Inactivation of Myxoviruses by Calcium Elenolate

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1975, p Copyright i 1975 Americn Society for Microbiology Vol. 8, No. 2 Printed in U.S.A Inctivtion of Myxoviruses by Clcium Elenolte HAROLD E. RENIS Experimentl Biology, The Upjohn Compny, Klmzoo, Michign Received for publiction 21 April 1975 Clcium elenolte inctivtes ll myxoviruses so fr tested. The ph of the rection mixture is less criticl for myxovirus inctivtion thn tht required for coxsckie A-21 virus; the myxoviruses re inctivted t brod spectrum of ph with the mximum ctivity occurring t ph below 7.0. The infectivity of the virus is more susceptible to the ction of clcium elenolte thn is either the neurminidse ctivity or the hemgglutinin. The inctivtion of Newcstle disese virus by clcium elenolte lso destroys the bility of the virus to induce interferon formtion in cell culture nd in mice. Clcium elenolte, monoterpine isolted from olive extrcts, is virucidl gent tht inctivtes severl viruses in vitro (11), reduces the virus titer from hmsters experimentlly infected with prinfluenz-3 virus (14), nd is well tolerted in rbbits nd in mn when given intrnslly (4). Recent studies showed tht the ribonucleic cid (RNA)- dependent deoxyribonucleic cid polymerse of murine leukemi viruses is inhibited by this gent (8). The structure nd totl synthesis of clcium elenolte hve been described (9, 10). The inctivtion of myxoviruses by clcium elenolte hs been further studied. The spectrum of viruses hs been extended so tht members of most of the groups of influenz nd prinfluenz viruses hve been included, s well s the conditions ffecting the inctivtion. In ddition, the effect of clcium elenolte on properties of myxoviruses other thn infectivity hve been investigted. The results of these studies re reported herein. MATERIALS AND METHODS Viruses. Influenz-A /Ann Arbor/1/57 (HIN2) ws supplied by G. E. Underwood s lyophilized llntoic fluid stock. Influenz-A /Hong Kong-Richrdson/68 (H3N2) ws supplied by Robert B. Couch, Bylor College of Medicine. Prinfluenz-1 (Sendi) TUC, isolted from hmsters, ws supplied by M. G. Soret. All other viruses were obtined from the Americn Type Culture Collection (ATCC). On receipt, ech influenz virus ws pssed by inoculting 1:100 dilution of the stock into the llntoic cvity of 9- to 11-dy-old embryonted eggs. After 48 h t 37 C, the inoculted eggs were chilled t 4 C for 8 to 18 h nd the llntoic fluids were pooled nd stored t - 70 C. The prinfluenz-3 virus (C-243, HA-1) ws originlly obtined from the ATCC nd hs undergone numerous pssges in these lbortories in HEP-2 cells. Prinfluenz-1 (Sendi), ATCC, ws grown in embryonted eggs. Prinfluenz-1 (C-35) nd prinfluenz-2 (CA virus) stocks were diluted in Hnks blnced slt solution nd used to infect monkey kidney monolyers. The fluids were hrvested fter 48 h nd used s the stock virus. Stocks of Newcstle disese virus (NDV) nd vesiculr stomtitis virus, Indin strin, were supplied by G. E. Underwood nd hve undergone numerous pssges in chicken kidney nd chicken embryo cell cultures, respectively. Egle miniml essentil medium contining 3% fetl bovine serum nd ntibiotics (BME-3 FBS) ws used for virus propgtion. Cells. Trypsinized suspensions of rhesus monkey kidney cells were purchsed from Microbiologicl Assocites, Inc. The cells on receipt were suspended in Melnicks A medium, supplemented with 2% fetl bovine serum, 0.5% nti-simin virus 5, nd 0.5% ntisimin virus 40 ser. Sixty-millimeter plstic petri pltes (Flcon) were inoculted with 106 cells/plte nd used within 1 week fter plnting. The techniques used to prepre monolyers of other cells hve been described (11). Virus ssys. (i) Plque ssy. Prinfluenz-3 virus ws ssyed by the plque method on monolyers of HEP-2 cells s described (11). Plques usully form within 48 h. The influenz viruses (PR-8, NWS, nd FM-1) nd NDV were ssyed by the plque method on chicken kidney monolyers using previously described methods (2, 11). Plques usully form within 4 dys. (ii) Quntittive hemdsorption. The method used hs been described by others (5, 7). Drined monolyers of rhesus monkey kidney cells were infected with 0.5 ml of virus (37 C for 30 min). BME-3 FBS (4.5 ml) ws dded nd the pltes were incubted t 37 C for 48 to 96 h. Freshly prepred guine pig erythrocytes (0.5% in sline) in 1.0 ml were dded to ech plte. The pltes were incubted t 4 C for 1 h. After wshing to remove the unttched erythrocytes, the dsorbed erythrocytes were lysed in 2 ml of 0.2 N NOH nd the opticl density ws determined 94 t 420 nm. (iii) Hemgglutinin (HA) titrtion. The method

2 VOL. 8, 1975 hs been previously described (11). The results re expressed s the reciprocl of the dilution showing positive hemgglutintion. (iv) Neurminidse ctivity. The ssy ws previously described (11) except tht the hydrolyzed N- cetyl-neurminic cid ws estimted by the method of Wrren (16) nd the colored product ws extrcted into BuOH-HCl (1). Interferon production nd titrtion. (i) In vitro. Monolyers of chicken embryo or L.2. cells were inoculted with 0.5 ml of NDV. After 60 min t 37 C, the excess virus ws removed nd the monolyers were fed with 5 ml of BME-3 FBS. The monolyers were incubted t 37 C for 24 h, then frozen nd thwed. The resulting fluids were djusted to ph 2 with HCl nd incubted t 4 C for 3 dys to destroy the infectivity of the virus. The fluids were djusted to ph 7, nd seril twofold dilutions were mde in BME-3 FBS. (ii) In vivo. Mle mice (Upj: TUC [ICR] SPF) (16 to 20 g) were injected with 0.2 ml of NDV into the til vein. Ten mice were included in ech group Eight hours fter injection, the blo(,d from ech group of mice ws hrvested nd pooled. The serum ws seprted from the clots. The interferon ctivity ws determined using seril twofold dilutions of the serum smples. The interferon titer ws determined with vesiculr stomtitis virus s the indictor virus by the plque reduction method of Wgner (15) using either chicken embryo monolyers or L92. cells for the estimtion of the interferon ctivity in the chicken kidney nd mouse systems, respectively. Drug. Stock solutions of clcium elenolte were prepred t 1 mg/ml in 0.9% NCl solution. The stock solution ws further diluted in sline to give the pproprite concentrtion. The concentrtions given re those of the diluted stocks. RESULTS Our erlier studies (11) showed tht clcium elenolte ws virucidl for certin members of the myxovirus group including influenz-a (PR-8), NDV, nd prinfluenz-3 virus. Since then we hve extended the spectrum of myxoviruses. All of the myxoviruses so fr studied (Tble 1) hve been found to be highly susceptible to inctivtion by clcium elenolte. The results of severl experiments in which different myxoviruses hve been incubted with different concentrtions of clcium elenolte re shown in Tble 2. The titers given re those which remined fter 30 min of incubtion t 37 C. In ll cses the high concentrtions (1.0 nd 0.5 mg/ml) of clcium elenolte destroyed ll infectivity s mesured by these methods. Clcium elenolte t 0.25 mg/ml inctivted 3 to 4 logs of infectivity with ll viruses, nd t mg/ml the infectivity ws decresed by 1.5 to 3 logs. The dt show tht influenz-a (PR-8) (HONI) ws susceptible to clcium elenolte INACTIVATION OF MYXOVIRUSES 195 TABLE 1. Myxoviruses inctivted by clcium elenolte Strin Influenz type A/NWS (HONI) A/PR/8/34 (HONI) A/FM/1/47 (HINI) A/Ann Arbor/1/57 (HIN2) A/Hong Kong/Richrdson/68 (H3N2) Influenz-B/Lee/40 Influenz-B/Mrylnd/1/59 Prinfluenz type Prinfluenz 1 (Sendi) ATCC Prinfluenz 1 (Sendi) TUC Prinfluenz 1 (C-35, HA-2) Prinfluenz 2 (CA, Greer) Prinfluenz 3 (C-243, HA-1) Pssge b CK Technique Equl volumes of clcium elenolte (1 mg/ml) nd virus suspension were incubted t 37 C for 30 min. CK, chicken kidney;, plque-forming units;, quntittive hemdsorption;, rhesus monkey kidney. 'The llntoic fluids were diluted 1:5 in 0.9% NCl prior to dding drug. regrdless of whether the stock ws prepred in llntoic fluid or chicken embryo kidney cell culture. The inctivtion of influenz-a (PR-8), influenz-a-1 (FM-1), nd prinfluenz-3 virus by clcium elenolte were studied t different ph's (Fig. 1). The results of vrying the ph of the incubtion mixture with these viruses indictes tht inctivtion occurs more rpidly t cid ph (below 6.5) thn t ph 7.0 or higher. The rte of inctivtion of influenz-a (PR-8) incubted with 0.1 mg of clcium elenolte per ml t different tempertures is shown in Fig. 2. At 0 C very little inctivtion occurred during the 2-h observtion period; t 25 C, it ppered tht the inctivtion ws somewht more rpid initilly. However, fter 2 h t 25 C clcium elenolte reduced the titer by bout 1.5 logs. The gretest inctivtion ws observed t 37 C. As with the 25 C study, the initil rte of inctivtion of virus (up to 30 min) is quite rpid, i.e., bout 1.5 logs of the virus ws inctivted during this time. After this initil period, the rection is somewht slower, so tht n dditionl 1.5 logs of virus re inctivted during the remining 1.5 h. The dt shown in Tble 3 re from study designed to determine the effect of clcium elenolte on other properties of myxoviruses in

3 196 RENIS TABLE 2. Inctivtion of influenz viruses by different concentrtions of clcium elenolte Viutte Influenztype T Influenz type Pssge 1Clcium elenolte Virus titer ~~(mg/mi) [(F/l A/NWS (HONI) 1.0 <10' 0.5 < < x 10' x 104 A/PR/8/34 (HONI) 1.0 <10' 0.5 <10' x 10' x x 106 A/PR/8/34 (HONI) CKs 1.0 <10' 0.5 <10' x 10l x 10' x 10l A/FM/1/47 (HINI) 1.0 < <10' x x x 10l A/Hong Kong/ 1.0 <10lb Richrdson/ < 10l (H3N2) x 10' x 10' x 10' Equl volumes of virus nd clcium elenolte were incubted t 37 C for 30 min. CK, chicken kidney;, plque-forming units. b The titers given re pproximte vlues obtined using the quntittive hemdsorption method for determining the infectivity. ddition to their infectivity. This study extends tht previously described (11) in which the infectivity of influenz virus ws shown to be more susceptible to clcium elenolte thn ws either the HA titer or the neurminidse ctivity of the virus prticle. The virus titers re lower thn usully encountered becuse the virus used in this study ws grown in medium free of serum or protein. During the 30-min incubtion with clcium elenolte the virus infectivity ws destroyed t ll concentrtions of clcium elenolte greter thn 0.12 mg/ml. The HA titer of the virus ws destroyed t drug concentrtions of 3.0 nd 0.6 mg/ml; wheres t 0.12 mg/ml, the HA titer ws nerly the sme s the untreted control. The neurminidse ctivity ws bout 10% of the control fter incubtion with 3.0 mg of clcium elenolte per ml; with lesser drug, the ctivity ws preserved. In ANTIMICROB. AGENTS CHEMOTHER. those smples incubted for longer time intervls (3.5 h) with clcium elenolte, the virus titer from the mg/ml drug concentrtion ws bout 1 log less thn the 30-min smple control, but the HA titer ws bout the sme s the nondrug-treted control. Prolonged incubtion for dditionl 3 h with 0.12 mg of clcium elenolte per ml destroyed t lest 75% of the HA. The enzyme levels for ll drug concentrtions observed t 3.5 h were lower thn the ctivity observed fter 30 min, indicting tht inctivtion ws due to drug nd/or therml irtctivtion. After 30 min of incubtion with 0.6 mg of clcium elenolte per ml, the neurminidse ctivity ws bout 25% of the control; when the incubtion ws extended to 3.5 h, the ctivity ws bout 13% of the control. These dt support the originl observtion tht the infectivity of influenz-a (PR-8) is more susceptible to clcium elenolte inctivtion thn is ci Lo U. I p. M 0 -J L ph FIG. 1. Influence of different ph on the inctivtion of influenz A/PR/8/34 (HONI) (-), influenz A/FM/1/47 (HINI) (-) nd prinfluenz-3 (C-243) (A) by clcium elenolte prepred in sline t 0.1 mg/ml. The solid symbols refer to the infectivity remining fter incubtion t 37 C for 30 min in sline, wheres the open symbols refer to the infectivity fter 37 C for 30 min in the presence of drug.

4 VOL. 8, 1975 INACTIVATION OF MYXOVIRUSES o E 5 U, 0 :D4 IL. 0 2J3 2i MINUTES FIG. 2. Influence of different tempertures on the inctivtion of influenz A/PR/8/34 (HONI) by clcium elenolte prepred in sline t 0.1 mg/ml. The infectivity in the sline-treted controls is shown by the open symbols, wheres the closed symbols refer to the infectivity in the drug-treted smples. TABLE 3. Effect of different concentrtions of clcium elenolte on the infectivity, HA titer, nd neurminidse ctivity of influenz-a/pr/8/34 (HONI) virus Clcium /0.5 ml HA titer Neurminidse elenolte 1 ctivityb (mg/ml) 30 min 3.5 h 30 min 3.5 h 30 min 3.5 h 3.0 < 10 <10' <2 < <10' <101 <2 < <10' <10' x 10' 3.6 x 10' x 10' 2.6 x 10' I I i Equl volumes of virus nd clcium elenolte were incubted t 37 C for the times indicted., plque-forming units. b Neurminidse ctivity expressed t AM NANA hydrolyzed from NANA-lctse x 102. either the HA or the neurminidse ctivity. NDV hs been used extensively to study interferon induction both in cell culture nd in mice (17). The present studies were crried out to determine the effect of clcium elenolte on the bility of NDV to induce interferon in vitro nd in vivo. NDV ws incubted with different concentrtions of clcium elenolte for 30 min or 3.5 h (Tble 4). The results indicte tht, like influenz-a (PR-8), the quntity of virus inctivted is incresed by incresing the incubtion time. For exmple, incubtion of NDV in the bsence of drug for 3.5 h reduced the virus titer bout 1 log when compred to the titer fter 30 min of incubtion. However, when clcium elenolte ws present t 0.12 mg/ml, the titer decresed by 2.5 log during the sme intervl. These dt lso indicte tht prolonged incubtion with clcium elenolte, t ll concentrtions studied, decreses the HA titer, nd further tht the HA titer is dependent on the concentrtion of clcium elenolte. The interferon-inducing cpcity of the NDV-clcium elenolte smples ws tested in three systems: (i) chicken embryo where extensive virus prolifertion occurs; (ii) L.29 cells where NDV growth is limited; nd (iii) in mice. The dt show tht, in chicken embryo cells, the interferon produced is relted to the infectivity of the virus; no detectble interferon could be observed in cultures which received virus tht hd been inctivted by clcium elenolte. As the incubtion proceeded from 30 min to 3.5 h, the quntity of interferon produced decresed with decresing infectivity. Interferon production ws similrly ffected in L,,29 cultures; i.e., s the infectivity of the virus decresed so did the interferon yield. The interferon titers from mice inoculted with the non-drug-treted virus ws high wheres the interferon levels decresed with decresing infectious virus. These dt in ll cses show tht prolonged incubtion of NDV with drug decresed the bility to induce interferon. DISCUSSION The studies reported herein show tht clcium elenolte inctivtes ll myxoviruses so fr tested. The dt further show tht the myx-

5 198 RENIS ANTIMICROB. AGENTS CHEMOTHER. TABLE 4. Effect of incubting NDVwith different concentrtions of clcium elenolte on the infectivity nd the interferon-inducing cpbility Interferon' U-14, 201D /0.5 ml HA (mg/mi) Chicken embryo L,2,9 Mouse serum 30 min 3.5 h 30 min 3.5 h 30 min 3.5 h 30 min 3.5 h 30 min 3.5 h 3.0 <101 <101 <2 <2 <2 <2 <2 <2 <10 < <101 <101 4 <2 <2 <2 <2 <2 <10 < x x <2 64 <2 10 < x x 10' ,024 2, x x ,024 2,048 1,024 See Tble 3 for detils of drug preprtion nd incubtion., Plque-forming units. Interferon titers expressed s the reciprocl of the dilution giving 50% fewer plques on treted monolyers compred to the controls. For detils, see text. oviruses re bout four times more susceptible to inctivtion by clcium elenolte thn is coxsckie A-21 virus (11). The inctivtion of myxoviruses differs from coxsckie A-21 virus with regrd to the effect of ph. Coxsckie A-21 inctivtion requires tht slightly lkline ph be employed for mximum inctivtion (11). Myxovirus inctivtion is quite different; inctivtion by clcium elenolte occurs t ny ph, but mximum inctivtion occurs t slightly cid ph. Similr ph curves were obtined with prinfluenz-3, influenz-a (PR-8), nd influenz-a-1 (FM-1). These dt suggest tht the mechnism whereby clcium elenolte inctivtes picornviruses is different from tht of myxovirus inctivtion. The inctivtion of influenz virus ws shown to be dependent upon the temperture of the rection mixture. Very little inctivtion occurred t 4 C, wheres t 25 nd 37 C the inctivtion ws biphsic. The initil inctivtion t both tempertures ppered to be mximl for the first 30 min, then the rte of inctivtion declined over the next 1.5 h. The quntity of virus inctivted ws much greter t 37 thn t 25 C. It ws found tht the infectivity ws the most susceptible of the virus properties to inctivtion by clcium elenolte. HA nd neurminidse ctivities were lso inctivted, but the concentrtion of drug required ws greter thn for inctivtion of infectivity. The interferoninducing cpcity of NDV ws lso decresed by clcium elenolte inctivtion. Interferon induction in three different systems (chicken embryo, L929 cells, nd mice) followed very closely the infectivity titer of the virus. Hirschmn (8) hs shown tht clcium elenolte inctivtes the RNA-dependent deoxyribonucleic cid polymerse of murine leukemi viruses but not the deoxyribonucleic cid or RNA polymerses of E. coli B. He ws unble to show tht clcium elenolte intercted with DNA of clf thymus or E. coli or yest RNA. From these studies he concluded tht clcium elenolte inhibited the enzyme itself rther thn the nucleic cid templte. He further suggests tht the inhibitory ctivity of clcium elenolte towrd the polymerse does not depend upon non-specific binding to the protein. These dt re consistent with our previous observtions (11) which showed tht clcium elenolte did not inctivte the infectious RNA of coxsckie A-21 virus nd suggested tht clcium elenolte intercts with the protein cot of the virus nd thereby renders the virus incpble of entering the cell. The interferon-inducing cpbility of NDV in chicken embryo cells is enhnced when the infectivity is destroyed by ultrviolet irrdition; however, continued irrdition results in loss of interferon induction (17). It hs been suggested tht functionl component of the virus prticle, either the nucleic cid (3, 6, 13) or the virion-ssocited RNA polymerse (12), is essentil for interferon induction. In L cells or mice, interferon induction by NDV is not ffected by ultrviolet irrdition (17). In the present study, the quntity of interferon induced, in ll of the systems studied, ppered to be relted to the number of infectious virus prticles contined in the inoculum nd lend dditionl support to the ide tht virus inctivted by clcium elenolte is incpble of entering the cell. The effect of clcium elenolte on the virion-ssocited RNA polymerse hs not been investigted. ACKNOWLEDGMENTS I wish to thnk Brbr A. Court, Frnk T. Mid, nd Emerson E. Eidson for their excellent technicl ssistnce.

6 VOL. 8, 1975 INACTIVATION OF MYXOVIRUSES 199 LITERATURE CITED 1. Aminoff, D Methods for the quntittive estimtion of N-cetylneurminic cid nd their ppliction to hydrolystes of silomucoids. Biochem. J. 81: Bbiker, H. A., nd R. Rott Plque formtion by influenz viruses in monolyers of chick kidney cells. J. Gen. Virol. 3: Dinzni, F., S. Ggnoni, C. E. Buckler, nd S. Bron Studies of the induction of interferon system by nonreplicting Newcstle Disese Virus. Proc. Soc. Exp. Biol. Med. 133: Elliott, G. A., D. A. Buthl, nd E. N. DeYoung Preliminry sfety studies with clcium elenolte, n ntivirlgent, p Antimicrob. Agents Chemother Finter, N. B Quntittive hemdsorption, new ssy technique. II. Assy of neutrlizing ntibodies to hemdsorbing viruses. J. Immunol. 98: Gndhi, S. S., nd D. C. Burke Interferon production by myxoviruses in chick embryo cells. J. Gen. Virol. 6: Goedemns, W. T., nd A. Peters Quntittive hemdsorption. A fst nd relible screening test for nti-myxovirl gents. Arch. Gesmte Virusforsch. 23: Hirschmn, S. Z Inctivtion of DNA-polymerses of murine leukemi viruses by clcium elenolte. Nture (London) 238: Kelly, R. C., nd I. Schletter Totl synthesis of dl-methyl elenolte. J. Am. Chem. Soc. 95: McKellr, F. A., R. C. Kelly, E. E. vn Tmelen, nd C. Dorschel Structure nd stereochemistry of elenolic cid. J. Am. Chem. Soc. 95: Renis, H. E In vitro ntivirl ctivity of clcium elenolte, p Antimicrob. Agents Chemother Sheff, E. T., A. Meger, nd D. C. Burke Fctors involved in the production of interferon by inctivted Newcstle Disese Virus. J. Gen. Virol. 17: Skehel, J. J., nd D. C. Burke Interferon production by Semliki Forest Virus inctivted with hydroxylmine. J. Gen. Virol. 3: Soret, M. G Antivirl ctivity of clcium elenolte on prinfluenz infection in hmsters, p Antimicrob. Agents Chemother Wgner, R. R Biologicl studies of interferon. I. Suppression of infection with Estern equine encephlomyelitis virus. Virology 13: Wrren, L The thiobrbituric cid ssy of silic cids. J. Biol. Chem. 234: Youngner, J. S., A. W. Scott, J. V. Hllum, nd W. R. Stinebring Interferon production by inctivted Newcstle Disese Virus in tell cultures nd in mice. J. Bcteriol. 92: Downloded from on September 16, 2018 by guest