Ann Revual Tospovirusses

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XMLPublishSM (2004/02/24)
10.1146/annurev.phyto.43.040204.140017
Annu. Rev. Phytopathol. 2005. 43:45989

doi: 10.1146/annurev.phyto.43.040204.140017
c 2005 by Annual Reviews. All rights reserved
Copyright
First published online as a Review in Advance on May 19, 2005
TOSPOVIRUS-THRIPS INTERACTIONS
Anna E. Whitfield,1 Diane E. Ullman,2 and
Thomas L. German1
Annu. Rev. Phytopathol. 2005.43:459-489. Downloaded from www.annualreviews.org
by Wageningen UR on 09/21/14. For personal use only.
Department of Entomology, University of Wisconsin, Madison, Wisconsin 53706;

email: aew@plantpath.wisc.edu, tlg@entomology.wisc.edu
2
Department of Entomology, University of California, Davis, California 95616;
email: deullman@ucdavis.edu
Key Words Bunyaviridae, insect vector, membrane glycoproteins, Thysanoptera,

Tomato spotted wilt virus
Abstract The complex and specific interplay between thrips, tospoviruses, and
their shared plant hosts leads to outbreaks of crop disease epidemics of economic
and social importance. The precise details of the processes underpinning the vectorvirus-host interaction and their coordinated evolution increase our understanding of
the general principles underlying pathogen transmission by insects, which in turn
can be exploited to develop sustainable strategies for controlling the spread of the
virus through plant populations. In this review, we focus primarily on recent progress
toward understanding the biological processes and molecular interactions involved in
the acquisition and transmission of Tospoviruses by their thrips vectors.
INTRODUCTION
Insects in the order Thysanoptera (commonly known as thrips) include many important direct crop pests and at least 10 species that transmit viruses in the genus
Tospovirus, the only plant-infecting genus in the family Bunyaviridae. Thrips
transmission of tospoviruses impacts a diverse number of food, fiber, and ornamental crops encompassing hundreds of plant species (14), resulting in crop disease
epidemics of worldwide economic and social significance. The biology that influences the coordinated evolution of thrips, tospoviruses, and their plant hosts
has provided an opportunity for scientists from many disciplines to address issues
of applied and basic significance since the early decades of the 1900s. Many
thrips that serve as tospovirus vectors have extensive plant host ranges. Both
tospoviruses and thrips vector species thrive in diverse climates, which explains
their worldwide importance.
The interest in the Thysanoptera, their role as tospovirus vectors, and the biology of tospoviruses is global. Growers and pest control advisors have recognized
that advances in our ability to manage thrips as direct pests and as vectors requires a broad and integrated research approach at the interface of entomology,
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ecology, systematics, and virology. To expand our knowledge of the fundamental principles underlying virus-vector relationships, scientists are beginning to
dissect the specific series of events governing the complex interaction between
thrips, tospoviruses, and their plant hosts. Recent progress toward understanding
the biological processes and molecular interactions that lead to virus acquisition,
movement within the vector, and transmission to plant hosts are covered in this
review.

TOSPOVIRUSES
The disease known as spotted wilt was first described in Australia in 1915 (12), and
thrips were implicated as vectors of the disease-causing agent several years later
(107). In 1930, Samuel et al. (121) demonstrated that a virus, which they named
Tomato spotted wilt virus (TSWV), was the causative agent of the disease. More
than 50 years later, Francki and colleagues (88) noted the similarity between TSWV
and viruses in the family Bunyaviridae: a large group of membrane-bound, mostly
arthropod-transmitted, animal-infecting viruses with tripartite negative-stranded
RNA genomes. They suggested that it was the first plant-infecting member of a
monotypic group of plant viruses (36). During the following two decades, data
on the molecular biology of TSWV (reviewed in 1, 41, 44, 93, 123, 124) and the
closely related Impatiens necrotic spot virus (INSV) (25, 73) confirmed (27) the
validity of the taxonomic status of these and related viruses.
The family Bunyaviridae includes animal-infecting viruses of the genera Orthobunyavirus, Hantavirus, Nairovirus, and Phlebovirus and the Tospovirus genus,
which consists of the plant-pathogenic, thrips-transmitted members of the family. TSWV is the type member of the genus that currently includes 14 species
(Table 1) separated primarily on the basis of the serological properties and amino
acid sequence identity of the viral structural proteins (23, 24). Typical of the family Bunyaviridae, TSWV has a genome consisting of three negative or ambisense
ssRNAs designated S (2.9 kb), M (4.8 kb), and L (8.9 kb). The RNAs have a
panhandle conformation created by base pairing of about 60 complementary nucleotides at the 3 and 5 ends of each strand (28). The core of the virion contains
ribonucleoproteins (RNPs) composed of the ssRNA components encapsidated by
the nucleoprotein (N) and a few copies of the viral RNA-dependent RNA polymerase (RdRp or L protein). The 80120-nm pleiomorphic virus particles are
formed by enclosure of the RNPs in a host-derived membrane studded with surface projections composed of two viral glycoproteins, GN and GC (Figure 1).
Although the details of the genomic organization and expression strategy (as
shown in Figure 2) of TSWV are well characterized, gene product function in
TSWV is just beginning to emerge. The ambisense 2.9-kb S RNA encodes a
52.4-kDa nonstructural protein (NSs, not present in the mature virus particle) in
the viral (v) sense and the 29-kDa N protein in the viral complementary (vc) sense
(29). Both proteins are expressed by translation of subgenomic RNA species that
possibly terminate at a long, stable intergenic hairpin structure (67). The tospovirus
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TABLE 1
Recognized Tospovirus species and their documented vectors
Tospovirus species

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Thrips vectors
Capsicum chlorosis virus (83)
Chrysanthemum stem necrosis virus (113)
Frankliniella occidentalis (95)

F. schultzei (94)
Groundnut bud necrosis virus (111)
F. schultzei (6)
Thrips palmi (70)
Scirtothrips dorsalis (6)
Groundnut ringspot virus (23)
F. occidentalis (156)
F. schultzei (156)
Impatiens necrotic spot virus (70)
Iris yellow spot virus (20)
T. tabaci (40)
Melon yellow spot virus (58)
T. palmi (57)
Peanut chlorotic fanspot virus (17)
S. dorsalis (17)
Peanut yellow spot virus (110)
S. dorsalis (109)
Tomato chlorotic spot virus (23)
F. intonsa (156)
F. schultzei (156)
Tomato spotted wilt virus (12)
F. bispinosa (151)
F. fusca (120)
F. intonsa (156)
F. schultzei (121)
T. setosus (65)
T. tabaci (107)
Watermelon bud necrosis virus (53)
T. palmi (92, 126)
Watermelon silver mottle virus (160)
T. palmi (161)
Zucchini lethal chlorotic virus (113)
F. zucchini (100)
NSs protein has been shown to function in suppression of RNA silencing during the
plant-infection phase of the virus life cycle. (13, 132). The NSs genes of the related
phleboviruses and orthobunyaviruses can influence the virulence of these viruses in
their animal hosts (11, 150), which has led scientists to speculate about the possible
role of the tospovirus counterpart in the thrips infection cycle. The nucleocapsid
protein (N) contributes to the viral replication cycle in a structural and, perhaps,
regulatory manner by participating in the complex interactions among the RNP
components leading to the initiation of viral RNA transcription and replication.
Consistent with its role in fulfilling this putative function, the N protein has been
shown to form dimers in the absence of RNA (56, 136) and to cooperatively bind
ssRNA but not dsRNA (114). Interestingly, mutated forms of N protein serve as
potent dominant-negative inhibitors of virus replication (118).
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Figure 2 Genomic organization and expression strategy of Tomato spotted wilt virus.
vRNA represents virion sense RNA, the predominate form of RNA in the virion. vcRNA
represents viral complementary sense RNA. Open boxes in the RNAs indicate open
reading frames expressible from either v or vc RNA. Stippled boxes at the 5 termini
of mRNAs represent nontemplated cap structures. The flexuous lines represent virusencoded proteins.
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The M RNA encodes a 33.6-kDa nonstructural protein (NSm) in the v sense

and a 127.4-kDa precursor to the two viral membrane glycoproteins, GN and
GC, in the vc sense. Using an expression strategy similar to that of the S RNA, the
NSm open reading frame (ORF) is expressed from a subgenomic RNA transcribed
from the vcRNA, and GN and GC are translated as a polyprotein from a single
ORF on a subgenomic mRNA transcribed from the vRNA (67). The resulting
GN /GC polyprotein is cleaved in the absence of other viral proteins to produce the
individual membrane components (2). The role of GN and GC in virus assembly
and transmission by thrips is treated in detail in the following sections. The role
of NSm in cell-to-cell movement is supported by its early expression profile, its
association with (68) and ability to alter the size exclusion limit of plasmodesmata
(129), and the observation that it forms tubules, a feature of other viral movement
proteins (30, 50, 128).
The L RNA is of entirely negative polarity, with one ORF located on the vc
strand corresponding to a primary translation product of 2875 amino acids, with
a predicted molecular mass of 331 kDa. This protein contains a cluster of nucleic
acid polymerase motifs (26) including the highly conserved serine-aspartic acidaspartic acid (SDD) element found in the RdRp of all segmented negative-strand
RNA viruses (134). Sequence and functional analyses of the L protein support the
role of a multifunctional viral RdRp that, by analogy to other negative-strand RNA
viruses, has NTPase, polymerase, nuclease, helicase, and polymerization activities.
In several negative-strand RNA virus families, these discrete functions are carried
out by individual proteins produced from unique mRNAs; however, the single
multifunctional protein organization of TSWV L RNA is characteristic of viruses
in the Orthobunyavirus, Hantavirus, and Phlebovirus genera of the Bunyaviridae
(134). Functional analysis has shown that an RdRp activity is associated with
detergent-disrupted TSWV virion preparations (3, 148) and that the L protein is
the source of this activity (15).
A brief overview of the replication cycle provides insight into the complex interplay transpiring between virus and host during the infection process. By analogy to
the animal-infecting members of the Bunyaviridae, it is reasonable to hypothesize
that tospoviruses infect insect cells by binding to a host cell receptor through the
mediation of a viral surface glycoprotein (Figure 3). A subsequent fusion event of
the viral and host membranes, possibly initiated by low pH (155), releases the genomic RNA segments in association with multiple copies of N protein (RNPs) into
the cytoplasm. The RNP serves as a template for transcription of viral mRNAs,
catalyzed by the virion-associated viral RdRp. Typical of segmented negativestrand RNA viruses, TSWV mRNAs are not polyadenylated and have eukaryotic
cap structures and nontemplated heterogenous sequences of host origin at their
5 ends. These are generated during a process, termed cap-snatching, during which
the viral polymerase cleaves some 1020 nucleotides along with the cap structure from a host mRNA and incorporates them into the 5 terminus of the newly
synthesized viral mRNA (32, 33, 69, 149). These messages then associate with
the host cell translational apparatus to produce the viral protein complement and
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Figure 3 Transmission electron micrographs showing the likely steps in virus fusion
and entry to the larval midgut of Frankliniella occidentalis. Protein A-gold was used
as the tag in all micrographs. (A) A thin section of a larval thrips midgut epithelial
cell. Virions ingested from an infected plant are labeled with polyclonal antibody
to the Tomato spotted wilt virus (TSWV) membrane glycoproteins. Inset shows a
labeled virion fusing with the apical membrane. [Reprinted with permission from the
American Phytopathological Society from (145, figure 1).] (B) Thin section of larval
thrips midgut epithelial cell immunostained with antibodies against TSWV membrane
glycoproteins and 15-nm Protein A-gold. The membrane just above a dense mass
is labeled, suggesting that TSWV membrane glycoproteins are bound to the apical
membrane. (C) Thin section adjacent to that shown in B immunostained with antibodies
against TSWV N protein and 15-nm Protein A-gold. Labeling of the mass suggests
that TSWV ribonucleoproteins or virions have entered the cell. ap, apical membrane;
c, cytoplasm; mv, microvilli; lu, lumen; v, virus; unmarked arrowhead, dense mass of
ribnucleoprotein; unmarked arrow, gold particle on apical membrane.
additional copies of the RdRp that generate full-length antigenomic RNAs. These
antigenomic RNAs, in turn, serve as templates for de novo synthesis of many
genomic RNA copies. The panhandle structure formed by the complementary 3
and 5 ends of each viral genomic segment most likely serve as promoters for
replication. Host proteins are then probably involved in the process of template
selection, stabilization of the polymerase complex, and catalytic events resulting
in extension of the RNA strands (4, 31, 84).
Tospovirus replication has been described in the context of a plant infection;
however, immunocytochemical and microscopic identification of TSWV NSs protein in cells of Frankliniella occidentalis established that viral replication also
occurs in the insect vector (141, 158). Therefore, in contrast to viruses that have
nonpersistent or circulative modes of transmission, adaptation and selection events
that influence the rate of virus replication, movement, assembly, and counterdefense strategies in the insect have an increased influence on transmission to plant
hosts. For example, the rate of virus replication in the midgut and the extent of
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virus migration from the midgut to the visceral muscle cells and the salivary glands
are crucial factors in the determination of vector competence (98). Consequently,
fully understanding the transmission process requires a detailed consideration of
the multiple points of interaction between virus and vector that govern entrance,
replication, movement within, and exit from the vector. Taken together, these steps
comprise the events culminating in the infection of a susceptible plant host.

COEVOLUTION OF THRIPS AND TOSPOVIRUSES

Although tospoviruses can be mechanically transmitted under experimental conditions, tospovirus dispersal and survival in nature depends on passage to plants by
thrips vectors. The dispersal and survival of TSWV depends upon the coexistence
of virus and vector populations under conditions in which their genetic and physiological make-up form a compatible vector-virus interaction. The environment and
the plant-host interactions with the virus and the insect also influence every phase
of the infection cycle. Thus, coevolution between thrips and tospoviruses must
greatly influence the observed variability between virus isolates, epidemics, and
even the emergence of new tospoviruses (Figure 4). Tremendous genetic variability
in tospovirus populations is provided by the high error rate inherent in RNA replication by the viral RdRp (21) and the reassortment of genomic segments between
different virus isolates in planta (108). Little is known about the thrips genome,
but all of the vector species are characterized by striking morphological diversity,
which suggests genetically variable populations (92). Tospovirus and thrips vector
populations certainly meet the basic requirements for rapid coevolution.
An example of likely coevolution between tospviruses and their vectors is provided by the altered status of Thrips tabaci as a vector of TSWV. Forty years ago
this species transmitted all known isolates of TSWV worldwide (119), but now
appears to be incapable of transmitting modern TSWV isolates (82, 156). New
vector-virus relationships have also arisen, possibly as a result of coevolutionary
events. For example, Thrips palmi can now transmit several newly emergent
tospoviruses in cucurbits (57, 92, 126), and F. bispinosa emerged as a vector
of TSWV (151), while Thrips tabaci emerged as a vector of Iris yellow spot virus
(IYSV) (40).
The precise mechanisms underlying the formation of these new vector-virus
relationships are not well understood, but certainly plant host-vector relationships
and thrips reproductive strategies play roles as well (16). Many plant hosts support mixed virus infection and multiple thrips species, which provide an arena for
genetic exchange. Reassortment of RNA segments from resistance-breaking or
thrips-transmissible isolates could endow nontransmissible isolates with resistancebreaking characteristics and new transmission qualities (108). This evidence
supports the notion that reassortment occurring in mixed infections could contribute to the appearance of isolates with a variety of new characteristics,
including the emergence of new vector species or reemergence of a vector like
T. tabaci.
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THRIPS LIFE CYCLE, REPRODUCTIVE STRATEGIES,

AND FEEDING BEHAVIOR
Thrips vector species are well equipped for their role as vectors of tospoviruses. The
thrips life cycle and its relevance to the tospovirus transmission cycle is illustrated
in Figure 5. Adult females lay eggs on plants and after eclosion, there are two
wingless larval stages that feed on plant leaves and flowering parts. The ensuing
pupal stages are nonfeeding and depending on the thrips species, this stage occurs
in the soil or on plants. Winged adults that strongly resemble the larvae emerge
and tend to disperse widely.
Most of the vector species have high fecundity and short reproductive cycles.
They are haplodiploid: Females are diploid and males are haploid. Thrips have two
reproductive strategies: Arrhenotokous thrips reproduce sexually and thelotokus
thrips reproduce asexually (16, 92). Knowledge of Thysanopteran reproductive
strategies can help us understand the genetic basis of transmission efficiency and
variability in tospovirus epidemics, e.g., thelotokus, and not arrhentokus, T. tabaci
populations are efficient vectors of certain tospoviruses (16).
The thrips species that transmit TSWV are polyphagous, piercing-sucking insects that feed on a variety of cell types (92, 138). Many thrips species feed on
pollen and flower structures, as well as on vegetative portions of plants (75, 138).
To feed, the insect punctures the leaf epidermis and ingests the cytoplasm from
mesophyll cells (138), either collapsing single cells or destroying several cells.
Intact plant organelles and virions were observed in thrips guts with the use of EM
(132, 139). Evidence from electronic monitoring suggests that the WFT has at least
two modes of feeding. They make many probes of short duration during which
they apparently salivate into and empty the contents of single or small groups of
plant cells probably just under the epidermal surface (63). Less often, probes of
much longer duration are made that consist of a short period of salivation followed
by what appears to be a long period of ingestion (51). This type of feeding tends to
be very destructive to leaves, with the damage extending through all or several leaf
cell layers (51, 63, 138). Sakimura (119) suggested that thrips were more likely to
transmit virus during brief shallow probes; however, more research in this area is
needed to define the impact of feeding behaviors on virus transmission to plants.
Virus Dissemination and Replication in Vector Thrips
To understand the specific events that lead to transmission of tospoviruses in nature,
it is critical to identify the anatomical features of the insect that mediate viral
movement and replication in the insect. Dissemination of the virus through the
insect illustrates its ability to bind and travel across host membranes. Here, we
describe the tissue systems and six membranes encountered by virions along their
path from the alimentary canal to the salivary glands (Figure 6). [For a more
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Figure 6 Thrips internal organs and their putative role in virus passage to the salivary
glands. This line drawing depicts the putative path of tospoviruses through the thrips and
illustrates the membrane barriers the virus must pass before successful inoculation of a
plant can occur. Tospoviruses enter the midgut lumen and move across the apical membrane of the brush border [1]. Tospoviruses replicate in the midgut and by an unknown
mechanism cross the basement membrane [2] into the visceral muscle cells where
replication continues as indicated by the presence of viroplasm [inset, 3]. The viruses
must then exit these cells across their basal membrane [inset, 4] and enter the salivary
gland [5]. Virions exit the salivary gland across the apical membrane [6] and flow with
the salivary secretions into the plant during thrips feeding. Hg, hindgut; Mc, mouthcone; Mg, midgut; E, esophagus; EF, efferent salivary duct; L, lumen; PSg, primary
salivary gland; s, cross sections of muscle; TSg, tubular salivary gland; VP, viroplasm;
DM, dense mass.
detailed description of thrips internal anatomy, see (138).] Upon ingestion of viral
particles, virions travel through the foregut into the midgut, the primary site of
TSWV-binding and entry into insect cells (8, 97, 139). A brush border of microvilli
extends into the midgut lumen and forms the first membrane barrier encountered
by the virus (see magnified inset in Figure 6, denoted by the number 1). Virus
particles move across the microvilli to the basal surface of the columnar epithelial
cells of the midgut, which is formed by the basement membrane, the next membrane
encountered by the virus. The virus exits the midgut epithelia (see magnified area
in Figure 6, denoted by the number 2) by crossing the basal membrane that is
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encircled by alternating series of longitudinal and circular muscle cells (90, 137,
141). TSWV has been observed in these muscle cells, and entry and exit from
these muscle cells presumably consitute the third and fourth membrane barriers
that virions must cross on their path to the salivary glands (7, 8, 97, 98, 101).
The primary salivary glands are thought to play a critical role in virus acquisition and transmission. Little is known about the basal membrane of these glands;
however, tospoviruses entering the salivary gland must traverse this membrane
(Figure 6, see magnified inset, the number 5). The lumen of each lobe is lined with
microvilli (Figure 6, see magnified inset, the number 6), and this represents the
last membrane the virus must cross in order for transmission to occur. Once inside
the salivary gland lumen, virions can move with saliva into a canal that leads to an
efferent salivary canal, a common salivary reservoir, and then a duct that ultimately
allows virus-laden saliva to exit the combined salivary-food canal.
As previously outlined, viral infection in larval thrips begins in the midgut after
which the virus replicates, spreads throughout the midgut, and subsequently infects
the muscle cells surrounding the midgut and the primary salivary glands (7, 97, 98,
135, 139, 141, 144, 145, 158). Presence of nonstructural proteins (NSs) and viral
inclusions in the insect support the contention that tospoviruses replicate in their
thrips vectors (141, 158) and the presence of viral inclusions in midgut epithelial
cells, muscles surrounding the alimentary canal, and the primary salivary glands of
thrips indicated that virus replicates in these tissues (141, 144, 145, 158). TSWV
glycoproteins were immunolocalized to membranes thought to be part of the Golgi
complex in thrips cells (145). Additionally, TSWV proteins were associated with
membrane-bound structures in midgut epithelial cells that appeared to be fusing
with basal membrane (145), and Nagata et al. (98) observed virions budding from
the basal side of infected midgut cells. Viral proteins were also associated with
vesicles that were similar in appearance to clathrin vesicles; furthermore, these
vesicles were often observed near vacuoles that also contained viral proteins (145).
It is likely that virions and/or viral proteins associated with secretory vesicles could
function in virus cell-to-cell movement via the exocytic pathway.
Models for Virus Movement in Thrips

Although contemporary electron microscopic evidence clearly shows virus infection of several thrips organs and tissues, little is known about the route virus takes
in the insect to eventually reach the salivary glands. Three hypothetical models
have been proposed to attempt to explain the mechanisms underlying tospovirus
movement from the thrips midgut to the salivary glands:
1. By analogy to other persistently transmitted viruses, virus could enter the insect gut, traverse the midgut and muscle cell barriers, and enter the hemocoel.
The virus would then circulate and may infect other organs, but ultimately
infect the salivary glands, allowing for successful transmission to plants. To
date, there is no direct evidence to support this hypothesis.
2. Examination of thrips anatomy showed that two structures connect the
midgut to the primary salivary glands: tubular salivary glands (138) and thin
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ligament-like structures (97). On the basis of immunolabeling of TSWV in

thrips, several authors propose that the ligament-like structures may serve as
a conduit for virus transport by connecting the midgut to the salivary glands
(8, 97, 98). Nagata et al. (97, 98) found that infection of the ligament-like
structure preceded salivary gland infection. Early infection sites in the salivary gland occurred at the point of contact with the ligament-like structure,
which led the authors to propose that the ligament-like tissue connects the
midgut to the salivary glands, thereby facilitating cell-to-cell spread of virus
from the midgut to the salivary gland. Transmission electron microscopic
evidence will be very useful in confirming the presence of virus in these
ligament-like structures. Although the tubular salivary glands would seem
to be convenient structures to support virus movement, there is no reported
evidence of virus infection in these structures (98; D.E. Ullman & D.M.
Westcot, unpublished data).
3. Based on thrips ontogeny, Moritz et al. (91) demonstrated that the proximity of organs changes during thrips development. They proposed that virus
movement from midgut tissues to the primary salivary glands occurs when
there is direct contact between membranes of the visceral muscle cells and the
primary salivary glands during larval development. These authors showed
that the primary salivary glands, midgut, and visceral muscles of the first
larval stage of F. occidentalis are compressed into one area of the thorax
where they lie in direct contact with one another through the early second
instar stage. It is proposed that virus moves from the midgut and muscles
to the salivary gland when these tissues lie in direct proximity to one another in the larval insect (91). As the insect grows, the brain and primary
salivary glands move forward, while the first midgut loop moves back into
the metathorax, resulting in spatial separation of these organs and preventing virus movement between these tissues. This hypothesis is compelling
because it is consistent with the evidence that only adults that feed as larval
stages can transmit virus.
Virus movement in the thrips vector is an exciting area of investigation. We
expect the near future to hold explanations for potential barriers to virus survival in
the hemolymph and the mechanisms of virus passage from the midgut and visceral
muscles to the primary salivary glands. Innovative techniques such as laser-capture
microdissection, immunohistochemistry, and real-time quantitative RT-PCR will
provide the needed tools to unravel the mysteries remaining in understanding the
route tospoviruses take while navigating the insect vector.
Thrips Stage-Dependent Acquisition of Tospovirus

Tospoviruses are transmitted in a persistent propagative fashion and are transstadially passed in their insect vector. Figure 5 illustrates the essential elements of
the tospovirus transmission cycle. The thrips-tospovirus relationship is unique because adult thrips can only transmit TSWV if acquisition occurs in the larval stages
(77). Larval acquisition of the virus is an essential determinant of adult vector
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competency, and furthermore, acquisition rates decrease as larval thrips develop

(147). For example, when first instar larvae were allowed to acquire TSWV, 47%
of the concomitant adults transmitted virus. In contrast, 12% of adults from cohorts acquiring virus as second instars were able to inoculate plants (97, 98,
147). Many plant species can be infected by polyphagous thrips adults as they
disperse and sample potential hosts; however, plant species that do not support
thrips development are dead ends for the virus and do not contribute to epidemic
development.
Adult thrips that feed on infected plants do not become viruliferous even if they
are allowed lengthy feeding on tospovirus-infected plants (119, 138, 146, 147).
EM observations by Ullman et al. (139) showed that virions were present in midgut
epithelial cells of adult F. occidentalis shortly after the acquisition access period
(AAP). The work of Ullman et al. (139) and Nagata et al. (97) with TSWV-MT2
and BR01, respectively, suggest that persistent infection of adult midgut cells is
a rare occurrence. Recent work with different isolates of TSWV shows that virus
can infect midgut cells (101) and, in some cases, spreads to muscle cells in insects
fed as adults (7). Ohnishi et al. (101) found that TSWV (originally isolated from
pepper in Japan) infected adult Thrips setosus midguts, and by three days postAAP, the infection spread through the insect midgut. By five days post-AAP, the
infection had diminished; furthermore, they found that virus was unable to spread
beyond the basal lamina of adult thrips midguts. These results suggest that the basal
lamina serves as a potential barrier to virus movement out of the insect midgut.
Other researchers, working with a TSWV isolate recently isolated from peanut in
Georgia, found that TSWV infected adult midguts (7). Adult F. occidentalis and
F. fusca sustained midgut infections and the virus also spread to the surrounding
muscle tissue. Virus was not found in ligament-like tissue or salivary glands, and
thrips given AAP as adults were unable to transmit. Importantly, all these studies
agree that adult thrips, whether they support midgut infection or not, are unable
to transmit TSWV and virus infection does not spread to the salivary gland unless
acquisition initially occurs during the larval stages of life. These experiments were
all performed with different isolates of TSWV, and these isolates varied in their
ability to initiate successful infection of adult midguts, escape the midgut, and
infect muscles. Virus genotype, thrips genotype, and environmental conditions
likely play a critical role in these differential interactions.
Vector specificity between thrips species and virus isolates does occur (99,
147). Working with four tospovirus species and four thrips species, Nagata et al.
(99) found that viruses persisted and perhaps replicated in insects that were unable
to transmit virus. Thrips fell into three categories: transmitters with detectable
levels of virus, nontransmitters with detectable virus, and nontransmitters without detectable virus. For example, Thrips palmi contained detectable amounts of
Chrysanthemum stem necrosis virus (CNSV) from larval stages to adulthood, but
they were unable to transmit this virus. These data provide evidence that virus enters and replicates in the insect, and one possible explanation for their inability to
transmit is that virus was unable to infect the salivary glands. In other virus-vector
combinations, virus did not persist and was not transmitted; Thrips tabaci did not
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contain detectable levels of Groundnut ringspot virus and Tomato chlorotic spot
virus and was unable to transmit either virus. Two possible explanations for these
observations are that virus replication was not supported in these insects or virus
was unable to enter midgut cells to initiate an infection. The barriers contributing
to vector specificity may well vary with vector species and virus isolate, as has
been observed in other virus-vector interactions, particularly the persistently transmitted Luteoviruses (47). Further work examining these incompatible interactions
will help us understand the differences between tospovirus vectors and nonvectors.
Is TSWV a Pathogen of Thrips?

Tospoviruses have an intimate association with their insect vectors. The effect of
virus infection on the insect has been the subject of much research, and recent
findings are strengthening our understanding of this intricate relationship. One
difficulty in comparing investigations of the impact of tospovirus infection on
thrips fitness lies in the genetic variability of the thrips populations and the genetic
diversity of tospovirus species, isolates, and populations. Two studies indicated
that TSWV and INSV, respectively, decreased thrips fitness (22, 115). In both of
these studies, thrips spent either their entire lives or a significant portion of their
larval development on infected plants. The pathogenicity of the viruses to thrips
could not be fully established because the nutritional status of infected plants may
have caused the decrease in fitness. Using the BR01 isolate of TSWV, Wijkamp
et al. (157) assessed the effect of infection on thrips developmental time, reproduction rate, and survival. Larvae were given a short AAP, reared to adulthood on
noninfected D. stramonium, and then moved to Petunia x hybrida Blue Magic
to assess their infection status. They found no significant differences between viruliferous, nonviruliferous, and control thrips (157). A serious concern in assessing
this investigation is that Petunia is not a host for thrips. Thus, the uniformly poor
fitness observed may have occurred because a nonhost plant was being used for the
investigation. Maris et al. (81) confirmed that TSWV-BR01 caused no difference
in mortality among thrips reared on leaf disks of noninfected and infected plants.
In addition, these authors found that development from egg to adult required 1 to
2 days less on TSWV-infected leaf disks. Medeiros et al. (85) found that TSWVBR01 infection of F. occidentalis appears to induce several genes that are characteristically initiated as part of the insect defense response to pathogens, providing
evidence that thrips mount an immune response to TSWV, or at least to isolate
BR01. In contrast to the findings of Marais et al. (81) and Wijkamp et al. (157),
experiments with another TSWV isolate yielded different results. Newly emerged
thrips were given a brief AAP, reared to adulthood on bean, and transmission assessed on datura leaf disks. Thrips that transmitted virus consistently until death
had shorter life spans and higher virus titers than nontransmitters. The latter group
did not differ significantly in their longevity from insects fed on noninfected plants.
(N.K.K. Kumar, A.E. Whitfield, T.L. German & D.E. Ullman, unpublished data).
Recent work provides clarification of these results and further insight into this
aspect of thrips-tospovirus relationships. Stumpf & Kennedy (130) examined the
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effect of TSWV isolate (CFL and RG2), temperature, and plant host on the insect
vector. They found that TSWV-infected F. fusca reared on infected foliage took
longer to develop into adults and were smaller than noninfected thrips also reared
on infected foliage, indicative of a direct effect of TSWV on thrips. Noninfected
thrips reared on noninfected leaves took longer to develop than noninfected thrips
on infected leaves, indicating that plant infection status also affected thrips. Noninfected females reared on virus-free host foliage were intermediate in development
time. The TSWV isolate also had an effect on the insects. CFL-infected females
took 0.6 days longer to reach the adult stage than females infected with RG2
and there was no effect on male developmental time. When taken together, it appears that TSWV isolates may differ in pathogenicity to thrips, as well as cause
diverse nutritional changes in plants. In addition, thrips may vary in their ability
to adapt to nutritional changes in their plant hosts and may have different defenses
against the virus isolates. These new research findings are beginning to explain
some of the complexity of the virus-vector relationship; discrepancies between
results achieved with different TSWV isolates and tospovirus species; and shed
light on the variability observed in the field epidemiology of the tospoviruses.
VIRUS ENTRY
Virus Entry Mechanisms
Virus entry into a host cell entails a complex series of events that culminate with the
fusion of viral and host membranes. Viruses bind to the molecules on the host cell,
and virion entry commonly occurs by one of two mechanisms, pH-independent
entry or pH-dependent entry. Virus entry via the pH-independent pathway begins
with virus attachment and is followed by direct fusion of viral membrane with host
plasma membrane. During pH-dependent entry, virus attachment is followed by
formation of a cellular compartment (e.g., endosome). Virions are engulfed by this
vesicle, and a change in compartmental pH or fusion with an acidic compartment
(e.g., lysosome) alters the conformation of the viral fusion protein. The subsequent
conformational change in the viral fusion protein exposes the fusion peptide or
fusion loop (89, 127). The fusion peptide inserts into the target membrane and
leads to the fusion of virion and host membranes and the subsequent release of the
virion contents into the cytoplasm (reviewed in 125).
Evidence indicates that some members of the Bunyaviridae enter cells by pHdependent receptor-mediated endocytosis (49, 55, 103). Researchers working with
Hantaan virus (HTN) showed that agents that disrupt clathrin-dependent endocytosis inhibited infection of cells; however, agents that disrupt caveolae-dependent
endocytosis did not inhibit infection of cells (55). These results were supported
with immunolabeling experiments that showed HTN localizing to endosomes and
later to lysosomes (55). Further support for pH-dependent entry comes from
studies of the Orthobunyaviruses. The envelope glycoprotein GC of La Crosse
virus (LAC) and California encephalitis (CE) undergoes a conformational change
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after exposure to acidic pH rendering it susceptible to protease cleavage, consistent with endocytic entry (46, 49, 103). Furthermore, conformation-specific
GC antibodies did not recognize GC after virions were incubated at acidic pH
(103). CE- and LAC-infected cells form syncytia (multinucleate cells) when the
extracellular pH is lowered, which indicates that virus entry is stimulated by low
pH (46, 49). These data provide strong evidence that bunyaviruses enter cells in
a pH-dependent process. Although the TSWV internalization pathway has not
been studied in such detail, by analogy to other members of the family Bunyaviridae, it is likely that TSWV enters thrips cells in a pH-dependent manner
and that the two membrane glycoproteins are involved in the entry process (154,
155).
TSWV Glycoproteins
Sequence analysis of the TSWV glycoprotein gene has provided insight into their
biology and function (Figure 7). The glycoproteins are encoded as a polyprotein
Figure 7 A schematic of the Tomato spotted wilt virus (TSWV) glycoprotein open
reading frame. The top box represents the polyprotein, with light gray and dark gray
boxes representing the low and medium areas of hydrophobicity, respectively. Putative
signal peptidase cleavage sites (S1 and S2), N- and O-linked glycosylation sites and
transmembrane domains are marked and were predicted as previously described (154).
The dark horizontal lines designated GN and GC represent the predicted mature proteins,
and numbers specify amino acid positions of the proposed N and C termini of the
proteins. Figure not drawn to scale.
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that is cleaved to generate the two mature glycoproteins. Analysis of the TSWV
(isolate MT2) polyprotein amino acid sequence revealed two hydrophobic domains that likely serve as signal peptides. The N-terminal hydrophobic domain
from residues 9 to 31 may serve as a signal sequence for GN and cleavage is predicted to occur at amino acid 35 (TDA-KV). The hydrophobic domain from amino
acid 438455 may be a signal peptide for GC and the predicted cleavage occurs
at residue 464 (SMA-QT). Similar, but not identical, hydrophobic regions and
cleavage sites were reported for another isolate of TSWV (61). If the hydrophobic
region from residue 438 to 455 serves as a signal peptide, then cleavage by a signal
peptidase could be the proteolytic process that separates the two glycoproteins, as
observed for HTN (78). Research on the expression and processing of the TSWV
GPs indicates that the polyprotein is cleaved when expressed in heterologous baculovirus and Semliki forest virus expression systems (2, 61, 153), indicating that
other TSWV proteins do not cleave the glycoprotein precursor. Protein sequencing
would define the exact ends of the glycoproteins and would confirm if the glycoprotein precursor is cleaved by a signal peptidase. Both GN and GC are anchored
in the viral membrane by hydrophobic regions. The hydrophobic regions spanning
amino acid residues 309 to 339 and 346 to 369 are likely membrane anchors for
GN . This is a particularly long hydrophobic region, and a similar region is seen in
other members of the Bunyaviridae (78). Residues 1062 to 1085 are predicted to
function as the GC membrane anchor. Based on sequence analysis, we can predict
that the glycoproteins have cytoplasmic tails of approximately 69 and 50 amino
acids for GN and GC , respectively. It is expected that these cytoplasmic tails interact with ribonucleoprotein complexes and play a vital role in virion packaging
and perhaps entry. There are nine possible N-glycosylation sites on the GP ORF;
however, it is expected that not all are used due to proximity to transmembrane regions and protein orientation. Biochemical analysis of the glycoproteins confirms
that they are glycosylated, but site usage and glycan role in pathogenesis has yet to
be described (2, 99, 153, 154). Processing and glycosylation of the glycoproteins
in infected thrips has not been examined.
In addition to playing a role in virus binding and entry, the GPs likely play
a role in virion assembly. Tospoviruses, like other bunyaviruses, bud from the
Golgi (60, 64, 74). In infected plants, assembled virions appear to be retrograde
transported to the ER (60). Based on evidence from mammalian expression of
the GPs, GN and GC efficiently travel from the ER to the Golgi as heterodimers
when expressed together (61). However, GC expressed alone is retained in the
ER, and GN expressed alone localizes to the Golgi. When GC is coexpressed with
GN , it is transported to and retained in the Golgi (61). These data suggest that GN
contains the Golgi targeting signal and GC localizes to the Golgi via an interaction
with GN . The processing pathway observed for TSWV GPs is consistent with the
pathway described for other viruses in the Bunyaviridae (18, 61, 105). Although
the TSWV GN Golgi localization signal has yet to be mapped, by analogy with
other GN proteins, it resides in the transmembrane domain and/or cytoplasmic tail
(42).
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Virus Entry-TSWV Glycoproteins

One of the most exciting areas of thrips-tospovirus research today is the investigation of TSWV entry into thrips midgut cells. The best-supported hypothesis of
virus acquisition involves the presence of a thrips midgut receptor that binds the
GPs. In line with this hypothesis, the subsequent fusion of the virion membrane
and a cellular membrane would allow the contents of the virion to enter the thrips
cell cytoplasm where it replicates. Previous findings support the hypothesis that
the TSWV GPs are determinants of thrips acquisition and probably serve as viral
attachment and fusion proteins. The two TSWV GPs decorate the surface of the
virion (Figure 1) and therefore are probably the first viral components that interact
with molecules in the thrips midgut. Isolates of TSWV that are serially, mechanically passed to plants generate mutations and deletions in the GP ORF and the
resulting viruses are not compromised in their ability to infect plants; however,
they are no longer thrips transmissible (96, 112). This loss of thrips transmissibility
indicates that the GPs play important roles in virus infection of thrips and that the
GPs are necessary for thrips acquisition.
For other members of the Bunyaviridae, the GPs are important in virus entry
(49, 80, 104, 131). Antibodies to GC (66, 104) and/or GN (76, 122) neutralize virus
infection. Another study revealed that when CE-infected cells were treated with
GC monoclonal antibody (MAb), virus fusion was inhibited without compromising virus attachment (49). Reassortment studies with hantaviruses and orthobunyaviruses have shown that virulence maps to the M RNA segment and that the
L segment is partly related to virulence (34, 48, 54). Mutations in both the GN and
GC protein have been identified that attenuate disease. Researchers studying HTN,
the prototype member of the genus Hantavirus, mapped a virulence determinant
to the GN protein transmembrane domain (34). Mutations at the carboxyl terminus
of HTN GN affect virulence, and antibodies that neutralize infection recognize this
same region (34, 76). Likewise, a mutation in the transmembrane region of HTN
GC reduced virulence in mice (52). A variant isolate of LAC was identified that was
restricted in its ability to infect the mosquito vector, and the change occurred in the
GC glycoprotein (131). These data indicate that both glycoproteins are important
in virulence of bunyaviruses. Studies with purified LAC GN and GC indicate that
pretreatment of a mosquito cell line with GN and GC inhibited LAC infection. In
contrast, pretreatment with GC alone inhibited infection of vertebrate cell lines,
suggesting that the GPs have unique roles in different hosts (80). Based on these
data, GPs are involved in virus attachment and entry into host cells whether it is a
vertebrate or invertebrate host. By analogy to other members of the Bunyaviridae,
the GP/thrips receptor hypothesis seems reasonable and is consistent with the role
of GPs in vector acquisition of bunyaviruses by other arthropod vectors.
Direct evidence from immunolabeling experiments with the anti-idiotypic antibodies (antibodies that mimic the epitope recognized by GN and GC monoclonal
antibodies) to TSWV GN and GC suggests that both proteins play a role in virus
acquisition by thrips. When GN and GC anti-idiotypic antibodies were incubated
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with dissected thrips, they specifically labeled the midgut, the expected location
of a cellular receptor (9). The GC anti-idiotype labeled the internal epithelial plasmalemma and GN anti-idiotype labeled the basal membrane of the midgut and
weakly labeled the internal membranes (9). When examined by TEM, GC antiidiotype labeled the apical plasmalemma of the microvilli lining the midgut cells
and receptosome-like structures in the cytoplasm of those cells (D.E. Ullman & S.
Kumm, unpublished data). Additionally, anti-idiotypic antibodies to GN and GC
also recognized a 50-kDa thrips protein, receptor candidate, in the gel overlay assay
(9, 86). These data provide support for the GP-receptor hypothesis; however, antiidiotypes mimic one epitope and may not reflect binding by a glycoprotein and/or
hetero- or homo-oligomeric protein complexes that may exist on the virion surface.
Several pieces of direct and indirect evidence suggest that the GN protein is involved in virus binding and/or entry. GN
contains an arginine-glycine-aspartic acid (RGD) motif near the N terminus, which
is characteristic of cell adhesion molecules (67), and could serve as a receptorbinding region. The RGD motif on GN is intriguing because this motif is known
to interact with -integrins on cell surfaces (106, 133). Several viruses have been
shown to bind -integrin receptors via RGD motifs in the context of their viral
attachment proteins (5, 35, 116). Moreover, hantaviruses use integrins as receptors
(38, 39). Research with LAC provides insight into possible TSWV GN participation in virus entry. Enzymatic removal of GC , but not GN , from LAC virions
resulted in an increased ability to bind mosquito midguts (79, 80). However, the
treated virions exhibited reduced binding to cultured mosquito and mammalian
cells (79, 80). This finding highlights the importance of GN in virus binding to
vectors and suggests that LAC GN may mediate attachment to insect midguts. In
support of the GN /vector interaction, sequence analysis of isolates of LAC with
different passage histories revealed that the GN coding sequence is more stable
than the GC coding sequence (10). These results provide evidence that GN plays
an important role in TSWV binding and/or entry into insect guts.
To directly determine the role(s) of GN in binding to thrips guts, we expressed
and purified a soluble, recombinant form of GN (GN -S). Because GN is an integral membrane protein, we expressed the ectodomain of GN from a recombinant
baculovirus in insect cells (SF21), thus creating a protein that was soluble in the absence of detergents (117). Soluble, recombinant proteins are essential in functional
studies with living organisms and cells in which membrane integrity is imperative
for determination of glycoprotein function. By expressing GN individually, we
examined its role in virus binding and entry in the absence of other viral proteins
(154). Thrips were fed purified protein and then cleared by feeding on a sucrose
solution so only proteins that were retained in the midgut were detected. We focused our study on the thrips midgut because it is the site of virus entry (139). As
stated earlier, the thrips midgut consists of a single layer of epithelial cells that
is surrounded by longitudinal and circular muscle cells (90). Midgut muscle and
epithelial cells have distinct labeling patterns when stained with Texas Red phalGN INVOLVEMENT IN VIRUS BINDING
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loidin. We found that GN -S could bind the midgut epithelial cells of larval thrips
without assistance from other TSWV proteins (Figure 8). The specificity of the
GN -S/thrips interaction was supported by the failure of another structural TSWV
protein, N, to bind thrips midgut epithelial cells. Furthermore, we demonstrated
that the viral attachment protein, glycoprotein B, from another enveloped virus,
Human cytomegalovirus (HCMV), did not bind to thrips guts (154).
Because GN -S bound larval thrips guts in a specific manner, we assayed it
for the ability to inhibit TSWV acquisition (154). Larval thrips were fed equal
amounts of purified virus, virus and GN -S, or virus and HCMV gB. Acquisition
was measured by detection of N protein in dissected midguts. We found that GN -S
inhibited TSWV acquisition by larval thrips (Figure 9), whereas HCMV gB did
not, again indicating a specific interaction between GN and thrips. Our findings
that GN -S binds larval thrips guts and inhibits TSWV acquisition provide evidence
that GN plays a role in virus binding and/or entry into the vector midgut, but do
not preclude a role for GC .
Accumulating evidence indicates that the
TSWV GC protein may serve as a fusion protein mediating entry into the insect vector cells (155). First, sequence comparison of orthobunyavirus GC, which
was shown to be involved in fusion (46, 49, 103), and TSWV GC suggests that the
protein may serve a similar role for both genera in virus entry (19, 67). Tospovirus
and orthobunyavirus GC proteins share a highly conserved domain in the core of
the GC coding region (19, 67). In TSWV-MT2, this conserved region encompasses
amino acid 674 to 727. This is notable because fusion peptides can be highly conserved within virus families (152). Second, GC contains hydrophobic domains that
could serve as a fusion peptide or loop following the pH-dependent conformational
change (refer to Figure 7). The orthobunyavirus-tospovirus conserved region overlaps with the area of low hydrophobicity from amino acid 465 to 783 and is close to
the smaller more hydrophobic region from residue 752 to 772. Third, we demonstrated that GC is cleaved at low pH, suggesting it undergoes a conformational
change consistent with it playing a role in pH-dependent endocytosis (155). A
pH-dependent cleavage of GC was observed by Western blot analysis. At neutral
and alkaline pH, the GC protein molecular mass was approximately 85 kDa, but at
acidic pH we observed an 85-kDa protein and a 72-kDa protein that reacted with
the GC MAb. In contrast to GC , we observed no change in the mobility of the GN
protein at acid, neutral, or high pH. These data are consistent with results obtained
with LAC and CE (46, 49) and provide evidence that the GN protein does not
undergo a pH-dependent conformational change, or if it does, it remains protease
resistant (155).
GC : A POSSIBLE FUSION PROTEIN?
The Search for a TSWV Receptor in Thrips

Thus far, researchers studying tospoviruses have been unable to identify a virus
receptor in thrips. This is not surprising because no virus receptor has been identified for an arthropod vector of any animal- or plant-infecting virus. Experimental
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approaches used to identify a thrips receptor include gel overlay assays of homogenized thrips and a thrips cDNA expression library (9, 86, 87). The work of
Bandla et al. (9) supports the hypothesis that GN and/or GC interact with a receptor
molecule in thrips. A 50-kDa protein in thrips (F. occidentalis) was identified as
a candidate TSWV receptor by gel overlay analysis (9). A consistent difference
in band intensity was observed between larval and adult thrips, a result that is
compatible with known TSWV-thrips biology (i.e., efficiency of virus acquisition
by larvae is reduced as the vector ages). Anti-idiotypic antibodies to GN and GC ,
as well as purified, recombinant forms of GN and GC recognized the 50-kDa thrips
protein in the gel overlay assay (9, 153). The putative 50-kDa thrips receptor was
immunoprecipitated with anti-idiotypic antibodies and antiGN /GC TSWV conjugate (86). Kikkert et al. (59) identified a 94-kDa protein in thrips that binds virus
in the gel overlay assay, but this protein was not present in the midgut of larval
thrips and may be involved in virus specificity in other insect tissues. The 50-kDa
protein has not been purified in a large enough quantity to sequence, largely due
to the small size of the insect. More research is needed to determine the specific
involvement of the 50- and 94-kDa proteins in virus infection.
A larval F. occidentalis cDNA library was constructed, expressed via phage
display, and screened for TSWV-binding proteins using a modified gel-overlay
assay (87). Several interesting proteins were identified using this assay, but after
expression and further analysis, the most promising candidate did not bind virus
(S.F. Hanson & T.L. German, unpublished data). The search for a receptor is
complicated by the likelyhood that the receptor exists on the surface of the thrips
gut. Cell surface proteins are often quite complex; for example, the receptor may
require chaperones to attain the proper three-dimensional structure necessary for
receptor-ligand interactions to occur, or it may be glycosylated and these glycans
may play a role in receptor-ligand interactions or stability of the receptor in the
milieu of the insect midgut.
Because attempts to identify a tospovirus receptor have been unfruitful, we
look to other members of the Bunyaviridae for enlightenment on the topic of virus
receptors. The only genus within the family Bunyaviridae for which a receptor has
been identified is the genus Hantavirus. Gavrilovskaya et al. (39) demonstrated
that antibodies to 3 integrins and 1 integrins respectively inhibit pathogenic
and nonpathogenic hantavirus infection of cells. Interestingly, the hantavirus GPs
do not contain an RGD motif, and RGD peptides have no effect on hantavirus
infection. They concluded that pathogenic and nonpathogenic hantaviruses use
different cellular receptors for virus entry, and they interact with integrins in an
RGD-independent manner (38, 39). Although integrins have been identified as
hantavirus receptors, their role, whether it be binding and/or entry, has not been
defined, and the role of GN and GC in viral attachment and/or fusion has not been
defined for the Hantavirus genus.
Based on the two genera that have been studied in any detail (Orthobunyavirus
and Hantavirus), it appears that closely related species can use the same receptor but members belonging to the same genus that are not closely related use a
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different receptor (39, 104). When LAC GC was used in blocking experiments,
the soluble protein was capable of blocking infection of closely related viruses
(California serogroup), but GC was unable to block infection by viruses from
different serogroups within the same genus (104). These results indicate that California serogroup bunyaviruses may share a common receptor, but other orthobunyaviruses use different receptors (104). Whether or not this finding applies to all
members of the Bunyaviridae is unresolved and may make finding receptors and/or
control strategies difficult. In addition to the -integrins that have been implicated
in hantavirus entry, a 30-kDa protein was identified in Vero-E6 cells that binds
Hantaan virus (62). Polyclonal antibodies generated to this protein reduce virus
infection by 70% (62). The molecular mass of this protein is much smaller than
that of integrins, approximately 100-kDa, suggesting that these viruses may bind
more than one receptor and/or that these proteins are involved in different steps of
the virus entry process.
Interestingly, integrins, the only identified receptor for a member of the family
Bunyaviridae, are a family of cell-adhesion receptors found in many eukaryotic
organisms. Integrins have not been identified in thrips, but are present in other
well-studied insects such as Drosophila melanogaster and Anopheles gambiae.
A novel V integrin was identified in D. melanogaster and found to be midgut
tissue specific (159). Since then, other integrins have been identified and have been
found to play an important role in control of epithelial cell morphogenesis (102),
and these receptors may play a role in arthropod cellular immune response (71). By
comparison to D. melanogaster, integrins may play a role in thrips development. If
a developmentally regulated integrin is implicated in thrips acquisition of TSWV,
it may explain the decrease in vectorial capacity as thrips mature.
CONCLUDING REMARKS
We are optimistic that in the next decade significant progress will be made in
understanding the complex biological processes involved in the transmission of
existing and emerging tospoviruses and their thrips vectors. The negative impact
of diseases caused by tospoviruses on a global scale will sustain internationally
collaborative research programs, and the utilization of new technologies will open
the doors to unique avenues of investigation. The field of genomics and proteomics
will provide a mechanism to identify genetic determinants of vector competence.
Comparative analysis of virus isolates and thrips species will lead to the discovery
of virulence factors and identify important barriers to transmission. Recombinant forms of viral proteins will continue to be useful for delving deeper into
mechanisms of tospovirus-thrips interactions and may become tools in the development of procedures to control both viral disease and thrips as pests. The
tospovirus-thrips relationship provides a rare opportunity to directly compare and
contrast pathogenicity in plant and animal hosts. Recent developments in our understanding of RNA silencing as a defense mechanism against viruses (and the
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corresponding counterdefense deployed by viruses) in their plant hosts will be extended to replication of viruses in their vectors. Clarifying the roles of viral genes
such as NSm, known to be involved in cell-to-cell movement in plants but with no
apparent corresponding function in the insect phase of replication, will increase
our understanding of animal virus replication mechanisms.
There are over 300, mainly arthropod-transmitted, viruses in the family Bunyaviridae alone. In addition to the devastating agricultural problems caused by
thrips and Tospoviruses, animal viruses in this family also cause acute debilitating diseases (Oropouche virus), encephalitis (La Crosse virus), acute respiratory
disease (Sin Nombre virus) or hemorrhagic fevers (Rift Valley fever virus). It is
reasonable to predict that research on the virus-vector relationship of Bunyaviruses
will lead to both an improved understanding of basic biological concepts and the
development of effective measures to lessen their impact on a wide range of human
activities.
ACKNOWLEDGMENTS
We thank Eileen Rendahl for graphic design assistance with figures and Dorith
Rotenberg for critical reading of the chapter. This work was supported by United
States Department of Agriculture grant 99-35303-8271 (T.L.G. and D.E.U.) and
by Hatch funds (WISO4316) (T.L.G.).
The Annual Review of Phytopathology is online at
http://phyto.annualreviews.org
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Figure 1 Diagram of TSWV virion. A double-layered membrane of host origin

(blue) is shown with the viral-encoded proteins GN and GC (green) projecting from
the surface in monomeric and dimeric configurations. The genomic RNA is presented as noncovalently closed circles in the form of a ribonucleoprotein (RNP) complex
created by its association with many copies of N protein (peach). A few copies of the
virion-associated RNA-dependent RNA polymerase (RdRp or L) are shown (purple)
in association with the RNPs.

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Figure 4 Conditions driving rapid coevolution between thrips vectors and tospoviruses include the genetic diversity that occurs in thrips populations, the potential
for them to carry and encounter mixtures of viral genotypes in plants, and the inherent genetic diversity that arises in RNA virus populations. The reassortment of the
viral genome allows for virus populations with new characteristics to arise, including new vector relationships and the ability to overcome host resistance. Graphic
design by Eileen Rendahl. Peanut photo courtesy of John Sherwood.
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Figure 5 Graphic representation of the thrips life cycle and the tospovirus transmission cycle. Thrips eggs are oviposited into plant tissue and within a few days the
first instar larvae emerge. Virus acquisition occurs solely during the larval stages
after which the virus is passed transstadially to the adult. The pupal stages are nonfeeding and do not move, although they do maintain virus infection. In nature,
Frankliniella occidentalis pupates in the soil. Many other vector species, e.g., Thrips
tabaci, pupate in the foliage. Adults emerge and have a tendency to disperse widely.
Only adult thrips (male and female) that acquired the virus during their larval stages
can transmit tospoviruses. Graphic design by Eileen Rendahl. Thrips and kalanchoe
photographs produced by Jack Kelley Clark.
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Figure 8 Soluble GN (GN-S) binds larval F. occidentalis guts in an in vivo binding

assay. Larval thrips were fed GN-S and after feeding, thrips guts were cleared for
two hours on a 5% sucrose solution. Thrips were then dissected, fixed in 4%
paraformaldehyde, and permeabilized. The guts were immunolabeled with 6xHis
MAb conjugated to Alexafluor 488 (green). Actin was stained with Texas Red phalloidin (red) and the visceral muscles and midgut epithelial cells are labeled. Staining
was visualized by confocal microscopy. (A) Thrips fed BSA, (B) thrips fed GN-S
showing that labeling is associated with midgut epithelial cell layers. The scale bar
is 50 m. [Reprinted from (154, figure 7) with permission from ASM Press.]
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Figure 9 In vivo thrips feeding experiment showing that soluble GN (GN-S) inhibits
acquisition of TSWV. Thrips were given two-hour acquisition access periods (AAP)
on (A) TSWV alone or (B) TSWV and GN-S. All treatments contained the same concentration of virus. Thrips were then allowed to feed on sucrose solution for two
hours to clear their guts after the AAP. Acquisition was measured by immunolabeling with TSWV nucleocapsid polyclonal antibody and secondary antibody conjugated to alexafluor 647 (blue). Actin was stained with Texas Red phalloidin (red).
Thrips guts were imaged with a confocal microscope. Insects fed the combination of
GN-S and TSWV had reduced amounts of virus in their guts when compared to
insects fed TSWV alone. The scale bar is 50 m.
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Annual Reviews
AR250-FM
Annual Review of Phytopathology

Volume 43, 2005

CONTENTS
FRONTISPIECE, Robert K. Webster
BEING AT THE RIGHT PLACE, AT THE RIGHT TIME, FOR THE RIGHT
REASONSPLANT PATHOLOGY, Robert K. Webster
FRONTISPIECE, Kenneth Frank Baker
KENNETH FRANK BAKERPIONEER LEADER IN PLANT PATHOLOGY,
R. James Cook
REPLICATION OF ALFAMO- AND ILARVIRUSES: ROLE OF THE COAT PROTEIN,
John F. Bol
RESISTANCE OF COTTON TOWARDS XANTHOMONAS CAMPESTRIS pv.
MALVACEARUM, E. Delannoy, B.R. Lyon, P. Marmey, A. Jalloul, J.F. Daniel,
J.L. Montillet, M. Essenberg, and M. Nicole
PLANT DISEASE: A THREAT TO GLOBAL FOOD SECURITY, Richard N. Strange
and Peter R. Scott
VIROIDS AND VIROID-HOST INTERACTIONS, Ricardo Flores,
Carmen Hernandez, A. Emilio Martnez de Alba, Jose-Antonio Dar`os,
and Francesco Di Serio
PRINCIPLES OF PLANT HEALTH MANAGEMENT FOR ORNAMENTAL PLANTS,
Margery L. Daughtrey and D. Michael Benson
THE BIOLOGY OF PHYTOPHTHORA INFESTANS AT ITS CENTER OF ORIGIN,
Niklaus J. Grunwald and Wilbert G. Flier
PLANT PATHOLOGY AND RNAi: A BRIEF HISTORY, John A. Lindbo
and William G. Doughtery
CONTRASTING MECHANISMS OF DEFENSE AGAINST BIOTROPHIC AND
NECROTROPHIC PATHOGENS, Jane Glazebrook
LIPIDS, LIPASES, AND LIPID-MODIFYING ENZYMES IN PLANT DISEASE
RESISTANCE, Jyoti Shah
PATHOGEN TESTING AND CERTIFICATION OF VITIS AND PRUNUS SPECIES,
Adib Rowhani, Jerry K. Uyemoto, Deborah A. Golino,
and Giovanni P. Martelli
MECHANISMS OF FUNGAL SPECIATION, Linda M. Kohn
xii
1
25
39
63
83
117
141
171
191
205
229
261
279
vii
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viii
11:17
Annual Reviews
AR250-FM
CONTENTS
PHYTOPHTHORA RAMORUM: INTEGRATIVE RESEARCH AND MANAGEMENT

OF AN EMERGING PATHOGEN IN CALIFORNIA AND OREGON FORESTS,
David M. Rizzo, Matteo Garbelotto, and Everett M. Hansen
309
COMMERCIALIZATION AND IMPLEMENTATION OF BIOCONTROL, D.R. Fravel
337
EXPLOITING CHINKS IN THE PLANTS ARMOR: EVOLUTION AND EMERGENCE

OF GEMINIVIRUSES, Maria R. Rojas, Charles Hagen, William J. Lucas,
and Robert L. Gilbertson
361
MOLECULAR INTERACTIONS BETWEEN TOMATO AND THE LEAF MOLD

PATHOGEN CLADOSPORIUM FULVUM, Susana Rivas
and Colwyn M. Thomas
REGULATION OF SECONDARY METABOLISM IN FILAMENTOUS FUNGI,
Jae-Hyuk Yu and Nancy Keller
TOSPOVIRUS-THRIPS INTERACTIONS, Anna E. Whitfield, Diane E. Ullman,
and Thomas L. German
HEMIPTERANS AS PLANT PATHOGENS, Isgouhi Kaloshian
and Linda L. Walling
RNA SILENCING IN PRODUCTIVE VIRUS INFECTIONS, Robin MacDiarmid
SIGNAL CROSSTALK AND INDUCED RESISTANCE: STRADDLING THE LINE
BETWEEN COST AND BENEFIT, Richard M. Bostock
GENETICS OF PLANT VIRUS RESISTANCE, Byoung-Cheorl Kang, Inhwa Yeam,
and Molly M. Jahn
BIOLOGY OF PLANT RHABDOVIRUSES, Andrew O. Jackson, Ralf G. Dietzgen,
Michael M. Goodin, Jennifer N. Bragg, and Min Deng
INDEX
Subject Index
ERRATA
An online log of corrections to Annual Review of Phytopathology chapters
may be found at http://phyto.annualreviews.org/
395
437
459
491
523
545
581
623
661

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26 Jul 2005 11:53

Annu. Rev. Phytopathol. 2005. 43:45989

Department of Entomology, University of Wisconsin, Madison, Wisconsin 53706;

Key Words Bunyaviridae, insect vector, membrane glycoproteins, Thysanoptera,

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Recognized Tospovirus species and their documented vectors

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Capsicum chlorosis virus (83)

Chrysanthemum stem necrosis virus (113)

Frankliniella occidentalis (95)

Groundnut bud necrosis virus (111)

Groundnut ringspot virus (23)

Impatiens necrotic spot virus (70)

Iris yellow spot virus (20)

Melon yellow spot virus (58)

Peanut chlorotic fanspot virus (17)

Peanut yellow spot virus (110)

Tomato chlorotic spot virus (23)

Tomato spotted wilt virus (12)

Watermelon bud necrosis virus (53)

T. palmi (92, 126)

Watermelon silver mottle virus (160)

Zucchini lethal chlorotic virus (113)

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The M RNA encodes a 33.6-kDa nonstructural protein (NSm) in the v sense

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COEVOLUTION OF THRIPS AND TOSPOVIRUSES

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THRIPS LIFE CYCLE, REPRODUCTIVE STRATEGIES,

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Models for Virus Movement in Thrips

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ligament-like structures (97). On the basis of immunolabeling of TSWV in

Thrips Stage-Dependent Acquisition of Tospovirus

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competency, and furthermore, acquisition rates decrease as larval thrips develop

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Is TSWV a Pathogen of Thrips?

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Virus Entry-TSWV Glycoproteins

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