Optimization of the blood culture pathway: a template for improved sepsis management and diagnostic antimicrobial stewardship.

Laboratory processing of blood cultures has remained static over the past 30 years, despite increasing antibiotic resistance and advances in analyser design. At the study hospital, siting the blood culture analyser in the blood sciences laboratory and optimizing the pre-analytical and analytic phases of blood culture management resulted in a reduction in the time taken to detect most blood culture isolates to <12h. Fifty percent of positive blood cultures containing Escherichia coli were definitively reported with antibiotic susceptibilities in <24h. More than 85% of blood cultures positive for E. coli had antibiotic susceptibilities reported within 36h of collection, compared with 66h at a comparator hospital.

Coronavirus reverse genetics by targeted RNA recombination.

Targeted RNA recombination was the first reverse genetics system devised for coronaviruses at a time when it was not clear whether the construction of full-length infectious cDNA clones would become possible. In its current state targeted RNA recombination offers a versatile and powerful method for the site-directed mutagenesis of the downstream third of the coronavirus genome, which encodes all the viral structural proteins. The development of this system is described, with an emphasis on recent improvements, and multiple applications of this technique to the study of coronavirus molecular biology and pathogenesis are reviewed. Additionally, the relative strengths and limitations of targeted RNA recombination and infectious cDNA systems are contrasted.

Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS.

The protein encoded by ORF 4 of mouse hepatitis virus (MHV) is not required for growth of some strains in tissue culture cells, but its role in pathogenesis in the murine host has not been defined previously in a controlled manner. MHV strain JHM causes acute and chronic neurological diseases in susceptible strains of rodents. To genetically manipulate the structural proteins of this and other strains of MHV, we have generalized an interspecies-targeted RNA recombination selection that was originally developed for the A59 strain of MHV. Using this approach, a recombinant MHV-JHM was constructed in which gene 4 was genetically inactivated. Virus lacking gene 4 expression replicated in tissue culture cells with similar kinetics to recombinant virus in which gene 4 expression was not disrupted. Both types of viruses exhibited similar virulence when analyzed in a murine model of encephalitis. These results establish a targeted recombination system for inserting mutations into MHV-JHM. Furthermore, the protein encoded by ORF 4 is not essential for growth in tissue culture cells or in the CNS of the infected host.

A study of primary care teaching comparing academic and community-based settings.

OBJECTIVE: To compare teaching activity and content between academic and community-based practices used in third year medical student primary care training. SETTING: Academic and community-based primary care practices participating in third-year internal medicine, family medicine, and primary care core clerkships. PARTICIPANTS: Five-hundred thirteen preceptor-student encounters involving 32 preceptors and 26 third-year medical students were evaluated. DESIGN: Student-preceptor pairs collected a convenience sample of data from shared patient encounters. Preceptors recorded the content of teaching interventions, and students independently documented learning points received for each clinical encounter. MEASUREMENTS AND MAIN RESULTS: Comparison of problem exposure, frequency and content of teaching interventions, and the effect of patient complexity and patient care workload on teaching frequency was made between the academic and community-based practices. Several small differences were found in the frequency of clinical problem exposure between the 2 settings. The frequency and focus of teaching interventions did not differ by practice type. Teaching by community-based preceptors tended to decrease with increased patient care workload, but increased in academically based practices. CONCLUSIONS: Although several differences exist between educational experiences in community- and academically based primary care practices, they appear to be minor and of minimal educational significance.

Evaluation of the role of heterogeneous nuclear ribonucleoprotein A1 as a host factor in murine coronavirus discontinuous transcription and genome replication.

Viruses with RNA genomes often capture and redirect host cell components to assist in mechanisms particular to RNA-dependent RNA synthesis. The nidoviruses are an order of positive-stranded RNA viruses, comprising coronaviruses and arteriviruses, that employ a unique strategy of discontinuous transcription, producing a series of subgenomic mRNAs linking a 5' leader to distal portions of the genome. For the prototype coronavirus mouse hepatitis virus (MHV), heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has been shown to be able to bind in vitro to the negative strand of the intergenic sequence, a cis-acting element found in the leader RNA and preceding each downstream ORF in the genome. hnRNP A1 thus has been proposed as a host factor in MHV transcription. To test this hypothesis genetically, we initially constructed MHV mutants with a very high-affinity hnRNP A1 binding site inserted in place of, or adjacent to, an intergenic sequence in the MHV genome. This inserted hnRNP A1 binding site was not able to functionally replace, or enhance transcription from, the intergenic sequence. This finding led us to test more directly the role of hnRNP A1 by analysis of MHV replication and RNA synthesis in a murine cell line that does not express this protein. The cellular absence of hnRNP A1 had no detectable effect on the production of infectious virus, the synthesis of genomic RNA, or the quantity or quality of subgenomic mRNAs. These results strongly suggest that hnRNP A1 is not a required host factor for MHV discontinuous transcription or genome replication.

Characterization of an essential RNA secondary structure in the 3' untranslated region of the murine coronavirus genome.

We have previously identified a functionally essential bulged stem-loop in the 3' untranslated region of the positive-stranded RNA genome of mouse hepatitis virus. This 68-nucleotide structure is composed of six stem segments interrupted by five bulges, and its structure, but not its primary sequence, is entirely conserved in the related bovine coronavirus. The functional importance of individual stem segments of this stem-loop was characterized by genetic analysis using targeted RNA recombination. We also examined the effects of stem segment mutations on the replication of mouse hepatitis virus defective interfering RNAs. These studies were complemented by enzymatic and chemical probing of the stem-loop. Taken together, our results confirmed most of the previously proposed structure, but they revealed that the terminal loop and an internal loop are larger than originally thought. Three of the stem segments were found to be essential for viral replication. Further, our results suggest that the stem segment at the base of the stem-loop is an alternative base-pairing structure for part of a downstream, and partially overlapping, RNA pseudoknot that has recently been shown to be necessary for bovine coronavirus replication.

Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier.

Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxy-terminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells.

Insertion of a new transcriptional unit into the genome of mouse hepatitis virus.

The subgenomic mRNAs of the coronavirus mouse hepatitis virus (MHV) are composed of a leader sequence, identical to the 5' 70 nucleotides of the genome, joined at distant downstream sites to a stretch of sequence that is identical to the 3' end of the genome. The points of fusion occur at intergenic sequences (IGSs), loci on the genome that contain a tract of sequence homologous to the 3' end of the leader RNA. We have constructed a mutant of MHV-A59 containing an extra IGS inserted into the genome immediately downstream of the 3'-most gene, that encoding the nucleocapsid (N) protein. We show that in cells infected with the mutant, there is synthesis of an additional leader-containing subgenomic RNA of the predicted size. Our study demonstrates that (i) an IGS can be a sufficient cis-acting element to dictate MHV transcription, (ii) the relative efficiency of an IGS must be influenced by factors other than the nucleotides immediately adjacent to the 5'AAUCUAAAC3' core consensus sequence or its position relative to the 3' end of the genome, (iii) a downstream IGS can exert a polar attenuating effect on upstream IGSs, and (iv) unknown factors prevent the insertion of large exogenous elements between the N gene and the 3' untranslated region of MHV. These results confirm and extend conclusions previously derived from the analysis of defective interfering RNAs.

Antibiotic susceptibility of Streptococcus pneumoniae in New Zealand.

AIMS: To determine the current antibiotic susceptibility patterns of Streptococcus pneumoniae from four centres in New Zealand. METHODS: Over a six-month period in 1997, 386 consecutive clinical isolates of S pneumoniae were collected by four laboratories (Auckland, Wellington, Hamilton and Christchurch) from general practice or inpatients. Susceptibility testing for seven antibiotics was performed by each centre using the Etest. RESULTS: Eighty-three-percent of isolates were penicillin susceptible, 12% showed intermediate resistance to penicillin and 5% were penicillin resistant. Overall, 93 and 91% of isolates were susceptible to amoxicillin/clavulanic acid and ceftriaxone, respectively. Erythromycin and tetracycline had similar rates of susceptibility (88 and 87%, respectively). Resistance to cotrimoxazole was common, with only 57% of isolates susceptible to this combination. No National Committee for Clinical Laboratory Standard (NCCLS) breakpoints were available for cefaclor to allow interpretation of the minimum inhibitory concentration data for this agent. Wellington had lower resistance rates than Auckland, Christchurch and Hamilton. Isolates from children had consistently higher resistance rates (two- to five-fold greater for beta-lactams and 1.2 to 1.3-fold for other agents) compared with isolates from adult patients. CONCLUSIONS: Resistance to multiple antibiotics among S pneumoniae is now evident in New Zealand, although rates varied between study centres. The overall rate of penicillin resistance is 5%, which is similar to that observed in many European and US cities but lower than the rates reported in badly affected areas (> 30%). These data suggest that amoxicillin (+/- clavulanic acid), erythromycin or tetracycline are appropriate agents for empirical use in less serious community acquired infections when S pneumoniae is suspected. Ceftriaxone, with or without vancomycin, should be considered in the empirical treatment of invasive, disease until sensitivities are known.

Evaluating students on an interdisciplinary primary care clerkship at the Pennsylvania State University College of Medicine.

With funding from The Robert Wood Johnson Foundation's Generalist Physician Initiative, the Pennsylvania State University College of Medicine created a community-based primary care clerkship in general pediatrics, general internal medicine, and family and community medicine, in which third-year students spend a month in a small town, rural area, or urban underserved medical community. In addition to linking students with preceptors who would teach the clinical skills essential to primary care practice, the medical school set out to teach and to evaluate knowledge, attitudes, and behaviors unique to primary care. This paper describes the three-part teaching tool/evaluation developed to address (1) a student's recognition of the characteristics of primary care (learning objectives assignment), (2) a student's ability to appreciate the multiple nonmedical factors influencing a patient's health and experience of illness (family project), and (3) a student's ability to solve clinical problems (clinical reasoning examination). The authors describe how these evaluation methods are linked with the clerkship's goals and objectives and how they yield a richer portrait of the student's performance than the traditional preceptor's evaluation alone can provide. They also discuss the relationship between students' performances on the primary care clerkship and their performances in other clinical clerkships. Similar clinical experiences in primary care should focus on features unique to primary care medicine in both teaching and evaluation.

Construction of a mouse hepatitis virus recombinant expressing a foreign gene.

The genome of the coronavirus mouse hepatitis virus (MHV) contains genes which have been shown to be nonessential for viral replication and which could, in principle, be used as sites for the introduction of foreign sequences. We have inserted heterologous genetic material into gene 4 of MHV in order (i) to test the applicability of targeted RNA recombination for site-directed mutagenesis of the MHV genome upstream of the N gene; (ii) to develop further genetic tools for mutagenesis of structural genes other than N; and (iii) to examine the feasibility of using MHV as an expression vector. A DI-like donor RNA vector containing the MHV S gene and all genes distal to S was constructed. Initially, a derivative of this was used to insert a 19-nucleotide tag into the start of ORF 4a of MHV-A59 using the N gene deletion mutant A1b4 as the recipient virus. Subsequently, the entire gene for the green fluorescent protein (GFP) was inserted in place of gene 4. This heterologous gene was shown to be expressed by recombinant viruses but not at levels sufficient to allow detection of fluorescence of viral plaques. Northern blot analysis of transcripts of GFP recombinants showed the expected displacement of the mobility, relative to those of wild-type, of all subgenomic mRNAs larger than mRNA5. An unexpected result of the Northern analysis was the observation that GFP recombinants also produced an RNA species the same size as that of wild-type mRNA4. RT-PCR analysis of the 5' end of this species revealed that it was actually a collection of mRNAs originating from a cluster of 10 different sites, none of which possessed a canonical intergenic sequence. The finding of these aberrant mRNAs, all of nearly the same size as wild-type mRNA4, suggests that long range structure of the MHV genome can sometimes be the sole determinant of the site of initiation of transcription.

An essential secondary structure in the 3' untranslated region of the mouse hepatitis virus genome.

The 3' untranslated regions (3' UTRs) of coronaviruses contain the signals necessary for negative strand RNA synthesis and may also harbor elements essential for positive strand replication and subgenomic RNA transcription. The 3' UTRs of mouse hepatitis virus (MHV) and bovine coronavirus (BCV) are more than 30% divergent. In an effort to learn what parts of these regions might be functionally interchangeable, we attempted to replace the 3' UTR of MHV with its BCV counterpart by targeted RNA recombination. Initially, we tried to substitute the 3' 267 nucleotides (nt) of the 301 nt MHV 3' UTR with the corresponding region of the BCV 3' UTR. This exchange did not yield viable recombinant viruses, and the donor DI RNA was shown to be unable to replicate with MHV as a helper virus. Subsequent analysis revealed that the entire BCV 3' UTR could be inserted into the MHV genome in place of the entire MHV 3' UTR. It resulted that the failure of the initial attempted substitution was due to the inadvertent disruption of an essential conserved bulged stem-loop secondary structure in the MHV and BCV 3' URTs immediately downstream of the N gene stop codon.

The pathogenesis of MHV nucleocapsid gene chimeric viruses.

A set of viruses in which various segments of the nucleocapsid (N) gene of MHV have been substituted with the corresponding segments of bovine coronavirus (BCV) by targeted recombination were analyzed for their biologic properties. Histology for organ pathology and plaque assay for viral titer analysis following intracerebral (IC) inoculation were studied. One chimeric virus (Alb85), in which only a small segment of the N gene was replaced, exhibited a phenotype similar to wild type MHV-A59. However, three of the chimeric viruses (Alb106, Alb112 and Alb100) produced acute encephalitis and demyelination but without hepatitis following IC inoculation. Intravenous (IV) and intrahepatic (IH) inoculations were able to restore the ability of these viruses to produce hepatitis. The common denominator of the three chimeric viruses with a different phenotype is a region between aa 306 and aa 386 in which 17 amino acids (aa) differences exist between the two strains. Thus this region may contain determinants which enable the virus to exit the brain and reach the blood stream.

Targeted recombination between MHV-2 and MHV-A59 to study neurotropic determinants of MHV.

MHV-A59 produces acute encephalitis, acute hepatitis and chronic demyelination in infected mice. MHV-2 produces only hepatitis and mild meningitis but without encephalitis or demyelination. We have previously studied a set of recombinant viruses between these two strains. The common denominator of viruses that produced encephalitis was a membrane (M) gene derived from MHV-A59. Thus to study the potential contribution of the M gene to acute encephalitis, chimeric viruses were produced in which the M gene of MHV-A59 was substituted with the M gene of MHV-2 by targeted recombination. A control virus was produced in which the M gene of A59 was recombined back into an A59 background. Viruses were then analyzed for their biologic properties and compared with the phenotypes of MHV-A59 and MHV-2 by histopathology and plaque assays for viral titers in organs following intracerebral (IC) inoculation. All three chimeric viruses had a phenotype similar to MHV-A59. Thus, the replacement of the M gene of MHV-A59 with that of MHV-2 is insufficient to produce a phenotype that lacks encephalitis similar to MHV-2.

Analysis of constructed E gene mutants of mouse hepatitis virus confirms a pivotal role for E protein in coronavirus assembly.

Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.

Coronavirus particle assembly: primary structure requirements of the membrane protein.

Coronavirus-like particles morphologically similar to normal virions are assembled when genes encoding the viral membrane proteins M and E are coexpressed in eukaryotic cells. Using this envelope assembly assay, we have studied the primary sequence requirements for particle formation of the mouse hepatitis virus (MHV) M protein, the major protein of the coronavirion membrane. Our results show that each of the different domains of the protein is important. Mutations (deletions, insertions, point mutations) in the luminal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain had effects on the assembly of M into enveloped particles. Strikingly, the extreme carboxy-terminal residue is crucial. Deletion of this single residue abolished particle assembly almost completely; most substitutions were strongly inhibitory. Site-directed mutations in the carboxy terminus of M were also incorporated into the MHV genome by targeted recombination. The results supported a critical role for this domain of M in viral assembly, although the M carboxy terminus was more tolerant of alteration in the complete virion than in virus-like particles, likely because of the stabilization of virions by additional intermolecular interactions. Interestingly, glycosylation of M appeared not essential for assembly. Mutations in the luminal domain that abolished the normal O glycosylation of the protein or created an N-glycosylated form had no effect. Mutant M proteins unable to form virus-like particles were found to inhibit the budding of assembly-competent M in a concentration-dependent manner. However, assembly-competent M was able to rescue assembly-incompetent M when the latter was present in low amounts. These observations support the existence of interactions between M molecules that are thought to be the driving force in coronavirus envelope assembly.

Bacterial expression and characterization of the mitochondrial outer membrane channel. Effects of n-terminal modifications.

Several forms of the voltage-dependent anion-selective channel (VDAC) have been expressed at high yield in Escherichia coli. Full-length constructs of the proteins of Neurospora crassa and Saccharomyces cerevisiae (ncVDAC and scVDAC) have been made with 20-residue-long, thrombin-cleavable, His6-containing N-terminal extensions. ncVDAC purified from bacteria or mitochondria displays a far-UV CD spectrum (in 1% lauryl dimethylamine oxide at pH 6-8) similar to that of bacterial porins, indicating extensive beta-sheet structure. Under the same conditions, the CD spectrum of bacterially expressed scVDAC indicates lower beta-sheet content, albeit higher than that of mitochondrial scVDAC under the same conditions. In phospholipid bilayers, the bacterially expressed proteins (with or without N-terminal extensions) form typical VDAC-like channels with stable, large conductance open states (4-4.5 nanosiemens in 1 M KCl) and voltage-dependent transitions to a predominant substate (about 2 nanosiemens). A variant of scVDAC missing the first eight residues and having no N-terminal extension also has been expressed in E. coli. The truncated protein has a CD spectrum similar to that of mitochondrial scVDAC, but its channel activity is abnormal, exhibiting an unstable open state and rapid transitions between multiple subconductance levels.