Mutagenesis and genome engineering of Epstein-Barr virus in cultured human cells by CRISPR/Cas9

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 nuclease (Cas9) system is a powerful genome-editing tool for both chromosomal and extrachromosomal DNA. DNA viruses such as Epstein–Barr virus (EBV), which undergoes episomal replication in human cells, can be effectively edited by CRISPR/Cas9. We have demonstrated targeted editing of the EBV genome by CRISPR/Cas9 in several lines of EBV-infected cells. CRISPR/Cas9-based mutagenesis and genome engineering of EBV provides a new method for genetic analysis, which has some advantages over bacterial artificial chromosome-based recombineering. This approach might also prove useful in the cure of EBV infection. In this chapter, we use the knockout of the BART promoter as an example to detail the experimental procedures for construction of recombinant EBV in human cells.postprin

Adenoviral delivery of RNA decoys restores cellular proapoptotic protein PUMA expression by silencing Epstein-Barr virus-encoded miR-BART5 in nasopharyngeal carcinoma cells

Poster Session 1 - Vaccines and Anti-Viral Therapeutics: no. 3.17Epstein-Barr virus (EBV) encodes 48 mature microRNAs that play important roles in viral maintenance and promote host cell survival by regulating viral transcripts expression, inhibiting apoptosis or facilitating to evade cell immune surveillance. We have previously shown that EBV-encoded miR-BART5 targets and downregulates cellular pro-apoptotic protein p53-upregulated modulator of apoptosis (PUMA) to promote cellular survival of EBV-infected nasopharyngeal carcinoma (NPC) cells. Since compromising miR-BART5 might induce apoptosis of EBV-infected NPC cell, in this study we have established an adenoviral expression system to deliver anti-miR-BART5 decoys to NPC cells. The anti-miR-BART5 decoys comprised 6 tandem repeats of miR-BART5 binding sites and their expression was driven by EBVEBER2 promoter. They were designed to serve as a competitive inhibitor of miR-BART5 to reverse miR-BART5's inhibitory effects on PUMA in EBV-infected NPC cells. The RNA polymerase III-dependent EBER2 promoter is particularly strong in ...postprin

Prognostic Factors for Transplant-Free Survival and Validation of Prognostic Models in Chinese Patients with Primary Biliary Cholangitis Receiving Ursodeoxycholic Acid

OBJECTIVES: We aimed to validate the prognostic models for primary biliary cholangitis (PBC) in Chinese patients receiving ursodeoxycholic acid (UCDA), and to compare their performances in predicting the long-term survival. METHODS: Chinese patients with PBC from a tertiary center were identified via electronic search of hospital medical registry. Risk factors associated with adverse events (liver transplantation or death from liver-related causes including hepatocellular carcinoma (HCC) and liver decompensation) were determined. Transplant-free survival was defined as survival free of liver-related death or transplantation. RESULTS: Of the 144 patients, 41 (28.5%) had baseline cirrhosis. The median age at diagnosis was 57.8 years. During a median follow-up of 7.0 years, 40 patients died (21 liver-related; 19 non-liver-related), 12 developed HCC, and 10 underwent transplantations. The 5-, 10-, and 15-year transplant-free survival probabilities were 91.0%, 78.1%, and 58.9%, respectively. Independent risk factors for adverse events were increasing age (hazard ratio (HR) 1.05), cirrhosis (HR 8.53), and suboptimal treatment response (HR 3.06). Aspartate aminotransferase/platelet ratio index at 1 year (APRI-r1) in combination with treatment response optimized the risk stratification. The performances of the GLOBE, UK-PBC scores, Rotterdam criteria, and APRI-r1 were comparable in predicting adverse events. The area under receiver operating curves within 5, 10, and 15 years were as follows-GLOBE score: 0.83, 0.85, and 0.85, respectively; UK-PBC score: 0.89, 0.83, and 0.79, respectively; Rotterdam criteria: 0.82, 0.76, and 0.80, respectively; APRI-r1: 0.80, 0.83, and 0.77, respectively. CONCLUSIONS: The UK-PBC, GLOBE scores, Rotterdam criteria, and APRI-r1 had good and comparable prognostic prediction values for Chinese PBC patients receiving UCDA.published_or_final_versio

Epidemiology and Natural History of Primary Biliary Cholangitis in the Chinese: A Territory-Based Study in Hong Kong between 2000 and 2015

OBJECTIVES: Studies on the epidemiology of primary biliary cholangitis (PBC) in the Chinese population are lacking. We aimed to determine the epidemiology of PBC in Hong Kong (HK) with a population of 7.3 million. METHODS: We retrieved data from the electronic database of the HK Hospital Authority, the only public healthcare provider in Hong Kong. PBC cases between 2000 and 2015 were identified by International Classification of Diseases (ICD)-9 code. We estimated the age-/sex-adjusted incidence rate and prevalence of PBC, and analyzed the adverse outcomes (hepatocellular carcinoma (HCC), liver transplantation, and death). RESULTS: One thousand and sixteen PBC patients aged >/=20 years were identified (female-to-male ratio 4:1; median age 60.6 years, interquartile range (IQR) 51.8-72.6 years; median follow-up 5.6 years, IQR 1.6-8.7 years). The average age/sex-adjusted annual incidence rate and prevalence were 8.4 per million person-years and 56.4 per million, respectively. Between 2000 and 2015, the age/sex-adjusted annual incidence rate increased from 6.7 to 8.1 per million person-years (Poisson P=0.002), while age/sex-adjusted prevalence increased from 31.1 to 82.3 per million (Poisson P<0.001). Fifty patients developed HCC, and 49 underwent liver transplantation. Case fatality risk decreased from 10.8 to 6.4% (Poisson P=0.003). The 5- and 10-year overall survival rates were 81.5 and 78.3%, whereas the transplant-free survival rates were 78.0% and 74.3%, respectively. Increasing age, cirrhosis and being treatment-naive were associated with lower transplant-free survival. CONCLUSIONS: There is a considerable increase in the incidence and prevalence of PBC in the Chinese population over the past 16 years, with significant morbidity and mortality.published_or_final_versio

Wisteria floribunda agglutinin-positive human Mac-2 binding protein predicts liver cancer development in chronic hepatitis B patients under antiviral treatment

AIM: The risk factors for hepatocellular carcinoma (HCC) development in chronic hepatitis B (CHB) patients with undetectable serum HBV DNA under nucleos(t)ide analogue (NA) therapy are not well defined. We aimed to examine the relationship between Wisteria floribunda agglutinin-positive human Mac-2 binding protein (WFA+-M2BP) and HCC development in these patients. RESULTS: There was a significant difference in the median levels of pre-treatment WFA+-M2BP between the HCC and control groups (0.67 vs 0.41 COI, respectively, p < 0.001). Among patients with cirrhosis, the median level of WFA+-M2BP was higher in HCC group than in control group (0.74 vs 0.47 COI, respectively, p = 0.014). Among patients without cirrhosis, the median level of WFA+-M2BP of HCC group was also higher (0.48 vs 0.28 COI, respectively, p = 0.002). With a cutoff value of 0.69, the AUROC of pre-treatment WFA+-M2BP to predict HCC development for the whole cohort was 0.70. With cutoff values of 0.69 and 0.34, the AUROCs to predict HCC were 0.67 and 0.77 for patients with and without cirrhosis, respectively. MATERIALS AND METHODS: Fifty-seven NA-treated patients with undetectable HBV DNA who developed HCC were compared with 57 controls (matched with demographics and treatment duration). WFA+-M2BP levels were measured, and expressed as cutoff index (COI). Subgroup analyses were also performed in patients with and without cirrhosis. CONCLUSIONS: A higher pre-treatment WFA+-M2BP level was associated with an increased risk of HCC development in patients with undetectable HBV DNA under NA therapy. Further longitudinal studies are required to examine the role of WFA+-M2BP as an accessory risk marker for HCC development.published_or_final_versio

Middle East respiratory syndrome coronavirus M protein suppresses type I interferon expression through the inhibition of TBK1-dependent phosphorylation of IRF3

published_or_final_versio

A man with a blistering eruption and tuberculosis.

published_or_final_versio

Clinical spectrum of paradoxical deterioration during antituberculosis therapy in non-HIV-infected patients

Paradoxical deterioration during antituberculosis therapy, defined as the clinical or radiological worsening of pre-existing tuberculous lesions or the development of new lesions in a patient who initially improves, remains a diagnostic dilemma. Although different clinical presentations of paradoxical response have been described, a systematic analysis of the entity in non-HIV-infected patients is lacking. Reported here are two cases of paradoxical deterioration in which sequential changes in lymphocyte counts and tuberculin skin test results are emphasized. In addition, 120 episodes of paradoxical response after antituberculosis treatment were reviewed. Of the total 122 episodes, 101 (82.8%) were associated with extrapulmonary tuberculosis. The median time from commencement of treatment to paradoxical deterioration was 60 days. The median time to onset of central nervous system manifestations (63 days) was longer than the time to onset of manifestations at other sites (56 days) (P=0.02). Development of new lesions in anatomical sites other than those observed at initial presentation was observed in 31 (25.4%) episodes. A surge in the lymphocyte count, accompanied by an exaggerated tuberculin skin reaction, was observed in our patients during the paradoxical deterioration, analogous to the findings in HIV-positive patients. Treatment of the paradoxical response included surgical intervention (60.7%) and administration of steroids (39.3%). The use of steroids appeared to be safe in this series, as 95% of the Mycobacterium tuberculosis isolates were susceptible to first-line antituberculosis therapy.postprin

Suppression of type I and type III interferon signalling by NSs protein of severe fever-with-thrombocytopenia syndrome virus through inhibition of STAT1 phosphorylation and activation

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen causing significant morbidity and mortality in Asia. NSs protein of SFTSV is known to perturb type I IFN induction and signalling, but the mechanism remains to be fully understood. Here, we showed the suppression of both type I and type III IFN signalling by SFTSV NSs protein is mediated through inhibition of STAT1 phosphorylation and activation. Infection with live SFTSV or expression of NSs potently suppressed IFN-stimulated genes but not NFκB activation. NSs was capable of counteracting the activity of IFN-α1, IFN-β, IFN-λ1 and IFN-λ2. Mechanistically, NSs associated with STAT1 and STAT2, mitigated IFN-β-induced phosphorylation of STAT1 at S727, and reduced the expression and activity of STAT1 protein in IFN-β-treated cells, resulting in the inhibition of STAT1 and STAT2 recruitment to IFN-stimulated promoters. Taken together, SFTSV NSs protein is an IFN antagonist that suppresses phosphorylation and activation of STAT1.postprin