J. gen. Virol. (1985), 65, 309-316. Printed in Great Britain
309
Key words: poliomyelitis virus/intertypic recombinant/neurovirulence/RNA- mutation
Neurovirulence of the Intertypic Poliovirus Recombinant v3/al-25:
Characterization of Strains Isolated from the Spinal Cord of Diseased
Monkeys and Evaluation of the Contribution of the 3' Half of the Genome
By V. I. A G O L , * S. G. D R O Z D O V , M. P. F R O L O V A , V. P. G R A C H E V ,
M. S. K O L E S N I K O V A , V. G. K O Z L O V , N. M. R A L P H , L. I, R O M A N O V A ,
E. A, T O L S K A Y A AND E. G. V I K T O R O V A
Institute of Poliomyelitis and Viral Encephalitides, The U.S.S.R. Academy o f Medical Sciences,
Moscow Region 142782, and Moscow State University, Moscow 119899, U.S.S.R.
(Accepted 8 October 1984)
SUMMARY
A tsRNA- intertypic recombinant, v3/al-25, which has the 5' and 3' halves of the
genome derived from the neurovirulent type 3 poliovirus strain 452/62 3D and the
attenuated type 1 poliovirus strain LSc-gr3, respectively, was previously shown to cause
severe paralytic poliomyelitis after intracerebral inoculation of monkeys. To ascertain
whether the illness was caused by the recombinant itself or by temperature-resistant
trRNA ÷ mutants that might have arisen in the inoculated monkeys, five independent
virus strains have been isolated from the spinal cord of the diseased animals. While two
of these isolates exhibited RNA ÷ and RNA -+ phenotypes, respectively, the other three
strains retained the parental R N A - character. Except for the R N A + strain, the RNase
T1 oligonucleotide maps of the genomes of all the isolates revealed only a minimal
deviation from the parental pattern. These results were interpreted to mean that v3/al25 is intrinsically neurovirulent despite the presence of a tsRNA- mutation(s) in the 3'
half of its genome. Nevertheless, this mutation, or other peculiarities of the 3' half of
the recombinant genome, may somewhat alleviate the pathogenicity of the virus. This
notion was inferred from the fact that, when used in a relatively small dose (about 103
p.f.u.), v3/al-25 appeared to exhibit a lower level of neurovirulence compared to either
the wild-type parent 452/62 3D, or a closely related intertypic recombinant having the
genome 3' half derived from a neurovirulent trRNA -+ type 1 poliovirus strain. The
problem of genetic determination of poliovirus neurovirulence and attenuation is
briefly discussed.
INTRODUCTION
Recently, we devised a method for constructing poliovirus recombinants that derived two
segments of the genome, each comprising roughly half of the RNA molecule, from parents
belonging to different serotypes (Tolskaya et al., 1983). Using the virulent and attenuated strains
of poliovirus type 1 and type 3, we constructed several recombinants of this kind and assayed
their neurovirulence by intracerebral inoculation of monkeys (Agol et al., 1983, 1984).
Irrespective of the origin of the 3' half of the genome, the recombinants that inherited the 5' half
from the neurovirulent parent were neurovirulent, whereas those that inherited a homologous
segment from the attenuated parent were not. These observations could be most simply interpreted to mean that the major determinants of neurovirulence of the recombinants studied were
located on the 5' halves of their genomes.
However, this interpretation, as already discussed (Agol et al., 1984), relied on several
assumptions. Thus, we assumed that the illness was caused by the recombinants themselves,
rather than by mutants that might have arisen (or been selected) in the central nervous system
(CNS). Such an assumption was particularly important for interpreting the results obtained with
0000-6236 (~) 1985 SGM
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310
V. I. AGOL AND OTHERS
the neurovirulent recombinant v3/al-25. The 5' and 3" halves of its genome were derived from
virulent type 3 (452/62 3D) and attenuated type 1 (LSc-gr3) strains, respectively, and the LScgr3-derived segment harboured a temperature-sensitive tsRNA- mutation(s) (Agol et al., 1984).
The possibility existed that the disease was actually due to temperature-resistant t r R N A ÷
mutants of v3/al-25, and not to the recombinant itself. To ascertain whether this was indeed the
case, we isolated several strains from the spinal cord of monkeys that developed paralytic poliomyelitis after intracerebral infection with v3/a 1-25. The isolates were compared to v3/a 1-25 with
respect to their ts and RNA phenotypes and by RNase T1 fingerprints of the genome. The
results reported here strongly suggest that v3/al-25 is intrinsically neurovirulent despite the
presence of a tsRNA- mutation(s) in the 3' half of its genome.
This does not mean, however, that the above mutation (or the entire 3' half of the v3/al-25
genome) is of no significance for the level of pathogenicity. In fact, we have noted previously
that the incubation period preceding the onset of clinical signs of poliomyelitis was, on average,
somewhat longer in monkeys inoculated with v3/al-25 than in monkeys inoculated with v3/v I-1
(the latter recombinant differed from the former by having the 3' half of the genome derived
from the virulent strain Mgr: Agol et al., 1984). Proceeding from this observation, we tentatively
suggested that the 3' half might modulate the level of neurovirulence although the latter was
largely determined by the 5' half of the recombinant genome. This suggestion has now been
verified by investigating the effect on poliomyelitis-inducing capacity of lower inoculation doses
of the two recombinants. When a dose of about 103 p.f.u, w a s used, v3/al-25 exhibited a
diminished level of virulence, as compared to v3/vl-1.
METHODS
Viruses. The origin and properties of poliovirus strains 452/62 3D, v3/vl-1 and v3/al-25 have been described
previously (Tolskaya et al., 1983; Agol et al., 1984). For the isolation of strains from the CNS of the virusinoculated monkeys, a I 0 ~ suspension of the tissue of the spinal cord lumbar enlargement was prepared in
physiological saline and inoculated, in a dose of 0.1 ml, into tubes with replicate monolayer cultures of monkey
kidney cells. Incubation of the infected cultures was carried out at 33 °C, 37 °C, 38.5 °C and 40 °C, and those
cultures which developed a cytopathic effect at the lowest temperature were chosen for further study. The isolated
strains were passed twice at 36 °C in HeLa cells before use.
Neurovirulence assay. Viral neurovirulence was tested by intracerebral inoculation of Cercopithecus aethiops
monkeys as described previously (Agol et al., 1984). To evaluate the severity of histological damage, two indices, S
(spread) and LS (lesion score), were used. Index S represented the number of levels of the CNS with specific histological lesions, the maximal value for a single monkey being 29 (the number of levels investigated). Index LS was
calculated as the average score of histological lesions (including zero scores) in one hemisection, the maximum
value being 4. It should be noted that evaluation of the lesions in the CNS of the virus-inoculated animals in the
present and previous (Agol et al., 1984) studies was done by different investigators; the absolute values of the LS
indices in these two reports should not, therefore, be directly compared.
Determination of viral RNA synthesis. Replicate monolayer cultures of HeLa cells (4 x 105 to 5 x 105 ceils) were
infected at an input multiplicity of about 100 p.f.u./cell and, after I h adsorption at room temperature and removal
of the unadsorbed virus, they were transferred to either 36.5 °C or 39.5 °C. At appropriate time intervals, 1 ~tg/ml
actinomycin D (Calbiochem) was added, followed after 15 rain by a mixture of 3H-labelled precursors (uridine,
adenosine and cytidine with specific activities of 20 to 30 Ci/mmol; 2.5 to 5 ~tCi/ml). The cultures were frozen 15
min later, and incorporation of label into TCA-insoluble material was determined. To evaluate the background
actinomycin D-resistant nucleoside incorporation, control cultures were infected with a guanidine-sensitive
poliovirus strain in the presence of I00 vtg/ml guanidine-HCl and were treated similarly to the above samples.
Oligonucleotide mapping of the viral genomes. RNA isolated from purified virus preparations was treated with
RNase T1, the resultant oligonucleotides were 5'-labelled using [~-32p]ATP and bacteriophage T4 polynucleotide
kinase and subjected to two-dimensional electrophoresis as described previously (Agol et al., 1984). It should be
pointed out that the relative intensity of some spots varied somewhat from experiment to experiment, but the
pattern itself was highly reproducible.
RESULTS
Virus isolation f r o m the central nervous system o f diseased monkeys
F i v e v i r u s s t r a i n s were i s o l a t e d f r o m t h e s p i n a l c o r d o f i n d i v i d u a l p a r a l y s e d m o n k e y s w h i c h
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Neurovirulence of polio recombinants
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label incorporation into the TCA-insoluble material was determined. The concentration of guanidineHC1 (the upper rightward
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X, and the newly acquired spots are arrowed; the black spot is shared by all of the five isolates but
appears to be absent from the parental genome,
Fig. 2. Oligonucleotide maps of the genomes of five isolates and of their parental virus v3/a 1-25. On the
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V. I. AGOL AND OTHERS
Table 1. Characterization of the diseased monkeys from which poliovirus strains were isolated
Isolate*
M4973
Dose of
v3/al-25
inoculated
(p.f.u.)
3 x l0 s
Incubation
period
(days)
11
Day of
killing
12
M4846
3 x 105
9
11
M4953
5 × 107
2
4
M4956
5 x 107
6
7
M4964
5 x 107
10
13t
Clinical and pathological findings
Paralysis of lower limbs and weakness of one of the upper
limbs; moderate to severe lesions at all the CNS levels
studied.
Partial paralysis of lower limbs and weakness of upper limbs;
moderate to severe lesions at all the CNS levels studied.
Partial paralysis of lower limbs; mild to moderate lesions at
many CNS levels.
Paralysis of lower limbs and weakness of upper limbs;
moderate to severe lesions at nearly all the CNS levels
studied.
Tetraplegy; moderate to severe lesions at nearly all the CNS
levels studied.
* Monkeys4973 and 4846 were from the experiment presented in Table 2 of this report, whereas monkeys 4953,
4956 and 4964 were from the experiment described in Table 4 of our previous study (Agol et al., 1984).
t Died.
Table 2. Effect of the inoculation dose on the poliomyelitis-inducing ability of recombinants
Histological lesions
r
Strain
452/62 3D
v3/vl-1
v3/al-25
Inoculation
dose
(p.f.u.)
2 x 105
2 × 103
4 x 105
4 x 103
3 x 105
3 x 103
Number
of monkeys
inoculated
3
3
7
5
6
4
Number of
monkeys with
histological
signs of
poliomyelitis
3
3
7
5
6
4
Spread*
(S)
27.0
26.7
27.3
23-0
26.7
10.7
Lesion
scoret
2-80
2.88
2.85
2-26
2.15
0.85
Number of
monkeys with
clinical
signs of
poliomyelitis
3
3
7
5
6
2
* The average number of levels of CNS with specific histological findings.
t The average score of histological lesions in one hemisection.
animals from which the viruses were isolated are briefly given in Table 1. Strains M4956, M4964
and M4973 were isolated from cultures incubated at 33 °C, while strains M4846 and M4953 were
isolated from cultures incubated at 37 °C. No virus strains could be isolated at 40 °C, except for
monkey 4964. Parallel plaque virus titration of the spinal cord suspensions revealed roughly l04
p.f.u./g tissue in monkeys 4964 and 4973, and about 102 p.f.u./g tissue in the remaining
monkeys.
Some phenotypic properties of the isolates
The ratio of efficiency of plaquing at 40 °C and 33 °C in HeLa cells was less than 10-s for all of
the isolates (as well as for the parental strain v3/al-25), except M4964, which formed
approximately the same n u m b e r of plaques at either temperature (data not shown). Thus, the
latter strain should be classified as tr (or ts +) whereas the remaining ones exhibited a ts
phenotype.
The ability of the isolates to synthesize virus-specific R N A at 39.5 °C was studied. The results
shown in Fig. 1 demonstrate that three strains, i.e. M4953, M4973 and M4846, like their parent
v3/al-25, failed to synthesize appreciable amounts of viral R N A at this temperature and all
should be assigned an R N A - phenotype. In this respect, these strains resembled LSc-gr3, the
donor of the 3' half of their genomes (see Agol et al., 1984). M4964, on the other hand, acquired
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Neurovirulence of polio recombinants
315
an R N A + phenotype. M4956 synthesized intermediate amounts of viral R N A at the restrictive
temperature and was assigned the RNA -+ phenotype.
Fingerprinting of the viral genomes
The RNase TI oligonucleotide maps of the genomes of the five isolates as well as of their
parental virus v3/a 1-25 are shown in Fig. 2. It is seen that the maps of the isolates resembled very
closely that of their parent. In fact, the fingerprints of four out of five isolates revealed only a
single spot common to all of them in addition to an apparently complete set of spots
characteristic of the fingerprint of v3/a 1-25. Strain M4964 was an exception: it exhibited a more
deviant pattern, with several parental oligonucleotides missing and new oligonucleotides
present (Fig. 2).
Neurovirulence studies
Our previous observations allowed us to consider the possibility that the 3' half of the genome
might modulate to some extent the expression of the pathogenicity of the recombinants (Agol et
al., 1984). In order to evaluate this modulating effect, we now compared the neurovirulence of
v3/al-25 and v3/vl-1 (two recombinants which shared the 5' segment of the RNA molecule but
inherited its 3' segment from attenuated and virulent parents, respectively) using lower
inoculation doses than previously (Agol et al., 1984). As shown in Table 2, v3/v-l, like its type 3
virulent parent 452/62 3D, expressed almost the same high level of pathogenicity irrespective of
the dose inoculated (in the range of 103 to 105 p.f.u.). On the other hand, v3/al-25, being also
highly virulent at 3 x 105 p.f.u., exerted a markedly milder effect at 3 x 103 p.f.u. At this latter
dose, v3/al-25 exhibited a reduced level of neurovirulence, as compared to v3/vl-l, in terms of
both the proportion of animals paralysed and the severity of morphological injuries in the
central nervous system.
DISCUSSION
Two points were addressed in this study. The first concerned the properties of the viruses
isolated from the CNS of monkeys paralysed after inoculation with the intertypic poliovirus
recombinant v3/al-25. In particular, we were interested to learn whether the isolates retained
the t s R N A - phenotype of the parental virus. A positive answer was obtained for three of the
five isolates studied. This fact supports our earlier contention that the existence of t s R N A mutation(s) is by itself not sufficient to endow the virus with a non-virulent phenotype.
Furthermore, all the three tsRNA- isolates (M4846, M4953 and M4973) appeared to have
undergone only a minimal genetic deviation from their parent. The remote possibility remains
that the changes in the genomes of the isolated viruses detected by fingerprinting (or even some
undetected mutations) are essential for neurovirulence. We consider it more likely, however,
that v3/al-25 itself is intrinsically neurovirulent.
At the same time, our results suggest that the loss of the t s R N A - character may confer a
selective advantage on the virus with regard to its ability to grow in the CNS. Thus, several
clones isolated from monkey no. 4964 all exhibited the tr phenotype (unpublished observations).
It seems possible, therefore, that the paralytic poliomyelitis in some monkeys inoculated with
v3/al-25 might have been caused by mutants which could have evolved prior to or during the
multiplication of the virus in the CNS. We would like to emphasize, however, that emergence of
such mutants does not appear to be a precondition for the manifestation of the disease.
The second point of this study concerned the possible contribution of the 3' half of the viral
genome to neurovirulence. The results showed that this part of the attenuated poliovirus type 1
genome when recombined with the 5' half of the virulent type 3 genome, may alleviate to some
extent the virulence of the recombinant. It is tempting to propose that this modulating effect is
caused by the tsRNA- mutation discussed above, present in the 3' half of the recombinant R N A
molecule.
Thus, the aggregate results obtained to date with intertypic (type 3 x type 1) poliovirus
recombinants support the notion that the 5' half of their genome contributes to neurovirulence
(attenuation) to a significantly greater extent than the 3' half does.
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316
V. I . A G O L
AND OTHERS
When considering the genetic determination of poliovirus neurovirulence in more general
terms, one may envisage two models. The first postulates the existence of a single (or a few)
property(ies) of the attenuated virus that specifically deprives it of the ability either to infect,
multiply in, and injure the target neural cells, or spread through the CNS. Virus strains
possessing this property will be attenuated irrespective of their reproductive capacity in other
host cells. Obligatory changes of this specific property are needed for the virus to be neurovirulent. In the framework of this model, the genetic locus responsible for this critical property
may be regarded as the major genetic determinant of attenuation (neurovirulence). Needless to
say, alterations in other regions of the genome that non-specifically either enhance or diminish
the reproductive capacity of virus strains may modulate the level of their pathogenicity. The
second model does not invoke any specific properties of poliovirus other than those involved in
its general reproductive capacity in sensitive cells of primate origin. Here, attenuated strains
differ from neurovirulent ones merely in the diminished yield of infectious progeny and,
possibly, reduced cytopathogenicity. For example, introduction of ts mutations into any region
of the vrial genome will be expected to result in an equal level of attenuation, provided these
mutations equally restrict the virus growth at the body temperature of the infected organism.
Data on the neurovirulence of poliovirus recombinants accumulated so far do not allow a clear
discrimination between these models. Nevertheless, the greater contribution of the 5' half of the
genome to the neurovirulence of the recombinants studied, as well as the relatively minor effect
of the strong tsRNA- mutation situated in the 3' half, suggest that a model implicating specific
viral properties in neurovirulence deserves serious attention. It is also appropriate to note that
among numerous mutations distributed over nearly the entire genome, of foot-and-mouth
disease virus, only a ts mutation in the capsid protein-coding region (probably a region coding
for polypeptide VP3) appeared to abolish the neurovirulent properties of the virus (McCahon et
al., 1981). On the other hand, a comparison of the partial nucleotide sequences of the genome of
a virulent type 3 poliovirus, its attenuated derivative and a virulent revertant of the latter failed
to reveal any changes in the amino acid sequence of the capsid polypeptides that could
unambiguously be linked to variation in neurovirulence (Stanway et al., 1983). It should be
remarked that major determinants of neurovirulence, if indeed located on the 5' half of the poliovirus genome, need not necessarily be related to virus-specific proteins; this segment of the
RNA molecule also encompasses a long untranslated region of potential regulatory importance
(Kitamura et al., 1981; Racaniello & Baltimore, 1981).
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