Progress in engineering application of human adenovirus / 生物工程学报

Human adenoviruses are widespread causative agent that induces respiratory diseases, epidemic keratoconjunctivitis and other related diseases. Adenoviruses are commonly used in experimental and clinical areas. It is one of the most commonly used virus vectors in gene therapy, and it has attracted a lot of attention and has a high research potential in tumor gene therapy and virus oncolytic. Here, we summarize the biological characteristics, epidemiology and current application of adenovirus, in order to provide reference for engineering application of adenovirus.

Characteristics of hepatitis C virus infection and genotype distribution among entry travelers at Tengchong Port / 中华传染病杂志

Objective To investigate the characteristics of epidemic and genotype/subtype distribution of hepatitis C virus (HCV) among entry travelers at Tengchong port,to provide references for HCV prophylaxis and treatment.Methods A total of 54 serum samples were collected from anti-HCV positive travelers at Tengchong port from June 2009 to June 2016.HCV NS5B gene was amplified using reverse transcription polyonerase chain reation (RT-PCR) and subsequently sequenced.Based on the obtained sequences and retrieved reference sequences,phylogenetic analysis was conducted to determine HCV genotype/subtype.Results HCV infection rate among entry travelers at Tengchong ports was 0.45 ％ (54/12 059).Forty five samples were successfully genotyped.Phylogenetically,HCV genotype 3b was revealed to be the predominant subtype (28.89 ％,13/45) in this population,followed by genotype 6n (20.0％,9/45),genotype 1b (17.78％,8/45),genotype 3a (13.33％,6/45),genotype 2a (11.11％,5/45),genotype 1a (2.22％,1/45) and genotype 6a (2.22％,1/45).The major genotype in Myanmar travelers was genotype 6,while in Chinese population,genotype 1 predominated.Genotype 6 in the population showed close phylogenetic relationship with strains prevalent in China and Southeast Asia.Genotype 3 was closely clustered with strains prevalent in China.Conclusions The distribution of HCV genotypes among entry travelers at Tengchong port is impacted by HCV epidemic strains both in Yunnan province and neighboring regions.This population serves as a transmitting media which may influence the epidemiological characteristics of HCV in Tengchong and neighboring areas.

Establishment of a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8] / 中国实验动物学报

Objective To explore the proliferation characteristics of primary small intestinal epithelial cells of tree shrews and the characteristics of human rotavirus(RV) G1P[8] infection to these cells,and establish a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8].Methods The primary small intestinal epithelial cells were obtained by collagenase Ⅺ and dispase I digestion from tree shrew.After purification and identification,the obtained primary small intestinal epithelial cells were infected with RV.Then,culture supernatants of infected cells were collected every 12 hours after infection.Viral titer and viral load were subsequently determined.Western blot and indirect immunofluorescence observation were used to detect the expression of RV protein VP6 in the primary cells.The infectivity of RV to the tree shrew primary cells was finally evaluated.Results After purification and identification of primary epithelial cells from the tree shrew,high purity above 90% primary tree shrew small intestinal epithelial cells was obtained.These primary small intestinal epithelial cells could be infected with RV virus by comparing the virus infectivity to primary renal cells,HCT116 cells and MA104 cells.The virus titer reached to 2.0×105TCID 50/mL at 72 h after infection.Using Western blot and indirect immunofluorescence observation,the specific viral protein of VP6 was determined to be expressed in the tree shrew primary small intestinal epithelial cells,and were located in the cytoplasm from days 1 to 5.Conclusions The separation,purification and cultivation methods of tree shrew primary small intestinal epithelial cells are successful,and the tree shrew model of RV-infected the tree shrew primary small intestinal epithelial cells is successfully established.

Establishment of a EV71 virus infection model of tree shrew primary renal cells / 中国实验动物学报

Objective To establish an enterovirus 71(EV71) infection model of tree shrew primary renal cells.Methods Tree shrew primary renal cells were obtained by trypsin digestion.After subculture and purification,EV71 virus was used to infect these primary cells.The culture supernatant of these EV71-infected cells was collected for virus titer detection at 1,2,4,6 and 8 days post-infection.The cells were collected for detection of EV71 VP1 protein by Western blot assay.Furthermore,the expression and location of VP1 protein in the infected cells were detected by indirect immunofluorescence assay.Vero cells were taken as positive control to evaluate the infectivity of EV71 virus to tree shrew primary renal cells.Results Morphologically,the cultured cells were proved to be majorly consisted of the primary renal cells after subculture and purification.The obtained primary cells were infected by EV71 virus.The virus titer was up to 1.3×106 TCID 50/mL during 48-96 h post-infection,proving that EV71 virus infected and proliferated in the tree shrew primary renal cells.Western blot showed that the viral VP1 protein was detected from infected primary cells at 2 to 8 d post infection.VP1 protein was also observed in the cytoplasm at 2 to 6 d post infection by indirect immunofluorescence.Compared with Vero cells,the infectivity of EV71 virus to tree shrew primary renal cells and its proliferation were confirmed.Conclusions Based on the successful establishment of cell culture of tree shrew primary renal cells,the infectivity to the obtained cells and proliferation of EV71 virus in the cells are confirmed.The model of EV71 virus-infected tree shrew primary renal cells is initially established.

Prokaryotic expression, purification, and polyclonal antibody production of a truncated recombinant rabies virus L protein

Background: rabies virus [RABV] is a deadly neurotropic virus that causes the disease of rabies in humans and animals. L protein is one of the large structural protein of rabies virus, which displays multiple enzymatic activities, and is required for viral transcription and replication

Objectives: a truncated L protein of Rabies virus is being cloned, expressed and purified to produce relevant polyclonal Antibody

Materials and Methods: the gene fragment of L protein of RABV was subcloned into prokaryotic expression vector pET- 28a and transformed into E. coli Rosetta DE3 host strain. The recombinant L protein of RABV was expressed and characterized by SDS-PAGE and western blot analysis using anti-his tag antibody. Mice were immunized with the purified recombinant L protein, the reaction of the anti-serum was checked by immunofluorescence and dot-blot, respectively

Results: the results of PCR and sequencing confirmed that the fragment of L gene of RABV was successfully cloned into the expression vector. The expression of recombinant L protein fragment induced by IPTG was confirmed by the band of 43 kDa in SDS-PAGE and western blot. The antiserum of purified L protein immunized mice was reacted with RABV infected N2a cells and suckling mouse brain tissue lysates

Conclusions: our data showed that the recombinant L protein produced by pET-28a vector was very successful, and the purified L protein could efficiently induce the antibody response in mice. The antiserum could recognize the virus in RABV infected cells and tissue very well

Prokaryotic expression of Hepatitis C Virus (HCV) NS3 protein and preparation of polyclonal antibody / 生物工程学报

To increase detection sensitivity and specificity on hepatitis C virus (HCV) is vital for prevention and controlling of the disease. To establish a more reliable detection method for HCV diagnosis, the full gene fragment of ns3 (non-structural protein of HCV) from recombinant plasmid of J6/JFH1 2a was amplified and then connected into the pET-28a prokaryotic expression vector, and the latter was subsequently transformed into Escherichia coli BL21 (DE3) to have the target protein expression. As a result, a protein with a molecular weight of 72 kDa was obtained and visualized in 10% SDS-PAGE. The purified NS3 protein was used as immunogen to inoculate BALB/c mice and the sera was collected after the fourth immunization. The antibody titer of serum is determined to be about 1:256000 with ELISA. Western blotting and indirect immunofluorescence analysis showed that the mouse polyclonal antibody could react specifically with the native NS3 protein in Huh 7.5.1 cells infected with HCV. These findings may provide basis for further preparation of monoclonal antibodies against NS3 and the development of related detection kit.

Evolution of hepatitis C virus quasispecies during natural disease progression of chronic hepatitis C and the clinical implications / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the variations of hepatitis C virus (HCV) quasispecies and the changes in their composition in untreated patients with chronic hepatitis C.</p><p><b>METHODS</b>Eleven patients chronic hepatitis C without previous specific anti-HCV treatment were tracked for disease progression and blood samples were collected at multiple time points. The major clinical parameters of liver function and viral load were tested. A fragment of HCV hypervariable region 1 (HVR1) was amplified and cloned, and the positive clones were sequenced and subsequently analyzed to determine the composition variation of HCV quasispecies during disease progression in relation to the major clinical parameters.</p><p><b>RESULTS</b>A total of 631 HVR1 sequences were acquired from the positive clones. The evolution of HCV HVR1 quasispecies in untreated chronic hepatitis C patients featured 3 patterns of variation in quasispecies composition, namely stable, fast and slow changes during the natural course of chronic hepatitis C. The genetic distance of the quasispecies was found to inversely correlated with ALT (R=-0.438, P=0.011) and AST level (R=-0.500, P=0.003), and sense mutation rate was also inversely correlated with ALT level (R=-0.387, P=0.026) and AST level (R=-0.410, P=0.018). No significant association was found between HCV load and any clinical or virological parameters.</p><p><b>CONCLUSION</b>Due to individual differences and immune pressure, HCV quasispecies can present with different patterns of evolution in the natural disease progression of chronic hepatitis C. HCV quasispecies evolution, due to its close correlation with the biochemical parameters, can be used to evaluate the severity and prognosis of chronic hepatitis C.</p>

Research progress of tree shrew models of viral hepatitis and modeling strategy / 中国实验动物学报

Viral hepatitis is a major liver disease caused by virus infection .Viral hepatitis is popular in China , mainly caused by hepatitis B and hepatitis C viruses .Experimental animal model is a necessary platform for the research on mechanism of viral infection and pathogenicity , for treatment and vaccine development .Up to date, a great progress in the development of viral hepatitis animal models has been achieved in spite of the most of findings are limited to hepatitis B and C.Here, we summarize the recent findings of viral hepatitis animal models , focusing on the tree shrew animal model and its modeling strategy .

Progress in application of tree shrew models in research on human viral diseases / 中国实验动物学报

Tree shrews get more and more concerns due to many of its physiological , biochemical and anatomical characteristics similar to those of human beings .Therefore, tree shrews models of human diseases such as viral diseases , neurological diseases and tumors attract more and more attention of researchers .In this article we will review the recent ad-vances in application of tree shrew models in research on human viral diseases .

Screening of hepatocyte proteins interacting with hepatitis B virus X protein using CytoTrap yeast two-hybrid technique / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen the hepatocyte proteins that interact with hepatitis B virus X protein (HBx).</p><p><b>METHODS</b>The recombinant plasmid pSos-HBx was constructed by inserting Sos-HBx fragment into the bait vector, and after sequence verification the plasmid was transformed into competent yeast cells. The expression and self-activation of Sos-HBx protein was detected in the yeast cells. The hepatocyte proteins interacting with the bait protein was screened with CytoTrap yeast two-hybrid technique.</p><p><b>RESULTS</b>The reconstructed plasmid harboring HBx gene expressed Sos-HBx protein in the yeast cells without self-activation of the protein. CytoTrap yeast two-hybrid system identified 6 hepatocyte proteins that interacted with HBx, including fibronectin 1, translationally controlled tumor protein, IQ motif and WD repeats 1, follistatin, orosomucoid 1, and disulfide isomerase family A member 3.</p><p><b>CONCLUSION</b>Six HBx-binding hepatocyte proteins have been identified using the CytoTrap yeast two-hybrid system, which provides clues for further investigation of the role of HBx protein in hepatitis and liver cancer.</p>

Study on human immunodeficiency virus-1 super-infection among high-risk populations in Myanmar / 中华传染病杂志

Objective To investigate the occurrence and the characteristics of human immunodeficiency virus (HIV)-1 co/super-infection among high-risk populations in Myanmar.Methods Forty-six HIV-1 positive plasma in Myanmar were collected. Possible cases with HIV-1 co/super infection were identified by discordant sequence results obtained with different polymerase chain reaction (PCR)/sequencing primers or by ambiguous readings in direct sequencing. HIV-1 quasispecies in plasma were then characterized by clonal sequence analysis of independent PCR-clones generated by TA cloning method. Thereafter, their phylogeny and recombinant structure were investigated. Results Co/super infection was identified in 3 (6.5 %) cases among the 46 screened HIV-1 positive patients.All of these three patients were heterosexuals and were co/super infected with CRF01_AE/subtype B′recombinants. Conclusions HIV-1 co/super infections are relatively common and provide a prerequisite for rapid generation of new recombinant forms in Myanmar.

Progress in Research of the Role of Hepatitis C Virus Protein in Cell Signal Transduction Pathway / 中国实验动物学报

Abnormal cell signal transduction is associated with the occurrence and development of human diseases. Some virus pathogenicity and infection mechanism are due to virus antigen protein acting on the host cell signal transduction pathway, leading to host cell signal transduction disorder. Hepatitis C virus (HCV) is a major pathogen of chronic hepatitis C, which causes cirrhosis and hepatocellular carcinoma. But the pathogenesis of HCV and persistent infection mechanism remain far from clear. HCV pathogenesis may be related to the HCV protein expression interfering host cell signal transduction pathways. The studies of hepatitis C virus proteins acting on host cell signal transduction pathways, not only help to clarify the impact of its pathogenic mechanisms, but also benefit to new drug design and development for new treatment methods. This article summarizes the recent progress in research on the effect of hepatitis C virus protein in cell signal transduction pathways in the past few years.

HIV and HCV: from Co-infection to Epidemiology, Transmission,Pathogenesis, and Treatment / 中国病毒学·英文版

Human immunodeficiency virus (HIV) is the infectious agent causing acquired immunodeficiency syndrome (AIDS), a deadliest scourge of human society. Hepatitis C virus (HCV) is a major causative agent of chronic liver disease and infects an estimated 170 million people worldwide,resulting in a serious public health burden. Due to shared routes of transmission, co-infection with HIV and HCV has become common among individuals who had high risks of blood exposures. Among hemophiliacs the co-infection rate accounts for 85%; while among injection drug users (IDU) the rate can be as high as 90%. HIV can accelerate the progression of HCV-related liver disease, particularly when immunodeficiency has developed. Although the effect of HCV on HIV infection is controversial,most studies showed an increase in mortality due to liver disease. HCV may act as a direct cofactor to fasten the progression of AIDS and decrease the tolerance of highly active antiretroviral therapy(HARRT). Conversely, HAART-related hepatotoxicity may enhance the progression of liver fibrosis.Due to above complications, co-infection with HCV and HIV-1 has imposed a critical challenge in the management of these patients. In this review, we focus on the epidemiology and transmission of HIV and HCV, the impact of the two viruses on each other, and their treatment.