Location of Cell-associated Virus

In document The growth cycle of poliovirus in cultured cells (Page 31-33)

EXPERIMENTAL RESULTS

III. Location of Cell-associated Virus

In an attempt to determine the location of cell-associated virus, in­ fected cells were treated with hyperimmune specific antiserum at dif­ ferent times during the cycle.

Cell monolayers were washed to remove free virus, and were treated

at 37° for 10 minutes with excess antibody. The antibody was then removed by washing several times with PBS. To exclude possible gen­ eral or local depletion of antibody, both the proportion of cells infected and the antiserum concentration were varied. Virus yields of mono- layers treated with antiserum were compared with monolayers sub­ jected to the same procedures, but with PBS substituted for antiserum.

G RO W TH CYCLE OF PO L IO V IR U S IN C U L T U R E D C E L L S. I I 105 T A B L E 3 In a c c e s s ib il it y o f Ce l l-a ss o c ia t e d Vir u s to Sp e c if ic An t ib o d y. Ex p e r im e n t s w it h Mo n o l a y e r Cu l t u r e s“ Tim e T re a tm e n t P laq u e co u n ts, cell-associated v iru s from d u p li­

c a te c u ltu re s T o ta l count 7.6 H ours P B S (C ontrol) 53, 60 113 A ntiserum 1:4 48, 44 92 A n tiseru m 1:20 52, 62 114 A ntiserum 1:100 39, 50 89 9.5 H ours P B S (C ontrol) 75, 61 136 A ntiserum 1:4 49, 66 115 A ntiserum 1:20 78, 88 166 A ntiserum 1:100 60, 76 136

“ A n tiseru m c o n tro l: S to ck v iru s m ixed w ith an equal volum e of 1:50 a n t i ­ serum , in c u b a te d fo r 5 m in u tes a t 37° an d assay ed a fte r d ilu tio n to 10~4, p ro ­ duced 11 p laq u es p e r c u ltu re .

V irus m ixed w ith an equal volum e of PB S , a n d in c u b a te d for 5 m in u tes a t 37°, produced 25 p laques p er c u ltu re a t a 10~5'7 d ilu tio n .

At th e s ta te d tim es p a irs of cell m onolayer c u ltu re s in fected a t an average adsorbed m u ltip lic ity of 0.08 were w ashed, tr e a te d w ith PB S o r d ilu te an tiseru m for 5 m in u tes, an d w ere th e n w ashed again to rem ove a n tib o d y . T he v iru s con­ te n t of th e cells w as th e n d eterm ined.

No reduction in virus content of the cells occurred as a result of treat­ ment with antiserum (Table 3).

Cell suspensions were treated with diluted antiserum for 10 minutes at 37°, and were then diluted 1:100 in PBS prior to disruption of the cells by freezing and thawing. Residual virus was assayed and compared with a second aliquot of the cell suspension treated in the same way but with the antiserum replaced by PBS. To show that the antiserum concentration was sufficient to neutralize the virus associated with the cells, a third aliquot of the suspension was frozen and thawed to re­ lease virus, before addition of the antiserum.

Results typical of these experiments are shown in Table 4.

With cell monolayers from which free virus had been removed by washing, no reduction in virus content occurred as a result of treatment with antiserum. With cell suspensions from which free virus had not been removed, the proportion of virus neutralized was essentially the

10 H O W E S

T A B L E 4

In a c c e s s ib il it y o f Ce l l-a s s o c ia t e d Vir u s to Sp e c if ic An t ib o d y. Ex p e r i­

m e n t s w it h Su s p e n d e d Ce l l s11

T re a tm e n t p rio r F in a l d ilu tio n P F U /m l X 104 Tim e to assay a t assay 11.1 H ours 12.1 H ours A n tib o d y -re sist-

a n t v iru s in suspension (A) 1. T re a te d w ith a n ti­ serum 2. D ilu te d lO“2 3. F ro zen a n d th aw ed 10-4 92 87 A n tib o d y -resist- a n t v iru s in fro ­ zen an d th aw ed suspension (B) 1. F rozen a n d th aw ed 2. T re a te d w ith a n ti­ serum 3.3 X I0-4 5.7 4 .5

T o ta l v iru s con- F ro zen an d th aw ed nr4 100 119 t e n t of su sp en ­

sion (C)

° Cells in fected a t an average ad so rb ed m u ltip lic ity of 0.9 w ere in c u b a te d in suspension, an d a t th e in d ic a te d tim es, a n tise ru m to a final d ilu tio n of 1:2,000 was a d d ed to one a liq u o t of th e in fe c te d cell suspension. A fter in c u b a tio n for 10 m in u tes a t 37°, th e suspension w as d ilu te d 1:100, frozen an d th aw ed to release cell-asso ciated v iru s, an d assay ed to give th e a m o u n t of a n tib o d y -re s is ta n t v iru s in th e suspension (A ). A second a liq u o t w as frozen an d th aw ed to release cell-asso ciated v iru s 10 m in u tes la te r , tr e a te d w ith a n tise ru m , an d assayed a fte r fu r th e r d ilu tio n (B). A th ird a liq u o t was tr e a te d as for th e first a n d a t th e sam e tim e, b u t w ith P B S in place of a n tise ru m , an d assaj^ed to give th e original v iru s c o n te n t of th e suspension (C).

same as the proportion of total virus present as free virus at this time. The presence of adequate amounts of antibody was shown by the neutralization of over 90 % of the virus produced by the cells, provided it was first released from the cells by a cycle of freezing and thawing.

In both types of experiment, therefore, the results show that cell- associated virus is inaccessible or resistant to antibody and is therefore intracellular.

In document The growth cycle of poliovirus in cultured cells (Page 31-33)