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1.
BMC Ophthalmol ; 21(1): 257, 2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112120

RESUMO

BACKGROUND: We report a case of chronic retinal necrosis (CRN) combined with cytomegalovirus (CMV) corneal endotheliitis. CASE PRESENTATION: An 80-year old man was diagnosed with CRN that developed after tube shunt surgery with vitrectomy for secondary glaucoma associated with CMV corneal endotheliitis. After the use of oral valganciclovir and panretinal photocoagulation, the retinal lesion resolved rapidly and he has maintained visual acuity better than before the onset of CRN. CONCLUSIONS: Use of oral valganciclovir, prophylactic panretinal photocoagulation for the non- perfusion area and vitrectomy were effective in maintaining the visual acuity for the patient with CRN.


Assuntos
Infecções por Citomegalovirus , Infecções Oculares Virais , Glaucoma , Idoso de 80 Anos ou mais , Antivirais/uso terapêutico , Humor Aquoso , Citomegalovirus/genética , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/tratamento farmacológico , DNA Viral , Epitélio Posterior , Infecções Oculares Virais/diagnóstico , Infecções Oculares Virais/tratamento farmacológico , Ganciclovir/uso terapêutico , Glaucoma/tratamento farmacológico , Glaucoma/cirurgia , Humanos , Masculino , Necrose/tratamento farmacológico
2.
BMC Ophthalmol ; 21(1): 194, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33933006

RESUMO

BACKGROUND: Cytomegalovirus (CMV) has been known to cause unilateral corneal endotheliitis with keratic precipitates and localized corneal edema, iridocyclitis, and secondary glaucoma. CMV endotheliitis is diagnosed based on clinical manifestations and viral examination using qualitative polymerase chain reaction (PCR) of the aqueous humor. CASE PRESENTATION: An 80-year-old woman was referred to our department for bullous keratopathy. Pigmented keratic precipitates were found in the right eye without significant anterior chamber inflammation. After 8 months there was inflammation relapse with mutton fat keratic precipitates and PCR on aqueous humor was performed, with negative results for CMV, herpes simplex virus, and varicella zoster virus. Keratic precipitates disappeared with steroid instillation, and Descemet-stripping automated endothelial keratoplasty (DSAEK) was performed for the right eye. CMV-DNA was positive at 6.0 × 102 copies/ GAPDH 105 copies in real time PCR of corneal endothelial specimen removed during DSAEK with negative results for all the other human herpes viruses. After diagnosis of CMV corneal endotheliitis, treatment with systemic and topical ganciclovir was initiated and there was resolution of symptoms. No recurrence of iridocyclitis or corneal endotheliitis was observed at 6 months follow up. CONCLUSIONS: This case report suggests that PCR should be performed using the endothelium removed during DSAEK for bullous keratopathy of an unknown cause, even if PCR for aqueous humor yields negative results.


Assuntos
Infecções por Citomegalovirus , Infecções Oculares Virais , Idoso de 80 Anos ou mais , Antivirais/uso terapêutico , Humor Aquoso , Citomegalovirus/genética , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/tratamento farmacológico , DNA Viral , Lâmina Limitante Posterior , Epitélio Posterior , Infecções Oculares Virais/diagnóstico , Infecções Oculares Virais/tratamento farmacológico , Feminino , Ganciclovir/uso terapêutico , Humanos , Resultados Negativos , Reação em Cadeia da Polimerase
3.
BMJ Case Rep ; 14(4)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931424

RESUMO

Cytomegalovirus (CMV) can cause recalcitrant recurrent keratouveitis and secondary glaucoma. We report a case of chronic recurrent anterior uveitis with secondary glaucoma presenting with acute visual loss and interface fluid 9 years after laser in situ keratomileusis. Based on clinical presentation, a viral aetiology was suspected. Aqueous tap was positive for CMV-DNA by real-time quantitative PCR of the aqueous humour. The patient was treated with systemic antivirals, topical corticosteroids and antiglaucoma medications. The interface fluid resorbed rapidly. The intraocular pressure (IOP) was controlled by trabeculectomy. There was no further corneal deterioration at 7-month follow-up and the IOP had also stabilised. We believe this is only the third reported case of CMV-related interface fluid syndrome. This case highlights the role of quantitative PCR analysis for establishing viral aetiology in recurrent unilateral hypertensive anterior uveitis and reports the unusual finding of interface fluid which resolved after starting systemic antiviral therapy.


Assuntos
Infecções por Citomegalovirus , Infecções Oculares Virais , Glaucoma , Ceratomileuse Assistida por Excimer Laser In Situ , Uveíte Anterior , Humor Aquoso , Citomegalovirus/genética , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/tratamento farmacológico , DNA Viral , Infecções Oculares Virais/diagnóstico , Infecções Oculares Virais/tratamento farmacológico , Glaucoma/tratamento farmacológico , Glaucoma/etiologia , Humanos , Pressão Intraocular , Ceratomileuse Assistida por Excimer Laser In Situ/efeitos adversos , Uveíte Anterior/diagnóstico , Uveíte Anterior/tratamento farmacológico , Uveíte Anterior/etiologia
4.
BMC Infect Dis ; 21(1): 386, 2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33902487

RESUMO

BACKGROUND: Congenital cytomegalovirus disease (cCMV) is common and can be fatal or cause severe sequelae. Circulating strains of cytomegalovirus carry a high number of variable or disrupted genes. One of these is UL146, a highly diverse gene with 14 distinct genotypes encoding a CXC-chemokine involved in viral dissemination. UL146 genotypes 5 and 6 lack the conserved ELR motif, potentially affecting strain virulence. Here, we investigate whether UL146 genotypes 5 and 6 were associated with congenital CMV infection. METHODS: Viral DNA was extracted and UL146 sequenced from 116 neonatal dried blood spots (DBS) stored in the Danish National Biobank since 1982 and linked to registered cCMV cases through a personal identifier. These sequences were compared to UL146 control sequences obtained from CMV DNA extracted from 83 urine samples from children with suspected bacterial urinary tract infections. RESULTS: Three non-ELR UL146 genotypes (5 and 6) were observed among the cases (2.6%) and two were observed among the controls (2.4%; P > 0.99). Additionally, no significant association with cCMV was found for the other 12 genotypes in a post-hoc analysis, although genotype 8 showed a tendency to be more frequent among cases with 12 observations against three (P = 0.10). All fourteen genotypes were found to have little intra-genotype variation. Viral load, gender, and sample age were not found to be associated with any particular UL146 genotype. CONCLUSIONS: No particular UL146 genotype was associated with cCMV in this nationwide retrospective case-control study. Associations between CMV disease and disrupted or polymorph CMV genes among immunosuppressed people living with HIV/AIDS and transplant recipients should be investigated in future studies.


Assuntos
Quimiocinas CXC/química , Quimiocinas CXC/genética , Infecções por Citomegalovirus/epidemiologia , Citomegalovirus/genética , Genótipo , Doenças do Recém-Nascido/epidemiologia , Proteínas Virais/química , Proteínas Virais/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Estudos de Casos e Controles , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/urina , Infecções por Citomegalovirus/virologia , DNA Viral/sangue , DNA Viral/genética , Dinamarca/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Doenças do Recém-Nascido/sangue , Doenças do Recém-Nascido/urina , Doenças do Recém-Nascido/virologia , Masculino , Polimorfismo Genético , Estudos Retrospectivos , Carga Viral
5.
Gene ; 781: 145541, 2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-33667607

RESUMO

Understanding how promoters work in non-host cells is complex. Nonetheless, understanding this process is crucial while performing gene expression modulation studies. This study began with the process of constructing a shuttle vector with CMV and OpIE2 promoters in a tandem arrangement to achieve gene expression in both mammalian and insect cells, respectively. In this system, inhibitory regions in the 5' end of the OpIE2 insect viral promoter were found to be blocking the activity of the CMV promoter in mammalian cells. Initially, the OpIE2 promoter was cloned downstream of the CMV promoter and upstream of the EGFP reporter gene. After introducing the constructed shuttle vector to insect and mammalian cells, a significant drop in the CMV promoter activity in mammalian cells was observed. To enhance the CMV promoter activity, several modifications were made to the shuttle vector including site-directed mutagenesis to remove all ATG codons from the downstream promoter (OpIE2), separating the two promoters to eliminate the effect of transcription interference between them, and finally, identifying some inhibitory regions in the OpIE2 promoter sequence. When these inhibitory regions were removed, high expression levels in insect and mammalian cells were maintained. In conclusion, a shuttle vector was constructed that works efficiently in both mammalian and insect cell lines in the absence of baculovirus infection or gene expression. Moreover, the shuttle vector can be used as a platform to further study the reason for this inhibition, which may give new insights about transcription and promoters' mode of action in both insect and mammalian hosts.


Assuntos
Baculoviridae/genética , Citomegalovirus/genética , Regulação Viral da Expressão Gênica , Vetores Genéticos , Regiões Promotoras Genéticas/genética , Animais , Sítios de Ligação , Simulação por Computador , DNA Recombinante/genética , DNA Recombinante/metabolismo , Células HEK293 , Células HeLa , Humanos , Células Sf9 , Fatores de Transcrição/metabolismo
6.
Science ; 372(6541)2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33766941

RESUMO

Strain 68-1 rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens elicit CD8+ T cells recognizing epitopes presented by major histocompatibility complex II (MHC-II) and MHC-E but not MHC-Ia. These immune responses mediate replication arrest of SIV in 50 to 60% of monkeys. We show that the peptide VMAPRTLLL (VL9) embedded within the RhCMV protein Rh67 promotes intracellular MHC-E transport and recognition of RhCMV-infected fibroblasts by MHC-E-restricted CD8+ T cells. Deletion or mutation of viral VL9 abrogated MHC-E-restricted CD8+ T cell priming, resulting in CD8+ T cell responses exclusively targeting MHC-II-restricted epitopes. These responses were comparable in magnitude and differentiation to responses elicited by 68-1 vectors but did not protect against SIV. Thus, Rh67-enabled direct priming of MHC-E-restricted T cells is crucial for RhCMV/SIV vaccine efficacy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/metabolismo , Vetores Genéticos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Fragmentos de Peptídeos/metabolismo , Vacinas contra a SAIDS/imunologia , Animais , Linhagem Celular , Citomegalovirus/genética , Epitopos de Linfócito T/imunologia , Fibroblastos/metabolismo , Vetores Genéticos/genética , Antígenos de Histocompatibilidade Classe I/genética , Ligantes , Macaca mulatta , Fragmentos de Peptídeos/genética , Transporte Proteico , Vírus da Imunodeficiência Símia
7.
Virology ; 558: 22-27, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33711560

RESUMO

Cytomegalovirus (CMV) promoter drives various gene expression and yields sufficient protein for further functional investigation. Receptor binding domain (RBD) on spike protein of the SARS_CoV2 is the most critical portal for virus infection. Thus native conformational RBD protein may facilitate biochemical identification of RBD and provide valuable support of drug and vaccine design for curing COVID-19. We attempted to express RBD under CMV promoter in vitro, but failed. RBD-specific mRNA cannot be detected in cell transfected with recombinant plasmids, in which CMV promoter governs the RBD transcription. Additionally, the pCMV-Tag2B-SARS_CoV2_RBD trans-inactivates CMV promoter transcription activity. Alternatively, we identified that both Chicken ß-actin promoter and Vaccinia virus-specific medium/late (M/L) promoter (pSYN) can highly precede SARS_CoV2 RBD expression. Our findings provided evidence that SARS_CoV2 RBD gene can be driven by Chicken ß-actin promoter or Vaccinia virus-specific medium/late promoter instead of CMV promoter, thus providing valuable information for RBD feature exploration.


Assuntos
COVID-19/virologia , Citomegalovirus/genética , Regiões Promotoras Genéticas , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Actinas/genética , Animais , Células CHO , Galinhas , Chlorocebus aethiops , Clonagem Molecular , Cricetulus , Células HEK293 , Humanos , Domínios Proteicos , Transcrição Genética , Vaccinia/genética , Células Vero
8.
Methods Mol Biol ; 2244: 39-50, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555581

RESUMO

Primary human diploid fibroblasts are used routinely to study host/pathogen interactions of human cytomegalovirus (HCMV). Fibroblasts' ease of culture and tremendous permissiveness for infection allow the study of all facets of infection, an abbreviated list of which includes ligand-receptor interactions, activation of cell signaling responses, and dysregulation of the cell cycle and DNA repair processes. Another advantage to fibroblasts' permissiveness for HCMV is the capability to grow high titer stocks of virus in them. This chapter will discuss the production of viral stocks of HCMV in primary human fibroblasts, commencing with culturing and infection of cells and continuing through harvest, titration (determining the infectious capacity of a particular virus preparation), and storage of viral stocks for use in downstream experiments.


Assuntos
Técnicas de Cultura de Células/métodos , Citomegalovirus/genética , Fibroblastos/virologia , Linhagem Celular , Células Cultivadas , Citomegalovirus/classificação , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/virologia , Diploide , Fibroblastos/citologia , Humanos , Modelos Biológicos , Replicação Viral
9.
Methods Mol Biol ; 2244: 19-38, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555580

RESUMO

Human cytomegalovirus is routinely isolated by inoculating fibroblast cultures with clinical specimens suspected of harboring HCMV and then monitoring the cultures for cytopathic effects characteristic of this virus. Initially, such clinical isolates are usually strictly cell-associated, but continued propagation in cell culture increases the capacity of an HCMV isolate to release cell-free infectious progeny. Once cell-free infection is possible, genetically homogenous virus strains can be purified by limiting dilution infections. HCMV strains can differ greatly with regard to the titers that can be achieved, the tropism for certain cell types, and the degree to which nonessential genes have been lost during propagation. As there is no ideal HCMV strain for all purposes, the choice of the most appropriate strain depends on the requirements of the particular experiment or project. In this chapter, we provide information that can serve as a basis for deciding which strain may be the most appropriate for a given experiment.


Assuntos
Técnicas de Cultura de Células/métodos , Citomegalovirus/genética , Tropismo Viral/genética , Citomegalovirus/classificação , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/virologia , Fibroblastos/citologia , Humanos , Projetos de Pesquisa , Tropismo Viral/fisiologia , Replicação Viral
10.
Methods Mol Biol ; 2244: 1-18, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555579

RESUMO

Human cytomegalovirus (HCMV) is a betaherpesvirus with a global seroprevalence of 60-90%. HCMV is the leading cause of congenital infections and poses a great health risk to immunocompromised individuals. Although HCMV infection is typically asymptomatic in the immunocompetent population, infection can result in mononucleosis and has also been associated with the development of certain cancers, as well as chronic inflammatory diseases such as various cardiovascular diseases. In immunocompromised patients, including AIDS patients, transplant recipients, and developing fetuses, HCMV infection is associated with increased rates of morbidity and mortality. Currently there is no vaccine for HCMV and there is a need for new pharmacological treatments. Ongoing research seeks to further define the complex aspects of HCMV pathogenesis, which could potentially lead to the generation of new therapeutics to mitigate the disease states associated with HCMV infection. The following chapter reviews the advancements in our understanding of HCMV pathogenesis in the immunocompetent and immunocompromised hosts.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Citomegalovirus/genética , Citomegalovirus/patogenicidade , DNA Viral/genética , Humanos , Hospedeiro Imunocomprometido/imunologia , Estudos Soroepidemiológicos
11.
Methods Mol Biol ; 2244: 133-158, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555586

RESUMO

To fully understand the function of cytomegalovirus (CMV) genes, it is imperative that they are studied in the context of infection. Therefore, the targeted deletion of individual viral genes and the comparison of these loss-of-function viral mutants to the wild-type virus allow for the identification of the relevance and role for a particular gene in the viral replication cycle. Targeted CMV mutagenesis has made huge advances over the past 20 years. The cloning of CMV genomes into Escherichia coli as bacterial artificial chromosomes (BAC) allows for not only quick and efficient deletion of viral genomic regions, individual genes, or single-nucleotide exchanges in the viral genome but also the insertion of heterologous genetic sequences for gain-of-function approaches. The conceptual advantage of this strategy is that it overcomes the restrictions of recombinant technologies in cell culture systems. Namely, recombination in infected cells occurs only in a few clones, and their selection is not possible if the targeted genes are relevant for virus replication and are not able to compete for growth against the unrecombined parental viruses. On the other hand, BAC mutagenesis enables the selection for antibiotic resistance in E. coli, providing selective growth advantage to the recombined genomes and thus clonal selection of viruses with even extremely poor fitness. Here we describe the methods used for the generation of a CMV BAC, targeted mutagenesis of BAC clones, and transfection of human cells with CMV BAC DNA in order to reconstitute the viral infection process.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Clonagem Molecular/métodos , Citomegalovirus/genética , Células Cultivadas , Escherichia coli/genética , Genes Virais/genética , Genoma Viral/genética , Humanos , Mutagênese/genética , Transfecção/métodos , Replicação Viral/genética
12.
Methods Mol Biol ; 2244: 199-211, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555588

RESUMO

Human cytomegalovirus (HCMV) entry into host cells is a complex process involving interactions between an array of viral glycoproteins with multiple host cell surface receptors. A significant amount of research has been devoted toward identifying these glycoprotein and cellular receptor interactions as the broad cellular tropism of HCMV suggests a highly regulated yet adaptable process that controls viral binding and penetration. However, deciphering the initial binding and cellular receptor activation events by viral glycoproteins remains challenging. The relatively low abundance of receptors and/or interactions with glycoproteins during viral entry, the hydrophobicity of membrane receptors, and the rapid degradation and recycling of activated receptors have complicated the analysis of HCMV entry and the cellular signaling pathways initiated by HCMV engagement to the host membrane. Here, we describe the different methodologies used in our laboratory and others to analyze the interactions between HCMV glycoproteins and host cellular receptors during the entry stage of the viral life cycle.


Assuntos
Técnicas de Cultura de Células/métodos , Citomegalovirus/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Linhagem Celular/virologia , Citomegalovirus/genética , Fibroblastos/metabolismo , Humanos , Cultura Primária de Células/métodos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus
13.
Methods Mol Biol ; 2244: 213-232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555589

RESUMO

Human cytomegalovirus (HCMV) is a large double-stranded DNA virus and member of the ß-herpesvirus family. HCMV is ubiquitous in the human population and causes lifelong infections. HCMV infection is associated with high morbidity and mortality in immunocompromised individuals and the virus is a major cause of virus-mediated congenital disease. There have been a number of HCMV entry receptors identified that use one of two viral receptor binding complexes, including the gH/gL/gO complex and the pentamer made up of gH/gL/UL128/UL130/UL131a. Cytomegaloviruses (CMVs) are typically host-restricted requiring the use of species-specific modeling and culture conditions. We use rat CMV (RCMV) to study CMV-accelerated vascular disease and chronic allograft rejection. RCMV encodes homologous versions of the entry complex proteins but their incorporation and copy number per virion are still unknown. In this methods article, we describe a novel approach of HiBiT tagging viral proteins in order to detect and quantify protein incorporation into particles. This method is independent of protein-specific antibodies and can be standardized using a commercially available HiBiT protein standard. Using bacterial artificial chromosome (BAC) recombineering, we have constructed two individual viruses containing a HiBiT tag fused to the C'-terminus of either the UL128 homolog (R129) or the UL130 homolog (R131). Viruses containing these mutations were rescued, purified and analyzed. Our data demonstrate that R129 and R131 are both incorporated into RCMV virions at equimolar ratios relative to genome copy number, supporting this antibody-free approach for quantifying viral protein incorporation and its application toward the identification of domains required for incorporation.


Assuntos
Medições Luminescentes/métodos , Proteínas Luminescentes/síntese química , Animais , Cromossomos Artificiais Bacterianos/genética , Citomegalovirus/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/genética , Ligação Proteica , Ratos , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Vírion/metabolismo , Internalização do Vírus
14.
Methods Mol Biol ; 2244: 159-197, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555587

RESUMO

All of the cytomegaloviruses discovered to date encode two or more genes with significant homology to G protein-coupled receptors (GPCRs). The functions of these cytomegalovirus GPCRs continue to be actively studied and it is clear that they exhibit numerous interesting functions in vitro and in vivo. In this chapter, we review the various methodologies that can be used to examine biochemical aspects of viral GPCR signaling in vitro, as well as examine the biological activity of these viral GPCRs in vitro and in vivo in virus infected cells using recombinant cytomegaloviruses.


Assuntos
Técnicas de Cultura de Células/métodos , Citomegalovirus/genética , Receptores Acoplados a Proteínas G/genética , Animais , Linhagem Celular/virologia , Citomegalovirus/metabolismo , Humanos , Cultura Primária de Células/métodos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
15.
Methods Mol Biol ; 2244: 247-264, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555591

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens enable virus-host genetic screens to be undertaken in a more robust manner than previously possible and has had a tremendous impact in the field of virus study. Researchers can take advantage of the power of CRISPR genetic screens to discover virus-host interaction genes including host receptors and signaling molecules (Bazzone et al., mBio 10 (1): e02734-18, 2019; E et al., Proc Natl Acad Sci U S A 116(14):7043-7052, 2019; McDougall et al., Curr Opin Virol 29:87-100, 2018; Savidis et al., Cell Rep 16(1):232-246, 2016). In principle, lysis of cells late in the virus infection cycle allows one to screen for essential genes using pooled single-guide RNAs (sgRNAs) that collective target an entire host cell genome simply by identifying mutant cells that are resistant to virus-induced cell death. Here we focus on using this technique on epithelial cells to identify host targets required for human cytomegalovirus (HCMV) infection.


Assuntos
Citomegalovirus/genética , Engenharia Genética/métodos , Interações entre Hospedeiro e Microrganismos/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células Epiteliais , Edição de Genes/métodos , Testes Genéticos/métodos , Humanos , Cultura Primária de Células , RNA Guia/genética , Viroses/genética
16.
Methods Mol Biol ; 2244: 301-342, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555594

RESUMO

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level by binding to sites within the 3' untranslated regions of messenger RNA (mRNA) transcripts. The discovery of this completely new mechanism of gene regulation necessitated the development of a variety of techniques to further characterize miRNAs, their expression, and function. In this chapter, we will discuss techniques currently used in the miRNA field to detect, express and inhibit miRNAs, as well as methods used to identify and validate their targets, specifically with respect to the miRNAs encoded by human cytomegalovirus.


Assuntos
Citomegalovirus/genética , Imunoprecipitação/métodos , MicroRNAs/análise , Regiões 3' não Traduzidas/genética , Northern Blotting/métodos , Expressão Gênica/genética , Regulação Viral da Expressão Gênica/genética , Humanos , MicroRNAs/genética , RNA Mensageiro/genética
17.
Methods Mol Biol ; 2244: 403-463, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555597

RESUMO

Human cytomegalovirus is the largest human herpesvirus and shares many core features of other herpesviruses such as tightly regulated gene expression during genome replication and latency as well as the establishment of lifelong persistence following infection. In contrast to stereotypic clinical syndromes associated with alpha-herpesvirus infections, almost all primary HCMV infections are asymptomatic and acquired early in life in most populations in the world. Although asymptomatic in most individuals, HCMV is a major cause of disease in hosts with deficits in adaptive and innate immunity such as infants who are infected in utero and allograft recipients following transplantation. Congenital HCMV is a commonly acquired infection in the developing fetus that can result in a number of neurodevelopmental abnormalities. Similarly, HCMV is a major cause of disease in allograft recipients in the immediate and late posttransplant period and is thought to be a major contributor to chronic allograft rejection. Even though HCMV induces robust innate and adaptive immune responses, it also encodes a vast array of immune evasion functions that are thought aid in its persistence. Immune correlates of protective immunity that prevent or modify intrauterine HCMV infection remain incompletely defined but are thought to consist primarily of adaptive responses in the pregnant mother, thus making congenital HCMV a potentially vaccine modifiable disease. Similarly, HCMV infection in allograft recipients is often more severe in recipients without preexisting adaptive immunity to HCMV. Thus, there has been a considerable effort to modify HCMV specific immunity in transplant recipient either through active immunization or passive transfer of adaptive effector functions. Although efforts to develop an efficacious vaccine and/or passive immunotherapy to limit HCMV disease have been underway for nearly six decades, most have met with limited success at best. In contrast to previous efforts, current HCMV vaccine development has relied on observations of unique properties of HCMV in hopes of reproducing immune responses that at a minimum will be similar to that following natural infection. However, more recent findings have suggested that immunity following naturally acquired HCMV infection may have limited protective activity and almost certainly, is not sterilizing. Such observations suggest that either the induction of natural immunity must be specifically tailored to generate protective activity or alternatively, that providing targeted passive immunity to susceptible populations could be prove to be more efficacious.


Assuntos
Vacinas contra Citomegalovirus/imunologia , Citomegalovirus/imunologia , Vacinação/métodos , Imunidade Adaptativa/imunologia , Anticorpos Antivirais/imunologia , Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/prevenção & controle , Suscetibilidade a Doenças , Feminino , Humanos , Imunidade Humoral/imunologia , Imunidade Inata/imunologia , Lactente , Masculino , Gravidez , Vacinas/imunologia , Vacinas/metabolismo , Vacinas/farmacologia
18.
ACS Sens ; 6(3): 815-822, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33529521

RESUMO

A human cytomegalovirus (HCMV) causes a persistent asymptomatic infection in healthy individuals and possesses unexpected dangers to newborn babies, immunocompromised people, and organ transplant recipients because of stealth transmission. Thus, an early and accurate diagnosis of HCMV infection is crucial for prevention of unexpected transmission and progression of the severe diseases. The standard method of HCMV diagnosis depends on serology, antigen test, and polymerase chain reaction-based nucleic acid detection, which have advantages for each target molecule. However, the serological test for an antibody is an indirect method assuming the past virus infection, and antigen and viral nucleic acid testing demand laborious, complex multistep procedures for direct virus detection. Herein, we present an alternative simple and facile fluorometric biosensor composed of a graphene oxide nanocolloid and fluorescent peptide nucleic acid (PNA) probe to detect the HCMV infection by simply monitoring the virally encoded microRNA as a new biomarker of lytic virus infection. We verify the sensing of HCMV-derived microRNA accumulated within 72 h after HCMV infection and examine the diagnosis of HCMV in living cells. We proceed with the time course and concentration-dependent investigation of hcmv-miRNA sensing in living cells as a direct method of HCMV detection at the molecular level on the basis of an intracellular hcmv-miRNA expression profile and graphene oxide nanocolloid-based simple diagnostic platform. The fluorometric biosensor enables the sequence-specific binding to the target HCMV miRNAs in HCMV-infected fibroblasts and shows the quantitative detection capability of HCMV infection to be as low as 4.15 × 105 immunofluorescence focus unit (IFU)/mL of the virus titer at 48 h post-infection with picomolar sensitivity for HCMV miRNA.


Assuntos
Infecções por Citomegalovirus , MicroRNAs , Citomegalovirus/genética , Infecções por Citomegalovirus/diagnóstico , Humanos , Lactente , Recém-Nascido , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase
19.
J Virol ; 95(9)2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33568511

RESUMO

Transposon-based insertional mutagenesis screens have assessed how disruption of numerous human cytomegalovirus (HCMV) open reading frames (ORFs) impacts in vitro viral replication. Insertional mutagenesis of the HCMV UL30 gene was previously found to substantially inhibit production of viral progeny. However, there are a number of putative UL30-associated ORFs, and it is unclear how they impact viral replication. Here, we report on the contributions of the eight UL30-associated ORFs to infection. We find that deletion of the canonically annotated UL30 ORF substantially reduces production of infectious virus at both high and low multiplicities of infection (MOI). This deletion likely has complex effects on viral replication, as we find that it reduces the expression of neighboring non-UL30-associated ORFs. Mutation of the initiating methionine of the canonical UL30 ORF indicated that it is dispensable for high- and low-MOI infection in the highly passaged AD169 strain, although it is important for low-MOI infection in the less-passaged TB40/E strain. Comutation of eight methionines in the UL30 region results in a low-MOI viral replication defect, as does mutation of the TATA box responsible for the most abundant UL30 transcript, which is found to be necessary for the accumulation of multiple UL30-associated protein isoforms during infection. In total, our data indicate the importance of the UL30-associated ORFs during low-MOI HCMV infection and further highlight the difficulty associated with the functional interrogation of broadly disruptive mutations: e.g., large deletions or transposon insertions.IMPORTANCE Viral genes and their products are the critical determinants of viral infection. Human cytomegalovirus (HCMV) encodes many gene products whose roles during viral infection have not been assessed. Elucidation of the contributions that various HCMV gene products make to infection provides insight into the infectious program, which could potentially be used to limit HCMV-associated morbidity, a major issue during congenital infection and in immunosuppressed populations. Here, we explored the role of HCMV's UL30-associated gene products and found that they are important for HCMV replication. Future work elucidating the mechanisms through which they contribute to viral infection could highlight novel avenues for therapeutic intervention.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus , Regulação Viral da Expressão Gênica , Fases de Leitura Aberta , Replicação Viral/genética , Linhagem Celular , Citomegalovirus/genética , Citomegalovirus/patogenicidade , DNA Viral , Fibroblastos , Genes Virais , Humanos
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