Effect of Sucrose Phosphate and Sorbitol on Infectivity of Enveloped Viruses During Storage

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1983, P. 658-662 Vol. 18, No. 3 0095-1137/83/090658-05$02.00/0 Copyright 1983, American Society for Microbiology Effect of Sucrose Phosphate and Sorbitol on Infectivity of Enveloped Viruses During CLIFFORD L. HOWELL' 2* AND MARJORIE J. MILLER2 School of Public Health' and Department ofpathology,2 University of California, Los Angeles, Medical Center, Los Angeles, California 90024 Received 18 April 1983/Accepted 10 June 1983 Cytomegalovirus and varicella-zoster virus recovery from sucrose phosphate (0.2 M SP) and 70% sorbitol (sorbitol) was compared after storage at -70, 4, and 20 C over time. Recovery from 0.2 M SP was uniformly better. More tissue culture infective doses and infectious foci were recovered in cell monolayers inoculated with 0.2 M SP virus stocks as compared with viruses stored in 70% sorbitol. Although both viruses were isolated from diluted fresh stocks (10-1 through 10-4), freezing diluted virus suspensions generally resulted in diminished recovery. Similar stabilizing effects on respiratory syncytial virus and herpes simplex virus type 1 infectivities were observed when stocks were preserved in 0.2 M SP, as compared with 70% sorbitol. Overall, 0.2 M SP was better than 70% sorbitol for stabilizing cytomegalovirus, varicella-zoster virus, respiratory syncytial virus, and herpes simplex virus type 1 infectivities under the conditions tested. The infectivities of many viruses decrease during transport and storage, resulting in isolation failures from clinical specimens and diminished titers in virus stocks. This phenomenon occurs more rapidly at 20 and 4 C than at ultralow temperatures. Lyophilization and freezing at low temperatures in protective substances have been the principal means of preserving most viruses (7, 10, 13). Since the former requires equipment not commonly found in most clinical laboratories and since some viruses are damaged by lyophilization, most diagnostic virologists find combining infected cell cultures with a cryoprotectant and freezing at -70 C or lower to be more convenient. Glycerol (14), dimethyl sulfoxide (33), albumin (6), balanced salt solutions plus gelatin (31), animal sera (14, 21, 23, 28), skim milk (31), sucrose (15, 17, 22), and sorbitol (25, 27, 28, 35) are among the more commonly used cryoprotectants. Unfortunately, none of these has proven to be uniformly satisfactory for all virus groups. Among the more frequently isolated viruses are those of the herpesvirus group, of which cytomegalovirus (CMV) and varicella-zoster virus (VZV) are the most labile. In many laboratories, the viruses are combined with sorbitol and stored at -70 C (4, 25, 28, 32, 35). Even so, CMV and VZV recovery is inconsistent, and sorbitol may be toxic for some cell cultures (19, 25). Grose et al. (11) have demonstrated enhanced recovery of VZV from cell-associated and cell-free virus suspensions lyophilized in buffered sucrose solutions. This correlated with the ability of sucrose to stabilize infectivity by preventing conformational changes in the viral envelope architecture. We sought to extend this observation by applying it to the storage of CMV and VZV stocks in clinical virology laboratories. Sorbitol and 0.2 M sucrose phosphate (0.2 M SP) were evaluated for suitability as both storage and transport media. CMV and VZV recovery and titer changes were compared after storage in these two media at different temperatures and over time. Herpes simplex virus type 1 (HSV1) and respiratory syncytial virus (RSV) were examined in a similar, but more limited manner. MATERIALS AND METHODS Cell cultures and media. CMV and VZV were propagated in human foreskin fibroblasts (Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.) and human embryonic lung fibroblasts, WI-38, (Flow Laboratories, Rockville, Md.), respectively, maintained on Eagle minimal essential medium supplemented with 5% fetal bovine serum, 7.5% NaHCO, and (per milliliter): 80 U penicillin, 80,ug of streptomycin, 0.04 mg of kanamycin, and 2 U of mycostatin. Virus stocks. CMV and VZV were isolated from clinical specimens and used at passage levels 2 and 3, respectively. After removing and saving infected cell culture fluids, monolayers exhibiting 75 to 100%o cytopathic effect were dispersed with a 0.05% trypsin- 0.02% EDTA solution (GIBCO Laboratories, Grand Island, N.Y.) (25, 34). Dispersed cells and culture fluids were combined, mixed with equal volumes of storage medium, dispensed into sterile screw-capped 2-ml vials (Wheaton Scientific, Millville, N.J.) and stored at 20, 4, and -70 C. RSV and HSV1 were isolated from clinical specimens and used at the second passage level. Both were propagated in WI-38, harvested at 3 to 4+ cytopathic effect, and stored in 658

VOL. 18, 1983 TABLE 1. VIRUS PRESERVATION 659 CMV recovery from 0.2 M SP and sorbitol after storage at -70 C Virus titers Log10 infectious foci/ml in: Log10 TCID.5ml in: (wks) 0.2 M SP Sorbitol 0.2 M M SP Sorbitol sorbitol ratio sorbitol ratio 0.2MrPbSrbtoo0.MaSP: 1 4.0 (100)a 3.2 (18) 6:1 4.5 (100) 3.9 (41.6) 2.4:1 2 4.5 (100) 3.4 (18) 6:1 5.2 (100) 4.7 (41.6) 2.4:1 6 4.2 (100) 2.5 (2.0) 50:1 5.2 (100) 4.3 (16.6) 8:1 8 3.2 (45) 2.6 (4.0) 11:1 3.3 (11) 3.0 (5.5) 2:1 a Numbers in parentheses represent percent recovery of control. the same manner as with CMV and VZV. media. Sorbitol was prepared and used as previously described (27, 35). Phosphate-buffered sucrose solution, 2x, was prepared by dissolving 149.24 g of sucrose, 2.436 g of K2HPO4 and 1.04 g of KH2PO4 in 1 liter of distilled water (3). All solutions were sterilized by filtration (Nalge Co., Rochester, N.Y.). Infectivity assays. Control and stock virus titrations were performed at designated intervals. Serial 10-fold dilutions were prepared in Hanks balanced salt solution, inoculated in triplicate onto WI-38 or human foreskin fibroblast monolayers, incubated at 35 C in stationary racks, and examined daily. Virus titers were expressed as 50% tissue culture infective doses (TCID50) (24) and as the number of infectious foci per milliliter. Although TCID50 has been commonly used to express virus concentration, it only indicates that 50% of inoculated cell cultures are positive; whereas, the quantitation of foci reflects the number of infectious virus particles in a given volume and permits positive cultures to be compared with regard to the extent of infection. RESULTS The effect of storage at -70 C in 0.2 M SP and sorbitol on CMV infectivity is shown in Table 1. Although the titer did not decrease after 6 weeks storage in the former, there was a 98% loss of CMV infectivity in sorbitol over the same period. By 56 days, almost one-half of the original CMV titer was recovered from 0.2 M SP, as compared with only 4% from sorbitol. Overall, 6 to 50 times more TCID50s and 2.5 to 8 times more infectious foci were observed in monolayers inoculated with stocks stored in 0.2 M SP. VZV titers decreased rapidly when stored in 0.2 M SP and sorbitol at -70 C (Table 2). Even so, preservation was better in 0.2 M SP. After 7 days, 18 times more TCID50s and 12 times the number of infectious foci were observed in monolayers inoculated with virus stocks stored in 0.2 M SP as compared with sorbitol. By the end of the observation period, VZV was recovered only from 0.2 M SP. Although CMV and VZV were readily recovered from diluted fresh stocks, (10-1 to 10-4), freezing diluted virus suspensions resulted in decreased recovery (Table 3). After storing CMV and VZV in 0.2 M SP and sorbitol at -70 C for 7 weeks, recovery was achieved only from the more concentrated stocks. CMV was isolated from 10-1 and 10-2 0.2 M SP stocks but only from the 10-1 sorbitol stock. VZV was recovered from the 10-1 0.2 M SP stock, but not from sorbitol. CMV and VZV titers decreased more rapidly at 20 and 4 C than at -70 C. More than 75% of CMV infectivity was lost after 24 h in sorbitol at 4 C and only 4% of the original titer remained after 3 days (Table 4). In contrast, CMV titers did not decrease after 24 h in 0.2 M SP at 4 C and almost 70% survival was observed at 72 h. Although CMV could not be isolated from sorbitol after 7 days, almost 50% of the original titer was recovered from 0.2 M SP. Furthermore, CMV could still be isolated from 0.2 M SP after 21 days of storage at 4 C. TABLE 2. VZV recovery from 0.2 M SP and sorbitol after storage at -700C Virus titers Log10 TCID50ml in: Log10 infectious foci/ml in: (wks) 0.2 M SP Sorbitol 0.2 M SP: 0.2 M SP Sorbitol 0.2 M SP: sorbitol ratio sorbitol ratio 1 3.2 (5.6)a 2.0 (0.3) 18:1 3.1 (10) 2.0 (0.8) 12:1 4 2.6(2.5) 1.8 (0.4) 6:1 2.8 (9.2) 1.9 (1.2) 8:1 6 2.8 (4.0) 2.0 (0.6) 6:1 2.8 (8.3) 1.9 (1.2) 7:1 7 2.6 (1.3) NVIb 2.2 (1.3) NVI a Numbers in parentheses represent percent recovery of control. b NVI, No virus isolated.

660 HOWELL AND MILLER TABLE 3. CMV and VZV recovery from diluted virus stocks, fresh and frozen, in 0.2 M SP or sorbitol and stored at -70 C Recovery of virusesa Stock CMV vzv virus dilution Frozen in: Frozen in: Fresh Fresh 0.2 M SP Sorbitol 0.2 M SP Sorbitol 10-1 + + + + + 10-2 + + 10-3 + 10-4 i+ -, v + 10-5 -I - I- +, Virus isolated; -,no virus isolated. When stored at 20 C for 24 h, more than half the original CMV titer was recovered from 0.2 M SP compared to 7% from sorbitol. A total of 15 and 10 times more infectious virus was isolated from 0.2 M SP stocks at 48 and 72 h, respectively, as compared with sorbitol. By 7 days, CMV was recovered only from 0.2 M SP. VZV titers were also more stable at 4 C than at 20 C (Table 5). When stored at 4 C for 24 and 48 h, 40% of the original VZV infectivity was retained in 0.2 M SP, whereas only 4% was recovered from sorbitol. After 7 days at 4 C, VZV was isolated only from 0.2 M SP. VZV could not be isolated from any sorbitol stocks held at 20 C, but could be recovered from 0.2 M SP stocks up to 72 h. Similar stabilizing effects were observed when RSV and HSV1 were stored in 0.2 M SP and sorbitol for 10 weeks at -70 C. Although there was no RSV or HSV1 titer loss when stored in 0.2 M SP, 82 and 94% decreases in infectivity, respectively, occurred in sorbitol. DISCUSSION Included among the many variables affecting the recovery of viruses from frozen storage are: virus type and concentration; use of cryoprotectant; rates of freezing and thawing; volume, size and type of storage container; temperature; ph; and osmolality. These factors interact interdependently to influence viral infectivity, which is best preserved when the environment in which viruses are stored is carefully controlled (7, 8, 18, 20). Enveloped viruses demonstrate greater lability during storage than those lacking envelopes (11, 33). Since viruses acquire their envelopes by budding through cellular membranes, the dynamics associated with deleterious changes which occur during freezing and thawing may affect both viruses and cells in a similar manner. The mechanisms of membrane and viral envelope damage appear to be caused by the forma- J. CLIN. MICROBIOL. TABLE 4. CMV recovery from 0.2 M SP and sorbitol after storage at 4 and 20 C CMV titersa time 0.2 M SP Sorbitol (days) 4 C 200C 40C 200C 1 3.4 (100) 3.1 (55.9) 2.7 (23.3) 2.2 (7.0) 2 3.2 (69.8) 2.9 (34.9) 2.1 (5.8) 1.7 (2.3) 3 3.2(69.8) 2.8(29.1) 1.9(3.7) 1.7(2.3) 7 3.0 (46.5) 2.3 (9.3) NVIb NVI 21 1.9 (3.5) NDC NVI NVI a Titers are expressed as log1o TCID50s. Numbers in parentheses represent percent recovery of control. b NVI, No virus isolated. c ND, Not done. tion of external and internal ice, excessive changes in ph and solute concentration, and ice recrystallization, during freezing and thawing. In fact, decreases in viral titer correlate with demonstrable changes in areas of the viral envelope presumed to be associated with viral infectivity (5, 11, 29). Cryoprotectants modify the physical and chemical characteristics of the external and internal environment of viruses so as to favor survival. Cooling small volumes of concentrated virus suspension and cryoprotectant, to ultralow temperatures in a stepwise fashion, promotes slow diffusion of water from the virion. This results in gradual shrinkage of the viral envelope, thereby reducing envelope damage and concomitant loss of infectivity. Cryoprotectants vary with respect to the viruses which they protect, toxicity for cell monolayers, concentration required, and ability to stabilize cell-free and cell-associated virus. Originally, 70% sorbitol (35) was used to enhance CMV recovery from frozen stocks and was subsequently applied to the preservation of virus in clinical specimens (9, 19). Further attempts to improve CMV recovery included freezing separate cell-free and cell-associated virus suspensions, usually with sorbitol (25, 32) and dimethyl sulfoxide (25), respectively. Although sorbitol has been the recommended cryoprotectant for TABLE 5. VZV recovery from 0.2 M SP and sorbitol after storage at 4 and 20 C VZV titersa time 0.2 M SP Sorbitol (days) 4 C 200C 4 C 200C 1 2.8 (40.0) 1.8 (4.0) 1.8 (4.0) NVIb 2 2.8 (40.0) 1.3 (1.3) 1.8 (4.0) NVI 3 2.3 (12.6) 1.8 (4.0) 1.8 (4.0) NVI 7 0.5 (0.2) NVI NVI NVI a Titers are expressed as log1o TCID50s. Numbers in parentheses represent percent recovery of control. b NVI, No virus isolated.

VOL. 18, 1983 CMV, the results of this study indicate 0.2 M SP to be substantially better at stabilizing CMV infectivity. Cell monolayers inoculated with 0.2 M SP stocks showed higher titers and produced more infectious foci than did those inoculated with sorbitol-preserved stocks. Limited stabilization of infectivity was observed when clinical specimens and infected cell cultures containing VZV were stored in skim milk and glucose (36). Although Rosanoff recovered VZV from stocks stored for 18 months at -70 C in glycerol and dimethyl sulfoxide, up to 99% of the original titer was lost (26). Increased stability was achieved by freezing cell-free virus in sorbitol (4, 28) and 10% glycerol (4) or cellassociated virus in 10% DMSO (28). Hondo et al. (15) and Grose et al. (11) achieved even better recovery of VZV from suspensions frozen and lyophilized in sucrose-containing media. Similarly, we observed improved recovery of cellassociated VZV from stocks stored in 0.2 M SP, as compared with sorbitol. Glycerol (14), skim milk (31), and sucrose (22) have been used to preserve crude and purified suspensions of HSV. Even though purified cellfree HSV was preserved for 1 year at -70 C employing only rapid freezing and thawing, use of a cryoprotectant such as glycerol improved virus survival after repeated freeze-thaw cycles (2). Although RSV is regarded as highly unstable and difficult to recover from frozen stocks and clinical specimens, Hambling demonstrated enhanced recovery of viruses from medium buffered at ph 7.5 and stored at -65 C (12). Sucrose solutions have also been used to maintain various concentrations of RSV over different temperatures and have proven suitable as transport media for clinical specimens (17). Although we observed stabilization of RSV and HSV1 in both 0.2 M SP and sorbitol, significantly more virus recovery was demonstrated in virus preparations preserved in 0.2 M SP. Often clinical specimens contain low and unpredictable levels of virus, depending upon when they are collected during the course of a disease and how they are handled before processing in the laboratory. Since it is common practice in many hospitals to hold specimens at ambient temperatures for various lengths of time, virus levels may decrease rapidly if specimens are not inoculated into cell culture soon after collection or placed in a protective environment. It is important to select a suitable transport-holding medium which will prevent excessive losses in viral infectivity, since even small to moderate titer losses can result in negative cultures. Although sorbitol has been recommended for the transport and holding of CMV and other enveloped viruses, titers may decrease rapidly. Indeed, we observed that a great VIRUS PRESERVATION 661 proportion of CMV and VZV infectivity was lost when stored in sorbitol, even for short periods, and that recovery of both viruses was significantly better from 0.2 M SP. Overall, 0.2 M SP was a better cryoprotectant and transport-holding medium for CMV and VZV than sorbitol and was effective for stabilizing the infectivity of RSV and HSV. We recommend its use in place of sorbitol in diagnostic virology laboratories. The utility of this medium is extended, since similar preparations have proven to be effective for stabilizing the infectivity of rickettsiae (3), chlamydiae (1, 16, 30), mycoplasma (30), and other viruses. The use of a single medium for both storage and transport of many different infectious agents saves time and expense and reduces additional manipulations of specimens in the laboratory. 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