Crimean-Congo hemorrhagic fever (CCHF) is a widespread arbovirus disease with a mortality rate of up to 40 % in humans. Despite the severity of the disease and the geographic distribution of CCHF, no vaccine or therapeutic agents are currently registered. The paper presents data on a recombinant rhabdovirus expressing a modified glycoprotein GPC of the CCHF virus, an isolate epidemiologically relevant for the Russian Federation.

Influenza A virus and SARS-CoV-2 virus have a high pandemic potential. Vaccination is an effective method of prevention, but existing vaccines cannot be quickly updated to match circulating virus variants. This paper describes a recombinant reassortant strain of influenza A virus expressing SARS-CoV-2 trimerized RBD, which can be used as a component of candidate multivalent vaccines.

Bluetongue, or catarrhal fever of sheep, is a viral vector-borne infection of ruminants, which is one of the economically significant arbovirus infections of animals. It is transmitted by blood-sucking insects of the Culicoides genus. Viral protein VP7 is a group-specific core protein of the virus, conservative for all known serotypes, and therefore represents the most suitable target for creation of diagnostic tests. To develop an immunochemical method for detecting viral infection, a panel of high-affinity monoclonal antibodies to VP7 has been obtained. The N -terminal fragment of VP7 expressed in E. coli and inactivated viral particles were used as immunogens. The resulting monoclonal antibodies are useful for detecting the virus in infected cells. As a species-specific antigen, a recombinant TrxA‑VP7_a protein has been created that contains spatial epitopes similar to those of the native viral antigen. With its help, promising antibodies were selected for diagnostics of the disease by competitive enzyme-linked immunosorbent assay, which allows the detection of specific antibodies to bluetongue virus in the sera of infected animals. Using competitive solid-phase ELISA, it has been shown that antibodies that interact most effectively with the TrxA-VP7_a protein (Bt14, Bt15, Bt18, Bt26, Bt33, Bt34, and Bt35) recognize close or overlapping VP7 epitopes. The interaction with the antigen of almost all the antibodies obtained was inhibited by reference specific sera against the bluetongue virus of 24 serotypes. The monoclonal antibody Bt14 showed maximum ability to block the interaction of VP7 with specific sera against 24 bluetongue virus serotypes. Thus, the resulting panel of monoclonal antibodies to VP7 of the bluetongue virus can be used to detect viral infection, as well as a component of kits for serological diagnosis of the disease.

ПОЛУЧЕНИЕ РЕКОМБИНАНТНОГО НУКЛЕОКАПСИДНОГО БЕЛКА ВИРУСА ЧУМЫ МЕЛКИХ

ПОЛУЧЕНИЕ РЕКОМБИНАНТНОГО НУКЛЕОКАПСИДНОГО БЕЛКА ВИРУСА ЧУМЫ МЕЛКИХ

of the translation products of recombinant plasmids pCI-neo/ASFV/p30, pCI-neo/ASFV/p54 and pCI-neo/ASFV/CD2v induced in adhesive cells (A-cells) after transfection. The antigenic activity of the recombinant proteins produced with these DNA constructs was compared in permanent cell line HEK-293T and swine leukocyte (SL) autologous primary cultures using a direct fluorescence technique. The highest expression of the antigen-active translation products in HEK-293T and SL cells transfected with pCI-neo/ASFV/p30, pCI-neo/ASFV/p54 or pCI-neo/ASFV/CD2v was observed on day 2. The peculiarity of the pig immunization schedule applied was that the animals were thrice immunized at a 14-day interval with autologous LS A cells transfected in vitro with the above recombinant plasmids. For this, as much as 90 cm 3 of LC cell culture was produced using blood samples from each of the animals No.No. 1-4. On day 2 of the culturing, 90 μ g of pCI-neo/ASFV/p30 (No. 1), pCI-neo/ASFV/p54 (No. 2) or pCI-neo/ASFV/CD2v (No. 3) was added thereto. The LS culture obtained from the pig No. 4 in a volume of 90 cm 3 was divided into three portions of 30 cm 3 , and each one was transfected with one of the three constructs (i.e., pCI-neo/ASFV/p30, pCI- neo/ASFV/p54 or pCI-neo/ASFV/CD2v) by adding 30 μ g of the plasmid DNA. Two days later, the pigs No. 1 to No. 4 were inoculated into the central auricular vein with about 10 7 autologous trans- fected A-cells of LS cultures. On days 14, 28 and 42, no antibody against ASFV proteins was detected in the blood of the immunized pigs using indirect solid-phase ELISA and immunoblotting. After the pigs were infected into the neck with 10 2 HAU 50 of an ASFV strain Mozambique-78 on day 42, the four pre-immunized pigs (No.No. 1-4) died

African swine fever virus (ASFV) isolated in 2016–2017 from domestic pigs and wild boars in eight regions of the Russian Federation has comparable biological characteristics: it is virulent, causes the death of animals with signs of acute and overactive forms without the manifestation of a complete symptom complex of clinical and pathologic symptoms, possesses hemadsorption and accumulates in the initial (swine leukocytes, SBM) and transplantable (A4C2/9k) cell cultures in titres 5.5–7.5 and 3.5–5.25 log HAU/ 50 cm 3 , respectively. The studied isolates belong to the eighth serotype and the second genotype. According to these indicators, the ASF virus is comparable with the virus isolates circulating in Russia since 2007.

The agent of African swine fever (ASF) is a large envelope virus (ASFV) belonging to family Asfarviridae and containing a double-stranded linear DNA of 170 to 190 kb in size coding for more than 150 proteins, most of which are involved in host-virus interactions (L.K. Dixon et al., 2004). Its virulent isolates cause a contagious hemorrhagic disease with 100 % mortality both among domestic pigs (Sus scrofa domesticus) and wild boars (Sus scrofa). The disease control is complicated by the lack of any specific preventive methods (R.J. Rowlands et al., 2008; D.A. Chapman et al., 2011; P. Rahimi et al., 2010). The attempts to protect pigs against ASF with experimental live and inactivated subunit vaccines developed by standard methods failed (S. Blome et al., 2014). This paper discusses immunological mechanisms to provide the specific defense base on potentially protective virus-specific proteins, and immunogenic and some protective properties of ASFV gene-based DNA constructs. Immune protection at ASF is due to cytotoxic T-lymphocytes (CTL) and antibody-dependent cellmediated cytotoxicity (ADCC) effectors against viral proteins located on infected monocyte/macrophage. There is a synergism of these effectors (A.D. Sereda, 2013). Based on i) the location, structure and functional properties of viral proteins, ii) the polypeptide specificity of blood antibodies after injecting pigs with ASFV attenuated or virulent strains, iii) the effects of pig immunization using purified proteins from infected cells or the recombinant proteins, and DNN constructs, p30, p54 and CD2v proteins are considered as potentially protective (S.D. Kollnberger et al., 2002; M.G. Barderas et al., 2001; J.G. Neilan et al., 2004). A significant disadvantage of the candidate DNA vaccine is a relatively low immune response, especially in large mammals. There were attempts of overcoming the problem using various strategies (J. Rajcani et al., 2005, M.A. Liu et al., 2006; L.H. van Drunen et al., 2004; J.A. Leifert et al., 2004). To target the lymphocytes expressing receptors CD48 and CD58 to the protein CD2 of the antigen presenting cells (APC), the secretory part (s) of ASFV protein HA (or CD2v) has been used (A. Brossay et al., 2003; K. Crosby et al., 2004). The addition of sHA gene to the DNA construct enhanced both humoral and cellular responses in pigs against fused recombinant proteins p30 and p54 (F. Ruiz-Gonzalvo et al., 1996). An increase in the humoral response due to targeting p30 and p54 fused to one chain of the antibody recognizing the invariant epitope of pig class II main histocompatibility complex (MHC) was demonstrated. However, the enhancement of the humoral immune response to p30 and p54 rather often resulted in earlier death of pigs infected with virulent strains. To stimulate the specific CD8+-T-cell responses, a pCMV-UbsHAPQ construct coding for antigenic determinants p30, p54 and sHA fused with cellular ubiquitin (Ub) was developed. The immunization using pCMV-UbsHAPQ did not induce an instrumentally determined antibody response though provided partially pig protection against ASFV challenge (J.M. Argilaguet et al., 2011). The potential of the DNA constructs was confirmed by pig immunization using ASFV DNA libraries (ASFVUblib) coding for viral genome short fragments combined with the cellular ubiquitin gene (A. Lacasta et al., 2014). In the 4029 clones, about 76 % of the viral genome (130 kb) were covered. As many as 60 % of ASFVUblib-immunized pigs survived after infection with an ASFV virulent strain. According to ELISA, none of the ASFVUblib-immunized pig had detectable specific antibodies to ASFV proteins prior to the challenge. The CD8+-T-cells comprised the only cell sub-

African swine fever (ASF) caused by African swine fever virus (ASFV) of Asfivirus genus, Asfarviridae family, can occur in peracute, acute, subacute, chronic or asymptomatic form. At early stages of epizootics, the infection usually occurs in its acute form eventually becoming chronic and/or asymptomatic. Seven to ten days post infection the survived pigs develop virus-specific antibodies which persist for a long time. An assumption is reasonable that in the near future, due to repeated passaging ASFV in wild boar populations in European countries, the ASFV isolates may appear which will cause chronic or asymptomatic rather than the acute forms of the disease. In our study we compared different tests to find those the most effective to reveal latent carriers when no apparent symptoms of the disease observed. Thus, our research was aimed at investigation of some special aspects of the laboratory diagnostics of chronic and asymptomatic forms of ASF. The chronic form of the disease was observed in a pig experimentally inoculated with an attenuated ASF virus Stavropol 01/08 A4S2/9k (at passage 33) at a dose of 106.0 HAU50. On day 5 to 7 post inoculation the signs typical of chronic forms of the infection were registered including depression and fever up to 40.5 С. The antiviral antibody was detected in the swine blood serum from day 7. After the animal was killed on day 21, a haemadsorption assay revealed ASF virus present at low titers in spleen and mandibular lymph node samples while in liver and lung samples it was not found. Based on the results of polymerase chain reaction (PCR), the viral DNA was determined in the mandibular lymph node sample only. Furthermore, immunoblotting assay identified ASF antibody titers of 1:20 to 1:160 in all the organs examined. The asymptomatic forms of ASF were observed in a wild boar yearling which has been intramuscularly inoculated with an attenuated ASF virus strain MK-200 at a dose of 107.0 HAU50. The antiviral antibody was observed in the wild boar serum from day 8. After the animal was killed on day 25, no pathological signs typical of ASF were found, nor was ASF virus found in the organ samples examined using haemadsorption assay or its DNA was detected in PCR. In immunoblotting assay, virus-specific antibodies were identified in liver, spleen, lung and mandibular lymph node samples at dilutions of 1:40 to 1:320. The opportunity of detecting antibodies in spleen, lung and/or liver samples facilitates the monitoring for ASF to be carried out under the infection control campaigns, especially with respect to wild boars shot in game husbandries. Animals sequentially infected with an ASFV low-virulent isolate and a virulent one can survive, in which case it is quite possible to diagnose the disease using both PCR and serological methods. For making laboratory diagnosis of ASF chronic and/or asymptomatic forms, as well as carrying out monitoring studies, serological methods are recommended.

This article is devoted to the development and evaluation of the immunoblotting test system for serological diagnosis of African swine fever (ASF), based on the highly purified recombinant p30 of ASF virus (ASFV) strain Stavropol 01/08 (Stavropol 2008), representative of the ASFV currently circulating in the Russian Federation. The main project stages are as follows: (i) cloning of the central hydrophilic region of the ASFV gene CP204L (p30) into a prokaryotic vector; (ii) expression and chromatographic purification of the recombinant product p30 with thioredoxin and poly-histidine site (p30e1_TrxA_6xHis); (iii) development of the immunoblotting test system (Rec p30-IB) using the highly purified recombinant p30; and (iv) evaluation of Rec p30-IB using sera and organ samples from domestic pigs and wild boars experimentally or naturally infected by ASFV. Testing of the Rec p30-IB showed the diagnostic specificity and sensitivity of the assay to be 98.75% and 100.00%, respectively. High sensitivity of the Rec p30-IB allowed the detection of ASFV-specific antibodies in samples of organs of the immune system and blood sera, collected from domestic pigs and wild boars, starting from 6 to 8 days post-infection, regardless of virus virulence, seroimmunotype and geographic origin of the samples (East Europe, South Europe, West Europe, Central and south-east Africa).

The capability of causing haemadsorption at African swine fever (ASF) virus (ASFV) reproduction in swine bone marrow cell cultures, leukocytes or continuous cells in the presence of swine erythrocytes is characteristic of the majority of the virus isolates (W. A. Malmquist, D. Hay, 1960).This trait is used for ASF diagnosis based on autohaemadsorption in porcine blood, the virus titration in cell culture, and selection of its attenuated variants in vitro (A.D. Sereda et al., 2014).The haemadsorption inhibition assay (HIA) in tandem with the bioassay using the disease-resistant pigs is applied for seroimmunotype-based classification of ASFV isolates (N.I.Mitin et al., 1985).The heterogeneity of an ASFV population for quantitative haemadsorption characteristic (like «dense», «moderate» or «loose») is a phenotypic trait of ASFV isolates, strains and/or variants (V.Makarov et al., 2016).Also, the proportion of the circumference of red blood cells as observed at their contact with infected macrophages serves as another quantifiable feature of haemadsorption.Some quantitative differences in HIA activity levels of swine blood sera are determined in the assays carried out using virulent reference variants and their attenuated derivatives, and the obtained results require some interpretation.The loss of ability to induce haemadsorption is not critical for ASFV reproduction and often accompanied by a decrease in the pathogen virulence levels.Hence, as a rule, attenuated ASFV variants are prepared through a selection by limiting dilution from populations of virulent isolates of the virus clones that are characterized by a reduced potential to induce haemadsorption (D.V. Kolbasov et al., 2014).In the course of the virus reproduction, haemadsorption precedes the exocytosis.Virions do not play a significant role in the mechanism of haemadsorption, nevertheless, their interaction with erythrocyte membranes promotes the virus dissemination throughout the swine organism and more effective introduction into the gut cells of ticks (L.K. Dixon et al., 2004).ASFV haemadsorbing potentiality is determined by highly glycosylated transmembrane protein CD2v (J.M. Rodríguez et al., 1993).Probably, nonhaemadsorbing avirulent isolates emerge as a result of some shift of the open reading frames for EP402R and EP153R encoding the CD2v and lectin-like proteins, respectively (D.A. Chapman et al., 2008).An assumption is made that the haemadsorption phenomenon is due to an interaction between carbohydrate residues of glycoproteins of ASFV oligosaccharides and lectin-like receptors of swine red blood cells.

African swine fever (ASF) caused by African swine fever virus (ASFV) of Asfivirus genus, Asfarviridae family, can occur in peracute, acute, subacute, chronic or asymptomatic form.At early stages of epizootics, the infection usually occurs in its acute form eventually becoming chronic and/or asymptomatic.Seven to ten days post infection the survived pigs develop virus-specific antibodies which persist for a long time.An assumption is reasonable that in the near future, due to repeated passaging ASFV in wild boar populations in European countries, the ASFV isolates may appear which will cause chronic or asymptomatic rather than the acute forms of the disease.In our study we compared different tests to find those the most effective to reveal latent carriers when no apparent symptoms of the disease observed.Thus, our research was aimed at investigation of some special aspects of the laboratory diagnostics of chronic and asymptomatic forms of ASF.The chronic form of the disease was observed in a pig experimentally inoculated with an attenuated ASF virus Stavropol 01/08 A 4 S 2 /9k (at passage 33) at a dose of 10 6.0 HAU 50 .On day 5 to 7 post inoculation the signs typical of chronic forms of the infection were registered including depression and fever up to 40.5 С.The antiviral antibody was detected in the swine blood serum from day 7.After the animal was killed on day 21, a haemadsorption assay revealed ASF virus present at low titers in spleen and mandibular lymph node samples while in liver and lung samples it was not found.Based on the results of polymerase chain reaction (PCR), the viral DNA was determined in the mandibular lymph node sample only.Furthermore, immunoblotting assay identified ASF antibody titers of 1:20 to 1:160 in all the organs examined.The asymptomatic forms of ASF were observed in a wild boar yearling which has been intramuscularly inoculated with an attenuated ASF virus strain MK-200 at a dose of 10 7.0 HAU 50 .The antiviral antibody was observed in the wild boar serum from day 8.After the animal was killed on day 25, no pathological signs typical of ASF were found, nor was ASF virus found in the organ samples examined using haemadsorption assay or its DNA was detected in PCR.In immunoblotting assay, virus-specific antibodies were identified in liver, spleen, lung and mandibular lymph node samples at dilutions of 1:40 to 1:320.The opportunity of detecting antibodies in spleen, lung and/or liver samples facilitates the monitoring for ASF to be carried out under the infection control campaigns, especially with respect to wild boars shot in game husbandries.Animals sequentially infected with an ASFV low-virulent isolate and a virulent one can survive, in which case it is quite possible to diagnose the disease using both PCR and serological methods.For making laboratory diagnosis of ASF chronic and/or asymptomatic forms, as well as carrying out monitoring studies, serological methods are recommended.

Control of African swine fever (ASF) is complicated by the lack of specific prevention medications.The attempts to obtain live attenuated vaccines by conventional methods were not promising, and the inactivated or subunit vaccines have not been developed so far (N.J. Petiska,