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1.
Int J Oral Sci ; 12(1): 3, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911577

RESUMO

High-risk human papillomaviruses (HPVs) are involved in the development of several human cancers, including oropharyngeal squamous cell carcinomas. However, many studies have demonstrated that HPV alone is not sufficient for the oncogenic transformation of normal human epithelial cells, indicating that additional cofactors are required for the oncogenic conversion of HPV-infected cells. Inasmuch as chronic inflammation is also closely associated with carcinogenesis, we investigated the effect of chronic exposure to tumor necrosis factor α (TNFα), the major proinflammatory cytokine, on oncogenesis in two immortalized oral keratinocyte cell lines, namely, HPV16-immortalized and human telomerase reverse transcriptase (hTERT)-immortalized cells. TNFα treatment led to the acquisition of malignant growth properties in HPV16-immortalized cells, such as (1) calcium resistance, (2) anchorage independence, and (3) increased cell proliferation in vivo. Moreover, TNFα increased the cancer stem cell-like population and stemness phenotype in HPV16-immortalized cells. However, such transforming effects were not observed in hTERT-immortalized cells, suggesting an HPV-specific role in TNFα-promoted oncogenesis. We also generated hTERT-immortalized cells that express HPV16 E6 and E7. Chronic TNFα exposure successfully induced the malignant growth and stemness phenotype in the E6-expressing cells but not in the control and E7-expressing cells. We further demonstrated that HPV16 E6 played a key role in TNFα-induced cancer stemness via suppression of the stemness-inhibiting microRNAs miR-203 and miR-200c. Overexpression of miR-203 and miR-200c suppressed cancer stemness in TNFα-treated HPV16-immortalized cells. Overall, our study suggests that chronic inflammation promotes cancer stemness in HPV-infected cells, thereby promoting HPV-associated oral carcinogenesis.


Assuntos
Carcinoma de Células Escamosas/genética , Papillomavirus Humano 16/metabolismo , MicroRNAs/metabolismo , Neoplasias Bucais/genética , Boca/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/virologia , Telomerase/genética , Fator de Necrose Tumoral alfa/metabolismo , Carcinogênese/genética , Carcinogênese/imunologia , Carcinoma de Células Escamosas/patologia , Transformação Celular Viral/genética , Regulação da Expressão Gênica , Genes Virais , Papillomavirus Humano 16/genética , Humanos , MicroRNAs/genética , Boca/virologia , Neoplasias Bucais/patologia , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Telomerase/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
2.
Gene ; 734: 144382, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31978513

RESUMO

Japanese macaque (Macaca fuscata) is an indigenous Old World monkey (OWM) species that inhabits the Japanese archipelago. There are two subspecies of Japanese macaque: Yakushima macaque (M. f. yakui) which inhabits Yakushima Island exclusively, and Hondo macaque (M. f. fuscata) which inhabits the mainland of Japan. Yakushima macaque is considered to be branched off from a certain parental macaque group that had inhabited the mainland of Japan. However, the process of sub-speciation of the Yakushima macaque is still unclear at present. In this study, to gain new insight into the process of sub-speciation of Japanese macaque, we utilized the simian foamy virus (SFV) as a marker. SFVs are found in virtually all primates except humans and undergo species-specific cospeciation with the hosts. The phylogenetic analysis of conserved regions of the env gene in SFVs remarkably resembled that of the OWMs with high statistical confidence. The phylogenetic analyses also indicated that there are four (1-4) genotypes among Asian OWMs investigated. SFVs derived from Asian OWMs except Yakushima macaque were classified as genotypes 1-3, whereas SFVs isolated from all Yakushima macaques and one Hondo macaque were classified as genotype 4. Interestingly, genotype 4 was firstly branched off from the rest of the genotypes, which might indicate that the macaques infected with genotype 4 SFV were derived from the "older" population of Japanese macaques. The high prevalence of genotype 4 SFVs among Yakushima macaque might reflect the possibility that they are a descendant of the population settled earlier, which has been geographically isolated in Yakushima Island.


Assuntos
/virologia , Vírus Espumoso dos Símios/classificação , Animais , Células Cultivadas , Genes Virais , Genes env , Integrases/genética , Tipagem Molecular , Filogenia , Vírus Espumoso dos Símios/genética , Vírus Espumoso dos Símios/isolamento & purificação , Especificidade da Espécie
3.
Appl Biochem Biotechnol ; 190(1): 293-304, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31346919

RESUMO

Cephalosporin C acylase (CCA) is the key enzyme in the production of 7-aminocephalosporanic acid (7-ACA) via a one-step enzymatic process. To improve the soluble expression level of CCA in recombinant Escherichia coli at elevated temperatures, a library of T7 promoter mutants was created by site-saturation mutagenesis, and a series of mutated promoters were subsequently screened. Green fluorescent protein (GFP) was fused to the C-terminus of CCA to facilitate library screening, and the expression of the CCA and GFP fusion proteins was investigated under the control of the T7 promoter. Twenty-four mutants were selected by detecting the fluorescence intensity of colonies on agar plates to form a library with different expression levels. The enzyme activities of the mutants were positively correlated with their fluorescence intensities. The highest enzyme activity among these mutant promoters was 1.3-fold higher than the enzyme activity resulting from the wild-type promoter when the cells were cultured at 32 °C for 16 h. In addition, the transcription and expression levels of several typical promoters were discussed, and the effects of GFP fusion on the enzyme activity of CCA were investigated.


Assuntos
Amidoidrolases/genética , Bacteriófago T7/genética , Cefalosporinas/metabolismo , Escherichia coli/genética , Ensaios de Triagem em Larga Escala , Mutação , Regiões Promotoras Genéticas , Amidoidrolases/metabolismo , Genes Virais , Proteínas de Fluorescência Verde/genética , Transcrição Genética
4.
Arch Virol ; 165(2): 515-517, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31863264

RESUMO

vB_BmeM-Goe8 is a phage preying on Bacillus megaterium. Its genome has a GC content of 38.9%, is 161,583 bp in size, and has defined ends consisting of 7436-bp-long terminal repeats. It harbours 11 genes encoding tRNAs and 246 coding DNA sequences, 66 of which were annotated. The particle reveals Myoviridae morphology, and the formation of a double baseplate upon tail sheath contraction indicates morphological relatedness to the group of SPO1-like phages. BLASTn comparison against the NCBI non-redundant nucleotide database revealed that Bacillus phage Mater is the closest relative of vB_BmeM-Goe8.


Assuntos
Fagos Bacilares/classificação , Fagos Bacilares/isolamento & purificação , Bacillus megaterium/virologia , Genes Virais , Genoma Viral , Myoviridae/classificação , Myoviridae/isolamento & purificação , Fagos Bacilares/genética , Fagos Bacilares/ultraestrutura , Composição de Bases , Análise por Conglomerados , Myoviridae/genética , Myoviridae/ultraestrutura , Filogenia , Análise de Sequência de DNA , Homologia de Sequência , Sequências Repetidas Terminais , Vírion/ultraestrutura
5.
Int J Cancer ; 146(1): 181-191, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31090066

RESUMO

Mechanisms of viral oncogenesis are diverse and include the off-target activity of enzymes expressed by the infected cells, which evolved to target viral genomes for controlling their infection. Among these enzymes, the single-strand DNA editing capability of APOBECs represent a well-conserved viral infection response that can also cause untoward mutations in the host DNA. Here we show, after evaluating somatic single-nucleotide variations and transcriptome data in 240 gastric cancer samples, a positive correlation between APOBEC3s mRNA-expression and the APOBEC-mutation signature, both increased in EBV+ tumors. The correlation was reinforced by the observation of APOBEC mutations preferentially occurring in the genomic loci of the most active transcripts. This EBV infection and APOBEC3 mutation-signature axis were confirmed in a validation cohort of 112 gastric cancer patients. Our findings suggest that APOBEC3 upregulation in EBV+ cancer may boost the mutation load, providing further clues to the mechanisms of EBV-induced gastric carcinogenesis. After further validation, this EBV-APOBEC axis may prove to be a secondary driving force in the mutational evolution of EBV+ gastric tumors, whose consequences in terms of prognosis and treatment implications should be vetted.


Assuntos
Citidina Desaminase/genética , DNA de Neoplasias/genética , Herpesvirus Humano 4/patogenicidade , Neoplasias Gástricas/virologia , Carcinogênese , Genes Virais , Herpesvirus Humano 4/genética , Humanos , Mutação , Neoplasias Gástricas/patologia
6.
PLoS Pathog ; 15(12): e1008210, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31834912

RESUMO

There are many documented examples of viral genes retained in the genomes of multicellular organisms that may in some cases bring new beneficial functions to the receivers. The ability of certain ichneumonid parasitic wasps to produce virus-derived particles, the so-called ichnoviruses (IVs), not only results from the capture and domestication of single viral genes but of almost entire ancestral virus genome(s). Indeed, following integration into wasp chromosomal DNA, the putative and still undetermined IV ancestor(s) evolved into encoding a 'virulence gene delivery vehicle' that is now required for successful infestation of wasp hosts. Several putative viral genes, which are clustered in distinct regions of wasp genomes referred to as IVSPERs (Ichnovirus Structural Protein Encoding Regions), have been assumed to be involved in virus-derived particles morphogenesis, but this question has not been previously functionally addressed. In the present study, we have successfully combined RNA interference and transmission electron microscopy to specifically identify IVSPER genes that are responsible for the morphogenesis and trafficking of the virus-derived particles in ovarian cells of the ichneumonid wasp Hyposoter didymator. We suggest that ancestral viral genes retained within the genomes of certain ichneumonid parasitoids possess conserved functions which were domesticated for the purpose of assembling viral vectors for the delivery of virulence genes to parasitized host animals.


Assuntos
Vírion/fisiologia , Vespas/genética , Vespas/virologia , Animais , Genes Virais/genética , Polydnaviridae/genética , Interferência de RNA
7.
BMC Evol Biol ; 19(1): 232, 2019 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-31878875

RESUMO

BACKGROUND: Inexpensive pathogen genome sequencing has had a transformative effect on the field of phylodynamics, where ever increasing volumes of data have promised real-time insight into outbreaks of infectious disease. As well as the sheer volume of pathogen isolates being sequenced, the sequencing of whole pathogen genomes, rather than select loci, has allowed phylogenetic analyses to be carried out at finer time scales, often approaching serial intervals for infections caused by rapidly evolving RNA viruses. Despite its utility, whole genome sequencing of pathogens has not been adopted universally and targeted sequencing of loci is common in some pathogen-specific fields. RESULTS: In this study we highlighted the utility of sequencing whole genomes of pathogens by re-analysing a well-characterised collection of Ebola virus sequences in the form of complete viral genomes (≈19 kb long) or the rapidly evolving glycoprotein (GP, ≈2 kb long) gene. We have quantified changes in phylogenetic, temporal, and spatial inference resolution as a result of this reduction in data and compared these to theoretical expectations. CONCLUSIONS: We propose a simple intuitive metric for quantifying temporal resolution, i.e. the time scale over which sequence data might be informative of various processes as a quick back-of-the-envelope calculation of statistical power available to molecular clock analyses.


Assuntos
Ebolavirus/genética , Genes Virais , Genoma Viral , Doença pelo Vírus Ebola/epidemiologia , Mapeamento Cromossômico , Surtos de Doenças , Doença pelo Vírus Ebola/transmissão , Doença pelo Vírus Ebola/virologia , Humanos , Cadeias de Markov , Filogenia , Sequenciamento Completo do Genoma
8.
Vet Microbiol ; 239: 108451, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31767095

RESUMO

The substantial genetic diversity exhibited by influenza A viruses of swine (IAV-S) represents the main challenge for the development of a broadly protective vaccine against this important pathogen. The consensus vaccine immunogen has proven an effective vaccinology approach to overcome the extraordinary genetic diversity of RNA viruses. In this project, we sought to determine if a consensus IAV-S hemagglutinin (HA) immunogen would elicit broadly protective immunity in pigs. To address this question, a consensus HA gene (designated H3-CON.1) was generated from a set of 1,112 H3 sequences of IAV-S recorded in GenBank from 2011 to 2015. The consensus HA gene and a HA gene of a naturally occurring H3N2 IAV-S strain (designated H3-TX98) were expressed using the baculovirus expression system and emulsified in an oil-in-water adjuvant to be used for vaccination. Pigs vaccinated with H3-CON.1 immunogen elicited broader levels of cross-reactive neutralizing antibodies and interferon gamma secreting cells than those vaccinated with H3-TX98 immunogen. After challenge infection with a fully infectious H3N2 IAV-S isolate, the H3-CON.1-vaccinated pigs shed significantly lower levels of virus in their nasal secretions than the H3-TX98-vaccinated pigs. Collectively, our data provide a proof-of-evidence that the consensus immunogen approach may be effectively employed to develop a broadly protective vaccine against IAV-S.


Assuntos
Genes Virais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae , Doenças dos Suínos , Vacinação/veterinária , Animais , Anticorpos Antivirais/sangue , Sequência Consenso/genética , Sequência Consenso/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Eliminação de Partículas Virais/imunologia
9.
Top Antivir Med ; 27(3): 111-121, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31634862

RESUMO

The 2019 edition of the IAS-USA drug resistance mutations list updates the Figure last published in January 2017. The mutations listed are those that have been identified by specific criteria for evidence and drugs described. The Figure is designed to assist practitioners in identifying key mutations associated with resistance to antiretroviral drugs, and therefore, in making clinical decisions regarding antiretroviral therapy.


Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação , Substituição de Aminoácidos , Fármacos Anti-HIV/uso terapêutico , Genes Virais/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Inibidores da Protease de HIV/farmacologia , Humanos , Inibidores da Transcriptase Reversa/farmacologia , Estados Unidos
10.
J Microbiol ; 57(11): 1033-1039, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31659688

RESUMO

Primary infections with the varicella-zoster virus (VZV) result in varicella, while latent reactivation leads to herpes zoster. Both varicella and zoster can be prevented by live attenuated vaccines. There have been reports suggesting that both clinical VZV strains and those in vaccine preparations are genetically polymorphic, containing mixtures of both wild-type and vaccine-type sequences at certain vaccine-specific sites. In this study, the genetic polymorphism of the VZV genome was examined by analyzing the frequencies of minor alleles at each nucleotide position. Next-generation sequencing of the clinical VZV strain YC02 passaged in an in vitro cell culture was used to identify genetically polymorphic sites (GPS), where the minor allele frequency (MAF) exceeded 5%. The number of GPS increased by 7.3-fold at high passages (p100) when compared to low passages (p17), although the average MAF remained similar. GPS were found in 6 open reading frames (ORFs) in p17, 35, and 54 ORFs in p60 and p100, respectively. GPS were found more frequently in the dispensable gene group than the essential gene group, but the average MAF was greater in the essential gene group. The most common two major/minor base pairs were A/g and T/c. GPS were found in all three passages at 16 positions, all located in the reiterated (R) region. The population diversity as measured by Shannon entropy increased in p60 and p100. However, the entropy remained unchanged in the R regions.


Assuntos
Técnicas de Cultura de Células , Herpesvirus Humano 3/genética , Polimorfismo Genético , Varicela/prevenção & controle , Genes Virais/genética , Variação Genética , Genoma Viral , Herpes Zoster/prevenção & controle , Humanos , Fases de Leitura Aberta , Vacinas Atenuadas , Sequenciamento Completo do Genoma
11.
Anal Bioanal Chem ; 411(28): 7451-7460, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31588523

RESUMO

Cervical cancer is the second most common cancer in the world's woman population with a high incidence in developing countries where diagnostic conditions for the cancer are poor. The main culprit causing the cancer is the human papillomavirus (HPV). HPV is divided into three major groups, i.e., high-risk (HR) group, probable high-risk (pHR) group, and low-risk (LR) group according to their potential of causing cervical cancer. Therefore, developing a sensitive, reliable, and cost-effective point-of-care diagnostic method for the virus genotypes in developing countries even worldwide is of high importance for the cancer prevention and control strategies. Here we present a combined method of isothermal recombinase polymerase amplification (RPA), lateral flow dipstick (LFD), and reverse dot blot (RDB), in quick point-of-care identification of HPV genotypes. The combined method is highly specific to HPV when the conserved L1 genes are used as targeted genes for amplification. The method can be used in identification of HPV genotypes at point-of-care within 1 h with a sensitivity of low to 100 fg of the virus genomic DNA. We have demonstrated that it is an excellent diagnostic point-of-care assay in monitoring the disease without time-consuming and expensive procedures and devices.


Assuntos
Southern Blotting/métodos , Genes Virais , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sequência de Aminoácidos , DNA Viral/análise , DNA Viral/normas , Humanos , Limite de Detecção , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Reprodutibilidade dos Testes
12.
PLoS Pathog ; 15(9): e1007936, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31504075

RESUMO

Wolbachia are the most widespread maternally-transmitted bacteria in the animal kingdom. Their global spread in arthropods and varied impacts on animal physiology, evolution, and vector control are in part due to parasitic drive systems that enhance the fitness of infected females, the transmitting sex of Wolbachia. Male killing is one common drive mechanism wherein the sons of infected females are selectively killed. Despite decades of research, the gene(s) underlying Wolbachia-induced male killing remain unknown. Here using comparative genomic, transgenic, and cytological approaches in fruit flies, we identify a candidate gene in the eukaryotic association module of Wolbachia prophage WO, termed WO-mediated killing (wmk), which transgenically causes male-specific lethality during early embryogenesis and cytological defects typical of the pathology of male killing. The discovery of wmk establishes new hypotheses for the potential role of phage genes in sex-specific lethality, including the control of arthropod pests and vectors.


Assuntos
Prófagos/genética , Prófagos/patogenicidade , Wolbachia/patogenicidade , Wolbachia/virologia , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Drosophila/embriologia , Drosophila/microbiologia , Drosophila/virologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/microbiologia , Drosophila melanogaster/virologia , Feminino , Genes Letais , Genes Virais , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Masculino , Prófagos/fisiologia , Razão de Masculinidade , Simbiose/genética , Simbiose/fisiologia , Proteínas Virais/genética , Proteínas Virais/fisiologia
13.
Acta Virol ; 63(3): 270-277, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507192

RESUMO

Orf, also called contagious ecthyma or contagious pustular dermatitis, is a significant zoonotic disease that primarily affects goat and sheep globally. Currently, the infection by orf virus (ORFV) has been observed in different host species worldwide, including China. Here, a suspected outbreak of orf infection in a goat farm in Anhui Province in 2018 was investigated. Through PCR, electron microscopy, and cell culture techniques, we confirmed that the outbreak was caused by ORFV. Consequently, the orf virus strain was named the AH/LA/2018 strain. The amplified and sequenced ORFV011 (B2L) and ORFV059 (F1L) genes were used to construct phylogenetic trees to elucidate the genetic characteristics of the ORFV and the molecular epidemiology of orf. The present study is the first systematic evolution analysis of the ORFV strain isolated in Anhui Province. The results of this study will be helpful to better understand the characteristics of ORFV, to help prevent and control the transmission of ORFV at an early stage in China. Keywords: Anhui Province; goat; orf virus; phylogenetic analysis.


Assuntos
Ectima Contagioso , Vírus do Orf , Filogenia , Animais , Células Cultivadas , China/epidemiologia , Ectima Contagioso/virologia , Genes Virais/genética , Doenças das Cabras/epidemiologia , Doenças das Cabras/virologia , Cabras , Microscopia Eletrônica de Transmissão , Vírus do Orf/classificação , Vírus do Orf/ultraestrutura , Reação em Cadeia da Polimerase , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/virologia
14.
BMC Infect Dis ; 19(1): 762, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477028

RESUMO

BACKGROUND: Avian influenza A (H5N6) virus poses a great threat to the human health since it is capable to cross the species barrier and infect humans. Although human infections are believed to largely originate from poultry contaminations, the transmissibility is unclear and only limited information was available on poultry environment contaminations, especially in Fujian Province. METHODS: A total of 4901 environmental samples were collected and tested for Avian Influenza Virus (AIV) from six cities in Fujian Province through the Fujian Influenza Surveillance System from 2013 to 2017. Two patient-related samples were taken from Fujian's first confirmed H5N6 human case and his backyard chicken feces in 2017. Chi-square test or Fisher's exact probability test was used to compare the AIV and the viral subtype positive rates among samples from different Surveillance cities, surveillance sites, sample types, and seasons. Phylogenetic tree analysis and molecular analysis were conducted to track the viral transmission route of the human infection and to map out the evolutions of H5N6 in Fujian. RESULTS: The overall positive rate of the H5 subtype AIVs was 4.24% (208/4903). There were distinctive differences (p < 0.05) in the positive rates in samples from different cities, sample sites, sample types and seasons. The viruses from the patient and his backyard chicken feces shared high homologies (99.9-100%) in all the eight gene segments. Phylogenetic trees also showed that these two H5N6 viruses were closely related to each other, and were classified into the same genetic clade 2.3.4.4 with another six H5N6 isolates from the environmental samples. The patient's H5N6 virus carried genes from H6N6, H5N8 and H5N6 viruses originated from different areas. The R294K or N294S substitution was not detected in the neuraminidase (NA). The S31 N substitution in the matrix2 (M2) gene was detected but only in one strain from the environmental samples. CONCLUSIONS: The H5 subtype of AIVs has started circulating in the poultry environments in Fujian Province. The patient's viral strain originated from the chicken feces in his backyard. Genetic reassortment in H5N6 viruses in Fujian Province was indicated. The H5N6 viruses currently circulating in Fujian Province were still commonly sensitive to Oseltamivir and Zanamivir, but the resistance against Amantadine has emerged.


Assuntos
Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Influenza Humana/epidemiologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia , Aves Domésticas/virologia , Animais , Embrião de Galinha , Galinhas/virologia , China/epidemiologia , Patos/virologia , Meio Ambiente , Microbiologia Ambiental , Genes Virais , Abrigo para Animais/normas , Humanos , Vírus da Influenza A/genética , Influenza Aviária/diagnóstico , Influenza Aviária/epidemiologia , Tipagem Molecular , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/transmissão , Filogenia , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/virologia , Fatores de Risco
15.
Cell Host Microbe ; 26(3): 347-358.e7, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31471273

RESUMO

Viral rebound upon stopping combined antiretroviral therapy poses a major barrier toward an HIV cure. Cellular and anatomical sources responsible for reinitiating viral replication remain a subject of ardent debate, despite extensive research efforts. To unravel the source of rebounding viruses, we conducted a large-scale HIV-STAR (HIV-1 sequencing before analytical treatment interruption to identify the anatomically relevant HIV reservoir) clinical trial. We collected samples from 11 participants and compared the genetic composition of (pro)viruses collected under treatment from different cellular and anatomical compartments with that of plasma viruses sampled during analytical treatment interruption. We found a remarkably heterogeneous source of viral rebound. In addition, irrespective of the compartment or cell subset, genetically identical viral expansions played a significant role in viral rebound. Our study suggests that although there does not seem to be a primary source for rebound HIV, cellular proliferation is an important driver of HIV persistence and should therefore be considered in future curative strategies.


Assuntos
Infecções por HIV/virologia , HIV-1/genética , Dispositivos de Acesso Vascular/virologia , Antirretrovirais/uso terapêutico , Medula Óssea/virologia , Proliferação de Células , Líquido Cefalorraquidiano/virologia , Feminino , Genes Virais , HIV-1/isolamento & purificação , Humanos , Cinética , Linfonodos/virologia , Tecido Linfoide/virologia , Masculino , Plasma , Carga Viral , Replicação Viral
16.
BMC Infect Dis ; 19(1): 778, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488066

RESUMO

BACKGROUND: A diagnostic method to simultaneously detect and discriminate porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3) in clinical specimens is imperative for the differential diagnosis and monitoring and control of PCVs in the field. METHODS: Three primer pairs were designed and used to develop a multiplex PCR assay. And 286 samples from 8 farms in Hubei province were tested by the developed multiplex PCR assay to demonstrate the accuracy. RESULTS: Each of target genes of PCV1, PCV2 and PCV3 was amplified using the designed primers, while no other porcine viruses genes were detected. The limit of detection of the assay was 10 copies/µL of PCV1, PCV2 OR PCV3. The results of the tissue samples detection showed that PCV1, PCV2 and PCV3 are co-circulating in central China. The PCV1, PCV2 and PCV3 singular infection rate was 52.4% (150/286), 61.2% (175/286) and 45.1% (129/286), respectively, while the PCV1 and PCV2 co-infection rate was 11.2% (32/286), the PCV1 and PCV3 co-infection rate was 5.9% (17/286), the PCV2 and PCV3 co-infection rate was 23.4% (67/286), and the PCV1, PCV2 and PCV3 co-infection rate was 1.7% (5/286), respectively, which were 100% consistent with the sequencing method and real-time PCR methods. CONCLUSIONS: The multiplex PCR assay could be used as a differential diagnostic tool for monitoring and control of PCVs in the field. The results also indicate that the PCVs infection and their co-infection are severe in Hubei province, Central China.


Assuntos
Infecções por Circoviridae/diagnóstico , Circovirus/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Animais , China , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/virologia , Circovirus/classificação , Circovirus/genética , Diagnóstico Diferencial , Genes Virais , Incidência , Tipagem Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/epidemiologia , Virologia/métodos
17.
Anal Chim Acta ; 1079: 171-179, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31387708

RESUMO

Recent study proves that the combination of loop mediated isothermal nucleic acid amplification (LAMP) with one-step strand displacement (OSD) is of great help to improve the sequence specificity during genetic detection. However, because OSD is incapable of signal amplification, the signal-to-noise ratio or the observable signal change may be usually not significant enough to satisfy practical usage. With the purpose to overcome this challenge, herein a more advanced and practical sensing principle is developed with the OSD replaced by an amplifiable nucleic acid circuit, hybridization chain reaction (HCR). The very contagious norovirus (NoV) was employed as the model target. Compared with LAMP-OSD, the LAMP-HCR can detect as few as 30 copies of NoV gene in 2% fecal samples with significantly enlarged signal change and signal-to-background ratio. Therefore, more reliable detection is achieved. Moreover, due to the high compatibility of HCR, the final LAMP-HCR products can be flexibly transduced into different types of readouts, including fluorescence, flow cytometer (FCM) and even a personal glucose meter (PGM). This further enlarges the operating environments for the detection from hospital labs, bedsides, to potential off-the-shelf devices in local pharmacies. Especially when using FCM or PGM, with the assistance of magnetic beads (MBs), the detection shows even higher tolerance capability to complicated biological matrices.


Assuntos
DNA Viral/análise , Citometria de Fluxo/métodos , Genes Virais , Norovirus/genética , DNA Viral/genética , Fezes/virologia , Humanos , Fenômenos Magnéticos , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes
18.
Microb Pathog ; 135: 103636, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377236

RESUMO

Plants deploy RNA silencing as a natural defence against invading viruses involving sequence-specific degradation of the viral RNAs. As a counter-defence strategy, viruses encode suppressor proteins that simultaneously target different steps of the silencing machinery. Tomato leaf curl Palampur virus (ToLCPalV) is a bipartite begomovirus in Geminiviridae family. It is responsible for significant reduction in the crop yield and quality. DNA-A of the virus encodes for six proteins whereas DNA-B codes for two proteins. In this study, all viral genes were screened for their role in suppression of green fluorescent protein (GFP) silencing in Nicotiana tabacum cv. Xanthi, employing agrobacterium based co-infiltration assay. The assay identified AC4 as a potential suppressor of RNA silencing. In addition, AC4 expression also suppressed virus-induced gene silencing (VIGS) of the phytoene desaturase (PDS) gene in N. benthamiana. Potato virus X (PVX) mediated transient expression of the AC4 in N. benthamiana showed enhanced symptoms that include downward leaf curling, leaf puckering and tissue necrosis. Further, N. benthamiana lines stably expressing AC4 showed severe developmental abnormalities. Mutational analysis suggested that glycine at 2nd position is essential for AC4 pathogenicity. Collectively, these findings demonstrate the role of ToLCPalV AC4 in viral pathogenesis, disease establishment and suppression of gene silencing.


Assuntos
Begomovirus/metabolismo , Doenças das Plantas/virologia , Interferência de RNA/fisiologia , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Begomovirus/genética , Coinfecção , Regulação Viral da Expressão Gênica , Genes Virais , Glicina/metabolismo , Proteínas de Fluorescência Verde , Oxirredutases/genética , Mutação Puntual , Potexvirus , Tabaco/virologia , Proteínas Virais/genética , Virulência
19.
Biotechnol Lett ; 41(10): 1121-1131, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31444662

RESUMO

OBJECTIVES: To analyze the effect of Ac25 on the proliferation of AcMNPV (Autographa californica multicapsid nucleopolyhedrovirus) progeny virus and its function in virogenic stroma. RESULTS: AcMNPV is a model of baculovirus and is the most widely studied baculovirus. Ac25, as a single-stranded DNA-binding protein, is involved in viral genomic DNA replication. Viral proliferation assay showed that AcMNPV progeny virus could not be produced when Ac25 was knocked out, which indicated it was crucial for BV production. Absolute quantitative PCR analysis indicated that Ac25 was able to promote replication of the AcMNPV genome in host Sf9 cells. It was also found that Ac25 could increase the transcription level of 38k and vp39 late expression genes, and inhibit host cell proliferation. CONCLUSION: Ac25 is highly accumulated in the nucleus and promotes progeny virus production by stimulating viral genome replication and up-regulating the expression of late genes. Two potential applications of vAc-Ac25-EGFP were proposed: an improved bac-to-bac eukaryotic protein expression systems and biopesticides.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Genes Virais , /genética , Proteínas Virais/metabolismo , Liberação de Vírus , Replicação Viral , Animais , Proteínas de Ligação a DNA/genética , Células Sf9 , Spodoptera , Proteínas Virais/genética
20.
ACS Appl Mater Interfaces ; 11(38): 34717-34724, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31469541

RESUMO

The CRISPR/Cas gene editing system has been successfully applied to combating bacteria, cancer, virus, and genetic disorders. While viral vectors have been used for the delivery of the CRISPR/Cas9 system, the time required for insert cloning, and virus packaging and standardization, hinders its efficient use. Additionally, the high molecular weight of the Cas9 endonuclease makes it not easy for packing into the vehicles. Herein we report the self-assembly of gold nanoclusters (AuNCs) with SpCas9 protein (SpCas9-AuNCs) under physiological conditions and the efficient delivery of SpCas9 into the cell nucleus. This assembly process is highly dependent on pH. SpCas9-AuNCs are stable at a higher pH but are disassembled at a lower pH. Significantly, this assembly-disassembly process facilitates the delivery of SpCas9 into cells and the cell nucleus, where the SpCas9 exerts its cleavage function. As a proof-of-concept, the assembled SpCas9-AuNCs nanoparticles are successfully used for efficient knockout of the E6 oncogene, restoring the function of tumor-suppressive protein p53 and inducing apoptosis in cervical cancer cells with little effect on normal human cells. The SpCas9-AuNCs are useful for sgRNA functional validation, sgRNA library screening, and genomic manipulation.


Assuntos
Proteína 9 Associada à CRISPR , Genes Virais , Ouro , Nanopartículas Metálicas , Oncogenes , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/farmacologia , Feminino , Ouro/química , Ouro/farmacologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia
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