Coinfection Of Cells Research Articles

Innovative dual system approach for selective eradication of cancer cells using viral-based delivery of natural bacterial toxin\u2013antitoxin system

The inactivation of p53, a tumor suppressor, and the activation of the RAS oncogene are the most frequent genetic alterations in cancer. We have shown that a unique E. coli MazF-MazE toxin–antitoxin (TA) system can be used for selective and effective eradication of RAS-mutated cancer cells. This out of the box strategy holds great promise for effective cancer treatment and management. We provide proof of concept for a novel platform to selectively eradicate cancer cells using an adenoviral delivery system based on the adjusted natural bacterial system. We generated adenoviral vectors carrying the mazF toxin (pAdEasy-Py4-SV40mP-mCherry-MazF) and the antitoxin mazE (pAdEasy-RGC-SV40mP-MazE-IRES-GFP) under the regulation of RAS and p53, resp. The control vector carries the toxin without the RAS-responsive element (pAdEasy-ΔPy4-SV40mP-mCherry-MazF). In vitro, the mazF-mazE TA system (Py4-SV40mP-mCherry-MazF+RGC-SV40mP-MazE-IRES-GFP) induced massive, dose-dependent cell death, at 69% compared to 19% for the control vector, in a co-infected HCT116 cell line. In vivo, the system caused significant tumor growth inhibition of HCT116 (KRASmut/p53mut) tumors at 73 and 65% compared to PBS and ΔPY4 control groups, resp. In addition, we demonstrate 65% tumor growth inhibition in HCT116 (KRASmut/p53wt) cells, compared to the other two control groups, indicating a contribution of the antitoxin in blocking system leakage in WT RAS cells. These data provide evidence of the feasibility of using mutations in the p53 and RAS pathway to efficiently kill cancer cells. The platform, through its combination of the antitoxin (mazE) with the toxin (mazF), provides effective protection of normal cells from basal low activity or leakage of mazF.

RSV and HMPV Infections in 3D Tissue Cultures: Mechanisms Involved in Virus-Host and Virus-Virus Interactions.

Respiratory viral infections constitute a global public health concern. Among prevalent respiratory viruses, two pneumoviruses can be life-threatening in high-risk populations. In young children, they constitute the first cause of hospitalization due to severe lower respiratory tract diseases. A better understanding of their pathogenesis is still needed as there are no approved efficient anti-viral nor vaccine against pneumoviruses. We studied Respiratory Syncytial virus (RSV) and human Metapneumovirus (HMPV) in single and dual infections in three-dimensional cultures, a highly relevant model to study viral respiratory infections of the airway epithelium. Our investigation showed that HMPV is less pathogenic than RSV in this model. Compared to RSV, HMPV replicated less efficiently, induced a lower immune response, did not block cilia beating, and was more sensitive to IFNs. In dual infections, RSV-infected epithelia were less permissive to HMPV. By neutralizing IFNs in co-infection assays, we partially prevented HMPV inhibition by RSV and significantly increased the number of co-infected cells in the tissue. This suggests that interference in dual infection would be at least partly mediated by the host immune response. In summary, this work provides new insight regarding virus-host and virus-virus interactions of pneumoviruses in the airway epithelium. This could be helpful for the proper handling of at-risk patients.

Apicomplexan co-infections impair with phagocytic activity in avian macrophages

Mixed infections of Toxoplasma gondii and Eimeria tenella are likely to occur frequently due to the high prevalence of both pathogens in free-ranging chickens. In this study, we investigated the co-occurrence of the two parasites in the same immune-competent host cell towards altered patterns of parasite-host interactions. Chicken blood monocyte–derived macrophages were co-infected with T. gondii RH tachyzoites and E. tenella Houghton sporozoites in vitro for 24 h. Through monitoring the uptake of pH-sensitive pHrodo™ Zymosan BioParticles (“Zymosan”) by macrophages, we created a three-dimensional model and to analyze quantitatively phagocytosis using confocal laser scanning microscopy. Assessments of parasite populations were performed by qPCR at 2, 6, 12, and 24 h post-infection (hpi). At 6 hpi, phagocytosis was inhibited in the E. tenella–infected cultures while no inhibition of phagocytosis was observed due to T. gondii. Phagocytosis activity revealed more complex interactions during co-infection. At 12 and 24 hpi, phagocytosis response to “Zymosan” was distinctly weaker in co-infected cells than in all other groups except for cells mono-infected with high doses of E. tenella at 24 hpi. By qPCR, significantly reduced numbers of both intracellular parasites were recorded (10-fold) in all infected groups at 2 hpi. At 12 hpi, the T. gondii population reached lowest values but dramatically increased by 24 hpi. Our data confirm that macrophage phagocytosis is involved in the control of invasion by apicomplexan parasites in chicken which particularly applies to E. tenella infection and it was able to be altered by the co-existing parasites.

Status of kinases in Epstein-Barr virus and Helicobacter pylori Coinfection in gastric Cancer cells

BackgroundHelicobacter pylori (H. pylori) and Epstein - Barr virus (EBV) plays a significant role in aggressive gastric cancer (GC). The investigation of genes associated with these pathogens and host kinases may be essential to understand the early and dynamic progression of GC.AimThe study aimed to demonstrate the coinfection of EBV and H. pylori in the AGS cells through morphological changes, expression of the kinase and the probable apoptotic pathways.MethodsGenomic DNA isolation of H. pylori and its characterization from clinical samples were performed. RT-qPCR of kinases was applied to scrutinize the gene expression of kinases in co-infected GC in a direct and indirect (separated through insert size 0.45 μm) H. pylori infection set up. Morphological changes in co-infected GC were quantified by measuring the tapering ends of gastric epithelial cells. Gene expression profiling of apoptotic genes was assessed through RT-qPCR.ResultsAn interleukin-2-inducible T-cell kinase (ITK) showed significant upregulation with indirect H. pylori infection. Moreover, Ephrin type-B receptor six precursors (EPHB6) and Tyrosine-protein kinase Fyn (FYN) showed significant upregulation with direct coinfection. The tapering ends in AGS cells were found to be extended after 12 h. A total of 24 kinase genes were selected, out of which EPHB6, ITK, FYN, and TYK2 showed high expression as early as 12 h. These kinases may lead to rapid morphological changes in co-infected gastric cells. Likewise, apoptotic gene expression such as APAF-1 and Bcl2 family genes such as BAD, BID, BIK, BIM, BAX, AND BAK were significantly down-regulated in co-infected AGS cells.ConclusionAll the experiments were performed with novel isolates of H. pylori isolated from central India, for the functional assessment of GC. The effect of coinfection with EBV was more profoundly observed on morphological changes in AGS cells at 12 h as quantified by measuring the tapering of ends. This study also identifies the kinase and apoptotic genes modulated in co-infected cells, through direct and indirect approaches. We report that ITK, EPHB6, TYK2, FYN kinase are enhanced, whereas apoptotic genes such as APAF-1, BIK, FASL, BAX are significantly down-regulated in AGS cells coinfected with EBV and H. pylori.

Hepatitis Delta Virus Infection Increases the Efficacy of HBV-Specific T Cell-Based Therapies

Hepatitis Delta virus (HDV) requires hepatitis B virus (HBV) to complete its infection cycle and causes a severe hepatitis with limited therapeutic options. To determine the prospect of T cell therapy in HBV/HDV co-infection, we first studied the HDV impact on viral antigen processing and presentation. Utilizing in vitro models of HBV/HDV co-infection, we demonstrated that HDV boosts HBV epitope presentation, both in HBV/HDV co-infected and neighboring mono-HBV infected cells through up-regulation of antigen processing pathway mediated by IFN-beta/lambda. This up-regulation was confirmed in liver biopsies of HBV/HDV patients. We then demonstrated in vitro and in HBV/HDV preclinical mouse model that HDV increases antiviral efficacy of HBV-specific T cell receptors engineered T cells. Thus, by unveiling the effect of HDV on HBV antigen presentation, we provide a framework to better understand HBV/HDV immune pathology, and we advocate the use of engineered HBV-specific T cells for the treatment of HBV/HDV co-infection.

Cytoplasmic factories, virus assembly, and DNA replication kinetics collectively constrain the formation of poxvirus recombinants.

Poxviruses replicate in cytoplasmic structures called factories and each factory begins as a single infecting particle. Sixty-years ago Cairns predicted that this might have effects on vaccinia virus (VACV) recombination because the factories would have to collide and mix their contents to permit recombination. We've since shown that factories collide irregularly and that even then the viroplasm mixes poorly. We’ve also observed that while intragenic recombination occurs frequently early in infection, intergenic recombination is less efficient and happens late in infection. Something inhibits factory fusion and viroplasm mixing but what is unclear. To study this, we’ve used optical and electron microscopy to track factory movement in co-infected cells and correlate these observations with virus development and recombinant formation. While the technical complexity of the experiments limited the number of cells that are amenable to extensive statistical analysis, these studies do show that intergenic recombination coincides with virion assembly and when VACV replication has declined to ≤10% of earlier levels. Along the boundaries between colliding factories, one sees ER membrane remnants and other cell constituents like mitochondria. These collisions don't always cause factory fusion, but when factories do fuse, they still entrain cell constituents like mitochondria and ER-wrapped microtubules. However, these materials wouldn’t seem to pose much of a further barrier to DNA mixing and so it’s likely that the viroplasm also presents an omnipresent impediment to DNA mixing. Late packaging reactions might help to disrupt the viroplasm, but packaging would sequester the DNA just as the replication and recombination machinery goes into decline and further reduce recombinant yields. Many factors thus appear to conspire to limit recombination between co-infecting poxviruses.

Infectious cell culture system for concurrent propagation and purification of Megalocytivirus ISKNV and nervous necrosis virus from Asian Sea bass (Lates calcarifer)

Megalocytivirus ISKNV and nervous necrosis virus (NNV) are two major viral pathogens affecting marine fish farms in Asia Pacific region. The present study reports an unexpected discovery of dual infections of both viruses in a single Asian sea bass (Lates calcarifer) farm experiencing ~50% cumulative fish mortality. A commercial Grunt Fin (GF) cell line was able to propagate concurrently two viruses from the clinical samples. Each virus was successfully purified from the co-infected cell culture, and its morphology was visualized by transmission electron microscopy (TEM). Subsequently, two viruses were identified as Megalocytivirus ISKNV and NNV from specific PCR assays using the extracted nucleic acids and sequencing of amplified PCR products. Approximately 104 and 103 copies of Megalocytivirus ISKNV and NNV were estimated in 200 ng DNA and RNA extracted from the co-infected cells. NNV propagation in GF cells was specifically confirmed using immunofluorescence microscopy. This is the first report for capability of NNV to propagate in the commercial GF cell line. This investigation opens up a promising in vitro model for studying concurrent infections of Megalocytivirus ISKNV and NNV and suggests a possible platform for concurrent production of Megalocytivirus ISKNV and NNV for bivalent vaccine.

Development of pathological process and activity of etiotropic drugs in cell culture under condition of viral co-infection

The aim of the study: to investigate the peculiarities of the development of the pathological process in cells in conditions of mixed viral infection and to study the effectiveness of antiviral drugs in this model. Materials and methods of research. A model of simultaneous mixed infection of MDBK cells with human adenovirus serotype 5 (HAdV-5) and herpes simplex virus type 1 (HSV-1) was developed. Mitochondrial activity, ultrastructure and the state of the cell population were studied using MTT assay, transmission electron microscopy and flow cytometry with propidium iodide dye. The intensity of virus reproduction in cells and their infectious titer were studied by the cytomorphology method. The level of the synthesis of the major proteins of associate viruses was analyzed using flow cytometry and the corresponding monoclonal antibodies. Results of the research. Co-infected cells demonstrated a lower rate of development of pathomorphological changes compared to mono-infections, related to the inhibition of the reproduction of associate viruses. It was found that the co-infection of cells with HSV-1 and HAdV-5 results in a decrease in the number of cells with virus-induced intranuclear inclusions of both viruses by up to 40 % and viruses titer by 1.6 lg and 2.6 lg, respectively. Inhibition of synthesis of major capsid protein and glycoproteins of the herpes virus by 83 % and 64 %, respectively, and a less pronounced decrease in the amount of adenovirus hexon protein (by 17 %) were also noted. It is shown that the mitochondrial activity of co-infected cells increases to 64 % in comparison with herpetic mono-infection. An analysis of the influence of co-infection on cell cycle revealed that the number of cells in G1 phase remained unchanged compared with both mono-infections, while the number of apoptotic cells compared with herpes infection was reduced by 24 %. An analysis of the officinal drugs Acyclovir and Ribavirin effectiveness in conditions of mixed infection showed a reduction in their antiviral activity against associate viruses by 1 to 2.3 lg compared to mono-infections. Conclusions. The presence of a specific innovative cellular model of mixed infection with known aspects of the course of associated infections allows it to be used for preclinical study of antiviral activity of compounds and to obtain new data of the role of viral-viral interactions in the development of inefficient application of antiviral agents in medical practice. Key words: mixed viral infection, cytomorphological changes, cell cycle, reproduction of associate viruses, and antiviral activity. For citation: Biliavska LO, Povnitsa OY, Pankivska YB, Zagorodnya SD. Development of pathological process and activity of etiotropic drugs in cell culture under condition of viral co-infection. Journal of the National Academy of Medical Sciences of Ukraine. 2019;25(4):476–87

Lipoarabinomannan from Mycobacterium indicus pranii shows immunostimulatory activity and induces autophagy in macrophages.

Mycobacterium indicus pranii (MIP) known for its immunotherapeutic potential against leprosy and tuberculosis is undergoing various clinical trials and also simultaneously being studied in animal models to get insight into the mechanistic details contributing to its protective efficacy as a vaccine candidate. Studies have shown potential immunomodulatory properties of MIP, the most significant being the ability to induce strong Th1 type of response, enhanced expression of pro-inflammatory cytokines, activation of APCs and lymphocytes, elicitation of M.tb specific poly-functional T cells. All of these form crucial components of host-immune response during M.tb infection. Also, MIP was found to be potent inducer of autophagy in macrophages which resulted in enhanced clearance of M.tb from MIP and M.tb co-infected cells. Hence, we further examined the component/s of MIP responsible for autophagy induction. Interestingly, we found that MIP lipids and DNA were able to induce autophagy but not the protein fraction. LAM being one of the crucial components of mycobacterial cell-wall lipids and possessing the ability of immunomodulation; we isolated LAM from MIP and did a comparative study with M.tb-LAM. Stimulation with MIP-LAM resulted in significantly high secretion of pro-inflammatory cytokines and displayed high autophagy inducing potential in macrophages as compared to M.tb-LAM. Treatment with MIP-LAM enhanced the co-localization of M.tb within the phago-lysosomes and increased the clearance of M.tb from the infected macrophages. This study describes LAM to be a crucial component of MIP which has significant contribution to its immunotherapeutic efficacy against TB.

Genetic and phenotypic characterization of recently discovered enterovirus D type 111.

Members of the species Enterovirus D (EV-D) remain poorly studied. The two first EV-D types (EV-D68 and EV-D70) have regularly caused outbreaks in humans since their discovery five decades ago but have been neglected until the recent occurrence of severe respiratory diseases due to EV-D68. The three other known EV-D types (EV-D94, EV-D111 and EV-D120) were discovered in the 2000s-2010s in Africa and have never been observed elsewhere. One strain of EV-D111 and all known EV-D120s were detected in stool samples of wild non-human primates, suggesting that these viruses could be zoonotic viruses. To date, EV-D111s are only known through partial genetic sequences of the few strains that have been identified so far. In an attempt to bring new pieces to the puzzle, we genetically characterized four EV-D111 strains (among the seven that have been reported until now). We observed that the EV-D111 strains from human samples and the unique simian EV-D111 strain were not phylogenetically distinct, thus suggesting a recent zoonotic transmission. We also discovered evidences of probable intertypic genetic recombination events between EV-D111s and EV-D94s. As recombination can only happen in co-infected cells, this suggests that EV-D94s and EV-D111s share common replication sites in the infected hosts. These sites could be located in the gut since the phenotypic analysis we performed showed that, contrary to EV-D68s and like EV-D94s, EV-D111s are resistant to acid pHs. We also found that EV-D111s induce strong cytopathic effects on L20B cells, a cell line routinely used to specifically detect polioviruses. An active circulation of EV-D111s among humans could then induce a high number of false-positive detection of polioviruses, which could be particularly problematic in Central Africa, where EV-D111 circulates and which is a key region for poliovirus eradication.

Co-infection with distinct Trypanosoma cruzi strains induces an activated immune response in human monocytes.

The aim of the study was to evaluate the immune response triggered by the first contact of human monocytes with two Tcruzi strains from distinct discrete typing units (DTUs) IV and V, and whether co-infection with these strains leads to changes in monocyte immune profiles, which could in turn influence the subsequent infection outcome. We evaluated the influence of in vitro single- and co-infection with AM64 and 3253 strains on immunological characteristics of human monocytes. Single infection of monocytes with AM64 or 3253 induced opposing anti-inflammatory and inflammatory responses, respectively. Co-infection was observed in over 50% of monocytes after 15hours of culture, but this percentage dropped ten-fold after 72hours. Co-infection led to high monocyte activation and an increased percentage of both IL-10 and TNF. The decreased percentage of co-infected cells observed after 72hours was associated with a decreased frequency of TNF-expressing cells. Our results show that the exacerbated response observed in co-infection with immune-polarizing strains is associated with a decreased frequency of co-infected cells, suggesting that the activated response favours parasite control. These findings may have implications for designing new Chagas disease preventive strategies.

Innovative dual system for selective eradication of cancer cells using exosomes carrying natural bacterial toxin-antitoxin (TA).

e14635 Background: Inactivation of P53 and activation of ras are frequent genetic alterations in cancer. We have shown in vitro and in vivo, that the TA system can selectively and effectively eradicate RAS-mutated cancer cells. Aim: Selective killing of cancer cells while sparing the normal cells based on tumor genetic signature. Methods: A “first generation” ΔE1/ΔE3 human type-5 adenoviral-vectors for gene delivery were designed and constructed to specifically target cancer cells. They are designated as "PY4-mazF-mCherry" (PY4, ras responsive element), "ΔPY4-mazF-mCherry" (control viruses) and "RGC-mazE-IRES-GFP" (RGC, P53 responsive element). Their potency was tested in vitro, by the enzymatic MTT assay, microscopic observation, colony formation assay and FACS analysis, and in a xenograft model of CRC. Next, we generated, small natural vesicles, exosomes, that directly targeted cancer through specific small antibody fragments against CD24 that is expressed in most cancer cells and rarely on normal cells. Results: The TA system ("PY4-mazF-mCherry"+"RGC-mazE-IRES-GFP") induced a massive cell death, in a dose-dependent manner in vitro, 69% as compared to 19% in control co-infected ("ΔPY4-mazF-mCherry"+"RGC-mazE-IRES-GFP") HCT116 CRC cells (mutated RAS and p53). In vivo, growth of HCT116-/- ( KRASmutand P53mut) and HCT116+/+ ( KRASmut and P53wt) tumors were significantly inhibited (70% and 65%, respectively). Conclusions: 1. Abusing the P53 genetic status and the activated Ras pathway holds promising effective and safe strategy to target tumor cells while sparing normal tissues. 2. It is a proof of concept for personalized cancer therapy based on the tumor genetic profile.

Enveloped viruses distinct from HBV induce dissemination of hepatitis D virus in vivo

Hepatitis D virus (HDV) doesn’t encode envelope proteins for packaging of its ribonucleoprotein (RNP) and typically relies on the surface glycoproteins (GPs) from hepatitis B virus (HBV) for virion assembly, envelopment and cellular transmission. HDV RNA genome can efficiently replicate in different tissues and species, raising the possibility that it evolved, and/or is still able to transmit, independently of HBV. Here we show that alternative, HBV-unrelated viruses can act as helper viruses for HDV. In vitro, envelope GPs from several virus genera, including vesiculovirus, flavivirus and hepacivirus, can package HDV RNPs, allowing efficient egress of HDV particles in the extracellular milieu of co-infected cells and subsequent entry into cells expressing the relevant receptors. Furthermore, HCV can propagate HDV infection in the liver of co-infected humanized mice for several months. Further work is necessary to evaluate whether HDV is currently transmitted by HBV-unrelated viruses in humans.

Characterising Birnaviridae replication and reassortment in vitro: virus factories derived from distinct input viruses form in the cytoplasm of co-infected cells and coalesce over time

The Birnaviridae family is comprised of non-enveloped viruses with a double-stranded RNA genome that is divided into two segments, A and B. Birnaviruses are responsible for major economic losses to the poultry and aquaculture industries, and reassortment complicates their epidemiology and control. However, little is known about the nature of theirreplication in cells, or the molecular mechanism underpinning reassortment. In order to address this, we rescued two recombinant infectious bursal disease (IBD) viruses, with either a GFP11 or Tetracysteine (TC) tag at the 3’ end of segment B (IBDV-GFP11 and IBDV-TC, respectively). DF-1 cells were either transfected with GFP1-10 prior to IBDV-GFP11 infection, or stained with ReAsH following IBDV-TC infection, which led to the apprearance of green or red foci in the cytoplasm, respectively. Foci co-localised with VP3 and dsRNA, suggesting these were virus factories (VFs). The average number of VFs significantly decreased from 60 to 5 per cell between 10 and 24 h post infection (P<0.01), while the average area significantly increased from 1.24 µm2 to 45.01 µm2 (P<0.01), suggesting VFs coalesce in the cytoplasm over time. Red, green and yellow foci were observed in the cytoplasm of co-infected cells, suggesting that VFs are initially derived from distinct input viruses prior to coalescence. Live cell imaging revealed that larger VFs were more static while smaller VFs were more mobile, and fusion events were observed. We speculate that VF coalescence is required for birnavirus reassortment, and current work is aimed at determining the cellular factors that drive coalescence.

The Cyclin-Dependent Kinase 5 Inhibitor Peptide Inhibits Herpes Simplex Virus Type 1 Replication

In order to evaluate the influence of CDK5 inhibitory peptide (CIP) on Human alphaherpesvirus 1 (HSV-1) replication, we constructed two recombinant adeno-associated-virus 2 (rAAV2) vectors encoding CIP fused with cyan-fluorescent-protein (CFP), with or without nuclear localization signal. A third vector encoding non-fused CIP and CFP was also constructed. HeLa and HEK 293T cells were infected with the rAAV-CIP vectors at multiplicity of infection (MOI) of 5000, in the absence or presence of a recombinant HSV-1 that encodes a yellow-fluorescent-protein (rHSV48Y; MOI = 1). Cells co-infected with rHSV48Y and rAAV vectors that did not express the CIP gene (rAAV-CFP-Neo) served as controls. At 24 h after infection, the effect of CIP on rHSV48Y replication was assessed by PCR, qRT-PCR, Western-blot, flow-cytometry, epifluorescence and confocal microscopy. We show that in cultures co-infected with rAAV-CFP-Neo, 27% of the CFP-positive cells present rHSV48Y replication compartments. By contrast, in cultures co-infected with CIP-encoding rAAV2 vectors and rHSV48Y only 6–20% of the cells positive for CIP showed rHSV48Y replication compartments, depending on the CIP variant. Flow-cytometry showed that less than 40% of the rHSV48Y/rAAV-CIP, and more than 75% of rHSV48Y/rAAV-CFP-Neo co-infected cells were positive for both transgene products. The microscopy and flow-cytometry data support the hypothesis that CIP is inhibiting HSV-1 replication.

Ubiquitin-Activating Enzyme E1 Inhibition Caused Acute Leukemia Cell Apoptosis By Affecting CHOP Pathway

Background: The ubiquitin proteinase system is involved in the pathogenesis of blood malignant tumors. Preliminary study of our group found that increased serum ubiquitin activase E1 (UBE1) fragment were in newly diagnosed and refractory & relapsed acute myeloid leukemia (AML), and its relative intensity was negatively correlated with AML's remission process and prognosis. The inhibition of UBE1 activity can promote AML cell apoptosis and may be related to the mechanism of endoplasmic reticulum stress. The current study is to clarify the role and mechanism of endoplasmic reticulation-derived transcription factor CHOP in apoptosis of leukemia cells induced by inhibiting ubiquitin activase E1 (UBE1) activity.Methods: UBE1-shRNA lentivirus carrier and CHOP-siRNA plasmid co-infected leukemia cells (U937, K562, NB4, THP-1). The protein expression of antiapoptosis molecules, endoplasmic reticulum stress protein and death receptors (Bcl-2, TRB3, ERO1α, DR5 and Bax) were detected by Western Blot. MTT and flow cytometry were used to detect cell proliferation inhibition and apoptosis. The model of leukemia cell bearing nude mouse was constructed. UBE1-shRNA and/or CHOP-siRNA were injected into the tumor. The tumor size, lifetime changes and tumor pathological changes were observed. Western Blot and RT-PCR were used to detect endoplasmic reticulum stress molecules and apoptosis molecules.Results: Infection of UBE1-shRNA significantly inhibited proliferation of leukemia cells (U937, K562, NB4, THP-1), while the inhibition of UBE1-shRNA and CHOP-siRNA co-transfected leukemia cells was decreased (p=0.032). The apoptosis rate in UBE1-shRNA leukemia cells was lower than that in UBE1-shRNA and CHOP-siRNA co-transfected leukemia cells (p=0.026). UBE1-shRNA can reduce the expression of apoptosis molecule Bcl-2 and increase the expression of TRB3, ERO1α, death receptor DR5, Bax protein. CHOP-siRNA can increase the expression of Bcl-2 in leukemia cells and reduce the expression of TRB3, ERO1α, death receptor DR5 and Bax protein. shRNA-UBE1 inhibited the leukemia cell xenograft, while siRNA-CHOP reversed the growth inhibition of shRNA-UBE1 on leukemia cell xenograft (p=0.002). A larger number of leukemia cells were degenerative necrosis in the UBE1-shRNA group comparing with the UBE1-shRNA and siRNA-CHOP co-transfected groups (p=0.023), and damaged membrane, different sizes, blurred outline, and nuclear pyknosis and karyorrhexis could be observed. The average survival time of mice with co-transfection of shRNA-UBE1 and siRNA-CHOP was shorter than the average survival time of mice with shRNA-UBE1 (p=0.016). Compared with the shRNA-UBE1 group, there was no difference in expression of endoplasmic reticulum stress molecules in co-transfection of UBE1-shRNA and siRNA-CHOP group, but TRB3, ERO1α, DR5, Bax expression were decreased, and Bcl-2 was increased (p<0.05).Conclusion: The inhibition of UBE1 activity can induce AML cell apoptosis by endoplasmic reticulum stress CHOP pathway. It will provide new clues for the treatment of acute myeloid leukemia.Disclosures: No relevant conflicts of interest to declare. DisclosuresNo relevant conflicts of interest to declare.

Fluorescent labelling of Beet necrotic yellow vein virus and Beet soil-borne mosaic virus for co- and superinfection experiments in Nicotiana benthamiana.

Infectious full-length clones of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV), both genus Benyvirus, were used for fluorescent labelling with the objective to study their interaction in coinfection and superinfection experiments. Fluorescent labelling was achieved by replacing a part of the RNA2 encoded coat protein read-through domain with either GFP or mRFP fluorescent marker proteins. This resulted in a translational fusion comprising the coat and the fluorescent protein. The labelled viruses were infectious and moved systemically in Nicotiana benthamiana, producing wild-type-like symptoms. Virus particles could be observed by electron microscopy, demonstrating that the viral read-through domain is dispensable for particle formation. Coinfection experiments revealed a spatial separation of differentially labelled populations of both identical and different Benyvirus species after N. benthamiana agro-inoculation. Identical observations were obtained when Tobacco rattle virus (TRV) was differentially labelled and used for coinfection. In contrast, coinfections of BSBMV with Potato virus X (PVX) or TRV resulted in many co-infected cells lacking spatial separation. Micro-projectile co-bombardment of N. benthamiana leaves revealed that two differently labelled populations of the same virus co-infected only a few cells before starting to separate. In superinfection experiments with N. benthamiana, BSBMV and BNYVV were unable to establish a secondary infection in plants that were previously infected with BNYVV or BSBMV. Taken together, this is the first work to describe the interaction between two economically important Benyviruses using fluorescence-labelled full-length clones.

Viral interference between low pathogenic avian influenza H9N2 and avian infectious bronchitis viruses in vitro and in ovo

BackgroundLow pathogenic avian influenza (LPAI) H9N2 and infectious bronchitis virus (IBV) are important pathogens of poultry, causing important economic losses for the sector. Replication interference between these two viruses was described using cell cultures (CC) and embryonated chicken eggs (ECE). Chicken embryo lung (CEL) and ECE were simultaneously or sequentially infected with IBV vaccine strain (H120) and LPAIV-H9N2 (A/Ck/TUN/145/2012) to evaluate viral interactionsin vitro and in ovo, respectively. Real-time RT-PCR was developed to specifically quantify both AIV and IBV genomes as well as viral gene copy numbers during mixed infections. The amount of IL-1 beta, in supernatants of co-infected cell cultures, was determined using an ELISA assay. ResultsQuantitative results of AIV and IBV co-infection showed that interferences between the two viruses yielded decreased viral growth. However, in the case of super-infection, the second virus, either AIV or IBV, induced a decrease in the growth of the first inoculated virus. ConclusionIt appears that either AIV or IBV has a negative impact on the other virus growth when they are inoculated simultaneously or sequentially. The ELISA results showed that higher level of secreted IL-1beta varies, depending on the viral interference conditions between both viruses, during mixed infections.

Matrix stiffness regulate apoptotic cell death in HIV-HCV co-infected hepatocytes: Importance for liver fibrosis progression

HIV-HCV co-infection causes rapid progression of liver fibrosis. These outcomes to liver cirrhosis can be improved, but not stopped by specific antiviral therapies. Due to high significance of HIV-HCV interactions for morbidity and mortality in co-infected patients, our attention was attracted to the multi-component pathogenesis of fibrosis progression as the transition to end-stage liver disease development. In this study, we hypothesize that increased matrix stiffness enhances apoptosis in HCV-HIV-co-infected hepatocytes and that capturing of apoptotic bodies (AB) derived from these infected hepatocytes by hepatic stellate cells (HSC) drives the fibrosis progression. As the source of viruses, JFH1 (HCV genotype 2a) and HIV-1ADA (either purified or containing in infected macrophage supernatants) were chosen. Using Huh7.5-CYP (RLW) cells and primary human hepatocytes mono-infected with HCV and HIV or co-infected, we have shown that both HCV and HIV RNA levels were increased in co-infected cells, which was accompanied by hepatocyte apoptosis. This apoptosis was attenuated by azidothymidine treatment. The levels of both infections and apoptosis were more prominent in primary hepatocytes cultured on substrates mimicking fibrotic stiffness (24 kPa-stiff) compared to substrates mimicking healthy liver (2.4 kPa-soft). The engulfment of AB from pathogen-exposed hepatocytes activated pro-fibrotic mRNAs in HSC. Overall, the increased matrix stiffness is not only a consequence of liver inflammation/fibrosis, but the condition that further accelerates liver fibrosis development. This is attributed to the switching of HSC to pro-fibrotic phenotype by capturing of excessive amounts of apoptotic HCV- and HIV-infected hepatocytes.

Aspartyl proteases in Candida glabrata are required for suppression of the host innate immune response

A family of 11 cell surface-associated aspartyl proteases (CgYps1–11), also referred as yapsins, is a key virulence factor in the pathogenic yeast Candida glabrata. However, the mechanism by which CgYapsins modulate immune response and facilitate survival in the mammalian host remains to be identified. Here, using RNA-Seq analysis, we report that genes involved in cell wall metabolism are differentially regulated in the Cgyps1–11Δ mutant. Consistently, the mutant contained lower β-glucan and mannan levels and exhibited increased chitin content in the cell wall. As cell wall components are known to regulate the innate immune response, we next determined the macrophage transcriptional response to C. glabrata infection and observed differential expression of genes implicated in inflammation, chemotaxis, ion transport, and the tumor necrosis factor signaling cascade. Importantly, the Cgyps1–11Δ mutant evoked a different immune response, resulting in an enhanced release of the pro-inflammatory cytokine IL-1β in THP-1 macrophages. Further, Cgyps1–11Δ–induced IL-1β production adversely affected intracellular proliferation of co-infected WT cells and depended on activation of spleen tyrosine kinase (Syk) signaling in the host cells. Accordingly, the Syk inhibitor R406 augmented intracellular survival of the Cgyps1–11Δ mutant. Finally, we demonstrate that C. glabrata infection triggers elevated IL-1β production in mouse organs and that the CgYPS genes are required for organ colonization and dissemination in the murine model of systemic infection. Altogether, our results uncover the basis for macrophage-mediated killing of Cgyps1–11Δ cells and provide the first evidence that aspartyl proteases in C. glabrata are required for suppression of IL-1β production in macrophages.