situations in the clinical management of this disease. PCR was considered useful in confirming the HSVE diagnosis in 3 atypical cases, and in the differentiation between virologic failure and postinfectious encephalitis in a patient with recurrence of symptoms. A case with typical HSVE clinical findings is also reported where PCR was negative and a temporal lobe lymphoma was diagnosed at autopsy. This last case is representative of the utility of PCR in the management of other diseases mimicking HSVE.

Application of the polymerase chain reaction to the diagnosis and management of neonatal herpes simplex virus disease. National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group.

Cerebrospinal fluid (CSF) specimens from 77 neonates with herpes simplex virus (HSV) disease were evaluated retrospectively by polymerase chain reaction (PCR). Samples were collected from 202 infants enrolled in a National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group trial that compared vidarabine with acyclovir for the treatment of neonatal HSV infection. HSV DNA was detected in the CSF of 26 (76%) of 34 infants with CNS disease, in 13 (93%) of 14 infants with disseminated infection, and in 7 (24%) of 29 with skin, eye, or mouth (SEM) involvement. One of the 7 PCR-positive SEM patients subsequently developed severe neurologic impairment. Eighteen (95%) of 19 infants with positive CSF PCR results after the completion of 10 days of antiviral therapy experienced significant morbidity or mortality. Application of PCR to neonatal HSV disease may provide additional information on which clinical decisions may be based, although its diagnostic utility outside the research setting is unproven.

A method for detection of HHV-6 antigens and its use for evaluating antiviral drugs.

A simple and reproducible method for detection of human herpesvirus 6 (HHV-6) antigens was developed using a dot blot assay in order to assess virus titer and to evaluate the effect of antiviral drugs against HHV-6. The titer of virus stocks obtained by the dot blot assay was the same as that determined by an immunofluorescence assay (IFA). This method was then applied to evaluate the effect of several antiviral drugs against HHV-6, including phosphonoformic acid (PFA), 9-(2-hydroxyethoxymethyl)guanine (ACV), 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine [(S)-HPMPC]. The end-point concentrations (EPC, which was determined visually) of DHPG and (S)-HPMPC were approximately 1 microgram/ml. These drugs were more effective than the others which had EPCs of approximately 16 micrograms/ml each. The EPC values of four drugs were almost similar to EC90 values determined by measuring density of each dot blot. Thus, the EPC values can be utilized to determine the efficacy of these drugs in the inhibition of HHV-6 replication. The block in virus replication was not due to toxic effect of these drugs on cord blood mononuclear cells (CBMCs). These results suggest that a dot blot method which detects HHV-6 antigens can be useful for titrating virus yield and evaluating antiviral drugs against HHV-6 replication.

Human herpesvirus 6 is associated with focal encephalitis.

Human herpesvirus 6 (HHV-6) is a cause of roseola infantum. Recent reports associate HHV-6 with cases of encephalitis; however, conclusive etiologic data do not exist. We evaluated clinical data and laboratory specimens obtained from patients with focal encephalitis of unknown etiology. Cerebrospinal fluid (CSF) specimens were tested by polymerase chain reaction for the presence of HHV-6 DNA. Selected samples were analyzed by DNA sequencing. We detected HHV-6 DNA in the CSF of nine of 138 patients. DNA sequencing revealed that group B strains of HHV-6 were present in those specimens that were analyzed. No significant differences could be demonstrated in clinical presentation, laboratory findings, or neurodiagnostic imaging results between the nine patients with confirmed HHV-6 infection and the 129 patients without evidence of HHV-6 infection. Neurological outcome for the nine HHV-6-infected patients varied from complete recovery without neurological deficit to death. Further prospective study is warranted.

Diagnosis of herpes simplex encephalitis: application of polymerase chain reaction to cerebrospinal fluid from brain-biopsied patients and correlation with disease. National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group.

Isolation of herpes simplex virus (HSV) from brain tissue after biopsy has been considered the reference standard for the diagnosis of herpes simplex encephalitis (HSE). During the evaluation of antiviral treatment of HSE, cerebrospinal fluid (CSF) was obtained from patients with clinical disease indicative of HSE who underwent diagnostic brain biopsy. HSV DNA was detected by polymerase chain reaction (PCR) in CSF of 53 (98%) of 54 patients with biopsy-proven HSE and was detected in all 18 CSF specimens obtained before brain biopsy from patients with proven HSE. Four of 19 CSF specimens were positive after 2 weeks of antiviral therapy. Positive results were found in 3 (6%) of 47 patients whose brain tissue was culture-negative. Detection of HSV DNA in the CSF correlated significantly with age and focal radiographic findings. Thus, PCR detection of HSV DNA should be the standard for diagnosis of HSE.

Herpes simplex virus infections of the central nervous system: therapeutic and diagnostic considerations.

Herpes simplex virus infections of the central nervous system remain a significant cause of morbidity and mortality, in spite of safe and efficacious antiviral therapy. Advances in the treatment of neonatal herpes and herpes simplex encephalitis with acyclovir have improved outcome. The application of polymerase chain reaction has allowed for the prompt and specific diagnosis of herpes simplex virus infections of the brain. This review summarizes our current knowledge on the pathogenesis, diagnosis, and treatment of herpes simplex virus infections of the brain. Opportunities for the future will be defined.

Rapid detection of herpes-simplex-virus DNA in cerebrospinal fluid of patients with herpes simplex encephalitis.

Herpes-simplex-virus (HSV) DNA in cerebrospinal fluid was amplified by use of the polymerase chain reaction and identified by hybridisation to a specific oligonucleotide probe. Specimens of cerebrospinal fluid (CSF) from 4 of 4 patients with herpes simplex encephalitis were positive for HSV DNA, whereas CSF specimens from 6 patients with other central-nervous-system infections were negative. This technique may expedite diagnosis of herpes simplex encephalitis.

An isoelectric focusing study in herpes simplex virus encephalitis.

To establish an early reliable diagnostic test for herpes simplex virus (HSV) type 1 encephalitis (HSVE), we used isoelectric focusing (IEF) and an IEF-overlay technique with radiolabeled HSV glycoprotein B (gB) to study 7 serum and 12 cerebrospinal fluid (CSF) samples from 12 patients with presumed or biopsy-proved HSVE. Blood-brain barrier damage and increased intra-blood-brain barrier IgG synthesis were detected in 5 of the 7 patients with HSVE. CSF oligoclonal bands were found in 6 of 11 patients. Using an IEF-overlay technique, we detected anti-gB antibody in all serum (7 of 7) and in 10 of 12 CSF samples. Anti-gB antibody was found in 4 of 6 CSF specimens obtained within the first week of disease (days 3 to 5) and in all samples collected later in the disease. The pH range of anti-gB antibody activity was broad (4.5 to 9.5), indicating a heterogeneous immune response to HSV. A hematogenous origin of the CSF antibody was suggested because anti-gB antibody appeared in serum before matched CSF and because both serum and matched CSF had a similar antibody IEF pattern. Local production of anti-gB antibody was suggested in some cases because of a greater prominence of anti-gB antibody in CSF than in matched sera and because CSF oligoclonal bands had anti-gB antibody activity. In contrast, only one of 6 CSF samples from patients with multiple sclerosis had gB antibody activity; in this case, anti-gB antibody activity did not correspond in isoelectric point location to oligoclonal bands.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro virucidal activity by components of a topical film-forming medication.

This study evaluated the in vitro virucidal activity of soluble components of a topical film-forming medication, Zilactin, against Herpes simplex virus type 1 (HSV-1). Using a standard plaque reduction assay, a 1:10 dilution of the experimental liquid resulted in total destruction of infectious HSV-1. The phenomenon could not be explained by ethanol concentration or lowering of pH, both of which had no effect on control samples. As employed in this assay, Zilactin diluted 1:10 did not demonstrate cyctotoxicity to the cell monolayer system. The implications of these findings on the design and implementation of clinical trials are discussed, with special reference made to the restrictions imposed by the natural history of clinical lesions caused by HSV-1.

Changing presentation of herpes simplex virus infection in neonates.

We compared the clinical presentation of 95 newborns with herpes simplex virus (HSV) infection from 1973 through 1981 (first period) with data from 196 newborns evaluated from 1982 through 1987 (second period). There was a significant change in the presentation of infection in these infants. From the first to the second period, the frequency of disseminated disease decreased from 50.5% to 22.9%, whereas the frequency of skin, eye, and mouth (SEM) diseases increased from 17.9% to 43.4% (P less than .001). The frequency of infants with central nervous system (CNS) disease remained relatively unchanged--31.6% versus 33.7%. We also compared the demographic and clinical characteristics of the infants and their mothers. For neonates with CNS or disseminated infection, disease duration and frequency of prematurity were significantly decreased in the second period, as was the frequency of skin vesicles for newborns with SEM or disseminated infection. These changes are most likely the consequence of recognizing and treating SEM infection before its progression to more-severe disease.