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CORD-19:12ce8410d1ca0fa6f438c4f22bd92af6f8b78f04 JSONTXT 7 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T1 0-64 Sentence denotes The Polypeptide Composition of Avian Infectious Bronchitis Virus
T2 66-74 Sentence denotes Abstract
T3 75-215 Sentence denotes Avian infections bronchitis virus grown in ovo was purified by differentiaI centrifugation and isopycnic sedimentation in density gradients.
T4 216-373 Sentence denotes The purified virus was analysed by SDS polyacrylamide gel electrophoresis and found to comprise up to sixteen polypeptides, four of which were glycopeptides.
T5 374-461 Sentence denotes BromelairL treatment of the particles removed three polypeptides and two glycopeptides.
T6 462-465 Sentence denotes 14"
T7 467-591 Sentence denotes The eoronaviruses have been classified as a separate virus group mainly or~ the basis of their distinctive morphology (12) .
T8 592-774 Sentence denotes There have been many studies [reviewed by McI~TosH (7) ] demonstrating that the growth of these viruses is insensitive to inhibitors of DNA metabolism or DNA dependent RNA synthesis.
T9 775-855 Sentence denotes For this reason the nature of the eoronavirus genome has been assumed to be RNA.
T10 856-950 Sentence denotes However, there have been few reports of the results of direct chemical analyses of the virion.
T11 951-1145 Sentence denotes There have been two reports (10, ll) of extraction and analysis of nucleic acid from purified virions of avian infectious bronchitis virus (IBV) and these indicated that the virus contains I~NA.
T12 1146-1435 Sentence denotes It has also been deduced from the appearance of the particles, the sensitivity of infectivity to ether and chloroform, and the mode of development of particles observed in thin sections of infected cells, that they contain lipid derived from the host as an integrM part of the outer shell.
T13 1436-1526 Sentence denotes However, there are no reports on extraction and anMysis of lipids from purified particles.
T14 1527-1727 Sentence denotes Although there are at least seven different, species in the eoronavirns genus (7) , only one t33oe, the human eoronavirus OCI3, has been subjected to electrophoretic analysis of the polypeptides (5) .
T15 1728-1944 Sentence denotes This report describes preliminary biochemical investigations of another coronavirus, avian infectious bronchitis virus. (a Massachusetts serotype) from the commercially available live vaccine, Bronvirin (Glaxo Ltd.).
T16 1945-2063 Sentence denotes Each of these stocks had a long history of passage in embryonated cMcken eggs before their receipt in this laboratory.
T17 2064-2155 Sentence denotes Primary chicken kidney cell cultures were used for production or infectivity assays of IBV.
T18 2156-2405 Sentence denotes The kidneys from 3-7 week old specific-pathogen-free chickens (Wickham Laboratories Ltd., ~Vickham, Hampshire) were dispersed by serial 5 minutes incubations at 37 ° C in 0.05 per cent trypsin (Difeo Laboratories) in phosphate buffered saline (PBSA:
T19 2406-2437 Sentence denotes Dulbecco's type A, Oxoid Ltd.).
T20 2438-2747 Sentence denotes The cells obtained were strained through nylon mesh and dispensed either in Eagle's Medium Basal (BME :'Wellcome Reagents Ltd. ) or in L-15 medium (Gibco), both media being supplemented with 8 per cent foetal calf serum, 4 per cent tryptose phosphate broth, 100 units/ml penicillin and 100 ~g/ml streptomycin.
T21 2748-2905 Sentence denotes The BME was used for cells grown in vented plastic dishes in a 5 per cent CO2 atmosphere, the L-15 used for growth of cells in closed glass bottles or tubes.
T22 2906-3081 Sentence denotes For the growth of virus, confluent cell cultures were changed into maintenance medium, which was similar in composition to the growth medium but without the foetal calf serum.
T23 3082-3263 Sentence denotes Estimations of virus infectivity were made by inoculating 0.2 ml aliquots of appropriate dilutions of virus into rolling tube cultures already containing 1 ml of maintenance medium.
T24 3264-3469 Sentence denotes Cytopathic effects on the cells were observed at 48 hours (strain Beaudette) or 72 hours (strain Massachusetts 4t and Connecticut) after inoculation, and infecti~dty measurements experssed as TCDs0/0.2 ml.
T25 3470-3584 Sentence denotes IBV-H t20 caused no distinct cytopathic effect and was not used in experiments requiring infectivity measurements.
T26 3585-3690 Sentence denotes IBV-Beaudette could also be estimated by plaque formation assays, but this method was not routinely used.
T27 3691-3749 Sentence denotes All cell culture and virus growth was performed at 37 o C.
T28 3750-3898 Sentence denotes Usually 104~105 TCD~0 of virus was inoculated (in 0.05--0.2 ml) into the allantoic cavity of 10-day old embryonated chicken eggs (Orchards Farm, Gt.
T29 3899-3954 Sentence denotes Missenden, Bucks) and incubated at 37 ° C for 24 hours.
T30 3955-4094 Sentence denotes After chilling at 4 ° C overnight the allantoic fluid was harvested and immediately clarified by centrifugation at 16,000 g for 20 minutes.
T31 4095-4160 Sentence denotes This and all subsequent operations were carried out at 00--4 ° C.
T32 4161-4218 Sentence denotes The virus was pelleted at 75,000g for 1 hour in a MSE No.
T33 4219-4298 Sentence denotes 59595 rotor and resuspended in PBSA with the aid of a glass Dounee homogeniser.
T34 4299-4448 Sentence denotes The resuspended virus was overlaid on a linear 25 to 55 per cent sucrose (w/w) in PBSA gradient and centrifuged overnight (12--16 hours) in a MSE No.
T35 4449-4472 Sentence denotes 59590 rotor at 90,000g.
T36 4473-4565 Sentence denotes The virus bands were collected, diluted 8-fold in PBSA and repelleted at 75,000g for 1 hour.
T37 4566-4837 Sentence denotes The pellets were resuspended in the same manner as before and layered on either (a) a linear 25--55 per cent sucrose (w/w) in PBSA gradient, or (b) a linear 25--55 per cent potassium sodium tartrate (w/w) in PBSA gradient and centrifuged overnight at 60,000g in a MSE No.
T38 4838-4850 Sentence denotes 59108 rotor.
T39 4851-5060 Sentence denotes Purified virus collected from the second gradient step was diluted in PBSA, deposited at 75,000g for 1 hour and dissolved in a small volume of 5 per cent sodium dodeeyl sulphate (SDS) at 37 ° C for 30 minutes.
T40 5061-5193 Sentence denotes 2-mercaptoethanol was added to a final concentration of 2 per cent and the solution incubated in a boiling water bath for 2 minutes.
T41 5194-5401 Sentence denotes The dissolved virus was dialysed overnight at room temperature against 5 m~ Tris, 38 mM glyeine buffer (pH 8.3) containing 1.0 per cent SDS, 0.I per cent 2-mercaptoethanol, 3.3 ~ Urea and 5 per cent sucrose.
T42 5402-5547 Sentence denotes After dialysis a trace amount of bromophenol blue was added and the sample reheated at 100 ° C for 1 minute immediately prior to eleetrophoresis.
T43 5548-5664 Sentence denotes Electrophoresis was carried out on cylindrical potyacrylarnide gels 12 cm × 0.65 cm diameter, cast in perspex tubes.
T44 5665-5799 Sentence denotes The acrylamide concentration was 10, 7.5 or 5 per cent (w/v), the acrylamide: bis-acrylamide ratio being constant at 37.5:1 by weight.
T45 5800-5987 Sentence denotes The gels also contained 0.1 per cent SDS, 0.5 ~'I urea, 0.03 per cent N, N, N', N'--tetramethy]ethylenediamine, 0.07 per cent ammonium persulphate and 0.375 5I Tris--I-IC1 buffer, pH 8.9.
T46 5988-6187 Sentence denotes Gels were pre-electrophoresed at 100V for 1 ~--2 hours, the electrophoresis buffer consisting of 25 m~ Tris, 192 m~ glycine, 0.1 per cent SDS, 0.5 ~ urea., 0.1 per cent 2-mereaptoacetic acid, pH 7.8.
T47 6188-6355 Sentence denotes If non-reducing conditions were required, the 2-mercaptoacetic acid was omitted----the buffer then being pH 8.3-and reductant omitted during preparation of the sample.
T48 6356-6523 Sentence denotes After pre-electrophoresis the sample was layered directly on the top of the gel in not more than 200 B1 voiume, and eleetrophoresis carried out at 100V for 4--8 hours.
T49 6524-6631 Sentence denotes In some experiments the system described by ~VEBE~ and Osso~ (t5), with phosphate buffer pI~ 7.0, was used.
T50 6632-6710 Sentence denotes After eleetrophoresis gels were stained for protein (9) or carbohydrate (16) .
T51 6711-6970 Sentence denotes The molecular weights of viraI peptides were determined by reference to the mobilities of fi-galactosidase, phosphorylaae A, transferrin, bovine serum albumin, pyruvate kinase, ovalburain, alcohol dehydrogenase (yeast), myoglobin and lysozyme in control gels.
T52 6971-7100 Sentence denotes Fractions from gradients were exhaustively dialysed against distilled water to remove the sucrose or tart.rate gradient material.
T53 7101-7158 Sentence denotes Concomitant changes in volume during dialysis were noted.
T54 7159-7246 Sentence denotes Estimations were carried out for RNA (8) , protein (6), I)NA (2) and carbohydrate (4) .
T55 7247-7354 Sentence denotes Alternatively protein and t{NA were estimated from the extinctions at 260 and 280 nm (E~6o and E2so) (13) .
T56 7355-7472 Sentence denotes IBV from the first sucrose gradient stage of the purification was incubated with bromelain (Sigma Chemical Co.) (3) .
T57 7473-7639 Sentence denotes After incubation, the mixture of virus and enzyme was diluted in ice-cold PBSA, pelleted, resuspended and centrifuged on a second sucrose gradient as described above.
T58 7640-7778 Sentence denotes On the first sucrose gradient stage the virus formed two distinct bands, Table 1 indicates the buoyant densities of the different strains.
T59 7779-7924 Sentence denotes The two bands were put on separate gradients for the second stage of the purification, and they retained their original density as a single band.
T60 7925-8068 Sentence denotes The various bands from both gradient stages were negatively stained in sodium phosphotungstate and examined with the electron microscope by Dr.
T61 8069-8071 Sentence denotes J.
T62 8072-8074 Sentence denotes D.
T63 8075-8226 Sentence denotes Almeida, Wellcome Research Laboratories, and were found to contain intact virus ; there was no detectable difference between the light and heavy forms.
T64 8227-8396 Sentence denotes A small proportion of the virus particles became degraded on tartrate gradients, so in subsequent purifications the tartrate was replaced by a secor~d sucrose gradient..
T65 8397-8505 Sentence denotes The infectivity of the purified virus fractions was measured, and a typical result is presented in Table 2 .
T66 8506-8688 Sentence denotes It, can be seen that both the light and heavy forms of the virus were i.niectious, although the heavy band was significantly more infectious with respect to protein and nucleic acid.
T67 8689-8866 Sentence denotes The second gradient step appears to have caused a loss of infectivity of the heavy form, and a reduced RNA: protein ratio of that band, suggesting a partiM loss of nucleic acid.
T68 8867-9023 Sentence denotes It was thought, that the virus might be contaminated by mycoplasmas, which are often present in hens eggs and which may be of similar size and density (if).
T69 9024-9105 Sentence denotes The infected allantoic fluids were therefore tested for viable mycoplasmas by 1~.
T70 9106-9108 Sentence denotes W.
T71 9109-9117 Sentence denotes BI~G~A~:
T72 9118-9134 Sentence denotes Miss Patricia M.
T73 9135-9188 Sentence denotes Furr of this Institute but the results were negative.
T74 9189-9264 Sentence denotes Purified IBV preparations were tested for avian leukosis virus (ALV) by Mr.
T75 9265-9267 Sentence denotes T.
T76 9268-9381 Sentence denotes Webster, Wellcome Research Laboratories, using the complement fixation test (COFAL) and these were also negative.
T77 9382-9605 Sentence denotes No particles resembling mycoplasmas, ALV or any other virus were ever seen in any of the electron microscopic examinations of purified virus. (13) . e Material from 1st sucrose gradient after rebanding on tartratc gradient.
T78 9606-9800 Sentence denotes It was not possible to obtain sufficient, purified virus to make a complete analysis of nucleic acid, protein, carbohydrate and lipid content and relate this to the dry weight of virus material.
T79 9801-9920 Sentence denotes However, it was possible to make some chemical determinations and relate these to the protein content of the particles.
T80 9921-10142 Sentence denotes The RNA: protein ratio ranged from 0.71--0.84 (5 batches of virus) when measured by chemical means (8, 6)--on measurement by extinctions, the ratios ranged from 0.036---0.069 (9 batches of virus), the average being 0.054.
T81 10143-10221 Sentence denotes The amounts of RNA determined by the two methods were in very close agreement.
T82 10222-10356 Sentence denotes The discrepancy in the RNA: protein ratio arises from the very different results obtained from the two methods for protein estimation.
T83 10357-10621 Sentence denotes As the method of LOWRY et at. (6) is rather sensitive to interference by a variety of compounds, including sugars, it is possible that the viral nucleic acid, carbohydrate and lipid have caused sufficient interference to render the result from this method invalid.
T84 10622-10754 Sentence denotes The carbo-hydrate: protein ratio ranged from 0.11--0.22 (3 batches of virus)--the protein being estimated by E260/E2s0 measurements.
T85 10755-10834 Sentence denotes The carbohydrate values were corrected for the RNA content of the preparations.
T86 10835-10908 Sentence denotes Attempts were also made to detect the presence of I)NA in purified virus.
T87 10909-11061 Sentence denotes Although trace levels were detected, these levels were within the calculated levels of interference in the diphenylamine test by the viral I~NA present.
T88 11062-11141 Sentence denotes I t was eoneluded that, there was probably no genuine I)NA in the preparations.
T89 11142-11336 Sentence denotes These results are the first direct chemical confirmation that the virion nucleic acid is indeed RNA, and also that the particles contain carbohydrate in addition to the nucleic acid and protein.
T90 11337-11389 Sentence denotes Other experiments in progress in this laboratory (S.
T91 11390-11405 Sentence denotes JOH~S0:N and R.
T92 11406-11408 Sentence denotes W.
T93 11409-11523 Sentence denotes BrNo~AM--unpublished results) have confirmed the presence of various lipids in purified preparations of the virus.
T94 11524-11770 Sentence denotes Initial experiments using the Tris-glycine buffered electrophoresis system gave much better resolution of the IBV polypeptides than that achieved with the phosphate system (Fig. 1 )--some of the bands visible in the former did not appear Fig. 1 .
T95 11771-11783 Sentence denotes PAGE of IBV.
T96 11784-11923 Sentence denotes A. phosphate buffer system, B. tHs-glyeine buffer system, 10 per cent aerylamide m both systems, identical samples loaded on all four gels.
T97 11924-11976 Sentence denotes P. stained for protein, C. stained for carbohydrate.
T98 11977-12173 Sentence denotes As the photographic process did not record the red bands as e/early as the blue, the positions of the carbohydrate bands seen with the naked eye have been marked alongside the appropriate gels It.
T99 12174-12176 Sentence denotes W.
T100 12177-12208 Sentence denotes BINc~nA~: at all in the latter.
T101 12209-12277 Sentence denotes Therefore all subsequent experiments were done by the former method.
T102 12278-12433 Sentence denotes Figure 2 illustrates typical densitometer tracings (made with a Joyce-Loebl Chromoscan) of gels which have been stained for either protein or carbohydrate.
T103 12434-12525 Sentence denotes There are up to sixteen peptides present, three or four of which appear to be glycosylated.
T104 12526-12716 Sentence denotes Although the profiles of the light and heavy forms appear to be dissimilar (Fig. 3a, b) , it may be seen that the same peptides are present in both, and only the relative proportions differ.
T105 12717-12799 Sentence denotes Examinations of many such profiles showed that this was a reproducible phenomenon.
T106 12800-12855 Sentence denotes No significant difference between strains was detected.
T107 12856-12970 Sentence denotes Numbers were assigned to the peptidcs on the basis of their molecular weights to assist in comparison of profiles.
T108 12971-13051 Sentence denotes The molecular weights determined from twelve experiments are listed in Table 3 .
T109 13052-13277 Sentence denotes When the separated light, and heavy forms of the virus were treated with bromelain and recentrifnged on sucrose gradients, it was found that both forms yielded identical single bands of virus at a new lighter density of 1.15.
T110 13278-13346 Sentence denotes These treated particles were examined by electron microscopy (by Dr.
T111 13347-13349 Sentence denotes S.
T112 13350-13426 Sentence denotes Patterson of this Institute) and were almost totally lacking in projections.
T113 13427-13510 Sentence denotes PAGE profiles of the virus before and after treatment are illustrated in Figure 3 .
T114 13511-13571 Sentence denotes I t can be seen that the bromelain removed for carbohydrate.
T115 13572-13597 Sentence denotes Gels run for 5 ~/2 hours.
T116 13598-13684 Sentence denotes Only those bands consistently found in every preparation have been labelled VP or VGP.
T117 13685-13893 Sentence denotes 0eeasionally VP 70 showed slight staining with PAS as seen here, but this was not reprodueible two of the glycopeptides (VGP180 and VGP83) and substantially reduced the proportions of VP 130, VP 106 and VP70.
T118 13894-14006 Sentence denotes As VP53 and VP49 usually tend to run as an overlapping pair, it is not possible to say whether any VP53 remains.
T119 14007-14134 Sentence denotes The relative proportions of the lower molecular weight peptides have considerably increased in relation to the prominent VP 49.
T120 14135-14270 Sentence denotes Three new peptides, designated XP 27, XP20 and XP18 (27,000, 20,000 and 18,000 daltons respectively) have also appeared in the profile.
T121 14271-14445 Sentence denotes It seems likely that these are cleavage products of some of the larger peptides, rather than contamination with the bromelain; when brome- heavy form gave identical pattern).
T122 14446-14579 Sentence denotes Gels run for 6 hours lain was electrophoresed in a parallel gel it gave several peaks, none of which coincided with the new peptides.
T123 14580-14803 Sentence denotes Also it was ealculated that about 90 per cent of the added bromelain would have had to remain adhering to the virus through the purification steps after incubation in order to yield the amounts detected of the new peptides.
T124 14804-14960 Sentence denotes These results suggest that VGP180, VP130, VP106, VGP83 and VP70 are accessible on the surface of the vMon, the remaining peptides presumably being internal.
T125 14961-15148 Sentence denotes Newcastle Disease virus (strain Hefts) and influenza virus (strain X31) were grown in ovo, purified by exactly the same techniques and electrophoresed in parallel to the IBV preparations.
T126 15149-15297 Sentence denotes Only the typical peptide bands of those viruses were detected, and there were no bands corresponding to any of the bands located in the IBV samples.
T127 15298-15499 Sentence denotes This was evidence that the purification scheme used was adequate for isolating pure specimens of other enveloped viruses of similar size, density and origin to IBV without host component contamination.
T128 15501-15642 Sentence denotes The prime question which arises when considering the observed polypeptide composition of any virus :is that of the purity of the preparation.
T129 15643-15750 Sentence denotes This is especially important when there are apparently a large number of peptides present, as in this ease.
T130 15751-15931 Sentence denotes The four main contaminants that may arise in purified preparations of most animal viruses are host cellular components, endogenous virus from those cells, mycoplasmas and bacteria.
T131 15932-16095 Sentence denotes The second and third possibilities have been tested for and excluded in this ease, and the fourth would have been very obvious in electron microscopic examination.
T132 16096-16244 Sentence denotes Also any significant level of bacterial or mycoplasmM contamination would have resulted in readily detectable quantities of DNA in the preparations.
T133 16245-16321 Sentence denotes This leaves the problem of possible contamination by host cellular peptides.
T134 16322-16528 Sentence denotes It is difficult to differentiate between host material that is an integral part of the virion and that which may have remained associated with the preparation through the various stages of the purification.
T135 16529-16719 Sentence denotes One approach to the problem would be a comparison of virus grown in different cell types on the assumption that the peptides contributed by the host would differ in the various preparations.
T136 16720-16842 Sentence denotes However, this would not resolve the question of whether it was an integral part of, or merely associated with, the virion.
T137 16843-17046 Sentence denotes This is not possible with IBV anyway, due to the very restricted selection of cells in which it may be grown--the strains used here were only capable of culture in ovo or in primary chicken kidney cells.
T138 17047-17155 Sentence denotes In the samples of purified virus used here, there were no major contaminants visible by electron microscopy.
T139 17156-17416 Sentence denotes Although there were occasional pieces of membrane, some clearly deriving from disintegrating virions, the relative amounts of virus particles and possible host components were such that any contribution of the latter to the analysis would have been negligible.
T140 17417-17718 Sentence denotes In some preparations examined there was a "contamination" by detached viral projections, mainly in the heavy band--as the amounts of these present varied from sample to sample, this could account for the difference in the relative proportions of the polypeptides observed in the light and heavy forms.
T141 17719-17808 Sentence denotes This leaves the possibility that host polypeptides may be an integral part of the virion.
T142 17809-18053 Sentence denotes It has been shown (1) that when IBV was treated with unheated rabbit antiserum raised against normal chicken cell membranes, holes appeared in the virus envelope, similar to those produced using rabbit antiserum raised against the virus itself.
T143 18054-18105 Sentence denotes Use of chicken antiserum did not cause these holes.
T144 18106-18187 Sentence denotes This suggested that host components were an essential part of the virus envelope.
T145 18188-18331 Sentence denotes A recent report (14) describes the I~NA content of the virus as being in one ribonuclease sensitive piece, of molecular weight 9 × 106 daltons.
T146 18332-18646 Sentence denotes If this represents the size of the IBV genome, then the virus would contain adequate coding capacity to account for all the peptides observed, assuming they are all independently coded and some are not cleavage products of others, eliminating the necessity to postulate the presence of peptides of cellular origin.
T147 18647-18761 Sentence denotes It is of interest to compare these results with the report on the polypeptides of the human coronavirus 0C43 (5) .
T148 18762-18929 Sentence denotes Although those authors only claim the presence of six or possibly seven polypeptides, examination of their gel tracings shows the presence of several more minor bands.
T149 18930-19057 Sentence denotes Also, they used a phosphate buffered pH 7.2 PAGE system very similar to that which gave comparatively poor resolution with IBV.
T150 19058-19146 Sentence denotes Thus the avian and human coronaviruses may not be as dissimilar as is at first apparent.