Download Differentiation of Rubella Virus Strains by Neutralization Kinetics

J. gen. Virol. (198o), 49, 423-426
423
Printed in Great Britain
Differentiation of Rubella Virus Strains by
Neutralization Kinetics
(Accepted 20 February ~98o)
SUMMARY
The neutralization of rubella virus was investigated and shown to proceed by first
order kinetics over the first Io to i5 min. A comparison of the rate constant of
neutralization (K) for six strains of rubella virus was carried out over this period of
the reaction. Two particularly antibody-sensitive isolates were detected. It was
noted that the three strains isolated from in utero infections were poorly neutralized
by heterologous antisera when compared to postnatal strains.
Although several highly sensitive serological tests have been used successfully for the
assay of rubella virus antibodies, including radio-immunoassay (Meurman et al. 1977),
enzyme immunoassay (Gravell et aL t977) and enhanced plaque neutralization (Sato et al.
979b), none are readily adaptable for the characterization of antigenic differences between
various rubella virus isolates or strains.
Simple haemagglutination inhibition has been applied to this type of analysis (Banatvala
& Best, I969), but neither this nor the kinetic haemagglutination inhibition test devised by
Best & Banatvala 097o) could detect a difference in the antigenic make-up of the various
strains of rubella virus examined. The neutralization test is, however, highly suitable for
comparative studies (Mandel, I978); Oxford (t 969) was able to demonstrate small antigenic
differences between strains of rubella virus using a 5o % infectious dose assay of surviving
virus. In the same year Fogel & Plotkin (I969) demonstrated some antigenic differences by
cross-neutralization tests employing a plaque reduction method. The development of a
highly reliable and reproducible method of plaque assay in GL-RK13 ceils (Gould et al.
197z), has made detailed investigation of the neutralization of the virus possible. This
report presents an examination of the neutralization kinetics of a standard strain and a
comparison of the rates of neutralization of six other strains of known history.
Six strains of rubella virus of varied history were selected for study (Table 1). Stocks of
virus were grown in Vero cells and stored at - 7 o °C. The standard virus used was a pool
of the RA27/3 strain grown in WI-38 cells, lyophilized and stored at - 2o °C. Veto cells
were obtained from the Public Health Laboratory Service at Colindale, U.K., and GL-RK13
cells from the National Institute of Health, Bethesda, U.S.A. The growth medium for both
cell cultures consisted of Eagle's MEM containing o.t 1% N a H C Q and 5 % foetal calf
serum, while maintenance medium contained o.22 % NaHCO~ and 28 % foetal calf serum.
Details of the plaque assay are given elsewhere (Gould et al. I972).
California strain rabbits received four successive intravenous inoculations of undiluted
virus at 7 to Io day intervals and were bled out from the heart I3 weeks after the first
inoculation. Rabbits receiving virulent virus were housed separately from those receiving
the attenuated strains and untreated rabbits housed with both failed to produce detectable
antibody to rubella virus at any time. Sera were separated and stored at - 2 o °C. The
standard pool of hyperimmune serum used was produced against the Edmunds strain of
rubella virus (Banatvala & Best, I969).
Dilutions of antiserum (2 ml) were mixed with equal volumes of virus suspension containing approx lo 33 p.f.u. The mixture was incubated in a sealed Bijoux bottle in a water
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424
T a b l e 1. K values for replicate tests on six systems of homologous antiserum and
antigen mixture
Antigen
Origin
Passage history
Mean K value
Mean NK*
Lesley
HPV-77
Cendehill
Dunning
Adult with rubella
Adult with rubella
Adult with rubella
Rubella syndrome
infant
Rubella syndrome
infant
Rubella-infected
conceptus
MK(3)
MK(78)
YRK(5~)
RKaa(4)
2'94 _ o
2"72 + 0-05
I'6O_+O'O3
2"5o ___o"I4
Ioo + o
1oo + 1-63
Ioo_+ 1"63
lOO+ 3'26
RKxa(3)
2.55 ---o.18
lOO_+4"31
H EK(4)
WI-38(32)
1.6o +_o"13
I oo _+8"16
Thomas
RA27/3
* Mean normalized K value.
Table 2. Normalized K values for each antiserum assayed separately with the
six different antigenic types
Antigens
Antisera
RA27/3
HPV-77
Cendehill
Dunning
Thomas
Lesley
RA27/3
HPV-77
Cendehill
Dunning
Thomas
Lesley
Ioo
53'3
73
67
66
3o
Io9"5
ioo
135
80
81
117
46
55"5
IOO
70
53
51
88
100
91
5o
57'5
66
61 '5
27
111
218
I45'5
210
102" 5
56
1(30
125
44
ioo
bath at 32.5 °C. Samples (o.I ml) were withdrawn at intervals and t i t r a t e d in m o n o l a y e r
cultures o f GL-RK13 cells. Mixtures o f p r e - i m m u n e serum with virus a n d s t a n d a r d s e r u m
with s t a n d a r d virus were similarly treated. T h e surviving virus was plotted on a l o g a r i t h m i c
scale against time on a linear scale and the rate o f neutralization per minute, the K value,
was calculated from the initial slope o f the curve where the reaction was a p p a r e n t l y first
order, i.e. a straight line passing t h r o u g h the origin (Dutbecco et al. 1956 ).
A c o m p a r i s o n o f the neutralization o f one antigen with different antisera is only possible
if the K values are n o r m a l i z e d (McBride, 1959), because the a n t i b o d y c o n t e n t o f each p o o l
o f a n t i s e r u m c a n n o t be standardized. N o r m a l i z e d K values represent the rate at which a
particular serum neutralized a h e t e r o l o g o u s virus as c o m p a r e d to the rate at which it
neutralized the h o m o l o g o u s virus, which is t a k e n as IOO. In any one test, one serum was
assayed against all the strains o f rubella virus to minimize variations between separate
experiments.
It was f o u n d t h a t a dilution o f 1/8 o f the s t a n d a r d a n t i s e r u m ( E d m u n d ) gave a reaction
curve with a s t a n d a r d p o o l o f R A 2 7 / 3 virus in which first o r d e r kinetics were observed for
a p p r o x i m a t e l y the first lo to 15 min. T h e results for thirteen replicate tests showed a g o o d
level o f reproducibility, with a s t a n d a r d deviation o f + 6-920 for the n o r m a l i z e d K value.
F o r all other investigations of the test sera, a dilution o f 1/8 was also used and t h e K values
determined for the first I o rain o f reaction time. T h e K values for all six h o m o l o g o u s
reactions, which were done in triplicate, showed very little v a r i a t i o n between replicates
(Table l).
T h e normalized K values for each antiserum d e t e r m i n e d with all the antigens u n d e r study
are given in T a b l e 2 and each value represents the m e a n o f two experiments. T w o strains o f
virus, HPV-77 and Lesley, a p p e a r e d to be p a r t i c u l a r l y sensitive to h e t e r o l o g o u s a n t i b o d y .
Thus, the n o r m a l i z e d K values for h e t e r o l o g o u s antisera with Lesley were, in all cases,
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higher than the homologous value. For HPV-77 virus, only two antisera, that is Dunning
and Thomas gave normalized K values less than IOO.With the other strains of virus, RAz7/3,
Cendehill, Dunning and Thomas, heterologous antisera often gave normalized K values
which were much less than Ion. In one instance with Thomas antiserum there was evidence
of identity with a heterologous antigen, Cendehill, but the relationship was not reciprocal.
The virus strains originating from cases of intra-uterine infection, RA27/3, Dunning and
Thomas, showed a reaction of identity only with homologous antisera whereas the other
strains, all isolated from post-natal infections we]'6 efficiently neutralized by some or all of
the heterologous antisera.
The mechanism and nature of the neutralization test is still unresolved (MandeI, I978),
but results reported here are in b.greement with Rawls et al. 0967), in that, for rubella virus,
neutralization proceeds by first order reaction over the first i o to 15 min. On this premise,
it is concluded that the varying K values recorded for different rubella virus isolates imply
some antigenic diversity and the most interesting feature of this diversity is the differentiation between viruses isolated from intra-uterine and post-natal infections. The apparent
differences between viruses isolated from in utero and post-natal infections deserves further
examination, if only to determine whether the prolonged in vivo growth of the former types
might result in the selection of variants sufficiently different antigenically from the parental
strain to account for the persistence of the virus. Such antigenic drift has already been
reported for equine infectious anaemia virus (Kono et al. I973) and Visna virus (Narayan
et al. I977).
There is no doubt that prolonged in vitro cell passage of rubella virus in cell culture may
lead to alterations in certain properties such as virulence of vaccine strains for man (Parkman
et al. 1966; Plotkin, i969), loss of ability of RA27/3 to exhibit intrinsic interference
(Kleiman & Carver, 1977) and diminished plaque size in two fresh isolates (Gould et al.
I972). In this regard, however, Sato et al. 0979a) found plaque characteristics of the vaccine
strains preserved after five serial subcultures, which confirmed a similar observation made
by Gould et al. (1972). The unique properties of Cendehill virus, such as the unusual pattern
of antigenicity for rabbits and monkeys (Oxford & Potter, I97O; Zygraich et aL t97I), the
delayed antibody response in man (Moffat et al. I972) and the characteristic plaque morphology (Gould et aL I972; Sato et al. I979a), may be due to the prolonged passage history in
rabbit kidney cells. This has not been investigated, however, but it would be of great
interest to compare the serological and other properties of the vaccine strains with the
original isolates from which they were developed.
Whether or not antigenic changes develop during passage of the virus in cell cultures has
not been shown although it is also worth noting that extensive passage of virus isolates in
cell culture did not seem to influence antigenic specificity or diversity, since Lesley virus,
with only three subcultures, was heterospecific as was HPV-77 virus after 83 subcultures and,
incidentally, both were from post-natal infections. Furthermore RA27/3 virus, isolated
from an intra-uterine infection and which had undergone 32 subcultures, was just as homospecific as Thomas virus, which also originated from an intra-uterine infection, with only
five subcultures.
Wellcome Research Laboratories
Beckenham, Kent, U.K.
~5
JANINE J. GOULD
MICHAEL BUTLER
V I R 49
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426
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