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1.
Microbiol Spectr ; 9(2): e0006421, 2021 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-34585975

RESUMO

Viruses of the phylum Nucleocytoviricota, or nucleo-cytoplasmic large DNA viruses (NCLDVs), undergo a cytoplasmic or nucleo-cytoplasmic cycle, the latter of which involves both nuclear and cytoplasmic compartments to proceed viral replication. Medusavirus, a recently isolated NCLDV, has a nucleo-cytoplasmic replication cycle in amoebas during which the host nuclear membrane apparently remains intact, a unique feature among amoeba-infecting NCLDVs. The medusavirus genome lacks most transcription genes but encodes a full set of histone genes. To investigate its infection strategy, we performed a time course RNA sequencing (RNA-seq) experiment. All viral genes were transcribed and classified into five temporal expression clusters. The immediate early genes (cluster 1, 42 genes) were mostly (83%) of unknown functions, frequently (95%) associated with a palindromic promoter-like motif, and often (45%) encoded putative nucleus-localized proteins. These results suggest massive reshaping of the host nuclear environment by viral proteins at an early stage of infection. Genes in other expression clusters (clusters 2 to 5) were assigned to various functional categories. The virally encoded core histone genes were in cluster 3, whereas the viral linker histone H1 gene was in cluster 1, suggesting they have distinct roles during the course of the virus infection. The transcriptional profile of the host Acanthamoeba castellanii genes was greatly altered postinfection. Several encystment-related host genes showed increased representation levels at 48 h postinfection, which is consistent with the previously reported amoeba encystment upon medusavirus infection. IMPORTANCE Medusavirus is an amoeba-infecting giant virus that was isolated from a hot spring in Japan. It belongs to the proposed family "Medusaviridae" in the phylum Nucleocytoviricota. Unlike other amoeba-infecting giant viruses, medusavirus initiates its DNA replication in the host nucleus without disrupting the nuclear membrane. Our RNA sequencing (RNA-seq) analysis of its infection course uncovered ordered viral gene expression profiles. We identified temporal expression clusters of viral genes and associated putative promoter motifs. The subcellular localization prediction showed a clear spatiotemporal correlation between gene expression timing and localization of the encoded proteins. Notably, the immediate early expression cluster was enriched in genes targeting the nucleus, suggesting the priority of remodeling the host intranuclear environment during infection. The transcriptional profile of amoeba genes was greatly altered postinfection.


Assuntos
Acanthamoeba castellanii/virologia , Núcleo Celular/virologia , Vírus de DNA/crescimento & desenvolvimento , Vírus de DNA/genética , Replicação Viral/genética , Sequência de Bases , Replicação do DNA/genética , Genoma Viral/genética , Histonas/genética , Família Multigênica/genética , RNA-Seq , Análise de Sequência de RNA , Transcriptoma/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
2.
Sci Rep ; 11(1): 5025, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33658544

RESUMO

Bioconversion of organic materials is the foundation of many applications in chemical engineering, microbiology and biochemistry. Herein, we introduce a new methodology to quantitatively determine conversion of biomass in viral infections while simultaneously imaging morphological changes of the host cell. As proof of concept, the viral replication of an unidentified giant DNA virus and the cellular response of an amoebal host are studied using soft X-ray microscopy, titration dilution measurements and thermal gravimetric analysis. We find that virions produced inside the cell are visible from 18 h post infection and their numbers increase gradually to a burst size of 280-660 virions. Due to the large size of the virion and its strong X-ray absorption contrast, we estimate that the burst size corresponds to a conversion of 6-12% of carbonaceous biomass from amoebal host to virus. The occurrence of virion production correlates with the appearance of a possible viral factory and morphological changes in the phagosomes and contractile vacuole complex of the amoeba, whereas the nucleus and nucleolus appear unaffected throughout most of the replication cycle.


Assuntos
Acanthamoeba/virologia , Vírus de DNA/ultraestrutura , DNA Viral/genética , Genoma Viral , Vírus Gigantes/ultraestrutura , Vírion/ultraestrutura , Acanthamoeba/ultraestrutura , Biomassa , Vírus de DNA/genética , Vírus de DNA/crescimento & desenvolvimento , Vírus de DNA/isolamento & purificação , DNA Viral/biossíntese , Vírus Gigantes/genética , Vírus Gigantes/crescimento & desenvolvimento , Vírus Gigantes/isolamento & purificação , Interações Hospedeiro-Patógeno/genética , Fagossomos/ultraestrutura , Fagossomos/virologia , Microbiologia do Solo , Termogravimetria , Vacúolos/ultraestrutura , Vacúolos/virologia , Vírion/genética , Vírion/crescimento & desenvolvimento , Replicação Viral , Microtomografia por Raio-X
3.
Virol J ; 16(1): 158, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842897

RESUMO

BACKGROUND: After the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genus Acanthamoeba; however other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated. METHODS: In this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays. RESULTS: We observed, through optical and IF microscopy, morphological changes in V. vermiformis cells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant. CONCLUSIONS: The results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.


Assuntos
Vírus de DNA/crescimento & desenvolvimento , Vírus Gigantes/crescimento & desenvolvimento , Replicação Viral , Antígenos Virais/análise , Vírus de DNA/ultraestrutura , Vírus Gigantes/ultraestrutura , Lobosea/virologia , Microscopia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Vírion/química , Vírion/ultraestrutura
4.
Viruses ; 11(11)2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31703327

RESUMO

Typical viral propagation involves sequential viral entry, uncoating, replication, gene transcription and protein synthesis, and virion assembly and release. Some viral proteins must be transported into host nucleus to facilitate viral propagation, which is essential for the production of mature virions. During the transport process, nuclear localization signals (NLSs) play an important role in guiding target proteins into nucleus through the nuclear pore. To date, some classical nuclear localization signals (cNLSs) and non-classical NLSs (ncNLSs) have been identified in a number of viral proteins. These proteins are involved in viral replication, expression regulation of viral genes and virion assembly. Moreover, other proteins are transported into nucleus with unknown mechanisms. This review highlights our current knowledge about the nuclear trafficking of cellular proteins associated with viral propagation.


Assuntos
Vírus de DNA/crescimento & desenvolvimento , Sinais de Localização Nuclear , Replicação Viral , Capsídeo/metabolismo , Núcleo Celular/metabolismo , Vírus de DNA/metabolismo , Genoma Viral , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Poro Nuclear/metabolismo , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/genética , Vírion/metabolismo , Montagem de Vírus , Internalização do Vírus
5.
Arch Virol ; 164(12): 3035-3043, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31602543

RESUMO

Seasonally recurrent outbreaks of mass mortality in Pacific oysters (Crassostrea gigas) caused by microvariant genotypes of ostreid herpesvirus 1 (OsHV-1) occur in Europe, New Zealand and Australia. The incubation period for OsHV-1 under experimental conditions is 48-72 hours and depends on water temperature, as does the mortality. An in vivo growth curve for OsHV-1 was determined by quantifying OsHV-1 DNA at 10 time points between 2 and 72 hours after exposure to OsHV-1. The peak replication rate was the same at 18 °C and 22 °C; however, there was a longer period of amplification leading to a higher peak concentration at 22 °C (2.34 × 107 copies/mg at 18 hours) compared to 18 °C (1.38 × 105 copies/mg at 12 hours). The peak viral concentration preceded mortality by 72 hours and 20 hours at 18 °C and 22 °C, respectively. Cumulative mortality to day 14 was 45.9% at 22 °C compared to 0.3% at 18 °C. The prevalence of OsHV-1 infection after 14 days at 18 °C was 33.3%. No mortality from OsHV-1 occurred when the water temperature in tanks of oysters challenged at 18 °C was increased to 22 °C for 14 days. The influence of water temperature prior to exposure to OsHV-1 and during the initial virus replication is an important determinant of the outcome of infection in C. gigas.


Assuntos
Crassostrea/fisiologia , Crassostrea/virologia , Vírus de DNA/crescimento & desenvolvimento , Frutos do Mar/virologia , Animais , Crassostrea/crescimento & desenvolvimento , Vírus de DNA/genética , DNA Viral/genética , Temperatura
6.
J Virol Methods ; 274: 113729, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31513859

RESUMO

Pathogenesis and reservoir host adaptation of animal and zoonotic viruses are poorly understood due to missing adequate cell culture and animal models. The bank vole (Myodes glareolus) and common vole (Microtus arvalis) serve as hosts for a variety of zoonotic pathogens. For a better understanding of virus association to a putative animal host, we generated two novel cell lines from bank voles of different evolutionary lineages and two common vole cell lines and assayed their susceptibility, replication and cytopathogenic effect (CPE) formation for rodent-borne, suspected to be rodent-associated or viruses with no obvious rodent association. Already established bank vole cell line BVK168, used as control, was susceptible to almost all viruses tested and efficiently produced infectious virus for almost all of them. The Puumala orthohantavirus strain Vranica/Hällnäs showed efficient replication in a new bank vole kidney cell line, but not in the other four bank and common vole cell lines. Tula orthohantavirus replicated in the kidney cell line of common voles, but was hampered in its replication in the other cell lines. Several zoonotic viruses, such as Cowpox virus, Vaccinia virus, Rift Valley fever virus, and Encephalomyocarditis virus 1 replicated in all cell lines with CPE formation. West Nile virus, Usutu virus, Sindbis virus and Tick-borne encephalitis virus replicated only in a part of the cell lines, perhaps indicating cell line specific factors involved in replication. Rodent specific viruses differed in their replication potential: Murine gammaherpesvirus-68 replicated in the four tested vole cell lines, whereas murine norovirus failed to infect almost all cell lines. Schmallenberg virus and Foot-and-mouth disease virus replicated in some of the cell lines, although these viruses have never been associated to rodents. In conclusion, these newly developed cell lines may represent useful tools to study virus-cell interactions and to identify and characterize host cell factors involved in replication of rodent associated viruses.


Assuntos
Arvicolinae , Linhagem Celular , Vírus de DNA/crescimento & desenvolvimento , Vírus de RNA/crescimento & desenvolvimento , Cultura de Vírus/métodos , Animais , Efeito Citopatogênico Viral , Replicação Viral
7.
Anal Chem ; 91(20): 12962-12970, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31509389

RESUMO

Electrospraying (ES) dissolved viral particles, followed by charge reduction and size analysis with a differential mobility analyzer (DMA), offers a flexible size-analysis tool for small particles in solution. The technique relies on pioneering work by Kaufman and colleagues, commercialized by TSI, and often referred to as GEMMA. However, viral studies with TSI's GEMMA have suffered from limited resolving power, possibly because of imperfections in either the instrument (DMA or charge reduction) or the sample solution preparation. Here, we explore the limits of the resolution achievable by GEMMA, taking advantage of (i) cleaner charge reduction methods and (ii) DMAs of higher resolving power. Analysis of the literature provides indications that mobility peak widths (fwhm) of 2% or less may be achieved by combining careful sample preparation with improved instrumentation. Working with purified PP7 bacteriophage particles small enough to be classifiable by existing high-resolution DMAs, we confirm that fairly narrow viral mobility peaks may be obtained (relative full width at half-maximum fwhm <5%). Comparison of spectra of a given apian virus sample obtained with TSI's GEMMA and our improved instrumentation confirms that one critical limitation is the DMA. This is further verified by narrow peaks from murine parvovirus, norovirus, and encephalomyelitis virus samples, obtained in our improved GEMMA with little sample preparation, directly from infected cell cultures. Classification of purified large (60 nm) coliphage PR772 particles leads to broad peaks, due to both viral degradation and limited intrinsic resolution of the DMAs used to cover the range of such large particles. We conclude that improved DMAs suitable for high-resolution analysis of particles larger than 30 nm need to be developed to determine the intrinsic mobility width of viral particles.


Assuntos
Infecções por Vírus de DNA/diagnóstico , Vírus de DNA/crescimento & desenvolvimento , Infecções por Vírus de RNA/diagnóstico , Vírus de RNA/crescimento & desenvolvimento , Espectrometria de Massas por Ionização por Electrospray/métodos , Vírion/isolamento & purificação , Vírion/fisiologia , Animais , Abelhas/virologia , Infecções por Vírus de DNA/virologia , Camundongos , Infecções por Vírus de RNA/virologia
8.
Viruses ; 11(9)2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31450758

RESUMO

Autophagy is a catabolic biological process in the body. By targeting exogenous microorganisms and aged intracellular proteins and organelles and sending them to the lysosome for phagocytosis and degradation, autophagy contributes to energy recycling. When cells are stimulated by exogenous pathogenic microorganisms such as viruses, activation or inhibition of autophagy is often triggered. As autophagy has antiviral effects, many viruses may escape and resist the process by encoding viral proteins. At the same time, viruses can also use autophagy to enhance their replication or increase the persistence of latent infections. Here, we give a brief overview of autophagy and DNA viruses and comprehensively review the known interactions between human and animal DNA viruses and autophagy and the role and mechanisms of autophagy in viral DNA replication and DNA virus-induced innate and acquired immunity.


Assuntos
Autofagia/fisiologia , Vírus de DNA , Imunidade Adaptativa , Adenoviridae/crescimento & desenvolvimento , Adenoviridae/imunologia , Adenoviridae/metabolismo , Animais , Autofagossomos/metabolismo , Vírus de DNA/crescimento & desenvolvimento , Vírus de DNA/imunologia , Vírus de DNA/metabolismo , Herpesviridae/crescimento & desenvolvimento , Herpesviridae/imunologia , Herpesviridae/metabolismo , Interações entre Hospedeiro e Microrganismos , Humanos , Evasão da Resposta Imune , Imunidade Inata , Lisossomos/metabolismo , Papillomaviridae/crescimento & desenvolvimento , Papillomaviridae/imunologia , Papillomaviridae/metabolismo , Fagocitose/fisiologia , Transdução de Sinais , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Replicação Viral/imunologia
9.
Nature ; 570(7760): 241-245, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31142834

RESUMO

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci in prokaryotes are composed of 30-40-base-pair repeats separated by equally short sequences of plasmid and bacteriophage origin known as spacers1-3. These loci are transcribed and processed into short CRISPR RNAs (crRNAs) that are used as guides by CRISPR-associated (Cas) nucleases to recognize and destroy complementary sequences (known as protospacers) in foreign nucleic acids4,5. In contrast to most Cas nucleases, which destroy invader DNA4-7, the type VI effector nuclease Cas13 uses RNA guides to locate complementary transcripts and catalyse both sequence-specific cis- and non-specific trans-RNA cleavage8. Although it has been hypothesized that Cas13 naturally defends against RNA phages8, type VI spacer sequences have exclusively been found to match the genomes of double-stranded DNA phages9,10, suggesting that Cas13 can provide immunity against these invaders. However, whether and how Cas13 uses its cis- and/or trans-RNA cleavage activities to defend against double-stranded DNA phages is not understood. Here we show that trans-cleavage of transcripts halts the growth of the host cell and is sufficient to abort the infectious cycle. This depletes the phage population and provides herd immunity to uninfected bacteria. Phages that harbour target mutations, which easily evade DNA-targeting CRISPR systems11-13, are also neutralized when Cas13 is activated by wild-type phages. Thus, by acting on the host rather than directly targeting the virus, type VI CRISPR systems not only provide robust defence against DNA phages but also prevent outbreaks of CRISPR-resistant phage.


Assuntos
Bacteriófagos/imunologia , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/imunologia , Endodesoxirribonucleases/metabolismo , Listeria/imunologia , Listeria/virologia , Bacteriófagos/genética , Bacteriófagos/crescimento & desenvolvimento , Sistemas CRISPR-Cas/genética , Vírus de DNA/genética , Vírus de DNA/crescimento & desenvolvimento , Vírus de DNA/imunologia , Listeria/genética , Listeria/crescimento & desenvolvimento , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Viral/genética , RNA Viral/metabolismo
10.
Antiviral Res ; 160: 48-54, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30339848

RESUMO

2'-Fluoro-2'-deoxycytidine (2'-FdC) was reported to inhibit various viruses in vitro, including Borna disease, hepatitis C, Lassa fever, influenza and certain herpes viruses, and is inhibitory to influenza viruses in mice. We investigated the antiviral activity of 2'-FdC against several unrelated bunyaviruses in 50% cytopathic effect (CPE) inhibition assays and, with viruses that cause limited CPE, 90% virus yield reduction (VYR) assays. La Crosse (LACV), Maporal, Punta Toro, Rift Valley fever (RVFV), and San Angelo viruses were inhibited in CPE assays at 2.2-9.7 µM concentrations. In VYR assays, Heartland and severe fever with thrombocytopenia syndrome (SFTSV) viruses were inhibited at 0.9 and 3.7 µM, respectively. In contrast, ribavirin inhibited these viruses at an average of 47 µM. Antiviral efficacy studies were also conducted in mice infected with RVFV, SFTSV, and LACV. Against RVFV, 2'-FdC (100 and 200 mg/kg/day) and ribavirin (100 mg/kg/day) treatments each delayed mortality by approximately 6 days compared to placebo. Liver, spleen, and serum viral titers were significantly reduced by antiviral treatments. 2'-FdC (100 and 200 mg/kg/day) prevented death in SFTSV-infected mice, but was not as effective as favipiravir (100 mg/kg/day) based on body weight loss during infection. The 100 mg/kg/day doses of 2'-FdC and favipiravir significantly reduced liver, spleen, and serum viral titers. 2'-FdC and ribavirin afforded no protection against LACV infection in mice, which is encephalitic and thus inherently more difficult to treat. Taken together, our data suggest that 2'-FdC may be a viable candidate for treating certain non-encephalitic bunyavirus infections such as those caused by phleboviruses.


Assuntos
Antivirais/administração & dosagem , Antivirais/farmacologia , Infecções por Bunyaviridae/tratamento farmacológico , Vírus de DNA/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Vírus de RNA/efeitos dos fármacos , Estruturas Animais/virologia , Animais , Peso Corporal , Efeito Citopatogênico Viral , Vírus de DNA/crescimento & desenvolvimento , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Camundongos , Testes de Sensibilidade Microbiana , Placebos/administração & dosagem , Vírus de RNA/crescimento & desenvolvimento , Análise de Sobrevida , Resultado do Tratamento , Carga Viral
11.
Curr Opin Virol ; 33: 7-12, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30015083

RESUMO

Viruses evolve rapidly in response to host defenses and to exploit new niches. Gene amplification, a common adaptive mechanism in prokaryotes, archaea, and eukaryotes, has also contributed to viral evolution, especially of large DNA viruses. In experimental systems, gene amplification is one mechanism for rapidly overcoming selective pressures. Because the amplification generally incurs a fitness cost, emergence of adaptive point mutations within the amplified locus or elsewhere in the genome can enable collapse of the locus back to a single copy. Evidence of gene amplification followed by subfunctionalization or neofunctionalization of the copies is apparent by the presence of families of paralogous genes in many DNA viruses. These observations suggest that copy number variation has contributed broadly to virus evolution.


Assuntos
Variações do Número de Cópias de DNA , Vírus de DNA/crescimento & desenvolvimento , Vírus de DNA/genética , Amplificação de Genes , Adaptação Biológica , Archaea/virologia , Bactérias/virologia , Eucariotos/virologia
12.
Annu Rev Virol ; 5(1): 141-164, 2018 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-29996066

RESUMO

Viral DNA genomes have limited coding capacity and therefore harness cellular factors to facilitate replication of their genomes and generate progeny virions. Studies of viruses and how they interact with cellular processes have historically provided seminal insights into basic biology and disease mechanisms. The replicative life cycles of many DNA viruses have been shown to engage components of the host DNA damage and repair machinery. Viruses have evolved numerous strategies to navigate the cellular DNA damage response. By hijacking and manipulating cellular replication and repair processes, DNA viruses can selectively harness or abrogate distinct components of the cellular machinery to complete their life cycles. Here, we highlight consequences for viral replication and host genome integrity during the dynamic interactions between virus and host.


Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , Vírus de DNA/crescimento & desenvolvimento , DNA Viral/biossíntese , Replicação Viral
13.
J Virol ; 92(16)2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29848589

RESUMO

The BAF-chromatin remodeling complex, with its mutually exclusive ATPases SMARCA2 and SMARCA4, is essential for the transcriptional activation of numerous genes, including a subset of interferon-stimulated genes (ISGs). Here, we show that C-terminally truncated forms of both SMARCA2 and SMARCA4 accumulate in cells infected with different RNA or DNA viruses. The levels of truncated SMARCA2 or SMARCA4 strongly correlate with the degree of cell damage and death observed after virus infection. The use of a pan-caspase inhibitor and genetically modified cell lines unable to undergo apoptosis revealed that the truncated forms result from the activity of caspases downstream of the activated intrinsic apoptotic pathway. C-terminally cleaved SMARCA2 and SMARCA4 lack potential nuclear localization signals as well as the bromo- and SnAC domain, with the latter two domains believed to be essential for chromatin association and remodeling. Consistent with this belief, C-terminally truncated SMARCA2 was partially relocated to the cytoplasm. However, the remaining nuclear protein was sufficient to induce ISG expression and inhibit the replication of vesicular stomatitis virus and influenza A virus. This suggests that virus-induced apoptosis does not occur at the expense of an intact interferon-mediated antiviral response pathway.IMPORTANCE Efficient induction of interferon-stimulated genes (ISGs) prior to infection is known to effectively convert a cell into an antiviral state, blocking viral replication. Additionally, cells can undergo caspase-mediated apoptosis to control viral infection. Here, we identify SMARCA2 and SMARCA4 to be essential for the efficient induction of ISGs but also to be targeted by cellular caspases downstream of the intrinsic apoptotic pathway. We find that C-terminally cleaved SMARCA2 and SMARCA4 accumulate at late stages of infection, when cell damage already had occurred. Cleavage of the C terminus removes domains important for nuclear localization and chromatin binding of SMARCA2 and SMARCA4. Consequently, the cleaved forms are unable to efficiently accumulate in the cell nucleus. Intriguingly, the remaining nuclear C-terminally truncated SMARCA2 still induced ISG expression, although to lower levels. These data suggest that in virus-infected cells caspase-mediated cell death does not completely inactivate the SMARCA2- and SMARCA4-dependent interferon signaling pathway.


Assuntos
Caspases/metabolismo , DNA Helicases/metabolismo , Vírus de DNA/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Proteínas Nucleares/metabolismo , Vírus de RNA/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Cromatina , Células HeLa , Humanos , Hidrólise
14.
Microbiol Mol Biol Rev ; 82(2)2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29592895

RESUMO

When a virus infects a host cell, it hijacks the biosynthetic capacity of the cell to produce virus progeny, a process that may take less than an hour or more than a week. The overall time required for a virus to reproduce depends collectively on the rates of multiple steps in the infection process, including initial binding of the virus particle to the surface of the cell, virus internalization and release of the viral genome within the cell, decoding of the genome to make viral proteins, replication of the genome, assembly of progeny virus particles, and release of these particles into the extracellular environment. For a large number of virus types, much has been learned about the molecular mechanisms and rates of the various steps. However, in only relatively few cases during the last 50 years has an attempt been made-using mathematical modeling-to account for how the different steps contribute to the overall timing and productivity of the infection cycle in a cell. Here we review the initial case studies, which include studies of the one-step growth behavior of viruses that infect bacteria (Qß, T7, and M13), human immunodeficiency virus, influenza A virus, poliovirus, vesicular stomatitis virus, baculovirus, hepatitis B and C viruses, and herpes simplex virus. Further, we consider how such models enable one to explore how cellular resources are utilized and how antiviral strategies might be designed to resist escape. Finally, we highlight challenges and opportunities at the frontiers of cell-level modeling of virus infections.


Assuntos
Vírus de DNA/crescimento & desenvolvimento , Vírus de RNA/crescimento & desenvolvimento , Animais , Vírus de DNA/patogenicidade , Genoma Viral/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Cinética , Modelos Teóricos , Vírus de RNA/patogenicidade , Proteínas Virais/genética , Viroses/virologia , Replicação Viral/fisiologia
15.
Food Environ Virol ; 10(1): 107-120, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29098656

RESUMO

Irrigation water is a doorway for the pathogen contamination of fresh produce. We quantified pathogenic viruses [human adenoviruses, noroviruses of genogroups I and II, group A rotaviruses, Aichi virus 1 (AiV-1), enteroviruses (EnVs), and salivirus (SaliV)] and examined potential index viruses [JC and BK polyomaviruses (JCPyVs and BKPyVs), pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV)] in irrigation water sources in the Kathmandu Valley, Nepal. River, sewage, wastewater treatment plant (WWTP) effluent, pond, canal, and groundwater samples were collected in September 2014, and in April and August 2015. Viruses were concentrated using an electronegative membrane-vortex method and quantified using TaqMan (MGB)-based quantitative PCR (qPCR) assays with murine norovirus as a molecular process control to determine extraction-reverse transcription-qPCR efficiency. Tested pathogenic viruses were prevalent with maximum concentrations of 5.5-8.8 log10 copies/L, and there was a greater abundance of EnVs, SaliV, and AiV-1. Virus concentrations in river water were equivalent to those in sewage. Canal, pond, and groundwater samples were found to be less contaminated than river, sewage, and WWTP effluent. Seasonal dependency was clearly evident for most of the viruses, with peak concentrations in the dry season. JCPyVs and BKPyVs had a poor detection ratio and correspondence with pathogenic viruses. Instead, the frequently proposed PMMoV and the newly proposed TMV were strongly predictive of the pathogen contamination level, particularly in the dry season. We recommend utilizing canal, pond, and groundwater for irrigation to minimize deleterious health effects and propose PMMoV and TMV as indexes to elucidate pathogenic virus levels in environmental samples.


Assuntos
Irrigação Agrícola , Vírus de DNA/crescimento & desenvolvimento , Monitoramento Ambiental/métodos , Vírus de Plantas/crescimento & desenvolvimento , Vírus de RNA/crescimento & desenvolvimento , Viroses/virologia , Poluição da Água/análise , Adenoviridae/genética , Adenoviridae/crescimento & desenvolvimento , Produtos Agrícolas/virologia , Vírus de DNA/genética , Enterovirus/genética , Enterovirus/crescimento & desenvolvimento , Humanos , Kobuvirus/genética , Kobuvirus/crescimento & desenvolvimento , Nepal , Norovirus/genética , Norovirus/crescimento & desenvolvimento , Vírus de Plantas/genética , Reação em Cadeia da Polimerase , Vírus de RNA/genética , Rios/virologia , Vírus do Mosaico do Tabaco/genética , Vírus do Mosaico do Tabaco/crescimento & desenvolvimento , Tobamovirus/genética , Tobamovirus/crescimento & desenvolvimento , Águas Residuárias/virologia , Água/normas
16.
In Vitro Cell Dev Biol Anim ; 53(10): 908-921, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29197034

RESUMO

The DSIR-HA-1179 coleopteran cell line has been identified as a susceptible and permissive host for the in vitro replication of the Oryctes nudivirus, which can be used as a biopesticide against the coconut rhinoceros beetle, pest of palms. The major challenge to in vitro large-scale Oryctes nudivirus production is ensuring process economy. This rests, among other requisites, on the use of low-cost culture media tailored to the nutritional and metabolic needs of the cell line, both in uninfected and infected cultures. The aim of the present study was to characterize the nutritional demands and the metabolic characteristics of the DSIR-HA-1179 cell line during growth and subsequent infection with Oryctes nudivirus in the TC-100 culture medium. Serum-supplementation of the culture medium was found to be critical for cell growth, and addition of 10% fetal bovine serum v/v led to a maximum viable cell density (16.8 × 105 cells ml-1) with a population doubling time of 4.2 d. Nutritional and metabolic characterization of the cell line revealed a trend of glucose and glutamine consumption but minimal uptake of other amino acids, negligible production of lactate and ammonia, and the accumulation of alanine, both before and after infection. The monitoring of virus production kinetics showed that the TC-100 culture medium was nutritionally sufficient to give a peak yield of 7.38 × 107 TCID50 ml-1 of OrNV at the 6th day post-infection in attached cultures of DSIR-HA-1179 cells in 25 cm2 T-flasks. Knowledge of the cell line's nutritional demands and virus production kinetics will aid in the formulation of a low-cost culture medium and better process design for large-scale OrNV production in future.


Assuntos
Besouros/citologia , Besouros/virologia , Vírus de DNA/patogenicidade , Vírus de Insetos/patogenicidade , Aminoácidos/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Proliferação de Células , Besouros/metabolismo , Meios de Cultura/farmacologia , Vírus de DNA/crescimento & desenvolvimento , Vírus de Insetos/crescimento & desenvolvimento , Cinética , Soro
17.
J Fish Dis ; 40(8): 1041-1054, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28025825

RESUMO

Cyprinid herpesvirus 3 (CyHV-3) is an alloherpesvirus, and it is the aetiological agent of koi herpesvirus disease. Although the complex morphogenic stages of the replication cycle of CyHV-3 were shown to resemble that of other members of the Herpesvirales, detailed analysis of the sequence and timing of these events was not definitively determined. This study describes these features through a time course using cyprinid cell cultures (KF-1 and CCB) infected with CyHV-3 (KHV isolate, H361) and analysed by transmission electron microscopy. Rapid viral entry was noted, with high levels of intracellular virus within 1-4 h post-infection (hpi). Intranuclear capsid assembly, paracrystalline array formation and primary envelopment of capsids occurred within 4 hpi. Between 1 and 3 days post-infection (dpi), intracytoplasmic secondary envelopment occurred, as well as budding of infectious virions at the plasma membrane. At 5-7 dpi, the cytoplasm contained cytopathic vacuoles, enveloped virions within vesicles, and abundant non-enveloped capsids; also there was frequent nuclear deformation. Several morphological features are suggestive of inefficient viral assembly, with production of non-infectious particles, particularly in KF-1 cells. The timing of this alloherpesvirus morphogenesis is similar to other members of the Herpesvirales, but there may be possible implications of using different cell lines for CyHV-3 propagation.


Assuntos
Infecções por Vírus de DNA/patologia , Vírus de DNA/crescimento & desenvolvimento , Doenças dos Peixes/patologia , Animais , Carpas , Linhagem Celular , Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/virologia , Microscopia Eletrônica de Transmissão/veterinária , Morfogênese
19.
Viruses ; 7(5): 2654-67, 2015 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-26008705

RESUMO

Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies.


Assuntos
Abelhas/virologia , Vírus de DNA/crescimento & desenvolvimento , Vírus de DNA/isolamento & purificação , Microsporídios/isolamento & purificação , Vírus de RNA/isolamento & purificação , Trypanosoma/isolamento & purificação , Animais , França , Trato Gastrointestinal/virologia , Suécia , Fatores de Tempo
20.
J Virol Methods ; 191(1): 16-23, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23578702

RESUMO

The development of rapid and effective methods to detect water- and food-borne enteric viruses is important for the prevention and control of mass infection. This study represents an attempt to develop a reliable cell culture-based detection system and optimize an effective and rapid protocol for the assaying of environmental samples for the presence of infectious enteric viruses. Six enteric viruses were used in this study: poliovirus, Coxsackie virus A9, Coxsackie virus B5, human rotavirus G1, hepatitis A virus, and adenovirus type 41. Among the cell lines from humans (A549, HeLa, HEK293, and HFF) and other primates (Vero, BS-C-1, FRhK-4, BGMK, and MA104), a cytopathic effect (CPE) analysis indicated that the MA104 cell line was the most optimal for use in the detection of infectious enteric viruses. Both the sensitivity and specificity of virus detection in MA104 cells were similar to or higher than those in standard BGMK cells. Next, a method was developed for the determination of the infectiousness of enteric viruses using the colorimetric thiazolyl blue (MTT) assay. This assay utilizes 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to yield % values based on colorimetric results. These results were compared with those from a conventional CPE-based TCID(50) assay, revealing no statistically significant difference between the two methods. The MTT% values in MA104 cells were comparable to those in BGMK cells. This MA104 cell-based MTT assay could substitute for the classical BGMK cell-based CPE assay for infectious enteric viruses.


Assuntos
Técnicas de Laboratório Clínico/métodos , Vírus de DNA/isolamento & purificação , Trato Gastrointestinal/virologia , Vírus de RNA/isolamento & purificação , Viroses/diagnóstico , Viroses/virologia , Animais , Linhagem Celular , Colorimetria/métodos , Vírus de DNA/crescimento & desenvolvimento , Humanos , Vírus de RNA/crescimento & desenvolvimento , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismo , Cultura de Vírus/métodos
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