RESUMEN
Cervical cancer screening in Brazil is opportunistic, based on cytology and offered for women aged 25-64 years, with low coverage (30%) and 70% of cancer diagnoses done in advanced stages, without impact on mortality. The current study reports 5-year first-round results of a population-based DNA-HPV testing screening program in a Brazilian city, which intended to be a model for transition to a more efficient program. Program flowchart is simple and current, indicating repetition of a negative test after five years. The first-round (October 2017-September 2022) screened 20,551 women by DNA-HPV testing with 58.7% coverage and 99.4% compliance with the program's targeted age range. Coverage increases to 77.8% when excluding the 'pandemic period'. The DNA-HPV testing was 87.2% negative with 6.2% colposcopy referrals and 84.8% colposcopies performed. A total of 258 high-grade precursor lesions and 29 cervical cancers (mean age = 41.4 years, 83% Stage I) were detected. As a reference, 41,387 cytology tests from the previous program (2012-2016) detected 36 cervical cancers (mean age = 52.0 years, p = 0.0005), with 67% in advanced stages (p < 0.0001). Organizing cervical cancer screening using DNA-HPV testing demonstrated good coverage, high age and colposcopy compliance, and detection of more precancerous lesions and cervical cancers 10 years in advance.
Asunto(s)
Detección Precoz del Cáncer , Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Humanos , Femenino , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/virología , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/epidemiología , Persona de Mediana Edad , Adulto , Detección Precoz del Cáncer/métodos , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/virología , Brasil/epidemiología , ADN Viral/genética , Colposcopía , Tamizaje Masivo/métodos , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Prevalencia , AncianoRESUMEN
While HC2 and GP5+/6+ PCR-EIA were pivotal in test validation of new HPV assays, they represent the first generation of comparator tests based upon technologies that are not in widespread use anymore. In the current guideline, criteria for second-generation comparator tests are presented that include more detailed resolution of HPV genotypes. Second-generation comparator tests should preferentially target only the 12 genotypes classified as carcinogenic (IARC-group I), and show consistent non-inferior sensitivity for CIN2+ and CIN3+ and specificity for ≤CIN1 compared to one of the first-generations comparators, in at least three validation studies using benchmarks of 0.95 for relative sensitivity and 0.98 for relative specificity. Validation should take into account used storage media and other sample handling procedures. Meta-analyses were conducted to identify the assays that fulfill these stringent criteria. Four tests fulfilled the new criteria: (1) RealTime High-Risk HPV Test (Abbott), (2) Cobas-4800 HPV test (Roche Molecular System), (3) Onclarity HPV Assay (BD Diagnostics), and (4) Anyplex II HPV HR Detection (Seegene), each evaluated in three to six studies. Whereas the four assays target 14 carcinogenic genotypes, the first two identify separately HPV16 and 18, the third assay identifies five types separately and the fourth identifies all the types separately.
Asunto(s)
Detección Precoz del Cáncer , Papillomaviridae , Infecciones por Papillomavirus , Sensibilidad y Especificidad , Neoplasias del Cuello Uterino , Femenino , Humanos , ADN Viral/genética , Detección Precoz del Cáncer/métodos , Genotipo , Pruebas de ADN del Papillomavirus Humano/métodos , Pruebas de ADN del Papillomavirus Humano/normas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Papillomaviridae/genética , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/virología , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/virologíaRESUMEN
Human papillomaviruses (HPVs) and herpesviruses are detected in patients with epithelial ovarian cancer (EOC). We sought to analyze the prevalence of HPV's 16 and 18, cytomegalovirus (CMV), and Epstein-Barr virus (EBV) DNA in peripheral blood, ovarian, and fallopian tube (FT) tissue samples collected from 97 EOC patients, including 71 cases of high-grade serous ovarian carcinoma (HGSOC), and from 60 women with other tumors or non-neoplastic gynecological diseases. DNA isolates were analyzed by PCR methods, including droplet digital PCR. The results demonstrate that (1) HPV16 DNA has been detected in one-third of the FT and tumor samples from EOCs; (2) the prevalence and quantity of HPV16 DNA were significantly higher in FT samples from HGSOCs, non-HGSOCs, and ovarian metastases than in those from non-neoplastic diseases; (3) CMV and EBV have been detected in approximately one-seventh of EOC samples. The results suggest that HPV16 might be a potential risk factor for EOC development.
Asunto(s)
Carcinoma Epitelial de Ovario , Trompas Uterinas , Papillomavirus Humano 16 , Neoplasias Ováricas , Infecciones por Papillomavirus , Humanos , Femenino , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/complicaciones , Factores de Riesgo , Carcinoma Epitelial de Ovario/virología , Carcinoma Epitelial de Ovario/patología , Persona de Mediana Edad , Trompas Uterinas/virología , Trompas Uterinas/patología , Adulto , Anciano , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/aislamiento & purificación , Neoplasias Ováricas/virología , Neoplasias Ováricas/patología , Herpesvirus Humano 4/aislamiento & purificación , Herpesvirus Humano 4/genética , Citomegalovirus/aislamiento & purificación , Citomegalovirus/genética , ADN Viral/genéticaRESUMEN
Lytic bacteriophages hold substantial promise in medical and biotechnological applications. Therefore a comprehensive understanding of phage infection mechanisms is crucial. CRISPR-Cas systems offer a way to explore these mechanisms via site-specific phage mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET to temporarily reduce phage DNA modifications, facilitating Cas nuclease cleavage and enhancing mutagenesis efficiency. This approach enables precise DNA targeting and seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for phage infection. Furthermore, by temporally removing DNA modifications, we elucidated the effects of these modifications on T4 phage infections without necessitating gene deletions. Our results present a strategy enabling the investigation of phage epigenome functions and streamlining the engineering of phages with cytosine DNA modifications. The described temporal modulation of the phage epigenome is valuable for synthetic biology and fundamental research to comprehend phage infection mechanisms through the generation of mutants.
Asunto(s)
Bacteriófagos , Sistemas CRISPR-Cas , ADN Viral , Epigenoma , ADN Viral/genética , Bacteriófagos/genética , Ingeniería Genética/métodos , Bacteriófago T4/genética , Mutagénesis Sitio-Dirigida/métodos , Escherichia coli/genética , Escherichia coli/virología , Genoma ViralRESUMEN
Herein, we report a target-triggered CRISPR/Cas12a assay by coupling lanthanide tagging and inductively coupled plasma mass spectrometry (ICP-MS) for highly sensitive elemental detection. Hepatitis B virus (HBV) DNA was chosen as a model analyte, and recombinase polymerase amplification (RPA) was used for target amplification. The double-stranded RPA amplicons containing a 5' TTTG PAM sequence can be recognized by Cas12a through a specific CRISPR RNA, activating the trans-cleavage activity of CRISPR/Cas12a and nonspecific cleavage of terbium (Tb)-ssDNA modified on magnetic beads (MBs). Following magnetic separation and acid digestion, the released Tb3+ ions were quantitated by ICP-MS and correlated to the concentration of HBV DNA. Taking advantage of the accelerated cleavage of Tb-ssDNA attached to the MB particles, RPA for target amplification, and ICP-MS for highly selective signal readout, this method permits the detection of 1 copy/µL of HBV DNA in serum with high specificity and holds great promise in the early diagnosis of viral infections or tumor development.
Asunto(s)
Sistemas CRISPR-Cas , ADN Viral , Virus de la Hepatitis B , Elementos de la Serie de los Lantanoides , Espectrometría de Masas , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , ADN Viral/genética , ADN Viral/análisis , Elementos de la Serie de los Lantanoides/química , Espectrometría de Masas/métodos , Sistemas CRISPR-Cas/genética , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , Recombinasas/metabolismoRESUMEN
BACKGROUND: Elephant endotheliotropic herpesvirus (EEHV) infection is the most common cause for lethal hemorrhagic disease in captive juvenile Asian elephants (Elephas maximus). Although EEHV1 is known as the most likely cause of fatal haemorrhagic disease in Asian elephants, EEHV5 was lately involved in lethal cases of haemorrhagic disease in captive elephants. CASE PRESENTATION: Here we report the first death of a four-year old Asian elephant diagnosed with EEHV5 in Germany. Molecular diagnosis yielded detection of EEHV5 DNA in all tested tissues. Histopathological examination revealed typical features of hemorrhagic disease in all examined organs. EEHV5 was sequenced from total DNA isolated from heart tissue by Illumina and Nanopore sequencing. Sequencing data showed 3,881 variants, distributed across the entire genome, compared to the published EEHV5 sequence. CONCLUSIONS: We have detected EEHV5 in a fatal disease case of a male Asian elephant. Whole genome sequencing revealed substantial differences of our DNA isolate compared to available EEHV5 sequences. This report of fatal haemorrhagic disease associated with EEHV5 infection should raise awareness for EEHV5 as an important elephant pathogen. Genome sequencing and downstream SNPs analysis will further encourage future research to understand genetic diversity, pathogenesis and virulence of EEHVs with respect to developing new diagnostic methods, prophylactic strategies, and implementation of surveillance and control measures.
Asunto(s)
Elefantes , Infecciones por Herpesviridae , Herpesviridae , Animales , Elefantes/virología , Infecciones por Herpesviridae/veterinaria , Infecciones por Herpesviridae/virología , Alemania , Masculino , Resultado Fatal , Herpesviridae/genética , Herpesviridae/aislamiento & purificación , Herpesviridae/clasificación , ADN Viral/genética , Genoma Viral/genética , Filogenia , Análisis de Secuencia de ADN , Variación Genética , Secuenciación Completa del GenomaRESUMEN
A novel virus, temporarily named "Arctic wolf parvovirus" (AWPV), was discovered in a pharyngeal metagenomic library derived from an Arctic wolf (Canis lupus arctos) in China. The genome sequence was assigned GenBase accession number C_AA071902.1. AWPV has a genome comprised of 4,920 base pairs with a nucleotide composition of 36.4% A, 23.4% T, 18.2% G, and 22.0% C, with a GC content of 40.2%. Its structure resembles parvoviruses, containing two open reading frames: the nonstructural (NS) region encoding replication enzymes and the structural (VP) region encoding capsid protein. Pairwise sequence comparison and phylogenetic analysis suggest AWPV may represent a novel species within the genus Protoparvovirus. This discovery enhances our understanding of mammalian virus ecology and potential future infectious diseases.
Asunto(s)
Genoma Viral , Sistemas de Lectura Abierta , Parvovirus , Filogenia , Lobos , Animales , Lobos/virología , Parvovirus/genética , Parvovirus/clasificación , Parvovirus/aislamiento & purificación , Proteínas de la Cápside/genética , Infecciones por Parvoviridae/virología , Infecciones por Parvoviridae/veterinaria , Composición de Base , China , ADN Viral/genéticaRESUMEN
Rapid and reliable detection method for African swine fever virus (ASFV) is proposed by surface-enhanced Raman spectroscopy (SERS). The ASFV target DNA can be specifically captured by sandwich hybridization between nanomagnetic beads and a SERS probe. Experimental results show that the significant Raman signal of the SERS probe with gold nanoparticles and a molecular reporter DTNB (5,5'-dimercapto-bis (2-nitrobenzoic acid)) can be adopted for detecting the hybridization chain reaction of ASFV DNA. The advantage of the SERS sandwich hybridization assay is the large response range from the single molecule level to 108 copies per mL, which not only can overcome the tedious time required for the amplification reaction but also provides a comparative method to polymerase chain reaction. Furthermore, real samples of African swine fever virus were detected from different subjects of swine fever virus including porcine reproductive respiratory syndrome virus and Japanese encephalitis virus. The proposed biosensor method can rapidly detect ASFV correctly within 15 min as a simple, convenient, low-cost detection approach. The biosensor can be used as a platform for the determination in biological, food, and environmental analytical fields.
Asunto(s)
Virus de la Fiebre Porcina Africana , Oro , Nanopartículas del Metal , Hibridación de Ácido Nucleico , Espectrometría Raman , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/genética , Espectrometría Raman/métodos , Nanopartículas del Metal/química , Animales , Oro/química , Técnicas Biosensibles/métodos , Porcinos , ADN Viral/análisis , ADN Viral/genética , Límite de Detección , Fiebre Porcina Africana/diagnóstico , Fiebre Porcina Africana/virologíaRESUMEN
Vibrio parahaemolyticus is a major seafood-borne zoonotic pathogen that causes gastroenteritis in humans and acute hepatopancreatic necrosis disease (AHPND) in shrimp. In this study, we isolated and characterized Vibrio phage vB_VpM-pA2SJ1, which infects clinical and AHPND-associated strains of V. parahaemolyticus. The phage genome is a linear dsDNA 51,054 bp in length with a G + C content of 43.7%, and it contains 89 open reading frames. Genome comparisons revealed basal similarity to other Vibrio phages, particularly Vibrio phage vB_VpP_1, with 84.2% identity and 46% coverage. Phylogenetic analysis based on the whole genome, the terminase large subunit, and the major capsid protein revealed that phage vB_VpM-pA2SJ1 did not cluster with other known phage families, thus indicating its uniqueness.
Asunto(s)
Bacteriófagos , Composición de Base , Genoma Viral , Sistemas de Lectura Abierta , Filogenia , Vibrio parahaemolyticus , Vibrio parahaemolyticus/virología , Vibrio parahaemolyticus/genética , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Bacteriófagos/clasificación , Animales , Penaeidae/virología , Penaeidae/microbiología , Vibriosis/microbiología , Vibriosis/virología , Vibriosis/veterinaria , Hepatopáncreas/virología , Hepatopáncreas/microbiología , Hepatopáncreas/patología , ADN Viral/genéticaRESUMEN
Despite scarcity of data, in recent years, human parvovirus B19 (PVB19) has been emerging as an important pathogen in acute encephalitis syndrome (AES). But, PVB19 virus is mostly looked for only after the exclusion of other common pathogens implicated in AES. Hence, this study was conducted to correlate clinical, radiological, and sequencing data to establish the crucial role of PVB19 in AES. Cerebrospinal fluid and/or serum samples were collected from AES patients as per WHO criteria and tested by ELISA, real-time PCR and bacterial culture sensitivity for various pathogens. PVB19 positive samples were subjected to sequencing. PVB19 attributed to 5% of total AES cases in the present study with fatalities in two of eight cases. Two isolates of PVB19 belonged to Genotype 1 A whereas one belonged to Genotype 3B. On multivariate analysis of predictive symptoms of PVB19 AES cases, blurring of vision (odds ratio [OR] 20.67; p = 0.001) was found to be significant independent predictor of PVB19 AES. Six of eight patients (two encephalitis specific and four nonspecific) had abnormal radiological findings. Hence, being an emerging viral pathogen, PVB19 should be included in the diagnostic algorithm of AES for prompt diagnosis and definitive management to prevent undesired neurological sequelae.
Asunto(s)
Infecciones por Parvoviridae , Parvovirus B19 Humano , Humanos , Parvovirus B19 Humano/genética , Parvovirus B19 Humano/aislamiento & purificación , Masculino , Femenino , Infecciones por Parvoviridae/virología , Infecciones por Parvoviridae/complicaciones , Niño , Adolescente , Adulto Joven , Preescolar , Genotipo , Adulto , Encefalopatía Aguda Febril/virología , Análisis de Secuencia de ADN , ADN Viral/líquido cefalorraquídeo , ADN Viral/genética , ADN Viral/sangre , Ensayo de Inmunoadsorción Enzimática , Encefalitis Viral/virología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The natural history of cervical cancer is closely linked to that of high-risk human papillomaviruses (HPV) infection. It is recognized that upon HPV DNA integration, partial or complete loss of the E2 open reading frame precludes expression of the corresponding protein, resulting in upregulation of the E6 and E7 viral oncoproteins. To better characterize HPV16 infection at the cervical level, viral load, viral DNA integration, and viral early transcript expression (E2, E5, and E6) were analyzed in a series of 158 cervical specimens representative of the full spectrum of cervical disease. Overall, the frequency of early transcript detection varied from 45% to 90% and tended to increase with lesion severity. In addition, the levels of E2, E5, and E6 transcript expression were slightly higher in high-grade lesions than in cervical specimens without abnormalities. Notably, early transcript expression was clearly associated with viral load, and no inverse correlation was found between the expression of E2 and E6 transcripts. No clear association was found between early transcript expression and HPV16 DNA integration, with the exception that samples with a fully integrated HPV16 genome did not harbor E2 or E5 transcripts. In conclusion, early HPV16 transcript expression appears to be associated with viral load rather than lesion grade. From a practical point of view, quantification of HPV16 early transcripts is difficult to translate into a relevant biomarker for cervical cancer screening.
Asunto(s)
Papillomavirus Humano 16 , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Carga Viral , Humanos , Femenino , Papillomavirus Humano 16/genética , Infecciones por Papillomavirus/virología , Proteínas Oncogénicas Virales/genética , Neoplasias del Cuello Uterino/virología , Integración Viral , Adulto , Persona de Mediana Edad , ADN Viral/genética , Anciano , Cuello del Útero/virología , Cuello del Útero/patologíaRESUMEN
BACKGROUND: Persistent infection with human papillomavirus (HPV) significantly contributes to the development of cervical cancer. Thus, it is urgent to develop rapid and accurate methods for HPV detection. Herein, we present an ultrasensitive CRISPR/Cas12a-based electrochemiluminescent (ECL) imaging technique for the detection of HPV-18 DNA. RESULT: The ECL DNA sensor array is constructed by applying black hole quencher (BHQ) and polymer dots (Pdots) co-labeled hairpin DNA (hpDNA) onto a gold-coated indium tin oxide slide (Au-ITO). The ECL imaging method involves an incubation process of target HPV-18 with a mixture of crRNA and Cas12a to activate Cas12a, followed by an incubation of the active Cas12a with the ECL sensor. This interaction causes the indiscriminate cleavage of BHQ from Pdots by digesting hpDNA on the sensor surface, leading to the restoration of the ECL signal of Pdots. The ECL brightness readout demonstrates superior performance of the ECL imaging technique, with a linear detection range of 10 fM-500 pM and a limit-of-detection (LOD) of 5.3 fM. SIGNIFICANCE: The Cas12a-based ECL imaging approach offers high sensitivity and a broad detection range, making it highly promising for nucleic acid detection applications.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas Electroquímicas , Mediciones Luminiscentes , Técnicas Electroquímicas/métodos , Mediciones Luminiscentes/métodos , Sistemas CRISPR-Cas/genética , Humanos , Técnicas Biosensibles/métodos , ADN Viral/análisis , ADN Viral/genética , Papillomavirus Humano 18/genética , Límite de Detección , Oro/química , Proteínas Asociadas a CRISPR , Proteínas Bacterianas , EndodesoxirribonucleasasRESUMEN
DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.
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ADN Viral , Genoma Viral , ADN Viral/genética , ADN Viral/metabolismo , ADN Viral/química , Microscopía por Crioelectrón , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Modelos Moleculares , Empaquetamiento del ADN , Ensamble de Virus/genética , Bacteriófagos/genética , Empaquetamiento del Genoma ViralRESUMEN
Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), particularly oropharyngeal squamous cell carcinoma (OPSCC), is an increasingly prevalent pathology worldwide, especially in developed countries. For diagnosing HPV in HNSCC, the combination of p16 immunohistochemistry (IHC) and polymerase chain reaction (PCR) offers high sensitivity and specificity, with p16 IHC being a reliable initial screen and PCR confirming HPV presence. Advanced techniques like next-generation sequencing (NGS) and RNA-based assays provide detailed insights but are primarily used in research settings. Regardless of HPV status, standard oncological treatments currently include surgery, radiation, and/or chemotherapy. This conventional approach does not account for the typically better prognosis of HPV-positive HNSCC patients, leading to increased chemo/radiation-induced secondary morbidities and reduced quality of life. Therefore, it is crucial to identify and detect HPV positivity and other molecular characteristics of HNSCC to personalize treatment strategies. This comprehensive review aims to summarize current knowledge on various HPV detection techniques and evaluate their advantages and disadvantages, with a focus on developing methodologies to identify new biomarkers in HPV-positive HNSCC. The review discusses direct and indirect HPV examination in tumor tissue, DNA- and RNA-based detection techniques, protein-based markers, liquid biopsy potentials, immune-related markers, epigenetic markers, novel biomarkers, and emerging technologies, providing an overall insight into the current state of knowledge.
Asunto(s)
Biomarcadores de Tumor , Neoplasias de Cabeza y Cuello , Infecciones por Papillomavirus , Carcinoma de Células Escamosas de Cabeza y Cuello , Humanos , Neoplasias de Cabeza y Cuello/virología , Neoplasias de Cabeza y Cuello/diagnóstico , Neoplasias de Cabeza y Cuello/genética , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/diagnóstico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Inmunohistoquímica , ADN Viral/genética , ADN Viral/análisis , Reacción en Cadena de la Polimerasa/métodos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genéticaRESUMEN
BACKGROUND AND AIMS: Antiviral therapy (AVT) is required in patients with newly diagnosed hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), if HBV DNA is detectable. We compared the risk of recurrence according to HBV replication activity at the curative treatment of HBV-related HCC. METHODS: Patients with HBV-related HCC who underwent surgical resection or radiofrequency ablation between 2013 and 2018 were enrolled in this retrospective cohort study. Patients were categorized into two groups according to HBV replication activity at the curative treatment of HBV-related HCC (group 1: patients who met the AVT indication for HBV-related HCC due to detectable HBV DNA but did not meet the AVT indication if without HCC; group 2: patients who met the AVT indication, regardless of HCC). RESULTS: In the entire cohort (n = 911), HCC recurred in 303 (33.3%) patients during a median follow-up of 4.7 years. After multivariate adjustment, group 2 showed a statistically similar risk of HCC recurrence (adjusted hazard ratio [aHR] = 1.18, P = 0.332) compared to that of group 1. In addition, group 2 showed statistically similar risks of early (< 2 years; aHR = 1.31) and late (≥ 2 years; aHR = 0.83) recurrence than that of group 1 (all P>0.05). Propensity score matching and inverse probability of treatment weighting analysis also yielded similar risks of HCC recurrence between the two groups (all P>0.05, log-rank tests). CONCLUSIONS: The risk of HCC recurrence in patients who received curative treatment for newly diagnosed HBV-related HCC was similar regardless of HBV replication activity, if AVT was properly initiated.
Asunto(s)
Carcinoma Hepatocelular , Virus de la Hepatitis B , Neoplasias Hepáticas , Recurrencia Local de Neoplasia , Replicación Viral , Humanos , Carcinoma Hepatocelular/virología , Carcinoma Hepatocelular/patología , Masculino , Neoplasias Hepáticas/virología , Neoplasias Hepáticas/patología , Femenino , Virus de la Hepatitis B/fisiología , Persona de Mediana Edad , Recurrencia Local de Neoplasia/virología , Estudios Retrospectivos , ADN Viral/genética , Anciano , Antivirales/uso terapéutico , Hepatitis B/complicaciones , Hepatitis B/virologíaRESUMEN
BACKGROUND: Viruses with double-stranded (ds) DNA genomes in the realm Duplodnaviria share a conserved structural gene module but show a broad range of variation in their repertoires of DNA replication proteins. Some of the duplodnaviruses encode (nearly) complete replication systems whereas others lack (almost) all genes required for replication, relying on the host replication machinery. DNA polymerases (DNAPs) comprise the centerpiece of the DNA replication apparatus. The replicative DNAPs are classified into 4 unrelated or distantly related families (A-D), with the protein structures and sequences within each family being, generally, highly conserved. More than half of the duplodnaviruses encode a DNAP of family A, B or C. We showed previously that multiple pairs of closely related viruses in the order Crassvirales encode DNAPs of different families. METHODS: Groups of phages in which DNAP swapping likely occurred were identified as subtrees of a defined depth in a comprehensive evolutionary tree of tailed bacteriophages that included phages with DNAPs of different families. The DNAP swaps were validated by constrained tree analysis that was performed on phylogenetic tree of large terminase subunits, and the phage genomes encoding swapped DNAPs were aligned using Mauve. The structures of the discovered unusual DNAPs were predicted using AlphaFold2. RESULTS: We identified four additional groups of tailed phages in the class Caudoviricetes in which the DNAPs apparently were swapped on multiple occasions, with replacements occurring both between families A and B, or A and C, or between distinct subfamilies within the same family. The DNAP swapping always occurs "in situ", without changes in the organization of the surrounding genes. In several cases, the DNAP gene is the only region of substantial divergence between closely related phage genomes, whereas in others, the swap apparently involved neighboring genes encoding other proteins involved in phage genome replication. In addition, we identified two previously undetected, highly divergent groups of family A DNAPs that are encoded in some phage genomes along with the main DNAP implicated in genome replication. CONCLUSIONS: Replacement of the DNAP gene by one encoding a DNAP of a different family occurred on many independent occasions during the evolution of different families of tailed phages, in some cases, resulting in very closely related phages encoding unrelated DNAPs. DNAP swapping was likely driven by selection for avoidance of host antiphage mechanisms targeting the phage DNAP that remain to be identified, and/or by selection against replicon incompatibility.
Asunto(s)
ADN Polimerasa Dirigida por ADN , Filogenia , Proteínas Virales , ADN Polimerasa Dirigida por ADN/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Evolución Molecular , Genoma Viral , Caudovirales/genética , Caudovirales/clasificación , ADN Viral/genética , Bacteriófagos/genética , Bacteriófagos/enzimología , Bacteriófagos/clasificación , Replicación del ADNRESUMEN
The rapid and specific identification and sensitive detection of human papillomavirus (HPV) infection is critical for preventing cervical cancer, particularly in resource-limited regions. In this work, we hope to propose a capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for point-of-care (POC) DNA testing. Using the thermal reversibility of DNA hydrogel and capillarity, the novel DNA hydrogel distance sensor can be rapidly and simply constructed by loading an ultra-thin CRISPR/Cas12a-responsive DNA-crosslinked hydrogel film at the end of the capillary tube. The target DNA-specific recombinase polymerase reaction (RPA) amplicons activate the trans-cleavage activity of the Cas12a enzyme, cleaving the crosslinked DNA in hydrogel film, and causing an increase of hydrogel's permeability. As a result, a sample solution containing target DNA travels into the capillary tube at a longer distance compared to the negative samples. Reading the solution traveling distance in capillary tubes, the novel sensor realizes target DNA detection without any special equipment. Benefiting from the exponential target amplification of RPA and multiple turnover response of trans-cleavage of CRISPR/Cas12a, the developed sensor can visually and specifically detect as low as 1 aM HPV 16 DNA within 30 min. These outstanding features, including exceptional sensitivity and specificity, simple and portable design, mild measurement conditions, quick turnaround time, and user-friendly read-out, make the novel distance sensor a promising option for POC diagnostic applications.
Asunto(s)
Técnicas Biosensibles , Sistemas CRISPR-Cas , ADN Viral , Hidrogeles , Infecciones por Papillomavirus , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Humanos , ADN Viral/análisis , ADN Viral/genética , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/diagnóstico , Hidrogeles/química , Endodesoxirribonucleasas/química , Límite de Detección , Papillomaviridae/genética , Proteínas Asociadas a CRISPR/química , Femenino , Proteínas Bacterianas/químicaRESUMEN
G-quadruplex (G4) structures are found in eukaryotic cell replication origins, but their role in origin function remains unclear. In this study G4 motifs are found in the lytic DNA replication origin (oriLyt) of human cytomegalovirus (HCMV) and recombinant viruses show that a G4 motif in oriLyt essential region I (ER-I) is necessary for viral growth. Replication assays of oriLyt-containing plasmids and biochemical/biophysical analyses show that G4 formation in ER-I is crucial for viral DNA replication. G4 pull-down analysis identifies viral DNA replication factors, such as IE2, UL84, and UL44, as G4-binding proteins. In enzyme-linked immunosorbent assays, specific G4-binding ligands inhibit G4 binding by the viral proteins. The Epstein-Barr virus oriLyt core element also forms a stable G4 that could substitute for the oriLyt ER-I G4 in HCMV. These results demonstrate that viral G4s in replication origins represent an essential structural element in recruiting replication factors and might be a therapeutic target against viral infections.
Asunto(s)
Citomegalovirus , Replicación del ADN , ADN Viral , G-Cuádruplex , Proteínas Inmediatas-Precoces , Origen de Réplica , Proteínas Virales , Replicación Viral , Citomegalovirus/genética , Citomegalovirus/fisiología , Humanos , Replicación Viral/genética , Origen de Réplica/genética , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Inmediatas-Precoces/genética , ADN Viral/genética , ADN Viral/metabolismo , Transactivadores/metabolismo , Transactivadores/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Unión ProteicaRESUMEN
BACKGROUND: Persistent infection with high-risk human papillomavirus (HR-HPV) plays a key role in the onset of cervical cancer. This study was designed to examine the epidemiological trends and genotype distribution of HPV from 2014 to 2023 in the plateau region of Southwest China. METHODS: The findings could offer valuable insights for clinical screening of cervical cancer and the formulation of HPV vaccination policies. This retrospective study analyzed 66,000 women who received HPV-DNA testing at the First People's Hospital of Qujing, Yunnan, China, between 2014 and 2023. The cohort consisted of 33,512 outpatients, 3,816 inpatients, and 28,672 individuals undergoing health examinations. Cervical cells were collected for DNA extraction, and PCR amplification along with Luminex xMAP technology were used to detect 27 HPV genotypes. The data analysis was conducted using GraphPad Prism and IBM SPSS Statistics 27 software. RESULTS: The overall HPV infection rate at the First People's Hospital of Qujing declined from 24.92% in 2014 to 16.29% in 2023, averaging 16.02%. Specific infection rates were 18.50% among outpatients, 12.97% among inpatients, and 13.53% for health examination attendees. The predominant high-risk HPV genotypes identified were HPV52 (2.61%), HPV16 (2.06%), HPV58 (1.81%), HPV53 (1.55%), and HPV39 (1.09%). Meanwhile, the most frequent low-risk HPV genotypes were HPV6 (1.30%), HPV61 (1.21%), and HPV11 (0.85%). In HPV-positive cases, the distribution of single, double, triple, and quadruple or more infections were 79.90%, 15.17%, 3.59%, and 1.33%, respectively. The proportions of pure LR-HPV, pure HR-HPV, and mixed infections were 22.16%, 67.82%, and 10.02%, respectively. Age-specific analysis revealed a bimodal distribution of HPV infection, with the infection rate rapidly decreasing from 44.02% in the ≤ 19 age group to 19.55% in the 20-29 age group and 13.84% in the 30-39 age group, followed by a gradual increase to 14.64% in the 40-49 age group, 16.65% in the 50-59 age group, and 22.98% in the ≥ 60 age group. The coverage rates of the three available vaccines are all below 50%. The results of this study indicated a declining trend in HPV prevalence in the plateau region of Southwest China over the period from 2014 to 2023, especially in the reduction of genotypes targeted by vaccines. CONCLUSION: There were significant variations in the genotypes prevalent among different age groups, years, and patient sources within the same region. The underwhelming vaccination rates emphasize the critical need for developing either a multivalent vaccine or a personalized vaccine that targets the HPV genotypes common in the Chinese population. Furthermore, vaccinating adolescents to curb HPV infection and ensuring regular cervical cancer screenings for postmenopausal women are crucial steps.
Asunto(s)
Genotipo , Papillomaviridae , Infecciones por Papillomavirus , Humanos , Femenino , Infecciones por Papillomavirus/epidemiología , Infecciones por Papillomavirus/virología , China/epidemiología , Adulto , Prevalencia , Persona de Mediana Edad , Estudios Retrospectivos , Adulto Joven , Papillomaviridae/genética , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Adolescente , Anciano , Neoplasias del Cuello Uterino/virología , Neoplasias del Cuello Uterino/epidemiología , ADN Viral/genética , Cuello del Útero/virologíaRESUMEN
T5 is a siphophage that has been extensively studied by structural and biochemical methods. However, the complete in situ structures of T5 before and after DNA ejection remain unknown. In this study, we used cryo-electron microscopy (cryo-EM) to determine the structures of mature T5 (a laboratory-adapted, fiberless T5 mutant) and urea-treated empty T5 (lacking the tip complex) at near-atomic resolutions. Atomic models of the head, connector complex, tail tube, and tail tip were built for mature T5, and atomic models of the connector complex, comprising the portal protein pb7, adaptor protein p144, and tail terminator protein p142, were built for urea-treated empty T5. Our findings revealed that the aforementioned proteins did not undergo global conformational changes before and after DNA ejection, indicating that these structural features were conserved among most myophages and siphophages. The present study elucidates the underlying mechanisms of siphophage infection and DNA ejection.