Vaibhav Tiwari

Comparative studies of cytokinins on in vitro propagation of Bacopa monniera

A mass in vitro propagation system for Bacopa monniera (L.) Wettst. (Scrophulariaceae), a medicinally important plant, has been developed. A range of cytokinins have been investigated for multiple shoot induction with node, internode and leaf explants. Of the four cytokinins (6-benzyladenine, thidiazuron, kinetin and 2-isopentenyladenine) tested thidiazuron (6.8 μM) and 6-benzyladenine (8.9 μM) proved superior to other treatments. Optimum adventitious shoot buds induction occurred at 6.8 μM thidiazuron where an average of 93 shoot buds were produced in leaf explants after 7 weeks of incubation. However, subculture of leaf explants on medium containing 2.2 μM benzyladenine yielded a higher number (129.1) of adventitious shoot buds by the end of third subculture. The percentage shoot multiplication (100%) as well as the number of shoots per explant remained the high during the first 3 subculture cycles, facilitating their simultaneous harvest for rooting. In vitro derived shoots were elongated on growth regulator-free MS medium and exhibited better rooting response on medium containing 4.9 μM IBA. After a hardening phase of 3 weeks, there was an almost 100% transplantation success in the field.

Virostatic potential of micro–nano filopodia-like ZnO structures against herpes simplex virus-1

Herpes simplex virus type-1 (HSV-1) entry into target cell is initiated by the ionic interactions between positively charged viral envelop glycoproteins and a negatively charged cell surface heparan sulfate (HS). This first step involves the induction of HS-rich filopodia-like structures on the cell surface that facilitate viral transport during cell entry. Targeting this initial first step in HSV-1 pathogenesis, we generated different zinc oxide (ZnO) micro-nano structures (MNSs) that were capped with multiple nanoscopic spikes mimicking cell induced filopodia. These MNSs were predicted to target the virus to compete for its binding to cellular HS through their partially negatively charged oxygen vacancies on their nanoscopic spikes, to affect viral entry and subsequent spread. Our results demonstrate that the partially negatively charged ZnO-MNSs efficiently trap the virions via a novel virostatic mechanism rendering them unable to enter into human corneal fibroblasts - a natural target cell for HSV-1 infection. The anti-HSV-1 activity of ZnO MNSs was drastically enhanced after creating additional oxygen vacancies under UV-light illumination. Our results provide a novel insight into the significance of ZnO MNSs as the potent HSV-1 inhibitor and rationalize their development as a novel topical agent for the prevention of HSV-1 infection.

Characterization of heparan sulphate 3-O-sulphotransferase isoform 6 and its role in assisting the entry of herpes simplex virus type 1

Heparan sulphate (HS) 3-O-sulphotransferase transfers sulphate to the 3-OH position of the glucosamine residue of HS to form 3-O-sulphated HS. The HS modified by 3-O-sulphotransferase isoform 3 binds to HSV-1 (herpes simplex virus type 1) gD (envelope glycoprotein D), and the resultant 3-O-sulphated HS serves as an entry receptor for HSV-1. In the present paper, we report the isolation and characterization of a novel HS 3-O-sulphotransferase isoform, designated HS 3-O-sulphotransferase isoform 6 (3-OST-6). Mouse 3-OST-6 gene was identified in the EST (expressed sequence tag) database and cloned into pcDNA3.1/Myc-His vector. A CHO (Chinese-hamster ovary) cell line that stably expresses 3-OST-6 (3OST6/CHO cells) was prepared. The disaccharide analysis of the HS isolated from 3OST6/CHO cells revealed that 3-OST-6 exhibits HS 3-O-sulphotransferase activity. Furthermore, 3OST6/CHO cells were susceptible to infection by HSV-1, but not by other alphaherpesviruses examined, suggesting that 3-OST-6 produces a specific entry receptor for HSV-1. Our results indicate that a new member of 3-OST family generates an entry receptor for HSV-1. The findings add to the growing body of evidence that HSV-1 entry is mediated by 3-O-sulphated HS generated by multiple members of 3-O-sulphotransferases.

Using a 3-O-sulfated heparin octasaccharide to inhibit the entry of herpes simplex virus type 1.

Heparan sulfate (HS) is a highly sulfated polysaccharide and is present in large quantities on the cell surface and in the extracellular matrix. Herpes simplex virus type 1 (HSV-1) utilizes a specialized cell surface HS, known as 3-O-sulfated HS, as an entry receptor to establish infection. Here, we exploit an approach to inhibiting HSV-1 infection by using a 3-O-sulfated octasaccharide, mimicking the active domain of the entry receptor. The 3-O-sulfated octasaccharide was synthesized by incubating a heparin octasaccharide (3-OH octasaccharide) with HS 3-O-sulfotransferase isoform 3. The resultant 3-O-sulfated octasaccharide has a structure of Delta UA2S-GlcNS6S-IdoUA2S-GlcNS6S-IdoUA2S-GlcNS3S6S-IdoUA2S-GlcNS6S (where Delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is D-glucosamine, and IdoUA is L-iduronic acid). Results from cell-based assays revealed that the 3-O-sulfated octasaccharide has stronger activity in blocking HSV-1 infection than that of the 3-OH octasaccharide, suggesting that the inhibition of HSV-1 infection requires a unique sulfation moiety. Our results suggest the feasibility of inhibiting HSV-1 infection by blocking viral entry with a specific oligosaccharide.

Prophylactic, therapeutic and neutralizing effects of zinc oxide tetrapod structures against herpes simplex virus type-2 infection

The attachment of Herpes simplex virus type-2 (HSV-2) to a target cell requires ionic interactions between negatively charged cell surface co-receptor heparan sulfate (HS) and positively charged residues on viral envelop glycoproteins, gB and gC. Effective blocking of this first step of HSV-2 pathogenesis demonstrates significant prophylactic effects against the viral disease; any in vitro therapeutic effects of blocking this interaction, however, are not clear. Here, we provide new evidence that zinc oxide tetrapod micro-nanostructures synthesized by flame transport approach significantly block HSV-2 entry into target cells and, in addition, demonstrate the potential to stop the spread of the virus among already infected cells. The zinc oxide tetrapods (ZnOTs) also exhibit the ability to neutralize HSV-2 virions. Natural target cells such as human vaginal epithelial and HeLa cells showed highly reduced infectivity when infected with HSV-2 virions that were pre-incubated with the ZnOTs. The mechanism behind the ability of ZnOTs to prevent, neutralize or reduce HSV-2 infection relies on their ability to bind the HSV-2 virions. We used fluorescently labeled ZnOTs and GFP-expressing HSV-2 virions to demonstrate the binding of the ZnOTs with HSV-2. We also show that the binding and hence, the antiviral effects of ZnOTs can be enhanced by illuminating the ZnOTs with UV light. Our results provide new insights into the anti-HSV-2 effects of ZnOT and rationalize their development as a HSV-2 trapping agent for the prevention and/or treatment of infection. The observed results also demonstrate that blocking HSV-2 attachment can have prophylactic as well as therapeutic applications.

Role for 3-O-Sulfated Heparan Sulfate as the Receptor for Herpes Simplex Virus Type 1 Entry into Primary Human Corneal Fibroblasts

ABSTRACT Herpes simplex virus type 1 (HSV-1) infection of the corneal stroma remains a major cause of blindness. Primary cultures of corneal fibroblasts (CF) were tested and found susceptible to HSV-1 entry, which was confirmed by deconvolution imaging of infected cells. Plaque assay and real-time PCR demonstrated viral replication and hence a productive infection of CF by HSV-1. A role for glycoprotein D (gD) receptors in cultured CF was determined by gD interference assay. Reverse transcription-PCR analysis indicated expression of herpesvirus entry mediator and 3-O-sulfated (3-OS) heparan sulfate (HS)-generating enzyme 3-O sulfotransferase 3 (3-OST-3) but not nectin-1 or nectin-2. Subsequently, HS isolated from these cells was found to contain two distinct disaccharides (IdoUA2S-AnMan3S and IdoUA2S-AnMan3S6S) that are representative of 3-OST-3 activity. The following lines of evidence supported the important role of 3-OS HS as the mediator of HSV-1 entry into CF. (i) Blockage of entry was observed in CF treated with heparinases. The same enzymes had significantly less effect on HeLa cells that use nectin-1 as the entry receptor. (ii) Enzymatic removal of cell surface HS also removed the major gD-binding receptor, as evident from the reduced binding of gD to cells. (iii) Spinoculation assay demonstrated that entry blockage by heparinase treatment included the membrane fusion step. (iv) HSV-1 glycoprotein-induced cell-to-cell fusion was inhibited by either prior treatment of cells with heparinases or by HS preparations enriched in 3-OS HS. Taken together, the data in this report provide novel information on the role of 3-OS HS in mediating infection of CF, a natural target cell type.

Micropropagation of Centella asiatica (L.), a valuable medicinal herb

A protocol is described for rapid and large-scale in vitro clonal propagation of the valuable medicinal herb Centella asiatica (L.) by enhanced axillary bud proliferation in nodal segments isolated from mature plants. Although bud break was dependent on BA supply, the synergistic combination of 22.2 μM BA and 2.68 μM NAA induced the optimum frequency (91%) of shoot formation as well as shoot number (4 to 5 shoots per node). Subculturing of nodal segments harvested from the in vitro derived axenic shoots on the multiplication medium enabled continuous production of healthy shoots with similar frequency. MS medium supplemented with 6.7 μM BA and 2.88 μM IAA was found most suitable for shoot elongation. Rooting was highest (90%) on full-strength MS medium containing 2.46 μM IBA. Micropropagated plants established in garden soil were uniform and identical to the donor plant with respect to growth characteristics. This micropropagation procedure could be useful for raising a stock of genetically homogenous plant material for field cultivation.

Herpes simplex virus type 1 infection induces oxidative stress and the release of bioactive lipid peroxidation by-products in mouse P19N neural cell cultures

To determine whether herpes simplex virus type 1 (HSV-1) infection causes oxidative stress and lipid peroxidation in cultured neural cells, mouse P19 embryonal carcinoma cells were differentiated into cells with neural phenotypes (P19N cells) by retinoic acid and were then infected with HSV-1. Cellular levels of reactive oxygen species (ROS) and the release of lipid peroxidation by-products into the tissue culture medium were then measured by the generation of fluorescent markers hydroxyphenyl fluorescein and a stable chromophore produced by lipid peroxidation products, malondialdehyde (MDA) and hydroxyalkenals (4-HAEs; predominantly 4-hydroxy-2-nonenal [HNE]), respectively. HSV-1 infection increased ROS levels in neural cells as early as 1 h post infection (p.i.) and ROS levels remained elevated at 24 h p.i. This viral effect required viral entry and replication as heat- and ultraviolet light-inactivated HSV-1 were ineffective. HSV-1 infection also was associated with increased levels of MDA/HAE in the culture medium at 2 and 4 h p.i., but MDA/HAE levels were not different from those detected in mock infected control cultures at 1, 6, and 24 h p.i. HSV-1 replication in P19N cells was inhibited by the antioxidant compound ebselen and high concentrations of HNE added to the cultures, but was increased by low concentrations of HNE. These findings indicate that HSV-1 infection of neural cells causes oxidative stress that is required for efficient viral replication. Furthermore, these observations raise the possibility that soluble, bioactive lipid peroxidation by-products generated in infected neural cells may be important regulators of HSV-1 pathogenesis in the nervous system.

A Role for Herpesvirus Entry Mediator as the Receptor for Herpes Simplex Virus 1 Entry into Primary Human Trabecular Meshwork Cells

ABSTRACT The human eye is an important target for infection with herpes simplex virus 1 (HSV-1). Damage to cells forming the trabeculum of the eye by HSV-1 infection could contribute to the development of glaucoma, a major blinding disease. Primary cultures of human trabecular meshwork cells were used as an in vitro model to demonstrate the ability of HSV-1 to enter into and establish a productive infection of the trabeculum. Blocking of entry by anti-herpesvirus entry mediator (HVEM) antibody implicated HVEM as the major receptor for HSV-1 infection.

Herpes simplex virus type 1 induces filopodia in differentiated P19 neural cells to facilitate viral spread.

Herpes simplex virus type-1 (HSV-1) is a neurotropic virus with significant potential as a viral vector for central nervous system (CNS) gene therapy. This study provides visual evidence that recombinant green fluorescent protein (GFP)-expressing HSV-1 travel down dendrites in differentiated P19 neuronal-like cells to efficiently reach the soma. The virus also promotes cytoskeletal rearrangements which facilitate viral spread in vitro, including often dramatic increases in dendritic filopodia. Viral movements, cell infection and filopodia induction were each reduced with the actin polymerization inhibitor cytochalasin D, suggesting the involvement of the actin cortex in these processes. The observation of neural cytoskeletal reorganization in response to HSV-1 may shed light on the mechanisms by which acute viral infection associated with herpes encephalitis produces cognitive deficits in patients.