Allantoic Fluid on Microporous Filters

Transcription

1 APPLIED AND ENVIRONENTAL ICROBIOLOGY, r. 1980, p /80/ /05 $02.00/0 Vol. 39, No. 3 Simple ethod for the Concentrtion of Influenz Virus from Allntoic Fluid on icroporous Filters SAGAR. GOYAL,* HENRY HANSSEN,t AND CHARLES P. GERBA Deprtment of Virology nd Epidemiology, Bylor College of edicine, Houston, Texs embrne filter dsorption-elution is n efficient method for concentrtion nd prtil purifiction of severl types of viruses from vrious queous solutions. For efficient virus dsorption to negtively chrged filters, the smple is djusted to 3.5 nd trivlent slts re dded before filtrtion. Since influenz virus is sensitive to extremes in, it cnnot be concentrted by ordinry filters. Zet Plus filters, which hve net positive chrge of up to 5 or 6, were evluted for the concentrtion of influenz virus from infectious llntoic fluids. Influenz virus efficiently dsorbed to Zet Plus filters t 6, nd ddition of slts ws not necessry. AdsoTbed virus ws eluted in smll volume of 2% bovine serum lbumin plus 1 NCl t 10. By this procedure, viruses in 100 ml of llntoic fluid were concentrted to finl volume of 8 ml, with n verge recovery efficiency of 71.0%. Severl different methods re vilble for the concentrtion of influenz virus from hrvested llntoic fluid. These include the following: highspeed centrifugtion (10), dsorption nd elution from erythrocytes (3), precipittion with methnol (1), dsorption nd elution from luminum phosphte gel (7), nd precipittion with polyethylene glycol 6000 (6) nd clcium phosphte (11). Veerrghvn nd Sreevlsn (13) compred severl different methods of concentrtion nd purifiction of influenz virus nd found tht two cycles of luminum phosphte tretment ws the best method, followed by ultrcentrifugtion with nd without methnol precipittion, erythrocyte dsorption, single-cycle luminum phosphte tretment, nd finlly the zinc oxide method (8). Although some of these methods re very efficient for recovery of influenz virus from llntoic fluid, they re time-consuming or require costly equipment or both. We hve reported previously on the use of Filterite filters for the concentrtion of poliovirus nd simin rotvirus from tissue culture hrvests (2). In tht method, the hrvest is first treted with Freon to remove orgnics tht interfere with virus dsorption (14), djusted to 3.5 nd luminum chloride, nd then pssed through series of microporous fiber glss filters. Under those conditions, the virus is dsorbed to the negtively chrged filters nd subsequently cn be eluted with smll volume of high- buffer. This membrne dsorption-elution methodology hs been used in t On leve from the Deprtment of icrobiology nd Prsitology, School of edicine, University of Antioqui, edellin, Colombi. 500 our lbortory for severl yers to concentrte nd detect smll numbers of virus in lrge volumes of wter nd wstewter (2, 4, 14; S.. Goyl nd C. P. Gerb, in B Dutk, ed., embrne Filtrtion, Applictions, Techniques, nd Problems, in press). The methodology could not be pplied to influenz virus, however, becuse it is very sensitive to extremes in (5), nd low is necessry to enhnce virus dsorption to negtively chrged filters. The loss in hemgglutintion (HA) titer upon cidifiction is believed to be due to the dsorption of virus to the precipitte tht forms under cidic conditions (5). Recently, Sobsey nd Jones (12) reported on the use of positively chrged membrne filters for the concentrtion of poliovirus from tp wter. The dvntge of using positively chrged filters is tht viruses (which re usully negtively chrged) cn dsorb to these filters t neutrl or nerly neutrl. The present study ws performed to evlute these positively chrged filters for concentrtion of influenz virus from infectious llntoic fluid. ATERIALS AND ETHODS Virus propgtion. The PR-8 strin of influenz virus, which ws obtined originlly from the Americn Type Culture Collection (Rockville, d.) nd since hs been mintined in the culture collection of this deprtment, ws used to inoculte 11-dy-old embryonted chicken eggs through the llntoic cvity. After 48 h of incubtion t 37 C, the eggs were chilled, nd the llntoic fluid of individul eggs ws collected, pooled, nd clrified of host cell debris by centrifugtion t 2,500 x g for 30 min t 4 C. Filters. Fiber glss filters (3.0- nd 0.45-tim porosity; Filterite Corp., Timonium, d.) were used to

2 VOL. 39, 1980 represent negtively chrged medi, wheres ceilulose-ditomceous erth-"chrge-modified" resin (series S, Zet Plus; AF CUNO, eriden, Conn.) ws used s positively chrged filter medium. The ltter filter is referred to s "30S" nd hs nominl porosity of 0.5,m. All filters were used s circulr disks housed in polypropylene or stinless steel holders of 47- or 25- mm dimeter. Virus concentrtion. Hrvested llntoic fluid ws filtered through the pproprite filters, nd influent nd effluent smples were ssyed for HA ctivity. The difference in the HA titers between the two smples ws tken to be the mount of virus dsorbed to the filter. The virus dsorbed to the filter ws eluted by slowly filtering the eluent through the filter. Severl different eluents were tested for elution efficiency s described below. The elutes were neutrlized immeditely fter elution. The HA ctivity of the neutrlized elute ws monitored to detect the mount of virus recovered from the filters. In some experiments, the infectivities of influent, effluent, nd elute smples were determined in ddition to the HA titer. Virus titrtion. Virus titrtion ws done by monitoring the HA ctivity of the smple to be tested. The HA test ws performed by using microtiter procedure nd 0.5% chicken erythrocytes (9). The results re reported s the reciprocl of the highest dilution giving positive HA. Virus infectivity ws determined by inoculting seril 10-fold dilutions of the smple into 11-dy-old embryonted chicken eggs nd determining the HA ctivity of the llntoic fluid of infected eggs. RESULTS We previously hve reported on the use of negtively chrged Filterite filters to concentrte cell culture hrvests of enteroviruses nd rotviruses (2). An ttempt ws mde to use these sme filters for the concentrtion of influenz virus from llntoic fluids. For this purpose, smples of infected llntoic fluid were djusted to different vlues nd then were pssed through combintion of 3- nd 0.45-,um Filterite filters. The results shown in Tble 1 indicte tht influenz virus ws inctivted t 5 or less. Also, the virus did not dsorb to Filterite TABLE 1. Adsorption of influenz virus to Filterite filters t different vlues HA titer Adsorption Twenty-milliliter smples of infected llntoic fluid were djusted to the indicted vlues nd were pssed through combintion of 3.0- nd jlm Filterite filters. CONCENTRATION OF INFLUENZA VIRUS 501 filters t ny tested except 6. At tht, 50% of the virus dsorbed to Filterite filters. Since lmost 100% of the virus could dsorb to positively chrged filters t tht, the Filterite filters were not tested further for concentrtion. They were used, however, to clrify the virus hrvest s indicted below. Since llntoic fluid contins high mount of minerls nd proteins nd is reltively viscid, filtrtion through the positively chrged filters is very difficult. To circumvent this problem, n ttempt ws mde to preclrify the llntoic fluid by pssing it through 3-jim-pore size Filterite filter. Since some virus did dsorb to the Filterite filters t 6 (Tble 1), the filters were treted with serum before they were used to clrify the virus hrvest (14). Wllis nd elnick (14) hve shown tht precoting the filters with norml fetl clf serum helps prevent loss of virus by dsorption to the filters. Tble 2 shows tht no dsorption occurred to serumcoted Filterite filters t ny vlue tested. There ws loss in titer, however, when the of the llntoic fluid ws lowered to 5.0. In subsequent experiments, the of the crude hrvest ws left undjusted (pproximtely 7.4 to 7.6) for the clrifiction step. Wheres only 3 ml of crude llntoic fluid could be pssed through 25-mm-dimeter type 30S filter t certin pressure, s much s 20 ml could be processed under similr conditions if the llntoic fluid hd been clrified through Filterite filter. Subsequent experiments therefore were performed with llntoic fluid clrified through 3-,um Filterite filter. The optimum conditions for virus dsorption to the 30S filters were exmined next. Smples of llntoic fluid preclrified through Filterite filters were djusted to vrious vlues by the use of 0.05 glycine buffer t 2 or 10. The llntoic fluid then ws pssed through the filter, nd influent nd effluent smples were ssyed TABLE 2. Adsorption of influenz virus to serumcoted Filterite filters t different vlues HA titer Adsorption Twenty milliliters of llntoic fluid ws djusted to the indicted vlues nd pssed through 3-nm Filterite filter tht hd been pretreted with 10%o fetl clf serum. Influent nd effluent smples were ssyed for HA ctivity.

3 502 GOYAL, HANSSEN AND GERBA for HA ctivity (Tble 3). ximum dsorption occurred t 6. Tble 3 shows tht the loss of HA titer upon cidifiction of the hrvest to 5.0 nd 4.0 ws not s gret s shown in Tbles 1 nd 2. The reson for this is tht crude virus hrvest ws used for the experiments in Tbles 1 nd 2, wheres preclrified virus ws used for the experiments in Tble 3. The mount of precipitte formed upon cidifiction of crude hrvest is greter thn tht formed upon cidifiction of clrified hrvest. Since the loss of virus upon cidifiction is believed to be due to virus dsorption on the resultnt precipitte (5), it is resonble to believe tht cidifiction of APPL. ENVIRON. ICROBIOL. clrified hrvest results in virus loss much less thn tht obtined by the use of crude hrvest. A number of different eluents were then tested to elute virus dsorbed to the filters (Tble 4). It is interesting to note tht beef extrct lone ws unble to elute the virus but could do so in the presence of sodium chloride. Other good eluents were 0.5 NCl ( 9), sline ( 11), nd mixture of equl volumes of 2% bovine serum lbumin nd 1 NCl t 10. Tble 5 shows the concentrtion of lrger volumes of influenz virus hrvest. A 50- to 100-ml mount of the virus hrvest could be concentrted by fctor of 5- to 10-fold with n verge recovery efficiency of 48% with 11 sline nd of 71% with the bovine serum lbumin nd sodium chloride mixture (Tble 5). It should be mentioned here tht the elute ws neutrlized immeditely fter the completion of the elution step. On severl occsions, the infectivity of the virus hrvest before nd fter concentrtion ws monitored by inocultion into embryonted eggs. Infectivity lwys prlleled the HA ctivity of the virus. TABLE 3. Optimum for virus dsorption to 30S filters HA titer Adsorption 8: DISCUSSION Smples of clrified llntoic fluid were djusted to the indicted vlues nd pssed through 25- embrne dsorption-elution technology is, mm-dimeter 30S filters. Influent nd effluent smples the method of choice for concentrting smll were ssyed for HA ctivity. mounts of cid-resistnt virus from lrge vol- TABLE 4. Evlution of different eluents for elution of influenz virus from 30S filters Eluent Totl HA ctivity Adsorption Totl HA c- (%) tivity in elute Recovery (%) 3% Beef extrct glycine Distilled wter, phosphte-buffered sline, % Beef extrct + 1 NCl (1: ), 9 2% Bovine serum lbumin NCl (1:1), NCl, 9 1, ,382 >100 Sline, 11 1, ,408 >100 Ten-milliliter mounts of clrified infectious llntoic fluid, 6, were pssed through 30S filters (25-mm size). The filters were eluted with 3-ml volumes of the indicted eluents. Elutes were neutrlized immeditely fter elution.

4 VOi. 39, 1980 CONCENTRATION OF INFLUENZA VIRUS 503 TABLE 5. Recovery of influenz virus from lrge volumes Totl HA ctivity Initil vol Finl vol Adsorption Totl HA c- Recovery.ution conditions (ml) (m1) Influent Ef- (%) tivity in elute (%) fluent , , vol of 2% bovine se , , rum lbumin , , NCl (1:1), , , , , Sline, , , The indicted mounts of llntoic fluid t 6 were pssed through 30S filters nd were eluted lter. Elutes were neutrlized immeditely fter elution. umes of wter nd wstewter (4; Goyl nd Gerb, in press). This method hs lso been used for the concentrtion of poliovirus nd simin rotvirus from infected cell culture fluids (2) nd for the concentrtion of coliphge hrvests (S.. Goyl, K. S. Zerd, nd C. P. Gerb, J. Virol. ethods, in press). Since the method involves mnipultion of the smple to low nd high vlues, it cnnot be pplied to the concentrtion of enveloped viruses, which re cid sensitive. According to Henderson et l. (5), cidifiction of the llntoic fluid results in the formtion of precipitte to which influenz virus dsorbs, resulting in loss of virus titer s mesured by HA ctivity. The influenz virus hemgglutinin ws shown to be cid resistnt by tretment with luminum sulfte nd by removl of precipittes formed during cidifiction. Becuse the use of positively chrged filters does not involve lrge fluctutions in (12), we decided to evlute these filters for the concentrtion of influenz virus from infectious llntoic fluid. The Zet Plus filters re unique in the sense tht they cn dsorb virus t neutrl or nerly neutrl vlues. Thus, quntittive dsorption ws chieved by pssing clrified llntoic fluid ( 6) through the 30S filters (Tble 3). Since dsorption ws 100% t 6 nd since no loss in HA ctivity ws detected t this, we decided to dsorb influenz virus to the filters t this. The dsorbed virus could be eluted from the filters by using two smll volumes of 2% bovine serum lbumin plus 1 NCl t 10 nd neutrlized subsequently with 0.05 glycine, 2. By this procedure, viruses in 100 ml of llntoic fluid were concentrted to finl volume of 8 ml, with n verge recovery efficiency of 71.0%. The verge recovery efficiency with sline t 11 ws 48% (Tble 5). It my be possible to concentrte influenz virus from lrger volumes thn those studied here, but lbortory resources limited us to 100-ml mounts. Also, the ddition of bovine serum lbumin to the finl concentrte my not be optiml for some experiments. If necessry, the finl concentrte cn be purified by density grdient centrifugtion. Since this method is convenient, inexpensive, nd time sving, it my be useful in the concentrtion of other enveloped viruses. ACKNOWLEDGENTS This study ws supported by reserch project R-805,933 from the Environmentl Protection Agency. H.H. is the recipient of Fulbright Scholrship. The technicl ssistnce of Jo Corbin nd Ann Wienmnn is grtefully cknowledged. LITERATURE CITED 1. Cox, H. R., J. vn der Scheer, S. Aiston, nd E. Bohnel The purifiction nd concentrtion of influenz virus by mens of lcohol precipittion. J. Immunol. 56: Frrh, S. R., S.. Goyl, C. P. Gerb, R. H. Conklin, C. Wllis, J. L elnick, nd H. L DuPont A simple method for concentrtion of enteroviruses nd rotviruses from cell culture hrvests using membrne filters. Intervirology 9: Frncis, T., Jr., nd J. E. Slk A simplified procedure for the concentrtion nd purifiction of influenz virus. Science 96: Gerb, C. P., S. R. Frrh, S.. Goyl, C. Wllis, nd J. L elnick Concentrtion of enteroviruses from lrge volumes of tp wter, treted sewge, nd sewter. Appl. Environ. icrobiol. 35: Henderson,., C. Wllis, nd J. L elnick Acid sensitivity of the influenz virus hemgglutinin. Appl. icrobiol. 25: Heywrd, J. T., R. A. Klims,. D. Stpp, nd J. F. Obijeski The rpid concentrtion nd purifiction of influenz virus from llntoic fluid. Arch. Virol. 55: iller, H. K., nd R. W. Schlesinger Differentition nd purifiction of influenz viruses by dsorption on luminum phosphte. J. Immunol. 75: Newton, N., nd R. E. Bevis Purifiction of niml viruses with Zn(OH)2. Virology 8: Plmer, D. F., L T. Colemn, W. R. Dowdle, nd G. C. Schild Advnced lbortory techniques for influenz dignosis. Center for Disese Control, Atlnt.

5 504 GOYAL, HANSSEN AND GERBA 10. Reimer, C. B., R. S. Bker, R.. vnfrnk, T. E. Newlin, C. B. Cline, nd N. G. Anderson Purifiction of lrge quntities of influenz virus by density grdient centrifugtion. J. Virol. 1: Slk, J. E The immunizing effect of clcium phosphte dsorbed influenz virus. Science 101: Sobsey,. D., nd B. L. Jones Concentrtion of APPL. ENVIRON. ICROBIOL. poliovirus from tp wter using positively chrged microporous filters. Appl. Environ. icrobiol. 37: Veerrghvn, N., nd T. Sreevlsn Evlution of some methods of concentrtion nd purifiction of influenz virus. Bull. W.H.O. 24: Wllis, C., nd J. L. elnick Concentrtion of enteroviruses on membrne filters. J. Virol. 1: