RÉSUMÉ
Objective: Our objective was to determine whether equine herpesviruses 1 (EHV-1) viral nucleic acids could be detected immediately after foaling from nasal and vaginal swabs, whole blood, and placental tissue of healthy mares. Animals procedure and results: Nasal and vaginal swabs, EDTA blood, and placental tissue (296 samples) were collected from 74 clinically healthy postpartum broodmares within 24 h after giving birth to live, clinically healthy foals. All samples were tested (PCR) for nucleic acids of neuropathogenic and non-neuropathogenic strains of EHV-1, and all were negative. Conclusion and clinical relevance: As EHV-1 was not detected in the immediate postpartum period in healthy mares with uncomplicated foaling, we inferred that EHV-1-positive samples from aborting mares and/or EHV-1 detection in fetal membranes indicate EHV-1-associated abortion.
Tests moléculaires pour l'herpèsvirus équin 1 (EHV-1) chez des juments poulinières post-partum en bonne santé. Objectif: Notre objectif était de déterminer si les acides nucléiques viraux de l'herpèsvirus équin 1 (EHV-1) pouvaient être détectés immédiatement après la mise bas à partir de prélèvements nasaux et vaginaux, de sang total et de tissus placentaires de juments saines. Animaux procédure et résultats: Des écouvillons nasaux et vaginaux, du sang EDTA et du tissu placentaire (296 échantillons) ont été prélevés sur 74 juments poulinières post-partum cliniquement saines dans les 24 heures suivant la naissance de poulains vivants et cliniquement sains. Tous les échantillons ont été testés (PCR) pour les acides nucléiques des souches neuropathogènes et non-neuropathogènes de l'EHV-1, et tous se sont révélés négatifs. Conclusion et pertinence clinique: Comme l'EHV-1 n'a pas été détecté dans la période post-partum immédiate chez des juments en bonne santé avec un poulinage sans complication, nous avons déduit que les échantillons positifs pour l'EHV-1 provenant de juments qui ont avorté et/ou la détection de l'EHV-1 dans les membranes foetales indiquent un avortement associé à l'EHV-1.(Traduit par Dr Serge Messier).
Sujet(s)
Infections à Herpesviridae , Herpèsvirus équin de type 1 , Maladies des chevaux , Période du postpartum , Animaux , Equus caballus , Herpèsvirus équin de type 1/isolement et purification , Femelle , Maladies des chevaux/virologie , Maladies des chevaux/diagnostic , Infections à Herpesviridae/médecine vétérinaire , Infections à Herpesviridae/virologie , Infections à Herpesviridae/diagnostic , Grossesse , Placenta/virologie , Vagin/virologie , Avortement chez les animaux/virologie , ADN viral/analyse , ADN viral/isolement et purification , Réaction de polymérisation en chaîne/médecine vétérinaireRÉSUMÉ
HBV covalently closed circular DNA (cccDNA) plays an important role in the persistence of hepatitis B virus (HBV) infection by serving as the template for transcription of viral RNAs. To cure HBV infection, it is expected that cccDNA needs either to be eliminated or silenced. Hence, precise cccDNA quantification is essential. Sample preparation is crucial to specifically detect cccDNA. Southern blot is regarded as the "gold standard" for specific cccDNA detection but lacks sensitivity. Here, we describe a rapid and reliable modified kit-based, HBV protein-free DNA extraction method as well as a novel enhanced sensitivity Southern blot that uses branched DNA technology to detect HBV DNA in cell culture and liver tissue samples. It is useful for both HBV molecular biology and antiviral research.
Sujet(s)
Technique de Southern , ADN circulaire , ADN viral , Virus de l'hépatite B , Virus de l'hépatite B/génétique , Virus de l'hépatite B/isolement et purification , Humains , ADN viral/génétique , ADN viral/isolement et purification , ADN circulaire/isolement et purification , ADN circulaire/analyse , ADN circulaire/génétique , Technique de Southern/méthodes , Hépatite B/virologie , Hépatite B/diagnostic , Foie/virologieRÉSUMÉ
Kaposi's sarcoma (KS) is a cancer affecting skin and internal organs for which the Kaposi's sarcoma associated herpesvirus (KSHV) is a necessary cause. Previous work has pursued KS diagnosis by quantifying KSHV DNA in skin biopsies using a point-of-care (POC) device which performs quantitative loop-mediated isothermal amplification (LAMP). These previous studies revealed that extracting DNA from patient biopsies was the rate limiting step in an otherwise rapid process. In this study, a simplified, POC-compatible alkaline DNA extraction, ColdSHOT, was optimized for 0.75 mm human skin punch biopsies. The optimized ColdSHOT extraction consistently produced 40,000+ copies of DNA per 5 µl reaction from 3 mg samples-a yield comparable to standard spin column extractions-within 1 h without significant equipment. The DNA yield was estimated sufficient for KSHV detection from KS-positive patient biopsies, and the LAMP assay was not affected by non-target tissue in the unpurified samples. Furthermore, the yields achieved via ColdSHOT were robust to sample storage in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer prior to DNA extraction, and the DNA sample was stable after extraction. The results presented in this study indicate that the ColdSHOT DNA extraction could be implemented to simplify and accelerate the LAMP-based diagnosis of Kaposi's sarcoma using submillimeter biopsy samples.
Sujet(s)
ADN viral , Herpèsvirus humain de type 8 , Techniques d'amplification d'acides nucléiques , Sarcome de Kaposi , Peau , Humains , ADN viral/génétique , ADN viral/isolement et purification , Herpèsvirus humain de type 8/isolement et purification , Herpèsvirus humain de type 8/génétique , Biopsie/méthodes , Peau/virologie , Peau/anatomopathologie , Sarcome de Kaposi/diagnostic , Sarcome de Kaposi/virologie , Sarcome de Kaposi/anatomopathologie , Sarcome de Kaposi/génétique , Techniques d'amplification d'acides nucléiques/méthodes , Systèmes automatisés lit malade , Techniques de diagnostic moléculaire/méthodesRÉSUMÉ
Quantitation of cytomegalovirus (CMV) DNA load in specimens other than blood such as bronchoalveolar lavages, intestinal biopsies, or urine has become a common practice as an ancillary tool for the diagnosis of CMV pneumonitis, intestinal disease, or congenital infection, respectively. Nevertheless, most commercially available CMV PCR platforms have not been validated for CMV DNA detection in these specimen types. In this study, a laboratory-developed test based on Alinity m CMV ("Alinity LDT") was evaluated. Reproducibility assessment using spiked bronchial aspirate (BAS) or urine samples showed low standard deviations of 0.08 and 0.27 Log IU/mL, respectively. Evaluating the clinical performance of Alinity LDT in comparison to a laboratory-developed test based on RealTime CMV ("RealTime LDT") showed good concordance across 200 clinical specimens including respiratory specimens, intestinal biopsies, urine, and stool. A high Pearson's correlation coefficient of r = 0.92, a low mean bias of -0.12 Log IU/mL, a good qualitative agreement of 90%, and a Cohen's kappa value of 0.76 (substantial agreement) were observed. In separate analyses of the sample types BAS, tracheal aspirates, bronchoalveolar lavage, biopsies, and urine, the assay results correlated well between the two platforms with r values between 0.88 and 0.99 and a bias <0.5 Log IU/mL. Overall, the fully automated, continuous, random access Alinity LDT yielded good reproducibility, high concordance, and good correlation to RealTime LDT in respiratory, gastrointestinal, and urine samples and may enhance patient management with rapid result reporting.IMPORTANCEIn transplant recipients, a major cause for morbidity and mortality is end-organ disease by primary or secondary CMV infection of the respiratory or gastrointestinal tract. In addition, sensorineural hearing loss and neurodevelopmental abnormalities are frequent sequelae of congenital CMV infections in newborns. Standard of care for highly sensitive detection and quantitation of the CMV DNA load in plasma and whole blood specimens is real-time PCR testing. Beyond that, there is a need for quantitative determination of CMV DNA levels in respiratory, gastrointestinal, and urinary tract specimens using a highly automated, random access CMV PCR assay with a short turnaround time to enable early diagnosis and treatment. In the present study, clinical performance of the fully automated Alinity m analyzer in comparison to the current RealTime LDT assay was evaluated in eight different off-label sample types.
Sujet(s)
Infections à cytomégalovirus , Cytomegalovirus , ADN viral , Tube digestif , Humains , Cytomegalovirus/isolement et purification , Cytomegalovirus/génétique , Infections à cytomégalovirus/diagnostic , Infections à cytomégalovirus/virologie , ADN viral/génétique , ADN viral/isolement et purification , Reproductibilité des résultats , Tube digestif/virologie , Charge virale/méthodes , Appareil respiratoire/virologie , Liquide de lavage bronchoalvéolaire/virologie , Sensibilité et spécificitéRÉSUMÉ
Emerging infectious diseases are a threat that contributes to the decline of global chelonian species. Herpesviruses are among the most impactful pathogens described in chelonians and are frequently associated with a range of presentations across hosts with the potential for severe morbidity and mortality. Trachemys herpesvirus 1 (TrHV1) has been reported in red-eared and yellow-bellied sliders (Trachemys scripta elegans and Trachemys scripta scripta, respectively) but is largely understudied. Invasive red-eared sliders may serve as a reservoir for transmission to sympatric native species. This study aimed to develop a sensitive and specific quantitative real-time PCR (qPCR) assay for the detection of TrHV1 DNA to aid in the characterization of the epidemiology of this virus in aquatic turtles. Two TaqMan-MGB FAM-dye labeled primer-probe sets were designed and evaluated using plasmid dilutions. The higher performing assay was specific for TrHV1 DNA and had a linear dynamic range of 1.0 × 107 to 1.0 × 101 copies per reaction with an R2 of 0.999, slope of -3.386, and efficiency of 97.39%. The limit of detection was 101 copies per reaction, and there was no loss of reaction efficiency in the presence of TrHV1-negative chelonian oral-cloacal DNA. Overall, the Trachemys herpesvirus 1 assay meets established criteria for acceptable qPCR assays and will be a valuable tool in characterizing the epidemiology of Trachemys herpesvirus 1 in chelonians.
Sujet(s)
Infections à Herpesviridae , Herpesviridae , Réaction de polymérisation en chaine en temps réel , Sensibilité et spécificité , Tortues , Animaux , Réaction de polymérisation en chaine en temps réel/méthodes , Tortues/virologie , Infections à Herpesviridae/médecine vétérinaire , Infections à Herpesviridae/diagnostic , Infections à Herpesviridae/virologie , Herpesviridae/génétique , Herpesviridae/isolement et purification , Herpesviridae/classification , ADN viral/génétique , ADN viral/isolement et purification , Amorces ADN/génétiqueRÉSUMÉ
The efficiency of PCR-based diagnostic assays can be impacted by the quality of DNA template, and anal samples can be particularly problematic due to the presence of faecal contaminants. Here, we compared the Quick-DNA Viral Kit (Zymo, Zymo Research, CA) and MagNA Pure 96 DNA and Viral NA Small Volume Kit (MP96, Roche) for use of the Seegene Anyplex II HPV28 assay (Anyplex28, Seegene) with anal samples. A total of 94 anal samples extracted using the MP96 and Zymo kits were tested via the Anyplex28, which detects high-risk human papillomavirus (HR-HPV, Panel A) and low-risk (LR-HPV, Panel B) HPV types. Testing the HR-HPV types (Panel A), 86 (91.5%) MP96 and 84 (89.4%) Zymo samples were deemed assessable. Overall agreement between the two methods was 87/94 (92.6%, 95% CI: 85.3-97.0) with the Kappa value of 0.678 (0.5-0.9). Of the 87 assessable samples, 50 (57.5%) were concordant, 34 (39.1%) partially concordant, and 10 (11.5%)discordant. In conclusion, the Anyplex28 produces comparable HPV genotyping results when using DNA extracts from either of these two methods.
Sujet(s)
ADN viral , Papillomaviridae , Infections à papillomavirus , Humains , ADN viral/génétique , ADN viral/isolement et purification , Infections à papillomavirus/diagnostic , Infections à papillomavirus/virologie , Papillomaviridae/génétique , Papillomaviridae/isolement et purification , Papillomaviridae/classification , Réaction de polymérisation en chaîne/méthodes , Canal anal/virologie , Trousses de réactifs pour diagnosticRÉSUMÉ
Mpox virus (MPXV) infection is difficult to distinguish from other (non-)infectious diseases. The etiology of rash can be differentiated by real-time polymerase chain reaction (rtPCR) on different types of samples. The study aims to provide experience with emerging MPXV diagnostics in a tertiary-level laboratory in Bosnia and Herzegovina. From July-December 2022, a total of 18 mpox suspected persons were tested. MPXV infection was confirmed by rtPCR in 10/18 (55.56 %) persons. The number of cases reached a peak in October 2022. The lowest median Crossing point (Cp) (xÌ = 29.76) was obtained from a swab of skin lesions in a viral transport medium (VTM). Evaluating the Cp values for the 7/9 mpox cases from whom paired swab samples from different anatomic sites were collected, higher positivity of skin lesion swabs in VTM was observed. In conclusion, our data highlighted the confirmatory role of rtPCR in the diagnosis of MPXV in skin lesion samples.
Sujet(s)
ADN viral , Réaction de polymérisation en chaine en temps réel , Humains , Bosnie-et-Herzégovine , Réaction de polymérisation en chaine en temps réel/méthodes , ADN viral/génétique , ADN viral/isolement et purification , Mâle , Femelle , Adulte , Orthopoxvirose simienne/diagnostic , Orthopoxvirose simienne/virologie , Orthopoxvirose simienne/épidémiologie , Adulte d'âge moyen , Adolescent , Jeune adulte , Centres de soins tertiaires , Enfant , Virus de la variole simienne/génétique , Virus de la variole simienne/isolement et purification , Peau/virologie , Techniques de diagnostic moléculaire/méthodesRÉSUMÉ
BACKGROUND: Human papillomavirus (HPV) is an oncogenic virus and the commonest sexually transmitted pathogen worldwide. Appropriate sampling is an important factor in infection management. This study aimed to compare the efficacy of cotton swabs (CS) and nylon-flocked swabs (NFS) in sampling for HPV-DNA PCR testing in male patients with genital warts. METHODS: The study included men with genital warts who presented to the urology outpatient clinic of Antalya Medical Park Hospital. Before wart treatment, multisite sampling of the penis and genital area was performed separately with CS and NFS. The samples were analyzed for HPV-DNA using real-time PCR. RESULTS: The study included 45 men with a mean age of 32.1 ± 8.6 years. At least one HPV type was detected in all 45 patients with NFS sampling and 44 patients with CS sampling (total HPV types detected: 106 and 84, respectively). NFS sampling detected 52 high-risk HPV types in 37 of the 45 patients, while CS sampling detected 37 high-risk types in 19 patients (p = 0.029). NFS sampling also detected a total of 54 low-risk HPV types in all 45 patients, versus 47 low-risk HPV types in 41 patients with CS sampling. Multiple HPV types were detected in 30 patients with NFS and 17 patients with CS (p = 0.001). CONCLUSION: NFS were more effective than CS for HPV-DNA testing in men with genital warts. NFS were superior to CS in detecting multiple-type HPV infection and high-risk HPV types. The use of NFS should be recommended for HPV-DNA PCR testing in men.
Sujet(s)
Condylomes acuminés , Nylons , Manipulation d'échantillons , Humains , Mâle , Adulte , Manipulation d'échantillons/méthodes , Condylomes acuminés/virologie , Condylomes acuminés/diagnostic , Infections à papillomavirus/diagnostic , Infections à papillomavirus/virologie , Fibre de coton , Jeune adulte , Papillomaviridae/isolement et purification , Papillomaviridae/génétique , ADN viral/analyse , ADN viral/isolement et purification , Adulte d'âge moyen , Réaction de polymérisation en chaine en temps réel/méthodesRÉSUMÉ
A next-generation sequencing (NGS) study identified a very high viral load of Torquetenovirus (TTV) in KD patients. We aimed to evaluate the feasibility of a newly developed quantitative species-specific TTV-PCR (ssTTV-PCR) method to identify the etiology of KD. We applied ssTTV-PCR to samples collected from 11 KD patients and 22 matched control subjects who participated in our previous prospective study. We used the NGS dataset from the previous study to validate ssTTV-PCR. The TTV loads in whole blood and nasopharyngeal aspirates correlated highly (Spearman's R = 0.8931, p < 0.0001, n = 33), supporting the validity of ssTTV-PCR. The ssTTV-PCR and NGS results were largely consistent. However, inconsistencies occurred when ssTTV-PCR was more sensitive than NGS, when the PCR primer sequences mismatched the viral sequences in the participants, and when the NGS quality score was low. Interpretation of NGS requires complex procedures. ssTTV-PCR is more sensitive than NGS but may fail to detect a fast-evolving TTV species. It would be prudent to update primer sets using NGS data. With this precaution, ssTTV-PCR can be used reliably in a future large-scale etiological study for KD.
Sujet(s)
Infections à virus à ADN , Maladie de Kawasaki , Réaction de polymérisation en chaîne , Virus torque teno , Virus torque teno/génétique , Virus torque teno/isolement et purification , Maladie de Kawasaki/virologie , Réaction de polymérisation en chaîne/méthodes , Séquençage nucléotidique à haut débit , Jeux de données comme sujet , Humains , Mâle , Femelle , Nourrisson , Enfant d'âge préscolaire , Enfant , Études prospectives , ADN viral/génétique , ADN viral/isolement et purification , Infections à virus à ADN/virologieRÉSUMÉ
BACKGROUND: Pseudorabies virus (PRV) was thought to only infect animals. Recent studies have shown that it can also infect human. CASE PRESENTATION: We report a case of pseudorabies virus encephalitis and endophthalmitis, diagnosed 89 days after onset, confirmed with intraocular fluid metagenomic next generation sequencing (mNGS) after the result of two cerebrospinal fluid (CSF) mNGS tests were negative. Although treatment with intravenous acyclovir, foscarnet sodium, and methylprednisolone improved the symptoms of encephalitis, significant diagnostic delay resulted in permanent visual loss. CONCLUSIONS: This case suggests that pseudorabies virus (PRV) DNA in the intraocular fluid may have a higher positivity than that in the CSF. PRV may persist in the intraocular fluid for an extended period and may thus require extended antiviral therapy. Patients with severe encephalitis and PRV should be examined with the focus on pupil reactivity and light reflex. A fundus examination should be performed in patients with a central nervous system infection, specifically, those in a comatose state, to help reduce eye disability.
Sujet(s)
Humeur aqueuse , Cécité , Encéphalite virale , Endophtalmie , Herpèsvirus porcin de type 1 , Maladie d'Aujeszky , Maladie d'Aujeszky/complications , Maladie d'Aujeszky/diagnostic , Maladie d'Aujeszky/traitement médicamenteux , Encéphalite virale/complications , Encéphalite virale/diagnostic , Encéphalite virale/traitement médicamenteux , Endophtalmie/diagnostic , Endophtalmie/traitement médicamenteux , Endophtalmie/virologie , Herpèsvirus porcin de type 1/génétique , Herpèsvirus porcin de type 1/isolement et purification , Métagénomique , Séquençage nucléotidique à haut débit , Retard de diagnostic , Humains , Mâle , Adulte d'âge moyen , Humeur aqueuse/virologie , Aciclovir/usage thérapeutique , Foscarnet/usage thérapeutique , Méthylprednisolone/usage thérapeutique , Antiviraux/usage thérapeutique , Cécité/virologie , ADN viral/isolement et purificationRÉSUMÉ
Rare CD4 T cells that contain HIV under antiretroviral therapy represent an important barrier to HIV cure1-3, but the infeasibility of isolating and characterizing these cells in their natural state has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Here we address this challenge using a microfluidic technology that isolates the transcriptomes of HIV-infected cells based solely on the detection of HIV DNA. HIV-DNA+ memory CD4 T cells in the blood from people receiving antiretroviral therapy showed inhibition of six transcriptomic pathways, including death receptor signalling, necroptosis signalling and antiproliferative Gα12/13 signalling. Moreover, two groups of genes identified by network co-expression analysis were significantly associated with HIV-DNA+ cells. These genes (n = 145) accounted for just 0.81% of the measured transcriptome and included negative regulators of HIV transcription that were higher in HIV-DNA+ cells, positive regulators of HIV transcription that were lower in HIV-DNA+ cells, and other genes involved in RNA processing, negative regulation of mRNA translation, and regulation of cell state and fate. These findings reveal that HIV-infected memory CD4 T cells under antiretroviral therapy are a distinctive population with host gene expression patterns that favour HIV silencing, cell survival and cell proliferation, with important implications for the development of HIV cure strategies.
Sujet(s)
Lymphocytes T CD4+ , Régulation de l'expression des gènes viraux , Infections à VIH , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Latence virale , Humains , Lymphocytes T CD4+/cytologie , Lymphocytes T CD4+/effets des médicaments et des substances chimiques , Lymphocytes T CD4+/métabolisme , Lymphocytes T CD4+/virologie , Prolifération cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , ADN viral/isolement et purification , Régulation de l'expression des gènes viraux/effets des médicaments et des substances chimiques , Infections à VIH/traitement médicamenteux , Infections à VIH/génétique , Infections à VIH/immunologie , Infections à VIH/virologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/effets des médicaments et des substances chimiques , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/isolement et purification , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/pathogénicité , Mémoire immunologique , Microfluidique , Nécroptose/effets des médicaments et des substances chimiques , Transduction du signal/effets des médicaments et des substances chimiques , Transcriptome/effets des médicaments et des substances chimiques , Latence virale/effets des médicaments et des substances chimiques , Antirétroviraux/pharmacologie , Antirétroviraux/usage thérapeutiqueRÉSUMÉ
Recently, a novel gammaherpesvirus, miroungine gammaherpesvirus 3 (MirGHV3), was described in two juvenile elephant seals (Mirounga angustirostris) with diffuse large B-cell lymphoma. We developed and validated a quantitative (q)PCR for rapid detection of MirGHV3 and investigated its potential association with lymphoma. We developed a duplex probe-hybridization qPCR with MirGHV3 DNA polymerase (pol) as the target gene. Each primer-probe combination was cross-validated against the others. Interference was not seen when they were run in the same well as a duplex assay. Twenty-three samples from seven northern elephant seals were tested using the duplex assay. Viral DNA was detected by the assay in 9 of 9 (100%) tissues affected by lymphoma and in 6 of 14 (43%) samples from tissues unaffected by lymphoma. There was a strong correlation between viral copies detected with each of the assays (P=0.0002). Viral load was significantly higher in tissues affected by lymphoma than in those unaffected (P<0.0001). Excluding the virus-negative samples, viral load was still significantly higher in tissues affected by lymphoma than in those unaffected (P=0.0004). This is consistent with a potential role of MirGHV3 in oncogenesis in northern elephant seals, although more studies are needed to determine this definitively. The qPCR developed has utility for further investigations of MirGHV3.
Sujet(s)
Gammaherpesvirinae , Lymphome B diffus à grandes cellules , Réaction de polymérisation en chaîne , Phoques , Infections à virus oncogènes , Animaux , Réaction de polymérisation en chaîne/méthodes , Réaction de polymérisation en chaîne/médecine vétérinaire , Phoques/virologie , Reproductibilité des résultats , Lymphome B diffus à grandes cellules/médecine vétérinaire , Lymphome B diffus à grandes cellules/virologie , Gammaherpesvirinae/génétique , Gammaherpesvirinae/isolement et purification , Infections à virus oncogènes/médecine vétérinaire , Infections à virus oncogènes/virologie , ADN viral/isolement et purification , Mâle , FemelleRÉSUMÉ
High-risk human papillomavirus (HR-HPV) testing has become an increasing important strategy in primary cervical cancer screening in recent years. It warrants the evaluation of molecular-based HPV tests for accuracy and efficacy of screening. The performance of Roche Cobas 4800 HPV test was validated and compared with Digene Hybrid Capture 2 (HC2) high-risk HPV DNA test for primary screening in a large Chinese screening cohort. Of 6345 women screened, overall agreement between Cobas and HC2 was 92.23% (95% CI: 91.57-92.89). The inter-assay agreement was correlated with the severity of underlying biology, with an increasing concordance found in samples with more severe abnormalities. Most of the discordant samples had the test signal strength closer to the test limits of the detection than concordant samples, reflecting a low viral load and infection of a cluster of low-risk HPV in these samples. The Cobas test demonstrated significantly higher specificity in identifying CIN2+/CIN3+ cases than HC2 test (66.46% vs 43.67% and 65.42% vs 42.86%, p<0.001), with comparable sensitivity in clinical evaluation. Increased specificity of Cobas test would accent women having the highest risk of developing CIN2+, with the potential to reduce unnecessary colposcopy referral in a screening population.
Sujet(s)
Dépistage précoce du cancer , Papillomaviridae , Infections à papillomavirus , Tumeurs du col de l'utérus , Chine , ADN viral/isolement et purification , Dépistage précoce du cancer/méthodes , Femelle , Humains , Papillomaviridae/génétique , Infections à papillomavirus/diagnostic , Sensibilité et spécificité , Tumeurs du col de l'utérus/diagnosticRÉSUMÉ
BACKGROUND: The potential risk and association of bovine leukemia virus (BLV) with human remains controversial as it has been reported to be both positive and negative in human breast cancer and blood samples. Therefore, establishing the presence of BLV in comprehensive human clinical samples in different geographical locations is essential. RESULT: In this study, we examined the presence of BLV proviral DNA in human blood and breast cancer tissue specimens from Japan. PCR analysis of BLV provirus in 97 Japanese human blood samples and 23 breast cancer tissues showed negative result for all samples tested using long-fragment PCR and highly-sensitive short-fragment PCR amplification. No IgG and IgM antibodies were detected in any of the 97 human serum samples using BLV gp51 and p24 indirect ELISA test. Western blot analysis also showed negative result for IgG and IgM antibodies in all tested human serum samples. CONCLUSION: Our results indicate that Japanese human specimens including 97 human blood, 23 breast cancer tissues, and 97 serum samples were negative for BLV.
Sujet(s)
Anticorps antiviraux , ADN viral , Virus de la leucémie bovine , Provirus , Anticorps antiviraux/isolement et purification , Sang/virologie , Tumeurs du sein/virologie , ADN viral/isolement et purification , Femelle , Humains , Immunoglobuline G/isolement et purification , Immunoglobuline M/isolement et purification , Japon , Virus de la leucémie bovine/génétique , Virus de la leucémie bovine/immunologie , Provirus/génétiqueRÉSUMÉ
Hepadnaviruses have been identified in several animal species. The hepadnavirus prototype, human hepatitis B virus (HBV), is a major public health problem associated with chronic liver diseases and hepatocellular carcinoma. Recently, a novel hepadnavirus, similar to HBV, was identified in domestic cats. Since several pathogens can be shared between cats and dogs, we hypothesized that dogs could also harbor hepadnaviruses and we tested a collection of canine sera with multiple molecular strategies. Overall, hepadnavirus DNA was identified in 6.3% (40/635) of canine serum samples, although the viral load in positive sera was low (geometric mean of 2.70 × 102 genome copies per mL, range min 1.36 × 102-max 4.03 × 104 genome copies per mL). On genome sequencing, the canine hepadnaviruses revealed high nucleotide identity (about 98%) and similar organization to the domestic cat hepadnavirus. Altered hepatic markers were found in hepadnavirus-positive dogs, although the role of hepadnavirus in canine health remains to be elucidated.
Sujet(s)
Chiens/virologie , Hepadnaviridae/isolement et purification , Animaux , ADN viral/sang , ADN viral/isolement et purification , Génome viral/génétique , Hepadnaviridae/génétique , Phylogenèse , Séquençage du génome entierRÉSUMÉ
Early detection of viral pathogens by DNA-sensors in clinical samples, contaminated foods, soil or water can dramatically improve clinical outcomes and reduce the socioeconomic impact of diseases such as COVID-19. Clustered regularly interspaced short palindromic repeat (CRISPR) and its associated protein Cas12a (previously known as CRISPR-Cpf1) technology is an innovative new-generation genomic engineering tool, also known as 'genetic scissors', that has demonstrated the accuracy and has recently been effectively applied as appropriate (E-CRISPR) DNA-sensor to detect the nucleic acid of interest. The CRISPR-Cas12a from Prevotella and Francisella 1 are guided by a short CRISPR RNA (gRNA). The unique simultaneous cis- and trans- DNA cleavage after target sequence recognition at the PAM site, sticky-end (5-7 bp) employment, and ssDNA/dsDNA hybrid cleavage strategies to manipulate the attractive nature of CRISPR-Cas12a are reviewed. DNA-sensors based on the CRISPR-Cas12a technology for rapid, robust, sensitive, inexpensive, and selective detection of virus DNA without additional sample purification, amplification, fluorescent-agent- and/or quencher-labeling are relevant and becoming increasingly important in industrial and medical applications. In addition, CRISPR-Cas12a system shows great potential in the field of E-CRISPR-based bioassay research technologies. Therefore, we are highlighting insights in this research direction.
Sujet(s)
Systèmes CRISPR-Cas/physiologie , ADN viral/isolement et purification , Techniques d'amplification d'acides nucléiques , Animaux , Techniques de biocapteur/méthodes , Techniques de biocapteur/tendances , COVID-19/virologie , ADN viral/analyse , Polluants environnementaux/analyse , Polluants environnementaux/isolement et purification , Contamination des aliments/analyse , Humains , Typage moléculaire/méthodes , Typage moléculaire/tendances , Techniques d'amplification d'acides nucléiques/méthodes , Techniques d'amplification d'acides nucléiques/tendances , SARS-CoV-2/génétique , Virologie/méthodes , Virologie/tendances , Maladies virales/classification , Maladies virales/diagnostic , Maladies virales/virologieRÉSUMÉ
Viruses are far more abundant than cellular microorganisms in the marine ecosystem. However, very few viruses have so far been isolated from marine sediments, especially hydrothermal vent sediments, hindering the understanding of the biology and ecological functions of these tiny organisms. Here, we report the isolation and characterization of a temperate bacteriophage, named PVJ1, which infects Psychrobacillus from a hydrothermal vent field in Okinawa Trough. PVJ1 belongs to the Myoviridae family of the order Caudovirales. The tailed phage possesses a 53,187 bp linear dsDNA genome, with 84 ORFs encoding structural proteins, genome replication, host lysis, etc. in a modular pattern. The phage genome is integrated into the host chromosome near the 3'-end of deoD, a gene encoding purine nucleoside phosphorylase (PNP). The phage integration does not appear to disrupt the function of PNP. The phage DNA is packaged by the headful mechanism. Release of PVJ1 from the host cell was drastically enhanced by treatment with mitomycin C. Phages encoding an MCP sharing significant similarity (≥70% identical amino acids) with that of PVJ1 are widespread in diverse environments, including marine and freshwater sediments, soils, artificial ecosystems, and animal intestines, and primarily infect Firmicutes. These results are valuable to the understanding of the lifestyle and host interactions of bacterial viruses at the bottom of the ocean.
Sujet(s)
Bacillaceae/virologie , Bactériophages/classification , Bactériophages/isolement et purification , Sédiments géologiques/virologie , Phylogenèse , Animaux , Bactériophages/génétique , Caudovirales/génétique , Caudovirales/isolement et purification , ADN viral/isolement et purification , Écosystème , Génome viral , Myoviridae/génétique , Myoviridae/isolement et purification , Cadres ouverts de lectureRÉSUMÉ
Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (±paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63-159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (<250 copies/million cells) and longer PCR amplicons (>150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials.
Sujet(s)
ADN viral/isolement et purification , Formaldéhyde , Techniques de diagnostic moléculaire/méthodes , Fixation tissulaire/méthodes , Humains , Rein , Foie , Poumon , Hybridation d'acides nucléiques , Réaction de polymérisation en chaine en temps réel , Analyse de séquence d'ADNRÉSUMÉ
BACKGROUND AND AIMS: Classical CD8 T cells are implicated for protective and pathogenic roles in chronic hepatitis B (CHB) infection. Recently, a subset of CD8 T cells expressing C-X-C chemokine receptor type 5 (CXCR5) and exhibiting features of TFH cells has been identified during chronic viral infections. However, in CHB, knowledge of their roles is limited. APPROACH AND RESULTS: We characterized circulating CD8+ CXCR5+/- cells and investigated their association with clinical and viral factors. We found that CHB infection did not influence the overall frequencies of CD8+ CXCR5+ cells whereas CD8+ CXCR5- cells were increased. However, among CHB, CD8+ CXCR5+ cells were higher in patients with low HBsAg and HBV-DNA levels, patients who were HBeAg negative and had high fibrosis scores, and these cells exhibited a significant association with HBsAg and HBV-DNA reduction. Contrarily, CD8+ CXCR5- cells were expanded and positively correlated with patients having high HBsAg, HBV-DNA, and alanine aminotransferase levels. CD8+ CXCR5+ cells express costimulatory molecules ICOS, OX40, CD40 ligand, inhibitory molecule programmed death 1, transcription factors B-cell lymphoma (BCL)-2, BCL-6, and signal transducer and activator of transcription 3, and are enriched in effector and central memory phenotype. Moreover, these cells are heterogeneous in nature given that they constitute different subsets of cytotoxic follicular T cells (TCF), including TCF1, TCF2, TCF17, and TCF22. Despite expressing high PD-1, CD8+ CXCR5+ cells are activated, proliferating, secreting more IFN-γ, IL-21, and IL-22, and have better cytolytic potential than CD8+ CXCR5- cells, which were inhibited after PD-1/PD-L1 blockade. CD8+ CXCR5+ cells are efficient in helping B cells in terms of plasmablasts and plasma cell generation. CONCLUSIONS: In conclusion, CD8+ CXCR5+ cells are enriched in effector phenotypes, produce HBV-specific cytokines despite increased PD-1, and are associated with HBsAg and HBV-DNA reduction. These cells competently support B-cell function, required for viral clearance, which may serve as potential therapeutic targets for CHB.
Sujet(s)
Lymphocytes T CD8+/immunologie , Hépatite B chronique , Récepteur-1 de mort cellulaire programmée/métabolisme , Récepteurs CXCR5/analyse , Adulte , Alanine transaminase/sang , ADN viral/isolement et purification , Femelle , Antigènes de surface du virus de l'hépatite B/sang , Virus de l'hépatite B/génétique , Virus de l'hépatite B/isolement et purification , Hépatite B chronique/sang , Hépatite B chronique/immunologie , Humains , Mémoire immunologique , Interleukines/sang , Mâle , Sous-populations de lymphocytes T/immunologie ,RÉSUMÉ
INTRODUCTION: Evidence on the biological danger associated with surgical plume is lacking. We examined whether surgical plume, generated by the energy devices ultrasonically activated scalpel (US) or electrocautery (EC) contains virus-related substances. MATERIAL AND METHODS: Experiment 1, ex-vivo model: Tumor mass of a hepatocellular carcinoma line was prepared in a Nod/SCID mouse. Surgical plume generated on the mass by US or EC was collected and detection of HBs gene fragment and antigens (HBsAg or AFP) was conducted. Experiment 2, clinical specimen: Detection of HBV-DNA and HBsAg was conducted following the collection of surgical plume generated from clinically obtained liver specimens from six HBV-associated hepatocellular carcinoma patients. RESULTS: Experiment 1: HBs gene fragment was detected in the solutions regardless of the device used. HBsAg was detected in US and EC solutions and AFP was also detected in a US solution. Experiment 2: HBV-DNA was detected in both devices, in all three cases whose preoperative serum HBV-DNA was positive. In the other serum-negative cases, HBV-DNA was not detected. While serum HBsAg was positive in five of six cases, it was not detected in any solution. CONCLUSIONS: DNA fragments or antigens of virus can exist in the surgical plume generated by EC or US.