Contrasting effects of rabbit and human platelets on chikungunya virus infectivity

Can. J. Microbiol. 1977.23:1237-1244. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 01/09/15. For personal use only.

M. A . CHERNESKY' A N D R. P. B. LARKE* Dcprrrtr~lolty f Ln11orrrtor:v M e d i c i t ~ rSt. , Josepll's Hospittrl, trrlcl Depcrrt~tr~tlt.~ ((fPet1itrtr.ic.strticl Pntlrology. Ftrcrrlty of Meclicit~e,McMnster Cltliversity, Hn17~ilto11, Otrt., Cnntrclcr LBN I YI Accepted May 18. 1977

CHERKESKY, M. A , , and R. P. B. L A R K E 1977. . Contrasting effects of rabbit and human platelets on chikung~lnyavirus infectivity. Can. J. Microbiol. 23: 1237-1244. Chikungunya virus infectivity was markedly stabilized in the presence of washed suspensions of human platelets but rapidly disappeared in similar prepalxtions of rabbit platelets. Supernatant fluids collected from human platelets had some stabilizing effect on chikungunya virus over a 6-day incubation period at 37°C. Rabbit platelet supernatant fluid had no virus-stabilizing effect. nor did it demonstrate any capacity to inactivate V ~ ~ L aI sS compared to whole rabbit platelet preparations. Thin-section electron microscopy demonstrated that chikungunya virus formed a n association with human platelets by becoming entlxppetl in platelet aggregates: during this process some of the platelets appeared to have undergone degranulation and lysis. Rabbit platelets exposed to chikungunya virus for 24 h demonstrated a considelxble amount of platelet degranulation and lysis but virus was not visualized either in association with platelet membranes o r within phagocytic vacuoles in the platelet cytoplasm. Human platelets, which appear to be more stable under these incubation conditions. may protect chikungllnya virus infectivity from heat inactivation by surrounding viruses with large platelet aggregates whereas rabbit platelets, which appear to be more fragile, d o not afford this type of protection. Thus, chikungunyavirus in the presence of rabbit platelets may become inactivated by heat o r may become bound irreversibly to membranes in such a fashion that infectivity assay and electron microscopy techniques may prove to be too insensitive for detection of virus. CHERNESKY, M. A., et R. P. B. L A R K E 1977. . Contrasting effects of rabbit and human platelets onchikungunyavirus infectivity. Can. J. Microbiol. 23: 1237-1244. L e pouvoir infectieux du virus chikungunya est nettement stabilise en presence de suspensions lavkes de plaquettes humaines mais disparait rapidement dans des preparations similaires de plaquettes de lapin. Les surnageants recueillis d e plaquettes humaines ont un certain effet stabilisateur sur le virus chikungunya pendant une incubation de 6 jours 37°C. Par contre le surnageant recueilli de plaquettes d e lapin n'a pas d'effet stabilisateur e t semble tout a fait incapable d'inactiver le virus comparativement a des suspensions completes d e plaquettes d e lapin. L'examen au microscope electronique de couches minces revele que le virus chikungunya forme un complexe avec les plaquettes humaines en se faisant piegerdans des aggregats plaquettaires. Durant ce processus il semble que quelques-unes des plaquettes subissent une degranulation et une lyse. Lorsque des plaquettes de lapin sont mises e n contact pendant 24 h avec le virus chikungunya, i l se produit une degranulation importante et une forte lyse des plaquettes, mais le virus n'est pas visible ni e n association avec les membranes plaquettaires ni a I'interieur des vacuoles phagocytaires du cytoplasme. Les plaquettes d'origine humaine apparemment plus stables dans ces conditions d'incubation pourraient proteger le pouvoir infectieux du virus chikungunya d e I'inactivation par la chaleur en enrobant le virus dans de gros aggrkgats plaquettaires alors que les plaquettes d e lapin, plus fragiles, ne peuvent pas fournir ce type de protection. Ainsi, en presence de plaquettes d e lapin, le virus chikungunya peut &re inactive par la chaleur ou se lier de f a ~ o nirreversible aux membranes de sorte que les mesures du pouvoir infectieux oG la microscopic Clectronique s'averent d e s techniques trop peu sensibles pour detecter le virus. [Traduit par le journal]

Introduction ~ ~ r o m ~ o c y t o p eisna~ prominent a feature of many viral infections, suggesting that viruses may interact with platelets so as to cause removal of

platelets from the circulation (3, 20). Although several reports have been published on the effect of viruses on blood platelets (7, 9, 10, 16), very little attention has been paid to the effect which late lets might exert on viral infectivity. Larke i n d Wheelock have reported a stabilizing effect washed On c h i k u n g u n ~ virus a pensions of human platelets (111, and strated an association between platelets and L-

'Author t o whom reprint requests should be sent. 2Present address: Department of Pediatrics and provincial Laboratory of Public Health, T h e University of Alberta, Edmonton, Alta., Canada.

Can. J. Microbiol. 1977.23:1237-1244. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 01/09/15. For personal use only.

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virus by centrifugation of virus-platelet mixtures at forces which do not deposit virus alone. This paper extends the observations of Larke and Wheelock using electron microscopy and reports a contrasting effect of washed suspensions of rabbit and human platelets on chikungunya virus infectivity.

Materials and Methods Cell C~rltrrres A continuous line of baby hamster kidney (BHK) cells, C13 clone, was cultured in Eagle's niininial essential medium (MEM) supplemented with 4% tryptose-phosphate broth (TPB, Difco), 10% unheated fetal calf serum (FCS), sodium bicarbonate (1.75 g/!), 1% glutamine, and penicillin (100 units/ml) and streptomycin (0.2 mg/ml). All cell culture vessels were incubated in a stationary position at 37°C in an atmosphere of 5% CO, in air. Virrrs Chikungunya virus, originally isolated from mosquitoes in Africa and passaged 170 times in the brains of suckling mice, was propagated in C13 cells. A retarded release method adapted from Waite and Pfefferkorn (18, 19) was used. After 24 h of growth, 32-oz bottles containing monolayer cultures of C13 cells were washed 5 times with phosphate-buffered saline (PBS). Inoculum (1.5 ml) containing lo6 tissue culture 50% infective doses (TCD,,) of chikungunya virus was applied to the monolayer and allowed to adsorb for 30 min at room temperature, after which 60 ml of BHK cell culture medium made hypotonic with the addition of one quarter the volume of steriledistilled water plus 10% FCS were added to the bottle. Bottles were incubated at 37°C for 17-20 h, after which the hypotonic supernatant was poured off and replaced by 12 ml of isotonic BHK medium without FCS. After incubating at room temperature for 20 min the supernatant fluid was collected. A second harvest of 12 ml was collected after incubating the bottle with isotonic medium at 37°C for 3 h. Immediately upon collection of viruscontaining supernatant fluids, the pH was adjusted to 8.5-9.0 with bicarbonate and transferred to polycarbonated centrifuge tubes. The fluids were first centrifuged at 9500g for 30 min in a Becknlan L3-50 ultracentrifuge, and the resulting supernates were centrifuged at 85 000 g for 1 h. Viral pellets were resuspended in borate saline pH 9.0 (0.5 nil per tube). Resuspension at 4°C overnight was followed by 2 rnin of sonication (12 kcycles per second) in an Artek Sonic 300 dismembrator in which a sonicating cup with a water jacket was attached. Each virus suspension was pooled, then stored as 0.5-ml aliquots in tightly sealed polystyrene tubes at -70°C. Chikungunya virus seed propagated, concentrated, and partially purified by when titrated in this method contained 1011~5TCDSo/ml C13 cells. Plateler Susperzsiorrs Human blood was collected from fasting, healthy donors and platelet suspensions were prepared by the method of Mustard et a/. (14). Rabbit blood was collected by carotid artery cannulation and platelets were prepared by the method of Ardlieet 01. (1,2). Blood was taken into acid citrate dextrose (ACD) anticoagulant (1 part to 6

parts blood) by means of a siliconized blood collection set (Abbott Laboratories). All procedures for preparation of platelet suspensions and sampling procedures were carried out at room temperature or 37°C and disposable polystyrene (Falcon Plastic) tubes and pipettes were used. For determination of the platelet concentration a sample of washed platelets was diluted in suspending fluid and counted by means of a hemocytometer viewed by phase microscopy. After the addition of virus, the final platelet concentration was about 500 000/mm3. Virus-Platelet Prepo~.ations Virus-platelet and control suspensions were incubated in loosely capped polystyrene tubes maintained in a n upright position at 37°C in an atmosphere of 5% CO, in air. Before sampling for virus, the suspensions were mixed by pipetting. For determination of the distribution of virus between platelets and fluid phase of a suspension, a sample was centrifuged at 1600 g for 30 rnin at room temperature, then the supernatant fluid was removed and filtered (mean pore diameter 0.22 pm, Millipore Corp) and the platelet pellet was resuspended to the original volume of the sample. All experiments were repeated 2 or more times and were performed in duplicate o n each occasion. Assay for Cllik~rngu~~ya Virrts Itlfectiuity Monolayer cultures of C13 cells were prepared in 16 x 125 mm glass culture tubes. Samples to be assayed for viral infectivity were diluted in a serial 10-fold manner in MEM; 0.1 ml of each virus dilution was then added to 0.9 ml of growth medium in each of 5 or 10 C13 culture tubes. Cultures were examined for cytopathic effects of virus on the 4th day of incubation at 37°C and the 50% end point was calculated by the Karber method. Differences in virus titer of more than 0.5 log,, between samples were considered to be significant. Electron Microscopy Platelet preparations were fixed in suspension with 1 o r 2% OsOl (Fisher) solution in 0.15 M sodium cacodylate buffer, pH 7.3, for 1-2 h, briefly washed with saline, and dehydrated in ethanol. After these specimens were embedded in SPURR low viscosity resin (Polysciences), sections of 80 nm were cut on a Reichert or LKB ultramicrotome. Specimens were mounted on 300-mesh grids and stained with uranyl acetate and lead citrate by the method of Venable and Coggeshall (22). Grids were observed in a Philips 300 electron microscope.

Results Effects ofPlatelets on Viral Infectivity Chikungunya virus infectivity was markedly stabilized in the presence of washed suspensions of human platelets but rapidly disappeared in the presence of rabbit platelets (Fig. 1). Virus titers declined from 1 0 6 ~ 5 ~ C D 5 0to / mundetectl able levels within 2 days after incubation at 37 "C with rabbit platelets. In contrast, after 6 days of incubation with human platelets 104~3TCD5,/ml of virus still remained in the incubation mixture.

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Can. J. Microbiol. 1977.23:1237-1244. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 01/09/15. For personal use only.

been incubated for 3 days in the presence of chikungunya virus.

DAYS AT

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FIG. I . Rates of inactivation of chikungunya virus in the presence of human ( 0 ) and rabbit (0) platelets. (Solid squares and circles represent platelet-free controls.)

Virus infectivity was gradually inactivated over a 6-day period in the presence of control platelet suspending fluids. The data presented in Fig. 1 is from a typical experiment. In search for a soluble factor which might be released Into platelet-suspending medium by rabbit or human platelets, platelet suspensions were incubated for 48 h, then centrifuged at 2500 g for 30 min. The resulting platelet-free supernates of both the rabbit and human preparations were then tested for their abilities to stabilize or inactivate virus. The results of a typical experiment are summarized in Fig. 2. Supernatant fluids collected from human platelets had some stabilizing effect on chikungunya virus infectivity over a 6-day incubation period at 37"C, but this virus stabilizing effect was not as great as that provided by a suspension containing intact human platelets (as illustrated in Fig. 1). Supernatant fluid collected from rabbit platelets had no virus infectivity stabilizing effect nor did it demonstrate any capacity to inactivate virus as compared to the whole platelet preparations represented in Fig. 1. A similar lack of virus-inactivating substance was demonstrated using supernatant fluids from rabbit platelets which had been disrupted by sonication or from platelets which had

Electron Microscopy Samples of each incubation mixture indicated in Fig. 1 were fixed with OsO, in suspension then centrifuged (1300 g for 15 min) at zero time and at 18 h after adding virus, along with control platelet preparations which did not contain virus. Figure 3 shows the appearance of rabbit platelets maintained overnight at 37°C in the absence of virus. The platelets are morphologically well preserved and contain a normal complement of storage granules, mitochondria, and vacuoles. Preparations viewed at zero time in both virusinfected and uninfected platelet preparations appeared similar to those in Fig. 3. Figure 4 shows a rabbit platelet suspension following overnight incubation at 37°C with chikungunya virus. A considerable amount of platelet degranulation and lysis has taken place, but chikungunya virus was not visualized either in association with platelet membranes or within phagocytic vacuoles in the rabbit platelet cytoplasm. Disruption of these preparations by sonication did not enable infectious virus to be detected in cell culture. Figure 5 shows the appearance of human

DAYS

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FIG. 2. Rates of inactivation of chikungunya virus in the presence of human ( 0 ) and rabbit (0) (platelet-free) platelet medium. (Solid squares and circles represent controls.)

C A N . J. MICKOBIOL. VOL. 23, 1977

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FIG. 3. Rabbit platelets maintained 18 h without virus. Platelets are disc-shaped and contain a normal complement of vacuoles (V), storage granules (SG), and mitochondria (M). ( x 10 000). FIG.4. Rabbit platelets incubated 18 h with chikungunya virus. Many of the platelets are lysed and degranulated. ( x 10 000).

Can. J. Microbiol. 1977.23:1237-1244. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 01/09/15. For personal use only.

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