MATRIX-BASED CONTROLLED RELEASE DELIVERY OF ACYCLOVIR FROM POLY-(ETHYLENE CO-VINYL ACETATE) RINGS.

Up to 85% of the US adult population carries herpes simplex virus type-1 (HSV-1), with a smaller percentage (22%) infected with HSV-2. Herpesviruses can survive in lytic phase, when the viruses are actively replicating, or in latency, when the virus is functionally dormant in ganglia. Among drugs to treat these infections is acyclovir (ACV). ACV exhibits poor oral bioavailability and a short in vivo half-life; only about 10-15% of ingested drug enters the bloodstream and its half-life is about 3 hours. With those disadvantages and the possibility of poor patient compliance, viral replication may not always be suppressed. To abrogate these shortcomings we propose local distribution via sustained drug release. We present a matrix-based antiherpetic ring, composed of poly(ethylene co-vinyl acetate), that releases ACV directly to the vaginal epithelium. A 30-day in vitro drug release trial showed that approximately 135 +/- 20 µg/day of ACV was consistently released. Rings were nontoxic in cell culture and suppressed primary HSV-1 and HSV-2 replication. We expect these data form the basis for novel interventions in human health, where new prophylactics and therapeutics against genital herpes are truly needed.

The human cytomegalovirus chemokine receptor homolog encoded by US27.

The genome of human cytomegalovirus contains four genes that encode chemokine receptor homologs. Of these, the protein encoded by US27 is of particular interest today because it is currently one of the viral G-protein coupled receptors that remain classified as an orphan receptor. This review explains what is presently understood about this glycoprotein: its biochemistry, its location in infected cells, and its apparent function. While there are still many questions that remain about these topics and more, we also discuss the utility of the US27-encoded glycoprotein as a new target for antiviral intervention.

Controlled release delivery of penciclovir via a silicone (MED-4750) polymer: kinetics of drug delivery and efficacy in preventing primary feline herpesvirus infection in culture.

BACKGROUND: Herpesviruses are ubiquitous pathogens that infect and cause recurrent disease in multiple animal species. Feline herpesvirus-1 (FHV-1), a member of the alphaherpesvirus family, causes respiratory illness and conjunctivitis, and approximately 80% of domestic cats are latently infected. Oral administration of famciclovir or topical application of cidofovir has been shown in masked, placebo-controlled prospective trials to reduce clinical signs and viral shedding in experimentally inoculated cats. However, to the authors' knowledge, other drugs have not been similarly assessed or were not safe or effective. Likewise, to our knowledge, no drugs have been assessed in a placebo-controlled manner in cats with recrudescent herpetic disease. Controlled-release devices would permit long-term administration of these drugs and enhance compliance. METHODS: We therefore engineered implantable cylindrical devices made from silicone (MED-4750) impregnated with penciclovir, for long-term, steady-state delivery of this drug. RESULTS: Our data show that these devices release penciclovir with a burst of drug delivery until the tenth day of release, then at an average rate of 5.063 ± 1.704 µg per day through the next 50 days with near zero-order kinetics (in comparison to MED-4750-acyclovir devices, which show the same burst kinetics and average 2.236 ± 0.625 µg/day thereafter). Furthermore, these devices suppress primary infection of FHV-1 in a cell culture system. CONCLUSIONS: The clinical deployment of these silicone-penciclovir devices may allow long-term treatment of FHV-1 infection with a single intervention that could last the life of the host cat.

Investigating the survival of herpes simplex virus on toothbrushes and surrogate phallic devices.

BACKGROUND: Herpes simplex virus-1 (HSV-1) is a member of the alphaherpesvirus (alphaherpesvirinae) subfamily, allowing it to affect a wide range of hosts. Herpes simplex virus-1 affects 3.7 billion people, or 67% of the population, under the age of 50. With a vast number of people infected by the virus, everyday objects are often contaminated with this agent. In this study we determined how long HSV-1 can remain viable on contaminated fomites. METHODS: Fomites were selected for their use near potentially contaminated orifices and variable frequency of sanitization. Toothbrushes and surrogate phallic devices (SPDs) were cut, sterilized, and contaminated. After contaminating the fomites, we collected samples over a 24 h period, then used plaque assays to determine viral titers at prescribed time points. RESULTS: The quantity of replication-competent virus present appears to decrease significantly 2 h post-contamination, then steadily declines over time, nearing zero at 24 h. CONCLUSIONS: Our findings suggest that different surfaces influence HSV-1 survival. Proper cleaning must be performed for these types of fomites, especially if shared in an environment where someone with active genital or oral herpes lesions uses one of these fomites shortly after someone else.

The World of Antiherpetic Vaccines and Drugs, 2022.

The world of antiherpetics has grown by leaps and bounds since the discovery of what would become the first antiherpetic drug in 1964 [...].

Current Drugs to Treat Infections with Herpes Simplex Viruses-1 and -2.

Herpes simplex viruses-1 and -2 (HSV-1 and -2) are two of the three human alphaherpesviruses that cause infections worldwide. Since both viruses can be acquired in the absence of visible signs and symptoms, yet still result in lifelong infection, it is imperative that we provide interventions to keep them at bay, especially in immunocompromised patients. While numerous experimental vaccines are under consideration, current intervention consists solely of antiviral chemotherapeutic agents. This review explores all of the clinically approved drugs used to prevent the worst sequelae of recurrent outbreaks by these viruses.

Plaquing of Herpes Simplex Viruses.

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can be difficult to perform, requiring focus on its specifications and refinement. It is an incredibly challenging method for new students to master, mainly because it requires meticulous attention to the most minute details. This demonstration of plaquing herpes simplex viruses should help those who have struggled with visualizing the method, especially its nuances, over the years. While this manuscript is based on the same principles of standard plaquing methodology, it differs in that it contains a detailed description of (1) how best to handle host cells to avoid disruption during the process, (2) a more useful viscous medium than agarose to limit the diffusion of virions, and (3) a simple fixation and staining procedure that produces reliably reproducible results. Furthermore, the accompanying video helps demonstrate the finer distinctions in the process, which are frequently missed when instructing others on conducting plaque assays.

Acyclovir, cidofovir, and amenamevir have additive antiviral effects on herpes simplex virus TYPE 1.

Herpes simplex virus-1 (HSV-1) affects a large portion of the global population and has been shown to cause more severe symptoms in immunocompromised patients. It is in immunocompromised populations that HSV-1 has shown to have higher rates of resistance to the most commonly used antiherpetics, such as acyclovir/valacyclovir/penciclovir/famciclovir. The development of drug resistance has forced research into new antiherpetic therapies, including combination drug therapies. One potential complication of multidrug therapies is the existence of drug-drug interactions; as more drugs are used in the therapy, those interactions tend to become more complicated. This study tested the combination of acyclovir/cidofovir/amenamevir, the last drug being a new antiherpetic that targets the helicase-primase complex to prevent replication of viral DNA, for multidrug intervention. We used the design of experiments (DOE) function in Minitab to analyze the drug-drug interactions in their ability to inhibit growth of HSV-1. The DOE software was unable to detect any significant drug-drug interactions among these three antiherpetics as dosed. This would imply that these drugs could be used in combination to suppress viral replication without synergistic or antagonistic effects. This study shows that this therapy holds potential for further study and that DOE software is a potentially useful tool for determining complex drug-drug interactions.

Pilot Study of the Safety and Tolerability of a Subconjunctival Penciclovir Implant in Cats Experimentally Infected with Herpesvirus.

PURPOSE: To assess safety and tolerability of a subconjunctival penciclovir implant in cats infected with feline herpesvirus type 1 (FHV-1). METHODS: Subconjunctival blank (n = 4 cats) or penciclovir-impregnated (n = 6) silicone implants were placed bilaterally in 10 normal, FHV-1-naive cats 7-8 days before viral inoculation. Outcomes included disease score, FHV-1 serology, conjunctival viral load, Schirmer tear tests (STT), tear film break-up times (TFBUTs), conjunctival histology, goblet cell density (GCD), body weight, tear and plasma penciclovir concentration, and corneal ulcer evaluation. RESULTS: Both groups had similar clinical and histologic disease scores, STT values, TFBUTs, GCD, FHV-1 titers, viral loads, and body weight changes. No ocular or systemic signs of toxicity were noted. Tear penciclovir concentration varied widely among cats and across time points. Tear penciclovir concentrations exceeded the lowest published half maximal inhibitory concentration (IC50) in 5/6 treated cats. Plasma penciclovir concentrations remained below 10 ng/mL. Cats with higher tear penciclovir concentrations at inoculation and/or time of peak disease had fewer corneal ulcers than cats in which tear penciclovir concentrations were inconsistent, low, or unrecordable. CONCLUSIONS: Subconjunctival blank and penciclovir-impregnated implants were well tolerated at the ocular surface and not associated with systemic toxicity, adverse effect, or appreciable plasma penciclovir concentrations. Tear penciclovir concentrations >IC50 were sometimes achieved, especially during burst release soon after implant placement. Further study is necessary to determine efficacy of locally delivered penciclovir when penciclovir concentration is consistently maintained above IC50. This will be especially useful in patients unable to receive systemic therapy.

Volatile Acid-Solvent Evaporation (VASE): Molecularly Homogeneous Distribution of Acyclovir in a Bioerodable Polymer Matrix for Long-Term Treatment of Herpes Simplex Virus-1 Infections.

Treatment for herpes simplex virus-1 and -2 (HSV-1 and -2) patients who suffer from recurrent outbreaks consists of multiple daily doses of the antiviral drugs acyclovir (ACV), penciclovir, or their more orally bioavailable derivatives valacyclovir or famciclovir. Drug troughs caused by missed doses may result in viral replication, which can generate drug-resistant mutants along with clinical sequelae. We developed a molecularly homogeneous mixture of ACV with the bioerodable polymer polycaprolactone. Through scanning electron microscopy, infrared spectroscopy, gel permeation chromatography, 1H NMR, and differential scanning calorimetry, our method of combining drug and polymer, termed Volatile Acid-Solvent Evaporation (VASE), does not compromise the integrity of polymer or drug. Furthermore, VASE creates materials that deliver therapeutic amounts of drug consistently for approximately two months. Devices with high enough drug loads diminish primary infection of HSV-1 in Vero cells to the same level as seen with a single dose of ACV. Our data will lead to further experiments in animal models, demonstrating efficacy in preventing reactivation of these viruses with a single intervention, and with other antiviral drugs amenable to such manipulation. Additionally, this type of treatment would leave no trace after its useful lifetime, as drug is released and polymer matrix is degraded in vivo.

The chemokine receptor homologue encoded by US27 of human cytomegalovirus is heavily glycosylated and is present in infected human foreskin fibroblasts and enveloped virus particles.

Human cytomegalovirus (HCMV), a member of the beta-herpesvirus family, encodes four homologues of cellular G protein-coupled receptors (GPCRs). One of these, the protein product of HCMV open reading frame (ORF) UL33, has been identified in HCMV-infected cells and virus particles and shown to be heat-aggregatable and N-glycosylated. Another, the product of ORF US28, has been functionally characterized as a beta-chemokine receptor. Here we report the use of RT-PCR, coupled in vitro transcription-translation, immunoprecipitation, and Western immunoassays to (i) show that RNA from the open reading frame US27 appears predominantly during the late phase of replication; (ii) identify the protein encoded by HCMV US27 in infected cells and enveloped virus particles; (iii) demonstrate that the US27-encoded protein is heterogeneously N-glycosylated and resolves as two species following treatment with peptide N-glycosidase F; and (iv) show that both the recombinant and deglycoylated infected cell US27 protein aggregate when heated in the presence of SDS prior to electrophoresis in polyacrylamide gels, a property which is abrogated with the addition of urea to sample buffer.

Development of an aciclovir implant for the effective long-term control of herpes simplex virus type-1 infection in Vero cells and in experimentally infected SKH-1 mice.

Human herpes simplex virus type-1 (HSV-1) is treatable with oral doses of an antiviral agent such as aciclovir (ACV), a drug that has poor bioavailability. An alternative for delivering ACV would employ a long-lived subcutaneous implant that would allow for near zero-order drug delivery kinetics. This study aimed to develop an implant composed of a matrix of silicone and ACV that is capable of sustained long-term release of ACV. Once the implants had been created, release of ACV from the implants was determined and quantified in vitro using a spectrophotometric assay for the drug. Solvent-exposed surface area of the implant (2.86 mm(2), 6.28 mm(2), 34.62 mm(2) and 100.48 mm(2)) had a significant effect on release kinetics, whereas temperature (37 degrees C, 25 degrees C and 4 degrees C) and pH (6.0, 7.0 and 8.0) did not. The implants were also used successfully to suppress HSV-1 (KOS)-induced cytopathic effect in cultured Vero cells. The implants protected HSV-1-infected SKH-1 mice from viral reactivation (n = 37; P = 0.0367) via ultraviolet light compared with mice that were untreated (n = 37). Furthermore, mice that received silicone-only implants had no lowered risk of reactivation (n = 34; P = 0.7268), demonstrating the antiviral efficacy of the ACV implants.

Silicone-acyclovir controlled release devices suppress primary herpes simplex virus-2 and varicella zoster virus infections in vitro.

Following initial infection, herpesviruses retreat into a permanent latent state with periodic reactivation resulting in an enhanced likelihood of transmission and clinical disease. The nucleoside analogue acyclovir reduces clinical symptoms of the three human alpha herpesviruses, HSV-1, HSV-2, and VZV. Long-term administration of acyclovir (ACV) can reduce the frequency and severity of reactivation, but its low bioavailability and short half-life require a daily drug regimen. Our lab is working to develop a subcutaneous delivery system to provide long-lasting, sustained release of ACV. Previously, we demonstrated that an implantable silicone (MED-4050) device, impregnated with ACV protected against HSV-1 both in vitro and in vivo. Here, we extend our in vitro observations to include protection against both HSV-2 and VZV. We also demonstrate protection against HSV-2 in vitro using MED-4750, a silicone polymer designed for long-term use in humans. When release of ACV from MED-4750 is quantitated on a daily basis, an initial burst of 5 days is observed, followed by a long period of slow release with near-zero-order kinetics, with an average daily release of 1.3923 ± 0.5908 µ g ACV over days 20-60. Development of a slow-release implant has the potential to significantly impact the treatment of human alpha herpesvirus infections.

Alternative assessment strategy and its impact on student comprehension in an undergraduate microbiology course.

Medical Microbiology is a content-intensive course that requires a large time commitment from the students. Students are typically biology or prenursing majors, including students headed for professional schools, such as medical school and pharmacy school. This group is somewhat diverse in terms of background science coursework, so it can be difficult to teach in a way that benefits all the students. Numerous changes have been implemented in our microbiology curriculum to address the different abilities of our students by altering assessment and teaching strategies. It was hypothesized that changing the assessment strategy from the traditional scheme of two or three exams and one final to a new model of seven or eight shorter exams would have a positive impact on student comprehension and retention. The quantity of material taught or expected of the students to learn did not change, but there was definitely an impact on them. Although 30.0% of students routinely did not pass microbiology in previous semesters, the new method of assessment resulted in only 9.63% not completing the semester successfully, as determined by earning a grade of C or better. There is some evidence from conversations and interviews with students that indicates a positive impact of this methodology on student attitude. Implementation of these changes in other courses and their current effectiveness will be examined in the future, with an eye towards more broadly applicable successful teaching techniques in the sciences, especially for nonmajors.