Acyclovir Resistance Research Articles

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

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

Efficacy and Safety of Amenamevir, a Helicase-Primase Inhibitor for the Treatment of Acyclovir-Resistant Herpes Simplex Virus 1 Keratitis.

The purpose of this study was to describe the efficacy and tolerance of amenamevir (AMNV), an inhibitor of the viral helicase-primase, for the treatment of recalcitrant herpes simplex keratitis (HSK) caused by acyclovir-resistant (ACVR) herpes simplex virus 1 strains. In this retrospective case series, 6 consecutive patients with HSK caused by an ACVR herpes simplex virus 1 strain with a failure of conventional antiviral therapy were included after having been treated with AMNV (there was no control group of comparable patients for whom previous treatment would have been continued despite its inefficacy). Medical files were assessed for clinical data including reason(s) for AMNV introduction (frequent recurrences despite appropriate preventive antiviral treatment and/or clinical resistance to suppressive antiviral treatment of an ongoing clinical relapse), genotypical resistance to herpes simplex virus 1 documentation, immune status, clinical types and number of HSK episodes before and during AMNV treatment, adverse effects observed during AMNV treatment, and best corrected visual acuity. Of 6 patients, 4 (66%) did not experience a single recurrence during AMNV therapy while 2 others had recurrences (1 over 24 months of treatment and 2 over 23 months, ie two-fold less frequently than with conventional preventive treatment). On the overall history of these 6 patients, AMNV appeared to be associated with a reduction in HSK recurrences, with a mean of only 0.02 ± 0.04 episodes/month during follow-up under AMNV as compared to 0.14 ± 0.04 episodes/month in the year preceding AMNV introduction (P = 0.03). Improvement in vision acuity was also observed (mean best corrected visual acuity 0.17 ± 0.12 logarithm of the minimum angle of resolution at the end of follow-up vs. 0.30 ± 0.35 before AMNV onset), albeit nonsignificant probably due to the limited number of patients (P = 0.38). Neither clinical nor biological adverse effects were observed while under AMNV during the follow-up (16.5 ± 5.8 months). Although there was no control group, AMNV may be a valuable option to reduce ACVR HSK recurrences.

Variant Analysis of the Thymidine Kinase and DNA Polymerase Genes of Herpes Simplex Virus in Korea: Frequency of Acyclovir Resistance Mutations.

The thymidine kinase (TK) and DNA polymerase (pol) genes of the herpes simplex viruses type 1 (HSV-1) and type 2 (HSV-2) are two important genes involved in antiviral resistance. We investigated the genetic polymorphisms of the HSV-TK and pol genes in clinical isolates from Korean HSV-infected patients using next-generation sequencing (NGS) for the first time in Korea. A total of 81 HSV-1 and 47 HSV-2 isolates were examined. NGS was used to amplify and sequence the TK and pol genes. Among the 81 HSV-1 isolates, 12 and 17 natural polymorphisms and 9 and 23 polymorphisms of unknown significance in TK and pol were found, respectively. Two HSV-1 isolates (2.5%) exhibited the E257K amino acid substitution in TK, associated with antiviral resistance. Out of 47 HSV-2 isolates, 8 natural polymorphisms were identified in TK, and 9 in pol, with 13 polymorphisms of unknown significance in TK and 10 in pol. No known resistance-related mutations were observed in HSV-2. These findings contribute to our understanding of the genetic variants associated with antiviral resistance in HSV-1 and HSV-2 in Korea, with frequencies of known antiviral resistance-related mutations of 2.5% and 0% in HSV-1 and HSV-2, respectively.

Genotypic testing improves detection of antiviral resistance in human herpes simplex virus

BackgroundAntiviral resistance in human herpes simplex viruses (HSV) remains a significant clinical challenge in immunocompromised populations. Although molecular tests have largely replaced viral culture for HSV diagnosis and molecular antiviral resistance testing is available for many viruses, HSV resistance testing continues to rely on phenotypic, viral culture-based methods, requiring weeks for results. Consequently, treatment of suspected HSV resistance remains largely empiric. MethodsWe used HSV whole genome sequencing and a database of previously characterized HSV acyclovir and foscarnet resistance mutations to evaluate the performance of genotypic antiviral resistance testing among 19 control strains compared to in-house plaque reduction assay (PRA) and 25 clinical isolates sent for reference lab PRA antiviral resistance testing. ResultsAmong control strains, 23/29 (79.3%) results were concordant, 5 (17.2%) were indeterminate, and 1 (3.4%) was discordant. Indeterminate results were caused by variants of uncertain significance (VUS), including mutations without published phenotypes and mutations with contradictory results. Among clinical isolates, 14/40 (35%) results were concordant, 17 (42.5%) were indeterminate, and 9 (22.5%) were discordant. All discordant results were in reportedly phenotypically-susceptible HSV-1 strains yet possessed resistance mutations. Three contained resistant subpopulations. 6/8 (75%) discordant phenotypes were concordant with resistant genotypes upon repeat PRA. ConclusionsThese data support the combination of genotypic and phenotypic testing to diagnose HSV resistance more accurately and likely more rapidly than phenotypic testing alone. Genotypic context of resistance mutations and the ability of viral strains to form plaques in culture may affect phenotypic resistance results, highlighting the limitations of PRA alone as a gold standard method.

Novel UL23 and UL30 substitutions in HSV1 and HSV2 viruses related to polymorphism or drug resistance

Data on herpes simplex virus (HSV) polymorphism as well as acyclovir (ACV) and foscarnet (FOS) resistance mutations are not exhaustive and may hinder accurate diagnosis by next-generation sequencing (NGS). Here, we report novel UL23 and UL30 substitutions for HSV1 and HSV2 identified in immunocompromised patients treated for hematological malignancies during the last 6 years of HSV resistance surveillance at the University Hospital of Lyon. For HSV1, 35 novel UL23 substitutions and 52 novel UL30 substitutions were identified. For HSV2, 2 novel UL23 substitutions and 12 novel UL30 substitutions were identified. These results allow to complete the database of HSV1 and HSV2 substitutions, related either to polymorphism or to ACV and FOS resistance.

Phylogenetic and Genomic Characterization of Whole Genome Sequences of Ocular Herpes Simplex Virus Type1 Isolates Identifies Possible Virulence Determinants in Humans.

There are limited data on the prevalence and genetic diversity of herpes simplex virus type 1 (HSV-1) virulence genes in ocular isolates. Here, we sequenced 36 HSV-1 ocular isolates, collected by the Bascom Palmer Eye Institute, a university-based eye hospital, from three different ocular anatomical sites (conjunctiva, cornea, and eyelid) and carried out a genomic and phylogenetic analyses. The PacBio Sequel II long read platform was used for genome sequencing. Phylogenetic analysis and genomic analysis were performed to help better understand genetic variability among common virulence genes in ocular herpetic disease. A phylogenetic network generated using the genome sequences of the 36 Bascom Palmer ocular isolates, plus 174 additional strains showed that ocular isolates do not group together phylogenetically. Analysis of the thymidine kinase and DNA polymerase protein sequences from the Bascom Palmer isolates showed multiple novel single nucleotide polymorphisms, but only one, BP-K14 encoded a known thymidine kinase acyclovir resistance mutation. An analysis of the multiple sequence alignment comprising the 51 total ocular isolates versus 159 nonocular strains detected several possible single nucleotide polymorphisms in HSV-1 genes that were found significantly more often in the ocular isolates. These genes included UL6, gM, VP19c, VHS, gC, VP11/12, and gG. There does not seem to be a specific genetic feature of viruses causing ocular infection. The identification of novel and common recurrent polymorphisms may help to understand the drivers of herpetic pathogenicity and specific factors that may influence the virulence of ocular disease.

Management of oral herpes simplex virus infections: The problem of resistance. A narrative review.

Herpes Simplex Virus (HSV) type 1 (HSV-1) and type 2 (HSV-2) are among the most common human viral pathogens, affecting several billion people worldwide. Although in healthy patients clinical signs and symptoms of HSV infection are usually mild and self-limiting, HSV-infections in immunocompromised patients are frequently more aggressive, persistent, and even life-threatening. Acyclovir and its derivatives are the gold standard antiviral drugs for the prevention and treatment of HSV infections. Although the development of acyclovir resistance is a rather uncommon condition, it may be associated with serious complications, especially in immunocompromised patients. In this review, we aim to address the problem of drug resistant HSV infection and discuss the available alternative therapeutic interventions. All relative studies concerning alternative treatment modalities of acyclovir resistant HSV infection published in PubMed between 1989 to 2022 were reviewed. Long-term treatment and prophylaxis with antiviral agents predisposes to drug resistance, especially in immunocompromised patients. Cidofovir and foscarnet could serve as alternative treatments in these cases. Although rare, acyclovir resistance may be associated with severe complications. Hopefully, in the future, novel antiviral drugs and vaccines will be available in order to avoid the existing drug resistance.

PKHB1 peptide induces antiviral effects through induction of immunogenic cell death in herpes simplex keratitis.

Herpes simplex keratitis (HSK) is a severe, infectious corneal disease caused by herpes simplex virus type 1 (HSV-1) infection. The increasing prevalence of acyclovir resistance, the side effects of hormonal drugs, and the ease of recurrence after surgery have made it crucial to develop new methods of treating HSK. HSV-1 evades the host immune response through various mechanisms. Therefore, we explored the role of the immunogenic cell death inducer PKHB1 peptide in HSK. After subconjunctival injection of PKHB1 peptide, we observed the ocular surface lesions and survival of HSK mice and detected the virus levels in tear fluid, corneas, and trigeminal ganglions. We found that PKHB1 peptide reduced HSV-1 levels in the eye and alleviated the severity of HSK. Moreover, it increased the number of corneal infiltrating antigen-presenting cells (APCs), such as macrophages and dendritic cells, and CD8+ T cells in ocular draining lymph nodes. We further observed that PKHB1 peptide promoted the exposure of calreticulin, as well as the release of ATP and high-mobility group box 1 in HSV-1-infected cells in vitro. Our findings suggested that PKHB1 peptide promoted the recruitment and maturation of APCs by inducing the release of large amounts of damage-associated molecular patterns from infected cells. APCs then phagocytized antigenic materials and translocated to the lymph nodes, triggering a cytotoxic T lymphocyte-dependent immune response that ultimately alleviated HSK.

Antiviral Activity of Syzygium polyanthum Extract against Herpes Simplex Virus-Type 1 (HSV-1)

Herpes simplex virus type 1 (HSV-1) is part of the alpha subfamily of human herpesviruses. Several antiviral medications can be prescribed for treating HSV-1 which is usually first line antiviral agents such as acyclovir (ACV). But there are several cases that have been reported on ACV resistance to HSV, thus, researchers tend to develop the antiviral agent from plants extract. Syzygium polyanthum is one of the plants that has been traditionally used as a drug which is rich in bioactive compounds, including essential oils, terpenoids, tannins, and flavonoids [1]. These phytochemical compounds, which have strong antioxidant action, can aid in the inhibition of viral genome replication which disables the viral lipid envelope [2].
 
 The objective of this study is to determine the cytotoxic concentration (CC50) and antiviral activity of S. polyanthum methanolic extract. Cytotoxicity was performed using the method described by [3]. The absorbance readings were analysed using microplate reader (Infinite M200 Pro). The 50% cytotoxicity concentration (CC50) was defined as the sample concentration that able to reduce 50% of cell viability compared to the untreated cells. Antiviral activity was performed by using plaque reduction assay with three different treatments [3] which are post- treatment, pre-treatment and virucidal assay.
 
 Based on Figure 1, cytotoxicity screening against Vero cells using MTT assay showed that the CC50 values for extract was 0.135 mg/mL which considered not in the range of cytotoxic compounds. Any CC50 or IC50 of a substance less than 4 µg/mL was considered an active cytotoxic effect [4].
 Based on Figure 2, post treatment assay showed the most anti-HSV-1 activity of S. polyanthum extract which the EC50 and SI value were 0.015mg/mL and 9.03, respectively. The extract showed to be slightly effective in reducing HSV-1 replication after 2-hour incubation which might be due to the inhibition in the early phase of the attachment. For pre-treatment, EC50 and SI values were 0.019 mg/mL and 7.12, which the extract showed to be moderately effective in inhibiting the attachment of HSV-1 before the replication can occur. This might occur as Vero cells bind to the extract that could interfere with the cell membrane's glycoprotein receptors which prevented HSV-1 from attaching to the cell surface [5]. The EC50 and SI value for virucidal assay was 0.018 mg/mL and 7.28 as the result showed moderately effective inhibited the viral replication. S. polyanthum has been shown to directly inactivate HSV-1 virions within 30 minutes of exposure in this study. An SI value greater than 10 (SI>10) indicates that any antimicrobial compound has the potency to become an antiviral treatment agent [6].
 In conclusion, the findings of this study demonstrate that S. polyanthum extract seems to have moderate antiviral effects and non-toxic to Vero cells.

Disseminated Herpes Zoster in Elderly with Human Immunodeficiency Virus (HIV) Infection: A Case Report

Background: Disseminated herpes zoster (HZ) is one of the complications of HZ in the form of the appearance of the main lesion accompanied by the spread of solitary vesicles on the body. This condition occurs in 2% of the general population and 15-30% of immunodeficient patients, such as the elderly and HIV infection.
 Case presentation: A 64-year-old man came with the complaint of rashes all over his body four days ago. Vesicles and erosions are multiple in 1 dermatome and are discrete and scattered throughout the body on dermatological examination. The diagnosis of disseminated HZ, in this case, was established based on history, physical examination, and Tzank examination. HZ spread is less common and is characterized by the appearance of more than 20 vesicles or more than 2 consecutive dermatomes. Old age and HIV infection are immunocompromised conditions causing reactivation of the varicella-zoster virus and other infections. The patient received oral acyclovir, vitamins B1, B6, and B12, salicylic powder and topical fusidic acid, antiretrovirals (ARVs), azithromycin, intraoral cotrimoxazole, ceftriaxone, and intravenous fluconazole for 10 days. Skin lesions improved in 10 days without complications.
 Conclusion: Disseminated HZ in HIV patients should be considered because of complications, recurrence, more difficult with a treat, and a higher risk of acyclovir resistance.

Swarms of chemically modified antiviral siRNA targeting herpes simplex virus infection in human corneal epithelial cells.

Herpes simplex virus type 1 (HSV-1) is a common virus of mankind and HSV-1 infections are a significant cause of blindness. The current antiviral treatment of herpes infection relies on acyclovir and related compounds. However, acyclovir resistance emerges especially in the long term prophylactic treatment that is required for prevention of recurrent herpes keratitis. Earlier we have established antiviral siRNA swarms, targeting sequences of essential genes of HSV, as effective means of silencing the replication of HSV in vitro or in vivo. In this study, we show the antiviral efficacy of 2´-fluoro modified antiviral siRNA swarms against HSV-1 in human corneal epithelial cells (HCE). We studied HCE for innate immunity responses to HSV-1, to immunostimulatory cytotoxic double stranded RNA, and to the antiviral siRNA swarms, with or without a viral challenge. The panel of studied innate responses included interferon beta, lambda 1, interferon stimulated gene 54, human myxovirus resistance protein A, human myxovirus resistance protein B, toll-like receptor 3 and interferon kappa. Our results demonstrated that HCE cells are a suitable model to study antiviral RNAi efficacy and safety in vitro. In HCE cells, the antiviral siRNA swarms targeting the HSV UL29 gene and harboring 2´-fluoro modifications, were well tolerated, induced only modest innate immunity responses, and were highly antiviral with more than 99% inhibition of viral release. The antiviral effect of the 2’-fluoro modified swarm was more apparent than that of the unmodified antiviral siRNA swarm. Our results encourage further research in vitro and in vivo on antiviral siRNA swarm therapy of corneal HSV infection, especially with modified siRNA swarms.

A Review of the Treatment of Herpes Simplex Virus-1 Encephalitis in Six Immunocompetent Patients.

IntroductionThe optimal treatment regimen for herpes simplex-1 (HSV-1) encephalitis is ill-defined. Current guidelines recommend the initiation of acyclovir in all suspected cases of encephalitis; however, there is limited research regarding the details of acyclovir treatment or the adjuvant use of corticosteroids. Specifically, there is a paucity of evidence-based guidelines detailing the optimal management of HSV-1 encephalitis in immunocompetent patients. In this study, we conducted a review of cases of immunocompetent patients with HSV-1 encephalitis to compare patterns in treatment and outcomes. MethodsA review of the literature was performed using PubMed using the terms herpes encephalitis, HSV, herpes zoster, and immunocompetent to identify cases of HSV-1 encephalitis in immunocompetent patients. The results were screened for cases describing the treatment regimen of HSV-1 encephalitis-positive, immunocompetent patients. ResultsSix cases were identified. All six patients were treated with acyclovir with one patient receiving adjuvant corticosteroid therapy. Additionally, three patients were found to have acyclovir resistance and were transitioned to foscarnet. Eventually, one patient expired, two patients recovered with chronic morbidities of varying severity, and three patients made a full recovery. DiscussionInconsistencies in the patient's disease course, therapeutic regimen, and comorbidities could all play a role in the varying case outcomes. While the optimal timing and composition of therapies in HSV-1 encephalitis in immunocompetent patients are still unclear, it seems the timely administration of antiviral treatment remains essential. Further research is needed to optimize HSV-1 encephalitis therapeutic regimens and improve patient outcomes.

Phenotypic and genotypic acyclovir resistance surveillance of genital herpes simplex virus 2 in South Africa

Acyclovir (ACV) is currently included in the syndromic management algorithm for genital ulcer disease in South Africa, and is the recommended first-line treatment for herpes simplex virus 2 (HSV-2). In the majority of cases, HSV-2 resistance to ACV is due to amino acid changes within the viral thymidine kinase (TK). Phenotypic and genotypic ACV resistance surveillance of HSV-2 derived from genital ulcer disease swab specimens was conducted at a primary healthcare facility in Johannesburg between 2018 and 2020. The objectives of this surveillance were to identify ACV resistance-associated mutations and polymorphisms in HSV-2 TK, and to determine the phenotypic ACV resistance profiles of the corresponding clinical HSV-2 isolates. Genotypic analysis of TK from 67 HSV-2 positive genital ulcer swabs revealed 48 specimens with TK mutations, conferring 113 nucleotide changes. No resistance-associated mutations were found, however, we identified nine known natural polymorphisms (R26H, A27T, S29A, G39E, N78D, L140F, T159I, R220K and R284S) and five amino acid changes of unknown significance (R18C, G39K, M70R, P75S and L263P). Phenotypic susceptibility testing of 52 cultivable HSV-2 isolates revealed all to be susceptible to ACV with IC50 values of <2 μg/ml. The five amino acid changes of unknown significance identified by genotypic testing were not correlated to phenotypic ACV resistance, and therefore grouped as natural polymorphisms. We did not detect any unknown or resistance-associated mutations in specimens that could not be phenotypically tested for ACV resistance. Our findings will supplement existing databases of HSV antiviral resistance-associated mutations and polymorphisms that could be used for genotypic ACV resistance screening.

Acyclovir-Resistant Herpes Simplex Virus 1 Keratitis: A Concerning and Emerging Clinical Challenge

To describe the clinical and virological profiles of patients with herpes simplex keratitis (HSK) caused by acyclovir-resistant (ACVR) strains of herpes simplex virus 1 (HSV-1). Multicenter retrospective case series. HSV-1 resistance to ACV was confirmed using sequencing of genes encoding HSV-1 thymidine kinase (TK) and DNA polymerase (DNA pol). Data were collected on the number of HSK episodes before and after the diagnosis of resistance, ocular findings including the type of HSK, immune status of patients, antiviral treatments, and HSV-1 genotypic resistance profiles. This study evaluated 18 HSK patients (13 male and 5 female, aged 66.8 ± 4.7 years) with ACVR HSV-1-positive ocular samples. Genotypic resistance testing was performed because of frequent recurrences despite adequate antiviral prophylaxis (AVP) (n=13, 72%), or poor response to suppressive antiviral therapy (n=5, 28%). Resistance mutations were found in the TK (n=15, 83%) or in the DNA pol gene (n=3, 17%). Prior to the diagnosis of resistance, the duration of disease was 29.8 ± 20.4 years, with more than 10 HSK recurrences in 15 patients (83%). The number of recurrences between the first episode and the diagnosis of resistance was significantly lower in immunocompromised patients (n=6, 33%) than in immunocompetent patients (n=12; 67%) (11.5 ± 4.9 vs 16.4 ± 1.9, P=.05). HSV-1 resistance to ACV must be suspected in HSK patients with recurrences despite AVP and/or in cases that respond poorly to a suppressive antiviral regimen. Immunocompromised patients and/or those with longstanding disease may be particularly at risk for developing resistance.

1060. Pritelivir in Immunocompromised Patients with Mucocutaneous Acyclovir-Resistant Herpes Simplex Virus-Infections – First Case Series

BackgroundHSV recurrences are usually managed effectively with existing antiviral drugs (nucleoside analogs such as acyclovir). However, in immunocompromised patients (e.g., malignancy, HIV, transplant), if lesions persist or recur while receiving antiviral treatment, acyclovir resistance should be suspected. In this population, there are limited treatment options. The helicase-primase inhibitor pritelivir is a novel oral antiviral, with a new mode of action and is active against both HSV-1 and HSV-2, including acyclovir and foscarnet-resistant strains. In this case series, we report the first clinical experiences with pritelivir in the treatment of immunocompromised patients with acyclovir resistant HSV infection.MethodsAll patient reported in this case series received pritelivir in a Phase 2 study. There were treated in an open-label design with a 400 mg pritelivir oral loading dose followed by a 100 mg oral maintenance dose daily for up to 28 days.ResultsOf the 23 patients, 11 had HIV infection and 12 had malignancy, transplant or an autoimmune disease. Of this cohort, 19 patients showed full resolution of their HSV-related lesions during the 28 day treatment period, while in 4 subjects lesions improved but did not completely heal during the observation period. Pritelivir was well tolerated without significant adverse effects.Reasons for incomplete lesion resolution during the 28 day treatment period, were extensive lesions in one patient, one patient with resistance development, and one patient with lesions in the oral cavity. Three patients subsequently experienced full resolution, while one patient required foscarnet due to CMV reactivation, necessitating early discontinuation.ConclusionPritelivir is a promising novel treatment option for patients with severe mucocutaneous HSV-1/2 infections that are resistant to acyclovir and foscarnet. An international Phase 3 study is underway to evaluate pritelivir efficacy in immunocompromised patients.Disclosures Joerg Albrecht, MD/PhD, Biogen (Scientific Research Study Investigator)Investigator for AiCuris (Scientific Research Study Investigator) Robin K. Avery, MD, Aicuris (Grant/Research Support)Astellas (Grant/Research Support)Chimerix (Research Grant or Support)Merck (Grant/Research Support)Oxford Immunotec (Grant/Research Support)Qiagen (Grant/Research Support)Takeda/Shire (Grant/Research Support) Roy F. Chemaly, MD, MPH, FACP, FIDSA, AiCuris (Grant/Research Support)Ansun Biopharma (Consultant, Grant/Research Support)Chimerix (Consultant, Grant/Research Support)Clinigen (Consultant)Genentech (Consultant, Grant/Research Support)Janssen (Consultant, Grant/Research Support)Karius (Grant/Research Support)Merck (Consultant, Grant/Research Support)Molecular Partners (Consultant, Advisor or Review Panel member)Novartis (Grant/Research Support)Oxford Immunotec (Consultant, Grant/Research Support)Partner Therapeutics (Consultant)Pulmotec (Consultant, Grant/Research Support)Shire/Takeda (Consultant, Grant/Research Support)Viracor (Grant/Research Support)Xenex (Grant/Research Support) Nicolas C. Issa, MD, AiCuris (Scientific Research Study Investigator)Astellas (Scientific Research Study Investigator)GSK (Scientific Research Study Investigator)Merck (Scientific Research Study Investigator) Camille Kotton, MD, Shire/Takeda (Advisor or Review Panel member) Camille Kotton, MD, UpToDate (Individual(s) Involved: Self): I write chapters on zoonoses for UpToDate., Independent Contractor Princy N. Kumar, MD, AMGEN (Other Financial or Material Support, Honoraria)Eli Lilly (Grant/Research Support)Gilead (Grant/Research Support, Shareholder, Other Financial or Material Support, Honoraria)GSK (Grant/Research Support, Shareholder, Other Financial or Material Support, Honoraria)Merck & Co., Inc. (Grant/Research Support, Shareholder, Other Financial or Material Support, Honoraria) Moti Ramgopal, MD FACP FIDSA, Abbvie (Scientific Research Study Investigator, Speaker’s Bureau)Gilead (Consultant, Scientific Research Study Investigator, Speaker’s Bureau)Janssen (Consultant, Scientific Research Study Investigator, Research Grant or Support, Speaker’s Bureau)Merck (Consultant, Scientific Research Study Investigator)ViiV (Consultant, Scientific Research Study Investigator, Speaker’s Bureau) Anna Wald, MD, MPH, Aicuris (Consultant)Crozet (Consultant)GSK (Scientific Research Study Investigator)Merck (Other Financial or Material Support, DSMB)Sanofi (Scientific Research Study Investigator)X-Vax (Consultant) Michael G. Ison, MD, MS, Celltrion, Inc. (Consultant)

Opposite effects of cytomegalovirus UL54 exonuclease domain mutations on acyclovir and cidofovir susceptibility

Acyclovir has weak activity against human cytomegalovirus (CMV). Despite some efficacy as prophylaxis, more potent anti-CMV drugs are preferred. Acyclovir resistance of CMV has been little studied. The viral UL97 kinase phosphorylates acyclovir, and cross-resistance of ganciclovir-resistant mutants is documented. However, UL54 exonuclease domain mutants may confer ganciclovir and cidofovir resistance by a mechanism that does not apply to acyclovir as an obligate chain terminator. To test for differential susceptibilities, 11 exonuclease domain mutants were tested for their 50% inhibitory concentrations (EC50s) of acyclovir in comparison with cidofovir. The 5 mutants with the highest cidofovir EC50s (>10-fold increased over wild type) all had acyclovir EC50s less than 20% of wild type. The relatively common N408K mutant had an acyclovir EC50 of 6 μM, comparable to that reported for wild type varicella-zoster virus. Several foscarnet-resistant UL54 mutants outside the exonuclease domains, some with low-grade ganciclovir/cidofovir cross-resistance, showed various degrees of acyclovir resistance. Based on these in vitro data, acyclovir may become a therapeutic option when a highly cidofovir-resistant exonuclease mutation is present without a simultaneous mutation in UL97.

Susceptibility Evaluation of Clinically Isolated HSV-1 Strains to Acyclovir: A Phenotypic and Genotypic Study

Background: Mutations in herpes simplex virus Thymidine kinase (TK, UL23) and DNA polymerase (pol, UL30) genes may confer resistance to acyclovir (ACV). Phenotypic resistance must be determined along with genotypic resistance to achieve complete acyclovir susceptibility. Objectives: The present study aimed to characterize HSV-1 clinical isolates from outpatients and organ transplant recipients in terms of phenotypic ACV resistance. Moreover, genotypic resistance to ACV was assessed through sequencing the viral TK and pol genes amplified from virus-infected cell DNA. Methods: Twenty-six HSV-1 clinical isolates collected between 2016 and 2019 were examined for drug susceptibility. The samples were collected from eyes, oropharyngeal, facial, and other skin parts of immunocompetent and immunocompromised individuals. Phenotypic susceptibility was determined by using three different concentrations of ACV. The results were expressed based on the ability of ACV in reducing viral plaques by 50%. Genotyping was carried out by polymerase chain reaction and sequencing of TK and pol genes. Results: All the strains were characterized as sensitive at 0.01 and 0.05 µg.ml-1 concentrations to ACV. Seventy percent inhibition was observed at ≥ 0.1 µg.mL-1 of ACV for three isolates (two from patients who received transplants and one from an outpatient). Nine natural polymorphisms were detected in the TK gene and 31 in the Pol gene. Furthermore, four susceptible-associated mutations in the DNA pol gene were analyzed. A substitution was encoded in the conserved region of the pol Exo III motif (M553L), and nine amino acid substitutions in TK were detected. The phylogenetic analysis of partial genome sequences revealed high diversity in the TK and pol genes of HSV-1. Conclusions: A higher number of mutations were observed in patients who received transplants and underwent long-term treatment compared with outpatients. The high genetic variability of HSV-1 TK and DNA pol was not associated with phenotypic resistance.

Antiviral susceptibility of recombinant Herpes simplex virus 1 strains with specific polymerase amino acid changes

Acyclovir (ACV) and penciclovir and their prodrugs are recommended for therapy or prophylaxis of Herpes simplex virus 1 (HSV-1) infections. Their administration, however, can lead to the emergence of resistant strains with altered viral thymidine kinase (TK) function, especially in immunocompromised patients. Furthermore, amino acid (aa) changes of the viral deoxyribonucleic acid polymerase (POL) may contribute to resistance to the aforementioned nucleoside analogues. Given this, treatment with foscarnet (FOS) or cidofovir (CDV) may represent an important alternative. Both drugs directly affect POL activity.Several aa changes of POL, such as L49I, E70K, L359I, E421V, P829S, T1121M, and M1226I, have been observed in ACV-resistant clinical strains which also carried relevant aa changes in their TK. Their contribution to ACV, FOS, and CDV resistance is not fully understood. In this study, these seven aa changes with unknown significance for ACV, FOS and CDV resistance were introduced separately into the POL of a recombinant HSV-1 strain rHSV-1(17+)Lox, equipped with or without information for expression of green fluorescent protein (GFP). The GFP-expressing variants were tested for susceptibility to ACV, FOS and CDV. An rHSV-1(17+)Lox GFP strain with the S724N change conferring resistance to ACV and FOS was generated and included as a control.Only the S724N change was confirmed to induce ACV and FOS resistance, whereas the other changes did not contribute to resistance. The underlying nucleotide substitutions of the POL gene should be therefore considered as natural polymorphism. These data will improve sequence-based prediction of antiviral susceptibility.

Isolation, characterization and acyclovir susceptibility of herpes simplex virus isolates among immunocompromised patients.

Introduction:Herpes simplex virus (HSV) Type 2 primarily causes genital herpes, while HSV Type 1 is responsible for oral and facial lesions. The objective of this study was to isolate and characterize HSV from herpetic lesions among human immunodeficiency virus (HIV) infected patients and to evaluate their acyclovir susceptibility pattern.Materials and Methods:Blister fluid and swabs from ulcers were collected from patients with clinical diagnosis of HSV infection among patients attending the HIV clinic of two tertiary care centers – Medical College, Kolkata, and School of Tropical Medicine, Kolkata. These samples were cultured in the Vero cell line. Growth of virus was noted by observing the characteristic cytopathic effect of HSV, which was further confirmed by immunofluorescence and polymerase chain reaction (PCR). These isolates were then subjected to the Vero cells with serial dilutions of acyclovir for determining the susceptibility pattern.Results:Among the 52 samples received, 8 (15.38%) showed growth of HSV. After confirmation by immunofluorescence and PCR, all seven isolates from genital samples were identified as HSV-2 and the lone isolate from oral lesion was confirmed as HSV 1. Out of the eight isolates, 25% showed resistance to acyclovir. The overall isolation rate was more from genital blister than genital ulcer which was 46.15% and 2.86%, respectively.Conclusion:HSV was isolated in 15.38% of cases of clinical herpes. There was a higher isolation rate of virus from blister fluid as compared to ulcer scrapings. Acyclovir resistance in 25% of cases is alarmingly high.

Herpes Simplex Virus Type 1 Clinical Isolates Respond to UL29-Targeted siRNA Swarm Treatment Independent of Their Acyclovir Sensitivity.

Acyclovir is the drug of choice for the treatment of herpes simplex virus (HSV) infections. Acyclovir-resistant HSV strains may emerge, especially during long-term drug use, and subsequently cause difficult-to-treat exacerbations. Previously, we set up a novel treatment approach, based on enzymatically synthesized pools of siRNAs, or siRNA swarms. These swarms can cover kilobases-long target sequences, reducing the likelihood of resistance to treatment. Swarms targeting the UL29 essential gene of HSV-1 have demonstrated high efficacy against HSV-1 in vitro and in vivo. Here, we assessed the antiviral potential of a UL29 siRNA swarm against circulating strains of HSV-1, in comparison with acyclovir. All circulating strains were sensitive to both antivirals, with the half-maximal inhibitory concentrations (IC50) in the range of 350–1911 nM for acyclovir and 0.5–3 nM for the UL29 siRNA swarm. Additionally, we showed that an acyclovir-resistant HSV-1, devoid of thymidine kinase, is highly sensitive to UL29 siRNA treatment (IC50 1.0 nM; Imax 97%). Moreover, the detected minor variations in the RNAi target of the HSV strains had no effect on the potency or efficacy of UL29 siRNA swarm treatment. Our findings support the development of siRNA swarms for the treatment of HSV-1 infections, in order to circumvent any potential acyclovir resistance.