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1.
BMC Infect Dis ; 20(1): 54, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31952510

RESUMO

BACKGROUND: Varicella is normally a self-limited childhood disease caused by varicella-zoster virus infection. However, it sometimes causes severe diseases, especially in immunocompromised individuals. We report a case of severe varicella in a young woman. CASE PRESENTATION: A 19-year-old woman presented to the emergency department with abdominal pain and a rash after taking methylprednisolone for 2 weeks for systemic lupus erythematosis. The laboratory data showed leukocytosis, thrombocytopenia, an elevated level of the liver transaminases and disseminated intravascular coagulation. Computed tomography of the abdomen revealed multiple air-fluid levels in the intestines. Hemorrhagic varicella was considered and antiviral therapy as well as immunoglobin were applied. Her condition deteriorated and she eventually died due to multi-organ failure and refractory shock. Next-generation sequencing performed on fluid from an unroofed vesicle confirmed the diagnosis of varicella. CONCLUSION: In its severe form, VZV infection can be fatal, especially in immunocompromised patients. Hemorrhagic varicella can be misdiagnosed by clinicians because of unfamiliar with the disease, although it is associated with a high mortality rate. In patients with suspected hemorrhagic varicella infection, antiviral therapies along with supportive treatment need to be initiated as soon as possible in order to minimize the case fatality rate.


Assuntos
Varicela/diagnóstico , Abdome/diagnóstico por imagem , Dor Abdominal/etiologia , Antivirais/uso terapêutico , Varicela/complicações , Varicela/tratamento farmacológico , Varicela/virologia , DNA Viral/química , DNA Viral/metabolismo , Feminino , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hospedeiro Imunocomprometido , Insuficiência de Múltiplos Órgãos/diagnóstico , Insuficiência de Múltiplos Órgãos/etiologia , Análise de Sequência de DNA , Tomografia Computadorizada por Raios X , Adulto Jovem
3.
BMC Infect Dis ; 19(1): 1046, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31822287

RESUMO

BACKGROUND: Congenital cytomegalovirus (cCMV) infection is the most common congenital infection and a leading cause of long-term neurological and sensory sequelae, the most common being sensorineural hearing loss (SNHL). Despite extensive research, clinical or laboratory markers to identify CMV infected children with increased risk for disease have not been identified. This study utilizes viral whole-genome next generation-sequencing (NGS) of specimens from congenitally infected infants to explore viral diversity and specific viral variants that may be associated with symptomatic infection and SNHL. METHODS: CMV DNA from urine specimens of 30 infants (17 asymptomatic, 13 symptomatic) was target enriched and next generation sequenced resulting in 93% coverage of the CMV genome allowing analysis of viral diversity. RESULTS: Variant frequency distribution was compared between children with symptomatic and asymptomatic cCMV and those with (n = 13) and without (n = 17) hearing loss. The CMV genes UL48A, UL88, US19 and US22 were found to have an increase in nucleotide diversity in symptomatic children; while UL57, UL20, UL104, US14, UL115, and UL35 had an increase in diversity in children with hearing loss. An analysis of single variant differences between symptomatic and asymptomatic children found UL55 to have the highest number, while the most variants associated with SNHL were in the RL11 gene family. In asymptomatic infants with SNHL, mutations were observed more frequently in UL33 and UL20. CONCLUSION: CMV genomes from infected newborns can be mapped to 93% of the genome at a depth allowing accurate and reproducible analysis of polymorphisms for variant and gene discovery that may be linked to symptomatic and hearing loss outcomes.


Assuntos
Infecções por Citomegalovirus/diagnóstico , Citomegalovirus/genética , DNA Viral/genética , Perda Auditiva Neurossensorial/diagnóstico , Criança , Citomegalovirus/classificação , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/virologia , DNA Viral/metabolismo , DNA Viral/urina , Feminino , Perda Auditiva Neurossensorial/complicações , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Filogenia , Análise de Componente Principal
4.
Emerg Microbes Infect ; 8(1): 1563-1573, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31672101

RESUMO

The episomal structures of all human bocavirus (HBoV) genotypes have been deciphered, including the circular genome of HBoV2 (HBoV2-C1). To discern the role of the circular HBoV2 genome, three distinct linearized HBoV2-C1 genomes were cloned into pBlueScript SKII(+) to obtain pBlueScript HBoV2 5043-5042 (retaining all secondary structures), pBlueScript-HBoV2 5075-5074 (retaining hairpin number 2 and the 5' terminal structure), and pBlueScript-HBoV2 5220-5219 (retaining only the 5' terminal structure at the 5' -genome end). The recombinant plasmids were separately transfected HEK293 cells, revealing that more HBoV2 DNA had accumulated in the pBlueScript HBoV2 5043-5042-transfected HEK293 cells at 72 h post-transfection, as determined by real-time PCR. However, more mRNA was transcribed by pBlueScript-HBoV2 5075-5074 than by the other constructs, as determined by dot-blot hybridization and RNAscope. No significant differences in NS1-70 protein expression were observed among the three HBoV2 genomic clones. However, electron microscopy showed that HBoV2 virus particles were only present in the pBlueScript HBoV2 5043-5042-transfected HEK293 cells. By using three hetero-recombinant HBoV2 genomic clones in HEK293 transfected cells, only the genome with intact secondary structures produced virus particles, suggesting that retaining these structures in a circular genome is important for HBoV2 DNA replication and virus assembly.


Assuntos
DNA Circular/genética , DNA Viral/genética , Bocavirus Humano/genética , Infecções por Parvoviridae/virologia , RNA não Traduzido/genética , Recombinação Genética , Montagem de Vírus , Replicação do DNA , DNA Circular/química , DNA Circular/metabolismo , DNA Viral/química , DNA Viral/metabolismo , Genoma Viral , Genômica , Genótipo , Células HEK293 , Bocavirus Humano/química , Bocavirus Humano/fisiologia , Humanos , Conformação de Ácido Nucleico , Filogenia , RNA não Traduzido/química , RNA não Traduzido/metabolismo
5.
Cell Host Microbe ; 26(4): 515-526.e6, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31585845

RESUMO

Type II CRISPR-Cas systems defend prokaryotes from bacteriophage infection through the acquisition of short viral DNA sequences known as spacers, which are transcribed into short RNA guides to specify the targets of the Cas9 nuclease. To counter the potentially devastating propagation of escaper phages with mutations in the target sequences, the host population acquires many different spacers. Whether and how pre-existing spacers in type II systems affect the acquisition of new ones is unknown. Here, we demonstrate that previously acquired spacers promote additional spacer acquisition from the vicinity of the target DNA site cleaved by Cas9. Therefore, CRISPR immune cells acquire additional spacers at the same time as they destroy the infecting virus. This anticipates the rise of escapers or related viruses that could escape targeting by the first spacer acquired. Our results thus reveal Cas9's role in the generation of immunological memories.


Assuntos
Sistemas CRISPR-Cas/genética , DNA Intergênico/genética , DNA Viral/metabolismo , RNA Guia/genética , Staphylococcus aureus/genética , Streptococcus thermophilus/genética , Bacteriófagos/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Staphylococcus aureus/imunologia , Staphylococcus aureus/virologia , Streptococcus thermophilus/imunologia , Streptococcus thermophilus/virologia
6.
Nat Commun ; 10(1): 4738, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31628321

RESUMO

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved. Here we investigate the dynamics of strand transfer and demonstrate that consecutive nucleoprotein intermediates interacting with a supercoiled target are increasingly stable, resulting in a net forward rate. Multivalent target interactions at discrete auxiliary interfaces render target capture irreversible, while allowing dynamic site selection. Active site binding is transient but rapidly results in strand transfer, which in turn rearranges and stabilizes the intasome in an allosteric manner. We find the resulting strand transfer complex to be mechanically stable and extremely long-lived, suggesting that a resolving agent is required in vivo.


Assuntos
Integrases/química , Provírus/genética , Retroviridae/genética , Spumavirus/genética , Integração Viral/genética , Cristalografia por Raios X , DNA Viral/química , DNA Viral/genética , DNA Viral/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Integrases/genética , Integrases/metabolismo , Substâncias Macromoleculares , Microscopia de Força Atômica , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Multimerização Proteica , Provírus/enzimologia , Retroviridae/enzimologia , Spumavirus/enzimologia
7.
BMC Infect Dis ; 19(1): 842, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615443

RESUMO

BACKGROUND: HPV test implementation as a primary screening tool has the potential to decrease cervical cancer incidence as shown by several studies around the world. However, in many low-resource settings, the HPV test introduction has been backed down mainly due to its price. In this study, we present a novel low-cost strategy involving simple devices and techniques for high-risk human papillomavirus (HR-HPV) detection. The analytical performance to detect HR-HPV infections of this novel strategy was assessed by comparing it with the Hybrid Capture 2 system (HC2), which is used as gold standard. METHODS: Paired-cervical samples were collected from 541 women assisting to gynecological services in an outpatient clinic. One sample was transported in the Hybrid Capture Standard Transport Medium for HR-HPV detection by the HC2. The second sample was transported on glass slide for detection by PCR-based techniques (GP-EIA, BSGP-EIA and pU 1 M-L/2R). RESULTS: The level of agreement between the PCR-based techniques and HC2 system was determined with the Cohen's kappa value. The kappa values between HC2 and GP-EIA, BSGP-EIA and pU 1 M-L/2R were 0.71 (CI 95% 0.63-0.78), 0.78 (CI 95% 0.71-0.84) and 0.63 (CI 95% 0.55-0.72), respectively. However, when the results from both BSGP-EIA and pU 1 M-L/2R were combined, the level of agreement with HC2 was increased to 0.82 (CI 95% 0.76-0.88), reflecting a very good agreement between the two HR-HPV detection strategies. Furthermore, the sensitivity of both techniques combined was also increased compared to the BSGP-EIA (88.7% vs 77.4%) and the pU (88.7 vs 60.9%) without penalizing the specificity obtained with the BSGP-EIA (95.1% vs 96.9%) and the pU (95.1% vs 96.5%). CONCLUSIONS: This novel strategy, combining two PCR-based techniques for HR-HPV detection, could be useful for cervical cancer screening in self-collected samples in low-income countries.


Assuntos
DNA Viral/análise , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Reação em Cadeia da Polimerase/métodos , Neoplasias do Colo do Útero/diagnóstico , Colo do Útero/patologia , DNA Viral/metabolismo , Detecção Precoce de Câncer , Feminino , Genótipo , Humanos , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase/economia , Kit de Reagentes para Diagnóstico , Risco , Sensibilidade e Especificidade , Análise de Sequência de DNA , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologia
8.
Elife ; 82019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31621580

RESUMO

Aedes aegypti transmit pathogenic arboviruses while the mosquito itself tolerates the infection. We examine a piRNA-based immunity that relies on the acquisition of viral derived cDNA (vDNA) and how this pathway discriminates between self and non-self. The piRNAs derived from these vDNAs are essential for virus control and Piwi4 has a central role in the pathway. Piwi4 binds preferentially to virus-derived piRNAs but not to transposon-targeting piRNAs. Analysis of episomal vDNA from infected cells reveals that vDNA molecules are acquired through a discriminatory process of reverse-transcription and recombination directed by endogenous retrotransposons. Using a high-resolution Ae. aegypti genomic sequence, we found that vDNAs integrated in the host genome as endogenous viral elements (EVEs), produce antisense piRNAs that are preferentially loaded onto Piwi4. Importantly, EVE-derived piRNAs are specifically loaded onto Piwi4 to inhibit virus replication. Thus, Ae. aegypti employs a sophisticated antiviral mechanism that promotes viral persistence and generates long-lasting adaptive immunity.


Assuntos
Aedes/virologia , Imunidade Inata , Vírus de RNA/crescimento & desenvolvimento , Vírus de RNA/imunologia , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/metabolismo , DNA Complementar/metabolismo , DNA Viral/metabolismo , Proteínas de Drosophila/metabolismo
9.
Elife ; 82019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31393262

RESUMO

Many viruses previously have been shown to have pressurized genomes inside their viral protein shell, termed the capsid. This pressure results from the tight confinement of negatively charged viral nucleic acids inside the capsid. However, the relevance of capsid pressure to viral infection has not been demonstrated. In this work, we show that the internal DNA pressure of tens of atmospheres inside a herpesvirus capsid powers ejection of the viral genome into a host cell nucleus. To our knowledge, this provides the first demonstration of a pressure-dependent mechanism of viral genome penetration into a host nucleus, leading to infection of eukaryotic cells.


Assuntos
Capsídeo/metabolismo , Núcleo Celular/virologia , DNA Viral/metabolismo , Células Eucarióticas/virologia , Herpesvirus Humano 1/fisiologia , Pressão Hidrostática , Internalização do Vírus , Animais , Linhagem Celular
10.
Nucleic Acids Res ; 47(16): 8337-8347, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31372632

RESUMO

DNA repair is critical for maintaining genomic integrity. Finding DNA lesions initiates the entire repair process. In human nucleotide excision repair (NER), XPC-RAD23B recognizes DNA lesions and recruits downstream factors. Although previous studies revealed the molecular features of damage identification by the yeast orthologs Rad4-Rad23, the dynamic mechanisms by which human XPC-RAD23B recognizes DNA defects have remained elusive. Here, we directly visualized the motion of XPC-RAD23B on undamaged and lesion-containing DNA using high-throughput single-molecule imaging. We observed three types of one-dimensional motion of XPC-RAD23B along DNA: diffusive, immobile and constrained. We found that consecutive AT-tracks led to increase in proteins with constrained motion. The diffusion coefficient dramatically increased according to ionic strength, suggesting that XPC-RAD23B diffuses along DNA via hopping, allowing XPC-RAD23B to bypass protein obstacles during the search for DNA damage. We also examined how XPC-RAD23B identifies cyclobutane pyrimidine dimers (CPDs) during diffusion. XPC-RAD23B makes futile attempts to bind to CPDs, consistent with low CPD recognition efficiency. Moreover, XPC-RAD23B binds CPDs in biphasic states, stable for lesion recognition and transient for lesion interrogation. Taken together, our results provide new insight into how XPC-RAD23B searches for DNA lesions in billions of base pairs in human genome.


Assuntos
Enzimas Reparadoras do DNA/química , Reparo do DNA , DNA Viral/química , Proteínas de Ligação a DNA/química , DNA/química , Dímeros de Pirimidina/química , Bacteriófago lambda/química , Bacteriófago lambda/genética , Sítios de Ligação , DNA/genética , DNA/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Difusão , Humanos , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Concentração Osmolar , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Dímeros de Pirimidina/metabolismo , Imagem Individual de Molécula
11.
Nat Commun ; 10(1): 3746, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31431626

RESUMO

Double-stranded DNA bacteriophages package their genome at high pressure inside a procapsid through the portal, an oligomeric ring protein located at a unique capsid vertex. Once the DNA has been packaged, the tail components assemble on the portal to render the mature infective virion. The tail tightly seals the ejection conduit until infection, when its interaction with the host membrane triggers the opening of the channel and the viral genome is delivered to the host cell. Using high-resolution cryo-electron microscopy and X-ray crystallography, here we describe various structures of the T7 bacteriophage portal and fiber-less tail complex, which suggest a possible mechanism for DNA retention and ejection: a portal closed conformation temporarily retains the genome before the tail is assembled, whereas an open portal is found in the tail. Moreover, a fold including a seven-bladed ß-propeller domain is described for the nozzle tail protein.


Assuntos
Bacteriófago T7/fisiologia , Proteínas do Capsídeo/ultraestrutura , Capsídeo/ultraestrutura , Empacotamento do DNA , Modelos Moleculares , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Viral/metabolismo , Domínios Proteicos
12.
BMC Infect Dis ; 19(1): 624, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307413

RESUMO

BACKGROUND: Two outbreaks of epidemic keratoconjunctivitis (EKC) occurred successively with an interval of 5 days in two primary boarding schools in Weixi Lisu Autonomous County, Diqing, and Tibetan Autonomous Prefecture, Yunnan. The aims of this study were to determine the intensity and characteristics of the outbreaks, as well as the clinical manifestations in the patients, the risk factors for infection and the pathogen responsible for the two outbreaks. METHODS: An outbreak investigation was conducted in two primary schools, and a case-control study including patients from the Weixi County Ethnic Primary School was performed. Relevant specimens were collected according to the case definition, and next-generation sequencing was employed to identify the pathogen. An epidemiological investigation method was used to analyse the related epidemiological characteristics, such as risk factors. The phylogenetic tree was constructed by MEGA 7.0. RESULTS: A total of 331 acute conjunctivitis cases, including probable cases of EKC, were reported in the two schools, and the attack rates were 30.59% (171/559, 95%CI: 26.76-34.42) and 20.41% (160/784, 95%CI: 17.58-23.24), respectively. Cases occurred in all grades and classes in both schools, and only one staff member in each school presented illness. The epidemics lasted for 54 days and 45 days, respectively. The patients had typical manifestations of EKC, such as acute onset, follicular hyperplasia, pseudomembrane formation, preauricular lymphadenopathy, corneal involvement and blurred vision, and a relatively long disease course (average 9.40 days, longest 23 days and shortest 7 days). The risk factor for infection was close contact with a patient or personal items contaminated by a patient. The pathogen responsible for the outbreaks was HAdV-8. The virus was highly similar to the 2016 HAdV-8 strain from Tibet, China. CONCLUSIONS: This study strongly suggests that HAdV-8 could lead to serious consequences. This is the second report of a HAdV-8-associated EKC outbreak in mainland China. Tibetan HAdV-8 might be circulating in southwest China; therefore, it is necessary to monitor the pathogens causing acute conjunctivitis in this area.


Assuntos
Infecções por Adenovirus Humanos/diagnóstico , Adenovírus Humanos/isolamento & purificação , Ceratoconjuntivite/diagnóstico , Infecções por Adenovirus Humanos/epidemiologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/classificação , Adenovírus Humanos/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , China/epidemiologia , DNA Viral/isolamento & purificação , DNA Viral/metabolismo , Surtos de Doenças , Feminino , Humanos , Ceratoconjuntivite/epidemiologia , Ceratoconjuntivite/virologia , Masculino , Filogenia , Fatores de Risco , Instituições Acadêmicas , Análise de Sequência de DNA , Adulto Jovem
13.
Nucleic Acids Res ; 47(13): 7130-7142, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31334814

RESUMO

Xenobiotic nucleic acids (XNA) are nucleic acid analogues not present in nature that can be used for the storage of genetic information. In vivo XNA applications could be developed into novel biocontainment strategies, but are currently limited by the challenge of developing XNA processing enzymes such as polymerases, ligases and nucleases. Here, we present a structure-guided modelling-based strategy for the rational design of those enzymes essential for the development of XNA molecular biology. Docking of protein domains to unbound double-stranded nucleic acids is used to generate a first approximation of the extensive interaction of nucleic acid processing enzymes with their substrate. Molecular dynamics is used to optimise that prediction allowing, for the first time, the accurate prediction of how proteins that form toroidal complexes with nucleic acids interact with their substrate. Using the Chlorella virus DNA ligase as a proof of principle, we recapitulate the ligase's substrate specificity and successfully predict how to convert it into an XNA-templated XNA ligase.


Assuntos
DNA Ligases/metabolismo , Proteínas Virais/metabolismo , Simulação por Computador , DNA Ligases/química , Vírus de DNA/enzimologia , DNA Viral/metabolismo , Desoxirribonuclease BamHI/metabolismo , Modelos Químicos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Moldes Genéticos , Proteínas Virais/química
14.
Oxid Med Cell Longev ; 2019: 5832410, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31360295

RESUMO

A growing number of studies reveal that oxidative stress is associated with viral infections or cancer development. However, there are few reports assessing the relationships between oxidative stress, viral infection, and carcinogenesis. The present study analyzed the level of total antioxidant status (TAS) as well as the activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in patients with oropharyngeal cancer both Epstein-Barr virus (EBV)-positive and EBV-negative in comparison with the control group. The correlations between these parameters and EBV type (wild-type LMP1 (wt-LMP1) or LMP1 with deletion (del-LMP1)), level of antibodies against EBV, the degree of tumor differentiation, and TNM classification were also investigated. Fresh frozen tumor tissue samples collected from 66 patients with oropharyngeal squamous cell carcinoma were tested using nested PCR assay for EBV DNA detection. Spectrophotometric methods were used to measure TAS values as well as SOD and GPx activities in homogenates of tissue, using diagnostic kits produced by Randox Laboratories. Sera from all individuals were investigated using ELISA method to detect the presence of Epstein-Barr virus capsid antigen (EBVCA) IgM and IgG, Epstein-Barr virus nuclear antigen (EBNA) IgG, and early antigen (EA) IgG antibodies. The level of TAS and activities of antioxidant enzymes (GPx and SOD) were significantly decreased in tissues with oropharyngeal cancer, particularly in EBV-positive cases. In 82.3% of patients, wt-LMP1 was detected. Significantly lower TAS, GPx, and SOD values were stated in patients infected with wild-type EBV. The presence of antibodies against early antigen (anti-EA) was detected in over 80% of patients, which suggests reactivation of EBV infection. The correlation between the degree of tumor differentiation and TN classification, especially in EBV-positive patients, was also observed. Determination of these parameters may be useful in evaluating tumor burden in patients with various stages of oropharyngeal cancer and could be an important prognostic factor. Future studies are needed to understand the role of EBV lytic reactivation induced by oxidative stress.


Assuntos
Antioxidantes/química , Infecções por Vírus Epstein-Barr/diagnóstico , Glutationa Peroxidase/metabolismo , Herpesvirus Humano 4/isolamento & purificação , Neoplasias Orofaríngeas/diagnóstico , Superóxido Dismutase/metabolismo , Idoso , Anticorpos Antivirais/sangue , Antioxidantes/metabolismo , DNA Viral/metabolismo , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/metabolismo , Feminino , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Orofaríngeas/complicações , Neoplasias Orofaríngeas/metabolismo
15.
Mol Cell ; 75(5): 1020-1030.e4, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31350119

RESUMO

Phage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution.


Assuntos
Bacteriófagos/fisiologia , DNA Viral/metabolismo , Escherichia coli/virologia , Ilhas Genômicas , Montagem de Vírus/fisiologia , DNA Viral/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
16.
BMC Gastroenterol ; 19(1): 110, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248389

RESUMO

BACKGROUND: Beta-herpesviruses are common opportunistic pathogens that cause morbidity after liver transplantation (LT). METHODS: Objective of the study was to evaluate the prevalence and correlation of herpesviruses in bile, blood and liver tissue and to investigate their association with biliary complications and retransplantation (re-LT) free survival after LT. The study design is a single-center case-control study. We performed quantative polymerase chain reaction (qPCR) for herpesvirus 1-8 DNA in bile, blood and liver tissue of 73 patients after first LT and analyzed their clinical courses retrospectively. RESULTS: The median follow-up was 48 months (range 2-102), during which a total of 16 patients underwent re-LT and 11 patients died. Of the patients, 46.5% received valganciclovir prophylaxis at the time of bile sample acquisition. Cytomegalovirus (CMV) (18.3%), human herpesvirus 6 (HHV-6) (34.2%), human herpesvirus 7 (HHV-7) (20.5%) and Epstein-Barr virus (EBV) (16.4%) were highly prevalent in bile after LT, while herpes simpex virus 1 and 2 (HSV-1, HSV-2), varicella-zoster virus (VZV) and human herpesvirus 8 (HHV-8) were not or rarely detected in bile. Valganciclovir prophylaxis did not reduce the prevalence of HHV-6 and HHV-7 in bile, but it did reduce the presence of CMV and EBV. The presence of HHV-6 in bile was associated with non-anastomotic biliary strictures (NAS) and acute cellular rejection (ACR). CONCLUSIONS: CMV, EBV, HHV-6 and HHV-7 are more prevalent in biliary fluid than in liver biopsy or blood serum after LT. HHV-6 and HHV-7 might be associated with biliary complications after LT. Biliary fluids might be an attractive target for routine herpesvirus detection.


Assuntos
Bile/virologia , DNA Viral/metabolismo , Infecções por Herpesviridae/epidemiologia , Herpesviridae/isolamento & purificação , Transplante de Fígado/efeitos adversos , Complicações Pós-Operatórias/virologia , Adulto , Idoso , Antivirais/uso terapêutico , Sangue/virologia , Estudos de Casos e Controles , Citomegalovirus/isolamento & purificação , DNA Viral/sangue , Feminino , Seguimentos , Infecções por Herpesviridae/complicações , Infecções por Herpesviridae/prevenção & controle , Humanos , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prevalência , Reoperação , Valganciclovir/uso terapêutico
17.
Vet Microbiol ; 233: 39-46, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176410

RESUMO

E5 protein, the major oncoprotein of bovine Deltapapillomavirus (BPV), was found to be expressed in 18 of 21 examined urothelial cancers of cattle. E5 oncoprotein was found to interact with p62 which was degraded through the autophagosome-lysosome pathway as well as LC3-II and appeared to be involved in the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α). Autophagy was morphologically documented by transmission electron microscope (TEM) through the detection of double-membrane autophagosomes and autolysosomes. Overexpression of Bag3 known to mediate selective autophagy was also demonstrated. Furthermore, Bag3 and BPV E5 oncoprotein were seen to co-localize with dynein and 14-3-3γ, which suggested that Bag3 could be involved in inducing the retrograde transport of BPV E5 along microtubules to aggresomes, perinuclear sites with high autophagic flux. Electron dense perinuclear structures consistent with aggresomes were also documented by TEM in urothelial cancer cells. Finally, Bag3 was found to also interact with synaptopodin 2 (Synpo2), which would seem to contribute to cargo degradation as it has been shown to facilitate autophagosome formation. This study provides mechanistic insights into the potential role(s) of autophagy in BPV disease, which can help to develop future treatment and control measures for BPV infection. Activation of autophagy correlates positively with BPV infection and may play a role in biological behavior of bladder cancer as urothelial carcinomas of cattle are known to be characterized by a relatively low rate of metastasis.


Assuntos
Autofagia , Papillomavirus Bovino 1/genética , Expressão Gênica , Proteínas Oncogênicas Virais/genética , Neoplasias da Bexiga Urinária/veterinária , Animais , Bovinos , Doenças dos Bovinos/virologia , DNA Viral/genética , DNA Viral/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Redes Reguladoras de Genes , Interações entre Hospedeiro e Microrganismos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Fosforilação , Neoplasias da Bexiga Urinária/virologia , Urotélio/virologia
18.
Hepatol Int ; 13(4): 431-439, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31177505

RESUMO

BACKGROUND: Data regarding the durability of HBV viral suppression with combination antiretroviral therapy (cART) containing tenofovir disoproxil fumarate (TDF) combined with lamivudine (3TC) or emtricitabine (FTC) in HIV/HBV-coinfected patients are scarce in hyperendemic areas of chronic HBV infection. METHODS: Between 2004 and 2016, HIV/HBV-coinfected Taiwanese with available baseline HBV DNA load were retrospectively reviewed. Determinations of plasma HBV DNA load, HBV serologic markers (HBsAg, anti-HBs, HBeAg, and anti-HBe), and liver function were performed after initiation of cART. Factors associated with time to undetectable HBV DNA load were explored. RESULTS: A total of 366 patients were included according to cART history: Group 1, 3TC as the only anti-HBV therapy (n = 73); Group 2, TDF-containing cART as initial therapy (n = 127); and Group 3, switch of 3TC-based to TDF-containing cART (n = 166). At year 5, HBV suppression was achieved in 77.8%, 95.7%, and 95.7% of Groups 1, 2 and 3, respectively. In multivariate Cox regression analysis, TDF ( ± 3TC or FTC) but not 3TC alone as initial anti-HBV therapy was significantly associated with HBV suppression (adjusted hazard ratio [aHR] 2.635; 95% CI 1.720-4.037), while HBeAg positivity at baseline was associated with failure to achieve HBV suppression (aHR 0.293; 95% CI 0.178-0.482). Loss of HBsAg occurred in 15 patients (4.1%), with 7 (1.9%) seroconversion to anti-HBs positivity, while HBeAg seroconversion occurred in 11 (16.9%) of 65 HBeAg-positive patients. CONCLUSIONS: TDF-containing cART achieved durable HBV viral suppression in HIV/HBV-coinfected patients and HBeAg positivity at baseline was associated with failure to achieve HBV suppression despite long-term TDF-containing cART.


Assuntos
Antivirais/administração & dosagem , Infecções por HIV/tratamento farmacológico , Hepatite B Crônica/tratamento farmacológico , Tenofovir/administração & dosagem , Adulto , DNA Viral/metabolismo , Substituição de Medicamentos , Quimioterapia Combinada , Feminino , Humanos , Lamivudina/administração & dosagem , Masculino , Estudos Retrospectivos , Resultado do Tratamento , Carga Viral
19.
PLoS Pathog ; 15(5): e1007691, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31107917

RESUMO

Cyclic GMP-AMP synthase (cGAS) senses viral DNA in the cytosol and then catalyzes synthesis of the second messenger cGAMP, which activates the ER-localized adaptor protein Mediator of IRF3 Activator (MITA) to initiate innate antiviral response. Human cytomegalovirus (HCMV) proteins can antagonize host immune responses to promote latent infection. Here, we identified HCMV UL42 as a negative regulator of cGAS/MITA-dependent antiviral response. UL42-deficiency enhances HCMV-induced production of type I interferons (IFNs) and downstream antiviral genes. Consistently, wild-type HCMV replicates more efficiently than UL42-deficient HCMV. UL42 interacts with both cGAS and MITA. UL42 inhibits DNA binding, oligomerization and enzymatic activity of cGAS. UL42 also impairs translocation of MITA from the ER to perinuclear punctate structures, which is required for MITA activation, by facilitating p62/LC3B-mediated degradation of translocon-associated protein ß (TRAPß). These results suggest that UL42 can antagonize innate immune response to HCMV by targeting the core components of viral DNA-triggered signaling pathways.


Assuntos
Antivirais/farmacologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Imunidade Inata/imunologia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Proteínas Virais/farmacologia , Citomegalovirus/efeitos dos fármacos , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , DNA Viral/genética , DNA Viral/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Transdução de Sinais
20.
PLoS One ; 14(5): e0217248, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31107918

RESUMO

The genome of Bacillus subtilis phage ϕ29 consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5' end (TP-DNA). ϕ29 DNA polymerase is the enzyme responsible for viral DNA replication, due to its distinctive properties: high processivity and strand displacement capacity, being able to replicate the entire genome without requiring the assistance of processivity or unwinding factors, unlike most replicases. ϕ29 single-stranded DNA binding protein (SSB) is encoded by the viral gene 5 and binds the ssDNA generated in the replication of the ϕ29 TP-DNA. It has been described to stimulate the DNA elongation rate during the DNA replication. Previous studies proposed residues Tyr50, Tyr57 and Tyr76 as ligands of ssDNA. The role of two of these residues has been determined in this work by site-directed mutagenesis. Our results showed that mutant derivative Y57A was unable to bind to ssDNA, to stimulate the DNA elongation and to displace oligonucleotides annealed to M13 ssDNA, whereas mutant Y50A behaved like the wild-type SSB.


Assuntos
Fagos Bacilares/genética , Fagos Bacilares/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Sequência de Bases , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA/genética , DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/química , Genoma Viral , Mutagênese Sítio-Dirigida , Tirosina/química , Proteínas Virais/química
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