Antiviral susceptibilities to ganciclovir, foscarnet, and cidofovir and sequencing of UL97 and DNA polymerase were done on 23 cytomegalovirus (CMV) isolates from 10 immunocompromised persons with end-organ CMV disease who were treated with ganciclovir alone or ganciclovir followed by foscarnet. Screening of UL97 for ganciclovir resistance mutations was done by restriction digest analysis. Of 14 isolates resistant to ganciclovir, 11 (79%) contained one or more UL97 mutations at codons known to confer resistance to this compound, and 10 (91%) had a concordant mutant pattern by restriction digest analysis. Of 9 isolates containing mutations in conserved regions of the DNA polymerase, 8 were resistant to ganciclovir, and 4 were cross-resistant to cidofovir. All isolates were susceptible to foscarnet. It is concluded that ganciclovir-resistant clinical CMV isolates may contain UL97 mutations, DNA polymerase mutations, or mutations in both genes. Ganciclovir therapy may select for CMV isolates that are cross-resistant to cidofovir.

Characterization of a ganciclovir-resistant cytomegalovirus strain from a patient with AIDS showed a histidine-to-glutamine change at residue 520 of UL97 (Q520 mutation). In anabolism studies, Q520 was associated with impaired phosphorylation of ganciclovir. Transfer of Q520 to a recombinant virus resulted in a ganciclovir-resistant phenotype.Resistance of clinical and laboratory cytomegalovirus (CMV) strains to ganciclovir has been associated with impaired phosphorylation of this compound in virus-infected cells (11). Characterization of ganciclovir-resistant CMV laboratory strains has demonstrated the presence of amino acid deletions (residues 590 to 593) or substitutions (residue 460) in conserved regions of the UL97 protein and/or point mutations in the DNA polymerase of the virus (2, 9, 10, 12, 13). In clinical CMV strains, resistance to ganciclovir has been associated with substitutions (residues 460, 594, and 595) or deletions (residue 595) in UL97 (1,4,14). We have identified a novel mutation in the UL97 protein of a ganciclovir-resistant CMV strain and demonstrated that this mutation is responsible for resistance to ganciclovir.(This work was presented in part at the 34th Interscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, Fla., 6 October 1994.) Viruses studied were from an AIDS patient with progressive CMV disease (retinitis and gastrointestinal disease) and included an early isolate obtained prior to ganciclovir therapy and a late isolate (C-9330) from a sample of rectal tissue obtained after 421 days of ganciclovir therapy. Ganciclovir susceptibilities were determined by a DNA hybridization method (3). A fragment of the catalytic domain of UL97 was amplified by PCR using viral DNA purified from fibroblasts infected with the patient's CMV isolates and primers designed on the basis of published data on strain AD169. Primer sequences were 5Ј-CATCGACGTTTCCACACAGAC-3Ј (Z2671, forward) and 5Ј-TTGCGCCGCCAGAATGAGCAG-3Ј (Z2672, reverse). PCRs were performed with Taq polymerase and consisted of 35 cycles at 95ЊC for 1 min, 55ЊC for 2 min, and 72ЊC for 1 min. Amplified products were filter purified and sequenced with a commercial kit (Prism Ready Reaction Dyedeoxy Terminator Cycle sequencing kit; Applied Biosystems) and sequencing primers Z2671 (forward), Z2828 (5Ј-ATCCGGATTACAACGAGCGCT-3Ј, forward), Z2672 (reverse), and Z2829 (5Ј-TAACATTCGCGCAGACGGTGC-3Ј, reverse). UL97 sequences were aligned with that of strain AD169 to determine whether mutations were present in the region analyzed. A recombinant virus (C-9330-5) was obtained in marker transfer experiments after cotransfection of MRC5 cells with full-length AD169 DNA and an 858-bp UL97 DNA fragment (encompassing codon 520) amplified by PCR from strain C-9330 using primers VS9714 (5Ј-ATGTTCTTGCGC CTTACGCA-3Ј, forward) and CT9729 (5Ј-CCATGCGCAC CTCGTCC-3Ј, reverse) (12). For ganciclovir anabolism studies, MRC5 cells infected with isolate C-9330 or the recombinant strain C-9330-5 were pulse-labeled with purified 8-14 C-ganciclovir, and ...

Background-CD133 (AC133) is a surface antigen that defines a broad population of stem cells, including myogenic and endothelial progenitors. CD133 ϩ cells are rare in adult tissues, and the factors that support their differentiation into mature angiomyogenic cells are not known. These hurdles have hampered the use of CD133 ϩ cells for therapeutic purposes. Because human fetal liver is a rich source of CD133 ϩ cells, we sought to identify the growth factors that promote codifferentiation of these cells into angiogenic and myogenic cells. Methods and Results-Human fetal liver CD133ϩ and CD133 Ϫ cell subpopulations were cultured with 5Ј-azacytidine or vascular endothelial growth factor (VEGF 165 ) and/or brain-derived nerve growth factor (BDNF

SUMMARYA virus designated bovine leukaemia virus (BLV), associated with leukaemia in cattle and previously demonstrated to induce the disease in sheep, was purified from chronically infected sheep cell cultures. Electrophoretic analysis showed a major protein of mol. wt. about 24ooo (p24) which reacted in gel diffusion and complement-fixation tests with sera from naturally infected cattle, experimentally infected sheep, and guinea pigs immunized with p24. BLV p24 has an isoelectric point of 8-6. Interspecies antigenic reactivities characteristic of mammalian Type C virus p3os were not detected in disrupted BLV or on p24. Sheep and guinea pig antisera to BLV, reactive with p24, also did not precipitate several Type C virus p3os in radioimmunoassays. BLV is also distinguished from Type C viruses and resembles mouse mammary turnout virus and Mason-Pfizer virus in having an RNA-dependent DNA polymerase which is preferentially active in the presence of Mg ++ when synthetic templates are used. Along with previously published morphological data, the above indicates that BLV is not a Type C virus as classically defined. Four hundred and forty one human sera from cancer patients and matched controls were non-reactive with disrupted BLV, BLV infected cells, and BLV p24 in complement-fixation tests.

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