The presence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and the permanent integration of HBV DNA into the host genome confers the risk of viral reactivation and hepatocellular carcinoma. Nucleoside/nucleotide analogs alone have little or no capacity to eliminate replicative HBV templates consisting of cccDNA or integrated HBV DNA. Recently, CRISPR/Cas9 technology has been widely applied as a promising genome-editing tool, and HBV-specific CRISPR-Cas9 systems were shown to effectively mediate HBV cccDNA disruption. However, the integrated HBV DNA fragments are considered as important pro-oncogenic properties and it serves as an important template for viral replication and expression in stable HBV cell line. In this study, we completely excised a full-length 3,175-bp integrated HBV DNA fragment and disrupted HBV cccDNA in a stable HBV cell line. In HBV-excised cell line, the HBV cccDNA inside cells, supernatant HBV DNA, HBsAg, and HBeAg remained below the negative critical values for more than 10 months. Besides, by whole genome sequencing, we analyzed off-target effects and excluded cell contamination. It is the first time that the HBV infection has been fully eradicated in a stable HBV cell line. These findings demonstrate that the CRISPR-Cas9 system is a potentially powerful tool capable of promoting a radical or “sterile” HBV cure.
Chronic hepatitis B infection remains incurable because HBV cccDNA can persist indefinitely in patients recovering from acute HBV infection. Given the incidence of HBV infection and the shortcomings of current therapeutic options, a novel antiviral strategy is urgently needed. To inactivate HBV replication and destroy the HBV genome, we employed genome editing tool CRISPR/Cas9. Specifically, we found a CRISPR/Cas9 system (gRNA-S4) that effectively targeted the HBsAg region and could suppress efficiently viral replication with minimal off-target effects and impact on cell viability. The mutation mediated by CRISPR/Cas9 in HBV DNA both in a stable HBV-producing cell line and in HBV transgenic mice had been confirmed and evaluated using deep sequencing. In addition, we demonstrated the reduction of HBV replication was caused by the mutation of S4 site through three S4 region-mutated monoclonal cells. Besides, the gRNA-S4 system could also reduce serum surface-antigen levels by 99.91 ± 0.05% and lowered serum HBV DNA level below the negative threshold in the HBV hydrodynamics mouse model. Together, these findings indicate that the S4 region may be an ideal target for the development of innovative therapies against HBV infection using CRISPR/Cas9.
BackgroundDelayed diagnosis of childhood Takayasu arteritis (TA) is common due to its atypical symptoms. The objective of the present study was to summarize the clinical features of childhood TA to raise awareness and improve management.MethodsEleven children diagnosed with TA at our hospital were enrolled. Clinical information, diagnosis, treatment, and outcome were then examined retrospectively. The Pediatric Vasculitis Activity Score (PVAS) and the Indian Takayasu Clinical Activity Score (ITAS2010) were used to assess disease activity.ResultsMale-to-female ratio was 4:7. The mean age was 9.4 (1.4–14) years and the average time to diagnosis was 40.6 days (12–90 days). All patients suffered from hypertension and few had immunologic abnormalities. Two patients had low levels of autoantibodies and one had elevated immunoglobulin E levels. Aberrant (elevated) laboratory parameters included erythrocyte sedimentation rate (ESR) (9/10 patients, 90.0%), protein excretion (8/9 patients, 88.9%), renin-angiotensin-aldosterone system (RAAS) activity (5/5 patients, 100.0%), and serum lipid levels (3/5 patients, 60%). The common onset patterns were headache with convulsions (27.2%) and kidney damage (27.2%). The abdominal aorta (81.8%) and renal artery (72.7%) were the most commonly involved vessels. At presentation, the mean PVAS and ITAS2010 scores were 12.1 (6–26)/63 and 9.7 (5–14)/57, respectively. All patients were treated with glucocorticoids and antihypertensive agents; two underwent renal artery stent placement.ConclusionThe diagnosis of TA should be considered in patients with pediatric hypertension and high expression of inflammatory markers or abnormal urine results. Doppler ultrasonography of major vessels may be helpful. PVAS and ITAS2010 both help to evaluate disease activity, and the PVAS is recommended for patients with kidney damage. Glucocorticoid and antihypertensive agents are effective. Interventional therapy can be an option for patients with persistent hypertension.Electronic supplementary materialThe online version of this article (doi:10.1186/s12969-017-0164-2) contains supplementary material, which is available to authorized users.
BackgroundBovine viral diarrhea virus (BVDV) is a pestivirus which infects both domestic animals and wildlife species worldwide. In China, cattle are often infected with BVDV of different genotypes, but there is very limited knowledge regarding BVDV infection in Chinese yaks and the genetic diversity of the virus. The objectives of this study were to detect viral infection in yaks in Qinghai, China and to determine the genotypes of BVDV based on analysis of the 5′untranslated region (5′UTR) and N-terminal protease (Npro) region.ResultsBetween 2010 and 2012, 407 blood samples were collected from yaks with or without clinical signs in six counties of Qinghai Province. Ninety-eight samples (24%) were found to be positive by reverse transcription polymerase chain reaction (RT-PCR) targeting a conserved region of BVDV-1 and BVDV-2. The nucleotide sequences of the 5′UTR and complete Npro region were determined for 16 positive samples. Phylogenetic reconstructions demonstrated that all 16 samples belong to subgenotypes BVDV-1b, BVDV-1d and BVDV-1q.ConclusionsThis study provides, for the first time, molecular evidence for BVDV infection in yaks in Qinghai involving multiple subgenotypes of BVDV-1. This may have occurred under three possible scenarios: interspecies transmission, natural infection, and the use of vaccines contaminated with BVDV. The results have important implications for yak production and management in China, and specifically indicate that unscientific vaccination practices should be stopped and bio-security increased.
Chlamydophila abortus is an important amphixenosis which in a wide range of animals, associated with reproductive disorders in yaks. In order to assess the prevalence of this infection in yaks in Qinghai, China, a cross-sectional study was carried out, and a total of 674 serum samples were collected from June to October 2012 in six counties, and antibodies to C. abortus were examined by indirect hemagglutination (IHA) test. The overall seroprevalence of C. abortus in yaks was 17.66 % (119/674), and the seroprevalence of antibodies to C. abortus in yaks ranged from 11.82 to 28.43 % among the six different areas, and the difference was statistically significant (P < 0.05). The seropositivity of C. abortus infection in different age groups varied from 16.33 to 18.49 %, and prevalence in yaks of ≥3 year (18.49 %) was slightly higher than that in yaks of <3 year, but the differences among the age groups were not statistically significant (P > 0.05). The seroprevalence of C. abortus infection in male yak (16.8 %) was slightly lower than that in females (17.85 %), and the difference was not statistically significant (P > 0.05). So far, this is the first systematic and comprehensive investigation of C. abortus infectionin in yaks in this area.
Red cell distribution width (RDW) has been proposed as an early prognosis marker with increased mortality in variety of pathophysiological conditions. We hypothesized that elevated RDW could be used in judging the severity of acute pancreatitis (AP). We retrospectively and prospectively studied 545 and 72 AP patients, who were admitted to the Shanghai Tenth People's Hospital of Tongji University, respectively. Compared with mild acute pancreatitis, significantly higher RDW was observed in patients with moderately severe acute pancreatitis and sever acute pancreatitis (14.03 ± 1.74% vs. 13.23 ± 1.23%, P < 0.000). RDW values were also found positively correlated with the patient's blood urea nitrogen (r = 0.120, P = 0.026), creatinine (r = 0.182, P = 0.000), age (r = 0.099, P = 0.028), and bedside index of severity in acute pancreatitis scoring system (r = 0.147, P = 0.001), and were negatively correlated with the serum albumin (r = -0.244, P = 0.000). The area under the receiver-operating characteristics was as follows-RDW: 0.677 (95% confidence interval [CI], 0.619-0.735, P < 0.000); combination of RDW and albumin: 0.693 (95% CI, 0.625-0.761, P < 0.000); and the optimal cutoff value for RDW to predict whether patients with AP should be in intensive care unit (ICU) was 13.55 with a sensitivity of 54.5% and a specificity of 73.6%. In the validation study, AP with RDW ≥ 13.55% had significantly higher ICU admission ratio than those with RDW < 13.55% (44.4% vs. 9.8%, P < 0.000). In conclusion, RDW is positively associated with AP severity, and is likely a useful predictive parameter of AP severity.
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