Biomedical subjects
T L Lewis
Publications and source records attributed to T L Lewis.
A conserved African swine fever virus IkappaB homolog, 5EL, is nonessential for growth in vitro and virulence in domestic swine.
An African swine fever virus (ASFV) gene with similarity to the cellular inhibitor of NFkappaB (IkappaB) was described in the pathogenic African isolate Malawi Lil-20/1 (ORF 5EL) and a cell-culture-adapted European virus, BA71V (ORF A238L). Recently, this gene was shown to be a functional IkappaB homolog capable of downregulating NFkappaB-regulated gene expression. This observation suggests the gene may be of significance to aspects of ASFV pathogenesis and virulence in domestic swine by interfering with a normal antiviral host response. Here we show, using nucleotide sequence analysis, that 5EL is highly conserved among various African and European pathogenic field isolates and that in all cases its similarity to IkappaB genes is limited to the presence of four low complexity ankyrin repeats in the ASFV gene. The 5EL gene of Malawi Lil-20/1 encodes a 28-kDa protein which was expressed early in virus-infected macrophage cell cultures with maximum levels observed at 3 to 5 hr postinfection. To study gene function, a Malawi Lil-20/1 5EL gene deletion mutant (Delta5EL) was constructed. Growth characteristics of Delta5EL in porcine macrophage cell cultures were indistinguishable from those of the parental virus. And, Delta5EL exhibited an unaltered parental Malawi Lil-20/1 disease and virulence phenotype in domestic swine. Thus, although highly conserved among ASFV isolates, 5EL is nonessential for growth in porcine macrophages in vitro and for viral virulence in domestic swine. A possible role for this gene in transmission of ASFV in nature, a setting which involves the cycling of ASFV between two highly adapted hosts, Ornithodoros ticks and warthogs or bush pigs, in sub-Saharan Africa is discussed.
Studies of the astrovirus signal that induces (-1) ribosomal frameshifting.
Human astroviruses use a (-1) ribosomal frameshift mechanism to regulate expression of the viral RNA-dependent RNA polymerase gene. To evaluate the efficiency of the astrovirus frameshift signal in cell culture, different regions of the frameshift signal were cloned into the rhesus rotavirus VP4 gene and expressed in an infection-transfection transient expression cell-culture system. The various astrovirus-VP4 constructs were transfected into BHK-21 cells infected with a recombinant vaccinia virus that expresses T7 RNA polymerase (vTF7-3). All constructs contain a T7 promoter, a picornavirus internal ribosome entry site, and a T7 terminator. Frameshifted and non-frameshifted proteins were distinguished by immunoprecipitation with monoclonal antibodies specific for either the VP4 amino- or carboxy-terminus. Frameshifting efficiency was calculated as the ratio of radioactive counts in the frameshifted protein to the total counts in both the frameshifted and nonframeshifted proteins as determined by Phosphorimager analysis. We found the efficiency of astrovirus frameshifting in intact cells to be 25-28%, significantly greater than the 5-7% efficiency reported previously in a cell-free uncoupled translation system. Since the transfected plasmid is expressed in the cytoplasm in the infection-transfection system, the frameshifting efficiency determined by this assay may be a more accurate reflection of the level of frameshifting for this RNA virus in which transcription and translation are likely coupled in the cytoplasm of infected cells. This hypothesis is supported by the observation that the level of astrovirus frameshifting is increased three-fold when evaluated in a cell-free coupled transcription-translation system. These studies also confirm in intact cells what was previously determined in cell-free studies: the shifty heptamer is an absolute requirement for astrovirus ribosomal frameshifting, but deletion of sequences downstream of the stem-loop that are potentially involved in pseudoknot formation does not affect the efficiency of frameshifting.
Astrovirus ribosomal frameshifting in an infection-transfection transient expression system.
Different regions of the human astrovirus frameshift signal were cloned into the rhesus rotavirus VP4 gene and evaluated in an infection-transfection transient expression cell culture system. BHK-21 cells, infected with a vaccinia virus that expresses T7 RNA polymerase (vTF7-3), were transfected with the various astrovirus-VP4 constructs. All constructs were driven by a T7 promoter and contained an internal ribosome entry site. Frameshifted and nonframeshifted protein products were immunoprecipitated with VP4 amino- and carboxy-terminal-specific monoclonal antibodies, and their ratios were determined by PhosphorImager analysis. The efficiency of frameshifting was 25 to 28%, significantly greater than the 5 to 7% efficiency reported previously in a cell-free translation system. Coupling of transcription and translation in a cell-free system yielded frameshifting efficiencies threefold greater than that of the uncoupled in vitro system. The presence of the shifty heptamer was an absolute requirement for frameshifting in both cell-free and intact-cell systems, while deletion of the potential downstream pseudoknot region did not affect the efficiency of frameshifting.
Role of ocular involvement in the prediction of visual development and clinical prognosis in Aicardi syndrome.
AIMS/BACKGROUND: This study was undertaken to document visual function and acuity in patients with Aicardi syndrome, and to determine whether there is any relation between ocular features of the syndrome exhibited at birth and later visual function. METHODS: Fourteen patients with Aicardi syndrome, all examined and followed by the same ophthalmologist, were reviewed between 1975 and 1992 and their ocular characteristics and visual acuity described. It was hypothesised that larger lacunae may be associated with poorer clinical outcome and therefore the relation between these two variables was investigated. RESULTS: Visual acuity as documented by Snellen, Sheridan-Gardner, preferential looking, or pattern visual evoked potential tests was in the normal to low normal range in six eyes of four patients. Visual function correlated significantly with macular appearance. Good visual function was preserved if the fovea appeared normal on funduscopic examination and was uninvolved by lacunae. The size of the largest chorioretinal lacuna also correlated significantly with clinical outcome: patients with large lacunae were more likely to be immobile and to have no language skills. CONCLUSION: It was concluded that good visual function in patients with Aicardi syndrome may be anticipated if the fovea is normal. Although many patients have severe psychomotor retardation, the presence of predominantly small chorioretinal lacunae may indicate a better prognosis for mobility and language development.
Celebrating 75 years at the Pennsylvania College of Optometry.
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An astrovirus frameshift signal induces ribosomal frameshifting in vitro.
Expression of the astrovirus RNA-dependent RNA polymerase has been hypothesized to be regulated by (-1) ribosomal frameshifting. Sequence analysis of the 70 nucleotide region between open reading frames 1a and 1b indicates the presence of a shifty heptamer consensus sequence and downstream sequences that may be needed for ribosomal frameshifting. We constructed four astrovirus cassettes that spanned this region and inserted each into the rhesus rotavirus VP4 gene. The constructs were expressed in an in vitro system, and products were immunoprecipitated by rotavirus amino and carboxy terminal-specific monoclonal antibodies. Ribosomal frameshifting, at an efficiency of 6-7%, was demonstrated in all constructs containing the shifty heptamer and stem-loop. Deletion of the downstream sequence potentially involved in pseudoknot formation did not affect frameshifting efficiency. However, deletion of the shifty heptamer resulted in no detectable frameshift activity.
Monoclonal antibodies for detection of Norwalk virus antigen in stools.
Monoclonal antibodies against the prototype 8FIIa strain of Norwalk virus were prepared and applied to an enzyme immunoassay (EIA) for detecting Norwalk virus in stool specimens. The monoclonal antibodies immunoprecipitated a 58-kDa protein which had been produced by in vitro transcription-translation of Norwalk virus cloned cDNA, and they reacted by EIA with recombinant Norwalk virus capsid protein at a sensitivity level of 1 ng/ml. The EIA detected virus in all tested samples from 15 different Norwalk virus-infected volunteers. No cross-reactions were seen in stools containing other caliciviruses or in stools containing rotaviruses, astroviruses, or enteric adenoviruses.
Increased yield of small DNA fragments purified by silica binding.
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Development of grating acuity in children treated for unilateral or bilateral congenital cataract.
PURPOSE: To study the development of grating acuity in children treated for dense congenital unilateral or bilateral cataract and to examine how variations in treatment affect grating acuity during early childhood. METHODS: The authors used optokinetic nystagmus (OKN), preferential looking (PL), or both to measure the grating acuity of children treated for congenital cataract in one eye (n = 63) or both eyes (n = 77) whenever possible from the time of treatment until 3 years of age. At each age, the authors compared patients' monocular acuity to that of children with no history of eye disorders. RESULTS: The OKN acuity of treated eyes did not improve with age and was abnormal by 12 months of age. In contrast, PL acuity improved with age, and acuity of most treated eyes was not outside normal limits until 24 to 30 months of age. Nonetheless, at 12 months and at 3 years of age, PL acuity correlated significantly with age at treatment in children who had bilateral cataract. In children who had unilateral cataract, PL acuity correlated significantly with the number of hours per day the good eye had been patched since treatment. Children whose good eye was patched fewer than 3 hours per day did significantly worse than children treated at a comparable age for bilateral congenital cataract. However, children whose good eye was patched at least 3 hours per day had PL acuities similar to those of children treated at a comparable age for bilateral congenital cataract. CONCLUSIONS: Children treated for congenital cataract show deficits in grating acuity, with the deficit apparent earlier in OKN acuity than in PL acuity. At least by 1 year of age, visual development has begun to be influenced by the age at treatment and, in children treated for unilateral cataract, by patching of the good eye.
Defining the scope of practice of optometry.
Defining the scope of practice of optometry is difficult, but important for the profession and optometric education. Even though the scope of practice can be defined in various ways and is constantly changing, the schools and colleges of optometry must have some sense of the boundaries of the profession in order to develop educational programs which will prepare students to practice well into the future. The use of three-dimensional diagrammatic models creates a visual representation of the elements which define the scope of practice. Additionally, these models can assist schools and colleges to improve their understanding of the various dynamics which must be considered in developing the most appropriate methods to analyze the competencies required for entry into the practice of optometry.
Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein.
We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase.
The nucleotide sequence of the 5'-untranslated region of bovine viral diarrhoea virus: its use as a probe in rapid detection of bovine viral diarrhoea viruses and border disease viruses.
A 289 bp cDNA fragment from the 5'-untranslated region (UTR) of 16 bovine viral diarrhoea virus (BVDV) isolates was amplified by reverse transcription and polymerase chain reaction, and sequenced by dideoxy DNA sequencing. The sequence showed greater than 90% homology between the isolates and BVDV NADL in this region, and greater than 97% homology within a 72 base sub-region (nt 314-386). The 289 bp fragment was then used as a probe for rapid detection of BVDV and border disease virus (BDV) from cell culture samples by dot-blot hybridization. This probe hybridized to 100% of BVDV isolates (n = 78) and 100% of BDV isolates (n = 9), but not to the uninfected BT cells or other bovine infectious agents. A shorter probe from the more conserved sub-region also was tested for hybridization with some of the isolates, and the results were similar to those using the longer probe. These results suggest that the 5'-UTR is highly conserved among BVDV and BDV isolates, and may be used as a potential probe for rapid detection of BVDV and BDV in clinical and cell culture samples from cattle and sheep.
Cloning and characterization of human astrovirus immunoreactive epitopes.
We report the cloning of antigenic, protein-coding regions of human astrovirus serotype 1 that appear to be common to most, if not all, serotypes of human astrovirus. Screening of lambda gt11 libraries identified three different but overlapping clones (A43, A35, and A1) and one independent clone (A14) that reacted with serum from a rabbit repeatedly immunized with purified astrovirus particles but not with its preimmunization serum. These clones were shown to be astrovirus specific. Of note, a radiolabeled probe representing the immunoreactive clones A43-A35-A1 hybridized exclusively to the 7.2-kb astrovirus genomic RNA, while a clone A14-specific probe hybridized with both the genomic and the 2.8-kb astrovirus subgenomic RNAs. This suggests that the immunoreactive epitopes, selected by antiserum to purified astrovirus particles, are encoded by the subgenomic RNA as well as other regions of the genomic RNA.
Diagnostic approaches for the detection of bovine viral diarrhea (BVD) virus and related pestiviruses.
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Sensitivity in the nasal and temporal hemifields in children treated for cataract.
PURPOSE: To determine if form-deprived aphakes, like normal infants, show especially poor sensitivity in the nasal visual field. The purpose of this article was also to examine the influence on their peripheral sensitivity of the timing and duration of deprivation, of whether deprivation was monocular or binocular, and of having patched the fellow nondeprived eye. METHODS: Static perimetry was used to measure intensity thresholds at 20 degrees in the nasal visual field and at 30 degrees in the temporal visual field in normal subjects (n = 20 7-year-old children, 20 8-year-old children, 12 9-year-old children, and 20 adults) and in 46 children treated for a dense and central cataract in one (n = 21) or both (n = 25) eyes. The deprivation began either at birth or after a normal early history, and the duration of deprivation varied widely among patients. Also tested were two adults who had been treated promptly for unilateral cataracts that had developed after the age of 40 years. RESULTS: In normal subjects, sensitivity was slightly higher at 20 degrees nasally than at 30 degrees temporally, with no developmental changes in sensitivity at either location. The deprived eyes of the children had losses in sensitivity at both locations but only children treated for unilateral congenital cataract had larger losses at 20 degrees nasally than at 30 degrees temporally. There were no significant effects on their sensitivity of the duration of deprivation or, in children treated for unilateral cataract, of patching of the nondeprived eye. In contrast, the two patients in whom cataracts did not develop until adulthood had normal sensitivity. CONCLUSIONS: Pattern deprivation interferes with the development of peripheral sensitivity at both 30 degrees temporally and 20 degrees nasally. Nasal sensitivity, which is slow to mature, is affected more by early monocular deprivation than by early binocular deprivation. The results are consistent with the hypothesis that unfair interocular competition during early infancy especially affects visual functions that are slow to mature.
Stereopsis after congenital cataract.
PURPOSE: The authors determined whether stereopsis can be demonstrated in children treated for congenital cataract after compensating for their amblyopia and strabismus. METHODS: A custom-made set of large stereograms was used to compensate for amblyopia and presented in a synoptophore to facilitate motor fusion. Each pair of stereograms contained five wide vertical bars of high contrast, of which two or three were in crossed disparity. The authors aligned the stimuli in the synoptophore, then decreased the disparity until the child could no longer identify which bars appeared to be "in front." RESULTS: In normal children (n = 25), stereo acuity on this test (Tytla-Lewis-Maurer-Brent [TLMB] test) correlated well with the Titmus and Randot results within the ranges measured by those tests. Seven children in whom a traumatic cataract had developed after 6 yr of age (four with no clinical stereopsis) had TLMB test stereo acuities ranging from 225 to 28 arc-sec. Of 30 children treated for congenital cataract with no clinical stereopsis, 5 regularly achieved a TLMB stereo acuity of up to 225 arc-sec, and 2 with Titmus stereo acuities up to 200 arc-sec, each had a TLMB stereo acuity of 112 arc-sec. These seven congenital cases (two unilateral and five bilateral) with measurable TLMB acuities are among those with the shortest deprivation, the highest minimum resolvable acuity, and the highest contrast sensitivity. CONCLUSIONS: By compensating for amblyopia and strabismus, stereopsis can be demonstrated in some form-deprived amblyopic patients.