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Zika virus (ZIKV) is one of several examples of an unprecedented pandemic spread and against which there is currently no suitable vaccine or treatment. Here, we constructed and characterized recombinant baculovirus-derived ZIKV-like particles (Zika VLPs) to study ZIKV-antibody interactions. These VLPs, uniquely consisted of the full-length ZIKV capsid (C), pre-membrane (prM), and envelope (E) proteins with either: a) the viral nonstructural NS2B and NS3 protease unit under one or two different promoters or b) an alternative host-cell furin protease encoding cleavage sequence inserted between the C and prM genes, together with lobster tropomyosin leader and honeybee signal sequences with one promoter for increased extracellular secretion. All these Zika VLPs displayed typical virion morphology in transmission electron microscopic analysis when expressed in both insect (Sf9) and mammalian (HEK293T) cells and no uncleaved prM glycoprotein was detected, as are present on immature virions. The importance of glycosylation of the E glycoprotein was shown by the effects on both polyclonal and monoclonal antibody reactions after these N-linked carbohydrate residues were disrupted by oxidation or enzymatic cleavage. Importantly, the construct which contained the host-cell furin protease cleavage sequence together with a lobster tropomyosin leader and honeybee signal sequences under one promoter produced higher Zika VLP titers and protein concentrations and which can now be tested as a superior construct in multifunctional diagnostic (ELISA and neutralization/antibody-dependent enhancement) assays and immunogenic assessments possibly leading to vaccine trials.

Introduction. African swine fever virus (ASF) is a large DNA virus that is the only member of the Asfarviridae family. The spread of the ASF virus in the territory of the Russian Federation, Eastern Europe and China indicates the ineffectiveness of existing methods of combating the disease and reinforces the urgent need to create effective vaccines. One of the most significant antigens required for the formation of immune protection against ASF is a serotype-specific CD2v protein. The purpose of the study. This study presents the results of immuno-informatics on the identification of B- and T-cell epitopes for the CD2v protein of the ASF virus using in silico prediction methods. Material and methods. The primary sequence of the CD2v protein of the ASFV virus strain Georgia 2007/1 (ID-FR682468) was analyzed in silico by programs BCPred, NetCTLpan, VaxiJen, PVS and Epitope Conservancy Analysis. Results. Using the BCPred and VaxiJen programs, 4 major B-cell immunogenic epitopes were identified. Analysis of the secretory region of ASF virus CD2v protein in NetCTLpan revealed 5 T-cell epitopes from the 32nd to the 197th position of amino acids that cross-link from the 1st to the 13th allele of the MHC-I of pig Discussion. This study presents the results in silico prediction to identify B- and T-cell epitopes of ASF virus CD2v protein. The soluble region of the CD2v protein can be included in the recombinant polyepitope vaccine against African swine fever. Conclusion. B- and T-cell epitopes in the secretory region of the CD2v protein (from 17 to 204 aa) of ASF virus were identified by in silico prediction. An analysis of the conservatism of the identified B- and T-cell epitopes allowed us to develop a map of the distribution of immune epitopes in the CD2v protein sequence.

The main criteria for current vaccines design are effectiveness, efficaciousness and safety. Increasing requirements for vaccine safety and purity push forward not only classical vaccine development, but also new generation vaccine technology, including sub-unit, recombinant, anti-idiotypic, DNA vaccines etc. This recombinant technology has already demonstrated its advantage, efficaciousness and safety in a large field of therapeutic and curative drug development for animal and human (S. Khan et al., 2016). In 2011, six novel drugs were created based on the new Fc-fusion protein technology. Most of the newly developed drugs affect receptor-ligand interactions, acting as antagonists by blocking direct receptor binding, i.e. Enbrel (etanercept; Amgen, USA), Zaltrap (aflibercept; Sanofi, France), Arcalyst (rilonacept; Regeneron, USA), or as agonists for direct stimulation of receptor function which augment immune response as Amevive (alefacept, Astellas, USA) does, or decrease immune response as Nplate (romiplostim; Amgen, USA) does. In this review, we pay attention to the most relevant results from the last few years for virus and bacterial vaccine designed based on Fc-fusion technology. The Fc-chimeras are hybrid sequences in which Fc-fragment of IgG (Fc-IgG) and targeted therapeutic protein are fused in an entire protein molecule (V. Pechtner et al., 2017). In this fusion, the hinge region of Fc-IgG is a flexible spacer between therapeutic protein and conservative part of IgG. It helps to minimize potential negative effect of two functional domains to each other. Therapeutic drugs based on Fc-fusion proteins are divided in three types, the receptor-Fc, peptide-Fc, and monomer-Fc. The Fc-fused proteins have tremendous therapeutic potential, since Fc domain in this molecules helps to specifically augment the pharmaco-dynamics values. Presence of Fc-domain in hybrid molecules prolongs half elimination of protein from plasma, which extends drug therapeutic activity and slows down kidney clearance for large molecules. Here, we summarize the most significant experimental data of Fc-fusion technology application against such pathogens as human immunodeficiency virus (D. Capon et al., 1989), Ebola virus (K. Konduru et al., 2011), Dengue virus (M.Y. Kim et al., 2018), influenza virus (L. Du et al., 2011), Mycobacterium tuberculosis (S. Soleimanpour et al., 2015), classical swine fever virus (Z. Liu et al., 2017). We also discuss the critical aspects of mechanism of action, drug design and Fc-fused protein production. Targeted activation of effector systems boosts protective potential of immuno-genic molecules and broadens its application. The interest of this review is focused on an application of Fc-fused proteins as potential vaccines against infectious human and animal diseases. We also briefly discuss the perspectives of Fc-fused antigens for novel effective medicine developments using African swine fever virus as an example.

Porcine epidemic diarrhea (PED) is a contagious viral disease in pigs, caused by the coronavirus porcine epidemic diarrhea virus (PEDV). PEDV infection results in significant mortality in piglets in unvaccinated herds. Like many others RNA viruses, PEDV has high evolutionary rate and is prone to genetic mutations. In this study, we analyzed the complete genome sequence of the recently sequenced isolate PEDV/Belgorod/dom/2008. A recombination event in S gene of PEDV/Belgorod/dom/2008 was detected. Pairwise identity analysis of the whole genome sequences revealed that PEDV/Belgorod/dom/2008 is an intermediate between PEDV and transmissible gastroenteritis virus (TGEV) strains. These results can be used for further analysis of the evolutionary variability, prevalence, and epidemiology of the porcine epidemic diarrhea virus.

Recent cancer immunotherapy breakthroughs have fundamentally changed oncology and revived the fading hope for a cancer cure. The immune checkpoint inhibitors (ICI) became an indispensable tool for the treatment of many malignant tumors. Alongside ICI, the application of oncolytic viruses in clinical trials is demonstrating encouraging outcomes. Dozens of combinations of oncolytic viruses with conventional radiotherapy and chemotherapy are widely used or studied, but it seems quite complicated to highlight the most effective combinations. Our review summarizes the results of clinical trials evaluating oncolytic viruses with or without genetic alterations in combination with immune checkpoint blockade, cytokines, antigens and other oncolytic viruses as well. This review is focused on the efficacy and safety of virotherapy and the most promising combinations based on the published clinical data, rather than presenting all oncolytic virus variations, which are discussed in comprehensive literature reviews. We briefly revise the research landscape of oncolytic viruses and discuss future perspectives in virus immunotherapy, in order to provide an insight for novel strategies of cancer treatment.

The recombination is one of the most frequently identified drivers of double-stranded DNA viruses evolution. However, the recombination events in African swine fever virus (ASFV) genomes have been poorly annotated. We hypothesize that the genetic determinants of ASFV variability are potential hot-spots for recombination. Here, we analyzed ASFV serotype-specific locus (C-type lectin (EP153R) and CD2v (EP402R)) in order to allocate the recombination breakpoints in these immunologically important proteins and reveal driving forces of virus evolution. The recombinations were found in both proteins, mostly among ASFV strains from East Africa, where multiple virus transmission cycles are notified. The recombination events were essentially associated with the domain organization of proteins. The phylogenetic analysis demonstrated the lack of clonal evolution for African strains which conclusively support the significance of recombinations in the serotype-specific locus. In addition, the signature of adaptive evolution of these two genes, pN/pS > 1, was demonstrated. These results have implications for the interpretation of cross-protection potential between evolutionary distant ASFV strains and strongly suggest that C-type lectin and CD2v may experience substantial selective pressure than previously thought.

Molecular epidemiology of viral infections traditionally based on the analysis of changes in individual genes or genetic markers. The analysis of the African swine fever virus (ASFV) genes encoding immunomodulatory proteins is an important tool for studying the diversity and evolution of the virus. In this work, we carried out a structural and phylogenetic analysis of the ASF virus immunomodulatory proteins 5EL (A238L gene), I14L (Dp71L gene), K11L (I329L gene). The degree of nucleotide substitutions of the ASFV concatenated genes A238L, I329L and Dp71L revealed purifying (stabilizing) selection at the nucleotide sequences level. The variability characteristic of the selected group of ASFV genes is of great interest for the genetic differences search in immunomodulatory proteins. The sequencing results of the A238L, I329L and Dp71L genes and their phylogenetic analysis showed that these genes are conservative among a large group of ASFV genes. The I329L gene is a genetic marker of common origin. The East African strains (Genotype X) of Dp71L gene have two forms: a long (184 amino acids) and a short (from 70 to 72 amino acids) and is formed by fusion of the 13L and 14L. All ASF virus Russian isolates isolated in 2016–2017 were identical to the reference strain ASFV/Georgia/wb/2007. Characterization of variability 5EL protein, I14L, K11L may be serve to identify target sites in the ASFV genome and to develop vaccines. The obtained data allow to evaluate the genetic diversity of the ASFV immunomodulatory proteins and the dynamics of their evolution, to predict the possible participation of the A238L, I329L and Dp71L genes in the virulence of various ASFV strains.

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/50cm3, 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.

Conventional methods, which quantitatively assess virus replication, are based on direct examination of viral cytopathic effect (CPE), which is time consuming, tedious and based on endpoint reading. The Real-Time Cell Analysis (RTCA) xCELLigence® system offers an alternative approach to evaluate virus-induced CPE, and here was evaluated as a means to dynamically assess CPE caused by African swine fever virus (ASFV). RTCA was used to identify optimum time for ASFV infection based on cell index (CI) and to evaluate ASFV CPE kinetics in COS-1 cells. Data indicated that the RTCA has tremendous potential to methodologically and quantitatively improve assays used to study efficiency of ASFV drug inhibitors and neutralizing antibodies.

The first notifications of the unknown disease of cattle appeared in September-October 2015 in North Caucasus region of Russia (Republic of North Ossetia-Alania). The clinical signs included watery discharge from eyes, apathy, loss of appetite, salivation, lameness and nodular skin lesions. Capripoxvirus genome was detected by real-time PCR in the tissue samples of sick animals. The aetiological agent was isolated in the primary cell cultures of lamb testis and goat testis, as well as in the continuous MDBK cell culture. Further sequencing of the GPCR gene and phylogenetic analysis showed the close genetic relationship of isolated capripoxvirus with a group of lumpy skin disease virus. Koch's postulates were fulfilled by the experimental infection of four calves with a suspension of tissue samples from sick animals.

African swine fever (ASF) is one of the most devastating diseases affecting the swine industry worldwide. No effective vaccine is currently available for disease prevention and control. Although live attenuated vaccines (LAV) have demonstrated great potential for immunizing against homologous strains of African swine fever virus (ASFV), adverse reactions from LAV remain a concern. Here, by using a homologous ASFV Congo strain system, we show passage-attenuated Congo LAV to induce an efficient protective immune response against challenge with the virulent parental Congo strain. Notably, only the parental challenge Congo strain was identified in blood and organs of recovered pigs through B602L gene PCR, long-range PCR, nucleotide sequencing and virus isolation. Thus, despite the great protective potential of homologous attenuated ASFV strain, the challenge Congo strain can persist for weeks in recovered pigs and a recrudescence of virulent virus at late time post-challenge may occur.

Lagoviruses belong to the Caliciviridae family. They were first recognized as highly pathogenic viruses of the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus) that emerged in the 1970–1980s, namely, rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), according to the host species from which they had been first detected. However, the diversity of lagoviruses has recently expanded to include new related viruses with varying pathogenicity, geographic distribution and host ranges. Together with the frequent recombination observed amongst circulating viruses, there is a clear need to establish precise guidelines for classifying and naming lagovirus strains. Therefore, here we propose a new nomenclature based on phylogenetic relationships. In this new nomenclature, a single species of lagovirus would be recognized and called Lagovirus europaeus. The species would be divided into two genogroups that correspond to RHDV- and EBHSV-related viruses, respectively. Genogroups could be subdivided into genotypes, which could themselves be subdivided into phylogenetically well-supported variants. Based on available sequences, pairwise distance cutoffs have been defined, but with the accumulation of new sequences these cutoffs may need to be revised. We propose that an international working group could coordinate the nomenclature of lagoviruses and any proposals for revision., This work was supported in part by a grant from the Agence Nationale de la Recherche (France), Calilago and by a grant from the Région des Pays de la Loire (France) Armina to JLP. It was performed within the framework of the ECALEP project selected during the 2nd joint call of the Animal Health and Welfare ERA-Net (Anihwa) initiative, a Coordination Action funded under the European Commission’s ERA-Net scheme within the Seventh Framework Programme (Contract No. 291815). The ECALEP project is funded by the Agence Nationale de la Recherche (France), the Ministry of Health, Depa for Veterinary Public Health, Nutrition and Food Safety (Italy) and the Research council FORMAS (Sweden). FCT-Foundation for Science and Technology, Portugal, supported the FCT Investigator grant of JA (ref. IF/01396/2013) and the Post-doc grant of AML (SFRH/BPD/115211/2016).

African swine fever virus introduction to naive swine population leads to high mortality and losses among susceptible animals. ASF epidemic in Russia (2007–to date) and lately in Eastern Europe highlights severe socio-economic consequences of this disease. The disease epidemiology is rather complex in endemic territories since many factors are involved in virus transmission. The disease control is only based on stamping-out policy and rapid virus diagnostics, since no effective and safe vaccine is available. This chapter focuses on African swine fever epidemiology, immunopathobiology and diagnostics with a brief overview of recent advances of ASF vaccine development.

African swine fever (ASF) is arguably the most dangerous and emerging swine disease worldwide. ASF is a serious problem for the swine industry. The first case of ASF in Russia was reported in 2007. We report an outbreak of ASF in Siberia, Russia, in 2017.

African swine fever virus (ASFV) is the only known DNA arbovirus, and the ability to replicate efficiently in both insect and mammalian cells is encoded in its viral genome. Despite having a relatively low overall genomic mutation rate, ASFV demonstrates genetic diversity in certain genes and complexity in gene content in other genomic regions, indicating that ASFV may exploit multiple mechanisms for diversification and acquire new phenotype characteristics. ASFV antigenic diversity is reflected in the ability to type cross-protective viruses together into serogroups, largely based on antibody-mediated inhibition of hemadsorption. Here we review ASFV genetic signatures of ASFV type specificity, genome variability, and the hemadsorption as a means of defining virus antigenic type, and how these may be used toward defining antigenic and phenotypic diversity that is problematic for development of vaccine solutions to ASF.

African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available, and progress is hindered by lack of knowledge concerning the extent of ASF virus (ASFV) strain diversity and the viral antigens conferring type-specific protective immunity in pigs. Previously, we demonstrated that ASFV serotype-specific proteins CD2v (EP402R) and/or C-type lectin (EP153R) are important for protection against homologous ASF infection. Here, we identified six discrete T-cell epitope regions present on CD2v and C-type lectin using IFN-γ ELISpot assay and PBMCs from ASF immune animals, indicating cellular reactivity to these proteins in the context of ASFV infection and protective immunity. Notably, three of the epitope regions map to previously described serotype-specific signature regions of these proteins. Improved understanding of ASFV protective antigens, relevant epitopes and their diversity in nature will facilitate ASFV subunit vaccine design and development.

African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens conferring type-specific protective immunity in pigs. Available data from vaccination/challenge experiments in pigs indicate that ASF protective immunity may be haemadsorption inhibition (HAI) serotype-specific. Recently, we have shown that two ASFV proteins, CD2v (EP402R) and C-type lectin (EP153R), are necessary and sufficient for mediating HAI serological specificity (Malogolovkin et al., 2015).. Here, using ASFV inter-serotypic chimeric viruses and vaccination/challenge experiments in pigs, we demonstrate that serotype-specific CD2v and/or C-type lectin proteins are important for protection against homologous ASFV infection. Thus, these viral proteins represent significant protective antigens for ASFV that should be targeted in future vaccine design and development. Additionally, these data support the concept of HAI serotype-specific protective immunity.

Discrimination between different field and vaccine strains of classical swine fever virus (CSFV) is crucial for meaningful disease diagnosis and epidemiological investigation. In this study, a rapid method for differentiating vaccine strains and outbreak CSFV isolates by combined RT-PCR and high-resolution melt (HRM) analysis has been developed. The assay is based on PCR amplification of short fragments from the most variable region of CSFVgene E2, followed by HRM analysis of amplicons. Real-Time PCR/HRM for CSFV detection and differentiation analysis has sensitivity comparable to RT-qPCR and genotyping resolution comparable to E2 nucleotide sequencing. This assay in one step enables rapid and sensitive identification and genotype discrimination of CSFV in field samples, and thus will be valuable for CSF outbreak response and disease control.

International audience; Novel HIV vaccine vectors and strategies are needed to control HIV/AIDS epidemic in humans and eradicate the infection. DNA vaccines alone failed to induce immune responses robust enough to control HIV-1. Development of lentivirus-based DNA vaccines deficient for integration and with a limited replication capacity is an innovative and promising approach. This type of vaccine mimics the early stages of virus infection/replication like the live-attenuated viruses but lacks the inconvenient integration and persistence associated with disease. We developed a novel lentivector DNA vaccine "CAL-SHIV-IN-" that undergoes a single round of replication in the absence of integration resulting in augmented expression of vaccine antigens in vivo. Vaccine gene expression is under control of the LTRs of a naturally attenuated lentivirus, Caprine arthritis encephalitis virus (CAEV) the natural goat lentivirus. The safety of this vaccine prototype was increased by the removal of the integrase coding sequences from the pal gene. We examined the functional properties of this lentivector DNA in cell culture and the immunogenicity in mouse models. Viral proteins were expressed in transfected cells, assembled into viral particles that were able to transduce once target permissive cells. Unlike the parental replication-competent SHIV-KU2 that was detected in DNA samples from any of the serial passage infected cells, CAL-SHIV-IN- DNA was detected only in target cells of the first round of infection, hence demonstrating the single cycle replication of the vaccine. A single dose DNA immunization of humanized NOD/SCID/beta 2 mice showed a substantial increase of IFN-gamma-ELISPOT in splenocytes compared to the former replication and integration defective Delta 4SHIV-KU2 DNA vaccine. (C) 2015 Elsevier Ltd. All rights reserved.

African swine fever virus (ASFV) causes highly lethal hemorrhagic disease among pigs, and ASFV's extreme antigenic diversity hinders vaccine development. We show that p72 ASFV phylogenetic analysis does not accurately define ASFV hemadsorption inhibition assay serogroups. Thus, conventional ASFV genotyping cannot discriminate between viruses of different virulence or predict efficacy of a specific ASFV vaccine.

We identified porcine epidemic diarrhea virus (PEDV) in stool samples from sick piglets in the Belgorod region of Russia. The complete coding genome sequence of 28,295 nucleotides (nt) of PEDV was generated. Compared to a prototype PEDV strain (DR13), an extreme number of mismatches in the S gene were revealed.

The nucleotide sequence of a section of the provirus pol gene of 18 bovine leukemia virus (BLV) isolates from animals from some farms in the Russian Federation is determined. A phylogenetic analysis showed that all investigated isolates belong to the same group as BLV isolates from Argentina, Australia, Brazil, and United States.

African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of ASF virus (ASFV) strain diversity and the viral antigens responsible for protection in the pig. Available data from vaccination/challenge experiments in pigs indicate ASF protective immunity is haemadsorption inhibition (HAI) serotype-specific. A better understanding of ASFV HAI serological groups and their diversity in nature, as well as improved methods to serotype ASFV isolates, is needed. Here, we demonstrated that the genetic locus encoding ASFV CD2v and C-type lectin proteins mediates HAI serological specificity and that CD2v/C-type lectin genotyping provides a simple method to group ASFVs by serotype, thus facilitating study of ASFV strain diversity in nature, and providing information necessary for eventual vaccine design, development and efficacious use.

Since the introduction of the virus into the Republic of Georgia in 2007 African swine fever (ASF) has become a large-scale epidemic involving the domestic pig population but wild boars are involved as well. From 2008 to 2009 the ASF epidemic affected wild and domestic pigs in all the southern regions of the Russian Federation (RF). The driving force of the epidemic in its initial stages was direct contact between infected wild boars and between wild boars and traditionally free-ranging domestic pigs in backyard farms. Driving forces of the epidemic at the its first stages was direct contact of infected wild boars between each other and with traditionally free ranged domestic pigs in backyard farms. The next stage developed due to illegal movement of pig products contaminated by African swine fever virus (ASFV) from affected regions and swill feeding, and inefficient implementation of measures to prevent and control ASF. From 2010 through 2012, ASF spread to other, previously unaffected regions of the RF. Most of outbreaks in the southern regions (Krasnodar, Stavropol, Rostov regions) are secondary. Currently, the disease situation observed in endemic areas of the RF, including the southern Krasnodar and Volgograd regions and the central Tver’ region, is very complicated. In 2012, a large number of outbreaks in domestic pigs and in wild boars were reported. The circulating ASFV is highly virulent and has maintained its virulence throughout the epidemic since its introduction in 2007. Considering the forces currently driving the ASF epidemic – circulation of ASF virus in wild boars, ineffectiveness of prevention and control measures, lack of common interest in eradicating the disease and absence of a nationally funded eradication program – continued outbreaks, including those in previously unaffected regions of the RF, can be expected.

African swine fever is one of the most important viral diseases of pigs and which caused significant economic damage on the pig production worldwide. Nowadays, it is still present on the African continent, in Transcaucasus countries (TCC), on Island of Sardinia and in Russia. Outbreaks of the disease have been reported in Russia for the last four years, affected especially the Southern Federal District of the country. Since 2010, a new outbreak area has been observed in the Northwestern Federal District.In order to study the evolution of African swine fever virus (ASFV) isolates, strains were collected in the Russian Federation from 2007 to 2011 and investigated by means of partial sequencing and fragment length polymorphism. In detail, 7 variable regions, namely B646L, E183L, I196L, B602L, I73R/I329R, I78R/I215L and KP86R were investigated. Phylogenetic analyses revealed 100% nucleotide identity of B646L and E183L gene sequences of all examined isolates. All isolates formed one genetic cluster within genotype II. Moreover, no amplified fragment length polymorphism (AFLP) was observed for B602L, I196L, I73R/I329R, and I78R/I215L genes. The flanking primers used to amplify the KP86R gene failed to amplify a product in all the isolates. The obtained data strongly suggests that only one ASFV virus variant caused the outbreaks from 2007 to 2011 in the territory of the Russian Federation. © 2012 Elsevier B.V.