Full-length Clone Research Articles

Recombination between African and Asian lineages of Zika virus in vitro and its consequences for viral phenotype

Recombination is a process of extensive genetic exchange that is known to contribute to virus evolution and has been frequently observed in positive-sense RNA viruses. Zika virus (ZIKV) is an emerging arbovirus of the family Flaviviridae with two distinct lineages – African and Asian. While some phylogenetic evidence suggests that recombination in the envelope-encoding region of the ZIKV genome has occurred during evolution, there has been no experimental evidence for ZIKV recombination to date. We conducted co-infections of mammalian and insect cells, using the prototype African ZIKV strain (MR766) and an Asian isolate from the 2015-16 ZIKV outbreak in Brazil (BeH819015), and used a recombinant-specific PCR assay to detect recombinant sequences from total cell RNA extracts. In brief, a 564bp fragment spanning the boundary between the structural and the non-structural genes of the viral genome was amplified using a primer pair consisting of an Asian-specific and an African-specific primer. A total of 24 individual sequences were screened. All were in-frame recombinants and they formed 10 unique junctions. Several of the detected recombinant sequences were chosen for construction of full-length infectious clones to test the viability and phenotype of the recombinant viruses. This study represents the first isolation of recombinant ZIKV sequences from co-infected cultured cells and demonstrates the capacity of ZIKV to recombine in an experimental system. Further investigation is required to better understand the evolutionary potential of this mechanism and its putative role in the emergence of ZIKV.

Construction and characterization of an infectious clone of Soybean mosaic virus isolate from Pinellia ternata

Soybean mosaic virus (SMV), one of the major viral diseases of Pinellia ternata (Thunb.) Breit., has had a serious impact on its yield and quality. The construction of viral infectious clones is a powerful tool for reverse genetics research on viral gene function and interaction between virus and host. To clarify the molecular mechanism of SMV infection in Pinellia ternata, it is particularly important to construct the SMV full-length cDNA infectious clone. Therefore, the infectious clone of Soybean mosaic virus Shanxi Pinellia ternata isolate (SMV-SXBX) was constructed in this study by Gibson in vitro recombination system, and the healthy Pinellia ternata leaves were inoculated by Agrobacterium infiltration, further through mechanical passage and RT-PCR, confirming that the 3' end of the SMV-SXBX infectious clone had a stable infectivity when it contained 56-nt of poly(A) tail. This method is not only convenient and efficient, but also avoids the instability of SMV infectious clones in Escherichia coli. The construction of SMV full-length infectious cDNA clones laid the foundation for further study on the molecular mechanism of SMV replication and pathogenesis.

MG-MLST: Characterizing the Microbiome at the Strain Level in Metagenomic Data.

The microbiome plays an important role in human physiology. The composition of the human microbiome has been described at the phylum, class, genus, and species levels, however, it is largely unknown at the strain level. The importance of strain-level differences in microbial communities has been increasingly recognized in understanding disease associations. Current methods for identifying strain populations often require deep metagenomic sequencing and a comprehensive set of reference genomes. In this study, we developed a method, metagenomic multi-locus sequence typing (MG-MLST), to determine strain-level composition in a microbial community by combining high-throughput sequencing with multi-locus sequence typing (MLST). We used a commensal bacterium, Propionibacterium acnes, as an example to test the ability of MG-MLST in identifying the strain composition. Using simulated communities, MG-MLST accurately predicted the strain populations in all samples. We further validated the method using MLST gene amplicon libraries and metagenomic shotgun sequencing data of clinical skin samples. MG-MLST yielded consistent results of the strain composition to those obtained from nearly full-length 16S rRNA clone libraries and metagenomic shotgun sequencing analysis. When comparing strain-level differences between acne and healthy skin microbiomes, we demonstrated that strains of RT2/6 were highly associated with healthy skin, consistent with previous findings. In summary, MG-MLST provides a quantitative analysis of the strain populations in the microbiome with diversity and richness. It can be applied to microbiome studies to reveal strain-level differences between groups, which are critical in many microorganism-related diseases.

Microbial ecology and biogeochemistry of hypersaline sediments in Orca Basin

In deep ocean hypersaline basins, the combination of high salinity, unusual ionic composition and anoxic conditions represents significant challenges for microbial life. We used geochemical porewater characterization and DNA sequencing based taxonomic surveys to enable environmental and microbial characterization of anoxic hypersaline sediments and brines in the Orca Basin, the largest brine basin in the Gulf of Mexico. Full-length bacterial 16S rRNA gene clone libraries from hypersaline sediments and the overlying brine were dominated by the uncultured halophilic KB1 lineage, Deltaproteobacteria related to cultured sulfate-reducing halophilic genera, and specific lineages of heterotrophic Bacteroidetes. Archaeal clones were dominated by members of the halophilic methanogen genus Methanohalophilus, and the ammonia-oxidizing Marine Group I (MG-I) within the Thaumarchaeota. Illumina sequencing revealed higher phylum- and subphylum-level complexity, especially in lower-salinity sediments from the Orca Basin slope. Illumina and clone library surveys consistently detected MG-I Thaumarchaeota and halotolerant Deltaproteobacteria in the hypersaline anoxic sediments, but relative abundances of the KB1 lineage differed between the two sequencing methods. The stable isotopic composition of dissolved inorganic carbon and methane in porewater, and sulfate concentrations decreasing downcore indicated methanogenesis and sulfate reduction in the anoxic sediments. While anaerobic microbial processes likely occur at low rates near their maximal salinity thresholds in Orca Basin, long-term accumulation of reaction products leads to high methane concentrations and reducing conditions within the Orca Basin brine and sediments.

The cloned SPI-1 type 3 secretion system can be functionally expressed outside Salmonella backgrounds

Due to its potential for use in bacterial engineering applications, we previously cloned the SPI-1 type 3 secretion system (T3SS) genes from the genome of Salmonella enterica serovar Typhimurium strain LT2. We have documented that this clone, while functionally expressed in S. Typhimurium strains, displays a severe expression defect in other Gram negative backgrounds including Escherichia coli. To address this issue, we compared SPI-1 DNA sequence across different backgrounds, fully sequenced the original SPI-1 clone, and cloned SPI-1 from other S. Typhimurium strains. In this process, we were able to successfully obtain SPI-1 clones that are functionally expressed in E. coli indicating the first such result for a full-length SP-1 T3SS clone. We discovered that the original cloning technique using a DNA homology-based capture method was the root of the expression defect and that the FRT-Capture technique is preferable over the homology-based method. This result paves the way for future studies and applications using cloned SPI-1 and other T3SS in non-Salmonella bacterial backgrounds.

HIV-1 Envelope and MPER Antibody Structures in Lipid Assemblies

SummaryStructural and functional studies of HIV envelope glycoprotein (Env) as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstitute a full-length Env clone into a nanodisc, complex it with a membrane-proximal external region (MPER) targeting antibody 10E8, and structurally define the full quaternary epitope of 10E8 consisting of lipid, MPER, and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapt the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with a peptidisc scaffold.

Construction of recombinant infectious clones of HCoV-OC43 expressing green fluorescent protein and viral rescue

Objective To investigate the feasibility of using recombinant infectious clones of human coronavirus OC43 (HCoV-OC43) as a vector for the expression of exogenous genes and to analyze the insertion sites. Methods Based upon pBAC-OC43FL, a full-length cDNA infectious clone of HCoV-OC43, three recombinant expression plasmids (pBAC-OC43-GFPΔNS2, pBAC-OC43-GFPΔNS12.9 and pBAC-OC43-N-GFP) were respectively constructed by replacing NS2 and NS12.9 genes with the reporter gene encoding the green fluorescent protein (GFP) and inserting the reporter gene after the N gene by using the overlapping-PCR and in vitro ligation. Reverse genetics techniques were used for viral rescue. All of the rescued virus strains were characterized by immunofluorescence assay (IFA) and Western blot (WB) assay after transfecting BHK-21 cells with the recombinant viruses. Results Two recombinant viruses, OC43-GFPΔNS2 and OC43-GFPΔNS12.9, could be successfully rescued by transfection the BHK-21 cells with pBAC-OC43-GFPΔNS2 and pBAC-OC43-GFPΔNS12.9 plasmids. The expressed GFP was observed in BHK-21 cells transfected with pBAC-OC43-GFPΔNS2 or pBAC-OC43-GFPΔNS12.9 plasmids, but not in the cells transfected with the pBAC-OC43-N-GFP plasmid. An efficient and stable expression of GFP was observed in the pBAC-OC43-GFPΔNS2 plasmid-transfected cells. The 10th generation of OC43-GFPΔNS2 virus was obtained after repeated freezing and thawing. The expression of GFP and N protein were detected in cells infected with the OC43-GFPΔNS2 virus after 10 passages. Conclusion The NS2 gene of HCoV-OC43 could be used as a promising insertion site of the pBAC-OC43FL infectious clone for the expression of exogenous genes. This study might provide a platform for further researches on the replication of HCoV-OC43 and the development of human coronavirus-based vectors. Key words: Human coronavirus OC43; Infectious clone; Viral rescue; Green fluorescent protein; Viral vector

Massively parallel interrogation and mining of natively paired human TCRαβ repertoires.

T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here, we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach uses massively parallel microfluidics to generate libraries of natively paired, full-length TCRαβ clones, from millions of primary T cells, which are then expressed in Jurkat cells. The TCRαβ-Jurkat libraries enable repeated screening and panning for antigen-reactive TCRs using peptide:MHC binding and cellular activation. We captured >2.9 million natively paired TCRαβ clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral antigen–reactive TCRs. We also mined a tumor-infiltrating lymphocyte (TIL) sample from a melanoma patient and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.

Cloning, Characterization and Expression Analysis of the Phosphate Starvation Response Gene, ClPHR1, from Chinese Fir

The study on the function and sequence of PHR1 (Phosphate Starvation Response gene 1) gene, which plays a central role in plant phosphorus (Pi) signal regulatory network, is of great significance to further study response mechanisms to Pi deficiency. In this work, the previously selected Pi-efficient Chinese fir clone M32 was used as research material to obtain the full-length sequence of ClPHR1 transcription factors in Chinese fir by RACE (Rapid Amplification of cDNA Ends) full-length cloning technique, and the structure, function and subcellular localization of ClPHR1 gene encoding protein were analyzed. The temporal and spatial expression characteristics of ClPHR1 transcription factors in Chinese fir under low Pi stress were also analyzed, and the overexpression of ClPHR1 gene in transgenic Arabidopsis thaliana was obtained to verify the function of ClPHR1 gene under low Pi stress. The results showed that the length of the ClPHR1 gene obtained by rapid amplification of cDNA ends technique was 1954 bp, of which 1512 bp was an open reading frame. ClPHR1 was predicted to be an unstable hydrophilic protein with only one possible transmembrane domain. The ClPHR1 gene had a highly conserved MYB-CC domain, which is similar to the PHR1 gene of other plants. Phylogenetic tree analysis showed that the sequence had high homology with PHR1 genes in the Prunus species. The ClPHR1 was expressed in all organs of Chinese fir, with the highest expression in the roots, followed by the leaves with the lowest expression in stems. ClPHR1 expression in roots was reduced dramatically at the beginning of Pi stress treatment and followed by an increase at 7days; in leaves, it increased dramatically at the beginning of Pi starvation treatment and showed a decreasing trend after 3 days; in stems, the expression level of ClPHR1 increased after 7 days of Pi stress treatment. The transient expression vector was introduced into plant cells, and it was found that ClPHR1 was located in the nucleus and was a MYB-CC transcription factor expressed in the cell nucleus. The ClPHR1 overexpression vector was constructed, and then introduced into Arabidopsis thaliana by agrobacterium infection inflorescence method. The expressions of Pi transporter genes, AtPHT1;1, AtPHT1;2, AtPHT1;8 and AtPHT1;9, was significantly higher in the overexpressing strain than that in the wild type strain. The results suggest that the ClPHR1 transcription factor could regulate the regulation of downstream Pi transporter gene and increase Pi utilization efficiency of the Chinese fir under Pi stress.

Engineering viable foot-and-mouth disease viruses with increased acid stability facilitate the development of improved vaccines

Foot-and-mouth disease virus (FMDV), the most acid-unstable virus among picornaviruses, tends to disassemble into pentamers at pH values slightly below neutrality. However, the structural integrity of intact virion is one of the most important factors that influence the induction of a protective antibody response. Thus, improving the acid stability of FMDV is required for the efficacy of vaccine preparations. According to the previous studies, a single substitution or double amino acid substitutions (VP1 N17D, VP2 H145Y, VP2 D86H, VP3 H142D, VP3 H142G, and VP1 N17D + VP2 H145Y) in the capsid were introduced into the full-length infectious clone of type O FMDV vaccine strain O/HN/CHN/93 to develop seed FMDV with improved acid stability. After the transfection into BSR/T7 cells of constructed plasmids, substitution VP1 N17D or VP2 D86H resulted in viable and genetically stable FMDVs, respectively. However, substitution VP2 H145Y or VP1 N17D + VP2 H145Y showed reverse mutation and additional mutations, and substitution VP3 H141G or VP3 H141D prevented viral viability. We found that substitution VP1 N17D or VP2 D86H could confer increased acid resistance, alkali stability, and thermostability on FMDV O/HN/CHN/93, whereas substitution VP1 N17D was observed to lead to a decreased replication ability in BHK-21 cells and mildly impaired virulence in suckling mice. In contrast, substitution VP2 D86H had no negative effect on viral infectivity. These results indicated that the mutant rD86H carrying substitution VP2 D86H firstly reported by us could be more adequate for the development of inactivated FMD vaccines with enhanced acid stability.

Novel calreticulin-nanoparticle in combination with focused ultrasound induces immunogenic cell death in melanoma to enhance antitumor immunity.

Rationale: Some studies have shown that the local activation of immunogenic cell death (ICD) by upregulating calreticulin (CRT) expression in solid tumors can improve antitumor effects. Although a promising approach, a key current challenge in ICD tumor therapy is the absence of a clinically translatable method for reproducibly inducing the CRT expression. Herein, we report a novel calreticulin-nanoparticle (CRT-NP) that enhances ICD and synergizes with focused ultrasound (FUS) to achieve local and systemic antitumor effects.Methods: Full-length clone DNA of calreticulin was encapsulated in NPs made from DOTAP and cholesterol. Three CRT-NP intratumoral injections of 20 µg each were given 2 days apart, and FUS heating (42-45°C, ~15min) was applied sequentially 24h after each injection to induce ICD. To investigate ICD specific immune effect, the splenocytes of mice vaccinated with CRT-NP (± FUS) treated B16F10 cells were evaluated ex-vivo for TRP-2 antigen specific immunity. Additionally, the long-term protection was evaluated by re-challenging with the melanoma cells in the flank regions of tumor bearing mice.Results: CRT-NP plus FUS (CFUS) upregulated CRT expression, expanded the population of melanoma TRP-2 specific functional CD4+ and CD8+ T cells and tumor-suppressing M1 phenotype, and increased PD-1 and PD-L1 marker expression in the T cells. Therapeutically, CFUS suppressed B16 melanoma growth by >85% vs. that seen in untreated controls, and >~50% vs. CRT-NP or FUS alone, and prevented tumor growth in distal untreated sites.Conclusions: CRT-NP amplifies the FUS and ICD therapeutic outcomes against melanoma, suggesting that the proposed combinatorial methodology may be clinically translatable.

Long-Term Cocirculation of Two Strains of Pepino Mosaic Virus in Tomato Crops and Its Effect on Population Genetic Variability.

Mixed viral infections are common in plants, and the evolutionary dynamics of viral populations may differ depending on whether the infection is caused by single or multiple viral strains. However, comparative studies of single and mixed infections using viral populations in comparable agricultural and geographical locations are lacking. Here, we monitored the occurrence of pepino mosaic virus (PepMV) in tomato crops in two major tomato-producing areas in Murcia (southeastern Spain), supporting evidence showing that PepMV disease-affected plants had single infections of the Chilean 2 (CH2) strain in one area and the other area exhibited long-term (13 years) coexistence of the CH2 and European (EU) strains. We hypothesized that circulating strains of PepMV might be modulating the differentiation between them and shaping the evolutionary dynamics of PepMV populations. Our phylogenetic analysis of 106 CH2 isolates randomly selected from both areas showed a remarkable divergence between the CH2 isolates, with increased nucleotide variability in the geographical area where both strains cocirculate. Furthermore, the potential virus-virus interaction was studied further by constructing six full-length infectious CH2 clones from both areas, and assessing their viral fitness in the presence and absence of an EU-type isolate. All CH2 clones showed decreased fitness in mixed infections and although complete genome sequencing indicated a nucleotide divergence of those CH2 clones by area, the magnitude of the fitness response was irrespective of the CH2 origin. Overall, these results suggest that although agroecological cropping practices may be particularly important for explaining the evolutionary dynamics of PepMV in tomato crops, the cocirculation of both strains may have implications on the genetic variability of PepMV populations.

Phylogenetic classification of a group of self-replicating RNAs that are common in co-infections with poleroviruses

Tombusvirus-like associated RNAs (tlaRNAs) are positive-sense single-stranded RNAs found in plants co-infected with viruses of the genus Polerovirus. TlaRNAs depend upon capsid proteins supplied in trans by the co-infecting polerovirus for vector transmission and intra-host systemic movement. Here, the full-length genomes of five tlaRNAs were determined using a combination of RT-PCR and next-generation sequencing, and evidence is provided for an additional tlaRNA associated with potato leafroll virus. Phylogenetic analyses based on conserved domains of the RdRp placed tlaRNAs as a monophyletic clade clustering with members of the family Tombusviridae and comprising three different subclades. Full-length clones of tlaRNAs from two of three subclades were confirmed to replicate autonomously, and each produces a subgenomic RNA during infection.

Development and application of a full-length infectious clone of potato virus Y isolate belonging to SYR-I strain

Potato virus Y (PVY) causes huge damage to potato and tobacco production worldwide. The complete genome sequence of GZ, a PVY isolate (strain SYR-I) from Guizhou province, China, was cloned into the binary vector pCambia0390. Three introns were individually inserted into the P3 and CI ORFs to produce plasmid pCamPVY-GZ. The plasmid could infect plants of Nicotiana benthamiana, N. tabacum via agroinfiltration and plants of pepper and potato by mechanical inoculation. The green fluorescence protein gene of Aequoria victoriae was cloned into the encoding regions between nuclear inclusion body ‘b’ and coat protein genes in pCamPVY-GZ to produce pCamPVY-GZ-GFP, which could infect plants of N. benthamiana, N. tabacum, potato and tomato, and produce green fluorescence in the systemic leaves of inoculated plants. Mutations were introduced to pCamPVY-GZ to make the lysine (K) 391 and glutamic acid (E)410 of helper component-proteinase to arginine (R) and asparagic acid (E), respectively. Unlike wild type PVY-GZ, the mutant PVY-K391R/E410D could not induce veinal necrosis in N. tabacum plants. With an interval of 14 days, mutant PVY-K391R/E410D could protect N. tabacum plants from the infection of severe PVY strain. The results presented here provide a promising alternate for the prevention of diseases caused by PVY.

Frequent occurrence of Mungbean yellow mosaic India virus in tomato leaf curl disease affected tomato in Oman

Next generation sequencing (NGS) of DNAs amplified by rolling circle amplification from 6 tomato (Solanum lycopersicum) plants with leaf curl symptoms identified a number of monopartite begomoviruses, including Tomato yellow leaf curl virus (TYLCV), and a betasatellite (Tomato leaf curl betasatellite [ToLCB]). Both TYLCV and ToLCB have previously been identified infecting tomato in Oman. Surprisingly the NGS results also suggested the presence of the bipartite, legume-adapted begomovirus Mungbean yellow mosaic Indian virus (MYMIV). The presence of MYMIV was confirmed by cloning and Sanger sequencing from four of the six plants. A wider analysis by PCR showed MYMIV infection of tomato in Oman to be widespread. Inoculation of plants with full-length clones showed the host range of MYMIV not to extend to Nicotiana benthamiana or tomato. Inoculation to N. benthamiana showed TYLCV to be capable of maintaining MYMIV in both the presence and absence of the betasatellite. In tomato MYMIV was only maintained by TYLCV in the presence of the betasatellite and then only at low titre and efficiency. This is the first identification of TYLCV with ToLCB and the legume adapted bipartite begomovirus MYMIV co-infecting tomato. This finding has far reaching implications. TYLCV has spread around the World from its origins in the Mediterranean/Middle East, in some instances, in live tomato planting material. The results here may suggest that begomoviruses which do not commonly infect tomato, such as MYMIV, could be spread as a passenger of TYLCV in tomato.

Molecular and biological characterization of Chilli leaf curl virus and associated Tomato leaf curl betasatellite infecting tobacco in Oman

BackgroundIn Oman tobacco (Nicotiana tabacum; family Solanaceae) is a minor crop, which is produced only for local consumption. In 2015, tobacco plants exhibiting severe downward leaf curling, leaf thickening, vein swelling, yellowing and stunting were identified in fields of tobacco in Suhar Al-Batina region, Oman. These symptoms are suggestive of begomovirus (genus Begomovirus, family Geminiviridae) infection.MethodsCircular DNA molecules were amplified from total DNA extracted from tobacco plants by rolling circle amplification (RCA). Viral genomes were cloned from RCA products by restriction digestion and betasatellites were cloned by PCR amplification from RCA product, using universal primers. The sequences of full-length clones were obtained by Sanger sequencing and primer walking. Constructs for the infectivity of virus and betasatellite were produced and introduced into plants by Agrobacterium-mediated inoculation.ResultsThe full-length sequences of 3 begomovirus and 3 betasatellite clones, isolated from 3 plants, were obtained. Analysis of the full-length sequences determined showed the virus to be a variant of Chilli leaf curl virus (ChiLCV) and the betasatellite to be a variant of Tomato leaf curl betasatellite (ToLCB). Both the virus and the betasatellite isolated from tobacco show the greatest levels of sequence identity to isolates of ChiLCV and ToLCB identified in other hosts in Oman. Additionally clones of ChiLCV and ToLCB were shown, by Agrobacterium-mediated inoculation, to be infectious to 3 Nicotiana species, including N. tabacum. In N. benthamiana the betasatellite was shown to change the upward leaf rolling symptoms to a severe downward leaf curl, as is typical for many monopartite begomoviruses with betasatellites.ConclusionsThe leaf curl disease of tobacco in Oman was shown to be caused by ChiLCV and ToLCB. This is the first identification of ChiLCV with ToLCB infecting tobacco. The study shows that, despite the low diversity of begomoviruses and betasatellites in Oman, the extant viruses/betasatellites are able to fill the niches that present themselves.

Occurrence and variability of begomoviruses associated with bhendi yellow vein mosaic and okra enation leaf curl diseases in south-western India.

Bhendi yellow vein mosaic disease (BYVMD) and Okra enation leaf curl disease (OELCuD) are common diseases of okra/bhendi [Abelmoschus esculentus (L.) Moench] affecting both pod yield and quality in the Indian subcontinent. BYVMD is caused by the infection of a begomovirus and associated betasatellite. In this study, we have made an attempt to investigate the diversity of begomoviral and the satellite sequences in okra samples showing BYVMD and OELCuD, by using a rapid PCR-based approach on 46 samples collected from 23 locations of Southern and Western India. We have also analyzed nine RCA-generated full-length begomoviral clones, some generated from the above samples displaying BYVMD and some OELCuD. By the PCR approach, we find the presence of begomovirus okra enation leaf curl virus (OELCuV) in most samples, irrespective of the disease being displayed (BYVMD or OELCuD). The nine apparently full-length sequences also show high identities with OELCuV and show instances of both intra-specific as well as intra-strainal recombination. We have also analyzed the begomoviral sequences associated with BYVMD and OELCuD from publicly available nucleotide sequence databases and show much higher sequence diversity amongst BYVMV, as compared to OELCuV. This is the first study which comprehensively demonstrates the presence of OELCuV in okra samples showing BYVMD and those showing OELCuD.

Assessment of the Accuracy of High-Throughput Sequencing of the ITS1 Region of Neocallimastigomycota for Community Composition Analysis.

Anaerobic fungi (Neocallimastigomycota) are common inhabitants of the digestive tract of large mammalian herbivores, where they make an important contribution to plant biomass degradation. The internal transcribed spacer 1 (ITS1) region is currently the molecular marker of choice for anaerobic fungal community analysis, despite its known size polymorphism and heterogeneity. The aim of this study was to assess the accuracy of high-throughput sequencing of the ITS1 region of anaerobic fungi for community composition analysis. To this end, full-length ITS1 clone libraries from five pure cultures, representing the ITS1 region size range, were Sanger sequenced to generate a reference dataset. Barcoded amplicons of the same five pure cultures, and four different mock communities derived from them, were then sequenced using Illumina HiSeq. The resulting sequences were then assessed in relation to either the reference dataset (for the pure cultures) or the corresponding theoretical mock communities. Annotation of sequences obtained from individual pure cultures was not always consistent at the clade or genus level, irrespective of whether data from clone libraries or high-throughput sequencing were analyzed. The detection limit of the high-throughput sequencing method appeared to be influenced by factors other than the parameters used during data processing, as some taxa with theoretical values >0.6% were not detected in the mock communities. The high number of PCR cycles used was considered to be a potential explanation for this observation. Accuracy of two of the four mock communities was limited, and this was speculated to be due to preferential amplification of smaller sized ITS1 regions. If this is true, then this is predicted to be an issue with only six of the 32 named anaerobic fungal clades. Whilst high-throughput sequencing of the ITS1 region from anaerobic fungi can be used for environmental sample analysis, we conclude that the accuracy of the method is influenced by sample community composition. Furthermore, ambiguity in the annotation of sequences within pure cultures due to ITS1 heterogeneity reinforces the limitations of the ITS1 region for the taxonomic assignment of anaerobic fungi. In order to overcome these issues, there is a need to develop an alternative taxonomic marker for anaerobic fungi.

Full-Length Infectious Clones of Two New Isolates of Tomato Mosaic Virus Induce Distinct Symptoms Associated with Two Differential Amino Acid Residues in 128-kDa Protein.

In 2017, two new tomato mosaic virus (ToMV) isolates were collected from greenhouses in Buyeo, Chungcheongnam-do, South Korea. Full-length cDNAs of the new ToMV isolates were cloned into dual cauliflower mosaic virus 35S and T7 promoter-driven vectors, sequenced and their pathogenicities investigated. The nucleotide sequences of isolates GW1 (MH507165) and GW2 (MH507166) were 99% identical, resulting in only two amino acid differences in nonconserved region II and the helicase domain, Ile668Thr and Val834Ile. The two isolates were most closely related to a ToMV isolate from Taiwan (KJ207374). Isolate GW1 (Ile668, Val834) induced a systemic hypersensitive response in Nicotiana benthamiana compared with the isolate GW2, which a single residue substitution showed was due to Val834.

Length of poly(A) tail affects transcript infectivity of three ZYMV symptom variants differing at only five amino acid positions

The incidence of plant virus diseases infecting important cucurbit vegetables in Korea has increased as new isolates have been introduced, associated with warming temperatures and vector movement caused by climate change. Transcript infectivity of full-length infectious clones of three new ZYMV isolates was dependent upon the length of the poly(A) tract; transcripts with 55 A residues were inefficiently infectious, whereas 60 A residues resulted in highly efficient infection and significantly reduced time to production of systemic symptoms. Sequences of isolates BR1 (MH042024), BR2 (MH042025), and BR3 (MH042026) showed 99% pair-wise identity and differed at only five amino acid positions in: HC-Pro (D134N in BR2), CI (F31 L in BR1), and 6 K2 (A24V in BR3), and two positions in NIb (T300S in BR2, H429Q in BR3). Cucurbita pepo plants inoculated with transcripts of clones with these amino acid differences showed symptoms that ranged from mild to severe. Phylogenetic analysis of these new ZYMV isolates with previously characterized isolates indicated that the new isolates had 87.8–97.5% identity to other ZYMV isolates and were most closely related to recent ZYMV isolates from Australia and Spain.