Hemophagocytic syndrome caused by primary herpes simplex virus 1 infection: report of a first case.

INTRODUCTION: Hemophagocytic syndrome represents a severe hyperinflammatory condition by activated macrophages. Leading viral triggering agents are Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenovirus. MATERIALS AND METHODS: We present a patient with Wegener's granulomatosis on azathioprine and prednisone medication, who developed a life-threatening hemophagocytic syndrome. Positive plasma polymerase chain reaction (PCR) with negative serology revealed a primary, disseminated infection with herpes simplex virus-1 as the triggering pathogen. After treatment with acyclovir, high-dose steroids, immunoglobulins, and etoposide, the patient recovered. CONCLUSION: Early diagnosis of potentially underlying infections of hemophagocytic syndrome influences the therapeutic approach. It is important to consider a variety of infectious agents, particularly in immunosuppressed individuals. The reported case emphasizes the importance of screening for herpes simplex virus 1.

In vitro activity of cycloSal-nucleoside monophosphates and polyhydroxycarboxylates against orthopoxviruses.

Because variola virus might be used as a pathogen in biological attacks, there is an urgent need to provide effective antiviral drugs for the treatment of orthopoxvirus infections. Thus, the aim of the present study was to test the antiviral activity of 3 pro-nucleotides of the acyclic nucleoside analogues aciclovir (ACV), 3 of penciclovir (PCV) and 38 of the cyclic nucleoside analogue brivudin (BVDU), on the basis of cycloSaligenyl-nucleoside monophosphate approach against vaccinia virus and cowpox virus in vitro. In further experiments, 13 synthetic humic acid-like polymers, so-called polyhydroxycarboxylates, were examined. Antiviral screening was performed by means of the plaque reduction assay and for quantification of the cytotoxicity of the test compounds the XTT-based tetrazolium reduction assay EZ4U was used. As result, three cycloSal-monophosphate derivatives of ACV proved to be potent inhibitors of both vaccinia virus and cowpox virus replication in vitro. Among the tested monophosphate derivatives of cycloSal-PCV and cycloSal-BVDU, selected substances showed a promising antiviral activity against vaccinia virus and cowpox virus. For the polyanionic compounds, no relevant antiviral activity was detected. In conclusion, by the delivery of nucleoside monophosphates from neutral, membrane-permeable prodrugs on the basis of the cycloSaligenyl-nucleotide concept, different ACV, PCV and BVDU derivatives can act as potent and selective inhibitors of orthopoxvirus replication. However, most of the cycloSal-monophosphate derivatives of BVDU had a higher cytotoxicity than their parent nucleosides.

Transformation of rabbit lymphocytes by an Epstein-Barr virus-related herpesvirus from Macaca arctoides.

Herpesvirus Macaca arctoides (HVMA), an Epstein-Barr virus (EBV)-related herpesvirus of macaque origin, induces malignant lymphomas in rabbits. To get more insights into the oncogenesis of the EBV/HVMA infection the aim of the present study was to prove the in vitro transforming ability of HVMA for rabbit lymphocytes as well as human umbilical cord blood lymphocytes. As a result, B-cell transformation could be demonstrated after infection with HVMA in all mononuclear cell samples of 20 rabbits. The transformation was evaluated microscopically and confirmed by the expression of EBV-related nuclear antigens. The transforming activity led to the establishment of permanent rabbit lymphoblastoid cell lines cultured up to more than 90 passages. The cell lines contained EBV-like HVMA-DNA. Interestingly, the transformed rabbit lymphocytes showed chromosomal abnormalities with a subtetraploid karyotype. The low extent of lytic cycle-dependent expression of virus capsid antigen in the established cell lines increased after treatment with the inducing agents iododeoxyuridine and mitomycin C. In contrast, no transformation could be induced after exposure of human umbilical cord blood lymphocytes to HVMA. The permanent rabbit lymphoblastoid cell lines provide a model for further studies on the role of EBV/HVMA in oncogenesis of lymphomas. In addition, it might be suitable for testing potential antiviral compounds in vitro.

Establishment and cidofovir sensitivity of a cell line from a heart transplant recipient with multiple cutaneous tumors.

A new cell line, designated as Tuwei00, is described. It originated from an Epstein-Barr virus-positive skin tumor biopsy of a heart transplant recipient, whose numerous cutaneous neoplasms were treated with the antiviral drug cidofovir what caused at least transient remissions. The cell line was established in vitro and maintained for more than 70 passages. Cells of early passages were characterized by a slower growth, the inability to form colonies and a higher sensitivity to cidofovir. After overcoming a crisis, the cells grew faster, to a higher density and were able to form adherent colonies from single cells as well as colonies in soft agar. Chromosome analysis showed diploidy/hyperdiploidy at the earlier and hypodiploidy at the later passages. Sensitivity to cidofovir was distinctly higher in early passages of Tuwei00 cells than in later passages and was characterized by distinct decline of cell survival after long term cidofovir exposure. Established normal human keratinocytes, HaCaT cells, which were checked for comparison, showed a low cidofovir sensitivity similar to late passage Tuwei00 cells.

Binding of a N,N'-bisheteryl derivative of dispirotripiperazine to heparan sulfate residues on the cell surface specifically prevents infection of viruses from different families.

N,N'-bisheteryl derivatives of dispirotripiperazine (DSTP) are a novel class of antiviral compounds with some of their representatives very effectively inhibiting the replication of herpes simplex virus type 1 (HSV-1) in cell culture. Using one representative of these compounds, the N,N'-bis(1-oxido[1,2,5]oxadiazolo[3,4-d]pyrimidin-7-yl)-3,12-diaza-6,9-diazonia(5,2,5,2)dispirohexadecane dichloride (DSTP 27), we here further tried to elucidate the molecular mechanisms responsible for the antiviral activity. The results from plaque reduction assays under a variety of conditions suggest that inhibition of HSV-1 strain Kupka replication by DSTP 27 occurs at the level of viral attachment by blockade of heparan sulfate (HS) structures on the cell surface that are used as viral receptors. In contrast to heparin and pentosan polysulfate, pretreatment of cells with DSTP 27 resulted in efficient inhibition of viral adsorption and replication persisting several hours after removal of the inhibitor. Specific binding of DSTP 27 to heparin was demonstrated in vitro. Titrations of gC-positive and gC-negative pseudorabies virus (PrV) mutants on HS-positive and HS-negative cell lines confirmed that inhibitory action of DSTP 27 is strictly HS dependent. Aside from HSV-1 Kupka and PrV, DSTP 27 efficiently inhibits growth of several HSV-1 and HSV-2 strains, among them aciclovir/foscarnet-resistant strains, human cytomegalovirus, human respiratory syncytial virus, and human immunodeficiency viruses known to attach to the cell surface via HS.

Pre-emptive therapy with rituximab for prevention of Epstein-Barr virus-associated lymphoproliferative disease after hematopoietic stem cell transplantation.

Epstein-Barr virus (EBV)-associated lymphoproliferative disease (LPD) is a life-threatening complication following hematopoietic stem cell transplantation (HSCT). Therefore, early diagnosis of EBV reactivation and pre-emptive therapy may be clinically useful. We report three patients who presented with an extremely high EBV load in peripheral blood mononuclear cells and plasma without evidence of EBV disease. Following pre-emptive therapy with a single dose of rituximab, a concordant decrease of EBV-genome copies and B lymphocytes was observed. In all three patients, no EBV-associated LPD occurred. We conclude that pre-emptive therapy with rituximab appears to be effective for prevention of EBV-associated LPD after HSCT.

Semiquantitative PCR analysis of Epstein-Barr virus DNA in clinical samples of patients with EBV-associated diseases.

The laboratory diagnosis of primary and reactivated Epstein-Barr virus (EBV) infection is based on serologic methods in immunocompetent patients. However, in immunocompromised patients, serologic data are difficult to interpret and do not often correlate with clinical data. In order to find a useful and practical marker for diagnosis of EBV-related diseases, a polymerase chain reaction (PCR) assay was established for semiquantitative detection of EBV sequences. The method was based on a nested PCR, using primers of the virus capsid antigen p23 region and an endpoint dilution. This method was carried out on 68 plasma samples, 68 samples of peripheral blood mononuclear cells and 5 cerebrospinal fluid samples of 39 patients with various diseases to evaluate the EBV-genome copy number. Samples from patients suffering from infectious mononucleosis served as positive controls for active EBV infection. In 5 patients with infectious mononucleosis, high copy numbers of EBV genomes in peripheral blood mononuclear cells were detected within a range of 1,000-40,000 copies in 10(5) peripheral blood mononuclear cells. In contrast, samples from 19 latently infected persons either showed low copy numbers (10-100 in 10(5) peripheral blood mononuclear cells) or were EBV PCR negative. Comparable results were observed in seven renal transplant patients without any symptoms. The practical value of the semiquantitative detection of EBV DNA was demonstrated in three bone marrow transplant recipients. Two developed a lymphoproliferative disease associated with extremely high amounts of EBV DNA in plasma (16,000 and 50,000 copies/ml, respectively) and peripheral blood mononuclear cells (100,000 and 6.5 million copies in 10(5) peripheral blood mononuclear cells, respectively). The high EBV load in plasma and peripheral blood mononuclear cells was reduced dramatically after successful antiviral therapy in one case. The third bone marrow transplant recipient developed an EBV-induced transverse myelitis with an increased number of EBV-genome copies in peripheral blood mononuclear cells and EBV-positive cerebrospinal fluid samples. After combined antiviral and immune therapy, the EBV-genome copy numbers decreased and the patient recovered completely. These data demonstrate a good correlation between semiquantitative detection of EBV genomes and clinical findings. The method is recommended for the diagnosis of EBV-associated diseases in patients after transplantation, as well as for monitoring the response to therapy.

Synthesis, hydrolysis and anti-EBV activity of a series of 3'-modified cycloSal-BVDUMP pronucleotides.

A series of cycloSal-BVDUMP phosphate triesters has been prepared. The prototype compound was 3-methyl-cycloSal-BVDUMP 2. Furthermore, a series of 3'-O-acyl-modified derivatives having carboxylic acids with different lipophilicity or a L-configurated alpha-amino acid (phenylalanine) was prepared. The hydrolysis properties in phosphate buffer PBS as well as in PBS containing pig liver esterase (PLE) will be described. Finally, the biological activity against EBV has been determined.

In vitro activity of polyhydroxycarboxylates against herpesviruses and HIV.

The antiviral activity of 17 polyhydroxycarboxylates derived from phenolic compounds was evaluated against herpesviruses and HIV. When present during virus adsorption several of the polymers exhibited potent activity against herpes simplex virus type 1 (HSV-1), HSV-2, thymidine kinase deficient HSV-1, human cytomegalovirus (HCMV) and HIV-1 and HIV-2 at concentrations that were not toxic to the host cells. A close correlation was found between the 50% inhibitory concentrations of the polyhydroxycarboxylates against HCMV-induced cytopathicity, their inhibitory effect on the expression of HCMV-specific immediate early antigens and their inhibitory effects on HCMV adsorption to the cells. The antiviral activity of the phenolic polymers was dependent on the presence of a sufficient number of carboxylic groups. The mechanism of antiviral action of the polyhydroxycarboxylates can thus be ascribed to inhibition of virus adsorption. This type of compound may have potential in a vaginal gel to prevent sexual transmission of HSV and HIV.

Inhibitory effect of cycloSaligenyl-nucleoside monophosphates (cycloSal-NMP) of acyclic nucleoside analogues on HSV-1 and EBV.

The in vitro antiviral activity of a new series of cycloSal-pro-nucleotides derived from the acyclic nucleoside analogues aciclovir and penciclovir against herpes simplex virus type 1 (HSV-1), thymidine kinase deficient (TK-) HSV-1, and Epstein-Barr virus (EBV) was evaluated. Using the XTT-based tetrazolium reduction assay EZ4U, the cycloSal derivatives were examined for their antiviral and cytotoxic effects in HSV-1 as well as HSV-1-TK--infected Vero cells. The anti-EBV activity was assessed by means of an EBV DNA hybridization assay using a digoxigenin-labeled probe specific for the Bam H1-W-fragment of the EBV genome and by measuring viral capsid antigen (VCA) expression in P3HR-1 cells by indirect immunofluorescence. Among the new cycloSal-phosphotriesters the three aciclovir monophosphates proved to be potent and selective inhibitors of HSV-1 replication, EBV DNA synthesis and EB-VCA expression. Of interest is the retention of activity of the aciclovir monophosphates in HSV-1-TK--infected cells. Particularly 3-methyl-cycloSal-aciclovir monophosphate retained the same effectiveness, as compared to the wild type virus strain. In contrast to the aciclovir pro-nucleotides the penciclovir cycloSal-phosphotriesters exhibited at best only a marginal antiviral effect on HSV and EBV replication.

Methods in Anti-HCMV Research.

The study of strategies for the treatment of human cytomegalovirus (HCMV) infection starts with the discovery of compounds that are potent and selective inhibitors of HCMV replication. Selectivity means that the window between the concentrations that block viral replication on the one hand and those that affect host cell functions on the other is sufficiently wide. Because HCMV induces an obvious cytopathic effect (CPE) in human fibroblast cultures, potential antiviral activity can easily be scored microscopically. For this type of assay, a relatively low input of virus should be used, so that the CPE does not appear within the first 2-3 d after infection. If too high a multiplicity of infection (MOI) is used, the input virus will result in a substantial amount of CPE shortly (i.e., within 24 h) after infection. As the CPE induced by this input virus will not be blocked by compounds that inhibibit new progeny virus formation, the use of a high MOI will mask a potential inhibitory effect of a given compound on HCMV replication. Scoring of CPE can be done either by counting the HCMV-induced plaques (although this is a rather labor-intensive method for large-scale screening purposes) or simply by scoring the CPE on a scale of 1-5 (or 10). Each assay should always be validated by using a reference compound with proven anti-HCMV activity (e.g., ganciclovir, cidofovir, foscarnet).

Successful treatment of Epstein-Barr virus-induced transverse myelitis with ganciclovir and cytomegalovirus hyperimmune globulin following unrelated bone marrow transplantation.

We report a patient who developed Epstein-Barr virus (EBV)-induced transverse myelitis 19 months after unrelated bone marrow transplantation (BMT). The disease was diagnosed by physical examination, serologic determinations, EBV-specific polymerase chain reaction in peripheral blood lymphocytes and cerebrospinal fluid, and characteristic magnetic resonance imaging scan of the spine. The patient was treated with ganciclovir and cytomegalovirus (CMV) hyperimmune globulin. He gradually improved and recovered completely within 4 weeks. This case suggests that ganciclovir and CMV hyperimmune globulin appear to be effective for the treatment of EBV-induced transverse myelitis in immunocompromised patients following BMT.

Chemistry and anti-herpes simplex virus type 1 evaluation of cycloSal-nucleotides of acyclic nucleoside analogues.

The synthesis of different cycloSal-phosphotriesters of the acyclic nucleoside analogues acyclovir (ACV), penciclovir (PCV) and T-penciclovir (T-PCV) as potential new lipophilic, membrane-soluble pronucleotides is described. The introduction of the cycloSal moiety was achieved by using reactive cyclic chlorophosphane reagents. In addition to the cycloSal-PCV monophosphate (MP) phosphotriesters, a second derivative bearing an acetyl group at the second primary alcohol function was prepared. In hydrolysis studies the cycloSal-ACVMPs showed the expected range of hydrolytic stability dependent on the substituent in the masking group (8-17 h). In contrast, the cycloSal-PCVMP derivatives exhibited a 11- to 15-fold increase in hydrolytic lability as compared to the corresponding cycloSal-ACVMP derivatives. We demonstrated that the free primary alcohol group is responsible for this rate acceleration because cycloSal-OAc-PCVMP, in which the hydroxyl group was blocked by acetylation, did not show the aforementioned acceleration. Unexpectedly, the hydrolysis product was not PCVMP but according to NMR and mass spectrometry it was cycloPCVMP (cPCVMP). The title compounds were evaluated in vitro for their ability to inhibit herpes simplex virus type 1 (HSV-1) and thymidine kinase-negative (TK-) HSV-1 replication in Vero cells. The cycloSal-ACVMP compounds exhibited high antiviral activity in HSV-1-infected cells. More importantly, one derivative retained all activity from the wild-type virus strain in HSV-1/TK(-)-infected Vero cells. The PCV derivatives were markedly less active. The reason for the failure of the cycloSal-PCVMPs seems to be due to the formation of cPCVMP instead of the desired PCVMP.

Inhibitory effects of novel nucleoside and nucleotide analogues on Epstein-Barr virus replication.

The anti-Epstein-Barr virus (EBV) activity of different classes of compounds was assessed by means of an EBV DNA hybridization assay using a digoxigenin-labelled probe specific for the BamHI W fragment of the EBV genome, as well as by measuring viral capsid antigen (VCA) expression after a 7 day incubation period of P3HR-1 producer cells with the test substances. Acyclovir, ganciclovir, cidofovir and zidovudine were included as reference compounds. Several compounds proved to be potent and selective inhibitors of EBV DNA synthesis and VCA expression. Of the new compounds that were evaluated for their anti-EBV activity, the highest efficacy (lowest EC50) and highest selectivity index (SI) were shown by the purine nucleoside analogue 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (S2242) (EC50 0.6 ng/ml; SI 600), the acyclic nucleoside phosphonate analogues 9-(2-phosphono -methoxyethyl)-6-dimethylaminopurine (EC50 1.1 micrograms/ml; SI 91), 9-(2-phosphonomethoxyethyl)-2- amino-6-benzhydrylaminopurine (EC50 1.3 micrograms/ml; SI 29), 7-(2-phosphonomethoxyethyl)-6-dimethyl-aminopurine (EC50 0.8 microgram/ml; SI 56), 9-(R)-(2-phosphonomethoxypropyl)-6-(2-dimethylaminoethyl)-aminopur ine (EC50 0.5 microgram/ml; SI 42), the 2',3'-dideoxythymidine derivative 3'-oximino-2',3'-dideoxythymidine (EC50 1.5 micrograms/ml; SI 65), and 1-(2,3- dideoxy-3-N-hydroxyamino-beta-D-threo-pentafuranyl)pentafuranos yl)thymine (EC50 4.1 micrograms/ml; SI > 24).

Use of the yellow fever virus vaccine strain 17D for the study of strategies for the treatment of yellow fever virus infections.

We have employed the attenuated vaccine strain 17D of yellow fever virus (YFV) to evaluate the inhibitory effect of a selected series of compounds on YFV in Vero cells. Use of the vaccine strain does not require high-level microbiological containment facilities and should allow extensive screening. In addition, YFV may serve as a model for other flaviviruses including hepatitis C virus (HCV), and thus strategies for the treatment of YFV infections may apply to flavivirus infections in general. In the present study, several compounds belonging to different classes of nucleoside analogues and polyanions were evaluated for their inhibitory effect on the replication of YFV. Compounds that are targeted at: (i) IMP dehydrogenase (ribavirin, EICAR, tiazofurin, selenazofurin and mycophenolic acid), (ii) OMP decarboxylase (pyrazofurin and 6-azauridine), (iii) CTP synthetase (carbodine and cyclopentenyl cytosine), (iv) dihydrofolate reductase (methotrexate) and the (v) sulfated polymers (dextran sulfate and PAVAS) proved inhibitory to the replication of YFV. Mycophenolic acid (EC50: 0.08 microgram/ml). EICAR (EC50: 0.8 microgram/ml) and methotrexate (EC50: 0.07 microgram/ml) were the most effective. The findings that EICAR and mycophenolic acid, despite their potent anti-YFV activity, had little or no effect on the replication of the bunyavirus Punta Toro or herpes simplex virus in Vero cells, indicates that their anti-YFV activity is rather specific and does not merely result from cytotoxicity. Inhibitors of S-adenosylhomocysteine hydrolase (SAH hydrolase) and thymidylate synthase were found to be devoid of anti-YFV activity.

Malignant lymphomas induced by an Epstein-Barr virus-related herpesvirus from Macaca arctoides--a rabbit model.

Animal models for Epstein-Barr virus (EBV) are restricted to some species of new-world monkeys which develop malignant lymphoid tumours or benign lymphoproliferative diseases after virus inoculation. Similar pathological features were induced in rabbits by the EBV-related herpesvirus of Macaca arctoides (HVMA). In this study 17 of 32 rabbits infected with varying amounts of HVMA produced from MAL-1 cells developed lymphoproliferative disorders. In 13 rabbits high-grade malignant lymphomas were detected, 4 rabbits revealed the histopathological feature of lymphoid hyperplasia. These lymphoproliferations were shown to be associated with HVMA by PCR and by the expression of EBV-like RNAs (EBER) in 14 and 10 cases, respectively. The homology in the polymerase gene region between DNA from EBV and HVMA, and from HVMA and the malignant tissue was found to be 94.8% and 100%, respectively. All the infected animals produced antibodies to antigens corresponding to early and late EBV proteins. By studying the HVMA expression in MAL-1 cells EBV-like proteins expressed in latency (EBNA1 and EBNA2) and in the lytic cycle (VCA, EA) were detected. Our findings suggested that HVMA caused a symptomatic infection in rabbits with pathological features that fit the conditions of an animal model suitable for testing antiviral drugs and vaccines against EBV.

Therapeutic effect of pyrophosphate analogues on cutaneous herpes simplex virus type 1 infection in guinea pigs.

We investigated the influence of disodium phosphonic formate (PFA-Na2) and trisodium thiophosphonic formate (TPFA-Na3), in comparison with acyclovir (Zovirax) and trisodium phosphonic formate (PFA-Na3) (Triapten) ointment, on the course of primary cutaneous herpes simplex virus infection in a guinea pig skin model. PFA-Na2 at 3.0%, TPFA-Na3 at 0.5% and PFA-Na3 at 0.5% as well as Triapten ointment (2.0% PFA-Na3) completely inhibited virus infection. Zovirax cream (5.0% acyclovir), applied five times (15 min., 4, 20, 24, and 28 h) after virus inoculation did not prevent virus infection. Similarly, application of Zovirax cream 5 times daily for 5 days did not prevent a vesicle formation following cutaneous herpes simplex virus infection of the guinea pig.