siRNAs encapsulated in recombinant capsid protein derived from Dengue serotype 2 virus inhibits the four serotypes of the virus and proliferation of cancer cells.

siRNA delivery potential of the Dengue virus capsid protein in cultured cells was recently reported, but target knockdown potential in the context of specific diseases has not been explored. In this study we have evaluated the utility of the protein as an siRNA carrier for anti Dengue viral and anti cancer applications using cell culture systems. We show that target specific siRNAs delivered using the capsid protein inhibit infection by the four serotypes of Dengue virus and proliferation of two cancer cell lines. Our data confirm the potential of the capsid for anti Dengue viral and anti cancer RNAi applications. In addition, we have optimized a fermentation strategy to improve the yield of Escherichia coli expressed D2C protein since the reported yields of E. coli expressed flaviviral capsid proteins are low.

Evaluation of target mRNA cleavage by aurorakinase B specific siRNA in prostate and hepatic cancer cells and its therapeutic potential in mouse models of liver cancer.

The anti proliferative potential of siRNA26, targeted to Aurora kinase B, in prostate cancer cells is known from a previous study from our laboratory. Here we first show that siRNA26 cleaves at the same position of the target mRNA in the prostate cancer and hepatocellular carcinoma cell lines, PC3 and HepG2 respectively. Aurorakinase B specific siRNA, but not a control siRNA, inhibited PC3 and HepG2 cell proliferation and cell migration. These effects correlated to RNA silencing of Aurorakinase B in both the cell lines. Intra-tumoral administration of HiPerfect complexed siRNA26 inhibited the growth of HepG2 xenografts in SCID mice. In an orthotopic setting, intravenous administration of HiPerfect encapsulated siRNA26 appeared to reduce the severity of multifocal lesions.

Infant mouse brain passaged Dengue serotype 2 virus induces non-neurological disease with inflammatory spleen collapse in AG129 mice after splenic adaptation.

AG129 mice are known to be permissive to infection by multiple serotypes of Dengue virus (DENV). There exists a concern that mouse passaged strains of the virus may induce neurological complications rather than increased vascular permeability in these mice, hence the use of human clinical isolates of the virus to develop the AG129 mouse model of Dengue disease with increased vascular permeability. The present study evaluated four mouse brain passaged DENV strains, each belonging to a different serotype and three of them having an original isolation history in India, for their suitability to serve as candidates to induce rapid lethal disease in AG129 mice. While all the viruses were able to establish a productive infection in the spleen, none of them induced paralysis despite their mouse brain passage history. Only the type-2 virus acquired the ability to induce a lethal disease after a single round of spleen to spleen passage, and became highly virulent after five more rounds. This apparently non-neurological lethal disease was characterized by high viral burden, elevated vascular permeability, serum TNF-α surge immediately before moribund stage, transient leukocytosis followed by severe leukopenia, lymphopenia throughout the course of the infection, and transient thrombocytopenia. The disease was also characterized by inflammatory splenic collapse during moribund stage, reminiscent of spontaneous splenic rupture reported in rare cases of severe Dengue in humans.

VEGF-C differentially regulates VEGF-A expression in ocular and cancer cells; promotes angiogenesis via RhoA mediated pathway.

Vascular angiogenesis is regulated by a number of cytokines of which vascular endothelial growth factor (VEGF)-A/and its receptor vascular endothelial growth factor receptor 2 (VEGFR2) play an indisputable role. Similarly lymphangiogenesis is regulated by VEGF-C and its receptor VEGFR3. Currently for treating vasculogenesis diseases such as proliferative retinopathies and cancer, a number of anti-VEGF-A therapies are approved for clinical use. Although clinical efficacies achieved are remarkable, they are found to be transitory in nature, followed by restoration of anti-VEGF therapy resistant angiogenesis. Recently the regulatory role of VEGF-C in initiating and potentiating neo-angiogenesis has been uncovered. Although the interactive nature of VEGF-A and C is known, the dynamics of their expression under knockdown conditions is yet to be established. Here in this study we have utilized siRNA to knockdown both VEGF-A and C either independently or in combination. Analysis of VEGF-A and C expression (only in cancer cell lines MCF7, A549 and H460 but not in the ocular cell line RPE19) has shown enhanced expression levels of VEGF-C with increase in knockdown of VEGF-A. However, VEGF-C knockdown has resulted in decreased expression levels of VEGF-A both in RPE19 and MCF7 cells in a dose dependent manner. In addition, VEGF-C knockdown also resulted in decreased expression of RhoA. Further, knockdown studies of RhoA even with supplementation of VEGF-C or A has resulted in decreased endothelial cell proliferation and stress fiber formation, indicating that VEGF-C does promote angiogenesis via RhoA mediated pathway.

Phylogenetic analysis of the species Theilovirus: emerging murine and human pathogens.

The Cardiovirus genus of the family Picornaviridae includes two distinct species, Encephalomyocarditis virus and Theilovirus. We now report the complete nucleotide sequences of three Theiler's murine encephalomyelitis virus (TMEV) strains (TO Yale, TOB15, and Vie 415HTR) and of Vilyuisk human encephalomyelitis virus (VHEV). This information, together with the recently reported sequences of divergent theiloviruses (Theiler's-like rat virus [TRV] and Saffold viruses 1 and 2 [SAFV-1 and SAFV-2]), enables an updated phylogenetic analysis as well as a reexamination of several gene products important in the pathogenesis of this emerging group of viruses. In the light of the known neurotropism of TMEV and the new human SAFV-1 and SAFV-2, the resulting data suggest the existence of theiloviruses that cause human central nervous system infections. Our phylogenetic analyses point to the classification of presently known theiloviruses into five types: TMEV, VHEV, TRV, SAFV-1, and SAFV-2.

Differential usage of carbohydrate co-receptors influences cellular tropism of Theiler's murine encephalomyelitis virus infection of the central nervous system.

Theiler's murine encephalomyelitis viruses (TMEV) are ubiquitous pathogens of mice, producing either rapidly fatal encephalitis (high-neurovirulence strains) or persistent central nervous system infection and inflammatory demyelination (low-neurovirulence strains). Although a protein entry receptor has not yet been identified, carbohydrate co-receptors that effect docking and concentration of the virus on the cell surface are known for both TMEV neurovirulence groups. Low-neurovirulence TMEV use alpha2,3-linked N-acetylneuramic acid (sialic acid) on an N-linked glycoprotein, whereas high-neurovirulence TMEV use the proteoglycan heparan sulfate (HS) as a co-receptor. While the binding of low-neurovirulence TMEV to sialic acid can be inhibited completely, only a third of the binding of high-neurovirulence TMEV to HS is inhibitable, suggesting that high-neurovirulence strains use another co-receptor or bind directly to the putative protein entry receptor. Four amino acids on the surface (VP2 puff B) of low-neurovirulence strains make contact with sialic acid through non-covalent hydrogen bonds. Since these virus residues are conserved in all TMEV strains, the capsid conformation of this region is probably responsible for sialic acid binding. A persistence determinant that maps within the virus coat using recombinant TMEV is also conformational in nature. Low-neurovirulence virus variants that do not bind to sialic acid fail to persist in the central nervous system of mice, indicating a role for sialic acid binding in TMEV persistence. Analysis of high-neurovirulence variants that do not bind HS demonstrates that HS co-receptor usage influences neuronal tropism in brain, whereas, the HS co-receptor use is not required for the infection of spinal cord anterior horn cells associated with poliomyelitis.

Theiler's virus persistence in the central nervous system of mice is associated with continuous viral replication and a difference in outcome of infection of infiltrating macrophages versus oligodendrocytes.

Theiler's murine encephalomyelitis virus (TMEV) infection of mice, in which persistent central nervous system (CNS) infection induces Th1 CD4+ T cell responses to both virus and myelin proteins, provides a relevant experimental animal model for MS. During persistence, >10(9) TMEV genome equivalents per spinal cord are detectable by real-time reverse transcription-polymerase chain reaction (RT-PCR). Because of the short half-life of TMEV (<1 day), continual viral replication is needed to sustain these very high TMEV copy numbers. An essential role for macrophages in TMEV persistence has been documented and, although limited by host anti-viral immune responses, TMEV nonetheless spreads during persistence to infect other cells, particularly oligodendrocytes, in which the infection is productive and lytic. Virus factors influencing persistence of TMEV are expression of the out-of-frame L* protein and use of sialic acid co-receptors.

Virus persistence in an animal model of multiple sclerosis requires virion attachment to sialic acid coreceptors.

Persistent Theiler's virus infection in the central nervous system (CNS) of mice provides a highly relevant animal model for multiple sclerosis. The low-neurovirulence DA strain uses sialic acid as a coreceptor for cell binding before establishing infection. During adaptation of DA virus to growth in sialic acid-deficient cells, three amino acid substitutions (G1100D, T1081I, and T3182A) in the capsid arose, and the virus no longer used sialic acid as a coreceptor. The adapted virus retained acute CNS virulence, but its persistence in the CNS, white matter inflammation, and demyelination were largely abrogated. Infection of murine macrophage but not oligodendrocyte cultures with the adapted virus was also significantly reduced. Substitution of G1100D in an infectious DA virus cDNA clone demonstrated a major role for this mutation in loss of sialic acid binding and CNS persistence. These data indicate a direct role for sialic acid binding in Theiler's murine encephalomyelitis virus persistence and chronic demyelinating disease.

Heparan sulfate-independent infection attenuates high-neurovirulence GDVII virus-induced encephalitis.

The high-neurovirulence Theiler's murine encephalomyelitis virus (TMEV) strain GDVII uses heparan sulfate (HS) as a coreceptor to enter target cells. We report here that GDVII virus adapted to growth in HS-deficient cells exhibited two amino acid substitutions (R3126L and N1051S) in the capsid and no longer used HS as a coreceptor. Infectious-virus yields in CHO cells were 25-fold higher for the adapted virus than for the parental GDVII virus, and the neurovirulence of the adapted virus in intracerebrally inoculated mice was substantially attenuated. The adapted virus showed altered cell tropism in the central nervous systems of mice, shifting from cerebral and brainstem neurons to spinal cord anterior horn cells; thus, severe poliomyelitis, but not acute encephalitis, was observed in infected mice. These data indicate that the use of HS as a coreceptor by GDVII virus facilitates cell entry and plays an important role in cell tropism and neurovirulence in vivo.

Amino acid substitutions in VP2 residues contacting sialic acid in low-neurovirulence BeAn virus dramatically reduce viral binding and spread of infection.

Theiler's murine encephalomyelitis viruses (TMEV) consist of two groups, the high- and low-neurovirulence groups, based on lethality in intracerebrally inoculated mice. Low-neurovirulence TMEV result in a persistent central nervous system infection in mice, leading to an inflammatory demyelinating pathology and disease. Low- but not high-neurovirulence strains use sialic acid as an attachment factor. The recent resolution of the crystal structure of the low-neurovirulence DA virus in complex with the sialic acid mimic sialyllactose demonstrated that four capsid residues make contact with sialic acid through noncovalent hydrogen bonds. To systematically test the importance of these sialic acid-binding residues in viral entry and infection, we mutated three VP2 puff B amino acids proposed to make contact with sialic acid and analyzed the consequences of each amino acid substitution on viral entry and spread. The fourth residue is in the VP3-VP1 cleavage dipeptide and could not be mutated. Our data suggest that residues Q2161 and G2174 are directly involved in BeAn virus attachment to sialic acid and that substitutions of these two residues result in the loss of or reduced viral binding and hemagglutination and in the inability to spread among BHK-21 cells. In addition, a gain of function-revertant virus was recovered with the Q2161A mutation after prolonged passage in cells.