J. gen. Virol. (1985), 66, 171-176.
Printed in Great Britain
171
Key words: tobravirus/TR V serotypes/PEB V/RNA sequence homology
Unequal Variation in the Two Genome Parts of Tobraviruses and Evidence
for the Existence of Three Separate Viruses
By D . J. R O B I N S O N *
AND B. D . H A R R I S O N
Scottish Crop Research Institute, lnvergowrie, Dundee DD2 5DA, U.K.
(Accepted 20 September 1984)
SUMMARY
Hybridization experiments using complementary D N A copies showed that 14
tobravirus isolates could be divided into three groups, comprising ten of tobacco rattle
virus (TRV) serotype I-II, one Of TRV serotype III, and three of pea early-browning
virus (PEBV), respectively. R N A from members of each group has much nucleotide
sequence homology with R N A of other members of the same group but little or none
with R N A from members of the two other groups. The ten TRV serotype I-II isolates
share extensive sequences in their RNA-1 species, but show much diversity in their
RNA-2 species and in the antigenic relatedness of their particles. However, homology
was found between the RNA-2 sequences of isolates that are serologically closely
related. Because strain CAM (TRV serotype III) is as different from isolates of TRV
serotype I-II in nucleotide sequence, antigenic specificity and biological properties as is
PEBV, it is now considered to be a separate virus and a return to its original name,
pepper ringspot virus, is recommended.
INTRODUCTION
The tobravirus group includes only two definitive members, tobacco rattle virus (TRV) and
pea early-browning virus (PEBV). The nucleoprotein particles of each of these viruses are of two
predominant lengths, referred to as long (L) and short (S). L particles of different isolates have
rather similar modal lengths: 185 to 197 nm for TRV (Harrison & Woods, 1966) and 200 to 220
nm for PEBV (Bos & van der Want, 1962; Harrison, 1966), whereas the S particles in different
isolates range from 50 nm to 115 nm in modal length. The genome consists of two singlestranded R N A species (RNA-1 and RNA-2) contained in L and S particles respectively
(Harrison & Robinson, 1978). The particle protein gene is in RNA-2 (S/inger, 1968).
A remarkably wide range of serological variation is found among TRV isolates. Isolates were
formerly separated into three serotypes (Harrison & Woods, 1966) but intergrading isolates are
known to occur in serotypes I and II, and it seemed better to combine these in an antigenically
somewhat heterogeneous group known as serotype I-II. However, the distinctness of serotype
III, represented by an isolate from Brazil, has been confirmed by subsequent work (Harrison &
Robinson, 1978). Two serotypes of PEBV have been reported, comprising British and Dutch
isolates respectively (Gibbs & Harrison, 1964; van Hoof, 1969). A Dutch isolate of PEBV is
serologically distantly related to a TRV serotype I-II isolate (Maat, 1963), but no such
relationship was detected using a British isolate of PEBV (Gibbs & Harrison, 1964).
Tests for nucleotide sequence homology, by hybridization with complementary D N A
(cDNA) copies, showed that the R N A - I species of two TRV serotype I-II strains are almost
identical, whereas that of a serotype III strain is quite different (Robinson, 1983). The RNA-2
species of the three strains had no detectable sequence homology with one another. It was
suggested that, within TRV serotype I-II, the nucleotide sequence of RNA-1, like its size, may
be strongly conserved, whereas the RNA-2 sequence may be very variable. Support for this
hypothesis came from tests on TRV isolates from narcissus, all of which were detected by
hybridization with c D N A to RNA-1 of TRV strain PRN, but fewer than half of which reacted
with antiserum to the same TRV strain (Harrison et al., 1983).
To get a more general picture of nucleotide sequence relationships within the tobravirus
group, nucleic acid hybridization experiments have now been done with 14 isolates of TRV and
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172
D.J.
ROBINSON AND B. D. HARRISON
P E B V , chosen for the diversity o f their o t h e r properties. T h e results confirm and e x t e n d
previous findings, and are c o n s i d e r e d to justify the s e p a r a t i o n o f T R V serotype I I I as a distinct
virus.
METHODS
Virus isolates. The following isolates of TRV were used. ORY, the yellow strain of Lister & Bracker (1969),
isolated from potato in Oregon, U.S.A. The culture used (OR3) had been freed of a mutant RNA-2 species
(Robinson, 1983). ORM and ORS, the mild and severe strains, respectively, of Lister & Bracker (1969), isolated
from the same source as ORY. SYM, a strain from spinach in England, characterized by Kurppa et al. (1981).
PRN, the type strain of TRV, originally isolated from potato in Scotland (Cadman & Harrison, 1959). HSN, an
isolate from tobacco in Japan (Tomaru & Nakata, 1967). NZP, an isolate from peony in New Zealand (Jones &
Young, 1978). RQ, an isolate obtained from a cucumber bait plant grown in soil from a field at SCRI (Cooper,
1972). RH, an isolate from Helianthus rigidus growing at Broughty Ferry, Scotland (R. I. Hamilton, unpublished
results). ASH, an isolate from Fraxinus mariesii growing in England (Cooper et al., 1983), kindly supplied by Dr
J. I. Cooper. CAM, the original serotype III strain, obtained from Bidens sp. in Brazil (Harrison & Woods, 1966).
The following isolates of PEBV were used. SP5, the type strain of the British serotype, originally obtained from
pea growing at Sporle, Norfolk, England (Gibbs & Harrison, 1964). SHE, an isolate obtained in 1970 from pea
grown in soil from Shernbourne, Norfolk. E116, a typical isolate of the Dutch serotype, recovered from pea seeds
kindly supplied by Dr L. Bos.
Isolates ORY, ORM, ORS, HSN, NZP, CAM and E l l 6 were held under licence from the Department of
Agriculture and Fisheries for Scotland.
Purification of virus particles and extraction of RNA. All isolates were propagated in Nicotiana clevelandii. Virus
particles were purified from leaf extracts in either 0-067 M-phosphate, pH 7.3 or 0-02 M-borate, pH 8-3, as described
for strain ORY by Robinson (1983). Some preparations were heated at 50 °C for 10 min and centrifuged at low
speed to remove residual impurities (Harrison & Woods, 1966). L particles of strain SYM were obtained as
described by Kurppa et al. (1981). RNA was extracted from virus particles as described by Robinson et al. (1983).
Complementary DNA-RNA hybridization. Preparation of 3H-labelled cDNA copies of unfractionated virus
RNA and of RNA-1, conditions for hybrid formation and conditions for assay using S1 nuclease were as described
by Robinson et al. (1980).
Serological tests. Immunosorbent electron microscopy (ISEM) was performed as described by Roberts &
Harrison (1979). Tube precipitin tests were done as described by Harrison & Woods (1966).
Electron microscopy. Lengths of virus particles were measured from electron micrographs of specimens stained
at pH 6-5, either with 2% ammonium molybdate after fixation in osmium tetroxide, or with 2% sodium
phosphotungstate, and were recorded and classified using an ID-TT-20 Tektronix digitizer linked to a 4051
graphic system.
RESULTS
Properties o f the lesser known virus isolates
M o s t of the isolates included in this study are already c h a r a c t e r i z e d but there is little or no
published i n f o r m a t i o n on others.
T R V isolate R Q has particles w i t h m o d a l lengths of 76 and 189 nm. It i n d u c e d s y m p t o m s
similar to those of strain P R N in Chenopodium amaranticolor, N. clevelandii and Phaseolus
vulgaris, and n e i t h e r isolate i n f e c t e d Pisum sativum or Vicia f a b a systemically. It caused
s y m p t o m s similar to, but s o m e w h a t less severe than, those o f strain P R N in N. tabacum cv.
S a m s u n N N . D e s p i t e these similarities, isolate R Q is not closely serologically related to strain
P R N . In tube p r e c i p i t i n tests, a n t i s e r u m to isolate R Q (homologous titre 1/1024) did not react at
dilutions o f 1/2 or greater w i t h particles o f strain P R N or strain C A M . I n similar tests,
a n t i s e r u m to P E B V strain SP5 (homologous titre 1/256) did not react (least dilution tested, 1/4)
w i t h R Q particles. H o w e v e r , the results of I S E M tests i n d i c a t e d that T R V isolates R H and A S H
are antigenically strongly related to isolate R Q (Table 1).
T R V isolate H S N has particles w i t h m o d a l lengths o f 65 and 188 n m a n d p r o d u c e d s y m p t o m s
similar to those of strain P R N in C. amaranticolor, N. tabacum cv. S a m s u n N N and P. vulgaris.
In tube precipitin tests w i t h P R N antiserum, a serological differentiation index (Van
R e g e n m o r t e l & Von W e c h m a r , 1970) o f 2 was found.
P E B V isolate S H E has particles w i t h m o d a l lengths of 103 and 216 n m and caused s y m p t o m s
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Variation in tobravirus genomes
173
Table 1. lmmunosorbent electron microscopy of strains R H and A S H using antisera to various
tobraviruses
Antiserum*
to strain
RQ
PRN
SYM
ORY
CAM
Control~"
t
Virus strain
"~
RH
ASH
47 000~2
1184
700
30
700
NT§
700
NT
700
yr
800
31
* Grids were coated with antiserum diluted 1000-fold.
t Untreated grids.
Figures are number of particles per 1000~tm2 of grid.
§ NT, Not tested.
similar to those of strain SP5 in N. clevelandii and P. vulgaris; both isolates infected P. sativum
and V. faba systemically. In tube precipitin tests, isolate SHE reacted with antiserum to strain
SP5 but was antigenically distinguishable (serological differentiation index about 3) from it.
Isolate SHE is therefore considered to belong to the British serotype of PEBV.
Sequence homology in RNA-1 of T R V isolates
Robinson (1983) found that the maximum extents of hybridization of eDNA copies of RNA-1
(cDNA-1) of strain SYM with RNA-1 of strain SYM and of strain ORY were the same. Similar
results were obtained when cDNA-1 of strain SYM was hybridized with unfractionated R N A of
strains SYM or ORY, to an Rot value of 0.5 tool.s/l; after correction for the S1 nuclease
resistance of eDNA in the absence of R N A (Gonda & Symons, 1978), 5 3 ~ of the c D N A
hybridized with R N A of strain SYM, and 5 2 ~ with R N A of strain ORY. In tests summarized
in Table 2, no hybridization was observed between cDNA-1 of strain SYM and unfractionated
R N A of strain CAM, but this c D N A did hybridize to R N A of all the other eight TRV isolates,
to extents indicating sequence homology (calculated according to Gonda & Symons, 1978) of
from 100 ~o (RNA of strain ORS) to 66 ~o (RNA of strain HSN). Thus, at least two-thirds, and in
some instances essentially all, of the sequences represented in eDNA-1 of strain SYM are shared
by all ten representatives of TRV serotype I-II.
Sequence homologies in unfractionated RNA
To obtain further information on the extent of genome homologies, eDNA copies were made
from unfractionated RNA preparations of TRV isolates SYM, ORY, PRN, RQ, ASH and
CAM, and of PEBV isolates SHE and Ell6, and each of these was hybridized with
unfractionated R N A of the whole range of representative tobravirus isolates. The apparent
percentage sequence homology for each combination is listed in Table 2. For each cDNA
preparation the apparent percentage sequence homology observed depends on the relative
proportions of copies of RNA-1 and RNA-2 in the preparation. Thus, although homology values
obtained with each cDNA preparation and different R N A preparations are directly
comparable, those obtained with different c D N A preparations are not. Furthermore, the two
ways of comparing each pair of isolates need not necessarily give the same value of apparent
percentage sequence homology. The 14 isolates clearly fall into three groups, containing
respectively the ten isolates of TRV serotype I-II, the sole representative of TRV serotype III,
and the three isolates of PEBV. Isolates within each group showed substantial sequence
homology with one another. Conversely, cDNA of isolates in each group hybridized only
slightly or not at all with RNA of isolates in the other two groups; the small extents of
hybridization recorded for some of these combinations were not detected consistently and it is
doubtful that they represent sequence homologies.
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174
D.J.
ROBINSON AND
B.
D.
HARRISON
Table 2. Apparent percentage sequence homology between RNA preparations from 14 tobraviruses
R N A of
strain
SYM
ORY
ORM
ORS
PRN
HSN
NZP
RQ
RH
ASH
CAM
SP5
SHE
E 116
e D N A to RNA-1
of TRV-SYM
100"
99
98
100
85
66
84
97
94
77
0
ND~"
ND
ND
r
SYM
100
63
61
59
67
63
54
67
49
44
1
0
0
0
e D N A to unfractionated R N A of strain
~
ORY
PRN
RQ
ASH
CAM
SHE
56
100
100
100
52
50
51
55
54
50
1
0
11
6
52
47
44
43
100
62
87
64
44
54
0
0
0
ND
41
39
37
38
41
44
41
100
75
65
0
0
1
ND
75
80
85
88
87
77
83
100
98
100
5
5
12
ND
2
7
5
7
3
3
2
1
3
0
100
4
6
6
0
0
2
0
0
0
0
0
1
0
0
90
100
59
E 116
5
0
7
ND
0
1
4
0
ND
ND
0
55
59
100
* Apparent percentage sequence homology, calculated according to Gonda & Symons (1978), in tests using
unfractionated R N A preparations hybridized to an Rot value of 0-5 tool. s/1. Each value is the mean of at least two
determinations, using the same preparation of e D N A and one or more preparations of RNA.
t ND, Not done.
Within TRV serotype I-II, isolates showed varying degrees of sequence homology. Using
e D N A of strain SYM, 63 ~ homology was observed with strain ORY, which shares essentially
all of its RNA-1 sequences and none of its RNA-2 sequences with strain SYM (Robinson, 1983).
None of the other TRV serotype I-II isolates showed significantly more homology with strain
SYM than did strain ORY, suggesting that they all have RNA-2 sequences substantially
different from those of strain SYM. R N A of strains ORY, ORM and ORS each hybridized with
c D N A of strain ORY to the same extent, showing that these three strains have very similar
sequences in both their RNA components. This suggests that they are derived by small-scale
mutational events from a common ancestor, which is consistent with their derivation from a
single field isolate and with their very close serological relationship (Lister & Bracker, 1969).
With R N A of the remaining serotype I-II isolates, e D N A to strain ORY showed between 50 and
56 ~o homology, suggesting that, like strain SYM, they have substantial homology in RNA-1 and
little or none in RNA-2. Similarly, with e D N A to isolates PRN, RQ and ASH, R N A of many of
the serotype I-II isolates showed similar degrees of homology. The homology revealed in
reactions of c D N A of strain P R N with R N A of isolate NZP was greater than that with R N A of
other isolates, suggesting the existence of some common sequences between the RNA-2 species
of isolates PRN and NZP, and consistent with their apparently close serological relationship
(Jones & Young, 1978). Another cluster of isolates that showed greater homology than most in
this serotype comprised isolates RQ, RH and ASH. The homology between these three isolates
seemed particularly great when e D N A to isolate ASH was used, but this preparation apparently
contained an unusually high proportion of copies of RNA-1, making it less discriminating of
differences among RNA-2 species. However, the grouping was also evident using e D N A to
isolate RQ, but seemed less close. This grouping fits well with the results of serological tests
(Table 1).
Among the PEBV isolates, a range of homologies similar to those among TRV serotype I-II
isolates was evident. The two examples of the British serotype, isolates SP5 and SHE, showed
9 0 ~ sequence homology, whereas isolate E l l 6 of the Dutch serotype showed less, but still
substantial, homology with these two.
DISCUSSION
The results reported here confirm the hypothesis (Robinson, 1983) that the nucleotide
sequence of RNA-1 is strongly conserved among isolates of TRV serotype I-II. There are
evidently much less strong constraints on variation in the sequence of RNA-2, which can differ
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Variation in tobravirus genomes
175
to such an extent that no common sequences are detectable by hybridization tests. However,
among the ten isolates studied, there are three groups that share some or all of their RNA-2
sequences. These groups comprise: (i) strains ORY, ORM and ORS; (ii) isolates P R N and
NZP; (iii) isolates RQ, RH and ASH. For each of these groups there is evidence of relatively
close serological relationships between their members (Lister & Bracker, I969; Jones & Young,
1978; and Table 1). Isolates SYM and HSN each stand somewhat apart from these three groups,
although isolate HSN is moderately closely related serologically to strain P R N and seems to
show some homology with its R N A - 2 .
The two PEBV isolates of the British serotype clearly share most of the sequences in both their
R N A species. Isolate E116 of the Dutch serotype has only partial homology with these two, and
it seems likely that this will prove to be in RNA-1. Indeed, a more extensive study of PEBV
strains (Robinson et al., 1984) shows a pattern of R N A sequence homologies similar to those of
TRV serotype I-II isolates.
On the basis of hybridization tests, strain CAM (TRV serotype III) is as distinct from TRV
serotype I-II as is PEBV, and there seems now to be little justification for classifying all
tobraviruses as isolates of two viruses. The existence of distant serological relationships between
isolates o f T R V serotype I-II and o f T R V serotype III (Harrison & Woods, 1966; Kurppa et at.,
1981) and between isolates of TRV serotype I-II and PEBV (Maat, 1963) could be taken as a
reason for considering all tobraviruses to be isolates of a single virus, but this would obscure the
practical distinction between PEBV isolates, which cause diseases in legumes, and other
tobravirus isolates, which cause diseases in non-legumes (Harrison & Robinson, 1978).
The idea that TRV serotype I-II and PEBV are distinct viruses is supported by the failure to
make pseudo-recombinants between them (Lister, 1968). Similarly, Frost et al. (1967) failed to
make pseudo-recombinants between isolates of TRV serotype I-II and serotype III, although
Lister (1969) apparently succeeded in making two pseudo-recombinants containing RNA-1 of a
serotype III strain and RNA-2 of strain ORY. However the tests used to identify these pseudorecombinants did not conclusively exclude the possibility that they were in fact variants of strain
ORY.
The pattern of nucleotide sequence homologies among tobraviruses, together with their
serological and biological properties, therefore suggests that they should be regarded as isolates
of three distinct viruses. For TRV serotype III isolates a return to the original name, pepper
ringspot virus (Kitajima et al., 1969; Kitajima & Costa, 1969), seems appropriate. Although
most Brazilian tobraviruses probably are isolates of this virus, strain CAM is the only one that
has been widely used in laboratory studies. Indeed, many studies on ' T R V ' have used strain
CAM, and it now seems unsafe to assume that properties of this strain are necessarily also
typical of those of TRV.
The pattern of sequence homologies among TRV serotype I-II isolates raises interesting
questions about their evolution. It seems certain that their RNA-1 species have derived from
a common ancestor, but much less certain that their RNA-2 species have done so. A model in
which the two genome parts have separate origins, and in which the event by which they became
associated occurred on numerous independent occasions and involved RNA-2 species from
different sources is easy to reconcile with the data.
In addition to those who provided isolates, we thank Mr I. M. Roberts for the immunosorbent electron
microscopy, and Ann Jenkins for technical assistance.
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