Efficacy of antiviral agents in feline herpetic keratitis: results of an in vitro study.

PURPOSE: To determine, by a plaque reduction assay, the in vitro efficacy of novel antiviral agents in the treatment of feline herpes virus 1 (FHV-1) keratitis in the domestic cat (Felis felis). MATERIALS AND METHODS: A standard plaque reduction assay was performed using a laboratory strain of FHV-1 and embryo-derived feline kidney cells to determine the in vitro efficacy of the antiviral drugs penciclovir (PCV), bromovinyldeoxyuridine (BVdU), and (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) and to compare these with the drugs acyclovir (ACV) and trifluorothymidine (TFT). Efficacy was assessed by determining the dose of drug at which 50% plaque reduction was noted (ED(50)). RESULTS: HPMPA was found to have greatest antiviral activity (ED(50) 0.07 microg/ml). ACV was least active (ED(50) 24 microg/ml), while TFT was active with an ED(50) of 5.7 microg/ml. PCV and BVdU had intermediate activity (ED(50) 1.6 and 1.7 microg/ml, respectively). CONCLUSIONS: This study suggests that the efficacy of HPMPA, BVdU, and penciclovir in cats with herpesviral keratitis should be determined in vivo as their efficacy in vitro was substantially greater than that of acyclovir, already shown to have demonstrable but limited clinical antiviral activity.

Pilot study to determine the feasibility of producing protease-resistant prion protein fragments by random PCR mutagenesis.

We report the results of a pilot study to determine the feasibility of using PCR random mutagenesis and in vitro transcription/translation to produce protease resistant full-length or truncated ovine prion proteins (PrP). Using this approach, we show the novel production of protease resistant recombinant ovine prion protein fragments isolated from a panel of seventy randomly mutated ovine PrP protein molecules. Protease resistance of the proteinase K (PK) digested fragments was present de novo within physiological conditions without the need for template-assisted conversion to protease resistance or the requirement of reductants, denaturants or acid pH reported to date. Four of the mutant proteins were truncated at their C-termini and all of these gave rise to digestion products which were protease resistant at significant PK concentrations and exposure times. All other mutant proteins translated as full length molecules and gave rise to PK-resistant products which showed a variability in their proteinase digestion profiles. We discuss the relevance of these finding to current research.

Performance of the enhanced Abbott AxSYM cardiac troponin I reagent in patients with heterophilic antibodies.

The presence of heterophilic antibodies in the serum of a small subpopulation of individuals continues to cause false results for modern-day immunoassays. In order to determine the frequency of heterophilic antibody (HA)-related false positives within our population of positive cardiac troponin I (cTnI) patients, we assayed 200 samples using the original in-house cTnI assay (Abbott AxSYM) and the Bayer ACS:180 cTnI, which we had previously observed to be more effective at blocking HA interference. Four samples were identified as false positives based on discordant results between the two assays, as well as the correction of the false positives by treatment of the samples with heterophilic antibody blocking reagent (HBR). An 'enhanced' version of the AxSYM cTnI reagent was designed to greatly reduce or eliminate HA interference, and has now replaced the original reagents. The present study shows that the enhanced reagent significantly reduced or eliminated much of the HA interference. Comparative studies between the enhanced cTnI reagent and the original Abbott AxSYM cTnI reagent showed excellent correlation and equivalent diagnostic concordance, when HA samples were excluded from the analysis.

Rapid expression of polymorphic ovine prion proteins and studies on their protease sensitivity.

We have used coupled and uncoupled in vitro transcription/translation to express rapidly aglycosyl ovine prion proteins from ovine genomic DNA genotyped for scrapie susceptible and nonsusceptible polymorphisms. Unlike previous in vitro studies of prion proteins, this method does not require cloning or laborious extractions. To our knowledge, this is the first report of ovine PrP expression at low (ng) levels under the control of an Escherichia coli promoter and ribosome binding site both coded for in the polymerase chain reaction primer. The rapidity of this approach could form the basis of a high throughput screening assay for PrP interactions, as proteins were expressed in a matter of hours from genomic DNA as the starting material. There was no difference observed in proteinase K sensitivity between prion translation products containing either scrapie susceptible or nonsusceptible polymorphisms.

Protective effects of equine herpesvirus-1 (EHV-1) glycoprotein B in a murine model of EHV-1-induced abortion.

In an attempt to determine if pregnant mice could be protected from abortion subsequent to challenge with equine herpesvirus-1 (EHV-1) in the mouse model of EHV-1 disease, female BALB/c mice were inoculated with baculovirus-expressed EHV-1 glycoprotein B (bac-gB), wild-type baculovirus (bac-wt), rabbit kidney (RK-13) or baby hamster kidney (BHK-21) cells. Using an ELISA, antibodies against EHV-1 were detected in the serum of mice following two injections of bac-gB and were enhanced by a third injection, after which low levels of neutralising antibody were also detected. After mating, mice in the bac-gB, bac-wt and RK-immunised groups were infected intranasally with 10(7) pfu of EHV-1 on day 16 of pregnancy. All challenged mice experienced body weight loss post-infection (pi). However, postnatally, the gB-immunised group demonstrated body weight gain which was not seen in the other groups. There were no maternal deaths in the gB-immunised group but 1/6 bac-wt-immunised and 3/6 RK-immunised mice died post-challenge. Litter survival rate was significantly higher (p < 0.001) for the gB-immunised dams (54%) than that of either the bac-wt-(9%) or RK-immunised (0%) dams and the mean body weight of young from the surviving bac-wt-immunised litter was significantly (p = 0.021) lower than either the gB-immunised group or the BHK-immunised unchallenged group at 10 days of age. The virus was not isolated from any foetus from a gB-immunised dam. However, the virus was detected in 9% of foetuses from bac-wt-immunised and 21% of foetuses from RK-immunised dams.

Human alpha-galactosidase A: high plasma activity expressed by the -30G-->A allele.

Human alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A) is the lysosomal exoglycosidase responsible for the hydrolysis of terminal alpha-galactosyl residues from glycoconjugates and is the defective enzyme causing Fabry disease (McKusick 301500). An unusally elevated level of plasma alpha-Gal A activity (> 2.5 times the normal mean) was detected in two unrelated normal males and the elevated activities were inherited as X-linked traits in their families. Sequencing of the alpha-Gal A coding region, intron/exon boundaries and 5'-flanking region from the proband identified a single mutation, a G-->A transition 30 nt upstream from the initiation of translation codon in exon 1. The -30G-->A mutation occurred in a putative NF kappa B/Ets consensus binding site that was recently shown to inhibit protein binding to the 5'-untranslated region of the gene, providing a possible explanation for its high activity. To further characterize the mutation, the mRNA and protein expressed by this variant allele were studied. Purified plasma and lymphoblast alpha-Gal A activity from individuals with the -30G-->A mutation had normal physical and kinetic properties. In vitro translation of mRNAs from the cloned normal and high plasma activity alleles resulted in similar levels of alpha-Gal A protein, indicating that this mutation did not enhance translation. These findings suggest that the -30G-->A mutation in the 5'-untranslated region of the alpha-Gal A gene enhances transcription, presumably by interfering with the binding of negatively-acting transcription factors which normally decrease alpha-Gal A expression in various cells. Preliminary studies of the frequency of the -30G-->A mutation in 395 unrelated normal males of mixed ancestry revealed two additional unrelated individuals who had high plasma enzymatic activity and the mutation, confirming the effect of this mutation on enzyme expression and suggesting that about 0.5% of normal individuals have high plasma alpha-Gal A activity due to this variant allele.

The pathogenesis of ED71, a defined deletion mutant of equine herpesvirus-1, in a murine intranasal infection model for equine abortion.

A series of mutants of equine herpesvirus-1 (EHV-1) which contain deletions in non-essential genes was previously characterized in a murine intranasal infection model. One mutant, ED71 which was shown to be attenuated in the model, was further characterized by inoculation into pregnant mice. Despite the attenuation previously reported, intranasal inoculation of pregnant mice resulted in premature parturition and the birth of dead or dying foetuses. Furthermore, mice inoculated before pregnancy with the same mutant, and subsequently challenged 14 days after conception with wild-type virus, were not protected from abortion.