Convenient Auto-Processing Vector Based on Bamboo Mosaic Virus for Presentation of Antigens Through Enzymatic Coupling

We have developed a new binary epitope-presenting CVP platform based on bamboo mosaic virus (BaMV) by using the sortase A (SrtA)-mediated ligation technology. The reconstructed BaMV genome harbors two modifications: 1) a coat protein (CP) with N-terminal extension of the tobacco etch virus (TEV) protease recognition site plus 4 extra glycine (G) residues as the SrtA acceptor; and 2) a TEV protease coding region replacing that of the triple-gene-block proteins. Inoculation of such construct, pKB5G, on Nicotiana benthamiana resulted in the efficient production of filamentous CVPs ready for SrtA-mediated ligation with desired proteins. The second part of the binary platform includes an expression vector for the bacterial production of donor proteins. We demonstrated the applicability of the platform by using the recombinant envelope protein domain III (rEDIII) of Japanese encephalitis virus (JEV) as the antigen. Up to 40% of the BaMV CP subunits in each CVP were loaded with rEDIII proteins in 1 min. The rEDIII-presenting BaMV CVPs (BJLPET5G) could be purified using affinity chromatography. Immunization assays confirmed that BJLPET5G could induce the production of neutralizing antibodies against JEV infections. The binary platform could be adapted as a useful alternative for the development and mass production of vaccine candidates.

Gene Rearrangements in the Mitochondrial Genomes of Whiteflies (Hemiptera: Aleyrodinae): Plesiomorphies, Synapomorphies and Autapomorphies

Mitochondrial genome rearrangements have been used for defining historical relationships, but there have been incidences of convergences at different taxonomic levels. Here, we sequenced complete mitogenome of Aleurodicus rugioperculatus (Aleyrodidae: Aleurodicinae) to examine gene rearrangements and phylogenetic relationships within the family Aleyrodidae. We identified five gene blocks (I-V) in the whitefly ancestor that are shared plesiomorphies retained in different whitefly lineages. Gene block I is conserved in all whiteflies except three species (Tetraleurodes acaciae and two Bemisia species). Conversely, we detected 83 derived gene boundaries within the family. Mapping these gene boundaries onto a phylogenetic tree revealed that 16 were symplesiomorphies for two subfamilies; 9 were synapomorphies at different taxonomic levels, and 28 autapomorphies for individual species. Bayesian Inference (BI) and Maximum Likelihood (ML) phylogenetic analyses yielded similar topologies supporting the monophyly of Aleyrodinae and Aleurodicinae. Exclusion of PCG third codon positions from phylogenetic analyses improved both node support and consistency with prior analyses. To understand the significance of gene order convergence on phylogeny of the whiteflies, more species-level data is required.

Signatures of mitonuclear coevolution in a warbler species complex

Divergent mitonuclear coadaptation could facilitate speciation. We investigate this possibility in two hybridizing species of warblers, Setophaga occidentalis and S. townsendi, in western North America. Inland S. townsendi harbor distinct mitochondrial DNA haplotypes from those of S. occidentalis. These populations also differ in several nuclear DNA regions. Coastal S. townsendi demonstrate mixed mitonuclear ancestry from S. occidentalis and inland S. townsendi. Of the few highly-differentiated chromosomal regions between inland S. townsendi and S. occidentalis, a 1.2 Mb gene block on chromosome 5 is also differentiated between coastal and inland S. townsendi. Genes in this block are associated with fatty acid oxidation and energy-related signaling transduction, thus linked to mitochondrial functions. Genetic variation within this candidate gene block covaries with mitochondrial DNA and shows signatures of divergent selection. Spatial variation in mitonuclear ancestries is correlated with climatic conditions. Together, these observations suggest divergent mitonuclear coadaptation underpins cryptic differentiation in this species complex.

Small RNA and transcriptome sequencing of a symptomatic peony plant reveals mixed infections with novel viruses

To identify the viruses in tree peony plants associated with the symptoms of yellowing, leaf rolling, stunted growth, and decline, high-throughput sequencing of small RNA and mRNA was conducted from a single symptomatic plant. Bioinformatic analyses and reconstruction of viral genomes indicated mixed viral infections involving cycas necrotic stunt virus (CNSV), apple stem grooving virus (ASGV), lychnis mottle virus (LycMoV), grapevine line pattern virus (GLPV), and three new viruses designated as peony yellowing-associated citrivirus (PYaCV, Citrivirus in Betaflexiviridae), peony betaflexivirus 1 (PeV1, unclassified in Betaflexiviridae), and peony leafroll-associated virus (PLRaV, Ampelovirus in Closteroviridae). PYaCV was 8,666 nucleaotides (nt) in length, comprising three open reading frames (ORFs) and shared 63.8–75.9% nucleotide sequence identity with citrus leaf blotch virus (CLBV) isolates. However, the ORF encoding the replication-associated protein (REP) shared 57% and 52% sequence identities at the nt and amino acid (aa) level, respectively, with other reported CLBV isolates, which were below the criterion for species classification within the family Betaflexiviridae. Recombination analysis identified putative recombination sites in PYaCV, which originated from CLBV. PeV1, only identified from the transcriptome data, was 8,124 nt in length with five ORFs encoding the REP (ORF1), triple gene block (TGB, ORF2–4) and coat protein (CP, ORF5) proteins. Phylogenetic analysis and sequence comparison showed that PeV1 clustered with an unassigned member, the garlic yellow mosaic-associated virus (GYMaV) within the Betaflexiviridae family, into a separate clade. Partial genome sequence analysis of PLRaV (12,545 nt) showed it contained seven ORFs encoding the partial polyprotein 1a, the RNA-dependent RNA polymerase (RdRp), two small hydrophobic proteins p11 and p6, HSP70h, p55, and a CP duplicate, which shared low aa sequence identity with Closteroviridae family members. Phylogenetic analysis based on the aa sequences of RdRp or HSP70h indicated that PLRaV clustered with grapevine leafroll-associated virus 1 (GLRaV-1) and GLRaV-13 in the Ampelovirus genus. Field investigation confirmed the wide distribution of these viruses, causing mixed infections of peony plants in Beijing.

A Survey Using High-Throughput Sequencing Suggests That the Diversity of Cereal and Barley Yellow Dwarf Viruses Is Underestimated

Worldwide, barley/cereal yellow dwarf viruses (YDVs) are the most widespread and damaging group of cereal viruses. In this study, we applied high-throughput sequencing technologies (HTS) to perform a virus survey on symptomatic plants from 47 cereal fields in Estonia. HTS allowed the assembly of complete genome sequences for 22 isolates of cereal yellow dwarf virus RPS, barley yellow dwarf virus GAV, barley yellow dwarf virus PAS (BYDV-PAS), barley yellow dwarf virus PAV (BYDV-PAV), and barley yellow dwarf virus OYV (BYDV-OYV). We also assembled a near-complete genome of the putative novel species BYDV-OYV from Swedish samples of meadow fescue. Previously, partial sequencing of the central part of the coat protein gene indicated that BYDV-OYV represented a putative new species closely related to BYDV-PAV-CN, which currently is recognized as a subtype of BYDV-PAV. The present study found that whereas the 3′gene block of BYDV-OYV shares the closest relationship with BYDV-PAV-CN, the 5′gene block of BYDV-OYV shows the closest relationships to that of BYDV-PAS. Recombination detection analysis revealed that BYDV-OYV is a parental virus for both. Analysis of complete genome sequence data indicates that both BYDV-OYV and BYDV-PAV-CN meet the species criteria of genus Luteovirus. The study discusses BYDV phylogeny, and through a systematic in silico analysis of published primers for YDV detection, the existing gaps in current diagnostic practices for detection of YDVs, proposing primer pairs based on the most recent genomic information for the detection of different BYDV species. Thanks to the rising number of sequences available in databases, continuous updating of diagnostic primers can improve test specificity, e.g., inclusivity and exclusivity at species levels. This is needed to properly survey the geographical and host distribution of the different species of the YDV complex and their prevalence in cereal/barley yellow dwarf disease epidemics.

Molecular Characterization of a Novel Wheat-infecting Virus in Family Betaflexiviridae

Wheat plants showing yellow mosaic in leaves and stunting were observed and collected from wheat fields in the Henan Province, China. Analyses of these plants through transmission electron microscopy showed that these plants contained two filamentous virus-like particles of 200–500 nm and 1000–1300 nm long, respectively. RNA-seq result unveiled a co-infection of wheat yellow mosaic virus (WYMV) and an unknown wheat-infecting virus. The complete genome sequence of the unknown virus is 8,410 nucleotide long, excluding its 3’ end poly (A) tail. This unknown virus has six open reading frames (ORFs). The ORF1 encodes a putative viral replication-associated protein, and the ORF2, 3 and 4 encode the triple gene block (TGB) proteins. The ORF5 and 6 encode the capsid protein (CP) and a protein with unknown function, respectively. Phylogenetic relationship analyses showed that this novel virus is evolutionarily related to viruses in the subfamily Quinvirinae, family Betaflexiviridae. It is, however, distinctly different from the viruses in other genera. Based on the species and genus demarcation criteria set by the International Committee on Taxonomy of Viruses (ICTV), we tentatively name this novel virus as wheat yellow stunt-associated betaflexivirus (WYSaBV). We also propose WYSaBV as a new member in a new genus in the family Betaflexiviridae.

Discovery of a Novel Member of the Carlavirus Genus from Soybean (Glycine max L. Merr.)

A novel member of the Carlavirus genus, provisionally named soybean carlavirus 1 (SCV1), was discovered by RNA-seq analysis of randomly collected soybean leaves in Illinois, USA. The SCV1 genome contains six open reading frames that encode a viral replicase, triple gene block proteins, a coat protein (CP) and a nucleic acid binding protein. The proteins showed highest amino acid sequence identities with the corresponding proteins of red clover carlavirus A (RCCVA). The predicted amino acid sequence of the SCV1 replicase was only 60.6% identical with the replicase of RCCVA, which is below the demarcation criteria for a new species in the family Betaflexiviridae. The predicted replicase and CP amino acid sequences of four SCV1 isolates grouped phylogenetically with those of members of the Carlavirus genus in the family Betaflexiviridae. The features of the encoded proteins, low nucleotide and amino acid sequence identities of the replicase with the closest member, and the phylogenetic grouping suggest SCV1 is a new member of the Carlavirus genus.

Opposing dominance within a color gene block is associated with maladaptive hybrid signal discordance

Color divergence is increasingly recognized as important for speciation in songbirds through its influence on social dynamics. However, the behavioral mechanisms underlying the eco-evolutionary feedback that acts across species boundaries is poorly understood. The hybrid zone between Setophaga occidentalis (SOCC) and S. townsendi (STOW) in the Cascade mountain ranges provides a natural observatory to test the interplay between genetics and social behaviour in maintaining species boundaries. Recently, we found that selection within a gene block underpinning color variation (ASIP-RALY) has maintained a stable and narrow hybrid zone. Here we investigated the social signaling roles of cheek darkness and flank streaking, two color traits linked to ASIP-RALY that reflect opposing dominance of SOCC and STOW alleles. We found that both traits act as honest badges of status, as they predicted male breeding quality. The opposing dominance effects of ASIP-RALY resulted in signal discordance for heterozygotes, which in turn was associated with inferior hybrid territorial performance, a fitness proxy quantified by vocal and physical responses of resident males to a decoy intruder. Taken together, this study highlights a potential behavioral mechanism underlying selection acting on a simple genetic architecture that has maintained a stable species boundary over decades despite significant gene flow.

Comparative Mitogenomic Analysis of Heptageniid Mayflies (Insecta: Ephemeroptera): Conserved Intergenic Spacer and tRNA Gene Duplication

Large intergenic spacers and tRNA gene duplications have been reported in several insect groups, although little is known about mitogenomes of mayflies. Here, we determined complete mitogenomes of ten heptageniid species and systemically analyzed their mitogenomic features. Both a conserved intergenic spacer (IGS) and trnM duplication were detected in those mitogenomes. The IGS, which was observed in heptageniids, could be further folded into a stable stem–loop structure. The tRNA gene duplication was found in almost all analyzed mitogenomes, and a unique gene block trnI-trnM-trnQ-trnM-ND2 was also discovered. Our analysis demonstrates that the heptageniid gene arrangement pattern can be explained by the tandem duplication-random loss (TDRL) model. Phylogenetic analyses using both Bayesian inference (BI) and maximum likelihood (ML) methods based on the nucleotide and amino acid sequence data recovered the genus Epeorus as monophyletic with strong support. Our results provide a better understanding of mitogenomic evolution in Heptageniidae, as well as novel molecular markers for species identification of mayflies.

Constructing Local Cell Specific Networks from Single Cell Data

ABSTRACTGene co-expression networks yield critical insights into biological processes, and single-cell RNA sequencing provides an opportunity to target inquiries at the cellular level. However, due to the sparsity and heterogeneity of transcript counts, it is challenging to construct accurate gene networks. We develop an approach that estimates cell-specific networks (CSN) for each cell using a method inspired by Dai et al. (7). Although individual CSNs are estimated with considerable noise, average CSNs provide stable estimates of network structure, which provide better estimates of gene block structures than traditional measures. The method, called locCSN, is based on a non-parametric investigation of the joint distribution of gene expression, hence it can readily detect nonlinear correlations, and it is more robust to distributional challenges. The individual networks preserve information about the heterogeneity of the cells and having repeated estimates of network structure facilitates testing for difference in network structure between groups of cells. The original CSN algorithm showed promise; however, it had shortcomings which locCSN overcomes. Additionally, we propose new downstream analysis methods using CSNs, to utilize more fully the information contained within them. Finally, to further our understanding of autism spectrum disorder we examined the evolution of gene networks in fetal brain cells and compared the CSNs of cells sampled from case and control subjects to reveal intriguing patterns in gene co-expression changes.