Bipartite Genome Research Articles

Evolution of a novel engineered tripartite viral genome of a torradovirus

ABSTRACT Viruses in the Secoviridae include monopartite and bipartite genomes, suggesting the possibility to study members of this family to experimentally address evolutionary transitions resulting in multipartitism. Torradoviruses are bipartite members of the family Secoviridae characterized by a genus-specific 5ʹ open reading frame (ORF), named P21, encoded by RNA2. Here, in a study originally intended to verify if P21 can function in trans, we attempted to provide P21 from a third P21-expressing construct under control of the 35S promoter and containing the 5ʹ- and 3ʹ- untranslated regions (UTRs) of wild type (WT) RNA2. When this construct was combined with an RNA2 with a complete deletion of the P21 coding region we verified that the P21 provided in trans cannot immediately complement the mutant, but occasional systemic infections in a limited number of the inoculated plants display the presence of a tripartite virus with an actively replicating P21-expressing RNA3. Furthermore, in all the systemically infected plants investigated in six distinct experiments, this replicating RNA3 accumulates deletions in a small region inside the original 3ʹ-UTR provided by the cDNA clone. Such tripartite virus, which we obtained through deconstructing the coding potential of the RNA2 in two distinct RNAs, can be transmitted mechanically and by whiteflies, is competent for virion formation, and its RNA3 is encapsidated. It can be mechanically transferred for eleven serial passages without losing its infectivity or showing major genomic rearrangements. Furthermore, mixing equal amounts of WT and tripartite virus inocula in the same leaf resulted in plants systemically infected only with the WT virus, showing that the tripartite virus has lower fitness than the WT. To our knowledge, this is the first example of an engineered tripartite viral genome becoming stable through artificial evolution in vivo, in plants. This tripartite system was also used to derive a stable viral vector to express GFP systemically in the context of viral infection.

Phylogeographic analysis of Begomovirus coat and replication-associated proteins.

Begomoviruses are globally distributed plant pathogens that significantly limit crop production. These viruses are traditionally described according to phylogeographic distribution and categorized into two groups: begomoviruses from the Africa, Asia, Europe and Oceania (AAEO) region and begomoviruses from the Americas. Monopartite begomoviruses are more common in the AAEO region, while bipartite viruses predominate in the Americas, where the begomoviruses lack the V2/AV2 gene involved in inter-cellular movement and RNA silencing suppression found in AAEO begomoviruses. While these features are generally accepted as lineage-defining, the number of known species has doubled due to sequence-based discovery since 2010. To re-evaluate the geographic groupings after the rapid expansion of the genus, we conducted phylogenetic analyses for begomovirus species representatives of the two longest and most conserved begomovirus proteins: the coat and replication-associated proteins. Both proteins still largely support the broad AAEO and Americas begomovirus groupings, except for sweet potato-infecting begomoviruses that form an independent, well-supported clade for their coat protein regardless of the region they were isolated from. Our analyses do not support more fine-scaled phylogeographic groupings. Monopartite and bipartite genome organizations are broadly interchanged throughout the phylogenies, and the absence of the V2/AV2 gene is highly reflective of the split between Americas and AAEO begomoviruses. We observe significant evidence of recombination within the Americas and within the AAEO region but rarely between the regions. We speculate that increased globalization of agricultural trade, the invasion of polyphagous whitefly vector biotypes and recombination will blur begomovirus phylogeographic delineations in the future.

First report of pepper yellow leaf curl Thailand virus infecting hot chili in central Vietnam and Laos.

Hot chili pepper (Capsicum annuum) cultivation has been on the rise in South East Asia to meet export demands. In Thailand, the top chili exporter in South East Asia, chili production has been severely hampered by pepper yellow leaf curl disease (YLCD) caused by the begomovirus pepper yellow leaf curl Thailand virus (PepYLCThV) (Chiemsombat et al., 2018; Suwor et al., 2021). In the neighbouring countries of Laos and Vietnam, a limited survey of chili fields (200 plants in total) in Savannakhet (Savannakhet University campus, n = 150), Laos and Quang Nam province (Ka Dang commune, Dong Giang district, n = 50), central Vietnam in 2023 led to the finding of eight plants (5 in Laos and 3 in Vietnam) exhibiting YLCD-like symptoms, which included bright yellow color in young leaves and leaf curl and mosaic chlorosis in mature leaves (Fig. S1). Total DNA was extracted from leaves of two symptomatic plants (one from Savannakhet and one from Quang Nam) using a cetyltrimethylammonium bromide-based DNA extraction protocol (Doyle & Doyle, 1987; Nguyen et al., 2023). Next, PCR were performed using newly designed PepYLCThV-specific primers based on PepYLCThV sequences in GenBank (Table 1). PCR products of expected sizes were observed in samples with disease symptoms, but not from DNA extracted from C. annuum (cv. VA.99999) grown at the Institute of Biotechnology in Thua Thien Hue, Vietnam (Fig. S2). The amplicons were Sanger sequenced (Apical Scientific, Selangor, Malaysia) and the complete bipartite genome sequence of two isolates ('Sava01' from Laos and 'QNam01' from Vietnam) were obtained. The sequences of the DNA-A component from isolates 'Sava01' (GenBank PP437580) and 'QNam01' (GenBank PP437581) exhibited the highest sequence identity of 99.2% and 94.7% with the PepYLCThV isolate 'ChiangDaoS1' (GenBank OM677627), respectively (Table 2). Conversely, the sequences of the DNA-B component from the isolates 'Sava01' (GenBank PP437579) and 'QNam01' (GenBank PP437582) exhibited the highest similarity of 91.8% and 90.9% with the PepYLCThV isolate 'KKN601' (GenBank MW715820), respectively (Table 2). These results confirmed the presence of PepYLCThV in hot chili pepper plants exhibiting YLCD-like symptoms in central Vietnam and Laos. Infectious clones of PepYLCThV DNA-A and DNA-B (isolate 'QNam01') were created based on the pLX-AS vector as described by Pasin (2022), and transformed into Agrobacterium tumefaciens EHA105. The resulting bacteria were cultured in LB broth containing rifampicin (25 μg/mL) and kanamycin (50 μg/mL) at 28°C and used for agroinoculation of Nicotiana benthamiana (n = 6) and C. annuum (cv. VA.99999, n = 6) (4-6 leaf plants) as described by Pasin (2022). In all N. benthamiana plants, agroinoculation with both DNA-A and DNA-B infectious clones caused stunted growth, severe leaf curl, with yellow and white patches 21 days post inoculation (Fig. S3). In C. annuum plants, symptom expression, which included leaf curl and stunted leaves with yellow mosaic patterns, was observed in two out of six inoculated plants six weeks postinoculation (Fig. S3). PCR assays confirmed the presence of PepYLCThV DNA in N. benthamiana and C. annuum symptomatic leaves (Fig. S4). To our knowledge, this is the first report of pepper yellow leaf curl Thailand virus in hot chili pepper in Laos and central Vietnam. Appropriate containment and management strategies should be developed and implemented to control the spread of this disease in hot chili pepper crops in both countries.

Evolution of an Extended Pathogenicity Motif in VP2 of Infectious Pancreatic Necrosis Virus Isolates from Farmed Rainbow Trout in Turkey.

Infectious pancreatic necrosis virus (IPNV) causes economic losses with a highly variable mortality rate worldwide, especially in rainbow trout. The virus has a double-stranded bi-partite RNA genome designated segment A and B. New complete genome sequences of nine rainbow trout isolates from Turkey were determined and subjected to phylogenetic analysis, identifying all as genotype 5 (serotype Sp). A time-dependent change in the extended pathogenicity motif of VP2 from P217T221A247 (PTA) to PTE P217T221E247 over a period of 10 years was identified. A wider analysis of 99 IPNV sequences from Turkey and Iran revealed the emergence of the motif PTE from 2007 to 2017, inducing significant morbidity in fry by 2013. In fact, displacement of the PTA motif, by the PTE motif in IPNV isolates appeared to be connected to a production peak of rainbow trout in 2013. An additional CAI analysis provided more evidence, indicating that rainbow trout culture in Turkey has an influence on the evolution of IPNV.

Rationally designed chromosome fusion does not prevent rapid growth of Vibrio natriegens

DNA replication is essential for the proliferation of all cells. Bacterial chromosomes are replicated bidirectionally from a single origin of replication, with replication proceeding at about 1000 bp per second. For the model organism, Escherichia coli, this translates into a replication time of about 40 min for its 4.6 Mb chromosome. Nevertheless, E. coli can propagate by overlapping replication cycles with a maximum short doubling time of 20 min. The fastest growing bacterium known, Vibrio natriegens, is able to replicate with a generation time of less than 10 min. It has a bipartite genome with chromosome sizes of 3.2 and 1.9 Mb. Is simultaneous replication from two origins a prerequisite for its rapid growth? We fused the two chromosomes of V. natriegens to create a strain carrying one chromosome with a single origin of replication. Compared to the parental, this strain showed no significant deviation in growth rate. This suggests that the split genome is not a prerequisite for rapid growth.

Molecular characterization of the complete genome of a novel ormycovirus infecting the ectomycorrhizal fungus Hortiboletus rubellus.

Advancements in high-throughput sequencing and the development of new bioinformatics tools for large-scale data analysis play a crucial role in uncovering virus diversity and enhancing our understanding of virus evolution. The discovery of the ormycovirus clades, a group of RNA viruses that are phylogenetically distinct from all known Riboviria members and are found in fungi, highlights the value of these tools for the discovery of novel viruses. The aim of this study was to examine viral populations in fungal hosts to gain insights into the diversity, evolution, and classification of these viruses. Here, we report the molecular characterization of a newly discovered ormycovirus, which we have named "Hortiboletus rubellus ormycovirus 1" (HrOMV1), that was found in the ectomycorrhizal fungus Hortiboletus rubellus. The bipartite genome of HrOMV1, whose nucleotide sequence was determined by HTS and RLM-RACE, consists of two RNA segments (RNA1 and RNA2) that exhibit similarity to those of previously studied ormycoviruses in their organization and the proteins they encode. The presence of upstream, in-frame AUG triplets in the 5' termini of both RNA segments suggests that HrOMV1, like certain other ormycoviruses, employs a non-canonical translation initiation strategy. Phylogenetic analysis showed that HrOMV1 is positioned within the gammaormycovirus clade. Its putative RNA-dependent RNA polymerase (RdRp) exhibits sequence similarity to those of other gammaormycovirus members, the most similarity to that of Termitomyces ormycovirus 1, with 33.05% sequence identity. This protein was found to contain conserved motifs that are crucial for RNA replication, including the distinctive GDQ catalytic triad observed in gammaormycovirus RdRps. The results of this study underscore the significance of investigating the ecological role of mycoviruses in mycorrhizal fungi. This is the first report of an ormycovirus infecting a member of the ectomycorrhizal genus Hortiboletus.

Genome comparisons reveal accessory genes crucial for the evolution of apple Glomerella leaf spot pathogenicity in Colletotrichum fungi.

Apple Glomerella leaf spot (GLS) is an emerging fungal disease caused by Colletotrichum fructicola and other Colletotrichum species. These species are polyphyletic and it is currently unknown how these pathogens convergently evolved to infect apple. We generated chromosome-level genome assemblies of a GLS-adapted isolate and a non-adapted isolate in C. fructicola using long-read sequencing. Additionally, we resequenced 17 C. fructicola and C. aenigma isolates varying in GLS pathogenicity using short-read sequencing. Genome comparisons revealed a conserved bipartite genome architecture involving minichromosomes (accessory chromosomes) shared by C.fructicola and other closely related species within the C. gloeosporioides species complex. Moreover, two repeat-rich genomic regions (1.61 Mb in total) were specifically conserved among GLS-pathogenic isolates in C. fructicola and C. aenigma. Single-gene deletion of 10 accessory genes within the GLS-specific regions of C. fructicola identified three that were essential for GLS pathogenicity. These genes encoded a putative non-ribosomal peptide synthetase, a flavin-binding monooxygenase and a small protein with unknown function. These results highlight the crucial role accessory genes play in the evolution of Colletotrichum pathogenicity and imply the significance of an unidentified secondary metabolite in GLS pathogenesis.

Bipartite viral RNA genome heterodimerization influences genome packaging and virion thermostability.

Multi-segmented viruses often multimerize their genomic segments to ensure efficient and stoichiometric packaging of the correct genetic cargo. In the bipartite Nodaviridae family, genome heterodimerization is also observed and conserved among different species. However, the nucleotide composition and biological function for this heterodimer remain unclear. Using Flock House virus as a model system, we developed a next-generation sequencing approach ("XL-ClickSeq") to probe heterodimer site sequences. We identified an intermolecular base-pairing site which contributed to heterodimerization in both wild-type and defective virus particles. Mutagenic disruption of this heterodimer site exhibited significant deficiencies in genome packaging and encapsidation specificity to viral genomic RNAs. Furthermore, the disruption of this intermolecular interaction directly impacts the thermostability of the mature virions. These results demonstrate that the intermolecular RNA-RNA interactions within the encapsidated genome of an RNA virus have an important role on virus particle integrity and thus may impact its transmission to a new host.IMPORTANCEFlock House virus is a member of Nodaviridae family of viruses, which provides a well-studied model virus for non-enveloped RNA virus assembly, cell entry, and replication. The Flock House virus genome consists of two separate RNA molecules, which can form a heterodimer upon heating of virus particles. Although similar RNA dimerization is utilized by other viruses (such as retroviruses) as a packaging mechanism and is conserved among Nodaviruses, the role of heterodimerization in the Nodavirus replication cycle is unclear. In this research, we identified the RNA sequences contributing to Flock House virus genome heterodimerization and discovered that such RNA-RNA interaction plays an essential role in virus packaging efficiency and particle integrity. This provides significant insight into how the interaction of packaged viral RNA may have a broader impact on the structural and functional properties of virus particles.

Unveiling mungbean yellow mosaic virus: molecular insights and infectivity validation in mung bean (Vigna radiata) via infectious clones.

Yellow mosaic disease (YMD) with typical symptoms of alternating bright yellow to green patches associated with stunting, downward cupping, and wrinkling has been observed in mung bean on agricultural farms in Coimbatore, Tamil Nadu, India. PCR using gene-specific primers indicated the presence of the yellow mosaic virus in symptomatic plants. Rolling circle amplification (RCA) followed by restriction digestion detected ~2.7 kb of DNA-A and DNA-B, allowing the identification of a bipartite genome. The full-length genome sequences were deposited in NCBI GenBank with the accession numbers MK317961 (DNA-A) and MK317962 (DNA-B). Sequence analysis of DNA-A showed the highest sequence identity of 98.39% to the DNA-A of mungbean yellow mosaic virus (MYMV)-Vigna radiata (MW736047), while DNA-B exhibited the highest level of identity (98.21%) to the MYMV-Vigna aconitifolia isolate (DQ865203) reported from Tamil Nadu. Recombinant analysis revealed distinct evidence of recombinant breakpoints of DNA-A within the region encoding the open reading frame (ORF) AC2 (transcription activation protein), with the major parent identified as MYMV-PA1 (KC9111717) and the potential minor parent as MYMV-Namakkal (DQ86520.1). Interestingly, a recombination event in the common region (CR) of DNA-B, which encodes the nuclear shuttle protein and the movement protein, was detected. MYMIV-M120 (FM202447) and MYMV-Vigna (AJ132574) were identified as the event's major and minor parents, respectively. This large variation in DNA-B led us to suspect a recombination in DNA-B. Dimeric MYMV infectious clones were constructed, and the infectivity was confirmed through agroinoculation. In future prospects, unless relying on screening using whiteflies, breeders and plant pathologists can readily use this agroinoculation procedure to identify resistant and susceptible cultivars to YMD.

Genomic and transcriptomic analyses of Phytophthora cinnamomi reveal complex genome architecture, expansion of pathogenicity factors, and host-dependent gene expression profiles.

Phytophthora cinnamomi is a hemibiotrophic oomycete causing Phytophthora root rot in over 5,000 plant species, threatening natural ecosystems, forestry, and agriculture. Genomic studies of P. cinnamomi are limited compared to other Phytophthora spp. despite the importance of this destructive and highly invasive pathogen. The genome of two genetically and phenotypically distinct P. cinnamomi isolates collected from avocado orchards in California were sequenced using PacBio and Illumina sequencing. Genome sizes were estimated by flow cytometry and assembled de novo to 140-141 Mb genomes with 21,111-21,402 gene models. Genome analyses revealed that both isolates exhibited complex heterozygous genomes fitting the two-speed genome model. The more virulent isolate encodes a larger secretome and more RXLR effectors when compared to the less virulent isolate. Transcriptome analysis after P. cinnamomi infection in Arabidopsis thaliana, Nicotiana benthamiana, and Persea americana de Mill (avocado) showed that this pathogen deploys common gene repertoires in all hosts and host-specific subsets, especially among effectors. Overall, our results suggested that clonal P. cinnamomi isolates employ similar strategies as other Phytophthora spp. to increase phenotypic diversity (e.g., polyploidization, gene duplications, and a bipartite genome architecture) to cope with environmental changes. Our study also provides insights into common and host-specific P. cinnamomi infection strategies and may serve as a method for narrowing and selecting key candidate effectors for functional studies to determine their contributions to plant resistance or susceptibility.

A novel bipartite negative-stranded RNA mycovirus of the order Bunyavirales isolated from the phytopathogenic fungus Fusarium sibiricum.

A novel negative-stranded RNA mycovirus was isolated from the phytopathogenic fungus Fusarium sibiricum strain AH32. This virus, tentatively named "Fusarium sibiricum coguvirus 1" (FsCV1), has a bipartite genome consisting of two RNA segments (RNA1 and RNA2). The negative-sense RNA1 is 6711 nt in length, encoding the RNA-dependent RNA polymerase (RdRp, p251) in the viral complementary (vc) strand. The ambisense RNA2 (1204 nt long) encodes two proteins from overlapping genes: the nucleocapsid protein (NP, p38) in the vc strand and a protein of unknown function (UFP, p36) in the viral (v) strand. In contrast to other Bunyavirales members, in FsCV1, the two open reading frames are separated by a long AU-rich intergenic region (IR). Sequence comparisons and phylogenetic analysis based on RdRp and NP sequences demonstrated that FsCV1 is a novel bipartite negative-stranded RNA mycovirus of the genus Coguvirus, family Phenuiviridae, order Bunyavirales.

Cell cycle-coordinated maintenance of the Vibrio bipartite genome.

To preserve the integrity of their genome, bacteria rely on several genome maintenance mechanisms that are co-ordinated with the cell cycle. All members of the Vibrio family have a bipartite genome consisting of a primary chromosome (Chr1) homologous to the single chromosome of other bacteria such as Escherichia coli and a secondary chromosome (Chr2) acquired by a common ancestor as a plasmid. In this review, we present our current understanding of genome maintenance in Vibrio cholerae, which is the best-studied model for bacteria with multi-partite genomes. After a brief overview on the diversity of Vibrio genomic architecture, we describe the specific, common, and co-ordinated mechanisms that control the replication and segregation of the two chromosomes of V. cholerae. Particular attention is given to the unique checkpoint mechanism that synchronizes Chr1 and Chr2 replication.

Molecular identification and development of an infectious cDNA clone of Trichosanthes kirilowii-infecting cucurbit mild mosaic virus

Trichosanthes kirilowii has been mainly grown for use in traditional Chinese medicine. In this study, cucurbit mild mosaic virus (CuMMV) belonging to the genus Fabavirus was identified from T. kirilowii plants. CuMMV possesses a segmented, bipartite linear single-stranded RNA genome composed of RNA1 and RNA2. Sequence analysis showed that each genomic segment shares the highest sequence similarity with those of CuMMV isolated from pumpkin. A full-length infectious cDNA clone of CuMMV was further constructed and was found to induce typical symptoms in T. kirilowii, Cucumis sativus, C. melo, Citrullus lanatus, and Cucurbita pepo. The sap inoculum derived from the infectious cDNA clone of CuMMV could be mechanically transmitted and reproduce similar symptoms in the tested plants. This is the first report on the construction of a biologically active, full-length infectious cDNA clone of CuMMV, which will provide a useful tool in understanding CuMMV-encoded proteins and plant-CuMMV interactions.

Cajanus scarabaeoides yellow mosaic virus, a new bipartite begomovirus causing yellow mosaic disease in Cajanus scarabaeoides (L.) Thouars in India.

Yellow mosaic disease of Cajanus scarabaeoides (L.) Thouars (CsYMD) was observed in up to 46% of C. scarabaeoides plants in the mungbean, urdbean and pigeon pea fields from 22 districts of Chhattisgarh State, India, during 2017-2019. The symptoms were characterized by yellow mosaic on green leaves and yellow discoloration of leaves in advanced stages of the disease. Severely infected plants showed shortened internodal length and reduced leaf size. CsYMD was transmissible to healthy C. scarabaeoides and Cajanus cajan by whitefly (Bemisia tabaci). The plants thus infected developed typical yellow mosaic symptoms on their leaves within 16 and 22 days of inoculation, respectively, suggesting a begomovirus etiology. Molecular analysis revealed that this begomovirus has a bipartite genome composed of DNA-A (2729 nucleotides) and DNA-B (2630 nucleotides). Sequence and phylogenetic analyses revealed that the nucleotide sequence of the DNA-A component had the highest identity of 81.1% with DNA-A of Rhynchosia yellow mosaic virus (RhYMV) (NC_038885) followed by mungbean yellow mosaic virus (MN602427) (75.3%). DNA-B had the highest identity of 74.0% with DNA-B of RhYMV (NC_038886). As per ICTV guidelines, this isolate had <91% nucleotide identity with DNA-A of any of the begomoviruses reported, hence, it is proposed as a new begomovirus species, tentatively named Cajanus scarabaeoides yellow mosaic virus (CsYMV). After agroinoculation with DNA-A and DNA-B clones of CsYMV, all Nicotiana benthamiana plants developed leaf curl symptoms along with light yellowing symptoms 8-10 days after inoculation (DAI), while ~60% of the C. scarabaeoides plants developed yellow mosaic symptoms similar to those observed in the field 18 DAI, thus fulfilling the Koch's postulates. From these agro-infected C. scarabaeoides plants, CsYMV was transmissible to healthy C. scarabaeoides plants by B. tabaci. Apart from these hosts, CsYMV also infected and caused symptoms in mungbean and pigeon pea.

Identification of two putative novel deltapartitiviruses and an enamovirus in coriander transcriptomes.

Coriander is a herbaceous spice and condiment crop also known for its medicinal properties. The present study identified two putative novel deltapartitiviruses and an enamovirus tentatively named as Coriandrum sativumdeltapartitivirus 1, 2 (CsDPV1, 2) and Coriandrum sativumenamovirus (CsEV) in the publicly available transcriptome-assembled contigs derived from coriander grown in India. CsDPV1 and 2 contained tripartite and bipartite genomes, respectively, with each genome segment encoding a single open reading frame (ORF). CsEV contained five ORFs encoding proteins P0, P1, P2, P3 and P5. Phylogenetic analysis revealed three distinct subgroups of deltapartitiviruses wherein CsDPV1 and 2 grouped in subgroup 3 and 1, respectively, whilst CsEV formed a distinct sub-clade within enamoviruses. Further, the presence of CsDPV2 in fruit samples of one of the cultivars from where the virus was identified was confirmed through RT-PCR assay and Sanger sequencing. The study highlights the need for further studies on understanding the importance and the biological properties of identified novel viruses.

Unraveling the devastating impact of Tomato Leaf Curl New Delhi Virus (ToLCNDV): understanding its characteristics, host range, and management strategies

Due to their stagnation, plants are frequently exposed to a variety of stresses, including both abiotic and biotic factors. Biotic stress in plants is due to many microbes that include viruses, fungi, bacteria, and parasitic organisms as plant pathogens. Plant viruses are one of those pathogens that frequently evolve and lower global agricultural productivity. Geminiviruses are the most damaging type of plant viruses. Tomato Leaf Curl New Delhi Virus (ToLCNDV) is a begomovirus belonging to the family Geminiviridae. This is a ssDNA virus having a bipartite genome with genomic length ranging between 2.6-2.7 kb. This virus was originally discovered in India in 1995 from tomato (Lycopersicon esculentum). It mostly infects solanaceous and cucurbitaceous crops and is a whitefly-transmitted, circulative and persistent virus. Its prominent symptoms are severe upward leaf curling, enations, stunting, puckering, yellow spots and yellow mosaic. This virus has devastating effects on the yield of different crops with yield losses ranging between 17.6-99.4 % depending upon the disease-causing factors. ToLCNDV-ES and ToLCNDV-OM are the most devastating strains of this virus. This comprehensive review will provide basic information on the characteristic features of ToLCNDV. The evolutionary mechanism of this virus is studied to know about the strain’s evolution with the passage of time on different host plants cultivated in several regions of the world. This review also covers the genome characteristics, geographical distribution, host range, transmission methods, genetic diversity, different molecular &amp; serological assays, yield losses, and the management of this virus.

Tomato golden net virus and tomato yellow net virus: two novel New World begomoviruses with monopartite genomes.

Two novel tomato-infecting begomoviruses were discovered via high-throughput sequencing in Brazil. Both viruses were also Sanger-sequenced and displayed DNA-A components phylogenetically related to New World bipartite begomoviruses. The names tomato golden net virus (ToGNV) and tomato yellow net virus (ToYNV) were proposed. The majority of the New World begomoviruses has bipartite genomes. However, extensive analyses revealed that ToGNV and ToYNV have monopartite genomes, because no cognate DNA-B components were detected. Hence, they may comprise a unique group of monopartite New World begomoviruses, which have enormous biological, molecular, and plant breeding interest.

Genome segment ratios change during whitefly transmission of two bipartite cassava mosaic begomoviruses

Cassava mosaic disease is caused by a complex of whitefly-transmitted begomoviruses, which often occur in co-infections. These viruses have bipartite genomes consisting of DNA-A and DNA-B that are encapsidated into separate virions. Individual viruses exist in plants and whitefly vectors as populations comprising both genome segments, which can occur at different frequencies. Both segments are required for infection, and must be transmitted for virus spread to occur. Cassava plants infected with African cassava mosaic virus (ACMV) and/or East African cassava mosaic Cameroon virus (EACMCV), in which the ratios of DNA-A:DNA-B titers differed between plants, were used to examine how titers of the segments in a plant relate to their respective probabilities of acquisition by whiteflies and to the titers of each segment acquired and subsequently transmitted by whiteflies. The probabilities of acquiring each segment of ACMV did not reflect their relative titers in the source plant but they did for EACMCV. However, for both viruses, DNA-A:DNA-B ratios acquired by whiteflies differed from those in the source plant and the ratios transmitted by the whitefly did not differ from one – the ratio at which the highest probability of transmitting both segments is expected.

Bipartite genome and structural organization of the parvovirus Acheta domesticus segmented densovirus

Parvoviruses (family Parvoviridae) are currently defined by a linear monopartite ssDNA genome, T = 1 icosahedral capsids, and distinct structural (VP) and non-structural (NS) protein expression cassettes within their genome. We report the discovery of a parvovirus with a bipartite genome, Acheta domesticus segmented densovirus (AdSDV), isolated from house crickets (Acheta domesticus), in which it is pathogenic. We found that the AdSDV harbors its NS and VP cassettes on two separate genome segments. Its vp segment acquired a phospholipase A2-encoding gene, vpORF3, via inter-subfamily recombination, coding for a non-structural protein. We showed that the AdSDV evolved a highly complex transcription profile in response to its multipartite replication strategy compared to its monopartite ancestors. Our structural and molecular examinations revealed that the AdSDV packages one genome segment per particle. The cryo-EM structures of two empty- and one full-capsid population (3.3, 3.1 and 2.3 Å resolution) reveal a genome packaging mechanism, which involves an elongated C-terminal tail of the VP, “pinning” the ssDNA genome to the capsid interior at the twofold symmetry axis. This mechanism fundamentally differs from the capsid-DNA interactions previously seen in parvoviruses. This study provides new insights on the mechanism behind ssDNA genome segmentation and on the plasticity of parvovirus biology.

The Potency of Cowpea Mosaic Virus Particles for Cancer In Situ Vaccination Is Unaffected by the Specific Encapsidated Viral RNA.

Plant virus nanoparticles can be used as drug carriers, imaging reagents, vaccine carriers, and immune adjuvants in the formulation of intratumoral in situ cancer vaccines. One example is the cowpea mosaic virus (CPMV), a nonenveloped virus with a bipartite positive-strand RNA genome with each RNA packaged separately into identical protein capsids. Based on differences in their densities, the components carrying RNA-1 (6 kb) denoted as the bottom (B) component or carrying RNA-2 (3.5 kb) denoted as the middle (M) component can be separated from each other and from a top (T) component, which is devoid of any RNA. Previous preclinical mouse studies and canine cancer trials used mixed populations of CPMV (containing B, M, and T components), so it is unclear whether the particle types differ in their efficacies. It is known that the CPMV RNA genome contributes to immunostimulation by activation of TLR7. To determine whether the two RNA genomes that have different sizes and unrelated sequences cause different immune stimulation, we compared the therapeutic efficacies of B and M components and unfractionated CPMV in vitro and in mouse cancer models. We found that separated B and M particles behaved similarly to the mixed CPMV, activating innate immune cells to induce the secretion of pro-inflammatory cytokines such as IFNα, IFNγ, IL-6, and IL-12, while inhibiting immunosuppressive cytokines such as TGF-β and IL-10. In murine models of melanoma and colon cancer, the mixed and separated CPMV particles all significantly reduced tumor growth and prolonged survival with no significant difference. This shows that the specific RNA genomes similarly stimulate the immune system even though B particles have 40% more RNA than M particles; each CPMV particle type can be used as an effective adjuvant against cancer with the same efficacy as native mixed CPMV. From a translational point of view, the use of either B or M component vs the mixed CPMV formulation offers the advantage that separated B or M alone is noninfectious toward plants and thus provides agronomic safety.