Search

ABSTRACTA collection of 68 Hafniastrains previously identified to the species level by 16S rRNA gene sequencing were investigated for simple phenotypic properties that could aid in their recognition in the clinical laboratory. Four tests, including malonate utilization, fermentation of salicin and d-arabinose, and expression of ß-glucosidase activity, correctly assigned each strain to either Hafnia alveior H. paralvei. Antibiotic susceptibility profiles were generated for 35 H. alveiand H. paralveiisolates using Etest strips for 24 antibiotics. All strains were susceptible to aminoglycosides, quinolones, carbapenems, and monobactams. Most of the Hafniaisolates had a colistin MIC of =2 µg/ml. Sequencing of an internal ampCgene fragment allowed genotypic differentiation of the two Hafniaspecies. Approximately 70% of the hafniae tested additionally produced a cytolytic toxin active on Vero cells which may play a role in gastroenteritis.

Human alkyladenine DNA glycosylase "flips" damaged DNA bases into its active site where excision occurs. Tyrosine 162 is inserted into the DNA helix in place of the damaged base and may assist in nucleotide flipping by "pushing" it. Mutating this DNA-intercalating Tyr to Ser reduces the DNA binding and base excision activities of alkyladenine DNA glycosylase to undetectable levels demonstrating that Tyr-162 is critical for both activities. Mutation of Tyr-162 to Phe reduces the single turnover excision rate of hypoxanthine by a factor of 4 when paired with thymine. Interestingly, when the base pairing partner for hypoxanthine is changed to difluorotoluene, which cannot hydrogen bond to hypoxanthine, single turnover excision rates increase by a factor of 2 for the wild type enzyme and about 3 to 4 for the Phe mutant. In assays with DNA substrates containing 1,N(6)-ethenoadenine, which does not form hydrogen bonds with either thymine or difluorotoluene, base excision rates for both the wild type and Phe mutant were unaffected. These results are consistent with a role for Tyr-162 in pushing the damaged base to assist in nucleotide flipping and indicate that a nucleotide flipping step may be rate-limiting for excision of hypoxanthine.

ABSTRACTWhile many herpes simplex virus (HSV) structural proteins are expressed with strict-late kinetics, the HSV virion protein 5 (VP5) is expressed as a “leaky-late” protein, such that appreciable amounts of VP5 are made prior to DNA replication. Our goal has been to determine if leaky-late expression of VP5 is a requirement for a normal HSV infection. It had been shown previously that recombinant viruses in which the VP5 promoter was replaced with promoters of other kinetic classes (including a strict late promoter) exhibited no alterations in replication kinetics or virus yields in vitro. In contrast, here we report that alterations in pathogenesis were observed when these recombinants were analyzed by experimental infection of mice. Following intracranial inoculation, a recombinant expressing VP5 from a strict-late promoter (UL38) exhibited an increased 50% lethal dose and a 10-fold decrease in virus yields in the central nervous system, while a recombinant expressing VP5 from an early (dUTPase) or another leaky-late (VP16) promoter exhibited wild-type neurovirulence. Moreover, following infection of the footpad, changing the expression kinetics of VP5 from leaky-late to strict-late resulted in 100-fold-less virus in the spinal ganglia during the acute infection than produced by either the parent virus or the rescued virus. These data indicate that the precise timing of appearance of the major capsid protein plays a role in the pathogenesis of HSV infections and that changing the expression kinetics has different effects in different cell types and tissues.

Herpes simplex virus type 1 (HSV-1) recombinant strain 17CREcontains a site-directed mutation in the 7-bp CRE consensus sequence located 38 nucleotides upstream of the transcription start site. Scarified mouse corneas received inoculations of 17syn+(parent), 17CRE, and rescue 17CREr. Slit lamp examination of herpetic lesions and tear film swabs containing infectious virus showed that 17CREhad the same acute phenotype as 17syn+and 17CREr. At 4 weeks, when the corneas had healed and latency was established, mice received hyperthermic shock. Eye swabs taken 24 h after hyperthermia showed that 17CREreactivated significantly less than 17syn+and 17CREr, while no significant differences were found in HSV-1 DNA genome copy numbers and latent virus in the trigeminal ganglia. These results are evidence that this CRE site in the LAT promoter facilitates ocular HSV-1 reactivation in mice.

Mutagenesis of a cyclic AMP response element (CRE) within the LAT promoter of HSV-1 reduces the ability of LAT expression to be induced in transient assays, but has only a minimal impact on reactivation of the virus inin vitrosystems. Here we show that a CRE mutation results in a significant reduction of adrenergically induced reactivationin vivoin the rabbit eye model. Spontaneous reactivation frequencies were also reduced. In addition, we demonstrate that this mutation has no effect on the amount of LAT expressed during latency when compared with the parent, 17syn+, and the rescuant. These results indicate a greater effect of CRE on induced reactivationin vivothan inin vitrosystems, but also suggest that the CRE in the LAT promoter is not autonomous in conducting the reactivation signal.