Thermal Inactivation of Rabies and Other Rhabdoviruses: Stabilization by the Chelating Agent

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1 INFECTION AND IMMUNITY, JUlY 1976, p Copyright 1976 American Society for Microbiology Vol. 14, No. 1 Printed in U.S.A. Thermal Inactivation of Rabies and Other Rhabdoviruses: Stabilization by the Chelating Agent Ethylenediaminetetraacetic Acid at Physiological Temperatures FRANK MICHALSKI, NANCY F. PARKS, FRANTISEK SOKOL,' AND H. FRED CLARK* The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania Received for publication 21 January 1976 Thermal inactivation of rabies and several other rhabdoviruses was studied using virus suspended in several different diluents. Rabies serogroup viruses were more stable than Kern Canyon or vesicular stomatitis viruses. Limited studies of two fish rhabdoviruses requiring low temperatures (<33 C) for replication indicated that they were not markedly more thermolabile than rabies virus. Bovine serum protein components in complex cell culture media stabilized virus at 56 C, but at temperatures of s37 C, sodium tris(hydroxymethyl)- aminomethane (NT) buffer containing ethylenediaminetetraacetic acid (EDTA) () was a much more efficient stabilizer of virus infectivity. Chelating agents EDTA and ethyleneglycol-bis-(,8-aminoethyl ether)tetraacetic acid were equally efficient in protection of rabies virus infectivity; the effect of each was lost when excess Ca2+ was added. Bovine serum in NT or buffers produced a thermostabilizing effect at 37 C not provided by the same serum concentration in complex cell culture media. Bovine serum was more efficient than EDTA in stabilizing virus infectivity during repeated cycles of freezing and thawing. We have previously described the thermoinactivation of several strains of fixed rabies virus and rabies ts mutants, propagated in cell culture and heated at 40.5 C (5, 8); other published reports on rabies virus thermoinactivation have dealt with virus propagated in mouse brain (21, 30). The analysis of thermoinactivation of vesicular stomatitis virus (VSV) has been restricted to one or a few temperatures and the influence of different suspending milieu on thermoinactivation has not been evaluated (11, 18, 19, 29). Because of the increasing importance of cell culture-propagated (concentrated or otherwise) rabies virus in vaccine production (33), we wished to determine the thermostability of such virus at several temperatures, in a variety of suspending media. As part of a continuing comparative study ofproperties of a variety of rhabdoviruses (2, 25, 28), we have also characterized the thermoinactivation of several other vertebrate rhabdoviruses. The diluents studied include solutions in standard use in our laboratory-complex cell culture media supplemented with bovine serum proteins, buffered salt solutions with or without I Deceased 25 May chelating agents, and buffered salt solutions supplemented with serum. Remarkable differences were noted in the thermostabilizing effects of protein in cell culture medium as compared with simple buffers. Differences in the effect of added cations (Mg2+ and Ca2+) on the thermostabilizing efficiency of ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol - bis(,8 - aminoethylether)tetraacetic acid (EGTA) was also documented. 135 MATERIALS AND METHODS Virus strains. Rabies virus strains ERA and Pitman-Moore (PM) (7), VSV strain Indiana, Lagos bat virus (3, 27) (Wistar clone 3) (9), Mokola virus (20, 27) (Wistar clone 1) (9), Kern Canyon virus (KCV; 23), spring viremia of carp virus (SVCV; 14), and pike fry rhabdovirus (PFV; 10) were used. All viral stocks were grown in BHK-21 cell monolayers infected at a cell multiplicity of approximately 1.0 and fed with Eagle minimal essential medium supplemented with 0.1% bovine serum albumin and twice the normal amount of sodium bicarbonate (MEM- 0.1 BSA). Mammalian rhabdovirus-infected cells were incubated at 33 C; SVCV and PFV were grown at 23 C (6). VSV was harvested after 24 h, KCV, SVCV, and PFV after 48 h, and rabies, Mokola, and Lagos viruses after 72 h. The viruses were titrated by a plaque technique in agarose-suspended BHK- 21/13S cells (26) except for SVCV and PFV, which

2 136 MICHALSKI ET AL. were titrated in BHK-21 cell monolayers (6). Rabies, Lagos, and Mokola virus plaques were read after 6 days at 35 C, KCV after 5 days at 35 C, VSV after 1 day at 35 C, and SVCV and PFV after 3 days at 23 C. Virus concentration. For released virus, the medium from infected cultures was harvested; for cellassociated virus, the cell sheet was washed three times with phosphate-buffered saline (; Dulbecco) (13) and frozen and thawed three times in MEM-0. 1 BSA. Virus preparations were clarified by low-speed centrifugation (20 min, 500 x g) and then sedimented at 36,000 x g for 1 h. The sediment, suspended overnight at 4 C in, was sonicated at 0 C at 1 A for 1 min with a Branson sonifier, again clarified by low-speed centrifugation, and then sedimented a second time. The sediment was again suspen4ed in or NT buffer [sodium tris(hydroxymethyl)aminomethane(tris)] at 4 C and then used immediately as the stock virus, concentrated approximately 100-fold. Diluents. Virus was suspended in (13), ph 7.4, MEM-0.1 BSA, ph 7.9, BHK medium (22) containing 6% tryptose phosphate broth and 2% fetal calf serum (BM-Sr2), ph 7.4, or buffer [0.13 M NaCl, 0.05 M Tris and M EDTA, ph 7.8]. Concentrated virus stocks in or NT buffer were diluted 1:100 in the test diluents prior to inactivation studies. Heat inactivation. The stock concentrated virus was diluted and held in an ice bath (no longer than 24 h) until the actual time of heating. Replicate 60- mm glass tubes (Wasserman) were filled with 1 ml ERA-BSA D I No E 0 2- z I~~~~~~~ o ~~~37~ INFECT. IMMUN. of the diluted virus, tightly stoppered, and exposed to test temperatures for variable periods of time. For harvest, the tubes were plunged into an ice bath, 1 ml of BM-Sr2 was added to stabilize the residual infectivity, and the sample was frozen at -70 C. Calculation of half-life. Virus inactivation rates were compared on the basis of half-lives of infectivity. For a given set of inactivation conditions, the log1,, of residual infectivities were plotted against time (see Fig. 1-3). The time required for 1 log1, decrease in titer was determined from the inactivation curve (the initial phase of two-phase curves was used); this time period was divided by 3.32 to give the time required for 1 log, decrease in titer (or one "half-life") (15). RESULTS Effect of temperature and diluent. A typical pattem of inactivation of the infectivity of rabies virus (strain ERA) suspended in our standard protein-containing virus growth medium (MEM-0.1 BSA) and exposed to various temperatures is shown in Fig. 1. The virus titer decreased more than 10')-fold in less than 15 min at 56 C, whereas less than a 102-fold decrease was noted during 15 days of observation at 4 C. Infectivity inactivation curves were either irregularly monophasic or biphasic, with declinations in the slope of the inactivation curve occurring after varying time intervals MINS DAYS FIG. 1. Thermal inactivation of rabies virus (ERA) suspended in MEM-O. % BSA. 4, 9, etc. represent imaginary values less than that of the point indicated. Inactivation curves were arbitrarily extended beyond the last point determined by a real value.

3 VOL. 14, 1976 THERMAL INACTIVATION OF RABIES VIRUS 137 a NT 4-0~~~~~~~~~~~~~~ 3 0 lil~~~~~~~~~ U 0~~~~~~~~~~~~~~~~~~~~~~E-S. a %~~~~~~~~~~~~~~~~~~~~~~~~~ 6 o.\ \~~~~~~~~~~~~~ME-OA 0 ~ ~ ~ ~ ~ ~~~~~~~~ 7' ' ' HOuRS FIG. 2. Thermal inactivation of (a) rabies virus (ERA) and (b) VSV at 37 C. released virus (RV) in ; 0, RV in MEM-Oi1% BSA; U, RV in ; 0, RV in BM-Sr2. The effect of the diluent upon the rate of thermal inactivation of rabies virus (released virus) and released VSV at 37 C is illustrated in Fig. 2a and b. It is clear that both viruses are protected against thermal inactivation much more efficiently by buffer (protein free) than by the other diluents, two of which contain protein additives. Cell-associated rabies virus (data not shown) was inactivated at a slightly more rapid rate than was released virus Ṫhe effect of the diluent upon the rate of thermal inactivation of these two viruses at 56 C (Fig. 3a and b) was markedly different. At this temperature each virus was protected more efficiently by protein-containing diluents than by either or buffers. After inactivation of the greatest portion of the virus infectivity within 5 to 10 min, a small fraction of thermostabile virus sometimes persisted for up to 30 min in protein-containing diluents only and in buffer in the case of VSV only. When rabies virus that had survived exposure to 56 C for 30 min in BM-Sr2 was regrown in BHK-21 cells and reheated at 56 C, rapid inactivation at a rate similar to that of the parental virus stock was obtained, indicating that the thermostable rabies virus population did not result from genetic variance. Half-life determinations indicating the relative effects of temperature and diluent upon the rates of inactivation of a variety of rhabdoviruses are listed in Table 1. The effect of the diluent on the thermostability of the vertebrate rhabdoviruses was remarkably similar. Virus suspended in diluents containing bovine serum proteins tended to best resist inactivation to heating at 56 C. At temperatures of 37 C or below, was uniformly the most protective diluent; at these temperatures, protein-containing diluents provided a lesser degree or no protection. Striking differences in the rate of thermal inactivation of the rabies (PM and ERA), rabies serogroup (Lagos and Mokola), and fish rhabdoviruses were not noted. VSV and KCV were inactivated at rates approximately twice those of the rabies serogroup viruses. A nonconcentrated preparation of rabies virus (PM) was more stable than concentrated virus regardless of diluent when heated at 56 C but not at lower temperatures. Identification of virus-thermostabilizing component of buffer. buffer, clearly

4 138 MICHALSKI ET AL. E 0- a. 2- superior to other test diluents in its thermostabilizing effect on rhabdoviruses tested at physiological temperatures, differed from other test diluents in its content of both Tris and EDTA. However, when rabies virus was heated at 37 C after suspension in Tris buffer with or without EDTA (Table 2), it became clear that the EDTA was the thermostabilizing component. If EDTA stabilizes rabies virus, it should be possible to titrate the effect in serially decreased concentrations of EDTA. The results of a series of experiments, carried out at 37 C, that demonstrate this effect are listed in Table 3. EDTA effectively protected rabies virus infectivity over a broad range of concentrations, from 0.1 to M, with an optimum efficiency observed at a concentration of 0.01 M. The protective effect of EDTA was completely lost only at a dilution to M concentration. An experiment with unconcentrated virus (experiment 3, Table 3) revealed that the protective effect of EDTA was not limited to virus possibly damaged during the manipulations included in our concentration procedure. Inasmuch as EDTA appeared to specifically protect rhabdoviruses, the assumption that such protection might be attributed to its cheaera-56' WI ~~0~,-. 3- <,4- %\@ o B"5r _ "1" a: U 5- INECT. IMMUN. lating activity for specific cations was tested in a virus-heating experiment in which Ca2+ and Mg2+ in various concentrations were added to virus preparations suspended in (0.001 M EDTA) or GTA (NT M concentration of the chelating agent EGTA) (Fig. 4). Concentrated rabies virus (ERA) in NT buffer was diluted 100-fold in NT,, GTA, or sodium citrate (SSC, 0.15 M NaCl, sodium citrate, ph 7.4) buffer. To replicate tubes of virus plus or GTA were added CaCl2, MgCI2, or equal amounts of CaCl2 and MgCl2 to give final concentrations of divalent cation that were equimolar, 10-fold less or 10- fold in excess of the concentration of the chelating agents. The virus tubes were heated at 37 C; samples were removed for virus titration at 24 and 48 h. All infectivity (104-fold reduction in titer) was lost in less than 24 h in virus suspensions in NT or SSC (data not shown) buffers. In the absence of added cations, EDTA and EGTA were equally effective in protecting virus against thermal inactivation. Furthermore, the virusprotective effect of each chelating agent was little affected by added Ca2+ or Mg2+ in amounts up to equimolar concentration. 0 o ) MINuTES FIG. 3. Thermal inactivation of (a) rabies virus (ERA) and (b) VSV at 56 C. Symbols: *, released virus (RV) in ; 0, RV in MEM-0.1 %BSA; 0, RV in ; O, RV in BM-Sr2; A, cell-associated virus (CAV) in ; A, CAV in MEM-O.1% BSA. I

5 VOL. 14, 1976 Rabies PM PM-NC Lagos Mokola VSV KCV SVCV THERMAL INACTIVATION OF RABIES VIRUS 139 TABLE 1. Effect of temperature and diluent on thermal inactivation of rhabdoviruses Half-lives of infectious titer" at: Virusb Diluent 56 C (min) 37 C (h) 33 C (h) 23 C (days) 4 C (days) Rabies ERA BM-Sr PFV BM-Sr2 NT NT < < < a A half-life = time for 1 log base2 decrease in titer during the initial phase of the thermal inactivation curve. Details of technique are given in Materials and Methods. b Concentrated released virus, except where designated otherwise. NC, Released virus not concentrated. ' Abbreviations:, (Dulbecco);, MEM-0.1% BSA; NT, Na Tris buffer;, Na Tris buffer with M EDTA; BM-Sr2, BHK medium with 2% fetal calf serum. TABLE 2. Effect of EDTA component of buffer on the inactivation of rabies virus (ERA) at 37 C Infectivity of rabies virus after heating in diluent Time of heating NT (h) Titer ph Titer (PFU/ml)a (PFU/ml) ph x x <5 x x a PFU, Plaque-forming units. (Added trace amounts of Ca2+ or Mg2+ appeared to slightly enhance the protective effect of EDTA.) Addition of a 10-fold excess of Ca2+ (or threefold excess of Ca2+ combined with a fivefold excess of Mg2+ [data not shown]) com pletely abolished the protective effect of EGTA and greatly reduced the protective effect of EDTA. Addition of excess Mg2+ reduced the protective effect of EDTA but had no effect on the efficiency of EGTA. Protective effect of serum in NT buffer. Standard proteinaceous virus diluents in use in this laboratory are formulated from complex cell culture media. As a further control on the specificity of the virus-protective effect noted with, the rate of inactivation of rabies virus was studied in NT and buffers with 2% serum added, as well as in the standard protein-containing diluent MEM (BHK) with 2 or 10% serum (Fig. 5). Surprisingly, 2% serum in NT buffer effectively stabilized rabies virus against inactivation at 37 C under conditions where 2 or 10%

6 140 MICHALSKI ET AL. INFECT. IMMUN. TABLE 3. Effect of EDTA concentration in buffer on the half-life of rabies virus (ERA)a at 37 C Expt 1 Expt 2 Expt 3 Diluentb (1:5) Half-life Diluent (1:15) Half-life Diluent (1:10) Half-life MEM (0.1% BSA) 3.3 NT 4.3 NT 2.1 NT 4.3 NT(0.1MEDTA) 21.2 NT (0.01 M EDTA) 79.5 NT (0.001 M EDTA) 17.3 NT (0.001 M EDTA) 11.5 NT (0.001 M EDTA) 15.8 NT ( M EDTA) 13.1 NT ( M EDTA) 9.6 NT ( M EDTA) 2.9 areleased virus. Concentrated virus in NT buffer was used in experiments 1 and 2. Unconcentrated virus in MEM-0.1 BSA was used in experiment 3. bthe ph of all diluents was 7.8. o GTA 20 C" 0-30 *~~~~~-mg"io.3m NT -1 % - NNT& T0Ec HOURS FIG. 4. Effect of added cations upon thermoinactivation of rabies virus suspended in media containing chelating agents (a) EDTA ( medium) or (b) EGTA (GTA medium). Chelating agents were present at 10-3M concentration; Ca2+ or Mg2+ (as chloride salts) was added at concentrations indicated on the figure prior to heating the virus at 37 C. serum added to BHK cell medium had no thermostabilizing effect. Very similar inactivation rates were observed in, NT + 2% serum, and + 2% serum. The thermostabilizing effects of EDTA and serum were apparently not additive in this system. Effect of EDTA and serum on rabies virus inactivation by freezing and thawing. Observations of thermostabilizing effects of diluents at different temperatures have suggested that different factors may account for thermal inactivation of viruses at 56 C and at lower, physiological temperatures (see below). Thus, an additional experiment was performed to determine whether NT buffer with EDTA and NT buffer with serum, equally effective in protecting rabies virus against inactivation at 37 C, HOURS could protect virus against loss of infectivity caused by freezing and thawing (Table 4). The results indicate that serum added to NT or completely protected virus against loss of infectivity after 10 cycles of freezing and thawing. The titer of virus suspended in alone decreased 5-fold after similar treatment but was a full 10-fold less than that in serumcontaining diluents because of an apparent increase in titer in such diluents. Virus in NT buffer alone declined 100-fold in titer during this treatment. DISCUSSION We have compared, by identical technique, the effect of heating, at assorted temperatures, upon the viability of rabies and several other

7 VOL. 14, 1976 rhabdoviruses propagated under similar conditions in BHK-21 cells. To our knowledge, this is the first report of a systematic study of thermoinactivation of rhabdoviruses. For each given combination of temperature and suspending diluent, it was determined that the halflives of rabies virus (strains ERA and PM) and the rabies serogroup Lagos bat and Mokola viruses were in general about twice those observed with VSV and KCV. Cdhsistent differences in thermolability among the four rabies serogroup viruses tested, which might have served as useful marker characteristics, were not observed. The fish rhabdoviruses SVCV and PFV, which show much lower optimum temperature for replication in BHK cells than do mammalian rhabdoviruses (6; H F. Clark, unpublished data), are not obviously more thermolabile than rabies virus. Biphasic inactivation curves were commonly observed with each virus when treated at 56 C in diluents other than or NT buffer. It was determined that diluents containing serum protein consistently afforded some protection to viruses heated at 56 C, whereas protein-free diluents did not. At temperatures of 37 C and below, provided a remarkable degree of protection for each virus, whereas the proteincontaining diluents based upon complex cell culture medium were totally inefficacious. These results suggest that different mechanisms of inactivation are operative at 56 C and at lower, physiological temperatures. A similar pattern detectable during heat inactivation of picornaviruses has previously been suggested based upon observation of: (i) two-component inactivation curves at high temperatures (12, 34); (ii) stabilization of infectivity by treatment with 1 M Mg2+, preferentially at high.temperatures (12); (iii) inactivation of infectivity of virus and nucleic acid at different rates at high z 0o j 22R w 5- -Sr 10 ERA-37 NT-Sr 2 ~~~~~~~~~~~ T HOURS FIG. 5. Inactivation at 37 C of rabies (ERA) virus suspended in NT buffer, with or without added EDTA and/or serum, or in BHK cell medium with added serum. THERMAL INACTIVATION OF RABIES VIRUS 141 TABLE 4. Inactivation ofrabies virus by freezing and thawinga Freeze and PFU/ml in diluent: thaw cycles NT NT-Sr2 -Sr2 5x 3.1 x l 1.1x104.2 x101.2 x 108 lox 2.5 x 10O 5.0 x x x 107 a Rabies virus (ERA) was concentrated by sedimentation and suspended in a minimum quantity of NT buffer. Concentrated virus was diluted 100-fold in test buffers and rapidly frozen and thawed 10 times, using a dry ice-ethanol bath and a 37 C water bath. Initial titer, 2.6 x 107 plaqueforming units (PFU)/ml. NT-Sr2 and -Sr2 are buffers with 2% fetal calf serum. temperature but at similar rates at lower temperature (12); and (iv) isolation of virus variants whose stability was increased at high temperature but unchanged at physiological temperatures (35). Dimmock (12) has suggested that these data are compatible with virus inactivation primarily by protein damage at the higher temperatures and by disruption of nucleic acid at physiological temperatures. Physical fragmentation of the ribonucleic acid genome of rhinoviruses inactivated by heating at 34.5 C has recently been demonstrated (17). The mechanism by which EDTA protects rhabdoviruses against heat inactivation has not been determined. The observation is in agreement with findings of others that substances with chelating properties, heparin in the case of rabies virus (30) and EDTA and bentonite in the case of a variety of viruses (1), protect infectivity. That a chelating agent might be expected to protect virus infectivity might also be expected from the reports of Wallis and Melnick that 1.0 M concentrations of Ca2+ or Mg2+ enhanced the thermolability of several types of ribonucleic acid or deoxyribonucleic acid viruses (31, 32) and from earlier reports that bacteriophage exhibited increased lability in media containing these ions in concentrations exceeding 0.15 M (4). Nevertheless, the fact that we observed protection of rhabdoviruses at EDTA concentrations as low as M suggests that we have observed a protective phenomenon based upon chelation of much lower concentrations of cations. The chelating effect may act by removing cations (i) that act as necessary cofactors for degradative enzymes, (ii) that non-enzymatically catalyze decomposition of virus components, or (iii) that poison endogenous virus enzymes necessary for replication. We do not know which ion is critically important; it is difficult to interpret the results of our experiment in which excess Mg2+ and Ca2+ were added to EDTA- or EGTA-protected virus suspensions. The efficient protective effect of EGTA, which allegedly complexes only Ca2+

8 142 MICHALSKI ET AL. and Ba2+ (24), suggests that Ca2+ may be the cnritical cation. Two contrary observations are: (i) the fact that excess Mg2+ greatly reduces the protective effect of EDTA despite the fact that Mg2+ is not capable of displacing Ca2+ from EDTA (16), and (ii) a slight protective effect of EDTA and EGTA persists in the presence of 5- to 10-fold excess concentrations of Ca2+, despite the fact that these chelating agents can complex cations only on an equimolar ratio (16). The latter observations suggest that the added Ca2+ and Mg2+ may eliminate the protective effect of the chelating agents by displacing some other cation, possibly present only in trace amounts. Serum added to NT buffer protects rabies virus at physiological temperatures under conditions where serum added to cell culture medium is totally without effect. Serum and EDTA protective effects in NT buffer are not additive. The explanation of these observations is uncertain. It is possible that serum itself exerts a minor chelating effect or that serum and EDTA may act in a similar nonspecific virus-complexing manner in simple diluents only. Final elucidation of the mechanism of the protective effect of EDTA and EGTA upon rhabdovirus will require study of the role played by other cations in the thermal inactivation of purified virus and the biochemical and morphological characterization of virions exposed to heat under chelating agent-protected and unprotected conditions. In the meantime, it is apparent that addition of EDTA or EGTA to the virus-suspending medium in minimal concentrations may be expected to very effectively stabilize the infectivity of rhabdoviruses undergoing storage for repaarch purposes or for use as live virus vaccines. Addition of serum to is indicated for preparations to be frozen. Cell culture medium is a poor milieu for the manipulation of rabies virus in the laboratory. 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