Herpesvirus Macaca arctoides (HVMA) has the propensity to transform macaque lymphocytes to lymphoblastoid cells (MAL-1). Inoculation of rabbits with cell-free virus-containing supernatant resulted in the development of malignant lymphomas and allowed isolation of immortalised HVMA-transformed rabbit lymphocytes (HTRL). In this study, the HVMA genome sequence (approx. 167 kbp), its organisation, and novel aspects of virus latency are presented. Ninety-one open reading frames were identified, of which 86 were non-repetitive. HVMA was identified as a Lymphocryptovirus closely related to Epstein–Barr virus, suggesting the designation as ‘Macaca arctoides gammaherpesvirus 1’ (MarcGHV-1). In situ lysis gel and Southern blot hybridisation experiments revealed that the MAL-1 cell line contains episomal and linear DNA, whereas episomal DNA is predominantly present in HTRL. Integration of viral DNA into macaque and rabbit host cell genomes was demonstrated by fluorescence in situ hybridisation on chromosomal preparations. Analysis of next-generation sequencing data confirmed this finding. Approximately 400 read pairs represent the overlap between macaque and MarcGHV-1 DNA. Both, MAL-1 cells and HTRL show characteristics of a polyclonal tumour with B- and T-lymphocyte markers. Based on analysis of viral gene expression and immunohistochemistry, the persistence of MarcGHV-1 in MAL-1 cells resemble the latency type III, whereas the expression pattern observed in HTRL was more comparable with latency type II. There was no evidence of the presence of STLV-1 proviral DNA in MAL-1 and HTRL. Due to the similarity to EBV-mediated cell transformation, MarcGHV-1 expands the available in vitro models by simian and rabbit cell lines.

Rabbit antithymocyte globulin (ATG) is commonly used before allogeneic hematopoietic stem cell transplantation (allo-HSCT) to prevent graft-versus-host disease. Studies comparing the effect of different ATG preparations and dosages on immune reconstitution and risk for Epstein–Barr virus (EBV)-mediated post-transplant lymphoproliferative disorder (PTLD) are rare.

Simvastatin, a cholesterol‐lowering drug, is reported to have immunomodulatory properties that attenuated acute lung injury independent of their major lipid lowering effects. Based on these reports, simvastatin is expected to be used for influenza prophylaxis and treatment. The present study evaluated the efficacy of simvastatin against influenza A/PR/8/34 virus infection in a murine model. In a first study, simvastatin was administered orally. To achieve high plasma levels, intraperitoneal application was used in a second study. Survival, body weight loss, viral titers in lung and trachea, and histologic lung injury were measured. Surprisingly, treatment with simvastatin resulted in lower survival rates and in more distinct body mass loss in comparison to virus‐infected control mice. Furthermore, the viral load in lungs and tracheas as well as histopathological lesions were not reduced by simvastatin. Overall, these results showed that simvastatin failed to protect mice against influenza virus infection. J Med. Virol. 85:1978–1982, 2013 . © 2013 Wiley Periodicals, Inc.

Infants with severe primary combined immunodeficiency (SCID) and children post-allogeneic hematopoietic stem cell transplantation (HSCT) are extremely susceptible to unusual infections. The lack of generic tools to detect disease-causing viruses among more than 200 potential human viral pathogens represents a major challenge to clinicians and virologists. We investigated retrospectively the causes of a fatal disseminated viral infection with meningoencephalitis in an infant with gamma C-SCID and of chronic gastroenteritis in 2 other infants admitted for HSCT during the same time period. Analysis was undertaken by combining cell culture, electron microscopy and sequence-independent single primer amplification (SISPA) techniques. Caco-2 cells inoculated with fecal samples developed a cytopathic effect and non-enveloped viral particles in infected cells were detected by electron microscopy. SISPA led to the identification of astrovirus as the pathogen. Both sequencing of the capsid gene and the pattern of infection suggested nosocomial transmission from a chronically excreting index case to 2 other patients leading to fatal infection in 1 and to transient disease in the others. Virus-specific, real-time reverse transcription polymerase chain reaction was then performed on different stored samples to assess the extent of infection. Infection was associated with viremia in 2 cases and contributed to death in 1. At autopsy, viral RNA was detected in the brain and different other organs, while immunochemistry confirmed infection of gastrointestinal tissues. This report illustrates the usefulness of the combined use of classical virology procedures and modern molecular tools for the diagnosis of unexpected infections. It illustrates that astrovirus has the potential to cause severe disseminated lethal infection in highly immunocompromised pediatric patients.

BACKGROUND Norovirus infection is the most common cause of acute self-limiting gastroenteritis. Only 3 cases of chronic norovirus infection in adult solid organ transplant recipients have been reported thus far. METHODS This case series describes 9 consecutive kidney allograft recipients with chronic norovirus infection with persistent virus shedding and intermittent diarrhea for a duration of 97-898 days. The follow-up includes clinical course, type of immunosuppression, and polymerase chain reaction for norovirus. Detailed molecular analyses of virus isolates from stool specimens over time were performed. RESULTS The intensity of immunosuppression correlated with the diarrheal symptoms but not with viral shedding. Molecular analysis of virus strains from each patient revealed infection with different variants of GII.4 strains in 7 of 9 patients. Another 2 patients were infected with either the GII.7 or GII.17 strain. No molecular evidence for nosocomial transmission in our outpatient clinic was found. Capsid sequence alignments from follow-up specimens of 4 patients showed accumulation of mutations over time, resulting in amino acid changes predominantly in the P2 and P1-2 region. Up to 25 amino acids mutations were accumulated over a 683-day period in the patient with an 898-day shedding history. CONCLUSION Norovirus infection may persist in adult renal allograft recipients with or without clinical symptoms. No evidence for nosocomial transmission in adult renal allograft recipients was found in our study. Molecular analysis suggests continuous viral evolution in immunocompromised patients who are unable to clear this infection.

Epstein‐Barr virus (EBV)‐associated post‐transplant lymphoproliferative disease is a life‐threatening complication following hematopoietic stem cell transplantation. A quantitative polymerase chain reaction to evaluate EBV‐genome copy numbers based on a nested polymerase chain reaction and an end‐point dilution was used. Applying this assay EBV load was prospectively screened weekly in 123 patients after transplantation. The results demonstrate that EBV reactivations with more than 1,000 EBV‐genome copies measured in 10 5 peripheral blood mononuclear cells were observed in 31 patients (25.2%). Three patients developed lymphoproliferative disease with extremely high EBV‐genome copies in peripheral blood mononuclear cells (>100,000 copies/10 5 cells) and plasma. After combined antiviral and immune therapy two of three patients showed a dramatic decrease of EBV load and survived, while the third patient died of lymphoma. A subclinical EBV reactivation was observed in 24 cases (19.5%) with EBV‐genome copies in 10 5 peripheral blood mononuclear cells ranging between 2,500 and mostly 10,000. After reduction of immunosuppression the EBV levels normalized. In four patients, the high copy number of ≥80,000 copies/10 5 peripheral blood mononuclear cells and plasma positivity prompted us to start pre‐emptive therapy with rituximab and cidofovir for prevention of lymphoproliferative disease. After drug administration the high EBV load was reduced remarkably. Ninety‐two patients (74.8%) who had ≤1,000 copies/10 5 peripheral blood mononuclear cells did not develop EBV‐associated lymphoproliferative disease. In conclusion, monitoring of EBV load is a sensitive and useful parameter in the surveillance of EBV reactivation for early intervention in EBV‐associated lymphoproliferative disease as well as for follow‐up of the efficacy of therapy. J. Med. Virol. 80:441–454, 2008. © 2008 Wiley‐Liss, Inc.

Measurement of viral load is important in predicting and monitoring of Epstein-Barr virus (EBV)-associated diseases especially in immunocompromised patients. The objectives of this study were the development of a LightCycler-based real-time PCR assay using primers and probes which recognize the virus capsid antigen p23-encoding region and its comparison to the semiquantitative PCR. The LightCycler protocol shows a high degree of specificity and inter- and intra-assay reproducibility. Concerning sensitivity, a good correlation between both methods was demonstrated for standard plasmid DNA, reference DNA isolated from the EBV-genome containing Namalwa cell line, and DNA extracted from plasma/cerebrospinal fluid (CSF). The detection limit was determined with 1 copy/microl eluate for the standard plasmid DNA and with 500 copies/ml plasma or CSF. For DNA derived from peripheral blood mononuclear cells (PBMCs), a decrease of sensitivity by factor 10-100 was found when larger amounts of background DNA (500 and 100 ng) were used presuming an inhibitory effect of cellular DNA. This was supported by running dilutions of the plasmid standard carried out with EBV-negative Ramos cell DNA. Thus, the cut-off level was estimated with 100-500 copies/10(5) PBMCs, when 50 or 10 ng total DNA were tested. The results indicate that the real-time PCR described here is a first line tool for the determination of viral load in plasma and CSF. Semiquantitative nested PCR is used for screening of PBMCs viral load. Positive specimens containing more than 500 copies/10(5) cells are measured for exact values by real-time PCR. To circumvent inhibitory effects of cellular DNA, measurements should be carried out generally with 50-10 ng DNA.

Pronucleotides represent a promising alternative to improve the biological activity of nucleoside analogs against different viral diseases and cancer chemotherapy. Moreover, pronucleotides are valuable tools for studies concerning nucleoside/nucleotide metabolism. The basic idea is to achieve nucleotide delivery into cells, bypassing limitations with intracellular formation of nucleotides from their nucleoside precursors. The cycloSaligenyl (cycloSal) concept is one of several pronucleotide systems reported so far but is the only approach in which a pronucleotide is cleaved successfully by simple but selective chemical hydrolysis. Beside others, for the nucleoside analog 2′,3′-dideoxy-2′,3′-didehydrothymidine (d4T), the application of the cycloSal approach improved antiviral potency. The basic concept, the chemistry, different structural modifications, and their effects on the antiviral potency of the cycloSal-d4T 5′-monophosphate triesters are discussed. The application of the approach to different biologically active nucleoside analogs against different targets is discussed. First results of a conceptual extension of the original cycloSal approach are summarized. Once the pronucleotides have passed the membrane, the aim is to trap the cycloSalphosphate triesters inside the cells. Therefore, enzyme-cleavable groups have been attached via a linker to the cycloSal moiety.

A series of 42 lipophilic bromovinyldeoxyuridine monophosphates (BVDUMPs) are presented as potential prodrugs of the antiviral agent (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU). The 5′-cycloSal-masking group technique has been applied to this cyclic nucleoside analogue to achieve delivery of the monophosphate of BVDU inside the target cells. The new substances have been tested for their antiviral activity against herpes simplex virus types 1 and 2 (HSV-1 and -2), thymidine kinase-deficient (TK−) HSV-1, varicella-zoster virus (VZV), human cytomegalovirus (HCMV) and Epstein–Barr virus (EBV). The XTT-based tetrazolium reduction assay EZ4U (for HSV), the plaque inhibition test (for VZV and HCMV) and a DNA hybridisation assay (for EBV) were used to assess antiviral activity. The results indicate that cycloSal-BVDUMP triesters proved to be potent and selective inhibitors of HSV-1 comparable with aciclovir. VZV replication was inhibited by very low concentrations, and two substances had a slightly better anti-VZV activity than the parent compound BVDU. No antiviral effect could be demonstrated against TK−-HSV-1, HSV-2 and HCMV, most likely owing to the lack of phosphorylation to BVDU diphosphate. Most remarkably, several cycloSal-BVDUMP triesters yielded promising anti-EBV activity whereas the parent compound BVDU was entirely inactive.

In neonates, herpes simplex virus (HSV) infections can lead to severe diseases associated with high mortality. We report a 6-day-old girl who developed clinical signs of fulminant hepatic failure accompanied by infectious-toxic shock and disseminated coagulopathy secondary to HSV type 1 (HSV-1) infection. The diagnosis was performed postmortem by demonstration of HSV-1 DNA in liver tissue as well as by retrospective detection of HSV-specific antibodies.

Previous studies have shown that cycloSaligenyl-monophosphate ( cycloSal-MP) derivatives of aciclovir (ACV), penciclovir (PCV) and brivudin (BVDU) can act as inhibitors of vaccinia virus and cowpox virus replication in vitro. The aim of the present study was to evaluate the inhibatory efficacy on DNA synthesis in vaccinia and cowpox viruses of several cycloSal-pro-nucleotides of ACV and BVDU, which have proven activity against pox viruses. Viral DNA was quantified in treated and non-treated virus-infected cells by semi-quantitative PCR on the basis of the haemag-glutinin protein gene of orthopoxviruses. As result, an inhibitory efficacy on vaccinia and cowpox virus DNA replication could be demonstrated for 3-methyl- cycloSal-ACVMP, 5-H- cycloSal-ACVMP, 6-chloro-7-ECM- cycloSal-3′-OH-BVDUMP, and 6-chloro-7-methyl- cycloSal-3′-OH-BVDUMP. At concentrations of 32–128 mg/ml, 3-methyl- cycloSal-ACVMP and 6-chloro-7-ECM- cycloSal-3′-OH-BVDUMP inhibited synthesis of viral DNA to a similar extent as the well-known inhibitors of pox viruses, cidofovir and 5-iodo-dUrd (deoxyuridine). When concentrations of 128 mg/ml were administered, both test substances diminished the amount of viral genome copies by ≥4 log 10 corresponding to ≥99.99% reduction. In conclusion, selected cycloSal-pro-nucleotide derivatives of ACV and BVDU can inhibit orthopoxviral DNA synthesis. The high inhibitory efficacy on both replication of viral DNA and infectious viral particles in cell cultures makes these compounds promising candidates for in vivo experiments.

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.

CycloSal-BVDUMP triesters 32-34 5-[(E)-2-bromovinyl]-2-deoxyuridine (BVDU 2) have been studied with regard to their potential anti-EBV activity. In addition to the 3-unmodified cycloSal-BVDUMP triesters 32a-f, the 3- hydroxyl function has been esterified with different aliphatic carboxylic acids (33a-g) and α-amino acids having natural and non-natural Cα-configuration (34a-m). In addition to the synthesis of these compounds, different physicochemical properties will be reported, i.e. lipophilicity and hydrolysis behaviour. It could be shown that BVDUMP and not 3,5- cyclic BVDUMP was delivered from most of the compounds by chemical hydrolysis in phosphate buffers at pH 6.8 and 7.3 as well as P3HR-1 cell extracts. Finally, the compounds were tested for their anti-EBV activity. As a result, the prototype compounds and particularly triesters 32c,d exhibited pronounced anti-EBV activity making these compounds promising candidates for further development. However, the 3-ester derivatives were devoid of any antiviral activity, while the 3-aminoacyl derivatives showed an antiviral activity, in dependence of the amino acid and the Cα- configuration. In addition, all cycloSal-BVDU phosphotriesters proved to be potent and selective inhibitors of herpes simplex virus type 1 replication. Several pronucleotide concepts will be briefly summarised but the cycloSal-pronucleotide system described in more detail is the only approach that showed an improvement in antiviral activity of the nucleoside analogue BVDU. Keywords: pronucleotides, cyclosal-prodrugs, ebv, hsv, anti-herpes virus drugs

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.

Epstein-Barr virus (EBV)-associated lymphoproliferative disease (LPD) is a serious complication following hematopoietic stem cell transplantation (HSCT). In 86 patients undergoing HSCT in a single center we prospectively performed a quantitative EBV-specific polymerase chain reaction in peripheral blood mononuclear cells (PBMC) weekly after HSCT. Cellular immune reconstitution was monitored by flow cytometric analysis on days 30 and 60 after transplantation. We observed an EBV reactivation (>103 EBV-genome copies/105 PBMC) only in patients who received antithymocyte globulin (ATG) for immunosuppression. 15 of 46 patients (33%) who received ATG experienced EBV reactivation. Interestingly, we found a similar incidence of EBV reactivation in 7 of 21 patients (33%) who received ATG (Fresenius) at a median total dose of 40 mg/kg and in 8 of 26 patients (31%) who received ATG (Sangstat) at a median total dose of 20 mg/kg. However, in patients with ATG (Fresenius) EBV reactivation was observed only in the range between 103 and 104 EBV-genome copies/105 PBMC and no EBV-associated LPD occurred. In contrast, in patients with ATG (Sangstat) EBV reactivation was always greater than 104 EBV-genome copies/105 PBMC. Median values for CD3+ cells were 157.7/μl versus 1.5/μl on day 30 and 327.0/μl versus 15.5/μl on day 60 in the group receiving ATG (Fresenius) or ATG (Sangstat), respectively. For CD3+CD4+ cells the corresponding numbers were 57.8/μl versus 0.5/μl on day 30 and 90.8/μl versus 10.0/μl on day 60. Median values for CD3+CD8+ cells were 89.4/μl versus 0.3/μl on day 30 and 237.3/μl versus 9.4/μl on day 60 in the group receiving ATG (Fresenius) or ATG (Sangstat), respectively. Patients with ATG (Sangstat) had significantly lower numbers of CD3+, CD3+CD4+, and CD3+CD8+ cells than patients with ATG (Fresenius) on day 30 (P <.001) and day 60 (P <.03). Four patients who received ATG (Sangstat) developed EBV-associated LPD at a median of 80 days after HSCT. The increase of EBV-genome copies occurred 1–5 weeks before the onset of LPD. After treatment of LPD 3 of 4 patients demonstrated a dramatic decrease of EBV-genome copies and survived, while the fourth patient died of lymphoma. All 4 patients with EBV-associated LPD presented with a high copy number (>104 EBV-genome copies/105 PBMC). In contrast, 79 patients who had a copy number lower than 104 EBV-genome copies/105 PBMC did not develop EBV-associated LPD (P <.001). This finding prompted us to start pre-emptive therapy in patients with a high copy number for prevention of EBV-associated LPD. Three patients developed a high viral load (>104 EBV-genome copies/105 PBMC). Pre-emptive therapy was performed with a single dose of rituximab (375 mg/m2) at a median of 52 days after HSCT. Following administration of rituximab a dramatic decrease of EBV-genome copies and B cells was observed. In all 3 patients no EBV-associated LPD occurred until 6 months after rituximab infusion. We conclude that in comparison to ATG (Fresenius) the use of ATG (Sangstat) as part of the conditioning regimen leads to a significant delay in T-cell reconstitution and to a significantly higher increase of EBV DNA load which predicts for EBV-associated LPD. Pre-emptive therapy with rituximab results in a dramatic decrease of EBV-genome copies and B cells and appears to be effective for preventing EBV-associated LPD.

Pronucleotides represent a promising alternative to improve the biological activity of nucleoside analogs against different viral diseases. Moreover, pronucleotides are valuable tools for studies concerning the nucleoside/nucleotide metabolism. The basic idea is to achieve nucleotide delivery into cells, bypassing limitations with intracellular formation of nucleotides from their nucleoside precursors. The cycloSal-concept is one of several pronucleotide systems reported so far but is the only approach in which a pronucleotide is cleaved successfully by a simple but selective chemical hydrolysis. Beside others, for the nucleoside analog d4T the application of the cycloSal-approach improved antiviral potency. In the first part, the basic concept, the chemistry, different structural modifications and their effects on the antiviral potency of the cycloSal-d4TMP triesters have been discussed in this review. In the second part, first results of a conceptional extension of the original cycloSal-approach will be summarized. Once the pronucleotides have passed the membrane, the aim is to trap the cycloSal-phosphate triesters inside the cells. Therefore, enzyme-cleavable groups have been attached via a linker to the cycloSal-moiety.

Epstein–Barr virus (EBV) infection and its relevance in post-transplant lymphoproliferative diseases (PTLDs) is an increasing area of concern. PTLD can differ clinically from a mononucleosis-like syndrome to malignant lymphoma. The incidence varies between < 1 and > 20% depending on different risk factors and the kind of transplant. Despite several treatment regimens, including reduction of immunosuppression, antiviral drugs, adoptive immunotherapy and administration of anti-CD20 monoclonal antibodies, the mortality rate is still high. Novel therapeutic strategies for managing PTLD use pharmacological induction of the viral thymidine kinase gene in tumour cells, the target of antivirals based on nucleoside analogues, followed by treatment with ganciclovir. Further treatment modalities include the development of vaccines and targeting of the latent EBV episomes. Prevention of PTLD by pre-emptive therapy based on molecular monitoring of EBV load will represent the main aim to reduce occurrence. This review examines patents and literature for the treatment of EBV-associated lymphoproliferative diseases.

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.