Prior induction of cellular antiviral pathways limits frog virus 3 replication in two permissive Xenopus laevis skin epithelial-like cell lines.

Frog virus 3 (FV3) causes mortality in a range of amphibian species. Despite the importance of the skin epithelium as a first line of defence against FV3, the interaction between amphibian skin epithelial cells and FV3 remains largely uncharacterized. Here, we used newly established Xenopus laevis skin epithelial-like cell lines, Xela DS2 and Xela VS2, to study the susceptibility and permissiveness of frog skin epithelial cells to FV3, and the innate immune antiviral and proinflammatory gene regulatory responses of these cells to FV3. Both cell lines are susceptible and permissive to FV3, yet do not exhibit appreciable transcript levels of scavenger receptors thought to be used by FV3 for cellular entry. Xela DS2 and Xela VS2 upregulate antiviral and proinflammatory cytokine transcripts in response to poly(I:C) but not to FV3 or UV-inactivated FV3. Poly(I:C) pretreatment limits FV3 replication and FV3-induced cytopathic effects in both cell lines. Thus, Xela DS2 and Xela VS2 can support FV3 replication, represent in vitro systems to investigate antiviral responses of frog skin epithelial cells, and can serve as novel tools for screening compounds that initiate effective antiviral programs to limit FV3 replication.

Distinct infection process of SARS-CoV-2 in human bronchial epithelial cell lines.

Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), leads to a series of clinical symptoms of respiratory and pulmonary inflammatory reactions via unknown pathologic mechanisms related to the viral infection process in tracheal or bronchial epithelial cells. Investigation of this viral infection in the human bronchial epithelial cell line (16HBE) suggests that SARS-CoV-2 can enter these cells through interaction between its membrane-localized S protein with the angiotensin-converting enzyme 2 molecule on the host cell membrane. Further observation indicates distinct viral replication with a dynamic and moderate increase, whereby viral replication does not lead to a specific cytopathic effect but maintains a continuous release of progeny virions from infected cells. Although messenger RNA expression of various innate immune signaling molecules is altered in the cells, transcription of interferons-α (IFN-α), IFN-ß, and IFN-γ is unchanged. Furthermore, expression of some interleukins (IL) related to inflammatory reactions, such as IL-6, IL-2, and IL-8, is maintained at low levels, whereas that of ILs involved in immune regulation is upregulated. Interestingly, IL-22, an IL that functions mainly in tissue repair, shows very high expression. Collectively, these data suggest a distinct infection process for this virus in respiratory epithelial cells, which may be linked to its clinicopathological mechanism.

The HIV Reservoir in Monocytes and Macrophages.

In people living with HIV (PLWH) who are failing or unable to access combination antiretroviral therapy (cART), monocytes and macrophages are important drivers of pathogenesis and progression to AIDS. The relevance of the monocyte/macrophage reservoir in PLWH receiving cART is debatable as in vivo evidence for infected cells is limited and suggests the reservoir is small. Macrophages were assumed to have a moderate life span and lack self-renewing potential, but recent discoveries challenge this dogma and suggest a potentially important role of these cells as long-lived HIV reservoirs. This, combined with new HIV infection animal models, has led to a resurgence of interest in monocyte/macrophage reservoirs. Infection of non-human primates with myeloid-tropic SIV implicates monocyte/macrophage activation and infection in the brain with neurocognitive disorders, and infection of myeloid-only humanized mouse models are consistent with the potential of the monocyte/macrophage reservoir to sustain infection and be a source of rebound viremia following cART cessation. An increased resistance to HIV-induced cytopathic effects and a reduced susceptibility to some antiretroviral drugs implies macrophages may be relevant to residual replication under cART and to rebound viremia. With a reappraisal of monocyte circulation dynamics, and the development of techniques to differentiate between self-renewing tissue-resident, and monocyte-derived macrophages in different tissues, a new framework exists to contextualize and evaluate the significance and relevance of the monocyte/macrophage HIV reservoir. In this review, we discuss recent developments in monocyte and macrophage biology and appraise current and emerging techniques to quantify the reservoir. We discuss how this knowledge influences our evaluation of the myeloid HIV reservoir, the implications for HIV pathogenesis in both viremic and virologically-suppressed PLWH and the need to address the myeloid reservoir in future treatment and cure strategies.

The possible role of cross-reactive dengue virus antibodies in Zika virus pathogenesis.

Zika virus (ZIKV) has been known for decades to circulate in Africa and Asia. However, major complications of a ZIKV infection have recently become apparent for reasons that are still not fully elucidated. One of the hypotheses for the seemingly increased pathogenicity of ZIKV is that cross-reactive dengue antibodies can enhance a ZIKV infection through the principle of antibody-dependent enhancement (ADE). Recently, ADE in ZIKV infection has been studied, but conclusive evidence for the clinical importance of this principle in a ZIKV infection is lacking. Conversely, the widespread circulation of ZIKV in dengue virus (DENV)-endemic regions raises new questions about the potential contribution of ZIKV antibodies to DENV ADE. In this review, we summarize the results of the evidence to date and elaborate on other possible detrimental effects of cross-reactive flavivirus antibodies, both for ZIKV infection and the risk of ZIKV-related congenital anomalies, DENV infection, and dengue hemorrhagic fever.

Western Zika Virus in Human Fetal Neural Progenitors Persists Long Term with Partial Cytopathic and Limited Immunogenic Effects.

The recent Zika virus (ZIKV) outbreak in the Western hemisphere is associated with severe pathology in newborns, including microcephaly and brain damage. The mechanisms underlying these outcomes are under intense investigation. Here, we show that a 2015 ZIKV isolate replicates in multiple cell types, including primary human fetal neural progenitors (hNPs). In immortalized cells, ZIKV is cytopathic and grossly rearranges endoplasmic reticulum membranes similar to other flaviviruses. In hNPs, ZIKV infection has a partial cytopathic phase characterized by cell rounding, pyknosis, and activation of caspase 3. Despite notable cell death, ZIKV did not activate a cytokine response in hNPs. This lack of cell intrinsic immunity to ZIKV is consistent with our observation that virus replication persists in hNPs for at least 28 days. These findings, supported by published fetal neuropathology, establish a proof-of-concept that neural progenitors in the developing human fetus can be direct targets of detrimental ZIKV-induced pathology.

A colorimetric bioassay for high-throughput and cost-effectively assessing anti-foot-and-mouth disease virus activity.

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal viruses. This virus is very sensitive to inhibition by type I interferons. Currently, a bioassay based on plaque reduction is used to measure anti-FMDV activity of porcine IFNs. The plaque reduction assay is tedious and difficult to utilize for high-throughput analysis. Using available FMDV susceptible bovine and porcine cells, we developed and tested a colorimetric assay based on cytopathic effect reduction for its ability to quantify FMDV-specific antiviral activity of bovine and porcine type I interferons. Our results show that this new method has significant advantages over other assays in terms of labor intensity, cost, high-throughput capability and/or anti-FMDV specific activity because of simpler procedures and direct measurement of antiviral activity. Several assay conditions were tested to optimize the procedures. The test results show that the assay can be standardized with fixed conditions and a standard or a reference for measuring antiviral activity as units. This is an excellent assay in terms of sensitivity and accuracy based on a statistical evaluation. The results obtained with this assay were highly correlated with a conventional virus titration method.

Characterization of cathepsin B gene from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.

The lysosomal cysteine protease cathepsin B of papain family is a key regulator and signaling molecule that involves in various biological processes, such as the regulation of apoptosis and activation of virus. In the present study, cathepsin B gene (Ec-CB) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CB cDNA was composed of 1918 bp and encoded a polypeptide of 330 amino acids with higher identities to cathepsin B of teleosts and mammalians. Ec-CB possessed typical cathepsin B structural features including an N-terminal signal peptide, the propeptide region and the cysteine protease domain which were conserved in other cathepsin B sequences. Phylogenetic analysis revealed that Ec-CB was most closely related to Lutjanus argentimaculatus. RT-PCR analysis showed that Ec-CB transcript was expressed in all the examined tissues which abundant in spleen, kidney and gill. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the mRNA expression of cathepsin B in E. coioides was up-regulated at 24 h post-infection. Subcellular localization analysis revealed that Ec-CB was distributed predominantly in the cytoplasm. When the fish cells (GS or FHM) were treated with the cathepsin B specific inhibitor CA-074Me, the occurrence of CPE induced by SGIV was delayed, and the viral gene transcription was significantly inhibited. Additionally, SGIV-induced typical apoptosis was also inhibited by CA-074Me in FHM cells. Taken together, our results demonstrated that the Ec-CB might play a functional role in SGIV infection.

Relative respiratory syncytial virus cytopathogenesis in upper and lower respiratory tract epithelium.

RATIONALE: Respiratory syncytial virus (RSV) is a major pathogen that primarily infects airway epithelium. Most infants suffer mild upper respiratory tract (URT) symptoms, whereas approximately one-third progress to lower respiratory tract (LRT) involvement. Despite the ubiquity of URT infection, little is known about the relative cytopathogenesis of RSV infection in infant URT and LRT. OBJECTIVES: This study aimed to compare RSV cytopathogenesis in nasal- and bronchial-derived epithelium from the same individuals using novel models derived from well-differentiated primary pediatric nasal (WD-PNECs) and bronchial epithelial cells (WD-PBECs). METHODS: WD-PNECs and WD-PBECs were generated from nasal and bronchial brushes, respectively, and mock-infected or infected with RSV BT2a. RSV tropism, infectivity, cytopathology, growth kinetics, cell sloughing, apoptosis, and cytokine and chemokine responses were determined. MEASUREMENTS AND MAIN RESULTS: RSV infection in both cultures was restricted to apical ciliated cells and occasional nonciliated cells but not goblet cells. It did not cause gross cytopathology. Infection resulted in apical release of progeny virus, increased apical cell sloughing, apoptosis, and occasional syncytia. RSV growth kinetics and peak titers were higher in WD-PBECs, coincident with higher ciliated cell contents, cell sloughing, and slightly compromised tight junctions. However, proinflammatory chemokine responses were similar for both cultures. Also, lambda IFNs, especially IL-29, were induced by RSV infection. CONCLUSIONS: RSV induced remarkably similar, albeit quantitatively lower, cytopathogenesis and proinflammatory responses in WD-PNECs compared with WD-PBECs that reproduce many hallmarks of RSV pathogenesis in infants. WD-PNECs may provide an authentic surrogate model with which to study RSV cytopathogenesis in infant airway epithelium.

[Comparison of three detection methods for neutralizing antibodies induced by Coxsackievirus A16 vaccine].

OBJECTIVE: To develop a comprehensive detection platform for immunoprotection of inactivated coxsackievirus A16(CA16) vaccine based on the neutralizing antibodies tested through competitive inhibition ELISA, micro-cytopathic effect neutralization test and neonatal mice challenge protection test. METHODS: The female BALB/c mice, SD rats and Hartley guinea pigs were inoculated intraperitoneally at 0, 4, 6, 8 weeks with the three vaccine candidates which were made from 3726, 4430 and 4432 virus absorbed on aluminium adjuvant. Immune sera were taken at 0, 4, 6, 8, 10 weeks and serum neutralizing antibodies were evaluated by competitive inhibition-ELISA, micro-cytopathic effect neutralization test and neonatal mice challenge protection test, respectively. The relationships of the three methods were analyzed by SPSS16.0 statistical software. RESULTS: The level of neutralizing antibodies reached the peak after the second booster. The correlation coefficient was 0.861 between competitive inhibition-ELISA and micro-cytopathic effect neutralization test, 0.8 between competitive inhibition-ELISA and neonatal mice challenge protection test and 0.89 between micro-cytopathic effect neutralization test and neonatal mice challenge protection test. CONCLUSION: A comprehensive detection system through competitive inhibition ELISA, micro-cytopathic effect neutralization test and neonatal mice challenge protection test measuring the neutralizing antibodies in immune sera has been constructed. The competitive inhibition-ELISA is superior to "golden standards" in speed, sensitivity, throughput and price, which may have great application prospect.

Caprine arthritis encephalitis virus (CAEV) replicates productively in cultured epididymal cells from goats.

The transmission of CAEV from male goats has not been well studied and the target cells that support viral replication are not well characterized. Epididymal epithelial cells (EECs) are important and play a key role in the fertility and motility of spermatozoa. During their transit, spermatozoa incorporate several EEC-produced proteins into their plasma membranes to stabilize them and prevent premature acrosomal reaction. This intimate interaction between spermatozoa and EECs may increase the likelihood of the infection of semen with CAEV if epididymal tissue is productively infected and sheds the virus into the duct. The aim of this study was to examine whether goat EECs are susceptible to CAEV infection in tissue culture. Cells were isolated from epididymides obtained from goats that were sampled from a certified-CAEV-free herd. Cultured cells were then inoculated with a molecularly-cloned isolate of CAEV (CAEV-pBSCA). Inoculated cells developed cytopathic effects (CPE), showing numerous multinucleated giant cells (MGC) in cell-culture monolayers. Expression of CAEV proteins was detected by immunofluorescence using an anti-p28, Gag-specific antibody. The culture medium of inoculated cells was shown to contain high titers (10(6) tissue culture infectious doses 50 per ml (TCID50/ml)) of infectious, cytopathic virus when assayed using indicator goat synovial membrane (GSM) cells. Our findings clearly demonstrate that cells of the buck genital tract are targets of CAEV and are thus a potential reservoir that sheds infectious CAEV into the semen of infected animals. These data suggest the use of sperm from CAEV-free goat males for artificial insemination in genetic selection programs to minimize CAEV dissemination.

Neurotropic arboviruses induce interferon regulatory factor 3-mediated neuronal responses that are cytoprotective, interferon independent, and inhibited by Western equine encephalitis virus capsid.

Cell-intrinsic innate immune responses mediated by the transcription factor interferon regulatory factor 3 (IRF-3) are often vital for early pathogen control, and effective responses in neurons may be crucial to prevent the irreversible loss of these critical central nervous system cells after infection with neurotropic pathogens. To investigate this hypothesis, we used targeted molecular and genetic approaches with cultured neurons to study cell-intrinsic host defense pathways primarily using the neurotropic alphavirus western equine encephalitis virus (WEEV). We found that WEEV activated IRF-3-mediated neuronal innate immune pathways in a replication-dependent manner, and abrogation of IRF-3 function enhanced virus-mediated injury by WEEV and the unrelated flavivirus St. Louis encephalitis virus. Furthermore, IRF-3-dependent neuronal protection from virus-mediated cytopathology occurred independently of autocrine or paracrine type I interferon activity. Despite being partially controlled by IRF-3-dependent signals, WEEV also disrupted antiviral responses by inhibiting pattern recognition receptor pathways. This antagonist activity was mapped to the WEEV capsid gene, which disrupted signal transduction downstream of IRF-3 activation and was independent of capsid-mediated inhibition of host macromolecular synthesis. Overall, these results indicate that innate immune pathways have important cytoprotective activity in neurons and contribute to limiting injury associated with infection by neurotropic arboviruses.

The impact of BVDV infection on adaptive immunity.

Bovine viral diarrhea virus (BVDV) causes immunosuppression of the adaptive immune response. The level of suppression of the adaptive immune response is strain dependent. The early events of antigen presentation require activation of toll-like receptors that results in the release of pro-inflammatory cytokines. Non-cytopathic (ncp) BVDV infection stimulates cytokines from macrophages in vitro but the effect of BVDV infection in vivo on macrophages or in vitro with monocytes is not clear. Antigen presentation is decreased and co-stimulatory molecules are down regulated. T-lymphocytes numbers are reduced following BVDV infection in a strain dependent manner. There is recruitment of lymphocytes to the bronchial alveolar space following cytopathic (cp) BVDV infection. Depletion of T-lymphocytes occurs in the lymphoid tissue and is strain dependent. BVDV cp T-lymphocyte responses appear to be primarily a T helper 1 response while the response following ncp BVDV induces a T helper 2 response. Cytotoxic T-lymphocytes (CTL), an important BVDV defense mechanism are compromised. The major neutralizing antigens are well characterized but cross-protection between strains is variable. PI animals have normal adaptive immune responses with the exception of the PI strain immunotolerance and mucosal disease may be a function of the level of gamma delta T cells.

Enterovirus-induced gene expression profile is critical for human pancreatic islet destruction.

AIMS/HYPOTHESIS: Virally induced inflammatory responses, beta cell destruction and release of beta cell autoantigens may lead to autoimmune reactions culminating in type 1 diabetes. Therefore, viral capability to induce beta cell death and the nature of virus-induced immune responses are among key determinants of diabetogenic viruses. We hypothesised that enterovirus infection induces a specific gene expression pattern that results in islet destruction and that such a host response pattern is not shared among all enterovirus infections but varies between virus strains. METHODS: The changes in global gene expression and secreted cytokine profiles induced by lytic or benign enterovirus infections were studied in primary human pancreatic islet using DNA microarrays and viral strains either isolated at the clinical onset of type 1 diabetes or capable of causing a diabetes-like condition in mice. RESULTS: The expression of pro-inflammatory cytokine genes (IL-1-α, IL-1-ß and TNF-α) that also mediate cytokine-induced beta cell dysfunction correlated with the lytic potential of a virus. Temporally increasing gene expression levels of double-stranded RNA recognition receptors, antiviral molecules, cytokines and chemokines were detected for all studied virus strains. Lytic coxsackievirus B5 (CBV-5)-DS infection also downregulated genes involved in glycolysis and insulin secretion. CONCLUSIONS/INTERPRETATION: The results suggest a distinct, virus-strain-specific, gene expression pattern leading to pancreatic islet destruction and pro-inflammatory effects after enterovirus infection. However, neither viral replication nor cytotoxic cytokine production alone are sufficient to induce necrotic cell death. More likely the combined effect of these and possibly cellular energy depletion lie behind the enterovirus-induced necrosis of islets.

Novel, real-time cell analysis for measuring viral cytopathogenesis and the efficacy of neutralizing antibodies to the 2009 influenza A (H1N1) virus.

A novel electronic cell sensor array technology, the real-time cell analysis (RTCA) system, was developed to monitor cell events. Unlike the conventional methods labeling the target cells with fluorescence, luminescence, or light absorption, the RTCA system allows for label-free detection of cell processes directly without the incorporation of labels. Here, we used this new format to measure the cytopathic effect (CPE) of the 2009 influenza A (H1N1) virus and the efficacy of neutralizing antibodies in human sera to this virus. The real-time dynamic monitoring of CPE was performed on MDCK cell cultures infected with the H1N1 virus, ranging from 5.50×10(2) to 5.50×10(7) copies/mL. The resulting CPE kinetic curves were automatically recorded and were both time and viral load dependent. The CPE kinetics were also distinguishable between different H1N1 stains, as the onset of CPE induced by the A/Shanghai/37T/2009 H1N1 virus was earlier than that of the A/Shanghai/143T/2009 H1N1 virus. Furthermore, inhibition of H1N1 virus-induced CPE in the presence of human specific anti-sera was detected and quantified using the RTCA system. Antibody titers determined using this new neutralization test correlated well with those obtained independently via the standard hemagglutination inhibition test. Taken together, this new CPE assay format provided label-free and high-throughput measurement of viral growth and the effect of neutralizing antibodies, illustrating its potential in influenza vaccine studies.

Propiedades biológicas de cepas de virus dengue serotipo 2 aisladas durante la epidemia en Santiago de Cuba, 1997 / Biological properties of dengue virus serotype 2 strains isolated from the epidemic occurred in Santiago de Cuba in 1997

Introducción: durante la epidemia cubana de dengue ocurrida en Santiago de Cuba, 1997, se evidenció un incremento de la severidad en el tiempo en términos de proporción de casos graves y muertes por dengue hemorrágico, que pudiera estar dado por la aparición de mutantes de escape a la neutralización, con mayor potencial virulento. Objetivos: estudiar algunas propiedades biológicas de cepas aisladas en diferentes momentos de la epidemia de Santiago de Cuba en 1997. MÚtodos: se estudiaron 9 cepas de DENV-2, se evaluó el efecto citopatogénico y el crecimiento viral en las líneas celulares C6/36 HT y Vero, tamaño de las placas virales, sensibilidad a la temperatura, neurovirulencia en ratones lactantes y la influencia del pH en la unión del virus a la célula, así como en el medio de multiplicación. Resultados: las cepas del final de la epidemia mostraron propiedades que las diferencian de las aisladas al inicio, entre las que se encuentran un mayor efecto citopatogénico en células C6/36 HT, títulos virales superiores y una mayor neurovirulencia en ratones lactantes. Por otra parte, en la unión del virus a la célula las cepas del inicio de la epidemia resultaron favorecidas por un pH ácido mientras que a las cepas del final de la epidemia las favoreció un pH básico. Conclusiones: pudo demostrarse que además de los cambios genotípicos observados previamente, existen diferencias fenotípicas entre cepas de distintos momentos de la epidemia, que pudieran estar asociados con diferencias en cuanto a la adecuación viral de estas y(o) en su potencial virulento(AU)

Introduction: during the Cuban epidemic that occurred in Santiago de Cuba in 1997, there was observed increasing severity in the course of time, in terms of proportion of serious dengue haemorrhagic cases and deaths that could be due to the emergence of escape mutants to neutralization with greater virulent potential. Objective: to study some biological attributes of a group of strains isolated at different points of time during the Santiago de Cuba epidemic in 1997. Methods: nine DENV-2 strains were studied. The cytopathogenic effect, the viral growth in C6/36 HT and VERO cell lines, the virus plaque sizes, the sensitivity to temperatures, the neurovirulence in newborn mice and the influence of the pH in the union of the virus to the cell as well as in the multiplication medium were all evaluated. Results: the strains isolated at the end of the epidemic differed from those of the beginning showing increased neurovirulence in newborn mice and higher viral titers and greater cytopathogenic effect in HT C6/36 cells. On the other hand, the virus and the cell union was favored by acid pH when testing strains from the beginning of the epidemic, whereas this union was favored by the basic PH in the strains isolated at the end of the epidemic Conclusions: the present study managed to show that in addition to the previously observed genotypical changes, there were phenotypical differences among the strains isolated at different points of time in the epidemic; all these aspects may be associated with differences in the viral fitness and/or in the virulent potential of these strains(AU)

Potent inhibition of human enterovirus 71 replication by type I interferon subtypes.

BACKGROUND: Enterovirus 71 (EV71) infection can induce a series of syndromes including herpangina, viraemia, hand-foot-and-mouth disease and even death. Outbreaks of EV71 infection have been reported periodically over the world and have caused a great number of casualties and a high medical expenditure. Some interferons (IFNs) have been used for the treatment of viral infections for decades; however, conventional IFNs only display mild anti-EV71 activities. No effective drug is currently available for the treatment of EV71 infection. Here, we aimed to investigate whether some IFN subtypes display potent anti-EV71 activities. METHODS: The antiviral activities of 17 type I IFNs were assayed in Vero cells using the cytopathic effect method. Cells were incubated with different concentrations of type I IFNs before or after virus infection. Viral replication was determined by quantitative real-time PCR (qRT-PCR). The expression levels of IFN downstream antiviral genes were also measured by qRT-PCR. RESULTS: Out of 17 type I IFNs, 4 IFNs (IFN-α4, IFN-α6, IFN-α14 and IFN-α16) displayed potent antiviral activity. Compared with conventional IFN-α2a, IFN-α14 displayed approximately 20× higher antiviral activity. The superior antiviral effect of IFN-α14 was caused by a strong induction of the downstream antiviral effectors. CONCLUSIONS: IFN-α14 and three other IFNs could be considered for the treatment of EV71 infection.

Protection from RNA and DNA viruses by IL-32.

Several studies have documented a proinflammatory role for IL-32, which induces IL-1α, IL-1ß, IL-6, TNF, and chemokines via NF-κB, p38MAPK, and AP-1. However, IL-32 also participates in the responses to infection with viruses such as HIV-1 and influenza. In this study, we explored these antiviral properties of IL-32. Vital staining assays demonstrated that low concentrations (5-10 ng/ml) of rIL-32γ protected epithelial WISH cells from vesicular stomatitis virus-induced cell death. By lactate dehydrogenase assays, treatment with IL-32γ resulted in a 3- to 4-fold decrease in viral load. Specific silencing of IL-32 revealed that the antiviral responses triggered by the synthetic analogs of ssRNA viruses (polyuridine) and dsRNA viruses (polyinosinic-polycytidylic acid) were significantly weaker (2- to 3-fold more virus) in WISH cells in the absence of IL-32. Importantly, we discovered that the polyinosinic-polycytidylic acid-induced increase in production of IFN-α in human PBMC was nearly completely abolished when IL-32 was silenced. Moreover, we observed that IL-32 antagonizes the DNA virus HSV-2 in epithelial Vero cells as well as in human umbilical cord endothelial cells, as production of HSV-2 increased 8-fold upon silencing of IL-32 (p < 0.001). Mechanistically, we found that IL-32 used the PKR-eIF-2α as well as the MxA antiviral pathways. Unexpectedly, a considerable part of the antiviral properties of IL-32 was not dependent on IFNs; specific blockade of IFN activity reduced the antiviral properties of IL-32 only moderately. In conclusion, these data suggest a central role for IL-32 in the immune response to RNA and DNA viruses, which may be exploitable for clinical use in the future.

Molecular cloning and antiviral activity of IFN-ß promoter stimulator-1 (IPS-1) gene in Japanese flounder, Paralichthys olivaceus.

The mitochondrial adaptor, IFN-ß promoter stimulator-1 (IPS-1), also known as MAVS/VISA/Cardif, plays a key role in the signal transduction of the RIG-1/MDA5 pathway to induce the production of interferons (IFNs) and other cytokines. In the present study, Japanese flounder (Paralichthys olivaceus) IPS-1 cDNA was cloned from Japanese flounder spleen using PCR-based methods. The full-length cDNA has 2235 nucleotides and encodes a polypeptide of 641 amino acids. The putative Japanese flounder IPS-1 protein contains an N-terminal CARD-like domain, a central proline-rich domain, a C-terminal transmembrane domain, and exhibits similarity to other teleost counterparts ranging from 20% to 34%. Semi-quantitative RT-PCR showed that Japanese flounder IPS-1 mRNA was expressed in all tissues examined. The expression level of flounder IPS-1 gene was unchanged in viral hemorrhagic septicemia virus (VHSV)-infected kidney as measured by quantitative real-time PCR (Q-PCR). In addition, Japanese flounder IPS-1-overexpressing cells were protected against hirame rhabdovirus (HIRRV) and VHSV infection as manifested by the delayed appearance of cytopathic effect (CPE) and decreased viral titers. Expression of IFN-inducible genes including Mx, ISG15 and IRF3 were also induced in the IPS-1-overexpressing cells. These results suggest that Japanese flounder IPS-1 is involved in the antiviral immunity as a one of the adaptors in fish IFN-activation pathway.

HSF1 overexpression enhances oncolytic effect of replicative adenovirus.

BACKGROUND: E1B55kD deleted oncolytic adenovirus was designed to achieve cancer-specific cytotoxicity, but showed limitations in clinical study. To find a method to increase its efficacy, we investigated the correlation between oncolytic effect of such oncolytic adenovirus Adel55 and intracellular heat shock transcription factor 1 (HSF1) activity. METHODS: In the present study, human breast cancer cell line Bcap37 was stably transfected with constitutively active HSF1 (cHSF1) or HSF1 specific siRNA (HSF1i) to establish increased or decreased HSF1 expression levels. Cytotoxicity of Adel55 was analyzed in these cell lines in vitro and in vivo. Furthermore, Adel55 incorporated with cHSF1 (Adel55-cHSF1) was used to treat various tumor xenografts. RESULTS: Adel55 could achieve more efficient oncolysis in cHSF1 transfected Bcap37 cells, both in vitro and in vivo. However, inhibition of HSF1 expression by HSF1i could rescue Bcap37 cell line from oncolysis by Adel55. A time course study of viral replication established a correlation between higher replication of Adel55 and cytolysis or tumor growth inhibition. Then, we constructed Adel55-cHSF1 for tumor gene therapy and demonstrated that it is more potent than Adel55 itself in oncolysis and replication in both Bcap37 and SW620 xenografts. CONCLUSIONS: cHSF1 enhances the Adel55 cell-killing potential through increasing the viral replication and is a potential therapeutic implication to augment the potential of E1B55kD deleted oncolytic adenovirus by increasing its burst.

Anti-West Nile virus activity of in vitro expanded human primary natural killer cells.

BACKGROUND: Natural Killer (NK) cells are a crucial component of the host innate immune system with anti-viral and anti-cancer properties. However, the role of NK cells in West Nile virus (WNV) infection is controversial, with reported effects ranging from active suppression of virus to no effect at all. It was previously shown that K562-mb15-41BBL (K562D2) cells, which express IL-15 and 4-1BBL on the K562 cell surface, were able to expand and activate human primary NK cells of normal peripheral blood mononuclear cells (PBMC). The expanded NK cells were tested for their ability to inhibit WNV infection in vitro. RESULTS: Co-culture of PBMC with irradiated K562D2 cells expanded the NK cell number by 2-3 logs in 2-3 weeks, with more than 90% purity; upregulated NK cell surface activation receptors; downregulated inhibitory receptors; and boosted interferon gamma (IFN-gamma) production by approximately 33 fold. The expanded NK (D2NK) cell has strong natural killing activity against both K562 and Vero cells, and killed the WNV infected Vero cells through antibody-dependent cellular cytotoxicity (ADCC). The D2NK cell culture supernatants inhibited both WNV replication and WNV induced cytopathic effect (CPE) in Vero cells when added before or after infection. Anti-IFN-gamma neutralizing antibody blocked the NK supernatant-mediated anti-WNV effect, demonstrating a noncytolytic activity mediated through IFN-gamma. CONCLUSIONS: Co-culture of PBMC with K562D2 stimulatory cells is an efficient technique to prepare large quantities of pure and active NK cells, and these expanded NK cells inhibited WNV infection of Vero cells through both cytolytic and noncytolytic activities, which may imply a potential role of NK cells in combating WNV infection.