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Viral-like particles are assembled from capsid protein structural subunits of different viruses and have ability to establish research in biomedicals, like construction of novel safety vaccines, gene therapy vectors by delivering systems for nucleic acids, small biomolecules and diagnostics. Papaya Mosaic Viral nanoparticals can provide as a vaccine candidate helps to increase the immunity by fusing the epitope based peptide antigen. Capripox viruses are the genus comprises Lymphy skin-disease, Sheep and Goat pox Viruses are notified by The World Animal Health Organization (OIE) based on their economic impotence act as a transboundary animal diseases viruses of sheep, goat, and cattle’s respectively. Plant viral based innovative vaccines have been emerged ineffective vaccine development. This research describes the engineering and development of a new vaccine candidate by display immunogenic peptide using the carrier capsid protein of Papaya Mosaic Virus. The Capripox virus P32 immunogenic protein is homologous of the vaccinia virus H3L gene displayed PapMV CP. The antigenicity of P32 protein epitope lowest score among epitopes C-terminally docked epitopes are EP6 > EP3 > EP8 as well the lowest score among epitopes N-terminally docked epitopes are EP8 > EP3 > EP6 presented on the N-terminus of PMV CP region which are found to be suitable for epitope display. And these modelled immunogenic peptide could be used to develop a viral like particles. Epitope based Antibody developed against immunogenic epitopic regions can contribute to a novel and robust protection from infection. As well might be used for developing cost effective detection kits for Transboundary animal disease viruses.

Cross-protection has been defined as the initial inoculation of a mild strain of a virus protecting plants against a severe strain. As the elucidation of the mechanisms that underlie this phenomenon evolves, we propose that this concept extends to the use of different species of viruses, where a mild species [Papaya mosaic virus (PapMV), a potexvirus] protects against other severe species [Papaya ringspot virus (PRSV), a potyvirus] in papaya plants.

An emerging virus isolated from papaya (Carica papaya) crops in northwestern (NW) Argentina was sequenced and characterized using next-generation sequencing. The resulting genome is 6667-nt long and encodes five open reading frames in an arrangement typical of other potexviruses. This virus appears to be a novel member within the genus Potexvirus. Blast analysis of RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes showed the highest amino acid sequence identity (67% and 71%, respectively) with pitaya virus X. Based on nucleotide sequence similarity and phylogenetic analysis, the name papaya virus X is proposed for this newly characterized potexvirus that was mechanically transmitted to papaya plants causing chlorotic patches and severe mosaic symptoms. Papaya virus X (PapVX) was found only in the NW region of Argentina. This prevalence could be associated with a recent emergence or adaptation of this virus to papaya in NW Argentina. Instituto de Patología Vegetal Fil: Cabrera Mederos, Dariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); Argentina Fil: Cabrera Mederos, Dariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Debat, Humberto Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Debat, Humberto Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); Argentina Fil: Torres, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Torres, Carolina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones en Bacteriología y Virología Molecular (IBaViM); Argentina Fil: Portal, Orelvis. Universidad Central “Marta Abreu” de Las Villas. Facultad de Ciencias Agropecuarias. Departamento de Biología; Cuba Fil: Portal, Orelvis. Universidad Central “Marta Abreu” de Las Villas. Facultad de Ciencias Agropecuarias. Centro de Investigaciones Agropecuarias; Cuba Fil: Jaramillo Zapata, Margarita. Universidad de San Pablo-T; Argentina Fil: Trucco, Veronica Milagros. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Trucco, Veronica Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); Argentina Fil: Flores, Ceferino Rene. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Yuto; Argentina Fil: Ortiz, Claudio Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Yuto; Argentina Fil: Badaracco, Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; Argentina Fil: Acuña, Luis Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Montecarlo; Argentina Fil: Nome Docampo, Claudia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Nome Docampo, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); Argentina Fil: Quito-Avila, Diego. Centro de Investigaciones Biotecnológicas del Ecuador. Escuela Superior Politécnica del Litoral; Ecuador Fil: Bejerman, Nicolas Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Bejerman, Nicolas Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); Argentina Fil: Castellanos Collazo, Onias. Fondo para la Investigación Científica y Tecnológica (FONCYT); Argentina Fil: Castellanos Collazo, Onias. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Sánchez-Rodríguez, Aminael. Universidad Técnica Particular de Loja. Departamento de Ciencias Biológicas; Ecuador Fil: Giolitti, Fabian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Giolitti, Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); Argentina

Chikungunya virus (CHIKV) infection is the cause of acute symptoms and chronic symmetrical polyarthritis associated with long-term morbidity and mortality. Currently, there is no available licensed vaccine or particularly useful drug for human use against CHIKV infection. This study was conducted to evaluate the efficacy of antibodies produced by papaya mosaic virus (PapMV) nanoparticles fused to E2EP3 peptide of CHIKV envelope as a recombinant CHIKV vaccine. PapMV, PapMV-C- E2EP3, and E2EP3-N-PapMV were produced in E. coli with an approximate size of 27 to 30 kDa. ICR mice (5 to 6 weeks of age) were injected subcutaneously with 25 micrograms of vaccine construct, and ELISA measured the titer of CHIKV specific IgG antibodies. The results showed that both recombinant proteins E2EP3-N-PapMV and PapMVC-E2EP3 were able to induce IgG antibodies production in immunized mice against CHIKV while immunization with recombinant PapMV showed no IgG antibodies induction. The neutralizing activity of the antibodies generated by either E2EP3-N-PapMV or PapMV-C-E2EP3 exhibited similar inhibition to CHIKV replication in Vero cells using the cells based antibody neutralizing assay and analyzed by plaque formation assay. This study showed the effectiveness of nanoparticles vaccine generated by fusing epitope peptide of CHIKV envelope to papaya mosaic virus envelope in inducing a robust immune response in mice against CHIKV. The data showed that levels of neutralizing antibodies correlate with a protective immune response CHIKV replication.

Background: The papaya mosaic virus (PapMV) vaccine platform is a rod-shaped nanoparticle made of the recombinant PapMV coat protein (CP) self-assembled around a noncoding single-stranded RNA (ssRNA) template. The PapMV nanoparticle induces innate immunity through stimulation of the Toll-like receptors (TLR) 7 and 8. The display of the vaccine antigen at the surface of the nanoparticle, associated with the co-stimulation signal via TLR7/8, ensures a strong stimulation of the immune response, which is ideal for the development of candidate vaccines. In this study, we assess the impact of where the peptide antigen is fused, whether at the surface or at the extremities of the nanoparticles, on the immune response directed to that antigen. Methods: Two different peptides from influenza A virus were used as model antigens. The conserved M2e peptide, derived from the matrix protein 2 was chosen as the B-cell epitope, and a peptide derived from the nucleocapsid was chosen as the cytotoxic T lymphocytes (CTL) epitope. These peptides were coupled at two different positions on the PapMV CP, the N- (PapMV-N) or the C-terminus (PapMV-C), using the transpeptidase activity of Sortase A (SrtA). The immune responses, both humoral and CD8+ T-cell-mediated, directed to the peptide antigens in the two different fusion contexts were analyzed and compared. The impact of coupling density at the surface of the nanoparticle was also investigated. Conclusions: The results demonstrate that coupling of the peptide antigens at the N-terminus (PapMV-N) of the PapMV CP led to an enhanced immune response to the coupled peptide antigens as compared to coupling to the C-terminus. The difference between the two vaccine platforms is linked to the enhanced capacity of the PapMV-N vaccine platform to stimulate TLR7/8. We also demonstrated that the strength of the immune response increases with the density of coupling at the surface of the nanoparticles.

Inactivated influenza vaccines efficacy is variable and often poor. We conducted a phase 1 trial (NCT02188810), to assess the safety and immunogenicity of a novel nanoparticle Toll-like receptor 7/8 agonist adjuvant (Papaya Mosaic Virus) at different dose levels combined with trivalent influenza vaccine in healthy persons 18&ndash, 50 years of age. Hemagglutination-inhibition assays, antibody to Influenza A virus nucleoprotein and peripheral blood mononuclear cells for measurement of interferon-gamma ELISPOT response to influenza antigens, Granzyme B and IFN&gamma, IL-10 ratio were measured. The most common adverse events were transient mild to severe injection site pain and no safety signals were observed. A dose-related adjuvant effect was observed. Geometric mean hemagglutination-inhibition titers increased at day 28 in most groups and waned over time, but fold-antibody responses were poor in all groups. Cell mediated immunity results were consistent with humoral responses. The Papaya Mosaic Virus adjuvant in doses of 30 to 240 &micro, g combined with reduced influenza antigen content was safe with no signals up to 3 years after vaccination. A dose-related adjuvant effect was observed and immunogenicity results suggest that efficacy study should be conducted in influenza antigen-na&iuml, ve participants.

Papaya ringspot virus (PRSV), a common potyvirus infecting papaya plants worldwide, can lead to either antagonism or synergism in mixed infections with Papaya mosaic virus (PapMV), a potexvirus. These two unrelated viruses produce antagonism or synergism depending on their order of infection in the plant. When PRSV is inoculated first or at the same time as PapMV, the viral interaction is synergistic. However, an antagonistic response is observed when PapMV is inoculated before PRSV. In the antagonistic condition, PRSV is deterred from the plant and its drastic effects are overcome. Here, we examine differences in gene expression by high-throughput RNA sequencing, focused on immune system pathways. We present the transcriptomic expression of single and mixed inoculations of PRSV and PapMV leading to synergism and antagonism. Upregulation of dominant and hormone-mediated resistance transcripts suggests that the innate immune system participates in synergism. In antagonism, in addition to innate immunity, upregulation of RNA interference-mediated resistance transcripts suggests that adaptive immunity is involved.

Rod-shaped virus-like nanoparticles (VLNP) made of papaya mosaic virus (PapMV) coat proteins (CP) self-assembled around a single stranded RNA (ssRNA) were showed to be a TLR7 agonist. Their utilization as an immune modulator in cancer immunotherapy was shown to be promising. To establish a clinical relevance in human for PapMV VLNP, we showed that stimulation of human peripheral blood mononuclear cells (PBMC) with VLNP induces the secretion of interferon alpha (IFNα) and other pro-inflammatory cytokines and chemokines. Plasmacytoid dendritic cells (pDCs) were activated and secreted IFN-α upon VLNP exposure. Monocyte-derived dendritic cells upregulate maturation markers and produce IL-6 in response to PapMV VLNP stimulation, which suggests the activation of TLR8. Finally, when co-cultured with NK cells, PapMV induced pDCs promoted the NK cytolytic activity against cancer cells. These data obtained with primary human immune cells further strengthen the clinical relevance of PapMV VLNPs as a cancer immunotherapy agent.

Papaya (Carica papaya L.) is susceptible to viral diseases caused by Papaya mosaic virus (PapMV) and Papaya ringspot virus (PRSV), which limit fruit production and affect economic yield. The symptoms produced by both the viruses are similar in early stages of infection and include vein and leaf chlorosis, which develop into mosaic at later stages of infection when leaf lamina can get reduced in size and distorted with a shoe-string aspect. Digital image analyses, such as fractal dimension (FD) and lacunarity (λ) were used here to examine papaya tissue after single and mixed infections of PRSV and PapMV. Morphological changes, such as hypoplasia, hyperplasia, and neoplasia are described in tissues with viral infections. Furthermore, we quantified these changes and suggest three ranges of tissue damage according to their λ values in rank 1 (0.01 to 0.39), rank 2 (0.4 to 0.79), and rank 3 (0.8 to 1). Our analyses suggest that in synergism and antagonism there is a competition of both viruses to occupy the same mesophyll cells, as indicated by their intermediate values of lacunarity.

Damicone, J. P., Edelson, J. V., Sherwood, J. L., Myers, L. D., and Motes, J. E. 2007. Effects of border crops and intercrops on control of cucurbit virus diseases. Plant Dis. 91:509-516. In five field trials over 3 years, control of aphid-transmitted, nonpersistent virus diseases on pumpkin, caused mostly by the potyviruses Watermelon mosaic virus (WMV) and Papaya ringspot virus type-W (PRSV-W), was achieved by intercropping with grain sorghum, as opposed to clean tillage. Reductions in disease incidence ranged from 43 to 96% (P ≤ 0.05). Surrounding pumpkin plots with borders of peanut, soybean, or corn was not effective. Borders of grain sorghum were effective, but disease control was generally less than for the intercrop treatment. Intercropping soybean and peanut with pumpkin reduced disease incidence by 27 to 60% (P ≤ 0.05), but disease control generally was less than for grain sorghum. Peak periods of alate aphid immigration generally preceded virus disease outbreaks by 7 to 14 days. However, alate landing rates, as measured in green tile traps, did not differ among treatments. Marketable yield was not increased by the intercrop treatments, and yield was reduced by up to 50% for the intercrop treatment with grain sorghum in two trials. The use of grass-selective herbicide applied along pumpkin rows, reduced seeding rates of the intercrops, or mowing did not alleviate the adverse effects of competition between pumpkin and the grain sorghum intercrop on yield.

The increasing use of plant viruses for the development of new vaccines and immunotherapy approaches poses questions regarding the mechanism by which the mammalian immune system recognizes these viruses. For example, although natural Abs (NA) and complement are key components of the innate immune system involved in the opsonization, phagocytosis, and destruction of microorganisms infecting mammals, their implication in plant virus recognition and immunogenicity is not well defined. In this study, we address the involvement of NA and the complement system in the activation of innate immunity through engagement of TLR7 with papaya mosaic virus (PapMV)-like nanoparticles. We demonstrate that NA, although binding to PapMV, are not involved in its recognition by the immune system. On the other hand, C3 strongly binds to PapMV nanoparticles and its depletion significantly reduces PapMV’s interaction with immune cells. Unexpectedly, however, we observed increased immune cell activation following administration of PapMV to complement-depleted mice. TLR7 activation by PapMV in the absence of C3 induced higher IFN-α production, resulting in superior immune cell activation and increased immunotherapeutic properties. In conclusion, in this study we established the involvement of the complement system in the recognition and the phagocytosis of PapMV nanoparticles and identified an unsuspected role for C3 in regulating the production of IFN-α following TLR7 activation.

Background: Nanoparticles derived from plant viruses possess fascinating structures, versatile functions and safe properties, rendering them valuable for a variety of applications. Papaya mosaic Virus-Like Particles (VLPs) are nanoparticles that contain a repetitive number of virus capsid proteins (PMV-CP) and are considered to be promising platforms for vaccine design. Previous studies have reported the antigenicity of PMV nanoparticles in mammalian systems. Materials and Methods: As experiments that concern vaccine development require careful design and can be time consuming, computational experiments are of particular importance. Therefore, prior to expressing PMV-CP in E. coli and producing nanoparticles, we performed an in silico analysis of the virus particles using software programs based on a series of sophisticated algorithms and modeling networks as useful tools for vaccine design. A computational study of PMV-CP in the context of the immune system reaction allowed us to clarify particle structure and other unknown features prior to their introduction in vitro. Results: The results illustrated that the produced nanoparticles can trigger an immune response in the absence of fusion with any foreign antigen. Conclusion: Based on the in silico analyses, the empty capsid protein was determined to be recognised by different B and T cells, as well as cells which carry MHC epitopes.

Influenza virus infections are a significant public threat and the best approach to prevent them is through vaccination. Because of the perpetual changes of circulating influenza strains, the efficacy of influenza vaccines rarely exceeds 50%. To improve the protection efficacy, we have designed a novel vaccine formulation that shows a broad range of protection. The formulation is made of the matrix protein 2 (M2e) and the nucleoprotein (NP) antigens. The multimerization of NP into nanoparticles improved significantly the immune response to NP. The combination of the NP nanoparticles with the PapMV-M2e nanoparticles enhances significantly the immune response directed to NP revealing the adjuvant property of the PapMV platform. The vaccine formulation combining these two types of nanoparticles protects mice from infectious challenges by two different influenza strains (H1N1 and H3N2) and is a promising influenza A vaccine capable to elicit a broad protection.

International audience; Although vaccination has been an effective way of preventing infections ever since the eighteenth century, the generation of therapeutic vaccines and immunotherapies is still a work in progress. A number of challenges impede the development of these therapeutic approaches such as safety issues related to the administration of whole pathogens whether attenuated or inactivated. One safe alternative to classical vaccination methods gaining recognition is the use of nanoparticles, whether synthetic or naturally derived. We have recently demonstrated that the papaya mosaic virus (PapMV)-like nanoparticle can be used as a prophylactic vaccine against various viral and bacterial infections through the induction of protective humoral and cellular immune responses. Moreover, PapMV is also very efficient when used as an immune adjuvant in an immunotherapeutic setting at slowing down the growth of aggressive mouse melanoma tumors in a type I interferon (IFN-I)-dependent manner. In the present study, we were interested in exploiting the capacity of PapMV of inducing robust IFN-I production as treatment for the chronic viral infection model lymphocytic choriomeningitis virus (LCMV) clone 13 (Cl13). Treatment of LCMV Cl13-infected mice with two systemic administrations of PapMV was ineffective, as shown by the lack of changes in viral titers and immune response to LCMV following treatment. Moreover, IFN-α production following PapMV administration was almost completely abolished in LCMV-infected mice. To better isolate the mechanisms at play, we determined the influence of a pretreatment with PapMV on secondary PapMV administration, therefore eliminating potential variables emanating from the infection. Pretreatment with PapMV led to the same outcome as an LCMV infection in that IFN-α production following secondary PapMV immunization was abrogated for up to 50 days while immune activation was also dramatically impaired. We showed that two distinct and overlapping mechanisms were responsible for this outcome. While short-term inhibition was partially the result of interleukin-1 receptor-associated kinase 1 degradation, a crucial component of the toll-like receptor 7 signaling pathway, long-term inhibition was mainly due to interference by PapMV-specific antibodies. Thus, we identified a possible pitfall in the use of virus-like particles for the systemic treatment of chronic viral infections and discuss mitigating alternatives to circumvent these potential problems.

Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

It has been shown by X-ray analysis that cores of coat proteins (CPs) from three potexviruses, flexible helical RNA-containing plant viruses, have similar α-helical structure. However, this similarity cannot explain structural lability of potexvirus virions, which is believed to determine their biological activity. Here, we used circular dichroism (CD) spectroscopy in the far UV region to compare optical properties of CPs from three potexviruses with the same morphology and similar structure. CPs from Alternanthera mosaic virus (AltMV), potato aucuba mosaic virus (PAMV), and potato virus X (PVX) have been studied in a free state and in virions. The CD spectrum of AltMV virions was similar to the previously obtained CD spectrum of papaya mosaic virus (PapMV) virions, but differed significantly from the CD spectrum of PAMV virions. The CD spectrum of PAMV virions resembled in its basic characteristics the CD spectrum of PVX virions characterized by molar ellipticity that is abnormally low for α-helical proteins. Homology modeling of the CP structures in AltMV, PAMV, and PVX virions was based on the known high-resolution structures of CPs from papaya mosaic virus and bamboo mosaic virus and confirmed that the structures of the CP cores in all three viruses were nearly identical. Comparison of amino acid sequences of different potexvirus CPs and prediction of unstructured regions in these proteins revealed a possible correlation between specific features in the virion CD spectra and the presence of disordered N-terminal segments in the CPs.

Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay’s specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%), 93/341 (27.3%), and 3/341 (0.9%), for PRSV, PLDMV, and PapMV, respectively, 59/341 (17.3%) of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya.

Bottle gourd or Lagenaria siceraria (Molina) Standl. (family: Cucurbitaceae) is an annual herbaceous crop grown worldwide. It is well-known for its use in Indian ayurvedic medicine. In India, during 2016 to 2017, total annual production of bottle gourd was 2,572,000 metric tons harvested from 157,000 ha (Government of India 2017). During December 2016 and February 2017, disease symptoms such as leaf mosaic and leaf curling were observed in the bottle gourd growing fields of sub-Himalayan West Bengal, India. In severe cases, blistering and deformation of leaf lamina were also observed. Twenty such symptomatic and three healthy-looking leaf samples were collected and analyzed using transmission electron microscopy. All symptomatic samples showed the presence of 500- to 700-nm-long filamentous rod-shaped particles resembling potexviruses (Kim et al. 2010). Total RNA was isolated from all the samples (Ghawana et al. 2011) and tested through reverse transcription polymerase chain reaction (RT-PCR) with Potex5 (5′-CAYCARCARGCMAARGAYGA-3′) and Potex2RC (5′-AGCATRGCNSCRTCYTG-3′) primers that specifically amplify the partial RNA-dependent RNA polymerase (RdRp) gene of the potexviruses (van der Vlugt and Berendsen 2002). RT-PCR conditions were set up as follows: initial incubation for 30 min at 50°C and initial denaturation for 5 min at 95°C; 35 cycles of denaturation at 95°C for 1 min, annealing at 51.5°C for 1 min, product extension at 72°C for 1 min; and final extension at 72°C for 10 min. The products were visualized under ultraviolet transilluminator after running on 1% agarose gel, and amplicon size was determined. All 20 infected samples produced an amplicon of ∼530 bp, whereas the three healthy-looking samples did not produce expected amplicons. All amplicons were cloned into pGEM-T vector (Promega, Madison, WI) and were sequenced. One such sequence of sample NE-02 was submitted to GenBank as Lagenaria mild mosaic virus (LaMMoV, accession no. MG752888) and showed 78% nucleotide (nt) identity and 91% amino acid (aa) identity with LaMMoV infecting bottle gourd in Myanmar (accession no. AB546335), as determined in a BLASTn analysis. To confirm the identity of the pathogen, another primer pair, RdRP1f (5′-CACCGGTGACTCAAAGCAAA-3′) and RdRP1r (5′-CCAATRGGCCTCTTTTCMAG-3′), was designed based on the genome of LaMMoV, papaya mosaic virus, and Alternanthera mosaic virus to amplify the RdRp gene of all the three potexviruses mentioned. For the primer set RdRP1f/RdRP1r, RT-PCR conditions were similar to those described above, except for the annealing step at 57°C for 1 min. RT-PCR conducted on all 20 symptomatic samples produced an amplicon of ∼810 bp, whereas none of the healthy-looking samples tested positive with the same primer pair. This new RT-PCR fragment of the sample NE-02 was also submitted to GenBank as LaMMoV (accession no. MH998222), because it showed 82% nt identity and 99% aa identity with LaMMoV (accession no. AB546335) following BLASTn analysis. The species demarcation criteria for the genus Potexvirus is less than 72% nt (or 80% aa) identity between their coat protein or RdRp genes (Adams et al. 2004). To the best of our knowledge, this is the first report of LaMMoV infecting bottle gourd in India. The finding of this study shall aid in developing effective management strategies.

Nanoparticles composed of the coat protein of a plant virus (papaya mosaic virus; PapMV) and a single-stranded RNA (ssRNA) trigger a strong innate immune stimulation in the lungs of the animals a few hours following instillation. A rapid recruitment of neutrophils, monocytes/macrophages and lymphocytes follows. This treatment was able to provide protection to an influenza challenge that lasts at least 5 days. Protection could be recalled for longer periods by repeating the instillations once per week for more than 10 weeks. The treatment also conferred protection to a lethal challenge with Streptococcus pneumoniae--the major cause of bacterial pneumonia. Finally, we also showed that the nanoparticles could be used to treat mice infected with influenza and significantly decrease morbidity. These data strengthen the potential for using PapMV nanoparticles as non-specific inducers of the innate immune response in lungs during viral pandemics or to combat bioterrorist attack.In this study, virus-like nanoparticles were utilized to induce innate immune responses in a mouse model. They were also demonstrated to provide enhanced immune responses during actual pneumonia and ongoing viral infection. Strategies like this may become very helpful in human applications, including bioterrorism countermeasures.

A new member of the genus Potexvirus was fully sequenced and characterized. The virus was isolated from babaco (Vasconcellea x heilbornii), a natural hybrid native to Ecuador. The virus contains a 6,692 nt long genome organized in five open reading frames in an arrangement typical of other potexviruses. Sequence comparisons revealed close relatedness with Papaya mosaic virus (PapMV), Alternathera mosaic virus (AltMV) and Senna mosaic virus (SenMV), exhibiting nucleotide identities up to 67% for the polymerase (Pol) and 68% for the coat protein (CP), with deduced amino acid identities of 70% and 72% for the Pol and CP, respectively. The presence of an AlkB domain, in the polymerase region, was observed. Terminal nucleotide sequences were conserved across potexviruses with characteristic motifs and predicted secondary structures at the 3’ UTR. Although serologically undistinguishable from PapMV and AltMV, the new virus showed differences in host range and symptom induction. The name babaco mosaic virus is proposed for this newly characterized Potexvirus. The complete genome sequence of the new virus has been deposited in NCBI GenBank under accession number MF978248.

Background The addition of an adjuvant to a vaccine is a promising approach to increasing strength and immunogenicity towards antigens. Despite the fact that adjuvants have been used in vaccines for decades, their mechanisms of action and their influence on the kinetics of the immune response are still not very well understood. The use of papaya mosaic virus (PapMV) nanoparticles-a novel TLR7 agonist-was recently shown to improve and broaden the immune response directed to trivalent inactivated flu vaccine (TIV) in mice and ferrets. Results We investigated the capacity of PapMV nanoparticles to increase the speed of the immune response toward TIV. PapMV nanoparticles induced a faster and stronger humoral response to TIV that was measured as early as 5 days post-immunization. The addition of PapMV nanoparticles was shown to speed up the differentiation of B-cells into early plasma cells, and increased the growth of germinal centers in a CD4+ dependent manner. TIV vaccination with PapMV nanoparticles as an adjuvant protected mice against a lethal infection as early as 10 days post-immunization. Conclusion In conclusion, PapMV nanoparticles are able to accelerate a broad humoral response to TIV. This property is of the utmost importance in the field of vaccination, especially in the case of pandemics, where populations need to be protected as soon as possible after vaccination. Electronic supplementary material The online version of this article (doi:10.1186/s12951-016-0200-2) contains supplementary material, which is available to authorized users.

International audience; The recent development of novel immunotherapies is revolutionizing cancer treatment. These include, for example, immune checkpoint blockade, immunomodulation, or therapeutic vaccination. Although effective on their own, combining multiple approaches will most likely be required in order to achieve the maximal therapeutic benefit. In this regard, the papaya mosaic virus nanoparticle (PapMV) has shown tremendous potential as (i) an immunostimulatory molecule, (ii) an adjuvant, and (iii) a vaccine platform through its intrinsic capacity to activate the innate immune response in an IFN-α-dependent manner. Here, we demonstrate that intratumor administration of PapMV significantly slows down melanoma progression and prolongs survival. This correlates with enhanced chemokine and pro-inflammatory-cytokine production in the tumor and increased immune-cell infiltration. Proportions of total and tumor-specific CD8(+) T cells dramatically increase following PapMV treatment whereas those of myeloid-derived suppressor cells (MDSC) concomitantly decrease. Moreover, systemic PapMV administration prevents metastatic tumor-implantation in the lungs. Importantly, PapMV also synergistically improves the therapeutic benefit of dendritic cell (DC)-based vaccination and PD-1 blockade by potentiating antitumor immune responses. This study illustrates the immunostimulatory potential of a plant virus-derived nanoparticle for cancer therapy either alone or in conjunction with other promising immunotherapies in clinical development.

A putative Potexvirus was detected from bottle gourd (Lagenaria siceraria) showing mosaic and mottle symptoms in Myanmar in 2007. The virus was designated Lagenaria mild mosaic virus (LaMMoV) and was further characterized. In artificial inoculation tests, infectivity of LaMMoV was limited to two families: Chenopodiaceae and Cucurbitaceae. The host range of LaMMoV differs from those of the two cucurbit-infecting potexviruses, Alternanthera mosaic virus (AltMV) and Papaya mosaic virus (PapMV). Sequence analyses of LaMMoV showed that the C-terminal 3,859 nucleotides, excluding the poly-A tail, includes the C-terminal region of an RNA-dependent RNA polymerase (RdRp), a triple gene block (TGB), a coat protein (CP), and a 3′ untranslated region (UTR), all of which are typical of potexviruses. Although LaMMoV is related closely to AltMV and PapMV, its nucleotide sequences differ from those of other previously reported potexviruses. Therefore, we report LaMMoV as a new species of the genus Potexvirus that occurs in the cucurbit bottle gourd.

With the emergence of highly virulent influenza viruses and the consequent risk of pandemics, new approaches to designing universal influenza vaccines are urgently needed. In this report, we demonstrate the potential of using a papaya mosaic virus (PapMV) platform carrying the universal M2e influenza epitope (PapMV-CP-M2e) as a candidate flu vaccine. We show that PapMV-CP-M2e virus-like particles (VLPs) can induce production in mice of anti-M2e antibodies that can recognize influenza-infected cells. PapMV-CP-M2e discs made of 20 coat protein (CP) subunits were shown to be poorly immunogenic compared to PapMV-CP-M2e VLPs composed of several hundred CP subunits. We also show that addition of either alum or PapMV-CP VLPs as adjuvant dramatically increased the immunogenicity of PapMV-CP-M2e-containing vaccine, and led to 100% protection against a challenge of 4LD(50) with the WSN/33 strain. These results show, for the first time, the potential of a recombinant plant virus protein to serve as both peptide delivery system and adjuvant in the crucial field of influenza vaccine development.

Fiber diffraction data have been obtained from Narcissus mosaic virus, a potexvirus from the family Flexiviridae, and soybean mosaic virus (SMV), a potyvirus from the family Potyviridae. Analysis of the data in conjunction with cryo-electron microscopy data allowed us to determine the symmetry of the viruses and to make reconstructions of SMV at 19 Å resolution and of another potexvirus, papaya mosaic virus, at 18 Å resolution. These data include the first well-ordered data ever obtained for the potyviruses and the best-ordered data from the potexviruses, and offer the promise of eventual high resolution structure determinations.

Papaya (Carica papaya L.), native to the South American continent, is an important horticultural crop cultivated across the tropical and subtropical regions of the globe. Papaya is rich source of vitamin-C and globally it is ranked fourth in total fruit production, next only to bananas, oranges and mangoes. India is the leading producer of papaya and both India and Brazil put together account for more than 50 % of global papaya production. Multiple pests and pathogens are known to inflict damage to papaya, of which viral diseases are the most damaging ones. Of all the viral diseases, papaya ring spot virus (PRSV) belonging to the Potyviridae family is most important one, followed by the viruses belonging to the Geminiviridae family causing leaf curl disease in papaya. Other viral diseases of papaya are Papaya meleira virus (PMeV), Papaya mosaic virus (PapMV), Papaya lethal yellowing virus (PLYV) and several other viruses are known to infect papaya, but may not be of economical significance. Management of viral diseases in papaya is very crucial to accomplish a good harvest, and of all the management practices, genetic engineering papaya for virus resistance is most promising and successful. The PRSV resistant transgenic papaya varieties “SunUp” and “Rainbow” developed by the University of Hawaii and extensively cultivated in the Hawaii islands of United States is the most successful field application of transgenic technology. Since there is significant sequence variation in the PRSV strains from different parts of the world and many more diverse range of viruses are known to infect papaya, there is an urgent need to develop region specific virus resistant papaya.

Plant-virus-based vaccines have emerged as a promising avenue in vaccine development. This report describes the engineering of an innovative vaccine platform using the papaya mosaic virus (PapMV) capsid protein (CP) as a carrier protein and a C-terminal fused hepatitis C virus (HCV) E2 epitope as the immunogenic target. Two antigen organizations of the PapMV-based vaccines were tested: a virus-like-particle (VLP; PapMVCP-E2) and a monomeric form (PapMVCP27–215-E2). While the two forms of the vaccine were both shown to be actively internalized in vitro in bone-marrow-derived antigen presenting cells (APCs), immunogenicity was demonstrated to be strongly dependent on antigen organization. Indeed, C3H/HeJ mice injected twice with the multimeric VLP vaccine showed a long-lasting humoral response (more than 120 days) against both the CP and the fused HCV E2 epitope. The antibody profile (production of IgG1, IgG2a, IgG2b, IgG3) suggests a Th1/Th2 response. Immunogenicity of the PapMV vaccine platform was not observed when the monomer PapMVCP-E2 was injected. These results demonstrate for the first time the potential of the PapMV vaccine platform and the critical function of multimerization in its immunogenicity.

Greenhouse-grown cucurbit crops in several Iranian regions were surveyed for the incidence of Cucumber mosaic virus (CMV), Squash mosaic virus (SqMV), Papaya ring spot virus-type W (PRSV-W), Watermelon mosaic virus-2 (WMV-2), Zucchini yellow mosaic virus (ZYMV), Cucumber necrosis virus (CuNV) and Tomato spotted wilt virus (TSWV) from September 2002 to June 2004. In all, 1,304 random and 1,085 symptomatic leaf or fruit samples were collected. Samples were analyzed for virus infection by enzyme-linked immunosorbent assay. CMV and ZYMV were the viruses most frequently detected, accounting for 21.2 and 18% of the infected plants, respectively. WMV-2 was detected with 4.3% incidence in 15 regions and TSWV with 1.25% incidence only in 2 regions. CuNV, SqMV, and PRSV-W were not detected in any samples. Double and triple infections involving different combinations of CMV, ZYMV, WMV-2, and TSWV were noted in 117 and 4 samples, respectively. Natural infection of cucumber with TSWV and ZYMV is reported for the first time from Iran. Of 16 plant species from 14 genera, growing in or around greenhouse facilities, 6 were found infected with ZYMV, TSWV, WMV-2, and CMV and may act as reservoirs of the viruses. Four species are reported as new hosts of these viruses.

The emergence of highly virulent influenza strains and the risks of pandemics as well as the limited efficiency of the current seasonal vaccines are important public health concerns. There is a major need for new influenza vaccines that would be broadly cross-protective. The ectodomain of matrix protein 2 (M2e) is highly conserved amongst different influenza strains and could be used as a broad spectrum antigen. To overcome its low immunogenicity we have fused a short peptide epitope derived from the human consensus sequence of M2e (amino acids 6-14, EVETPIRNE) to the N-terminus of papaya mosaic virus coat protein. The fusion harboring coat proteins were assembled around a single stranded RNA into virus-like particles (PapMV-sM2e). The resulting PapMV-sM2e rod-shaped particle was stable and indistinguishable from regular PapMV particles. A single intramuscular immunization with PapMV-sM2e was sufficient to mount appreciable levels of CD4 dependent M2e specific total IgG and IgG2a antibody in mice sera. PapMV-sM2e proved to be self-adjuvanting since the addition of PapMV as an exogenous adjuvant did not result in significantly improved antibody titers. In addition, we confirmed the adjuvant property of PapMV-sM2e using the trivalent inactivated flu vaccine as antigen and demonstrated that the newly engineered nanoparticles areas efficacious as an adjuvant than the original PapMV nanoparticles. Upon infection with a sub-lethal dose of influenza, PapMV-sM2e vaccinated animals were completely protected from virus induced morbidity and mortality. Mice immunized with decreasing amounts of PapMV-sM2e and challenged with a more stringent dose of influenza virus displayed dose-dependent levels of protection. Seventy percent of the mice immunized once with the highest dose of PapMV-sM2e survived the challenged. The survival of the mice correlated mainly with the levels of anti-M2e IgG2a antibodies obtained before the infection. These results demonstrate that PapMV-sM2e can be an important component of a broadly cross-reactive influenza vaccine.

Potexvirus isolates from creeping phlox (Phlox stolonifera) and trailing portulaca (Portulaca grandiflora) obtained from commercial nurseries were found to react strongly with polyclonal antiserum against Papaya mosaic virus (PapMV). Cloning and sequencing of a phlox isolate from Pennsylvania revealed that the coat protein (CP) gene was c.97% identical at the amino acid level to the CP gene sequence of Alternanthera mosaic virus (Geering and Thomas, 1999), and only c.75% to the CP of PapMV. The full genomic sequence of the phlox isolate was then determined. The regions to the 5' of the CP gene were found to be more closely related to (but distinct from) PapMV than to any other characterized potexvirus. This phlox potexvirus isolate was therefore named AltMV-PA. The host range of AltMV-PA was found to be similar to that of the Australian isolate, AltMV-Au. The 3'-terminal portion of the genome of the portulaca potexvirus isolate, and of additional potexvirus isolates from two phlox cultivars from commercial nurseries in Maryland, were also cloned and sequenced. The sequences of these isolates were similar to those of AltMV-Au and AltMV-PA, and the isolates were designated AltMV-Po (Portulaca), AltMV-SP (creeping phlox 'Sherwood Purple'), and AltMV-BR (creeping phlox 'Blue Ridge'). Each of the phlox and portulaca isolates reacted strongly with antiserum to AltMV-Au, and also with anti-PapMV antiserum. The host range of AltMV includes several ornamentals; these findings suggest that AltMV may be quite widespread, and may be mis-diagnosed as PapMV.

Vibrational Raman optical activity (ROA), measured as a small difference in the intensity of Raman scattering from chiral molecules in right and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the aqueous solution structure of proteins. On account of the large number of structure-sensitive bands in protein ROA spectra, multivariate analysis techniques such as non-linear mapping (NLM) are especially favourable for determining structural relationships between different proteins. Here NLM is used to map a dataset of 80 polypeptide, protein and virus ROA spectra, considered as points in a multidimensional space with axes representing the digitized wavenumbers, into readily visualizable two and three-dimensional spaces in which points close to or distant from each other, respectively, represent similar or dissimilar structures. Discrete clusters are observed which correspond to the seven structure classes all alpha, mainly alpha, alphabeta, mainly beta, all beta, mainly disordered/irregular and all disordered/irregular. The average standardised ROA spectra of the proteins falling within each structure class have distinct features characteristic of each class. A distinct cluster containing the wheat protein A-gliadin and the plant viruses potato virus X, narcissus mosaic virus, papaya mosaic virus and tobacco rattle virus, all of which appear in the mainly alpha cluster in the two-dimensional representation, becomes clearly separated in the direction of increasing disorder in the three-dimensional representation. This suggests that the corresponding five proteins, none of which to date has yielded high-resolution X-ray structures, consist mainly of alpha-helix and disordered structure with little or no beta-sheet. This combination of structural elements may have functional significance, such as facilitating disorder-to-order transitions (and vice versa) and suppressing aggregation, in these proteins and also in sequences within other proteins. The use of ROA to identify proteins containing significant amounts of disordered structure will, inter alia, be valuable in structural genomics/proteomics since disordered regions often inhibit crystallization.