ANNUAL REPORT - Department of Biotechnology

Delhi. The protocol for 4-colour whole blood flow
cytometry has been standardized. Forty HIV infected
Indian patients and 15 normal individuals for T cell
activation and coreceptor expression were analysed
using the protocol. Indian HIV patients were found to
have a marked decrease in percentage of activated
CD4 T (CD4+HLA-DR+) cells expressing CCR5. The
expression of CCR5 on CD14+ monocytes or CD8+
T cells is not down regulated. HIV infection induced T
cell activation in Indian HIV patients alters both the
percentage and expression of CCR5 on activated
CD4 T cells. The protocol for IFN-γ Elispot has been
standardized and used to test immodominant
epitopes in 4 HIV infected patients. Attempts were
made to isolate HIV-1 by co-culture methods from an
Indian patient with a skin cancer and seropositive
positive for HIV on routine testing as it was unique for
two reasons: (a) it would be the earliest case from
India, and (b) it showed long-term non-progression.
These observations suggest that the virus may be a
CXCR4 utilizing virus. This itself would be a unique
observation as all Indian isolates have so far been
reported to be CCR5-tropic.
SARS
Towards studies on functional analysis of SARS
virus proteins for understanding pathogenesis at
ICGEB, New Delhi, polyclonal antibodies to the X1
protein have been generated and tested. Protease
protection study suggested that the topology of X1 is
such that its N-terminus is towards the luman of the
vesicle and C-terminus is towards the cytoplasm,
thus exposing its cytoplasmic domain to trypsin. It
was also found that X1 and Caveolin are present in
the same biochemical fractions, supporting the case
for their interaction.
Studies on cloning and characterization of the SARS
virus structural proteins and their biomolecular
interactions at ICGEB, New Delhi indicated that s
phase inhibitory activity of the N protein may have
major significance during viral pathogenesis. Also,
the 3a accessory protein of SARS-CoV is an RNA
binding protein and specifically interacts with the 5'
UTR of its viral RNA using a 92 amino-acid
DBT Annual Report 2006-07
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interaction domain. It has been found that ORF6 may
play a role in virus replication as observed in
interaction between SARS-CoV accessory protein
and nsp8.
Work on reagents for Immunological detection of
SARS associated Coronavirus (SARS-CoV) at
DUSC, New Delhi led to development of a sandwitch
ELISA using high affinity anti N MAb to capture intact
N and purified anti-N polyclonal antibody to reveal
captured N-protein. This assay detected 10 pg of Nprotein.
The same will be evaluated for detecting Nprotein
in SARS-CoV infected persons. At AIIMS,
New Delhi a real time RT-PCR has been developed
for SARS virus.
HEV
Studies were carried on development of nonradioactive
antigen specific reporter release
cytotoxicity assay and analysis of cytotoxicity against
HEV proteins at AIIMS, New Delhi. Bicistronic
eukaryotic HBV core/HEV reporter expression
vectors were generated to develop and validate the
non-radioactive antigen specific CTL assay using
HBVcore as target antigen were checked in HepG2
cell line for the expression of both antigen and
reporter proteins. The expression of the reporter
proteins were transiently transfected in non-adherent
mouse A20 cell line but transfection efficiency was
found to be low. Bicistronic HBVcore/HEV (RdRp,
ORF2, and ORF3) reporter pLXSN retroviral
constructs were made and tested for expression in
HepG2 cell line. These vectors were transfected into
retroviral packaging cell line to generate viral
particles and were able to infect human HepG2 and
mouse NIH3T3 cell lines. Neomycin selectable
HBVcore reporter retroviral vector containing
packaging cell lines were generated to produce
retroviral particles.
HCV
Studies on inhibition of HCV RNA translation and
replication using small RNAs jointly at IISc.,
Bangalore and DUSC, New Delhi demonstrated the