First report of Passion fruit green spot virus in yellow Passion fruit (Passiflora edulis f. flavicarpa) in Casanare, Colombia.

Passiflora edulis, commonly known as passion fruit, is a vine species of passionflower native to South America. In Colombia, yellow passion fruit (P. edulis f. flavicarpa) is the most important species in terms of net production and local consumption. Recently two brevipalpus transmitted cileviruses, (i) passion fruit green spot virus (PfGSV) and (ii) hibiscus strain of citrus leprosis virus C2 (CiLV-C2H) were detected in passion fruit in Brazil and Hawaii, respectively (Ramos-González et al., 2020, Olmedo-Velarde et al., 2022). CiLV-C2H infects both citrus and hibiscus in Colombia (Roy et al., 2015, 2018) but there was no report of PfGSV elsewhere apart from Brazil and Paraguay (Costa-Rodrigues et al., 2022). Apart from emerging begomovirus diseases, five major viruses are known to infect passion fruit in Colombia: soybean mosaic virus (SMV), cowpea aphid-borne mosaic virus, passion fruit yellow mosaic virus, cucumber mosaic virus, and a tentative Gulupa bacilliform badnavirus A (Cardona et al., 2022). Current findings of CiLV-C2H in passion fruit and PfGSV in hibiscus motivated us to investigate the possibilities of cilevirus infection in passion fruit in Colombia. During surveys, along with healthy yellow passion fruit leaves, five symptomatic plant samples from Meta and three from Casanare were collected before sent to the Molecular Plant Pathology Laboratory at Beltsville, MD under APHIS permit. Passion fruit samples from Meta showed leaf mottling, rugose mosaic, and leaf distortion, whereas leaf variegation, chlorotic spots, yellowing, green spots in senescent leaves and green vein banding were observed in the Casanare samples (Supp. Fig. 1). Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen, USA). To know the potential cilevirus infection in these samples, three PfGSV specific (Ramos-González et al. 2020) and a CiLV-C2 generic primer pairs (Olmedo-Velarde et al. 2021) were used in the RT-PCR assays. All five passion fruit samples from Meta failed to produce either CiLV-C2 or CiLV-C2H or PfGSV amplicon whereas all three Casanare samples successfully amplified 321, 244 and 299 nts of PfGSV-RNA1 and -RNA2 amplicons using C13F/C13R, C6F/C6R and C8F/C8R primers, respectively. Bi-directional amplicon sequencing followed by BlastN analysis revealed ≥99% nt identity with the PfGSV-RNA1 (MK804173) and -RNA2 (MK804174) genome sequences. An optimized ribo-depleted library preparation protocol was utilized to prepare two cDNA libraries using the RNA extracts of a PfGSV suspected positive (Casanare) and a negative (Meta) samples (Chellappan et al., 2022). HTS libraries of Casanare and Meta samples resulted in 22.7 to 29.5 million raw reads, respectively. After adapter trimming and filtering, clean reads were mapped to the Arabidopsis thaliana reference genome and unmapped reads were de novo assembled (Chellappan et al., 2022). BlastN analysis from the assembled contigs identified 1-3 contigs corresponding to PfGSV-RNA1 and -RNA2, respectively, from Casanare sample whereas 3 contigs of SMV were identified in Meta passion fruit sample. No other virus sequence was obtained from either of the libraries. Assembled contigs covered 99.33% of the RNA1 and 94.42% of the RNA2 genome, with read depths of 64,474 and 119,549, respectively. Meta sample contigs (OP564897) covered >99% of the SMV genome, which shared >99% nt identity with the Colombian SMV isolates (KY249378, MW655827). Both RNA-1 (OP564895) and -2 (OP564896) segments of the Casanare isolate shared 99% nt identity with PfGSV isolate (MK804173-74). Our discovery identified PfGSV in Colombia, for the first-time outside Brazil and Paraguay. The findings of PfGSV in yellow passion fruit increases the potential threat and possibility of PfGSV movement via Brevipalpus sp. from passion fruit to other hosts.

Desymmetrization of pibrentasvir for efficient prodrug synthesis.

A novel and practical desymmetrization tactic is described to access a new class of pibrentasvir prodrugs. The homotopic benzimidazoles of pibrentasvir (PIB) are differentiated via a one-pot di-Boc/mono-de-Boc selective N-Boc protection and formaldehyde adduct formation sequence, both enabled by crystallization-induced selectivity. The first step represents the only known application of the Horeau principle of statistical amplification for C 2-symmetric polyheterocycle regioselective functionalization. The resulting versatile intermediate is employed in the high-yielding preparation of several pibrentasvir prodrug candidates.

Enhanced Serologically Based Detection of Liberibacters Associated with Citrus Huanglongbing.

'Candidatus Liberibacter spp.' are associated with the most devastating disease of citrus Huanglongbing (HLB). In previous work, we established an in situ tissue print method for the detection of 'Ca. L. asiaticus' (CLas) in sweet orange. We optimized the protocol by preincubation of the anti-Omp antibody with 5% (w/v) extract of healthy rough lemon. This simple process eliminated cross reactions between citrus and the antibody. The optimized protocol enhanced the application of the polyclonal antibody, and we demonstrate detection of CLas from all parts of the world, including isolates from Japan, Thailand, Vietnam, Pakistan, Saudi Arabia, Brazil, the United States, and a selection of strains from China representative of the diversity extant there. The assay also was used to detect four isolates of 'Ca. L. africanus' (CLaf) representative of the diversity present in South Africa. The corresponding outer membrane genes of representative isolates were cloned and sequenced. The coding sequences were highly conserved, and isolates of CLas and CLaf shared 53.8 to 55.9% identity between species at the amino acid level. The optimized protocol is efficient for recognition of both CLas and CLaf in phloem cells of different citrus tissues regardless of geographic origin of the HLB samples. The method is simple and scales well to match the urgent need for accurate, sensitive, and high-throughput screening of HLB bacteria, and may play an important role especially for plant inspection and quarantine programs.

Canine olfactory detection of a vectored phytobacterial pathogen, Liberibacter asiaticus, and integration with disease control.

Early detection and rapid response are crucial to avoid severe epidemics of exotic pathogens. However, most detection methods (molecular, serological, chemical) are logistically limited for large-scale survey of outbreaks due to intrinsic sampling issues and laboratory throughput. Evaluation of 10 canines trained for detection of a severe exotic phytobacterial arboreal pathogen, Candidatus Liberibacter asiaticus (CLas), demonstrated 0.9905 accuracy, 0.8579 sensitivity, and 0.9961 specificity. In a longitudinal study, cryptic CLas infections that remained subclinical visually were detected within 2 wk postinfection compared with 1 to 32 mo for qPCR. When allowed to interrogate a diverse range of in vivo pathogens infecting an international citrus pathogen collection, canines only reacted to Liberibacter pathogens of citrus and not to other bacterial, viral, or spiroplasma pathogens. Canines trained to detect CLas-infected citrus also alerted on CLas-infected tobacco and periwinkle, CLas-bearing psyllid insect vectors, and CLas cocultured with other bacteria but at CLas titers below the level of molecular detection. All of these observations suggest that canines can detect CLas directly rather than only host volatiles produced by the infection. Detection in orchards and residential properties was real time, ∼2 s per tree. Spatiotemporal epidemic simulations demonstrated that control of pathogen prevalence was possible and economically sustainable when canine detection was followed by intervention (i.e., culling infected individuals), whereas current methods of molecular (qPCR) and visual detection failed to contribute to the suppression of an exponential trajectory of infection.

Anesthesia and Cognitive Outcome in Elderly Patients: A Narrative Viewpoint.

Better ways to manage preoperative, intraoperative and postoperative care of surgical patients is the bailiwick of anesthesiologists. Although we care for patients of all ages, protecting the cognitive capacity of elderly patients more frequently requires procedures and practices that go beyond routine care for nonelderly adults. This narrative review will consider current understanding of the reasons that elderly patients need enhanced care, and recommendations for that care based on established and recent empirical research. In that latter regard, unless and until we are able to classify anesthetic neurotoxicity as a rare complication, the first-do-no-harm approach should: (1) add anesthesia to surgical intervention on the physiological cost side of the cost/benefit ratio when making decisions about whether and when to proceed with surgery; (2) minimize anesthetic depth and periods of electroencephalographic suppression; (3) limit the duration of continuous anesthesia whenever possible; (4) consider the possibility that regional anesthesia with deep sedation may be as neurotoxic as general anesthesia; and (5) when feasible, use regional anesthesia with light or no sedation.

Reassortment of Genome Segments Creates Stable Lineages Among Strains of Orchid Fleck Virus Infecting Citrus in Mexico.

The genus Dichorhavirus contains viruses with bipartite, negative-sense, single-stranded RNA genomes that are transmitted by flat mites to hosts that include orchids, coffee, the genus Clerodendrum, and citrus. A dichorhavirus infecting citrus in Mexico is classified as a citrus strain of orchid fleck virus (OFV-Cit). We previously used RNA sequencing technologies on OFV-Cit samples from Mexico to develop an OFV-Cit-specific reverse transcription PCR (RT-PCR) assay. During assay validation, OFV-Cit-specific RT-PCR failed to produce an amplicon from some samples with clear symptoms of OFV-Cit. Characterization of this virus revealed that dichorhavirus-like particles were found in the nucleus. High-throughput sequencing of small RNAs from these citrus plants revealed a novel citrus strain of OFV, OFV-Cit2. Sequence comparisons with known orchid and citrus strains of OFV showed variation in the protein products encoded by genome segment 1 (RNA1). Strains of OFV clustered together based on host of origin, whether orchid or citrus, and were clearly separated from other dichorhaviruses described from infected citrus in Brazil. The variation in RNA1 between the original (now OFV-Cit1) and the new (OFV-Cit2) strain was not observed with genome segment 2 (RNA2), but instead, a common RNA2 molecule was shared among strains of OFV-Cit1 and -Cit2, a situation strikingly similar to OFV infecting orchids. We also collected mites at the affected groves, identified them as Brevipalpus californicus sensu stricto, and confirmed that they were infected by OFV-Cit1 or with both OFV-Cit1 and -Cit2. OFV-Cit1 and -Cit2 have coexisted at the same site in Toliman, Queretaro, Mexico since 2012. OFV strain-specific diagnostic tests were developed.

Transcriptomic analyses reveal physiological changes in sweet orange roots affected by citrus blight.

BACKGROUND: Citrus blight is a very important progressive decline disease of commercial citrus. The etiology is unknown, although the disease can be transmitted by root grafts, suggesting a viral etiology. Diagnosis is made by demonstrating physical blockage of xylem cells that prevents the movement of water. This test was used to identify symptomatic trees from four commercial groves in Florida. Total RNA extracts of phloem-enriched scaffold root tissues were prepared from seven trees that failed to take up water and from one healthy tree. These RNA extracts were used for transcriptomic analyses using paired end RNA-Seq from an Illumina 2500 system. The expression of transcripts annotated as polyprotein of citrus endogenous pararetrovirus were estimated by both RT-qPCR and RNA-Seq. RESULTS: Transcripts from seven RNA-Seq libraries from trees affected by citrus blight were compared to a control tree. 129-148 million RNA fragments (two paired-end reads/fragment) were generated per library and were mapped to the sweet orange reference genome. In response to citrus blight stress, genes encoding aquaporins, proteins with water channel activity and several cellulose synthase genes were down-regulated, whereas genes involved in lignin and glucosinolate biosynthesis were up-regulated. Transcripts encoding proteins in pathways of carbohydrate metabolism, nucleotide synthesis, signaling, hormone metabolism, secondary metabolism, transport, and biotic stress pathways were overwhelmingly down regulated in all libraries. CONCLUSION: Reduced water intake and xylem plugging were observed in the trees tested and the changes in their transcriptome were analyzed. Plants adapted to reduced water flow by regulating primary and secondary metabolism, nuclear transport and hormone associated pathways. The patterns of energy generation, transcription, translation and protein degradation were consistent with irreversible decline. The down regulation of cellulose synthase transcripts and up regulation of transcripts related to lignin production likely lead to an imbalance in the pathways leading to wood formation, and may lead to the blockage of the xylem vessels seen as the cardinal symptom of citrus blight. Transcripts of a pararetrovirus were elevated in the transcriptome of roots used in this study.

Synthesis of ABBV-168, a 2'-Bromouridine for the Treatment of Hepatitis C.

ABBV-168 is a dihalogenated nucleotide under investigation for the treatment of hepatitis C virus. Three synthetic routes aimed at achieving the stereoselective installation of the C2' gem-Br,F substitution and subsequent Vorbruggen glycosylation were explored to prepare the penultimate nucleoside intermediate. Development culminated in a route to ABBV-168 featuring a de novo chromatography-free furanose synthesis, protecting group-directed Vorbruggen glycosylation, and highly selective phosphoramidation to furnish the API.

Asymmetric Synthesis of Remote Quaternary Centers by Copper-Catalyzed Desymmetrization: An Enantioselective Total Synthesis of (+)-Mesembrine.

Catalytic asymmetric syntheses of remote quaternary stereocenters have been developed by copper-catalyzed 1,4-hydrosilylation of γ,γ-disubstituted cyclohexadienones. A variety of cyclohexenones have been synthesized in good yield and excellent enantioselectivity. Versatile 2-silyloxy diene intermediates bearing γ,γ-disubstituted all carbon stereogenic centers can be isolated from the mild reaction conditions. The utility of this strategy is exemplified in a catalytic asymmetric total synthesis of (+)-mesembrine.

Complete Nucleotide Sequence of a Novel Hibiscus-Infecting Cilevirus from Florida and Its Relationship with Closely Associated Cileviruses.

The complete nucleotide sequence of a recently discovered Florida (FL) isolate of hibiscus-infecting cilevirus (HiCV) was determined by Sanger sequencing. The movement and coat protein gene sequences of the HiCV-FL isolate are more divergent than other genes of the previously sequenced HiCV-HI (Hawaii) isolate.

Transcriptional analysis of sweet orange trees co-infected with 'Candidatus Liberibacter asiaticus' and mild or severe strains of Citrus tristeza virus.

BACKGROUND: Citrus worldwide is threatened by huanglongbing (HLB) and tristeza diseases caused by 'Candidatus Liberibacter asiaticus' (CaLas) and Citrus tristeza virus (CTV). Although the pathogens are members of the α-proteobacteria and Closteroviridae, respectively, both are restricted to phloem cells in infected citrus and are transmitted by insect vectors. The response of sweet orange to single infection by either of these two pathogens has been characterized previously by global gene expression analysis. But because of the ubiquity of these pathogens where the diseases occur, co-infection by both pathogens is very common and could lead to increased disease severity based on synergism. We therefore co-inoculated sweet orange trees with CaLas and either a mild or a severe strain of CTV, and measured changes of gene expression in host plants. RESULTS: In plants infected with CaLas-B232, the overall alteration in gene expression was much greater in plants co-inoculated with the severe strain of CTV, B6, than when co-infected with the mild strain of CTV, B2. Plants co-infected with CaLas-B232 and either strain of CTV died but trees co-infected with CTV-B2 survived much longer than those co-infected with CTV-B6. Many important pathways were perturbed by both CTV-B2/CaLas-B232 and/or CTV-B6/CaLas-B232, but always more severely by CTV-B6/CaLas-B232. Genes related to cell wall modification and metal transport responded differently to infection by the pathogens in combination than by the same pathogens singly. The expressions of genes encoding phloem proteins and sucrose loading proteins were also differentially altered in response to CTV-B2 or CTV-B6 in combination with CaLas-B232, leading to different phloem environments in plants co-infected by CaLas and mild or severe CTV. CONCLUSIONS: Many host genes were expressed differently in response to dual infection as compared to single infections with the same pathogens. Interactions of the pathogens within the host may lead to a better or worse result for the host plant. CTV-B6 may exert a synergistic effect with CaLas-B232 in weakening the plant; on the other hand, the responses activated by the mild strain CTV-B2 may provide some beneficial effects against CaLas-B232 by increasing the defense response of the host.

Co-infection of Sweet Orange with Severe and Mild Strains of Citrus tristeza virus Is Overwhelmingly Dominated by the Severe Strain on Both the Transcriptional and Biological Levels.

Citrus tristeza is one of the most destructive citrus diseases and is caused by the phloem-restricted Closterovirus, Citrus tristeza virus. Mild strain CTV-B2 does not cause obvious symptoms on indicators whereas severe strain CTV-B6 causes symptoms, including stem pitting, cupping, yellowing, and stiffening of leaves, and vein corking. Our laboratory has previously characterized changes in transcription in sweet orange separately infected with CTV-B2 and CTV-B6. In the present study, transcriptome analysis of Citrus sinensis in response to double infection by CTV-B2 and CTV-B6 was carried out. Four hundred and eleven transcripts were up-regulated and 356 transcripts were down-regulated prior to the onset of symptoms. Repressed genes were overwhelmingly associated with photosynthesis, and carbon and nucleic acid metabolism. Expression of genes related to the glycolytic, oxidative pentose phosphate (OPP), tricarboxylic acid cycle (TCA) pathways, tetrapyrrole synthesis, redox homeostasis, nucleotide metabolism, protein synthesis and post translational protein modification and folding, and cell organization were all reduced. Ribosomal composition was also greatly altered in response to infection by CTV-B2/CTV-B6. Genes that were induced were related to cell wall structure, secondary and hormone metabolism, responses to biotic stress, regulation of transcription, signaling, and secondary metabolism. Transport systems dedicated to metal ions were especially disturbed and ZIPs (Zinc Transporter Precursors) showed different expression patterns in response to co-infection by CTV-B2/CTV-B6 and single infection by CTV-B2. Host plants experienced root decline that may have contributed to Zn, Fe, and other nutrient deficiencies. Though defense responses, such as, strengthening of the cell wall, alteration of hormone metabolism, secondary metabolites, and signaling pathways, were activated, these defense responses did not suppress the spread of the pathogens and the development of symptoms. The mild strain CTV-B2 did not provide a useful level of cross-protection to citrus against the severe strain CTV-B6.

Characterization and purification of proteins suitable for the production of antibodies against 'Ca. Liberibacter asiaticus'.

The citrus disease huanglongbing (HLB), which is caused by 'Candidatus Liberibacter asiaticus' (CaLas), is one of the most devastating pathogens of citrus, and with no effective method of control, poses a serious threat to citrus production throughout the world. In a previous study we described the production of single chain antibodies against several CaLas proteins that provide the basis for efficient and accurate detection of CaLas in citrus tissues. The isolation of a sufficient amount of purified antigen is a key step in the production of functional antibodies. The current report details purification procedures for six protein antigens used to select recombinant and produce polyclonal antibodies. These proteins include a flagellar biosynthesis protein (FlhA), a dinucleoside polyphosphate hydrolase (InvA), a portion of the major outer membrane protein (OmpA), a component of type IV pilus (CapB), the polysialic acid capsule expression protein (KpsA) and the outer membrane efflux protein (TolC). Results of purification under completely native or denatured conditions were not satisfactory. Therefore different hybrid purification conditions were optimized for each of the different proteins. The results of bioinformatic analysis also indicated that the six proteins contained a great diversity of potential antigenic epitopes, which varied in number, and that the antigenic clusters were not uniformly distributed throughout the proteins. The purified proteins are useful for the development of highly specific antibodies capable of differentiating specific strains of Liberibacter.

Immune Tissue Print and Immune Capture-PCR for Diagnosis and Detection of Candidatus Liberibacter Asiaticus.

'Candidatus Liberibacter asiaticus' (CaLas), associated with citrus Huanglongbing (HLB), is a non culturable member of the α-proteobacteria. In this study serologically based methods for the detection of CaLas were developed. An anti-outer membrane protein A (OmpA) polyclonal antibody previously produced (in our laboratory) was highly effective for the detection of CaLas from citrus tissues in a simple tissue printing format. The antibody was also used to capture bacteria from periwinkle extracts. About 80% of all field samples analyzed tested positive with both immune tissue printing and qPCR; whereas 95% were positive with at least one of these two methods. When asymptomatic citrus tissues were tested, the tissue printing method gave a higher rate of detection (83%) than the qPCR method (64%). This is consistent with a lower concentration of CaLas DNA, but a higher proportion of viable cells, in the asymptomatic tissues. The immune tissue printing method also highlights the detail of the spatial distribution of 'Ca. Liberibacter asiaticus' in diseased citrus tissues. Both the immune capture PCR and immune tissue printing methods offer the advantages of low cost, high throughput, ease of scaling for multiple samples and simplicity over current PCR-based methods for the detection of 'Ca. Liberibacter asiaticus'.