Synthesis and biological evaluation of novel rhodanine-based structures with antiviral activity towards HHV-6 virus.

An increased awareness of diseases associated with Human herpesvirus 6 (HHV-6) infection or reactivation has resulted in a growing interest in the evaluation of the best treatment options available for the clinical management of HHV-6 disease. However, no compound has yet been approved exclusively for HHV-6 infection treatment. For this reason, the identification of anti-HHV6 compounds provides a valuable opportunity for developing efficient antiviral therapies. A possible target for antiviral drugs is the virus-cell fusion step. In this study, we synthetized potential fusion intermediates inhibitors based on the rhodanine structure. The obtained derivatives were tested for cytotoxicity and for antiviral activity in human cells infected with HHV6. Level of infection was monitored by viral DNA quantification at different time points up to 7 days post infection. Among the synthetized derivatives, 9e showed a significative inhibitory effect on viral replication that lasted over 7 days, probably attributable to the particular combination of hydrophilic and hydrophobic substituents to the rhodanine moiety. Our results support the use of these amphipathic fusion inhibitors for the treatment of HHV-6 infections.

Effects of Prophylactic Foscarnet on Human Herpesvirus-6 Reactivation and Encephalitis in Cord Blood Transplant Recipients: A Prospective Multicenter Trial with an Historical Control Group.

Cord blood transplantation (CBT) is a distinct risk factor for human herpesvirus-6 (HHV-6) reactivation and HHV-6 encephalitis. In a prospective multicenter trial we investigated the effects of prophylactic foscarnet (90 mg/kg i.v. infusion from days 7 to 27 after CBT) on the occurrence of HHV-6 reactivation, HHV-6 encephalitis, and acute graft-versus-host disease (aGVHD) in CBT recipients. Between 2014 and 2016, 57 patients were included in a foscarnet-prophylaxis group. Outcomes were compared with an historical control group who received CBT between 2010 and 2014 (standard-treatment group, n = 63). The cumulative incidence of high-level HHV-6 reactivation, defined as plasma HHV-6 DNA ≥ 104 copies/mL, at 60 days after CBT was significantly lower in the foscarnet-prophylaxis group than in the standard-treatment group (18.3% versus 57.3%, P < .001). Multivariate analysis revealed that myeloablative preconditioning and standard treatment were significant risk factors for high-level HHV-6 reactivation. The cumulative incidence of HHV-6 encephalitis at 60 days after CBT was not different between the groups (foscarnet-prophylaxis group, 12.4%; standard-treatment group, 4.9%; P = .14). The cumulative incidences of grades II to IV and grades III to IV aGVHD at 60 days after CBT were not different between the groups (grades II to IV aGVHD: foscarnet-prophylaxis group, 42.0%; standard-treatment group, 40.5%; P = .96; grades III to IV aGVHD: foscarnet-prophylaxis group, 14.5%; standard-treatment group, 14.5%; P = 1.00). In the setting of this study foscarnet significantly suppressed systemic HHV-6 reactivation in CBT recipients but failed to prevent the development of HHV-6 encephalitis. Suppression of HHV-6 reactivation by foscarnet did not show any effects against the incidence of aGVHD.

HHV-6 in liver transplantation: A literature review.

Human herpesvirus 6 (HHV-6A and HHV-6B) can cause primary infection or reactivate from latency in liver transplant recipients, which can result in a variety of clinical syndromes, including fever, hepatitis, encephalitis and higher rates of graft dysfunction as well as indirect effects including increased risks of mortality, CMV disease, hepatitis C progression and greater fibrosis scores. Although HHV-6 infection is currently diagnosed by quantifying viral DNA in plasma or blood, biopsy to demonstrate histopathological effects of HHV-6 remains the gold standard for diagnosis of end-organ disease. HHV-6 reactivation may be restricted to the infected organ with no evidence of active infection in the blood. HHV-6 infections in liver transplant patients are mostly asymptomatic, but clinically significant tissue-invasive infections have been treated successfully with ganciclovir, foscarnet or cidofovir. Inherited chromosomally integrated HHV-6 (ciHHV-6), in either the recipient or the donor organ, may create confusion about systemic HHV-6 infection. Recipients with inherited ciHHV-6 may have an increased risk of opportunistic infection and graft rejection. This article reviews the current scientific data on the clinical effects, risk factors, pathogenesis, diagnosis and treatment of HHV-6 infections in liver transplant recipients.

Successful treatment and FDG-PET/CT monitoring of HHV-6 encephalitis in a non-neutropenic patient: case report and literature review.

Human herpesvirus (HHV)-6 reactivation is associated with severe forms of encephalitis among patients undergoing hematopoietic stem cell transplantation. Cases in non-neutropenic patients are uncommon. The efficacy of ganciclovir and other compounds that are used for the treatment of HHV-6 encephalitis remains suboptimal and linked to toxicity. Valganciclovir, the oral prodrug of ganciclovir, could be practical to treat outpatients, but it is not commonly used for severe cases. We report a case of HHV-6 encephalitis in a non-neutropenic patient successfully treated with valganciclovir and undergoing therapeutic drug monitoring in plasma and in the cerebrospinal fluid. Resolution of infectious foci was documented by cerebral MRI and F18-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT). A review of the literature on HHV-6 encephalitis is also reported.

Antiviral drug resistance in herpesviruses other than cytomegalovirus.

The discovery of acyclovir (ACV), a nucleoside analogue, more than 30 years ago, represents a milestone in the management of HSV and VZV infections. The modest activity of ACV against CMV prompted the development of another nucleoside analogue, ganciclovir, for the management of systemic and organ-specific CMV diseases. Second-line agents such as the pyrophosphate analogue foscarnet and the nucleotide analogue cidofovir have been approved subsequently. In contrast to ACV and ganciclovir, the latter drugs do not require selective phosphorylation by viral protein kinases to be converted into their active forms. Since the introduction of these antivirals, the emergence of drug-resistant mutants has been constantly reported particularly in severely immunocompromised patients such as bone marrow and solid organ transplant recipients as well as HIV-infected individuals. In this manuscript, we discuss the characteristics of the antiviral agents currently approved for the management of HSV, VZV and CMV diseases. In recent years, the resistance of CMV to antiviral drugs has been extensively reviewed. The emergence of antiviral drug resistance is also observed with other members of the Herpesviridae family, namely HSV-1, HSV-2, VZV and HHV-6, which are the focus of this review. More specifically, we describe the laboratory methods for assessing drug susceptibilities, the frequency and clinical significance of drug-resistant infections and their management.

Synthesis and antiviral activities of methylenecyclopropane analogs with 6-alkoxy and 6-alkylthio substitutions that exhibit broad-spectrum antiviral activity against human herpesviruses.

Methylenecyclopropane nucleosides have been reported to be active against many of the human herpesviruses. The most active compound of this class is cyclopropavir (CPV), which exhibits good antiviral activity against human cytomegalovirus (HCMV), Epstein-Barr virus, both variants of human herpesvirus 6, and human herpesvirus 8. CPV has two hydroxymethyl groups on the methylenecyclopropane ring, but analogs with a single hydroxymethyl group, such as the prototypical (S)-synguanol, are also active and exhibit a broader spectrum of antiviral activity that also includes hepatitis B virus and human immunodeficiency virus. Here, a large set of monohydroxymethyl compounds with ether and thioether substituents at the 6 position of the purine was synthesized and evaluated for antiviral activity against a range of human herpesviruses. Some of these analogs had a broader spectrum of antiviral activity than CPV, in that they also inhibited the replication of herpes simplex viruses 1 and 2 and varicella-zoster virus. Interestingly, the antiviral activity of these compounds appeared to be dependent on the activity of the HCMV UL97 kinase but was relatively unaffected by the absence of thymidine kinase activity in HSV. These data taken together indicate that the mechanism of action of these analogs is distinct from that of CPV. They also suggest that they might be useful as broad-spectrum antiherpesvirus agents and may be effective in the treatment of resistant virus infections.

Evaluation of Epstein-Barr virus, human herpesvirus 6 (HHV-6), and HHV-8 antiviral drug susceptibilities by use of real-time-PCR-based assays.

Evaluation of candidate antiviral drugs against Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and HHV-8 is hampered by the lack of convenient laboratory assays. We developed real-time quantitative PCR assays performed on supernatants of lymphoma cell lines and determined the 50% inhibitory concentrations (IC50s) of nucleoside, nucleotide, and pyrophosphate analogues against these herpesviruses.

Early human herpesvirus type 6 reactivation after allogeneic stem cell transplantation: a large-scale clinical study.

This study investigated the impact of human herpesvirus type 6 (HHV6) reactivation within 100 days of allogeneic stem cell transplantation (allo-SCT) on patient outcomes. HHV6 plasma loads were monitored weekly by quantitative PCR. Of 235 consecutive patients, 112 (48%) had an early positive HHV6 PCR test (group A) and 123 (52%) did not (group B). HHV6 reactivation was less frequent in patients who received reduced-intensity conditioning (P = .028). In group A, only 6 patients (5%) were asymptomatic; the most common clinical manifestations were fever (n = 60), skin rash (n = 57), diarrhea (n = 51), pulmonary complications (n = 19), and neurologic disorders (n = 12). Compared with the patients in group B, those in group A experienced delayed platelet engraftment (P = .003) and more frequent grade II-IV acute graft-versus-host disease (GVHD) (47% versus 30% in group B; P = .009). In multivariate analysis, the most important factors influencing the development of grade II-IV acute GVHD development were early HHV6 reactivation (P = .03) and unrelated donor status (P < .001). HHV6 reactivation adversely influenced 6-month survival (P = .04). Of the 38 evaluable patients receiving antiviral treatment, 34 had a significantly decreased HHV6 load. Our findings indicate that HHV6 reactivation after allo-SCT is associated with delayed platelet engraftment, early posttransplantation mortality, and the development of acute GVHD. Careful monitoring of HHV6 by PCR is warranted during the early posttransplantation period.

Confirmation of the low clinical effect of human herpesvirus-6 and -7 infections after renal transplantation.

Human herpesvirus-6 and -7 (HHV-6 and HHV-7) may lead to pathological manifestations in renal transplant recipients. The aim of this study was to investigate beta-herpesvirus infections in 50 adult kidney transplant recipients after transplantation to examine the effect, interactions, and pathogenic consequences of infection and the effect of immunosuppressive regimens and Human cytomegalovirus (HCMV) prophylaxis with VACV. Beta-herpesviruses loads in the blood of 50 adult kidney transplant recipients over a 6-month period after transplantation and 198 blood donors were determined using polymerase chain reaction. The rate of HHV-6 detection in peripheral mononuclear cells (PBMCs) was higher in patients with end-stage renal disease and during the post-transplantation follow-up than in healthy subjects (33% and 68% vs. 12%, respectively). The detection rate of HHV-7 in PBMCs was similar between patients, both before grafting and during the follow-up for transplant recipients (69% and 88%, respectively), and healthy subjects (78%), and correlated with the number of lymphocytes. HCMV in plasma was detected only in patients during the post-transplant period (24%). VACV prophylaxis had no negative effect on the replication of HHV-6 or HHV-7, and univariate analyses demonstrated associations between HHV-6 infection and acute graft rejection [Odds ratio (OR) = 2.94, 95% confidence interval (CI), 1.05-8.2, P = 0.04], and between HHV-7 infection and cholestasis [OR = 2.61 (95% CI, 1.08-6.3), P = 0.03]. Immunosuppressive regimens had no effect on beta-herpesviruses infections. This study revealed the differing behavior of HCMV, HHV-6, and HHV-7 in kidney transplant recipients, and confirmed the association of HHV-6 with graft rejection.

Safety of pre-engraftment prophylactic foscarnet administration after allogeneic stem cell transplantation.

Human herpesvirus-6 (HHV-6) is a major cause of limbic encephalitis with a dismal prognosis after allogeneic hematopoietic stem cell transplantation (SCT). Because our previous trial of preemptive therapy with foscarnet sodium (phosphonoformic acid; PFA) failed to prevent HHV-6 encephalitis, we conducted a prospective study to examine the safety of prophylactic PFA administration and elucidate the changes in the plasma HHV-6 DNA levels in the early post-SCT period. Plasma HHV-6 DNA was measured thrice weekly from day 6. PFA, 90 mg/kg/day, was administered from days 7 to 21 after bone marrow or peripheral blood SCT and to day 25 after umbilical cord blood transplantation. Of the 10 patients enrolled, 2 dropped out of the study, 1 because of early death, and 1 with a low glomerular filtration rate. Grade 3 or greater adverse events occurred in 9 of the 10 prophylactic PFA patients and in 7 of the 10 control patients who had clinical backgrounds similar to the study subjects and underwent SCT during the same period. Neurological disorders developed in none of the study subjects but in 4 of the 10 control patients, including 2 with HHV-6 encephalitis. HHV-6 reactivation occurred in 3 of the 10 study subjects. The prophylactic PFA regimen was thus safe and it may reduce the risk of limbic encephalitis, but is not considered to be potent enough to prevent HHV-6 reactivation.

Benzimidazole analogs inhibit human herpesvirus 6.

Several benzimidazole nucleoside analogs, including 1H-ß-D-ribofuranosyl-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and 1H-ß-L-ribofuranosyl-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir [MBV]), inhibit the replication of human cytomegalovirus. Neither analog inhibited the related betaherpesvirus human herpesvirus 6 (HHV-6). Additional analogs of these compounds were evaluated against both variants of HHV-6, and two L-analogs of BDCRB had good antiviral activity against HHV-6A, as well as more modest inhibition of HHV-6B replication.

Cyclopropavir inhibits the normal function of the human cytomegalovirus UL97 kinase.

Cyclopropavir (CPV) is active against human cytomegalovirus (CMV), as well as both variants of human herpesvirus 6 and human herpesvirus 8. The mechanism of action of CPV against CMV is similar to that of ganciclovir (GCV) in that it is phosphorylated initially by the CMV UL97 kinase, resulting in inhibition of viral DNA synthesis. Resistance to CPV maps to the UL97 kinase but is associated primarily with H520Q mutations and thus retains good antiviral activity against most GCV-resistant isolates. An examination of CMV-infected cultures treated with CPV revealed unusual cell morphology typically associated with the absence of UL97 kinase activity. A surrogate assay for UL97 kinase activity confirmed that CPV inhibited the activity of this enzyme and that its action was similar to the inhibition seen with maribavir (MBV) in this assay. Combination studies using real-time PCR indicated that, like MBV, CPV also antagonized the efficacy of GCV and were consistent with the observed inhibition of the UL97 kinase. Deep sequencing of CPV-resistant laboratory isolates identified a frameshift mutation in UL27, presumably to compensate for a loss of UL97 enzymatic activity. We conclude that the mechanism of action of CPV against CMV is complex and involves both the inhibition of DNA synthesis and the inhibition of the normal activity of the UL97 kinase.

Ganciclovir-resistant human herpesvirus-6 encephalitis in a liver transplant patient: a case report.

Human herpesvirus-6 (HHV-6) was initially isolated in 1986 from patients with lymphoproliferative disorders (Ablashi et al. 1987). Since that time, two genetically distinct variants were sequenced, HHV-6A and HHV-6B (Ablashi and Balachandran 1991). Both variants have been linked with neurologic disease (Crawford et al. 2007). HHV-6 encephalitis has been well described in literature, typically presenting with confusion, coma, seizure, and headache. The majority of HHV-6 encephalitis has been limited to post-transplant recipients (Singh and Paterson 2000). Encephalitis due to HHV-6 infection has been reported in two liver transplant recipients (Massih and Razonable 2009 and Montejo et al. 2002). Although there has been in vitro studies regarding the potential resistance patterns for HHV-6 virus, there has been only one clinic case report supporting these findings (Isegawa et al. 2009). We describe the first case of ganciclovir-resistant HHV-6 encephalitis in a post-liver transplant patient.

Does human bocavirus infection depend on helper viruses? A challenging case report.

A case of severe diarrhoea associated with synergistic human bocavirus type 1 (HBoV) and human herpes virus type 6 (HHV6) is reported. The case supports the hypotheses that HBoV infection under clinical conditions may depend on helper viruses, or that HBoV replicates by a mechanism that is atypical for parvoviruses, or that HBoV infection can be specifically treated with cidofovir.

A case of drug-induced hypersensitivity syndrome due to carbamazepine.

The patient was a 51-year-old man who had been prescribed carbamazepine for right third-branch trigeminal neuralgia. He had stopped taking the medication after the neuralgia resolved. When the neuralgia recurred, he resumed medication, and about 1 month later he developed fever, fatigue, cervical lymphadenopathy, generalized skin flushing, facial edema and perioral vesicles, and was admitted to Ichikawa General Hospital, Tokyo Dental College. Oral findings showed reddening and erosion of the buccal mucosa. Routine laboratory examination revealed leukocytosis and hepatic dysfunction. Human herpesvirus 6 antibody titer remarkably increased during development of eruptions. These findings led to a diagnosis of drug-induced hypersensitivity syndrome. Carbamazepine was discontinued, and prednisolone (30 mg/day) was started and tapered based on improvement of symptoms. Because skin symptoms recurred after he was discharged 15 days after admission, the dose of prednisolone was increased and the symptoms finally disappeared. The patient has experienced no further recurrence.

Occupational trichloroethylene hypersensitivity syndrome with human herpesvirus-6 and cytomegalovirus reactivation.

Patients having a generalised rash with severe liver dysfunction associated with exposure to trichloroethylene (TCE) have been reported mainly in Asian countries. However, no case has been reported in Japan since the 1990s. Here, we describe a case of hypersensitivity syndrome (HS) caused by TCE in a 30-year-old Japanese man. The patient developed a rash, fever and liver dysfunction 21 days after he had been exposed to TCE at his workplace. Serum human herpesvirus (HHV)-6 and cytomegalovirus (CMV) DNA were detected 4 and 7 weeks, respectively, after the onset; the IgG antibody titres to HHV-6 and CMV were significantly elevated 6 and 9 weeks, respectively, after the onset. Patch testing was positive for the metabolites of TCE (i.e. trichloroethanol, trichloroacetic acid and chloral hydrate) but not for TCE itself; these results suggest that the TCE metabolites induced this disease. Human leucocyte antigen-B*1301, which has been reported to be strongly associated with TCE-induced HS, was identified in this patient. In addition, the clinical findings, laboratory data and period of virus reactivation after onset were quite similar to those of drug-induced hypersensitivity syndrome (DIHS). We also review TCE-induced HS from the viewpoint of the similarity to DIHS in this article.

Amoxicillin-induced flare in patients with DRESS (Drug Reaction with Eosinophilia and Systemic Symptoms): report of seven cases and demonstration of a direct effect of amoxicillin on Human Herpesvirus 6 replication in vitro.

Amoxicillin is known to induce exanthema in patients with EBV-induced infectious mononucleosis. It is widely recognized that the reactivation of herpesviruses, including HHV-6 (Human Herpesvirus 6) and EBV (Epstein Barr virus) is associated with DRESS (Drug Reaction with Eosinophilia and Systemic Symptoms). We report 7 cases of amoxicillin-induced flare in patients with DRESS induced by other drugs and investigate whether amoxicillin may have a direct effect on HHV-6 replication in vitro. 7 cases of DRESS with amoxicillin-induced flare were retrospectively analysed. The influence of amoxicillin on HHV-6 HST strain replication was studied in vitro in a human T lymphoblastoid MT4 cell line. The viral replication was quantified by immunofluorescence assay and by real-time polymerase chain reaction (PCR). Comparisons were performed using the Student's t test. Amoxicillin-induced flare was observed in 7 patients with DRESS induced by other drugs. In two cases HHV-6 reactivation was studied and was demonstrated by PCR. Amoxicillin neither modified cell viability nor cell proliferation for the range of tested concentrations. Amoxicillin increased the replication of HHV-6 at 25 microg*mL-1 and 50 microg*mL-1. Amoxicillin may induce a flare of DRESS, possibly by acting directly on herpesvirus replication.

Inhibition of herpesvirus replication by 5-substituted 4'-thiopyrimidine nucleosides.

A series of 4'-thionucleosides were synthesized and evaluated for activities against orthopoxviruses and herpesviruses. We reported previously that one analog, 5-iodo-4'-thio-2'-deoxyuridine (4'-thioIDU), exhibits good activity both in vitro and in vivo against two orthopoxviruses. This compound also has good activity in cell culture against many of the herpesviruses. It inhibited the replication of herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus with 50% effective concentrations (EC(50)s) of 0.1, 0.5, and 2 microM, respectively. It also inhibited the replication of human cytomegalovirus (HCMV) with an EC(50) of 5.9 microM but did not selectively inhibit Epstein-Barr virus, human herpesvirus 6, or human herpesvirus 8. While acyclovir-resistant strains of HSV-1 and HSV-2 were comparatively resistant to 4'-thioIDU, it retained modest activity (EC(50)s of 4 to 12 microM) against these strains. Some ganciclovir-resistant strains of HCMV also exhibited reduced susceptibilities to the compound, which appeared to be related to the specific mutations in the DNA polymerase, consistent with the observed incorporation of the compound into viral DNA. The activity of 4'-thioIDU was also evaluated using mice infected intranasally with the MS strain of HSV-2. Although there was no decrease in final mortality rates, the mean length of survival after inoculation increased significantly (P < 0.05) for all animals receiving 4'-thioIDU. The findings from the studies presented here suggest that 4'-thioIDU is a good inhibitor of some herpesviruses, as well as orthopoxviruses, and this class of compounds warrants further study as a therapy for infections with these viruses.