Journal
of General Virology (1999), 80, 1339–1346. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Recombination in RNA viruses and in virus-resistant transgenic
plants
Rachid Aaziz and Mark Tepfer
Laboratoire de Biologie Cellulaire, INRA-Versailles, 78026 Versailles cedex, France
Introduction
Recombination in RNA viruses is a general phenomenon,
and is considered to play a major role as a driving force in virus
variability and thus in virus evolution. An ever increasing
number of RNA viruses has been shown to undergo RNA
recombination, whether under natural or experimental conditions. More than 30 years after its discovery, the mechanisms
of viral RNA recombination are only now beginning to be
elucidated. Recent reports strongly suggest that RNA recombination is linked to virus replication, and that it occurs by
a copy-choice mechanism. The detection of recombination
between host transcripts and infecting viral RNA genomes has
given rise to new concerns about the release of virus-resistant
transgenic crops, since recombination could generate viruses
with properties different from the infecting strain. We will
describe here current knowledge of recombination in RNA
viruses, and then will present results showing recombination in
transgenic virus-infected plants, and finally will discuss to what
extent this may lead to increased variability in plant RNA
viruses.
Recombination in RNA viruses
Adaptation of living organisms to a changing environment
through evolution requires a compromise between genetic
variation and phenotypic selection, and has generated the
considerable variability that we encounter every day. In the
virus world, and particularly in that of RNA viruses, high
variability is observed, and is thought to be due to three main
evolutionary forces : mutation, reassortment (for viruses with a
segmented genome) and recombination (Roossinck, 1997 ;
Domingo & Holland, 1997). Viral RNA replication is characterized by a high mutation rate, due to the lack of
proofreading-repair of viral RNA-dependent RNA polymerases (RdRp). This, in conjunction with short replication
times and a high multiplicity, leads towards a dynamic mutant
population, a unique feature of RNA viruses termed virus
Author for correspondence : Mark Tepfer.
Fax j33 1 30 83 30 99. e-mail mark!versailles.inra.fr
quasi-species and corresponding to a swarm of sequence
variants (Holland & Domingo, 1998). However, genetic
divergence is restricted by the necessity to maintain a
functional viral RNA genome and by environmental selective
pressures. By allowing the spread of new mutations, both
reassortment and recombination increase genetic variability
and favour the creation of variant viruses that may be best
adapted to withstand future environmental selective pressure
on the virus population. In addition, RNA recombination is
thought to rescue viral genomes by repairing mutation errors
in essential viral genes or in structures that could be introduced
during RNA replication (Lai, 1992 ; Carpenter & Simon, 1996).
Until the 1960s, genetic recombination was considered to
be a feature confined exclusively to DNA molecules. DNA
viruses, prokaryotes and eukaryotes undergo DNA recombination during sexual reproduction, such as in meiosis or in
bacterial conjugation. Likewise, DNA recombination has been
reported during mitosis of somatic cells in cases of gene
conversion. Recombination between DNA molecules will not
be treated further here (for review see Osman & Subramani,
1998), nor will we discuss recombination in retroviruses, since
their mode of replication is quite different from that of RNA
viruses, and recombination is an integral part of this process
(for review see Mansky, 1998).
RNA recombination was originally observed in poliovirus
by Hirst (1962) and Ledinko (1963). By co-infecting HeLa cells
with two distinct strains of type 1 poliovirus carrying single
mutations, these authors were able to obtain recombinant
viruses that had inherited the two mutations from both
parental strains. Since then, recombination in RNA viruses has
been abundantly documented, and the list of recombinant
viruses continues to grow. Two types of analysis provide
evidence of viral RNA recombination. One is based on
phylogenetic analysis of RNA viral genomes. In this case,
when different areas of a viral genome are apparently more
closely related to different viruses, this suggests that recombination has led to a genomic rearrangement at some time
during the evolution of these viruses. Indeed, as nucleotide
sequences of an increasing number of RNA viruses have been
determined, a growing amount of evidence for natural RNA
recombination is being reported. Insertions of cellular sequences into viral RNA genomes, presumed to arise through
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R. Aaziz and M. Tepfer
Table 1. Direct laboratory evidence of recombination in RNA viruses
Virus group
Animal
Picornavirus
Coronavirus
Alphavirus
Orthomyxovirus
Nodavirus
Plant
Bromovirus
Carmovirus
Tobamovirus
Alfamovirus
Tombusvirus
Cucumovirus
Potyvirus
Bacteria
Virus
Reference*
Poliovirus
Foot-and-mouth disease virus
Murine hepatitis virus
Infectious bronchitis virus
Sindbis virus
Influenza A virus
Flock house virus
Hirst (1962)
McCahon et al. (1985)
Lai et al. (1985)
Kottier et al. (1995)
Weiss & Schlesinger (1991)
Bergmann et al. (1992)
Li & Ball (1993)
Brome mosaic virus
Cowpea chlorotic mottle virus
Turnip crinkle virus
Tobacco mosaic virus
Alfalfa mosaic virus
Cucumber necrotic virus, tomato
bushy stunt virus DI-RNA†
Cucumber mosaic virus, tomato
aspermy virus
Zucchini yellow mosaic virus
Bujarski & Kaesberg (1986)
Allison et al. (1990)
Cascone et al. (1990)
Beck & Dawson (1990)
van der Kuyl et al. (1991)
White & Morris (1994)
Qβ
φ6
Munishkin et al. (1988)
Onodera et al. (1993)
Ferna! ndez-Cuartero et al. (1994)
Gal-On et al. (1998)
* Only the first reported references are indicated.
† DI-RNA, Defective interfering RNA.
RNA recombination, have also been cited (Mayo & Jolly,
1991 ; Masuta et al., 1992 ; Lai, 1992). In addition, defective
interfering RNAs, which are also generated by RNA recombination, have been reported in both plant and animal
RNA viruses (Lazzarini et al., 1981 ; Fields & Winter, 1982 ;
Inoue-Nagata et al., 1997). These examples illustrate the major
role of recombination in RNA virus evolution. The second
type of evidence is from the abundant direct laboratory
experimental data. By making use of either non-replicative (for
animal viruses) or movement-defective (for plant viruses)
variant viruses, functional recombinant viruses restored
through RNA recombination have been obtained in several
laboratories. Thus, many RNA viruses have been shown to
undergo RNA recombination (Table 1).
Based on the similarity of parental RNA molecules, two
types of RNA recombination have been defined (Lai, 1992).
Homologous RNA recombination (HR) occurs between two
similar or related RNA molecules at precisely comparable
(precise HR) or divergent (imprecise or aberrant HR) matched
crossover sites. In contrast, non-homologous recombination
(NHR) occurs between dissimilar or unrelated RNA molecules.
This nomenclature is somewhat unsatisfactory for two reasons.
First, the term homologous generates a semantic problem,
since it signifies that the parental molecules share common
ancestry, which is not necessarily the case, for instance, if
BDEA
recombination occurs in short segments of sequence identity in
otherwise unrelated genes. Second, it is not informative about
the mechanism by which recombination occurs. In an attempt
to alleviate these problems, an alternative classification was
proposed recently (Nagy & Simon, 1997). This classification is
based both on the mechanism generating recombinants and on
the nature of the recombinant products, and defines three
classes of RNA recombination. Similarity-essential recombination occurs when sequence similarity between parental
RNA molecules is essential in the recombination event.
Depending on the end-products, two types are distinguished :
precise and imprecise similarity-essential recombination.
Similarity-non-essential recombination occurs when sequence
similarity of parental RNA molecules is not apparently
required ; other RNA determinants such as secondary structure,
heteroduplex formation, etc. determine the recombination
event. Similarity-assisted recombination combines the characteristics of the two above-mentioned classes. This novel
classification system may also prove to be temporary, and in
particular may need to be reconsidered as new light is shed on
the mechanisms of RNA recombination. In spite of its flaws, we
will continue to use Lai’s system here, since it is the more
widely accepted, and since in many cases the mechanism of
recombination is unknown.
Two mechanistic models are generally proposed to account
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Review : RNA recombination and transgenic plants
for RNA recombination : (i) a cleavage–ligation model, which
supposes a breakage at two distinct sites of the same or
different precursor RNA molecules, followed by ligation of the
two cleaved RNA molecules, and (ii) an RdRp-mediated copychoice model, in which recombination would occur during
RNA synthesis if the RdRp pauses (on the donor strand) and
switches to another site on the same template or to another
one (acceptor strand) to resume nascent RNA synthesis
(Cooper et al., 1974). Studying in vitro RNA recombination of
Qβ bacteriophage, Chetverin et al. (1997) exclusively observed
non-homologous recombinants, which they proposed to be
generated by a mechanism of ligation–transesterification.
However, this is the only example supporting the ligation
model. In contrast, in vivo recombination studies of the same
bacteriophage disagree with this model (Palasingam & Shaklee,
1992). These authors showed the existence of NHR, but also
HR, which would have most likely been obtained by a
template-switching mechanism. In addition, despite numerous
attempts, Ramaswamy et al. (1995) could not demonstrate
possible ligation activity that could be involved in RNA
recombination of non-replicative RNA molecules of Sindbis
alphavirus. Thus, nearly all the existing evidence supports the
RdRp-mediated template-switching model. Consistent with
this is the finding that RNA replication is necessary for
poliovirus RNA recombination (Kirkegaard & Baltimore,
1986). Furthermore, studies on brome mosaic bromovirus
(BMV) have shown that mutations in viral proteins 1a and 2a,
which are components of the BMV replicase, affected viral
RNA recombination (Nagy et al., 1995 ; Figlerowicz et al., 1997,
1998). Although RdRp-mediated template-switching is the
most accepted model, it has not yet been formally demonstrated. Considering the difficulties, it is unlikely that formal
proof of template switching will be obtained in in vivo
recombination experiments. There is more likelihood of success
with a cell-free system, such as poliovirus (Molla et al., 1991),
or Qβ (Biebricher & Luce, 1992) and φ6 (Qiao et al., 1997)
bacteriophages. Recent elegant studies with turnip crinkle
tombusvirus replicase show that it may be possible to dissect
the mechanism of RNA recombination in vitro with this system
as well (Nagy & Simon, 1998 a, b).
In almost all experimental studies, recombinant viruses
were recovered under conditions of strong selection pressure.
As a result, only those of notably higher fitness were detected,
and those negatively selected were lost. Therefore, the
frequency of recombination is nearly always underestimated,
and perhaps some mechanisms of recombination still remain
unknown. For example, previous studies have identified a
recombinational hot-spot region in mouse hepatitis coronavirus (Banner et al., 1990). However, in the absence of selection,
initial recombination events were randomly distributed, but
after several passages of the recombinant viruses in tissue
culture, recombination sites became localized to a restricted
region corresponding to the initially identified hot spot (Banner
& Lai, 1991). This study suggests that RNA recombination is
more frequent than previously thought, and also that the
recombinant viruses obtained may reflect the results of
selective pressure rather than the mechanism of recombination
itself.
It would appear that different RNA viruses do not
recombine with the same frequency or efficiency. Detailed
phylogenetic studies on potyviral strains gave evidence of
numerous recombination events between strains, whereas
when cucumoviruses were analysed, no evidence of recombination was observed (Revers et al., 1996 ; Candresse et al.,
1997). Although this may in part be due to differences in the
efficiency of detection in different virus systems, it is tempting
to speculate on the existence of processes regulating RNA
recombination that could be determined either by the virus
itself (the RdRp and\or sequence-specific or structural signals
in the viral RNA) or by the host. In particular, nothing is
currently known about the role of host factors in RNA
recombination.
Recombination in virus-resistant transgenic
(VRT) plants
The first VRT plants were obtained 13 years ago (PowellAbel et al., 1986). They represent the first application of the
concept of pathogen-derived resistance (Sanford & Johnston,
1985). Since then, numerous transgenic crops tolerant or
resistant to a wide range of viruses have been developed
(Beachy, 1997). Different genomic sequences were used,
including viral genes encoding coat proteins (CP), replicases,
defective movement proteins, proteases or helper components
(Lomonossoff, 1995). CP-mediated resistance, which is the
most frequently used strategy, has been used to confer
resistance to viruses in at least 13 RNA virus genera, including
23 distinct virus species (Grumet, 1995). Three CP-mediated
resistant plants have recently been approved for commercial
release in the USA and more VRT crops will soon be
deregulated (White, 1999). The increasing interest in VRT
plants is understandable, since they offer numerous potential
agronomical and ecological benefits. This is clearly the case
when no corresponding natural host resistance genes have
been identified, and also when introgression of such a natural
resistance gene into commercial crops would be too lengthy a
process, or when it would have undesirable effects on
genetically complex plants that are vegetatively propagated.
VRT plants should also allow reduction of the amounts of
pesticide used to eliminate virus vectors.
Nevertheless, in order to take advantage of these benefits
under the best conditions, it is appropriate to examine VRT
plants carefully from the point of view of biosafety. Due to
possible interactions in transgenic plants between products of
the viral transgene, whether RNA or protein, and an incoming
virus, three main potential ecological risks should be considered : synergism, heteroencapsidation and recombination
(Tepfer, 1993 ; Robinson, 1996). For some species, there is also
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R. Aaziz and M. Tepfer
a risk of transgene flow from VRT plants to neighbouring wild
relatives (Fuchs & Gonsalves, 1997). Two types of alterations
in populations of viruses or neighbouring plants can be
distinguished. The first involves phenotypic alterations of
VRT plants or infecting viruses, such as an increase in symptom
severity due to a virus\transgene synergism or the spread of a
non-transmissible virus after heteroencapsidation. This kind of
risk, of course, remains restricted to the vicinity of the VRT
crop, and will disappear without persistent effects on the
environment if VRT plant use is halted. The second involves
genotypic modifications, which in essence are expected to be
durable, and may involve either plant (transgene flow) or viral
(RNA recombination) genomes (for review see Tepfer &
Bala! zs, 1997).
RNA recombination in virus-infected transgenic plants
could generate novel viruses with biological properties distinct
from those of the parental strain. To investigate this phenomenon, one strategy is to inoculate a movement-defective virus
onto transgenic plants constitutively expressing a gene encoding the corresponding protein in non-defective form, and
then look for potential recombinant systemic viruses. To date,
four experimental systems concerning this phenomenon have
been reported, including only three plant viruses, cauliflower
mosaic caulimovirus (CaMV), cowpea chlorotic mottle bromovirus (CCMV) and tomato bushy stunt tombusvirus (TBSV).
Recombinant CaMVs were first obtained after transgenic
Brassica napus expressing CaMV ORF VI was agroinfected
with a CaMV isolate lacking ORF VI (Gal et al., 1992). ORF VI
of CaMV D4 strain determines systemic movement in
Nicotiana bigelovii (Schoelz et al., 1986). Transgenic N. bigelovii
expressing the D4 ORF VI inoculated with CM1841-CaMV, a
strain naturally non-systemic in solanaceous hosts, also
generated recombinant CaMVs (Schoelz & Wintermantel,
1993). In these two examples, strong evidence suggests that
recombinant viruses arose by two template-switching events
between viral RNA and transcripts from the virus-derived
transgene. However, since CaMV is a pararetrovirus (with a
DNA step in its virus cycle), homologous DNA–DNA
recombination cannot be excluded, since it has already been
shown to occur in CaMV (Vaden & Melcher, 1990 ; Gal et al.,
1991). It should also be noted that copy-choice recombination
in CaMV as described above is due to the viral reverse
transcriptase, and that as a consequence this system may be
rather dissimilar to what occurs in recombination mediated by
an RNA virus RdRp.
Greene & Allison (1994, 1996) have elegantly demonstrated recombination between an isolate of CCMV, crippled
by deletion of the 3h one-third of the CP gene, and transgenic
N. benthamiana containing the 3h two-thirds of the CP gene and
the 3h non-coding region of the CCMV genome. Recombinant
progeny became systemic on N. benthamiana, following the
restoration of a functional CCMV CP gene. The presence of
different mutation markers in the systemic virus unequivocally
demonstrated that RNA recombination took place between
BDEC
the transgene mRNA and the challenging movement-defective
viral genome. Sequence analysis of recombinant viral RNAs
revealed an imprecise HR type, with numerous modifications
flanking the putative crossover site. When the recombinant
strains were tested on a range of host plants, four out of seven
displayed novel symptoms (Allison et al., 1997), but when coinoculated with wild-type virus, none of them was more fit
than the parental strain (Allison et al., 1999).
All the above examples correspond to conditions of high
selection pressure, i.e. the viral inoculum was movementdefective, and therefore only the systemic recombinant viruses
were detected and isolated. However, since defective viruses
are at most minor components of natural virus strains,
searching for recombinant viruses in VRT plants infected with
wild-type viruses under conditions of little or no selection
pressure is more pertinent to risk assessment. In more recent
work, recombinant CaMV has been isolated from transgenic
plants under conditions of moderate selection pressure. Here
inoculation of transgenic N. bigelovii plants expressing D4CaMV ORF VI with W260-CaMV, which is systemic in
solanaceous hosts, generated recombinant viruses that had a
distinct competitive advantage in N. bigelovii compared to the
parental W260 strain (Wintermantel & Schoelz, 1996).
More recently, recombinant viruses have also been recovered under conditions of moderate selection pressure from
plants expressing RNA virus sequences. When plants expressing a gene encoding the TBSV CP were inoculated with
virus defective for the CP gene, Borja et al. (1999) observed
high frequency recovery of virus with a restored CP gene,
which must have been generated by at least two crossover
events. The sequence of the recombinant viruses was identical
to the wild-type, and thus resulted from precise HR. In this
case, the CP-defective viral inoculum was systemic, but was at
a selective disadvantage relative to the restored wild-type
recombinant viruses.
Potential risks associated with recombination
in VRT plants
If a strategy could be developed to eliminate the possibility
of recombination, while taking full advantage of virus-derived
resistance, this would make any detailed evaluation of potential
risks associated with recombination unnecessary. One such
strategy has been proposed in which the resistance observed in
certain plants expressing viral sequences is due to induction of
degradation of both the transgene mRNA and the homologous
viral RNA by a mechanism similar to that of post-transcriptional gene silencing (for review see Baulcombe, 1996).
This type of RNA-mediated resistance could present certain
advantages from a biosafety perspective. In particular, since
little or no transgene-derived RNA accumulates, the likelihood
of its recombination with the RNA of infecting viruses would
be expected to be extremely low. There may, however, also be
flaws in this type of resistance. In particular, it has been shown
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recently that infection by certain viruses blocks post-transcriptional gene silencing, and thus could be expected to cause
breakdown of RNA-mediated resistance (Anandalakshmi et al.,
1998 ; Be! clin et al., 1998 ; Brigneti et al., 1998 ; Kasschau &
Carrington, 1998). In addition, Jakab et al. (1997) have shown
that when plants are inoculated with two potato potyvirus Y
strains, one of which is targeted by RNA-mediated resistance,
this provides selection pressure in favour of recombination
between the viral genomes, resulting in replacement of the
targeted sequence. Thus, in any case, the implications of
recombination in plants expressing viral sequences clearly
merit close examination.
In discussions of risk assessment, two distinct but interdependent elements are generally distinguished, namely
frequency and hazard (Hull, 1994). In the case of recombination
in VRT plants, evaluation of the frequency of recombination is
quite difficult, in particular since the frequency is expected to
be low. As discussed in the previous section, almost all
recombinant viruses that have been observed reflect the postrecombination selection process, rather than recombination
alone. Therefore, recombination frequency in the absolute
sense is generally underestimated. Another important point is
that infection of VRT plants by a related incoming virus
corresponds in many ways to a frequently encountered natural
situation, viz. simultaneous infection of non-transgenic plants
by two related viruses. Consequently, to evaluate risks of RNA
recombination in virus-infected VRT plants, one must compare
the frequency of its occurrence in VRT plants to that in doubly
infected non-transgenic plants. In addition, determination of
these frequencies must be carried out under conditions of little
or no selection pressure.
Mixed infections have not been extensively studied, except
regarding the synergy that can occur between two distantly
related viruses (Vance et al., 1995), and co-infection of a given
host plant by two closely related viruses can be difficult to
achieve. For example, in the case of two cucumoviruses,
cucumber mosaic virus (CMV) and tomato aspermy virus
(TAV), successful co-infection of Nicotiana hosts is the
exception, and generally CMV excludes TAV (Stackey &
Francki, 1990 ; our unpublished data). It is thus not surprising
that reports of RNA recombination in mixed infections of wildtype viruses are rare, if not non-existent. Indeed, RNA
recombination between two different viruses requires that they
undergo RNA replication at the same time and at the same
place, even down to the level of subcellular localization. In
contrast, in VRT plants the virus-derived transgene is constitutively expressed in all cells, which could favour the
occurrence of RNA recombination between a viral transgene
mRNA and an invading viral genome. One of the prerequisites
for study of recombination during mixed infections is to
develop sensitive techniques for detection of recombinant
molecules in the presence of a large excess of non-recombinant
parental RNAs. Recently, using a sensitive RT–PCR assay,
recombination events between wild-type CMV and TAV have
been observed in doubly infected plants (our unpublished
data).
In spite of the generally perceived importance of the
frequency factor in risk assessment, it can be argued cogently
that in the case of recombination in VRT plants, considering
the enormous scale of the proposed future commercial releases,
even extremely rare recombination events could have a
significant impact. If this is the case, then it is particularly
important to examine closely the other element of the risk
equation, namely potential hazard. In this case, the essential
question is : will recombination between transgene mRNAs
and infecting viruses lead to the creation of strains with novel
and more damaging properties ?
To investigate the hazard element of the potential risk
associated with recombination in VRT plants, one approach is
to create recombinant viruses in vitro by exchanging RNA
segments between different strains or between different related
viruses and then to evaluate their biological properties, such as
symptom severity or expansion of host range. Several groups
have created recombinant cucumoviruses, and have reported
often striking changes in symptomatology (Ding et al., 1996 ;
Sala! nki et al., 1997 ; Carre' re et al., 1999). However, it is
premature to conclude that more virulent recombinant cucumoviruses are likely to appear in transgenic plants, since in none
of the above cases was the fitness of the recombinants tested
relative to that of the parental viruses. In this regard it is
significant that, as mentioned above, the recombinant CCMV
strains recovered in transgenic plants under conditions of high
selection pressure, although expressing novel symptoms, were
less fit than the parental virus (Allison et al., 1999). There is,
however, one report of a recombinant cucumovirus created in
the laboratory that proved to have a selective advantage
relative to the parental strains (Ferna! ndez-Cuartero et al.,
1994). However, this may be considered to be something of a
laboratory freak, since it appeared spontaneously during
several years of maintenance in the greenhouse of a pseudorecombinant strain composed of CMV RNAs 1 and 2 and TAV
RNA3, and in fact several recombination events have apparently occurred.
For a thorough evaluation of potential risks associated
with recombination between viral sequences transcribed from
a transgene and the genome of an infecting virus, several areas
of uncertainty remain. The clearest experimental results would
be obtained from a detailed comparison of doubly infected
non-transgenic plants and singly infected transgenic ones
under conditions of minimal selection pressure, although such
an experiment is difficult to perform. Not only is this the only
way to approach an evaluation of the frequency of recombination under these two situations, but it would also
clarify whether the nature of the recombinants created is
similar. This latter point is particularly important, since if a
similar array of recombinants is generated initially in these two
situations, then one would expect that selection pressure, due
for instance to virus movement or to transmission to other
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R. Aaziz and M. Tepfer
hosts, would lead to equivalent virus populations. In this case,
one could conclude that indeed plants expressing viral
sequences do not present risks different from those already
present naturally in a pre-transgenic world. On the other hand,
if there are distinct differences in the types of recombinants
that occur under these two situations, then an evaluation of the
fitness of the recombinants occurring in transgenic plants
would be appropriate.
mutants with a host transgene. Molecular Plant–Microbe Interactions 12,
153–162.
R. Aaziz was supported by an INRA doctoral fellowship. This
research was in part financed by a grant from the French Ministry of
Research.
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Carpenter, C. D. & Simon, A. E. (1996). In vivo restoration of
biologically active 3h ends of virus-associated RNAs by nonhomologous
RNA recombination and replacement of a terminal motif. Journal of
Virology 70, 478–486.
Carre' re, I., Tepfer, M. & Jacquemond, M. (1999). Recombinants of
cucumber mosaic virus (CMV) : determinants of host range and
symptomatology. Archives of Virology 144, 365–379.
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