Vladimíra Ďurmanová

Multiorgan distribution of human influenza A virus strains observed in a mouse model.

Multiorgan spread and pathogenesis of influenza infection with three human influenza A viruses was studied in mice. Mouse-adapted viruses A/Dunedin/4/73(H3N2), A/Mississippi/1/85(H3N2), and A/PR/8/34(H1N1) differed considerably in virulence (p.f.u./LD50): 79,000 p.f.u. for Dunedin, 5,000 p.f.u. for Mississippi, and 65 p.f.u. for PR/8, which qualified Dunedin as low virulent, Mississippi as intermediate, and PR/8 as highly virulent. All three viruses were detected in lungs, heart, and thymus by cultivation and RT-PCR. Moreover, vRNA of all viruses was found in liver and spleen, of Dunedin and PR/8 also in kidneys and that of Dunedin and Mississippi in blood. Only vRNA of Dunedin was demonstrated in brain. Lung damage accompanied by histopathological changes and thymus reduction were most extensive after infection with the highly virulent virus PR/8. We assume that the ability to spread to multiple organs may be a more common property of influenza viruses in mammalian hosts than previously believed.

Early expression of herpes simplex virus (HSV) proteins and reactivation of latent infection

During the last decade, new data accumulated describing the early events during herpes simplex virus 1 (HSV-1) replication occurring before capsid formation and virion envelopment. The HSV virion carries its own specific transcription initiation factor (α-TIF), which functions together with other components of the cellular transcriptase complex to mediate virus-specific immediate early (IE) transcription. The virus-coded IE proteins are the transactivator and regulatory elements modulating early transcription and subsequent translation of nonstructural virus-coded proteins needed mainly for viral DNA synthesis and for the supply of corresponding nucleoside components. They also cooperate at the late transcription and translation of the viron (capsid, tegument and envelope) proteins. In addition, the transactivator, IE proteins down-regulate their own transcription, while others facilitate viral mRNA processing or interfere with the presentation of newly synthesized virus antigens. Establishment of latency is closely related to the transcription of a separate category of transcripts, termed latency-associated (LAT). Formation of LATs occurs mainly in nondividing neurons which are metabolically less active and express lower levels of cellular transcription factors (nonpermissive cells). Expression of the stable non-spliced (2 kb), and especially of stable spliced (1.5 and 1.45 kb) LATs is a prerequisite for HSV reactivation. Different HSV genomes (from various HSV strains) do not undergo IE transcription at the same rate. Restricted IE transcription and the absence of viral DNA synthesis favors LAT formation and persistence of the silenced genome. Uneven levels of LAT expression and differences in the metabolic state of carrier neurons infleunce the reactivation competence. Under artificial or natural activation conditions, sufficient amounts of IE transactivator proteins and proteins promoting nucleoside metabolism are synthesized even in the absence of the viral α-TIF facilitating reactivation.

Transcription at Early Stages of Herpes Simplex Virus 1 Infection and during Reactivation

Objective: The kinetics of immediate early (IE) and early (E) herpes simplex virus 1 (HSV-1) mRNA transcription was followed in explanted trigeminal ganglia from rabbits with established latency. Methods: The expression of IE and E mRNAs was first assessed in infected Vero cells by RT-PCR and then in explanted trigeminal ganglia by nested RT-PCR. Results: In infected Vero cells, IE mRNAs [for infected cell protein (ICP) 0, ICP4 and ICP27] were first detected 1–2 h post-inoculation (p.i.), peaking at 3 h p.i. The transcription of E mRNAs [for thymidine kinase (TK), RR1 and UL9], which were first detected from 3 h p.i., peaked between 5 and 10 h p.i. In explanted ganglia, the ICP0, ICP4 and ICP27 mRNAs were first detected after 4 h in culture. This was followed by the appearance of TK mRNA at 8 h and then by the UL9 mRNA, detected from 12 h post-explantation. A further E mRNA (RR1), as well as the late gC mRNA, were first observed after 24 h in culture. Moreover, ICP4 mRNA could be found in non-cultured ganglia. Conclusions: During reactivation of latent HSV-1 in explanted ganglia, the onset of ICP0 and ICP27 transcription at 4 h in culture was followed by TK transcription (at 8 h). Thus, in the rabbit reactivation model, ICP0 gene transcription rather than ICP4 transcription represents the relevant indicator of latency reactivation.

Developments in herpes simplex virus vaccines: Old problems and new challenges

Vaccination has remained the best method for preventing virus spread. The herpes simplex virus (HSV) candidate vaccines tested till now were mostly purified subunit vaccines and/or recombinant envelope glycoproteins (such as gB and gD). In many experiments performed in mice, guinea pigs and rabbits, clear-cut protection against acute virus challenge was demonstrated along with the reduction of the extent of latency, when established in the immunized host. The immunotherapeutic effect of herpes vaccines seems less convincing. However, introduction of new adjuvants, which shift the cytokine production of helper T-cells toward stimulation of cytotoxic T-cells (TH1 type cytokine response), reveals a promising development. Mathematical analysis proved that overall prophylactic vaccination of seronegative women, even when eliciting 40–60 % antibody response only, would reduce the frequency of genital herpes within the vaccinated population. Even when partially effective, immunotherapeutic vaccination might represent a suitable alternative of chronic chemotherapy in recurrent labial and genital herpes.

Characterization of MICA gene polymorphism of HLA complex in the Slovak population

Background: The function of the MHC class I polypeptide-related sequence A (MICA) gene, which belongs to the MHC class I chain-related genes, is to trigger cytolysis of target cells mediated by NKG2D receptor recognition in NK (Natural Killer) cells and CD8 T-lymphocytes. The MICA gene has a high degree of polymorphism, especially observed in exons 2–5. MICA allelic diversity has been reported in association with some autoimmune diseases such Behcets disease, psoriasis and diabetes, as well as with organ rejections. Aim: The aim of this study was to analyse MICA gene polymorphism in the Slovak population, to establish frequencies of MICA alleles and to compare the results with those found in other Western Eurasian populations. No such study has been performed previously in the Slovak population. Subjects and methods: This study examined DNA samples from 124 unrelated Slovak individuals (51 women and 73 men with an average age of 40.3 years) using direct sequencing of MICA exons 2–5. Allele and genotype frequencies were calculated by direct counting and statistical analysis was carried out using Arlequin software. Results: This study identified 15 out of 71 MICA alleles. The most frequent allele was MICA*008 (37.1%) followed by alleles MICA*002 (16.5%) and MICA*009 (11.3%). The rarest alleles were MICA*027, MICA*006 (both 0.8%) and MICA*057 (0.4%), respectively. The most frequent genotypes were 008/008 and 008/002, both with a frequency of 13.7%. Exon 5 microsatellite polymorphism screening revealed five MICA alleles, namely A4, A5, A5.1, A6 and A9. The most frequent was allele A5.1 (37.1%) and the rarest A5 (8.1%). Finally it was found that haplotype MICA*008 A5.1 was the most frequent (37.1%). Conclusion: A comparison of these results with those reported in the literature revealed similarity in MICA polymorphism to that found in other Western Eurasian populations. The data will be useful for further association studies on MICA polymorphism and its function.

Epstein-barr virus (HHV-4) inoculation to rabbits by intranasal and oral routes results in subacute and/or persistent infection dissimilar to human disease

Objective: We report the infection of New Zealand white rabbits with Epstein-Barr virus (EBV). Methods: EBV prepared in B95-8 (producer) cells was inoculated to rabbits by combined intranasal and oral routes. Blood and white blood cell (WBC) samples were taken before infection, then on days 8, 28 and 98 post-infection (p.i.). Results: Administration of either 3 × 108 (group A, 11 rabbits) or 1 × 109 (group B, 10 rabbits) EBV DNA copies per animal induced subacute and/or persistent infection. The IgG antibodies in plasma were detected by ELISA as well as by immunoblot (IB). The IB bands showed mainly antibodies to the BZRF1/Zta transactivation polypeptide (69.2%), the p54 early protein (53.4%) and to the p23 capsid protein (35.8%). No anti-EBNA1 antibody was detected throughout. Viral DNA could be detected by PCR in WBCs and/or spleen of 7 out of 21 infected rabbits (30%), while 60-80% of them showed serologic response. The transiently present EBV DNA was accompanied by LMP1 antigen. Conclusions: Rabbits developed persistent EBV infection in the absence of EBNA1 antibodies and by the lack of typical infectious mononucleosis-like syndrome. The absence of EBNA1 antibody may reflect the lack of EBNA1 in B cells of EBV-inoculated rabbits.

Donor non-specific MICA antibodies in renal transplant recipients

Despite recent advances in solid organ transplantations, an antibody mediated rejection caused by donor specific antibodies is still a major problem in kidney graft survival. Besides HLA-induced humoral response, antibodies against MICA antigens have recently attracted attention because of their possible role in graft rejection. The aim of our study was to establish whether renal recipients produce antibodies against MICA molecules due to the transplantation and if they are specific for MICA antigens of the donors. MICA antibody screening was performed in 124 kidney recipient sera. 22 sera, that were found to be MICA antibody positive, were further examined for MICA antibody profiles and compared with donor MICA alleles. The analysis of MICA antibody positive sera showed mostly more complex reactivity patterns. A significant fraction of patient sera (59%) reacted not only with the donor MICA antigens, but also with other MICA patterns. A match between antibody specificities and MICA antigens was observed in 41% of renal recipients only. On the other hand, as much as in 36% of recipient sera were detected antibodies against their own MICA molecules. We did not prove a complete correlation between the recipient MICA antibody specificities and MICA antigens of the donor. We assume that MICA antibody induction occurs not only due to the allogeneic stimulation itself but also due to other factors that need to be elucidated.

A simple procedure for expression and purification of selected non-structural (α and β) herpes simplex virus 1 (HSV-1) proteins

Abstract The expression and isolation of herpes simplex virus 1 (HSV-1) immediate early (α) IE63 (ICP27) and of the early (β) thymidine kinase (Tk) polypeptides in Escherichia coli JM 109 cells transformed with the PinPoint Xa-1 (Promega) plasmid construct carrying either the HSV-1 UL54 or UL23 genes are described. The resulting biotinylated fusion protein(s) could be easily induced and were purified in appropriate amounts by means of a monomeric avidin-conjugated resin ( SoftLink™ Soft Release Avidin Resin , Promega) provided that: (1) the exponential growth of the selected transformed cells was monitored carefully; (2) the post-induction harvest interval was properly chosen; and (3) the period for adsorption to the avidin resin suitably adjusted. The isolated protein(s), although partially digested in the case of the IE63 polypeptide, were suitable antigen(s) for immunization of various animal species. Co-purification of trace amounts of endogenous biotinylated protein(s) produced in E. coli was eliminated by shortening the duration of adsorption to the avidin resin.

Immune response and cytokine production following immunization with experimental herpes simplex virus 1 (HSV-1) vaccines

Balb/c mice were immunized with the recombinant fusion protein gD1/313 (FpgD1/313 representing the ectodomain of HSV-1 gD), with the non-pathogenic ANGpath gE-del virus, with the plasmid pcDNA3.1-gD expressing full-length gD1 and with the recombinant immediate early (IE) HSV-1 protein ICP27. Specific antibodies against these antigens (as detected by ELISA) reached high titers with the exception of the DNA vaccine. High-grade protection against challenge with the virulent strain SC16 was found following immunization with the pcDNA3.1-gD plasmid and with the gE-del virus. Medium grade, but satisfactory protection developed after immunization with the FpgD1/313 and minimum grade protection was seen upon immunization with the IE/ICP27 polypeptide. A considerable response of peripheral blood cells (PBL) and splenocytes in the lymphocyte transformation test (LTT) was found in mice immunized with FpgD1/313, with the pcDNA3.1-gD plasmid and with the live ANGpathgE-del virus. For lymphocyte stimulation in vitro, the FpgD1/313 antigen was less effective than the purified gD1/313 polypeptide (cleaved off from the fusion protein); both proteins elicited higher proliferation at the 5 μg per 0.1 mL dose than at the 1 μg per 0.1 mL dose. The secretion of Th type 1 (TNF, IFN-γ and IL-2) and Th type 2 (IL-4 and IL-6) cytokines was tested in the medium fluid of purified PBL and splenocyte cultures; their absolute values were expressed in relative indexes. The PBL from FpgD1/313 immunized mice showed increased secretion of both TH1 (TNF) as well as TH2 (IL-4) cytokines (7–10-fold, respectively). Splenocytes from FpgD1/313 immunized mice showed a significant (23-fold) increase in IL-4 production.

Herpes Simplex Virus 1 and 2 Vaccine Design: What can we Learn from the Past?

This chapter is devoted to the topics of not yet marketed HSV vaccine, which is still in the focus of interest, especially from the point of immunotherapeutic use. To under‐ stand the principles of vaccination strategies (prophylactic and/or immunotherapeu‐ tic), the pathogenesis of herpes simplex virus 1 (HSV-1) and/or HSV-2 infections in animal models is briefly outlined. Even when both herpesviruses may spread via bloodstream, which is especially true in the immunocompromised host, the main route of their transmission is along peripheral nerves. Both viruses establish latency in ganglion cells, and after reactivation, they spread along axons back to the site of primary infection. Since neither the establishment of latency nor its reactivation can be fully controlled by virus-neutralizing antibodies, the outcome of immune response greatly depends on the activity of cytotoxic CD8+ T lymphocytes. The majority of important antigenic epitopes is located in envelope glycoproteins (such as gB, gD, gE, gC and gG) that are related to virus adsorption and penetration into susceptible cells. The HSV-1 and/or HSV-2 experimental vaccines designed so far were either purified virion products derived from infected cells (subunit vaccines), purified recombinant immunogenic herpes simplex virus HSV-coded proteins (especially gD), and/or attenuated live viruses lacking some of virulence tools (such as gH and/or gE). We bring a comprehensive overview of the efficacy of experimental HSV-1/HSV-2 vaccines and discuss our own data. In conclusion, we believe in the continued demand of HSV-1 and HSV-2 vaccines, at least for their immunotherapeutic use, suggesting unified evalua‐ tion criteria for clinical trials to reach consent at their interpretation.