SEROTYPE-SPECIFIC AND HAEMADSORPTION PROTEIN OF THE AFRICAN SWINE FEVER VIRUS

This review presents comparative results of simultaneously conducted studies on proteins responsible for the haemadsorption and serotype-specific properties of African swine fever virus (ASFV). An ASFV gene EP402R (or LMW8-DR) encoding protein CD2v homologous to murine, human or porcine T-cell adhesive receptor was found. The CD2v was shown to be directly involved into a haemadsorption process, and expressed in ASFV-infected cells as a glycoprotein with a molecular weight of approximately 105-110 kDa. In the presence of a glycosylation inhibitor, tunicamycin, its molecular weight is about 42 kDa. In ASFV-infected cells labeled with 3H-glucosamine or 14C-sodium acetate, a virus-specific major glycoprotein with a molecular weight of 110-140 kDa (gp 110-140) was identified using radioimmunoprecipitation assay. Using ASFV reference strains belonging to seroimmunotypes I-IV and the corresponding antisera active in haemadsorption inhibition assay (HADIA), we determined that gp 110-140 defines the serotype specificity. Genotyping on the basis of the genetic locus encoding the CD2v and a C-type lectin protein also showed a concurrence with the grouping of ASFV isolates and strains based on their seroimmunotypes. Immunization of pigs with the gp 110-140 within liposomes, or a recombinant haemagglutinin (CD2v) protected 67 to 100% of animals from death due to their subsequent infection with homologous virulent ASFV strains. Based on the physico-chemical and biological characteristics of the gp 110-140 and CD2v it is suggested that they are one and the same virus-specific glycoprotein crucial for induction of the immunological protection against ASF. Key words: ASFV; seroimmunotypes; serotype; glycoproteins; gp 110-140; CD2v; protectivity   SEROTIPNO-SPECIFIČEN IN HEMADSORPCIJSKI PROTEIN VIRUSA AFRIŠKE PRAŠIČJE KUGE A.D Povzetek: Pregledni članek predstavlja primerjavo rezultatov sočasno izvedenih raziskav o beljakovinah, ki so pomembne za hemadsorpcijo in serotipno specifične lastnosti virusa afriške prašičje kuge (ASFV; iz angl. african swine fever virus). Pri virusu ASFV je bil odkrit gen EP402R (imenovan tudi LMW8-DR), ki kodira beljakovino CD2v, ki je homologna glodavskemu, človeškemu in prašičjemu T-celičnemu adhezivnemu receptorju. Pokazalo se je, da je CD2v neposredno vpletena v proces hemadsorpcije in je izražena v celicah, okuženih z ASFV kot glikoprotein z molekulsko maso okrog 105-110 kDa. V prisotnosti zaviralca glikozilacije tunicamicina je njegova molekulska masa približno 42 kDa. V celicah, okuženih z ASFV, označenih s 3H-glukozaminom ali 14C-natrijevim acetatom, je bil s testom radioimunoprecipitacije ugotovljen virusno specifični osrednji glikoprotein z molekulsko maso 110-140 kDa (gp 110-140). Z uporabo referenčnih sevov ASFV, ki pripadajo seroimunotipom I-IV, in ustreznim antiserumom, ki so bili aktivni pri preizkusu zaviranja hemadsorpcije (HADIA), smo ugotovili, da gp 110-140 določa specifičnost serotipa. Genotipizacija na osnovi genskega lokusa, ki kodira CD2v in C-tip lektinske beljakovine, je pokazala soizražanje s skupino izolatov in sevov ASFV na podlagi njihovih seroimunotipov. Imunizacija prašičev z gp 110-140 v liposomih ali z rekombinantnim hemaglutininom (CD2v) je zaščitila od 67 do 100 odstotkov živali pred smrtjo zaradi njihove naknadne okužbe z virulentnimi sevi ASFV. Na podlagi fizikalno-kemičnih in bioloških značilnosti beljakovin gp 110-140 in CD2v menimo, da gre za isti virusni glikoprotein, ki je ključnega pomena za vzpodbuditev imunološke zaščite pred ASF. Ključne besede: ASFV; seroimunotipi; serotip; glikoproteini; gp 110-140; CD2v; zaščita

TNFR1 Absence Is Not Crucial for Different Types of Cell Reaction to TNF: A Study of the TNFR1-Knockout Cell Model

Background: One of the mechanisms regulating the biological activity of tumor necrosis factor (TNF) in cells is the co-expression of TNFR1/TNFR2 receptors. A model with a differential level of receptor expression is required to evaluate the contribution of these mechanisms. Aim: The development of a cellular model to compare the effects of TNF on cells depending on the presence of both receptors and TNFR2 alone. Methods: TNFR1 absence modifications of ZR-75/1 and K-562 cell lines were obtained by TNFR1 knockout. The presence of deletions was confirmed by Sanger sequencing, and the absence of cell membrane receptor expression was confirmed by flow cytometry. The dose-dependent effect of TNF on intact and knockout cells was comparatively evaluated by the effect on the cell cycle, the type of cell death, and the profile of expressed genes. Results: Knockout of TNFR1 resulted in a redistribution of TNFR2 receptors with an increased proportion of TNFR2+ cells in both lines and a multidirectional change in the density of expression in the lines (increased in K562 and decreased in ZR75/1). The presence of a large number of cells with high TNFR2 density in the absence of TNFR1 in the K562 cells was associated with greater sensitivity to TNF-stimulating doses and increased proliferation but did not result in a significant change in cell death parameters. A twofold increase in TNFR2+ cell distribution in this cell line at a reduced expression density in ZR75/1 cells was associated with a change in sensitivity to low cytokine concentrations in terms of proliferation; an overall increase in cell death, most pronounced at standard stimulating concentrations; and increased expression of the lymphocyte-activation gene groups, host–pathogen interaction, and innate immunity. Conclusions: The absence of TNFR1 leads to different variants of compensatory redistribution of TNFR2 in cellular models, which affects the type of cell response and the threshold level of sensitivity. The directionality of cytokine action modulation and sensitivity to TNF levels depends not only on the fraction of cells expressing TNFR2 but also on the density of expression