Claire Deback

Genotypic Characterization of UL23 Thymidine Kinase and UL30 DNA Polymerase of Clinical Isolates of Herpes Simplex Virus: Natural Polymorphism and Mutations Associated with Resistance to Antivirals

ABSTRACT The molecular mechanisms of herpes simplex virus (HSV) resistance to antiviral drugs interfering with viral DNA synthesis reported so far rely on the presence of mutations within UL23 (thymidine kinase [TK]) and UL30 (DNA polymerase) genes. The interpretation of genotypic antiviral resistance assay results requires the clear distinction between resistance mutations and natural interstrain sequence variations. The objectives of this work were to describe extensively the natural polymorphism of UL23 TK and UL30 DNA polymerase among HSV-1 and HSV-2 strains and the amino acid changes potentially associated with HSV resistance to antivirals. The sequence analysis of the full-length UL23 and UL30 genes was performed. Ninety-four drug-sensitive clinical isolates (43 HSV-1 and 51 HSV-2) and 3 laboratory strains (KOS, gHSV-2, and MS2) were studied for natural polymorphism, and 25 clinical isolates exhibiting phenotypic traits of resistance to antivirals were analyzed for drug resistance mutations. Our results showed that TK and DNA polymerase are highly conserved among HSV strains, with a weaker variability for HSV-2 strains. This study provided a precise map of the natural polymorphism of both viral enzymes among HSV-1 and HSV-2 isolates, with the identification of 15 and 51 polymorphisms never previously described for TK and DNA polymerase, respectively, which will facilitate the interpretation of genotypic antiviral-resistant testing. Moreover, the genotypic characterization of 25 drug-resistant HSV isolates revealed 8 new amino acid changes located in TK and potentially accounting for acyclovir (ACV) resistance.

Exhausted cytotoxic control of Epstein-Barr virus in human lupus.

Systemic Lupus Erythematosus (SLE) pathology has long been associated with an increased Epstein-Barr Virus (EBV) seropositivity, viremia and cross-reactive serum antibodies specific for both virus and self. It has therefore been postulated that EBV triggers SLE immunopathology, although the mechanism remains elusive. Here, we investigate whether frequent peaks of EBV viral load in SLE patients are a consequence of dysfunctional anti-EBV CD8+ T cell responses. Both inactive and active SLE patients (n = 76 and 42, respectively), have significantly elevated EBV viral loads (P = 0.003 and 0.002, respectively) compared to age- and sex-matched healthy controls (n = 29). Interestingly, less EBV-specific CD8+ T cells are able to secrete multiple cytokines (IFN-γ, TNF-α, IL-2 and MIP-1β) in inactive and active SLE patients compared to controls (P = 0.0003 and 0.0084, respectively). Moreover, EBV-specific CD8+ T cells are also less cytotoxic in SLE patients than in controls (CD107a expression: P = 0.0009, Granzyme B release: P = 0.0001). Importantly, cytomegalovirus (CMV)-specific responses were not found significantly altered in SLE patients. Furthermore, we demonstrate that EBV-specific CD8+ T cell impairment is a consequence of their Programmed Death 1 (PD-1) receptor up-regulation, as blocking this pathway reverses the dysfunctional phenotype. Finally, prospective monitoring of lupus patients revealed that disease flares precede EBV reactivation. In conclusion, EBV-specific CD8+ T cell responses in SLE patients are functionally impaired, but EBV reactivation appears to be an aggravating consequence rather than a cause of SLE immunopathology. We therefore propose that autoimmune B cell activation during flares drives frequent EBV reactivation, which contributes in a vicious circle to the perpetuation of immune activation in SLE patients.

Herpes simplex encephalitis : from virus to therapy.

Herpes simplex virus (HSV) is the cause of herpes simplex encephalitis (HSE), a devastating human disease which occurs in 2-4 cases per million/year. HSE results either from a primary infection or virus reactivation, in accordance with the common pattern of HSV infection which is a chronic lifelong process. However its pathophysiology remains largely unknown and its poor prognosis is in contrast with the usually good tolerance of most clinical herpetic manifestations. HSE is due to HSV type 1 (HSV-1) in most cases but HSV type 2 (HSV-2) may be also implicated, especially in infants in the context of neonatal herpes. Polymerase chain reaction detection of HSV DNA in cerebrospinal fluid is the diagnosis of choice for HSE. Acyclovir, a nucleoside analogue which inhibits viral DNA polymerase activity, is the reference treatment of HSE while foscarnet constitutes an alternative therapy and the efficacy of cidofovir is currently uncertain in that context. The emergence of HSV resistance to acyclovir, a phenomenon which is mainly observed among immunocompromised patients, is a current concern although no case of HSE due to an acyclovir-resistant HSV strain has been reported to date. Nevertheless the identification and development of novel therapeutic strategies against HSV appears to be a non dispensable objective for future research in virology.

Resistance pattern of cytomegalovirus (CMV) after oral valganciclovir therapy in transplant recipients at high-risk for CMV infection.

In transplant recipients, cytomegalovirus (CMV) resistance to antivirals causes an increasing problem. Here we report the clinical, therapeutic, and virological characteristics of 11 cases of CMV resistance among transplant recipients at high-risk for CMV infection and receiving valganciclovir as a prophylactic, preemptive or maintenance therapy. Active CMV infection was monitored by viral DNA quantification in whole blood, and CMV resistance was assessed by UL97 and UL54 viral gene sequencing. For 10 patients, ganciclovir resistance detected after valganciclovir therapy was associated with one mutation within UL97 phosphotransferase located at codons 460 and 592-603, which constitutes a similar pattern of resistance to what has been reported previously in AIDS patients treated with valganciclovir. For the last patient, two mutations in UL97 and UL54 genes were identified. The start of valganciclovir maintenance treatment after an intravenous curative treatment while CMV DNA is still detectable in peripheral blood might represent a risk factor for the emergence of CMV resistance. The possible emergence of CMV resistance in transplant recipients at high-risk for CMV infection who receive valganciclovir therapy should be taken into account. Among those patients, CMV infection has to be closely monitored in order to detect promptly the emergence of drug-resistance.

Successful treatment of aciclovir and foscarnet resistant Herpes simplex virus lesions with topical imiquimod in patients infected with human immunodeficiency virus type 1

Aciclovir (ACV)‐resistant Herpes simplex virus type‐2 (HSV‐2) infections are observed commonly in patients also infected with HIV‐1. The use of foscarnet (FOS) in these patients may also lead to resistance. This situation can become a difficult therapeutic challenge. Four cases of patients infected with HIV and with mucocutaneous HSV‐2 resistant to ACV and FOS are reported. These patients were treated successfully with topical 5% imiquimod. Imiquimod treatment also appeared to delay the time to recurrence of HSV lesions. J. Med. Virol. 84:194–197, 2012.

Microsatellite analysis of HSV-1 isolates: from oropharynx reactivation toward lung infection in patients undergoing mechanical ventilation.

BACKGROUND According to recent reports, herpes simplex virus type 1 (HSV-1) induces bronchopneumonitis (BPn) in immunocompetent patients undergoing prolonged mechanical ventilation (MV), whose respiratory functions deteriorate with a poor outcome. HSV-1 BPn is associated with HSV symptomatic or symptomless reactivation in the oropharynx. OBJECTIVES We sought to systematically and genetically characterize HSV-1 strains isolated from immunocompetent patients receiving prolonged MV and to characterize the genetic relationship of strains sequentially isolated from oropharyngeal samples (OPS) and broncho-alveolar liquids (BAL) to determine the natural course of HSV BPn. STUDY DESIGN In this molecular epidemiological study, microsatellite technology was used to determine genetic relationships between 211 HSV-1 strains isolated from OPS and/or BAL from 106 patients receiving MV. RESULTS Microsatellite haplotypes of HSV-1 strains sequentially isolated from the same individual were identical, and HSV-1 isolates from the lung were genetically indistinguishable from strains isolated from the oral cavity. Each patient was characterized by their own HSV-1 microsatellite haplotype, and no nosocomial transmission of strains between patients was observed. CONCLUSION Our results demonstrate that, in patients who receive MV, the HSV-1 pulmonary infection results from the reactivation of genetically related HSV-1 in the oropharynx, which progressively infects the lower respiratory tract.

Management of multidrug-resistant CMV infection in immunocompromised patients: case report of a heart-transplant recipient and review of the literature.

Cytomegalovirus (CMV) remains a leading cause of morbidity after solid organ transplantation. The efficiency of antivirals for the treatment of CMV infections may be hampered because of the emergence of CMV resistance to antivirals. The development of CMV multidrug resistance, which remains uncommon but does occur, constitutes a clinically challenging complication and may contribute to difficult therapeutic management and adverse clinical outcome. We report here the observation of the emergence of a multidrug-resistant CMV infection in a heart-transplant recipient and review the literature on similar cases to identify the potential strategies for the successful management of CMV multidrug resistance among immunocompromised patients.

Dynamics of cytomegalovirus populations harbouring mutations in genes UL54 and UL97 in a haematopoietic stem cell transplant recipient

We characterised by pyrosequencing, the dynamics of cytomegalovirus populations harbouring mutations A594V in gene UL97 and A834P and Q578H in gene UL54 in a haematopoietic stem cell transplant recipient. Unexpected re-emergence of A594V and decrease of A834P under CMX001 were shown to depend on both the selection pressure exerted by the antiviral treatments and the immune response.

Testing the susceptibility of human herpesviruses to antivirals

Herpesviruses cause chronic lifelong infections in humans and may cause life-threatening diseases in immunosuppressed patients. Antiviral drugs targeted to viral DNA polymerase, such as acyclovir, penciclovir, ganciclovir, foscarnet and cidofovir, are currently available and have been proven to be efficient against clinical symptoms of herpesvirus infections. The resistance of herpesviruses to these drugs is associated with specific mutations of viral genes encoding either DNA polymerase or enzymes phosphorylating nucleoside analogs. Resistance is detected and characterized by means of specific susceptibility assays, which can be classified as phenotypic, genetic and functional. These tests are used both to investigate novel antiviral compounds and look for the emergence of resistant viruses in treated patients in case of clinical failure. Although susceptibility assays are often time consuming and present some limitations regarding the interpretation of their results, their use in the monitoring of antiherpetic treatments should be promoted and improved, in parallel to the development of novel efficient drugs.

Detection of Pandemic (H1N1) 2009 Virus in Patients Treated with Oseltamivir

To the Editor: In April 2009, an influenza outbreak caused by a novel strain of influenza virus A (H1N1) was identified in Mexico. The rapid spread of this new virus among humans led the World Health Organization to raise the phase of pandemic alert to 6. We report results from the 2 virology laboratories from university hospitals that were involved in the surveillance network of pandemic (H1N1) 2009 in Paris at the beginning of the outbreak in France. Patients exhibiting influenza-like illness (i.e., fever, sore throat, cough, asthenia, headache, myalgia) and who recently had traveled to countries where the pandemic (H1N1) 2009 outbreak had started (i.e., Mexico, United States, Canada, Japan) were hospitalized. Symptoms began either the day before or the day of hospitalization. Nasal-swab specimens were collected at admission by using the Virocult system (ELITech; Salon-de-Provence, France), and treatment with oseltamivir was started (75 mg, 2×/day). Pandemic (H1N1) 2009 infection was diagnosed by using rapid test QuickVue Influenza A+B (Quidel, San Diego, CA, USA) and real-time reverse transcription–PCR (RT-PCR) assays from the French National Influenza Centers or the US Centers for Disease Control and Prevention (1). In the case of a positive result, influenza virus in nasal secretions from patients was monitored daily by RT-PCR until viral genomes became undetectable. From April 24 through June 7, 2009, nasal swab specimens from 234 persons (132 men; median age of all patients 33 years) were processed; pandemic (H1N1) 2009 infection was confirmed for 17 men and 15 women (median age 33 years) by RT-PCR. Results of the Quidel rapid tests were available for 27 specimens, with positive results for 9 (33% sensitivity). However, no positive result was observed with the Quidel rapid tests among the nasal-swab specimens with negative RT-PCR results (100% specificity). Influenza virus detection in nasal secretions was monitored for 16 patients who had laboratory-confirmed pandemic (H1N1) 2009 infection and were treated with oseltamivir. Viral detection by RT-PCR was absent 2 to >5 days after antiviral treatment began (Figure). Significant differences were not found in sex and age of the patients (data not shown). Figure Duration of pandemic (H1N1) 2009 excretion in nasal swabs from patients treated with oseltamivir. The number of days from start of oseltamivir treatment to achievement of negative results of reverse transcription–PCR (RT-PCR) is indicated for ... These preliminary virologic data obtained during the first 6 weeks of pandemic (H1N1) 2009 in France confirm the poor sensitivity of the Quidel test toward this new virus, as recently reported (2). Further studies are needed to evaluate the performances of other rapid tests. Hayden et al. (3) demonstrated that treatment with oseltamivir significantly reduced duration of viral shedding among patients infected with seasonal influenza virus A (H1N1), in comparison with a placebo group: 1.5–2.5 days vs. 3.5–5.5 days (p = 0.003). In our study, surprisingly, PCR results for sequential nasal swab specimens from 16 patients infected by pandemic (H1N1) 2009 and treated with oseltamivir were negative within 3 days after therapy for only 9 (56%); indeed, for 3 (19%) patients, viral genome could be detected >5 days after antiviral treatment began. These data raise questions about potential virus transmission during antiviral treatment and the possible resistance of pandemic (H1N1) 2009 to oseltamivir. This latter point is now under study.