Curcumin Inhibits Replication of Human Parainfluenza Virus Type 3 by Affecting Viral Inclusion Body Formation.

Human Parainfluenza Virus Type 3 (HPIV3) is one of the main pathogens that cause acute lower respiratory tract infections in infants and young children. However, there are currently no effective antiviral drugs and vaccines. Herein, we found that a natural compound, curcumin, inhibits HPIV3 infection and has antiviral effects on entry and replication of the virus life cycle. Immunofluorescence and western blotting experiments revealed that curcumin disrupts F-actin and inhibits viral inclusion body (IB) formation, thus inhibiting virus replication. Curcumin can also downregulate cellular PI4KB and interrupt its colocalization in viral IBs. This study verified the antiviral ability of curcumin on HPIV3 infection and preliminarily elucidated its influence on viral replication, providing a theoretical basis for antiviral drug development of HPIV3 and other parainfluenza viruses.

Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infection.

Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2'-deoxyuridine (EdU) labelling of herpes simplex virus 1 (HSV-1) DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs) rapidly entrapped viral DNA (vDNA) leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16) and the induction of interferon stimulated gene (ISG) expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK)-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote vDNA release from PML-NBs and the onset of HSV-1 lytic replication.

Nucleocapsid of Tomato spotted wilt tospovirus forms mobile particles that traffic on an actin/endoplasmic reticulum network driven by myosin XI-K.

A number of viral proteins from plant viruses, other than movement proteins, have been shown to traffic intracellularly along actin filaments and to be involved in viral infection. However, there has been no report that a viral capsid protein may traffic within a cell by utilizing the actin/endoplasmic reticulum (ER) network. We used Tomato spotted wilt tospovirus (TSWV) as a model virus to study the cell biological properties of a nucleocapsid (N) protein. We found that TSWV N protein was capable of forming highly motile cytoplasmic inclusions that moved along the ER and actin network. The disruption of actin filaments by latrunculin B, an actin-depolymerizing agent, almost stopped the intracellular movement of N inclusions, whereas treatment with a microtubule-depolymerizing reagent, oryzalin, did not. The over-expression of a myosin XI-K tail, functioning in a dominant-negative manner, completely halted the movement of N inclusions. Latrunculin B treatment strongly inhibited the formation of TSWV local lesions in Nicotiana tabacum cv Samsun NN and delayed systemic infection in N. benthamiana. Collectively, our findings provide the first evidence that the capsid protein of a plant virus has the novel property of intracellular trafficking. The findings add capsid protein as a new class of viral protein that traffics on the actin/ER system.

Thiazolides, a new class of antiviral agents effective against rotavirus infection, target viral morphogenesis, inhibiting viroplasm formation.

Rotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented double-stranded RNA genome, exhibit a unique morphogenetic pathway requiring the formation of cytoplasmic inclusion bodies called viroplasms in a process involving the nonstructural viral proteins NSP5 and NSP2. In these structures the concerted packaging and replication of the 11 positive-polarity single-stranded RNAs take place to generate the viral double-stranded RNA (dsRNA) genomic segments. Rotavirus infection is a leading cause of gastroenteritis-associated severe morbidity and mortality in young children, but no effective antiviral therapy exists. Herein we investigate the antirotaviral activity of the thiazolide anti-infective nitazoxanide and reveal a novel mechanism by which thiazolides act against rotaviruses. Nitazoxanide and its active circulating metabolite, tizoxanide, inhibit simian A/SA11-G3P[2] and human Wa-G1P[8] rotavirus replication in different types of cells with 50% effective concentrations (EC50s) ranging from 0.3 to 2 µg/ml and 50% cytotoxic concentrations (CC50s) higher than 50 µg/ml. Thiazolides do not affect virus infectivity, binding, or entry into target cells and do not cause a general inhibition of viral protein expression, whereas they reduce the size and alter the architecture of viroplasms, decreasing rotavirus dsRNA formation. As revealed by protein/protein interaction analysis, confocal immunofluorescence microscopy, and viroplasm-like structure formation analysis, thiazolides act by hindering the interaction between the nonstructural proteins NSP5 and NSP2. Altogether the results indicate that thiazolides inhibit rotavirus replication by interfering with viral morphogenesis and may represent a novel class of antiviral drugs effective against rotavirus gastroenteritis.

Aurintricarboxylic acid is a potent inhibitor of influenza A and B virus neuraminidases.

BACKGROUND: Influenza viruses cause serious infections that can be prevented or treated using vaccines or antiviral agents, respectively. While vaccines are effective, they have a number of limitations, and influenza strains resistant to currently available anti-influenza drugs are increasingly isolated. This necessitates the exploration of novel anti-influenza therapies. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the potential of aurintricarboxylic acid (ATA), a potent inhibitor of nucleic acid processing enzymes, to protect Madin-Darby canine kidney cells from influenza infection. We found, by neutral red assay, that ATA was protective, and by RT-PCR and ELISA, respectively, confirmed that ATA reduced viral replication and release. Furthermore, while pre-treating cells with ATA failed to inhibit viral replication, pre-incubation of virus with ATA effectively reduced viral titers, suggesting that ATA may elicit its inhibitory effects by directly interacting with the virus. Electron microscopy revealed that ATA induced viral aggregation at the cell surface, prompting us to determine if ATA could inhibit neuraminidase. ATA was found to compromise the activities of virus-derived and recombinant neuraminidase. Moreover, an oseltamivir-resistant H1N1 strain with H274Y was also found to be sensitive to ATA. Finally, we observed additive protective value when infected cells were simultaneously treated with ATA and amantadine hydrochloride, an anti-influenza drug that inhibits M2-ion channels of influenza A virus. CONCLUSIONS/SIGNIFICANCE: Collectively, these data suggest that ATA is a potent anti-influenza agent by directly inhibiting the neuraminidase and could be a more effective antiviral compound when used in combination with amantadine hydrochloride.

Protective action of salicylic acid against bean yellow mosaic virus infection in Vicia faba leaves.

In this study, morphological, ultrastructural and physiological modifications of faba bean (Vicia faba cv Giza 461) leaves in response to bean yellow mosaic virus (BYMV) infection and salicylic acid (SA) treatments were examined. Under BYMV stress, leaves showed symptoms including severe mosaic, mottling, crinkling, size reduction and deformations. Three weeks after virus inoculation, photosynthetic rate, pigment contents and transpiration rate were significantly reduced in response to BYMV infection. Ultrastructural investigations of BYMV-infected leaves demonstrated that most chloroplasts with increased stromal area became spherical in shape and some lost their envelopes, either partially or totally. The internal structures of chloroplast, grana and thylakoids were dilated. Two kinds of inclusions were detected in BYMV-infected leaves: straight or slightly curved bands sometimes coiled or looped at the end, and electron opaque crystals with varied shapes. BYMV-infected cells showed lower chloroplast number in comparison to the control. Spraying of SA on faba bean leaves helped to reduce or prevent the harmful effects produced after virus infection. Application of 100 microM SA three days before inoculation restored the metabolism of infected leaves to the levels of healthy controls. SA treatment improved plant health by increasing the photosynthesis rates, pigment contents and levels of other parameters studied similar to control values. Moreover, SA treatment increased plant resistance against BYMV. This was observed through induction of chloroplast number, reduction in percentage of infected plants, decrease in disease severity and virus concentration of plants treated with SA prior to BYMV inoculation. Cells of SA-treated samples showed well-developed chloroplasts with many starch grains and well-organized cell organelles. The present results provide an overview of the negative effects on faba bean leaves due to BYMV infection from physiological and subcellular perspectives. Also, a role of SA involved in induction of resistance against BYMV infection in bean plants is discussed.

Interferon-induced alterations in the pattern of parainfluenza virus 5 transcription and protein synthesis and the induction of virus inclusion bodies.

Although parainfluenza virus 5 (simian virus 5 [SV5]) circumvents the interferon (IFN) response by blocking IFN signaling and by reducing the amount of IFN released by infected cells, its ability to circumvent the IFN response is not absolute. The effects of IFN on SV5 infection were examined in Vero cells, which do not produce but can respond to IFN, using a strain of SV5 (CPI-) which does not block IFN signaling. Thus, by infecting Vero cells with CPI- and subsequently treating the cells with exogenous IFN, it was possible to observe the effects that IFN had on SV5 infection in the absence of virus countermeasures. IFN rapidly (within 6 h) induced alterations in the relative levels of virus mRNA and protein synthesis and caused a redistribution of virus proteins within infected cells that led to the enhanced formation of virus cytoplasmic inclusion bodies. IFN induced a steeper gradient of mRNA transcription from the 3' to the 5' end of the genome and the production of virus mRNAs with longer poly(A) tails, suggesting that the processivity of the virus polymerase was altered in cells in an IFN-induced antiviral state. Additional evidence is presented which suggests that these findings also apply to the replication of strains of SV5, parainfluenza virus type 2, and mumps virus that block IFN signaling when they infect cells that are already in an IFN-induced antiviral state.

[Intralesional cidofovir injections for recurrent laryngeal papillomatosis: first results]. / Intraläsionale Cidofovir-Injektion bei rezidivierender Larynxpapillomatose--Erste Ergebnisse.

BACKGROUND: The treatment of recurrent laryngeal papillomatosis still presents an important therapeutic problem. This primarily benign disease of the larynx is caused by an infection with the human papilloma virus (HPV) and forms epithelial neoplastic papillomas. Therapy in larynx obstructing papillomatosis usually requires laser ablation. Cidofovir acts virustatically as an nucleosidanalogon. Currently pilot studies investigate the effectiveness of intralesional Cidofovir injection. PATIENTS AND METHODS: The present study covers the period from October 2001 to March 2003. Seven patients, age of five to 70 years, were treated with intralesional injections of Cidofovir after microlaryngoscopic ablation of laryngeal papillomas. In all patients papillomatosis was confirmed histologically and a clinical-phoniatric examination and a photo documentation pre- and postoperatively was carried out. We treated patients to a maximum of six sessions. RESULTS AND CONCLUSIONS: After three to six sessions of laser ablation of the papillomas and intralesional injections with Cidofovir a distinct papilloma reduction could be observed in all patients and in two cases a complete remission was achieved. The follow-up period of seven to 15 months revealed no recurrent laryngeal papillomatosis. The majority of patients showed a defined voice improvement. There were no local or systemic side-effects caused by the virustatic drug. Intralesional injection of Cidofovir appears to develop into a promising adjuvant therapy option in recurrent laryngeal papillomatosis. First results of the study seem to achieve a considerable reduction of the previously high rate of recurrence of laryngeal papillomatosis.

Adenovirus infection of a renal allograft.

One month after renal transplantation, a 60-year-old man developed acute allograft dysfunction associated with gross hematuria and dysuria. Urinary cytological examination showed viral inclusion-bearing epithelial cells. A renal transplant biopsy specimen showed granulomatous interstitial nephritis, tubular necrosis, and ground glass-like intranuclear viral inclusion bodies in tubular cells caused by an adenovirus (ADV) infection. A reduction in baseline immunosuppressive therapy resulted in rapid normalization of allograft function and ultimately viral clearance. We report this case not only to illustrate an exceptional manifestation of an ADV infection in a renal allograft, but also to highlight the beneficial effect of reduction in immunosuppressive therapy on viral replication and clinical outcome.

Fine structure of cells infected with human cytomegalovirus after treatment with 9-(1,3-dihydroxy-2-propoxymethyl)guanine.

Infection with human cytomegalovirus (CMV) is characterized by cytological changes which are readily visualized by electron microscopy using ultrathin sections of infected cells. Treatment of such cells with 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), a potent inhibitor of CMV, is effective when initiated at early or late times after infection and the response to such treatment has been studied by fine structural analysis. Inhibition of viral DNA synthesis by DHPG treatment (50 microM) late in virus infection resulted in a cessation of virus growth accompanied by a lack of development and possible regression in skein-like intranuclear inclusions together with a depletion in cytoplasmic dense bodies. Such changes were accompanied by the appearance of nuclear dense bodies. These were also present when virus growth was reduced (5 microM-DHPG) rather than completely inhibited (50 microM-DHPG) by treatment initiated from the time of infection. The nuclear bodies were predominantly of a reticular type structure after the early treatment but mainly of a homogeneous form when virus growth was interrupted at late times. Their presence appeared to be connected with the ability of infected cells to initiate the synthesis of late proteins and their morphology may relate to the extent of such protein synthesis. Unlike cytoplasmic dense bodies, provisional findings on the characterization of the nuclear bodies suggested that the 69K matrix protein was not present in abundance.

Simultaneous activation by 5-azacytidine of intracisternal R particles and murine intracisternal-A particle related sequences in Syrian hamster cells.

5-Azacytidine treatment of Syrian hamster (Mesocricetus auratus) cells, BHK21-cl.13, and primary embryo fibroblasts, activates the production of intracisternal type R particles (IRP) as ascertained by electron microscopy scanning and counting. Efficiency of the activation is dose- and time of treatment-dependent. The transcription of mouse intracisternal A particle (IAP) related sequences, which have been described in Syrian hamster genomic DNA, is increased at the same time. This correlates with demethylation of hamster IAP related genes, as shown by restriction of high molecular weight DNA from 5-azacytidine treated cells with methylation sensitive enzymes, and subsequent electrophoresis and molecular hybridization of the fragments with specific DNA probes. In 5-azacytidine treated cells derived from other hamster species (Cricetulus griseus and C. migratorius), intracisternal R particles were not induced, nor were mouse IAP-related RNAs detected.

Interaction of Chlamydia trachomatis with human genital epithelium in culture.

Primary cultures of human endometrial and ectocervical epithelial cells were examined as a new model system to study genital infection by Chlamydia trachomatis. Initial studies demonstrated that these cells were indeed susceptible to chlamydial infection. Inocula, adjusted to produce inclusions in 50 to 80% of equivalent numbers of standard McCoy cells, resulted in infection rates of approximately 15 to 30% for the columnar cells of the endometrium and 5 to 10% for the squamous cells of the ectocervix. Exposure of cultures to DEAE-dextran and centrifugation-assisted inoculation, manipulations reported to enhance infection of HeLa and McCoy cells, did not alter the number of inclusion-positive genital cells. Addition of cycloheximide to the post-inoculation culture medium slightly increased numbers of inclusion-bearing cells while growth of genital cells in hormone-supplemented medium resulted in a variable effect on inclusion development and a significant reduction in the association of radiolabelled organisms with these cells. The basis for the different levels of infection in McCoy versus genital cell cultures was revealed by immunofluorescence analysis of chlamydial association with host cells immediately after inoculation. Chlamydiae failed to adhere to many cells in the genital cell cultures while adherence to McCoy cells was uniform. In addition, the association of radiolabelled C. trachomatis was significantly lower with genital cells than with McCoy cells. Finally, culture conditions were defined which markedly inhibited inclusion development without an immediate loss of chlamydial growth potential. This investigation indicates that primary genital cell cultures are susceptible to chlamydial infection and will be valuable for studies on the nature of C. trachomatis interactions with natural human target cells.

Tubuloreticular inclusions in peripheral blood mononuclear cells related to systemic therapy with alpha-interferon.

Tubuloreticular inclusions (TRI) developed within the endoplasmic reticulum of peripheral blood mononuclear cells (PBMC) sampled from eight patients with chronic type B hepatitis during cycles of therapy with DNA-recombinant human alpha-interferon (rIFN alpha A). Each cycle of therapy consisted of a series of six intramuscular injections (triweekly) of a fixed dose of rIFN alpha A (from 18 to 68 X 10(6) IU/dose). In PBMC examined by transmission electron microscopy, TRI were absent prior to therapy and developed during therapy in all cases. Peak serum levels of alpha-interferon (320 to 960 IU/ml) were achieved within 12 hours. At 24 hours. TRI were detected in 0.5 to 6.5% of PBMC sections, and they persisted in 1.4 to 6.8% of sections examined at 48 hours. After five sequential interferon doses, TRI were observed in 1.6 to 9.8% of PBMC sections. TRI could no longer be detected at 5 to 16 days after cessation of rIFN alpha A, but they reappeared during subsequent cycles of therapy. Subpopulations of the PBMC with TRI were differentiated by immunoelectron microscopy utilizing a battery of anti-Leu monoclonal antibodies: surface markers of T cells, helper/inducer or cytotoxic/suppressor T cell subsets, natural killer cells, or B-cells were identified by direct or indirect procedures utilizing avidin and biotinylated peroxidase. In cases analyzed with multiple monoclonal antisera, TRI were expressed in all of the major PBMC subpopulations. Monocytes with TRI were demonstrated by the endogenous peroxidase reaction. TRI were not found in circulating polymorphonuclear granulocytes. Lymphocytes isolated from healthy donors and exposed to rIFN alpha A (100 IU/ml), for 48 to 72 hours in vitro, developed TRI in proportions of PBMC sections (2.3 to 8.4%) comparable to those observed in the interferon-treated patients. Stimulation of lymphocytes with concanavalin A, for 72 hours before rIFN alpha A exposure, enhanced formation of TRI which could then be found in T blasts. Stimulation of donor lymphocytes with Sendai virus, a potent inducer of alpha-interferon, also resulted in formation of TRI by 48 hours. This suggested that lymphocytotrophic virus infections could exercise a primary role in the natural pathogenesis of TRI.

Butyrate increases type-A and type-R virus-like particles in BKV-transformed hamster spleen cells.

A cell line derived from hamster spleen transformed by human papovavirus BK was adapted to grow in 5 mM butyrate. Ultrastructurally, the butyrate-adapted cells were found to contain large numbers of type-R and intracytoplasmic type-A virus-like particles (VLP). In contrast, the unadapted cells contained only rare VLP. Treatment of the unadapted cells with nontoxic and toxic concentrations of butyrate for 24 and 48 hr failed to induce VLP, indicating the importance of butyrate adaptation in the induction of VLP. The increase in type-R VLP was stable after removal of the adapted cells from butyrate; however, the increase in type-A VLP was not.

Effect of aluminum chloride and zinc sulfate on Autographa california nuclear polyhedrosis virus (ACNPV) replication in cell culture.

When IPL-SF-21AE III continuous insect cell line was grown and maintained in IPL-41 insect cell culture medium supplemented with 16 microM of AlCl3 or 0.24 microM of ZnSO4 . 7H2O, or both metallic salts, and then infected with Autographa california nuclear polyhedrosis virus, virus replication was increased significantly. The yield of polyhedral inclusion bodies (PIB) was enhanced up to 121%. Synthesis of cell-free nonoccluded virus was increased to 365% when infectivity was assayed by the plaque method. Newly applied electron microscopic quantitation and stereological techniques also revealed a significant increase in virus particles (VP) and in amount and size of PIB as well as number of VP per PIB.

Reversibility of the transformed and neoplastic phenotype. I. Progressive reversion of the phenotype of X-ray-transformed C3H/10T1/2 cells under prolonged treatment with interferon.

X-ray transformed mouse C3H/10T1/2 cells were cultivated and passaged in the continuous presence of partially purified mouse interferon. This prolonged interferon treatment resulted in a stepwise progressive reversion of the transformed phenotype to the non-transformed phenotype. Thus interferon-treated cells displayed an epithelioid morphology, grew to a lower cell density, and were no longer tumorigenic. Reversion to the non-transformed phenotype was, however, stable only as long as interferon was continuously present in the culture medium. When interferon was removed, the cells "back reverted" to the transformed phenotype. Our results suggest that interferon induced a reversion of the transformed phenotype in the entire cell population rather than a selection of an interferon resistant cell population. C-type viral particles and significant levels of reverse transcriptase were present in transformed cells, but neither present in the parental 10T1/2 cells nor in interferon-treated cells. When interferon was removed form the culture medium, viral particles and reverse transcriptase activity were again detected. It is possible, therefore, that interferon induces reversion through its antiviral activity, or that it induces reversion by its effects on cell function and structure, independently of any antiviral effect. Inhibition of cell multiplication per se does no appear to be sufficient to induce reversion, since cycloheximide inhibited cell multiplication; however, even after ten passages, it did not affect tumorigenicity. Our results suggest the possibility that interferon may act in vivo not only by inhibiting tumor cell multiplication but also by inducing a reversion. Patients with some tumors may therefore benefit from long-term interferon treatment.

Virus-like particles induced by bromodeoxyuridine in melanoma and neuroblastoma of Xiphophorus.

5-Bromodeoxyuridine (BrdUrd) injected into the muscular tissue of fish bearing melanoma or neuroblastoma induces the production of virus-like particles in these tumors. The particles in the melanoma are morphologically similar to papovaviruses of polyoma-type, those in the neuroblastoma resemble oncornaviruses of B- and C-type.

Effect of bromodeoxyuridine and interferon on cellular and viral functions in human prostatic cells.

Some of the human prostatic cells in culture apparently produce oncornavirus-like particles. Bromodeoxyuridine does not enhance the production of these particles. On the contrary, this drug depresses such production. This depression is likely to be due to the cytotoxic effects of bromodeoxyuridine for these cells. These results can be interpreted to suggest that the human prostatic cells used in this study do not contain endogenous oncornavirus genetic information that is inducible. Purified human interferon inhibits the production of oncornavirus-like particles by these prostatic cells. It is also inhibits the rate of cellular DNA synthesis in these cells. These results are consistent with the notion that the inhibitory effects of interferon are mediated through its effect on cellular biosynthetic machinery.