Human Cytomegalovirus Isolates Research Articles

Aciclovir selects for ganciclovir‐cross‐resistance of human cytomegalovirus in vitro that is only in part explained by known mutations in the UL97 protein

Phenotypically, ganciclovir-resistant human cytomegalovirus strains could be selected by aciclovir as effectively as by ganciclovir in vitro. Three clinical human cytomegalovirus isolates with different sensitivities against ganciclovir, aciclovir, foscarnet, and cidofovir, but without any mutation in the viral UL97 protein known to confer ganciclovir resistance, were propagated each in duplicate in the presence of ganciclovir or aciclovir. After drug selection, all 12 strains were less susceptible to ganciclovir (increase of 50% focus reduction dose between 2.1– and 31.5-fold) but were still sensitive to foscarnet and cidofovir; 7/12 exhibited a ganciclovir-resistant phenotype with a 50% focus reduction dose >30 μM, and in 6 out of these typical mutations in the UL97 coding region could be found by genotyping. All four strains selected from one isolate carried the identical UL97 mutation at amino acid position 460 (methionine to valine). The decreased sensitivity to ganciclovir and aciclovir in the other strains could neither be attributed to known UL97 mutations nor to mutations in the viral polymerase (UL54), which have been reported to induce resistance. J. Med. Virol. 65:70–76, 2001. © 2001 Wiley-Liss, Inc.

Human cytomegalovirus a sequence and UL144 variability in strains from infected children

Human cytomegalovirus (HCMV) displays genetic polymorphisms. This variability may contribute to strain-specific tissue tropism and disease expression in HCMV-infected humans. To determine strain variability in a sequence and UL144 gene regions, 51 low-passage isolates from 44 HCMV-infected children were studied. Isolates were obtained from 28 healthy children attending child care centers in Iowa and from 16 congenitally infected infants born in Texas. Isolates demonstrated substantial nucleotide variation in each gene region. Phylogenetic analysis of a sequence variability allowed 39 isolates to be grouped into six clades. The largest clade contained 16 isolates with > or = 95% nucleotide homology. Forty-eight of the 49 HCMV isolates yielding UL144 amplicons was grouped according to the clades described a few years ago [Lurain et al. (1999) Journal of Virology 73:10040-10050]. No linkage was observed among a sequence, UL144, and glycoprotein B (gB; UL55) polymorphisms. Four Texas and 11 Iowa isolates displayed > or = 95% sequence homology for a sequence and UL144 regions and possessed identical gB genotypes. No relationship between UL144 polymorphisms and outcome of congenital HCMV infection was observed. These data indicate that HCMV strains circulating among young children have UL144 polymorphisms similar to those of HCMV strains excreted by immunocompromised adults. Identification of conserved nucleotide sequences among Iowa and Texas children suggests genetic stability and biologic importance of these gene regions.

Aciclovir selects for ganciclovir‐cross‐resistance of human cytomegalovirus in vitro that is only in part explained by known mutations in the UL97 protein*

Phenotypically, ganciclovir-resistant human cytomegalovirus strains could be selected by aciclovir as effectively as by ganciclovir in vitro. Three clinical human cytomegalovirus isolates with different sensitivities against ganciclovir, aciclovir, foscarnet, and cidofovir, but without any mutation in the viral UL97 protein known to confer ganciclovir resistance, were propagated each in duplicate in the presence of ganciclovir or aciclovir. After drug selection, all 12 strains were less susceptible to ganciclovir (increase of 50% focus reduction dose between 2.1- and 31.5-fold) but were still sensitive to foscarnet and cidofovir; 7/12 exhibited a ganciclovir-resistant phenotype with a 50% focus reduction dose >30 microM, and in 6 out of these typical mutations in the UL97 coding region could be found by genotyping. All four strains selected from one isolate carried the identical UL97 mutation at amino acid position 460 (methionine to valine). The decreased sensitivity to ganciclovir and aciclovir in the other strains could neither be attributed to known UL97 mutations nor to mutations in the viral polymerase (UL54), which have been reported to induce resistance.

Infrequent Occurrence of Natural Mutations in the pp65495–503Epitope Sequence Presented by the HLA A∗0201 Allele among Human Cytomegalovirus Isolates

To determine if mutations of an immunodominant HLA-restricted cytomegalovirus (CMV) peptide sequence occur in nature, the sequence corresponding to the HLA A*0201-specific peptide CMVpp65(495-503) was determined in 50 human CMV isolates. Rare mutations were detected; 6 of 50 were silent mutations at the amino terminus of the peptide, while 3 of 50 were mutations of the native methionine residue to isoleucine (M499I). The observed M499I mutation in three isolates decreased cytolytic targeting.

Human Cytomegalovirus Replicates Abortively in Polymorphonuclear Leukocytes after Transfer from Infected Endothelial Cells via Transient Microfusion Events

Using a recently developed model for in vitro generation of pp65-positive polymorphonuclear leukocytes (PMNLs), we demonstrated that PMNLs from immunocompetent subjects may harbor both infectious human cytomegalovirus (HCMV) and viral products (pp65, p72, DNA, and immediate-early [IE] and pp67 late mRNAs) as early as 60 min after coculture with human umbilical vein endothelial cells (HUVEC) or human embryonic lung fibroblasts (HELF) infected with a clinical HCMV isolate (VR6110) or other wild-type strains. The number of PMNLs positive for each viral parameter increased with coculture time. Using HELF infected with laboratory-adapted HCMV strains, only very small amounts of viral DNA and IE and late mRNAs were detected in PMNLs. A cellular mRNA, the vascular cell adhesion molecule-1 mRNA, which is abundantly present in both infected and uninfected HUVEC, was detected in much larger amounts in PMNLs cocultured with VR6110-infected cells than in controls. Coculture of PMNLs with VR6110-infected permissive cells in the presence or absence of RNA, protein, and viral DNA synthesis inhibitors showed that only IE genes were transcribed in PMNLs during coculture. Synthesis of IE transcripts in PMNLs was also supported by the finding that only the copy number of IE mRNA (and not the DNA or the pp67 mRNA) per infected PMNL increased markedly with time, and the pp67 to IE mRNA copy number ratio changed from greater than 10 in infected HUVEC to less than 1 in cocultured PMNLs. Fluorescent probe transfer experiments and electron microscopy studies indicated that transfer of infectious virus and viral products from infected cells to PMNLs is likely to be mediated by microfusion events induced by wild-type strains only. In addition, HCMV pp65 and p72 were both shown to localize in the nucleus of the same PMNLs by double immunostaining. Two different mechanisms may explain the virus presence in PMNLs: (i) one major mechanism consists of transitory microfusion events (induced by wild-type strains only) of HUVEC or HELF and PMNLs with transfer of viable virus and biologically active viral material to PMNLs; and (ii) one minor mechanism, i.e., endocytosis, occurs with both wild-type and laboratory strains and leads to the acquisition of very small amounts of viral nucleic acids. In conclusion, HCMV replicates abortively in PMNLs, and wild-type strains and their products (as well as cellular metabolites and fluorescent dyes) are transferred to PMNLs, thus providing evidence for a potential mechanism of HCMV dissemination in vivo.

In vitro activities of methylenecyclopropane analogues of nucleosides and their phosphoralaninate prodrugs against cytomegalovirus and other herpesvirus infections.

Human cytomegalovirus (HCMV) infection does not generally cause problems in the immunocompetent adult but can result in severe clinical disease in the fetus, neonate, and immunocompromised host. Ganciclovir (GCV), the agent currently used to treat most HCMV infections, has resulted in much therapeutic success; however, efficacy remains suboptimal. Therefore, there is still a need to develop new compounds for use against HCMV infections. In the present study, several Z- and E-series methylenecyclopropane analogues and their phosphoroalaninate prodrugs were tested initially for activity against HCMV, strain AD169, and murine cytomegalovirus (MCMV) in vitro. Many were found to exhibit efficacy comparable to that of GCV against HCMV in plaque assays and were active against MCMV as well. The compounds were also tested for efficacy against herpes simplex virus types 1 and 2, varicella-zoster virus, and Epstein-Barr virus, and some had levels of activity that were comparable to that of acyclovir. In addition, the compounds synguanol (QYL-438) and 2-amino-6-cyclopropylamino analogue (QYL-769) were chosen for further evaluation and were found to be effective against additional laboratory and clinical isolates of HCMV and GCV-resistant isolates. QYL-438 and QYL-769 were found to be nontoxic in human and mouse fibroblasts and were considerably less toxic than GCV in granulocyte macrophage CFUs and erythroid burst-forming units. These results provide evidence for the high activity of some of these methylenecyclopropane analogues against various herpesviruses, particularly HCMV, in tissue culture and suggest that further evaluation is warranted to determine their potential for use in future clinical studies.

15 Novel and potent anti-EBV compound, L-I-OddU dependents it's activity on EBV thymidine : Kinase Toshihiko Kira 1)2), Shiro Shigeta 1), Yung-Chi Cheng 2) 1)2)FuKushima Medical University, Dep. Microbiology, Hikarigaoka 1, Fukushima, Japan 960-1295 2)Yale University, Dep. Pharmacology, 333 Cedar St., New Haven, CT, 16520

The human cytomegalovirus (HCMV) gene UL97 product was shown to play an important role in phosphorylation of ganciclovir (GCV) in HCMV-infected cells. The deletion of the 4 amino-acid sequence AACR confers resistance to a laboratory mutant. The aim of this study was to develop a rapid and simple method to detect mutations within the 12 base pair (bp) fragment encoding AACR, from isolates and clinical specimens (urine, bronchoalveolar lavage, cerebral spinal fluid samples). A target region encompassing this 12 bp sequence was amplified by a single-step PCR assay from HCMV isolates and reference strains, and a two-step procedure from clinical specimens. Reaction products were submitted to restriction enzyme analysis and dot-blot hybridization assay. Two biotinylated probes were used: one probe (DL) overlapping the 12 bp region; and a control probe with similar length and GC content. Hybridization was performed under conditions allowing the detection of one bp deletion (HCMV strain susceptibility to GCV was determined by a rapid late antigen synthesis reduction assay.) The control probe hybridized to the UL97 sequence amplified from all 23 tested isolates and the reference strains. The DL probe gave a positive signal with GCV-susceptible strains; no signal was obtained for five out of seven resistant isolates, and for a laboratory mutant derived from the strain AD169. Restriction analysis of amplification products showed different patterns suggesting this region can be involved in various DNA changes. The results from assays directly performed on 10 clinical samples correlated with GCV strain susceptibility determination. This rapid and simple method for detecting mutations in this region of the gene UL97 could be a useful tool for identification of some GCV-resistant strains from isolates and directly from clinical specimens.

Human intestinal endothelium shows high susceptibility to cytomegalovirus and altered expression of adhesion molecules after infection.

Human cytomegalovirus (HCMV) causes gastro intestinal disease with ulcerations, apparently as a consequence of cytopathic damage to endothelial cells (EC) and subsequent microvascular obliteration. In this study we showed that cultured human intestinal microvascular endothelial cells (HIMEC) are much more susceptible to HCMV infection than human umbilical vein endothelial cells (HUVEC). When both cell types were challenged with a clinical isolate of HCMV (10 pfu per cell), 30% of HIMEC expressed HCMV immediate early proteins, but only 10% of HUVEC. Enhanced susceptibility was also reflected in the expression of early and late HCMV proteins. In addition, the interleukin-1beta (IL-1beta)-induced cellular expression of adhesion molecules differed between HIMEC and HUVEC after HCMV-infection. E-selectin was unaffected in HUVEC but increased in HIMEC, whereas vascular cell adhesion molecule (VCAM)-1 was increased in HUVEC but decreased in HIMEC. Furthermore, HCMV-infection enhanced the expression of intercellular adhesion molecule (ICAM)-1 in both cell types. In conclusion, the enhanced susceptibility to HCMV infection observed in HIMEC and the elevated expression of E-selectin and ICAM-1 observed in these cells may provide an indication to the liability of developing gastrointestinal HCMV disease and may have a possible relevance to the survival of intestinal transplants.

147 Phenotypic and genotypic resistance pattern of human cytomegalovirus isolates obtained from vitreous and urine samples of AIDS patients with CMV-retinitis

147 Phenotypic and genotypic resistance pattern of human cytomegalovirus isolates obtained from vitreous and urine samples of AIDS patients with CMV-retinitis

A novel peptide aldehyde with activity against human cytomegalovirus in two different in vivo models.

Novel peptide aldehydes (PAs) were identified as potent inhibitors of human cytomegalovirus (HCMV) in vitro. Although these compounds were highly effective against HCMV, they did not exhibit any activity against murine cytomegalovirus (MCMV). The purpose of this study was to test the antiviral activity of PA 8 as a representative of this novel class of inhibitors against HCMV in vivo. Because of the strict species specificity of HCMV we had to use two artificial animal models. In the first model, HCMV-infected human cells were entrapped into agarose plugs and transplanted into mice. In the second model, SCID mice were transplanted with human tissues that were subsequently infected with a clinical isolate of HCMV. In these two models the antiviral activity of PA 8 was clearly demonstrated, ganciclovir only being slightly superior in its in vivo antiviral activity.

Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome.

Following extensive propagation in fibroblasts, human cytomegalovirus (HCMV) loses tropism for a number of otherwise natural host cells, in particular, endothelial cells. In this study, the hypothesis was tested that loss of endothelial tropism is associated with the appearance of genomic variants. Initial quantitative focus expansion assays on endothelial monolayers demonstrated that, while the laboratory strains AD169 and Towne failed to form detectable foci, 29 out of 30 recent clinical HCMV isolates had the potential to expand in endothelial cell culture. By long-term adaptation in fibroblast cultures, nonendotheliotropic strains could be selected from clinical HCMV isolates, while long-term endothelial-adapted strains of the same isolates retained both fibroblast tropism and endothelial tropism. Such differentially adapted isolate pairs always displayed genomic differences in restriction fragment length analyses. Coinfection of endothelial cells by two nonendotheliotropic HCMV strains yielded an endotheliotropic recombinant HCMV variant combining portions of the genomes of both parental viruses. When DNA purified from various isolates was transfected into fibroblasts, progeny virus retained the specific tropism of parental virus from which the DNA was isolated. These findings demonstrate that endothelial tropism is an inherent property of most clinical HCMV isolates and is determined by the viral genome. Although the specific determinants of HCMV cell tropism are still unknown, this study provides the first evidence for a genetic contribution.

Translational effects of mutations and polymorphisms in a repressive upstream open reading frame of the human cytomegalovirus UL4 gene.

The human cytomegalovirus (HCMV) gpUL4 mRNA contains a 22-codon upstream open reading frame (uORF2), the peptide product of which represses downstream translation by blocking translation termination at its own stop codon and by causing ribosomes to stall on the mRNA. A distinctive feature of this unusual mechanism is its strict dependence on the uORF2 peptide sequence. To delineate sequence elements that function in the inhibitory mechanism, deletions and missense mutations affecting the previously uncharacterized amino-terminal region of uORF2 were analyzed in transient-transfection and infection assays. These experiments identified multiple codons in this region that are necessary for inhibition of downstream translation by uORF2 and, in conjunction with previous results, demonstrated that amino acids dispersed throughout the uORF2 peptide participate in the repressive mechanism. In contrast to the highly conserved carboxy terminus, the amino-terminal portion of the uORF2 peptide is polymorphic. A survey of uORF2 sequences in HCMV clinical isolates revealed that although most have uORF2 sequences that are predicted to retain the uORF2 inhibitory activity, approximately 15% contain polymorphisms at codons that are essential for full inhibition by uORF2. Consistent with predictions based on analyses of engineered mutations, two viral isolates with uORF2 sequences that do not inhibit downstream translation in transfection assays expressed much more gpUL4 protein but similar levels of UL4 mRNA compared to the levels produced by the prototypic laboratory strain HCMV (Towne) and another clinical isolate with an inhibitory variant uORF2. These results demonstrate that uORF2 is polymorphic in sequence and repressive activity and suggest that the uORF2 regulatory mechanism, although prevalent among natural HCMV isolates, is not absolutely essential for viral replication.

Rapid detection by reverse hybridization of mutations in the UL97 gene of human cytomegalovirus conferring resistance to ganciclovir.

BACKGROUND OF STUDY: Diseases due to human cytomegalovirus (HCMV) infection constitute a major threat in marrow and solid organ transplant recipients. Ganciclovir (GCV) is widely used in prophylaxis and pre-emptive therapy of active HCMV infection. Resistance to ganciclovir (GCV) may arise at variable frequency under GCV therapy and is conferred by mutations (i) in the UL97 gene (codons 460, 520, and 591-607) encoding a phosphotransferase which is essential for monophosphorylation of GCV and, to a lesser extent, (ii) in the UL54 gene coding for the DNA polymerase of HCMV. The purpose was to develop a rapid assay to screen for emerging GCV resistance mutations in the UL97 gene of HCMV whereby avoiding virus isolation and nucleotide sequencing procedures. A nested PCR (nPCR) amplifying UL97 codons 450-672 was developed. Nested amplicons were subsequently sequenced directly. Oligonucleotides for use in a reverse hybridization assay were designed to detect relevant non-synonymous mutations at codons UL97 460, 520, 603 and 607. Strain AD169 served as a wild-type control. UL97-specific nPCR amplicons were obtained from 18 EDTA blood samples of ten transplant recipients receiving GCV for more than 30 days. In three consecutive samples from a single patient a GCV resistance mutation at codon 603 (C-->W) was detected. In addition, two out of four cell culture-adapted HCMV isolates known to exhibit GCV resistance in vitro revealed mutations at codons 460 (M-->V) and 607 (C-->Y), respectively. By reverse hybridization a discrimination of single nucleotide changes at codons 460, 520, 603 and 607 was possible whereby matching exactly the results of the nucleotide sequence analysis for all 23 amplicons examined. Reverse hybridization appeared to be a rapid and convenient alternative to nucleotide sequencing when screening the UL97 gene of HCMV for selected markers of GCV resistance.

INHIBITION OF CYTOMEGALOVIRUS IN VITRO AND IN VIVO BY THE EXPERIMENTAL IMMUNOSUPPRESSIVE AGENT LEFLUNOMIDE

825 Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) continues to complicate the clinical course of many allograft recipients. Enhancement of viral activity by requisite immune suppression frequently engages the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide, an inhibitor of protein tryrosine kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent with demonstrated effectiveness against acute and chronic rejection in animal models. We have recently discovered that the active metabolite of this agent, A77 1726, inhibits the production of multiple isolates of human CMV in both human fibroblasts and endothelial cells in vitro. Viral plaque assays demonstrated a consistent dose-dependent reduction of virus yield over a range of concentrations equivalent to those which inhibit in vitro immune activation. Unlike anti-CMV drugs currently in use, A77 1726 has no effect upon viral DNA synthesis, as demonstrated by CMV-specific DNA dot blot analysis and direct biochemical assay of viral DNA polymerase activity. Rather, as indicated by transmission electron microscopy, this agent appears to act at the level of virion assembly by interfering with nucleocapsid tegumentation, possibly by inhibition of tegument protein phosphorylation. Likely as a consequence of this unique mechanism of action, A77 1726 is equally effective against drug-resistant isolates. To determine the effectiveness of leflunomide in the control of viral load in vivo, groups of immunodeficient nude rats were inoculated with rat CMV (RCMV, Maastricht strain, 105 plaque-forming units/animal, IP) and treated with either leflunomide (15 mg/kg/day for 14 days), ganciclovir (10 mg/kg/day for 5 days), or drug-free vehicle. Experiments also included uninfected control animals. Following euthanization at 14 days post-inoculation, viral infection was confirmed by histologic observation of typical cytomegalic ductal epithelium in salivary glands, and further verified by immunohistochemical detection of RCMV antigens in multiple tissues. Plaque assay of tissue homogenates prepared from salivary gland, spleen, and lung demonstrated 75-99% reduction in virus yield in organs harvested from leflunomide-treated animals, and 85-99% reduction in those harvested from ganciclovir-treated animals. No virus was ever recovered from uninfected control rats. Thus in addition to its in vitro antiviral activity, leflunomide is capable of reducing viral load in vivo. These findings imply that leflunomide, an effective immunosuppressive agent, shows potential to concurrently attenuate a major complication of immunosuppression, CMV disease, by a novel mechanism of antiviral activity.

Synthesis and antiviral activity of 6-benzoyl-benzoxazolin-2-one and 6-benzoyl-benzothiazolin-2-one derivatives.

The synthesis and antiviral activity of an original series of 6-benzoyl-benzoxazolin-2-one and 6-benzoyl-benzothiazolin-2-one derivatives are described. Several compounds were found to have a selective inhibitory activity against human cytomegalovirus (HCMV) and varicella-zoster virus (VZV) in vitro, being inactive against a variety of other DNA and RNA viruses. 6-(3-fluorobenzoyl)benzoxazolin-2-one, 6-(3-fluorobenzoyl)benzothiazolin-2-one, 6-(3-bromobenzoyl)benzothiazolin-2-one, 6-(3-iodobenzoyl)benzothiazolin-2-one, 3-methyl-6-(3-fluorobenzoyl)benzothiazolin-2-one, 3-benzyl-6-benzoyl-benzothiazolin-2-one, 3-benzyl-6-(3-fluorobenzoyl)benzothiazolin-2-one and 3-benzoyl-6-(3-fluorobenzoyl)benzothiazolin-2-one were the most active of the series against HCMV and VZV with a selectivity index (CC50/IC50) ranging from 10 to 20. They displayed similar activity against thymidine kinase competent (TK+) and deficient (TK-) VZV strains, and also proved to be active against clinical HCMV isolates that were resistant to ganciclovir (GCV). Time-of-addition experiments revealed a site of interaction with the HCMV replicative cycle that may be close or similar to that of GCV and cidofovir (HPMPC). The compounds showed poor, if any, activity against herpes simplex virus type 1 (HSV-1) and HSV-2, and were not inhibitory against human immunodeficiency virus and other DNA and RNA viruses. Therefore, these compounds may represent a novel lead for the development of specific HCMV and VZV drugs.

Strain-specific neutralization of human cytomegalovirus isolates by human sera.

Induction of an effective antibody response against human cytomegalovirus (HCMV) is an important defense mechanism since it is potentially capable of neutralizing infectious viruses. We have analyzed the extent of HCMV strain-specific neutralization capacity in human sera. Nine recent HCMV isolates and their corresponding sera were investigated in cross-neutralization assays. We observed differences, independent of the overall neutralization capacity, in the 50% neutralization titers of the sera against individual strains, differences that ranged from 8-fold to more than 60-fold. For one isolate, complete resistance to neutralization by two human sera was observed. The neutralization capacity of human sera was not influenced by the presence of various concentrations (up to 100-fold excess) of noninfectious envelope glycoproteins, an inherent contamination of virus preparations from recent HCMV isolates. This indicated that the decisive parameter for neutralization is the titer of the neutralizing antibodies and that neutralization is largely independent of the concentration of virus. Analysis with transplant patients revealed that during primary infection strain-specific and strain-common antibodies are produced asynchronously. Thus, our data demonstrate that the induction of strain-specific neutralizing antibodies is a common event during infection with HCMV and that it might have important implications for the course of the infection and the development of anti-HCMV vaccines.

Strong conservation of the constitutive activity of the IE1/2 transcriptional control region in wild-type strains of human cytomegalovirus.

The IE1/2 transcriptional control region of human cytomegalovirus (HCMV) drives the expression of the HCMV major immediate-early genes (UL123-122), which encode proteins crucial for initiation of the virus replicative cycle. Nucleotide sequence polymorphism in this region of the viral genome could account for variations in the replication of HCMV wild-type strains. In order to test this hypothesis, the constitutive transcription-enhancing activity of the IE1/2 transcriptional control region derived from 12 clinical isolates of HCMV was compared. This was done by PCR amplification of the respective elements followed by cloning up-stream of a beta-globin reporter gene. After transient expression in various cell types, including human teratocarcinoma cell lines, and quantification of RNA levels, the activating function of this complex cis-element was shown to be strongly conserved. This was mirrored by high nucleotide sequence conservation, even within the so-called modulator region. This strong evolutionary conservation of sequence and of transcription-enhancing function strengthens the assumption that the IE1/2 transcriptional control region plays an essential role in initiation of the HCMV replicative cycle.

Coinfection of the immunocompromised but not the immunocompetent host by multiple human cytomegalovirus strains.

Coinfection by multiple human cytomegalovirus (HCMV) strains was investigated in immunocompetent individuals and AIDS patients. Thirty HCMV maternal and fetal or newborn isolate pairs from 9 cases of congenital HCMV infection as well as 36 HCMV isolates and 2 PCR-HCMV-positive CSF samples from 13 AIDS patients were tested by restriction fragment length polymorphism analysis of multiple genome regions. Results from the group of congenital infections showed that: i) all the 9 women with primary HCMV infection presumably harboured a single HCMV strain; ii) all strains were genetically unrelated; iii) isolates from infected fetuses or newborns consisted of a single strain apparently indistinguishable from the maternal strain; iv) no strain variations were observed in isolates from different body sites or in sequential isolates from newborns up to 8 months after birth. Results from the AIDS patient group demonstrated that; i) all patients were infected by unrelated strains; ii) 6/13 (46.1%) patients were coinfected by 2 or more HCMV strains; iii) in a single patient two different HCMV strains were detected in blood and urine, respectively, whereas a mixture of the two was found in the pharynx; iv) 4 patients showed the sequential appearance of a mixed virus population or different strains suggesting sequential reinfections.

Anti-herpesvirus activity profile of 4′-thioarabinofuranosyl purine and uracil nucleosides and activity of 1- β- d-2′-fluoro-4′-thioarabinofuranosyl guanine and 2,6-diaminopurine against clinical isolates of human cytomegalovirus

Newly synthesized 4′-thio- and 2′-fluoro-4′-thioarabinofuranosyl purine and pyrimidine nucleosides were compared with the corresponding 4′-oxo type arabinosyl nucleosides for anti-herpesvirus and anti-cell proliferative potencies. 4′-Thioarabinosyl- and 2′-fluoro-4′-thioarabinofuranosyl 5-substituted uracils had selective antiviral activities, but were not superior to 4′-oxo nucleosides, except for the activity of 5-ethyl-uracil 4′-thio nucleosides against herpes simplex virus. Furthermore, 4′-thio substituted derivatives of sorivudine (BV-araU) and related compounds, and 2′-fluoro-5-methyl-arabinosyluracil exhibited reduced activity against varicella-zoster virus compared with the parent compounds. The 4′-thioarabinosyluracils, except for 5-methyluracil derivatives, were inactive against human cytomegalovirus (HCMV). 4′-Thioarabinofuranosyl guanine and diaminopurine had the most potent anti-HCMV and anti-proliferative activities, whereas arabinosyl guanine and diaminopurine had only marginal antiviral activity. 2′-Fluoro-4′-thioarabinofuranosyl derivatives of guanine (4′-thio-FaraG) and 2,6-diaminopurine (4′-thio-FaraDAP), however, had particularly high activity against all herpesviruses tested with anti-proliferative activity equipotent to that of arabinosyl guanine and diaminopurine. 4′-Thio- and 2′-fluoro-4′-thioarabinofuranosyladenines exhibited biological activities similar to that of arabinosyladenine. Both 4′-thio-FaraG and 4′-thio-FaraDAP had a 6-fold lower ED 50 than ganciclovir against clinical isolates of HCMV. A ganciclovir-resistant isolate, obtained from a patient who had received long-term ganciclovir-treatment, was susceptible to 4′-thio-FaraG and 4′-thio-FaraDAP.

A simplified assay for screening of drug resistance of cell-associated cytomegalovirus strains.

Conventional phenotypic drug resistance determination of cell-free clinical human cytomegalovirus (HCMV) isolates is usually very laborious and may take 8-12 weeks, since serially passages of slowly growing viral isolates in tissue cultures are required to obtain a sufficient viral titer for an appropriate inoculum. Rapid screening of a large number of samples would therefore only be possible if simplified, less work-intensive methods are employed. The aim of this work was to develop an assay which speeds up the whole procedure of phenotypic drug resistance determination. Steps of the classical plaque reduction assay should be simplified or omitted, but on the other hand, the assay should be reliable and reproducible. Twenty-six clinical HCMV isolates from 20 immunocompromised patients (ten pre-treatment and 16 post-treatment with ganciclovir) were tested for drug susceptibility with the simplified plaque reduction assay. Most isolates were tested at least twice in independent assays. Virus titration could be avoided by using four different doses of cell-associated virus from the secondary culture for coculture susceptibility testing. Drug susceptibility values were determined by plaque titration and Probit analysis. All clinical HCMV isolates tested showed a mean ganciclovir ID50 value of 1.98 microM, (range 0.2-12.2; median 0.95) and a mean foscarnet ID50 value of 92.4 microM (range 35.7-181; median 81). All except one isolate were classified ganciclovir sensitive when compared to ID50 values of two ganciclovir resistant control stains (53.7 +/- 6.4 and 12.7 +/- 0.9 microM) and the sensitive laboratory strain Towne (2.1 +/- 0.8 microM). Repeated tests of individual isolates were reproducible, although the infectivity of the inoculum has not been determined prior of the assay. The mean time which elapsed between receipt of the clinical specimen and read-out of the assay was circa 4 weeks. Phenotypic resistance testing of HCMV isolates following to this protocol drastically reduces expenditure of time and work. The assay allows reliably the discrimination of HCMV isolates as drug resistant or sensitive according to the recent classification criteria of the AIDS Clinical Trials Group (ACTG). The simple handling and uncomplicated calibration of this assay facilitates the screening of large specimen numbers and renders drug susceptibility determination of HCMV more accessible to diagnostic routine use.