Identification and construction of replicon vectors of Japanese encephalitis virus (strain SA14-14-2) / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>In order to lay the groundwork for studying the novel vaccine Identified.</p><p><b>METHODS</b>(1) Two replicons were constructed. One's prM/E gene was deleted completely (Full AprM/E Replicon), the other's prM/E gene was deleted partially (213 bp of C terminal of E gene was reserved; Partial delta prM/E Replicon), and the deleted parts was replaced as the MCS. (2) Replicons RNA were which will use the JEV as the vector, replicon vectors of JEV was constructed and transfected into BHK-21 cell. After 24, 48, 72, 96 h, method of real-time PCR was used to identify Replicons' replication ability. (3) YFP gene was inserted into the MCS of those two replicons. Their RNA was transfected into BHK-21 cell. Expression of YFP was tested by the fluorescence microscopy and flow cytometer.</p><p><b>RESULTS</b>(1) After the two replicons RNA were transfected into BHK-21 cell, as time went by, the quantity of RNA increased. (2) After RNA of the replicons with YFP were transfected into BHK-21 cell, increasing trend of fluorescent signal and rate of YFP positive cell was observed and tested.</p><p><b>CONCLUSION</b>Full delta prM/E Replicon and Partial delta prM/E Replicon have the ability to duplicate itself and express the foreign protein.</p>

Construcion of a chimeric Japanese encephalits virus/dengue virus-2 / 病毒学报

The prM/E gene of DV2 was cloned into the JEV (SA14-14-2 strain) replicon vector which had been constructed previously, and the resulting recombinant plasmid was named pPartialdeltaprM/E. The constructed chimeric clone was linearized and then was transcripted into RNA in vitro. The produced RNA was transfected into BHK-21 cells. Five to seven days later, CPE could be observed on the transfected BHK-21cells, and then the supernatant containing the chimeric virus was collected. The Supernatant was inoculated to BHK-1 cells and C6/36 cells, respectively. CPE could be observed about 4 days post the infection of C6/36cell with the chimeric virus. The results from RT-PCR, IFA, Western blot showed that the virus contained the chimeric RNA and the envelop protein of DV2. However, the chimeric virus could not be passaged in BHK-21 cell. The successful construction of the infectious clone JE/DEN-2 laid the basis for the further research of the DV vaccine.

SARS patients-derived human recombinant antibodies to S and M proteins efficiently neutralize SARS-coronavirus infectivity / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection.</p><p><b>METHODS</b>By using phage display technology, we constructed a naive antibody library from convalescent SARS patient lymphocytes. To obtain the neutralizing antibody to SARS virus surface proteins, the library panning procedure was performed on purified SARS virions and the specific Fab antibody clones were enriched by four rounds of repeated panning procedure and screened by highthroughput selection. The selected Fab antibodies expressed in the periplasma of E. coli were soluble and further purified and tested for their binding properties and antiviral function to SARS virus. The functional Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell systems and their neutralizing activities were further determined.</p><p><b>RESULTS</b>After four rounds of the panning, a number of SARS-CoV virus-targeted human recombinant Fab antibodies were isolated from the SARS patient antibody library. Most of these were identified to recognize both natural and recombinant SARS spike (S) proteins, two Fab antibodies were specific for the virus membrane (M) protein, only one bound to SARS-CoV nucleocapsid protein. The SARS-CoV S and M protein-targeted Fab or IgG antibodies showed significant neutralizing activities in cytopathic effect (CPE) inhibition neutralization test, these antibodies were able to completely neutralize the SARS virus and protect the Vero cells from CPE after virus infection. However, the N protein-targeted Fab or IgG antibodies failed to neutralize the virus. In addition, the SARS N protein-targeted human Fab antibody reacted with the denatured N proteins, whereas none of the S and M protein specific neutralizing antibodies did. These results suggested that the S and M protein-specific neutralizing antibodies could recognize conformational epitopes which might be involved in the binding of virions to cellular receptors and the fusion activity of the virus.</p><p><b>CONCLUSION</b>The SARS-CoV spike protein and membrane proteins are able to elicite efficient neutralizing antibodies in SARS patients. The neutralizing antibodies we generated in this study may be more promising candidates for prophylaxis and treatment of SARS infection.</p>

Using recombinant antigens of Hantavirus to study the kinetics of serum IgA, IgG, IgM antibodies in the acute-phase of hemorrhagic fever renal syndrome / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To observe the features of serum specific IgA, IgG, IgM antibodies in the acute phase of hemorrhagic fever renal syndrome (HFRS).</p><p><b>METHODS</b>The nucleocapsid (NP) protein and glycoproteins (GP) of Hantavirus were expressed by recombinant baculovirus, and used as ELISA antigens to test 61 serial sera of 14 acute phase HFRS patients.</p><p><b>RESULTS</b>Seoul like virus RNA were detected from 11 of 14 patients. An early and strong IgA, IgG and IgM antibody response to recombinant NP (rNP) was observed in almost all HFRS cases. The titers of antibody to rNP was apparently higher than that to Rgp. In the early stage, titer of IgG antibody elevated most drastically among all the three classes of antibodies to rNP, followed by IgM and IgA antibody responses. The elevation trend of IgM and IgA antibodies to rNP stayed nearly at the same level, but the IgA titers to rNP were apparently higher than that of IgM. Among the antibodies to rGP, IgA changed distinctly greater than IgG. The elevation trend of IgM could be found during first week after the onset, and the titers dropped gradually after the second week. IgM antibodies of one case who was viral RNA positive were not detected at early stage, but IgA titers were high. The only severe case of the 14 patients kept the lower IgA, IgG and IgM during the whole acute phase.</p><p><b>CONCLUSION</b>HFRS patients kept an early and strong humoral response to NP and GPs in acute phase of HFRS.IgA could be used together with IgM to improve the diagnostic accuracy.</p>