Lipidation of a bioactive cyclotide-based CXCR4 antagonist greatly improves its pharmacokinetic profile in vivo.

The CXCR4 chemokine is a key molecular regulator of many biological functions controlling leukocyte functions during inflammation and immunity, and during embryonic development. Overexpression of CXCR4 is also associated with many types of cancer where its activation promotes angiogenesis, tumor growth/survival, and metastasis. In addition, CXCR4 is involved in HIV replication, working as a co-receptor for viral entry, making CXCR4 a very attractive target for developing novel therapeutic agents. Here we report the pharmacokinetic profile in rats of a potent CXCR4 antagonist cyclotide, MCo-CVX-5c, previously developed in our group that displayed a remarkable in vivo resistance to biological degradation in serum. This bioactive cyclotide, however, was rapidly eliminated through renal clearance. Several lipidated versions of cyclotide MCo-CVX-5c showed a significant increase in the half-life when compared to the unlipidated form. The palmitoylated version of cyclotide MCo-CVX-5c displayed similar CXCR4 antagonistic activity as the unlipidated cyclotide, while the cyclotide modified with octadecanedioic (18-oxo-octadecanoic) acid exhibited a remarkable decrease in its ability to antagonize CXCR4. Similar results were also obtained when tested for its ability to inhibit growth in two cancer cell lines and HIV infection in cells. These results show that the half-life of cyclotides can be improved by lipidation although it can also affect their biological activity depending on the lipid employed.

Development of flow cytometry-based Zika virus detection assay.

Standard assays based on ELISA and RT-PCR have been widely used to detect flaviviral infections, including the Zika virus (Zika). Despite their simple, unique, and sensitive features, RT-PCR and ELISA-based assays cannot meet the requirements of high-throughput screening of bulk samples during an outbreak. Several research groups around the world are working on the development of rapid, multiplex, and sensitive assays to overcome the limitations of standard assays used in viral detection. Recent advances in flow cytometry have led to remarkable progress in its use as a basic analysis tool in laboratories. Here, we used the advantages of flow cytometry to develop a Zika detection assay using recombinant Zika envelope (E) protein. The E protein-based flow cytometry assay was able to detect anti-Zika E antibodies from Zika-infected patients, Zika-infected mice, and mice immunized with recombinant Zika E protein. We report the development of the first flow cytometry-based diagnostic assay that can be used for Zika detection. Its rapid turnaround time and ability to detect antibodies from Zika-infected patients can be used to improve the diagnostic accuracy of Zika detection. Keywords: Flavivirus; Zika virus; E protein; NS-1 protein; flow cytometry; ELISA; RT-PCR.

HLA-E-restricted HIV-1-specific CD8+ T cell responses in natural infection.

CD8+ T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E-restricted CD8+ T cell responses in HIV infection, however, remains unknown. In this study, CD8+ T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E-restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8+ T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8+ T cells. Importantly, bulk CD8+ T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E-restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E-restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8+ T cell responses in HIV infection.

A DNA Vaccine Protects Human Immune Cells against Zika Virus Infection in Humanized Mice.

A DNA vaccine encoding prM and E protein has been shown to induce protection against Zika virus (ZIKV) infection in mice and monkeys. However, its effectiveness in humans remains undefined. Moreover, identification of which immune cell types are specifically infected in humans is unclear. We show that human myeloid cells and B cells are primary targets of ZIKV in humanized mice. We also show that a DNA vaccine encoding full length prM and E protein protects humanized mice from ZIKV infection. Following administration of the DNA vaccine, humanized DRAG mice developed antibodies targeting ZIKV as measured by ELISA and neutralization assays. Moreover, following ZIKV challenge, vaccinated animals presented virtually no detectable virus in human cells and in serum, whereas unvaccinated animals displayed robust infection, as measured by qRT-PCR. Our results utilizing humanized mice show potential efficacy for a targeted DNA vaccine against ZIKV in humans.

Combination of IL-10 and IL-2 induces oligoclonal human CD4 T cell expansion during xenogeneic and allogeneic GVHD in humanized mice.

IL-10 is a crucial anti-inflammatory cytokine which can also exert a seemingly divergent immunostimulatory effects under certain conditions. We found high levels of the cytokine in a xenogeneic GVHD model where NOD-scid IL2rγcnull (NSG) mice were transplanted with human PBMCs in presence of IL-2. Presence of exogenous IL-10 altered the kinetics of IL-2 induced human T cell reconstitution in vivo, showing an initial delay, followed by rapid expansion. Further, compared to IL-2 alone, treatment with IL-2 in combination with IL-10 increased survival in most animals and completely protected ∼20% of mice from GVHD. Additionally, IL-2 induced expansion of both CD4+ and CD8+ xenoreactive T cells whereas a combination of IL-2 and IL-10 resulted in selective expansion of CD4+ T cells only. TCR Vß repertoire analysis of CD4+ T cells showed that in contrast to IL-2 alone, simultaneous presence of both cytokines drastically reduced the Vß repertoire of the expanded CD4+ T cells. Highly restricted Vß usage was also observed when the cytokine combination was tested in an allogeneic GVHD model where NOD-scid IL2rγcnull mice expressing HLA-DR4 (NSG-DR4) were transplanted with purified CD4+ T cells from HLA-DR4 negative donors. Taken together, our results demonstrate that IL-10 can profoundly modulate the subset composition and repertoire of responding T cells during GVHD.

Gene therapy with plasmids encoding IFN-ß or IFN-α14 confers long-term resistance to HIV-1 in humanized mice.

Because endogenous interferon type I (IFN-I) produced by HIV-1 infection might complicate the analysis of therapeutically administered IFN-I, we tested different humanized mouse models for induction of IFN-I during HIV-1 infection. While HIV-1 induced high levels of IFN-α in BLT mice, IFN-I was undetectable following infection in the Hu-PBL mouse model, in which only T cells expand. We therefore tested the effect of treatment with Pegylated IFN-2 (pegasys), in Hu-PBL mice. Pegasys prevented CD4 T cell depletion and reduced the viral load for 10 days, but the effect waned thereafter. We next expressed IFN-I subsets (IFN-α2, -α6, -α8, -α14, and -ß) in Hu-PBL mice by hydrodynamic injection of plasmids encoding them and 2 days later infected the mice with HIV-1. CD4 T cell depletion was prevented in all subtypes of IFN-I-expressing mice by day 10. However, at day 40 post-infection, protection was seen in IFN-ß- and IFN-α14-expressing mice, but not the others. The viral load followed an inverse pattern and was highest in control mice and lowest in IFN-ß- and IFN-α14-expressing mice until day 40 after infection. These results show that gene therapy with plasmids encoding IFN-ß and -α14, but not the commonly used -α2, confers long-term suppression of HIV-1 replication.

A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death.

West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the ER-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death.

Targeting DNA vaccines to myeloid cells using a small peptide.

Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able to prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WNV-neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses.

Multiplexing seven miRNA-Based shRNAs to suppress HIV replication.

Multiplexed miRNA-based shRNAs (shRNA-miRs) could have wide potential to simultaneously suppress multiple genes. Here, we describe a simple strategy to express a large number of shRNA-miRs using minimal flanking sequences from multiple endogenous miRNAs. We found that a sequence of 30 nucleotides flanking the miRNA duplex was sufficient for efficient processing of shRNA-miRs. We inserted multiple shRNAs in tandem, each containing minimal flanking sequence from a different miRNA. Deep sequencing of transfected cells showed accurate processing of individual shRNA-miRs and that their expression did not decrease with the distance from the promoter. Moreover, each shRNA was as functionally competent as its singly expressed counterpart. We used this system to express one shRNA-miR targeting CCR5 and six shRNA-miRs targeting the HIV-1 genome. The lentiviral construct was pseudotyped with HIV-1 envelope to allow transduction of both resting and activated primary CD4 T cells. Unlike one shRNA-miR, the seven shRNA-miR transduced T cells nearly abrogated HIV-1 infection in vitro. Additionally, when PBMCs from HIV-1 seropositive individuals were transduced and transplanted into NOD/SCID/IL-2R γc(-/-) mice (Hu-PBL model) efficient suppression of endogenous HIV-1 replication with restoration of CD4 T cell counts was observed. Thus, our multiplexed shRNA appears to provide a promising gene therapeutic approach for HIV-1 infection.

IL-10 exacerbates xenogeneic GVHD by inducing massive human T cell expansion.

Although patients with GVHD have elevated serum levels of IL10, whether its role is protective or pathogenic remains unclear. Here, we used a humanized mouse model to study the role of IL-10 in GVHD. When human PBMCs were engrafted in NOD-scid IL2rγc(null) mice expressing human IL-10, the T cells underwent massive expansion resulting in lethality by day 21, whereas control mice survived for at least 40 days. Histopathology of the liver showed extensive mononuclear cell infiltration in IL-10 expressing but not in control mice. Corresponding to their aggressiveness, the T cells in the IL-10 group exhibited predominantly an effector memory phenotype (CD45RO(+)CD27(-)) while in control mice, the T cells were of transitional memory phenotype (CD45RO(+)CD27(+)). Further, IL-10 receptor blocking antibody was able to protect the animals from GVHD. Since our results demonstrate a direct pathogenic role for IL-10, blockade of IL-10 signaling may provide a therapeutic option for GVHD.

CCR5 Gene Editing of Resting CD4(+) T Cells by Transient ZFN Expression From HIV Envelope Pseudotyped Nonintegrating Lentivirus Confers HIV-1 Resistance in Humanized Mice.

CCR5 disruption by zinc finger nucleases (ZFNs) is a promising method for HIV-1 gene therapy. However, successful clinical translation of this strategy necessitates the development of a safe and effective method for delivery into relevant cells. We used non-integrating lentivirus (NILV) for transient expression of ZFNs and pseudotyped the virus with HIV-envelope for targeted delivery to CD4(+) T cells. Both activated and resting primary CD4(+) T cells transduced with CCR5-ZFNs NILV showed resistance to HIV-1 infection in vitro. Furthermore, NILV transduced resting CD4(+) T cells from HIV-1 seronegative individuals were resistant to HIV-1 challenge when reconstituted into NOD-scid IL2rγc null (NSG) mice. Likewise, endogenous virus replication was suppressed in NSG mice reconstituted with CCR5-ZFN-transduced resting CD4(+) T cells from treatment naïve as well as ART-treated HIV-1 seropositive patients. Taken together, NILV pseudotyped with HIV envelope provides a simple and clinically viable strategy for HIV-1 gene therapy.

Long-term engraftment of human natural T regulatory cells in NOD/SCID IL2rγc(null) mice by expression of human IL-2.

Regulatory T cells are essential to maintain immune homeostasis and prevent autoimmunity. Therapy with in vitro expanded human nT(Regs) is being tested to prevent graft versus host disease, which is a major cause for morbidity and mortality associated with hematopoietic stem cell transplantation. Their usefulness in therapy will depend on their capacity to survive, migrate appropriately and retain suppressive activity when introduced into a transplant recipient. The lack of a suitable animal model for studying the in vivo reconstitutive capability of human nT(Regs) is a major impediment for investigating the behavior of adoptively transferred nT(Regs)in vivo. We show that injection of a plasmid encoding human IL-2 is necessary and sufficient for long term engraftment of in vitro expanded nT(Regs) in NOD-SCID IL2rγc(null) mice. We also demonstrate that these in vivo reconstituted T(Regs) traffic to different organs of the body and retain suppressive function. Finally, in an IL-2 accelerated GVHD model, we show that these in vivo reconstituted T(Regs) are capable of preventing severe xenogenic response of human PBMCs. Thus, this novel 'hu-T(Reg) mouse' model offers a pre-clinical platform to study the in vivo function and stability of human nT(Regs) and their ability to modulate autoimmune diseases and GVHD.

Human macrophage and dendritic cell-specific silencing of high-mobility group protein B1 ameliorates sepsis in a humanized mouse model.

Hypersecretion of cytokines by innate immune cells is thought to initiate multiple organ failure in murine models of sepsis. Whether human cytokine storm also plays a similar role is not clear. Here, we show that human hematopoietic cells are required to induce sepsis-induced mortality following cecal ligation and puncture (CLP) in the severely immunodeficient nonobese diabetic (NOD)/SCID/IL2Rγ(-/-) mice, and siRNA treatment to inhibit HMGB1 release by human macrophages and dendritic cells dramatically reduces sepsis-induced mortality. Following CLP, compared with immunocompetent WT mice, NOD/SCID/IL2Rγ(-/-) mice did not show high levels of serum HMGB1 or murine proinflammatory cytokines and were relatively resistant to sepsis-induced mortality. In contrast, NOD/SCID/IL2Rγ(-/-) mice transplanted with human hematopoietic stem cells [humanized bone marrow liver thymic mice (BLT) mice] showed high serum levels of HMGB1, as well as multiple human but not murine proinflammatory cytokines, and died uniformly, suggesting human cytokines are sufficient to induce organ failure in this model. Moreover, targeted delivery of HMGB1 siRNA to human macrophages and dendritic cells using a short acetylcholine receptor (AchR)-binding peptide [rabies virus glycoprotein (RVG)-9R] effectively suppressed secretion of HMGB1, reduced the human cytokine storm, human lymphocyte apoptosis, and rescued humanized mice from CLP-induced mortality. siRNA treatment was also effective when started after the appearance of sepsis symptoms. These results show that CLP in humanized mice provides a model to study human sepsis, HMGB1 siRNA might provide a treatment strategy for human sepsis, and RVG-9R provides a tool to deliver siRNA to human macrophages and dendritic cells that could potentially be used to suppress a variety of human inflammatory diseases.

Ago-2-mediated slicer activity is essential for anti-flaviviral efficacy of RNAi.

RNA interference can be mediated by fully complementary siRNA or partially complementary miRNA. siRNAs are widely used to suppress viral replication and the fully complementary siRNA bound Ago-2 in the RISC is known to degrade the target RNA. Although other argonaute proteins lacking slicer activity can also bind oligonucleotides with both si and miRNA structures, whether they can also contribute to antiviral effects is not entirely clear. We tested si and miRNA structured oligos for target repression in dual luciferase assays as well as for inhibition of Dengue and West Nile virus replication in ES cells expressing individual Ago proteins. In luciferase assays, both fully complementary and partially complementary oligos effectively repressed their targets in all individual Ago expressing cell lines, although the efficacy with fully complementary oligos was higher in Ago-2+ cells. However, partially complementary oligos had no effect on virus replication in any cell line, while fully complementary siRNAs were highly effective in Ago-2 expressing, but not in cells expressing other Ago proteins. This occurred irrespective of whether the target sequences were located in the coding region or 3'UTR of the virus. We conclude that Ago-2 slicer activity is essential for anti-viral efficacy of siRNAs and miRNA-mediated translational repression/transcript destabilization is too weak to suppress the abundantly expressed flaviviral proteins.

Silencing early viral replication in macrophages and dendritic cells effectively suppresses flavivirus encephalitis.

West Nile (WN) and St. Louis encephalitis (SLE) viruses can cause fatal neurological infection and currently there is neither a specific treatment nor an approved vaccine for these infections. In our earlier studies, we have reported that siRNAs can be developed as broad-spectrum antivirals for the treatment of infection caused by related viruses and that a small peptide called RVG-9R can deliver siRNA to neuronal cells as well as macrophages. To increase the repertoire of broad-spectrum antiflaviviral siRNAs, we screened 25 siRNAs targeting conserved regions in the viral genome. Five siRNAs were found to inhibit both WNV and SLE replication in vitro reflecting broad-spectrum antiviral activity and one of these was also validated in vivo. In addition, we also show that RVG-9R delivers siRNA to macrophages and dendritic cells, resulting in effective suppression of virus replication. Mice were challenged intraperitoneally (i.p.) with West Nile virus (WNV) and treated i.v. with siRNA/peptide complex. The peritoneal macrophages isolated on day 3 post infection were isolated and transferred to new hosts. Mice receiving macrophages from the anti-viral siRNA treated mice failed to develop any disease while the control mice transferred with irrelevant siRNA treated mice all died of encephalitis. These studies suggest that early suppression of viral replication in macrophages and dendritic cells by RVG-9R-mediated siRNA delivery is key to preventing the development of a fatal neurological disease.

Japanese encephalitis virus utilizes the canonical pathway to activate NF-kappaB but it utilizes the type I interferon pathway to induce major histocompatibility complex class I expression in mouse embryonic fibroblasts.

Flaviviruses have been shown to induce cell surface expression of major histocompatibility complex class I (MHC-I) through the activation of NF-kappaB. Using IKK1(-/-), IKK2(-/-), NEMO(-/-), and IKK1(-/-) IKK2(-/-) double mutant as well as p50(-/-) RelA(-/-) cRel(-/-) triple mutant mouse embryonic fibroblasts infected with Japanese encephalitis virus (JEV), we show that this flavivirus utilizes the canonical pathway to activate NF-kappaB in an IKK2- and NEMO-, but not IKK1-, dependent manner. NF-kappaB DNA binding activity induced upon virus infection was shown to be composed of RelA:p50 dimers in these fibroblasts. Type I interferon (IFN) production was significantly decreased but not completely abolished upon virus infection in cells defective in NF-kappaB activation. In contrast, induction of classical MHC-I (class 1a) genes and their cell surface expression remained unaffected in these NF-kappaB-defective cells. However, MHC-I induction was impaired in IFNAR(-/-) cells that lack the alpha/beta IFN receptor, indicating a dominant role of type I IFNs but not NF-kappaB for the induction of MHC-I molecules by Japanese encephalitis virus. Our further analysis revealed that the residual type I IFN signaling in NF-kappaB-deficient cells is sufficient to drive MHC-I gene expression upon virus infection in mouse embryonic fibroblasts. However, NF-kappaB could indirectly regulate MHC-I expression, since JEV-induced type I IFN expression was found to be critically dependent on it.

Targeted delivery of small interfering RNA to human dendritic cells to suppress dengue virus infection and associated proinflammatory cytokine production.

Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide (DC3) fused to nona-D-arginine (9dR) residues (DC3-9dR) delivers siRNA and knocks down endogenous gene expression in heterogenous DC subsets, (monocyte-derived DCs [MDDCs], CD34(+) hematopoietic stem cell [HSC])-derived Langerhans DCs, and peripheral blood DCs). Moreover, DC3-9dR-mediated delivery of siRNA targeting a highly conserved sequence in the dengue virus envelope gene (siFvE(D)) effectively suppressed dengue virus replication in MDDCs and macrophages. In addition, DC-specific delivery of siRNA targeting the acute-phase cytokine tumor necrosis factor alpha (TNF-alpha), which plays a major role in dengue pathogenesis, either alone or in combination with an antiviral siRNA, significantly reduced virus-induced production of the cytokine in MDDCs. Finally to validate the strategy in vivo, we tested the ability of the peptide to target human DCs in the NOD/SCID/IL-2Rgamma(-/-) mouse model engrafted with human CD34(+) hematopoietic stem cells (HuHSC mice). Treatment of mice by intravenous (i.v.) injection of DC3-9dR-complexed siRNA targeting TNF-alpha effectively suppressed poly(I:C)-induced TNF-alpha production by DCs. Thus, DC3-9dR can deliver siRNA to DCs both in vitro and in vivo, and this delivery approach holds promise as a therapeutic strategy to simultaneously suppress virus replication and curb virus-induced detrimental host immune responses in dengue infection.

Nonclassical MHC-I and Japanese encephalitis virus infection: induction of H-2Q4, H-2T23 and H-2T10.

Nonclassical MHC Class 1b antigens differ from classical MHC class 1a antigens in having a restricted polymorphism as well as varied surface expression in different cell types. They have been hypothesized to play a role in bridging adaptive and innate immune responses. We examined the effects of JEV infection on the expression of classical MHC class 1a and nonclassical MHC class 1b genes in five different cell lines. Among the nonclassical genes, H-2Q4 was induced in H-6 hepatoma, primary astrocytes, mouse embryo fibroblasts, L929 and 3T3 cells. H-2T23 and H-2T10 genes were not induced in H-6 and 3T3, respectively, but were induced in the other cell lines examined. Both H-2Q4 encoded Qb1 and H-2T23 encoded Qa-1(b) antigens were induced on the cell surface upon JEV infection in primary astrocytes and mouse embryonic fibroblasts. Classical MHC-I genes and the genes associated with antigen presentation such as Tap1, Tap2, Tapasin, Lmp2, Lmp7 and Lmp10 as well as type 1 (alpha/beta) IFNs were induced in all cell lines. However, IFNgamma was not induced. Further, induction of H-2Q4 and H-2T23 by JEV was independent of NF-kappaB but type 1 IFN dependent while H-2T10 was dependent on NF-kappaB and type 1 IFN independent. Thus, while classical MHC genes were induced by JEV in all cell lines tested despite high levels of constitutive expression in L929 and 3T3, nonclassical genes were not inducible in all cell lines tested and involved different mechanisms of induction.

Induction of classical and nonclassical MHC-I on mouse brain astrocytes by Japanese encephalitis virus.

Infection with Flaviviruses upregulates the cell surface expression of MHC-I, MHC-II, ICAM-1 (CD54), VCAM-1 (CD106) and TAP proteins. Although all these studies have been confirmed using West Nile virus and other Flaviviruses, there are few reports that have examined the effects of Japanese encephalitis virus (JEV) infection directly on nonclassical and classical MHC expression in astrocytes. We show in this report that JEV infection of mouse brain astrocytes results in induction of the nonclassical MHC Class Ib genes, H-2T23, H-2Q4 and H-2T10 in addition to MHC-I, Type I (alpha/beta) IFNs, TAP-1, TAP-2, Tapasin, LMP-2, LMP-7 and LMP-10 but not IFNgamma, CD80, CD86 and MHC-II genes. The increased cell surface expression of these antigens as well as induction of the genes mentioned above as measured by RT-PCR suggests that JEV infection may lead to the induction of classical MHC Class Ia as well as nonclassical MHC Class Ib molecules.