Biennial Report 2011–2012

Department of Eukaryote Genetic Engineering
HBoV1-4 antigens in yeast expression system has not yet been
reported. , In the current study, the capsid proteins VP1 and
VP2 of HBoV1were expressed in yeast S.cerevisiae. Electron
microscopy demonstrated that both purified recombinant proteins
self assembled into virus-like particles (VLPs) exhibiting
the typical icosahedral appearance of parvoviruses with a
diameter of approximately 20 nm. The yield and stability of
HBoV1 VP2 protein was significantly higher in comparison to
VP1, therefore VP2 was chosen for the developing of an immunoassay
and generation of monoclonal antibodies. HBoV1
VP2 VLPs were stable in yeast and were easily purified by caesium
chloride gradient ultracentrifugation. Therefore, yeast
expression system proved to be simple, efficient and cost-effective,
suitable for high-level production of HBoV1 VP2 as
VLPs. Four monoclonal antibodies (MAbs) of IgG1 subtype
were generated by immunization of mouse with recombinant
HBoV1 VP2 VLPs. Three of them specifically recognized
only HBoV1 VP2 protein; one MAb was cross-reactive with
HBoV2 and HBoV4 VP2 proteins. Recombinant HBoV1
VP2 VLPs and VP2-specific MAbs were employed to develop
serological assays to detect virus-specific IgG antibodies in human
serum specimens.
Human PARV4
Human parvovirus 4 (PARV4) is a recently discovered new
member of the Parvoviridea family not closely related to any
of the known human parvoviruses. PARV4 is widely distributed
in intravenous drug users, particularly in those co-infected
with human immunodeficiency virus and hepatitis C. PARV4
has been isolated from plasma of individuals with symptoms
of acute viral infection, however, till now PARV4 has not been
associated with any disease and its prevalence in human population
is not yet clearly established. In the current study, the
major capsid protein VP2 of PARV4 was generated in yeast
Sacharomyces cerevisiae and used for serological detection of virus-specific
IgG and IgM in the sera of low risk individuals.
One-hundred seventy serum specimens obtained from patients
with acute respiratory diseases were tested for PARV4-specific
IgG and IgM antibodies. Sixteen (9.4%) seropositive individuals
were diagnosed, including 10 IgG positive, 12 IgM positive
and 6 both IgG and IgM positive. Six of 16 sero-positive
A
B
Figure 2. Electron micrograph of CsCl purified VLPs. Scale bar 200nm.
A - HBoV1 VP2; B - HBoV1.
PhD student Kotryna Kvederavičiūtė and
bioengineer Justinas Šimulis
MAb
Optical density (OD450) values by an indirect ELISA
clone HBoV1 VP2 HBoV2 VP2 HBoV3 VP2 HBoV4 VP2 B19 BPV1
4C2 3.7260 0.0105 0.0320 0.0160 0.0055 0.0085
9G12 1.0770 0.0085 0.0040 0.0115 0.0025 0.0055
12C1 3.0281 0.0106 0.0076 0.0121 0.0051 0.0036
15C6 3.3491 3.4706 0.0166 3.0376 0.0071 0.0081
Table. The cross-reactivity of MAbs rose against yeast-expressed HBoV1 VP2 VLPs with recombinant VP2 proteins of related human parvoviruses.
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