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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.

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., Highlights • Zika DNA vaccine elicits protective antibody response in humanized DRAG mice. • Human myeloid cells and B cells are targets of Zika virus. A Zika virus vaccine requires testing in human immune cells to determine its effectiveness. In this communication, we report that a DNA vaccine encoding Zika prM and E protein is able to elicit protective neutralizing antibodies against Zika virus in humanized DRAG mice, and that human myeloid cells and B cells are primary targets of Zika virus.

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 endoplasmic reticulum-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death.

// Sojan Abraham 1 , Jang-Gi Choi 1,2 , Nora M. Ortega 1 , Junli Zhang 1 , Premlata Shankar 1 and N. Manjunath Swamy 1 1 Center of Emphasis in Infectious Disease, Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, TX, USA 2 KM Application Center, Korea Institute of Oriental Medicine, Dong-gu, Daegu, Republic of Korea Correspondence to: N. Manjunath Swamy, email: // Keywords : interferon, HIV-1, hydrodynamic injection, Immunology and Microbiology Section, Immune response, Immunity Received : May 20, 2016 Accepted : October 02, 2016 Published : October 06, 2016 Abstract 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 -β) 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-β- 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-β- and IFN-α14-expressing mice until day 40 after infection. These results show that gene therapy with plasmids encoding IFN-β and -α14, but not the commonly used -α2, confers long-term suppression of HIV-1 replication.

Flaviviruses have been shown to induce cell surface expression of major histocompatibility complex class I (MHC-I) through the activation of NF-κB. 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-κB in an IKK2- and NEMO-, but not IKK1-, dependent manner. NF-κB 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-κB activation. In contrast, induction of classical MHC-I (class 1a) genes and their cell surface expression remained unaffected in these NF-κB-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-κB for the induction of MHC-I molecules by Japanese encephalitis virus. Our further analysis revealed that the residual type I IFN signaling in NF-κB-deficient cells is sufficient to drive MHC-I gene expression upon virus infection in mouse embryonic fibroblasts. However, NF-κB could indirectly regulate MHC-I expression, since JEV-induced type I IFN expression was found to be critically dependent on it.

The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors are used. To overcome this limitation, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles for transient exposure and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific single guide RNAs (sgRNAs). We then constructed an 'all in one virus' to express sgRNAs in association with pre-packaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using an sgRNA-targeting HIV long terminal repeat, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared with continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy applications.

Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein antigens 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, that 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 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 antigen 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 WN neutralizing antibodies 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.

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.

Japanese encephalitis virus (JEV) is a flavivirus that is one of the major causes of encephalitis in Eastern and Southern Asia with recent observations marking its spread to newer geographical regions. Flaviviruses, in general consitutute a global health threat since many of them are endemic in large parts of the Americas, Africa and Australia in addition to Asia and suitable vaccines are unavailable for many of them. JEV causes serious inflammation of the brain, which may lead to permanent brain damage, and has a high mortality rate. More than three billion people live in JE endemic areas and JEV is estimated to cause 45,000 human cases of disease and 10000 deaths per year (Solomon and Winter, 2004; van den Hurk et al., 2009). The occurrence of encephalitic epidemics in humans due to this virus have been recognized from 1870s in Japan where it was termed Japanese B encephalitis to distinguish it from type A encephalitis caused by sleeping sickness. The first prototype Nakayama strain was isolated and established in 1935 from the brain of a fatal case from Tokyo, Japan. This was closely followed by virus isolation from Culex tritaeniorrhynchus in 1936 confirming earlier suspicions that mosquitoes served as the transmission vector (Solomon et al., 2000). JEV transmission occurs through a zoonotic cycle involving mosquitoes as an important intermediate maintenance and replicative vector. Vertebrates, chiefly pigs and ardeid birds act as amplifying hosts. Only those animals that develop high viraemias are significant in this natural enzootic cycle. Humans are infected only coincidently when bitten by an infected mosquito and are dead end hosts. Human to human transmission is yet to be documented, possibly due to the presence of transient viraemia. The absence of high serum viraemia prevents the virus from being picked up during mosquito bites. Pigs play a major role in the transmission cycle with respect to human infection since they are often bred close to humans whereas herons, egrets and other ardeid birds are important for maintenance and overwintering of this virus. Among the other vertebrates, horses also develop central nervous system (CNS) infections and are considered as dead end hosts. Rodents are relatively refractory to infections while amphibians, reptiles and bats can also be infected (Mackenzie et al., 2004). JEV does not cause encephalitis in pigs and birds although it is

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-α), 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-2Rγ −/− 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-α effectively suppressed poly(I:C)-induced TNF-α 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 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, IFN\gamma was not induced. Further, induction of H-2Q4 and H-2T23 by JEV was independent of NF-\kappa B but type 1 IFN dependent while H-2T10 was dependent on NF-\kappa B 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.

Infection with Flaviviruses upregulates the cell surface expression of MHC-I,MHC-II, ICAM- I (CD54),VCAM- I (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 IFN gamma, CD80, CD86 and MHC-II genes. The increased cel I 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 la as well as nonclassical MHC Class lb molecules.

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.

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.