RESUMO
While suppressive antiretroviral therapy can effectively limit HIV-1 replication and evolution, it leaves behind a residual pool of integrated viral genomes that persist in a state of reversible nonproductive infection, referred to as the HIV-1 reservoir. HIV-1 infection models were established to investigate HIV-1 latency and its reversal; recent work began to probe the dynamics of HIV-1 latency reversal at single-cell resolution. Signals that establish HIV-1 latency and govern its reactivation are complex and may not be completely resolved at the cellular and regulatory levels by the aggregated measurements of bulk cellular-sequencing methods. High-throughput single-cell technologies that characterize and quantify changes to the epigenome, transcriptome, and proteome continue to rapidly evolve. Combinations of single-cell techniques, in conjunction with novel computational approaches to analyze these data, were developed and provide an opportunity to improve the resolution of the heterogeneity that may exist in HIV-1 reactivation. In this review, we summarize the published single-cell HIV-1 transcriptomic work and explore how cutting-edge advances in single-cell techniques and integrative data-analysis tools may be leveraged to define the mechanisms that control the reversal of HIV-1 latency.
Assuntos
HIV-1/genética , HIV-1/fisiologia , Análise de Célula Única/métodos , Ativação Viral , Latência Viral/genética , Latência Viral/fisiologia , Linfócitos T CD4-Positivos/virologia , Perfilação da Expressão Gênica , Infecções por HIV/virologia , Humanos , Análise de Sequência de RNARESUMO
To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.
Assuntos
Betacoronavirus/genética , Colorimetria/métodos , Infecções por Coronavirus/diagnóstico , Programas de Rastreamento/economia , Pneumonia Viral/diagnóstico , RNA Viral/análise , Betacoronavirus/isolamento & purificação , COVID-19 , Colorimetria/economia , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/metabolismo , SARS-CoV-2 , Carga ViralRESUMO
Human adenovirus type 55 (HAdV-B55) is a recently identified acute respiratory disease (ARD) pathogen in HAdV species B with a recombinant genome between renal HAdV-B11 and respiratory HAdV-B14. Since HAdV-B55 first appeared in China school in 2006, no more ARD cases associated with it had been reported until 2011, when there was an outbreak of adult severe community-acquired pneumonia (CAP) in Beijing, China. Reported here is the bioinformatics analysis of the re-emergent HAdV-B55 responsible for this outbreak. Recombination and protein sequence analysis re-confirmed that this isolate (BJ01) was a recombinant virus with the capsid hexon gene from HAdV-B11. The selection pressures for the three capsid proteins, i.e., hexon, penton base, and fiber genes, were all negative, along with very low non-synonymous (dN) and synonymous (dS) substitutions/site (<0.0007). Phylogenetic analyses of the whole genome and the three major capsid genes of HAdV-B55 revealed the close phylogenetic relationship among all HAdV-B55 strains. Comparative genomic analysis of this re-emergent HAdV-B55 strain (BJ01; 2011) with the first HAdV-B55 strain (QS-DLL; 2006) showed the high genome identity (99.87%), including 10 single-nucleotide non-synonymous substitutions, 11 synonymous substitutions, 3 insertions, and one deletion in non-coding regions. The major non-synonymous substitutions (6 of 10) occurred in the protein pVI in its L3 region, which protein has different functions at various stages of an adenovirus infection, and may be associated with the population distribution of HAdV-B55 infection. No non-synonymous substitutions were found in the three major capsid proteins, which proteins are responsible for type-specific neutralizing antibodies. Comparative genomic analysis of the re-emergent HAdV-B55 strains associated with adult severe CAP revealed conserved genome and capsid proteins, providing the foundation for the development of effective vaccines against this pathogen. This study also facilitates the further investigation of HAdV-B55 epidemiology, molecular evolution, patterns of pathogen emergence and re-emergence, and the predication of genome recombination between adenoviruses.
RESUMO
Human adenoviruses (HAdVs) are highly contagious pathogens causing acute respiratory disease (ARD), among other illnesses. Of the ARD genotypes, HAdV-7 presents with more severe morbidity and higher mortality than the others. We report the isolation and identification of a genome type HAdV-7d (DG01_2011) from a recent outbreak in Southern China. Genome sequencing, phylogenetic analysis, and restriction endonuclease analysis (REA) comparisons with past pathogens indicate HAdV-7d has re-emerged in Southern China after an absence of twenty-one years. Recombination analysis reveals this genome differs from the 1950s-era prototype and vaccine strains by a lateral gene transfer, substituting the coding region for the L1 52/55â kDa DNA packaging protein from HAdV-16. DG01_2011 descends from both a strain circulating in Southwestern China (2010) and a strain from Shaanxi causing a fatality and outbreak (Northwestern China; 2009). Due to the higher morbidity and mortality rates associated with HAdV-7, the surveillance, identification, and characterization of these strains in population-dense China by REA and/or whole genome sequencing are strongly indicated. With these accurate identifications of specific HAdV types and an epidemiological database of regional HAdV pathogens, along with the HAdV genome stability noted across time and space, the development, availability, and deployment of appropriate vaccines are needed.
Assuntos
Infecções por Adenovirus Humanos/epidemiologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Doenças Transmissíveis Emergentes , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Adenovírus Humanos/classificação , China/epidemiologia , Biologia Computacional , Surtos de Doenças , Ordem dos Genes , Genes Virais , Genoma Viral , Humanos , Tipagem Molecular , Mutação , Filogenia , Polimorfismo de Nucleotídeo Único , Proibitinas , Recombinação Genética , Seleção GenéticaRESUMO
OBJECTIVE: To construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics. METHODS: The gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157: H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD600 value and Giemsa staining. RESULTS AND CONCLUSION: We established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.
Assuntos
Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Deleção de Genes , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Primers do DNA , Reação em Cadeia da PolimeraseRESUMO
Human adenoviruses (HAdVs), especially HAdV-B3, -E4 and -B7, are associated with Acute Respiratory Disease in Chinese children, and occasionally in adults. In order to establish and document the profiles of the respiratory adenovirus pathogen among children in Guangzhou, Southern China, a rapid, simple and practical method for identification and typing of respiratory adenoviruses was developed and evaluated. One pair of universal PCR primers was designed according to the conserved region of the hexon gene, which can detect not only HAdV-B3, -E4 and -B7, but also HAdV-B14, -F40 and -F41, with a specific 300bp PCR product. Three pairs of type-specific PCR primers were also designed according to the hypervariable regions of the hexon gene to type HAdV-B3, -E4 and -B7 by three independent PCR reactions, making it easy to optimize the PCR conditions. By using this method, one hundred throat swab specimens collected during Oct 2010 to Dec 2011 and suspected of being positive for adenoviral infection were identified and typed for adenoviruses. Of these samples, fifty-five were adenovirus-positive. The most common HAdV type was HAdV-B3, identified in 92.7% of samples, which is not only consistent with the data reported in 2004-2006, but also consistent with the recent report in Hangzhou, eastern China, indicating that HAdV-B3 has been circulating in Guangzhou, and maybe in eastern China, for many years. The method for the respiratory adenovirus identification and typing we developed is rapid, simple and practical, which has a potential in the real-time surveillance of circulating adenovirus strains and also to provide etiological evidence for the adenovirus-relative disease control and prevention in China.
Assuntos
Infecções por Adenovirus Humanos/diagnóstico , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Adenovírus Humanos/patogenicidade , Adenovírus Humanos/classificação , Adenovírus Humanos/isolamento & purificação , Linhagem Celular , China , DNA Viral/genética , Humanos , Reação em Cadeia da Polimerase , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologiaRESUMO
Enterohemorrhagic Escherichia coli (EHEC) employs a type III secretion system (TTSS) to export the translocator and effector proteins required for mucosal colonization. As an important bacterial effector protein in locus of enterocyte effacement four, the EspF protein causes F-actin filament aggregations to form attaching and effacing (A/E) lesions, and induces the destruction of brush-border microvilli and cytoskeletal rearrangements to form pedestals. However, the molecular pathogenesis of A/E lesions due to EHEC O157:H7 infection is unclear. In this study, we constructed an espF-deficient mutant (ΔespF) with a 162-bp deletion in the N-terminal domain by using overlap extension PCR. The results showed that EHEC EspF translocated into intestinal epithelial cells, targeted mitochondria and induced apoptosis. The ΔespF mutant, compared to EHEC prototype Guangzhou strain, had lower cell attachment and effacement abilities, lower caspase-9/3 and lactate dehydrogenase levels, lower bacterial adhesion, weaker mitochondria apoptosis, and a higher mouse survival rate. Our results demonstrate the probable function of the EspF N-terminal domain, which targets mitochondria and binds mitochondria heat shock protein 70 to induce cell apoptosis via A/E lesions. These findings may be invaluable in clarifying the molecular pathogenesis of EspF of EHEC O157:H7.
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Infecções por Escherichia coli/metabolismo , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Domínios e Motivos de Interação entre Proteínas , Animais , Apoptose/genética , Aderência Bacteriana/genética , Sistemas de Secreção Bacterianos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Interações Hospedeiro-Patógeno , Peptídeos e Proteínas de Sinalização Intracelular , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Mutação , Virulência/genéticaRESUMO
Human adenovirus type 55 (HAdV-B55) is an acute respiratory disease (ARD) pathogen first completely characterized in China (2006). This is a unique Trojan horse microbe with the virus neutralization attribute of a renal pathogen and the cell tropism and clinical attributes of a respiratory pathogen, bypassing herd immunity. It appeared to be an uncommon pathogen, with earlier putative, sporadic occurrences in Spain (1969) and Turkey (2004); these isolates were incompletely characterized using only two epitopes. Reported here is the genome of a second recent isolate (China, 2011), indicating that it may occur more frequently. The availability of this HAdV-B55 genome provides a foundation for studying adenovirus molecular evolution, the dynamics of epidemics, and patterns of pathogen emergence and re-emergence. These data facilitate studies to predict genome recombination between adenoviruses, as well as sequence divergence rates and hotspots, all of which are important for vaccine development and because HAdVs are used for epitope and/or gene delivery vectors.
Assuntos
Adenoviridae/genética , Infecções por Adenovirus Humanos/genética , Infecções Respiratórias/genética , China , Biologia Computacional/métodos , DNA Viral/genética , Epitopos/química , Vetores Genéticos , Genoma , Genoma Viral , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Recombinação Genética , Infecções Respiratórias/virologiaRESUMO
Emergent pathogens may be examined rapidly at high resolution on a molecular level using genomics, allowing an understanding of their evolution. China is a unique environment for studying pathogens, having a large, dense, and generally closed population. Human adenovirus type 14 (HAdV-14) was originally identified as an acute respiratory disease (ARD) pathogen in The Netherlands (1955), with a second isolation in England (1957). Since then, few reports of this virus appeared until an ARD pathogen with a similar genome caused multiple outbreaks in the United States (2006 to 2009). This report presents the first genome of HAdV-B14 isolated in China (2010). As China experienced two recent outbreaks of an emergent ARD pathogen, HAdV-B55, containing much of the HAdV-B14 genome, the availability of this HAdV-B14 sequence will facilitate studies of the epidemiology of these pathogens, as well as provide a foundation for studying adenovirus evolution and the genesis of emergent pathogens. These observations may be invaluable in predicting possible recombination between wild-type viruses and adenoviral gene delivery vectors, including adenovirus vaccines.