Low Multiplicity Of Infection Research Articles

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.

Enrichment Culture but Not Metagenomic Sequencing Identified a Highly Prevalent Phage Infecting Lactiplantibacillus plantarum in Human Feces.

Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) is increasingly used as a probiotic to treat human diseases, but its phages in the human gut remain unexplored. Here, we report its first gut phage, Gut-P1, which we systematically screened using metagenomic sequencing, virus-like particle (VLP) sequencing, and enrichment culture from 35 fecal samples. Gut-P1 is virulent, belongs to the Douglaswolinvirus genus, and is highly prevalent in the gut (~11% prevalence); it has a genome of 79,928 bp consisting of 125 protein coding genes and displaying low sequence similarities to public L. plantarum phages. Physiochemical characterization shows that it has a short latent period and adapts to broad ranges of temperatures and pHs. Furthermore, Gut-P1 strongly inhibits the growth of L. plantarum strains at a multiplicity of infection (MOI) of 1e-6. Together, these results indicate that Gut-P1 can greatly impede the application of L. plantarum in humans. Strikingly, Gut-P1 was identified only in the enrichment culture, not in our metagenomic or VLP sequencing data nor in any public human phage databases, indicating the inefficiency of bulk sequencing in recovering low-abundance but highly prevalent phages and pointing to the unexplored hidden diversity of the human gut virome despite recent large-scale sequencing and bioinformatics efforts. IMPORTANCE As Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) is increasingly used as a probiotic to treat human gut-related diseases, its bacteriophages may pose a certain threat to their further application and should be identified and characterized more often from the human intestine. Here, we isolated and identified the first gut L. plantarum phage that is prevalent in a Chinese population. This phage, Gut-P1, is virulent and can strongly inhibit the growth of multiple L. plantarum strains at low MOIs. Our results also show that bulk sequencing is inefficient at recovering low-abundance but highly prevalent phages such as Gut-P1, suggesting that the hidden diversity of human enteroviruses has not yet been explored. Our results call for innovative approaches to isolate and identify intestinal phages from the human gut and to rethink our current understanding of the enterovirus, particularly its underestimated diversity and overestimated individual specificity.

Establishment of an In Vitro Model of Pseudorabies Virus Latency and Reactivation and Identification of Key Viral Latency-Associated Genes.

Alphaherpesviruses infect humans and most animals. They can cause severe morbidity and mortality. The pseudorabies virus (PRV) is a neurotropic alphaherpesvirus that can infect most mammals. The PRV persists in the host by establishing a latent infection, and stressful stimuli can induce the latent viruses to reactivate and cause recurrent diseases. The current strategies of antiviral drug therapy and vaccine immunization are ineffective in eliminating these viruses from the infected host. Moreover, overspecialized and complex models are also a major obstacle to the elucidation of the mechanisms involved in the latency and reactivation of the PRV. Here, we present a streamlined model of the latent infection and reactivation of the PRV. A latent infection established in N2a cells infected with the PRV at a low multiplicity of infection (MOI) and maintained at 42 °C. The latent PRV was reactivated when the infected cells were transferred to 37 °C for 12 to 72 h. When the above process was repeated with a UL54-deleted PRV mutant, it was observed that the UL54 deletion did not affect viral latency. However, viral reactivation was limited and delayed. This study establishes a powerful and streamlined model to simulate PRV latency and reveals the potential role of temperature in PRV reactivation and disease. Meanwhile, the key role of the early gene UL54 in the latency and reactivation of PRV was initially elucidated.

The diversity of Plasmodium falciparum isolates from asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo

BackgroundUnderstanding Plasmodium falciparum population diversity and transmission dynamics provides information on the intensity of malaria transmission, which is needed for assessing malaria control interventions. This study aimed to determine P. falciparum allelic diversity and multiplicity of infection (MOI) among asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo (DRC).MethodsA total of 438 DNA samples (248 asymptomatic and 190 symptomatic) were characterized by nested PCR and genotyping the polymorphic regions of pfmsp1 block 2 and pfmsp2 block 3.ResultsNine allele types were observed in pfmsp1 block2. The K1-type allele was predominant with 78% (229/293) prevalence, followed by the MAD20-type allele (52%, 152/293) and RO33-type allele (44%, 129/293). Twelve alleles were detected in pfmsp2, and the 3D7-type allele was the most frequent with 84% (256/304) prevalence, followed by the FC27-type allele (66%, 201/304). Polyclonal infections were detected in 63% (95% CI 56, 69) of the samples, and the MOI (SD) was 1.99 (0.97) in P. falciparum single-species infections. MOIs significantly increased in P. falciparum isolates from symptomatic parasite carriers compared with asymptomatic carriers (2.24 versus 1.69, adjusted b: 0.36, (95% CI 0.01, 0.72), p = 0.046) and parasitaemia > 10,000 parasites/µL compared to parasitaemia < 5000 parasites/µL (2.68 versus 1.63, adjusted b: 0.89, (95% CI 0.46, 1.25), p < 0.001).ConclusionThis survey showed low allelic diversity and MOI of P. falciparum, which reflects a moderate intensity of malaria transmission in the study areas. MOIs were more likely to be common in symptomatic infections and increased with the parasitaemia level. Further studies in different transmission zones are needed to understand the epidemiology and parasite complexity in the DRC.

Coliphage cocktails for controlling antimicrobial-resistant Escherichia coli: emphasizing polyphage and multihost interactions at different levels of multiplicity of infection.

This study elucidates the in-vitro bactericidal effectiveness of polyphage cocktail combinations of 2, 4, 6, 8, and 10 individual coliphages against a cocktail of 20 AMR Escherichia coli. Different polyphage cocktails viz., 45 two-phage combinations, 28 four-phage combinations, 15 six-phage combinations, 6 eight-phage combinations, and 1 ten-phage combination were formulated using a pool of ten coliphages that were isolated from two different geographical locations (East and West coasts of India). The different polyphage cocktails were tested at four different levels of Multiplicity of Infection (MOI) viz., MOI-1, MOI-10, MOI-100, and MOI-1000. All the 2, 4, 6, 8, and 10-phage cocktails were found to be effective in controlling the growth of a cocktail of 20 AMR bacteria when tested at MOI-1000 and MOI-100 but variations in antibacterial activity were observed at lower MOIs of 10 and 1. The ten coliphage cocktail showed lytic activity against 100% of AMR E. coli from farmed brackish water shrimp, 96% of laboratory collection of AMR E. coli, 92% of AMR E. coli from farmed freshwater fish, and 85% of AMR E. coli from market shrimp. Polyphage cocktails of 2, 4, 6, 8, and 10 coliphages applied at an MOI of 1000 effectively suppressed the growth of antimicrobial-resistant E. coli. The results indicated phage-phage synergy in the lytic activity of several coliphage combinations at higher MOIs of 1000 and 100 while phage-phage antagonism was evidenced at lower MOIs of 10 and 1.

Mucin and Agitation Shape Predation of Escherichia coli by Lytic Coliphage.

The ability of bacteriophage (phage), abundant within the gastrointestinal microbiome, to regulate bacterial populations within the same micro-environment offers prophylactic and therapeutic opportunities. Bacteria and phage have both been shown to interact intimately with mucin, and these interactions invariably effect the outcomes of phage predation within the intestine. To better understand the influence of the gastrointestinal micro-environment on phage predation, we employed enclosed, in vitro systems to investigate the roles of mucin concentration and agitation as a function of phage type and number on bacterial killing. Using two lytic coliphage, T4 and PhiX174, bacterial viability was quantified following exposure to phages at different multiplicities of infection (MOI) within increasing, physiological levels of mucin (0-4%) with and without agitation. Comparison of bacterial viability outcomes demonstrated that at low MOI, agitation in combination with higher mucin concentration (>2%) inhibited phage predation by both phages. However, when MOI was increased, PhiX predation was recovered regardless of mucin concentration or agitation. In contrast, only constant agitation of samples containing a high MOI of T4 demonstrated phage predation; briefly agitated samples remained hindered. Our results demonstrate that each phage-bacteria pairing is uniquely influenced by environmental factors, and these should be considered when determining the potential efficacy of phage predation under homeostatic or therapeutic circumstances.

CRISPR-Cas provides limited phage immunity to a prevalent gut bacterium in gnotobiotic mice.

Many bacteria and archaea harbor the adaptive CRISPR-Cas system, which stores small nucleotide fragments from previous invasions of nucleic acids via viruses or plasmids. This molecular archive blocks further invaders carrying identical or similar nucleotide sequences. However, few of these systems have been confirmed experimentally to be active in gut bacteria. Here, we demonstrate experimentally that the type I-C CRISPR-Cas system of the prevalent gut bacterium Eggerthella lenta can specifically target and cleave foreign DNA in vitro by using a plasmid transformation assay. We also show that the CRISPR-Cas system acquires new immunities (spacers) from the genome of a virulent E. lenta phage using traditional phage assays in vitro but also in vivo using gnotobiotic (GB) mice. Both high phage titer and an increased number of spacer acquisition events were observed when E. lenta was exposed to a low multiplicity of infection in vitro, and three phage genes were found to contain protospacer hotspots. Fewer new spacer acquisitions were detected in vivo than in vitro. Longitudinal analysis of phage-bacteria interactions showed sustained coexistence in the gut of GB mice, with phage abundance being approximately one log higher than the bacteria. Our findings show that while the type I-C CRISPR-Cas system is active in vitro and in vivo, a highly virulent phage in vitro was still able to co-exist with its bacterial host in vivo. Taken altogether, our results suggest that the CRISPR-Cas defense system of E. lenta provides only partial immunity in the gut.

Trichomonas tenax induces barrier defects and modulates the inflammatory cytotoxicity of gingival and pulmonary epithelial cells.

Trichomonas tenax is a single-cell flagellated anaerobic organism, commensal in the human oral cavity. Although a previous study indicated that T. tenax could cause cell damage and phagocytose host epithelial cells, its pathological effects on gum cells remain unknown. Furthermore, several case reports have detected T. tenax in several patients with empyema and/or pleural effusion, which may have been aspirated from the oral cavity. However, the cytotoxic effects and immune responses of alveolar cells are unknown. Therefore, we aimed to determine the cytotoxic and immune effects of T. tenax on gums and pulmonary cell lines. The cytopathic effect and lactate dehydrogenase (LDH) cytotoxicity assays were used to determine the level of cell damage in gum and lung epithelial cells. Western blot was used to determine the disruption of cell junctions. Finally, epithelial cell cytokines were measured using ELISA to elucidate the immune response to T. tenax. We found that T. tenax induced a cytotoxic effect on gum epithelial cells by disrupting cell junctions; however, it hardly triggered cellular damage in alveolar A549 cells and mucoepidermoid NCI-H292 cells. Furthermore, T. tenax induced the production of IL-6 at a low multiplicity of infection (MOI) in gum, A549, and NCI-H292 cells. Our results suggest that T. tenax can trigger gingival cell cytotoxicity, disrupt cell junctions, and induce IL-6 production in gingival and pulmonary cell lines.

Transcriptome analysis of Sf9 insect cells during production of recombinant Adeno-associated virus.

The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as an alternative time- and cost-efficient production platform for recombinant Adeno-associated virus (AAV) for gene therapy. However, a better understanding of the underlying biological mechanisms of IC-BEVS is fundamental to further optimize this expression system toward increased product titer and quality. Here, gene expression of Sf9 insect cells producing recombinant AAV through a dual baculovirus expression system, with low multiplicity of infection (MOI), was profiled by RNA-seq. An 8-fold increase in reads mapping to either baculovirus or AAV transgene sequences was observed between 24 and 48h post-infection (hpi), confirming a take-over of the host cell transcriptome by the baculovirus. A total of 336 and 4784 genes were identified as differentially expressed at 24 hpi (vs non-infected cells) and at 48 hpi (vs. infected cells at 24 hpi), respectively, including dronc, birc5/iap5, and prp1. Functional annotation found biological processes such as cell cycle, cell growth, protein folding, and cellular amino acid metabolic processes enriched along infection. This work uncovers transcriptional changes in Sf9 in response to baculovirus infection, which provide new insights into cell and/or metabolic engineering targets that can be leveraged for rational bioprocess engineering of IC-BEVS for AAV production.

Human 3D Airway Tissue Models for Real-Time Microscopy: Visualizing Respiratory Virus Spreading.

Our knowledge about respiratory virus spreading is mostly based on monolayer cultures that hardly reflect the complex organization of the airway epithelium. Thus, there is a strong demand for biologically relevant models. One possibility to study virus spreading at the cellular level is real-time imaging. In an attempt to visualize virus spreading under somewhat more physiological conditions, Calu-3 cells and human primary fibroblasts were co-cultured submerged or as air-liquid interface (ALI). An influenza A virus (IAV) replicating well in cell culture, and carrying a red fluorescent protein (RFP) reporter gene was used for real-time imaging. Our three-dimensional (3D) models exhibited important characteristics of native airway epithelium including a basement membrane, tight junctions and, in ALI models, strong mucus production. In submerged models, first fluorescence signals appeared between 9 and 12 h post infection (hpi) with a low multiplicity of infection of 0.01. Virus spreading further proceeded in the immediate vicinity of infected cells. In ALI models, RFP was found at 22 hpi and later. Consequently, the progression of infection was delayed, in contrast to the submerged model. With these features, we believe that our 3D airway models can deliver new insights in the spreading of IAV and other respiratory viruses.

Identifying Novel Regulators of Erythroid Maturation and Proliferation Using a Genome-Scale CRISPR Activation Screen

Red blood cells (RBCs) provide the essential function of oxygen delivery to the body's tissues and organs. Though RBC transfusion is a lifesaving therapeutic intervention, RBCs are a resource often in shortage, particularly in lower-income countries. This is especially true considering that 47% of blood donations are collected in high-income countries, which house only 19% of the global population. Furthermore, the RBC shelf life is ~6 weeks; therefore, even regions with adequate stocks could readily enter periods of shortage. To alleviate these shortages, the development of a methodology for the efficient, scalable, and safe production of RBCs ex vivo is needed. To identify novel genes that enhance erythroid differentiation/ proliferation, we performed a functional pooled genome-wide CRISPR activation (CRISPRa) screen using the human erythroid cell line HUDEP-2. In this screen, we obtained the CRISPRa v2 library (Addgene: #1000000078), expanded it in competent bacterial cells, isolated pooled plasmids, and confirmed adequate expansion of the library without significant drifts in small guide RNAs (sgRNAs). Subsequently, we prepared pooled lentiviral stocks from the expanded plasmid library. This viral library delivers a VP64 activation domain fused to a catalytically dead Cas9 (dCas9-VP64), a blasticidin resistance cassette, and one of 3 unique sgRNAs targeting virtually every coding gene in the human genome (the library consists of 70,290 sgRNAs). In parallel, we generated a clonal HUDEP-2 cell line that stably expresses the transcriptional activator complex MPH (MS2-P65-HSF1), hereafter referred to as HUDEP-2-MPH cell line. We confirmed adequate growth and differentiation of the latter cell line, as well as validated MPH expression/function. The HUDEP-2-MPH clonal cell line was subsequently transduced at a low multiplicity of infection (~0.3) with the lentiviral CRISPRa library. Transduced cells were selected and subsequently underwent synchronized erythroid differentiation. Cells were harvested at 2 time points: 1) D0 of differentiation (corresponding to proerythroblasts) and 2) D10 of differentiation, sorting for orthochromatic erythroblasts using cell surface markers. We harvested genomic DNA from both populations, sequenced the sgRNAs using NextGen sequencing, and compared the abundance of the sgRNAs between both cell populations. This experiment was done in biological triplicates, while maintaining >200x sgRNA coverage at all times. As expected, the screen identified known regulators of erythroid differentiation and/or proliferation, including EPOR and KIT, confirming the validity of the screen. Notably, this screen also identified several novel candidate genes that appear to either promote or reduce the efficiency of terminal erythroid maturation and/or proliferation. The top ranked gene was IGF2BP3, which encodes the Insulin-like growth factor 2 mRNA-binding protein 3. In preliminary validation studies, we used 3 independent sgRNAs to activate IGF2BP3 in HUDEP-2-MPH cells. Compared to cells transduced with control sgRNAs, increased expression of IGF2BP3 mRNA (by ~20 fold) resulted in a 3-fold increase in cell expansion during differentiation. Additionally, HUDEP-2-MPH cells transduced with IGF2BP3 activating sgRNAs appeared to exhibit enhanced differentiation capacity, with ~67% of cells displaying Band3 expression and CD49d downregulation at day 12 of differentiation (compared to ~30% of cells transduced with control sgRNAs). These findings were further confirmed by morphologic evaluation of cytospins prepared from cells at day 12 of differentiation. We are currently further validating the impact of increased IGF2BP3 expression on erythroid differentiation in CD34+ cells differentiated into erythroid cells in vitro. Additionally, we are evaluating the role of other novel genes (identified in our screen) on erythroid maturation and proliferation. Targeting key genes found in this screen is anticipated to improve the efficiency of erythroid maturation. These findings have important implications for the ability to generate sufficient numbers of mature RBCs ex vivo, which could be used to balance the worldwide supply and demand of RBCs for transfusions.

Complete deletion of Ectromelia virus p28 impairs virus genome replication in a mouse strain, cell type, and multiplicity of infection-dependent manner

p28 is a poxvirus-encoded E3 ubiquitin ligase that possesses an N-terminal KilA-N domain and a C-terminal RING domain. In Ectromelia virus (ECTV), disruption of the p28 RING domain severely attenuated virulence in A strain mice, which normally succumb to ECTV infection. Moreover, this mutant virus exhibited dramatically reduced genome replication and impaired factory formation in A strain mice peritoneal macrophages (PMs) infected at high multiplicity of infection (MOI) These defects were not observed in PMs isolated from C57BL/6 mice which survive ECTV infection, demonstrating that p28 functions in a context-specific manner. To further investigate p28 function, we completely deleted the p28 gene from ECTV (ECTV-Δp28). In contrast to previous findings, we found that the ECTV-Δp28 virus exhibited severely compromised virus production and genome replication in PMs isolated from A strain mice only when infected at low MOI. This defect was minimal in bone marrow-derived macrophages and two cell lines derived from A strain mice. Furthermore, this low MOI defect in virus production was also observed in PMs isolated from the susceptible BALB/c mouse strain, but not PMs isolated from C57BL/6 mice. Taken together, our data demonstrate that the requirement for ECTV p28 to establish a productive infection depends on the MOI, the cell type, as well as the mouse strain.

Histone deacetylase inhibitors enhance oncolytic herpes simplex virus therapy for malignant meningioma

Approximately 20% of meningiomas are not benign (higher grade) and tend to relapse after surgery and radiation therapy. Malignant (anaplastic) meningioma (MM) is a minor subset of high-grade meningioma that is lethal with no effective treatment options currently. Oncolytic herpes simplex virus (oHSV) is a powerful anti-cancer modality that induces both direct cell death and anti-tumor immunity, and has shown activity in preclinical models of MM. However, clinically meaningful efficacy will likely entail rational mechanistic combination approaches. We here show that epigenome modulator histone deacetylase inhibitors (HDACi) increase anti-cancer effects of oHSV in human MM models, IOMM-Lee (NF2 wild-type) and CH157 (NF2 mutant). Minimally toxic, sub-micromolar concentrations of pan-HDACi, Trichostatin A and Panobinostat, substantively increased the infectability and spread of oHSV G47Δ within MM cells in vitro, resulting in enhanced oHSV-mediated killing of target cells when infected at low multiplicity of infection (MOI). Transcriptomics analysis identified selective alteration of mRNA processing and splicing modules that might underlie the potent anti-MM effects of combining HDACi and oHSV. In vivo, HDACi treatment increased intratumoral oHSV replication and boosted the capacity of oHSV to control the growth of human MM xenografts. Thus, our work supports further translational development of the combination approach employing HDACi and oHSV for the treatment of MM.

Hepatitis D virus-induced interferon response and administered interferons control cell division-mediated virus spread.

Besides HBV-dependent de novo infection, cell division-mediated spread contributes to HDV persistence and dampens the effect of antivirals that abrogate de novo infection. Nonetheless, the combination of these antivirals with interferons (IFNs) showed strong synergism in recent clinical trials, implying a complementary mode-of-action of IFNs. Therefore, we investigated the effect of IFN response on cell division-mediated HDV spread. Cells infected with HDV were passaged to undergo cell division. The effect of the IFN response was evaluated by blocking HDV-induced IFN activation, by applying different IFN treatment regimens, and by adjusting HDV infection doses. Cell division-mediated HDV spread was highly efficient following infection of HuH7NTCP cells (defective in IFN production), but profoundly restricted in infected IFN-competent HepaRGNTCP cells. Treatment with IFN-α/-λ1 inhibited HDV spread in dividing HuH7NTCP cells, but exhibited a marginal effect on HDV replication in resting cells. Blocking the HDV-induced IFN response with the JAK1/2 inhibitor ruxolitinib or knocking down MDA5 augmented HDV spread in dividing HepaRGNTCP cells. The virus-induced IFN response also destabilized HDV RNA in dividing cells. Moreover, the effect of exogenous IFNs on cell division-mediated HDV spread was more pronounced at low multiplicities of infection with weak virus-induced IFN responses. Both HDV-induced IFN response and exogenous IFN treatment suppress cell division-mediated HDV spread, presumably through acceleration of HDV RNA decay. Our findings demonstrate a novel mode-of-action of IFN, explain the more pronounced effect of IFN therapy in patients with lower HDV serum RNA levels, and provide insights for the development of combination therapies. Chronic hepatitis D is a major health problem. The causative pathogen hepatitis D virus (HDV) can propagate through viral particle-mediated infection and the division of infected cells. Although viral particle-dependent infection can be blocked by recently developed drugs, therapies addressing the cell division route have not been reported. Taking advantage of relevant cell culture models, we demonstrate that the widely used immune modulator interferon can efficiently suppress HDV spread through cell division. This work unveils a new function of interferon and sheds light on potentially curative combination therapies.