The genome of a bunyavirus cannot be defined at the level of the viral particle but only at the scale of the viral population.

Bunyaviruses are enveloped negative or ambisense single-stranded RNA viruses with a genome divided into several segments. The canonical view depicts each viral particle packaging one copy of each genomic segment in one polarity named the viral strand. Several opposing observations revealed nonequal ratios of the segments, uneven number of segments per virion, and even packaging of viral complementary strands. Unfortunately, these observations result from studies often addressing other questions, on distinct viral species, and not using accurate quantitative methods. Hence, what RNA segments and strands are packaged as the genome of any bunyavirus remains largely ambiguous. We addressed this issue by first investigating the virion size distribution and RNA content in populations of the tomato spotted wilt virus (TSWV) using microscopy and tomography. These revealed heterogeneity in viral particle volume and amount of RNA content, with a surprising lack of correlation between the two. Then, the ratios of all genomic segments and strands were established using RNA sequencing and qRT-PCR. Within virions, both plus and minus strands (but no mRNA) are packaged for each of the three L, M, and S segments, in reproducible nonequimolar proportions determined by those in total cell extracts. These results show that virions differ in their genomic content but together build up a highly reproducible genetic composition of the viral population. This resembles the genome formula described for multipartite viruses, with which some species of the order Bunyavirales may share some aspects of the way of life, particularly emerging properties at a supravirion scale.

New Technologies for Studying Negative-Strand RNA Viruses in Plant and Arthropod Hosts.

The plant viruses in the phylum Negarnaviricota, orders Bunyavirales and Mononegavirales, have common features of single-stranded, negative-sense RNA genomes and replication in the biological vector. Due to the similarities in biology, comparative functional analysis in plant and vector hosts is helpful for understanding host-virus interactions for negative-strand RNA viruses. In this review, we will highlight recent technological advances that are breaking new ground in the study of these recalcitrant virus systems. The development of infectious clones for plant rhabdoviruses and bunyaviruses is enabling unprecedented examination of gene function in plants and these advances are also being transferred to study virus biology in the vector. In addition, genome and transcriptome projects for critical nonmodel arthropods has enabled characterization of insect response to viruses and identification of interacting proteins. Functional analysis of genes using genome editing will provide future pathways for further study of the transmission cycle and new control strategies for these viruses and their vectors.

Disruption of vector transmission by a plant-expressed viral glycoprotein.

Vector-borne viruses are a threat to human, animal, and plant health worldwide, requiring the development of novel strategies for their control. Tomato spotted wilt virus (TSWV) is one of the 10 most economically significant plant viruses and, together with other tospoviruses, is a threat to global food security. TSWV is transmitted by thrips, including the western flower thrips, Frankliniella occidentalis. Previously, we demonstrated that the TSWV glycoprotein GN binds to thrips vector midguts. We report here the development of transgenic plants that interfere with TSWV acquisition and transmission by the insect vector. Tomato plants expressing GN-S protein supported virus accumulation and symptom expression comparable with nontransgenic plants. However, virus titers in larval insects exposed to the infected transgenic plants were three-log lower than insects exposed to infected nontransgenic control plants. The negative effect of the GN-S transgenics on insect virus titers persisted to adulthood, as shown by four-log lower virus titers in adults and an average reduction of 87% in transmission efficiencies. These results demonstrate that an initial reduction in virus infection of the insect can result in a significant decrease in virus titer and transmission over the lifespan of the vector, supportive of a dose-dependent relationship in the virus-vector interaction. These findings demonstrate that plant expression of a viral protein can be an effective way to block virus transmission by insect vectors.

Variation in Tomato spotted wilt virus titer in Frankliniella occidentalis and its association with frequency of transmission.

Tomato spotted wilt virus (TSWV) is transmitted in a persistent propagative manner by Frankliniella occidentalis, the western flower thrips. While it is well established that vector competence depends on TSWV acquisition by young larvae and virus replication within the insect, the biological factors associated with frequency of transmission have not been well characterized. We hypothesized that the number of transmission events by a single adult thrips is determined, in part, by the amount of virus harbored (titer) by the insect. Transmission time-course experiments were conducted using a leaf disk assay to determine the efficiency and frequency of TSWV transmission following 2-day inoculation access periods (IAPs). Virus titer in individual adult thrips was determined by real-time quantitative reverse transcriptase-PCR (qRT-PCR) at the end of the experiments. On average, 59% of adults transmitted the virus during the first IAP (2 to 3 days post adult-eclosion). Male thrips were more efficient at transmitting TSWV multiple times compared with female thrips of the same cohort. However, females harbored two to three times more copies of TSWV-N RNA per insect, indicating that factors other than absolute virus titer in the insect contribute to a successful transmission event. Examination of virus titer in individual insects at the end of the third IAP (7 days post adult-eclosion) revealed significant and consistent positive associations between frequency of transmission and virus titer. Our data support the hypothesis that a viruliferous thrips is more likely to transmit multiple times if it harbors a high titer of virus. This quantitative relationship provides new insights into the biological parameters that may influence the spread of TSWV by thrips.

A cryptic promoter in potato virus X vector interrupted plasmid construction.

BACKGROUND: Potato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV-2) VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation). RESULTS: A cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR. CONCLUSION: It is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but in E. coli) can interrupt the downstream work.

Methods for effective real-time RT-PCR analysis of virus-induced gene silencing.

We applied real-time RT-PCR to the analysis of Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) of the phytoene desaturase (PDS) gene in Nicotiana benthamiana and tomato. Using a combination of direct measurement and mathematical assessment, we evaluated three plant genes, ubiquitin (ubi3), elongation factor-1 alpha (EF-1), and actin, for use as internal reference transcripts and found that EF-1 and ubi3 were least variable under our experimental conditions. Primer sets designed to amplify the 5' or 3' regions of endogenous PDS transcripts in tomato yielded similar reductions in transcript levels indicating a uniform VIGS-mediated degradation of target RNA. By measuring the ratio of the abundance of the PDS insert transcript to the TRV coat protein RNA, we established that the PDS insert within TRV was stable in both hosts. VIGS in N. benthamiana resulted in complete photo-bleaching of all foliar tissue compared to chimeric bleaching in tomato. PDS transcript levels were decreased eleven- and seven-fold in photobleached leaves of N. benthamiana and tomato, respectively, while sampling tomato leaflets on the basis of age rather than visible bleaching resulted in only a 17% reduction in PDS coupled with a large leaf-to-leaf variation. There was a significant inverse relationship (r2=76%, P=0.01) between the relative abundance of CP RNA and the amount of PDS transcript in rTRV::tPDS-infected tomato suggesting that virus spread and accumulation are required precursors for successful VIGS in this host.

Tospovirus-thrips interactions.

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 vector-virus-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.

Tomato spotted wilt virus glycoprotein G(C) is cleaved at acidic pH.

Tomato spotted wilt virus (TSWV) is a plant-infecting member of the family Bunyaviridae. TSWV encodes two envelope glycoproteins, G(N) and G(C), which are required for virus infection of the arthropod vector. Other members of the Bunyaviridae enter host cells by pH-dependent endocytosis. During this process, the glycoproteins are exposed to conditions of acidic pH within endocytic vesicles causing the G(C) protein to change conformation. This conformational change renders G(C) more sensitive to protease cleavage. We subjected TSWV virions to varying pH conditions and determined that TSWV G(C), but not G(N), was cleaved under acidic pH conditions, and that this phenomenon did not occur at neutral or alkaline pH. This data provides evidence that G(C) changes conformation at low pH which results in altered protease sensitivity. Furthermore, sequence analysis of G(C) predicts the presence of internal hydrophobic domains, regions that are characteristic of fusion proteins. Like studies with other members of the Bunyaviridae, this study is the first step towards characterizing the nature of cell entry by TSWV.

Expression and characterization of a soluble form of tomato spotted wilt virus glycoprotein GN.

Tomato spotted wilt virus (TSWV), a member of the Tospovirus genus within the Bunyaviridae, is an economically important plant pathogen with a worldwide distribution. TSWV is transmitted to plants via thrips (Thysanoptera: Thripidae), which transmit the virus in a persistent propagative manner. The envelope glycoproteins, G(N) and G(C), are critical for the infection of thrips, but they are not required for the initial infection of plants. Thus, it is assumed that the envelope glycoproteins play important roles in the entry of TSWV into the insect midgut, the first site of infection. To directly test the hypothesis that G(N) plays a role in TSWV acquisition by thrips, we expressed and purified a soluble, recombinant form of the G(N) protein (G(N)-S). The expression of G(N)-S allowed us to examine the function of G(N) in the absence of other viral proteins. We detected specific binding to thrips midguts when purified G(N)-S was fed to thrips in an in vivo binding assay. The TSWV nucleocapsid protein and human cytomegalovirus glycoprotein B did not bind to thrips midguts, indicating that the G(N)-S-thrips midgut interaction is specific. TSWV acquisition inhibition assays revealed that thrips that were concomitantly fed purified TSWV and G(N)-S had reduced amounts of virus in their midguts compared to thrips that were fed TSWV only. Our findings that G(N)-S binds to larval thrips guts and decreases TSWV acquisition provide evidence that G(N) may serve as a viral ligand that mediates the attachment of TSWV to receptors displayed on the epithelial cells of the thrips midgut.

Interaction Between Tomato spotted wilt virus N Protein Monomers Involves Nonelectrostatic Forces Governed by Multiple Distinct Regions in the Primary Structure.

ABSTRACT The ambisense RNA genome of Tomato spotted wilt virus (TSWV) isby interaction with numerous copies of the virus encoded nucleocapsid (N) protein to form a subvirion structure called a ribonucleo-protein (RNP). RNPs are central to the viral replication cycle because they, and not free viral RNA, serve as templates for viral gene expression and genome replication. N protein monomers bind to viral RNA molecules in a cooperative manner. We have examined regions of the N protein that are involved in the N-N interactions that likely contribute to the cooperative binding of N to viral RNA. We created random and alanine scanning mutants of N and then screened the mutants for defects in N-N interaction using reverse and forward yeast two-hybrid assays. Our experiments identified residues in three distinct regions of the primary structure of the protein, residues 42 to 56, 132 to 152, and in the C-terminal 26 amino acids, that contribute to N-N dimerization or multimerization.interactions between N monomers mediated by the residues we identified are of a nonelectrostatic nature.

Association of L protein and in vitro tomato spotted wilt virus RNA-dependent RNA polymerase activity.

We have previously described an in vitro assay for RNA-dependent RNA polymerase activity in virions of tomato spotted wilt virus (TSWV). Here we report antibody inhibition of virion-associated RNA synthesis in vitro with an L-protein-specific polyclonal antibody raised against the carboxy-terminus of the L protein. In contrast, RNA synthesis was not inhibited by a heterologous antiserum and was unaffected by antiserum raised against an internal portion of the L protein. Our results directly associate the TSWV L protein, the putative viral polymerase, with RNA synthesis functions in vitro.