Morphogenetic study of human adenovirus type 41 in 293TE cells / 病毒学报

To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41.

Characterization of Marburg virus morphology / 病毒学报

Ebola virus (EBOV) and Marburg virus (MARV) belong to the family Filoviridae. Filoviruses cause severe filovirus hemorrhagic fever (FHF) in humans, with high case fatality rates, and represent potential agents for bioterrorism and biological weapons. It is necessary to keep surveillance of filoviruses, even though there is no report of their isolation and patients in China so far. To characterize MARV morphology, the Lake Victoria marburgvirus--Leiden was stained negatively and observed under a transmission electron microscope which is one of important detection methods for filoviruses in emergencies and bioterrorism. MARV showed pleomorphism, with filamentous, rod-shaped, cobra-like, spherical, and branch-shaped particles of uniform diameter but different lengths. Pleomorphism of negatively stained MARV is summarized in this article, so as to provide useful information for possible electron microscopic identification of filoviruses in China.

Characterization of fibrillous inclusion body (FIB) of human adenovirus type 41 (Ad41) using immunoelectron microscopy / 病毒学报

To investigate the components of fibrillous inclusion body (FIB), which was formed in packaging cells during the replication of human adenovirus type 41 (Ad41), Ad41 long fiber knob (LFK) and short fiber knob (SFK) proteins were expressed in prokaryote respectively and then used to immunize BALI mice for preparation of anti-LFK serum and anti-SFK sera. The activity and specificity of anti-LFK and an ti-SFK sera were confirmed with Western blot, indirect immunofluorescence assay (IFA) and immunonegative staining, suggesting these sera could be applied in immuno-colloidal gold labelling electron microscopy (EM). 293TE cells were infected with wild-type Ad41. Ultrathin sections of infected cells were made, and labelled with immuno-colloidal gold technique using anti-Ad41 sera, anti-LFK sera, anti-SFK sera, or anti-fiber monoclonal antibody 4D2, respectively. The labelled sections were observed under EM, and the results demonstrated that both Ad41 long fiber protein and short fiber protein were included FIB.

Establishment of localization ultrathin section for cytopathic cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a localization ultrathin section method through which target cytopathic cells could be sectioned in situ.</p><p><b>METHODS</b>Lab-Tek Chamber slide system (177402) was selected as resin embedding mould. Cells infected with Human adenovirus type 5 (Ad5) or A/HN/SWL3/ 2009 (H1N1) influenza virus were embedded in situ as models. Target cytopathic cells were exposed by trimming, sectioned and observed under transmission electron microscope (TEM).</p><p><b>RESULTS</b>Target cells could be sectioned in situ and virus particles could be found easily on sections.</p><p><b>CONCLUSION</b>A localization ultrathin sectioning method was established and this technique could be applied in virus detection in cytopathic cells to improve TEM detection efficiency.</p>

The genetic stability of recombinant adenovirus expressing human rotavirus VP6 gene which used Ad41 as vector / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the genetic stability of non-replicating recombinant adenovirus which used Ad41 as vector and could express VP6 gene of group A rotavirus during continous passage, in order to develop the vaccine of rotavirus.</p><p><b>METHODS</b>The recombinant adenovirus rvAd41-VP6 (o) was prepared by our laboratory early, it then was continuously propagated on 293TE7 cells for 14 passages. After that samples of the infected cells were collected at every 2 passages for the detection of the integration of the VP6 gene by PCR, and the expression of the target protein was detected by Western Blot analysis.</p><p><b>RESULTS</b>Analysis by PCR revealed that, there was stable integration of specific VP6 gene in the rvAd41-VP6 (o), Western Blot analysis confirmed that rvAd41-VP6 (o) could stably expressed the group-specific antigen structural protein VP6 (o), and it had preferable genetic stability.</p><p><b>CONCLUSION</b>The recombinant adenovirus rvAd41-VP6 (o) which could stably express the VP6 (o) gene had favorable biological property in vitro, and it has provided a basis for further research of animal immunization.</p>

Optimization of the codon strengthened the human rotavirus VP6 antigen's serum immune responses and protective responses in mice model / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To observe the serum immune responses and protection in mice model of the recombinant adenovirus vector mediated human rotavirus VP6 gene expression through coden optimization (rvAdVP6(o)) in comparison with the wild type (rvAdVP6).</p><p><b>METHODS</b>6-8 week female BALB/c mice were randomly grouped and immunized three times intranasally with 10(8) TCID50 rvAdVP6(o) and rvAdVP6, respectively, then detect the serum IgG level against rotavirus induced by rvAdVP6(o) and rvAdVP6. The amount of sheding viral antigens in feces was detectd after mice rotavirus was taked orally.</p><p><b>RESULTS</b>The serum IgG level against rotavirus induced by rvAdVP6(o) was higher than that of rvAdVP6 after three times of immunization. The immunized mice shed lower amount of viral antigens in feces as compared with the rvAdVP6.</p><p><b>CONCLUSION</b>The recombinant adenovirus which encode optimized human rotavirus VP6 proteins (rvAdVP6(o)) could induce stronger serum immune and protective responses against the challenge of the rotavirus than the wild type (rvAdVP6) at the same immunizing dosage.</p>

Expression and purification of a secreted form of fusion glycoprotein of human respiratory syncytial virus encoded by recombinant baculovirus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the expression and purification of a secreted form of fusion glycoprotein (sF) of human respiratory syncytial virus (RSV) encoded by recombinant baculovirus.</p><p><b>METHODS</b>According the ORF of F protein, a pair of specific primers was designed and PCR technique was exploited to amplify the gene of sF in which the gene sequence of the transmembrane and cytoplasmic tail domains were replaced by a C-terminal six-histidine tag. Then, a recombinant baculovirus encoding sF-His was constructed, and transfected into sf9 insect cells by Lipofectamine cellfectine reagent. Finally, the expressed sF was purified by Ni2+ -affinity chromatograph.</p><p><b>RESULTS</b>The gene encoding sF-His was obtained. The resulting construct of recombinant baculovirus is capable of expressing sF protein. The concentration of Ni2+ -affinity chromatograph purified sF is 1.084 mg/ml with the purity of no less than 90%.</p><p><b>CONCLUSION</b>Baculovirus expression system is a good method for large scale of preparation of sF. The purified F paves the way for the development of potential RSV vaccine and diagnostic kit, etc.</p>

Construction and identification of replication deficient recombinant adenovirus encoding F gene of subgroup A human respiratory syncytial virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>A strain of replication deficient recombinant adenvirus encoding fusion glycoprotein (F) of subgroup A human respiratory syncytial virus (RSV) was constructed and the expression of F was identified.</p><p><b>METHODS</b>The F gene was obtained from pGEM3zf-F with Xho I and Hind III, cloned into adenoviruse shuttle vector pShuttle-CMV,and then the resulting pShuttle-CMV/F was transformed into E. coli BJ5183/p with pAdeasy-1 to produce pre-adenoviral plasmid encoding F by homologous recombination. This resultant plasmid was linearized by digestion with Pac I and transfected into 293 packaging cells to generate FGAd-F. Finally, the expression of F protein was identified by Western Blot analysis.</p><p><b>RESULTS</b>FGAd/F was successfully constructed, and the expression of RSV F protein was identified by Western Blot.</p><p><b>CONCLUSION</b>We have obtained a strain of replication-defective adenovirus FGAd/F encoding RSV F protein, which can be used further to investigate its protective efficacy against RSV infection in vivo.</p>

Improved expression of human rotavirus G1VP7 and G3VP7 antigens in the recombinant adenoviruses by codon optimization / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To increase the recombinant adenovirus vector mediated human rotavirus G1VP7 and G3VP7 gene expression through coden optimization.</p><p><b>METHODS</b>We have artificially synthesized two genes of group A human rotavirus that encode G1VP7 and G3VP7 according to the human biased codon. The modified genes were transfected into 293 cells using adenovirus vectors and the gene products, the respective proteins were produced. The expression level of optimized genes and wild type genes was detected by Western Blot.</p><p><b>RESULTS</b>A remarkable increase of the expression level of optimized G1VP7 and G3VP7 genes in comparison with the wild type control.</p><p><b>CONCLUSION</b>The coden optimization indeed help increasing the recombinant adenovirus mediated human rotavirus gene expression, which indicated the potential applicationof such recombinant adenoviruses in the development of adenoviral-vectored rotavirus vaccines.</p>

The genetic stability of non-replicating recombinant adenoviruses on 293 cells during continuous passage / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the genetic stability during the serial passages of non-replicating recombinant adenoviruses based on the novel AdEasy system.</p><p><b>METHODS</b>Four non-replicating adenoviruses expressing rotavirus antigens, which were generated by the AdEasy system, were used as models and continuously propagated on 293 cells for 20 passages. Samples of the infected cells were collected at every 5 passages for the PCR analysis of the inserted rotavirus genes, replication-competent adenoviruses (RCAs) as well as the Western blot examination of the expression of the certain rotavirus genes.</p><p><b>RESULTS</b>The adenoviruses can be stably propagated on 293 cells. The existence and the stable expression of inserted rotavirus genes were able to be detected during the serial passage. The RCAs were not found within the first 10 passages, but the rvAdG2VP7(o) at passage 15 and the rvAdG2VP7(o) and rvAdG1VP7(o) at passage 20.</p><p><b>CONCLUSION</b>The non-replicating adenoviruses showed promising genetic stability during the continuous passage on 293 cells. The RCAs normally will not be detectable within 10 continuous passages. The results indicated the potential of such recombinant adenoviruses in the research and development of the gene therapies and adenoviral-vectored vaccines.</p>

Effects of Newcastle disease virus on the mitochondria of human gastric carcinoma BGC-823 cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore changes in structure and function of the mitochondria of human gastric carcinoma BGC-823 cells after Newcastle disease virus (NDV) infection.</p><p><b>METHODS</b>Electron microscopy was applied to observe the structure of mitochondria; Rhodamine 123 staining was used to determine the mitochondrial membrane potential; the activity of Na(+)-K(+)-ATPase and Ca(2+)-ATPase were also determined and the release of cytochrome C was detected by Western blotting.</p><p><b>RESULTS</b>The structure of mitochondria in the tumor cells infected with NDV changed distinctly. In the infected group the activity of mitochondrial Na(+)-K(+)-ATPase and Ca(2+)-ATPase significantly declined (P < 0.01), and compared with control cells, mitochondrial trans-membrane potential was decreased. NDV infection induced the decrease of cytochrome C levels.</p><p><b>CONCLUSION</b>The effects of NDV infection on the structure and functions of mitochondria of human gastric carcinoma BGC-823 cells might play a role in the oncolysis of NDV.</p>

Improved replication of enteric adenovirus type 41 in Hep2 cell line expressing E1B55K / 病毒学报

Adenovirus type 40 and 41 (Ad40, Ad41), which belong to human adenovirus subgroup F, are called fastidious adenoviruses due to their property of poor growth in cultured cell lines in vitro The effect of expression of exogenous E1B55K in Hep2 on Ad41 replication in this cell line was investigated. E1B55K gene was amplified by PCR with DNA extracted from Ad41-positive feces supernatant as template. Eukaryotic expression plasmid (pcDNA3) carrying E1B55K was constructed, purified, and transferred into Hep2 cell. Expression of E1B55K in G418-resistant clones was assayed by RT-PCR, and one clone named as Hep2-E1B4#4 could produce more Ad41 progenies when compared with other clones by the method of inducing complete cytopathic effect (CPE) in 293 cells. Infection of equivalent Ad41 caused more significant cytopathic effect (CPE) in Hep2-E1B#4 than that in the control cells of Hep2 or Hep2-DNA3, also suggesting enhanced viral replication in Hep2-E1B#4. The titer of Ad41 was further determined by method of immunocytochemical staining, and semi-quantity PCR was employed to compare the copy number of Ad41 genome DNA. The results showed that the yield of Ad41 in Hep2-E1B#4 was more than 9 times of that in control cells when equal amount of seed viruses were incubated, and the copy number of Ad41 genome increased 4 times in the raw extract from the infected Hep2-E1B#4 when compared with that from control cells. In conclusion, E1B55K gene transfer improved the ability of Hep2 in packaging Ad41, and the Hep2-E1B#4 cell line, which expressed E1B55K constitutively, would be helpful in isolation, cultivation and amplification of Ad41.

Dynamics of in vitro amyloid fiber formation of yeast prion protein Sup35NM / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the dynamics of amyloid fiber formation of yeast (Saccharomyces cerevisiae) prion protein Sup35NM under the native condition to provide materials and clues for the elucidation of amyloid fiber formation.</p><p><b>METHODS</b>The Sup35NM gene was cloned and expressed in E. coli. The recombinant Sup35NM protein was purified under denaturing conditions through Nickel-Sepharose chromatography. Aliquots were removed at designated time points for transmission electron microscopy (TEM), circular dichroism (CD) spectra, protease K resistance assay, as well as thioflavin T (ThT) binding assay.</p><p><b>RESULTS</b>The Sup35NM expressed and purified under denaturing conditions. The morphological alteration of the Sup35NM in PBS (pH7.4) during the protein aggregation and amyloid fiber formation was visualized by TEM. The CD assay showed that the course of amyloid fiber formation underwent a conformational shift from alpha-helix to beta-sheet. The fibers had higher capacity of resistance to protease K digestion compared to the monomers. ThT fluorescence assay displayed a rapid growth phase before reaching a final equilibrium phase during the fiber formation, and the higher concentration of Sup35NM could greatly accelerate the fiber formation in vitro.</p><p><b>CONCLUSION</b>Yeast prion protein Sup35NM forms amyloid readily under native conditions in vitro. The dynamics of Sup35NM amyloid formation may provide supporting evidences for the nucleating polymerization models of amyloid fiber formation.</p>

Prevalence of anti-human parvovirus B19 IgG antibody among blood donors in Jilin province / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the prevalence of the parvovirus B19 infection among the blood donors in Jilin province to provide the basic data to evaluate the epidemics of B19 virus in China.</p><p><b>METHODS</b>Indirect ELISA was used to detect IgG antibody against parvovirus B19 in the sera from blood donors.</p><p><b>RESULTS</b>In a total of 184 serum samples, IgG antibody was detected in 55.43% samples, antibody positive rate in female was significantly higher than that in male (P<0.05) and the positive rate peaked at 35-45 years age group.</p><p><b>CONCLUSION</b>These data illustrate that the prevalence of the B19 antibody in blood donors of Jilin province was high, and it is therefore necessary to detect the B19 DNA to ensure the blood safety.</p>

Improved Expression of Immunotoxin DT_(386)-GMCSF in E.coli / 中国生物工程杂志

The development of immunotoxin DT386-GMCSF, a fusion protein which bears the N-terminal 386 amino acids of diphtheria toxin and human granulocyte-macrophage colony-stimulating factor (GM-CSF) and targets the GM-CSF receptor (GM-CSFR), has provided a promising alternative therapy to the acute myeloid leukemia (AML). However, the poor expression of the protein in E.coli is still a bottleneck which limits the industrial production. To identify the critical down-regulating factors on the expression of DT386-GMCSF, a series of truncated mutants of DT386-GMCSF at the C-terminal of GM-CSF were generated and expressed in E.coli. The results showed that the encoding sequences for the L114 of the GM-CSF dramatically impact the expression of DT386-GMCSF. On this basis, a serial of mutants integrating amino acid substitutes were generated. The results revealed that the expression level of the mutant DF123GVT, which harbors the amino acids 1-123 of GM-CSF whose L114L115V116 was substituted with G114V115T116, was evidently higher than that of the DT386-GMCSF, whereas the specific cytotoxicity to blast recovered from mice injected with HL60, a cell line highly expresses the GM-CSFR, was similar. These results have provided an important basis for the future development of the immunotoxins targeting the GM-CSFR.

Development and Application of RNAi Interference Library in Functional Genomics / 中国生物工程杂志

RNA interference ( RNAi) is a process in which double-stranded RNA ( dsRNA) induces specific postranscriptional silencing of homologous transcripts. Systematic silencing of genes on a genome-wide scale using RNAi library targeting many thousands of genes provides a powerful research tool in functional genomics. RNAi libraries, which may be derived from plasmids cloning, virus packaging, PCR amplification, chemically synthesis or enzymatic digestion, have been successfully used to identify gene function, dissect signaling pathway and discover drug targets, etc. Promising progresses have been made in this field. The development, application and problems of RNAi library methodology and future prospects were reviewed.

Immunogenicity of Different Genetic Type Rotavirus NSP4 in Mice / 中国生物工程杂志

NSP4, as the diarrhea-related protein of rotavirus, is becoming an attractive candidate for vaccine development. To compare the immunogenicity of NSP4 from different genetic groups, we constructed eukaryotic expression plasmids comprising the NSP4 genes from four different genetic types using the pCI vector. The recombinant vectors were designated as pCI-97B6, pCI-97S36, pCI-97S34 and pCI-97SZ8, respectively. Following the conformation of the transient expression of the constructs in 293 cells, the plasmids were respectively subjected to the 5 round i. m. inoculation of BALB/c mice. The specific antibodies against NSP4 as well as the IgG1/IgG2a subclasses of immunoglobulin in mice sera were examined with indirect ELJSA after each immunization. The results showed that the immunization of plasmids expression NSP4s could elicit not only humoral but also cellular immunity, but the humoral immune response is dominant. There is a difference of immunogenecity among the NSP4 of different genetic type. Further studies were needed to focus on the relationship between the immunogenicity and protection effect.

Receptor-binding ability of fragments 260-600 and 397-796 of SARS-associated coronavirus spike protein / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the interaction between the host cell and the truncated S fragments to identify the receptor-binding domain of the spike (S) protein of SARS-associated coronavirus (SARS-CoV).</p><p><b>METHODS</b>Two different fragments S260-600 and S397-796 of the SARS-CoV S protein were expressed in Escherichia coli (E.coli) using a pET expression vector, respectively. The two recombinant proteins were separately verified by Western blot, purified by nickel-affinity chromatography, and incubated with Vero cells, a susceptible cell line of SARS-CoV infection, for cell binding assay. After the sequential probing with sera from convalescent SARS-patients and FITC-labeled anti-human IgG, the cells were analyzed by flow cytometry. The NIH 3T3 cell, a non-permissive cell line of SARS-CoV infection, was used as controls.</p><p><b>RESULTS</b>The recombinant proteins S260-600 and S397-796 were efficiently expressed in an insoluble form in E.coli. The appropriate expression of the proteins was confirmed by Western blotting using both SARS patients' sera and anti-6 x histidine antibody. The flow cytometry results showed that the both proteins were able to bind Vero cells, but the binding ability of S260-600 was somewhat stronger than that of S397-796. In contrast, the S260-600 protein did not bind NIH3T3 cells.</p><p><b>CONCLUSION</b>Both S260-600 and S397-796 exhibited different receptor binding activity. The S260-600 fragment probably contains the important receptor binding domain and could be a potential candidate for the development of SARS vaccine and anti-SARS therapeutics.</p>

Preparation and characterization of monoclonal antibodies against SARS-associated coronavirus nucleocapsid protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain monoclonal antibodies (McAbs) against severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) nucleocapsid (N) protein to develop diagnostic test for SARS and study the pathogenesis of the disease.</p><p><b>METHODS</b>BALB/c mice were immunized with purified N protein of SARS-CoV. Hybridoma cell lines secreting monoclonal antibodies against SARS-associated coronavirus nucleocapsid were established after cell fusion with mouse splenic cells and SP2/0 cells. The specificity of the McAbs obtained was examined by Western blot and indirect fluorescence assay. Epitopes reacted with the McAbs were preliminarily located through Western blot by expressing truncated N proteins.</p><p><b>RESULTS</b>After cell fusion and three rounds of cell cloning, six hybridoma cell lines secreting monoclonal antibodies specifically against SARS-CoV nucleocapsid were obtained. Western blot and indirect fluorescence assay showed that the McAbs reacted specifically with nucleocapsid protein and SARS-CoV. Among the six McAbs, three recognize the epitopes located in the N-terminus of the protein, whereas the others reacted with those located in the C-terminus.</p><p><b>CONCLUSION</b>The anti-SARS-CoV nucleocapsid McAbs were developed and these McAbs may be useful in the development of diagnosis assays and basic research of SARS.</p>