Alternanthera Mosaic Virus Research Articles

Assessment of a Structurally Modified Alternanthera Mosaic Plant Virus as a Delivery System for Sarcoma Cells

The virions of plant viruses and their structurally modified particles (SP) represent valuable platforms for recombinant vaccine epitopes and antitumor agents. The possibility of modifying their surface with biological compounds makes them a tool for developing medical biotechnology applications. Here, we applied a new type of SP derived from virions and virus-like particles (VLP) of Alternanthera mosaic virus (AltMV) and well-studied SP from Tobacco mosaic virus (TMV). We have tested the ability of SP from AltMV (AltMV SPV) and TMV virions also as AltMV VLP to bind to and penetrate Ewing sarcoma cells. The adsorption properties of AltMV SPV and TMV SP are greater than those of the SP from AltMV VLP. Compared to normal cells, AltMV SPV adsorbed more effectively on patient-derived sarcoma cells, whereas TMV SP were more effective on the established sarcoma cells. The AltMV SPV and TMV SP were captured by all sarcoma cell lines. In the established Ewing sarcoma cell line, the effectiveness of AltMV SPV penetration was greater than that of TMV SP. The usage of structurally modified plant virus particles as a platform for drugs and delivery systems has significant potential in the development of anticancer agents.

Development of novel species-specific and genus-specific primers for the detection of Babaco Mosaic Virus (BabMV).

Babaco is a hybrid cultivar native to the Andean region of Ecuador and Colombia, commercially attractive for its fruit. Babaco production in Ecuador faces losses from plant pathogens like babaco mosaic virus (BabMV), an RNA virus that causes chlorosis, leaf mottling, and deformation. Phylogenetic studies link BabMV to papaya mosaic virus (PapMV), alternanthera mosaic virus, and senna mosaic virus. To address this threat, we developed novel species-specific primers to detect BabMV targeting a 165bp region of the coat protein (CP). Genus-specific primers were designed to validate the species-specific primers and attest their ability to discriminate between BabMV and its closest relatives. These primers targeted a 175bp fragment of the CP region. The most effective sets of primers were chosen for reverse transcription polymerase chain reaction (RT-PCR) and SYBR® Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) in symptomatic and asymptomatic babaco plants. Among 28 plants tested, 25 were positive and 3 were negative for BabMV using species-specific and genus-specific primers in RT-PCR and RT-qPCR, while the PapMV positive control was detected with the genus-specific primers and was negative for the species-specific primers. These primers represent a valuable molecular tool for detecting BabMV, potentially enhancing crop management.

Survey of Viruses Infecting Basella alba in Hawaii.

Malabar spinach plants (Basella alba, Basellaceae) with leaves exhibiting symptoms of mosaic, rugosity, and malformation were found in a community garden on Oahu, HI in 2018. Preliminary studies using enzyme-linked immunosorbent assay and reverse-transcription (RT)-PCR identified Basella rugose mosaic virus (BaRMV) in symptomatic plants. However, nucleotide sequence analysis of RT-PCR amplicons indicated that additional potyviruses were also present in the symptomatic Malabar spinach. High-throughput sequencing (HTS) analysis was conducted on ribosomal RNA-depleted composite RNA samples of potyvirus-positive plants from three locations. Assembled contigs shared sequences similar to BaRMV, chilli veinal mottle virus (ChiVMV), Alternanthera mosaic virus (AltMV), Basella alba endornavirus (BaEV), broad bean wilt virus 2 (BBWV2), and Iresine viroid 1. Virus- and viroid-specific primers were designed based on HTS sequencing results and used in RT-PCR and Sanger sequencing to confirm the presence of these viruses and the viroid. We tested 63 additional samples from six community gardens for a survey of viruses in Malabar spinach and found that 21 of them were positive for BaRMV, 57 for ChiVMV, 21 for AltMV, 19 for BaEV, and 14 for BBWV2. This is the first characterization of the virome from B. alba.

Influence of the structure of the coat protein N-terminal segment in potato virus X and alternanthera mosaic virus on the structure and physico-chemical properties of virions

The amino acid sequences of coat proteins (CPs) of potexviruses such as potato virus X (PVX) and alternanthera mosaic virus (AltMV) share about 40% sequence identity. However, the N-terminal CP domains of these virions differ both in length (the N-terminal CP domain of PVX is longer by 28 residues, ∆N = 28), and in amino acid sequence. In this work, we determined the effect of the N-terminal CP domain on the structure and physicochemical properties of the entire PVX and AltMV virions. It was shown that the melting point of PVX samples is 10-12°C higher than that of AltMV preparations; the circular dichroism spectra of these viruses also differ significantly. Spatial alignment of the existing high-resolution potexvirus CP structures showed that the RMSD value between Cα-atoms was the largest for the N-terminal domains of the two compared models. From computer simulations the ∆N-terminal CP domain of PVX is completely disordered. According to synchrotron small-angle X-ray scattering (SAXS) data, the structure of CP of PVX and AltMV virions differs, in particular, CP PVX has a larger size of crystallinity regions and, therefore, is more ordered. Using SAXS, virion diameters and helix parameters in solution are calculated. The influence of the conformation and localization of the N-terminal domain of PVX CP relative to the surface of the virion on its structure was revealed. Presumably, the increased thermal stability of PVX virions compared to AltMV is provided by the elongated N-terminal domains (ΔN = 28), which ensures additional contact between the adjacent CP subunits in the PVX virion.

Effect of the Coat Protein N-Terminal Domain Structure on the Structure and Physicochemical Properties of Virions of Potato Virus X and Alternanthera Mosaic Virus

The amino acid sequences of the coat proteins (CPs) of the potexviruses potato virus X (PVX) and alternanthera mosaic virus (AltMV) share ~40% identity. The N-terminal domains of these proteins differ in the amino acid sequence and the presence of the N-terminal fragment of 28 residues (ΔN peptide) in the PVX CP. Here, we determined the effect of the N-terminal domain on the structure and physicochemical properties of PVX and AltMV virions. The circular dichroism spectra of these viruses differed significantly, and the melting point of PVX virions was 10-12°C higher than that of AltMV virions. Alignment of the existing high-resolution 3D structures of the potexviral CPs showed that the RMSD value between the Cα-atoms was the largest for the N-terminal domains of the two compared models. Based on the computer modeling, the ΔN peptide of the PVX CP is fully disordered. According to the synchrotron small-angle X-ray scattering (SAXS) data, the structure of CPs from the PVX and AltMV virions differ; in particular, the PVX CP has a larger portion of crystalline regions and, therefore, is more ordered. Based on the SAXS data, the diameters of the PVX and AltMV virions and helix parameters in solution were calculated. The influence of the conformation of the PVX CP N-terminal domain and its position relative to the virion surface on the virion structure was investigated. Presumably, an increased thermal stability of PVX virions vs. AltMV is provided by the extended N-terminal domain (ΔN peptide, 28 amino acid residues), which forms additional contacts between the adjacent CP subunits in the PVX virion.

Stimulated Raman Scattering in Aqueous Alternanthera Mosaic Virus and Potato Virus X Suspensions in the Frequency Range of 20–60 GHz

Stimulated Raman Scattering in Aqueous Alternanthera Mosaic Virus and Potato Virus X Suspensions in the Frequency Range of 20–60 GHz

Stimulated Raman Scattering in Suspensions of Alternanthera Mosaic Virus and Potato Virus X in the Frequency Range of 1–15 GHz

Stimulated Raman Scattering in Suspensions of Alternanthera Mosaic Virus and Potato Virus X in the Frequency Range of 1–15 GHz

First Report of Bean common mosaic virus Infecting Heavenly Bamboo (Nandina domestica) in China.

Heavenly bamboo (Nandina domestica) is an evergreen ornamental plant with worldwide distribution. In May 2018, seven out of twenty N. domestica plants showing virus-like symptoms, such as yellow mosaic and curling, were observed in Lin'an, Zhejiang province. To determine the causal agent, a small RNA library was constructed using the Small RNA v1.5 Sample Prep Kit (Illumina, San Diego, USA) with total RNA extracted from leaves of a symptomatic plant. The library was sequenced by the Solexa platform at BGI Genomics (Shenzhen, China). A total number of 21,071,675 high-quality reads of 17-28 nucleotides (nt) in length remained after trimming adapter sequences and quality control. Reads were assembled using Velvet 0.7.31 and Oases 0.2.07 with the k-mer value of 17 (Schulz et al. 2012). BlastN and BlastX search against the GenBank viral nonredundant sequence databases revealed fifty-six contigs homologous to bean common mosaic virus (BCMV; genus Potyvirus; family Potyviridae). No contig homologous to the genomic sequence of other plant-infecting viruses was identified. These contigs were further assembled into a 9,315-nt fragment by SeqMan Pro 7.1.0 in Lasergene package (DNASTAR, Madison, WI), which covered 92.68% of the genome of BCMV strain CT (BCMV-CT; GenBank accession no. KM076650). The genome of this BCMV isolate (BCMV-NTZ1) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using primers designed based on assembled contigs with the Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Beijing, China) and the FirstChoice® RLM-RACE Kit (Invitrogen, Carlsbad, USA), respectively. Amplicons were cloned and Sanger sequenced with three independent clones per amplicon. The genome is 10,052 nt in length excluding the poly-A tail (Genbank accession no. MZ670770) and shared the highest nt sequence identities with BCMV-CT (88.46%). The putative polyprotein shared 93.36% amino acid (aa) sequence identity with that of BCMV-CT. BCMV-NTZ1 also clustered with BCMV-CT in phylogenetic trees based on BCMV full genomes and aa sequences of coat protein. Five-leaf-stage seedlings of Nicotiana tabacum, N. benthamiana, Glycine max(Linn.) Merr., and Capsicum frutescens were mechanically inoculated with sap of BCMV-infected N. domestica leaves at fifteen plants per species. Seedlings of G. max developed virus-like (mosaic and leaf deformity) symptoms (7/15) at 15 days post-inoculation, while other plants remained symptomless throughout the experiment. Subsequent RT-PCR on all the plants using primers 27F1/14Rter and sequencing confirmed the presence and absence of BCMV-NTZ1 in all symptomatic G. max seedlings and other asymptomatic indicator plants, respectively. Subsequent RT-PCR survey further confirmed the association of BCMV with symptomatic heavenly bamboo samples but not asymptomatic plants (7/20). To the best of our knowledge, this is the first report of BCMV naturally infecting heavenly bamboo in China. N. domestica is susceptible to many viruses, e.g., cucumber mosaic virus, plantago asiatica mosaic virus, nandina stem pitting virus, apple stem grooving virus, and alternanthera mosaic virus (Barnett et al. 1973; Ahmed et al. 1983; Hughes et al. 2002, 2005; Tang et al. 2010; Wei et al. 2015). Our results indicate that N. domestica can also serve as an overwinter reservoir for BCMV and special attention should be paid to the damage it may cause.

Thermal remodelling of Alternanthera mosaic virus virions and virus-like particles into protein spherical particles.

The present work addresses the thermal remodelling of flexible plant viruses with a helical structure and virus-like particles (VLPs). Here, for the first time, the possibility of filamentous Alternanthera mosaic virus (AltMV) virions’ thermal transition into structurally modified spherical particles (SP) has been demonstrated. The work has established differences in formation conditions of SP from virions (SPV) and VLPs (SPVLP) that are in accordance with structural data (on AltMV virions and VLPs). SP originate from AltMV virions through an intermediate stage. However, the same intermediate stage was not detected during AltMV VLPs’ structural remodelling. According to the biochemical analysis, AltMV SPV consist of protein and do not include RNA. The structural characterisation of AltMV SPV/VLP by circular dichroism, intrinsic fluorescence spectroscopy and thioflavin T fluorescence assay has been performed. AltMV SPV/VLP adsorption properties and the availability of chemically reactive surface amino acids have been analysed. The revealed characteristics of AltMV SPV/VLP indicate that they could be applied as protein platforms for target molecules presentation and for the design of functionally active complexes.

A Replicating Viral Vector Greatly Enhances Accumulation of Helical Virus-Like Particles in Plants.

The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ-HT). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs.

NOVEL APPROACH FOR DESIGNING ROTAVIRUS VACCINE CANDIDATE BASED ON TWO PLANT VIRUSES

Rotavirus А (genus Rotavirus, family Reoviridae) is still the main cause of viral gastroenteritis in children under 5 years. Existing attenuated vaccines have serious disadvantages, including the risk of potential reversion to pathogenic form, and side effects, the most dangerous of which is intussusception. Moreover, they occurred to be less effective in developing countries, where the most rotavirus-associated deaths are recorded. The development of an effective recombinant rotavirus A vaccine is an actual assignment; herewith the selection of effective and safe adjuvant is the key point for that. Plant viruses are very promising for innovative vaccine designing; they possess high immunostimulating properties, safe for humans and mammals and can serve as a carrier for pathogens’ epitopes. Here we suggest an approach for rotavirus A vaccine development that involves two plant viruses: Alternanthera mosaic virus (AltMV) and Tobacco mosaic virus (TMV) as simultaneously epitope carriers and adjuvants. Spherical particles (SPs) generating by the heating of tobacco mosaic virus were used as an adjuvant and platform for presentation of obtained in our previous study chimeric recombinant protein ER6, which is an AltMV coat protein (CP) fused with the epitope RV14 (RLSFQLMRPPNMTP) of rotavirus A antigen VP6. Epitope RV14 are able to induce protective immune response and is conservative for the majority of rotavirus A strains therefore its usage gives hope to the successful overcoming of one of the main difficulties in rotavirus A vaccine development: wide serological diversity. In present work, effective adsorption of ER6 on the SPs surface leading to the SPs-ER6 complex formation without loss of ER6 antigenic specificity was demonstrated. Two immune antisera with specificity to RV14 epitope within ER6 were obtained. The first serum was obtained via anti-ER6 sera depletion with AltMV CP and recombinant AltMV CP (AltMV rCP), which was expressed in Escherichia coli but did not contain RV14 sequence. The second serum was obtained by a direct immunization with synthetic peptide RLSFQLMRPPNMTP. These sera were utilized for studying RV14 within SPs-ER6 complexes. By means of immunofluorescent microscopy, SPs-ER6 complexes were demonstrated to interact with both depleted serum and anti-RV14 serum. Therefore, rotavirus epitope was confirmed to keep its ability to interact with antibodies within obtained complexes. Considering unique adjuvant properties of spherical particles and characteristics of selected epitope obtained SPs-ER6 complexes can be thought as a promising component for recombinant rotavirus A vaccine. Moreover, it can be hoped that the suggested in present work approach, involving the usage of TMV SPs as a platform and adjuvant for chimeric AltMV CP, containing pathogen’s epitope, will be useful not only for rotavirus A vaccine development but for designing of vaccines against other pathogens of humans or farm animals.

НОВЫЙ ПОДХОД К РАЗРАБОТКЕ КАНДИДАТНОЙ ВАКЦИНЫ ПРОТИВ РОТАВИРУСА НА ОСНОВЕ ДВУХ ВИРУСОВ РАСТЕНИЙ

Rotavirus А (genus Rotavirus, family Reoviridae) is still the main cause of viral gastroenteritis in children under 5 years. Existing attenuated vaccines have serious disadvantages, including the risk of potential reversion to pathogenic form, and side effects, the most dangerous of which is intussusception. Moreover, they occurred to be less effective in developing countries, where the most rotavirus-associated deaths are recorded. The development of an effective recombinant rotavirus A vaccine is an actual assignment; herewith the selection of effective and safe adjuvant is the key point for that. Plant viruses are very promising for innovative vaccine designing; they possess high immunostimulating properties, safe for humans and mammals and can serve as a carrier for pathogens’ epitopes. Here we suggest an approach for rotavirus A vaccine development that involves two plant viruses: Alternanthera mosaic virus (AltMV) and Tobacco mosaic virus (TMV) as simultaneously epitope carriers and adjuvants. Spherical particles (SPs) generating by the heating of tobacco mosaic virus were used as an adjuvant and platform for presentation of obtained in our previous study chimeric recombinant protein ER6, which is an AltMV coat protein (CP) fused with the epitope RV14 (RLSFQLMRPPNMTP) of rotavirus A antigen VP6. Epitope RV14 are able to induce protective immune response and is conservative for the majority of rotavirus A strains therefore its usage gives hope to the successful overcoming of one of the main difficulties in rotavirus A vaccine development: wide serological diversity. In present work, effective adsorption of ER6 on the SPs surface leading to the SPs-ER6 complex formation without loss of ER6 antigenic specificity was demonstrated. Two immune antisera with specificity to RV14 epitope within ER6 were obtained. The first serum was obtained via anti-ER6 sera depletion with AltMV CP and recombinant AltMV CP (AltMV rCP), which was expressed in Escherichia coli but did not contain RV14 sequence. The second serum was obtained by a direct immunization with synthetic peptide RLSFQLMRPPNMTP. These sera were utilized for studying RV14 within SPs-ER6 complexes. By means of immunofluorescent microscopy, SPs-ER6 complexes were demonstrated to interact with both depleted serum and anti-RV14 serum. Therefore, rotavirus epitope was confirmed to keep its ability to interact with antibodies within obtained complexes. Considering unique adjuvant properties of spherical particles and characteristics of selected epitope obtained SPs-ER6 complexes can be thought as a promising component for recombinant rotavirus A vaccine. Moreover, it can be hoped that the suggested in present work approach, involving the usage of TMV SPs as a platform and adjuvant for chimeric AltMV CP, containing pathogen’s epitope, will be useful not only for rotavirus A vaccine development but for designing of vaccines against other pathogens of humans or farm animals.

Suppression of Arabidopsis Mediator Subunit-Encoding MED18 Confers Broad Resistance Against DNA and RNA Viruses While MED25 Is Required for Virus Defense.

Mediator subunits play key roles in numerous physiological pathways and developmental processes in plants. Arabidopsis Mediator subunits, MED18 and MED25, have previously been shown to modulate disease resistance against fungal and bacterial pathogens through their role in jasmonic acid (JA) signaling. In this study, Arabidopsis mutant plants of the two Mediator subunits, med18 and med25, were tested against three ssRNA viruses and one dsDNA virus belonging to four different families: Turnip mosaic virus (TuMV), Cauliflower mosaic virus (CaMV), Alternanthera mosaic virus (AltMV), and Cucumber mosaic virus (CMV). Although both subunits are utilized in JA signaling, they occupy different positions (Head and Tail domain, respectively) in the Mediator complex and their absence affected virus infection differently. Arabidopsis med18 plants displayed increased resistance to RNA viral infection and a trend against the DNA virus, while med25 mutants displayed increased susceptibility to all viruses tested at 2 and 14 days post inoculations. Defense marker gene expression profiling of mock- and virus-inoculated plants showed that med18 and med25 mutants exhibited an upregulated SA pathway upon virus infection at 2 dpi for all viruses tested. JA signaling was also suppressed in med18 plants after virus infection, independent of which virus infected the plants. The upregulation of SA signaling and suppression of JA signaling in med18 may have led to more targeted oxidative burst and programmed cell death to control viruses. However, the susceptibility exhibited by med25 mutants suggests that other factors, such as a weakened RNAi pathway, might play a role in the observed susceptibility. We conclude that MED18 and MED25 have clear and opposite effects on accumulation of plant viruses. MED18 is required for normal virus infection, while MED25 is important for defense against virus infection. Results from this study provide a better understanding of the role of Mediator subunits during plant-virus interactions, viral disease progression and strategies to develop virus resistant plants.

A Recombinant Rotavirus Antigen Based on the Coat Protein of Alternanthera Mosaic Virus.

—Thanks to their strong immunostimulating properties and safety for humans, plant viruses represent an appropriate basis for the design of novel vaccines. The coat protein of Alternanthera mosaic virus can form virus-like particles that are stable under physiological conditions and have adjuvant properties. This work presents a recombinant human rotavirus A antigen based on the epitope of rotavirus structural protein VP6, using Alternanthera mosaic virus coat protein as a carrier. An expression vector containing the gene of Alternanthera mosaic virus (MU strain) coat protein fused to the epitope of rotavirus protein VP6 was designed. Immunoblot analysis showed that the chimeric protein was effectively recognized by commercial polyclonal antibodies to rotavirus and therefore is a suitable candidate for development of a vaccine prototype. Interaction of the chimeric recombinant protein with the native coat protein of Alternanthera mosaic virus and its RNA resulted in the formation of ribonucleoprotein complexes that were recognized by anti-rotavirus antibodies.

Рекомбинантный антиген ротавируса, полученный на основе белка оболочки вируса мозаики альтернантеры

Thanks to their strong immunostimulating properties and safety for humans, plant viruses represent an appropriate basis for the design of novel vaccines. The coat protein of Alternanthera mosaic virus can form virus-like particles that are stable under physiological conditions and have adjuvant properties. This work presents a recombinant human rotavirus A antigen based on the epitope of rotavirus structural protein VP6, using Alternanthera mosaic virus coat protein as a carrier. An expression vector containing the gene of Alternanthera mosaic virus (MU strain) coat protein fused to the epitope of rotavirus protein VP6 was designed. Immunoblot analysis showed that the chimeric protein was effectively recognized by commercial polyclonal antibodies to rotavirus and therefore is a suitable candidate for development of a vaccine prototype. Interaction of the chimeric recombinant protein with the native coat protein of Alternanthera mosaic virus and its RNA resulted in the formation of ribonucleoprotein complexes that were recognized by anti-rotavirus antibodies.

First Report of Lagenaria mild mosaic virus Infecting Bottle Gourd in India

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.

First Report of Alternanthera Mosaic Virus Infecting Epiphyllum spp.

Epiphyllum spp. (orchid cactus) is a group of epiphytic plants in the cactus family grown as flowering ornamentals. Symptoms of virus infections are frequently observed in them (Chessin 2002). In 2015, 13 Epiphyllum hybrids (seven with chlorotic ring spots, mottle, or mosaic symptoms and six without any symptoms) were tested by RT-PCR/PCR using specific or group primers for cactus mild mottle virus, cactus virus X, impatiens necrotic spot virus, saguaro cactus virus, zygocactus virus X, badnavirus, and potyvirus, respectively. None of these viruses were detected from a hybrid ‘Dragon Heart’ showing irregular chlorotic patches on its stems. This plant was tested in subsequent high-throughput sequencing analysis for identification of viruses and viroids. Total RNAs were extracted from stem tissue using an RNeasy Plant Mini Kit (Qiagen, Germantown, MD) and subjected to Illumina NextSeq 500 sequencing (SeqMatic, Fremont, CA). Analysis of the RNA reads of 75 nucleotides (nt) revealed the presence of alternanthera mosaic virus (AltMV, 213,038 reads, 2,445x) and a new cavemovirus in the Dragon Heart plant. Infection of AltMV-DH was confirmed by RT-PCR using AltMV-detF6c (5′-TCACYCAGGAACAGATGRACG-3′) and AltMVR6 (5′-TAGGTCTACAGAAGCGGAAGC-3′). To obtain the genomic sequence of this AltMV isolate, RT-PCR was conducted using six overlapping primer pairs designed according to the alignment of the AltMV type species (GenBank no. NC_007731) and AltMV contig obtained in this study. The RT-PCR amplicons were cloned into a pGEM-T easy vector (Promega, Fitchburg, WI) and sequenced (MCLAB, South San Francisco, CA). The complete genomic sequence of 6,633 nt (GenBank no. MH423501) was assembled from six overlapped amplicon sequences. BLAST search showed that this isolate had the highest nucleotide sequence identity of 94 to 95% with four AltMV isolates available in the GenBank databases. Stem tips from the Dragon Heart plant were grafted onto three plants of hybrid ‘King’s Ransom’, a hybrid that tested negative for the nine viruses included in this study. Three grafted plants showed mottling symptoms 6 months after inoculation. AltMV was detected in new growth of all three grafted plants, indicating the virus was graft transmissible. A total of 30 additional Epiphyllum hybrids were tested by RT-PCR, and AltMV was detected from four symptomatic hybrids (‘Clarabelle’, ‘Mark Twin’, ‘Professor Ebert’, and ‘Over the Top’) and two asymptomatic hybrids (‘Cream and Gold’ and ‘Montrose’). AltMV is a member of the genus Potexvirus in the family Alphaflexiviridae. The virus was first reported from Alternanthera pungens in Australia (Geering and Thomas 1999) and has been found from plants of at least eight families in the United States, Europe, and Brazil (Hammond and Reinsel 2015). To our knowledge, this is the first report of AltMV in orchid cactus. It is not clear if AltMV alone causes symptoms in Epiphyllum spp., because two of the positive hybrids were asymptomatic, but the infected cactus may serve as a reservoir from which AltMV may spread to other hosts. We are currently characterizing the new cavemovirus detected in the hybrid Dragon Heart.

Antimicrobial activity of bacteriophage derived triple fusion protein against Staphylococcus aureus.

The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases is a promising approach to combat bacterial infections. In our study, we constructed a 2 kb-triple-acting fusion gene (TF) encoding the N-terminal amidase-5 domain of streptococcal LambdaSA2 prophage endolysin (D-glutamine-L-lysin endopeptidase), a mid-protein amidase-2 domain derived from the staphylococcal phage 2638A endolysin (N-acetylmuramoyl-L-alanine amidase) and the mature version (246 residues) of the Staphylococcus simulans Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus. The TF gene was expressed in Nicotiana benthamiana plants using the non-replicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT and the replicating Alternanthera mosaic virus (AltMV)-based pGD5TGB1L8823-MCS-CP3 vector, and in Escherichia coli using pET expression vectors pET26b+ and pET28a+. The resulting poor expression of this fusion protein in plants prompted the construction of a TF gene codon-optimized for expression in tobacco plants, resulting in an improved codon adaptation index (CAI) from 0.79 (TF gene) to 0.93 (TFnt gene). Incorporation of the TFnt gene into the pEAQ-HT vector, followed by transient expression in N. benthamiana, led to accumulation of TFnt to an approximate level of 0.12 mg/g of fresh leaf weight. Antimicrobial activity of purified plant- and bacterial-produced TFnt proteins was assessed against two strains of Gram-positive Staphylococcus aureus 305 and Newman. The results showed that plant-produced TFnt protein was preferentially active against S. aureus 305, showing 14% of growth inhibition, while the bacterial-produced TFnt revealed significant antimicrobial activity against both strains, showing 68 (IC50 25 µg/ml) and 60% (IC50 71 µg/ml) growth inhibition against S. aureus 305 and Newman, respectively. Although the combination of codon optimization and transient expression using the non-replicating pEAQ-HT expression vector facilitated production of the TFnt protein in plants, the most functionally active antimicrobial protein was obtained using the prokaryotic expression system.

Alternanthera mosaic potexvirus: Several Features, Properties, and Application.

Alternanthera mosaic virus (AltMV) is a typical member of the Potexvirus genus in its morphology and genome structure; still it exhibits a number of unique features. They allow this virus to be considered a promising object for biotechnology. Virions and virus-like particles (VLPs) of AltMV are stable in a wide range of conditions, including sera of laboratory animals. AltMV VLPs can assemble at various pH and ionic strengths. Furthermore, AltMV virions and VLPs demonstrate high immunogenicity, enhancing the immune response to the target antigen thus offering the possibility of being used as potential adjuvants. Recently, for the first time for plant viruses, we showed the structural difference between morphologically similar viral and virus-like particles on AltMV virions and VLPs. In this review, we discuss the features of AltMV virions, AltMV VLP assembly, and their structure and properties, as well as the characteristics of AltMV isolates, host plants, infection symptoms, AltMV isolation and purification, genome structure, viral proteins, and AltMV-based vectors.

Translational Cross-Activation of the Encapsidated RNA of Potexviruses

We had shown the genomic RNA of potexviruses potato virus X and the alternanthera mosaic virus to be inaccessible in vitro to ribosomes while in intact virion form, but the RNAs can be translationally activated following the binding of movement protein 1 (MP1) to virus particles. Here, we present the results of the follow-up study targeting two more potexvirus species - the Narcissus mosaic virus and the Potato aucuba mosaic virus. We found encapsidated potexviral RNA to share common translational features in vitro and the MP1 to be potent over homological virions of its own species and over heterological virions of other species, as well exhibiting selective specificity. Reciprocal cross-activation is observed among viral species phylogenetically either close or distant. There is direct evidence that MP1 binding to the end of the virion is necessary, but not sufficient, for translational activation of encapsidated RNA.