iNTERACTIQN Q? NC-NHEUYEALSZED M‘éfi NESTRALEZ‘EE AWAN‘ WE‘EQTEGSS ERONCHETEfi Vififig WWH 'z'E-ifi CHICKEN EMBRYQ RES-REY CELL E: EN'E‘RY EWCQ THE GEL; EL WRAL QEGEA’JAfiGR T516333 gov {'Em Degree 0‘? DE. 9‘ MICHEGAN STAYS UNEYERSETY Mark F. Sfiinski E959 LIBRARY Michigan State University This is to certify that the thesis entitled Interaction of nonneutralized and neutralized avian infectious bronchitis virus with the chicken embryo kidney cell. presented by Mark F. Stinski has been accepted towards fulfillment of the requirements for Ph. D. degree in Microbiology C. H. Cunningham .7 " /Kumucw mcHHmEmm 30mm mom Umumoflccfi mum mcoflumcfifiumumc H500 mo mwmcmu 0cm mammz .Uwum mzu Mom consuflumnzm .m.w mm .Amunc Rmv mmm £uw3 usn mannaafiwm Uwummuu mmB A00 Houucou mDHH> UmNflHmuusm: one .EswUmE Hmmm £ua3 Ummmaum>o mumB A00 msnw> cwuflamnusmc Ho A40 msnfl> UwNHHmuuswccoc nuHB maamo .coflum>fluomwn wave can .cowumuwamuusmc .COHumHomUm Hmuw> How mmuscwooum cumpcmum Gnu Hmum¢ .>mH Umuflamnuswclumasaawomuuxm mo coflum>wuomwn Uflom kn consummfi mm A00 oe 0cm .Amv mm .Aév um um Uxmo £uw3 NmHmEoo aconHuGMI>mH mnu mo cofluumnmucH .m mnsmflm 22 m m h a 2.: ON. 00. om nu ow nv on n. o a a q i _ a q , 0 $04, W/W/W\W\T+\%/W 1 ID 0/ . I 00/)IW/Mdlvv3w Til/Tillie. _ .. \U\IV 0.N muhazi 0m. 00. 00 n# 00 9.. on o. o .fi . Wv/W d u u a q u a Q 1 I) 0" . . ..d ( all/W/ "I NVJH 0.N mmh32_2 0m. 00. Om nb 00 m? on n. 0 i 4 a a I _ 4 Ti: H . lw N .0 III%/// ///@\\\WIIJWV_ W T n w/w/ 4 M. $/* ma ( ,T\\ J 0. N l———<}——1 v——4—-‘ L——_L— I) N TT. Iii/T \V\OKM 25 .83 as: 83228 83 0.552808 2: E E8 35. Ease; 28 E 86268.. mes m2; 8262.8: or: 526562me EB 5:888 BE .2 8:88: 5:885 Ecucam e: .23 332855.. o .8 w an m .3 cu o .3 x .mm m .5 0mm 4 w N .3 m .3 OR .EE 8H .55 8 .55 o 33832.8 US$82 m2; 80:er e. £08358 :8 - >2 so: @825 B 83283.6 28 3 8.988 was mzozuflg Emu 00¢ 05 :0 0:5. 0:0 Sagan—Em“ no 5th .n Amanda... Figure 4. 24 Adsorption of IBV to CEKC and the effect of acid treatment on adsorbed virus. Cells were inoculated with IBV and incubated at 570. At various intervals of time the cells were washed for 10 sec. with either glycine-HCl buffer (0.1 N), pH 2.0, (--0-—) or PBS (5% nbcs), pH 7.5, (-O-) followed immediately by two washings with PBS (5% nbcs), pH 7.5. MEAN p.f. u. /ml.(log,01 25 2.5 I“ O I in I __ l 4 l I 'o l l l l l l 1 l . 5 IS 50 45 60 75 9O l05 MINUTES FOR ADSORPTION AT 37" l20 26 When the same eXperiment was done at 40, less adsorption occurred and approximately 6% of the virus dissociated from the cell. Reactiygtion of neutralized IBV and the inability» to reneutralize the virus with horse anti-chicken globulin antibody Neutralized virus that was reactivated by papain or exposure to pH 4.0, still had antibody attached and subse- quently, could be reneutralized with antidy globulin anti- body (51). To determine if chicken globulins were still associated with IBV after exposure of neutralized virus to pH 2.0, CEKC were inoculated with 1.2 x 102 p.f.u./ml. Viral adsorption, neutralization, and acid reactivation were according to the standard procedures. Cultures were then divided into 5 groups to which was added PBS (5% nbcs), normal horse serum, and horse anti-chicken globulin antibody, respectively. After 50 min. at 40, the cultures were washed with PBS (5% nbcs), pH 7.5, and overlayed with agar medium. The addition of horse anti-chicken globulin antibody to a mixture of virus and antiserum increases the amount of neutralization. Approximately 42% of the neutralized virus was reactivated by the acid treatment. Since the reactivated virus could not be reneutralized by horse anti-chicken globu- lin antibody, it was concluded that acid treatment at pH 2.0 dissociated antibody from the virus. Similar results were obtained with the controls, PBS (5% nbcs) and normal horse serum (Table 2). 27 55.58: 25 2: .3 5695 :2. 82.. 29:56 3..., 53.2355: 2:... £558: 265 2: .3 BEES»: 5: «5; 29:50 2:... 82.2335: 2:: 23583. .N .55 H $52.55 533 .05 ON... 85.... 8; 5.3. 55.5 57:5. o N 6m v .8 m o o w: .6255 5353 w .3 .6825 5:52: C0x0_£0I=Cc mmhoc + me0_r_0I=CD mmhoc + m3.__> “03023001 mac; umNZDE—sz H o o .e ._ __ . em .. .. N O m .0? EDme 09.0...— EFCOC + w .mw qumm mmhoz _0E._0C + m:.:> “0302300”— m3.__> “53—0.:sz A o o .3 __ .. m .a : : N 0 mg .muacoxom. mma + w .8 .3933. mmn. + m3.__> Umuo>=ucmm m3.__> “003—0.:sz ~ COZDNZDob—Jowm .3: “CQEHDQLH cochZDmeZ a... «copra—00:. .9.me e c030>=0001 o\o 0\0 55:5 5383 5.25:5 $5.. 5.; m2; 2: 56:55.. 8 5.5:. U5 >2 5N__o:5c .0 5.6255. N NAMES. DISCUSSION Although viruses in general, including IBV reported in the present study, attach to cells at 40, they do not enter the cells and remain vunerable to antibody (25,55,59, 44). However, IBV and other viruses (20,59) which enter cells at 25 to 570 are invulnerable to antibody. It was possible to detect neutralized IBV at the cell surface by dissociating the antibody from the virus at pH 2.0 with accompanying reactivation of the virus. Acid treatment of chicken embryo kidney cells (CEKC) did not dissociate IBV from the cell, inactivate the viability of the cell, or interfere with the subsequent replication of IBV. The inability to detect chicken globulins attached to the virus after exposure to pH 2.0 indicated that antibody had dissociated from the virus. Even though neutralized poliovirus was reactivated by papain or exposure to pH 4.0, antibody was still associated with the virus (51). Because reactivation of neutralized IBV decreased linearly with time at 57 and 250, but not at 40, it was pro- posed that neutralized IBV was either eluting from the cells or merging with the cytoplasmic membrane. However, it was not possible to detect significant elution of neutralized 28 29 virus. If neutralized virus merged with the cytoplasmic membrane, it could not be reactivated because the antibody would no longer be readily accessible to the acid. There- fore, it is proposed that neutralized IBV enters cells at 25 and 570 as does nonneutralized IBV. Alternative hypotheses for the decrease in reactivation of neutralized virus are (i) a stable bond formed between antibody and virus or (ii) a large number of antibodies reacted with the virus and thus prevented complete reactiva- tion by acid. These interpretations are not considered adequate because the majority of IBV, neutralized in vitro by high concentrations of antibody for a long incubation period, could be reactivated at pH 2.0 (54). Entry of neutralized poliovirus into HeLa cells is reported to occur at 570 (55,58). In these experiments, the rate of entry into the cells at 570 occurred immediately and was exponential for 50 min. followed by a decrease in the rate of the reaction. In contrast, entry of neutralized IBV at 570 also occurred immediately but the rate of the reaction was slower and remained linear during the 120 min. of the experiments. Entry of nonneutralized envelope viruses such as herpes simplex (45), influenza (44), and Sendai (45) involves a digestion of the viral envelOpe and the host cell cytOplasmic membrane at the point of contact. If heterotypic antibody is added after viral adsorption, it blocks digestion. 50 However, the virus-antibody complexes are pinocytized ig.£939 by the host cell (44). Since the entry rate constant of nonneutralized IBV is at a greater magnitude than that for neutralized IBV, it is possible that the mechanism of entry is different for the two types of virions. Entry of IBV without antibody is probably by digestion of the viral envelope at the cytoplasmic membrane as is the case for other envelOped viruses (45,44,45). However, IBV with anti- body may be pinocytized ip_£2£g_by the host cell. Studies with a virus that does not possess an envelope, poliovirus, have indicated that virus with antibody entered cells at the same rate and to the same degree as virus with- out antibody (58). In contrast, antibody effects the rate of entry of a virus containing an envelOpe such as IBV. With Newcastle disease virus, antibody prevents entry into the cell by influencing viral elution (55). Homotypic antibody reacts only with the viral projections of IBV and does not react with the viral envelope (6). Therefore, antibody attached to IBV projections either reduced the rate of entry or influenced the mechanism of entry. Several reports have indicated that antibody may also attach to nonspecific areas of viruses and consequently, the virus can enter the cell and replicate (5,29,51,57). This report emphasizes that antibody effects the interaction of enveloped viruses with the host cell and supports the inference that viruses, neutralized after adsorption, will enter the host 51 cell. To delineate these phenomena, the interaction of neutralized IBV with the CEKC was studied by using 32P- labeled virus as described in the following paper. SUMMARY The interaction of nonneutralized and neutralized avian infectious bronchitis virus (IBV) with the chicken embryo kidney cell (CEKC) was studied. Biologically homotypic antiserum containing only 7 S (IgG) antibody was used for neutralization. Entry of IBV into the cell, as measured by insensitivity of virus-cell complexes to antibody, occurred at 57 and 250 but not at 40. To study entry of neutralized virus, IBV was adsorbed and neutralized at 40 and then the cells were incubated at either 4 (control), 25, or 570. Extracellular neutralized IBV was detected by dissociating antibody from the virus at pH 2.0 for 10 sec. which subse- quently reactivated the virus. This treatment did not dis- sociate the virus from the cell or injure the cell. Since it was not possible to detect any significant elution of neutral- ized virus, it was concluded that neutralized IBV merged with the cytoplasmic membrane at 57 and 250 but the neutral- ized‘virus remained extracellular at 40. When the neutralized virus merged with the cytOplasmic membrane it was not acces- sible to the acid and consequently it could not be reactivated. The kinetics of entry of virus without antibody was at a faster rate than that for virus with antibody. 52 INTERACTION OF NONNEUTRALIZED AND NEUTRALIZED AVIAN INFECTIOUS BRONCHITIS VIRUS WITH THE CHICKEN EMBRYO KIDNEY CELL II. Viral Degradation INTRODUCTION After avian infectious bronchitis virus (IBV) adsorbed to the chicken embryo kidney cells (CEKC) at 40, the sub- sequent addition of antibody neutralized the virus. Never- theless, the virus with attached antibody entered the cell at 25 and 570 but did not replicate (55). Studies with poliovirus indicated that virus with antibody also entered cells at 570 (58). In contrast, viruses neutralized before inoculation generally do not enter cells (50,55,45,50). The effect of antibody on the interaction of virus with the host cell is variable. Antibody may either stimulate viral elution from the cell (50,55) or SUppress elution (57, 58). Intracellular virus with antibody attached is only partially uncoated (50) and the viral nucleic acid is not released (50,58) or is uncoated with release and subsequent degredation of the viral nucleic acid (16,58). The present paper describes the effect of antibody on the interaction of 32P-labeled IBV with the CEKC and the degradation of virus without and with antibody. 55 SCI P.‘ PH‘ ~ ORDER NO . DATE IS SUE W. 0. N0 . YEAR l—LL l n I n 3 -, 8 scaog m/Uk S. U - N°’ P°3‘ P.o. N0. (953 L39 fictffiéo’l/m959fl07/Total PHYS. Desc.J/Doa/< bd- LC C/R SCH. 1 2 25 B PUBL. N0. NO.PAGES PRICE 1 P03. AUTHOR NAME (LAST, FIRST, MI.) 1-2 25 31 33 36 37 I P 39 57 Format It 16 22 32 35 50 54 59 77 Format B 2.1 (9,909.40! . 7o . . .. beWSKI. Mfimjx’. .E. . . . . Micro and Abst. PD00421 Publ. Abst. only PD00421 PD00422 PD00422 Pages in PD00433 PDOO434 Positive Copies to 7- P I? /)/)Q , ,' Qbs+,f; // 7‘ L//; Jr 3M c/r p. as rec'd made by ed. stripped by ed. "a" pp, by author by ed. nnbc __\L. 00 Lt. & dk.type NOCbg I Pap. prnt. — NOC ‘ Mntd. illus. .— Glos. photo __ Ditto Carbon Color photo CONDITIION: /. A$ Foreig METHODS Plaque assay of avian infectious bronchitis virus (IBV) in chicken embryo kidney cells (CEKC), standard procedures for viral adsorption and neutralization at 4°, and the preparation of IgG neutralizing antibody were described in the preceding paper (55). 32P-labe1ing,o§_;§y, Twenty—four hr old CEKC cultures in supplemented medium 199 were washed with phOSphate-free Hanks' (PFH) solution at pH 7.0. Citrate, 0.001 M, was sub- stituted for the phosphates in Hanks' balanced salt solution and the pH was adjusted with 1.4% NaHCOa. To remove residue phosphates, the cells were incubated at 57° for 24 hrs in phosphate-free medium 199.* During this period the phosphate- free medium was replaced twice. The stock virus was dialyzed for 72 hrs against PFH and then 2 X 107 p.f.u./ml. were added to the cells. After incu— bation at 570 for 90 min., the inoculum was decanted. Phosphate—free medium supplemented with 5% newborn calf serum dialyzed for 72 hrs against PFH and with 32? as carrier free orthophosphate, 0.02 mC./ml., was added to the cells. *- Grand Island Biological Co., Grand Island, New York, U.S.A. 54 55 Infected cultures were incubated approximately 72 hrs and then frozen and thawed 5 times in the medium. The mix- ture of medium and cell lysate was collected and is referred to as the "crude" virus preparation. Purification of virus. The crude virus was centrifuged at 10,000g_for 50 min. and then the SUpernatant fluid was collected and centrifuged at 78,0003 for 2 hrs. After dis- carding the supernatant fluid, the sediment was washed, resuspended to original volume in 0.02 M phosphate buffer, pH 7.2, and subjected to a second cycle of low and high speed centrifugation. -Sediments were washed and suspended to 1/10 the original volume in 0.02 M—phosphate buffer, pH 7.2. Two milliliters of virus were placed on a diethylaminoethyl (DEAE)-cellulose column (1 X 10 cm.) and the column was washed with 0.02 M-phosphate buffer until the radioactivity in the effluent was at background count. Virus was eluted from the column with 0.45 M-NaCl in 0.02 M-phosphate buffer, pH 7.2. The eluate was collected in successive 2 ml. amounts, dialyzed against PFH for 72 hrs, and tested for both infectivity (p.f.u.) and radioactivity (counts/min.). Fractions with 1 X 103 p.f.u./ml. or greater were pooled and when necessary concentrated by dialysis against polyethylene glycol until the radioactivity was 1 X 104 counts/min./ml. or greater. After concentration, the viral preparation was dialyzed against phosphate buffered saline solution free of magnesium and calcium, pH 7.5. 56 Protein was determined by the Folin phenol test (35). Density gradient centrifugation after DEAE-cellulose chromatography was not necessary. Radiochemical procedures. Radioactivity was measured in a Mark I Nuclear Chicago liquid scintillation counter using the solvent system described by Bray (7). To determine the acid-soluble radioactivity, crystalline bovine albumin and then trichloroacetic acid were added to the samples with final concentrations of 0.2% and 10%, respectively. The reaction mixtures were held at 40 for 2 hrs and then the precipitates were sedimented by centrifugation at 2,2003 for 1 hr. A sample of the supernatant fluid, 0.5 ml., was removed and the precipitate was resusPended in the remaining supernatant fluid. The pH was adjusted to neutrality with 1 N—NaOH and radioactivity was measured. After determining the amount of radioactivity in the SUpernatant fluid and the precipitate, the per cent acid-soluble activity was calculated. Samples labeled with 32F were classified as whole virus (acid-insoluble and RNase resistant), intact RNA (acid- insoluble but RNase sensitive), or degraded viral material (acid-soluble). Equal volumes of sample and RNase, 20 ug./ml. in Tris buffer (0.1 M), pH 7.5, or sample and Tris buffer were mixed and incubated at 57° for 15 min. Acid-soluble radioactivity before RNase treatment was considered to be that of degraded viral material. After RNase treatment, 37 radioactivity that was acid—insoluble was considered to be that of whole virus and the increase in acid-soluble activ- ity due to RNase was attributed to intact RNA. RESULTS Differential centrifugation and DEAE- cellulose chromatography for purifi- gggion of 32P-1abeled_;§y Although 99% of the extraneous radioactivity was re- moved by centrifugation, 63% of the original infectious virus was recovered. The increase in specific activity, p.f.u./counts/min., and specific infectivity, p.f.u./mg. protein, indicated that nonviral material was being removed by differential centrifugation (Table 1). After DEAE- cellulose chromatography, there was a direct relationship between the concentration of virus, p.f.u./ml., and the amount of radioactivity, counts/min./ml. (Table 1). Even though there were differences in the viral concentrations, the specific activity in the respective fractions remained essentially the same. Washing the DEAE column thoroughly before elution of the virus with 0.45 M-NaCl, removed some infectious virus and a large amount of viral aggregates remained on the column after elution. Consequently, the yield of infectious virus was low. Only 0.6% of the original radioactivity was recovered. Protein in the respective fractions was not sufficient for accurate determination. 58 59 If a mixture of labeled non—infected CEKC medium, cell lysate, and non—labeled virus were subjected to the same purification procedure, there was little 32F radioactivity associated with the virus. Ethyl ether fractionization and sodium dodecyl sulfate solubilization of 32P- labeled IBV Equal volumes of purified virus and ethyl ether were mixed and incubated at 250 for 2 hrs in closed tubes that were shaken vigorously every 50 min. The mixture was centrifuged at 6003 for 15 min. and the radioactivity of the ether layer, ether-water interface, and water layer was determined. Layers such as these with influenza virus have been reported to contain lipid, lipoprotein, and nonlipid, respectively (28). Radioactivity of the nonlipid was con- sidered to be associated mainly with the viral RNA. A small amount of the 32F was in the lipid fraction but greater amounts were in the lipoprotein and nonlipid frac- tions (Table 2A). To substantiate the amount of 32P associated with the viral lipid and lipoprotein, purified virus was treated with 0.4% sodium dodecyl sulfate (SDS) at 250 for 15 min. The control was virus in phosphate buffered saline without magnesium or calcium. Infectious virus was completely inactivated by 0.4% SDS and approximately 50% of the 32P was soluble in trichloro- acetic acid (Table 2B). Approximately the same amount of 32F 4O 2 6me d mo; 2239: ”.23 on: 0 93:8 9.6QO :89 2:: $58 838968 8582. mm .0 3m SN 8 S .o mow 0R 3 Om .o 02 A coo B . on .o mmm 08 2 R .o OS 4 RN 2 R .o com A 08 3 9 .o o3 A CE 3 R .0 RN .N 03 A 2 me .o omm .m oom .H 2 cm .9 one .q 8m .N : on .o oom .m coo .N 2 mm .0 08 .N coo .v 0 me G 0mm .0 8m .q m om .o 08 .o 2: .v N om; oo .o So cow 0 9888mm 6: 986mg Sc m; ”595:: Emacs Suzi med MSG NS x 2 .w 2 .3 08 x 2 .m 02 x S .m 92-2 EEESCS .53 EN #5563 38 .2 N2 x «m A S .m me x em .N 02 x 8 e 996 E - €2.53 38 we n2 x 8 .o 2 .o N2 x R .m 02 x 8 .m 9:: - E2859: :8 .2 m2 x mm .N om .o 08 x mm .N 02 x om N $20 2685 an: .3 .a .9 1 999581.: .e .9 l .E: £5558. . .E: .3 .e .9 298m £2695 9.68m 53:2 £58m €258€om £2635 >2 8.221 s 8:852 9 mm 2852385 8938.33 US 8233::ch EEEmEQ H Em¢9 41 m .Nmo .8 o .55 m_ .2 0mm 6 mmn. E memo: .o N 3% .N m3 x H 4 .59 m_ 18 omm 6 mm: $328.22. .25 .3 .e .m 39> Co #558: 9588952 mmmeo 5532328 mom .m H .mwo .3 99.52. m «we .Ne £28893 Two .m 29.. amm .x. 5:8: 8958821 Em 28 .< .>m: 3232.; mm to Am: cozcuzgsm 383m Summon EDEOm was As CREE—.5309: .653 325 N Emda 42 in the viral lipid and lipoprotein was detected by both ethyl ether fractionization and treatment with SDS. Adsorption of 32p-labeled IBV to CEKC After incubating one group of cells at 40 and another group at 570 for 50 min., the cells were inoculated with 2 X 104 p.f.u./ml. of 32P-labeled IBV. At certain time intervals, the amount of unadsorbed virus at 40 and at 570 was determined by infectivity and radioactivity (21,52,57). Adsorption was exponential and approximately 20 and 50% of the inoculum adsorbed in 5 min. at 4 and 57°, respectively (Fig. 1). Lesser adsorption occurred at 4°. At the respec— tive temperatures, the kinetics of adsorption when measured by infectivity or radioactivity were identical (Fig. 1). Effect of temperature on the distribution of ffgin CEKC with nonneutralized and neutral- ized 33P-labeled IBV Using 1 X 104 p.f.u./ml. of 32P-labeled IBV, viral adsorption and neutralization were according to the standard incubation procedure. Cells with nonneutralized and neutralized 32P-labeled IBV were washed 5 times with phos- phate buffered saline (PBS), pH 7.5, to remove unadsorbed virus and then 1 ml. of PBS was added. Groups of CEKC cul- tures were incubated at 40 (control) and at either 25 or 57°. At 15 min. intervals, the extracellular fluid was re- moved and the cells were suspended in 0.5 ml. of PBS. The 32F radioactivity associated with the cells and in the extracellular fluid was determined. 45 At 570 for 75 min., the 32F cell-associated activity decreased linearly and simultaneously the activity in the extracellular fluid increased (Fig. 2A). In contrast, the 32F cell-associated activity decreased slightly at 250 and in the 40 control, there was no change in the 32P cell- associated activity. At all temperatures, there was no difference in the interaction of nonneutralized and neutral— ized IBV with the host cell (Fig. 2A, B, C). The extracellular fluids were tested for elution of 32P, and degraded viral material. At 570, infectious virus, approximately 1% of the adsorbed infectious virus eluted from the cells with nonneutralized virus (Table 5). It is possible that some of the adsorbed infectious virus was no longer infectious after elution. The majority of the radio- activity eluted was probably due to acid-insoluble virus and viral lipoprotein components. Approximately, i’of the activity was due to degraded viral material. At the 5% level of significance there were no significant differences (P > 0.10) between nonneutralized or neutralized IBV with respect to the per cent 32P eluted or the per cent degraded viral material in the extracellular fluid (Table 5). Antibody did not enhance elution of virus. Viruses with antibody were apparently degraded at the same rate as viruses without anti— body. At 250 for 120 min., the small amount of 32P activity in the extracellular fluid was mainly acid-insoluble. 44 Figure 1. Adsorption of 32P-labeled IBV to CEKC. The per cent unadsorbed virus at 4 ( 57O ( ----- ) was determined by 32F (0,A) and ) and infectivity (0,A). 45 OOIIIA— A O. A—A 3 O C A A A-- ’ o o A O O - A --z-- 0 O A A ~- 00 44s _ \oo \ oo\2\ OQIIIIIHHHHMWHH\._ - p p . . p O O 0 O O 0 0 O O O m. 9 8 7 6 5 4 3 2 I. Qmmtbmdvss ”at: .x. l05 IZO 90 MINUTES 30 45 60 75 I5 O 46 .ham>auommmmu .AUV 03 can .Amv mm .Adv hm um Umumnsocw ouo3 UMMO mo mmsoum .coflumu Ifiamuusmc Ucm :oHumHomUm Hmnw> How muscoooum coflumnsocfl UHmUcmum map Hmumd .>mH UmHonHImNn A0.0V cmNHHmuusmc Ho A<. .323 1.3.3 H.m3..> 89.9582 8 28:32. 89.85% 89.23.38 .:02 -coz 9.3.8-98 8.6 92. 8H3: mNm .N. 8:... m39> R .3 9.88.929: aNm .N. 39895 .N. 3 39.2 89.253: 25 39.8325: .>mH H.989-..Nm 5.; 8.8 92. .9389 .2; 885% ES .n. .m3..> 3969:. Ho :93: Nm m names 49 Qegradation of nonneutralized and neutral- ized SZP-labeled IBV in CEKC Using 1 X 104 p.f.u./ml. of 32P-labeled IBV, viral adsorption and neutralization were according to the standard incubation procedure and the cells were washed as previously described. The cells were incubated at either 40 (control) or 570. At certain time intervals, the extracellular fluid was removed and the cells were washed with PBS, suspended in 0.5 ml. of double glass-distilled water, and lyzed by 4 cycles of freezing and thawing. The cells were then analyzed for acid-insoluble RNase resistant, acid-insoluble RNase sensi- tive, and acid-soluble material as previously described. Freezing and thawing did not damage the virus. There was no increase in the 32F acid-soluble activity in the 40 controls. However, approximately 5% of the activity was solubilized by 570 for 15 min. and subsequently there was a 5-6% increase in RNase-sensitive material. Therefore, some of the viral particles were probably damaged at 57°. No cell-associated degraded viral material was detected at time zero. The RNase-sensitive material at this time was prob— ably due to externally adsorbed-virus damaged at 570 (Fig. 5). At 57°, the same amount of whole virus and degraded viral material was associated with cells containing non- neutralized or neutralized virus. Maximum intracellular degradation of virus without and with antibody did not occur until after 120 min. After 60 min. at 570, the amount of acid-insoluble, RNase sensitive material remained at Figure 5. 50 Degradation of 32P-labeled IBV, nonneutralized ( ) and neutralized ( ----- ) in CEKC at 57°. The cells were analyzed for acid-insoluble, RNase resistant (A,A), acid-insoluble, RNase sensitive (0.0), and acid-soluble material (II,E]). % counts/min /ml. ASSOCIATED WITH THE CEKC 51 MINUTES L I20 52 approximately 12% in cells with nonneutralized virus but decreased to 1% in cells with neutralized virus (Fig. 5). This difference in the amount of acid-insoluble RNase sensitive material was most apparent at a time when the majority of neutralized virus was intracellular. At 250 only a small amount, 8%, of degraded viral material was detected and there were no differences between nonneutralized and neutralized virus. DISCUSSION Studies on the interaction of neutralized 32P-labeled avian infectious bronchitis virus (IBV) with the chicken embryo kidney cell (CEKC) established certain similarities as well as dissimilarities with other viruses (16,50,57,58, 55). Antibody had no effect on elution of IBV from CEKC. In contrast, antibody initiates elution of Newcastle disease virus from HeLa cells (55). However, with poliovirus the opposite occurs in that there is less elution of virus with antibody than without antibody (57,58). It was previously proposed (55) that neutralized IBV enters CEKC by merging with the cytoplasmic membrane at 57 and 25°. The present results confirm that neutralized IBV enters CEKC at 570 and is subsequently degraded. Similar results have been reported with neutralized viruses of rabbitpox (50), vaccinia (16), and polio (58). At 250 IBV remains cell-associated and very little degradation occurs. It is possible that at 250 additional time might be required for degradation or the viral lipoprotein is actually removed from the virus but remains associated with the cytoplasmic membrane and is consequently acid-insoluble. At 250 polio— virus is not uncoated (59). 55 54 Even though it was established that IBV with antibody enters at a slower rate than IBV without antibody (55), there were no significant differences in the rate of degradation at 57°. Approximately the same per cent extra— cellular or intracellular degraded IBV material was detected. However, there was a difference in the amount of RNase sensi- tive material. In cells with neutralized virus, a decrease in RNase sensitive material occurred and consequently, little or none could be detected after 180 min. A decrease in the amount of intact RNA from neutralized 82P—labeled poliovirus occurs within 15 min. at 570 (58). However, experiments with 3H-labeled vaccinia DNA indicated that the viral DNA from neutralized virus is degraded within 1 to 2 hrs at 570 (16). Studies with 32P-labeled poliovirus (58) suggested that antibody may either suppress a reaction necessary for the release of intact viral RNA or stimulate a reaction which rapidly degrades the released RNA. Of the two hypotheses, the latter was considered to be a more favorable explanation of the results. The results obtained with neutralized IBV confirm that degradation occurs but further studies are necessary to differentiate between the degradation of virus without and with antibody. It is possible that the nucleoprotein of nonneutralized IBV escapes from the virus at the cytoplasmic membrane as does that of viruses such as herpes simplex (45), influenza (44), and Sendai (45). With antibody attached, IBV may be 55 pinocytized by the host cell and completely degraded within pinocytic vesicles by lysosomal enzymes. The RNase in pinocytic vesicles could destroy the viral RNA and conse- quently prevent replication. According to Morgan and Rose (44), heterotypic antibody prevented digestion of influenza viral envelope at the cytoplasmic membrane but the virus- antibody complexes appeared to enter the cell by pinocytosis. However, the accumulation of degraded viral material in the extracellular fluid suggested that homotypic antibody did not prevent digestion of the IBV enve10pe. Neutralized vaccinia virus was pinocytized and then degraded within cyto- plasmic vesicles (16). According to Berry and Almeida (6), biologically homo— typic antibody reacts with the viral projections but not with the envelope of IBV. In the present investigation homo- typic antibody was also used. Neutralized IBV entered the CEKC but no replication occurred. Therefore, it is hypo- thesized that the attachment of antibody to the viral projec- tions of IBV either inhibits the uncoating of the viral nucleoprotein or influences the degradation of the viral RNA. The latter hypothesis is more favorable. SUMMARY There was an identical relationship between adsorption of infectious virus and radioactivity of 3‘°'P-labeled avian infectious bronchitis virus (IBV) purified by centrifuga- tion and chromatography. .Antibody did not enhance or sup- press elution of IBV from chicken embryo kidney cells (CEKC). The distribution of 32F from nonneutralized and neutralized labeled IBV in CEKC changed rapidly at 570 but not at 25 or 4°. At 57°, virus without antibody was degraded at the same rate as virus with antibody. Even though virus without and with antibody entered cells at 25°, there was very little detectable degradation. 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