Svetlana Kozireva. Anti-sense polynucleotides as inhibitors of

of Svetlana Kozireva's Doctoral work "Anti-sense polyoucleotides as inhibitors of
bovine leukemia virus replication" submitted to the 2 Promotion and Habilitation
Council of the University of Latvia (biological sciences).
The present work was carried out in the Laboratory of Oncovirology of the August
Kirchenstein Institute of Microbiology and Virology from 1990 to 1997 and is
presented as a series of publications. The aim of the present study was the investigation
of the antiviral effects of anti-sense (as) polynucleotides on bovine leukemia virus
replication in vitro and in vivo. In accordance with the aim, the main objectives were:
1) to optimize the model of experimental BLV infection in rabbits;
2) to study antiviral activity of asRNA genes targeted at LTR R-U5 and pX regions of
the viral genome in cells persistently infected with BLV;
3) to investigate antiviral activity of asRNA gene selected in vitro in transgenic rabbits
challenged by BLV.
BLV infection in rabbits was studied in animals inoculated with: FLK culture
supernatant, FLK culture cells and the native leukocytes of BLV-infected cow. The
earliest production of the antibodies to the main BLV proteins (gp51 and p24), as well
as the highest level of the antibodies, were detected in sera of rabbits inoculated with
native leukocyte of BLV-infected cow. The changes of haematological indices in these
rabbits were also considerably stronger than in animals inoculated with the other types
of BLV-containing material. Thus, the native leukocytes appear to be the optimal
virus-containing material for creation of an experimental model of BLV infection in
The antiviral effect of asRNA genes targeted at BLV LTR R-U5 and pX regions on
BLV replication in vitro was investigated in a clone of FLK culture (D12) persistently
infected with BLV. In all clones carrying the asRNA genes, downregulation of BLV
replication was observed. Expression of as-sequences in the persistently infected cells
resulted in lower viral RNA levels and significant reductions of reverse transcriptase
activity, concentration of BLV p24 and amounts of viral particles. The degree of the
inhibition was strongly dependent on the target site selected in the virus genome. More
efficient inhibition of BLV replication was observed in cells expressing as-sequence
complementary to LTR R-U5 region of BLV. The expression of the as-sequence
targeted at BLV pX region also led to reduction of BLV replication, although the
degree of the inhibition was less. There was evidence of reduced tumorigenicity of
cells carrying the asRNA gene targeted at BLV pX region.
The antiviral activity of the asRNA gene complementary to BLV LTR R-U5 region in
vivo was studied in transgenic rabbits carrying the as-sequence in their genome which
were challenged by BLV. In non-transgenic rabbits infected with native leukocytes of
BLV-infected cow, typical retroviral infection developed, accompanied by the
appearance of antibodies to BLV gp51 and high titres of antibodies to BLV p24, as
well as by an increase in the total amount of leukocytes and changes in the proportion
of T-- and B-cell subpopulation of lymphocytes (increase in relative and absolute
amounts of B-lymphocytes and decrease in relative and absolute amounts of
T-lymphocytes) Two control infected rabbits died with clinical symptoms of
pneumonia (the percentage of lymphocytes in these rabbits was
significantly reduced). The BLV-infected transgenic rabbits also displayed some
typical haematological changes (increase in the total number of leukocytes and
percentage of lymphocytes) in the early stages of BLV infection. However, from 910
months post-inoculation, these haematological indices in the transgenic animals were
in the ranges of control age limits. For transgenic rabbits, only low levels of antibodies
to BLV p24 were observed, and in first generation of transgenic infected animals, the
antibodies to the protein had almost disappeared by 7-9 months post-infection. This
observation indicate that synthesis of p24 in transgenic rabbits was strongly inhibited.
The relative and absolute amounts of T- and B-lymphocytes in transgenic rabbits were
shown to be near to the norm and no mortal cases were registered in these animals. All
the data indicate that the antiBLV asRNA gene confers enhanced resistance to BLV
infection in transgenic rabbits compared to nontransgenic animals. Expression of
as-transgene was detected during 30 months postinfection.
The actuality of the present work is defined by the need for development and
investigation of new effective methods for prophylaxis and therapy of viral infections,
since the high percentages of human and animal diseases in Latvia and in other
countries have viral etiology. Although great strides have been made in the irradication
of viral illness, the control of the infections which are associated not only with virus
itself but also with cellular gene activation, such as ones caused by retroviruses of
HTLV-BLV group, remain problematic.
The results of the study indicate that:
1) BLV replication in persistently infected cells is inhibited by as-nucleic acids
targeted at the LTR R-U5 and pX regions of the viral genome;
2) the efficacy of the antiviral activity of as-nucleic acids strongly depends on the
target site in the viral genome;
3) the BLV LTR R-U5 region is the most promising target for asRNA-mediated
inhibition of the viral replication;
4) the asRNA gene targeted at BLV pX region lowers the tumorigenicity of cells
persistently infected with BLV;
5) the asRNA gene targeted at BLV LTR R-U5 region has high antiviral activity in
vitro and is able to confer enhanced resistance to BLV infection in vivo;
6) the efficacy of inhibition of BLV replication is directly associated with the
persistence period and the level of expression of asRNA in transgenic rabbits.
The results presented in the work not only confirm that as-polynucleotides are
promising and perspective agents to control retrovirus infections such as proliferative
disorder caused by BLV, but also create a basis for future selection and investigation
of these antiviral agents.
The results of this work are presented in the 4 papers and have been reported at 5
international conferences, 1 international seminar and at local seminars.