Cytopathic Cell Research Articles

Antiviral effects against EV71 of pimprinine and its derivatives isolated from Streptomyces sp.

BackgroundThe pimprinine family of compounds represent very important and promising microbial metabolites for drug discovery. However, their ability in inhibiting viral infections has not yet been tested.MethodsThe antiviral activity of the pimprinine family of compounds was evaluated by determining the cytopathic effect (CPE), cell viability or plaque-forming unit (PFU), and virus yield. The mechanism of action against EV71 was determined from the virucidal activity, and effective stage and time-of-addition assays. The effects on EV71 replication were evaluated further by determining viral RNA synthesis, protein expression and cells apoptosis using the SYBR Green assays, immunofluorescence assays and flow cytometric assays, respectively.ResultsPimprinethine, WS-30581 A and WS-30581 B inhibited EV71-induced CPE, reduced progeny EV71 yields, as well as prevented EV71-induced apoptosis in human rhabdomyosarcoma (RD) cells. These compounds were found to target the early stages of the EV71 replication in cells including viral RNA replication and protein synthesis. They also showed antiviral activity against ADV-7, and were slightly active against CVB3, HSV-1 and H1N1 with a few exceptions. Pimprinine was slightly active or inactive against all the viruses tested. The mechanisms by which these compounds act against the viruses tested may be similar to that demonstrated for EV71.ConclusionThe data described herein demonstrate that the pimprinine family of compounds are inhibitors effective against the replication of EV71 and ADV-7, so they might be feasible therapeutic agents for the treatment of viral infections.

Nonstructural protein 1 (NS1)-mediated inhibition of c-Abl results in acute lung injury and priming for bacterial co-infections: insights into 1918 H1N1 pandemic?

Nonstructural protein 1 (NS1) proteins from avian influenza viruses like the 1918 pandemic NS1 are capable of inhibiting the key signaling integrator c-Abl (Abl1), resulting in massive cytopathic cell alterations. In the current study, we addressed the consequences of NS1-mediated alteration of c-Abl on acute lung injury and pathogenicity in an in vivo mouse model. Comparing isogenic strains that differ only in their ability to inhibit c-Abl, we observed elevated pathogenicity for the c-Abl-inhibiting virus. NS1-mediated blockade of c-Abl resulted in severe lung pathology and massive edema formation and facilitated secondary bacterial pneumonia. This phenotype was independent of differences in replication and immune responses, defining it as an NS1 virulence mechanism distinct from its canonical functions. Microarray analysis revealed extensive downregulation of genes involved in cell integrity and vascular endothelial regulation. NS1 protein-mediated blockade of c-Abl signaling drives acute lung injury and primes for bacterial coinfections revealing potential insights into the pathogenicity of the 1918 pandemic virus.

Sulfated galactans isolated from the red seaweed Gracilaria fisheri target the envelope proteins of white spot syndrome virus and protect against viral infection in shrimp haemocytes

The present study was aimed at evaluating an underlying mechanism of the antiviral activity of the sulfated galactans (SG) isolated from the red seaweed Gracilaria fisheri against white spot syndrome virus (WSSV) infection in haemocytes of the black tiger shrimp Penaeus monodon. Primary culture of haemocytes from Penaeus monodon was performed and inoculated with WSSV, after which the cytopathic effect (CPE), cell viability and viral load were determined. Haemocytes treated with WSSV-SG pre-mix showed decreased CPE, viral load and cell mortality from the viral infection. Solid-phase virus-binding assays revealed that SG bound to WSSV in a dose-related manner. Far Western blotting analysis indicated that SG bound to VP 26 and VP 28 proteins of WSSV. In contrast to the native SG, desulfated SG did not reduce CPE and cell mortality, and showed low binding activity with WSSV. The current study suggests that SG from Gracilaria fisheri elicits its anti-WSSV activity by binding to viral proteins that are important for the process of viral attachment to the host cells. It is anticipated that the sulfate groups of SG are important for viral binding.

One Potential Oncolytic Adenovirus Expressing Lipocalin-2 for Colorectal Cancer Therapy

Colorectal cancer is an aggressive malignancy with a high mortality rate; however, effective therapies are currently lacking. Cancer-targeting gene-virotherapy (CTGVT) has been proposed to be a promising strategy for cancer therapy. The purpose of this study was to investigate the antitumor activity of the oncolytic adenovirus harboring Lipocalin-2 (ZD55-Lipocalin-2, an example of CTGVT) in colorectal cancer. ZD55-Lipocalin-2 was generated by deleting E1B55-KD and inserting the Lipocalin-2 gene. Its cytopathic effects and cell growth inhibition were detected in vitro, and antitumor effects were examined in a nude mouse model of human colorectal cancer xenografts. Results showed that ZD55-Lipocalin-2 significantly inhibited the colorectal cancer growth by selective cytolysis, inducing apoptosis and decreasing the microvessel density in tumors. The anticancer potential of ZD55-Lipocalin-2 showed stronger than that of the isolated Lipocalin-2 gene therapy or isolated ZD55 oncolytic adenovirus therapy. ZD55-Lipocalin-2 may serve as a potential anticancer agent for colorectal cancer treatment.

Over-expression of mitochondrial antiviral signaling protein inhibits coxsackievirus B3 infection by enhancing type-I interferons production

BackgroundRecent studies have revealed that Mitochondrial Antiviral Signaling (MAVS) protein plays an essential role in the inhibition of viral infection through type I interferon (IFN) pathway. It has been shown that 3C (pro) cysteine protease of coxsackievirus B3 (CVB3) cleaves MAVS to inhibit type I IFNs induction. Other workers also found that MAVS knock-out mice suffered CVB3 susceptibility and severe histopathological change. Accordingly,our experiments were designed to explore the protection of over-expressing MAVS against CVB3 infection and the possible mechanism.ResultsIn this study, HeLa cells (transfected with MAVS constructs pre- or post- exposure to CVB3) were used to analyze the function of exogenous MAVS on CVB3 infection. The results revealed that though CVB3 infection induced production of type I IFNs, viral replication and cell death were not effectively inhibited. Similarly, exogenous MAVS increased type I IFNs moderately. Morever, we observed robust production of type I IFNs in CVB3 post-infected HeLa cells thereby successfully inhibiting CVB3 infection, as well formation of cytopathic effect (CPE) and cell death. Finally, introduction of exogenous MAVS into CVB3 pre-infected cells also restricted viral infection efficiently by greatly up-regulating IFNs.ConclusionsIn summary, exogenous MAVS effectively prevents and controls CVB3 infection by modulating and promoting the production of type I IFNs. The IFNs level in MAVS over-expressing cells is still tightly regulated by CVB3 infection. Thus, the factors that up-regulate MAVS might be an alternative prescription in CVB3-related syndromes by enhancing IFNs production.

Abstract 333: Chemokine and Autophagy-Related Genes in Novel In Vivo and In Vitro Models for Viral Myocarditis

Picornavirus infection is one of leading causes of myocarditis in humans. Theiler's murine encephalomyelitis virus (TMEV) belongs to the genus Cardiovirus , the family Picornaviridae . While TMEV has been used as a mouse model for multiple sclerosis (MS), TMEV can also cause myocarditis in mice. We found that the susceptibilities of mouse strains to myocarditis differed from susceptibilities to the MS model. In the heart, we detected viral replication and inflammation. However, it is unclear whether virus itself or immune cells damaged the heart. To clarify the role of direct virus infection in myocarditis we used the cardiomyocyte cell line, HL-1, which retains a differentiated cardiomyocyte phenotype and contractile activity. We infected HL-1 with TMEV and monitored cytopathic effect (CPE) and cell viability. At 8 hours post infection (hpi), HL-1 cells developed CPE with decreased contractile activities, while cell death was not obvious. We detected 54% and 93% of cell death at 12 and 24 hpi, respectively, while no cell death was observed in mock-infection. We conducted microarray analyses to compare mRNA expression patterns between TMEV and mock-infected groups, using an Affymetrix GeneChip Mouse 1.0 ST Array. Principal component analysis (PCA) for microarray data clearly separated TMEV- and mock-infected groups (PC1 proportion of variance, 59%). TMEV infection induced high expression of several interferon-associated and chemokine genes, particularly Cxcl10/IP-10 . Cxcl10 is a potent chemoattractant for activated T cells and NK cells, and its potential roles have been associated with immune cells in other myocarditis models. Our current results demonstrated that virus infection alone could induce Cxcl10 in cardiomyocytes without immune cells. We also found upregulation of autophagy-related 12 gene ( Atg12 ). In general, autophagy can contribute to eradication of intracellular pathogens including viruses. However, since induction of autophagy by picornavirus has been shown to enhance virus replication, autophagy may also play a detrimental role in TMEV-induced myocarditis. Our novel in viv o and in vitro models of myocarditis are powerful tools to dissect the role of viral and immune pathogenesis of the heart, complementing each other.

Screening of anti-dengue activity in methanolic extracts of medicinal plants

BackgroundDengue fever regardless of its serotypes has been the most prevalent arthropod-borne viral diseases among the world population. The development of a dengue vaccine is complicated by the antibody-dependent enhancement effect. Thus, the development of a plant-based antiviral preparation promises a more potential alternative in combating dengue disease.MethodsPresent studies investigated the antiviral effects of standardised methanolic extracts of Andrographis paniculata, Citrus limon, Cymbopogon citratus, Momordica charantia, Ocimum sanctum and Pelargonium citrosum on dengue virus serotype 1 (DENV-1).ResultsO. sanctum contained 88.6% of total flavonoids content, an amount that was the highest among all the six plants tested while the least was detected in M. charantia. In this study, the maximum non-toxic dose (MNTD) of the six medicinal plants was determined by testing the methanolic extracts against Vero E6 cells in vitro. Studies also determined that the MNTD of methanolic extract was in the decreasing order of M. charantia >C. limon >P. citrosum, O. sanctum >A. paniculata >C. citratus. Antiviral assay based on cytopathic effects (CPE) denoted by degree of inhibition upon treating DENV1-infected Vero E6 cells with MNTD of six medicinal plants showed that A. paniculata has the most antiviral inhibitory effects followed by M. charantia. These results were further verified with an in vitro inhibition assay using MTT, in which 113.0% and 98.0% of cell viability were recorded as opposed to 44.6% in DENV-1 infected cells. Although methanolic extracts of O. sanctum and C. citratus showed slight inhibition effect based on CPE, a significant inhibition was not reflected in MTT assay. Methanolic extracts of C. limon and P. citrosum did not prevent cytopathic effects or cell death from DENV-1.ConclusionsThe methanol extracts of A. paniculata and M. charantia possess the ability of inhibiting the activity of DENV-1 in in vitro assays. Both of these plants are worth to be further investigated and might be advantageous as an alternative for dengue treatment.

Encephalomyocarditis Virus 2A Protein Is Required for Viral Pathogenesis and Inhibition of Apoptosis

The encephalomyocarditis virus (EMCV), a Picornaviridae virus, has a wide host spectrum and can cause various diseases. EMCV virulence factors, however, are as yet ill defined. Here, we demonstrate that the EMCV 2A protein is essential for the pathogenesis of EMCV. Infection of mice with the B279/95 strain of EMCV resulted in acute fatal disease, while the clone C9, derived by serial in vitro passage of the B279/95 strain, was avirulent. C9 harbored a large deletion in the gene encoding the 2A protein. This deletion was incorporated into the cDNA of a pathogenic EMCV1.26 strain. The new virus, EMCV1.26Δ2A, was capable of replicating in vitro, albeit more slowly than EMCV1.26. Only mice inoculated with EMCV1.26 triggered death within a few days. Mice infected with EMCV1.26Δ2A did not exhibit clinical signs, and histopathological analyses showed no damage in the central nervous system, unlike EMCV1.26-infected mice. In vitro, EMCV1.26Δ2A presented a defect in viral particle release correlating with prolonged cell viability. Unlike EMCV1.26, which induced cytopathic cell death, EMCV1.26Δ2A induced apoptosis via caspase 3 activation. This strongly suggests that the 2A protein is required for inhibition of apoptosis during EMCV infection. All together, our data indicate that the EMCV 2A protein is important for the virus in counteracting host defenses, since Δ2A viruses were no longer pathogenic and were unable to inhibit apoptosis in vitro.

Abstract 2862: Bioengineered oncolytic adenovirus induces autophagic cell death through an E2F1-microRNA-7-epidermal growth factor receptor axis

Abstract Autophagy is known to have a cytoprotective role under various cellular stresses, such as deprivation of nutrients, hypoxia and interruption of growth signaling. In contrast, autophagy also results in robust cell death as an important safeguard mechanism that protects the organism against invading pathogens and unwanted cancer cells. Recent accumulating evidences have suggested the implication of autophagy on the oncolytic adenovirus-mediated cell death in human cancer cells. However, the molecular mechanism underlying oncolytic adenovirus-mediated autophagic cell death remains unclear. We previously generated a genetically engineered oncolytic adenovirus (Telomelysin®: OBP-301) that replicates and induces autophagic cell death against human cancer cells in a telomerase-dependent manner. As microRNA (miRNA), a post-transcriptional regulator of gene expression, has been recently shown to regulate diverse cell fates including autophagy, we hypothesized that OBP-301 infection induces autophagic cell death through miRNA modulation in human cancer cells. In this study, we attempted the role of miRNA on the OBP-301-mediated autophagic cell death in human cancer cells. We used 3 human cancer cell lines (H1299, A549 and T.Tn), that showed different sensitivities to OBP-301. OBP-301 infection upregulated miR-7 expression in OBP-301-sensitive human cancer cell lines (H1299 and A549), but not OBP-301-resistant human cancer cell line (T.Tn). Expression levels of miR-7 upregulated after OBP-301 infection was significantly associated with OBP-301-mediated cytopathic activity and autophagic cell death, which was confirmed by western blot for autophagy-related proteins (LC3-I/II, Atg5 and p62). We also revealed that OBP-301-mediated miR-7 upregulation mainly depended on enhanced expression of the E2F1 protein after OBP-301 infection. Furthermore, ectopic expression of miR-7 in human cancer cell lines suppressed cell viability and subsequently induced autophagy through inhibition of epidermal growth factor receptor (EGFR) expression. OBP-301 infection efficiently suppressed EGFR expression, which was significantly associated with miR-7 upregulation and cytopathic activity, in human cancer cell lines. Our results suggest that oncolytic adenovirus OBP-301 induces autophagic cell death through an E2F1-miR-7-EGFR pathway in human cancer cells, providing a novel insight into the molecular mechanism of an anticancer virotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2862. doi:10.1158/1538-7445.AM2011-2862

Rethinking the Pathogenesis of Asthma

Asthma research has focused primarily on allergic pathways on the basis that the majority of asthma is associated with atopy, the recruitment of the Th2-type T cell, the cytokines, and the chemokines that are released on exposure to allergens, and the IgE pathway. However, despite considerable investment by industry, targeting these pathways has not resulted in new treatments being developed beyond blockade of cysteinyl leukotrienes and IgE and improvements in inhaled corticosteroids and beta(2)-adrenoceptor bronchodilators. Increasingly, it is recognized that asthma is a heterogeneous disorder, and while important, allergen sensitization is only one component of the disease, with many other environmental and genetic factors playing a role. In addition, these factors act locally on a susceptible airway epithelium that is both structurally and functionally deficient. It may be worthwhile to focus on increasing the resilience of the airways to environmental insults in addition to improving strategies that modify adaptive immunity or suppress inflammation.

Differential Modulation of TNF-α–Induced Apoptosis by Neisseria meningitidis

Infections by Neisseria meningitidis show duality between frequent asymptomatic carriage and occasional life-threatening disease. Bacterial and host factors involved in this balance are not fully understood. Cytopathic effects and cell damage may prelude to pathogenesis of isolates belonging to hyper-invasive lineages. We aimed to analyze cell–bacteria interactions using both pathogenic and carriage meningococcal isolates. Several pathogenic isolates of the ST-11 clonal complex and carriage isolates were used to infect human epithelial cells. Cytopathic effect was determined and apoptosis was scored using several methods (FITC-Annexin V staining followed by FACS analysis, caspase assays and DNA fragmentation). Only pathogenic isolates were able to induce apoptosis in human epithelial cells, mainly by lipooligosaccharide (endotoxin). Bioactive TNF-α is only detected when cells were infected by pathogenic isolates. At the opposite, carriage isolates seem to provoke shedding of the TNF-α receptor I (TNF-RI) from the surface that protect cells from apoptosis by chelating TNF-α. Ability to induce apoptosis and inflammation may represent major traits in the pathogenesis of N. meningitidis. However, our data strongly suggest that carriage isolates of meningococci reduce inflammatory response and apoptosis induction, resulting in the protection of their ecological niche at the human nasopharynx.

Thiazolidinedione use and the risk of fractures

<b>Background</b> Although coronavirus disease 2019 (COVID-19) causes significant morbidity, mainly from pulmonary involvement, extrapulmonary symptoms are also major components of the disease. Kidney disease, usually presenting as acute kidney injury (AKI), is particularly severe among patients with COVID-19. It is unknown, however, whether such injury results from direct kidney infection with COVID-199s causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or from indirect mechanisms. <b>Methods</b> Using <i>ex vivo</i> cell models, we sought to analyze SARS-Cov2 interactions with kidney tubular cells and assess direct tubular injury. These models comprised primary human kidney epithelial cells (derived from nephrectomies and grown as proliferating monolayers) and more quiescent three-dimensional kidney spheroids. <b>Results</b> We demonstrated that viral entry molecules and high baseline levels of type 1 interferon-related molecules were present in monolayers and kidney spheroids. Although both models support viral infection and replication, they did not exhibit a cytopathic effect and cell death, outcomes that were strongly present in SARS-CoV-2-infected controls (Vero E6 cultures). A comparison of monolayer and spheroid cultures demonstrated higher infectivity and replication of SARS-Cov-2 in actively proliferating monolayers, although the spheroid cultures exhibited higher levels of ACE2. Monolayers exhibited elevation of some tubular injury molecules—including molecules related to fibrosis (COL1A1 and STAT6) and dedifferentiation (SNAI2)—as well as a loss of cell identity, evident by reduction in megalin (LRP2). The three-dimensional spheroids were less prone to such injury. <b>Conclusions</b> SARS-CoV-2 can infect kidney cells without a cytopathic effect. AKI-induced cellular proliferation may potentially intensify infectivity and tubular damage by SARS-CoV-2, suggesting that early intervention in AKI is warranted to help minimize kidney infection.

Effect of caffeine on the multiplication of DNA and RNA viruses

The present study examined the effect of caffeine on RNA and DNA viruses, revealing that it inhibits the multiplication of both. In the presence of caffeine, the progeny virus yield of both herpes simplex virus type 1 (HSV-1) and poliovirus decreased with increasing concentrations of the reagent, although HSV-1 was much more sensitive than poliovirus. The influenza virus was not affected by caffeine at the same concentrations. None of the viruses were directly inactivated by caffeine at the tested concentrations. Characterization of the mode of action of caffeine against HSV-1 infection revealed that the addition of the reagent at 10 h post-infection significantly affected the formation of progeny virus, indicating that caffeine can inhibit the multiplication of HSV-1 during the step(s) following the completion of viral DNA replication and the formation of nucleocapsids. In addition, the reagent selectively enhanced the cytopathic effects and cell death of the infected cells over uninfected cells, suggesting that the antiviral action of caffeine against HSV-1 is, at least in part, the result of accelerated degeneration of the infected cells.

Potential Effect of Acacia arabica. on Peste des Petits Ruminants. Virus Replication

An aqueous preparation from Acacia arabica. var. indica. Benth (Mimosaceae) (locally known as “babul”) leaves (BExt) was assessed for its in vitro. antiviral activity using peste des petits ruminants. virus (PPRV) as a test model in the Vero cell system. Cytopathic effect (CPE) inhibition, virus titration, cell ELISA, sandwich-ELISA (s-ELISA), and PCR assays were used to determine the antiviral effects (at maximum noncytotoxic concentrations 150 and 200 μg/mL) against PPRV, and in vitro. cytotoxicity assays established the relative safety concentration of the BExt for the cells. BExt inhibited viral infectivity drastically in terms of decreased virus titer and antigen load in a dose-dependent manner either when added to cell monolayers postinfection or when preincubated with virus before adsorption on the cells. Inhibition of cell-free and cell-associated PPRV during replication in presence of BExt in Vero cells, using a multistep growth curve experiment, were assessed by s-ELISA. BExt (200 μg/mL) completely inhibited PPRV replication in Vero cells that were infected with PPRV at 0.01 multiplicity of infection. Incubation of PPRV with BExt (150 and 200 μg/mL) followed by infection had a virucidal effect on subsequent progeny virus yield by a 3 log10 TCID50 reduction. This indicates that active principle(s) of BExt either inactivated the virus or inhibited the viral release. Real-time PCR data based on nucleoprotein gene showed 196.7-and 770.6-fold reduction in the viral load in the presence of BExt concentrations of 150 and 200 μag/mL, respectively, indicating the efficacy of BExt in inhibiting PPRV multiplication. These data suggest that extracts of A. arabica. could be a potential natural antiviral agent for management of PPR disease and also a possible addition in the traditional phyto-antiviral repertoire for viral disease control.

Transdominant Inhibition of Bovine Viral Diarrhea Virus Entry

Bovine viral diarrhea virus (BVDV) is a positive-strand RNA virus and a member of the genus Pestivirus in the family Flaviviridae. To identify and characterize essential factors required for BVDV replication, a library expressing random fragments of the BVDV genome was screened for sequences that act as transdominant inhibitors of viral replication by conferring resistance to cytopathic BVDV-induced cell death. We isolated a BVDV-nonpermissive MDBK cell clone that harbored a 1.2-kb insertion spanning the carboxy terminus of the envelope glycoprotein 1 (E1), the envelope glycoprotein E2, and the amino terminus of p7. Confirming the resistance phenotype conferred by this library clone, naïve MDBK cells expressing this fragment were found to be 100- to 1,000-fold less permissive to both cytopathic and noncytopathic BVDV infection compared to parental MDBK cells, although these cells remained fully permissive to vesicular stomatitis virus. This restriction could be overcome by electroporation of BVDV RNA, indicating a block at one or more steps in viral entry prior to translation of the viral RNA. We determined that the E2 ectodomain was responsible for the inhibition to BVDV entry and that this block occurred downstream from BVDV interaction with the cellular receptor CD46 and virus binding, suggesting interference with a yet-unidentified BVDV entry factor.

Inhibition of henipavirus infection by Nipah virus attachment glycoprotein occurs without cell-surface downregulation of ephrin-B2 or ephrin-B3

Nipah virus (NiV) and Hendra virus (HeV) are newly identified members of the family Paramyxoviridae and have been classified in the new genus Henipavirus based on unique genetic characteristics distinct from other paramyxoviruses. Transgenic cell lines were generated that expressed either the attachment protein (G) or the fusion protein (F) of NiV. Functional expression of NiV F and G was verified by complementation with the corresponding glycoprotein, which resulted in the development of syncytia. When exposed to NiV and HeV, expression of NiV G in Crandall feline kidney cells resulted in a qualitative inhibition of both cytopathic effect (CPE) and cell death by both viruses. RT-PCR analysis of surviving exposed cells showed a complete absence of viral positive-sense mRNA and genomic negative-sense viral RNA. Cells expressing NiV G were also unable to fuse with cells co-expressing NiV F and G in a fluorescent fusion inhibition assay. Cell-surface staining for the cellular receptors for NiV and HeV (ephrin-B2 and ephrin-B3) indicated that they were located on the surface of cells, regardless of NiV G expression or infection by NiV. These results indicated that viral interference can be established for henipaviruses and requires only the expression of the attachment protein, G. Furthermore, it was found that this interference probably occurs at the level of virus entry, as fusion was not observed in cells expressing NiV G. Finally, expression of NiV G by either transient transfection or NiV infection did not alter the cell-surface levels of the two known viral receptors.

Sindbis Virus Nonstructural Protein nsP2 Is Cytotoxic and Inhibits Cellular Transcription

Replication of alphaviruses in vertebrate cells strongly affects cell physiology and ultimately leads to development of a cytopathic effect (CPE) and cell death. Sindbis virus (SIN) replication causes major changes in cellular macromolecular synthesis, in which the strong downregulation of transcription of cellular mRNAs and rRNAs plays a critical role. SIN nonstructural protein nsP2 was previously proposed as one of the main regulators of virus-host cell interactions, because point mutations in the carboxy-terminal part of nsP2 could make SIN and other alphaviruses and replicons less cytopathic and capable of persisting in some vertebrate cell lines. These mutants were incapable of inhibiting transcription and downregulating a viral stress-induced cell response. In the present work, we demonstrate that (i) SIN nsP2 is critically involved in CPE development, not only during the replication of SIN-specific RNAs, but also when this protein is expressed alone from different expression cassettes; (ii) the cytotoxic effect of SIN nsP2 appears to be at least partially determined by its ability to cause transcriptional shutoff; (iii) these functions of SIN nsP2 are determined by the integrity of the carboxy-terminal peptide of this protein located outside its helicase and protease domains, rather than by its protease activity; and (iv) the cytotoxic activity of SIN nsP2 depends on the presence of this protein in a free form, and alterations in P123 processing abolish the ability of nsP2 to cause CPE.

Peroxisome proliferator‐activated receptor gamma ligands inhibit the replication of respiratory syncytial virus

Respiratory syncytial virus (RSV) is worldwide the major cause of morbidity and life-threatining lower respiratory tract disease in infants. However, there exist no promising antiviral treatment. Recently, peroxisome proliferator-activated receptor-γ (PPARγ) has been shown to modulate the virus growth of HIV. We hypothesized, therefore, that activation of PPARγ might interfere with the replication of RSV in human lung epithelial cells (A549), the target for RSV infection. Specific activation of PPARγ was induced with the endogenous agonist 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), the synthetic thiazolidinedione compounds ciglitazone and troglitazone, and Fmoc-Leu, respectively. All PPARγ agonists under study subsequently added to RSV-infection reduced the virus-induced cytotoxicity. However, only the PPARγ agonists ciglitazone, troglitazone, and Fmoc-Leu, respectively, totally abolished the cytopathic cell damage up to 72 h postinfection. Simultaneously, all PPARγ agonists markedly inibited the cell surface expression of the viral G and F protein on RSV-infected A549 cells. The reduced viral G protein expression correlated with a diminished G protein mRNA amount determined by RT-PCR. Simultaneously, a diminished RSV N gene expression was demonstrated by real-time RT-PCR. Theses changes led to a 1000fold reduced release of infectious progeny virus. Our in vitro data suggest that especially the synthetic PPARγ agonists Fmoc-Leu and the thiazolidinediones ciglitazone and troglitazone may have a significantly protective impact on human lung epithelial cells in the course of RSV infection.

Nipah virus RNA synthesis in cultured pig and human cells

Nipah virus infection of porcine stable kidney cells (PS), human neuronal cells (SK-N-MC), human lung fibroblasts cells (MRC-5), and human monocytes (THP-1) were examined. Rapid progression of cytopathic effects (CPE) and cell death were noted in PS cell cultures treated with Nipah virus, followed by MRC-5, SK-N-MC, and THP-1 cell cultures, in descending order of rapidity. Significant increase in the intracellular Nipah virus RNA occurred beginning at 24 hr PI in all the infected cells. Whereas, the extracellular release of Nipah virus RNA increased significantly beginning at 48 and 72 hr PI for the infected MRC-5 cells and PS cells, respectively. No significant release of extracellular Nipah virus RNA was detected from the Nipah virus-infected SK-N-MC and THP-1 cells. At its peak, approximately 6.6 log PFU/microl of extracellular Nipah virus RNA was released from the Nipah virus-infected PS cells, with at least a 100-fold less virus RNA was recorded in the Nipah virus-infected SK-N-MC and THP-1. Approximately 15.2% (+/-0.1%) of the released virus from the infected PS cell cultures was infectious in contrast to approximately 5.5% (+/-0.7%) from the infected SK-N-MC cells. The findings suggest that there are no differences in the capacity to support Nipah virus replication between pigs and humans in fully susceptible PS and MRC-5 cells. However, there are differences between these cells and human neuronal cells and monocytes in the ability to support Nipah virus replication and virus release.

Lymphocytopathogenic activity in vitro correlates with high virulence in vivo for BVDV type 2 strains: Criteria for a third biotype of BVDV

Two biotypes of bovine viral diarrhea viruses (BVDV), cytopathic (cp) and noncytopathic (ncp), are recognized based on their activity in cultured epithelial cells. Biotype does not correlate to virulence in acute infections as BVDV strains associated with severe acute BVD outbreaks are all noncytopathic based on their growth characteristics in cultured epithelial cells. Previous studies have shown that acute infections with highly virulent BVDV result in depletion of cells in lymphoid tissues. In this study, flow cytometry demonstrated that infection with a highly virulent BVDV strain was associated with a pronounced reduction in circulating white blood cells (WBC) and increased numbers of apoptotic and necrotic circulating WBC in vivo. Infection with low virulence BVDV did not result in a significant increase in death of circulating WBC. Thus, there appeared to be a correlation between depletion of circulating WBC and virulence. To study the interaction of BVDV strains with lymphoid cells in the laboratory, we developed an in vitro model that used a bovine lymphoid cell line (BL-3 cells). Using this model, it was found that while BVDV strains are segregated into two biotypes based on their activity in cultured epithelial cells, they may be segregated into three biotypes based on their activity in cultured lymphoid cells. These three biotypes are noncytopathogenic (no obvious effects on the viability of either cultured epithelial or lymphoid cells), cytopathogenic (cytopathic effect and cell death in both cultured epithelial and lymphoid cells within 48 h of infection) and lymphocytopathogenic (no effect on cultured epithelial cells, however, cell death in cultured lymphoid cells is observed within 5 days of infection). The proposed lymphocytopathic biotype correlates with high virulence in acute infections in vivo. Cell death caused by the lymphocytopathogenic biotype was not associated with changes typically seen with cytopathic viruses grown in cultured epithelial cells (e.g. changes in processing of the NS2/3 protein observed within 24 h post infection, crenation and breakdown of cell integrity within the first 48 h post infection). These data suggest that the cytopathic effect induced in cultured lymphoid cells by a ncp highly virulent BVDV strain may occur by a different mechanism than the cytopathic effect induced by cp BVDV strains.