A Nomogram Based on Consolidation Tumor Ratio Combined with Solid or Micropapillary Patterns for Postoperative Recurrence in Pathological Stage IA Lung Adenocarcinoma.

BACKGROUND: Patients with pathological stage IA lung adenocarcinoma (LUAD) are at risk of relapse. The value of the TNM staging system is limited in predicting recurrence. Our study aimed to develop a precise recurrence prediction model for stage IA LUAD. MATERIALS AND METHODS: Patients with pathological stage IA LUAD who received surgical treatment at Zhongshan Hospital Fudan University were retrospectively analyzed. Multivariate Cox proportional hazards regression models were used to create nomograms for recurrence-free survival (RFS). The predictive performance of the model was assessed using calibration plots and the concordance index (C-index). RESULTS: The multivariate Cox regression analysis revealed that CTR (0.75 < CTR ≤ 1; HR = 9.882, 95% CI: 2.036-47.959, p = 0.004) and solid/micropapillary-predominance (SMPP; >5% and the most dominant) (HR = 4.743, 95% CI: 1.506-14.933, p = 0.008) were independent prognostic factors of RFS. These risk factors were used to construct a nomogram to predict postoperative recurrence in these patients. The C-index of the nomogram for predicting RFS was higher than that of the eighth T-stage system (0.873 for the nomogram and 0.643 for the eighth T stage). The nomogram also achieved good predictive performance for RFS with a well-fitted calibration curve. CONCLUSIONS: We developed and validated a nomogram based on CTR and SMP patterns for predicting postoperative recurrence in pathological stage IA LUAD. This model is simple to operate and has better predictive performance than the eighth T stage system, making it suitable for selecting further adjuvant treatment and follow-up.

High expression of the classical swine fever virus (CSFV) envelope protein E2 by a single amino acid mutation and its embedded in the pseudorabies virus (PRV) vector for immunization.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases in pigs. Most pig farms in China are vaccinated against these two diseases. Gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live attenuated viral vector vaccines. It has been reported that recombinant PRV can express a truncated E2 protein (1-338 aa), but it has not been reported that recombinant PRV can express a full-length E2 protein. We constructed nine groups of E2 proteins with different expression forms and found that the E2 protein could be expressed in vitro only when the transmembrane region of E2 was removed and the signal peptide was added. Analysis of the transmembrane region of E2 revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating an amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant of E2 (E2FL-muta3 or E2FL-muta4) could be expressed. The expressed full-length mutant E2 could also localize to the cell membrane. Mice immunized with a PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4 developed specific cellular immunity to the E2 protein and stimulated higher levels of E2 antibody than mice immunized with a PRV vector expressing truncated E2. After immunizing the rabbits, the lethal challenge by PRV-ZJ2013 and the febrile response elicited by CSFV were simultaneously prevented. These results suggest that rPRV-dTK/gE-E2FL-muta4 is a promising bivalent vaccine against CSFV and PRV infections.

8-Prenyl daidzein and 8-prenyl genistein from germinated soybean modulate inflammatory response in activated macrophages.

Soy isoflavones have been shown to have anti-inflammatory properties; however, the anti-inflammatory effects of isoflavone metabolites produced during soybean germination remain unclear. We found that the daidzein and genistein derivatives, 8-prenyl daidzein (8-PD) and 8-prenyl genistein (8-PG), demonstrated a more potent effect than daidzein and genistein on repressing inflammatory responses in macrophages. Although IkB protein levels were unaltered, 8-PD and 8-PG repressed nuclear factor kappa B (NF-κB) activation, which was associated with reduced ERK1/2, JNK, and p38 MAPK activation and suppressed mitogen- and stress-activated kinase 1 phosphorylation. Inflammatory responses induced by the medium containing hypertrophic adipocyte secretions were successfully suppressed by 8-PD and 8-PG treatment. In the ex vivo study, 8-PD and 8-PG significantly inhibited proinflammatory C-C motif chemokine ligand 2 (CCL2) secretion from the adipose tissues of mice fed a long-term high-fat diet. The data suggest that 8-PD and 8-PG could regulate macrophage activation under obesity conditions.

Expression of PD-L1 through evolution phase from pre-invasive to invasive lung adenocarcinoma.

BACKGROUND: This study evaluated programmed cell death-ligand 1 (PD-L1) expression from pre-invasive adenocarcinoma to invasive lung adenocarcinoma, aimed to investigate the potential association of PD-L1 pathway with lung adenocarcinoma early evolution. METHODS: We evaluated PD-L1 expression in 1123 resected lung specimens of adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma (IAC) of stage IA1-IA3. PD-L1 expression was defined based on the proportion of stained tumor cells using the tumor proportion score: < 1% (negative), ≥ 1% (positive) and ≥ 50% (strongly positive). Correlations between PD-L1 expression and T stage, pathological subtype, adenocarcinoma grade, spread through air space (STAS), vascular invasion, lymphatic invasion and driven genes were analyzed. RESULTS: There was almost no PD-L1 expression in AIS or MIA. However, PD-L1 expression was correlated with invasiveness of lung adenocarcinoma. The percentages of PD-L1 positive in IA1-IA3 were 7.22%, 11.29%, and 14.20%, respectively. The strongly positive rates of PD-L1 were 0.38%, 1.64%, and 3.70% in IA1-IA3, respectively. PD-L1 expression and positive rate were also associated with poor pathological subtype and poor biological behavior, such as adenocarcinoma Grade 3, micropapillary or solid dominant subtype, STAS and vascular invasion. Finally, PD-L1 positive rate seems also corrected with driven gene ALK, ROS-1 and KRAS. CONCLUSIONS: PD-L1 expression was positively correlated with the emergence of invasiveness and poor pathological subtype or biological behavior of early-stage lung adenocarcinoma. PD-L1 pathway may be involved in the early evolution of lung adenocarcinoma from AIS to IAC.

Recombinant pseudorabies virus (PRV) expressing stabilized E2 of classical swine fever virus (CSFV) protects against both PRV and CSFV.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases of pigs. Most pig farms in China are immunized against these two diseases. Here, we describe a stabilized E2 protein as an immunogen inserted into the PRV genome as a bivalent live virus-vectored vaccine. The E2 protein has 48 variant sites, there are 2-5 candidate amino acids per variant site, and the relative energy contribution of each amino acid to E2 energy was calculated. Combined substitutions of amino acids at the neighbor variant site (neighbor substitution) were performed to obtain the E2 protein sequence with the lowest energy (stabilized E2). Multiple amino acid substitutions at 48 variant sites were performed, and the results were consistent with neighbor substitutions. The stabilized E2 sequence was obtained, and its energy decreased by 22 Rosetta Energy Units (REUs) compared with the original sequence. After the recombinant PRV expressing stabilized E2 of CSFV was constructed, the secretion efficiency of stabilized E2 was increased by 2.97 times, and the thermal stability was increased by 10.5 times. Immunization of mice resulted in a 2-fold increase in antibody production, and a balanced antibody level against subtype 1.1 and subtype 2.1d E2 was achieved. In rabbits immunized, the lethal challenge of PRV-ZJ and the fever response induced by CSFV could be prevented simultaneously. These findings suggest that rPRV-muta/287aaE2 is a promising bivalent vaccine against CSFV and PRV infections.

Isolation and Pathogenicity of a Novel Goose Astrovirus from Overfed Adult Landaise Geese in China.

Goose astrovirus (GAstV) is an important pathogen causing visceral gout and high mortality in goslings, which has broken out and spread across China. In 2021, a disease characterized by urate deposition on the visceral surface and 30% mortality occurred in commercial adult Landaise geese in Zhejiang Province, China. A systematic study identified an infecting astrovirus, designated ZJCX, that was efficiently isolated from a diseased goose with a chicken hepatocellular carcinoma cell line (LMH). In contrast to other GAstVs originating from goslings, ZJCX caused cytopathogenic effects in LMH cells, and the crystalline arrangement of viral particles was observed through transmission electron microscopy. Indeed, phylogenetic analysis and nucleotide homology comparison revealed that ZJCX isolate belongs to the genotype II cluster of GAstVs and displays 97.8-98.4% identity with other GAstV II strains. However, several specific mutations occurred in the polyprotein and capsid protein regions. Moreover, a pathogenicity assessment of ZJCX with a gosling model was conducted, and typical visceral gout was reproduced and led to 18% mortality. The viral loads of ZJCX in the blood, kidney, and liver were detected with specific primers after inoculation, which demonstrated that the kidney and liver presented viral loads peaking at seven days post-inoculation (dpi). Biochemical parameter examination showed that AST, ALT, γ-GT, UA, and BUN levels were significantly increased by GAstV, whereas body weight was reduced. Overall, this study indicated that the GAstV isolate could infect adult geese, and the results regarding the viral loads and biochemical parameters induced by ZJCX provide insight into GAstV pathogenicity.

Novel epitopes identified from Tembusu virus NS3 protein induce cytotoxic T lymphocyte response.

Since 2010, Tembusu virus (TMUV) has spread widely in China, causing huge economic losses to the poultry industry. Due to the infectious and zoonotic nature of flaviviruses, their potential threat to public health is of great concern. Cellular immune responses usually play a critical role in combating viral infections. To study the molecular basis of cell immunity induced by TMUV, 14 cytotoxic T lymphocyte (CTL) epitope peptides of TMUV antigen E, NS1 and NS3 were predicted by bioinformatics tools. Their abilities to induce cellular immune responses were determined by IFN-γ ELISpot assay, and 4 peptides were found to exhibit highly significant responses upon stimulation. In addition, the cytotoxic activity induced by the epitope peptides was assessed by lactate dehydrogenase (LDH) release assay. Finally, among these peptides, we identified two murine TMUV NS3-derived H-2d-restricted CTL epitopes in BALB/c mice, which could be used to further study of epitope vaccines against TMUV infection.

Proteomic analysis of host cellular proteins co-immunoprecipitated with duck enteritis virus gC.

Duck enteritis virus (DEV), the causative agent of duck viral enteritis, causes a contagious, lethal viral disease in Anseriformes (waterfowls). In virus infection, host-virus interaction plays a crucial role in virus replication and pathogenesis. In our previous study, mRFP was fused with the C-terminus of DEV glycoprotein C (gC) to construct a fluorescent-tag DEV virus rgCRFP. In the current study, fluorescent fusion protein (gC-mRFP) was used as the proteomic probe. Co-immunoprecipitation and mass spectrometric analysis of proteins from rgCRFP-infected chicken embryo fibroblasts using commercial anti-RFP antibody led to the identification of a total of 21 gC interacting host proteins. Out of these 21 proteins, the interaction of seven host proteins (GNG2, AR1H1, PPP2CA, UBE2I, MCM5, NUBP1, HN1) with DEV gC protein was validated using membrane-bound split-ubiquitin yeast two-hybrid system (MbYTH) and bimolecular fluorescence complementation (BiFC) analyses. It indicated direct interaction between these proteins with DEV gC protein. This study has furthered the current understanding of DEV virus infection and pathogenesis. SIGNIFICANCE: gC is an crucial glycoprotein of duck enteritis virus that plays an important role in the viral life cycle. Uncovering the interaction between virus-host is very important to elucidate the pathogenic mechanism of the virus. In this study, host factors interacting with DEV gC have been discerned. And seven host proteins (GNG2, AR1H1, PPP2CA, UBE2I, MCM5, NUBP1, HN1) have been further validated to interact with DEV gC using MbYTH and BiFC analyses. These outcomes could shed light on how DEV manipulates the cellular machinery, which could further our understanding of DEV pathogenesis.

Performance and potential mechanism of transformation of polycyclic aromatic hydrocarbons (PAHs) on various iron oxides.

The abiotic transformation of polycyclic aromatic hydrocarbons (PAHs) is significantly impacted by soil components, especially inorganic redox species like iron oxides. In this study, the catalytic activities of three types of iron oxides in PAHs degradation without light irradiation were evaluated using a combination of experimental techniques. The results showed that α-Fe2O3 possessed the highest transformation rate for anthracene (ANT), with a reaction rate constant (Kobs) up to 0.28 d-1, followed by Fe3O4 (Kobs = 0.06 d-1) and α-FeOOH (Kobs = 0.06 d-1). X-ray photoelectron spectroscopy (XPS) characterization suggested that α-Fe2O3 had the highest oxygen vacancy concentration, which was conducive to the adsorption of O2 by α-Fe2O3, providing sufficient adsorbed oxygen species. Oxygen vacancy contributed to the exposure of Fe(III), and accordingly, more active sites were created that were responsible for ANT degradation. According to these results, two possible pathways for the degradation of PAHs on iron oxides can be concluded: (1) direct oxidation by Fe(III) and (2) oxidation by the O2•- generated onto oxygen vacancies. This study provides significant insights into the environmental fate of PAHs on iron oxides, and raises the possibility that iron oxides may be used as catalytic materials in the remediation PAHs-contaminated soil.

Comorbidity and its impact on 1590 patients with COVID-19 in China: a nationwide analysis.

BACKGROUND: The coronavirus disease 2019 (COVID-19) outbreak is evolving rapidly worldwide. OBJECTIVE: To evaluate the risk of serious adverse outcomes in patients with COVID-19 by stratifying the comorbidity status. METHODS: We analysed data from 1590 laboratory confirmed hospitalised patients from 575 hospitals in 31 provinces/autonomous regions/provincial municipalities across mainland China between 11 December 2019 and 31 January 2020. We analysed the composite end-points, which consisted of admission to an intensive care unit, invasive ventilation or death. The risk of reaching the composite end-points was compared according to the presence and number of comorbidities. RESULTS: The mean age was 48.9 years and 686 (42.7%) patients were female. Severe cases accounted for 16.0% of the study population. 131 (8.2%) patients reached the composite end-points. 399 (25.1%) reported having at least one comorbidity. The most prevalent comorbidity was hypertension (16.9%), followed by diabetes (8.2%). 130 (8.2%) patients reported having two or more comorbidities. After adjusting for age and smoking status, COPD (HR (95% CI) 2.681 (1.424-5.048)), diabetes (1.59 (1.03-2.45)), hypertension (1.58 (1.07-2.32)) and malignancy (3.50 (1.60-7.64)) were risk factors of reaching the composite end-points. The hazard ratio (95% CI) was 1.79 (1.16-2.77) among patients with at least one comorbidity and 2.59 (1.61-4.17) among patients with two or more comorbidities. CONCLUSION: Among laboratory confirmed cases of COVID-19, patients with any comorbidity yielded poorer clinical outcomes than those without. A greater number of comorbidities also correlated with poorer clinical outcomes.

Optimized Expression of Duck Tembusu Virus E Gene Delivered by a Vectored Duck Enteritis Virus In Vitro.

In our previous study, a recombinant duck enteritis virus (DEV) delivering codon-optimized E gene (named as E-ch) of duck Tembusu virus (DTMUV) optimized referring to chicken's codon bias has been obtained based on the infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus vaccine strain pDEV-EF1, but the expression level of E-ch in recombinant virus rDEV-E-ch-infected cells was very low. To optimize DTMUV E gene expression delivered by the vectored DEV, different forms of E gene (collectively called EG) including origin E gene (E-ori), truncated E451-ori gene, codon-optimized E-dk gene optimized referring to duck's codon bias, as well as the truncated E451-ch and E451-dk, Etpa-ori and Etpa-451-ori, which contain prefixing chick TPA signal peptide genes, were cloned into transfer vector pEP-BGH-end, and several recombinant plasmids pEP-BGH-EG were constructed. Then the expression cassettes pCMV-EG-polyABGH amplified from pEP-BGH-EG by PCR were inserted into US7/US8 gene intergenic region of pDEV-EF1 by two-step Red/ET recombination, 7 strain recombinant mutated BAC clones pDEV-EG carrying different E genes were constructed. Next, the recombinant viruses rDEV-EG were reconstituted from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed that E or E451 protein is expressed in rDEV-E-ori, rDEV-E-ch, rDEV-Etpa-ori, rDEV-E451-ori, rDEV-E451-dk, and rDEV-E451-ch-infected CEFs, and protein expression level in rDEV-E451-dk-infected CEFs is the highest. These studies have laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.

Video-assisted thoracoscopic surgery for the treatment of mediastinal lymph node tuberculous abscesses.

The mediastinal lymph node tuberculous abscesses (MLNTAs) are secondary to mediastinal tuberculous lymphadenitis. Surgical excision is often required when cold abscesses form. This study was aimed to examine video-assisted thoracoscopic surgery (VATS) for the treatment of MLNTA. Clinical data of 16 MLNTA patients who were treated in our hospital between December 1, 2013 and December 1, 2015 were retrospectively analyzed. All of the patients underwent the radical debridement and drainage of abscesses, and intrathoracic lesions were removed by VATS. They were also administered the intensified anti-tuberculosis treatment (ATT), and engaged in normal physical activity and follow-up for 3 to 6 months. The results showed that VATS was successfully attempted in all of the 16 MLNTA patients and they all had good recovery. Two patients developed complications after surgery, with one patient developing recurrent laryngeal nerve injury, and the other reporting poor wound healing. It was concluded that VATS is easy to perform, and safe, and has high rates of success and relatively few side-effects when used to treat MLNTA.

Nine susceptibility loci for hepatitis B virus-related hepatocellular carcinoma identified by a pilot two-stage genome-wide association study.

Previous studies have indicated that complex interactions among viral, environmental and genetic factors lead to hepatocellular carcinoma (HCC). To identify susceptibility alleles for hepatitis B virus (HBV)-related HCC, the present study conducted a pilot two-phase genome-wide association study (GWAS) in 660 Han Chinese individuals. In phase 1, a total of 500,447 single-nucleotide polymorphisms (SNPs) were genotyped in 50 HCC cases and 50 controls using Affymetrix GeneChip 500k Array Set. In phase 2, 1,152 SNPs were selected from phase 1 and genotyped in 282 cases and 278 controls using the Illumina GoldenGate platform. The prior probability of HCC in control subjects was assigned at 0.01, and false-positive report probability (FPRP) was utilized to evaluate the statistical significance. In phase 1, one SNP (rs2212522) showed a significant association with HCC (Pallele=5.23×10-8; ORallele=4.96; 95% CI, 2.72-9.03). In phase 2, among 27 SNPs with unadjusted Pallele<0.05, 9 SNPs were associated with HCC based on FPRP criteria (FPRP <0.20). The strongest statistical evidence for an association signal was with rs2120243 (combined ORallele=1.76; 95% CI, 1.39-2.22; P=2.00×10-6), which maps within the fourth intron of VEPH1. The second strongest statistical evidence for an association was identified for rs1350171 (combined ORallele=1.66; 95% CI, 1.33-2.07; P=6.48×10-6), which maps to the region downstream of the FZD4 gene. The other potential susceptibility genes included PCDH9, PRMT6, LHX1, KIF2B and L3MBTL4. In conclusion, this pilot two-phase GWAS provides the evidence for the existence of common susceptibility loci for HCC. These genes involved various signaling pathways, including those associated with transforming growth factor ß, insulin/phosphoinositide 3 kinase, Wnt and epidermal growth factor receptor. These associations must be replicated and validated in larger studies.

Efficacy of video-assisted thoracoscopic surgery for 29 patients with tuberculosis-destroyed lung.

BACKGROUND: Incompleteness of interlobar fissures and pleural adhesions, common in tuberculous destroyed lung (TDL), are among "technical contraindications" for video-assisted thoracoscopic surgery (VATS). The efficacy and safety of VATS in the treatment of TDL, has not yet been detailed in. OBJECTIVE: The objective of the present study is to observe the efficacy and safety of VATS in the management of TDL. METHODS: A retrospective review of anatomic lobectomy by VATS on 29 cases of TDL was performed in the Department of Thoracic Surgery of Wuhan Medical Treatment Center between October 2010 and October 2013. RESULTS: All the 29 surgeries by VATS were successfully completed. No death case was reported. Operative duration of VATS was 75~400 min, with an average of 185.4 min; intraoperative amount of bleeding ranged 50 to 2300 ml, with an average of 575.6 ml; the incidence of postoperative complication was 21.4% (6/28). Postoperative complications occurred in 6 cases, among which there were 2 cases of persistent postoperative pulmonary air leak, 2 cases of pleural effusion, one case of thoracic hemorrhage and one case of arrhythmia complicated with left heart failure. There was one patient who was converted from VATS to open thoracic surgery half-way. CONCLUSION: Our results have shown the efficacy, safety and a breakthrough in the "technical contraindications" of VATS in the management of TDL.

Risk factors for major adverse events of video-assisted thoracic surgery lobectomy for lung cancer.

AIMS: The purpose of this study was to identify the risk factors for major adverse events of VATS (Video-Assisted Thoracic Surgery) lobectomy for primary lung cancer. METHODS: 1806 Patients (1032 males, 57.1%) planned to undergo VATS lobectomy for stage IA-IIIA lung cancer from July 2007 to June 2012. The Thoracic Morbidity and Mortality Classification TM&M system was used to evaluate the presence and severity of complications. Postoperative complications were observed during a 30-day follow up. Univariate and multivariate analysis were used to analyze the independent risk factors for major adverse events. RESULTS: Successful rate of VATS lobectomy was 97.6% (1763/1806). Major complications occurred in 129 patients (7.3%), with a mortality of 0.3% (5/1763). Pulmonary complications contribute up to 90.7% of the major complications and 80% of mortality. Logistic regression indicated that comorbidities, elder age ≥70y, operative time ≥240min and hybrid VATS were predictors for major adverse events (P<0.05). Hybrid and converted VATS lobectomy result in higher major adverse events compared with complete VATS, 15.1%, 20.9% and 7.4% respectively (P=0.013). CONCLUSIONS: The overall complication rate and mortality of VATS lobectomy are low, while major complications sometimes occur. Pulmonary complications are the most common major complications and cause of mortality. Age ≥70y, comorbidities, operative time ≥240min and Hybrid VATS are predictors of major adverse events.

Association of hepatitis B virus pre-S deletions with the development of hepatocellular carcinoma in Qidong, China.

BACKGROUND/AIM: To investigate the roles of mutations in pre-S and S regions of hepatitis B virus (HBV) on the progression of hepatocellular carcinoma (HCC) in Qidong, China. METHODS: We conducted an age matched case-control study within a cohort of 2387 male HBV carriers who were recruited from August, 1996. The HBV DNA sequence in pre-S/S regions was successfully determined in 96 HCC cases and 97 control subjects. In addition, a consecutive series of samples from 11 HCC cases were employed to evaluate the pre-S deletion patterns before and after the occurrence of HCC. RESULTS: After adjustment for age, history of cigarette smoking and alcohol consumption, HBeAg positivity, pre-S deletions, pre-S2 start codon mutations, and T53C mutation were significantly associated with HCC, showing adjusted odds ratios (ORs) from 1.914 to 3.199. HCC patients also had a lower frequency of T31C mutation in pre-S2 gene, compared with control subjects (0.524; 95% CI 0.280-0.982). HBV pre-S deletions were clustered mainly in the 5' end of pre-S2 region. Multivariate analysis showed that pre-S deletions and pre-S2 start codon mutations were independent risk factors for HCC. The OR (95% CI) were 2.434 (1.063-5.573) and 3.065 (1.099-8.547), respectively. The longitudinal observation indicated that the pre-S deletion mutations were not acquired at the beginning of HBV infection, but that the mutations occurred during the long course of liver disease. CONCLUSION: Pre-S deletions and pre-S2 start codon mutations were independently associated with the development of HCC. The results also provided direct evidence that pre-S deletion mutations were not acquired from the beginning of infection but arose de novo during the progression of liver disease.

Effect of combined mutations in the enhancer II and basal core promoter of hepatitis B virus on development of hepatocellular carcinoma in Qidong, China.

AIM: To investigate the roles of mutations in enhancer II (Enh II), basal core promoter (BCP) and precore (PC) regions of hepatitis B virus (HBV) in the progression of hepatocellular carcinoma (HCC) in Qidong, China. METHODS: We conducted a case-control study within a cohort of 2387 male HBV carriers who were recruited between August and September 1996. The HBV DNA sequence was determined in 152 HCC and 131 chronic hepatitis patients. Mutation exchanges during follow up in 115 cases were compared with 108 controls with serum samples taken during a similar length of follow up. In addition, a longitudinal study was conducted in 22 cases in which serial serum samples were available before HCC. RESULTS: After adjustment for age, history of cigarette smoking and alcohol consumption, hepatitis B e-antigen positivity, T1653, V1753 and T1762/A1764 double mutations were associated with risk of HCC. Multivariate analysis showed that T1653, V1753 and T1762/A1764 double mutations were independent risk factors of HCC. Moreover, a significant biological gradient of HCC risk by number of mutations in Enh II/BCP regions was observed. Paired samples analysis indicated that the increased HCC risk for at-risk sequence mutations were attributable to the persistence of these mutations, but not a single time point mutation. The longitudinal observation demonstrated a gradual combination of mutations in Enh II/BCP regions accumulated during the development of HCC. CONCLUSION: T1653, V1753 and T1762/A1764 double mutations were independent risk factors of HCC. The effect of combined mutations in Enh II/BCP regions increased the risk and persistence of at-risk sequence mutations and was critical for HCC development.

Complete genome sequence of a novel flavivirus, duck tembusu virus, isolated from ducks and geese in china.

Duck tembusu virus (DTMUV) is an emerging agent that causes a severe disease in ducks. We report herein the first complete genome sequences of duck tembusu virus strains YY5, ZJ-407, and GH-2, isolated from Shaoxing ducks, breeder ducks, and geese, respectively, in China. The genomes of YY5, ZJ-407, and GH-2 are all 10,990 nucleotides (nt) in length and encode a putative polyprotein of 3,426 amino acids. It is flanked by a 5' and a 3' noncoding region (NCR) of 94 and 618 nt, respectively. Knowledge of the whole sequence of DTMUV will be useful for further studies of the mechanisms of virus replication and pathogenesis.

Combined pre-S deletion and core promoter mutations related to hepatocellular carcinoma: A nested case-control study in China.

AIM: To investigate the roles of biomedical factors, hepatitis B virus (HBV) DNA levels, genotypes, and specific viral mutation patterns on the progression of hepatocellular carcinoma (HCC) in Qidong, China. METHODS: A total of 2387 males (aged 20-65 years) who were seropositive for the hepatitis B surface antigen (HBsAg), but had not been diagnosed with HCC, were recruited to a community-based HCC screening study from August, 1996. Evaluation of virological parameters at recruitment was determined for 196 HCC patients during 10 years of follow-up and 323 controls. RESULTS: After adjustment for age at recruitment, history of cigarette smoking and alcohol consumption, alanine aminotransferase (ALT) elevation, alpha-fetoprotein (AFP) levels >20 ng/mL, hepatitis B e antigen positive, HBV DNA levels ≥4.00 log(10) copies/mL, pre-S deletion, T1653 mutation, T1762/A1764 double mutations, and T1766 and/or A1768 mutations were associated with subsequent risk of HCC. A significant biological gradient of HCC risk by HBV DNA levels from less than 2.69 log(10) copies/mL to 6.00 log(10) copies/mL or greater was observed. HBV with a complex mutation combination pattern (pre-S deletion, T1762/A1764 double mutations, and T1766 and/or A1768 mutations) rather than a single mutation was associated with the development of HCC. The longitudinal observation demonstrated a gradual combination of pre-S deletion, T1762/A1764 double mutations, and T1766 and/or A1768 mutations during the development of HCC. CONCLUSIONS: AFP levels >20 ng/mL, high HBV DNA levels, pre-S deletion, and T1762/A1764 double mutations at recruitment were independent risk factors of HCC. Combination of pre-S deletion and core promoter mutations increased the risk of HCC.

Minor structural protein VP2 in rabbit hemorrhagic disease virus downregulates the expression of the viral capsid protein VP60.

Rabbit hemorrhagic disease virus (RHDV) has two structural proteins: the major capsid protein VP60 and the minor capsid protein VP2. VP2 is speculated to play an important role in the virus life cycle. To investigate the effect of VP2 on VP60 expression, three types of experiment (baculovirus-insect cell system, mammalian-luciferase assay system and in vitro coupled transcription/translation system) were used to express VP60 alone or co-expressed with VP2. Both forms of VP60 were able to form virus-like particles in insect cells. Western blot analysis and dual-luciferase assays demonstrated that the presence of VP2 results in downregulation of the expression of VP60 in vivo. Real-time RT-PCR of mRNA levels showed that downregulation of VP60 occurs at the transcriptional level. The ability of the viral minor structural protein VP2 to regulate capsid protein levels may contribute to effective virus infection.