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Biology of Japanese encephalitis virus |
| Principal Investigator : Sudhanshu Vrati
Project
Associates/Assistants Ph D Students Japanese
encephalitis virus (JEV) is a member of the Flaviviridae family of
animal viruses that contains several other medically important viruses such as
Dengue and Yellow fever. JEV is a major cause of human encephalitis and is
responsible for considerable mortality and morbidity in India. Frequent
epidemics of JEV are being reported from various parts of India and the virus
has become endemic in several parts of the country. Different aspects of JEV
biology are being studied in our laboratory, some of which are outlined below. A.
Development of a plasmid DNA-based vaccine for JEV The
objective of this work is to develop plasmid DNA-based immunization procedures
for vaccination against JEV. To this end, we intend to make recombinant
plasmids carrying various structural and non-structural protein genes of JEV
under the control of a strong eukaryotic promoter. These plasmid constructs
will be evaluated for their potential to generate protective immunity in
experimental animals against JEV. In
order to improve the efficacy of the plasmid DNA immunization against JEV,
methods need to be employed that would enhance neutralizing antibody titers.
In order to improve the uptake of the DNA in animal, we have adsorbed the
plasmid DNA on cationic microparticles and have compared its immunogenicity
with the naked plasmid DNA. We used plasmid pMEa for these studies. As seen in
the ELISA, the microparticle-adsorbed DNA induced higher titers of anti-JEV
antibodies when compared to those induced by the naked DNA. No difference,
however, was seen in JEV neutralization titers. The microparticle-adsorbed DNA
induced mixed Th1-Th2 kind of immune responses as opposed to Th1 type of
immune responses elicited by the naked DNA. B.
Development of a tissue culture-derived vaccine against JEV A
mouse brain derived vaccine using the Japanese Nakayama strain of JEV is
currently being produced in limited amounts at the Central Research Institute,
Kasauli. However, the cost of this vaccine precludes its large-scale usage for
mass vaccination as may be required for the effective control of the virus
activity in the field. In order to bring down the cost of vaccine production,
efforts are being made to grow the virus to high titers in tissue cultured
cells. The
virus grown in vero cells cultured on microcarriers in a spinner flask was
formalin-inactivated and its immunogenicity and protective efficacy in mice
were tested in comparison with the commercially available vaccine. Our studies
indicated that formalin-inactivated, vero cell-cultured JEV induced high
levels of protective immunity in mice. Virus inactivation with formalin at 22°C,
which required shorter incubation period, was found to be as good or better to
virus inactivation at 4°C for generating high titers of anti-JEV antibodies.
Similarly, the 22°C-inactivated virus generated JEV neutralizing antibody
titers as good or higher than those induced by the 4°C-inactivated virus.
Thus for the vaccine production, inactivation of JEV with formalin at 22°C
would be a preferred method as it is faster and does not require cold room
storage. C.
Interaction of cellular proteins with JEV RNA JEV
genome is a plus-sense single-stranded RNA of ~11 kilo bases. A minus-sense
RNA template is generated during virus replication, which is then copied to
produce a large number of plus-sense genomic RNA molecules. Based on the amino
acid sequence homologies with other replicases, a couple of viral proteins
have been speculated to be involved in replication of the JEV genome. However,
we do not know if any of the cellular proteins also are needed for viral
replication. We are, therefore, studying cellular proteins that interact with
JEV genome sequences, which are likely to be involved in viral replication. One
of the proteins interacting with the 3'-NCR of JEV was identified to be the La
protein of ~50 kDa. Out of the 408 amino acids of the protein, the N-terminal
segment of 208 amino acids was found to bind the the 3'-NCR of JEV. D.
Development of an oral JEV vaccine Human
adenoviruses cause mild, benign infections and in recent years these viruses
are increasingly being used for human gene therapy and receombinant vaccine
development. From the vaccine point of view, the added advantage that
adenoviruses present is that they can be delivered orally. We are developing
adenovirus recombinats that synthesise JEV E protein as potential vaccine
candidate for immunization against Japanese encephalitis. We will also be
developing transgenic plants that synthesize JEV E protein and study their
potential in animals for oral immunization against JEV. Replication-defective
recombinant adenoviruses (RAds) were constructed that synthesized the
pre-membrane and envelope (E) proteins of Japanese encephalitis virus.
Recombinant virus RAdEa synthesized Ea, the membrane-anchored E protein, and
RAdEs synthesized Es, the secretory E protein. Compared with RAdEs, RAdEa
replicated poorly in HEK 293A cells and synthesized lower amounts of E
protein. Oral immunization of mice with RAds generated low titers of anti-JEV
antibodies that had only little JEV neutralizing activity. Intra-muscular (IM)
immunization of mice with both RAds generated high titers of anti-JEV
antibodies. Interestingly, RAdEa induced only low titers of JEV neutralizing
antibodies, however, these were significantly higher in case of RAdEs
immunization. Splenocytes from mice immunized IM with RAds secreted large
amounts of interferon-g
and moderate amounts of interleukin-5 in presence of JEV and showed cytotoxic
activity against JEV-infected cells. Mice immunized IM with RAdEs showed
complete protection against lethal dose of JEV given intra-cerebral. Transgenic
tobacco plants have been developed that contain the cDNA encoding JEV E
protein in its full-length membrane-anchored or the truncated secretory forms
under the control of the CaMV promoter. The tobacco plants are being used as
model system to test the integrity of our gene constructs and their ability to
express protein in plants. Subsequently, we plan to develop transgenic plants
that could be consumed uncooked. E.
Development of DNA molecules to inhibit JEV replication JEV
is a RNA virus which actively replicates in the host brain leading to the
development of encephalitis. There is no specific treatment available to deal
with JEV infection as no drugs are known that specifically inhibit JEV
replication. We will attempt to design molecules that could be used for
inhibiting JEV replication. DNAzymes
are short oligodeoxynucleotides that have the capability to cleave RNA
molecules at defined site in a sequence-specific manner. The 3'- and the
5'-NCRs of JEV genome play crucial role in JEV replication. We have designed
DNAzymes that can specifically cleave JEV RNA within the 3’- and the 5'-NCR.
These oligonucleotides were found to inhibit JEV replication in cultured
macrophage cells.
Publications
Original
peer-reviewed articles 1. Kaur R, Rauthan M and Vrati S (2004) Immunogenicity in mice of a cationic microparticle-adsorbed plasmid DNA encoding Japanese encephalitis virus envelope protein. Vaccine (in press). 2. Appaiahgari MB and Vrati S (2004) Immunogenicity and protective efficacy in mice of a formaldehyde-inactivated Indian strain of Japanese encephalitis virus grown in vero cells. Vaccine (in press). 3. Viswanathan P, Venkaiah B, Kumar MS, Rasheedi S, Vrati S, Bashyam MD and Hasnain SE (2003) The homologous region sequence (hr1) of Autographa californica multinucleocapsid polyhedrosis virus can enhance transcription from non-baculoviral promoters in mammalian cells. J Biol Chem 278:52564-52571. 4. *Rath A., Batra D, Kaur R, Vrati S and Gupta SK (2003) Characterization of immune response in mice to plasmid DNA encoding dog zona pellucida glycoprotein-3. Vaccine 21:1913-1923. (*in press last year, since published)
Reviews/Proceedings 1. Kaur R and Vrati S (2003) Development of recombinant vaccine against Japanese encephalitis. J Neurovirol 9:421-431.
Patents 1. Vrati S and Appaiahgari A (2003) Vaccine for Japanese encephalitis and its process thereof. Indian patent application # 1130/DDEL/2003. 2. Vrati S (2003) Recombinant adenovirus-based Japanese encephalitis virus vaccine. Indian patent application # 1516/DEL/2003. |