VIROLOGY 26, 630-637 (1965)

A Factor Capable of Enhancing Virus Replication Appearing in Parainfluenza Virus Type 1 (HVJ1)-Infected Allantoic Fluid

NOBUO KATO, A K I K O OKADA, ANn F U J I T O OTA

Department of Bacteriology, Nagoya University School of Medicine, Nagoya, Japan

Accepted May 7, 1965

A factor was released into the allantoic fluid of eggs infected with HVJ, which was able to enhance hemagglutinin production by HVJ and PR8 virus in roller tube cul- tures of fragments of shell with chorioallantoic membrane attached. The enhancing factor could be distinguished from virus particles and S antigen. It appears that it is a nonviral cellular product formed along with the multiplication of HVJ.

The enhancement of hemagglutinin production was obtained either by preincu- bation of the cultures with the enhancing factor at 37 ° for 24 hours before challenge, or by its addition into the cultures simultaneously with challenge.

The enhancing factor appeared in allantoic fluid within 6 hours after infection with 1@ to 105 EID~0 of HVJ and reached a maximal level at 18 hours. The enhancing activity of the fluid declined rapidly after 36 hours of infection, probably owing to the appearance of interferon. At the time that the enhancing factor was released into allantoic fluid and maintained its maximal level, virus titers in the allantoic fluid were increasing approximately at a linear slope.

INTRODUCTION

Many virus-cell systems have been shown to produce interferon (Ho, 1962; Wagner, 1963; Isaacs, 1963). However, no report has appeared on a substance which is produced by cells as a reaction to virus and which is capable of increasing the extent of virus replication. Recently we have found that such a factor is present in allantoic fluids of eggs, infected with HVJ and harvested in the early stages of infection. The present re- port is concerned with this finding.

MATERIALS AND METHODS

Virus. The Nagoya 1-60 strain of HVJ Was used. This virus was isolated originally from laboratory mice (Kato et al., 1961). Unless otherwise stated, egg-adapted HVJ which had been carried through more than 100 egg passages was employed. In one ex- periment mentioned in the text the same strain of HVJ in its 8th egg passage was used

1 Hemagglutinating virus of Japan; synonym: Sendal virus.

as the challenge virus. Other viruses used were the PR8 strain of influenza virus A and Sindbis virus. Sindbis virus, which was kindly supplied by Dr. I. Nagata, Depart- ment of Virology, Cancer Research Insti- tute, Nagoya University School of 5~[edicine, was propagated in chick embryo cell cul- tures.

Cultures of pieces of chorioallantoic mem- branes. Cultures of small pieces of shell with chorioallantoic membrane attached were prepared using a modification of the method described by Ishida et al. (1958). Eleven- to 13-day eggs were used. The egg contents were poured out and the inner surfaces of the deembryonated eggs were washed in two changes of 0.01 M phosphate-buffered saline at pH 7.4 (PBS). Small pieces of shell with membrane attached (1 X 2 cm 2) were cut out of deembryonated eggs, and the shell- membrane pieces were washed in PBS. Seven to 9 pieces were taken from each egg. The shell-membrane pieces were placed in test tubes, 1 piece in each tube. The volume

630

A VIRAL GROWTH ENHANCING FACTOt~ 631

of test and control materials or media was 1 ml in each tube. The assay method of inter- feron described by Lindenmann et al. (1957) was followed, and six tubes were used for each experimental group. The tubes were rotated in a roller drum at 37 ° (12 revolu- tions per hour). Media used were Hanks' solution not containing glucose, Hanks' solution, and Hanks' solution supplemented with 0.1% yeast extract and 0.5 % lactal- bumin hydrolyzate (YLH). A challenge dose of HVJ and PR8 virus was 1 HA unit per tube.

Hemagglutinin (HA) titration. HA titra- ~ions were performed with a 0.5 % suspen- sion of chicken erythrocytes. Serial twofold dilutions (0.5 ml) of the material were made in PBS and 0.5 ml of the erythrocyte sus- pension was added. The mixtures were shaken and allowed to sediment at 4% The highest dilution giving either complete or partial but definite agglutination was taken as the end point. The results of the experi- ments are given in the tables as geometric mean HA titers of a group of six tubes (Lindenmann et al., 1957), the titers being expressed as the log2 of the dilution at the hemagglutination end point. A mean differ- ence of 2 log2 units between experimental and control groups was found to be signifi- cant at the 1% level (Isaacs and Linden- mann, 1957).

Infectivity titration. Infectivity titration of HVJ was performed as follows. Serial ten- fold dilutions of the test material were made in PBS and 0.5 ml of each dilution were inoculated into the allantoie cavity of 9- to 10-day eggs. After 86 hours' incubation, allantoic fluids were harvested and tested for the presence of HA. Titers were calcu- luted in terms of EID56 according to the method of Reed and Muench (1938).

Complement fixation (CF) technique. Com- plement fixing V and S antigens of HVJ were determined by testing twofold dilutions of the test material with standard V and S sera. Twofold dilutions of the antigen (0.1 ml), 4 units of serum (0.1 ml), and 2 units of com- plement (0.1 ml) were incubated for 1 hour at 37 °, after which 1.5 % sheep erythrocytes sensitized with 2 units of hemolysin (0.15 ml) was added. Dilutions of all materials

were made with Veronal-buffered saline. The system was incubated at 37 ° for 30 min- utes. The highest dilution of the material tested exhibiting more than 75 % fixation of complement was considered to contain 1 unit of antigen. Detailed procedures for preparation of standard V and S sera will be described elsewhere (Kato, in preparation). In brief, V antiserum was produced in rab- bits by three intravenous injections of 3 ml of ultraviolet-inactivated purified vaccine containing 10,240 HA units per milliliter. The virus suspension for the vaccine was ob- tained from infected allantoic fluid by one cycle of adsorption to and elution from chicken erythrocytes and two cycles of differential eentrifugation. Anti-V titers of the sera obtained 7-10 days after the last injection were 640 to 1280. Since the sera contained CF antibodies to normal tissue components of chorioallantoic membranes, they were absorbed before use with minced membranes. S antiserum was produced in guinea pigs by an intramuscular and intra- peritoneal injections of 1.5 ml of S antigen mixed with equal volumes of Freund's ad- iuvant. S antigen was prepared from 20 % emulsions of chorioallantoic membranes in- fected with HVJ. Anti-S titers of the sera obtained 16-21 days after the last injection had high anti-S titers (256-512). Since the sera also contained V and chorioallantois antibodies, they were absorbed before use with purified V antigen and then with chorio- allantoic membranes.

Interferon assay by the plaque method. Pri- mary chick embryo cell cultures were pre- pared from 10- to 12-day-old decapitated embryos by trypsinization (0.25%). Cells were suspended in YLH supplemented with 10 % calf serum. Of the cell suspension con- raining 5 X 106 cells per milliliter, 5 ml was added into each 50 ml-bottle; the bottle was stoppered and incubated at 37 ° . The cell sheet usually contained about 1 X 107 cells when used 16-18 hours later. Interferon was assayed as follows. Chick embryo cell cul- tures were incubated at 37 ° for 3 hours in duplicate with 2.0 ml of material to be tested diluted in YLH supplemented with 2 % calf serum. The fluids were decanted and the cultures were washed once with Hanks' solu-

632 KATO, OKADA, AND OTA

tion and challenged with approximately 100 P FU of Sindbis virus contained in 0.1 ml. After incubation at 37 ° for 1 hour, the cul- tures were overlaid with 5 ml of overlay me- dium, and plaques were counted after 48 hours. Overlay medium for plaque assay consisted of Hanks' solution, 0.5 % lactal- bumin hydrolyzate, 0.1% yeast extract, 1% Noble agar, 1% calf serum, and 1:30,000 neutral red. The interferon titers were ex- pressed as the reciprocal of the dilution of interferon which reduced the plaque count by 5O %.

Preparation of materials to be tested for en- hancing and interferon activities. Allantoic fluids harvested at intervals from eggs in- fected with HVJ were examined. The fluids were centrifuged at 80,000 g for 1 hour. The supernatants were collected and rendered free of infectious virus by heating at 60 ° for 30 minutes. Heating at60 ° for 30 minutes completely destroyed the infectivity and HA contained in the supernatant fluids (see text). Normal allantoic fluid was obtained from uninfected 11- to 13-day eggs and treated in the same way as the infected allan= toic fluids.

RESULTS

Effect of Preincubation with Supernatants of HVJ-Infected Allantoic Fluids on Repli- cation of H V J in Chorioallantoic Mem- branes

The allantoie fluids were harvested from eggs at intervals of 6-12 hours after infection with 104 to 10 ~ EIDs0 of ItVJ. The fluids were centrifuged at 80,000 g for 1 hour, and the supernatant fluids obtained were heated at 60 ° for 30 minutes to inactivate residual vi- rus. Heating for this time completely de- stroyed any infectivity and HA that re- mained in the supernatant fluids, although S antigen was not inactivated (Table 1).

Groups of roller tube cultures of shell- membrane pieces were preincubated for 24 hours with the heated supernatants of in- fected and normal allantoic fluids. The heated supernatants of infected fluids were obtained from allantoie fluids harvested at intervals of 6-12 hours after infection with HVJ. The tubes of the control group were preincubated for 24 hours with PBS. At the

TABLE 1

DECR~ASn O~ "VIRUS TITERS IN A I-IV J-INFECTED ALLANTOIC ]~LUID 2~FTEI~ ULTRACENTRIFUGATION

AND ~tEATING

Material EID~o HA CFV CFS

Infected allantoic 10 ~-s 4096 32 8 fluid harvested at 72 hours

Supernatant after 105.5 16 0 2 centrifugation at 80,000 g for 1 hour

Supernatant 0 0 0 2 heated at 60 ° for 30 minutes

end of preineubation the fluids were de- canted, the cultures were washed with PBS, infected with 1 HA unit of HVJ, and 1 ml of YLH medium was added. After incubation at 37 ° for 84 hours the HA titers of the culture fluids were determined. A difference of mean yield of HA was estimated by subtracting the mean logs of a group of control tubes from that of groups of experimental tubes. The results of four experiments employing different lots of allantoic fluids are plotted in Fig. 1. I t can be seen that enhancement of HA production was obtained when the cul- tures were preincubated with the superna- tants of the allantoic fluids harvested in the early stages of infection. The enhancing ac- t ivity of the fluids appeared within 6 hours after infection, reached a maximal level at 18 hours, and declined rapidly after 36 hours. After 60 hours of infection, definite inter- fering activity appeared in the fluids. At 48 hours after infection the fluids had neither enhancing nor interfering activity. HA pro- duction in the cultures preincubated with normal allantoic fluid was nearly equal to tha t in the control cultures preincubated with PBS.

Curves of Growth of HVJ and of Interferon Production in Allantoic Fluid of Eggs

As described in the previous section, allantoic fluids harvested in the early stages of infection showed enhancing activity for HA production, and fluids harvested in the later stages showed interfering activity. To

A VIRAL GROWTH ENHANCING FACTOR 633

-3

4 • ,4

• 0 • , ~ , - - ~ . . . . . .

o • ,e ~ o m

• . - ' - - I k . . :

-4

NJF lr2 2~1 3~6 418 610 7'2 84 - - HOURS AFTER INOCULATION

FIG. 1. The appearance of viral growth en- hancing and interfering activities in the allantoic fluids of eggs infected with 104 to l0 s EIDs0 of HVJ. Allantoie fluids were harvested at various times after infection with HVJ. The time on the abscissa represents the time after infection when the allantoic fluids were harvested. Cultures of shell-membrane pieces were preineubated for 24 hours with the heated supernatants of the in- fected and normal allantoic fluids. The cultures were then washed and infected with 1 HA unit of HVJ. V/V0 (represented by filled circles) was esti- mated by subtracting the mean HA yield (logs) of a group of control tubes which were preincubated with PBS from that of groups of experimental tubes. NF, normal allantoic fluid harvested from uninfected eggs. The dashed line was drawn through an average (represented by open circles) of all determinations.

s tudy the relationship with time of the ap- pearance of these effects, of virus growth and of interferon production, the allantoic fluids harvested at various intervals after inlet- tion with 10 ~ EIDs0 of HVJ were assayed for EIDs0, HA, CFV and S antigens, and interferon. The results are shown in Fig. 2. I t can be seen that interferon first appeared in the allantoic fluid at 60 hours after infee- tion, at which time the titers of infectivity, HA, and CFV antigen had already reached their maximal levels. The interferoa produc- tion curve obtained here corresponds well with the time of appearance of interfering activity which is shown in Fig. 1. I t is likely therefore that the demonstrated reduction of HA yield in cultures preineubated with the allantoic fluids harvested after 60 hours of infection is attributable to interferon con-

e HA -=

12 24 36 48 60 72 84 HOURS AFTER iNOCULATION

FIG. 2. Growth curves for HVJ and for inter- feron production in atlantoie fluids. Ten-day eggs were inoculated with 106 EIDso of HVJ.

rained in the fluids. At the time that the en- hancing activity of the fluids was increasing and maintained its maximal level (see Fig. 1), virus titers in the allantoic fluid were in- creasing approximately at a linear slope. CFS antigen was first detectable at 36 hours and reached a maximal level at 72 hours after infection. Allantoie fluids in which en- hancing activity was maximal therefore did not contain CFS antigen, or contained only a trace of it.

Effect of Preincubation with the Enhancing Fluid on Replication of H V J in Chorioal- lantoic Membranes Nourished with De- ficient Media

Our strain of HVJ does not produce any HA in cultures of shell-membrane pieces nourished with Hanks' solution not contain- ing glucose. When Hanks ' solution is used as medium, HA production occurs, but it is much lower than when YLH medium is used. We next tested whether enhancement of HA production could be obtained even in these deficient media, when the cultures were pretreated with the enhancing fluid. In this experiment a heated supernatant of the allantoic fluid harvested at 24 hours after in- fection with HVJ was employed as the en- haneing fluid. Three groups of the cultm~es were preincubated for 24 hours with the en- hancing fluid, and three other groups were preincubated with normal altantoic fluid and served as the controls. At the end of preincu- bation the fluids were decanted, the cultures

634 KAT0, 0KADA, AND 0TA

TABLE 2

EFFECT OF PREINCUBATION WITH THE ENHANCING

FLUID ON REPLICATION OF HVJ IN CHORIO-

ALLANTOIC MEMBRANES NOURISHED WITH DEFICIENT MEDIA

Material for preincuba-

tion ~

Normal fluid

Enhancing fluid

Normal fluid

Enhancing fluid

Normal fluid

Enhancing fluid

Medium after challenge

Hanks' solution not containing glucose

Hanks' solution not containing glucose

Hanks' solution

Hanks' solution

YLH

YLH

Mean yield of HA b (log2)

<0

3.4

4.0

7.2

7.2

9.5

TABLE 3

EFFECT OF PREINCUBATION WITH THE ENHANCING

FLUID ON REPLICATION OF EARLY EGG

PASSAGE HVJ IN CHORIOALLANTOIC

M E M B R A N E S a

Mean yield of HA c V/V0 c Material for preincubation b (log2) (log2)

Normal fluid <0 Enhancing fluid 4.0

HVJ in its 8th egg passage was used as the > 3.4 challenge virus.

6 See text. Expressed as in Table 2.

3.2

2.3

trol cultures, and accordingly the difference between the HA yields in the enhanced and control goups was somewhat decreased. In other experiments it was found tha t addition of calf serum to Y L H at levels of 0.5-5.0 % did not affect the HA yield in both enhanced and control cultures.

See text. b Geometric mean HA titer (log2) of each

group. Estimated by subtracting the mean log2 of a

group of the cultures preincubated with normal fluid from that of a group of the cultures pre- incubated with the enhancing fluid.

were washed and infected with 1 H A unit of HVJ, and 1 ml of medium was added. Media for experimental and control cul- tures were Hanks ' solution not containing glucose, Hanks ' solution and YLH, re- spectively. After incubation at 37 ° for 84 hours the HA titers of the culture fluids were determined. The results of a typical experi- ment are shown in Table 2. In the cultures preineubated with normal fluid for 24 hours before challenge and nourished with Hanks ' solution not containing glucose after chal- lenge, H A production could not be detected. In contrast, when the cultures were prein- cubated with the enhancing fluid for 24 hours before challenge, significant amounts of H A were produced even in such deficient media. When Hanks ' solution was used as medium, H A production was detectable in control cul- tures and enhancement of HA yield was again obtained. When Y L H was used as me- dium, the H A yield was high even in con-

Effect of Preincubation with the Enhancing Fluid on Replication of an Early Egg Passage Virus of H V J

Our strain of H V J has a low degree of in- fectivity for the allantoie cavity in the early phases of allantoic passage (Kato et al., 1961). Virus tha t has been carried through less than 10 egg passages cannot produce any H A in cultures of shell-membrane pieces. An experiment was carried out to examine whether the enhancing fluid is able to pro- mote replication of such an early egg passage HVJ in cultures of shell-membrane pieces. The enhancing fluid was prepared as de- scribed in the previous section. The control fluid was normal allantoie fluid. Two groups of roller tube cultures of shell-membrane pieces were preineubated for 24 hours with the enhancing and normal fluids, re- spectively. One H A unit of the 8th passage virus of H V J was inoculated into each tube. Y L H was used as medium, and incubation was carried out for 96 hours after challenge. Other experimental conditions were the same as in the previous section. At the end of the incubation period the H A titers of the culture fluids were determined. The results are shown in Table 3. I t can be seen tha t while H A production could not be detected

A V I R A L G R O W T H E N H A N C I N G F A C T O I ~ 635

T A B L E 4

EFFECT OF PREINCUBATION WITH THE ENHANCING FLUID ON REPLICATION OF INFLUENZA

VIR~JS (PR8) IN CHORIOALLANTOIC MEMBR~NES

Mean lVfaterialfor yield of V/Vo ~

Expt. no. ~ preincubation b HA ~ (log~) (log2)

1 N o r m a l f luid 5 .3 - - E n h a n c i n g f luid 8 .3 3 .0

N o r m a l f luid 1 .2 E n h a n c i n g f luid 5 .0 3 .8

M e d i u m a f t e r cha l l enge : H a n k s ' s o l u t i o n in e x p e r i m e n t 1; H a n k s ' s o l u t i o n n o t c o n t a i n i n g g lucose in e x p e r i m e n t 2.

b See t ex t . E x p r e s s e d as in T a b l e 2.

in the control cultures, significant amounts of HA were produced in the cultures prein- eubated with the enhancing fluid. The yield of HA, however, was markedly less than the yield obtained by the fully adapted HVJ in the cultures nourished with YLH medium (see Table 2). I t appears therefore that the enhancing fluid is able to facilitate to some extent the replication of HVJ which is not fully adapted to the allantoic cells.

Effect of Preincubation with the Enhancing Fluid on Replication of Influenza Virus in Chorioallantoic Membranes

In the experiments described to this point, the effect of enhancing fluids was tested with HVJ as the challenge virus. The effect of these enhancing fluids on replication of in- fluenza virus (PR8) was next examined. The experimental conditions were similar to those described in the previous sections except tha t both Hanks' solution and Hanks' solution not containing glucose were used as media, and incubation was carried out for 60 hours after challenge. According to Ishida et al. (1958), a good yield of PR8 virus is obtained in roller tube cultures of shell-membrane pieces when Hanks' solution is used as me- dium. The results of a typical experiment are shown in Table 4. I t can be seen that the results are analogous to those obtained with HVJ (see Table 2).

Effect of the Enhancing and Normal Fluids When Added Simultaneously with Chal- lenge

In this experiment equal volumes (0.5 ml) of the enhancing or normal fluids and medium were added to cultures of shell-mem- brane pieces simultaneously with challenge with 1 HA unit of PR8 virus. Equal volumes of PBS and medium were added to the con- trol cultures. Hanks' solution was used as the medium. Incubation was carried out for 60 hours. The results of a typical experiment are shown in Table 5. I t can be seen that en- hancement of HA production was obtained as before. When normal allantoic fluid was used, however, a marked reduction of HA yield occurred. This reduction of HA yield seems to be due to an HA inhibitor contained in normal allantoic fluid. Hemagglutination inhibition (HI) titers were determined on normal and enhancing fluids. The procedures for the HI test were the same as those de- scribed previously (Kato et al., 1961). As Table 6 shows, normal allantoic fluids con- tained an HA inhibitor of PR8 virus, whereas the inhibitor could not be detected in the en- hancing fluid. The inhibitor was stable to heat at 60 ° for 30 minutes.

In view of the fact that normal allantoie fluid contains HA inhibitor, and that the pieces of membrane in the cultures may themselves produce the inhibitor, the ques- tion arose whether the enhancing fluid con- tains some material which is able ~o inacti- vate normal inhibitor. To examine the ques- tion the following experiment was carried out. Equal volumes of the enhancing fluid and normal fluid were mixed and then incu-

T A B L E 5

EFFECT OF THE ENHANCING AND NORM.4L FLUIDS ON REPLICATION OF P R 8 V i r u s WHEN ADDED

SIMULTAN~]OUSLy ~;VITH CHALLENGE

Mean yield V/V0 c Material a of HA b (log2) (log2)

PBS (control) 5.6 - -

N o r m a l f luid 3 .0 - - 2 . 6 E n h a n c i n g f luid 8 .8 3 .2

See t ex t . b E x p r e s s e d as in T a b l e 2. c E x p r e s s e d as in Fig . 1.

636 KATO, OKADA, AND OTA

TABLE 6 EFFECT OF INCUBATION WITH THE ENHANCING

FLUID ON HA INHIBITOI~ (HI) CONTAINED IN NORMAL ALLANTOIC FLUID

HI titer Material per ml b

Normal fluid 32 Enhancing fluid <2 Normal fluid + PBS a incubated at 16

37 ° for 24 hours Normal fluid + enhancing fluid a in- 16

eubated at 37 ° for 24 hours

a Equal volumes of the two materials were mixed.

b HI t i ter against 4 HA units of PR8 virus.

bated at 37 ° for 24 hours. As the control, equal volumes of PBS and normal fluid were mixed and incubated in the same way. At the end of the incubation period HI titers of the mixtures were determined. The re- sults are shown in Table 6. It can be seen that the enhancing fluid could not inactivate the normal inhibitor.

DISCUSSION

This paper presents evidence for the re- lease of a viral growth-enhancing factor into the allantoie fluid of eggs infected with HVJ. The enhancing factor can be distinguished from the activity of virus particles, since the enhancing fluids employed in the present studies contained no infectivity and HA ac- tivity. The enhancing fluids did contain a trace of CFS antigen, but the time of release of CFS antigen into the allantoie fluids bore no relationship to the time for the release of the enhancing factor. I t is likely therefore that the enhancing factor is a nonviral ma- terial formed by the interaction of HVJ with the allantoie cells. We propose the term "en- hancer" for this material.

The release of enhancer into allantoie fluid occurred in the early stages of infec- tion. The enhancer activity of the fluid de- elined rapidly after 36 hours of infection and was followed by the appearance of inter- fering activity. It is likely that the appear- ance of interfering activity was due to the release of interferon into allantoie fluid. Interferon activity appeared first in allantoie

fluid at 60 hours after infection, when virus titers in the allantoie fluid had already reached maximal levels. This situation is consistent with that found with influenza virus (WS) as reported by Wagner (1960). In recent experiments, it has been found that enhancer activity can be completely counter- acted by large amounts of interferon. In addition, and as might be expected, inter- feron activity can be counteracted by en- hancer (Kato, Okada, and Ota, in prepara- tion). It is clear that since both types of activities are induced by the virus, and since they counteract each other, the measure- ment of each activity will be affected by the presence of the other activity. For example infected allantoic fluids taken at late times may still have considerable enhancer ac- tivity, but this is counteracted by interferon activity. The decline in enhancer activity may therefore be illusory to some extent. Similar arguments apply to the measurement of interferon activity. Early fluids may have interferon which does not show itself be- cause of the presence of enhancer activity.

Other cell-virus systems in which one virus enhanced the replication of another virus have been reported (Kumagai et al., 1961; Matumoto et al., 1961; Frothingham, 1963; Hermodsson, 1963). In these reports the enhancing effect appeared to be associ- ated with the infectious virus particles since no effect was obtained when virus particles were removed by eentrifugation or were in- activated by antiviral serum, heat, or ultra- violet irradiation. However, no other report has appeared so far on a cellular product which is formed as a reaction to virus and which is able to enhance the replication of virus.

A C K N O W L E D G M E N T

The authors are grateful to Professor K. Ogasa- wara for his encouragement throughout the work.

R E F E R E N C E S

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A VIRAL GROWTH ENHANCING FACTOR 637

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