Low Multiplicity Of Infection Research Articles

Robust differential expression testing for single-cell CRISPR screens at low multiplicity of infection.

Single-cell CRISPR screens (perturb-seq) link genetic perturbations to phenotypic changes in individual cells. The most fundamental task in perturb-seq analysis is to test for association between a perturbation and a count outcome, such as gene expression. We conduct the first-ever comprehensive benchmarking study of association testing methods for low multiplicity-of-infection (MOI) perturb-seq data, finding that existing methods produce excess false positives. We conduct an extensive empirical investigation of the data, identifying three core analysis challenges: sparsity, confounding, and model misspecification. Finally, we develop an association testing method - SCEPTRE low-MOI - that resolves these analysis challenges and demonstrates improved calibration and power.

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high-titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and efficient gene transfer to human CD34+ hematopoietic stem and progenitor cells (HSPC). This LVV contains locus control region cores expressing an anti-sickling βAS3-globin gene and two microRNA adapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbAAS3 + HbF), while concurrently reducing sickle hemoglobin (HbS). The reduction in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34+ cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo. With high titer and enhanced transduction of HSPC at low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to reduce costs and help improve accessibility to gene therapy for SCD.

Accumulation Dynamics of Defective Genomes during Experimental Evolution of Two Betacoronaviruses.

Virus-encoded replicases often generate aberrant RNA genomes, known as defective viral genomes (DVGs). When co-infected with a helper virus providing necessary proteins, DVGs can multiply and spread. While DVGs depend on the helper virus for propagation, they can in some cases disrupt infectious virus replication, impact immune responses, and affect viral persistence or evolution. Understanding the dynamics of DVGs alongside standard viral genomes during infection remains unclear. To address this, we conducted a long-term experimental evolution of two betacoronaviruses, the human coronavirus OC43 (HCoV-OC43) and the murine hepatitis virus (MHV), in cell culture at both high and low multiplicities of infection (MOI). We then performed RNA-seq at regular time intervals, reconstructed DVGs, and analyzed their accumulation dynamics. Our findings indicate that DVGs evolved to exhibit greater diversity and abundance, with deletions and insertions being the most common types. Notably, some high MOI deletions showed very limited temporary existence, while others became prevalent over time. We observed differences in DVG abundance between high and low MOI conditions in HCoV-OC43 samples. The size distribution of HCoV-OC43 genomes with deletions differed between high and low MOI passages. In low MOI lineages, short and long DVGs were the most common, with an additional cluster in high MOI lineages which became more prevalent along evolutionary time. MHV also showed variations in DVG size distribution at different MOI conditions, though they were less pronounced compared to HCoV-OC43, suggesting a more random distribution of DVG sizes. We identified hotspot regions for deletions that evolved at a high MOI, primarily within cistrons encoding structural and accessory proteins. In conclusion, our study illustrates the widespread formation of DVGs during betacoronavirus evolution, influenced by MOI and cell- and virus-specific factors.

Evaluation of Lentiviral Based Gene Delivery System in Adherent and Suspension in vitro Cell Models

Lentiviruses are a highly robust gene delivery system capable of in vitro and in vivo gene transfer into multiple cell types. Recent fourth-generation lentiviral systems have been designed for enhanced safety, however, the increased recombination events required to produce infective lentiviral particles may reduce production efficiency. A set protocol for all types of target cells is not recommended and optimization of conditions for gene transfer into different target cells is required. In this study, we aim to evaluate the efficiency and reproducibility of lentiviral production using a fourth-generation lentiviral packaging system and identify optimal parameters for successful transduction in two different cell models, adherent and suspension cells. Lentiviral production, effect of viral volume, sustained gene expression and transduction adjuvants on adherent and suspension gene- cell models were evaluated. Transfection and transduction efficiency of lentiviruses was evaluated by fluorescence microscopy and flow cytometry. This study demonstrates that production of green fluorescent protein (GFP)- lentiviruses using the fourth-generation lentiviral packaging is consistent and reproducible. Optimal transduction of adherent cell types is achieved at lower multiplicity of infection (MOI) compared to suspension cells and produces GFP-expressing cells with higher intensity. Expression of GFP is sustained in all cell types over multiple passages. Polycation DEAE-dextran was determined to improve transduction in suspension cells, however, provides similar transduction efficiency as polybrene in adherent cells. In conclusion, fourth generation lentiviral system reproducibly generates high titre lentiviruses capable of infecting multiple cell types, however transduction protocols for different cell types require further optimization.

Formation of Mycobacterium abscessus colonies in cellular culture in an in vitro infection model

Mycobacterium abscessus is one of the most important nontuberculous mycobacteria that cause lung diseases. In vitro infection models developed to analyze the immune response are frequently based on the addition of mycobacteria to mononuclear cells or neutrophils from peripheral blood. An important requirement of these assays is that most cells phagocytose mycobacteria, only accomplished by using large multiplicities of infection (1 or more bacteria per cell) which may not adequately reflect the inhalation of a few mycobacteria by the host. We propose modifications that try to mimic some of the conditions in which immune cells deal with mycobacteria. For the preparation of the inoculum mycobacteria are grown in solid media followed by preparation to a single cell suspension. Multiplicities of infection (number of bacteria per cell) are below 0.01. Serum-free cellular media is used to allow the growth of M. abscessus. After several days of incubation Bacterial Colonies in Cellular Culture (BCCC) develop, which are enumerated directly under an inverted microscope. These colonies may represent biofilm formation during chronic infections.•Low multiplicity of infection (below 0.01 bacteria per cell) reflects more realistically conditions encountered by immune cells in the lungs.•The surface of mycobacteria prepared for infection assays that are grown in solid media are less affected than that of mycobacteria grown in liquid media with detergents.•Colony formation in the infected cells may reflect the aggregation and biofilm formation in the lungs during chronic infection.

Isolation and Characterization of a Novel Aeromonas salmonicida-Infecting Studiervirinae Bacteriophage, JELG-KS1.

Representatives of the bacterial genus Aeromonas are some of the most notorious aquaculture pathogens associated with a range of diseases in different fish species. As the world forges toward the post-antibiotic era, alternative options for combating bacterial pathogens are needed. One such alternative option is phage biocontrol. In this study, a novel podophage-JELG-KS1-infecting Aeromonas salmonicida was retrieved from wastewater along with its host strain. The genome of the JELG-KS1 phage is a 40,505 bp dsDNA molecule with a GC% of 53.42% and 185 bp direct terminal repeats and encodes 53 predicted proteins. Genomic analysis indicates that JELG-KS1 might represent a novel genus within the subfamily Studiervirinae. Podophage JELG-KS1 is a strictly lytic phage without any identifiable virulence or AMR genes that quickly adsorbs onto the surface of host cells to initiate a 48 min long infectious cycle, resulting in the release of 71 ± 12 JELG-KS1 progeny virions per infected cell. JELG-KS1 effectively lyses its host population in vitro, even at very low multiplicities of infection. However, when challenged against a panel of Aeromonas spp. strains associated with diseases in aquaculture, JELG-KS1 shows host-specificity that is confined only to its isolation strain, immediately compromising its potential for Aeromonas spp. biocontrol in aquaculture.

Abstract P37: Decoding Cancer Dependency and Molecular Pathways of Long Noncoding RNAs with a Novel CRISPR-Cas13d Platform

Abstract Long noncoding RNAs (lncRNAs) constitute a significant portion of the human transcriptome, yet their precise role in tumor cells remains elusive. In our study focusing on multiple myeloma (MM), we employed the CRISPR-Cas13d endonuclease's RNA-targeting capabilities to establish an innovative functional transcriptomics screening platform. This platform facilitated the comprehensive identification of tumor-promoting lncRNAs (tp-lncRNAs) in MM cells and provided mechanistic insights. Utilizing RNA-seq data from 319 MM patients, we identified 5,304 expressed lncRNAs. Subsequently, we designed and implemented a pooled lentiviral library incorporating nine single-guide RNAs (sgRNAs) per identified lncRNA, alongside 500 non-targeting sgRNAs as negative controls and nine sgRNAs targeting established protein-coding oncogenes in MM (e.g., IRF4) as positive controls. This library was introduced at a low multiplicity of infection (MOI < 0.3) across five MM cell lines, each expressing a Cas13d-GFP fusion protein. After three weeks, deep sequencing and MAGeCK assessed sgRNA depletion/enrichment in MM cells. Our findings revealed ~600 tp-lncRNAs upon which MM cell lines demonstrated significant growth dependency, constituting around 12% of the identified lncRNA transcriptome. Notably, this surpassed the previous estimation of 3–5%, with considerable overlap across MM cell lines. The dependency score of numerous tp-lncRNAs rivaled or exceeded that of the positive control IRF4. To validate dependencies, we employed a library of 3,500 sgRNAs to target 314 tp-lncRNAs in vitro and in xenograft models. This approach was also applied to a panel of six non-MM cancer cell lines, identifying a broad tumor-dependency on >59 lncRNAs and a specific MM-dependency on a smaller subset of lncRNAs. We explored the sub-cellular localization and site-of-function of tp-lncRNAs using a Cas13d variant accumulating either in the cytosol or in the nucleus, coupled with sub-cellular RNA-seq and single-molecule RNA FISH. Confirming the functionality and nuclear enrichment of most tp-lncRNAs, we identified >20 hits providing dependency in the cytosol. Our investigation also unveiled multiple transcript variants within the lncRNA transcriptome, originating from the same lncRNA genes but differing in sequences, structures, and likely functions. Crucially, our screening platform precisely identified tp-lncRNA isoform(s) conferring dependencies. Finally, we combined short-term Cas13d screens with single-cell RNA-seq in H929 MM cells. Analysis of over 20,000 cells for gRNA and transcript expression enabled us to delineate molecular pathways linked to individual perturbations of over 30 lncRNAs in a single experiment. The modulated pathways included therapeutically relevant ones, such as the unfolded protein response or mitochondrial homeostasis, further suggesting a potential therapeutic benefit from targeting tp-lncRNAs. In summary, our novel platform offers a comprehensive delineation of tumor dependency on lncRNAs in MM, providing valuable insights to guide further mechanistic inquiries. Citation Format: Eugenio Morelli, Anil Aktas-Samur, Domenico Maisano, Claire Gao, Na Liu, Vanessa Favasuli, Marcello Turi, Pietro Folino, Liang Chen, Francesca Barello, Mariateresa Fulciniti, Annamaria Gulla, Kenneth Anderson, Mehmet Kemal Samur, Nikhil Munshi. Decoding Cancer Dependency and Molecular Pathways of Long Noncoding RNAs with a Novel CRISPR-Cas13d Platform [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P37.

Discovery of a Novel Antiviral Effect of the Restriction Factor SPOC1 against Human Cytomegalovirus.

The chromatin-remodeler SPOC1 (PHF13) is a transcriptional co-regulator and has been identified as a restriction factor against various viruses, including human cytomegalovirus (HCMV). For HCMV, SPOC1 was shown to block the onset of immediate-early (IE) gene expression under low multiplicities of infection (MOI). Here, we demonstrate that SPOC1-mediated restriction of IE expression is neutralized by increasing viral titers. Interestingly, our study reveals that SPOC1 exerts an additional antiviral function beyond the IE phase of HCMV replication. Expression of SPOC1 under conditions of high MOI resulted in severely impaired viral DNA replication and viral particle release, which may be attributed to inefficient viral transcription. With the use of click chemistry, the localization of viral DNA was investigated at late time points after infection. Intriguingly, we detected a co-localization of SPOC1, RNA polymerase II S5P and polycomb repressor complex 2 (PRC2) components in close proximity to viral DNA in areas that are hypothesized to harbor viral transcription sites. We further identified the N-terminal domain of SPOC1 to be responsible for interaction with EZH2, a subunit of the PRC2 complex. With this study, we report a novel and potent antiviral function of SPOC1 against HCMV that is efficient even with unrestricted IE gene expression.

A cytoprotective role for optineurin during mycobacterial infection of macrophages

Autophagy has emerged as a critical innate immune mechanism for host elimination of intracellular pathogens, however, the role of the autophagy receptor Optineurin during mycobacterial infection is not fully understood. To address this lacuna, we infected bone marrow-derived macrophages (BMDMs) derived from Optn+/+ and Optn−/− mice with Mycobacterium smegmatis, and observed the infection outcome at sequential time points. While low multiplicity of infection (MOI) did not show any significant difference between BMDMs from the two groups, at high MOI Optn−/− mice-derived BMDMs showed significantly lower colony forming unit counts, as well as lower cell counts at 12 h and 24 h post-infection. Quantification of cell numbers and nuclear morphologies at various time points post-infection indicated a markedly higher cell death in the Optineurin-deficient macrophages. Optineurin-deficient BMDMs showed significantly lower levels of the autophagosomal protein LC3-II upon infection, indicating a potential role for Optineurin in regulating autophagy during mycobacterial infection. Moreover, when stimulated by bacterial LPS, Optineurin deficient macrophages, showed altered levels of the inflammatory cytokine pro-IL-1β. These observations taken together suggest a novel regulatory role for Optineurin during mycobacterial infection. Its deficiency leads to an impairment in macrophage responses, directly impacting the pathophysiology of infection.

Regulation of human neutrophil IL-1β secretion induced by Escherichia coli O157:H7 responsible for hemolytic uremic syndrome.

Shiga-toxin producing Escherichia coli (STEC) infections can cause from bloody diarrhea to Hemolytic Uremic Syndrome. The STEC intestinal infection triggers an inflammatory response that can facilitate the development of a systemic disease. We report here that neutrophils might contribute to this inflammatory response by secreting Interleukin 1 beta (IL-1β). STEC stimulated neutrophils to release elevated levels of IL-1β through a mechanism that involved the activation of caspase-1 driven by the NLRP3-inflammasome and neutrophil serine proteases (NSPs). Noteworthy, IL-1β secretion was higher at lower multiplicities of infection. This secretory profile modulated by the bacteria:neutrophil ratio, was the consequence of a regulatory mechanism that reduced IL-1β secretion the higher were the levels of activation of both caspase-1 and NSPs, and the production of NADPH oxidase-dependent reactive oxygen species. Finally, we also found that inhibition of NSPs significantly reduced STEC-triggered IL-1β secretion without modulating the ability of neutrophils to kill the bacteria, suggesting NSPs might represent pharmacological targets to be evaluated to limit the STEC-induced intestinal inflammation.

A newly isolated Pseudomonas otitidis phage, vB_PotS-PotUPM1 from tilapia in Malaysia

A new Pseudomonas species, Pseudomonas otitidis was first known as an otic infectious agent in human. The presence of an inherent metallo-β-lactamase gene and its recent emergence in aquaculture underscore its potential zoonotic pathogenicity. The present study is aimed to characterize P. otitidis NK1 and its bacteriophage vB_PotS-PotUPM1 (PotUPM1), which were isolated from an infected tilapia fish farm in Malaysia. P. otitidis NK1 showed beta-hemolysis on blood agar and is resistant to several antibiotics. Its genome size was 6067,534 bp, with an average nucleotide identity of less than 99 % to all P. otitidis genome assemblies in GenBank, making it a novel strain of P. otitidis. The phage PotUPM1 has a latent period of 45 min and a burst size of 405 phage particles per bacterium. It effectively inhibited the growth of P. otitidis NK1 even at a low multiplicity of infection of 0.001 up to 9-hour post-inoculation. Transmission electron microscopy showed that it has a prolate head and a long, non-contractile tail, resembling those in the Siphoviridae family. Based on bioinformatic analysis, PotUPM1 has no resistance or virulence genes and is a lysogenic phage based on the presence of an integrase gene in the phage genome. Similarity analysis showed that PotUPM1 may represent a novel viral species with less than 95 % nucleotide sequence similarity to other characterized phages. The findings of this study could provide an insight on the occurrence of P. otitidis in the aquaculture industry and lay a basis for the application of phage or phage-derived proteins against pathogenic P. otitidis to alleviate antimicrobial resistance.

A Genome Scale CRISPR Screen Identifies Novel Genes That Regulate Erythropoietin Production

Erythropoietin (EPO) is a plasma glycoprotein that binds bone marrow erythroid progenitors, stimulating their proliferation and differentiation. EPO is secreted into the circulation by hepatocytes and specialized kidney peritubular fibroblasts, which contribute ~20% and ~80% of the total plasma EPO level, respectively. EPO production is induced during hypoxia, due to enhanced stability of the transcription factor hypoxia-inducible factor (HIF). However, increased HIF activity results in enhanced expression of angiogenesis and oncogenesis promoting genes, raising concerns about clinical strategies that promote EPO production via increasing HIF levels. To define novel regulators of EPO production, we developed a genome-wide functional screen that provides a quantifiable and selectable readout of EPO production. In order to perform such a screen, we generated a reporter HEP3B cell line with homozygous insertion of coding sequences for p2A and eGFP at the endogenous EPO locus. This HEP3B EPO-p2A-eGFP (HEG) cell line expresses equivalent levels of EPO and eGFP proteins, both translated from the same mRNA molecule. Treatment of HEG cells with the HIF stabilizing molecule DMOG results in increased EPO expression, which is abolished by deletion of the hypoxia-responsive-element in the EPO locus. These findings demonstrate the utility of this reporter cell line to study the regulation of EPO production. To identify novel genes that regulate EPO production, we mutagenized the HEG cell line with the GeCKO-v2 CRISPR/Cas9 knock-out library, which delivers a puromycin resistance cassette, SpCas9, and a pooled collection of 123,411 single guide RNAs (sgRNAs) that include 6 independent sgRNAs targeting every human gene. Transduction was performed at a low multiplicity of infection (MOI ~0.3), so that most infected cells receive 1 sgRNA to delete 1 gene only. Puromycin selection was applied from days 1-4 post-transduction and cells were subsequently recovered for 8 days. Cells were then treated with DMOG for 24 hours or left untreated. Cells with increased (top ~10%) or decreased (bottom ~10%) eGFP fluorescence were isolated. This screen was done in biological triplicates. Genes for which sgRNAs are enriched/depleted in eGFP high versus eGFP low DMOG-treated cells likely impact EPO expression independently of the HIF pathway. In contrast, genes for which sgRNAs are enriched/depleted in eGFP high versus eGFP low untreated cells may impact EPO expression in either a HIF dependent or independent manner. As expected, sgRNAs targeting VHL were found to be among the most significantly enriched sgRNAs in eGFP-high versus eGFP-low cells under no treatment conditions. Also as anticipated, sgRNAs targeting genes known to promote EPO production ( HNF4A, WT1, RXRA, and CREBBP) were enriched in eGFP-low versus eGFP-high cells under either DMOG-treated or untreated conditions. To refine our findings, we generated a custom library targeting the top 1,200 candidate regulators of EPO production nominated by the genome-scale screen. Using this library, we performed a secondary CRISPR screen (in biological triplicates) as above. Owning to the 10-fold smaller size of the custom library, the secondary screen was performed with average coverage of >150x throughout the entire screen (including the sorted cell populations). Indeed, the statistical confidence obtained with the secondary screen was superior than that achieved in the genome scale screen. Using a false discovery rate (FDR) cutoff of 1%, gene-level analysis identified 100 and 79 candidate genes whose targeting was associated with increased or decreased EPO production respectively, under either untreated or DMOG-treated conditions. A high degree of concordance was observed for regulators of EPO expression between both screens, performed with and without DMOG treatment. To identify genes that regulate EPO production independent of HIF, we evaluated several of the top genes for which sgRNAs were enriched in eGFP high vs. eGFP low cells treated with DMOG. Using 3 independent sgRNAs targeting each of these candidates, we found 2 genes ( ZNF574 and RBM18), that when deleted, result in increased EPO mRNA, even following DMOG treatment. Additional validation experiments as well as studies aimed to define the role of these 2 genes in the regulation of EPO production are ongoing.

Identification of CNOT4 As a Positive Regulator of Erythroid Cell Growth Using a Genome Wide CRISPR Knock-out Screen

Red blood cells, also known as erythrocytes, are the principle means of oxygen delivery to the body, and inadequate or disordered erythrocyte production is a major cause of human disease. While the cell-extrinsic factors that control erythrocyte production have been well studied, our understanding of the cell-intrinsic regulation of this process is incomplete. To identify genes that regulate erythroid proliferation and/or maturation, we performed a genome-scale CRISPR knock-out screen in the immortalized human erythroid cell line, HUDEP-2. HUDEP-2 cells can be maintained at the pro-erythroblast stage and then sequentially differentiated into basophilic, polychromatic, and orthochromatic erythroblasts by changing culture conditions, which permits separate evaluation of the impact of genetic perturbations on pro-erythroblast viability and terminal erythroid differentiation capacity. To perform our screen, we used the commercially available GeCKO-v2 library, which includes 6 sgRNAs targeting virtually every protein-coding gene in the human genome. This library was packaged into lentiviral particles, each expressing Cas9, an sgRNA, and a puromycin resistance gene. HUDEP-2 cells were transduced with the pooled GeCKO-v2 library at low multiplicity of infection to ensure delivery of only one sgRNA per cell. Following puromycin selection, pro-erythroblasts were collected prior to differentiation and orthochromatic erythroblasts were sorted at day 12 of differentiation (selecting for cells that downregulated CD49d). By identifying sgRNAs that were under-represented in orthochromatic erythroblasts compared to the proerythroblasts, we identified several genes that when deleted lead to impaired erythroid differentiation. One of these genes was CNOT4, which encodes a RING E3 ligase member of the CCR4-NOT complex, a conserved transcriptional regulator. This gene has been previously reported to positively regulate JAK/STAT signaling, which is known to play a key role in erythroid growth and differentiation. Additionally, only 30 out of 1095 cell lines tested in the Depmap portal exhibit diminished growth following CNOT4 deletion, suggesting a rather specific impact on erythroid cells. To define the role of CNOT4 in erythropoiesis, we electroporated human CD34+ hematopoietic stem and progenitor cells (HSPCs) with a ribonucleoprotein (RNP) complex consisting of Cas9 protein and CNOT4-targeting sgRNA. Three unique CNOT4-targeting sgRNAs were tested. Following RNP electroporation, cells were differentiated using a four-phase erythroid differentiation culture protocol and assessed for growth, indel frequencies, and erythroid differentiation capacity. For all three sgRNAs tested, CNOT4 disruption resulted in 10-fold reduced proliferation over the course of differentiation compared to cells electroporated with non-targeting sgRNAs. Erythroid differentiation, assessed by flow cytometry using CD49d and CD233 expression, was identical in CNOT4 deleted and control cells. However, evaluation of cytospins revealed reduced cell size with diminished cytoplasm to nuclear ratio in CNOT4 deficient compared to control cells. Notably, the findings above were validated in erythroid cells differentiated from HSPCs harvested from two independent donors. Taken together, these findings suggest a novel role for CNOT4 and/or the CCR4-NOT complex in erythroid differentiation. Studies are ongoing to determine the mechanism by which CNOT4 disruption negatively impacts erythropoiesis and to examine if CNOT4 overexpression results in enhanced proliferation/differentiation of erythroid cells.

Interaction of Mesonivirus and Negevirus with arboviruses and the RNAi response in Culex tarsalis-derived cells

BackgroundMosquito-specific viruses (MSVs) comprise a variety of different virus families, some of which are known to interfere with infections of medically important arboviruses. Viruses belonging to the family Mesoniviridae or taxon Negevirus harbor several insect-specific viruses, including MSVs, which are known for their wide geographical distribution and extensive host ranges. Although these viruses are regularly identified in mosquitoes all over the world, their presence in mosquitoes in Germany had not yet been reported.MethodsA mix of three MSVs (Yichang virus [Mesoniviridae] and two negeviruses [Daeseongdong virus and Dezidougou virus]) in a sample that contained a pool of Coquillettidia richiardii mosquitoes collected in Germany was used to investigate the interaction of these viruses with different arboviruses in Culex-derived cells. In addition, small RNA sequencing and analysis of different mosquito-derived cells infected with this MSV mix were performed.ResultsA strain of Yichang virus (Mesoniviridae) and two negeviruses (Daeseongdong virus and Dezidougou virus) were identified in the Cq. richiardii mosquitoes sampled in Germany, expanding current knowledge of their circulation in central Europe. Infection of mosquito-derived cells with these three viruses revealed that they are targeted by the small interfering RNA (siRNA) pathway. In Culex-derived cells, co-infection by these three viruses had varying effects on the representative arboviruses from different virus families (Togaviridae: Semliki forest virus [SFV]; Bunyavirales: Bunyamwera orthobunyavirus [BUNV]; or Flaviviridae: Usutu virus [USUV]). Specifically, persistent MSV co-infection inhibited BUNV infection, as well as USUV infection (but the latter only at specific time points). However, the impact on SFV infection was only noticeable at low multiplicity of infection (MOI 0.1) and at specific time points in combination with the infection status.ConclusionsTaken together, these results are important findings that will lead to a better understanding of the complex interactions of MSVs, mosquitoes and arboviruses.Graphical

Repression of the major immediate early promoter of human cytomegalovirus allows transcription from an alternate promoter.

Following infection, the human cytomegalovirus (HCMV) genome becomes rapidly associated with host histones which can contribute to the regulation of viral gene expression. This can be seen clearly during HCMV latency where silencing of the major immediate early promoter (MIEP), normally responsible for expression of the key lytic proteins IE72 and IE86, is mediated by histone methylation and recruitment of heterochromatin protein 1. Crucially, reversal of these histone modifications coupled with histone acetylation drives viral reactivation which can be blocked with specific histone acetyltransferase inhibitors (HATi). In lytic infection, a role for HATi is less clear despite the well-established enhancement of viral replication observed with histone deacetylase inhibitors. Here we report that a number of different broad-acting HATi have a minor impact on viral infection and replication during lytic infection with the more overt phenotypes observed at lower multiplicities of infection. However, specific analyses of the regulation of major immediate early (MIE) gene expression reveal that the HATi C646, which targets p300/CBP, transiently repressed MIE gene expression via inhibition of the MIEP but by 24 h post-infection MIE gene expression was rescued due to compensatory activation of an alternative IE promoter, ip2. This suggested that silencing of the MIEP promoted alternative ip2 promoter activity in lytic infection and, consistent with this, ip2 transcription is impaired in cells infected with a recombinant HCMV that does not auto-repress the MIEP at late times of infection. Furthermore, inhibition of the histone methyltransferases known to be responsible for auto-repression is similarly inhibitory to ip2 transcription in wild-type infected cells. We also observe that these discrete transcriptional activities of the MIEP and ip2 promoter are also reflected in reactivation; essentially in cells where the MIEP is silenced, ip2 activity is easier to detect at very early times post-reactivation whereas in cells where robust activation of the MIEP is observed ip2 transcription is reduced or delayed. Finally, we observe that inhibition of pathways demonstrated to be important for reactivation of HCMV in dendritic cells, e.g. in response to IL-6, are preferentially important for activation of the MIEP and not the ip2 promoter. Together, these data add to the hypothesis that the existence of multiple promoters within the MIE region of HCMV can drive reactivation in a cell type- and ligand-specific manner and also suggest that inter-dependent regulatory activity between the two promoters exists.

A Bacteriophage Cocktail Reduces Five Relevant Salmonella Serotypes at Low Multiplicities of Infection and Low Temperatures.

Salmonella are important pathogenic bacteria and, following Campylobacter, they are the second most common cause of bacterial foodborne infections worldwide. To reduce the presence of bacteria along the food chain, the application of bacteriophages (phages) may be a promising tool. In this study, the lytic properties of six phages against five relevant Salmonella serotypes (S. Enteritidis, S. Typhimurium, S. Infantis, S. Paratyphi B and S. Indiana) were analyzed. Three phages were able to lyse all five serotypes. We determined the lytic potential of each phage on indicator strains in vitro at room temperature (RT) and at 37 °C using low multiplicities of infection (MOIs). Most phages reduced their host more efficiently at RT than at 37 °C, even at the lowest MOI of 0.001. Following this, the lytic activity of a cocktail comprising five phages (MOI = 0.1) was examined with each of the five serotypes and a mix of them at RT, 15, 12, 10, 8 and 6 °C. All cultures of single serotypes as well as the mixture of strains were significantly reduced at temperatures as low as 8 °C. For single serotypes, reductions of up to 5 log10 units and up to 2.3 log10 units were determined after 6 h (RT) and 40 h (8 °C), respectively. The mixture of strains was reduced by 1.7 log10 units at 8 °C. The data clearly suggest that these phages are suitable candidates for biocontrol of various Salmonella serotypes under food manufacturing conditions.

Characterization of bacteriophage vB_KleM_KB2 possessing high control ability to pathogenic Klebsiella pneumoniae

Klebsiella pneumoniae is a widespread pathogen of several human diseases. The emergence of multidrug-resistant K. pneumoniae makes the treatment of these diseases a significant challenge. The application of bacteriophages is a potential approach for dealing with the emergence of multidrug-resistant pathogenic bacteria. This study isolates a novel bacteriophage vB_KleM_KB2 that infects the multidrug-resistant clinical isolates of K. pneumoniae. The bacteriophage exhibits a short latent period of 10 min, and can effectively lyse the bacterium within 60 min. Notably, the bacteriophage can completely inhibit the growth of the host bacterium at the initial concentration of 107 CFU/mL with a low multiplicity of infection of 0.001, which proves its high lytic activity. Furthermore, the bacteriophage shows high environmental tolerances, which can facilitate the practical application of the bacteriophage. Analysis of the bacteriophage genome shows that the bacteriophage possesses a novel genome sequence and can represent a new bacteriophage genus. Considering the high lytic activity, short latent period, high stability, and novel genetic background, bacteriophage vB_KleM_KB2 enriches the bacteriophage library and provides a new alternative for controlling the diseases caused by multidrug-resistant pathogenic K. pneumoniae.

WSSV latency is maintained by a dynamic balance of host immune factors and disturbed by exposure to bacterial infection and low salinity

Shrimp is one of the primary aquaculture species worldwide. The white spot syndrome virus (WSSV) is a highly lethal, stress-dependent virus that has led to enormous economic losses in shrimp aquaculture. WSSV can trigger a persistent infection in shrimp (latency), which can be reactivated to become an acute infection following various stimuli. However, the molecular mechanisms underlying the WSSV latency establishment and the induction of reactivation are poorly understood. A WSSV latency model using low multiplicity infection via intramuscular injection was developed in this study. This research examined the effects of multiple host factors and stimuli on WSSV latency. These effects generated latency reactivation in this model. The outcomes revealed that low multiplicity infection via intramuscular injection could be applied to establish WSSV latency in shrimp. Besides, the expression levels of host genes IRF, Dorsal and Relish were found to be substantially correlated with viral loads during WSSV latent infection in vivo. The knockdown of IRF resulted in elevated viral loads and rendered the reactivation of WSSV latency to acute infection that caused mass mortality among shrimp. Furthermore, several bacterial infection and low salinity stress could induce WSSV transitions from latent to acute infection. Noteworthily, the expression levels of Dorsal and Relish, which are considered to promote the replication of WSSV, are upregulated following bacterial infection and low salinity stress. These results indicated that a delicate balance between host and WSSV interaction regulates the latency establishment. This balance could be broken by several stimuli, such as bacterial infection and low salinity stress, which will provide insights into developing management strategies for WSSV prevention and control.

A closed, autologous bioprocess optimized for TCR-T cell therapies.

Autologous cell therapy has proven to be an effective treatment for hematological malignancies. Cell therapies for solid tumors are on the horizon, however the high cost and complexity of manufacturing these therapies remain a challenge. Routinely used open steps to transfer cells and reagents through unit operations further burden the workflow reducing efficiency and increasing the chance for human error. Here we describe a fully closed, autologous bioprocess generating engineered TCR-T cells. This bioprocess yielded 5-12 × 10e9 TCR-expressing T cells, transduced at low multiplicity of infections, within 7-10 days, and cells exhibited an enriched memory T-cell phenotype and enhanced metabolic fitness. It was demonstrated that activating, transducing, and expanding leukapheresed cells in a bioreactor without any T-cell or peripheral blood mononuclear cell enrichment steps had a high level of T-cell purity (~97%). Several critical process parameters of the bioreactor, including culturing at a high cell density (7e6 cells/mL), adjusting rocking agitations during phases of scale-up, lowering glycolysis through the addition of 2-deoxy- d-glucose, and modulating interleukin-2 levels, were investigated on their roles in regulating transduction efficiency, cell growth, and T-cell fitness such as T-cell memory phenotype and resistance to activation-induced cell death. The bioprocess described herein supports scale-out feasibility by enabling the processing of multiple patients' batches in parallel within a Grade C cleanroom.

Impact of dual-baculovirus infection on the Sf9 insect cell transcriptome during rAAV production using single-cell RNA-seq.

The insect cell-baculovirus expression vector system (IC-BEVS) has shown to be a powerful platform to produce complex biopharmaceutical products, such as recombinant proteins and virus-like particles. More recently, IC-BEVS has also been used as an alternative to produce recombinant adeno-associated virus (rAAV). However, little is known about the variability of insect cell populations and the potential effect of heterogeneity (e.g., stochastic infection process and differences in infection kinetics) on product titer and/or quality. In this study, transcriptomics analysis of Sf9 insect cells during the production of rAAV of serotype 2 (rAAV2) using a low multiplicity of infection, dual-baculovirus system was performed via single-cell RNA-sequencing (scRNA-seq). Before infection, the principal source of variability in Sf9 insect cells was associated with the cell cycle. Over the course of infection, an increase in transcriptional heterogeneity was detected, which was linked to the expression of baculovirus genes as well as to differences in rAAV transgenes (rep, cap and gfp) expression. Noteworthy, at 24 h post-infection, only 29.4% of cells enclosed all three necessary rAAV transgenes to produce packed rAAV2 particles, indicating limitations of the dual-baculovirus system. In addition, the trajectory analysis herein performed highlighted that biological processes such as protein folding, metabolic processes, translation, and stress response have been significantly altered upon infection. Overall, this work reports the first application of scRNA-seq to the IC-BEVS and highlights significant variations in individual cells within the population, providing insight into the rational cell and process engineering toward improved rAAV2 production in IC-BEVS.