Transmissible gastroenteritis virus (TGEV), an enteric coronavirus of swine, is a potent inducer of alpha interferon (IFN-α) both in vivo and in vitro. Incubation of peripheral blood mononuclear cells with noninfectious viral material such as inactivated virions or fixed, infected cells leads to early and strong IFN-α synthesis. Previous studies have shown that antibodies against the virus membrane glycoprotein M blocked the IFN induction and that two viruses with a mutated protein exhibited a decreased interferogenic activity, thus arguing for a direct involvement of M protein in this phenomenon. In this study, the IFN-α-inducing activity of recombinant M protein expressed in the absence or presence of other TGEV structural proteins was examined. Fixed cells coexpressing M together with at least the minor structural protein E were found to induce IFN-α almost as efficiently as TGEV-infected cells. Pseudoparticles resembling authentic virions were released in the culture medium of cells coexpressing M and E proteins. The interferogenic activity of purified pseudoparticles was shown to be comparable to that of TGEV virions, thus establishing that neither ribonucleoprotein nor spikes are required for IFN induction. The replacement of the externally exposed, N-terminal domain of M with that of bovine coronavirus (BCV) led to the production of chimeric particles with no major change in interferogenicity, although the structures of the TGEV and BCV ectodomains markedly differ. Moreover, BCV pseudoparticles also exhibited interferogenic activity. Together these observations suggest that the ability of coronavirus particles to induce IFN-α is more likely to involve a specific, multimeric structure than a definite sequence motif.

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Antimicrobial therapy of infections with Staphylococcus aureus can pose a challenge due to slow response to therapy and recurrence of infection. These treatment difficulties can partly be explained by intracellular survival of staphylococci, which is why the intracellular activity of antistaphylococcal compounds has received increased attention within recent years. The intracellular activity of plectasin, an antimicrobial peptide, against S. aureus was determined both in vitro and in vivo. In vitro studies using THP-1 monocytes showed that some intracellular antibacterial activity of plectasin was maintained (maximal relative efficacy [E max ], 1.0-to 1.3-log reduction in CFU) even though efficacy was inferior to that of extracellular killing (E max , >4.5-log CFU reduction). Animal studies included a novel use of the mouse peritonitis model, exploiting extra-and intracellular differentiation assays, and assessment of the correlations between activity and pharmacokinetic (PK) parameters. The intracellular activity of plectasin was in accordance with the in vitro studies, with an E max of a 1.1-log CFU reduction. The parameter most important for activity was fC peak /MIC, where fC peak is the free peak concentration. These findings stress the importance of performing studies of extra-and intracellular activity since these features cannot be predicted from traditional MIC and killing kinetic studies. Application of both the THP-1 and the mouse peritonitis models showed that the in vitro results were similar to findings in the in vivo model with respect to demonstration of intracellular activity. Therefore the in vitro model was a good screening model for intracellular activity. However, animal models should be applied if further information on activity, PK/pharmacodynamic parameters, and optimal dosing regimens is required.

The bovine rotavirus VP2 protein is the major component of the core and forms the most internal layer surrounding the dsRNA genome. We have constructed recombinant baculoviruses expressing truncated VP2 proteins. The nucleic acid binding activity of these truncated proteins was tested by North-Western blotting experiments with single-stranded and double-stranded probes. The nucleic acid binding domain in VP2 was localized between amino acids 1 to 132. Recombinant proteins bound single-stranded and double-stranded nucleic acids, but showed less affinity for doublestranded RNA and DNA. Interactions of VP2 with the genome were investigated in viral single-shelled particles by u.v.-cross-linking. In these experiments, only VP2 protein bound the genomic RNA in purified singleshelled particles.

Linezolid exerts only a weak intracellular activity against the strains of S. aureus tested, even though, in contrast to most other antibiotics, its potency does not appear impaired in comparison with the extracellular activity.

49Previous work in our laboratory have provided evidence that the membrane glycoprotein M ofTGEV is centrally involved in efficient induction of alpha interferon (IFN-a) synthesis by non-immune peripheral blood mononuclear cells incubated with fixed, TGEV-infected cells or inactivated virions. Here we report recent completion of studies initiated to get a better understanding of the nature of the interferogenic determinant(s). Transfected cells expressing TGEV M together with the minor structural component E (formerly called sM) were found to trigger IFN-a. synthesis. Co-expression of these two proteins was shown to be necessary and sufficient for assembly and release of pseudoparticles resembling TGEV virions. Purified pseudoparticles exhibited an interferogenic activity close to that of authentic virions. Chimeric recombinant particles expressing BCV M ectodomain also induced IFN. Examination of cell cultures infected by viruses representative of the three Nidovirales genera revealed that the capacity to act as an efficient IFN-a inducer is a common feature of viral particles of the corona virus genus. Altogether these data bring new insights regarding the putative nature of the viral structure involved in IFN-a. induction.

Antibiotic treatment ofBoth models showed (i) the relative maximal efficacy (1-log-unit reduction in the numbers of CFU) of DCX intracellularly and (ii) the equal relative potency of DCX intra-and extracellularly, with the MIC being a good indicator of the overall response in both situations. Discordant results, based on data obtained different times after dosing, were obtained from the two models when the extracellular activity of DCX was measured, in which the in vitro model showed a considerable reduction in the number of CFU from that in the original inoculum (3-log-unit decrease in the number of CFU after 24 h), whereas the extracellular CFU reduction achieved in vivo after 4 h did not exceed 1 log unit. Multiple dosing of DCX in vivo revealed increased extra-and intracellular efficacies (2.5 log and 2 log units of reduction in the numbers of CFU after 24 h, respectively), confirming that DCX is a highly active antistaphylococcal antibiotic. PK/PD analysis revealed that fT MIC is the index that is the most predictive of the outcome of infection both intra-and extracellularly.Staphylococcus aureus is a major cause of both communityand hospital-acquired infections (28, 30), which range from simple and uncomplicated skin and wound infections (2, 24) to more serious and life-threatening situations such as pneumonia (15, 36), endocarditis (16, 37), osteomyelitis (13, 25), and meningitis (34). S. aureus infections often show poor and slow responses to therapy, with recurrences and ensuing mortality (8,9,27,37,38,46). These responses could be caused by the ability of the bacteria to invade and survive inside cells (5,10,21,22,31,32). Intracellular antimicrobial activity depends on both drug-and bacterium-related factors (penetration, accumulation, subcellular bioavailability, expression of activity in the local environment, and the state of responsiveness of the organisms [42,44]). In general, intracellular antimicrobial activity is markedly impaired compared to the activity seen in broth or the extracellular milieu (3, 39, 45), although we know about situations in which the opposite is true (7). Thus, the direct assessment of antibiotic activity in the pertinent models is warranted. Several in vitro models with either human or animal cells have been developed to study the intracellular activities of antibiotics (3,6,14,21,35,41), and a corresponding in vivo model (a modified version of a murine peritonitis model) has recently been described (39). We have now combined these models and report here our results obtained by using dicloxacillin (DCX) as a prototype of antistaphylococcal ␤-lactam antibiotics. Isoxazolyl penicillins have usually been preferred for the treatment of methicillin-susceptible S. aureus (MSSA) infections (2,20,26,30). DCX has been the main choice in Denmark and many other countries due to its stability against penicillinases, low level of toxicity, and availability for both oral and intravenous administration (19). We examined the intra-and extracellular time-and concentration-kill relationshi...

Objectives: Treatment of chronic or recurrent Staphylococcus aureus infections may require using antibiotics with activity against intracellular multiresistant organisms. Quinupristin/dalfopristin (3:7) has been examined in this context. Results: erm(A)-positive strains were all susceptible to quinupristin/dalfopristin (except strain CM05), with MICs not adversely influenced by acid pH or by the MRSA, VISA or VRSA character of the strain. In concentrationresponse experiments, quinupristin/dalfopristin showed similar patterns for all strains (except strain CM05), with a .3 log 10 cfu decrease in broth and a 1.3 [erm(A) strain] to 2.6 [fully susceptible, vat(B) and msr(A/B) strains] log 10 cfu decrease for intracellular bacteria at the maximal extracellular concentration tested (25 mg/L). Maximal extracellular and intracellular activity was obtained for a quinupristin/dalfopristin ratio of 3:7. For strain CM05, quinupristin/dalfopristin was static in all conditions.Conclusions: Based on historical comparisons with rifampicin, fluoroquinolones, lipoglycopeptides and other antistaphylococcal drugs with a large accumulation in eukaryotic cells, quinupristin/dalfopristin appears to be one of the most active antibiotics against intracellular S. aureus studied in this model so far, largely irrespective of its resistance phenotype.