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The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) 3CL protease is a promising target for inhibition of viral replication by interaction with a cysteine residue (Cys145) at its catalytic site. Dalcetrapib exerts its lipid-modulating effect by binding covalently to cysteine 13 of a cholesteryl ester transfer protein. Because 12 free cysteine residues are present in the 3CL protease, we investigated the potential of dalcetrapib to inhibit 3CL protease activity and SARS-CoV-2 replication. Molecular docking investigations suggested that dalcetrapib-thiol binds to the catalytic site of the 3CL protease with a delta G value of −8.5 kcal/mol. Dalcetrapib inhibited both 3CL protease activity in vitro and viral replication in Vero E6 cells with IC50 values of 14.4 ± 3.3 μM and an EC50 of 17.5 ± 3.5 μM (mean ± SD). Near-complete inhibition of protease activity persisted despite 1000-fold dilution after ultrafiltration with a nominal dalcetrapib-thiol concentration of approximately 100 times below the IC50 of 14.4 μM, suggesting stable protease–drug interaction. The inhibitory effect of dalcetrapib on the SARS-CoV-2 3CL protease and viral replication warrants its clinical evaluation for the treatment of COVID-19.

Intravenous ketamine is an effective treatment of bipolar depression. One of its most important side-effects is a transient altered state of consciousness commonly referred to as dissociation. These states can be anxiety-provoking, distressing and even treatment-limiting, warranting research into mitigation strategies. In this article, we present two cases that demonstrate the potential of adjunctive music to diminish the distress associated with ketamine-induced dissociation - though not necessarily its degree - in bipolar 1 disorder. Both patients suffering from severe depression underwent their first ketamine infusion without music and opted for music with subsequent infusions. They reported that music significantly improved the tolerance of their dissociative symptoms, thereby reducing distress and facilitating subsequent treatments. Both patients achieved remission from their highly treatment-resistant depressive episodes following six ketamine infusions. This is the first report of music's benefits on ketamine for bipolar 1 depression, though there is precedence in the scientific literature on 'psychedelics' where the use of music in combination with medication-induced altered states has been studied. The principles regarding music selection that have resulted from this paradigm may be applicable to the use of ketamine in unipolar and bipolar depression. The optimal use of music with ketamine warrants further research.

Amino acid substitutions conferring resistance of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) to foscarnet (PFA) are located in the genes UL30 and UL54, respectively, encoding the DNA polymerase (pol). In this study, we analyzed the impact of substitutions located in helix K and region II that are involved in the conformational changes of the DNA pol. Theoretical substitutions were identified by sequences alignment of the helix K and region II of human herpesviruses (susceptible to PFA) and bacteriophages (resistant to PFA) and introduced in viral genomes by recombinant phenotyping. We characterized the susceptibility of HSV-1 and HCMV mutants to PFA. In UL30, the substitutions I619K (helix K), V715S, and A719T (both in region II) increased mean PFA 50% effective concentrations (EC(50)s) by 2.5-, 5.6-, and 2.0-fold, respectively, compared to the wild type (WT). In UL54, the substitution Q579I (helix K) conferred hypersusceptibility to PFA (0.17-fold change), whereas the substitutions Q697P, V715S, and A719T (all in region II) increased mean PFA EC(50)s by 3.8-, 2.8- and 2.5-fold, respectively, compared to the WT. These results were confirmed by enzymatic assays using recombinant DNA pol harboring these substitutions. Three-dimensional modeling suggests that substitutions conferring resistance/hypersusceptibility to PFA located in helix K and region II of UL30 and UL54 DNA pol favor an open/closed conformation of these enzymes, resulting in a lower/higher drug affinity for the proteins. Thus, this study shows that both regions of UL30 and UL54 DNA pol are involved in the conformational changes of these proteins and can influence the susceptibility of both viruses to PFA.

Letermovir (LTV) is approved for the prophylaxis of human cytomegalovirus (HCMV) infection in adult seropositive recipients of an allogeneic hematopoietic stem cell transplant. Here, we report on the in vitro activity of LTV against a large panel of clinical HCMV isolates and recombinant viruses with different drug susceptibility phenotypes to currently-approved DNA polymerase inhibitors or maribavir. No pre-existing mutations conferring resistance to LTV were detected by Sanger sequencing in clinical HCMV isolates susceptible or resistant to DNA polymerases inhibitors. The susceptibility of LTV against the different recombinant HCMV mutants with amino acid substitutions in the UL97 kinase or in the UL54 DNA polymerase was similar to that of the wild type virus. LTV was also effective against recombinant HCMV harboring UL97 mutations conferring resistance to maribavir.

This study aimed at understanding the impact of different substitutions at codon 715 localized in the region II of the palm domain of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) DNA polymerases (pol). Here, we report a new theoretical mutation V715S that confers resistance of HSV-1 to foscarnet/acyclovir (5.6- and 9.2-fold increases EC50 values compared to wild type, respectively) and of HCMV to foscarnet/ganciclovir (2.8- and 2.9-fold increases in EC50 values compared to wild type, respectively). To further analyze the importance of this amino acid, we investigated the impact of the already known mutations V715M and V715G on the replicative capacities and drug susceptibilities of both viruses as well as on the activity and drug inhibition of the DNA pol. The V715G recombinant HSV-1 mutant was resistant to foscarnet and acyclovir (3.4- and 4.6-fold EC50 increase, respectively) whereas the V715M mutant was susceptible to foscarnet and resistant to acyclovir (3.4-fold EC50 increase). The V715G recombinant HCMV mutant did not grow and the V715M mutant was resistant to foscarnet (3.7-fold EC50 increase) and susceptible to ganciclovir. Finally, we showed by three-dimensional modeling that the differential impact of these mutations on the viral replicative capacity and drug resistance profile was related to different hydrophobic local environments for V715 in the DNA pol of the two viruses. Furthermore, we hypothesize that the DNA pol of HSV-1 is more tolerant to changes at this residue compared to that of HCMV because of a more hydrophobic environment stabilizing the region.

Background Zika virus, a previously neglected mosquito-borne virus, is prompting worldwide concern because of its connection with congenital defects, Guillain-Barré syndrome, meningoencephalitis and myelitis in infected individuals. However, no specific antiviral therapy is available at present. In this study, we investigated the in vitro susceptibility of geographically and temporally distinct Zika viruses against the RNA polymerase inhibitors, favipiravir (T-705) and ribavirin. Methods The in vitro activity of each drug and a 1:1 mixture combination was assessed against five geographically and temporally distinct Zika strains by plaque reduction assay (PRA), the gold standard phenotypic method. Results We showed that both drugs exhibit in vitro inhibitory activity against five different Zika strains isolated in different years and continents, with mean 50% inhibitory concentration (IC50) values of 35 ±14 and 35 ±20 μM, respectively, by PRA. We did not observe a synergistic effect when both drugs were combined at the equimolar concentration (IC50 =33 ±11 μM). Conclusions These results indicate that T-705 has the potential to be used in patients with complicated diseases and/or those individuals presenting with significant comorbidities.

DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the α-like DNA polymerase family. In spite of similarities in structure and function, the RB69 enzyme is relatively resistant to foscarnet, requiring the mutation V478W in helix N to promote the closed conformation of the enzyme to make it susceptible to the antiviral. Here, we generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility and viral replicative capacity. The mutation W781V in HSV-1 induced resistance to foscarnet, acyclovir, and ganciclovir (3-, 14-, and 3-fold increases in the 50% effective concentrations [EC 50 s], respectively). The recombinant HCMV mutant harboring the W780V mutation was slightly resistant to foscarnet (a 1.9-fold increase in the EC 50 ) and susceptible to ganciclovir. Recombinant HSV-1 and HCMV mutants had altered viral replication kinetics. The apparent inhibition constant values of foscarnet against mutant UL30 and UL54 DNA polymerases were 45- and 4.9-fold higher, respectively, than those against their wild-type counterparts. Structural evaluation of the tryptophan position in the UL54 DNA polymerase suggests that the bulkier phenylalanine (fingers domain) and isoleucine (N-terminal domain) could induce a tendency toward the closed conformation greater than that for UL30 and explains the modest effect of the W780V mutation on foscarnet susceptibility. Our results further suggest a role of the tryptophan in helix N in conferring HCMV and especially HSV-1 susceptibility to foscarnet and the possible contribution of other residues localized at the interface between the fingers and N-terminal domains. IMPORTANCE DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the α-like DNA polymerase family. However, the RB69 DNA polymerase is relatively resistant to the broad-spectrum antiviral agent foscarnet. The mutation V478W in helix N of the fingers domain caused the enzyme to adopt a closed conformation and to become susceptible to the antiviral. We generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility. The W781V mutation in HSV-1 induced resistance to foscarnet, whereas the W780V mutation in HCMV slightly decreased drug susceptibility. This study suggests that the different profiles of susceptibility to foscarnet of the HSV-1 and HCMV mutants could be related to subtle conformational changes resulting from the interaction between residues specific to each enzyme that are located at the interface between the fingers and the N-terminal domains.

Selection of human cytomegalovirus variants in the presence of ganciclovir or foscarnet led to 18 DNA polymerase mutations, 14 of which had not been previously studied. Using bacterial artificial chromosome technology, each of these mutations was individually transferred into the genome of a reference strain. Following reconstitution of infectious viral stocks, each mutant was assessed for its drug susceptibility and growth kinetics in cell culture. Computer-assisted three-dimensional (3D) modeling of the polymerase was also used to position each of the mutations in one of four proposed structural domains and to predict their influence on structural stability of the protein. Among the 10 DNA polymerase mutations selected with ganciclovir, 7 (P488R, C539R, L545S, V787L, V812L, P829S, and L862F) were associated with ganciclovir resistance, whereas 2 (F595I and V946L) conferred only foscarnet resistance. Among the eight mutations selected with foscarnet, only two (T552N and S585A) conferred foscarnet resistance, whereas four (N408D, K500N, L802V, and L957F) had an impact on ganciclovir susceptibility. Surprisingly, the combination of mutations, some of which were not associated with resistance for a specific antiviral, resulted in increasing resistance effects. 3D modeling suggested that none of the mutated residues were directly involved in the polymerase catalytic site but rather had an influence on drug susceptibility by modifying the structural flexibility of the protein. Our study significantly adds to the number of DNA polymerase mutations conferring in vitro drug resistance and emphasizes the point that evaluation of individual mutations may not accurately reflect the phenotype conferred by multiple mutations.

Background. In a phase III controlled trial IMproved Protection Against Cytomegalovirus in Transplantation (IMPACT) comparing 200 with 100 days of valganciclovir prophylaxis in 318 cytomegalovirus D+/R— kidney transplant recipients, an equal number of patients (n=3 per arm) had known ganciclovir resistance mutations detected during viral breakthrough. In addition, many other viral sequence variants were observed that were of unknown significance for ganciclovir resistance. Recombinant phenotyping was performed to determine whether the previously uncharacterized genotypic changes affected ganciclovir susceptibility, especially in those receiving the longer duration of prophylaxis. Methods. Sequences encoding individual amino acid substitutions in the UL97 kinase or UL54 DNA polymerase gene were transferred by recombination into a cloned cytomegalovirus laboratory strain, followed by reporter-based yield reduction phenotypic assay of the resulting virus for ganciclovir susceptibility. Results. Twenty-six uncharacterized amino acid substitutions were detected, 2 in UL97 and 24 in UL54. All 10 substitutions in the 200-day arm and 9 of 17 substitutions in the 100-day arm (prioritized based on location and conservation) were selected for phenotyping; one substitution was detected in both subsets. Results were generated for nine of ten 200-day and eight of nine 100-day substitutions, with no substitution demonstrating a significant reduction in ganciclovir susceptibility. The two remaining amino acid substitutions, both in UL54, were not evaluated because of poor viral viability. Conclusion. Phenotypic evaluation of previously uncharacterized viral genotypes in the 200-day valganciclovir prophylaxis group showed no evidence of an increased incidence of genotypic ganciclovir resistance when compared with those in the 100-day prophylaxis group.

BACKGROUND: Primary herpes simplex virus (HSV) infection usually involves one mucosal region.OBJECTIVE: To describe an unusual disseminated HSV-1 infection involving two mucosal sites in a healthy man.RESULTS: Primary HSV infection involved oral and genital mucosa and was associated with viremia, hepatitis and rash. Phylogenetic analysis of genital and oral viruses revealed that the patient was infected by a single HSV-1 strain.CONCLUSION: Use of polymerase chain reaction detection techniques for HSV may identify viremic patients in the absence of obvious immunosuppression.

Background Amphipathic DNA polymers are promising therapies for the prevention of HIV and genital herpes infections. Recent studies on a panel of such compounds indicated potent activity against HIV binding and entry. This current study was conducted to explore the anti-herpes simplex virus (HSV) activity of the same panel of compounds and to determine their mechanism of activity. Methods The anti-HSV activity of a 40-nucleotide degenerate polymer (REP 9), a 40-nucleotide polycytidine amphipathic DNA polymer (REP 9C) and an analogue lacking amphipathic activity (Randomer 3) were compared in plaque reduction assays in the absence or presence of human genital tract secretions; the mechanisms of anti-HSV activity were explored. Results REP 9 inhibited HSV infection 10,000-fold, whereas Randomer 3 displayed no anti-HSV activity. The antiviral activity was independent of sequence but was dependent on size: the most potent activity was observed for analogues of 40 nucleotides in length. Mechanistic studies indicated that REP 9 and REP 9C blocked HSV-2 binding and entry, were active when added post-entry, inhibited viral gene expression and blocked HSV-induced apoptosis. Confocal microscopy studies showed rapid delivery of fluorescently tagged REP 9 and REP 9C into human epithelial cells, and delivery was significantly greater in infected cells as compared with uninfected cells. REP 9 exhibited no cytotoxicity and retained anti-HSV activity in the presence of cervicovaginal secretions and when virus was introduced in seminal plasma. Conclusions REP 9 and REP 9C represent a novel class of antiviral agents that act by multiple mechanisms. These compounds warrant further development for systemic or topical delivery for the prevention and treatment of HIV and HSV.

Background Few studies have performed sequential evaluation of the herpes simplex virus (HSV) load using quantitative PCR during episodes of herpes labialis. Objective To determine HSV viral load kinetics during recurrences of herpes labialis. Study design Twenty-two subjects were monitored by daily swabs during recurrences of herpes labialis. A real-time PCR detecting both HSV-1 and HSV-2 was used to quantify the viral load. Results Median duration of HSV-1 shedding was 60 h and 48 h by PCR and culture, respectively. No HSV-2 DNA was detected in that study. Peak viral DNA load (123.6 copies/cell) occurred at 48 h with no virus detected beyond 96 h of onset of symptoms. Conclusions These viral load kinetics data could be used as surrogate markers of antiviral activity in future clinical trials.

A system of reverse genetics was used to generate influenza A/H1N1 viruses harbouring neuraminidase (NA) mutations previously associated with resistance to NA inhibitors in various viral subtypes. The His274Tyr and Glu119Gln mutants were rescued whereas the Arg292Lys and Glu119→Gly, Val, Ala or Asp mutants could not be generated. In NA inhibition assays, the His274Tyr mutant was resistant to oseltamivir (430-fold over wild-type) and BCX-1812 (50-fold) but was sensitive to zanamivir. A similar trend was seen when the mutant was evaluated by plaque reduction assay (PRA). The Glu119Gln mutant expressed a low level of resistance to oseltamivir (ninefold) and zanamivir (fourfold) in NA inhibition assay but was only marginally resistant to oseltamivir (fourfold) in PRA. The replication capacity of both mutants, in particular that of the His274Tyr virus, was impaired when compared with the wild-type virus in vitro.

Background: Human cytomegalovirus (HCMV) infections cause significant morbidity in immunocompromised hosts. Resistance to ganciclovir is predominantly associated with alterations in the HCMV UL97 kinase and, more occasionally, with mutations in the HCMV DNA polymerase gene. Objectives: The aim of this study was to investigate the impact of two different mutations found at the same UL97 codon on drug susceptibility and viral replicative capacity. Mutation V466G was observed in a solid organ transplant recipient whereas mutation V466M was observed in a patient with AIDS. Study design: Two HCMV UL97 mutations, V466M and V466G, were transferred to recombinant viruses using a bacterial artificial chromosome system. Susceptibility testing of the recombinant wild-type and mutant viruses was performed using a standard plaque reduction assay. Replication kinetics of recombinant viruses was investigated using a yield assay. Results: Mutant V466G was resistant to ganciclovir and had significant replicative defect whereas mutant V466M was drug susceptible and had unaltered replication kinetics. Furthermore, mutant V466G formed small viral plaques with intracellular inclusions. Conclusions: To our knowledge, this is the first report of such contrasting phenotypes for drug susceptibility and replicative capacity for HCMV mutations found at the same codon of the UL97 gene.

In the present study, the relationship between hyperlipidemia induced by ingestion of animal protein and tissue lipid peroxidation was examined. Male Golden Syrian hamsters were fed for 27 days cholesterol-enriched (0.088 wt%) semi-purified diets having either 15 wt% casein (CAS) or egg white (EGG) as the protein source. Hamsters fed the CAS diet showed higher concentrations of serum total cholesterol and triglycerides as compared with EGG-fed hamsters. The change in total serum cholesterol and triglyceride concentrations over the 27-day dietary period was significantly greater in the CAS-fed hamsters than in the EGG group. Lipid peroxidation as measured by lipid hydroperoxides (LPO) was increased in the serum and liver of CAS-fed relative to the EGG-fed hamsters. Liver concentrations of thiobarbituric acid reactive substances, another index of lipid peroxidation, were unaffected by dietary protein composition. When the two dietary treatments were grouped together, a positive correlation was observed between serum LPO and serum apolipoprotein B (r=0.52; P P

Human herpesvirus 6 (HHV-6) and human cytomegalovirus (HCMV) are major opportunistic pathogens in solid organ transplant (SOT) recipients. The use of antivirals for the treatment of HCMV disease can result in the development of drug resistance mutations in HCMV and also potentially in HHV-6.The emergence of HHV-6 drug resistance mutations was evaluated in SOT recipients at the onset of HCMV disease and following treatment with ganciclovir (GCV) or valganciclovir (VGCV).Detection of HHV-6 was performed by real-time PCR from whole blood samples serially obtained from SOT recipients treated for HCMV disease with an induction dose of intravenous GCV or oral VGCV for 21 days followed by VGCV maintenance for 28 days in both arms. Baseline and last positive HHV-6 samples were tested for mutations in the genes encoding the protein kinase (U69) and the DNA polymerase (U38).The rate of HHV-6 viraemia among SOT patients with HCMV disease at baseline was 3.2% (5/155). All isolates belonged to the HHV-6B species. Mutations L213I and Y479H were detected at baseline and at later times in the U69 kinase. Mutation L213I was previously reported as polymorphism whereas the role of mutation Y479H in drug resistance is unknown. Mutations D854E and E855Q found in the DNA polymerase were known as natural variants.The incidence of HHV-6 viraemia in SOT recipients with established HCMV disease before initiation of antiviral therapy was low. Treatment with GCV or VGCV did not induce the emergence of HHV-6 drug resistance mutations.

Previous research has linked hyperlipidemia with increased serum concentrations of lipid peroxidation products; however, a specific association between diet-induced oxidative stress and hyperlipidemia has not been studied. In the present study, the relationship between tissue lipid peroxidation and hyperlipidemia induced by ingestion of fish oil was examined. In Experiment 1, male Golden Syrian hamsters were fed semipurified diets composed of 1.6 wt% safflower oil plus 15.0 wt% of either butterfat (BF), safflower oil (SAFF), or high-cholesterol menhaden oil [MHO(H-CHOL)] semipurified diets for 27 d. The cholesterol contents of the diets were adjusted to 0.088%. The MHO(H-CHOL)-fed hamsters exhibited higher serum concentrations of total cholesterol, triglycerides, apolipoprotein B, and lipid peroxides when compared to the BF and SAFF diet groups. In a further study (Experiment 2), hamsters were fed for 27 d three dietary treatments: (i) MHO(H-CHOL) with no vitamin E content; (ii) a low-cholesterol menhaden oil containing high concentrations of vitamin E (2.5 mg tocopherol/g oil or dietary concentrations of 375 mg/kg) [MHO(L-CHOL) + E]; and (iii) the MHO(L-CHOL + E) with added cholesterol (595 mg/kg) [MHO(L-CHOL) + CHOL + E] to match the cholesterol content of the MHO(H-CHOL). The MHO(L-CHOL) + E and MHO(L-CHOL) + CHOL + E diet groups showed lower concentrations of serum cholesterol, triglycerides, and hepatic lipid peroxides than the MHO(H-CHOL)-treated group. Moreover, in contrast to the hypercholesterolemia caused by the MHO(H-CHOL) feeding, the MHO(L-CHOL)+ E and MHO(L-CHOL) + CHOL + E diets did not show a serum cholesterol-elevating action. This study supports the hypothesis that oxidative stress in the Syrian hamster could play a causal role in dietary-induced hyperlipidemia which can be inhibited by high vitamin E intake.

Background The IMPACT study was a randomized, double-blind study comparing 100 to 200 days of VGCV prophylaxis (900 mg once daily) in D+/R− kidney transplant recipients. Although extending the duration of prophylaxis resulted in a significant reduction in confirmed cytomegalovirus (CMV) disease (100-day: 36.8% vs 200-day: 16.1% 1 ), the consequence of extending the duration of prophylaxis on the development of viral resistance remains unknown. Objective To determine whether extending valganciclovir prophylaxis from 100 days to 200 days increased the incidence of ganciclovir resistance. Study design Genotypic analysis of CMV UL97 and UL54 was conducted on virus isolated from patients meeting the predefined resistance analysis criteria (RAC). Results A greater number of patients met the RAC in the 100 day prophylaxis arm (50/163; 31%) compared to the 200 day prophylaxis arm (22/155; 14%). Sequence data were successfully generated for all 200-day patients and 48/50 100-day patients. Three patients in each treatment arm (100 day: 3/163 (1.8%) vs 200 day: 3/155 (1.9%)) had a single known valganciclovir resistance mutation detected (100 day: UL97 gene: M460V, C592G twice; 200 day: UL97 gene: C603W, M460V and UL54 gene: P522S). Overall, a resistance mutation was more likely to be detected if the patient met the RAC during prophylaxis (5/12 (42%)) compared to post-prophylaxis (1/58 (2%)). All six patients with known ganciclovir resistance mutations cleared the virus; three cleared virus without treatment and three cleared virus following treatment. Conclusions Extending valganciclovir prophylaxis from 100 days to 200 days did not significantly affect the incidence of ganciclovir resistance.

Background Drug-resistant cytomegalovirus (CMV) infections can cause significant morbidity among high-risk transplant recipients. Objectives The aims of this study were to determine the incidence and clinical consequences of CMV mutations conferring ganciclovir resistance in pediatric solid organ transplant (SOT) patients who received valganciclovir oral solution or tablets for prophylaxis of CMV disease. Recombinant CMV mutants were also generated to assess the role of two UL97 mutations of unknown significance. Study design Genotypic resistance mutations and CMV viral load were sought in blood samples from pediatric SOT recipients who received valganciclovir prophylaxis for 100 days. Recombinant viruses containing novel CMV UL97 mutations were generated using a bacterial artificial chromosome containing the CMV genome to assess ganciclovir susceptibility. Results Overall, four known resistance UL97 mutations were observed in blood samples from 2 of 46 patients during the study with no development of CMV disease. Two UL97 changes (M615V and V466G) of unknown significance and one UL97 mutation (C603R) associated with ganciclovir resistance, but not yet confirmed by marker transfer, were also detected. Recombinant viruses containing these novel mutations were generated to assess ganciclovir susceptibility. The M615V recombinant virus was susceptible to ganciclovir while the V466G and C603R mutant viruses displayed 3.5-fold and 3.6-fold decreases in susceptibility, respectively. Conclusions The low incidence of ganciclovir resistance-associated mutations and the absence of clinical consequences associated with drug-resistant viruses observed in this pilot study should encourage the design of larger clinical trials aimed at evaluating the efficacy of valganciclovir prophylaxis and treatment in the pediatric setting.

Background Human metapneumovirus (hMPV) is a major respiratory viral pathogen in young children, elderly individuals and immunocompromised patients. Despite its major effects related to bronchiolitis, pneumonia and its potential role in recurrent wheezing episodes, there is still no commercial treatment or vaccine available against this paramyxovirus. Methods We tested a therapeutic strategy for hMPV that was based on RNA interference. Results An hMPV genome-wide search for small interfering RNAs (siRNAs) by computational analysis revealed 200 potentially effective 21-mer siRNAs. Initial screening with a luciferase assay identified 57 siRNAs of interest. Further evaluation of their inhibitory potential against the four hMPV subgroups by quantitative real-time reverse transcriptase PCR and plaque immunoassay identified two highly potent siRNAs with 50% inhibitory concentration (IC50) values in the subnanomolar range. siRNA45 targets the nucleoprotein messenger RNA (mRNA) and had IC50 values 50 values between 0.090– Conclusions We successfully identified two highly efficient siRNAs against hMPV targeting essential components of the hMPV replication complex.

Clinical consequences of ganciclovir resistant cytomegalovirus (CMV) infections were studied during 2 large prophylactic trials consisting of 100 days of valganciclovir or ganciclovir prophylaxis in solid organ transplant (SOT) recipients. The first one involved 301 high-risk (CMV donor seropositive/recipient seronegative) SOT recipients excluding lung transplants followed for 12 months, whereas the second one involved 80 lung transplant patients evaluated over 6 months. Among the 7 patients (4 non-lung and 3 lung transplant patients) carrying viruses with known ganciclovir-resistance [corrected] mutations in blood, adverse clinical outcome was only observed in the lung transplant recipients. Additionally, no CMV resistance mutations were observed in non-lung transplant patients receiving valganciclovir.

We previously reported the absence of CMV UL97 (kinase) gene resistance mutations up to 12 months post-transplant following 100 days of valganciclovir prophylaxis, and a low incidence of resistance mutations following 100 days of oral ganciclovir prophylaxis in a prospective multicenter study in solid organ transplant recipients excluding lung transplants. Herein, we report UL54 (DNA polymerase) gene sequencing results for all patients with previous UL97 PCR-positive samples (n = 99) in our study. One UL54 resistance mutation (L545S known to confer ganciclovir and cidofovir resistance) was detected in a routine day-100 sample from an asymptomatic patient who received oral ganciclovir. Notably, this CMV UL54 mutation occurred in the absence of a UL97 mutation. Additionally, new UL54 variants were observed. Thus, emergence of CMV UL54 mutations in the absence of UL97 mutations is a rare but possible event that is not necessarily associated with detrimental clinical outcome in solid organ transplant recipients.

The emergence of resistance to amantadine in influenza A viruses has been shown to occur rapidly during treatment as a result of single-amino-acid substitutions at position 26, 27, 30, 31, or 34 within the transmembrane domain of the matrix-(M)-2 protein. In this study, reverse genetics was used to generate and characterize recombinant influenza A (H1N1) viruses harboring L26F, V27A, A30T, S31N, G34E, and V27A/S31N mutations in the M2 gene. In plaque reduction assays, all mutations conferred amantadine resistance, with drug concentrations resulting in reduction of plaque number by 50% (IC 50 s) 154- to 3,300-fold higher than those seen for the wild type (WT). M2 mutants had no impairment in their replicative capacities in vitro on the basis of plaque size and replication kinetics experiments. In addition, all mutants were at least as virulent as the WT in experimentally infected mice, with the highest mortality rate being obtained with the recombinant harboring a double V27A/S31N mutation. These findings could help explain the frequent emergence and transmission of amantadine-resistant influenza viruses during antiviral pressure in the clinical setting.

We investigated the emergence of cytomegalovirus (CMV) ganciclovir-resistance mutations in 301 high-risk solid-organ transplant (SOT) recipients after oral prophylaxis, for 100 days, with either valganciclovir or ganciclovir. For patients treated with ganciclovir, the incidence of CMV UL97 mutations was 1.9% (2/103) at the end of prophylaxis and 6.1% (2/33) for patients with suspected CMV disease up to 1 year after transplantation. No resistance mutations were detected in samples from valganciclovir-treated patients. Dual polymerase (UL54) and UL97 resistance mutations were not seen. Valganciclovir was associated with negligible risk of resistance and thus constitutes a useful alternative to ganciclovir prophylaxis for CMV in high-risk SOT recipients.

Forty-two influenza A and 23 influenza B isolates collected from untreated subjects during the 1999-2000 influenza season in Canada were tested for their susceptibility to three neuraminidase inhibitors (zanamivir, oseltamivir carboxylate and RWJ-270201 or BCX-1812) using a chemiluminescent neuraminidase assay. Influenza B isolates were less susceptible than A viruses to all tested drugs. RWJ-270201 was the most potent drug against both influenza A(H3N2) (mean IC(50): 0.60 nM) and B (mean IC(50): 0.87 nM) viruses. Oseltamivir carboxylate was more active than zanamivir for influenza A(H3N2) isolates (mean IC(50): 0.73 vs. 2.09 nM) whereas it was less potent against B viruses (mean IC(50): 11.53 vs. 4.15 nM).

The degree of sialylation has been shown previously to modulate the process of human immunodeficiency virus type-1 (HIV-1) infection by affecting the interaction between the virus and CD4-expressing target cells. In the present study, we investigated whether HIV-1 replication cycle was affected by neuraminidase (NA) derived from the human influenza (flu) virus. We first demonstrate that the level of HIV-1-mediated syncytium formation was greatly enhanced in the presence of purified flu NA. Pretreatment of established monocytic and lymphocytic cell lines as well as primary mononuclear cells with purified flu NA augmented also the process of virus infection. A comparable up-regulating effect was observed when using several strains of UV-inactivated whole flu virus, thereby suggesting that virus-anchored NA enzymes positively modulate the HIV-1 life cycle. Furthermore, flu NA-mediated positive effect on HIV-1 biology was abrogated with zanamivir, a specific flu NA inhibitor. Our results provide a new model allowing the investigation of the potential benefit of using NA inhibitors in the treatment of HIV-1-infected patients suffering from coinfection with NA-bearing pathogens.

In this study, we compared antigenic (hemagglutination inhibition (HI) assay) and molecular (sequencing of the hemagglutinin (HA1) gene) characterization of influenza isolates collected in the Province of Quebec (Canada) during the last three flu seasons (1997–2000). Twenty-three isolates were tested by a standard HI assay and 37 by sequencing of the HA1 gene for their homology to the A/H3N2 vaccine strains A/Wuhan/359/95 (1997–1998) and A/Sydney/5/97 (1998–1999 and 1999–2000). By HI, two isolates were antigenically similar to A/Wuhan/359/95 (both from 1997 to 1998), 16 were similar to A/Sydney/5/97 (1997–2000) and no conclusions could be inferred for the other five isolates due to identical HI titers for the two vaccine strains ( n =4) or insufficient viral titer ( n =1). Sequence analysis revealed that four isolates from 1997 to 1998 were related to A/Wuhan/359/95 whereas the others ( n =4) from 1997 to 1998, as well as all isolates from 1998 to 1999 ( n =18) and 1999 to 2000 ( n =11) were closer to A/Sydney/5/97. The mean number of amino acid differences for the 33 A/Sydney/5/97-like isolates compared with the homologous vaccine strain was 6.3 (1.9%), 9.2 (2.8%) and 13.6 (4.1%) for those collected in 1997–1998, 1998–1999, and 1999–2000, respectively. Phylogenetic analysis confirmed that a progressive drift occurred among our A/H3N2 influenza isolates over the last three flu seasons. Furthermore, it revealed that isolates collected during the last two flu seasons were in fact more related to A/Panama/2007/99 (2000–2001 vaccine strain) than to A/Sydney/5/97. Our studies suggest that molecular analysis of the HA1 gene should complement the HI assay for a more accurate analysis of influenza A virus drift.

Phenotypic and molecular studies were conducted to characterize multiple influenza A isolates recovered from an immunocompromised patient who died of viral and fungal pneumonitis. The recovery of amantadine-resistant isolates was correlated with the detection of 2 drug-resistant M2 variants (codons 27 and 31) in combination with a wild-type virus. The mutant viruses persisted within the viral population in variable proportions >1 month after cessation of antiviral therapy. These results confirm animal studies reported elsewhere regarding the genetic stability of influenza M2 mutants and their potential for transmission in humans.

The antiviral and clinical effects of inhaled zanamivir (10 mg twice daily for 5 days, started within the first or second day of a flulike illness) were evaluated in a randomized, placebo-controlled trial during the 1997-1998 influenza season in Canada. Pharyngeal secretions were collected with swabs every 12 h during 6 days, and symptoms were self-evaluated twice daily during 14 days. After only 12 h of treatment (1 dose), median virus titers decreased by 1.0 log10 TCID50/mL in the zanamivir group (n=17), compared with a 0. 42-log10 increase in the placebo group (n=10; P=.08). This was associated with a 4.5-day (47.4%) reduction in the median time to alleviation of all significant flu symptoms in the zanamivir recipients (P=.03 after adjusting for the initial virus titer and the time between onset of symptoms and treatment). Resistance to zanamivir was not detected in virus isolates by either phenotypic or genotypic assays.

Amphipathic DNA polymers are promising therapies for the prevention of HIV and genital herpes infections. Recent studies on a panel of such compounds indicated potent activity against HIV binding and entry. This current study was conducted to explore the anti-herpes simplex virus (HSV) activity of the same panel of compounds and to determine their mechanism of activity.The anti-HSV activity of a 40-nucleotide degenerate polymer (REP 9), a 40-nucleotide polycytidine amphipathic DNA polymer (REP 9C) and an analogue lacking amphipathic activity (Randomer 3) were compared in plaque reduction assays in the absence or presence of human genital tract secretions; the mechanisms of anti-HSV activity were explored.REP 9 inhibited HSV infection 10,000-fold, whereas Randomer 3 displayed no anti-HSV activity. The antiviral activity was independent of sequence but was dependent on size: the most potent activity was observed for analogues of 40 nucleotides in length. Mechanistic studies indicated that REP 9 and REP 9C blocked HSV-2 binding and entry, were active when added post-entry, inhibited viral gene expression and blocked HSV-induced apoptosis. Confocal microscopy studies showed rapid delivery of fluorescently tagged REP 9 and REP 9C into human epithelial cells, and delivery was significantly greater in infected cells as compared with uninfected cells. REP 9 exhibited no cytotoxicity and retained anti-HSV activity in the presence of cervicovaginal secretions and when virus was introduced in seminal plasma.REP 9 and REP 9C represent a novel class of antiviral agents that act by multiple mechanisms. These compounds warrant further development for systemic or topical delivery for the prevention and treatment of HIV and HSV.

Immunocompromised patients are at risk of developing primary infections and reactivations of human herpesvirus 6 (HHV-6), HHV-8, and Epstein-Barr virus (EBV), which can lead to severe and sometimes life-threatening diseases ([1][1][–][2][3][3]). Therefore, effective antiviral drugs are needed to

To evaluate the role of inhibitory substances produced by bacteria in the oral cavity, we estimated, by a deferred test on Todd-Hewitt agar enriched with hemin and vitamin K, the proportion of bacteria that inhibited or stimulated the growth of Streptococcus mutans and Porphyromonas gingivalis, from the saliva of 109 patients (54 males and 55 females) attending our dental clinics. The patients, aged from 8 to 75 years old (mean: 31 +/- 18 years), were randomly selected whatever the reason for their visit. The results, evaluated with the Spearman rank test, indicated that there was no statistically significant (P > 0.05) correlation between the proportion of salivary bacteria inhibiting or stimulating P. gingivalis with the Community Periodontal Index of Treatment Needs (CPITN), the number of carious, missing and filled teeth, or with the decayed, missing and filled teeth index (DMFT). Also, no statistically significant correlation was observed between the proportion of salivary bacteria stimulating the growth of S. mutans and the above mentioned health indexes. However, a statistically significant (P < 0.005) negative correlation was found between the percentage of cultivated bacteria that inhibit S. mutans and the percentage of untreated carious teeth as well as with the CPITN. The results thus indicate a possible role for inhibitory substances produced by bacteria in the maintenance of oral health.