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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 flavivridae 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. Molecular determinants of JEV virulence ThisThis
project aims at identifying the genetic determinants of JEV virulence by
studying the genetic and biological phenotypes of naturally occurring isolates
of the virus that show marked change in their mouse virulence compared to the
virulent prototype JaOArS982 strain. An Indian isolate of
JEV, which shows significantly altered biological properties in
tissue-cultured cells and mice is being studied at the genetic level. We wish
to construct an infectious clone of JEV that would allow us to rescue defined
mutations in viral proteins believed to be important for virulence and
pathogenesis. We have divided the complete genome of JEV into four parts,
which are to be cloned independently. Subsequently these will be assembled
into a single clone encoding the complete genome of JEV. This sequence would
then be placed under the bacteriophage T7 promoter so that RNA representing
the JEV genome could be transcribed from it. We have cloned one of the genome
segments so far. Cloning of the second segment is progressing. B. Development
of naked DNA based vaccine for JEV The objective of
this work is to develop naked 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. Plasmid DNAs capable
of synthesizing the membrane-anchored or the secretory form of JEV envelope
protein together with the pre-membrane protein, were delivered to mice by
intra-muscular injection or using a gene gun. Immunogenecity and the
protective efficacy of these plasmids were compared with a formaline-inactivated,
commercial JEV vaccine. Intra-muscular plasmid immunization induced anti-E
antibody response similar to the vaccine. The gene gun, however, induced lower
antibody response. The two forms of the E protein induced similar antibody
titers by a given DNA delivery mode. Both plasmids generated high titers of
JEV neutralizing antibodies although these titers were lower than those
induced by the vaccine. Of the plasmid immunizations, gene gun delivery of
plasmid expressing the anchored E protein consistently gave higher
neutralization activity. Spleens from mice immunized with the plasmids
contained JEV-specific memory cytotoxic T lymphocytes that were not seen in
the vaccine-immunized mice. Intra-muscular plasmid inoculation generated
higher cytotoxic activity than the gene gun based DNA delivery. Intra-muscular
DNA immunization resulted in a Th-1 type of immune response while the gene gun
immunizations induced Th-2 type responses based on the isotype of the anti-JEV
antibodies and the profile of the cytokines secreted by the spleen cells from
the immunized mice. Vaccine inoculation induced a mixed Th-1, Th-2 response.
Interestingly, compared to the secretory E, the anchored protein heavily
skewed the immune response to a Th-1 or Th-2 type depending on the route of
immunization. In an intra-cerebral JEV challenge model, mice immunized with
plasmid DNAs showed about 60% protection compared to over 90% protection
afforded by the formaline-inactivated JEV vaccine. Form of the E protein or
the route of DNA delivery did not affect the level of protection. C. Development
of a tissue culture derived vaccine against JEV The cost of the presently available JEV vaccine
precludes its large-scale usage for mass vaccination as may be required for
the effective control of the virus activity in 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. Several methods for purifying JEV from
the tissue culture supernatant were tested. Mice immunizations have been
carried out with tissue culture grown, formaline-inactivated JEV to test the
potential of this virus for vaccine preparation. D. Interaction of cellular proteins
with JEV RNA JEV genome is a plus-sense single-stranded RNA
of ~11 kb. A minus-sense RNA template is generated during virus replication,
which is then copied to produce lots 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 are also
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. Screening of the mouse brain cDNA library with JEV RNA
probe has identified a couple of more proteins that have potential to bind
with JEV RNA. An 80-kDa protein has been identified which is a transmembrane
transporter involved in neurotransmission. We are attempting to get the
full-length clone of the protein so that it could be synthesized in large
amounts for further characterization. E. Use of a self assembling plant virus protein
for presenting antigenic peptides from JEV Peptide vaccines have a number of potential
advantages in terms of production, safety and stability. Studies have shown
that presentation of peptides in a highly ordered aggregate form can result in
enhanced immune response. We are, therefore, investigating the use of a
self-polymerizing coat protein from Johnson grass mosaic virus (JGMV) for
presenting antigenic peptides from JEV. JGMV is an RNA virus whose particles
are 11 nm thick long flexous rods of about 800 nm. A 33 kDa protein of JGMV is
the only protein found in the virion coat. cDNA coding for the coat protein
was amplified and cloned under the control of the T7 promoter for its
overexpression in E.coli. The protein expressed in large amounts and was shown
to self assemble in long rod shaped structures. Vectors have been made for
expressing the JEV peptides fused to the coat protein. Publications Original peer-reviewed articles 1.
*Ta M and Vrati S (2000) Mov34 protein from mouse brain interacts with
the 3’-non-coding-region of Japanese encephalitis virus. J Virol
74:5108-5115 (*in press last year, since published). 2.
Vrati S (2000) Comparison of the genome sequences and the phylogenetic
analyses of the GP78 and the Vellore P20778 isolates of Japanese encephalitis
virus from India. J Biosci 25:257-262. |