Drug targets in herpes simplex and Epstein Barr Virus infections.

Herpes simplex virus (HSV) and Epstein Barr Virus (EBV) are Herpesviridae. Although infections are often subclinical, HSV can cause mild to severe diseases, especially in immunocompromised patients. EBV infections are also often asymptomatic but this virus may be associated to carcinoma in immunocompetent patients and to severe diseases in immunocompromised patients. These viruses establish latency, in neuronal cells for HSV and in B-cells for EBV, and may reactivate, with or without symptoms. There are few drugs licensed for the treatment of HSV infections. Most of them target the viral DNA polymerase, in which acyclovir remains the reference treatment some thirty years after its discovery. The emergence of resistant viral strains, mainly in immunocompromised patients, highlights the crucial need for the development of new anti-herpes drugs that can inhibit infection by both wild-type viruses and acyclovir-resistant strains. No antiviral drug has been yet licensed for EBV. Therapies mainly rely on control of immunosuppression and/or immunotherapies. Antiviral drug such as acyclovir and ganciclovir have been used with limited impact except when used with drugs that induce EBV lytic cycle. Over the last few years, significant efforts have been made to set up a range of strategies for the identification of potential new antiviral drugs. For HSV, and more strikingly for EBV, there is a crucial need for antiviral drug active on latent virus. One alternative is to develop drugs with different mechanisms of action. The present article reviews potential targets, viral and cellular, for which specific inhibitors have been identified.

Differential susceptibility of adenovirus clinical isolates to cidofovir and ribavirin is not related to species alone.

BACKGROUND: We have previously reported that human adenovirus (HAdV) reference strains clearly show species-dependent resistance to ribavirin, whereas different species of HAdV are equally sensitive to cidofovir. All the serotypes tested were susceptible to cidofovir, whereas only serotypes from species C were sensitive to ribavirin. Here, we aimed to extend these investigations to clinical isolates. METHODS: In vitro, we tested 126 isolates obtained from 65 patients included in a European survey of HAdV infection. RESULTS: Among the 126 isolates tested, all presented cidofovir 50% inhibitory concentration (IC50) in the same range as the HAdV 5 reference strain. Regarding ribavirin, all isolates from species C (79 tested) showed an IC50 comparable with previously reported results for reference strains; however, 24/32, 2/6 and 3/3 tested isolates from species A, B and D, respectively, were shown to have a ribavirin IC50 comparable with the HAdV 5 reference strain (species C), contrary to previous observations for reference strains of the same species. Among patients who were treated with cidofovir for disseminated HAdV infection, > or = 4 sequential isolates could be obtained from 9 patients; no variation in cidofovir susceptibility could be detected. CONCLUSIONS: Cidofovir is active in vitro in all HAdV clinical isolates. Ribavirin was revealed to be active on most HAdV isolates from species A, B and D, and in all isolates from species C. Finally, no resistance to cidofovir became apparent in sequential isolates obtained from treated patients.

Genotypic detection of acyclovir-resistant HSV-1: characterization of 67 ACV-sensitive and 14 ACV-resistant viruses.

Infections due to herpes simplex virus (HSV) resistant to acyclovir (ACV) represent an important clinical concern in immunocompromised patients. In order to switch promptly to an appropriate treatment, rapid viral susceptibility assays are required. We developed herein a genotyping analysis focusing on thymidine kinase gene (TK) mutations in order to detect acyclovir-resistant HSV in clinical specimens. A total of 85 HSV-1 positive specimens collected from 69 patients were analyzed. TK gene could be sequenced directly for 81 clinical specimens (95%) and 68 HSV-1 specimens could be characterized as sensitive or resistant by genotyping (84%). Genetic characterization of 67 susceptible HSV-1 specimens revealed 10 polymorphisms never previously described. Genetic characterization of 14 resistant HSV-1 revealed 12 HSV-1 with either TK gene additions/deletions (8 strains) or substitutions (4 strains) and 2 HSV-1 with no mutation in the TK gene. DNA polymerase gene was afterwards explored. With this rapid PCR-based assay, ACV-resistant HSV could be detected directly in clinical specimens within 24 h.

Influenza burden in febrile infants and young children in a pediatric emergency department.

BACKGROUND: In France, epidemiologic data in children in ambulatory settings are scarce. We aimed to measure the burden of influenza in young children. METHODS: Febrile children younger than 36 months were consecutively recruited in a pediatric emergency department during the 2002 epidemic peak. Virology analysis and follow-up were systematic. RESULTS: During calendar weeks 3 to 6, 2002, 575 children were recruited; 49% were positive: A/H3N2 in 44% and B in 5%. Prevalence rate was 57% in 12- to 35-month-old children and 39% in infants younger than 12 months. The main clinical pictures were nonrespiratory in one third of them. One of 8 patients had a complication. One of 10 patients was hospitalized, and the estimated specific hospitalization rate for the study period was 237 of 100,000 in the general population among infants younger than 12 months. Forty-two percent of children (n = 110) were prescribed antibiotics and at least 34% of them were inappropriate (n = 89). Median length of disease was 8 days, and 25% of the children had not fully recovered by day 11. Only one child had been previously vaccinated of 65 with chronic conditions. Both epidemic strains were covered by the vaccine. CONCLUSIONS: Health outcomes showed that influenza disease burden in young French children is similar to that observed in North America. An active vaccination strategy would have strongly reduced the burden of influenza and lowered antibiotic use. Continuous efforts are needed to reach requirements of our influenza vaccination policy.

Resistance of herpes simplex virus type 1 to acyclovir: thymidine kinase gene mutagenesis study.

By site-directed mutagenesis, we investigate the role of six mutations of herpes simplex virus type 1 thymidine kinase (TK) gene in the acquisition of resistance to acyclovir (ACV). TK activity was not impaired by substitutions located at codons 17, 161 and 374 and these mutations were thus related to TK gene polymorphism. Mutations His105Pro, Leu364Pro and Asp162Ala lead to the loss of TK activity that could result in ACV-resistance.

Human parainfluenza virus type 4 infections: a report of 20 cases from 1998 to 2002.

BACKGROUND: Human Parainfluenza Viruses (HPIV) type 4 are responsible for respiratory infections. Unlike HPIV types 1-3, they are associated with mild infections and appear to be infrequent. Thus, they often go undetected. STUDY DESIGN: From 1998 to 2002, in 20 respiratory samples of hospitalised patient, we isolated viruses presenting a large syncytial cytopathic effect when inoculated on LLC-MK2 cells. Most of the patients (16/20) were young infants and all of them presented with respiratory infections. RESULTS: We detected 18 cases during autumn and winter, 1 case during spring and 1 during summer. We could not identify these viruses using the panel of routine assays. Samples were then analysed by specific HPIV 4 RT-PCR and IF assays. All the samples were scored positive with both methods. CONCLUSION: We conclude that HPIV 4 infections are probably underestimated. Their role in viral respiratory infections should be carefully investigated using techniques adapted to their detection and culture.

In vitro susceptibility of adenovirus to antiviral drugs is species-dependent.

Adenovirus infections are a frequent and serious complication following allogeneic haematopoietic stem cell transplantation (HSCT). The antiviral drugs cidofovir and ribavirin have been used as first-line therapy for disseminated infections with variable results. In the present study, in vitro susceptibility to these two drugs was evaluated on HEp-2 cells in adenovirus reference strains representing serotypes of each of the six species and in clinical isolates. Susceptibility to cidofovir was comparable between species with inhibition of replication of all tested serotypes in a narrow dose range (IC50=17-81 microM). However, susceptibility to ribavirin was highly dependent on the species. Serotypes from species A, B, D, E and F were all resistant to ribavirin (IC50=396 to >500 microM). Only replication of serotypes from species C was inhibited by ribavirin (IC50=48-108 microM). This species-dependent susceptibility of adenovirus to ribavirin was confirmed in clinical isolates. When tested on other cell lines (PLC, A549 and 293), all species were revealed to be resistant to ribavirin. If our in vitro findings are predictive of virological responses in vivo, these results suggest that ribavirin would not be effective for management of non-C species adenovirus infections after HSCT.

S-adenosyl methionine decarboxylase activity is required for the outcome of herpes simplex virus type 1 infection and represents a new potential therapeutic target.

All the available antiherpetic drugs are directed against viral proteins. Their extensive clinical use has led to the emergence of resistant viral strains. There is a need for the treatment of herpes infections due to resistant strains, especially for immunocompromised patients. To design new kinds of drugs, we have developed a strategy to identify cellular targets. Herpes simplex virus type 1 (HSV-1) infection is concomitant to a repression of most host protein synthesis. However, some cellular proteins continue to be efficiently synthesized. We speculated that some of them could determine the outcome of infection. Since two polyamines, spermidine and spermine, are components of the HSV-1 virions, we investigated whether enzymes involved in their synthesis could be required for viral infection. We show that inhibition of S-adenosyl methionine decarboxylase, a key enzyme of the polyamine metabolic pathway, prevents HSV-1 infection. Inhibition of polyamine synthesis prevents infection of culture cells with HSV-1 laboratory strains as well as clinical isolates that are resistant to the conventional antiviral drugs acyclovir and foscarnet. Our data provide the opportunity to develop molecules with a novel mechanism of action for the treatment of herpes infection.

HSV excretion after bone marrow transplantation: a 4-year survey.

BACKGROUND: Herpes simplex virus (HSV) oral excretions are common after bone marrow transplantation (BMT). OBJECTIVE: We report a 4-year systematic survey of HSV excretions in an adult population who underwent BMT (289 transplantations). STUDY DESIGN: Patients received either intravenous ACV treatment when mucositis occurred or systematic intravenous ACV prophylaxis from initiation of the BMT conditioning until the end of aplasia. All patients were followed up for HSV excretions. RESULTS: Twenty-eight patients (9.7%) excreted HSV. The occurrence of HSV excretions was similar in both allogeneic and autologous transplant patients. The incidence was significantly lower when ACV was systematically used after transplantation (2.5%) compared to when ACV was implemented for mucositis (12%). ACV-resistant HSV was detected in three patients who received allogeneic transplantation, representing 27% of allogeneic recipients excreting HSV. CONCLUSION: HSV infection prophylaxis with high dose of intravenous ACV resulted in a decreased incidence of HSV excretion. Nevertheless, the risk of emergence of ACV resistance, especially among allogeneic transplant patients, appears to be identical whatever the route and dose of ACV prophylaxis.

Natural polymorphism of cytomegalovirus DNA polymerase lies in two nonconserved regions located between domains delta-C and II and between domains III and I.

We described the natural polymorphism of cytomegalovirus DNA polymerase in 42 unrelated isolates susceptible to ganciclovir, foscarnet, and cidofovir. All variations, including an eight-amino-acid deletion, were located between domains delta-C and II and between domains III and I, suggesting that these specific residues are not involved in enzymatic functions.

Cytomegalovirus and adenovirus infections and diseases among 75 paediatric unrelated allogeneic bone marrow transplant recipients.

Viral infections remain a major complication of allogeneic bone marrow transplantation. A population of children who underwent unrelated allogeneic bone marrow transplantation in a single centre has been followed-up for viral infections and diseases. We describe the detection of cytomegalovirus (CMV) and adenovirus among 75 children transplanted between 1989 and 2000. CMV was detected among 22 patients (29%) and adenovirus among 19 patients (25%); they were associated with clinical diseases in 10 and 8 patients, respectively. Four patients had adenovirus and CMV coinfection. The obvious risk factor for CMV infection is seropositivity of the recipient prior to transplantation. Adenovirus is detected significantly more frequently when conditioning regimen includes anti-thymocyte or anti-lymphocyte globulin. Diseases associated with adenovirus have been correlated with a significantly higher mortality rate, stressing the need for the implementation of a systematic virological survey for this virus and for the evaluation of therapeutic protocols including new molecules.

Herpes simplex virus resistance to antiviral drugs.

Herpes simplex virus (HSV) infections are efficiently treated with antiviral drugs such as acyclovir (ACV). However, resistance has been reported, mainly among immunocompromised patients (prevalence around 5%) and particularly allogeneic bone marrow transplant patients (prevalence reaching 30%). Resistance to ACV is associated with mutations on one of the two viral enzymes involved in the ACV mechanism of action: thymidine kinase (TK) and DNA polymerase. In 95% of the cases, ACV resistance is associated with a mutation in the TK gene as this enzyme is not essential for viral replication, unlike viral DNA polymerase, which is rarely involved in resistance. Strains resistant to ACV are almost always cross-resistant to other TK-dependent drugs such as penciclovir and famciclovir. Resistant infections can be managed by foscarnet or cidofovir but both are more toxic than ACV. These drugs also inhibit viral DNA polymerase but they are active on most ACV-resistant HSV as they do not depend on TK; nevertheless virus resistant to ACV because of a mutation in the DNA polymerase may be cross-resistant to these molecules. Published data on genetic characterization of resistant clinical isolates point out hot spots in viral TK and DNA polymerase genes. TK mutations associated with resistance are either insertion or deletion (codons 92 and 146 of TK gene) or substitution (codon 176-177, 336 of TK gene). DNA polymerase mutations are mainly located in conserved sites of the enzyme. A high level of gene polymorphism has also been reported for these genes, especially for TK. These results are useful for the development of rapid genotypic assays for the detection of mutations associated with resistance.

A screening dye-uptake assay to evaluate in vitro susceptibility of herpes simplex virus isolates to acyclovir.

The widespread use of acyclovir (ACV) could increase the prevalence of herpes simplex virus (HSV) ACV-resistant isolates, and a screening assay are thus important for routine surveillance of the ACV susceptibility of HSV. A screening dye-uptake assay was developed, based on the conventional dye-uptake assay [J. Biol. Stand. 14 (1986) 201]. The susceptibility of HSV was measured by testing two virus dilutions (10(-1) and 10(-2)) against two ACV concentrations (5 and 10 microM) on Vero cells and expressed as a reduced percentage of viral replication. The reproducibility was evaluated with HSV1 and HSV2 ACV-sensitive and ACV-resistant reference strains introduced as controls in successive series. The dye-uptake by Vero cells, the growth capacity of the HSV strains and the reduction of the viral replication in the presence of acyclovir varied by less than 14, 20 and 30%, respectively. This assay allowed the detection of a heterogenous population containing as few as 20% of ACV-resistant strain. The screening test was applied to 500 HSV isolates in a prospective study, and over 95% of the HSV isolates tested were characterised using a single test. This test appeared to be half the cost and much easier to carry out than the conventional dye-uptake assay, and consequently is well suited for large scale surveillance.

Detection of measles vaccine in the throat of a vaccinated child.

Measles vaccine is widely used, most often in association with mumps and rubella vaccines. We report here the case of a child presenting with fever 8 days after vaccination with a measles-mumps-rubella vaccine. Measles virus was isolated in a throat swab taken 4 days after fever onset. This virus was then further genetically characterised as a vaccine-type virus. Fever occurring subsequent to measles vaccination is related to the replication of the live attenuated vaccine virus. In the case presented here, the vaccine virus was isolated in the throat, showing that subcutaneous injection of an attenuated measles strain can result in respiratory excretion of this virus.