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1.  Mapping of Neural Pathways that Influence Diaphragm Activity and Project to the Lumbar Spinal Cord in Cats 
During breathing, the diaphragm and abdominal muscles contract out of phase. However, during other behaviors (including vomiting, postural adjustments, and locomotion) simultaneous contractions are required of the diaphragm and other muscle groups including abdominal muscles. Recent studies in cats using transneuronal tracing techniques showed that in addition to neurons in the respiratory groups, cells in the inferior and lateral vestibular nuclei (VN) and medial pontomedullary reticular formation (MRF) influence diaphragm activity. The goal of the present study was to determine if neurons in these regions have collateralized projections to both diaphragm motoneurons and the lumbar spinal cord. For this purpose, the transneuronal tracer rabies virus was injected into the diaphragm and the monosynaptic retrograde tracer Fluoro-Gold (FG) was injected into the Th13-L1 spinal segments. A large fraction of MRF and VN neurons (median of 72 and 91%, respectively) that were infected by rabies virus were dual-labeled by FG. These data show that many MRF and VN neurons that influence diaphragm activity also have a projection to the lumbar spinal cord, and thus likely are involved in coordinating behaviors that require synchronized contractions of the diaphragm and other muscle groups.
doi:10.1007/s00221-010-2197-3
PMCID: PMC2864334  PMID: 20186399
Vomiting; locomotion; respiration; rabies virus; transneuronal tracer
2.  FMR1 CGG repeat length predicts motor dysfunction in premutation carriers 
Neurology  2007;70(16 Pt 2):1397-1402.
Background
Fragile X–associated tremor/ataxia syndrome (FXTAS) is a recently described, underrecognized neurodegenerative disorder of aging fragile X mental retardation 1 (FMR1) premutation carriers, particularly men. Core motor features are action tremor, gait ataxia, and parkinsonism. Carriers have expanded CGG repeats (55 to 200); larger expansions cause fragile X syndrome, the most common heritable cause of mental retardation and autism. This study determines whether CGG repeat length correlates with severity and type of motor dysfunction in premutation carriers.
Methods
Persons aged ≥50 years with a family history of fragile X syndrome underwent structured videotaping. Movement disorder neurologists, blinded to carrier status, scored the tapes using modified standardized rating scales. CGG repeat length analyses for women incorporated the activation ratio, which measures the percentage of normal active chromosome X alleles.
Results
Male carriers (n = 54) had significantly worse total motor scores, especially in tremor and ataxia, than age-matched male noncarriers (n = 51). There was a trend toward a difference between women carriers (n = 82) and noncarriers (n = 39). In men, increasing CGG repeat correlated with greater impairment in all motor signs. In women, when activation ratio was considered, increasing CGG correlated with greater ataxia.
Conclusions
CGG repeat size is significantly associated with overall motor impairment in premutation carriers. Whereas this association is most pronounced for men and covers overall motor impairment—tremor, ataxia, and parkinsonism—the association exists for ataxia among women carriers. This is the first report of a significant correlation between the premutation status and a motor feature of fragile X–associated tremor/ataxia syndrome in women.
doi:10.1212/01.wnl.0000281692.98200.f5
PMCID: PMC2685188  PMID: 18057320
3.  Mechanical and cold hypersensitivity in nerve-injured C57BL/6J mice is not associated with fear-avoidance- and depression-related behaviour 
British journal of anaesthesia  2007;98(6):816-822.
Background
Neuropathic pain is associated with significant co-morbidity, including anxiety and depression, which impact considerably on the overall patient experience. However, pain co-morbidity symptoms are rarely assessed in animal models of neuropathic pain. To improve the clinical validity of a widely used rodent model of traumatic peripheral neuropathy, we have investigated fear-avoidance- and depression-related behaviours in nerve-injured and sham-operated mice over a 4 week period.
Methods
Male C57BL/6J mice were subjected to partial sciatic nerve ligation (PSNL) or sham surgery and were assessed on days 7, 14, and 28 after operation. Withdrawal thresholds to punctate mechanical and cooling stimuli were measured. Mice were tested on the novel open-field and elevated plus-maze tests for fear-avoidance behaviour, and on the tail suspension test for depression-related behaviour.
Results
Hypersensitivity to punctate mechanical and cool stimuli was evident up to day 28 after PSNL. However, there was no change in fear-avoidance- or depression-related behaviours regardless of interval after-surgery.
Conclusion
These data demonstrate that pain behaviour in nerve-injured C57BL/6J mice was not associated with alterations in emotion-related behaviours.
doi:10.1093/bja/aem087
PMCID: PMC2656645  PMID: 17478455
mouse; pain, chronic; pain, neuropathic; pain, psychological variables; research, animal
4.  In Vitro Survey of Triazole Cross-Resistance among More than 700 Clinical Isolates of Aspergillus Species▿  
Journal of Clinical Microbiology  2008;46(8):2568-2572.
Few data exist to describe in vitro patterns of cross-resistance among large collections of clinical Aspergillus isolates, including those of species other than Aspergillus fumigatus. We examined 771 Aspergillus spp. clinical isolates collected from 2000 to 2006 as part of a global antifungal surveillance program (553 A. fumigatus, 76 A. flavus, 59 A. niger, 35 A. terreus, and 24 A. versicolor isolates and 24 isolates of other Aspergillus species). Antifungal susceptibility testing was performed by the Clinical and Laboratory Standards Institute (CLSI) M38-A broth dilution method with itraconazole (ITR), posaconazole (POS), ravuconazole (RAV), and voriconazole (VOR). We examined the potential for cross-resistance by using measures of correlation overall and by species. For most Aspergillus isolates (from 88% of isolates for ITR to 98% of isolates for VOR and POS), MICs of each triazole were ≤1 μg/ml. When all 771 isolates were examined, there were statistically significant correlations for all six triazole-triazole pairs. For A. fumigatus, the strongest correlations seen were those between VOR and RAV MICs (r = 0.7) and ITR and POS MICs (r = 0.4). Similarly, for A. flavus, only VOR and RAV MICs and ITR and POS MICs demonstrated statistically significant positive correlations. We have demonstrated correlations among triazole MICs for Aspergillus, which for the most common species (A. fumigatus and A. flavus) were strongest between VOR and RAV MICs and ITR and POS MICs. However, Aspergillus species for which MICs of VOR or POS were >2 μg/ml remain extremely rare (<1% of isolates).
doi:10.1128/JCM.00535-08
PMCID: PMC2519461  PMID: 18562581
5.  Further characterisation of a rat model of varicella zoster virus (VZV)-associated pain 
Neuroscience  2006;144(4):1495-1508.
Persistent herpes zoster-associated pain is a significant clinical problem and an area of largely unmet therapeutic need. Progress in elucidating the underlying pathophysiology of zoster-associated pain and related co-morbidity behaviour, in addition to appropriately targeted drug development has been hindered by the lack of an appropriate animal model. This study further characterises a recently developed rat model of zoster-associated hypersensitivity and investigates (a) response to different viral strains; (b) relationship between viral inoculum concentration (‘dose’) and mechanical hypersensitivity (‘response’); (c) attenuation of virus-associated mechanical hypersensitivity by clinically useful analgesic drugs; and (d) measurement of pain co-morbidity (anxiety-like behaviour) and pharmacological intervention in the open field paradigm (in parallel with models of traumatic peripheral nerve injury). VZV was propagated on fibroblast cells before subcutaneous injection into the glabrous footpad of the left hind limb of adult male Wistar rats. Control animals received injection of uninfected fibroblast cells. Hind-limb reflex withdrawal thresholds to mechanical, noxious thermal and cooling stimuli were recorded at specified intervals post-infection. Infection with all viral strains was associated with a dose-dependent mechanical hypersensitivity but not a thermal or cool hypersensitivity. Systemic treatment with intraperitoneal (i.p.) morphine (2.5mg/kg), amitriptyline (10mg/kg), gabapentin (30mg/kg), (S)-(+)-ibuprofen (20mg/kg) and the cannnabinoid WIN55,212-2 (2mg/kg) but not the antiviral, acyclovir (50mg/kg), was associated with a reversal of mechanical paw withdrawal thresholds. In the open field paradigm, virus-infected and nerve-injured animals demonstrated an anxiety-like pattern of ambulation (reduced entry into the central area of the open arena) which was positively correlated with mechanical hypersensitivity. This may reflect pain-related comorbidity. Further, anxiety-like behaviour was attenuated by acute i.p. administration of gabapentin (30mg/kg) in nerve-injured, but not virus-infected animals. This model will prove useful in elucidating the pathophysiology of zoster-associated pain and provide a tool for pre-clinical screening of analgesic drugs.
doi:10.1016/j.neuroscience.2006.11.029
PMCID: PMC2394505  PMID: 17197105
zoster-associated pain; postherpetic neuralgia; neuropathy; analgesia; open field; anxiety-like behaviour
6.  Neural progenitor cells from postmortem adult human retina 
Background: Given the presence of neural progenitor cells (NPC) in the retina of other species capable of differentiating into multiple neural components, the authors report the presence of NPC in the adult human retina. A resident population of NPC suggests that the retina may constitutively replace neurons, photoreceptors, and glia.
Methods: Adult human postmortem retinal explants and cell suspensions were used to generate cells in tissue culture that display the features of NPC. The phenotype of cells and differentiation into neurons was determined by immunocytochemistry. Dividing cells were labelled with 5-bromo-2-deoxyuridine (BrdU) and neurospheres were generated and passaged.
Results: Cells labelled with nestin, neurofilament M (NFM), rhodopsin, or glial fibrillary acidic protein (GFAP) grew out from explant cultures. BrdU labelling of these cells occurred only with basic fibroblast growth factor (FGF-2). Dissociated retina and pars plana generated primary neurospheres. From primary neurospheres, NPC were passaged to generate secondary neurospheres, neurons, photoreceptors, and glia. BrdU labelling identified dividing cells from neurospheres that differentiated to express NFM and rhodopsin.
Conclusion: The adult human retina contains NPC and may have the potential to replace neurons and photoreceptors. This has implications for the pathogenesis and treatment of retinal disorders and degenerations, including glaucoma, and those disorders associated with retinal scarring.
doi:10.1136/bjo.2004.057687
PMCID: PMC1772454  PMID: 15615756
progenitor cells; postmortem adult human retina
7.  Quantification of the rat spinal microglial response to peripheral nerve injury as revealed by immunohistochemical image analysis and flow cytometry 
Journal of Neuroscience Methods  2007;164(2):207-217.
Microgliosis is implicated in the pathophysiology of several neurological disorders, including neuropathic pain. Consequently, perturbation of microgliosis is a mechanistic and drug development target in neuropathic pain, which highlights the requirement for specific, sensitive and reproducible methods of microgliosis measurement. In this study, we used the spinal microgliosis associated with L5 spinal nerve transection and minocycline-induced attenuation thereof to: (1) evaluate novel software based semi-quantitative image analysis paradigms for the assessment of immunohistochemical images. Microgliosis was revealed by immunoreactivity to OX42. Several image analysis paradigms were assessed and compared to a previously validated subjective categorical rating scale. This comparison revealed that grey scale measurement of the proportion of a defined area of spinal cord occupied by OX42 immunoreactive cells is a robust image analysis paradigm. (2) Develop and validate a flow cytometric approach for quantification of spinal microgliosis. The flow cytometric technique reliably quantified microgliosis in spinal cord cell suspensions, using OX42 and ED9 immunoreactivity to identify microglia.
The results suggest that image analysis of immunohistochemical revelation of microgliosis reliably detects the spinal microgliosis in response to peripheral nerve injury and pharmacological attenuation thereof. In addition, flow cytometry provides an alternative approach for quantitative analysis of spinal microgliosis elicited by nerve injury.
doi:10.1016/j.jneumeth.2007.04.013
PMCID: PMC2726922  PMID: 17553569
Microglia; Nerve injury; Spinal cord; OX42; ED9; Immunohistochemistry; Flow cytometry; Image analysis
8.  Use of Fluconazole as a Surrogate Marker To Predict Susceptibility and Resistance to Voriconazole among 13,338 Clinical Isolates of Candida spp. Tested by Clinical and Laboratory Standards Institute-Recommended Broth Microdilution Methods▿  
Clinical laboratories frequently face the problem of delayed availability of commercially prepared approved reagents for performing susceptibility testing of new antimicrobials. Although this problem is encountered more often with antibacterial agents, it is also an issue with antifungal agents. A current example is voriconazole, a new triazole antifungal with an expanded spectrum and potency against Candida spp., Aspergillus spp., and other opportunistic fungal pathogens. The present study addresses the use of fluconazole as a surrogate marker to predict the susceptibility of Candida spp. to voriconazole. Reference broth microdilution MIC results for 13,338 strains of Candida spp. isolated from more than 200 medical centers worldwide were used. Voriconazole MICs and interpretive categories (susceptible, ≤1 μg/ml; susceptible dose dependent, 2 μg/ml; resistant, ≥4 μg/ml) were compared with those of fluconazole by regression statistics and error rate bounding analyses. For all 13,338 isolates, the absolute categorical agreement was 91.6% (false susceptible or very major error [VME], 0.0%). Since voriconazole is 16- to 32-fold more potent than fluconazole, the performance of fluconazole as a surrogate marker for voriconazole susceptibility was improved by designating those isolates with fluconazole MICs of ≤32 μg/ml as being susceptible to voriconazole, resulting in a categorical agreement of 97% with 0.1% VME. Clinical laboratories performing antifungal susceptibility testing of fluconazole against Candida spp. can reliably use these results as surrogate markers until commercial FDA-approved voriconazole susceptibility tests become available.
doi:10.1128/JCM.01551-06
PMCID: PMC1828974  PMID: 17079501
9.  Global Trends in the Antifungal Susceptibility of Cryptococcus neoformans (1990 to 2004) 
Journal of Clinical Microbiology  2005;43(5):2163-2167.
The antifungal susceptibilities of 1,811 clinical isolates of Cryptococcus neoformans obtained from 100 laboratories in 5 geographic regions worldwide between 1990 and 2004 were determined. The MICs of amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole, and ravuconazole were determined by the National Committee for Clinical Laboratory Standards broth microdilution method. Isolates were submitted to a central reference laboratory (University of Iowa) from study centers in Africa (5 centers, 395 isolates), Europe (14 centers, 102 isolates), Latin America (14 centers, 82 isolates), the Pacific region (7 centers, 50 isolates), and North America (60 centers, 1,182 isolates). Resistance to amphotericin B, flucytosine, and fluconazole was ≤1% overall. Susceptibility to flucytosine (MIC, ≤4 μg/ml) ranged from 35% in North America to 68% in Latin America. Similarly, only 75% of isolates from North America were susceptible to fluconazole (MIC, ≤8 μg/ml) compared to 94 to 100% in the other regions. Isolates remained highly susceptible to amphotericin B (99% susceptibility at a MIC of ≤1 μg/ml) over the entire 15-year period. Susceptibility to flucytosine (MIC, ≤4 μg/ml) increased from 34% in 1990 to 1994 to 66% in 2000 to 2004. Susceptibility to fluconazole (MIC, ≤8 μg/ml) increased from 72% in 1990 to 1994 to 96% in 2000 to 2004. Voriconazole, posaconazole, and ravuconazole all were very active (99% of isolates susceptible at MIC of ≤1 μg/ml) against this geographically diverse collection of isolates. We conclude that in vitro resistance to antifungal agents used in the treatment of cryptococcosis remains uncommon among isolates of C. neoformans from five broad geographic regions and has not increased over a 15-year period.
doi:10.1128/JCM.43.5.2163-2167.2005
PMCID: PMC1153799  PMID: 15872236
10.  Diverse Hepatitis C Virus Glycoproteins Mediate Viral Infection in a CD81-Dependent Manner 
Journal of Virology  2004;78(16):8496-8505.
We recently reported that retroviral pseudotypes bearing the hepatitis C virus (HCV) strain H and Con1 glycoproteins, genotype 1a and 1b, respectively, require CD81 as a coreceptor for virus-cell entry and infection. Soluble truncated E2 cloned from a number of diverse HCV genotypes fail to interact with CD81, suggesting that viruses of diverse origin may utilize different receptors and display altered cell tropism. We have used the pseudotyping system to study the tropism of viruses bearing diverse HCV glycoproteins. Viruses bearing these glycoproteins showed a 150-fold range in infectivity for hepatoma cells and failed to infect lymphoid cells. The level of glycoprotein incorporation into particles varied considerably between strains, generally reflecting the E2 expression level within transfected cells. However, differences in glycoprotein incorporation were not associated with virus infectivity, suggesting that infectivity is not limited by the absolute level of glycoprotein. All HCV pseudotypes failed to infect HepG2 cells and yet infected the same cells after transduction to express human CD81, confirming the critical role of CD81 in HCV infection. Interestingly, these HCV pseudotypes differed in their ability to infect HepG2 cells expressing a panel of CD81 variants, suggesting subtle differences in the interaction of CD81 residues with diverse viral glycoproteins. Our current model of HCV infection suggests that CD81, together with additional unknown liver specific receptor(s), mediate the virus-cell entry process.
doi:10.1128/JVI.78.16.8496-8505.2004
PMCID: PMC479078  PMID: 15280458
11.  Further Standardization of Broth Microdilution Methodology for In Vitro Susceptibility Testing of Caspofungin against Candida Species by Use of an International Collection of More than 3,000 Clinical Isolates 
Journal of Clinical Microbiology  2004;42(7):3117-3119.
The influence of test variables on in vitro susceptibility testing of caspofungin was examined with 694 isolates of Candida albicans including seven laboratory-derived glucan synthesis mutants. The conditions providing the greatest separation between the mutant strains and the clinical isolates were RPMI medium, MIC end point criterion of partial inhibition, and incubation for 24 h. These testing conditions were then applied to 3,322 isolates of Candida spp. (3,314 clinical isolates and eight glucan synthesis mutants). Among the 11 isolates for which caspofungin MICs were ≥2 μg/ml, eight were accounted for by the glucan synthesis mutants. The MICs for >99% of isolates were ≤1 μg/ml, and thus these isolates were differentiated from strains with reduced in vitro and in vivo susceptibilities to caspofungin.
doi:10.1128/JCM.42.7.3117-3119.2004
PMCID: PMC446304  PMID: 15243069
12.  Cross-Resistance between Fluconazole and Ravuconazole and the Use of Fluconazole as a Surrogate Marker To Predict Susceptibility and Resistance to Ravuconazole among 12,796 Clinical Isolates of Candida spp. 
Journal of Clinical Microbiology  2004;42(7):3137-3141.
Cross-resistance within a class of antimicrobial agents is a problem that is often encountered with antibacterial agents, and it is also an issue with antifungal agents. A current example is ravuconazole, a new triazole antifungal with an expanded spectrum and potency against Candida spp., Aspergillus spp., and other opportunistic fungal pathogens. The present study addresses the issue of cross-resistance between fluconazole and ravuconazole and the use of fluconazole as a surrogate marker to predict the susceptibility of Candida spp. to ravuconazole. Reference broth microdilution MIC results for 12,796 strains of Candida spp. isolated from more than 200 medical centers worldwide were used. Ravuconazole MICs and tentative interpretive categories (susceptible, ≤1 μg/ml; resistant, ≥2 μg/ml) were compared with those of fluconazole by using regression statistics and error rate bounding analyses. For all 12,796 isolates, the absolute categorical agreement rate was 92.5% (rate of false-susceptible results, or very major errors [VME], 0.1%). Ravuconazole was active (MIC, ≤1 μg/ml) against 99.9% of the fluconazole-susceptible isolates, 96% of the fluconazole-susceptible dose-dependent isolates, and 49% of the fluconazole-resistant isolates, including 99% of the Candida krusei isolates. Since ravuconazole is 16- to 32-fold more potent than fluconazole, the performance of fluconazole as a surrogate marker for ravuconazole susceptibility was improved by designating those isolates with fluconazole MICs of ≤32 μg/ml susceptible to ravuconazole, resulting in a categorical agreement rate of 98.3%, with a VME rate of 0.3% (99 and 0.4%, respectively, when C. krusei was omitted). Cross-resistance between fluconazole and ravuconazole applies most directly to fluconazole-resistant Candida glabrata and is variable among other species of Candida. Fluconazole may serve as a surrogate marker to predict the susceptibility of Candida spp. to ravuconazole.
doi:10.1128/JCM.42.7.3137-3141.2004
PMCID: PMC446250  PMID: 15243072
13.  Evaluation of the NCCLS M44-P Disk Diffusion Method for Determining Susceptibilities of 276 Clinical Isolates of Cryptococcus neoformans to Fluconazole 
Journal of Clinical Microbiology  2004;42(1):380-383.
We evaluated the NCCLS M44-P fluconazole disk diffusion method in comparison with the NCCLS M27-A2 broth microdilution method for determining the susceptibility of 276 isolates of Cryptococcus neoformans. Disk diffusion testing was performed using Mueller-Hinton agar supplemented with 2% glucose and 0.5 μg of methylene blue/ml. Among the 276 isolates, 259 (93.8%) were susceptible, 16 (5.8%) were susceptible—dose dependent, and 1 (0.4%) was resistant to fluconazole as determined by the NCCLS broth microdilution method. The overall categorical agreement between the two methods was 86%, with 0% very major errors, 2% major errors, and 12% minor errors. The disk diffusion method using Mueller-Hinton agar supplemented with glucose and methylene blue appears to be a useful approach for determining the fluconazole susceptibility of C. neoformans.
doi:10.1128/JCM.42.1.380-383.2004
PMCID: PMC321716  PMID: 14715784
14.  trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication. 
Journal of Virology  1997;71(12):9608-9617.
Mutational analysis of the nonstructural protein 1 (NS1) of yellow fever virus (YF) has implicated it in viral RNA replication. To further explore this observation, we sought a method for uncoupling NS1 function from NS1 expression and processing as part of the large YF polyprotein. Here we describe a strategy for providing NS1 in trans, utilizing a noncytopathic Sindbis virus vector. Replication of a defective YF genome containing a large in-frame deletion of NS1 was dependent on functional expression of NS1. Recovered mutant virus was shown to contain the deletion and was neutralized by YF-specific antiserum. Complemented mutant virus increased in titer with kinetics similar to those of parental YF 17D but peaked at lower titers. trans-complementation has allowed us to derive high-titer, helper-free stocks of YF defective in NS1 with which to further characterize the role of this gene product in RNA replication. The first cycles of RNA replication were analyzed by using a sensitive strand-specific RNase protection assay. We document these events for mutant and wild-type viruses in the presence or absence of complementation. These data strongly suggest a role for NS1 prior to or at initial minus-strand synthesis.
PMCID: PMC230269  PMID: 9371625
15.  Phosphorylation of the hepatitis C virus NS5A protein in vitro and in vivo: properties of the NS5A-associated kinase. 
Journal of Virology  1997;71(10):7187-7197.
NS5A derived from a hepatitis C virus (HCV) genotype 1b isolate has previously been shown to undergo phosphorylation on serine residues (T. Kaneko, Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno, Biochem. Biophys. Res. Commun. 205:320-326, 1994). In this report, phosphorylation of NS5A derived from HCV isolates of the 1a and distantly related 2a genotypes is demonstrated. Phosphoamino acid analysis of NS5A from the 1a isolate indicated that phosphorylation occurs predominantly on serine, with a minor fraction of threonine residues also being phosphorylated. NS5A phosphorylation was observed in diverse cell types, including COS-1, BHK-21, HeLa, and the hepatoma cell line HuH-7. Phosphorylation of a glutathione S-transferase (GST)/HCV-H NS5A fusion protein was also demonstrated in an in vitro kinase assay. This activity seemed to be highest when the pH of the reaction was neutral or slightly alkaline and displayed a preference for Mn2+ over Mg2+, with an optimum concentration of approximately 10 mM Mn2+. Somewhat surprisingly, in vitro phosphorylation of NS5A was inhibited by the addition of > or = 0.25 mM Ca2+ to reaction buffer containing Mn2+ and/or Mg2+. Comparison of phosphopeptide maps of NS5A phosphorylated in vitro and in cultured cells showed that most of the phosphopeptides comigrated, suggesting that one or more kinases involved in NS5A phosphorylation in vivo and in vitro are the same. The effects of various kinase inhibitors on NS5A phosphorylation were consistent with a kinase activity belonging to the CMGC group of serine-threonine kinases. The development of an in vitro kinase assay for NS5A phosphorylation should facilitate identification of kinase(s) responsible for its phosphorylation and of phosphorylation sites which may influence the function of NS5A in HCV propagation.
PMCID: PMC192058  PMID: 9311791
16.  Secondary structure determination of the conserved 98-base sequence at the 3' terminus of hepatitis C virus genome RNA. 
Journal of Virology  1997;71(10):7345-7352.
The RNA genome of hepatitis C virus (HCV) terminates with a highly conserved 98-base sequence. Enzymatic and chemical approaches were used to define the secondary structure of this 3'-terminal element in RNA transcribed in vitro from cloned cDNA. Both approaches yielded data consistent with a stable stem-loop structure within the 3'-terminal 46 bases. In contrast, the 5' 52 nucleotides of this 98-base element appear to be less ordered and may exist in multiple conformations. Under the experimental conditions tested, interaction between the 3' 98 bases and upstream HCV sequences was not detected. These data provide valuable information for future experiments aimed at identifying host and/or viral proteins which interact with this highly conserved RNA element.
PMCID: PMC192079  PMID: 9311812
17.  Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication. 
Journal of Virology  1997;71(7):5312-5322.
Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication.
PMCID: PMC191768  PMID: 9188600
18.  Immunization with nonstructural proteins promotes functional recovery of alphavirus-infected neurons. 
Journal of Virology  1997;71(5):3415-3419.
The encephalitic alphaviruses are useful models for understanding virus-neuron interactions. A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in mice by infecting motor neurons and inducing apoptosis of these nonrenewable cells. Antibodies to the surface glycoproteins suppress virus replication, but other recovery-promoting components of the immune response have not been recognized. We assessed the effect on the outcome of NSV-induced encephalomyelitis of immunization of mice with nonstructural proteins (nsPs) by using recombinant vaccinia viruses. Mice immunized with vaccinia virus expressing nsPs and challenged with NSV initially developed paralysis similar to unimmunized mice but then recovered neurologic function. Mice preimmunized with vaccinia virus expressing structural proteins were completely protected from paralysis. Mice immunized with vaccinia virus alone showed paralysis with little evidence of recovery. Vaccinia virus expressing only nsP2 was as effective as vaccinia virus expressing all the nsPs. Protection provided by immunity to nsPs was not associated with a reduction in virus replication or with improved antibody responses to structural proteins. Protection could not be passively transferred with nsP immune serum. The depletion of T cells at the time of NSV infection decreased protection. The data show that antiviral immune responses can improve the ability of neurons to survive infection and to recover function without altering virus replication.
PMCID: PMC191486  PMID: 9094611
19.  Genetic determinants of Sindbis virus neuroinvasiveness. 
Journal of Virology  1997;71(4):2636-2646.
After peripheral inoculation of mice, Sindbis virus replicates in a variety of tissues, leading to viremia. In some cases, the virus can enter the central nervous system (CNS) and cause lethal encephalitis. The outcome of infection is age and virus strain dependent. Recently, two pairs of Sindbis virus variants differing in neurovirulence and neuroinvasiveness were derived by limited serial passaging in mouse brain. Two early passage isolates (SVA and SVB) were neurotropic but did not cause lethal encephalitis. SVB, but not SVA, was neuroinvasive. A second independent pair of isolates (SVN and SVNI), which had undergone more extensive mouse brain passaging, were highly neurotropic and caused lethal encephalitis. Only SVNI could reach the brain after peripheral inoculation. From these isolates, virion RNAs were obtained and used to construct full-length cDNA clones from which infectious RNA transcripts could be recovered. The strains recovered from these clones were shown to retain the appropriate phenotypes in weanling mice. Construction and analysis of recombinant viruses were used to define the genetic loci determining neuroinvasion. For SVB, neuroinvasiveness was determined by a single residue in the E2 glycoprotein (Gln-55). For SVNI, neuroinvasive loci were identified in both the 5' noncoding region (position 8) and the E2 glycoprotein (Met-190). Either of these changes on the SVN background was sufficient to confer a neuroinvasive phenotype, although these recombinants were less virulent. To completely mimic the SVNI phenotype, three SVNI-specific substitutions on the SVN background were required: G at position 8, E2 Met-190, and Lys-260, which by itself had no effect on neuroinvasion. These genetically defined strains should be useful for dissecting the molecular mechanisms leading to Sindbis virus invasion of the CNS.
PMCID: PMC191385  PMID: 9060616
20.  Genetic analysis of the yellow fever virus NS1 protein: identification of a temperature-sensitive mutation which blocks RNA accumulation. 
Journal of Virology  1997;71(1):291-298.
The flavivirus NS1 protein is a highly conserved nonstructural glycoprotein that is capable of eliciting protective immunity. NS1 homodimers are secreted from virus-infected mammalian cells, but the protein is also present at the plasma membrane and in the lumen of intracellular vesicles. Based on these properties, it has been speculated that NS1 may function in virus maturation or release. To gain further insight into NS1 function, we used clustered charged-amino-acid-to-alanine mutagenesis to create 28 clustered substitutions in the NS1 protein of yellow fever virus. To screen for conditional mutations, full-length RNAs containing each mutation were assayed for plaque formation at 32 and 39 degrees C after RNA transfection. We found that 9 mutations were lethal, 18 allowed plaque formation at both temperatures, and 1, ts25, was strongly heat sensitive and was unable to form plaques at 39 degrees C. Lethal mutations clustered in the amino-terminal half of NS1, whereas those leading to impaired replication relative to the parent were distributed throughout the protein. High-multiplicity infections at 39 degrees C demonstrated that ts25 was defective for RNA accumulation, leading to depressed viral protein synthesis and delayed virus production. Although ts25 secreted less NS1 than did the parent, temperature shift experiments failed to demonstrate any temperature-dependent differences in polyprotein processing, NS1 stability and secretion, or release of infectious virus. The ts lesion of ts25 was shown to be due to a single alanine substitution for Arg-299, a residue which is conserved among flaviviruses. These results argue that NS1 plays an essential but as yet undefined role in flavivirus RNA amplification.
PMCID: PMC191050  PMID: 8985349
21.  Formation of native hepatitis C virus glycoprotein complexes. 
Journal of Virology  1997;71(1):697-704.
The hepatitis C virus (HCV) glycoproteins (E1 and E2) interact to form a heterodimeric complex, which has been proposed as a functional subunit of the HCV virion envelope. As examined in cell culture transient-expression assays, the formation of properly folded, noncovalently associated E1E2 complexes is a slow and inefficient process. Due to lack of appropriate immunological reagents, it has been difficult to distinguish between glycoprotein molecules that undergo productive folding and assembly from those which follow a nonproductive pathway leading to misfolding and aggregation. Here we report the isolation and characterization of a conformation-sensitive E2-reactive monoclonal antibody (H2). The H2 monoclonal antibody selectively recognizes slowly maturing E1E2 heterodimers which are noncovalently linked, protease resistant, and no longer associated with the endoplasmic reticulum chaperone calnexin. This complex probably represents the native prebudding form of the HCV glycoprotein heterodimer. Besides providing a novel reagent for basic studies on HCV virion assembly and entry, this monoclonal antibody should be useful for optimizing production and isolation of native HCV glycoprotein complexes for serodiagnostic and vaccine applications.
PMCID: PMC191102  PMID: 8985401
23.  Differential cytotoxic T-lymphocyte responsiveness to the hepatitis B and C viruses in chronically infected patients. 
Journal of Virology  1996;70(10):7092-7102.
Cytotoxic T lymphocytes (CTL) are thought to control hepatitis B virus (HBV) infection, since they are readily detectable in patients who clear the virus whereas they are hard to detect during chronic HBV infection. In chronic hepatitis C virus (HCV) infection, however, the virus persists in the face of a CTL response. Indeed, most infected patients respond to one or more HCV-1 (genotype 1a)-derived CTL epitopes in the core, NS3, and NS4 proteins, and the CTL response is equally strong in patients infected by different HCV genotypes, suggesting broad cross-reactivity. To examine the effect of the HCV-specific CTL response in patients with chronic hepatitis C on viral load and disease activity, we quantitated the strength of the multispecific CTL response against 10 independent epitopes within the HCV polyprotein. We could not detect a linear correlation between the CTL response and viral load or disease activity in these patients. However, the CTL response was stronger in the subgroup of patients whose HCV RNA was below the detection threshold of the HCV branched- chain DNA assay than in branched-chain-DNA-positive patients. These results suggest that the HCV-specific CTL response may be able to control viral load to some extent in chronically infected patients, and they indicate that prospective studies in acutely infected patients who successfully clear HCV should be performed to more precisely define the relationship between CTL responsiveness, viral clearance, and disease severity in this infection.
PMCID: PMC190761  PMID: 8794355
25.  Identification of a highly conserved sequence element at the 3' terminus of hepatitis C virus genome RNA. 
Journal of Virology  1996;70(6):3363-3371.
Previous reports suggest that the hepatitis C virus (HCV) genome RNA terminates with homopolymer tracts of either poly(U) or poly(A). By ligation of synthetic oligonucleotides followed by reverse transcription-PCR, cDNA cloning, and sequence analysis, we determined the 3'-terminal sequence of HCV genome RNA. Our results show that the HCV 3' nontranslated region consists of four elements (positive sense, 5' to 3'): (i) a short sequence with significant variability among genotypes, (ii) a homopolymeric poly(U) tract, (iii) a polypyrimidine stretch consisting of mainly U with interspersed C residues, (iv) a novel sequence of 98 bases. This latter nucleotide sequence is not present in human genomic DNA and is highly conserved among HCV genotypes. The 3'-terminal 46 bases are predicted to form a stable stem-loop structure. Using a quantitative-competitive reverse transcription-PCR assay, we show that a substantial fraction of HCV genome RNAs from a high- specific-infectivity inoculum contain this 3'-terminal sequence element. These results indicate that the HCV genome RNA terminates with a highly conserved RNA element which is likely to be required for authentic HCV replication and recovery of infectious RNA from cDNA.
PMCID: PMC190207  PMID: 8648666

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