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1.
J Fish Dis ; 44(10): 1531-1542, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34287959

RESUMO

The koi sleepy disease of carp caused by the carp oedema virus (CEV) was observed on farms and in ponds in France since the 2010s. Samples of CEV collected in France over a period of eight years were characterized at the molecular level by sequencing the partial p4a gene. All the sequences, except one, fell into two well-defined genogroups. Sequences obtained from CEV detected in common carp generally clustered in genogroup I and sequences from CEV detected in the koi were assigned to genogroup II. A particular sample was different to the others and represented a putative new genogroup possibly arose from a recombination event between a genogroup II sequence and one from an unknown genogroup. Compared with sequences from CEV of other countries, most of the French sequences exhibited high degree of DNA identities with those published previously, indicating identical sources of viruses. The sequence diversity suggests multiple introductions of the viruses in France. Among the French sequences, two genogroup-specific molecular markers were identified. One was an insertion/deletion identified within a microsatellite and other was a group of single nucleotide polymorphisms. CEV seems to generate genetic diversity via diverse mechanisms: substitutions, indels and recombination events.


Assuntos
Carpas , Doenças dos Peixes/virologia , Variação Genética , Infecções por Poxviridae/veterinária , Poxviridae/genética , Animais , França , Infecções por Poxviridae/virologia
2.
J Virol ; 95(16): e0085221, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34076488

RESUMO

Poxviruses are exceptional in having a complex entry-fusion complex (EFC) that is comprised of 11 conserved proteins embedded in the membrane of mature virions. However, the detailed architecture is unknown and only a few bimolecular protein interactions have been demonstrated by coimmunoprecipitation from detergent-treated lysates and by cross-linking. Here, we adapted the tripartite split green fluorescent protein (GFP) complementation system in order to analyze EFC protein contacts within living cells. This system employs a detector fragment called GFP1-9 comprised of nine GFP ß-strands. To achieve fluorescence, two additional 20-amino-acid fragments called GFP10 and GFP11 attached to interacting proteins are needed, providing the basis for identification of the latter. We constructed a novel recombinant vaccinia virus (VACV-GFP1-9) expressing GFP1-9 under a viral early/late promoter and plasmids with VACV late promoters regulating each of the EFC proteins with GFP10 or GFP11 attached to their ectodomains. GFP fluorescence was detected by confocal microscopy at sites of virion assembly in cells infected with VACV-GFP1-9 and cotransfected with plasmids expressing one EFC-GFP10 and one EFC-GFP11 interacting protein. Flow cytometry provided a quantitative way to determine the interaction of each EFC-GFP10 protein with every other EFC-GFP11 protein in the context of a normal infection in which all viral proteins are synthesized and assembled. Previous EFC protein interactions were confirmed, and new ones were discovered and corroborated by additional methods. Most remarkable was the finding that the small, hydrophobic O3 protein interacted with each of the other EFC proteins. IMPORTANCE Poxviruses are enveloped viruses with a DNA-containing core that enters cells following fusion of viral and host membranes. This essential step is a target for vaccines and therapeutics. The entry-fusion complex (EFC) of poxviruses is unusually complex and comprised of 11 conserved viral proteins. Determination of the structure of the EFC is a prerequisite for understanding the fusion mechanism. Here, we used a tripartite split green fluorescent protein assay to determine the proximity of individual EFC proteins in living cells. A network connecting components of the EFC was derived.


Assuntos
Poxviridae/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Animais , Linhagem Celular , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ligação Proteica , Vírus Vaccinia/genética , Vírus Vaccinia/metabolismo , Vírus Vaccinia/fisiologia , Proteínas Virais de Fusão/genética
3.
Front Immunol ; 12: 689302, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34177946

RESUMO

Salmon Gill Poxvirus Disease (SGPVD) has emerged as a cause of acute mortality in Atlantic salmon (Salmo salar L.) presmolts in Norwegian aquaculture. The clinical phase of the disease is associated with apoptotic cell death in the gill epithelium causing acute respiratory distress, followed by proliferative changes in the regenerating gill in the period after the disease outbreak. In an experimental SGPV challenge trial published in 2020, acute disease was only seen in fish injected with hydrocortisone 24 h prior to infection. SGPV-mediated mortality in the hydrocortisone-injected group was associated with more extensive gill pathology and higher SGPV levels compared to the group infected with SGPV only. In this study based on the same trial, SGPV gene expression and the innate and adaptive antiviral immune response was monitored in gills and spleen in the presence and absence of hydrocortisone. Whereas most SGPV genes were induced from day 3 along with the interferon-regulated innate immune response in gills, the putative SGPV virulence genes of the B22R family were expressed already one day after SGPV exposure, indicating a potential role as early markers of SGPV infection. In gills of the hydrocortisone-injected fish infected with SGPV, MX expression was delayed until day 10, and then expression skyrocketed along with the viral peak, gill pathology and mortality occurring from day 14. A similar expression pattern was observed for Interferon gamma (IFNγ) and granzyme A (GzmA) in the gills, indicating a role of acute cytotoxic cell activity in SGPVD. Duplex in situ hybridization demonstrated effects of hydrocortisone on the number and localization of GzmA-containing cells, and colocalization with SGPV infected cells in the gill. SGPV was generally not detected in spleen, and gill infection did not induce any corresponding systemic immune activity in the absence of stress hormone injection. However, in fish injected with hydrocortisone, IFNγ and GzmA gene expression was induced in spleen in the days prior to acute mortality. These data indicate that suppressed mucosal immune response in the gills and the late triggered systemic immune response in the spleen following hormonal stress induction may be the key to the onset of clinical SGPVD.


Assuntos
Anti-Inflamatórios/farmacologia , Doenças dos Peixes/imunologia , Hidrocortisona/farmacologia , Imunidade nas Mucosas/efeitos dos fármacos , Infecções por Poxviridae/imunologia , Salmo salar/imunologia , Animais , Antígenos CD4/genética , Antígenos CD4/imunologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Brânquias/imunologia , Brânquias/virologia , Granzimas/genética , Granzimas/imunologia , Interações Hospedeiro-Patógeno , Interferon gama/genética , Interferon gama/imunologia , Membrana Mucosa/imunologia , Poxviridae/genética , Salmo salar/genética , Salmo salar/virologia
4.
Eur J Med Chem ; 221: 113485, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-33965861

RESUMO

Currently, smallpox, caused by the variola virus belonging to the poxvirus family, has been completely eradicated according to the WHO. However, other representatives of poxviruses, such as vaccinia virus, cowpox virus, ectromelia virus, monkeypox virus, mousepox virus and others, remain in the natural environment and can infect both animals and humans. The pathogens of animal diseases, belonging to the category with a high epidemic risk, have already caused several outbreaks among humans, and can, in an unfavorable combination of circumstances, cause not only an epidemic, but also a pandemic. Despite the fact that there are protocols for the treatment of poxvirus infections, the targeted design of new drugs will increase their availability and expand the arsenal of antiviral chemotherapeutic agents. One of the potential targets of poxviruses is the p37 protein, which is a tecovirimat target. This protein is relatively small, has no homologs among proteins of humans and other mammals and is necessary for the replication of viral particles, which makes it attractive target for virtual screening. Using the I-TASSER modelling and molecular dynamics refinement the p37 orthopox virus protein model was obtained and its was confirmed by ramachandran plot analysis and superimposition of the model with the template protein with similar function. A virtual library of adamantane containing compounds was generated and a number of potential inhibitors were chosen from virtual library using molecular docking. Several compounds bearing adamantane moiety were synthesized and their biological activity was tested in vitro on vaccinia, cowpox and mousepox viruses. The new compounds inhibiting vaccinia virus replication with IC50 concentrations between 0.133 and 0.515 µM were found as a result of the research. The applied approach can be useful in the search of new inhibitors of orthopox reproduction. The proposed approach may be suitable for the design of new poxvirus inhibitors containing cage structural moiety.


Assuntos
Adamantano/farmacologia , Antivirais/farmacologia , Desenho de Fármacos , Proteínas de Membrana/antagonistas & inibidores , Poxviridae/efeitos dos fármacos , Proteínas do Envelope Viral/antagonistas & inibidores , Adamantano/síntese química , Adamantano/química , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Proteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismo
5.
Viruses ; 13(3)2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33802414

RESUMO

Mass mortality events of common carp (Cyprinus carpio, carp) associated with carp edema virus (CEV) alone or in coinfections with koi herpesvirus (KHV), is an emerging issue. Despite recent outbreaks of CEV in wild carp populations, the host range of North American species has not been well studied. To that end, we intensively sampled carp (n = 106) and co-habiting native fish species (n = 5 species; n = 156 total fish) from a CEV-suspect mass-mortality event of carp in a small Minnesota lake (Lake Swartout). Additionally, fecal and regurgitant samples (n = 73 each) from double-crested cormorants (Phalacrocorax auritus, DCCO) were sampled to test the potential of DCCO to act as a vector for virus transmission. CEV was confirmed to be widespread in the Lake Swartout carp population during the outbreak with high viral loads and histological confirmation, suggesting that CEV was the cause of the mortality event. There were no detections of CEV in any native fish species; however, DCCO regurgitant and fecal samples were positive for CEV DNA. In addition, three CEV-positive and one CEV + KHV-positive mortality events were confirmed with no observed mortality or morbidity of non-carp species in other lakes. This study provides evidence that CEV infection and disease may be specific to carp during mortality events with mixed-species populations, identifies DCCO as a potential vector for CEV, and further expands the known range of CEV, as well as coinfections with KHV, in North America.


Assuntos
Coinfecção/veterinária , Surtos de Doenças , Doenças dos Peixes/epidemiologia , Infecções por Herpesviridae/veterinária , Infecções por Poxviridae , Poxviridae , Animais , Carpas , Coinfecção/epidemiologia , Doenças dos Peixes/virologia , Infecções por Herpesviridae/epidemiologia , Especificidade de Hospedeiro , Lagos , Minnesota/epidemiologia , Poxviridae/isolamento & purificação , Poxviridae/fisiologia , Infecções por Poxviridae/epidemiologia , Infecções por Poxviridae/veterinária
6.
Viruses ; 13(3)2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806696

RESUMO

During 2019, five carcasses of juvenile Egyptian fruit bats (Rousettus aegyptiacus) were submitted to the Kimron Veterinary Institute. These bats exhibited typical poxvirus like lesion plaques of different sizes on the skin, abdomen and the ventral side of the wings. Clinical and histopathological findings suggested a poxvirus infection. Infectious virus was isolated from skin swabs, skin tissue and tongue of the dead bats and was further confirmed to be a Poxvirus by molecular diagnosis using PCR with pan-chordopoxviruses primers. All the dead bats were found positive for two Poxvirus genes encoding a metalloproteinase and DNA dependent DNA polymerase. In this study, a novel real time quantitative PCR (qPCR) assay was established to further confirmed the presence of specific poxvirus viral DNA in all pathologically tested tissues. Moreover, according to sequence analysis, the virus was found to be highly similar to the recently discovered Israeli Rousettus aegyptiacus Pox Virus (IsrRAPXV).


Assuntos
Quirópteros/virologia , DNA Viral/isolamento & purificação , Infecções por Poxviridae/virologia , Poxviridae/isolamento & purificação , Animais
7.
Vet Dermatol ; 32(3): 283-e77, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33728715

RESUMO

BACKGROUND: Interleukin (IL)-2 stimulates antitumour immunity and is successfully used for the treatment of different neoplasias. HYPOTHESIS/OBJECTIVES: Canarypox virus locally expressing feline IL-2 is safe and can be used to treat equine sarcoids. ANIMALS: Twenty horses of different breeds with a median age of eight years (interquartile range 6.0-13.3 years) and a total number of 59 sarcoids were included in the study. METHODS: In this prospective clinical trial, sarcoids were injected twice seven days apart, with a recombinant canarypox virus expressing feline IL-2. Complete blood counts (CBC) and fibrinogen levels were measured before treatment and on days 1, 2, 7 and 8. RESULTS: Complete regression was achieved in eight horses (40%) and partial regression in two horses (10%). No change in sarcoid size was observed in two horses (10%) and the disease progressed in five horses (25%). Sarcoids of three horses (15%) showed initial response followed by tumour growth. There were no significant changes in CBC and fibrinogen levels after either injection. One horse developed a mild fever the day after each injection, which subsided without treatment the following day. CONCLUSIONS: Treatment of equine sarcoids with recombinant canarypox virus expressing feline IL-2 seems to be a safe therapy option. Although the expression of IL-2 after vector injection and its biological activity in horses were not proven in this study, the treatment resulted in regression and partial regression in 50% of the cases. Further studies are necessary to verify these findings and to establish a treatment protocol.


Assuntos
Doenças do Gato , Doenças dos Cavalos , Poxviridae , Sarcoidose , Neoplasias Cutâneas , Animais , Gatos , Doenças dos Cavalos/terapia , Cavalos , Interleucina-2/genética , Sarcoidose/veterinária , Neoplasias Cutâneas/terapia , Neoplasias Cutâneas/veterinária
8.
Emerg Infect Dis ; 27(4): 1177-1180, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33754985

RESUMO

We obtained the complete sequence of a novel poxvirus, tentatively named Brazilian porcupinepox virus, from a wild porcupine (Coendou prehensilis) in Brazil that had skin and internal lesions characteristic of poxvirus infection. The impact of this lethal poxvirus on the survival of this species and its potential zoonotic importance remain to be investigated.


Assuntos
Infecções por Poxviridae , Poxviridae , Brasil , Genômica , Humanos , Filogenia
9.
Arch Virol ; 166(6): 1729-1733, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33745070

RESUMO

Anthrax is endemic in Georgia, as are multiple zoonotic poxviruses. Poxvirus-associated infections share some clinical manifestations and exposure risks with anthrax, and so it is important to distinguish between the two. With this in mind, an archived collection of anthrax-negative DNA samples was retrospectively screened for poxviruses, and of the 148 human samples tested, 64 were positive. Sequence analysis confirmed the presence of orf virus, bovine papular stomatitis virus, and pseudocowpox virus. This study provides evidence of previously unrecognized poxvirus infections in Georgia and highlights the benefit of the timely identification of such infections by improving laboratory capacity.


Assuntos
Infecções por Poxviridae/virologia , Poxviridae/genética , República da Geórgia/epidemiologia , Humanos , Filogenia , Poxviridae/isolamento & purificação , Infecções por Poxviridae/epidemiologia , Estudos Retrospectivos
12.
J Fish Dis ; 44(7): 939-947, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33591616

RESUMO

Mortality in wild fish populations represents a challenging issue for public fish health inspectors. When a single fish species is involved, an infective aetiology is frequently suspected, with focus on viral notifiable diseases. However, other viral agents not subjected to regulation and causing mortality in common carp have been reported such as carp edema virus (CEV). In mid-June 2020, a severe common carp mortality was observed in an artificial lake in north-east of Italy. Sleepy fish were noted some days before the beginning of the mortality itself, which lasted several days and involved over 340 adult specimens. During the outbreak, water temperature was around 15°C, water quality was normal, and no adverse meteorological events were reported in the area. Four specimens, which showed severe cutaneous hyperaemia and increased mucus production on skin and gills, were tested by bacteriological methods and virological analysis targeting the main carp pathogens. Molecular analysis performed on gills, kidney and brains from all the fish analysed resulted positive for CEV, which, based on anamnestic information and laboratory findings, was considered the responsible for the mortality event herein described.


Assuntos
Carpas/virologia , Doenças dos Peixes/mortalidade , Infecções por Poxviridae/veterinária , Poxviridae/classificação , Animais , Animais Selvagens , Doenças dos Peixes/epidemiologia , Doenças dos Peixes/virologia , Itália/epidemiologia , Filogenia , Infecções por Poxviridae/epidemiologia , Infecções por Poxviridae/mortalidade , Infecções por Poxviridae/virologia , Proteínas Virais/genética
13.
Viruses ; 13(2)2021 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-33572619

RESUMO

Emerging viral disease is a significant concern, with potential consequences for human, animal and environmental health. Over the past several decades, multiple novel viruses have been found in wildlife species, including reptiles, and often pose a major threat to vulnerable species. However, whilst a large number of viruses have been described in turtles, information on poxvirus in cheloniids remains scarce, with no molecular sequence data available to date. This study characterizes, for the first time, a novel poxvirus, here tentatively designated cheloniid poxvirus 1 (ChePV-1). The affected cutaneous tissue, recovered from a green sea turtle (Chelonia mydas) captured off the Central Queensland coast of Australia, underwent histological examination, transmission electron microscopy (TEM), DNA extraction and genomic sequencing. The novel ChePV-1 was shown to be significantly divergent from other known poxviruses and showed the highest sequence similarity (89.3%) to avipoxviruses (shearwater poxvirus 2 (SWPV2)). This suggests the novel ChePV-1 may have originated from a common ancestor that diverged from an avipoxvirus-like progenitor. The genome contained three predicted unique genes and a further 15 genes being truncated/fragmented compared to SWPV2. This is the first comprehensive study that demonstrates evidence of poxvirus infection in a marine turtle species, as well as a rare example of an avipoxvirus crossing the avian-host barrier. This finding warrants further investigations into poxvirus infections between species in close physical proximity, as well as in vitro and in vivo studies of pathogenesis and disease.


Assuntos
Doenças Transmissíveis Emergentes/veterinária , Infecções por Poxviridae/veterinária , Tartarugas/virologia , Animais , Austrália , Doenças Transmissíveis Emergentes/virologia , Filogenia , Poxviridae/classificação , Poxviridae/genética , Poxviridae/isolamento & purificação , Infecções por Poxviridae/virologia
14.
Sci Rep ; 11(1): 1798, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469067

RESUMO

Success in smallpox eradication was enabled by the absence of non-human reservoir for smallpox virus. However, other poxviruses with a wider host spectrum can infect humans and represent a potential health threat to humans, highlighted by a progressively increasing number of infections by (re)emerging poxviruses, requiring new improved diagnostic and epidemiological tools. We describe here a real-time PCR assay targeting a highly conserved region of the poxvirus genome, thus allowing a pan-Poxvirus detection (Chordopoxvirinae and Entomopoxvirinae). This system is specific (99.8% for vertebrate samples and 99.7% for arthropods samples), sensitive (100% for vertebrate samples and 86.3% for arthropods samples) and presents low limit of detection (< 1000 DNA copies/reaction). In addition, this system could be also valuable for virus discovery and epidemiological projects.


Assuntos
Poxviridae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA Viral/genética , Genes Virais , Humanos , Limite de Detecção , Filogenia , Poxviridae/genética
15.
J Fish Dis ; 44(4): 371-378, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33460151

RESUMO

The importance of world aquaculture production grows annually together with the increasing need to feed the global human population. Common carp (Cyprinus carpio) is one of the most important freshwater fish in global aquaculture. Unfortunately, carp production is affected by numerous diseases of which viral diseases are the most serious. Koi herpesvirus disease (KHVD), spring viraemia of carp (SVC), and during the last decades also koi sleepy disease (KSD) are currently the most harmful viral diseases of common carp. This review summarizes current knowledge about carp edema virus (CEV), aetiological agent causing KSD, and about the disease itself. Furthermore, the article is focused on summarizing the available information about the antiviral immune response of common carp, like production of class I interferons (IFNs), activation of cytotoxic cells, and production of antibodies by B cells focusing on anti-CEV immunity.


Assuntos
Imunidade Adaptativa , Carpas , Doenças dos Peixes/imunologia , Imunidade Inata , Infecções por Poxviridae/veterinária , Poxviridae/fisiologia , Animais , Doenças dos Peixes/virologia , Infecções por Poxviridae/imunologia , Infecções por Poxviridae/virologia
16.
Viruses ; 12(12)2020 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-33291286

RESUMO

The presence of carp edema virus (CEV) was confirmed in imported ornamental koi in Chiang Mai province, Thailand. The koi showed lethargy, loss of swimming activity, were lying at the bottom of the pond, and gasping at the water's surface. Some clinical signs such as skin hemorrhages and ulcers, swelling of the primary gill lamella, and necrosis of gill tissue, presented. Clinical examination showed co-infection by opportunistic pathogens including Dactylogyrus sp., Gyrodactylus sp. and Saprolegnia sp. on the skin and gills. Histopathologically, the gill of infected fish showed severe necrosis of epithelial cells and infiltrating of eosinophilic granular cells. Electron microscope examination detected few numbers of virions were present in the cytoplasm of gill tissue which showed an electron dense core with surface membranes worn by surface globular units. Molecular detection of CEV DNA from gill samples of fish was performed by polymerase chain reaction (PCR) and confirmed by nested-PCR. Phylogenetic analyses revealed that CEV isolate had 99.8% homology with the CEV isolated from South Korea (KY946715) and Germany (KY550420), and was assigned to genogroup IIa. In conclusion, this report confirmed the presence of CEV infection of koi Cyprinus carpio in Chiang Mai province, Thailand using pathological and molecular approaches.


Assuntos
Carpas/virologia , Doenças dos Peixes/virologia , Poxviridae , Animais , Biópsia , Genes Virais , Brânquias/patologia , Brânquias/virologia , Filogenia , Reação em Cadeia da Polimerase , Poxviridae/classificação , Poxviridae/genética , Poxviridae/ultraestrutura , Tailândia
17.
Front Immunol ; 11: 567348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33154747

RESUMO

We conducted a large genome-wide association study (GWAS) of the immune responses to primary smallpox vaccination in a combined cohort of 1,653 subjects. We did not observe any polymorphisms associated with standard vaccine response outcomes (e.g., neutralizing antibody, T cell ELISPOT response, or T cell cytokine production); however, we did identify a cluster of SNPs on chromosome 5 (5q31.2) that were significantly associated (p-value: 1.3 x 10-12 - 1.5x10-36) with IFNα response to in vitro poxvirus stimulation. Examination of these SNPs led to the functional testing of rs1131769, a non-synonymous SNP in TMEM173 causing an Arg-to-His change at position 232 in the STING protein-a major regulator of innate immune responses to viral infections. Our findings demonstrate differences in the ability of the two STING variants to phosphorylate the downstream intermediates TBK1 and IRF3 in response to multiple STING ligands. Further downstream in the STING pathway, we observed significantly reduced expression of type I IFNs (including IFNα) and IFN-response genes in cells carrying the H232 variant. Subsequent molecular modeling of both alleles predicted altered ligand binding characteristics between the two variants, providing a potential mechanism underlying differences in inter-individual responses to poxvirus infection. Our data indicate that possession of the H232 variant may impair STING-mediated innate immunity to poxviruses. These results clarify prior studies evaluating functional effects of genetic variants in TMEM173 and provide novel data regarding genetic control of poxvirus immunity.


Assuntos
Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , Infecções por Poxviridae/genética , Infecções por Poxviridae/imunologia , Poxviridae/imunologia , Alelos , Suscetibilidade a Doenças , Efeito Fundador , Expressão Gênica , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Imunidade Inata/genética , Fenômenos Imunogenéticos , Ligantes , Proteínas de Membrana/metabolismo , Modelos Biológicos , Fosforilação , Infecções por Poxviridae/virologia , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
18.
Vaccine ; 38(49): 7774-7779, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33164801

RESUMO

Virus-like particles (VLPs) are considered useful tools for vaccine development because they induce an immune response and are safe. In addition, VLPs may be useful as a platform for the presentation of foreign antigens to elicit immune responses. In this study, we aimed to produce a chimeric VLP composed of L1 protein of bovine papillomavirus type 6 (BPV6-L1) that can display an entire foreign protein on its surface. Based on prediction of the conformational structure of VLP of BPV6-L1 (BPV6-VLP), candidate insertion sites for the foreign protein into BPV6-VLP were identified. Fusion proteins of BPV6-L1 and EGFP as a model foreign protein were constructed and produced. Only the fusion protein in which EGFP was inserted between amino acids 136 and 137 of BPV6-L1 self-assembled into VLPs and did not exhibit hindrance of the conformation of EGFP. Chimeric BPV6-VLP-immunized mice produced specific IgG against both BPV6 and EGFP. This is the first demonstration of the production of an immunogenic, bivalent, chimeric BPV6-VLP incorporating an entire protein for stable surface display. Thus, immunogenic chimeric BPV6-VLP may constitute a promising vaccine platform.


Assuntos
Poxviridae , Vacinas de Partículas Semelhantes a Vírus , Animais , Camundongos , Papillomaviridae/genética , Vacinas de Partículas Semelhantes a Vírus/genética
19.
PLoS Pathog ; 16(11): e1009069, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33253317

RESUMO

Insects are known to host a wide variety of beneficial microbes that are fundamental to many aspects of their biology and have substantially shaped their evolution. Notably, parasitoid wasps have repeatedly evolved beneficial associations with viruses that enable developing wasps to survive as parasites that feed from other insects. Ongoing genomic sequencing efforts have revealed that most of these virus-derived entities are fully integrated into the genomes of parasitoid wasp lineages, representing endogenous viral elements (EVEs) that retain the ability to produce virus or virus-like particles within wasp reproductive tissues. All documented parasitoid EVEs have undergone similar genomic rearrangements compared to their viral ancestors characterized by viral genes scattered across wasp genomes and specific viral gene losses. The recurrent presence of viral endogenization and genomic reorganization in beneficial virus systems identified to date suggest that these features are crucial to forming heritable alliances between parasitoid wasps and viruses. Here, our genomic characterization of a mutualistic poxvirus associated with the wasp Diachasmimorpha longicaudata, known as Diachasmimorpha longicaudata entomopoxvirus (DlEPV), has uncovered the first instance of beneficial virus evolution that does not conform to the genomic architecture shared by parasitoid EVEs with which it displays evolutionary convergence. Rather, DlEPV retains the exogenous viral genome of its poxvirus ancestor and the majority of conserved poxvirus core genes. Additional comparative analyses indicate that DlEPV is related to a fly pathogen and contains a novel gene expansion that may be adaptive to its symbiotic role. Finally, differential expression analysis during virus replication in wasps and fly hosts demonstrates a unique mechanism of functional partitioning that allows DlEPV to persist within and provide benefit to its parasitoid wasp host.


Assuntos
Entomopoxvirinae/genética , Genoma Viral/genética , Genômica , Poxviridae/genética , Simbiose , Vespas/virologia , Animais , Entomopoxvirinae/fisiologia , Feminino , Masculino , Poxviridae/fisiologia , Replicação Viral/genética
20.
Viruses ; 12(11)2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33198108

RESUMO

Repurposing of approved drugs that target host functions also important for virus replication promises to overcome the shortage of antiviral therapeutics. Mostly, virus biology including initial screening of antivirals is studied in conventional monolayer cells. The biology of these cells differs considerably from infected tissues. 3D culture models with characteristics of human tissues may reflect more realistically the in vivo events during infection. We screened first, second, and third generation epidermal growth factor receptor (EGFR)-inhibitors with different modes of action and the EGFR-blocking monoclonal antibody cetuximab in a 3D cell culture infection model with primary human keratinocytes and cowpox virus (CPXV) for antiviral activity. Antiviral activity of erlotinib and osimertinib was nearly unaffected by the cultivation method similar to the virus-directed antivirals tecovirimat and cidofovir. In contrast, the host-directed inhibitors afatinib and cetuximab were approx. 100-fold more efficient against CPXV in the 3D infection model, similar to previous results with gefitinib. In summary, inhibition of EGFR-signaling downregulates virus replication comparable to established virus-directed antivirals. However, in contrast to virus-directed inhibitors, in vitro efficacy of host-directed antivirals might be seriously affected by cell cultivation. Results obtained for afatinib and cetuximab suggest that screening of such drugs in standard monolayer culture might underestimate their potential as antivirals.


Assuntos
Antivirais/farmacologia , Técnicas de Cultura de Células , Descoberta de Drogas/métodos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Poxviridae/efeitos dos fármacos , Poxviridae/fisiologia , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Esferoides Celulares , Replicação Viral
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