Predicting the Evolutionary Variability of the Influenza A Virus.

The influenza A virus remains one of the most common and dangerous human health concerns due to its rapid evolutionary dynamics. Since the evolutionary changes of influenza A viruses can be traced in real time, the last decade has seen a surge in research on influenza A viruses due to an increase in experimental data (selection of escape mutants followed by examination of their phenotypic characteristics and generation of viruses with desired mutations using reverse genetics). Moreover, the advances in our understanding are also attributable to the development of new computational methods based on a phylogenetic analysis of influenza virus strains and mathematical (integro-differential equations, statistical methods, probability-theory-based methods) and simulation modeling. Continuously evolving highly pathogenic influenza A viruses are a serious health concern which necessitates a coupling of theoretical and experimental approaches to predict the evolutionary trends of the influenza A virus, with a focus on the H5 subtype.

Immunization with influenza A NP-expressing vaccinia virus recombinant protects mice against experimental infection with human and avian influenza viruses.

Two-fold immunization of Balb/c mice with a vaccinia virus recombinant expressing the NP protein of influenza A/PR8/34 (H1N1) virus under the control of a strong synthetic promoter induced specific antibodies and protected animals against low-dose challenge by mouse-adapted heterosubtypic variants of human A/Aichi2/68 (H3N2) and avian A/Mallard/Pennsylvania/10218/84 (H5N2) influenza virus strains. The surviving immunized animals had lower anti-hemagglutinin antibody titers compared to non-immunized mice. There was no difference in viral titers in lungs of immunized and non-immunized animals that succumbed to the infection. In order to try to increase immune system presentation of NP-protein-derived peptides, and thereby increase their immunogenicity, we constructed another vaccinia-based NP-expressing recombinant containing a rapid proteolysis signal covalently bound to the NP protein. This sequence, derived from the mouse ornithine decarboxylase gene has been shown to increase degradation of various proteins. However, we found that when used as part of a recombinant NP, this signal neither increased its proteolytic degradation, nor was it more efficient in the induction of a protective response against influenza infection.

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein.

BACKGROUND: Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol. RESULTS: The heterologous prime-boost vaccination protocol, using a VV recombinant and bacterial plasmid, both containing the NS1 TBE virus protein gene under the control of different promoters, achieved a high level of protection in mice against lethal challenge with a highly pathogenic TBE virus strain. No signs of pronounced TBE infection were detected in the surviving animals. CONCLUSION: Heterologous prime-boost vaccination protocols using recombinant VV and bacterial plasmids could be used for the development of flavivirus vaccines.

Vaccinia virus recombinant expressing gene of tick-borne encephalitis virus non-structural NS1 protein elicits protective activity in mice.

A vaccinia virus recombinant containing the non-structural tick-borne encephalitis virus (TBEV) NS1 gene was developed. The recombinant expressed native dimeric form of the NS1 protein in infected cells and protected mice against lethal infection with TBEV.

Genetic instability of vaccinia virus containing artificially duplicated genome regions.

A double recombinant of vaccinia virus (W-lacZ/J-tk/F) was obtained, which contains two inverted copies of the virus tk gene, separated by 45 kb: (i) the native copy located in the HindIII J fragment of the virus genome was inactivated due to insertion of E. coli lacZ gene; (ii) the second active copy was artificially inserted into the HindIII F fragment. The virus expressing both thymidine kinase and beta-galactosidase (tk+lac+ phenotype) was cloned. Due to the presence of duplicated inverted sequences of the tk gene in the virus genome extensive recombination was observed leading to genetic heterogeneity of the virus population. The population consisted mainly of the virions with the tk+lac- (77%) and tk+lac+ (23%) phenotypes. Passages in the presence of BUdR revealed minor fractions of the tk-lac+ and tk-lac- phenotypes. Structural analysis of DNA isolated from virions confirmed the genetic heterogeneity of the virus population. Nine different HindIII fragments were detected containing HindIII F, J and (or) lacZ sequences. The structure of these fragments indicates that predominantly two types of recombination events occur in the population: (i) translocation of the lacZ gene between duplicated sequences of the tk gene or displacement of lacZ by tk via intergenome and intragenome double crossing over; (ii) inversion of a 45 kb sequence in the conserved region of the genome between duplicated sequences of the tk gene due to a intragenome single crossing over.

Heterogeneity of hepatitis B virus surface antigen in sera of patients with acute hepatitis B infection in relative content of the antigenic determinants a and preS2.

Using five ELISA variants we have analyzed the ratios of the antigenic determinants 'a' (a-D), preS2 (preS2-D) and the polymerized human serum albumin binding domains (PHSA-BD) in the hepatitis B virus surface antigen (HBsAg) particles contained in sera of three patients with acute viral hepatitis B. Quantitative relations between the a-D, preS2-D and PHSA-BD were shown to differ in these sera. Population of the HBsAg particles in each of the sera appeared heterogeneous in respect to the PHSA-BD activity. The HBsAg particles with high affinity PHSA-BD prevailed in one serum. An additional class of particles with low affinity to PHSA or complete lack of the PHSA-BD was present in the remaining sera. The HBsAg particles carrying the high affinity PHSA-BD were characterized by an elevated (and almost constant for the three sera) surface density of the preS2-D. The results have led us to the conclusion that the existence of the preS2-D on the surface of the HBsAg particles is necessary but insufficient for the high affinity binding of the HBsAg to PHSA. A model for the interaction between the preS2-D carrying HBsAg particles and PHSA is presented.

Immunogenicity of recombinant vaccinia viruses expressing hepatitis B virus surface antigen in mice.

The ability of different recombinant vaccinia viruses (RVV) expressing hepatitis B virus surface antigen (HBsAg) to induce anti-HBs and anti-vaccinia virus responses has been analyzed in mice. The RVVs tested differed with regard to the original vaccinia virus strain used as vector and the site of insertion of a foreign gene. It was found that the immunological responses to RVVs based on the WR strain were higher than those to RVVs based on the Lister strain. The immunological response was also quantitatively affected by the viral TK phenotype. The presence of the preS2 region in HBsAg gene, inserted into the RVV genome, led to the induction of anti-preS2 antibodies but decreased the antibody response to the S-portion of HBsAg.

Viable double vaccinia virus recombinants with the non-inducible phage T7 expression system.

Double vaccinia virus recombinants expressing both the T7 RNA polymerase gene, controlled by a weak early poxvirus PF promoter, and the Escherichia coli beta-galactosidase gene, controlled by the phage T7 promoter, have been obtained. The viability of the double recombinants depended on the T7 RNA polymerase expression level. If the T7 RNA polymerase gene was inserted into a recombinant already containing the beta-galactosidase gene, the efficiency of formation of the double recombinants was significantly higher compared to that for the reverse insertion order. The negative effect of the phage T7 terminator on beta-galactosidase expression in cells infected with the recombinant viruses has been shown. The dynamics and levels of beta-galactosidase formation by different vaccinia virus recombinants have been studied.

Two vaccinia virus recombinants expressing HBsAg with different concentration of A- and pre-S2 antigenic determinants.

Comparative studies of two vaccinia virus (VV) recombinants expressing the hepatitis B virus (HBV) surface antigen (HBsAg) including the pre-S2 region (M-protein) showed that the L-pre-S2/15 recombinant expressed 5-fold more HBsAg as determined by the content of a-determinant than the recombinant v137. However, both recombinants expressed comparable amounts of the pre-S2 antigenic determinant as assessed by enzyme immunoassay with monoclonal antibodies. According to our calculations, one HBsAg unit expressed by the recombinant v137 contained 7-9 times more pre-S2 antigen than did one HBsAg unit expressed by the L-pre-S2/15 recombinant. Binding of pre-S2 region to polymerized human serum albumin was shown not to be an efficient assay at low pre-S2 concentration. HBsAg expressed by the v137 recombinant was less extensively secreted from cells as compared to that expressed by L-pre-S2/15 recombinant. Both recombinants induced the production of antibodies to the pre-S2 antigenic determinant in rabbits. L-pre-S2/15 induced anti-HBsAg a-determinant antibody as well.

Complementation of cell and serum growth factors during proliferation of tumour cells in semisolid medium.

Foetal bovine serum (FBS) and the preparation Ultraser (LKB, Sweden) contain more growth factors (GF) necessary to promote proliferation of cells in soft agar than the sera from newborn and adult animals. It was found that non-dialysable GFs are produced during growth of cells in semisolid medium. The dependence of colony-forming efficiency (CFE) on plating cell dose (PCD) was studied in 11 cell lines at varying concentrations of FBS. Cell proliferation depended on both serum and cell GFs. The minimum serum concentration at which CFA is not dependent on PCD can be selected for each cell line and, therefore, serum GFs completely compensate for the lack of cell GFs. Serum GFs within a certain range are, conversely, complemented by cell GFs: the minimum plating cell dose capable of compensating for the lack of serum GFs can be determined for each cell line.

Properties of the retrovirus RPMI8226V.

The retrovirus designated RPMI8226V (isolated from human myeloma cells RPMI8226) has been characterized with respect to its morphological, biochemical and immunological properties as well as to find the most permissive cells for its propagation. The RPMI8226V with its morphological, biochemical and immunological characteristics was not found to be related to any known avian and mammalian oncoviruses. The most permissive cells for its propagation were bat (CCL88) and mink (CCL64).

In vivo selection of two agents differing in hepatoma-inducing activity from strain MC29 avian leukosis virus.

MC29 virus induces acute leukemia (myelocytomatosis) and primary tumors of liver (hepatomas) in turkey poults. By in vivo passages two viruses were selected; designated "liver" and "bone marrow" variants, differing in hepatoma-inducing activity. The "liver" variant induces hepatoma and acute leukemia, the "bone marrow" variant induces acute leukemia. The variants differ in leukemogenic activity. The "bone marrow" variant induces high grade leukocytosis, while the "liver" variant causes lymphocytosis and heteropenia. The variants also induce the appearance of primitive myeloid cells in the blood. The kinetics of accumulation of viral gs protein (p27) and the presence of viruses inducing hepatoma and leukemia were studied in organs of infected turkeys. The differences between the variants showed no relationship with their selective reproduction capacity in liver and/or bone marrow cells. The results obtained suggest that different viral particles are responsible for the induction of leukemia and hepatoma.

Radioimmunoassay for major internal structural protein (p21) of the bovine leukemia virus; antibodies detection and viruses identification.

The major internal protein of bovine leukemia virus (BLV p24) was isolated using ion exchange chromatography on phosphocellulose and gel filtration. The specificity of the BLV p24 isolated was checked by both the radioimmunoprecipitation (RIP) and the competitive radioimmunoassay (RIA). No cross reactivity between BLV p25 and the mammalian viruses of type C and D and the retrovirus isolated from human myeloma cells RPMI8226 [8, 17] was detected. The analysis of 429 leukemia-suspected bovine blood sera resulted in the detection of 165 (38.5%) positive and 264 (61.5%) negative blood sera. A correlation of the results of radioimmunoprecipitation reaction of major internal protein p24 and immunodiffusion test on the glycoprotein antigen of BLV was observed.

A new virion precipitation test for oncovirus envelope antigens which detects common antigenic determinants in mammalian type-C viruses and Mason-Pfizer monkey virus.

A method for the study of oncovirus envelope antigens was developed, bases on the precipitation of intact virions by a double antibody technique. The amount of precipitated virus was then measured as reverse transcriptase activity. The method was designated the virion precipitation test (VPT). It has been used for titration of antibodies to envelope antigens of oncoviruses. The study of envelop antigens of 11 different oncoviruses permitted their differentiation into the following groups: (1) murine type-C viruses: (2) feline type-C viruses; (3) simian type-C viruses; (4) the RD-114/BEV group; (5) Mason-Pfizer monkey virus (M-PMV); (6) bovine leukemia virus; (7) avian type-C viruses; (8) mouse mammary tumor virus. No common antigenic determinants were detected in the last three groups. Mammalian type-C viruses (RD-114, NIH-MuLV, G-MuLV) had common antigenic determinants in the envelope, as demonstrated with an anti-RD-114 serum. Mammalian type-C viruses also shared antigenic determinants with M-PMV. The relationship of type-C viruses to M-PMV decreased in the following order: RD-114--NIH-MuLV--G-MuLV. It was also shown that the endogenous xenotropic feline RD-114 virus was more closely related to xenotropic NIH-MuLV than to ecotropic G-MuLV. The nature of the common antigenic determinants, as demonstrated by VPT on the surface of mammalian type-C viruses and M-PMV, and their significance for the concept of oncovirus evolution are discussed.

A rapid neutralization test for antibodies to bovine leukemia virus, with the use of rhabdovirus pseudotypes.

Two rhabdoviruses, vesicular stomatitis (type Indiana) and Chandipura viruses, formed pseudotype particles with envelope antigens provided by bovine leukemia virus (BLV). The pseudotypes are infectious for calf, human, mink, and rat cells, but the most sensitive indicator proved to be the Vero cells. Infectivity of the pseudotypes was increased by DEAE-dextran present during adsorption. Sera of spontaneously infected cattle contained high titers (some over 1/10,000) of antibodies neutralizing the pseudotypes, whereas sera of cattle from uninfected herds possessed no neutralizing activity in 1/10 dilution. The neutralization of these pseudotypes can serve as a rapid and sensitive test for the detection of antibodies in the cattle infected with BLV.

Radioimmunoassay of type D oncovirus from continuous J-96 cells.

Radioimmunoassay of J-96 virus and an extract of J-96 cells in the homologous and heterologous systems aimed at detecting antigenic determinants of p25 of Mason-Pfizer virus, as well as group-specific and interspecies antigenic determinants p30 of Rauscher leukaemia virus demonstrated that (1) J-96 virus contains a major internal protein immunologically identical with p25 protein of Mason-Pfizer virus based on the antigenic determinants detectable by the radioimmunoassay used; and (2) no interspecies antigenic determinants characteristic of the major internal protein of mammalian type C viruses were detectable in the J-96 virus or the J-96 cell extract.

Pseudotype particles of vesicular stomatitis virus with surface antigens of bovine leukaemia virus--VSV (BLV) -- as a sensitive probe for detecting antibodies in the sera of spontaneously infected cattle.

Phenotypically mixed particles containing the genome of vesicular stomatitis virus (VSV) and envelope antigen corresponding to bovine leukaemia virus (BLV) -- the VSV (BLV) pseudotypes -- can be employed as a rapid, specific and sensitive probe for detecting BLV-neutralizing antibodies in bovine sera.

Phenotypic mixing of vesicular stomatitis virus and D-type oncornavirus.

After mixed infection of cells with vesicular stomatitis virus (VSV, thermolabile mutant tl 17) and oncornavirus type D, phenotypically mixed virions are formed containing the VSV genome and oncornavirus and VSV envelope. The virions are thermostable and have serologic characteristics of both viruses.