**[Viral Interactions in Viral Coat Protein Transgenic
Plants:]{.smallcaps}**

**[A Literature Review]{.smallcaps}**

Virus infection is a serious problem in agricultural production.
Virtually every plant species is susceptible to infection by at least
one of more than 500 known plant viruses (Waterhouse et al. 2001). Plant
viruses create economic losses for a vast variety of crops by reducing
yields and negatively affecting the quality of the crop (Tolin 1991).
Growers may need to use several control methods during a crop season in
an attempt to prevent viral infection and dissemination, primarily by
planting virus-free material for mechanically transmitted viruses. For
vector-transmitted viruses, control measures have often focused on
chemical insecticides, fungicides, and nematicides to reduce the
population of vectors that transmit viruses from plant to plant.
However, control of vectors is not always feasible or effective as a way
to control virus transmission (OECD Environment Directorate 1996). In
another common control strategy, plants are infected with a mild form of
a virus to confer resistance to a more virulent form. This method has
serious limitations as well. In some cases, the development of resistant
cultivars can be the only viable means of virus control. Plants
developed through conventional breeding techniques offer some degree of
virus resistance. However, breeding for resistance has not been
successful for the majority of field crops that are severely affected by
viruses (Tolin 1991). More recent transgenic methods for creating
resistant varieties incorporate viral DNA into the plant's genome to
confer effective resistance to infection by that virus as well as others
of similar sequence.

Many different types of transgenes have been used to confer resistance
to viral infection including viral replicase, movement protein, and
nuclear inclusion genes as well as nonviral sequences from a variety of
species (Tepfer 2002). However, the most common type of transgenes used
to confer virus resistance are viral coat protein (VCP) genes (White
2000). VCPs encapsidate the viral nucleic acid and are thought to be
important in nearly every stage of viral infection including
replication, movement throughout an infected plant, and transport from
plant to plant (Callaway et al. 2001). Given their central role in viral
infection, the interaction of a VCP transgene with the genome of an
invading virus could potentially exacerbate viral disease through a
change in the disease characteristics or transmission properties of that
virus. Continued development and testing of new VCP-transgenic plants
has facilitated evaluation of such potential impacts in the 18 years
since the first one was created (Abel et al. 1986). This review
evaluates the current scientific understanding of the potential risks
associated with viral interactions in VCP-transgenic plants.

**Viral Interactions**

Mixed viral infections can be extremely common in crops and other plants
(for review see Hammond et al. 1999). In natural, mixed infections,
viral genomes from different strains and/or different species
simultaneously infect the same plant and thus have opportunities to
interact. In spite of many opportunities for interaction in nature, such
events rarely lead to any detectable adverse outcome (Falk & Bruening
1994). However, such *in planta* interactions do have the potential to
result in a virus that causes increased agricultural or other
environmental damage. For example, the epidemic of severe cassava mosaic
disease in Uganda is thought to be due to the combination and/or
sequential occurrence of several phenomena including recombination,
psuedorecombination, and/or synergy among cassava geminiviruses (Pita et
al. 2001). VCP-transgenic plants potentially raise concerns because
every virus infection is essentially a mixed infection with respect to
the CP gene (de Zoeton 1991). The pertinent question is whether the
risks associated with VCP-transgenic plants may be either greater in
degree or different in kind than are presented by natural, mixed
infections (Tepfer 2002).

Recombination, heterologous encapsidation, and synergy are discussed
individually to evaluate each viral interaction as it occurs under
natural conditions, its potential to occur in VCP-transgenic plants, and
ways that the frequency of its occurrence could be reduced if warranted.
Then the limited number of field evaluations of viral interactions in
VCP-transgenic plants are discussed. Finally, the ecological
significance of these studies is put into context by considering whether
viral interactions in transgenic plants could occur at an increased
frequency or be unlike those that occur in natural, mixed infections.

**Recombination**

*Recombination under natural conditions*

Recombination plays a significant role in virus evolution. Evidence of
past recombination having led to the creation of new DNA and RNA viruses
has been found in a number of different groups including bromoviruses
(Allison et al. 1989), caulimoviruses (Chenault & Melcher 1994),
luteoviruses (Gibbs & Cooper 1995), nepoviruses (Le Gall et al. 1995),
cucumoviruses (Masuta et al. 1998), and geminiviruses (Pita et al. 2001;
Zhou et al. 1997). Sequence analysis of viruses from the family
Luteoviridae indicated that this family has evolved via both intra- and
interfamilial recombination (Moonan et al. 2000).

The propensity to recombine and thus the significance of recombination
varies within and across virus groups (Worobey & Holmes 1999), possibly
due to the degree of dissimilarity the viral replicase can tolerate
(Hammond et al. 1999). Sequence analysis of the CP and 3' untranslated
region (UTR) of 109 potyvirus isolates revealed intraspecies
recombination led to viable recombinants in four out of the eight
species represented (Revers et al. 1996).

Recombination is more likely to occur and thus more likely to result in
viable recombinants that have the appropriate transcription recognition
signals the more closely related the viruses are. However, experiments
suggest that recombination between even unrelated viruses may prove
significant. A pseudorecombinant strain created by experimentally
combining regions of the cucumber mosaic virus (CMV) and tomato aspermy
cucumovirus (TAV) genomes was found to have more severe symptoms than
either of the parentals, although the recombinant wasn't able to move
beyond infection of the initially infected cells (Salánki et al. 1997).
Experiments have also shown interspecific recombination between CMV and
TAV under conditions in which recombinants would not be expected to have
any particular fitness advantage (Aaziz & Tepfer 1999). In another
example, alteration of the host range of tobacco mosaic virus (TMV)
occurred when a chimeric virus expressed the CP from alfalfa mosaic
virus (AMV) instead of its own (Spitsin et al. 1999).

In addition to virus-virus recombination, recombination has also been
found to occur between virus and plant host RNA. Sequence analysis of
the 5' terminal sequence of potato leafroll virus (PLRV) suggests that
it arose via recombination with host mRNA (Mayo & Jolly 1991). Evidence
suggests that such recombination events can affect virus virulence (for
review see Rubio et al. 1999).

Recombination only rarely leads to the persistence of a new, viable
virus. Whether a recombinant virus survives in the field depends on how
well it is able to compete with other viruses at all stages of the
infective cycle: transmission, gene expression, replication, and
assembly of new virions (Falk & Bruening 1994). An analysis of CMV
isolates in natural populations showed that while mixed infections are
common, viable recombinants were very rarely recovered. Such results
suggest that recombinants are infrequent or are at a selective
disadvantage relative to parental viruses (Fraile et al. 1997). However,
a virus that has altered transmission patterns from parental varieties
might be able to invade a new niche and thus escape such intense
competition (Gibbs 1994). Laboratory experiments have demonstrated that
some RNA-RNA recombinant viruses are potentially able to have increased
fitness, possibly due to increased replication efficiency or increased
stability of the viral RNA (Fernández-Cuartero et al. 1994).

*Laboratory experiments of recombination in transgenic plants with viral
transgenes*

Like a plant host genome, viral transgenes would be available for
recombination with infecting viruses, and portions of the transgene
could thus be incorporated into the replicating virus. Laboratory
experiments with pseudorecombinant transcripts of papaya ringspot virus
(PRSV) have shown that recombinant viruses that theoretically could be
produced in field-grown transgenic papaya would be able to affect the
virulence of the infecting strains (Chiang et al. 2001). Viruses that
are pathogens of the host plant would be available for recombination
with transgenes. In addition, other viruses that are generally not
pathogens may also be available if the plant resists infection by that
virus by restricting its movement while still allowing its replication
(Greene & Allison 1994). The theoretical possibility exists that
recombination between a replication-competent but nonpathogenic virus
and a transgene could convert the virus to a pathogen (Allison et al.
1996).

Several laboratory experiments have investigated the potential for
recombination between the RNA of viral genes in a transgenic plant and
an infecting RNA virus of the same type:

-   Transgenic *Nicotiana benthamiana* containing the red clover
    necrotic mosaic virus cell-to-cell movement protein transgene were
    inoculated with infectious transcripts of the virus. Sequence
    analysis of virus found in noninoculated, systemically infected
    leaves showed that the infecting virus had recombined with the
    cell-to-cell movement protein transgene to yield a virus capable of
    systemic infection (Lommel & Xiong 1991).

-   Transgenic *Nicotiana benthamiana* containing the 3' 2/3 of the
    cowpea chlorotic mottle virus (CCMV) CP gene were inoculated with a
    CCMV deletion mutant lacking the 3' 1/3 of the gene such that
    recombination within the central 1/3 region of the gene would allow
    restoration of a virus capable of systemic infection. Three percent
    of inoculated plants produced viable recombinants, each from a
    separate aberrant homologous recombination event, in spite of
    experimental conditions designed to favor precise homologous
    recombination (Greene & Allison 1994).

-   Transgenic *Nicotiana benthamiana* containing the CP coding sequence
    from tomato bushy stunt virus (TBSV) were inoculated with CP mutants
    of TBSV. Double recombination events restored wild-type virus
    phenotype in up to 20% of the plants (Borja et al. 1999).

-   Transgenic *Nicotiana benthamiana* containing the CP coding region
    of TMV was inoculated with a CP deletion mutant. Recombinant RNA was
    detected in 32% of the infected plants, but encapsidated recombinant
    virions were not detected. No recombinant RNA was detected when the
    region investigated was a gene nonessential for infection rather
    than the CP gene (Adair & Kearney 2000).

-   Transgenic *Nicotiana benthamiana* containing the CP coding region
    of plum pox virus (PPV) with either a complete or a partially
    deleted 3' UTR were inoculated with CP mutants of an infectious
    full-length clone of PPV. Recombination produced viable PPV, but
    only when the transgenic plants contained the complete 3' UTR
    (Varrelmann et al. 2000).

Similar experiments show that recombination between the RNA of viral
transgenes and infecting DNA viruses can also occur. The transgenic
plants used in these experiments (detailed below) actually show no
resistance to the DNA virus whose coat protein they contain, and
attempts to develop transgenic DNA virus-resistant plants in general
have had little success (Hammond et al. 1999). Thus, the relevance of
the recombination events observed in these plants as an indicator of
environmental concern might be questioned.

-   Transgenic *Brassica* were infected with a defective CaMV genome. A
    gene that complements the defective region was inserted into the
    plants such that recombination between virus and transgene could
    produce a functional copy of the virus genome that would enable
    virus replication and spread. Although CaMV is a DNA virus, it
    replicates through an RNA intermediate. Both RNA and DNA
    recombination events appeared to be responsible for generation of
    viable recombinants in four out of 12 inoculated plants (Gal et al.
    1992).

-   Transgenic *Nicotiana bigelovii* containing a transgene from an
    infective strain of CaMV were inoculated with isolates of a CaMV
    strain that do not infect *N. bigelovii* systemically.
    Complementation but not recombination occurred with one isolate, and
    recombination but not complementation occurred in eight out of ten
    plants of another isolate. Recombination between the CaMV transgene
    and infecting viruses was able to alter the symptomatology and
    expand the host range of the infecting virus strain (Schoelz &
    Wintermantel 1993).

-   Transgenic *Nicotiana bigelovii* containing a transgene from CaMV
    were inoculated with CaMV strains that could infect *N. bigelovii*
    systemically, so recombinant viruses would be expected to have
    little selective advantage over the parentals. Recombinant viruses
    were recovered in three out of 23 transgenic plants inoculated with
    one isolate and three out of 32 plants inoculated with another.
    Under strong selection pressure for recombination, recombinants were
    recovered from all 24 transgenic plants tested (Wintermantel &
    Schoelz 1996).

-   Several transgenic *Nicotiana benthamiana* lines containing the CP
    coding sequence from African cassava mosaic virus (ACMV; a
    geminivirus) were inoculated with a CP deletion mutant of ACMV.
    Recombinant progeny were recovered from transgenic lines only when
    they contained geminiviral noncoding sequence on both sides of the
    CP transgene. The frequency of recombinants varied from \<1% to 67%
    of plants in a given line (Frischmuth & Stanley 1998).

Experimental evidence thus demonstrates that recombination can occur
between viral transgenes and invading viruses. However, to facilitate
the detection of recombinants, most of these experiments were conducted
under conditions of high selective pressure, i.e., the virus was not
viable unless a recombination event occurred. The selective pressure is
usually likely to be much weaker under normal field conditions. Parental
viruses will outnumber the new recombinant and will be competent in all
of the functions needed for propagation. The relevance of experiments
demonstrating recombination under conditions of strong selection is thus
unclear beyond documenting that recombination between viral genomes and
transgenes can occur.

Although the laboratory experiments outlined above artificially
establish a high selection pressure for recombinants that is unlikely to
occur in nature, such selection might arise in transgenic plants when
viruses partially overcome the engineered resistance, as they have been
known occasionally to do (Carrington et al. 2001). If the VCP transgene
only partially eliminated an infecting virus, recombinant virions that
acquired a CP gene from a different infecting virus would be able
completely to evade the resistance conferred by the VCP transgene. Such
recombination events among different infecting viruses could occur in
the absence of the transgene. However, the transgene could create a
selective environment that would favor persistence of recombinants and
thus increase their potential to be epidemiologically significant (Jakab
et al. 1997).

The potential environmental impact of any recombinant viruses that might
arise in VCP-transgenic plants and the likelihood of their arising need
to be evaluated in the context of the events that occur in
non-transgenic plants. The recombinants that arise in transgenic plants
are theoretically unlikely to be qualitatively different from those that
could arise in natural, mixed infections if the transgene is from a
virus that normally infects the plant and is expressed in the same cells
that normally are infected. Under such circumstances the potential new
viruses that could be created through recombination are expected in
theory to be the same in transgenic and nontransgenic plants (Roossinck
1997).

*Reducing the frequency of recombination in transgenic plants*

All evidence suggests that recombination among RNA viruses occurs via
template switching by the viral replicase during replication such that a
hybrid molecule is formed (AIBS 1995). Modifications of CP transgenes
may reduce the possibility of their participation in recombination. A
number of ways have been suggested, including:

-   Remove the 3' untranslated region (UTR) in the CP mRNA transcript
    (Teycheney et al. 2000). Inclusion of this region may enable
    replication to begin on the mRNA transcript and then switch to the
    RNA of the invading virus. Removal of this region would necessitate
    two separate template-switching events to form a successful
    recombinant and thus reduce its likelihood of occurrence (Greene &
    Allison 1994). Experiments with CCMV demonstrated that deletions in
    the 3' UTR did indeed reduce the recovery of recombinant viruses
    (Greene & Allison 1996). Since functional resistance is still
    conferred by constructs containing a CP lacking the 3' UTR, this
    region may not be necessary.

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-   Reduce the extent of shared sequence similarity between the
    infecting virus and the transgene to reduce the opportunities for
    homologous recombination (Nagy et al. 1999).

-   exclude any sequences containing replicase recognition sites that
    are potential sites of recombination and any sequences known or
    thought to be recombination hotspots, e.g., promoters for genomic
    and subgenomic RNA synthesis (Miller et al. 2000).

-   Avoid potential hairpin structures in the transgene that might
    function as acceptor structures for the replicase complex (Nagy et
    al. 1998).

-   Insert GC-rich sequences downstream of any AU-rich region thought to
    be important for conferring resistance (Hammond et al. 1999).
    AU-rich regions were found to be associated with imprecise
    homologous recombination in brome mosaic virus (BMV; Nagy & Bujarski
    1996), and insertion of GC-rich sequences in BMV was found to reduce
    the incidence of recombination in spite of increasing the total
    amount of sequence identity (Nagy & Bujarski 1998). The
    applicability of these results to other viruses has yet to be
    demonstrated.

-   Use the smallest viral fragment that confers effective resistance,
    as longer fragments provide larger recombination targets (Nagy et
    al. 1999). In addition, longer segments are more likely to encode
    functional modules that could be incorporated into another virus as
    a single unit, thus increasing the chance that recombinants would be
    viable.

**Heterologous encapsidation**

*Heterologous encapsidation under natural conditions*

For many viruses, transmission from plant to plant occurs by insect
vectors and each virus tends to be transmitted by only one type of
insect (Callaway et al. 2001). The CP, possibly in conjunction with
other viral factors, is essential for transmission and responsible for
conferring the high degree of specificity. Heterologous encapsidation
occurs when the capsid protein subunits of one virus surround the
nucleic acid of a different virus, thus potentially changing its vector
specificity.

Most evidence of heterologous encapsidation is derived from laboratory
or greenhouse studies. The high frequency of mixed infections suggests
the potential for heterologous encapsidation to occur in nature is
great, but most mixed infections do not lead to heterologous
encapsidation, and those virus interactions that do occur are very
specific (Falk et al. 1995). Heterologous encapsidation is however known
to be a regular occurrence among some plant viruses. Its frequency
depends on the viruses involved and is more likely to occur among close
relatives (Tepfer 1993). An expansion of aphid vector specificity due to
heterologous encapsidation was first observed in plants infected with
two different isolates of barley yellow dwarf luteovirus (BYDV; Rochow
1970) and was later shown to be a general phenomenon among these viruses
in natural populations of several plant species (Creamer & Falk 1990).
Heterologous encapsidation was also shown to occur in potyviruses. An
isolate of zucchini yellow mosaic virus (ZYMV) that is normally
non-aphid transmissible due to a transmission-deficient CP was found to
be aphid transmissible due to heterologous encapsidation when in a mixed
infection with another potyvirus, papaya ringspot virus (Bourdin & Lecoq
1991). Heterologous encapsidation may be an important route of disease
transmission for viruses that have no CP. For example, potato spindle
tuber viroid is transmitted only by foliar contact or botanical seed
unless encapsidated by the coat protein of PLRV which renders it aphid
transmissible (Querci et al. 1997).

Heterologous encapsidation is considered a possible environmental
concern because if the new vector has a host range different from the
original vector, the virus may be spread to new plant varieties that can
support its replication but that it would not have infected if not for
the heterologous encapsidation (de Zoeton 1991). Such concerns are
largely mitigated by several factors. If replication is possible in the
new host, it would cause the virus to be encapsidated by its own CP,
thus limiting the epidemiological consequences of heterologous
encapsidation. Secondly, the vector transmission of the encapsidating
virus may require regions of the genome other than the CP for effective
transmission, so heterologous encapsidation could not lead to a change
in vector specificity (Robinson 1996). Thirdly, the vector may have a
limited host range in the area where the crop is to be grown such that
it would be unlikely to transmit a heterologous virus to a novel host.
Rather, the vector would transmit virus only to the same plant that the
virus is already able to infect (Robinson 1996).

However, under certain limited circumstances heterologous encapsidation
theoretically might still have environmental or agricultural impacts.
For one, a virus may become available for transmission by new potential
vectors that feed on the new host but not the virus' original host
(Hammond et al. 1999). Through such a mechanism, theoretically both the
host and vector range of a virus may be expanded, and the change of both
plant host and vector could make detection of heterologous encapsidation
difficult. In addition, with a high enough frequency of vector
transmission to a new host due to heterologous encapsidation, secondary
spread among new plant hosts might not be required for the phenomenon to
affect them. Due to the lack of a proofreading mechanism during
replication of RNA viruses, they are thought to exist as "quasispecies"
in which each viral genome differs by a few nucleotides from a consensus
sequence. After expansion to a new host, rapid selection of variants
best adapted to the new environment might lead to the evolution of a new
virus (Hammond et al. 1999). No direct evidence of such events exists,
but their occurrence is suggested by the appearance of "new" viruses in
areas that have had recent agricultural expansions. When previously wild
areas are cultivated, novel interactions may occur among potential
vectors, viruses in the local natural vegetation, and crops previously
unexposed to these viruses (Hammond et al. 1999).

*Laboratory experiments of heterologous encapsidation in transgenic
plants with viral transgenes*

Experimental studies have shown that the protein from VCP genes in
transgenic plants has the ability to encapsidate even unrelated
infecting viruses:

-   Transgenic tobacco plants engineered with the CP of TMV were
    infected with strains of TMV defective for the CP gene. The
    transgenic CP was able to complement the defective TMV and enable
    long-distance transport (Osbourn et al. 1990).

-   Transgenic tobacco plants engineered with the CP gene of AMV were
    infected with CMV. Heterologous encapsidation was observed to occur
    between these two unrelated viruses in one third of plants at a
    frequency of about 5 x 10^-7^ (Candelier-Harvey & Hull 1993).

-   Transgenic tobacco plants engineered with the CP of PPV were
    infected with a strain of ZYMV that is non-aphid transmissible due
    to the lack of a functional CP gene. Aphid transmission of ZYMV was
    found to occur, most likely by heterologous encapsidation in the PPV
    CP (Lecoq et al. 1993).

-   Transgenic tobacco plants engineered with the CP gene of PPV were
    infected with various potyviruses and with viruses from other
    groups. Newly formed potyvirus particles were found to contain PPV
    CP, but no other viral particles did (Maiss et al. 1994).

Heterologous encapsidation can only occur in transgenic plants if the
coat protein is expressed. Therefore, in transgenic VCP plants that
express very little CP (i.e., those relying on post-transcriptional gene
silencing to effect resistance), any effects associated with
heterologous encapsidation would be minor except in cases of resistance
breakdown. In addition, as with recombination, as long as the VCP
inserted in the transgenic plant is from a virus that normally infects
the plant in the area where it is planted, the outcome of any
heterologous encapsidation that may occur is expected to be
qualitatively the same in transgenic plants as in natural, mixed
infections.

*Reducing the frequency and/or impact of heterologous encapsidation in
transgenic plants*

Steps can be taken to reduce the likelihood of heterologous
encapsidation and/or vector transmission occurring. Specific locations
within the CP gene of several viruses have been shown to affect aphid
transmission:

-   Amino acid changes in the CP of CMV differentially reduce the
    efficiency of transmission by two different aphid species (Perry et
    al. 1998).

-   The readthrough domain (RTD) of the CP plays a key role in
    determining aphid transmission specificity in PLRV (Jolly & Mayo
    1994), BYDV (Chay et al. 1996), and beet western yellows luteovirus
    (BWYV; Brault et al. 1995; Reinbold et al. 2001; Bruyère et al.
    1997; Brault et al. 2000).

-   Point mutations in the major capsid protein (P3) of BWYV affect
    aphid transmission (Brault et al. 2003).

-   Amino acid changes in a conserved loop structure of the CP of CMV
    affect aphid transmission (Liu et al. 2002).

-   A three amino acid sequence, asp-ala-gly (DAG) is conserved in
    aphid-transmissible strains of potyviruses including tobacco vein
    mottling virus (Atreya et al. 1991) and ZYMV (Gal-On et al. 1992).
    Mutations in this region in either virus render it
    non-aphid-transmissible. The particular context in which this amino
    acid triplet is found also appears to be important in determining
    aphid transmissibility in TVMV (López-Moya et al. 1999).

Experiments suggest that VCP gene modifications could reduce the
frequency of heterologous encapsidation and/or vector transmission.
Mutations in the two assembly motifs (RQ and D) of the CP of PPV were
found to suppress heterologous encapsidation, particle assembly, and
complementation without affecting resistance to viral infection
(Varrelmann & Maiss 2000). Likewise, the CP gene of PPV was modified or
truncated in order to reduce any potential impacts of heterologous
encapsidation: either the DAG triplet was deleted or the first 420
nucleotides of the PPV CP gene were removed (Jacquet et al. 1998b). Both
transgenic lines were resistant to PPV infection, indicating that the
full-length CP region is not needed for resistance. Further experiments
confirmed that such changes in the CP did effectively mitigate the
potential for heterologous encapsidation to have any effect (Jacquet et
al. 1998a). However, investigation of the DAG motif in begomoviruses
revealed that it is not the precise determinant of whitefly transmission
of this virus. Rather, amino acids 123 to 149 of the CP are minimally
required for transmission, and amino acids 149 to 174 contribute to
efficient transmission (Höhnle et al. 2001).

If changes that resulted in loss of aphid transmissibility were to be
incorporated into the VCP genes used in transgenic plants, then
heterologous encapsidation would be extremely unlikely to change vector
specificity, since the CP would no longer confer any specificity at all.
Experiments with various potyviruses showed that the degree of
heterologous encapsidation failed to correlate with the degree of
resistance conferred by different CP constructs (Hammond & Dienelt
1997), suggesting that such strategies could be deployed without
compromising product efficacy. However, while particular changes in the
CP are effective at reducing any potential impacts associated with
heterologous encapsidation, variation among viruses may preclude a
single generic solution that could be used for all.

**Synergy**

*Synergy under natural conditions*

In addition to the creation of a novel virus through recombination and
the alteration of transmission properties of an existing virus through
heterologous encapsidation, viral interactions may cause increased
disease severity through alteration of the replication and/or movement
efficiency of a virus. In a synergistic disease the severity of two
viruses together is greater than expected based on the severity of each
alone. When potato virus X (PVX) is coinfected with a number of
potyviruses including TVMV, TEV, and pepper mottle virus, the disease
symptoms are considerably worsened and PVX accumulates to a greater
concentration (Vance et al. 1995). CMV and TAV cause no synergistic
disease in double infections, but an interspecies hybrid was
significantly more virulent than either parent in all plant species
tested (Ding et al. 1996). The increased severity was most likely due to
a synergistic interaction between TAV and the protein produced from the
CMV portion of the hybrid. A listing of reported viral synergisms has
been compiled (OECD Environment Directorate 1996).

Synergistic interactions are extremely common among the luteoviruses,
but the CP is considered much less likely to be responsible for
synergism than other regions of the viral genome, for example the
polymerase gene (Miller et al. 1997). Within potyviruses, the 5'
proximal 1/3 of the viral genome is thought to contain the factors that
mediate synergism, including the 5' UTR and the coding region for the 5'
N-terminal portion of the viral polyprotein (Pruss et al. 1997).

*Synergy in transgenic plants with viral transgenes*

Synergy in transgenic plants is generally an agroeconomic rather than
environmental concern. Any negative effects are expected primarily to
affect the transgenic crop itself which would be quickly abandoned once
such effects were detected. Temporarily increased viral loads caused by
synergistic disease could result in limited damage to nearby
conventional crops if the transgenic crop were already deployed in large
field acreages when synergistic disease was discovered (Miller et al.
1997). However, synergistic interactions can be evaluated in transgenic
plants before deployment by experimental inoculation with all of the
viruses likely to be encountered in the field (Robinson 1996).
Developers have a strong incentive to undertake such efforts to ensure
the efficacy of their product after deployment.

*Reducing the frequency of synergy in transgenic plants*

As with heterologous encapsidation, constructs can be engineered to
reduce the likelihood of synergy. Particular transgenes known to
participate in synergistic interactions could be avoided or defective
copies of genes could be used. Stacking multiple resistances within the
same plant such that it will have reduced viral loads for all of its
normal pathogens has also been suggested (Palukaitis 2000). The benefits
of this approach may however be outweighed by the concerns associated
with stacking in VCP-transgenic plants (discussed below).

**Field evaluations of viral interactions in transgenic plants with
viral transgenes**

The many laboratory experiments outlined above that investigated the
potential viral interactions in VCP-transgenic plants are only one part
of evaluating potential environmental impacts of such events.
Consideration of the potential impact under natural, field conditions
evaluated in the context of the likelihood of any such event occurring
is equally important (García-Arenal et al. 2000). Relatively few field
studies have been conducted to address these questions, but those that
have been done have found no significant impact associated with
deployment of VCP-transgenic plants beyond natural background events. A
six-year experiment searched for and failed to find evidence of
interactions involving viral transgenes in 25,000 transgenic potato
plants transformed with various PLRV CP constructs. Both greenhouse and
field tests failed to show any change in the type or severity of disease
symptoms, and all viruses isolated were previously known to infect the
plants and had the expected transmission characteristics (Thomas et al.
1998).

An experiment with transgenic melon and squash expressing CP genes of an
aphid-transmissible strain of CMV failed to find evidence that either
recombination or heterologous encapsidation enabled spread of an aphid
non-transmissible strain of CMV in the field (Fuchs et al. 1998). A
similar experiment with transgenic squash expressing CP genes of an
aphid-transmissible strain of watermelon mosaic virus (WMV) showed that
an aphid non-transmissible strain of ZYMV was not detected in
nontransgenic fields but was transmitted to 2% of plants in transgenic
fields, likely due to heterologous encapsidation. However, this rate of
transmission failed to lead to the development of an epidemic of ZYMV in
transgenic squash fields (Fuchs et al. 1999).

In an experiment to assess the biological and genetic diversity of
California CMV isolates before and after deployment of transgenic melon
containing the CMV CP gene showed that the only CMV isolate to show
significant sequence changes after infecting the transgenic squash was
not the result of recombination (Lin et al. 2003). The only field
experiment to directly assess the effect of recombination in a
VCP-transgenic plant found no detectable grapevine fanleaf virus (GFLV)
recombinants containing the inserted CP sequence over the course of a
four-year study (Vigne et al. 2004).

The limited number of field evaluations thus indicate that the likely
environmental consequences of viral interactions in VCP-transgenic
plants are minimal. However, large acreages of VCP-transgenic plants
grown over many years may provide increased opportunity for rare events
to occur that are unlikely to be detected in experimental studies
(Miller et al. 1997), so consideration of theoretical outcomes and their
likelihood of occurrence may be necessary to facilitate a complete
evaluation of the risk concerns.

**Is the frequency of viral interactions in transgenic plants different
than in natural, mixed infections?**

Few experimental studies have addressed the relative degree of risk
directly, mostly because it is hard under any circumstances to determine
the frequency of viral interactions that lead to a measurable effect
given how rare these events are. Some characteristics of VCP-transgenic
plants suggest that the frequency of interactions may be lower than in
natural, mixed infections. Other characteristics suggest that the
frequency may be higher (see below).

One factor that may *decrease* the frequency of interactions in
transgenic systems is that the cellular concentration of viral RNA
transcripts expressed from transgenes will be orders of magnitude lower
than the concentration of viral RNA commonly found in natural, mixed
infections (Allison et al. 1996). The concentration of infecting viral
RNA from the target virus will also be considerably reduced,
particularly when the mechanism of resistance relies on
post-transcriptional gene silencing to remove all viral RNA transcripts
with homology to the transgene (Rovere et al. 2002). However, the
significance of these observations is difficult to interpret. While it
is known that greater concentrations of RNA will provide greater
opportunity for interactions, meaningful values for high or low
concentrations are unavailable (AIBS 1995).

The frequency of interactions in transgenic systems may be *increased*
because promoters currently used in VCP-transgenic plants cause
constitutive expression of transgenes at developmental stages that might
otherwise be unaffected by viral infection and often in tissues that the
virus does not normally infect (Allison et al. 2000). For example,
luteoviruses are normally expressed only in phloem tissue, but the
cauliflower mosaic virus (CaMV) promoter drives expression of luteoviral
CP in all plant cells. Some evidence suggests that in natural infections
different viruses have different temporal or spatial expression patterns
that would limit their interactions (Gibbs 1994; Hull 1994; Aaziz &
Tepfer 1999). Viruses must simultaneously replicate in the same cellular
compartment for their RNA to be able to interact. However, when a virus
invades a cell, it often replicates and then moves to other cells within
the plant. The RNA remaining in the initially infected cell becomes
encapsidated and is no longer available for interactions with another
invading virus (Allison et al. 2000).

How these competing factors balance out to affect the relative frequency
of viral interactions in transgenic versus nontransgenic plants is thus
likely to vary with the virus, the plant, and the mechanism of
resistance. Whether the nature of the interactions that do occur may be
different in VCP-transgenic plants than expected in natural, mixed
infections must also be considered.

**When could viral interactions in transgenic plants be unlike those
likely to occur in natural, mixed infections?**

Another key question for evaluating the significance of the above
studies is whether the viral interactions in transgenic plants may be
unlike those likely to occur in non-transgenic plants. The potential
risks associated with viral interactions in VCP transgenic plants are
similar to those that arise with natural, mixed infections.
Recombination, heterologous encapsidation, and synergy may occur in both
cases, although a comparison of frequencies is difficult. Theoretically,
the hazards associated with these phenomena are expected to be similar
whether these events occur in transgenic or nontransgenic plants.
However, under certain circumstances, transgenic plants may in theory
provide opportunities for unique interactions that would not be expected
to occur in a natural, mixed infection:

-   Transgenic multiresistances: A plant may be engineered to resist
    infection from multiple viruses by incorporation of several CPs into
    the same plant. While generally, a transgenic plant that is
    resistant to a particular virus will be planted only where the virus
    is a problem, with stacked resistances, the likelihood of doing
    otherwise increases. For example, a cucurbit resistant to CMV, WMV2,
    and ZYMV has been created (Fuchs et al. 1997). Such a plant would
    probably be used in all areas where ZYMV is prevalent, including
    tropical areas where WMV2 never or only rarely occurs (Hammond et
    al. 1999). WMV2 could then interact with a local strain that would
    otherwise have had no opportunity for interaction with it.

-   Heterologous resistance: A plant may be resistant to infection by a
    particular virus in spite of having the CP of another virus
    incorporated into its genome. For example, VCP genes from LMV were
    used to provide resistance to PVY in tobacco which is not infected
    by LMV (Dinant et al. 1993). In such plants, LMV might have a new
    opportunity to interact with viruses that infect tobacco.

-   Plants may be engineered with VCP genes from an exotic strain of a
    virus that may be more virulent or have other properties different
    from endemic isolates. Under certain circumstances, the desire that
    transgenes be from an endemic isolate may be balanced by other
    concerns. For example, when the costs of an exotic viral invasion
    would be particularly devastating, incorporation of a viral
    transgene intended to prevent such establishment might be warranted
    (Hammond et al. 1999). In addition, identification of local virus
    isolates may not always be possible, especially given that the
    spectrum of isolates in a given area will change over time. Even the
    definition of what an endemic isolate is can be complicated by
    international trade in seed and vegetative propagating material.
    Nevertheless, thorough evaluation of a VCP-transgenic plant requires
    knowledge of the plant viruses that are present in the release
    environment and that naturally infect the host (Robinson 1996).

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-   Plants may express VCP genes in cells and/or tissues that the virus
    does not normally infect (as discussed above).

-   Plants may be engineered with VCP genes that have been altered such
    that they do not resemble any that exist in nature. Any interactions
    involving such VCP genes would thus be novel.

Such situations may present the opportunity for novel viral interactions
in VCP-transgenic plants, i.e., interactions between portions of two or
more different viruses not expected to occur in a mixed infection found
in nature. Such situations may be avoided in designing and deploying
VCP-transgenic plants. However, if avoidance is impossible or
undesirable, strategies such as those discussed above to prevent the
occurrence of viral interactions would greatly reduce the frequency of
all interactions, whether novel or not.

**Conclusion**

While the nature of the potential risks posed by novel interactions
among viral genomes in transgenic plants is relatively well understood,
data to evaluate the actual impact and likelihood of such events under a
wide range of natural conditions for different VCP-transgenic plants are
sparse. Further experimental data may help to provide a more complete
picture that would allow a careful evaluation of the likelihood of
adverse impacts and the subsequent evaluation of the risks and benefits
of deploying VCP-transgenic plants. Although current evidence suggests
that the risks are unlikely to be much greater than those found in
natural, mixed infections (Hammond et al. 1999; Bruening & Falk 1994),
steps may be taken when designing products to minimize any potential
impacts.

**References**