Establishment of internally controlled real-time PCR for the detection of human parvovirus B19 DNA in serum / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a TaqMan-based Real-time PCR assay with internal control for the detection of parvovirus B19 DNA in human serum.</p><p><b>METHODS</b>Two DNA fragments in length of 113 bp each were artificially synthesized, cloned into T vector and used as standard DNA or internal control, respectively. One pair of primers and two probes were included in the Real-time PCR. The probes were labeled with different fluoresceins and could bind B19 DNA or internal control, respectively. Precision and specificity of the method were evaluated. Specimens of human serum were examined by this assay to find B19 DNA.</p><p><b>RESULTS</b>The standard curve was constructed using the quantified standard B19 DNA. The Real-time PCR method was established. It was stable according to precision evaluation by the intra- and inter-assay and specific without any evident cross-reaction with human hepatitis B virus (HBV). Among 160 samples of human serum, B19 DNA was detected in 2 with a concentration of 2.1 x 10(5) Geq/ml and 3.6 x 10(3) Geq/ml, respectively.</p><p><b>CONCLUSION</b>The Real-time PCR for B19 DNA detection was developed successfully, in which the internal control was helpful to exclude false-negative results.</p>

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>

The construction of SCID-Hu IC mice model and application in rAd5HPV16L1-E7 vaccine / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct human-SCID chimeric mice through implantation of mononuclear cells from human cord blood and study the immunoreaction of SCID-Hu IC mice immunized with rAd5HPV16L1-E7 vaccine.</p><p><b>METHODS</b>(1) Experiment groups were injected with the suspension of mononuclear cells from human cord blood through a tail vein; the control ones were injected with non serum RPMI 1640 medium. Eight weeks after implantation, blood was collected and human serum IgG level in the mice were tested, and human CD45, CD3 and CD19 were determined. (2) SCID-Hu IC mice were divided into two groups: in group A the mice were immunized intraperitoneally with rAd5HPV16L1-E7 virus and in group B the mice were immunized through nasal drip with rAd5HPV16L1-E7 virus. At the end of fourth week, the serum specific IgG antibody to rAd5HPV16L1-E7 virus, IFN-gamma in culture medium of spleen lymphocyte and T-lymphocyte propagation were tested.</p><p><b>RESULTS</b>(1) In the experiment groups, the number of mice positive for human IgG was 10/15, the average values of CD45, CD3 and CD19 were (9.39+/-4.21), (3.25+/-3.99) and (1.69+/-0.75), respectively. In the control ones, the human IgG, CD45, CD3 and CD19 were negative. (2) The results in the experiment groups showed that the IFN-gamma and T-lymphocyte stimulated by HPV16 protein were higher than those in the non-stimulated group (P less than 0.05).</p><p><b>CONCLUSION</b>(1) The results indicated that the construction of human-SCID chimaera through the implantation of mononuclear cells from human cord blood into SCID mice was successful. They also indicated that the reconstructed SCID-Hu IC mice has the ability to produce immune response against rAd5HPV16L1-E7 recombinant virus.</p>

Establishment and preliminary characterization of hybridoma cell lines secreting monoclonal antibodies against Prion Proteins / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain monoclonal antibodies (McAbs) which can be widely used to detect mammalian prions (PrP) and to develop diagnostic tests for screening transmissile spongiform encephalopathies (TSE) as well as for studying pathogenesis of prion-related diseases.</p><p><b>METHODS</b>BALB/c mice were immunized separately with bovine PrP peptide 29-48 (BoP1) and 89-108 (BoP2) coupled to keyhole limpt hemocyan. Two hybridoma cell lines secreting monoclonal antibodies against these peptides were established by cell fusion and 2 to 3 rounds of cell cloning. The reactions of the McAbs to the recombinant bovine (Bo)PrP(25-242), human (Hu)PrP(23-231) and hamster (Ha) PrP (23?231) were tested separately by Western blotting.</p><p><b>RESULTS</b>Through cell fusion, two hybridoma cell lines secreting McAbs against BoP1 and BoP2, designated D11 and D8 accordingly, were identified by ELISA and cell cloning. The McAbs produced by these cell lines reacted well with the recombinant PrP proteins; (Bo) PrP (25-242), (Hu) PrP (23-231), and (Ha) PrP (23-231), respectively.</p><p><b>CONCLUSIONS</b>Two McAbs reacting with bovine, human and hamster PrPs were successfully generated, they are potential to be used to detect PrPs in mammals and to study the mechanism of pathogenesis of TSE.</p>

Inhibition effects of rhubarb ethanol extract on herpes simplex virus infection in vivo / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To know the anti-viral effects of rhubarb ethanol extract (REE) on herpes simplex virus(HSV) infection in vivo.</p><p><b>METHODS</b>BALB/c mice inoculated from tail vein with 0.15 ml of HSV (TCID50=10(3)) were injected hypodermically with REE next day. After divided into seven groups, three groups of mice were given different doses of REE respectively and the other groups as controls. Pathological sections from the liver, spleen, kidney were made at different times of postinfection, and their pathological changes were observed under microscope; the virus titers in viscera were assayed by using plaque formation technique and the rhubarb inhibitions to the infection of HSV in vivo?were observed.</p><p><b>RESULTS</b>No toxic response to mice were observed for REE injected hypodermically; no pathological changes were observed in different therapy groups of spleens. And those in livers and kidneys at medium- and high-dosed groups disappeared quickly. The effect of low-dosed group was equal to that of positive control group, acyclovir(ACV); the results of the titer tests showed that the virus decreased rapidly by using REE, especially in the medium- and high-dosed groups which were much more marked than the low-dosed group; Q test of the data showed that total mean value had statistical significance (F=49.1459, P<0.01); moreover there were statistical significance between therapy groups (ACV, DH1, DH2, DH3) and non-therapy groups (VC) (P<0.01 ) and between DH2, DH3 and DH1 (P<0.01); no statistical significance were found between DH1, DH2 or DH3 and ACV (P>0.05). Results show that as to the effect of decreasing the average of the total titer, rhubarb is as effective as ACV; furthermore, the medium- and high-dosed groups are superior to the low-dosed group.</p><p><b>CONCLUSIONS</b>REE has significant anti-viral effect on HSV in vivo; there will be a wide application foreground of it in clinical usage.</p>

Genetic characterization of the non-structural protein NSP4 from epidemic strains of human rotavirus in China / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To clarify the features of gene variation among epidemic strains of human rotavirus NSP4 in China.</p><p><b>METHODS</b>SP4 genes from 27 epidemic strains of human rotavirus isolated in different area of China in recent years were amplified with RT-PCR, the resulted cDNAs were cloned and sequenced. The sequences of full length cDNAs were compared with 10 rotavirus NSP4 sequences available in the GenBank using the Clustal x 1.8 TreeView32 and DNA Star softwares. The G serotype of VP7 was analysed by PCR.</p><p><b>RESULTS</b>The homology of the amino acid among the 27 rotavirus strains isolated in China was 81.7%-99.4%. Based on the variation of amino acid sequence, the virus strains can be divided into two groups, represented by Wa and KUN with the homology of 92.0%-99.4% and 92.0%-98.9% within each group, respectively. The diversity between the two groups were 16.6%-21.0%. The Wa group could further be separated into three subgroups, according to the diversity between those strains and the characterization in the highly variable domain. The association between VP7 serotype and NSP4 genotype was not strong.</p><p><b>CONCLUSIONS</b>The NSP4 gene of human rotavirus epidemic strains in China can be divided into Wa and KUN two groups, Wa group is the main group and contains three subgroups possessing characteristic amino acid sites. Samples isolated in the same year but not in the same area shared higher homology.</p>