J. gen. Virl. (I974), 25, 351-357 Printed in Great Britain 35I Plaque Frmatin by Influenza Viruses in the Presence f Trypsin By G. APPLEYARD AND H. B. MABER Micrbilgical Research Establishment, Prtn, Nr Salisbury, Wiltshire, U.K. (Accepted I2 August I974) SUMMARY Eight strains f influenza virus were titrated as plaque-frming units in bth mnlayers and suspensins f chick embry cells. In the absence f trypsin, satisfactry plaques were frmed nly by the A/WSN strain f virus. When trypsin was included in the verlay medium f cell mnlayers, all the influenza virus strains tested prduced plaques, and the plaque infectivities f all but ne strain were clse t the egg infectivities. Using cells suspended in agar in the presence f trypsin, fur strains gave plaque infectivities indistinguishable frm egg infectivities, tw strains frmed plaques with rather lw efficiency and tw strains did nt prduce plaques. Plaque frmatin was als enhanced by pr~.ase r subtilisin. A preliminary investigatin was made f the mde f actin f trypsin. INTRODUCTION Many types f investigatin n influenza viruses are hindered by the lack f a cnvenient plaquing prcedure. A few strains f virus, ntably A/WSN and fwl plague, readily prduce plaques in primary cultures f chick embry cells, and these have received a pssibly disprprtinate amunt f study. But mst influenza viruses frm plaques, if they d s at all, nly in cell cultures that are less widely available. In the curse f develping an infectivity assay fr the influenza virus recmbinant A]BEL[42-Singapre[I[57 (Schild, McCahn & Kendal, I97O), we nted that the frmatin f plaques in chick cell cultures was greatly enhanced by the inclusin f trypsin in the verlay medium. A similar bservatin was made by Came, Pascale & Shimnaski (I968), wh used pancreatin t facilitate plaque frmatin by influenza viruses, and recently Tbita & Kilburne (I974) reprted the frmatin f plaques by tw strains f type B influenza virus in the presence f trypsin. We describe here tw methds f plaque assay which are based n this phenmenn. All strains f influenza virus that were tested frmed plaques by at least ne methd, and mst culd be titrated with a sensitivity equal t that fund in eggs. METHODS Viruses. The influenza virus used fr mst f this study was the recmbinant A/BEL/42- Singapre/I/57 (HON2) f Schild et al. (I97O). Other virus strains used were A/WSN (HONI), A/BELl42 (HONI), A/PR]8134 (HONI), A/Singapre]I/57 (H2N2), A/England/ 42/72 (H3N2), MRC2 (a recmbinant f A/England]42[72 and A[PR]8]34, having the surface antigens f A]England]42]72 but grwing well in eggs) and B/Lee/4. Viruses were grwn in the allantic cavity f I I-day embrynated hens' eggs and were stred as infected allantic fluids at - 70 C. All were well adapted t grwth in eggs, except fr the A/England/ 42]72 strain which had been passed nce in mnkey kidney cells and six times in eggs since islatin.
352 G. APPLEYARD AND H. B. MABER Chick embry cells. Cell suspensins were prepared by trypsinizatin f I i-day chick embrys as described by Beltn & Garrick 0972). Plaque assay prcedures. (I) In mnlayers: with the exceptin f strain A/WSN, the viruses used in this study frmed plaques either prly r nt at all in chick embry cell mnlayers when these were incubated withut trypsin. The fllwing prcedure gave the best results. Plastic Petri dishes, 5 cm in diam., were swn with 5 I~ chick cells in 5 ml Medium I99 cntaining 0"4 % bicarbnate and 0"5 % bvine serum albumin. On the fllwing day, the medium was aspirated frm the cultures and. t ml samples f virus suspensins diluted in phsphate buffered saline, ph 7"4, plus 1% bvine serum albumin were allwed t adsrb at rm temperature fr I h. The cultures were verlaid with 5 ml Medium I99 plus 0.5% Difc!nagar n. I and i/zg/ml DEAE-dextran and were incubated in air at 36 C. Plaques frmed by the A/WSN strain f virus were stained by verlaying after 3 days with 2 ml agar medium cntaining 0"05 % neutral red and were cunted n the fllwing day. Plaques frmed by ther virus strains werl prly revealed by staining with neutral red. A mre satisfactry prcedure was t lift ff the agar after abut 6 days, rinse the mnlayers with ethyl alchl and allw them t dry at rm temperature. Plaques culd then be cunted as paque fci mst easily visible under semi-darkgrund illuminatin. When trypsin was t be included in the medium, a 1% slutin f crystallized trypsin (Armur Pharmaceutical Cmpany Ltd.) in IO -3 ~-HC1 was added t the verlay medium t give a cncentratin f I #g[ml. After incubatin at 36 C fr 3 t 6 days, plaques were stained by the additin f a neutral red verlay and were cunted n the fllwing day. (2) In suspensin:.i ml samples f diluted virus were added t I a chick cells, suspended in 2. 5 ml Medium I99 in I z bttles. Duble strength agar medium, cnsisting f 2"5 ml Medium 199 plus 1% Inagar n. I and 20/~g/ml DEAE-dextran, was added t each bttle, mixed thrughly and pured int 5 cm diam. Petri dishes. When trypsin was used, it was added t the duble strength agar medium at IO/zg/ml, giving a final cncentratin in the cultures f 5/~g/ml. Cultures were incubated fr 3 t 6 days, and the plaques were stained with neutral red as described fr assays in mnlayers. Egg infectivity assays. Grups f IO t 25 I I-day hens' eggs were inculated in the allantic cavity with.i ml samples f diluted virus suspensin. After incubatin at 36 C fr 2 days, the eggs were tested fr infectin by the ability f their allantic fluids t agglutinate chicken red cells. The infectivities f virus suspensins were expressed as lgl (EID/ml), which were btained by subtracting -I6 frm estimates f lgl (EIDs/ml) and then runding t ne decimal place. Unless therwise stated, infectivities were derived frm the cmbined results f several tests, using a ttal f at least 8 eggs inculated with virus dilutins that gave an infectin rate between 20 % and 80 %. RESULTS Plaque assay in cell mnlayers When the A[BEL[42-Singapre/i]57 strain f virus was titrated in chick cell mnlayers withut trypsin, the plaque infectivity was nly abut 5% f the egg infectivity (Table I). Plaques were very indistinct when the mnlayers were stained with neutral red 6 days after infectin (Fig. Ia), and they were smetimes difficult t cunt even when the alchl 'staining' methd was used. Hwever, when trypsin was included at I #g/ml in the verlay medium, small plaques were visible 2 days after infectin and by 4 days they were clearly defined and abut 4 mm in diam. (Fig. ib). The plaque infectivity in the presence f trypsin was indistinguishable frm the egg infectivity (Table I).
Influenza virus plaque frmatin 353 Table L Replicate titratins f strain A/BEL/42-Singapre/I/57 influenza virus in eggs and chick embry cell mnlayers lgl (p.f.u.]ml.) ). With lg10 Withut trypsin Expt. (EID/ml)* trypsin I Izg/ml I 8'9 7"7 9"0 2 9-0 7"5 8-8 3 8"7 7"6 9"0 4 9"0 7"8 8"9 * Only 20 t 30 eggs per titratin. Fig. I. Influenza virus plaques in mnlayers f chick embry cells. (a) A/BEL/42-Singapre[I/57 withut trypsin, 6 days after infectin; (b) A/BEL/42-Singapre/I/57 with l #g/ml trypsin, inculure I/2O f that in (a), 4 days after infectin; (c) A/WSN withut trypsin, 4 days after infectin. Mnlayers under an agar verlay cntaining trypsin were rather easily damaged, and micrscpic examinatin shwed that the ceils were runded and nly lsely attached t the Petri dish surface. Nevertheless, cncentratins f trypsin up t I #g/ml were tlerated; these higher cncentratins did nt further enhance plaque frmatin. Trypsin at levels between i and 5 #g/ml had less effect n plaque frmatin and, smewhat surprisingly, caused the mnlayers t becme extremely fragile. Belw I #g/ml, trypsin did nt affect plaque frmatin. In cntrast t the influenza virus strain A/BEL/42-Singapre/I]57, strain A/WSN prduced large plaques in the absence f trypsin (Fig. I c), and the plaque infectivity apprximated t the egg infectivity (Table 2). Trypsin increased the size f A/WSN plaques but nt their number. Other virus strains behaved like A[BEL/42-Singapre[I[57, frming plaques with difficulty r nt at all in the absence f trypsin. When trypsin was added t the verlay medium, strains A/BEL]42 and A]PR]8]34 prduced large, clear plaques similar t thse f strain A/BEL/42-Singapre/i/57; strains A[Singapre/~/57, MRC2 and A/England/42/72 als frmed plaques but these were smaller and develped mre slwly (Table 3)- The plaque infectivities f all these viruses, except fr A/England[42172 and pssibly A/Singapre/I 57, were nt significantly different frm their egg infectivities (Table 2). The nly type B influenza virus t be used, B/Lee/4, prduced plaques bth with and withut trypsin, but trypsin increased the plaque infectivity abut tenfld t the level f infectivity fund in eggs. 24 VIR 25
354 G. APPLEYARD AND H. B. MABER Table 2. Infectivity titratins f influenza viruses in eggs and chick embry cell cultures In mnlayers lgl (p.f.u./ml)*-lgl (EID/ml) In suspensin e ~ e lgx N Plus N Plus Virus strain (EID/ml) trypsin trypsin trypsin trypsin A/WSN (0 8"6 - " i - 0-2 NTt NT (2) 9"0 +0"2 +0.2 +.I +0"2 A/BEL/42-Sing/I/57 8'9 - t'3. - 2.8 + " t A/BEL/42 ([) 8"7 -- 0"9 - " t NT NT (2) 9"3 --0-8 +q --2"7 +0.2 A[PR[8134 9'0 - I'6-0"3-2"9 - 'I A/Sing/I/5 7 (I) 8"7 --:~ --0"5 -- --0"8 (2) 8"9 -- - 0"4 -- - '8 MRCz (I) 9"0 -- -q -- -- (2) 8"8 -- +0'I -- -- A/England/42~72 8" -- - 0"9 -- -- B/Lee/4 (t) 8"6 -- I '0 -- 0"I -- 2"9 -- 0"8 (2) 9" -- 1.6 0'0 -- 2'6 --O+5 * Mean f at least three titratins. t Nt titrated. :~ N plaques. Table 3. Plaque frmatin by influenza viruses in the presence f trypsin r Apprximate plaque diam. (mm) x In mnlayers In suspensin A &-~ r ~ r Virus strain at 4 days at 6-7 days at 4 days at 6-7 days A/WSN 6 5 A/BEL/42-Sing/r/57 4 3 A/BEL/42 4 3 A]PR]8]34 3 3 A/Singapre/t/57 z I MRC2 2 --* A/England/42/72 I -- B/Lee/4 I I * N plaques. Plaque assay in cell suspensins In the absence f trypsin, nly the A/WSN strain f virus frmed gd plaques by the suspended cell technique (Fig. 2a); as in mnlayers, the plaque infectivity was similar t the egg infectivity (Table 2). Strains A/BEL[42-Singapre/t[57, A/BEL[42, A/PR[8/34 and B]Lee]4 als frmed plaques, but these were very small and indistinct and the infectivity was less than 1% f that in eggs (Table z). When trypsin, 5/zg/ml, was included in the medium f suspensin cultures, mst virus strains prduced gd plaques (Tables 2 and 3)- Strains A[BEL/42-Singapre/i/57, A/BEL] 41 and A[PR/8/34 all frmed clear plaques at least 3 mm in diam., with infectivities which did nt differ frm thse in eggs r mnlayer cultures. Plaques prduced by the A/Singapre/I/57 and B/Lee/4 strains were smaller and the infectivities were nly I % t 2 % f thse in eggs. The MRC2 and A/England/42~72 strains failed t prduce plaques in sus-
Influenza vlrus plaque frmatin 3 55 Fig. 2. Influenza virus plaques in suspensins f chick embry cells. (a) AIWSN withut trypsin, 4 days after infectin; (b) A/BEL/42-Singapre/I/57 with 5 #g/ml trypsin, 4 days after infectin; (c) A/WSN with 5 #g/ml trypsin, 4 days after infectin. pensin even in the presence f trypsin. With strain A/WSN, trypsin increased the size, but nt the number, f plaques in suspensin (Fig. 2c; Table 2). Further studies n the actin f trypsin Effect f trypsin inhibitr T exclude the pssibility that plaque enhancement was caused by sme impurity in the trypsin preparatin, a slutin f trypsin cntaining 0"5 mg/ml was mixed with varius cncentratins f sybean trypsin inhibitr (Calbichem Ltd) and was then used at i/~g/ml in the mnlayer plaque assay f A[BEL[42-Singapre[I/57 virus. The enhancing actin f the trypsin was greatly reduced by '5 mg/ml f trypsin inhibitr and ablished by I mg/ml. Hence, trypsin itself was respnsible fr the effect n plaque frmatin. Pre-incubatin f medium with trypsin T test whether trypsin might act by destrying an inhibitr in the medium, agar verlay medium was incubated with trypsin (50 #g/ml) at 36 C fr 24 h. The medium was then heated at IOO C t remelt the agar and t inactivate the trypsin, and was used as verlay fr the plaque assay f A/BEL[42-Singapre/I[57 virus. The trypsin-treated medium did nt enhance plaque frmatin, althugh it still permitted satisfactry plaque frmatin if further trypsin was added. Therefre trypsin did nt act by remving a pre-existing virus inhibitr frm the medium. Time f actin f trypsin Trypsin was added t cultures r its actin was halted at varius stages f the plaque assay prcedure. In ne experiment, chick cell mnlayers were expsed t trypsin at IO #g/ml in Medium 199 fr 5 h at 36 C befre infecting with A[BEL/42-Singapre/I/57 virus; the cultures were then incubated under verlay medium withut trypsin. This pretreatment f the cultures with trypsin did nt enhance plaque frmatin. In the secnd experiment, replicate suspensin cultures were infected with abut 5 p.f.u. f A/BEL[42-Singapre[I/57 virus and incubated either with r withut trypsin (5 #g/ml) in the medium. At daily intervals, an verlay cntaining sybean trypsin inhibitr was added t grups f cultures with trypsin and an verlay cntaining trypsin was added t grups withut trypsin. Plaques were cunted 4 days after the additin f trypsin. Sme cultures cntaining trypsin were als stained at daily intervals t fllw the nrmal curse f plaque 24-2
356 G. APPLEYARD AND H. B. MABER Table 4. Time f actin f trypsin n plaque frmatin by strain A/BEL[42- Singapre[I/57 influenza virus in chick embry cell suspensins Treatment f cultures, Plaque frmatin t Day f ~ Day f additin f trypsin additin f trypsin inhibitr Day f cunting Mean diam. (mm) Mean* cunt t I" I 2 3 4 -- 2 I 37 -- 3 2 52 -- 4 3½ 45 -- 5 5 SC~t -- 6 6 SC 4 -- I 4 ½ I8 2 4 2 50 4 3½ 45 -- 5 4 39 6 3½ 34 -- 7 4 33 -- 8 4 26 Frm three cultures. ~f Identical grups f cultures. :~ Semicnfluent due t large size f plaques. develpment. The results (Table 4) shwed that trypsin inhibitr halted plaque develpment when added as late as 2 days after infectin, indicating that trypsin was needed cntinuusly during the perid f plaque frmatin. Further, plaques develped even when trypsin was nt added t the cultures until 4 days after inculatin f virus, and their final numbers were nly slightly reduced cmpared with cntrl cultures. This suggests that trypsin was nt required fr the initial infectin f chick cells but it was required t enable the infectin t spread sufficiently t prduce plaques. Effect f ther prteases Sme ther prtelytic enzymes were tested fr their ability t enhance plaque frmatin by the A[BEL[42-Singapre/I/57 strain f virus in suspended cell cultures. Prnase (Kch- Light Labratries Ltd) at I/zg/ml and subtilisin (Sigma Chemical Cmpany) at I/zg/ml were apprximately as effective as trypsin at 5/zg/ml. Prnase, which is a mixture f prteases btained frm Streptmyces griseus, was fractinated n a clumn f Sephadex G-75 by Dr J. D. Oram, and the plaque enhancing activity was fund t be assciated with a fractin having trypsin-like specificity. Tw ther enzymes, c~-chymtrypsin (Wrthingtn Bichemical Crpratin) and thermlysin (Calbichem Ltd) had n plaque enhancing actin at cncentratins frm I t I/zg]ml. The enhancing activity f prnase (I/zg/ml) was ablished by the inclusin f sybean trypsin inhibitr (t/zg/ml) in the medium, but that f subtilisin (t/zg/ml) was unaffected by trypsin inhibitr at cncentratins up t 80/zg/ml. DISCUSSION The A/WSN strain f influenza virus is well knwn t frm plaques in mnlayer cultures f chick embry cells, and it is nw evident that the virus can be titrated with equal efficiency in chick cell suspensins in agar. The ability f ther strains f influenza virus t prduce
Influenza virus plaque frmatin 357 plaques in cultures f chick cells in the presence f trypsin shuld allw these readily available cells t be used mre widely in studies requiring the titratin f plaque islatin f influenza viruses. Fr virus strains that frmed plaques bth in mnlayers and in cell suspensins, the latter methd was mre cnvenient; n preliminary preparatin f mnlayers was required, n prblem arse frm fragility f the mnlayers, and the plaques tended t be clearer and mre easily cunted than in mnlayers. The mechanism by which trypsin enhances plaque frmatin might relate t sme aspects f the replicatin f influenza viruses. The results reprted here shw nly that trypsin was required during the whle perid f plaque develpment and therefre presumably acted n successive virus grwth cycles. We are investigating this prblem further. Came et al. (I965) suggested that pancreatin prduced its effect by an actin n the hst cells. Hwever, we have fund that the infectivity f influenza virus grwn in chick cell cultures was increased abut tenfld by incubatin with trypsin. Hence, the enzyme may facilitate the develpment f plaques by acting n the released virus particles, as was described fr revirus by Spendlve & Schaffer (i965). We wish t thank Mrs J. C. Frder fr skilled technical assistance. Mst f the influenza virus strains were kindly prvided by Dr A. S. Beare r Dr G. C. Schild. This wrk was supprted by a grant frm the Department f Health and Scial Security. REFERENCES BELTON, F. C. & GARRIOCK, O. J. (I972). A semiautmatic methd fr the prductin f primary cell suspensins. Jurnal f Applied Chemistry and Bitechnlgy 22, 335-34I. CAME, P. E., PASCALE, E. & SHIMONASKI, G. (I968). Effect f pancreatin n plaque frmatin by influenza viruses. Archly flit gesamte Virusfrschung 23, 346-352. SCHILD, G. C., McCAHON, D. & KENDAL, A. P. (I970)- Immunlgical and structural studies n a recmbinant influenza A virus. In Bilgy f Large RNA Viruses, 638-654. Edited by R. D. Barry & B. W. J. Mahy. Lndn and New Yrk: Academic Press. SPENDLOVE, R.S. & SCHAFFER, F.L. 0965). Enzymatic enhancement f infectivity f revirus. Jurnal f Bacterilgy 89, 597-62. TOBITA, K. & KILBOURNE, E. D. (1974). Genetic recmbinatin fr antigenic markers f antigenically different strains f influenza B virus. Jurnal f Virlgy x3, 347-352. (Received I9 June I974)