Infection Assays Research Articles

Structure-based design of a SARS-CoV-2 Omicron-specific inhibitor

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) introduced a relatively large number of mutations, including three mutations in the highly conserved heptad repeat 1 (HR1) region of the spike glycoprotein (S) critical for its membrane fusion activity. We show that one of these mutations, N969K induces a substantial displacement in the structure of the heptad repeat 2 (HR2) backbone in the HR1HR2 postfusion bundle. Due to this mutation, fusion-entry peptide inhibitors based on the Wuhan strain sequence are less efficacious. Here, we report an Omicron-specific peptide inhibitor designed based on the structure of the Omicron HR1HR2 postfusion bundle. Specifically, we inserted an additional residue in HR2 near the Omicron HR1 K969 residue to better accommodate the N969K mutation and relieve the distortion in the structure of the HR1HR2 postfusion bundle it introduced. The designed inhibitor recovers the loss of inhibition activity of the original longHR2_42 peptide with the Wuhan strain sequence against the Omicron variant in both a cell-cell fusion assay and a vesicular stomatitis virus (VSV)-SARS-CoV-2 chimera infection assay, suggesting that a similar approach could be used to combat future variants. From a mechanistic perspective, our work suggests the interactions in the extended region of HR2 may mediate the initial landing of HR2 onto HR1 during the transition of the S protein from the prehairpin intermediate to the postfusion state.

Identification and validation of novel non-nucleoside class of molecules inhibiting the dengue virus replication

There is currently no drug available to treat mosquito-borne dengue. The C-terminal RNA-dependent RNA polymerase (RdRp) domain in the non-structural type 5 (NS5) protein of the dengue virus (DENV) is essential for viral RNA synthesis and replication, and therefore, it is an attractive target for the anti-dengue drug development. We report herein the discovery and validation of two novel non-nucleoside classes of small molecules as DENV RdRp inhibitors. Firstly, using the refined X-ray structure of the DENV NS5 RdRp domain (PDB-ID: 4V0R), we conducted docking, binding free-energy studies, and short-scale molecular dynamics simulation to investigate the binding sites of known small molecules that led to the optimized protein–ligand complex. Subsequently, protein structure-based screening of a commercial database (∼500,000 synthetic compounds), pre-filtered for the drug-likeness, led to the top-ranked 171 molecules, which was then subjected to structural diversity analysis and clustering. This process led to six structurally distinct and best-scored compounds that were procured from the commercial vendor, and then subjected to the in vitro testing in the MTT and dengue infection assays. It revealed two unique and structurally unique compounds, KKR-D-02 and KKR-D-03, exhibiting 84 and 81% reductions, respectively, in DENV copy number in repeated assays in comparison to the virus-infected cell controls. These active compounds represent novel scaffolds for further structure-based discovery of novel candidate molecules for the intervention of dengue.Communicated by Ramaswamy H. Sarma

Varicella Zoster Virus infects mucosal associated Invariant T cells.

Mucosal Associated Invariant T (MAIT) cells are innate-like T cells that respond to conserved pathogen-derived vitamin B metabolites presented by the MHC class I related-1 molecule (MR1) antigen presentation pathway. Whilst viruses do not synthesize these metabolites, we have reported that varicella zoster virus (VZV) profoundly suppresses MR1 expression, implicating this virus in manipulation of the MR1:MAIT cell axis. During primary infection, the lymphotropism of VZV is likely to be instrumental in hematogenous dissemination of virus to gain access to cutaneous sites where it clinically manifests as varicella (chickenpox). However, MAIT cells, which are found in the blood and at mucosal and other organ sites, have yet to be examined in the context of VZV infection. The goal of this study was to examine any direct impact of VZV on MAIT cells. Using flow cytometry, we interrogated whether primary blood derived MAIT cells are permissive to infection by VZV whilst further analysing differential levels of infection between various MAIT cell subpopulations. Changes in cell surface extravasation, skin homing, activation and proliferation markers after VZV infection of MAIT cells was also assessed via flow cytometry. Finally the capacity of MAIT cells to transfer infectious virus was tested through an infectious center assay and imaged via fluorescence microscopy. We identify primary blood-derived MAIT cells as being permissive to VZV infection. A consequence of VZV infection of MAIT cells was their capacity to transfer infectious virus to other permissive cells, consistent with MAIT cells supporting productive infection. When subgrouping MAIT cells by their co- expression of a variety cell surface markers, there was a higher proportion of VZV infected MAIT cells co-expressing CD4+ and CD4+/CD8+ MAIT cells compared to the more phenotypically dominant CD8+ MAIT cells, whereas infection was not associated with differences in co-expression of CD56 (MAIT cell subset with enhanced responsiveness to innate cytokine stimulation), CD27 (co-stimulatory) or PD-1 (immune checkpoint). Infected MAIT cells retained high expression of CCR2, CCR5, CCR6, CLA and CCR4, indicating a potentially intact capacity for transendothelial migration, extravasation and trafficking to skin sites. Infected MAIT cells also displayed increased expression of CD69 (early activation) and CD71 (proliferation) markers. These data identify MAIT cells as being permissive to VZV infection and identify impacts of such infection on co- expressed functional markers.

Application of Size Exclusion Chromatography with Multiangle Light Scattering in the Analytical Development of a Preclinical Stage Gene Therapy Program.

To provide safe recombinant adeno-associated viruses (rAAV) to patients, scalable manufacturing processes are required. However, these processes may introduce impurities that impact the performance and quality of the final drug product. Empty rAAV capsids are product-related impurities. Regulatory guidance requires that accurate analytical methods be implemented early in product development to measure the level of empty capsids. A process confirmation vector, produced from 200 L production, was used to develop and optimize a size exclusion chromatography with UV and multiangle light scattering (SEC-MALS) method. Vector produced from a 500 L production was used to assess the full-to-empty ratio using the following analytical methods: sedimentation velocity analytical ultracentrifugation (SV-AUC), droplet digital PCR (ddPCR) with capsid enzyme-linked immunosorbent assay (ELISA), bulk absorbance at 260/280 nm, cryogenic electron microscopy, and SEC-MALS. This test article was used for a 30-day, non-Good Laboratory Practices animal study that assessed biodistribution of the product (STRX-330). SEC-MALS outperformed the other methods and correlated well with SV-AUC values of full-to-empty particles. In addition, SEC-MALS agreed with ddPCR and ELISA measurements for vector genomes/mL and capsid particles/mL, respectively. SEC-MALS was linear, accurate, and precise while achieving chromatography quality control (QC) recommendations. Compared to other stability-indicating assays, SEC-MALS performed similarly to ddPCR, capsid ELISA, and infectivity assays in accelerated stress studies. In response to alkaline, but not acidic stress, SEC-MALS indicated distinct changes in the DNA content of the monomer Adeno-associated viruses (AAV) peak for STRX-330, which was supported by ddPCR data. Conversely, acidic treatment resulted in more aggregated vector, but did not impact the DNA content. This work indicates that SEC-MALS is a valuable analytical tool in the analytical development and QC testing of AAV. In addition, this work suggests that SEC-MALS can provide fundamental understanding of AAV in response to environmental stress. This may impact steps of the manufacturing process to minimize conditions that reduce performance.

Transmission of stimulus-induced epigenetic changes through cell division is coupled to continuous transcription factor activity.

Trained immunity, or innate immune memory, has been attributed to the long-term retention of stimulus-induced histone post-translational modifications (PTMs) following clearance of the initial stimulus. Yet, it remains unknown how this epigenetic memory can persist for months in dividing cells given the lack of any known mechanism for stimulus-induced histone PTMs to be directly copied from parent to daughter strand during DNA replication. Here, using time course RNA-seq, ChIP-seq, and infection assays, we find that trained macrophages are transcriptionally, epigenetically, and functionally re-programmed for at least 14 cell divisions after stimulus washout. However, the epigenetic changes observed after multiple rounds of cell division do not result from the self-sustained propagation of stimulus-induced epigenetic changes through cell division. Instead, long-lasting epigenetic differences between trained and non-trained cells are always coupled with changes in transcription factor (TF) activity, emphasizing the central role played by TFs, and gene expression changes more broadly, in driving the transmission of stimulus-induced epigenetic changes across cell divisions.

Importance, Applications and Features of Assays Measuring SARS-CoV-2 Neutralizing Antibodies

More than three years ago, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused the unforeseen COVID-19 pandemic with millions of deaths. In the meantime, SARS-CoV-2 has become endemic and is now part of the repertoire of viruses causing seasonal severe respiratory infections. Due to several factors, among them the development of SARS-CoV-2 immunity through natural infection, vaccination and the current dominance of seemingly less pathogenic strains belonging to the omicron lineage, the COVID-19 situation has stabilized. However, several challenges remain and the possible new occurrence of highly pathogenic variants remains a threat. Here we review the development, features and importance of assays measuring SARS-CoV-2 neutralizing antibodies (NAbs). In particular we focus on in vitro infection assays and molecular interaction assays studying the binding of the receptor binding domain (RBD) with its cognate cellular receptor ACE2. These assays, but not the measurement of SARS-CoV-2-specific antibodies per se, can inform us of whether antibodies produced by convalescent or vaccinated subjects may protect against the infection and thus have the potential to predict the risk of becoming newly infected. This information is extremely important given the fact that a considerable number of subjects, in particular vulnerable persons, respond poorly to the vaccination with the production of neutralizing antibodies. Furthermore, these assays allow to determine and evaluate the virus-neutralizing capacity of antibodies induced by vaccines and administration of plasma-, immunoglobulin preparations, monoclonal antibodies, ACE2 variants or synthetic compounds to be used for therapy of COVID-19 and assist in the preclinical evaluation of vaccines. Both types of assays can be relatively quickly adapted to newly emerging virus variants to inform us about the magnitude of cross-neutralization, which may even allow us to estimate the risk of becoming infected by newly appearing virus variants. Given the paramount importance of the infection and interaction assays we discuss their specific features, possible advantages and disadvantages, technical aspects and not yet fully resolved issues, such as cut-off levels predicting the degree of in vivo protection.

Host Cell Rap1b mediates cAMP-dependent invasion by Trypanosoma cruzi.

Trypanosoma cruzi cAMP-mediated invasion has long been described, however, the detailed mechanism of action of the pathway activated by this cyclic nucleotide still remains unknown. We have recently demonstrated a crucial role for Epac in the cAMP-mediated invasion of the host cell. In this work, we gathered evidence indicating that the cAMP/Epac pathway is activated in different cells lines. In accordance, data collected from pull-down experiments designed to identify only the active form of Rap1b (Rap1b-GTP), and infection assays using cells transfected with a constitutively active mutant of Rap1b (Rap1b-G12V), strongly suggest the participation of Rap1b as mediator of the pathway. In addition to the activation of this small GTPase, fluorescence microscopy allowed us to demonstrate the relocalization of Rap1b to the entry site of the parasite. Moreover, phospho-mimetic and non-phosphorylable mutants of Rap1b were used to demonstrate a PKA-dependent antagonistic effect on the pathway, by phosphorylation of Rap1b, and potentially of Epac. Finally, Western Blot analysis was used to determine the involvement of the MEK/ERK signalling downstream of cAMP/Epac/Rap1b-mediated invasion.

Polycaprolactone nanofibers colonized by HeLa cells: an alternative for in vitro investigation of fungal infection.

Introduction: In vitro 3D equivalent tissues can be used for studies of fungal infections. Objectives: To develop 3D electrospun nanofibers using polycaprolactone (PCL) colonized by HeLa cells as a possible in vitro model for the investigation of fungal infection. Materials & methods: A PCL solution was synthesized and electrospun. HeLa cells were cultured on the nanostructured PCL scaffolds, forming a 3D structure. Physicochemical, biological and Candida albicans infection assays were performed in this model. Results: The nanostructured PCL scaffolds showed favorable physicochemical characteristics and allowed the colonization of HeLa cells, which showed indications of extracellular matrix production. Conclusions: Fungal infection was evidenced in the 3D nanostructured PCL scaffolds, being viable, economical and compatible to study fungal infections in vitro.

Assessment of esxA, hsp18, and 16S transcript expression as a measure of Mycobacterium leprae viability: A comparison with the mouse footpad assay

Objectives Shepard’s mouse footpad (MFP) assay is the definitive method for ascertaining Mycobacterium leprae viability; however, this technique is laborious and prolonged. Alternate methods that alleviate burden and time while maintaining sensitivity would benefit clinical and experimental studies. We compared a molecular viability assay (MVA), which measures normalized hsp18 and esxA transcript expression, with growth in the MFP as a determinant of M. leprae viability. Methods Receiver operating characteristic curve analyses were performed to determine viability cut-off values for M. leprae expression of hsp18, esxA, and 16S transcripts. To verify these values, immunocompetent BALB/c mice were inoculated in the footpads with a high dose of viable M. leprae. Footpads were harvested on Day 1 and at various timepoints up to 12 months post infection. M. leprae viability was determined by MVA or by sub-inoculating bacilli into passage mice per Shepard’s MFP assay. Results Cut-off levels for expression of each transcript for M. leprae viability were established with high confidence (CI = 99%, P < 0.0001). In the high-dose infection assay, M. leprae exhibited a significant decrease in transcript detection by three months post-infection. Expression of hsp18 and esxA transcripts by M. leprae correlated with bacterial growth in the MFP assay. In contrast, 16S transcripts were detected in M. leprae populations confirmed dead by the MFP assay. Conclusion The MVA, which is a sensitive, specific, and rapid viability indicator, was directly validated by the MFP assay. This study confirms the accuracy of the MVA for determining M. leprae viability.

Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells.

Coronavirus disease (COVID-19) is an infectious disease caused by the SARS coronavirus 2 (SARS-CoV-2) virus. Direct assessment, detection, and quantitative analysis using high throughput methods like single-cell RNA sequencing (scRNAseq) is imperative to understanding the host response to SARS-CoV-2. One barrier to studying SARS-CoV-2 in the laboratory setting is the requirement to process virus-infected cell cultures, and potentially infectious materials derived therefrom, under Biosafety Level 3 (BSL-3) containment. However, there are only 190 BSL3 laboratory facilities registered with the U.S. Federal Select Agent Program, as of 2020, and only a subset of these are outfitted with the equipment needed to perform high-throughput molecular assays. Here, we describe a method for preparing non-hazardous RNA samples from SARS-CoV-2 infected cells, that enables scRNAseq analyses to be conducted safely in a BSL2 facility-thereby making molecular assays of SARS-CoV-2 cells accessible to a much larger community of researchers. Briefly, we infected African green monkey kidney epithelial cells (Vero-E6) with SARS-CoV-2 for 96 hours, trypsin-dissociated the cells, and inactivated them with methanol-acetone in a single-cell suspension. Fixed cells were tested for the presence of infectious SARS-CoV-2 virions using the Tissue Culture Infectious Dose Assay (TCID50), and also tested for viability using flow cytometry. We then tested the dissociation and methanol-acetone inactivation method on primary human lung epithelial cells that had been differentiated on an air-liquid interface. Finally, we performed scRNAseq quality control analysis on the resulting cell populations to evaluate the effects of our virus inactivation and sample preparation protocol on the quality of the cDNA produced. We found that methanol-acetone inactivated SARS-CoV-2, fixed the lung epithelial cells, and could be used to obtain noninfectious, high-quality cDNA libraries. This methodology makes investigating SARS-CoV-2, and related high-containment RNA viruses at a single-cell level more accessible to an expanded community of researchers.

Development of a novel double-antibody sandwich quantitative ELISA for detecting SADS-CoV infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteric alphacoronavirus that can cause acute diarrhea, vomiting, dehydration, and death of newborn piglets. In this study, we developed a double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) for detection of SADS-CoV by using an anti-SADS-CoV N protein rabbit polyclonal antibody (PAb) and a specific monoclonal antibody (MAb) 6E8 against the SADS-CoV N protein. The PAb was used as the capture antibodies and HRP-labeled 6E8 as the detector antibody. The detection limit of the developed DAS-qELISA assay was 1ng/mL of purified antigen and 101.08TCID50/mL of SADS-CoV, respectively. Specificity assays showed that the developed DAS-qELISA has no cross-reactivity with other swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV). Three-day-old piglets were challenged with SADS-CoV and collected anal swab samples which were screened for the presence of SADS-CoV by using DAS-qELISA and reverse transcriptase PCR (RT-PCR). The coincidence rate of the DAS-qELISA and RT-PCR was 93.93%, and the kappa value was 0.85, indicating that DAS-qELISA is a reliable method for applying antigen detection of clinical samples. KEY POINTS: • The first double-antibody sandwich quantitative enzyme-linked immunosorbent assay for detection SADS-CoV infection. • The custom ELISA is useful for controlling the SADS-CoV spread.

Inactivation of SARS-CoV-2 on Surfaces by Cold-Plasma-Generated Reactive Species.

A Cold Atmospheric Plasma (CAP) apparatus was designed and developed for SARS-CoV-2 killing as evaluated by pseudotyped viral infectivity assays. The reactive species generated by the plasma system was fully characterized by using Optical Emission Spectroscopy (OES) measurement under given conditions such as plasma power, flow rate, and treatment time. A variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS) were identified from plasma plume with energies of 15-72 eV in the frequency range between 500-1000 nm. Systematic virus killing experiments were carried out, and the efficacy of CAP treatment in reducing SARS-CoV-2 viral infectivity was significant following treatment for 8 s, with further enhancement of killing upon longer exposures of 15-120 s. We correlated killing efficacy with the reactive species in terms of type, intensity, energy, and frequency. These experimental results demonstrate effective cold plasma virus killing via ROS and RNS under ambient conditions.

A High Viral Load in Urine Correlates With Acute Kidney Injury and Poor Outcomes in Hospitalized Patients With Severe Fever With Thrombocytopenia Syndrome: A Noninvasive and Convenient Prognostic Marker.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with an extensive geographical distribution and high mortality rate. To date, the role of SFTS virus (SFTSV) in urine is still elusive. We aimed to explore the relationship between urinary bunyavirus and acute kidney injury (AKI) and mortality in patients with SFTS. Urine samples were collected from 102 patients to quantify SFTSV load in urine (U-SFTSV). Patient renal function was evaluated on admission. Receiver operating characteristic (ROC) curve and logistic regression analysis were performed to evaluate the predictive value of U-SFTSV. Viral infectivity assays in Vero cells were performed from 10 urine samples. The U-SFTSV level was positively correlated with SFTSV load in plasma (r = 0.624) and indicators of renal damage. The U-SFTSV level was identified as an independent risk factor for SFTS-associated AKI (odds ratio, 3.631; P = .019). The U-SFTSV showed great value in predicting the fatal outcome of SFTS patients with high area under curve (0.881). The Kaplan-Meier survival comparison showed that patients with U-SFTSV levels greater than 6379 copies/mL were at a higher risk of death within 28 days after onset. In addition, 4 urine samples with high U-SFTSV levels were infectious. Our large cohort study identified that the U-SFTSV level is a novel convenient and noninvasive predictive biomarker for incidence of AKI and poor outcome of patients with SFTS. Urine specimens could be a source of SFTSV infection in humans.

Diversity and pathogenicity of species of Botrytis, Cladosporium, Neopestalotiopsis and Pestalotiopsis causing flower diseases of macadamia in Australia

AbstractFlower diseases threaten macadamia production, but there is a limited understanding of their aetiology. This study was undertaken to establish the ecology of the causal agents in commercial orchards in Australia. Raceme samples at different developmental stages were collected from 42 commercial macadamia orchards during the 2018–2020 flowering seasons. Morphological and molecular phylogenetic analyses of the isolates obtained from the samples with symptoms revealed that 16 fungal species, including two Botrytis spp., six Cladosporium spp., six Neopestalotiopsis spp. and two Pestalotiopsis spp., were associated with the flower blights. Twelve of the fungal species were identified as new records in macadamia. A comparison of the fungal genera showed that Neopestalotiopsis spp. (48%) and Cladosporium spp. (47%) were the most prevalent in the majority of macadamia production regions and were obtained from all four raceme developmental stages. The genus Botrytis was more commonly distributed (16%) in the production areas where humidity was high and was mostly isolated from the mature developmental raceme stages. Based on in planta infection assays under field conditions, the fungal pathogens varied in aggressiveness on macadamia racemes, with B. cinerea, B. macadamiae, C. cladosporioides, C. devikae, N. drenthii, N. macadamiae, N. maddoxii, N. vheenae, P. macadamiae and P. telopeae as the most aggressive and C. angulosum and C. proteacearum as the least aggressive. This study improves our understanding of the aetiology and distribution of the causal agents of flower blight complex in macadamia.

Early antitermination in the atypical coliphage mEp021 mediated by the Gp17 protein

The coliphage mEp021 belongs to a phage group with a unique immunity repressor, and its life cycle requires the host factor Nus. mEp021 has been classified as non-lambdoid based on its specific characteristics. The mEp021 genome carries a gene encoding an Nλ-like antiterminator protein, termed Gp17, and three nut sites (nutL, nutR1, and nutR2). Analysis of plasmid constructs containing these nut sites, a transcription terminator, and a GFP reporter gene showed high levels of fluorescence when Gp17 was expressed, but not in its absence. Like lambdoid N proteins, Gp17 has an arginine-rich motif (ARM), and mutations in its arginine codons inhibit its function. In infection assays using the mutant phage mEp021ΔGp17::Kan (where gp17 has been deleted), gene transcripts located downstream of transcription terminators were obtainedonly when Gp17 was expressed. In contrast to phage lambda, mEp021 virus particle production was partially restored (>1/3 relative to wild type) when nus mutants (nusA1, nusB5, nusC60, and nusE71) were infected with mEp021 and Gp17 was overexpressed. Our results suggest that RNA polymerase reads through the third nut site (nutR2), which is more than 7.9 kbp downstream of nutR1.

A Single Dose, Thermostable, Trivalent Human Papillomavirus Vaccine Formulated Using Atomic Layer Deposition

Formulations of human papillomavirus (HPV) 16, 18, and 31 L1 capsomere protein antigens were spray dried to obtain glassy microspheres that were then coated by atomic layer deposition (ALD) with nanometer-thin protective layers of alumina. Spray-drying was used to formulate human papillomavirus (HPV) 16, 18, and 31 L1 capsomere protein antigens within glassy microspheres to which nanoscopic protective layers of alumina were applied using ALD. Suspensions of alumina-coated, capsomere-containing microparticles were administered in a single dose to mice. ALD-deposited alumina coatings provided thermostability and a delayed in vivo release of capsomere antigens, incorporating both a prime and a boost dose in one injection. Total serotype-specific antibody titers as well as neutralizing titers determined from pseudovirus infectivity assays were unaffected by incubation of the ALD-coated vaccines for at 4, 50, or 70 °C for three months prior to administration. In addition, even after incubation for three months at 70 °C, single doses of ALD-coated vaccines produced both higher total antibody responses and higher neutralizing responses than control immunizations that used two doses of conventional liquid formulations stored at 4 °C.

Flavescence dorée phytoplasma enters insect cells by a clathrin-mediated endocytosis allowing infection of its insect vector

To perform its propagative and circulative cycle into its insect vector, the flavescence dorée phytoplasma invades different cell types. Clathrin-mediated endocytosis is used by a wide range of bacteria to infect eukaryote cells. Among the insect proteins interacting with the phytoplasma adhesin VmpA, we identified the adaptor protein complex AP-1 and AP-2 suggesting that phytoplasmas could enter the insect cells via clathrin-mediated endocytosis. By infection assays of insect cells in culture, we showed that phytoplasmas entry into Drosophila S2 cells was more efficient than infection of the Euva cell line developed from the insect vector Euscelidius variegatus. Chlorpromazine, cytochalasin D and knockdown of clathrin heavy chain (chc) gene expression using RNA interference inhibited entry of phytoplasmas into S2 cells. During invasion of S2 cells, phytoplasmas were observed very closed to recombinant GFP-labelled clathrin light chain. To verify the role of clathrin in the insect colonization by phytoplasmas, RNAi was performed via artificial feeding of chc dsRNA by the vector E. variegatus. This decreased the expression of chc gene in the midgut and heads of E. variegatus. The chc lower expression correlated to a decreased of midgut and salivary gland cells colonization after the insects had ingested phytoplasmas from infected plants. In conclusion, results indicate that clathrin is important for the FD phytoplasma to enter insect cells and colonize its insect vector.

Effect of the pseudomonas metabolites HQNO on the Toxoplasma gondii RH strain in vitro and in vivo

Toxoplasmosis is a widespread disease in humans and animals. Currently, toxoplasmosis chemotherapy options are limited due to severe side effects. There is an urgent need to develop new drugs with better efficacy and few side effects. HQNO, a cytochrome bc1 and type II NADH inhibitor in eukaryotes and bacteria, possesses extensive bioactivity. In this study, the cytotoxicity of HQNO was evaluated in Vero cells. The in vitro effects of HQNO were determined by plaque assay and qPCR assay. To determine the in vivo effect of HQNO, pharmacokinetic experiments and in vivo infection assays were performed in mice. The changes in tachyzoites after HQNO exposure were examined by transmission electron microscopy (TEM), MitoTracker Red CMXRos staining, ROS detection and ATP detection. HQNO inhibited T. gondii invasion and proliferation with an EC50 of 0.995 μM. Pharmacokinetic experiments showed that the Cmax of HQNO (20 mg/kg·bw) was 3560 ± 1601 ng/mL (13.73 μM) in healthy BALB/c mouse plasma with no toxicity in vivo. Moreover, HQNO induced a significant decrease in the parasite burden load of T. gondii in mouse peritoneum. TEM revealed alterations in the mitochondria of T. gondii. Further assays verified that HQNO also decreased the mitochondrial membrane potential (ΔΨm) and ATP levels and enhanced the level of reactive oxygen species (ROS) in T. gondii. Hence, HQNO exerted anti-T. gondii activity, which may be related to the damage to the mitochondrial electron transport chain (ETC).

LRRC15 mediates an accessory interaction with the SARS-CoV-2 spike protein.

The interactions between Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and human host factors enable the virus to propagate infections that lead to Coronavirus Disease 2019 (COVID-19). The spike protein is the largest structural component of the virus and mediates interactions essential for infection, including with the primary angiotensin-converting enzyme 2 (ACE2) receptor. We performed two independent cell-based systematic screens to determine whether there are additional proteins by which the spike protein of SARS-CoV-2 can interact with human cells. We discovered that in addition to ACE2, expression of LRRC15 also causes spike protein binding. This interaction is distinct from other known spike attachment mechanisms such as heparan sulfates or lectin receptors. Measurements of orthologous coronavirus spike proteins implied the interaction was functionally restricted to SARS-CoV-2 by accessibility. We localized the interaction to the C-terminus of the S1 domain and showed that LRRC15 shares recognition of the ACE2 receptor binding domain. From analyzing proteomics and single-cell transcriptomics, we identify LRRC15 expression as being common in human lung vasculature cells and fibroblasts. Levels of LRRC15 were greatly elevated by inflammatory signals in the lungs of COVID-19 patients. Although infection assays demonstrated that LRRC15 alone is not sufficient to permit viral entry, we present evidence that it can modulate infection of human cells. This unexpected interaction merits further investigation to determine how SARS-CoV-2 exploits host LRRC15 and whether it could account for any of the distinctive features of COVID-19.

Membrane binding is involved in the action of broadly neutralizing influenza antibodies.

Influenza poses a major health issue globally. Characterized by annual epidemics with mild to severe symptoms and even a significant number of deaths, the virus has historically resulted in substantial economic and societal problems in the world. Neutralizing antibodies that bind to the membrane-proximal region of hemagglutinin (HA) provide considerable protection against the infection by blocking the fusion of virus to the host cell. Here, using an array of advanced modeling and simulation techniques employed to a very large molecular assembly, for the first time, we develop a high-resolution structural model of full-length, antibody-bound HA in a native viral membrane and describe key molecular interactions that determine the binding affinity of the antibody to the viral membrane. The model allowed us to identify key residues in the antibody that interact with and bind to the membrane. Guided by the simulation predictions, the virus neutralization activity of the antibody was evaluated by mutagenesis experiments and infectivity assays. In alignment with the simulation results, the experiments show that mutating the predicted basic residues located away from the antigen binding site in the antibody decreases the neutralizing activity by up to an order of magnitude. Based on the results, we propose that the direct interaction of membrane-proximal antibodies with the viral membrane plays a role in their neutralizing activity against the virus. Introducing additional basic side chains enhancing phospholipid-binding is expected to further stabilize the HA-antibody. Given the rapid evolution of the influenza virus, the developed model provides a framework for rational design and development of more effective therapeutic antibodies.