Allele replacement: an application that permits rapid manipulation of herpes simplex virus type 1 genomes.

Herpes simplex virus (HSV) is a new platform for gene therapy. We cloned the human herpesvirus HSV-1 strain F genome into a bacterial artificial chromosome (BAC) and adapted chromosomal gene replacement technology to manipulate the viral genome. This technology exploits the power of bacterial genetics and permits generation of recombinant viruses in as few as 7 days. We utilized this technology to delete the viral packaging/cleavage (pac) sites from HSV-BAC. HSV-BAC DNA is stable in bacteria and the pac-deleted HSV-BAC (p45-25) is able to package amplicon plasmid DNA as efficiently as a comparable pac-deleted HSV cosmid set when transfected into mammalian cells. Moreover, the utility of bacterial gene replacement is not limited to HSV, since most herpesviruses can be cloned as BACs. Thus, this technology will greatly facilitate genetic manipulation of all herpesviruses for their use as research tools or as vectors in gene therapy.

Homopolymer mutational hot spots mediate herpes simplex virus resistance to acyclovir.

In the majority of cases, the mechanism underlying the resistance to acyclovir (ACV) of herpes simplex viruses (HSVs) is thymidine kinase (TK) deficiency. Plaque isolates from eight ACV-resistant (ACVr) clinical isolates from AIDS patients, of which five reactivated, were sequenced to determine the genetic lesion within the tk gene conferring resistance and whether this may have correlated with reactivation potential. Mutations were clustered within two homopolymer nucleotide stretches. Three plaque isolates (1737-14, 90-150-3, and 89-650-5) had insertion mutations within a stretch of 7 guanosines, while two isolates (89-063-1 and 89-353-1) had frameshift mutations within a stretch of 6 cytosines (a deletion and an insertion, respectively). Mutations resulted in premature termination codons, and the predicted 28- and 32-kDa truncated TK products were detected by Western blot analysis of virus-infected cell extracts. The repair of one homopolymer frameshift mutation (in isolate 1737-14) restored TK activity, demonstrating that this mutation is the basis of TK deficiency. Of the five reactivated isolates, four were TK deficient and contained frameshift mutations while the fifth retained TK activity because of its altered-TK or Pol- phenotype. These data demonstrate that the majority of ACVr clinical isolates contain frameshift mutations within two long homopolymer nucleotide stretches which function as hot spots within the HSV tk gene and produce nonfunctional, truncated TK proteins.

Comparative in vitro activity of FK-037, a new cephalosporin antibiotic.

The in vitro activity of FK-037, a novel parenteral oxime-type cephalosporin, was compared with that of cefepime, cefpirome, ceftazidime, imipenem, and gentamicin against a total of 668 recent clinical isolates. Minimum inhibitory concentrations were determined by a standard agar dilution procedure, and all isolates were tested at two inocula (10(4) and 10(6) colony forming units). FK-037 inhibited 90% of isolates of Escherichia coli, Klebsiella species, Proteus mirabilis, P. vulgaris, Morganella morganii, Serratia marcescens, Providencia stuartii, Citrobacter freundii, Salmonella typhi, Shigella sonnei, Yersinia enterocolitica, Aeromonas species, and Haemophilus influenzae at < or = 1 microgram/ml. FK-037 was less active against Enterobacter species, Acinetobacter species, and Pseudomonas species, requiring 16 micrograms/ml to inhibit 90% of isolates, and was inactive against Xanthomonas maltophilia. FK-037 inhibited 90% of methicillin-susceptible Staphylococcus aureus at < or = 1 microgram/ml and 90% of methicillin-resistant S. aureus at < or = 8 micrograms/ml.