Multigenic family 110 (1 L-5-6 L) of African swine fever virus modulate cytokine genes expression in vitro.

BACKGROUND: African swine fever (ASF) is a viral disease that affects pigs and wild boars providing economic burden in swine industry. METHODS AND RESULTS: In this study, we investigated the effect of deleting the ASFV multigene family 110 (MGF110) fragment (1 L-5-6 L) on apoptosis modulation and the expression of proinflammatory cytokines. Gene expression in swine peripheral blood macrophages infected with either the parental "Volgograd/14c" strain or the gene-deleted "Volgograd/D(1L-5-6L) MGF110" strain was analyzed. Caspase-3 activity was 1.15 times higher in macrophages infected with the parental ASFV strain compared to the gene-deleted strain. Gene expression analysis of Caspase-3 (Cas-3), Interferon-A (IFN-A), Tumor Necrosis Factor A (TNF-A), B-cell Lymphoma-2 (Bcl-2), Nuclear Factor Kappa B (NF-kB), Interleukin-12 (IL-12), and Heat Shock Protein-70 (HSP-70) using RT-qPCR at various time points after infection revealed significant differences in expression profiles between the strains. The peak expression of cytokines (except NF-kB) occurred at 24 h post-infection with the "Volgograd/D(1L-5-6L) MGF110" strain. In samples infected with the ASFV "Volgograd/14c" strain, the most intense expression was observed at 72 and 96 h, except for Bcl-2 and NF-kB, which peaked at 6 h post-infection. The cytokine expression trend for the "Volgograd/D(1L-5-6L) MGF110" strain was more stable with higher expression values. CONCLUSION: The expression trend for the parental strain increased over time, reaching maximum values at 72 and 96 h post-infection, but the overall expression level was lower than that of the gene-deleted strain. These findings suggest that deleting the multigene family 110 members (1 L-5-6 L) contributes to ASFV attenuation without affecting virus replication kinetics.

Comparative analysis of two novel complete genomes of myxoma virus vaccine strains.

Myxoma virus (MYXV) is a double-stranded DNA-containing virus of the family Poxviridae, genus Leporipoxvirus. MYXV is an important model virus for evolutionary and immunological research and a promising oncolytic. In this study, we sequenced and analyzed two complete genomes of MYXV virus vaccine strains B-82 and Rabbivac-B, which are widely used for vaccine production in Russia. Here, we first show that MYXV vaccine strains B-82 and Rabbivac-B share a common origin with the American recombinant MYXV MAV vaccine strain. In addition, our data suggest that the MYXV B-82 and Rabbivac-B strains contain a number of genes at the 5' and 3' ends that are identical to the virulent MYXV Lausanne strain. Several unique genetic signatures were identified in the M013L, M017L, M023, and M121R genes, helping to achieve high genetic resolution between vaccine strains. Overall, these findings highlight the evolutionary flexibility of certain genes in the MYXV genome and provide insights into the molecular epidemiology of the virus and subsequent vaccine development.

Recent Advances in Gene Therapy for Hemophilia: Projecting the Perspectives.

One of the well-known X-linked genetic disorders is hemophilia, which could be hemophilia A as a result of a mutation in the F8 (factor VIII) gene or hemophilia B as a result of a mutation in the F9 (factor IX) gene, leading to insufficient levels of the proteins essential for blood coagulation cascade. In patients with severe hemophilia, factor VIII or factor IX activities in the blood plasma are considerably low, estimated to be less than 1%. This is responsible for spontaneous or post-traumatic bleeding episodes, or both, leading to disease complications and death. Current treatment of hemophilia relies on the prevention of bleeding, which consists of expensive lifelong replacement infusion therapy of blood plasma clotting factors, their recombinant versions, or therapy with recombinant monoclonal antibodies. Recently emerged gene therapy approaches may be a potential game changer that could reshape the therapeutic outcomes of hemophilia A or B using a one-off vector in vivo delivery and aim to achieve long-term endogenous expression of factor VIII or IX. This review examines both traditional approaches to the treatment of hemophilia and modern methods, primarily focusing on gene therapy, to update knowledge in this area. Recent technological advances and gene therapeutics in the pipeline are critically reviewed and summarized. We consider gene therapy to be the most promising method as it may overcome the problems associated with more traditional treatments, such as the need for constant and expensive infusions and the presence of an immune response to the antibody drugs used to treat hemophilia.

Optimization strategies and advances in the research and development of AAV-based gene therapy to deliver large transgenes.

Adeno-associated virus (AAV)-based therapies are recognized as one of the most potent next-generation treatments for inherited and genetic diseases. However, several biological and technological aspects of AAV vectors remain a critical issue for their widespread clinical application. Among them, the limited capacity of the AAV genome significantly hinders the development of AAV-based gene therapy. In this context, genetically modified transgenes compatible with AAV are opening up new opportunities for unlimited gene therapies for many genetic disorders. Recent advances in de novo protein design and remodelling are paving the way for new, more efficient and targeted gene therapeutics. Using computational and genetic tools, AAV expression cassette and transgenic DNA can be split, miniaturized, shuffled or created from scratch to mediate efficient gene transfer into targeted cells. In this review, we highlight recent advances in AAV-based gene therapy with a focus on its use in translational research. We summarize recent research and development in gene therapy, with an emphasis on large transgenes (>4.8 kb) and optimizing strategies applied by biomedical companies in the research pipeline. We critically discuss the prospects for AAV-based treatment and some emerging challenges. We anticipate that the continued development of novel computational tools will lead to rapid advances in basic gene therapy research and translational studies.

Enhanced Zika virus-like particle development using Baculovirus spp. constructs.

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.

RNA-Seq Analysis of Trans-Differentiated ARPE-19 Cells Transduced by AAV9-AIPL1 Vectors.

Inherited retinal disorders (IRD) have become a primary focus of gene therapy research since the success of adeno-associated virus-based therapeutics (voretigene neparvovec-rzyl) for Leber congenital amaurosis type 2 (LCA2). Dozens of monogenic IRDs could be potentially treated with a similar approach using an adeno-associated virus (AAV) to transfer a functional gene into the retina. Here, we present the results of the design, production, and in vitro testing of the AAV serotype 9 (AAV9) vector carrying the codon-optimized (co) copy of aryl hydrocarbon receptor-interacting protein like-1 (AIPL1) as a possible treatment for LCA4. The pAAV-AIPL1co was able to successfully transduce retinal pigment epithelium cells (ARPE-19) and initiate the expression of human AIPL1. Intriguingly, cells transduced with AAV9-AIPL1co showed much less antiviral response than AAV9-AIPL1wt (wild-type AIPL1) transduced. RNA-sequencing (RNA-seq) analysis of trans-differentiated ARPE-19 cells transduced with AAV9-AIPL1co demonstrated significant differences in the expression of genes involved in the innate immune response. In contrast, AAV9-AIPL1wt induced the prominent activation of multiple interferon-stimulated genes. The key part of the possible regulatory molecular mechanism is the activation of dsRNA-responsive antiviral oligoadenylate synthetases, and a significant increase in the level of histone coding genes' transcripts overrepresented in RNA-seq data (i.e., H1, H2A, H2B, H3, and H4). The RNA-seq data suggests that AAV9-AIPL1co exhibiting less immunogenicity than AAV9-AIPL1wt can be used for potency testing, using relevant animal models to develop future therapeutics for LCA4.

Development of Recombinant Oncolytic rVSV-mIL12-mGMCSF for Cancer Immunotherapy.

Anti-cancer therapy based on oncolytic viruses (OVs) is a targeted approach that takes advantage of OVs' ability to selectively infect and replicate in tumor cells, activate the host immune response, and destroy malignant cells over healthy ones. Vesicular stomatitis virus (VSV) is known for its wide range of advantages: a lack of pre-existing immunity, a genome that is easily amenable to manipulation, and rapid growth to high titers in a broad range of cell lines, to name a few. VSV-induced tumor immunity can be enhanced by the delivery of immunostimulatory cytokines. The targeted cytokine delivery to tumors avoids the significant toxicity associated with systemic delivery while also boosting the immune response. To demonstrate this enhanced effect on both tumor growth and survival, a novel recombinant VSV (rVSV)-mIL12-mGMCSF, co-expressing mouse IL-12 (interleukin-12) and GM-CSF (granulocyte-macrophage colony-stimulating factor), was tested alongside rVSV-dM51-GFP (rVSV-GFP) that was injected intratumorally in a syngeneic in vivo C57BL/6 mouse model infused subcutaneously with B16-F10 melanoma cells. The pilot study tested the effect of two viral injections 4 days apart and demonstrated that treatment with the two rVSVs resulted in partial inhibition of tumor growth (TGII of around 40%) and an increased survival rate in animals from the treatment groups. The effect of the two VSVs on immune cell populations will be investigated in future in vivo studies with an optimized experimental design with multiple higher viral doses, as a lack of this information presents a limitation of this study.

Optogenetic technologies in translational cancer research.

Gene and cell therapies are widely recognized as future cancer therapeutics but poor controllability limits their clinical applications. Optogenetics, the use of light-controlled proteins to precisely spatiotemporally regulate the activity of genes and cells, opens up new possibilities for cancer treatment. Light of specific wavelength can activate the immune response, oncolytic activity and modulate cell signaling in tumor cells non-invasively, in dosed manner, with tissue confined action and without side effects of conventional therapies. Here, we review optogenetic approaches in cancer research, their clinical potential and challenges of incorporating optogenetics in cancer therapy. We critically discuss beneficial combinations of optogenetic technologies with therapeutic nanobodies, T-cell activation and CAR-T cell approaches, genome editors and oncolytic viruses. We consider viral vectors and nanoparticles for delivering optogenetic payloads and activating light to tumors. Finally, we highlight herein the prospects for integrating optogenetics into immunotherapy as a novel, fast, reversible and safe approach to cancer treatment.

African Swine Fever Virus Hemadsorption Inhibition Assay.

Adsorption of red blood cells (hemadsorption) on the surface of macrophages infected with African swine fever virus (ASFV) is a unique phenomenon allowing to determine virus infectious titer in hemadsorption unit (HAU) and differentiate virus strains phenotypically. In the meantime, hemadsorption of particular ASFV strain can by inhibited by homologous anti-ASFV serum containing antibody to the serogroup-specific virus protein (CD2v). Here, we describe a hemadsorption inhibition assay (HADIA) to phenotype ASFV strains to one of the known nine serogroups using blood-derived swine macrophages. The HADIA is a powerful method in the ASFV immunopathology and vaccine research since it provides additional antigenic and phenotypic characteristics of virus strains that can't be defined by other assays.

Combinatorial Approaches for Cancer Treatment Using Oncolytic Viruses: Projecting the Perspectives through Clinical Trials Outcomes.

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.

Recombination shapes African swine fever virus serotype-specific locus evolution.

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.

Identifying climate-sensitive infectious diseases in animals and humans in Northern regions.

BACKGROUND: General knowledge on climate change effects and adaptation strategies has increased significantly in recent years. However, there is still a substantial information gap regarding the influence of climate change on infectious diseases and how these diseases should be identified. From a One Health perspective, zoonotic infections are of particular concern. The climate in Northern regions is changing faster than the global average. This study sought to identify climate-sensitive infectious diseases (CSIs) of relevance for humans and/or animals living in Northern regions. Inclusion criteria for CSIs were constructed using expert assessments. Based on these principles, 37 potential CSIs relevant for Northern regions were identified. A systematic literature search was performed in three databases using an explicit stepwise approach to determine whether the literature supports selection of these 37 potential CSIs. RESULTS: In total, 1275 nominated abstracts were read and categorised using predefined criteria. Results showed that arthropod vector-borne diseases in particular are recognised as having potential to expand their distribution towards Northern latitudes and that tick-borne encephalitis and borreliosis, midge-borne bluetongue and the parasitic infection fasciolosis can be classified as climate-sensitive. Many of the other potential CSIs considered are affected by extreme weather events, but could not be clearly classified as climate-sensitive. An additional literature search comparing awareness of climate influences on potential CSIs between 1997-2006 and 2007-2016 showed an increase in the number of papers mentioning effects of climate change. CONCLUSIONS: The four CSIs identified in this study could be targeted in a systematic surveillance programme in Northern regions. It is evident that climate change can affect the epidemiology and geographical range of many infectious diseases, but there were difficulties in identifying additional CSIs, most likely because other factors may be of equal or greater importance. However, climate-ecological dynamics are constantly under change, and therefore diseases may fall in or out of the climate-sensitive definition over time. There is increasing awareness in the literature of the effects of climate change on infectious diseases over time.

Genetic and antigenic diversity of African swine fever virus.

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.

Molecular characteristics of a novel recombinant of porcine epidemic diarrhea virus.

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.

Identification of T-cell epitopes in African swine fever virus CD2v and C-type lectin proteins.

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.

Real-time analysis of the cytopathic effect of African swine fever virus.

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.

African Swine Fever Virus, Siberia, Russia, 2017.

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.

Complete Genome Sequence of a Porcine Epidemic Diarrhea Virus Isolated in Belgorod, Russia, in 2008.

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.

Proposal for a unified classification system and nomenclature of lagoviruses.

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.

Virulent strain of African swine fever virus eclipses its attenuated derivative after challenge.

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.