Replication of poliovirus in primate cell cultures maintained under anaerobic conditions

VIROLOGY

4, 216-223 (1957)

Replication of Poliovirus in Primate Cell Cultures Maintained under Anaerobic Conditions* GEORGE E. GIFFORD~ AND JEROME T. SYVE~~TON Department

of Bacteriology

and Zmmunology,

Cniuersit~/

of Minnesota,

Minneapolis Accepted .Jwne II,

1957

Poliovirus, Type 1 (Mahoney strain), was show to replicate in HeLa cells maintained anaerobically. While the latent period of virus development under anaerobic condit,ions compared to aerobic conditions was increased, the yield of virus per destroyed cell was similar. Infection by poliovirus did not significantly alter either oxygen consumption by HeLa cells maintained aerobically, or anaerobic a.cid production until cytopathology was evident. Absence of oxygen did not affect ahsorption of poliovirus by cells.

A dependence on aerobic respiration of host cells for synthesis of new virus has been shown for several animal cell-virus systems. Under anaerobic conditions, virus production was markedly reduced or abolished in cultures of influenza virus and minced chick embryo (Magi11 and Francis, 1936), influenza virus and chorioallantoic membrane (Ackermann, 1951), feline pneumonitis virus and yolk sac (Moulder et al., 1953), or Theiler’s GD VII mouse encephalomyelitis virus and minced mouse brain (Pearson, 1950). To date, no exceptions to these findings for animal viruses have been reported in the literature. The present study was concerned with poliovirus production by HeLa cells and monkey kidney cells infected under anaerobic conditions. MATERIALS

ANI) METHOIX3

Vi?“%3 Poliovirus, Type 1 (Mahoney strain), was employed as a pool of supernatant fluid from HeLa cell cultures of the sixty-fourth virus passage. Virus suspensions were stored in ampules at -70”. 1 Aided t)y a Grant from The National Foundation for Infantile Paralysis, Inc. 2 Present address: Department of Microbiology, Universit,y of Florida College of Medicine, Gainesville, Florida. 216

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HeLa cells were grown essentially according to the method of Syverton and Scherer (1954), in 20 % human serum (HuS-20) and 80 % Hanks’s balanced salt solution (H-80) with yeast extract (Difco “Yeastolate”) added to a final concentration of 0.1% (Robertson et al., 1955). For virus assay by the plaque technique, cells were trypsinized, dispensed, and established in a medium consisting of 20 % calf serum (CaS-20) and 80 % Hanks’s balanced salt solut,ion (H-80) containing 0.1 % yeast extract. Media in manometric st.udies consisted of 90% modified maintenance solut,ion and 10 % chicken serum (MMS-90, ChS-10) (Gifford et al., 1957) with 0.01 31 sodium hydrogen phosphite and 0.01 M tris (hydroxymethyl) aminomethane (THAN) as supplementary buffers. Monkey kidney cells were trypsinized by Bodian’s procedure (Bodian, 1956) and cultivated in 5 % calf serum in Hanks’s solut,ion supplemented with 0.5 % lsctalbumin hydrolyzate. Manometric Uethod Oxygen uptake and carbon dioxide production were measured by the direct, method in the conventional Warburg respirometer (Gifford et al., 1954). Duplicate flasks were employed for each analysis. Krebs’s modification of Pardee’s carbon dioxide buffer was employed in oxygen uptake st’udies to maintain an equilibrium concentration of 2% carbon dioxide in the gas phase. For establishment of anaerobiosis, nitrogen (passed over hot. copper to remove trace oxygen) containing 2% carbon dioxide was allowed to pass t.hrough t,he syst.em for 1 hour while the flasks were shaken. Flasks were incubated an additional hour for the cells to consume possible residual dissolved oxygen before virus was tipped carefully from the side arm into the main compartment. Accumulative oxygen consumption or carbon dioxide production was expressed as microliters. Estimation of t,he bicarbonate present at the end of each experiment was determined manometrically (Umbreit, 1945).

Initially, virus assays were done by t,he usual tube dilution procedures and most’ probable numbers of infectious units expressed as TCIU (tissue culture infectious unit,s). Later, infectious virus was estimated quantitatively as plaque-forming units (P&V) by a modification of the techniclrw of Dulhecao and Vogt (1954). Icor mass production of assay cultures, 5-ml aliquot,s of trypsinizod HeLa cell suspension containing

218

GIFFORD

AND

SYVERTON

200,000 cells/ml in CaS-20, H-80 with 0.1% yeast extract, were dispensed into 30 X GO-mm screw-capped, square, soft-glass bottles.3 These bottles usually contained a complete monolayer of cells by the following day. Medium was decanted and 0.5 ml of each virus dilution added to each of two bottle cultures. Bottles were capped and tipped gently from side to side and end to end so as to spread the virus inoculum over all of the monolayer. During incubat,ion at 37” for 1.5 hours, the bottles were tipped every 30 minutes to redistribute the fluid over the cell monolayer. Agar-medium overlay, 4.0 ml, was added to each bottle and mixed with the virus inoculum by gentle tipping five to six times. The overlay was prepared by adding 1 part molten (55”) 3 % Difco Special Agar (“Noble”) in distilled water to 3 parts of medium; the medium, heated in a water bath to 55”, was ChS-20, MMS-80 with 0.1% yeast extract and 0.13% sodium bicarbonate. Bottles were incubated for 18 to 72 hours or until development of plaques. Agar-st,ain overlay, 3.0 ml (2 % agar in distilled wat.er with 0.01 % ireutral red), was added to each bottle to stain the cells. The st,iffer agar prevented the init’ial overlay from sliding when bottles were inverted for visual or microscopic examination of plaques. Bottles were incubated for an additional hour to allow penetration of the neutral red to the cell monolayer and were stored at 4’ for 12 to 24 hours to allow intensification of the staining reaction. Virus titers were expressed as PFU per milliliter. RESULTS

HeLa cells, approximately 200,000 in 3.0 ml THAM-phosphite buffered MMS-90, ChS-10, were dispensed intoWarburg flasks. The flasks were attached to manometers, placed in the bath at 35” and gassed for 1 hour with either air or nitrogen (treated to remove trace oxygen) containing 2 % carbon dioxide. After an additional hour for equilibration, approximately 40 TCIU of poliovirus were added to each flask from the side arm. Incubation was continued for 50 hours: after this time supernatant fluid from anaerobic cultures showed a titer of 105.7 TCIIJ /ml and that from aerobic cultures, a titer of 107.0 TCIU/ml. The reason(s) for the decreased titer of virus under anaerobic conditions was investigated. The manometric assay syst,em was exploited for analysis of aerobic and anaerobic virus production, in the following manner. The oxygen consumption of maint,aining Hel,a cells was known 3 Twin

Cit.y Bot,tle Co., Minneapolis,

Minnesota.

Chtalog

No. 271

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to be co&ant wit.h time and directly proportional t,o live cell number (Gifford et al., 1954, 1957). To HeLa cell cultures in Warburg flasks, sufficient virus was added at. zero time for one cycle of virus replicaCon t.o destroy a significant proportion of t,he cells. A change in rate of oxygen uptake or in COr released revealed: (a) the time of cell destruction, and hence the approximate lengt,h of the latent period; a.nd (b) the proport,ion of cells dest,royed with each cycle of virus replication, by comparison of the altered course of oxygen uptake or COr production by infected cell cultures with t,he unahered course of uninfect,ed control cultures. The angle of divergence was proportional to t,he fraction of cell population destroyed. Figure I shows the results of a representative experiment. Warhurg flasks containing approximat,ely 900,000 HeLa cells in 3.0 ml of THA3Cphosphit.e buffered MMS-90, ChS-IO were gassed for 1 hour with eit,her air or t,reated nitrogen containing 2 % carbon dioxide, Nasks were allowed to equilibrate for an additional hour before virus was Cpped from the side arm into the reaction vessel with cells. Sufficient virus was CO2 Production l

Control

0

Infected

220

GIFFOHD

AND

SYVEllTON

added to destroy a significant proportion of the cells after one cycle of replication. Carbon dioxide production or oxygen consumpt.ion was measured at intervals of 30 minutes. Manometric estimation (Vmbreit, 1945) of the bicarbonate remaining in culture medium at the completion of the experiment indicated that the 225 ,ul of carbon dioxide produced by the anaerobic uninfected culture apparently were released from bicarbonate due to acid production. The linear accumulation of produced carbon dioxide, shown in Pg. 1 indicates anaerobic maiutenance of the HeLa cells. It is also apparent that prior to initiat,ion of cell destruction the oxygen uptake of the infect.ed aerobic cultures, a.ild t.he acid production of the infected anaerobic cultures, were not affect,ed. The time until destruction of infected cells incubated aerobically was approximately 7.5 hours, compared to approximately 11 hours until destruction of cells incubated anaerobically. This delay in cytopathogeuicity under anaerobic conditions explains, at least in part., the decreased yield of poliovirus. Absence of oxygen had no great effect on absorption of poliovirus by HeLa cells, because equal angels of initial divergence as previously defined indicate that equal fractions of the cell populat.ions were destroyed following the primary cycle of virus replicatioii in aerobic and anaerobic cultures, and hence that about. t.he same number of cells in each case were initially infected. Nxamination of the angles of divergence between oxygen-consumption or carbon dioxide production curves for control and infected cultures indicates t,hatj in t,he anaerobic infected culture only a single cycle of cell destruct.ion orcurred, while in the aerobic infected culture a second cyrle of cell destruct)ion followed the first. It can be inferred that simiIa,r cycles of virus replication are reprcsented. The effect of anaerobiosis on poliovirus replication by HeLa cells was investigated further by analysis of virus yield per destroyed cell. The number of destroyed cells was estimat,ed from manometric data and virus yield by plaque count. Table 1 summarizes t,hree separat.e experiments. The minimum total yield of poliovirus in Hella cultures maintained aerobically in each experiment, exceeded that, obtained anaerobically. However, when the PPIr obtained under either condit’ion were divided by the manometrically estimated number of destroyed cells, t,he resultant minimum yield of Plcti per dcst,royed c&e11 was similar. It was concluded that. HeLa ~~~11s maintained wider anaerobic conditions produced a.s much virus per infected cell as did <
REPLICATION

OF

221

POLIOVIRUS

A1Wlge minimum yield/cell destroyed - .-. 1.

K .‘C’

1.2i

0.75

0.59

li”

192

xj, AiP

1.27 1.27

0.75 0.75

0.50 0.5!)

18 31

126 309

Air

0.75

0.65

35

312

NT”

1.16 .__0.81

1.08

1.33

34c

5-t

N2 Air*

0.81 O.Sl

1.08 1.08

1.33 1.33

28 100

53 2%

Air

O.Sl

I.08

2.00

100

302

Np

1.35

7.50

5.55

16”

40

&? AiP

1.35 1.35

i.50 7.50

5.55 5.55

27 100

67 314

N2

1.13

7.50

6.6-I

100

250

721)

740 2.

___215

373 .__ 3.

--.

234

2%

(’ With ZOJOcarlxm diosidc. L 20 hours post-infection. C 18.5 hours post-infection C117 hours post-infection.

port,ion of cells was infected initially in each case, virus replication was suficient,ly faster under aerobic condit,ions for infection and destruction of a secord lot of cells. Therefore, a higher net yield of virus was obtained in aerobic cultures. The fact t,hat virus production per unit t.ime in aerobic cultures eseeeded anaerobic production during each int,erval of the tota. period of incubat,ion as shown for monkey kidney cells in Table 2 does not. conflict, wit,h t.he above interpretat.ion, since animal cells do not, release \:irus abruptly (Dulbecco and Vogt, 19.54; I,wofY et al., I 9%). Replication of polio\+rus by monkey kidney cells under anaerobic conditions (Table 2) provided cont?rmat,ory evidence of anaerobic poliovirus replication.

222

GIFFORD

AND

SYVERTON

TABLE REPLICATION

OF

POLIOVIRUS

IN

ANAEROBIC

AND

2

MOSI
AEROBIC

KIDNEY

Total Expehny

t

Gas phase”

N2

-_ 2.

virus

yield

106 PFU __-__

HOWS

Is2 x2 N2 Air Air Air Air -__.-

UNDER

Vir~;inpoEF;;lulum 8.5

1.

CELLS

CONDITIONS

__--

x2 N2 N!Z Air Air Air Air

1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6

0.8 0.6 2.2 0.6 -

2.0 2.0 2.0 2.0 2.0 2.0 2.0

-

-

12

22

26

-

-

-

-

___ 1.1 1.6 4.2 3.3 -

7.7 2.8 23.7 36.8 -~ .~~ -

-12.0 14.0 90.0 117.0

u N2 and sir b0t.h with a(?0Oz. DISCUSSION

Poliovirus, Type 1 (Mahoney strain) was shown to replicate in HeLa and monkey kidney cells that were maintained anaerobically. The latent period of virus development under anaerobic conditions compared with that under anaerobic conditions was increased, but the yield of poliovirus per destroyed cell was essentially uualtered. These results contradict other published findings with animal viruses and the generally held view that synthesis of virus by animal cells depends on oxidativc metabolism. It is possible that poliovirus may be an exception. Alternatively, since product,ion of virus is apparemly an inherently cellular process, induced but not carried out by original virus, ability or inability to produce virus anaerobically may reflect a more general property of cells. It is hypothesized that mammalian cells can, like bacterial cells, be classified as obligate aerobes or facultative anaerobes. Cells like those of the chorioallantoic membrane of the developing chick embryo would be classified as obligate aerobes. This tentat.ive description is substantiated by recent findings of Tamm (1956) who noted macroscopic damage

REPLICATION

OF

POLIOVIRUS

223

of chorioallant,oic membranes under anaerobic conditions. Inability t,o synthesize virus anaerobically, then, is not surprising. HeLa cells, or monkey kidney cells for which similar resuhs have been found, appear to be facult,ative because anaerobically t,hey cont,inue to maintain and met’abolize. Since they remain active, they can synthesize virus. A longer Iat,ent period for anaerobic \rirus development can be explained by the usual greater efficiency of farultati\re cells under aerobic condit,ions.

ACKERX\IANS,W. W. (1951). Concerning the relation of the Krehs c,ycle to virus propagation. J. Riol. Chem. 189, 421-428. Boorah-, I>. (1956). Simplified met,hod of dispersion of m0nke.v kidney cells with t rylwin. C-iroloq1(2, 575-576. I)I.LBE(:(Y), I~., and VOGT, RI. (19543). Plaque formation and isolation of pure lines with poliomyelitis viruses. .1. Ezptl. Med. 99, 167-182. I)r~~,sscco, R., and VU(:T, &I. (1954h). One-step growth curve of Western equine cllcephalom?lelit.is virus on chicken etnbryo cells grown in vitro and analysis of virus yields from single cells. J. Eqtl. iTfed. 99, 183-199. (~IFFORD, 6. I?., ROBERTSON. H. E., and SYVERTON, ,J. T. (1954). Application of manomet.ric method to testing chemical agents in vitro for interference with poliomyelitis virus synthesis. Proc. Sot. E.&l. Biol. Xed. 86, 515-522. GIFFORD, G. E., ROBERTSON, H. E., and SYVERTON, J. T. (1957). Metabolism of HeI,:t cells: Methodology and media ev:tluation. J. Cellular rind Comp. Physiol. In press. I,WOFV, h., ~)r~r.sscco, It., VOGT, hf., and Lnos~, M. (1955). Kinetics of the reIe:isc of poliomyelitis virus from single cells. Virology 1, 128-139. MAGILI., T. I’., end FRANUS, T., JR. (1936). Studies with human influenza virus cultivated in nrt.ifici;tl medium. J. Exptl. Mad. 63, 893-811. MOI~IAER, J. W., MCCORMWK, 13. R. S., and ITATANI, M. K. (1953). Energy requirements for synt,hesis of feline pneumonitis virus in isolated chick embryo yolk s&c. J. I~jbclio~s Diseases 93, 140-149. I'EARSOK, H. 1<. (1950). Factors affecting t,he propagation of Theiler’s GDVII mouse encel~h:~lom~elitis virus in tissue cultures. J. Iwta~nol. 64, 447-154. ROBERTWS, H. I<., L)RUNXER, Ii. T., and SYVERTON, J. T. (1955). Propagation if& ??lro of poliom),elit is viruses. VII. pH change of HeI,a cell cult~ures for assay. Pror,. Sot. Erptl. Rio/. Med. 88, 119-122. SYVERTOS, J. T., :~rlct SCHERER, W. F. (195-t). The application of mammalian cells in continuorw crdtwe for :~ss,~ysin virology. ~lnn. N. Y. ilcnd. Sci. 58, 1056-1071. T~wr, I. (1956). Select,ive chemical inhibition of influenza B virus multiplicat.ion. J. Bncfwiol. 72, 12-53. UMBREIT, W. W. (1!?15). The use of bicarbonate buffers for measuring acid prodllct.ion nntlcr anaerobic conditions. In :Unnotnet& ‘Techniques and Tissue Uc,tcboli.st~, Second I