Adaptation of feeding behaviors on two Brassica species by colonizing and noncolonizing Bemisia tabaci (Hemiptera: Aleyrodidae) NW whiteflies.

Bemisia tabaci New World (NW) (Gennadius) (Hemiptera: Aleyrodidae), a whitefly in the B. tabaci species complex, is polyphagous on many plant species. Yet, it has been displaced, albeit not entirely, by other whitefly species. Potential causes could include issues with adaptation, feeding, and the colonization of new-hosts; however, insights that would help clarify these possibilities are lacking. Here, we sought to address these gaps by performing electropenetrography (EPG) recordings of NW whiteflies, designated "Napus" and "Rapa," reared on 2 colony hosts, Brassica napus and B. rapa, respectively. Analysis of 17 probing and pathway (pw) phase-related EPG variables revealed that the whiteflies exhibited unique probing behaviors on their respective colony hosts, with some deterrence being encountered on B. rapa. Upon switching to B. rapa and B. napus, the probing patterns of Napus and Rapa whiteflies, respectively, adapted quickly to these new-hosts to resemble that of whiteflies feeding on their colony hosts. Results for 3 of the EPG variables suggested that B. rapa's deterrence against Napus whitefly was significant prior to the phloem phase. This also suggested that adaptation by Rapa whitefly improved its pw probing on B. rapa. Based on analysis of 24 phloem phase-related EPG variables, Napus and Rapa whiteflies performed equally well once they entered phloem phase and exhibited comparable phloem acceptability on both the colony- and new-hosts. These findings demonstrate that NW whiteflies reared on a colony host are highly adaptable to feeding on a new host despite encountering some deterrence during the nonphloem phases in B. rapa plant.

Antimicrobial activity of compounds identified by artificial intelligence discovery engine targeting enzymes involved in Neisseria gonorrhoeae peptidoglycan metabolism.

BACKGROUND: Neisseria gonorrhoeae (Ng) causes the sexually transmitted disease gonorrhoea. There are no vaccines and infections are treated principally with antibiotics. However, gonococci rapidly develop resistance to every antibiotic class used and there is a need for developing new antimicrobial treatments. In this study we focused on two gonococcal enzymes as potential antimicrobial targets, namely the serine protease L,D-carboxypeptidase LdcA (NgO1274/NEIS1546) and the lytic transglycosylase LtgD (NgO0626/NEIS1212). To identify compounds that could interact with these enzymes as potential antimicrobials, we used the AtomNet virtual high-throughput screening technology. We then did a computational modelling study to examine the interactions of the most bioactive compounds with their target enzymes. The identified compounds were tested against gonococci to determine minimum inhibitory and bactericidal concentrations (MIC/MBC), specificity, and compound toxicity in vitro. RESULTS: AtomNet identified 74 compounds that could potentially interact with Ng-LdcA and 84 compounds that could potentially interact with Ng-LtgD. Through MIC and MBC assays, we selected the three best performing compounds for both enzymes. Compound 16 was the most active against Ng-LdcA, with a MIC50 value < 1.56 µM and MBC50/90 values between 0.195 and 0.39 µM. In general, the Ng-LdcA compounds showed higher activity than the compounds directed against Ng-LtgD, of which compound 45 had MIC50 values of 1.56-3.125 µM and MBC50/90 values between 3.125 and 6.25 µM. The compounds were specific for gonococci and did not kill other bacteria. They were also non-toxic for human conjunctival epithelial cells as judged by a resazurin assay. To support our biological data, in-depth computational modelling study detailed the interactions of the compounds with their target enzymes. Protein models were generated in silico and validated, the active binding sites and amino acids involved elucidated, and the interactions of the compounds interacting with the enzymes visualised through molecular docking and Molecular Dynamics Simulations for 50 ns and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA). CONCLUSIONS: We have identified bioactive compounds that appear to target the N. gonorrhoeae LdcA and LtgD enzymes. By using a reductionist approach involving biological and computational data, we propose that compound Ng-LdcA-16 and Ng-LtgD-45 are promising anti-gonococcal compounds for further development.

Data-driven subclassification of ANCA-associated vasculitis: model-based clustering of a federated international cohort.

BACKGROUND: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a heterogenous autoimmune disease. While traditionally stratified into two conditions, granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), the subclassification of ANCA-associated vasculitis is subject to continued debate. Here we aim to identify phenotypically distinct subgroups and develop a data-driven subclassification of ANCA-associated vasculitis, using a large real-world dataset. METHODS: In the collaborative data reuse project FAIRVASC (Findable, Accessible, Interoperable, Reusable, Vasculitis), registry records of patients with ANCA-associated vasculitis were retrieved from six European vasculitis registries: the Czech Registry of ANCA-associated vasculitis (Czech Republic), the French Vasculitis Study Group Registry (FVSG; France), the Joint Vasculitis Registry in German-speaking Countries (GeVas; Germany), the Polish Vasculitis Registry (POLVAS; Poland), the Irish Rare Kidney Disease Registry (RKD; Ireland), and the Skåne Vasculitis Cohort (Sweden). We performed model-based clustering of 17 mixed-type clinical variables using a parsimonious mixture of two latent Gaussian variable models. Clinical validation of the optimal cluster solution was made through summary statistics of the clusters' demography, phenotypic and serological characteristics, and outcome. The predictive value of models featuring the cluster affiliations were compared with classifications based on clinical diagnosis and ANCA specificity. People with lived experience were involved throughout the FAIRVASVC project. FINDINGS: A total of 3868 patients diagnosed with ANCA-associated vasculitis between Nov 1, 1966, and March 1, 2023, were included in the study across the six registries (Czech Registry n=371, FVSG n=1780, GeVas n=135, POLVAS n=792, RKD n=439, and Skåne Vasculitis Cohort n=351). There were 2434 (62·9%) patients with GPA and 1434 (37·1%) with MPA. Mean age at diagnosis was 57·2 years (SD 16·4); 2006 (51·9%) of 3867 patients were men and 1861 (48·1%) were women. We identified five clusters, with distinct phenotype, biochemical presentation, and disease outcome. Three clusters were characterised by kidney involvement: one severe kidney cluster (555 [14·3%] of 3868 patients) with high C-reactive protein (CRP) and serum creatinine concentrations, and variable ANCA specificity (SK cluster); one myeloperoxidase (MPO)-ANCA-positive kidney involvement cluster (782 [20·2%]) with limited extrarenal disease (MPO-K cluster); and one proteinase 3 (PR3)-ANCA-positive kidney involvement cluster (683 [17·7%]) with widespread extrarenal disease (PR3-K cluster). Two clusters were characterised by relative absence of kidney involvement: one was a predominantly PR3-ANCA-positive cluster (1202 [31·1%]) with inflammatory multisystem disease (IMS cluster), and one was a cluster (646 [16·7%]) with predominantly ear-nose-throat involvement and low CRP, with mainly younger patients (YR cluster). Compared with models fitted with clinical diagnosis or ANCA status, cluster-assigned models demonstrated improved predictive power with respect to both patient and kidney survival. INTERPRETATION: Our study reinforces the view that ANCA-associated vasculitis is not merely a binary construct. Data-driven subclassification of ANCA-associated vasculitis exhibits higher predictive value than current approaches for key outcomes. FUNDING: European Union's Horizon 2020 research and innovation programme under the European Joint Programme on Rare Diseases.

An engineered citrus tristeza virus (T36CA)-based vector induces gene-specific RNA silencing and is graft transmissible to commercial citrus varieties.

A protein-expressing citrus tristeza virus (CTV)-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus induced gene silencing (VIGS) system intended for use with studies of California citrus. VIGS constructs engineered with a truncated Citrus macrophylla (Cm) PHYTOENE DESATURASE (PDS) gene sequence in the sense or anti-sense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two non-synonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3 were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 week-post-inoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8 to 12 month-post-inoculation (mpi) window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 mpi, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.

Epstein-Barr virus reactivation induces divergent abortive, reprogrammed, and host shutoff states by lytic progression.

Viral infection leads to heterogeneous cellular outcomes ranging from refractory to abortive and fully productive states. Single cell transcriptomics enables a high resolution view of these distinct post-infection states. Here, we have interrogated the host-pathogen dynamics following reactivation of Epstein-Barr virus (EBV). While benign in most people, EBV is responsible for infectious mononucleosis, up to 2% of human cancers, and is a trigger for the development of multiple sclerosis. Following latency establishment in B cells, EBV reactivates and is shed in saliva to enable infection of new hosts. Beyond its importance for transmission, the lytic cycle is also implicated in EBV-associated oncogenesis. Conversely, induction of lytic reactivation in latent EBV-positive tumors presents a novel therapeutic opportunity. Therefore, defining the dynamics and heterogeneity of EBV lytic reactivation is a high priority to better understand pathogenesis and therapeutic potential. In this study, we applied single-cell techniques to analyze diverse fate trajectories during lytic reactivation in two B cell models. Consistent with prior work, we find that cell cycle and MYC expression correlate with cells refractory to lytic reactivation. We further found that lytic induction yields a continuum from abortive to complete reactivation. Abortive lytic cells upregulate NFκB and IRF3 pathway target genes, while cells that proceed through the full lytic cycle exhibit unexpected expression of genes associated with cellular reprogramming. Distinct subpopulations of lytic cells further displayed variable profiles for transcripts known to escape virus-mediated host shutoff. These data reveal previously unknown and promiscuous outcomes of lytic reactivation with broad implications for viral replication and EBV-associated oncogenesis.

Computable phenotype for real-world, data-driven retrospective identification of relapse in ANCA-associated vasculitis.

OBJECTIVE: ANCA-associated vasculitis (AAV) is a relapsing-remitting disease, resulting in incremental tissue injury. The gold-standard relapse definition (Birmingham Vasculitis Activity Score, BVAS>0) is often missing or inaccurate in registry settings, leading to errors in ascertainment of this key outcome. We sought to create a computable phenotype (CP) to automate retrospective identification of relapse using real-world data in the research setting. METHODS: We studied 536 patients with AAV and >6 months follow-up recruited to the Rare Kidney Disease registry (a national longitudinal, multicentre cohort study). We followed five steps: (1) independent encounter adjudication using primary medical records to assign the ground truth, (2) selection of data elements (DEs), (3) CP development using multilevel regression modelling, (4) internal validation and (5) development of additional models to handle missingness. Cut-points were determined by maximising the F1-score. We developed a web application for CP implementation, which outputs an individualised probability of relapse. RESULTS: Development and validation datasets comprised 1209 and 377 encounters, respectively. After classifying encounters with diagnostic histopathology as relapse, we identified five key DEs; DE1: change in ANCA level, DE2: suggestive blood/urine tests, DE3: suggestive imaging, DE4: immunosuppression status, DE5: immunosuppression change. F1-score, sensitivity and specificity were 0.85 (95% CI 0.77 to 0.92), 0.89 (95% CI 0.80 to 0.99) and 0.96 (95% CI 0.93 to 0.99), respectively. Where DE5 was missing, DE2 plus either DE1/DE3 were required to match the accuracy of BVAS. CONCLUSIONS: This CP accurately quantifies the individualised probability of relapse in AAV retrospectively, using objective, readily accessible registry data. This framework could be leveraged for other outcomes and relapsing diseases.

A comprehensive framework for the delimitation of species within the Bemisia tabaci cryptic complex, a global pest-species group.

Identifying cryptic species poses a substantial challenge to both biologists and naturalists due to morphological similarities. Bemisia tabaci is a cryptic species complex containing more than 44 putative species; several of which are currently among the world's most destructive crop pests. Interpreting and delimiting the evolution of this species complex has proved problematic. To develop a comprehensive framework for species delimitation and identification, we evaluated the performance of distinct data sources both individually and in combination among numerous samples of the B. tabaci species complex acquired worldwide. Distinct datasets include full mitogenomes, single-copy nuclear genes, restriction site-associated DNA sequencing, geographic range, host speciation, and reproductive compatibility datasets. Phylogenetically, our well-supported topologies generated from three dense molecular markers highlighted the evolutionary divergence of species of the B. tabaci complex and suggested that the nuclear markers serve as a more accurate representation of B. tabaci species diversity. Reproductive compatibility datasets facilitated the identification of at least 17 different cryptic species within our samples. Native geographic range information provides a complementary assessment of species recognition, while the host range datasets provide low rate of delimiting resolution. We further summarized different data performances in species classification when compared with reproductive compatibility, indicating that combination of mtCOI divergence, nuclear markers, geographic range provide a complementary assessment of species recognition. Finally, we represent a model for understanding and untangling the cryptic species complexes based on the evidence from this study and previously published articles.

CLINICAL FEATURES AND SURGICAL OUTCOMES OF SCLERAL BUCKLE SURGERY FOR PRIMARY RHEGMATOGENOUS RETINAL DETACHMENT: Moorfields Buckle Study.

PURPOSE: Long-term study to evaluate the clinical and surgical outcomes of scleral buckle (SB) surgery for primary rhegmatogenous retinal detachment (RRD) at a large tertiary eye center. METHODS: Noncomparative, retrospective case series of 589 eyes of 569 patients with primary RRD who underwent SB surgery between 2004 and 2022 with a median follow-up of 6 months. The main outcome measures were best-corrected visual acuity, surgical outcomes, complications, and classification of RRD. RESULTS: At baseline, 447/589 (76.1%) round hole RRD, and 133/589 (22.7%) retinal dialysis RRD. Overall primary SB success rate was 83.7% for all retinal detachment subtypes, with round hole retinal detachment 84.8% and dialysis RRD 81.2%. Overall, the baseline best-corrected visual acuity was 0.42 logarithm of the minimum angle of resolution (logMAR) and the final best-corrected visual acuity was 0.26 logMAR ( P < 0.0001). In macula-off RRD, the best-corrected visual acuity significantly improved from 0.79 to 0.48 logMAR ( P < 0.0001). In patients with macula-on RRD, it improved from 0.19 to 0.12 logMAR ( P = 0.014). Binary logistic regression showed registrar surgeon grade (odds ratio [OR] 0.09, 95% confidence interval [CI] 0.01-0.55), and partial or complete posterior vitreous detachment (OR 0.21, 95% CI 0.10-0.49) was associated with reduced odds of primary success. Higher surgical failure was associated with low pre-fellowship SB surgeon experience ( P = 0.024). CONCLUSION: Favorable visual and functional outcomes have been reported in a large series of SB for primary retinal detachment, mainly for patients with round hole RRD and retinal dialysis RRD.

Discovery of New Broad-Spectrum Anti-Infectives for Eukaryotic Pathogens Using Bioorganometallic Chemistry.

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens, including fungal infections. Herein, we show that the simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from that of the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and neglected tropical diseases (NTDs) targeted for elimination by 2030.

Discovery of New Broad-Spectrum Anti-Infectives for Eukaryotic Pathogens Using Bioorganometallic Chemistry.

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens including fungal infections. Since these diseases target the most vulnerable communities who are disadvantaged by health and socio-economic factors, new agents should be, if possible, easy-to-prepare to allow for commercialization based on their low cost. In this study, we show that simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and NTDs targeted for elimination by 2030. Overall, the discovery of these new compounds with broad-spectrum activity opens new avenues for the development of treatments for several current human infections, either caused by fungi or by parasites, including other NTDs, as well as newly emerging diseases. ONE-SENTENCE SUMMARY: Simple derivatives of the well-known antifungal drug fluconazole were found to be highly effective in vivo against fungal infections, and also potent against the parasitic nematode Brugia, which causes lymphatic filariasis and against Trichuris, one of the soil-transmitted helminths that infects millions of people globally.

High-throughput Sequencing to Identify Bacteremia in Children with Cancer.

Current methods to diagnose bacteremia are limited. In this pilot study of children with cancer presenting with fever, we determined the concordance between a novel high-throughput sequencing platform called BacCapSeq and blood culture. High-throughput sequencing had modest concordance with blood culture. Discordant organisms included those with both unlikely or potential clinical relevance.

Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor (rhu GM-CSF) as Adjuvant Therapy for Invasive Fungal Diseases.

Background: Sargramostim (yeast-derived, glycosylated recombinant human granulocyte-macrophage colony-stimulating factor [rhu GM-CSF]) augments innate and adaptive immune responses and accelerates hematopoietic recovery of chemotherapy-induced neutropenia. However, considerably less is known about its efficacy as adjunctive immunotherapy against invasive fungal diseases (IFDs). Methods: The clinical courses of 15 patients with pediatric malignancies and IFDs treated adjunctively with sargramostim at a single institution were analyzed in a retrospective cohort review. Further, a systematic review of published reports of rhu GM-CSF for IFDs was also conducted. Results: Among 65 cases, 15 were newly described pediatric patients and 50 were previously published cases of IFDs treated with rhu GM-CSF. Among the newly reported pediatric patients, IFDs were caused by Candida spp., Trichosporon sp., and molds (Aspergillus spp., Rhizopus sp., Lichtheimia sp., and Scedosporium sp). Twelve (80%) were neutropenic at baseline, and 12 (80%) were refractory to antifungal therapy. Among 12 evaluable patients, the overall response rate was 92% (8 [67%] complete responses, 3 [25%] partial responses, and 1 [8%] stable). Treatment is ongoing in the remaining 3 patients. Among 50 published cases (15 Candida spp., 13 Mucorales, 11 Aspergillus spp., 11 other organisms), 20 (40%) had baseline neutropenia and 36 (72%) were refractory to standard therapy before rhu GM-CSF administration. Consistent with responses in the newly reported patients, the overall response rate in the literature review was 82% (40 [80%] complete responses, 1 [2%] partial response, and 9 [18%] no response). Conclusions: Sargramostim may be a potential adjunctive immunomodulator for selected patients with hematological malignancies and refractory IFDs.

A balance between vector survival and virus transmission is achieved through JAK/STAT signaling inhibition by a plant virus.

Viruses pose a great threat to animal and plant health worldwide, with many being dependent on insect vectors for transmission between hosts. While the virus-host arms race has been well established, how viruses and insect vectors adapt to each other remains poorly understood. Begomoviruses comprise the largest genus of plant-infecting DNA viruses and are exclusively transmitted by the whitefly Bemisia tabaci. Here, we show that the vector Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway plays an important role in mediating the adaptation between the begomovirus tomato yellow leaf curl virus (TYLCV) and whiteflies. We found that the JAK/STAT pathway in B. tabaci functions as an antiviral mechanism against TYLCV infection in whiteflies as evidenced by the increase in viral DNA and coat protein (CP) levels after inhibiting JAK/STAT signaling. Two STAT-activated effector genes, BtCD109-2 and BtCD109-3, mediate this anti-TYLCV activity. To counteract this vector immunity, TYLCV has evolved strategies that impair the whitefly JAK/STAT pathway. Infection of TYLCV is associated with a reduction of JAK/STAT pathway activity in whiteflies. Moreover, TYLCV CP binds to STAT and blocks its nuclear translocation, thus, abrogating the STAT-dependent transactivation of target genes. We further show that inhibition of the whitefly JAK/STAT pathway facilitates TYLCV transmission but reduces whitefly survival and fecundity, indicating that this JAK/STAT-dependent TYLCV-whitefly interaction plays an important role in keeping a balance between whitefly fitness and TYLCV transmission. This study reveals a mechanism of plant virus-insect vector coadaptation in relation to vector survival and virus transmission.

The association between ambient UVB dose and ANCA-associated vasculitis relapse and onset.

BACKGROUND: The aetiology of ANCA-associated vasculitis (AAV) and triggers of relapse are poorly understood. Vitamin D (vitD) is an important immunomodulator, potentially responsible for the observed latitudinal differences between granulomatous and non-granulomatous AAV phenotypes. A narrow ultraviolet B spectrum induces vitD synthesis (vitD-UVB) via the skin. We hypothesised that prolonged periods of low ambient UVB (and by extension vitD deficiency) are associated with the granulomatous form of the disease and an increased risk of AAV relapse. METHODS: Patients with AAV recruited to the Irish Rare Kidney Disease (RKD) (n = 439) and UKIVAS (n = 1961) registries were studied. Exposure variables comprised latitude and measures of ambient vitD-UVB, including cumulative weighted UVB dose (CW-D-UVB), a well-validated vitD proxy. An n-of-1 study design was used to examine the relapse risk using only the RKD dataset. Multi-level models and logistic regression were used to examine the effect of predictors on AAV relapse risk, phenotype and serotype. RESULTS: Residential latitude was positively correlated (OR 1.41, 95% CI 1.14-1.74, p = 0.002) and average vitD-UVB negatively correlated (0.82, 0.70-0.99, p = 0.04) with relapse risk, with a stronger effect when restricting to winter measurements (0.71, 0.57-0.89, p = 0.002). However, these associations were not restricted to granulomatous phenotypes. We observed no clear relationship between latitude, vitD-UVB or CW-D-UVB and AAV phenotype or serotype. CONCLUSION: Our findings suggest that low winter ambient UVB and prolonged vitD status contribute to AAV relapse risk across all phenotypes. However, the development of a granulomatous phenotype does not appear to be directly vitD-mediated. Further research is needed to determine whether sufficient vitD status would reduce relapse propensity in AAV.

A Short Series of Case Reports of COVID-19 in Immunocompromised Patients.

Immunocompromised individuals are at risk of prolonged SARS-CoV-2 infection due to weaker immunity, co-morbidities, and lowered vaccine effectiveness, which may evolve highly mutated variants of SARS-CoV-2. Nonetheless, limited data are available on the immune responses elicited by SARS-CoV-2 infection, reinfections, and vaccinations with emerging variants in immunocompromised patients. We analyzed clinical samples that were opportunistically collected from eight immunocompromised individuals for mutations in SARS-CoV-2 genomes, neutralizing antibody (NAb) titers against different SARS-CoV-2 variants, and the identification of immunoreactive epitopes using a high-throughput coronavirus peptide array. The viral genome analysis revealed two SARS-CoV-2 variants (20A from a deceased patient and an Alpha variant from a recovered patient) with an eight amino-acid (aa) deletion within the N-terminal domain (NTD) of the surface glycoprotein. A higher NAb titer was present against the prototypic USA/WA1/2020 strain in vaccinated immunocompromised patients. NAb titer was absent against the Omicron variant and the cultured virus of the 20A variant with eight aa deletions in non-vaccinated patients. Our data suggest that fatal SARS-CoV-2 infections may occur in immunocompromised individuals even with high titers of NAb post-vaccination. Moreover, persistent SARS-CoV-2 infection may lead to the emergence of newer variants with additional mutations favoring the survival and fitness of the pathogen that include deletions in NAb binding sites in the SARS-CoV-2 surface glycoprotein.

Human Polyomavirus 9-An Emerging Cutaneous and Pulmonary Pathogen in Solid Organ Transplant Recipients.

IMPORTANCE: We describe the first report to our knowledge of cutaneous and systemic pathogenicity of human polyomavirus 9 in solid organ transplant recipients. OBJECTIVE: Three solid organ transplant recipients developed a widespread, progressive, violaceous, and hyperkeratotic skin eruption. All died from pulmonary and multiorgan failure around 1 year from onset of the rash. Routine clinical diagnostic testing could not identify any causative agent; therefore, samples and autopsies were investigated for novel pathogens using high-throughput sequencing. DESIGN, SETTING, AND PARTICIPANTS: This case series, including 3 solid organ transplant recipients who developed characteristic pink, violaceous, or brown hyperkeratotic papules and plaques throughout the body, was conducted at the Columbia University Medical Center. Lesional skin biopsies were collected from all 3 patients and subjected to high-throughput illumina sequencing for identification of microbial pathogens. Human polyomavirus 9 was identified in lesional skin biopsies. We subsequently collected ocular swabs, oral swabs, urine samples, and blood samples from patients, and organ tissues at autopsy in 1 patient. We investigated these samples for the presence of human polyomavirus 9 using in situ hybridization and quantitative polymerase chain reaction (PCR) assays. MAIN OUTCOMES AND MEASURES: A description of the clinical and pathologic findings of 3 patients. RESULTS: This case series study found that human polyomavirus 9 was detected in the skin biopsies of all 3 patients by a capture-based high-throughput sequencing method platform (VirCapSeq-VERT). Human polyomavirus 9 was also detected in blood, oral, ocular swabs, and urine by real-time polymerase chain reaction (PCR) assay. In situ hybridization and quantitative PCR assays were performed on the skin biopsies from 3 patients and lung autopsy of 1 patient, which showed the presence of human polyomavirus 9 messenger RNA transcripts, indicating active viral replication and pathogenesis in the skin and lungs. CONCLUSIONS AND RELEVANCE: Human polyomavirus 9 was associated with the widespread cutaneous eruption. All 3 patients had progression of cutaneous disease, accompanied by clinical deterioration, pulmonary failure, and death. One patient underwent autopsy and human polyomavirus 9 was identified in the lungs and paratracheal soft tissue. These findings suggest that human polyomavirus 9 may be associated with cutaneous and possibly pulmonary infection and death in solid organ transplant recipients.

Compassionate-use pocapavir and immunoglobulin therapy for treatment of rituximab-associated enterovirus meningoencephalitis.

A 71-year-old woman previously on rituximab treatment for rheumatoid arthritis presented with 2 years of progressive neurologic symptoms. She was found to have persistent hypogammaglobulinemia and B cell depletion despite rituximab discontinuation a year prior. MRI revealed diffuse meningeal enhancement along the entire neuroaxis. LP showed a CSF lymphocytic pleocytosis, elevated protein, and presence of enterovirus by PCR. The patient was hospitalized several times for progressive clinical and radiologic decline, though she had transient improvements following treatment with immunoglobulin therapy. Her CSF remained positive for enterovirus PCR for at least 12 months. Though two brain biopsies were non-diagnostic, pan-Enterovirus was ultimately identified using a high-throughput next-generation sequencing technique. She was treated with compassionate-use pocapavir with clinical stabilization at 4-month follow-up; however, she expired 8 months later from a bacterial pneumonia.

Isolation and Transfection of Citrus Protoplasts with Citrus Exocortis Viroid.

Viroids are RNA-based infectious agents that are single-stranded, covalently closed circular, non-coding, and naked. Unlike RNA viruses, which at least encode proteins for replication, encapsidation, and movement, lack of protein-coding capacity of viroids makes them completely reliant on host for replication and movement. The high genetic diversity in viroids is believed to be due to the absence of proof-reading activity of the host RNA polymerases, the large population size, and the rapid rate of replication. Protoplasts are viable plant cells that are prepared by enzymatic removal of cell walls. Plant protoplasts provide a synchronous single-cell system for studying early events of viroid infection such as replication and genetic diversity at the cellular level. A simple and efficient method to isolate and transfect citrus protoplasts with transcript RNA of citrus exocortis viroid is described in this chapter.

SARS-CoV-2 Sequence Analysis during COVID-19 Case Surge, Liberia, 2021.

In June 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases surged in Liberia. SARS-CoV-2 sequences from patients hospitalized during March-July 2021 revealed the Delta variant was in Liberia in early March and was dominant in June, irrespective of geography. Mutations and deletions suggest multiple SARS-CoV-2 Delta variant introductions.

Plasticity of the lettuce infectious yellows virus minor coat protein (CPm) in mediating the foregut retention and transmission of a chimeric CPm mutant by whitefly vectors.

Transmission of the crinivirus, lettuce infectious yellows virus (LIYV), is determined by a minor coat protein (CPm)-mediated virion retention mechanism located in the foregut of its whitefly vector. To better understand the functions of LIYV CPm, chimeric CPm mutants engineered with different lengths of the LIYV CPm amino acid sequence and that of the crinivirus, lettuce chlorosis virus (LCV), were constructed based on bioinformatics and sequence alignment data. The 485 amino acid-long chimeric CPm of LIYV mutant, CPmP-1, contains 60 % (from position 3 to 294) of LCV CPm amino acids. The chimeric CPm of mutants CPmP-2, CPmP-3 and CPmP-4 contains 46 (position 3 to 208), 51 (position 3 to 238) and 41 % (position 261 to 442) of LCV CPm amino acids, respectively. All four mutants moved systemically, expressed the chimeric CPm and formed virus particles. However, following acquisition feeding of the virus preparations, only CPmP-1 was retained in the foreguts of a significant number of vectors and transmitted. In immuno-gold labelling transmission electron microscopy (IGL-TEM) analysis, CPmP-1 particles were distinctly labelled by antibodies directed against the LCV but not LIYV CPm. In contrast, CPmP-4 particles were not labelled by antibodies directed against the LCV or LIYV CPm, while CPmP-2 and -3 particles were weakly labelled by anti-LIYV CPm but not anti-LCV CPm antibodies. The unique antibody recognition and binding pattern of CPmP-1 was also displayed in the foreguts of whitefly vectors that fed on CPmP-1 virions. These results are consistent with the hypothesis that the chimeric CPm of CPmP-1 is incorporated into functional virions, with the LCV CPm region being potentially exposed on the surface and accessible to anti-LCV CPm antibodies.