DNA Load Research Articles

Presence of HIV-1 G-to-A mutations linked to APOBEC editing is more prevalent in non-B HIV-1 subtypes and is associated with lower HIV-1 reservoir.

APOBEC3 editing activity contributes to sequences variation and viral diversification. We aimed to characterize virological and clinical factors associated with G-to-A mutations and stop codons in the HIV-1 reservoir, markers of APOBEC3 footprints, in order to better understand HIV-1 diversity among virologically suppressed HIV-1-infected patients. Immuno-virological and clinical factors were compared between 92 patients harbouring G-to-A mutations and stop codons (APOBEC+) in the reverse transcriptase gene and 92 patients without G-to-A mutations (APOBEC-) and stop codons in their DNA genotypes. Patients were predominantly men (74.5%) and were mostly infected by B-subtype (69.0%), with 44.1% and 55.9% in APOBEC+ and APOBEC- groups, respectively. At time of HIV DNA genotypes, the total cell-associated HIV-1 DNA load was 2.34 log10 copies/106 cells (IQR 1.85-2.67) and 33.2% of them had a detectable ultrasensitive plasma viral load. Hypermutated sequences were identified in 28.2% of the APOBEC+ group. The median total cell-associated HIV-1 DNA level was significantly lower in APOBEC+ than APOBEC- group: 2.13 log10 copies/106 cells (IQR 1.60-2.60) versus 2.52 log10 copies/106 cells (IQR 2.19-2.71) (P < 0.001), respectively. Presence of G-to-A mutations and stop codon was independently associated with HIV-1 subtype non-B (P = 0.017). These results show an independent association between the presence of G-to-A mutations and stop codons with HIV-1 subtype non-B and low proviral DNA that could be explained by the APOBEC3 footprints and restriction of DNA synthesis and integration. However, further investigations are needed to study the contribution of Vif amino acid variability among HIV-1 subtypes.

Hepatic microbiome in healthy lean and obese humans

Background & AimsDysbiosis of the gut microbiota in response to an energy-rich Western diet and the potential leak of bacteria and/or bacterial products from the intestine to the liver is perceived as a potential risk factor for the development of non-alcoholic fatty liver disease (NAFLD). We investigated the microbiome in liver biopsies from healthy lean and obese individuals and compared it with their blood microbiome.MethodsWe examined liver biopsies from 15 healthy lean and 14 obese individuals (BMI of 18.5–25 and 30–40 kg/m2, respectively). Bacterial 16S ribosomal DNA (rDNA) was analysed by quantitative polymerase chain reaction (qPCR) and 16S metagenomic sequencing targeting the hypervariable V3–V4 region. Metagenomic analysis was performed using the linear discriminant analysis effect size (LEfSe) algorithm. Data are medians with IQRs in brackets.ResultsHistology revealed hepatic steatosis in 13 obese individuals and in 2 lean individuals. A robust signal from qPCR revealed significantly higher amounts of bacterial rDNA copies in liver samples from obese individuals compared with those from lean individuals (148 [118–167] vs. 77 [62–122] 16S copies/ng DNA, p <0.001). Liver biopsies from the obese group were characterised by lower alpha diversity at the phylum level (Shannon index 0.60 [0.55–0.76] vs. 0.73 [0.62–0.90], p = 0.025), and metagenomic profiling revealed a significantly higher proportion of Proteobacteria in this group (81.0% [73.0–82.4%] vs. 74.3% [68.4–78.4%], p = 0.014).ConclusionsWe provide evidence for the presence of bacterial rDNA in the healthy human liver. Based on differences in the hepatic microbiome between obese individuals and healthy lean individuals, we suggest that changes in the liver microbiome could constitute an additional risk factor for the development of NAFLD.Lay summaryNon-alcoholic fatty liver disease (NAFLD) has become the most common liver disease globally, and new evidence suggests that obesity is associated with a disturbed gut bacterial composition, which may influence the development of NAFLD. We examined the composition of bacterial DNA in liver biopsies from healthy lean and obese individuals and found a different composition of bacterial DNA in liver biopsies from the obese group. We propose that the increased bacterial DNA load in the livers of obese individuals could constitute an early risk factor for the progression of NAFLD.Clinical trial numberNCT02337660

Resistant Sources and Genetic Control of Resistance to ToLCNDV in Cucumber.

Tomato leaf curl New Delhi virus (ToLCNDV) is a severe threat for cucurbit production worldwide. Resistance has been reported in several crops, but at present, there are no described accessions with resistance to ToLCNDV in cucumber (Cucumis sativus). C. sativus var. sativus accessions were mechanically inoculated with ToLCNDV and screened for resistance, by scoring symptom severity, tissue printing, and PCR (conventional and quantitative). Severe symptoms and high load of viral DNA were found in plants of a nuclear collection of Spanish landraces and in accessions of C. sativus from different geographical origins. Three Indian accessions (CGN23089, CGN23423, and CGN23633) were highly resistant to the mechanical inoculation, as well as all plants of their progenies obtained by selfing. To study the inheritance of the resistance to ToLCNDV, plants of the CGN23089 accession were crossed with the susceptible accession BGV011742, and F1 hybrids were used to construct segregating populations (F2 and backcrosses), which were mechanically inoculated and evaluated for symptom development and viral load by qPCR. The analysis of the genetic control fit with a recessive monogenic inheritance model, and after genotyping with SNPs distributed along the C. sativus genome, a QTL associated with ToLCNDV resistance was identified in chromosome 2 of cucumber.

Association of an IFN-γ variant with susceptibility to chronic hepatitis B by the enhancement of HBV DNA replication

Interferon gamma (IFN-γ) is a crucial cytokine in host immune response to hepatitis B virus (HBV) infection. This study aimed to determine whether a functional polymorphism +874T/A in IFN-γ gene linked to high and low producer phenotypes [IFN-γ (+874Thigh → Alow)] may alter the outcomes of chronic HBV infection in Tunisian population.The +874T/A was analysed by ARMS-PCR method in the group of 200 patients chronically infected with HBV and 200 healthy controls.We observed that minor +874A allele, minor +874AA and +874TA genotypes were significantly more frequent in the chronic hepatitis B group in comparison to the control group [49 vs. 31%, P < 10−4; 24 vs. 13%, P < 10−4; 52 vs. 38%, P < 10−4; respectively]. Besides, they were associated with susceptibility to hepatitis B infection [OR = 2.15, 3.87 and 2.84, respectively].The minor +874A allele and +874AA genotype were statistically more representative in the sub-group of patients with high viral DNA load when compared with the sub-group of patients with low HBV DNA load [(57% vs. 43%, P = 0.003, OR = 1.79); (33% vs. 14%, P = 0.003, OR = 3.59), respectively].Collectively, our study suggests an association between the IFN-γ +874T/A SNP and persistence of HBV by the enhancement of HBV DNA replication.

Associations Between Sputum Torque Teno Virus Load and Lung Function and Disease Severity in Patients With Chronic Obstructive Pulmonary Disease

Purpose: Viral load of Torque Teno virus (TTV) is elevated in immunosuppressed patients. The weakened immune response is typical in chronic obstructive pulmonary disease (COPD) patients. However, the relationship between TTV and COPD is still unknown.Patients and Methods: We enrolled 91 patients admitted to hospitals with acute exacerbation of COPD (AECOPD) between January 2017 and August 2017 (ClinicalTrials.gov ID, NCT03236480). Sputum samples were gathered during hospitalization and the 120-day follow-up. TTV distribution and genogroups were assessed, and the associations between viral loads and clinical parameters were analyzed.Results: TTV DNA was detected in 95.6% of COPD patients, and the viral load was nearly invariable at the stable and exacerbation states. Most TTV DNA-positive patients carried four distinct genotypes. Sputum load of TTV was positively associated with RV/TLC (r = 0.378, p = 0.030), and negatively correlated with FEV1/pre and FEV1/FVC (r = −0.484, −0.432, p = 0.011, 0.024, respectively). Neutral correlation between the TTV DNA load and COPD assessment test (CAT) scores (r = 0.258, p = 0.018) was observed.Conclusion: Sputum loads of TTV DNA could be a novel indicator for lung function and disease severity assessment in COPD patients.

Innate Immune Modulation Induced by EBV Lytic Infection Promotes Endothelial Cell Inflammation and Vascular Injury in Scleroderma

Microvascular injury is considered an initial event in the pathogenesis of scleroderma and endothelial cells are suspected of being the target of the autoimmune process seen in the disease. EBV has long been proposed as a trigger for autoimmune diseases, including scleroderma. Nevertheless, its contribution to the pathogenic process remains poorly understood. In this study, we report that EBV lytic antigens are detected in scleroderma dermal vessels, suggesting that endothelial cells might represent a target for EBV infection in scleroderma skin. We show that EBV DNA load is remarkably increased in peripheral blood, plasma and circulating monocytes from scleroderma patients compared to healthy EBV carriers, and that monocytes represent the prominent subsets of EBV-infected cells in scleroderma. Given that monocytes have the capacity to adhere to the endothelium, we then investigated whether monocyte-associated EBV could infect primary human endothelial cells. We demonstrated that endothelial cells are infectable by EBV, using human monocytes bound to recombinant EBV as a shuttle, even though cell-free virus failed to infect them. We show that EBV induces activation of TLR9 innate immune response and markers of vascular injury in infected endothelial cells and that up-regulation is associated with the expression of EBV lytic genes in infected cells. EBV innate immune modulation suggests a novel mechanism mediating inflammation, by which EBV triggers endothelial cell and vascular injury in scleroderma. In addition, our data point to up-regulation of EBV DNA loads as potential biomarker in developing vasculopathy in scleroderma. These findings provide the framework for the development of novel therapeutic interventions to shift the scleroderma treatment paradigm towards antiviral therapies.

High Prevalence of Preexisting HBV Polymerase Mutations in Pregnant Women Does Not Limit the Antiviral Therapy Efficacy.

Background HBV-resistant mutants in treatment-naïve patients may lead to antiviral treatment failure. It is not clear if HBV mutants are present in pregnant women and about the influence of the preexisting mutants on the short-term antiviral therapy during pregnancy. Method We enrolled 73 pregnant women with high HBV DNA load and telbivudine (TBV) treatment during pregnancy in this retrospective study. The UDPS was used to detect the HBV mutations before and after the TBV treatment. Results Before TBV treatment, the complexity of HBV quasispecies of all subjects was 0.40 ± 0.09; 41.1% (30/73) and 53.4% (39/73) subjects had rtM204I/V and rtN236 T/A detected, respectively; and 9.6% (7/73) patients had more than 20% frequency mutation of rtM204I/V, which was also similar with high frequency of rtN236 T/A mutation (41.1% vs. 53.4%, P=0.136; frequencies >20%: 9.6% vs. 5.5%, P=0.347). After TBV treatment, 71.2% (52/73) subjects had HBV DNA load ≥ 103 IU/mL at delivery. Among them, 75.0% of patients with rtM204I positive had HBV DNA load ≥103 IU/mL at delivery, which was comparable with the subjects without rtM204I (75.0% vs. 70.8%, P=0.710). No changes were found in the frequencies and the complexity of HBV quasispecies of rtM204I mutation after the TVB treatment. Conclusion The prevalence of preexisting drug-resistant mutations among pregnant women was high using UPDS. However, the preexisting HBV mutation had limited influence on the efficacy of short-term TBV treatment, and TBV treatment during late pregnancy seemed not to increase the risk of emerging HBV-resistant mutants.

Transforming growth factor β as a possible independent factor in chronic hepatitis B.

To investigate the association between immune-cell-related cytokines and the development of chronic hepatitis B (CHB), patients with chronic hepatitis B virus (HBV) infection in the immunotolerant (IT) phase (n = 30) or hepatitis B envelope antigen (HBeAg)-positive CHB (n = 250) were enrolled in this study. Serological indicators and plasma cytokine levels were measured at the time of enrollment. The results showed that there were significant differences in the median age of the patients (27 vs. 31 years), alanine aminotransferase levels (ALT, 29.85 vs. 234.70 U/L), alanine aminotransferase levels (AST, 23.40 vs. 114.90 U/L), HBsAg levels (4.79 vs. 3.88 log10 IU/ml), HBeAg levels (1606.36 vs. 862.47 S/CO), and the HBV DNA load (8.17 vs. 6.71 log10 IU/ml) between the IT and CHB groups (all P < 0.01). The median values of Fms-like tyrosine kinase 3 ligand (FLT3-L), interferon-gamma (IFN-γ), interleukin- 17A (IL-17A), and transforming growth factor beta (TGF-β1) were significantly higher in the IT group than in the CHB group (FLT3-L, 41.62 vs. 27.47 pg/ml; IFN-γ, 42.48 vs. 33.18 pg/ml; IL-17A, 15.66 vs. 8.90 pg/ml; TGF-β1, 4921.50 vs. 2234 pg/ml; all P < 0.01). The median IFN-α2, TGF-β3 and IL-10 levels in the IT group were significantly lower than those in the CHB group (IFN-α2, 15.24 vs. 35.78 pg/ml, P = 0.000; TGF-β3, 131.69 vs. 162.61 pg/ml, P = 0.025; IL-10, 5.02 vs. 7.9 pg/ml, P = 0.012). Multivariate logistic regression analysis indicated that TGF-β 1 (OR = 0.999, 95% CI 0.999-1.000, P < 0.001) and TGF-β2 levels (OR = 1.008, 95%CI 1.004-1.012, P < 0.001) were modestly but significantly associated with the incidence of CHB. The results suggest that TGF-β level might be an independent factor related to the occurrence of CHB.

Relationship between C genotype HBV mutation and intrauterine transmission

Objective: To analyze the virus genome mutation of mothers with C genotype HBV and explore its relationship with HBV intrauterine transmission. Methods: A total of 399 mothers carrying HBV and their newborns hospitalized in the obstetrics department of the Third People's Hospital of Taiyuan from 2011 to 2013 were selected. Necessary information about mothers and children was obtained through a questionnaire survey and medical records. HBV DNA and HBV serological markers were detected by quantitative fluorescence PCR and electrochemiluminescence. Within 24 hours after birth and before active/passive immunization, those with positive HBsAg and/or HBV DNA in femoral venous blood were determined as HBV intrauterine transmission. According to the requirements of cloning and sequencing, mothers' HBV DNA load should be ≥106 IU/ml. Among 54 cases of HBV intrauterine transmission, 22 pairs of mothers and their newborns meeting the requirements of cloning and sequencing were used as the intrauterine transmission group. The same number of mothers and their newborns without intrauterine transmission was selected as the random seed method's control group. After PCR amplification of HBV DNA, gene cloning, and sequencing, the gene mutation analysis of mothers with C genotype HBV was performed. Results: Among the 44 samples, 39 (88.63%, 39/44) were genotype C, 2 were genotype B, and 3 were mixed genotype B, and C. A total of 406 clone beads from 42 mothers with C genotype HBV were analyzed for gene mutation, including 204 in the intrauterine transmission group and 202 in the control group. The base substitution mutation rate of PreS1, S, C, and P regions in the HBV intrauterine transmission group were significantly lower than those in the control group (χ2 ranged from 8.67 to 40.73, P<0.05). The mutation rate of base deletion in PreC and X regions in the HBV intrauterine transmission group was lower than that in the control group (χ2 values were 17.82 and 34.78, P<0.001). Two clones in the X region had 31 bp insertion mutations between nt1644 and nt1645, and two clones had 27 bp insertion mutations between nt1649 and nt1650, all of which took place in the control group. Conclusions: The base substitution mutations in the PreS1, S, C, and P segments of the HBV genome in mothers with C genotype HBV were associated with the occurrence of intrauterine transmission of HBV. Deletion mutations in the PreC region, insertion and deletion mutations in the X region may reduce intrauterine transmission risk.

Resolved Hepatitis B: Achieved or Imaginary Wellbeing?

Aim. Assessment of the clinical impact of previous hepatitis B infection (PHB).Key points. PHB is characterized by the presence of viral DNA in the organism (including intrahepatic cccDNA and integrated DNA). Possible virus persistence in the PHB patient's hepatocytes potentiates the agent transmission risk via haemotransfusion, organ transplantation and haemodialysis. Occult HBV infection in PHB individuals can reactivate at background immunosuppressive or chemotherapies. PHB with chronic liver diseases of various aetiology significantly rises the risk of cirrhosis and hepatic cancer. The PHB association with autoimmune liver diseases and extrahepatic gastrointestinal cancer needs a careful research to confirm the possible involvement of hepatitis B virus in morbid genesis.Conclusion. No clinical signs of acute or chronic disease, HBsAg clearance and negative viral DNA load in blood of PHB individuals do not necessarily imply a complete disease eradication.PHB elicitation improves accuracy of the overall prognosis, reduces the virus transmission risk and prevents the reactivation of HBV infection.

Risk analysis for hepatocellular carcinoma in patients with chronic hepatitis B-associated cirrhosis complicated by type 2 diabetes mellitus: a 5-year prospective cohort study

To elucidate the risk and synergistic factors of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B-associated cirrhosis (CHB-Cir) complicated by type 2 diabetes (T2DM). The patients with CHB-Cir who were followed up in Hepatology Center of Nanfang Hospital from June 2010 to June 2019 were divided based on their T2DM status into two cohorts matched for gender, age, HBeAg status and HBV DNA load: CHB-Cir with T2DM group (observation group) and CHB-Cir without T2DM group (control group). All the patients were followed up at a 6-month interval, and the cases with complete clinical data and follow-up data for more than 2 years were included in the analysis. Kaplan- Meier method was used to compare the cumulative incidence of HCC between the two groups. A Cox proportional hazard regression model was used to analyze the relationship between T2DM and the risk of HCC in these patients. A total of 467 patients with a mean follow-up time of 4.4±1.62 years were included in the analysis, including 203 in the observation group and 264 in the control group. Sixty-nine and forty-eight new HCC cases occurred in the observation group and control group, respectively, showing a significantly higher incidence rate of HCC in the observation group (P < 0.001). The cumulative incidence of HCC in the observation group was significantly higher than that in the control group (P < 0.001), with a relative risk of 2.096 (P < 0.01). After adjustment for age (≥40 years), family history of liver cancer, previous antiviral therapy, elevated cholesterol and elevated LDL cholesterol, T2DM remained an independent risk factor for HCC in CHB-Cir patients (P=0.000). T2DM is an independent risk factor for HCC, and the risk of HCC increases by more than two folds in CHB-Cir patients complicated by T2DM, suggesting the clinical significance of early interventions of diabetes to reduce the risk of HCC in CHB-Cir patients.

Efficacy of traditional herbal medicine versus transcatheter arterial chemoembolization in postsurgical patients with hepatocellular carcinoma: A retrospective study

BackgroundRecurrence is a major obstacle to improve the prognosis of hepatocellular carcinoma (HCC) patients. Transcatheter arterial chemoembolization (TACE) has been routinely used as an adjuvant therapy in treating HCC, but efficacy of TACE in preventing the recurrence of HCC remains unsatisfactory. This study aimed to compare the efficacy of a traditional herbal medicine (THM) therapy and TACE in preventing tumor recurrence and improving survival in postsurgical patients with HCC. Materials and methodsA total of 1506 HCC patients were enrolled from January 2008 to June 2017, including 262 patients who received THM therapy and 1244 patients who were treated with TACE. All patients were followed up until the occurrence of outcome event or June 30th, 2019. The recurrence-free survival (RFS) and overall survival (OS) were calculated by the Kaplan-Meier method, and the differences of RFS and OS between THM group and TACE group were analyzed by the log-rank test. Factors affecting the RFS or OS among these patients were assessed by the Cox proportional hazard regression model. A nomogram was built with the factors based on the Cox regression analysis to predict the prognosis. ResultsThe 1-, 3-, and 5-year RFS were 91.0%, 68.3%, and 49.7%, respectively, in the THM group and 79.4%, 38.6%, and 19.3%, respectively, in the TACE group. The RFS in the THM group was significantly higher than that of the TACE group (P = 6.2 × 10−11). The 1-, 3-, and 5-year OS were significantly improved in the THM group as compared to those in the TACE group (94.3%, 65.2%, and 41.4% vs. 82.7%, 46.0%, and 25.4%, P = 2.2 × 10−11). Multivariate analysis revealed that serum AFP level ≥400 ng/mL, HBV DNA load ≥500 copies/mL, TNM stage III-IV, tumor diameter ≥5 cm, presence of MVI, and multiple tumor nodules were independent risk factors for RFS, while complete tumor encapsulation and THM therapy were protective factors for RFS and OS. The nomogram demonstrated good accuracy in predicting RFS and OS, with the adjusted C-index of 0.748 and 0.796, respectively. ConclusionThe efficacy of THM therapy was superior to that of TACE in preventing recurrence and improving survival for HCC patients after hepatectomy.

Experimental infection of indigenous North African goats with goatpox virus

BackgroundGoatpox is a viral disease caused by infection with goatpox virus (GTPV) of the genus Capripoxvirus, Poxviridae family. Capripoxviruses cause serious disease to livestock and contribute to huge economic losses. Goatpox and sheeppox are endemic to Africa, particularly north of the Equator, the Middle East and many parts of Asia. GTPV and sheeppox virus are considered host-specific; however, both strains can cause clinical disease in either goats or sheep with more severe disease in the homologous species and mild or sub-clinical infection in the other. Goatpox has never been reported in Morocco, Algeria or Tunisia despite the huge population of goats living in proximity with sheep in those countries. To evaluate the susceptibility and pathogenicity of indigenous North African goats to GTPV infection, we experimentally inoculated eight locally bred goats with a virulent Vietnamese isolate of GTPV. Two uninfected goats were kept as controls. Clinical examination was carried out daily and blood was sampled for virology and for investigating the antibody response. After necropsy, tissues were collected and assessed for viral DNA using real-time PCR.ResultsFollowing the experimental infection, all inoculated goats displayed clinical signs characteristic of goatpox including varying degrees of hyperthermia, loss of appetite, inactivity and cutaneous lesions. The infection severely affected three of the infected animals while moderate to mild disease was noticed in the remaining goats. A high antibody response was developed. High viral DNA loads were detected in skin crusts and nodules, and subcutaneous tissue at the injection site with cycle threshold (Ct) values ranging from 14.6 to 22.9, while lower viral loads were found in liver and lung (Ct = 35.7 and 35.1). The results confirmed subcutaneous tropism of the virus.ConclusionClinical signs of goatpox were reproduced in indigenous North African goats and confirmed a high susceptibility of the North African goat breed to GTPV infection. A clinical scoring system is proposed that can be applied in GTPV vaccine efficacy studies.

Coordinated Expression of HPV-6 Genes with Predominant E4 and E5 Expression in Laryngeal Papilloma.

Laryngeal papilloma (LP) associated with human papillomavirus (HPV)-6 or -11 infection shows aggressive growth. However, the detailed molecular mechanism of virus-driven tumorigenesis has not been uncovered fully. HPV-6 viral gene expression and dynamic alterations were investigated with in situ localization of viral DNA and RNA in 13 patients with HPV-6-infected laryngeal papilloma. The average viral load was 4.80 × 105 ± 1.86 × 105 copies/ng DNA. E4, E5a, and E5b mRNAs accounted for 96% of the expression of 9 mRNAs. The alteration of viral DNA load during recurrence paralleled the mRNA expression levels, and the expression of all mRNAs showed a similar curve. E4, E5a, and E5b were expressed in the middle to upper part of the epithelium and were co-expressed in the same cells. E4 immunohistochemistry demonstrated an extensively positive reaction in the upper cell layer in accordance with E4 mRNA expression. These results suggest that individual viral genes are coordinately expressed for viral replication, virus release, and immunosurveillance avoidance. The newly developed E4-specific monoclonal antibody can be applied to further functional studies and clinical applications such as targeted molecular therapies.

Factors and virological significance of hepatitis B virus pregenomic RNA status after 5 years of antiviral therapy

ObjectivesTo investigate the factors and virological significance of serum hepatitis B virus (HBV) pregenomic RNA (pgRNA) status after long-term antiviral therapy with nucleos(t)ide analogues (NAs) in patients with chronic hepatitis B (CHB). MethodsIn total, 51 treatment-naïve patients with CHB were included in the study. Clinical data were collected at baseline, during 5 years and at year 10 of NA treatment. Serum HBV pgRNA status of 51 patients was determined at year 5. ResultsAt year 5, 45% of the patients remained positive for HBV pgRNA. There were significant differences in baseline hepatitis B e antigen (HBeAg) status, HBV DNA load and hepatitis B surface antigen (HBsAg) levels between patients testing positive and negative for HBV pgRNA at year 5. Serum HBV pgRNA status and serum HBV DNA load were correlated after 5 years of NA treatment (r = 0.347, P = 0.013). Being HBV pgRNA positive at year 5 was an independent risk factor for sustainedly undetectable HBV DNA after 10 years of NA treatment (odds ratio 13.638, 95% confidence interval 1.32–140.81; P = 0.028). Furthermore, HBV pgRNA status at year 5 was associated with HBV DNA re-appearance at year 10 (P = 0.009). ConclusionsHBV pgRNA status at year 5 can reveal HBV covalently closed circular DNA (cccDNA) activity, and HBV pgRNA positivity after long-term antiviral therapy may indicate higher transcriptional activity of HBV cccDNA. Long-term dynamic monitoring of HBV pgRNA should be considered.

Large Extracellular Vesicle Characterization and Association with Circulating Tumor Cells in Metastatic Castrate Resistant Prostate Cancer.

Simple SummaryNon-invasive, liquid biopsies are an attractive means for tumor diagnosis and monitoring. In addition to DNA and cells, tumors have an increased propensity compared to normal cells to shed vesicles. These large extracellular vesicles, which are believed to be more frequent in aggressive cancers, carry tumor DNA and proteins and can thus be informative sources for diagnosis and prognosis. In this study, we developed a method to identify and molecularly characterize large extracellular vesicles in parallel to circulating tumor cells at a single-cell/single-vesicle level. We show that the number of large extracellular vesicles correlates to and exceeds the number of circulating tumor cells, and that analysis of tumor-derived large extracellular vesicles hence increases the sensitivity of the liquid biopsy assay.Liquid biopsies hold potential as minimally invasive sources of tumor biomarkers for diagnosis, prognosis, therapy prediction or disease monitoring. We present an approach for parallel single-object identification of circulating tumor cells (CTCs) and tumor-derived large extracellular vesicles (LEVs) based on automated high-resolution immunofluorescence followed by downstream multiplexed protein profiling. Identification of LEVs >6 µm in size and CTC enumeration was highly correlated, with LEVs being 1.9 times as frequent as CTCs, and additional LEVs were identified in 73% of CTC-negative liquid biopsy samples from metastatic castrate resistant prostate cancer. Imaging mass cytometry (IMC) revealed that 49% of cytokeratin (CK)-positive LEVs and CTCs were EpCAM-negative, while frequently carrying prostate cancer tumor markers including AR, PSA, and PSMA. HSPD1 was shown to be a specific biomarker for tumor derived circulating cells and LEVs. CTCs and LEVs could be discriminated based on size, morphology, DNA load and protein score but not by protein signatures. Protein profiles were overall heterogeneous, and clusters could be identified across object classes. Parallel analysis of CTCs and LEVs confers increased sensitivity for liquid biopsies and expanded specificity with downstream characterization. Combined, it raises the possibility of a more comprehensive assessment of the disease state for precise diagnosis and monitoring.

Comparative study of IS711 and bcsp31-based polymerase chain reaction (PCR) for the diagnosis of human brucellosis in whole blood and serum samples

BackgroundClinical diagnosis of human brucellosis (HB) is often difficult due to non-specific symptoms. Immunological tests have been the most common method used in HB diagnosis, but molecular methods based on quantitative polymerase chain reaction (qPCR) have largely replaced these diagnostic methods. The aim of this study was to validate a HB diagnostic qPCR method; assessing different target Brucella genes, and the influence of biological matrices (serum vs. whole blood) on analytical parameters. Material and methodsTwo target genes, IS711 and bcsp31, for HB molecular diagnosis were evaluated, together with biological matrix type (whole blood and serum) using samples spiked with Brucella abortus. In addition, diagnostic parameters of this qPCR method were evaluated in paired whole blood and serum samples from patients with suspected HB. ResultsBoth genes could be potential diagnostic targets, but IS711 showed a lower limit of detection. In spiked matrix experiments, whole blood showed a lower limit of detection than serum after probit regression (224 vs. 3681 CFU/mL) and ANOVA analysis showed a significant (p < 0.001) difference between the Cq of whole blood at all dilutions and that of serum. In 12 paired clinical samples, no serum samples and only one whole blood sample tested positive for Brucella using this qPCR detection method. ConclusionsThis standardized qPCR-based Brucella detection method could improve diagnosis of HB, serving as a rapid, highly sensitive, and specific test. Whole blood is better suited to qPCR-based HB diagnosis due to the presence of higher target DNA loads in this matrix, compared to serum.

Immediately early 2 (IE-2) and DNA polymerase SiRNA as virus-specific antiviral against novel transplacental cytomegalovirus strain ALL-03 in vitro.

This study investigated the suitability of siRNA targeting specific genes that cause inhibition of virus replication in vitro especially for the virus that capable of crossing placenta and we employed a novel transplacental rat cytomegalovirus that mimics infection in human. Six unique siRNAs with three each targeting different regions of IE2 (ie2a, ie2b and ie2c) and DNA polymerase (dpa, dpb and dpc) were prepared and tested for antiviral activities. The efficacy as an antiviral was determined in in-vitro by measuring TCID50 virus titer, severity of virus-induced cytopathic effect (CPE), intracellular viral genome loads by droplet digital PCR, the degree of apoptosis in siRNA-treated cells and relative expression of viral mRNA in infected Rat Embryo Fibroblast (REF) cells. Remarkably, the siRNAs: dpa, dpb and IE2b, significantly reduced virus yield (approximately >90%) compared to control group at day 18 post infection (p.i). Changes in CPE indicated that DNA polymerase siRNAs were capable of protecting cells against CMV infection at day 14 p.i with higher efficiency than GCV (at the concentration of 300pmol). Gene expression analysis revealed a marked down regulation of the targeted DNA polymerase gene (73.9%, 96.0% and 90.7% for dpa, dpb and dpc siRNA, respectively) and IE2 gene (50.8%, 49.9% and 15.8% for ie2a, ie2b and ie2c siRNA, respectively) when measured by RT-qPCR. Intracellular viral DNA loads showed a significant reduction for all the DNA polymerase siRNAs (dpa: 96%, dpb: 98% and dpc:92) compared to control group (P<0.05). In conclusion, this study clearly highlighted the feasibility of RNAi as an alternative antiviral therapy that could lead to controlling the CMV infection.

Tick-Tattoo: DNA Vaccination Against B. burgdorferi or Ixodes scapularis Tick Proteins.

Introduction Borrelia burgdorferi sensu lato (sl) is the causative agent of Lyme borreliosis. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines. DNA tattoo vaccination with B. afzelii strain PKo OspC in mice has proven to be fully protective against B. afzelii syringe challenge and induces a favorable humoral immunity compared to recombinant protein vaccination. Alternatively, several recombinant protein vaccines based on tick proteins have shown promising effect in tick-bite infection models. In this study, we evaluated the efficacy of DNA vaccines against Borrelia OspC or tick antigens in a tick-bite infection model.MethodWe vaccinated C3H/HeN mice with OspC using a codon-optimized DNA vaccine or with recombinant protein. We challenged these mice with B. burgdorferi sensu stricto (ss)-infected Ixodes scapularis nymphs. Subsequently, we vaccinated C3H/HeN mice with DNA vaccines coding for tick proteins for which recombinant protein vaccines have previously resulted in interference with tick feeding and/or Borrelia transmission: Salp15, tHRF, TSLPI, and Tix-5. These mice were also challenged with B. burgdorferi ss infected Ixodes scapularis nymphs.ResultsDNA tattoo and recombinant OspC vaccination both induced total IgG responses. Borrelia cultures and DNA loads of skin and bladder remained negative in the mice vaccinated with OspC DNA vaccination, except for one culture. DNA vaccines against tick antigens Salp15 and Tix-5 induced IgG responses, while those against tHRF and TSLPI barely induced any IgG response. In addition, Borrelia cultures, and DNA loads from mice tattooed with DNA vaccines against tick proteins TSLPI, Salp15, tHRF, and Tix-5 were all positive.ConclusionA DNA tattoo vaccine against OspC induced high specific IgG titers and provided near total protection against B. burgdorferi ss infection by tick challenge. In contrast, DNA tattoo vaccines against tick proteins TSLPI, Salp15, tHRF, and Tix-5 induced low to moderate IgG titers and did not provide protection. Therefore, DNA tattoo vaccination does not seem a suitable vaccine strategy to identify, or screen for, tick antigens for anti-tick vaccines. However, DNA tattoo vaccination is a straightforward and effective vaccination platform to assess novel B. burgdorferi sl antigen candidates in a relevant tick challenge model.

Human Microbiota and Breast Cancer-Is There Any Relevant Link?-A Literature Review and New Horizons Toward Personalised Medicine.

Breast cancer (BC) is the most common malignancy and the second cause of cancer-specific death in women from high-income countries. Recently, gut microbiota dysbiosis emerged as a key player that may directly and/or indirectly influence development, treatment, and prognosis of BC through diverse biological processes: host cell proliferation and death, immune system function, chronic inflammation, oncogenic signalling, hormonal and detoxification pathways. Gut colonisation occurs during the prenatal period and is later diversified over distinct phases throughout life. In newly diagnosed postmenopausal BC patients, an altered faecal microbiota composition has been observed compared with healthy controls. Particularly, β-glucuronidase bacteria seem to modulate the enterohepatic circulation of oestrogens and their resorption, increasing the risk of hormone-dependent BC. Moreover, active phytoestrogens, short-chain fatty acids, lithocholic acid, and cadaverine have been identified as bacterial metabolites influencing the risk and prognosis of BC. As in gut, links are also being made with local microbiota of tumoural and healthy breast tissues. In breast microbiota, different microbial signatures have been reported, with distinct patterns per stage and biological subtype. Total bacterial DNA load was lower in tumour tissue and advanced-stage BC when compared with healthy tissue and early stage BC, respectively. Hypothetically, these findings reflect local dysbiosis, potentially creating an environment that favours breast tumour carcinogenesis (oncogenic trigger), or the natural selection of microorganisms adapted to a specific microenvironment. In this review, we discuss the origin, composition, and dynamic evolution of human microbiota, the links between gut/breast microbiota and BC, and explore the potential implications of metabolomics and pharmacomicrobiomics that might impact BC development and treatment choices toward a more personalised medicine. Finally, we put in perspective the potential limitations and biases regarding the current microbiota research and provide new horizons for stronger accurate translational and clinical studies that are needed to better elucidate the complex network of interactions between host, microorganisms, and drugs in the field of BC.