Argonaute 5-mediated antiviral defense and viral counter-defense in Nicotiana benthamiana.

The argonaute (AGO) family proteins play a crucial role in preventing viral invasions through the plant antiviral RNA silencing pathway, with distinct AGO proteins recruited for specific antiviral mechanisms. Our previous study revealed that Nicotiana benthamiana AGO5 (NbAGO5) expression was significantly upregulated in response to bamboo mosaic virus (BaMV) infection. However, the roles of NbAGO5 in antiviral mechanisms remained to be explored. In this research, we examined the antiviral functions of NbAGO5 in the infections of different viruses. It was found that the accumulation of NbAGO5 was induced not only at the RNA but also at the protein level following the infections of BaMV, potato virus X (PVX), tobacco mosaic virus (TMV), and cucumber mosaic virus (CMV) in N. benthamiana. To explore the antiviral mechanism and regulatory function of NbAGO5, we generated NbAGO5 overexpression (OE-NbAGO5) and knockout (nbago5) transgenic N. benthamiana lines. Our findings reveal that NbAGO5 provides defense against BaMV, PVX, TMV, and a mutant CMV deficient in 2b gene, but not against the wild-type CMV and turnip mosaic virus (TuMV). Through affinity purification and small RNA northern blotting, we demonstrated that NbAGO5 exerts its antiviral function by binding to viral small interfering RNAs (vsiRNAs). Moreover, we observed that CMV 2b and TuMV HC-Pro interact with NbAGO5, triggering its degradation via the 26S proteasome and autophagy pathways, thereby allowing these viruses to overcome NbAGO5-mediated defense. In addition, TuMV HC-Pro provides another line of counter-defense by interfering with vsiRNA binding by NbAGO5. Our study provides further insights into the antiviral RNA interference mechanism and the complex interplay between NbAGO5 and plant viruses.

First Report of Distinct Bamboo mosaic virus (BaMV) Isolates Infecting Bambusa funghomii in Vietnam and the Identification of a Highly Variable Region in the BaMV Genome.

New isolates of the Bamboo mosaic virus (BaMV) were identified in Bambusa funghomii bamboo in Vietnam. Sequence analyses revealed that the Vietnam isolates are distinct from all known BaMV strains, sharing the highest sequence identities (about 77%) with the Yoshi isolates reported in California, USA. Unique satellite RNAs were also found to be associated with the BaMV Vietnam isolates. A possible recombination event was detected in the genome of BaMV-VN2. A highly variable region was identified in the ORF1 gene, in between the methyl transferase domain and helicase domain. These results revealed the presence of unique BaMV isolates in an additional bamboo species in one more country, Vietnam, and provided evidence in support of the possible involvement of environmental or host factors in the diversification and evolution of BaMV.

Voltage-dependent anion channel proteins associate with dynamic Bamboo mosaic virus-induced complexes.

Infection cycles of viruses are highly dependent on membrane-associated host factors. To uncover the infection cycle of Bamboo mosaic virus (BaMV) in detail, we purified the membrane-associated viral complexes from infected Nicotiana benthamiana plants and analyzed the involved host factors. Four isoforms of voltage-dependent anion channel (VDAC) proteins on the outer membrane of mitochondria were identified due to their upregulated expression in the BaMV complex-enriched membranous fraction. Results from loss- and gain-of-function experiments indicated that NbVDAC2, -3, and -4 are essential for efficient BaMV accumulation. During BaMV infection, all NbVDACs concentrated into larger aggregates, which overlapped and trafficked with BaMV virions to the structure designated as the "dynamic BaMV-induced complex." Besides the endoplasmic reticulum and mitochondria, BaMV replicase and double-stranded RNAs were also found in this complex, suggesting the dynamic BaMV-induced complex is a replication complex. Yeast two-hybrid and pull-down assays confirmed that BaMV triple gene block protein 1 (TGBp1) could interact with NbVDACs. Confocal microscopy revealed that TGBp1 is sufficient to induce NbVDAC aggregates, which suggests that TGBp1 may play a pivotal role in the NbVDAC-virion complex. Collectively, these findings indicate that NbVDACs may associate with the dynamic BaMV-induced complex via TGBp1 and NbVDAC2, -3, or -4 and can promote BaMV accumulation. This study reveals the involvement of mitochondrial proteins in a viral complex and virus infection.

Stable Display of Artificially Long Foreign Antigens on Chimeric Bamboo mosaic virus Particles.

Plant viruses can be genetically modified to generate chimeric virus particles (CVPs) carrying heterologous peptides fused on the surface of coat protein (CP) subunits as vaccine candidates. However, some factors may be especially significant in determining the properties of chimeras. In this study, peptides from various sources and of various lengths were inserted into the Bamboo mosaic virus-based (BaMV) vector CP N-terminus to examine the chimeras infecting and accumulating in plants. Interestingly, it was found that the two different strains Foot-and-mouth disease virus (FMDV) VP1 antigens with flexible linker peptides (77 or 82 amino acids) were directly expressed on the BaMV CP, and the chimeric particles self-assembled and continued to express FMDV antigens. The chimeric CP, when directly fused with a large foreign protein (117 amino acids), can self-fold into incomplete virus particles or disks. The physicochemical properties of heterologus peptides N-terminus, complex strand structures of heterologus peptides C-terminus and different flexible linker peptides, can affect the chimera accumulation. Based on these findings, using plant virus-based chimeras to express foreign proteins can increase their length limitations, and engineered plant-made CVP-based vaccines have increasing potential for further development as novel vaccines.

Argonaute 5 family proteins play crucial roles in the defence against Cymbidium mosaic virus and Odontoglossum ringspot virus in Phalaenopsis aphrodite subsp. formosana.

The orchid industry faces severe threats from diseases caused by viruses. Argonaute proteins (AGOs) have been shown to be the major components in the antiviral defence systems through RNA silencing in many model plants. However, the roles of AGOs in orchids against viral infections have not been analysed comprehensively. In this study, Phalaenopsis aphrodite subsp. formosana was chosen as the representative to analyse the AGOs (PaAGOs) involved in the defence against two major viruses of orchids, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). A total of 11 PaAGOs were identified from the expression profile analyses of these PaAGOs in P. aphrodite subsp. formosana singly or doubly infected with CymMV and/or ORSV. PaAGO5b was found to be the only one highly induced. Results from overexpression of individual PaAGO5 family genes revealed that PaAGO5a and PaAGO5b play central roles in the antiviral defence mechanisms of P. aphrodite subsp. formosana. Furthermore, a virus-induced gene silencing vector based on Foxtail mosaic virus was developed to corroborate the function of PaAGO5s. The results confirmed their importance in the defences against CymMV and ORSV. Our findings may provide useful information for the breeding of traits for resistance or tolerance to CymMV or ORSV infections in Phalaenopsis orchids.

Transmission of Bamboo mosaic virus in Bamboos Mediated by Insects in the Order Diptera.

Bamboo mosaic virus (BaMV), a member of the genus Potexvirus, is the major threat to bamboo cultivation. Similar to most potexviruses, the transmission of BaMV by insect vectors has not been documented previously. However, field observations of BaMV disease incidences suggested that insect vectors might be involved. In this study, we aimed to investigate the possibility of insect-mediated transmission of BaMV among bamboo clumps, in order to provide further insights into the infection cycles of BaMV for the development of effective disease management measures. From the major insects collected from infected bamboo plantations, BaMV genomic RNAs were detected inside the bodies of two dipteran insects, Gastrozona fasciventris and Atherigona orientalis, but not in thrips (Scirtothrips dorsalis). Artificial feeding assays using green fluorescent protein-tagged BaMV virions revealed that BaMV could enter the digestive systems and survive in the regurgitant and excretion of the dipterans. BaMV RNA could be retained in the dipterans for up to 4 weeks. Insect-mediated transmission assays indicated that both dipterans could transmit BaMV to bamboo seedlings through artificially created wounds with low infection efficiency (14 - 41%), suggesting that the dipterans may mediate the transmission in a mechanical-like manner. These results demonstrated that dipterans with sponge-like mouthparts may also serve as vectors for at least one potexvirus, BaMV, among bamboo plants. The finding suggested that dipteran insect control should be integrated into the disease management measures against BaMV infections.

A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system.

Plasma haptoglobin concentrations are elevated in patients with coronary artery disease.

Inflammation underlies the development and progression of coronary artery plaques. Haptoglobin (Hp) is an acute phase protein, the synthesis of which is increased during inflammation. The aim of this study was to investigate plasma Hp concentrations and phenotype in patients with coronary artery disease (CAD). We recruited 359 patients with fixed luminal stenosis ≥50% in at least one coronary artery (CAD group) and 83 patients with luminal stenosis ≤40%, normal ejection fraction, and normal regional wall motion (control group). Plasma Hp concentrations were measured using a phenotype-specific enzyme-linked immunosorbent assay. Hp phenotype was determined by native polyacrylamide gel electrophoresis. Plasma lipid concentrations were measured. Plasma Hp concentrations were significantly higher in the CAD compared with the control group (262.4±144.2 vs 176.0±86.7 ng/mL, P<0.001); however, there was no between group difference in the distribution of Hp phenotype (1-1 = 7.5% vs 7.2%; 2-1 = 40.4% vs 42.2%; 2-2 = 52.1% vs 50.6%). Stepwise multivariate logistic regression revealed that high Hp concentrations (odds ratio [OR] = 5.865), male sex (OR = 3.689), hypertension (OR = 2.632), diabetes mellitus (OR = 3.300), and low-density lipoprotein concentrations (OR = 1.480) were independently associated with CAD (all P<0.05). Hp phenotype was not associated with CAD. Plasma Hp concentrations were significantly correlated with the severity of luminal stenosis (r = 0.236, P<0.001). Our findings suggest that plasma Hp concentrations may be elevated in patients with CAD. There does not appear to be any relationship between Hp phenotype and CAD.

Induction of protective immunity in chickens immunized with plant-made chimeric Bamboo mosaic virus particles expressing very virulent Infectious bursal disease virus antigen.

Very virulent Infectious bursal disease virus (vvIBDV) causes a highly contagious disease in young chickens and leads to significant economic loss in the poultry industry. Effective new vaccines are urgently needed. Autonomously replicating plant virus-based vector provides attractive means for producing chimeric virus particles (CVPs) in plants that can be developed into vaccines. In this study, we demonstrate the potential for vaccine development of Bamboo mosaic virus (BaMV) epitope-presentation system, where the antigen from vvIBDV VP2 was fused to the N-terminus of BaMV coat protein. Accordingly, an infections plasmid, pBIBD2, was constructed. Inoculation of the recombinant BaMV clone pBIBD2 enabled the generation of chimeric virus, BIBD2, and stable expression of IBDV VP2 antigen on its coat protein. After intramuscular immunization with BIBD2 CVPs, chickens produced antibodies against IBDV and were protected from vvIBDV (V263/TW strain) challenges. These results corroborate the feasibility of BaMV-based CVP platform in plants for the development and production of vaccines against IBDV.