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1.
Sci Rep ; 11(1): 17758, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493784

RESUMO

DNA viruses can exploit host cellular epigenetic processes to their advantage; however, the epigenome status of most DNA viruses remains undetermined. Third generation sequencing technologies allow for the identification of modified nucleotides from sequencing experiments without specialized sample preparation, permitting the detection of non-canonical epigenetic modifications that may distinguish viral nucleic acid from that of their host, thus identifying attractive targets for advanced therapeutics and diagnostics. We present a novel nanopore de novo assembly pipeline used to assemble a misidentified Camelpox vaccine. Two confirmed deletions of this vaccine strain in comparison to the closely related Vaccinia virus strain modified vaccinia Ankara make it one of the smallest non-vector derived orthopoxvirus genomes to be reported. Annotation of the assembly revealed a previously unreported signal peptide at the start of protein A38 and several predicted signal peptides that were found to differ from those previously described. Putative epigenetic modifications around various motifs have been identified and the assembly confirmed previous work showing the vaccine genome to most closely resemble that of Vaccinia virus strain Modified Vaccinia Ankara. The pipeline may be used for other DNA viruses, increasing the understanding of DNA virus evolution, virulence, host preference, and epigenomics.


Assuntos
Vírus Defeituosos/genética , Epigenoma , Genoma Viral , Sequenciamento por Nanoporos , Orthopoxvirus/genética , Sinais Direcionadores de Proteínas/genética , Análise de Sequência de DNA/métodos , Vírus Vaccinia/genética , Proteínas Virais/genética , Vacinas Virais , Motivos de Aminoácidos , Sequência de Aminoácidos , Vírus de DNA/genética , Anotação de Sequência Molecular , Orthopoxvirus/imunologia , Deleção de Sequência , Software , Especificidade da Espécie , Emirados Árabes Unidos , Vacinas Atenuadas
2.
Emerg Infect Dis ; 27(10): 2570-2577, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34352194

RESUMO

Cowpox virus (CPXV) has an animal reservoir and is typically transmitted to humans by contact with infected animals. In 2017, CPXV infection of a pregnant woman in France led to the death of her fetus. Fetal death after maternal orthopoxvirus (smallpox) vaccination has been reported; however, this patient had not been vaccinated. Investigation of the patient's domestic animals failed to demonstrate prevalence of CPXV infection among them. The patient's diagnosis was confirmed by identifying CPXV DNA in all fetal and maternal biopsy samples and infectious CPXV in biopsy but not plasma samples. This case of fetal death highlights the risk for complications of orthopoxvirus infection during pregnancy. Among orthopoxviruses, fetal infection has been reported for variola virus and vaccinia virus; our findings suggest that CPXV poses the same threats for infection complications as vaccinia virus.


Assuntos
Varíola Bovina , Orthopoxvirus , Animais , Varíola Bovina/diagnóstico , Varíola Bovina/epidemiologia , Varíola Bovina/veterinária , Vírus da Varíola Bovina/genética , Feminino , Morte Fetal , Feto , França/epidemiologia , Humanos , Adulto Jovem
3.
J Virol ; 95(19): e0101221, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34260287

RESUMO

Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MVs are further wrapped and secreted to spread infection within the host. However, late during infection, MVs accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here, we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titers. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate limiting for EV production. The A26 gene is conserved within vertebrate poxviruses but is strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfill different functions represents a novel level of complexity in mammalian viruses with major impacts on evolution, adaptation, and transmission. IMPORTANCE Chordopoxviruses are mammalian viruses that uniquely produce a first type of virion adapted to spread within the host and a second type that enhances transmission between hosts, which can take place by multiple ways, including direct contact, respiratory droplets, oral/fecal routes, or via vectors. Both virion types are important to balance intrahost dissemination and interhost transmission, so virus maturation pathways must be tightly controlled. Here, we provide evidence that the abundance and kinetics of expression of the viral protein A26 regulates this process by preventing formation of the first form and shifting maturation toward the second form. A26 is expressed late after the initial wave of progeny virions is produced, so sufficient viral dissemination is ensured, and A26 provides virions with enhanced environmental stability. Conservation of A26 in all vertebrate poxviruses, but not in those transmitted exclusively via biting arthropods, reveals the importance of A26-controlled virus maturation for transmission routes involving environmental exposure.


Assuntos
Regiões Promotoras Genéticas , Vírus Vaccinia/fisiologia , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Chordopoxvirinae/genética , Chordopoxvirinae/metabolismo , Engenharia Genética , Humanos , Orthopoxvirus/genética , Orthopoxvirus/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Vírus Vaccinia/genética , Ensaio de Placa Viral , Proteínas Virais/genética
5.
PLoS Pathog ; 17(1): e1009183, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33444388

RESUMO

The antiviral protein kinase R (PKR) is an important host restriction factor, which poxviruses must overcome to productively infect host cells. To inhibit PKR, many poxviruses encode a pseudosubstrate mimic of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), designated K3 in vaccinia virus. Although the interaction between PKR and eIF2α is highly conserved, some K3 orthologs from host-restricted poxviruses were previously shown to inhibit PKR in a species-specific manner. To better define this host range function, we compared the sensitivity of PKR from 17 mammals to inhibition by K3 orthologs from closely related orthopoxviruses, a genus with a generally broader host range. The K3 orthologs showed species-specific inhibition of PKR and exhibited three distinct inhibition profiles. In some cases, PKR from closely related species showed dramatic differences in their sensitivity to K3 orthologs. Vaccinia virus expressing the camelpox virus K3 ortholog replicated more than three orders of magnitude better in human and sheep cells than a virus expressing vaccinia virus K3, but both viruses replicated comparably well in cow cells. Strikingly, in site-directed mutagenesis experiments between the variola virus and camelpox virus K3 orthologs, we found that different amino acid combinations were necessary to mediate improved or diminished inhibition of PKR derived from different host species. Because there is likely a limited number of possible variations in PKR that affect K3-interactions but still maintain PKR/eIF2α interactions, it is possible that by chance PKR from some potential new hosts may be susceptible to K3-mediated inhibition from a virus it has never previously encountered. We conclude that neither the sensitivity of host proteins to virus inhibition nor the effectiveness of viral immune antagonists can be inferred from their phylogenetic relatedness but must be experimentally determined.


Assuntos
Antivirais/antagonistas & inibidores , Especificidade de Hospedeiro , Orthopoxvirus/classificação , Orthopoxvirus/fisiologia , Infecções por Poxviridae/virologia , Replicação Viral , eIF-2 Quinase/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Antivirais/metabolismo , Células HeLa , Humanos , Fosforilação , Filogenia , Infecções por Poxviridae/genética , Infecções por Poxviridae/metabolismo , Homologia de Sequência , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
6.
Immunity ; 54(2): 247-258.e7, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33444549

RESUMO

The vaccine strain against smallpox, vaccinia virus (VACV), is highly immunogenic yet causes relatively benign disease. These attributes are believed to be caused by gene loss in VACV. Using a targeted small interfering RNA (siRNA) screen, we identified a viral inhibitor found in cowpox virus (CPXV) and other orthopoxviruses that bound to the host SKP1-Cullin1-F-box (SCF) machinery and the essential necroptosis kinase receptor interacting protein kinase 3 (RIPK3). This "viral inducer of RIPK3 degradation" (vIRD) triggered ubiquitination and proteasome-mediated degradation of RIPK3 and inhibited necroptosis. In contrast to orthopoxviruses, the distantly related leporipoxvirus myxoma virus (MYXV), which infects RIPK3-deficient hosts, lacks a functional vIRD. Introduction of vIRD into VACV, which encodes a truncated and defective vIRD, enhanced viral replication in mice. Deletion of vIRD reduced CPXV-induced inflammation, viral replication, and mortality, which were reversed in RIPK3- and MLKL-deficient mice. Hence, vIRD-RIPK3 drives pathogen-host evolution and regulates virus-induced inflammation and pathogenesis.


Assuntos
Vírus da Varíola Bovina/fisiologia , Varíola Bovina/imunologia , RNA Interferente Pequeno/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Vírus Vaccinia/metabolismo , Proteínas Virais/metabolismo , Animais , Evolução Molecular , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Inflamação , Camundongos , Camundongos Knockout , Necroptose/genética , Orthopoxvirus , Filogenia , Proteínas Quinases/genética , Proteólise , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Análise de Sequência de RNA , Proteínas Virais/genética , Replicação Viral
7.
Expert Rev Anti Infect Ther ; 19(3): 331-344, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32882158

RESUMO

INTRODUCTION: Tecovirimat (TPOXX®; ST-246) was approved for the treatment of symptomatic smallpox by the USFDA in July of 2018 and has been stockpiled by the US government for use in a smallpox outbreak. While there has not been a reported case of smallpox since 1978 it is still considered a serious bioterrorism threat. AREAS COVERED: A brief history of smallpox from its proposed origins as a human disease through its eradication in the late 20th century is presented. The current smallpox threat and the current public health response plans are described. The discovery, and development of tecovirimat through NDA submission and subsequent approval for treatment of smallpox are discussed. Google Scholar and PubMed were searched over all available dates for relevant publications. EXPERT OPINION: Approval of tecovirimat to treat smallpox represents an important milestone in biosecurity preparedness. Incorporating tecovirimat into the CDC smallpox response plan, development of pediatric liquid and intravenous formulations, and approval for post-exposure prophylaxis would provide additional health security benefit.Tecovirimat shows broad efficacy against orthopoxviruses in vitro and in vivo and could be developed for use against emerging orthopoxvirus diseases such as monkeypox, vaccination-associated adverse events, and side effects of vaccinia oncolytic virus therapy.


Assuntos
Antivirais/administração & dosagem , Benzamidas/administração & dosagem , Isoindóis/administração & dosagem , Varíola/tratamento farmacológico , Animais , Antivirais/farmacologia , Benzamidas/farmacologia , Bioterrorismo/prevenção & controle , Humanos , Isoindóis/farmacologia , Orthopoxvirus/efeitos dos fármacos , Orthopoxvirus/isolamento & purificação , Infecções por Poxviridae/tratamento farmacológico , Infecções por Poxviridae/virologia
8.
Mol Cancer Ther ; 20(1): 173-182, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33262221

RESUMO

Although it is known that oncolytic viruses can inflame and recruit immune cells to otherwise immunosuppressed tumor microenvironments, the influence of the antiviral immune response on antitumor immunity is less clear across viral platforms and tumor types. CF33 is a recombinant orthopoxvirus backbone effective against colon cancer. We tested derivatives of CF33 with and without immune-checkpoint inhibition (anti-PD-L1) in mouse models of colon cancer. Results showed that the efficacy of CF33 backbone with J2R deletion (single-deleted) against colon cancer is not altered by additional deletion of F14.5L in vitro or in vivo CF33 infection upregulated PD-L1 expression on tumor cells and led to an increased influx of lymphocytes and macrophages in tumors. Also, the levels of active CD8+ (IFNγ+) T cells in the virus-treated tumors were higher than those in control-treated tumors. Furthermore, a combination of CF33 derivatives with anti-PD-L1 resulted in durable tumor regression and long-term survival, resistant to tumor rechallenge. Analysis of immune cells from the treated mice showed that tumor-specific T cell activation occurred more robustly in tumors treated with the virus and that T cells were more strongly activated against the virus than against tumor, in an MHC-I-dependent manner. Our findings warrant further studies on the role of cross-priming of T cells against viral and tumor antigens, in the overall success of viroimmunotherapy.


Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/virologia , Apresentação Cruzada/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunidade , Orthopoxvirus/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Neoplasias do Colo/tratamento farmacológico , Apresentação Cruzada/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Memória Imunológica/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Recombinação Genética/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia
10.
Genome Biol ; 21(1): 286, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33272280

RESUMO

According to a recent article published in Genome Biology, Duggan and coworkers sequenced and partially assembled five genomes of smallpox vaccines from the nineteenth century. No information regarding the ends of genomes was presented, and they are important to understand the evolutionary relationship of the different smallpox vaccine genomes during the centuries. We re-assembled the genomes, which include the largest genomes in the vaccinia lineage and one true horsepox strain. Moreover, the assemblies reveal a diverse genetic structure in the genome ends. Our data emphasize the concurrent use of horsepox and horsepox-related viruses as the smallpox vaccine in the nineteenth century.


Assuntos
Orthopoxvirus , Vacina Antivariólica , Varíola , Guerra Civil Norte-Americana , Genômica , Humanos , Varíola/prevenção & controle , Vacina Antivariólica/genética , Estados Unidos
11.
Genome Biol ; 21(1): 287, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33272282

RESUMO

We thank Brinkmann and colleagues for their correspondence and their further investigation into these American Civil War Era vaccination strains. Here, we summarize the difficulties and caveats of work with ancient DNA.


Assuntos
Orthopoxvirus , Vacina Antivariólica , Varíola , Guerra Civil Norte-Americana , Genômica , Humanos , Orthopoxvirus/genética , Varíola/prevenção & controle , Estados Unidos
12.
Viruses ; 12(12)2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33317132

RESUMO

Akhmeta virus is a zoonotic Orthopoxvirus first identified in 2013 in the country of Georgia. Subsequent ecological investigations in Georgia have found evidence that this virus is widespread in its geographic distribution within the country and in its host-range, with rodents likely involved in its circulation in the wild. Yet, little is known about the pathogenicity of this virus in rodents. We conducted the first laboratory infection of Akhmeta virus in CAST/EiJ Mus musculus to further characterize this novel virus. We found a dose-dependent effect on mortality and weight loss (p < 0.05). Anti-orthopoxvirus antibodies were detected in the second- and third-highest dose groups (5 × 104 pfu and 3 × 102 pfu) at euthanasia by day 10, and day 14 post-infection, respectively. Anti-orthopoxvirus antibodies were not detected in the highest dose group (3 × 106 pfu), which were euthanized at day 7 post-infection and had high viral load in tissues, suggesting they succumbed to disease prior to mounting an effective immune response. In order of highest burden, viable virus was detected in the nostril, lung, tail, liver and spleen. All individuals tested in the highest dose groups were DNAemic. Akhmeta virus was highly pathogenic in CAST/EiJ Mus musculus, causing 100% mortality when ≥3 × 102 pfu was administered.


Assuntos
Doenças dos Animais/virologia , Infecção Laboratorial/veterinária , Orthopoxvirus/fisiologia , Infecções por Poxviridae/veterinária , Doenças dos Animais/diagnóstico , Doenças dos Animais/mortalidade , Animais , Feminino , Camundongos , Testes Sorológicos , Carga Viral
13.
Sci Rep ; 10(1): 19307, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33168908

RESUMO

A vaccine for smallpox is no longer administered to the general public, and there is no proven, safe treatment specific to poxvirus infections, leaving people susceptible to infections by smallpox and other zoonotic Orthopoxviruses such as monkeypox. Using vaccinia virus (VACV) as a model organism for other Orthopoxviruses, CRISPR-Cas9 technology was used to target three essential genes that are conserved across the genus, including A17L, E3L, and I2L. Three individual single guide RNAs (sgRNAs) were designed per gene to facilitate redundancy in rendering the genes inactive, thereby reducing the reproduction of the virus. The efficacy of the CRISPR targets was tested by transfecting human embryonic kidney (HEK293) cells with plasmids encoding both SaCas9 and an individual sgRNA. This resulted in a reduction of VACV titer by up to 93.19% per target. Following the verification of CRISPR targets, safe and targeted delivery of the VACV CRISPR antivirals was tested using adeno-associated virus (AAV) as a packaging vector for both SaCas9 and sgRNA. Similarly, AAV delivery of the CRISPR antivirals resulted in a reduction of viral titer by up to 92.97% for an individual target. Overall, we have identified highly specific CRISPR targets that significantly reduce VACV titer as well as an appropriate vector for delivering these CRISPR antiviral components to host cells in vitro.


Assuntos
Sistemas CRISPR-Cas , Dependovirus/genética , Monkeypox/terapia , Orthopoxvirus/metabolismo , RNA Guia/metabolismo , Varíola/terapia , Antivirais , Proteínas de Bactérias/metabolismo , Edição de Genes/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Monkeypox/virologia , Plasmídeos/metabolismo , Varíola/virologia , Transfecção , Vírus Vaccinia
14.
Open Vet J ; 10(2): 144-156, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32821659

RESUMO

Background: Camelpox is the most infectious and economically important disease of camelids that causes significant morbidity and mortality rates. Several live attenuated vaccines against Camelpox virus (CMLV) are produced worldwide by passaging field isolates in cell culture. Sequence of a high passage Saudi isolate of CMLV was previously found closely resembled Vaccinia virus (VACV). Aim: To determine whether other high cell culture passage CMLV isolates are genetically resemble VACV and further to explore the possible mechanism of the resemblance. Methods: We performed polymerase chain reaction and DNA sequence analysis of A-type inclusion body protein (ATIP), L1R, and open reading frame (ORF) 185 genes on different cell culture passage levels of a field isolate, two high passage vaccines, wild-type, and reference strains of CMLV. Results: We demonstrate that additional two high passage attenuated vaccine candidate from Sudan and UAE likewise contain sequences resembling VACV more than CMLV. Furthermore, sequence analysis of the ATIP gene of selected virus passages in cell culture revealed that the shift to VACV-like occurred between passage 11 and 20 and up to the 10th passage the genome still resembles wild-type virus. This observation was further confirmed by recombination analysis which indicated recombination events at ATIP and ORF185 genes occurred at higher passages. Conclusion: We confirmed that the cell culture passage CMLV turns to resemble VACV after cell culture passage and concluded that the resemblance may not be a result of contamination or misidentification as previously thought but could be due to recombination events that occurred during the passage process.


Assuntos
Camelus/virologia , Orthopoxvirus/imunologia , Infecções por Poxviridae/veterinária , Vacinas Atenuadas/genética , Vírus Vaccinia/genética , Animais , Técnicas de Cultura de Células/veterinária , Fases de Leitura Aberta/genética , Orthopoxvirus/genética , Reação em Cadeia da Polimerase/veterinária , Infecções por Poxviridae/prevenção & controle , Infecções por Poxviridae/virologia , Análise de Sequência de DNA/veterinária
15.
Viruses ; 12(8)2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32717784

RESUMO

Camelpox is a viral contagious disease of Old-World camelids sustained by Camelpox virus (CMLV). The disease is characterized by mild, local skin or severe systemic infections and may have a major economic impact due to significant losses in terms of morbidity and mortality, weight loss, and low milk yield. Prevention of camelpox is performed by vaccination. In this study, we investigated the composition of a CMLV-based, live-attenuated commercial vaccine using next-generation sequencing (NGS) technology. The results of this analysis revealed genomic sequences of Modified Vaccinia virus Ankara (MVA).


Assuntos
Orthopoxvirus/genética , Filogenia , Vírus Vaccinia/genética , Vacinas Virais/genética , Sequenciamento Completo do Genoma , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Vacinas Atenuadas/genética
16.
Viruses ; 12(7)2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32629851

RESUMO

Although orthopoxviruses (OPXV) are known to encode a majority of the genes required for replication in host cells, genome-wide genetic screens have revealed that several host pathways are indispensable for OPXV infection. Through a haploid genetic screen, we previously identified several host genes required for monkeypox virus (MPXV) infection, including the individual genes that form the conserved oligomeric Golgi (COG) complex. The COG complex is an eight-protein (COG1-COG8) vesicle tethering complex important for regulating membrane trafficking, glycosylation enzymes, and maintaining Golgi structure. In this study, we investigated the role of the COG complex in OPXV infection using cell lines with individual COG gene knockout (KO) mutations. COG KO cells infected with MPXV and vaccinia virus (VACV) produced small plaques and a lower virus yield compared to wild type (WT) cells. In cells where the KO phenotype was reversed using a rescue plasmid, the size of virus plaques increased demonstrating a direct link between the decrease in viral spread and the KO of COG genes. KO cells infected with VACV displayed lower levels of viral fusion and entry compared to WT suggesting that the COG complex is important for early events in OPXV infection. Additionally, fewer actin tails were observed in VACV-infected KO cells compared to WT. Since COG complex proteins are required for cellular trafficking of glycosylated membrane proteins, the disruption of this process due to lack of individual COG complex proteins may potentially impair the virus-cell interactions required for viral entry and egress. These data validate that the COG complex previously identified in our genetic screens plays a role in OPXV infection.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Orthopoxvirus/fisiologia , Infecções por Poxviridae/metabolismo , Infecções por Poxviridae/virologia , Internalização do Vírus , Proteínas Adaptadoras de Transporte Vesicular/genética , Glicosilação , Complexo de Golgi , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Mutação , Orthopoxvirus/genética , Infecções por Poxviridae/genética
17.
Vopr Virusol ; 65(1): 49-56, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32496721

RESUMO

INTRODUCTION: Currently, new directions in cancer therapy are actively developing, one of which is oncolytic immunotherapy. This approach would be to use of viruses as cancer specific cytolytic agents capable of stimulating both the tumor-specific and non-specific immune response. The objective paper was obtain a recombinant vaccinia virus containing genes encoding immunostimulating molecules and study oncolytic and immunostimulating properties of recombinant virus. MATERIAL AND METHODS: MTT test, ELISA, methods of transient dominant selection. RESULTS: The recombinant vaccinia virus (L-IVP_oncoB) were obtained with deletion of the gene encoding thymidine kinase and had an integrated gene encoding GM-CSF. Also the virus have deletion of the gene encoding viral growth factor and integrated genes encoding synthetic tumor-specific polyepitopic immunogens. It was shown that the modifications made to the viral genome did not affect the growth characteristics of the virus when cultured on CV-1 and 4647 cell cultures, and the cytopathogenic efficacy of the virus was determined in relation to cancer cultures of cells of various genesis. In in vivo experiment, it was revealed that the polyepitopic construct in the genome L-IVP_oncoB is able to initiate a change in the profile of cytokines. DISCUSSION: The obtained data characterized L-IVP_oncoB as a promising cytopathogenic and immunostimulating agent and showed the need for further study of its properties as means of oncolytic immunotherapy. CONCLUSION: The basic experiments on the evaluation of the biological properties of the obtained L-IVP_oncoB, which are necessary for the characterization of the oncolytic virus, have been carried out.


Assuntos
Neoplasias da Mama/terapia , Vírus Oncolíticos/genética , Vírus Vaccinia/genética , Replicação Viral/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/virologia , Linhagem Celular Tumoral , Feminino , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Humanos , Imunoterapia , Orthopoxvirus/genética , Poxviridae/genética , Replicação Viral/imunologia
19.
Biologicals ; 65: 39-41, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32334926

RESUMO

Camelpox virus is the causative agent of Camelpox, a highly contagious disease of camels. A high passage Camelpox virus strain has previously been reported to contain several genes which more closely resemble Vaccinia, a virus species with no known natural host, encompassing various strains that show high inter-strain genomic variation. In this study, we demonstrate that yet another high passage, live attenuated vaccine, comprising a different strain of Camelpox virus, contains genomic sequences that match a differing strain of Vaccinia virus. These results are discussed in the context of hypotheses put forward to explain the unknown origins of Vaccinia virus, suggesting further studies to elucidate evolutionary trajectories of Orthopoxviruses through passaging.


Assuntos
Orthopoxvirus/genética , Vacinas Atenuadas/genética , Vírus Vaccinia/genética , Animais , Camelus , DNA Viral , Infecções por Poxviridae/prevenção & controle , Infecções por Poxviridae/veterinária , Inoculações Seriadas , Vacinas Atenuadas/uso terapêutico
20.
PLoS Pathog ; 16(4): e1008505, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32320436

RESUMO

The wild-derived inbred CAST/EiJ mouse, one of eight founder strains in the Collaborative Cross panel, is an exceptional model for studying monkeypox virus (MPXV), an emerging human pathogen, and other orthopoxviruses including vaccinia virus (VACV). Previous studies suggested that the extreme susceptibility of the CAST mouse to orthopoxviruses is due to an insufficient innate immune response. Here, we focused on the low number of natural killer (NK) cells in the naïve CAST mouse as a contributing factor to this condition. Administration of IL-15 to CAST mice transiently increased NK and CD8+ T cells that could express IFN-γ, indicating that the progenitor cells were capable of responding to cytokines. However, the number of NK cells rapidly declined indicating a defect in their homeostasis. Furthermore, IL-15-treated mice were protected from an otherwise lethal challenge with VACV or MPXV. IL-15 decreased virus spread and delayed death even when CD4+/CD8+ T cells were depleted with antibody, supporting an early protective role of the expanded NK cells. Purified splenic NK cells from CAST mice proliferated in vitro in response to IL-15 and could be activated with IL-12/IL-18 to secrete interferon-γ. Passive transfer of non-activated or activated CAST NK cells reduced VACV spread but only the latter completely prevented death at the virus dose used. Moreover, antibodies to interferon-γ abrogated the protection by activated NK cells. Thus, the inherent susceptibility of CAST mice to orthopoxviruses can be explained by a low level of NK cells and this vulnerability can be overcome either by expanding their NK cells in vivo with IL-15 or by passive transfer of purified NK cells that were expanded and activated in vitro.


Assuntos
Interleucina-15/farmacologia , Células Matadoras Naturais/imunologia , Orthopoxvirus/imunologia , Infecções por Poxviridae/imunologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Feminino , Imunidade Inata/efeitos dos fármacos , Interferon gama/imunologia , Interleucina-15/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Orthopoxvirus/efeitos dos fármacos , Orthopoxvirus/patogenicidade , Infecções por Poxviridae/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Baço/efeitos dos fármacos , Baço/patologia , Baço/virologia , Vírus Vaccinia/imunologia
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