Rev. sci. tech. Off. int. Epiz., 1989, 8 (3), 747-764.

Effect of formaldehyde and binary ethyleneimine

(BEI) on the integrity of
foot and mouth disease virus capsid
M.M. RWEYEMAMU, O. UMEHARA, W. GIORGI, R. MEDEIROS,
D. LUCCA NETO and M. BALTAZAR *

Summary: Formaldehyde treatment of foot and mouth disease (FMD) virus

caused a statistically significant (p < 0.05) reduction in the concentration of
140S particles (26-72%) for 5 of the 6 South American vaccine virus strains
tested. Formaldehyde also reduced the stability of the virus; after storage at
4°C for 6 months, there was a decay of 33-85% in the concentration of 140S
particles.
In contrast, samples treated with BEI did not undergo a significant reduction
in 140S immediately after inactivation or after storage. The efficiency of
infectivity inactivation by BEI was influenced by its concentration and
temperature. No significant reduction in 140S was observed at 36°C in 24 hours.

KEYWORDS: Aphthovirus - Ethyleneimines - Foot and mouth disease -

Formaldehyde - Inactivation - Vaccines.

INTRODUCTION

It is commonly accepted that the immunogenicity of foot and mouth disease (FMD)
vaccines is dependent on the presence of the intact capsid, as either the nucleocapsid
with a sedimentation rate of 140S or the natural empty particle which sediments at
75S (10, 15, 20, 37, 40, 44). F o r the majority of strains, the principal immunogenic
component is the 140S particle. Several studies have shown a direct relationship
between the dose of 140S antigen in vaccine lots a n d the rate, height and persistence
of virus neutralising antibody in cattle after b o t h primary and secondary vaccination
(8, 9, 29, 30, 34, 39). Hence many modern F M D vaccine manufacturing establishments
include the quantification of 140S antigen in the battery of quality control tests during
production (5, 2 1 , 22, 26, 3 1 , 34, 38, 42).

There are conflicting reports in the literature on the effect of commonly used virus
inactivators, formaldehyde and aziridine c o m p o u n d s , on the integrity of F M D virus
capsid and thereby o n immunogenicity. Formaldehyde has been largely discredited
as an inactivator, mainly because of non-linear inactivation kinetics leading t o residual
infective virus which, sometimes, is detectable for several weeks after completion of

* Pfizer International Inc., Laboratorios Pfizer Ltda., Rodovia Presidente Dutra Km 225, CEP 07010
Guarulhos, Sâo Paulo, Brazil.
748

the " i n a c t i v a t i o n " period of 48 hours (12, 19, 41). There have been several instances
of F M D outbreaks considered to have been associated with the use of formaldehyde-
inactivated vaccines (7). In contrast, the aziridine c o m p o u n d s acetylethyleneimine
(12), ethyleneimine (17) and binary ethyleneimine (3) have been associated with first-
order virus inactivation kinetics and have, generally, resulted in innocuous vaccines.
Such compounds fulfil a recent recommendation by the O I E for inactivated F M D
vaccines (33).

Nevertheless, some reports in the literature have claimed that both formaldehyde
(27) and aziridine compounds (36) are able to destroy the F M D virus capsid and
thereby reduce vaccine immunogenicity. A t Pirbright (32, 36), a hybrid inactivation
procedure involving sequential treatment of virus with formaldehyde and A E I was
devised in order to preserve the 140S particle. As far as we know, the method is not
widely employed by F M D vaccine manufacturers.

In the present study we investigated the effect of formaldehyde and binary

ethyleneimine on South American strains of F M D virus capsid when the c o m p o u n d s
were used either singly or in sequence.

MATERIALS A N D METHODS

Virus strains
The strains of F M D V used in this study were all of South American origin: O
Campos/Brasil-58, A Cruzeiro/Brasil-55, A Venceslau/Brasil-76, A Bagé/Brasil-76,
A Castellano/Argentina-87 and C Indaial/Brasil-71. They had been adapted to growth
in B H K suspension cells (14) for vaccine production. For this study each virus was
grown in BHK suspension cells either in a spinner culture of 2 litre volume or in a
3000 litre fermenter. Virus was harvested at m a x i m u m cytopathic effect (c.p.e.),
usually after 16 to 24 h.

Inactivation with formaldehyde

The virus suspension was adjusted to p H 8.0 with glycine buffer and a temperature
of 26°C in a water-bath fitted with a magnetic stirrer. Then 1:10 formalin in distilled
water was added to the virus suspension to a final concentration of 0.06% v / v
formalin. Periodically the virus-containing flask was manually inverted in order to
bring virus in the air space above the liquid level (less than 10% of total flask capacity)
into contact with inactivator. Inactivation proceeded for 48 h under magnetic agitation.

Inactivation with BEI

The procedure adopted was similar to that described by Bahnemann (3). BEI was
generated by treating 0.1M 2-bromoethylamine hydrobromide with 0.2N N a O H for
1 hour at 37°C. This solution was then added to the virus suspension to give the desired
concentration of BEI, i.e. 1% v / v for 1mM BEI; 2 % v / v for 2 m M BEI, etc.
Periodically, the virus-containing bottle was inverted in order to inactivate virus in
the air space above the liquid level. Temperature and time of inactivation were as
specified under " R e s u l t s " . Excess BEI was neutralised at the end of the inactivation
period with 0 . 1 % w / v sodium thiosulphate.
 749

Inactivation kinetics and innocuity testing

For measuring inactivation kinetics, samples were collected at hourly intervals
up to 4 h after adding inactivator. The inactivator was neutralised and samples titrated
for surviving virus infectivity by using the microcolour test (28). The kinetics constant,
slope and half-life were calculated.
At 24 and 48 hours after commencement of inactivation, samples were collected
for innocuity testing. After neutralisation, each sample was inoculated onto duplicate
Roux flasks of BHK monolayers maintained on a serum-free medium. The cultures
were examined for c.p.e. and complement fixing antigen over three serial passages
(2, 16).

Quantification of 140S
Test virus samples were first concentrated tenfold by using saturated a m m o n i u m
sulphate either at 4 ° C overnight or at r o o m temperature (ca. 25°C) for two hours,
and resuspended in 0.04M phosphate buffer. Then the concentration of the 140S
particles was assayed by sucrose density gradient using the methods of Barteling and
Meloen (5) and Doel et al. (21). Briefly, the sample was treated with 0 . 0 0 1 % (w/v)
bovine ribonuclease at 36°C for 10 min to degrade any free R N A or ribosome, and
then 1 ml of this was layered onto a preformed, linear 10-30% (w/v) sucrose density
gradient. The gradients were centrifuged at 40,000 r p m using the Beckmann SW 41
Ti rotor in the Beckmann L8-55M ultracentrifuge for 60 min. Then the gradients
were scanned at 259 n m wavelength using the Beckmann DU-7U spectrophotometer
fitted with a continuous 0.2 cm pathlength flow-cell. The area under the peak was
calculated and used to determine the concentration of 140S particles using the
following formula:

140S µ g / m l = FRxPAxFSDxlOOO
S x P L x E x W x 10
where FR = flow rate in m l / m i n
2
PA = area under the peak in c m
FSD = full scale absorbence optical density unit setting
S = speed of the chart recorder in c m / m i n
PL = pathlength of the flow cell in cm
E = extinction point for F M D V , i.e. 78.8 at 259 nm
W = sample volume (ml) applied to gradient
(N.B. value divided by 10 to allow for the concentration factor)
Under our conditions the method gives a coefficient of variation of 12% for inter-
test and 5 % for intra-test variation.

Statistical analysis
The statistical analysis of data was carried out using the R S / 1 programme of BBN
Software Products Corporation (Cambridge, USA) operating in the D O S m o d e of
IBM on the Microtec X T 2002 microcomputer. The level of rejection of the null
hypothesis was set at 0.05.
750

RESULTS

Formaldehyde versus BEI on F M D V 140S particle

Two series of experiments were carried out. In the first the virus harvest was divided
into four lots: one lot was inactivated with formaldehyde (0.06% v / v formalin); the
second was inactivated with two doses of 1mM BEI 24 hours apart; the third was
treated for the first 24 hours with formaldehyde and then, additionally, with 1mM
BEI; the fourth lot was left as a live virus control. The p H of all 4 lots was adjusted
to p H 8.0 and they were all incubated under agitation in the same water-bath at 26°C
for a total of 48 hours. For each virus strain 4-6 replicate tests were thus carried out.
The results of this set of experiments are summarised in Table I a-d and Figure
1, from which it is evident that the reduction in 140S level due to BEI treatment was
less than 2 5 % and statistically insignificant for any of the tested strains. In contrast,
formaldehyde, either alone or in combination with BEI, reduced 140S by up to 7 2 % .
This reduction was evidently primarily due to the action of formaldehyde. By one­
way analysis of variance the reduction in 140S level caused by formaldehyde was shown
to be significant for strains O Campos and A Cruzeiro (p < 0.05).

TABLE la

Effect of inactivators on concentration of 140S antigen

(µg/ml) in vaccine virus harvests of strain O Campos
Lot no. Live virus Formalin Formalin/BEI BEI ImM
xl/26°C/48h 26°C/48h x2/26°C/48h
1 0.900 0.510 0.630 0.860
2 1.400 0.830 0.590 1.180
3 0.950 0.310 0.860 0.940
4 2.280 1.180 0.860 1.860
5 1.860 0.880 1.130 1.830
Mean 1.478 0.742 0.814 1.334
STDEV 0.593 0.339 0.217 0.481
SEM 0.265 0.152 0.970 0.215

Anova oneway: p = 0.036

TABLE lb

Effect of inactivators on concentration of 140S antigen

(ng/ml) in vaccine virus harvests of strain A Cruzeiro
Lot no. Live virus Formalin Formalin/BEI BEI ImM
x l/26°C/48h 26°C/48h x 2/26°C/48h
1 0.330 0.010 0.010 0.430
2 0.400 0.070 0.010 0.330
3 0.610 0.210 0.050 0.720
4 0.570 0.010 0.070 0.520
5 0.570 0.010 0.070 0.480
6 0.760 0.250 0.500 0.410
Mean 0.540 0.093 0.118 0.482
STDEV 0.154 0.109 0.189 0.133
SEM 0.063 0.045 0.077 0.054

Anova oneway: p = 0.001

 751

TABLE le

Effect of inactivators on concentration of 140S antigen

(¡xg/ml) in vaccine virus harvests of strain A Venceslau

Lot no. Live virus Formalin Formalin/BEI BEI ImM

x l/26°C/48h 26°C/48h X 2/26°C/48h

1 0.240 0.110 0.070 0.210

2 0.700 0.170 0.140 0.400
3 0.300 0.010 0.140 0.260
4 0.990 0.200 0.500 0.860

Mean 0.558 0.122 0.212 0.432

STDEV 0.530 0.084 0.194 0.296
SEM 0.177 0.042 0.097 0.148

Anova oneway: p = 0.112

TABLE Id

Effect of inactivators on concentration of 140S antigen

(¡jLg/ml) in vaccine virus harvests of strain C Indaial

Lot no. Live virus Formalin Formalin/BEI BEI ImM

x l/26°C/48h 26°C/48h x 2/26°C/48h

1 1.330 0.950 0.710 1.000

2 1.500 0.470 0.720 1.030
3 0.750 0.710 0.650 0.710
4 1.400 1.200 1.170 1.380

Mean 1.245 0.832 0.812 1.030

STDEV 0.337 0.314 0.240 0.274
SEM 0.169 0.157 0.120 0.137

Anova oneway: p = 0.185

In the second set of experiments a direct comparison was m a d e of formaldehyde

plus BEI treatment versus BEI alone. For each strain 8-10 replicate sets of inactivations
were carried out. The results are summarised in Table II and Figure 2. The statistical
analysis hypothesis investigated was whether the residual 140S concentration was
significantly lower after formaldehyde than after BEI treatment, as the first set of
experiments had already indicated a destructive effect for formaldehyde. Therefore,
a one-tailed t-test was applied to the data.

It was demonstrated for five of the six South American virus strains tested that
formaldehyde significantly reduced the concentration of 140S particles.

Effect of formaldehyde on stability of stored virus

Pairs of eight lots of harvested virus inactivated either with BEI alone ( I m M x 2
for 48 hours) or formaldehyde plus BEI (as described above) were stored at 3-6°C
for six m o n t h s . They were assayed for 140S content at the beginning and end of the
storage period.
 752

FIG. 1
Effect of inactivators on concentration of 140S antigen (%)
in FMD vaccine virus harvests
 753

A-Cast
•- t— Un oo Os oo ON NO en un

BEI
o O t - en en o en ON
r- r- o so CN ON o r- S en
^ H CN CN CN CN ^ CN d o

rmal.
•Cast
NO OO NO un NO NO •sl- en un (--
00 o NO oo ON r— en ON -* ON
OO ON as en oo NO un
o o o CN CN rt
d d
z<
A-Bagé

NO un un 00 O NO en en un
BEI

o as O C*N O NO un oo en

p values: O-Camp = 0.0005; A-Cruz = 0.0011; A-Venc = 0.0275; C-Inda = 0.0334; A-Bagé = 0.3410; A-Cast = 0.018
as Un CN o O r— O CN en
o ^ ^ d d
Formal.
A-Bagé

o r - m oo NO m CN ON oo
un un un oo CN oo en
Effect of formaldehyde and BEI on FMDV 140S concentration

en ON o r— OO oo CN m
o O o d S*
~
C-Inda

1.380
1.030

1.120
0.388
1.000

0.710

0.148
BEI

1.587
1.359
1.508

0.417
rmal.
Inda

o o o o NO r- oo o eN en
CN un NO CN o NO ON
r- r- NO ON NO o en p- CN o
o O o o O 'O d d d
TABLE II

A-Venc

o O o o o NO CN NO en oo
BEI

O ND NO oo ON en ON en r»
CN CN OO o en en en CN o
O o O o o O o d d d d d
Formal.
A-Venc

O o O o en r- m
010'

010'
010

o vo
r—
o un
NO
o CN ten un
O
o o O o o O o o o d d d
A-Cruz

o o o o o O oo ON un un
BEI

en en CN CN oo ON CN un un
en r-; en O
o O o "°.O Ö O o O d d d
Formal.
A-Cruz

o o o o o
010'
010'

ON CN
un t - r- o CN NO
O o o un o CN r-
o
o o Ö o o O o © d d d
a
O-Cam

o o O o o o ON O un ON CN
BEI

NO oo NO en NO OO
00 Os OO oo NO ON un
o O ^ CN O " —' — 1
o o

• a
O-Cam
Formal

o O o o o O CN OO oo NO r-
en ON NO NO en un un NO oo en en NO
NO un OO oo r- oo r- r-- CN o
One-tailed t-test.

O o O o o o o o o o O o
STDEV
Mean

SEM

O) o CN C*N un NO r— oo ON o
en e
 754

(O/u)
UOIJBJJIISOUCO S u r i
FIG. 2

Effect of formaldehyde and BEI on FMDV 140S concentration

 755

The results obtained are summarised in Table III. For BEI inactivated samples,
there was no significant d r o p in the 140S level over the study period. However, the
formaldehyde-treated samples had shown a 33-85% reduction in 140S level; this decay
was shown to be statistically significant by the one-tailed t-test (p < 0.05).

TABLE III

Effect of BEI or formalin on stability of FMDV 140S

Virus BEI-start BEI Percent Formalin- Formalin Percent

lot no. µg/ml) 6 months residua] start 6 months residual
(µg/ml) (µg/ml) (µg/ml)

O-504 1.670 1.839 100.00 0.705 0.286 40.57

O-505 2.119 1.925 90.84 0.852 0.406 47.65
O-506 0.941 1.150 100.00 0.461 0.308 66.81
A-V-260 0.394 0.449 100.00 0.163 0.070 42.94
A-V-263 0.192 0.417 100.00 0.067 0.010 14.93
C-IN-291 1.508 1.744 100.00 0.966 0.354 36.65
C-IN-293 1.587 1.593 100.00 1.008 0.659 65.38
A-Cr-146 0.348 0.362 100.00 0.249 0.100 40.16

Values > 100% recorded as 100%

One-tailed t-test: stored BEI-treated samples n.s. loss (p = 0.599); formald. samples decay significant
(p = 0.0410)

Effect of temperature of BEI inactivation on 140S particles

FMDV inactivation with BEI is usually carried out at 35-37°C with a concentration
of 1.0-1.5mM applied either once for 24 hours or repeated for an additional 24 hour
period (i.e. total 48 h). A recent report by Bahnemann et al. (4) implied that the virus
breaks down under these conditions, and these authors adopted a higher dose of BEI
(3mM) at 26°C for only 24 h.

Two sets of experiments were carried out to investigate this. In the first set, for
each virus strain, 5-15 lots of harvest were inactivated in parallel either by one dose
of 3mM BEI at 26°C for 48 hours or by 2 doses each of 1mM BEI at 36°C with
the second dose after the initial 24 hours. Results of 140S assay are shown in Table
IV and Figure 3 from which it can be deduced that no significant difference was
detected between the lower BEI dose at 36°C and the higher BEI dose at 26°C
(p > 0.3).

In the second set of experiments comparison was made of 3mM BEI at 26°C with
3mM at 36°C for a total period of 48 h. In each case a single dose was applied. Each
virus strain was thus tested in ten replicates with different virus harvests. Results are
summarised in Table V. There was no significant difference in the level of 140S
detected after inactivation at either temperature (p > 0.7), although in general there
was a tendency for slightly lower values at 36°C after 48 hours than at 2 6 ° C .

Efficiency of inactivation of virus infectivity by different BEI procedures

BEI was tested in concentrations of l-3mM at 26°C or 36°C over 48-hour periods.
Samples for inactivation kinetics titration were collected at times 0, 1, 2, 3, 4 hours
 756

TABLE IV

Effect of BEI dose and temperature on concentration of FMDV 140S (µg/ml)

One tailed t-test

p values for O-Camp = 0.3441; A-Cruz = 0.3302; A-Venc = 0.6817; C-Inda = 0.3800
 757

FIG. 3
Effect of temperature and BEI dose on F M D V 140S concentration
 758

TABLE V

Effect of temperature of FMD V inactivation with 3mM BEI on 140S (µg/ml)

c/3
Campos Campos Campos Cruzeiro Cruzeiro Cruzeiro Vences. Vences. Vences. Indaial Indaial Indaial

n
26°C/48h 36°C/24h 36°C/48h 26°C/48h 36°C/24h 36°C/48h 26°C/48h 36°C/24h 36°C/48h 26°C/48h 36°C/24h 36°C/48h

1.299 1.102 0.954 0.469 0.499 0.304 0.645 0.537 0.450 0.777 0.775 0.965
1.528 1.495 1.578 0.034 0.164 0.099 0.704 0.655 0.778 0.645 0.613 0.833

m
0.840 0.903 0.835 0.506 0.512 0.475 0.010 0.034 0.016 1.412 1.296 1.282
1.055 0.968 0.820 0.413 0.317 0.311 0.944 1.218 1.127 0.792 0.615 0.706
2.226 1.590 1.458 0.328 0.468 0.424 0.781 0.589 0.673 1.648 1.836 1.562

NO
0.582 0.592 0.638 0.265 0.253 0.288 0.681 0.552 0.745 1.977 1.904 1.821
1.107 0.834 0.795 0.098 0.056 0.061 1.344 1.149 0.558 2.113 1.552 1.799

q
oo

OO
1.701 1.755 1.644 0.690 0.635 0.664 0.992 1.182 1.351 1.412 1.319

ON
2.694 2.263 2.316 0.427 0.325 0.346 0.044 0.071 0.098 0.226 0.188 0.261

2
m
OO
OO

2.000 2.010 1.934 0.723 0.684 0.746 1.370 1.221 1.007 1.120 0.919

Mean 1.503 1.351 1.297 0.395 0.391 0.372 0.760 0.718 0.685 1.195 1.131 1.147
o
o
>o

STDEV 0.660 0.554 0.569 0.225 0.203 0.218 0.465 0.463 0.422 0.610 0.570
SEM 0.209 0.175 0.180 0.071 0.064 0.069 0.147 0.146 0.134 0.193 0.180 0.159

Anova oneway
p values for O-Camp = 0.728; A-Cruz = 0.966; A-Vences = 0.932; C-Inda = 0.966
 759

and for innocuity testing in B H K monolayer cells at 24 and 48 h o u r s . The results

are summarised in Tables VI and VII.

TABLE V I

Inactivation kinetics of FMD V by BEI

Mean half-life in minutes

Virus strain lmM/26°C 2mM/26°C 3mM/26°C lmM/36°C 2mM/36°C

0 Campos 70.59 39.87 33.79 36.55 16.33

A Venceslau 71.79 30.20 34.27 35.28 23.60
A Cruzeiro 83.24 77.59 119.00 75.62 24.61
C Indaial 185.94 53.39 34.93 28.18 13.30
Overall mean 107.24 44.90 51.09 41.17 18.31
STDEV 72.60 22.41 43.61 17.36 4.98
N 13.00 17.00 15.00 13.00 10.00

Overall mean and standard deviation refer to total n observations

TABLE V I I

Innocuity results of FMD V inactivation by BEI

(Positive/total)

Virus lmMx2 2mM/26°C 3mM/26°C 3mM/26°C lmMx2 2mM/36°C

strain 26°C/48h 48h 24h 48h 36°C/48h 24h

O Campos 1/6 2/6 3/11 0/34 3/19 0/25

A Venceslau 1/13 1/6 3/7 2/23 0/2 0/19
A Cruzeiro 0/2 0/2 1/6 0/17 0/9 0/13
C Indaial 1/4 0/4 0/4 0/12 0/7 0/10
Total 3/23 3/18 7/28 2/86 3/37 0/67
Percent 13 16 25 2 8 0

Positive = virus detected

When BEI inactivation was performed at 26°C, regardless of concentration, or

at a concentration of 1mM regardless of temperature, there-was a risk of incomplete
inactivation of virus infectivity. Procedures at 36°C with BEI doses of 2 m M or above
were found to be safe in terms of F M D V innocuity.

DISCUSSION

Aziridine c o m p o u n d s are efficient inactivators of F M D virus for vaccine

manufacture (24, 35). However, workers at Pirbright demonstrated that certain strains
of serotype SAT 2 were particularly labile to acetylethyleneimine, and that this
destructive effect of A E I could be overcome by pre-fixing the virus with formaldehyde
760

(32, 36). It seemed possible, therefore, that the stability of F M D V vaccines would
be improved by always treating virus harvests with formaldehyde before complete
inactivation with an aziridine c o m p o u n d , such as BEI. However, several studies had
indicated that formaldehyde itself might be deleterious to some strains of F M D V .
For example, Wild and Brown (44) observed that although formaldehyde-inactivated
F M D virus sedimented at 140S, its immunising activity for guinea pigs was much
lower t h a n the A E I inactivated preparation. Girard et al. (27) found that by using
BEI, instead of formalin, to inactivate a strain of type O virus from Turkey, there
was a marked improvement in the potency for cattle of the corresponding vaccine.
Adamowicz et al. (1) demonstrated that formaldehyde degraded the 140S particles
of types O and C virus but not type A . In a more recent study, Ferris et al. (25) found
that formaldehyde had a variable effect on 140S particles, depending on virus strain:
it stabilised a strain of serotype SAT 2, had little influence on the 140S of some strains,
produced a reduction of 140S for some and totally degraded other strains (e.g. A
France 1/68). In several cases these authors also observed that pretreatment of virus
with formaldehyde resulted in antigens of reduced immunogenicity for guinea pigs.

Most of these studies, however, were based on a limited number of observations

and often there was no statistical analysis of the observed effect. In the present study,
an attempt was m a d e to obtain data from statistically analysable sets of replicates.
By so doing, we have shown that 5 of 6 South American strains of FMDV, of serotypes
O , A , C, suffered a significant reduction in 140S compared with intact virus or virus
inactivated with BEI. The exception was strain A Bagé, which was not significantly
susceptible to the action of formaldehyde. Further, we have shown that upon storage
at 4 ° C , formaldehyde-treated 140S particles degrade significantly over six m o n t h s
in contrast to BEI-treated virus. Ferris et al. (25) found that only strain C Resende
decayed upon storage at 4 ° C . But in agreement with our results is the work of Czelleng
et al. (18) who found that of the virus strains studied, there was only 3-30% residual
complement fixing activity after a storage period of five weeks. Therefore, it can
be concluded that far from being a general stabilising agent, formaldehyde can often
degrade the immunising antigen (140S particles) of F M D V .
These observations would seem t o be at variance with the general experience by
users of formalin who claim high potency for formaldehyde-inactivated antigen. The
apparent anomaly is probably explicable on the following grounds. Firstly, such claims
m a y express sentiment more t h a n experimental evidence. Secondly, because of the
curvilinear inactivation kinetics by formaldehyde, it is quite probable that some
residual live virus in the vaccine could contribute to the potency of antigens inactivated
only with formaldehyde. Thirdly, it can be argued that adsorption of virus onto
aluminium hydroxide before inactivation might preserve the virus capsid from the
destructive effects of formaldehyde. However, in their re-evaluation of F M D
inactivation by formaldehyde, Barteling and Woortmeyer (6) did not confirm this
preserving effect of aluminium hydroxide. It should be noted that with adsorbed
antigen, it is difficult to quantify effects on 140S owing to the inefficiency of
quantitative elution of virus from aluminium hydroxide (23, 4 1 , 43). A further
explanation of why the deleterious effects of formaldehyde on virus capsid may not
become readily apparent in routine potency tests, lies in the fact that the relationship
between antigen dose and antibody response for F M D vaccines is sigmoid rather than
linear (9, 39). The effect of changes in antigen content can be masked by responses
being in the upper plateau of the sigmoid curve.
Finally our studies with BEI have shown that concentration (l-3mM) and
temperature of virus inactivation (26°C or 36°C) are important for complete
 761

inactivation of infectivity, but probably not as critical as indicated by the observation

of Bahnemann (4) in terms of 140S particle integrity. We have not been able to confirm
his observation of thermal lability of South American F M D V strains at 36°C within
24 h o u r s . Our results agree with those of Brown and others (11, 13) who showed
that at 37°C F M D virus loses infectivity on account of in situ R N A degeneration,
but without breakdown of the capsid into sub-units. It is probable that culture
constituents or environmental conditions, other t h a n temperature, or the quality of
the primary chemical, bromoethylamine, or the efficiency of its conversion to BEI
may be more critical in the effect of inactivation on the 140S particle t h a n simply
temperature (26°C or 36°C) per se. It is important, therefore, that these parameters
be standardised within the production environment of a vaccine manufacturer.

*
* *

EFFET DU FORMOL ET DE L'ÉTHYLÈNE-IMINE BINAIRE (EIB) SUR L'INTÉGRITÉ

DE LA CAPSIDE DU VIRUS APHTEUX. - M.M. Rweyemamu, O. Umehara, W. Giorgi,
R. Medeiros, D. Lucca Neto et M. Baltazar.

Résumé : Le traitement du virus aphteux par le formol a provoqué une réduction

statistiquement significative (p < 0,05) de la concentration des particules 140S
(26 à 72%) chez cinq des six souches sud-américaines de virus vaccinal testées.
Le formol a également réduit la stabilité du virus ; après stockage à 4°C pendant
six mois, la concentration des particules 140S a baissé de 33 à 85%.
En revanche, pour les échantillons traités par l'éthylène-imine binaire (EIB),
il n'y a pas eu de réduction significative des particules 140S, ni immédiatement
après l'inactivation ni après stockage. L'efficacité de l'inactivation du pouvoir
infectant par l'EIB a été influencée par la concentration du produit et par la
température. Aucune réduction significative des 140S n'a été observée en 24
heures à 36°C.

MOTS-CLÉS : Aphthovirus - Ethylène-imines - Fièvre aphteuse - Formol -

Inactivation - Vaccins.

*
* *

EFECTO DEL FORMOL Y DEL ETILENOIMINO BINARIO (EIB) EN LA INTEGRIDAD

DE LA CAPSIDA DEL VIRUS AFTOSO. - M.M. Rweyemamu, O. Umehara, W. Giorgi,
R. Medeiros, D. Lucca Neto y M. Baltazar.

Resumen: El tratamiento del virus aftoso con formol ha provocado una

reducción estadísticamente significativa (p < 0,05) de la concentración de
partículas 140S (del 26 al 72%) en cinco de las seis cepas sudamericanas de virus
vacunal probadas. El formol también redujo la estabilidad del virus. Así, al
cabo de 6 meses de almacenamiento a 4 °C, la concentración de las partículas
140S había disminuido entre un 33 y un 85%.
En cambio, para las muestras tratadas con etilenoimino binario (EIB), no
hubo disminución significativa de las partículas 140S ni inmediatamente después
de la inactivación ni después del almacenamiento. La eficacia de la inactivación
del poder infectante por el EIB fue influenciada por la concentración del
producto y por la temperatura. No se observó ninguna reducción significativa
de las 140S en 24 horas a 36°C.
762

PALABRAS CLAVE: Aftovirus - Etilenoiminos - Fiebre aftosa - Formol -

Inactivación - Vacunas.

*
* *

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