2000 Vol.15(2)
Abstract：Two promoters．LTR (1ong terminal repeat)and IP (internal promoter)，exist in
genomes of foamy viruses．Ge1l transfection and transient expression assay demonstrated that!the
basal activity of BFV (bovine foamy virus)IP is much higher than that of BFV LTR；transacti．
vator of BF 一Borf-1，which activates gene expression directed by BFV LTR，aLso hmctions on
BFV IP with an activation foId higher than tha t on LTR ．The results suggest that BFv IP and
LTR may regulate viral gene expression by difierent mechan isms，and that Bod-1 ma y stimulate
BFV IP and LTR in diferent ways．In addition．an in vivo DNA compe tition assay demonstrat
ed that a comm on transcxiption factor may be involved in both mechanisms of the two promoters ．
In order to investigate the stress response in the cells infected with Hantaan virus and the relationship between the expression of HSP70 and the virus replication, the Vero E6 cells infected with Hantaan virus (A9 strain) were detected by immunohistochemistry and in situ hybridization. The results show that the expression of HSP70 can be induced in the cells 4 hours after Hantaan virus infection and lasted until the fifth days after infection. The distribution of HSP70 also varied. It suggests that the expression of HSP70 can be induced directly by Hantaan virus.
In order to investigate the stress response in the cells infected with Hantaan virus and the relationship between the expression of HSP70 and the virus replication, the Vero E6 cells infected with Hantaan virus (A9 strain) were detected by immunohistochemistry and in situ hybridization. The results show that the expression of HSP70 can be induced in the cells 4 hours after Hantaan virus infection and lasted until the fifth days after infection. The distribution of HSP70 also varied. It suggests that the expression of HSP70 can be induced directly by Hantaan virus.
HCMVs in blood leukocytes and in serum from the same blood donor were examined by polymerase chain reaction (PCR) and DNA hybridization technique. In the mean time, HCMV IgM and HCMV IgG in the serum were examined by ELISA. Two hundred blood leukocyte samples and 200 serum samples were examined in a successive two years period. HCMV positive rates for the blood leukocytes by PCR in the different years were 63% and 70% while the positive rates by DNA hybridization were 42% and 50%, respectively. HCMV postive rates for the serum by PCR in the different years were 49% and 53% while the positive rates by DNA hybridization were 33% and 39%, respectively. A 5% HCMV IgM positive rate was found in the two years while 54% and 58% HCMV IgG positive rates were found in the two different years.
The HIV 1 p24 gene (gag gene) was amplified by polymerase chain reaction (PCR) and cloned into an E.coli expression vehicle pQE30 between BamH I and Kpn I sites. After induction with IPTG, the transformed E.coli strain M15 expressed the HIV 1 p24 gene efficiently. The recombinant p24 protein was expressed as a soluble protein, composing 20% of the total protein. After onestep purification with Ni NTA + affinity chromatography, the protein was purified to almost homogeneity. When applied to ELISA, the p24 protein exhibited good specific reactivity with sera of HIV infected individuals. The antibodies against recombinant p24 protein from HIV infected plasma were purified and confirmed with HIV Western blot kit that the purified antibodies can only react with HIV 1 p24 antigen. It suggests that the recombinant HIV 1 p24 protein is suitable for assembling the HIV diagnostic kits.
The target fragment of HGV genes including C, E1, E2, NS2 and partial NS3 regions was microinjected into the pronuclei of mouse fertilized eggs according to the standard methods. Ten mice containing the HGV genes were identified by the screening of polymerase chain reaction with the primers in NS3 region. The integrated HGV genes were vertically transmitted to the offspring, and the F 1 heterozygous mice from three founders were detected positive. The results of RT PCR indicated that the HGV genes can be transcripted in blood monocytes and hepatocytes. The conventional pathological observation suggested that the transgenic mice had the cloudy swelling, steatosis, hydropic change and inflammatory cellular infiltration. However, serum ATL level showed no significant difference between transgenic and normal mice.
In order to understand the characteristics and rules of HIV diversity when passaged in vitro , the systematic study on the variation of env gene of HIV strain introduced from American in 1988 was carried out. Taking all the following strains as the objects: the 96 strain derived from the primary strain by continuous freeze thaw usage in laboratory for more than eight years; the 42?h strain derived from primary strain by serial passaging with continuous changing of host cell in MT4 and HelaCD4+ and the strains sampled from serial passaged primary strain in MT4 cell every ten generations, The pre DNA fragments of these HIV env gene were amplified by nested PCR. The fragment of 1?200 bp originated from the V1 V5 region was analyzed. The subtypes of these HIV strains were identified by Heteroduplexes Mobility Assay (HMA). The main results were: (1) the env gene diversity of all the strains were indistinctive, all the nucleotide homogeneous were greater than 92% and all the gene diversity distance were .
From the sera of 3 Chinese patients with hepatitis C, 3 new nucleotide sequences of the 5 non coding region (5NCR) of hepatitis C virus (HCV) were found using reverse transcription polymerase chain reaction (RT PCR) and DNA recombinant and sequencing techniques. Comparing with previously published HCV corresponding sequences, these new sequences have some consecutive nucleotides deleted or inserted. Sequence YS225 1 has 18 nucleotides deletion between the original position 174～-155 compared with the corresponding sequence of the prototype, HCV 1 (gene 1a); while sequence YS203 4 and sequence YS242 1 respectively contain extra 28 nucleotides and 40 nucleotides insertion between the original position -56～-55. These nucleotides are the repeating sequences existing in the forward sense (original position -83～-56). In sequence YS242 1, there is a sequence containing 11 nuclotides (corresponding to the original position -76～-56) repeated twice. In addition, variations of the nucleotides among these sequen.
Insect Sf 9 cells were infected with constructed recombinant virus AcMNPV OCC - GST 6xHis Etp 28, which could produce GST fusion protein and the supernatant of 72 h pi cell lysate was examined by SDS PAGE. The results showed that GST 6xHis Etp 28 fusion protein of 53 kDa was expressed in insoluble status. After the sodium salt of the alkylanionic detergent sarkosyl was added to insect cell lysis buffer to a final concentration of 1.5%, and the final concentration of TritonX 100 was increased from 1% to 2%, at least 1/3 of GST 6xHis Etp 28 appeared to be soluble, which was examined by SDS PAGE. Then soluble GST 6xHis Etp 28 was purified by affinity chromatography using glutathione agarose.
The development of Dendrolimus punctatus wenshanensis Cytoplasm Polyhedrosis Virus (DpwCPV) insecticide is reported. The formulation is an oil in water emulsion and the concentration of polyhedrosis is 2.510 9 PIB/mL. The auxiliary materials come from the agriculture products. It is safety and the viral insecticide contained no any pathogenic bacteria. The average mortality is 85.5% when the second instar larvae were infected by 10 6 PIB/mL DpwCPV.
The RNA4 intergenic regions of two isolates of rice stripe virus (RSV) in China (de signated as RSV JN and RSV YL) were amplified by RT PCR and cloned into pGEM T easy vector. Sequence analysis showed that they were all 654 bp in length, and shared only 92% identity at the nucleotide level. Computer assisted folding analysis showed that the intergenic region was rich in A and U residues where two distant hairpin structures could be formed. One was highly conserved and stable, but the other was rather unstable because of bases variation. Therefore, there were molecular variation among isolates of rice stripe virus in China.
The Antiviral substance was induced from blastulae embryonic cell line of crucian carp (CAB) by ultra violet inactivated grass carp reovirus (GCRVuv). This substance was sensitive to trypsin and stable at 56 ℃ pH 211. The activity of this substance was influenced by target cell density, incubation temperature and time with CAB cells, but not decreased by treatment of the special antibody of GCRV. This substance couldnt kill virus directly, but showed anti GCRV and anti STIV activity in various cultured fish cells. Inhibition of viral replication in cells was dependent on the process of cellular RNA transcription and protein synthesis. These characteristics consist with IFN alpha/beta of mammals, and the substance is a kind of crucian carp interferon.
Some multiplication properties of the Lapinized Chinese strain of Classical Swine Fever Virus (CSFV C strain) in primary bovine testicular cells were studied. One CSFV C strain which can be detected by immunofluorescence technique was found and a new method in titering called flurescent plaque formation unit (F PFU) was developed. It took about 5 days for all cells of primary bovine testis to be infected by CSFV and the titre of progeny virions in medium was 10 5 F PFU/ml from then on. The cell types of cultured bovine testicular cells changed along with cell passages. The mechanism of some phenomena in vaccine production was showed and some factors which effect the multiplication of CSFV C strain in primary bovine testicular cells were studied. Aknowledgements on the multiplication of CSFV C strain in primary bovine testicular cells were enriched and a new way to raise the titer of CSFV was suggested.
The glycoprotein I gene (gI) of vv +MDV strain 648A was amplified by PCR, cloned and sequenced, its DNA and amino acid sequences were compared to published sequences of vMDV strain GA and vvMDV strain RB1B. The results indicated that the gI sequence of 648A was identical to the published 761 bases of 5 side of ORF of strain RBIB, but there were 8 bases and then 7 amino acids different between GA and 648A strains. All these base changes located at the 5 end of gI, about the one third of the 3 end was identical in different pathotypes.
The morphogenesis of the Grass Carp Reovirus (GCRV) was studied in CIK cells. The CIK cells were infected by the GCRV at MOI (multiplicity of infection) of 5 10 PFU/Cell. It was demonstrated that the subviral particles without outer layer proteins could be seen in the cytoplasm at 4 hours post infection. At 8 hours post infection many viroplasms could be found in the cytoplasm of the cells. And there were many subviral particles without outer layer proteins in them. As the infection progressed, the mature virus, which is about 72 nm in diameter produced after the subviral particles were transiently enveloped during 12 16 hours post infection. During its replication, at 8 hour post infection inclusion bodies were formed, in which there were many immature and mature viruses. And the progeny virions were released after the inclusion bodies lysised at 16 20 hours post infection .
The RNA of dengue 2 virus (New Guinea C strain, NGC) was extracted from small amount of dengue 2 virus culture supernatant. The large NS3 gene fragment was amplified from RNA of dengue 2 virus with RT PCR method. The amplified NS3 gene fragment was cloned directly into the pUC18 plasmid vector. The recombinant plasmid was identified by agarose gel electrophoresis analysis after digestion with restriction endonuclease and nested PCR method. The results showed that the amplified NS3 gene fragment was about 1978 bp in length; the recombinant plasmid contained NS3 gene of dengue 2 virus. The method of amplified large fragment of dengue virus from small amount of dengue virus culture supernatant was established.
A 1.66 kb expected S1 gene fragment of avian infectious brochitis virus (IBV) H strain was amplified by RT PCR. Its PCR/RFLP pattern was similar to IBV D 41 strain and M 41 strain when digested using BstYI, HaeⅢ and EcoRI respectively. IBV H strain was thought as Massachusetts serotype.
Based on the known RNA sequence of rice grassy stunt virus , IRRI isolate (RGSV IR), the cDNA of nucleocapsid protein (NCP) gene was obtained by RT PCR, with genomic RNAs of Shaxian isolate (RGSV SX) as template. The cDNA was then cloned into pGEM T vector and sequenced. The results showed that the nucleotide sequence between the two isolates was 99.4% identical. A bacterial expression plasmid pGTNCP which produced a fusion protein with molecular weight of about 62?kD was constructed using the cDNA clone and vector pGEX 2T. Western blot analysis showed that the fusion protein reacted strongly with antibodies raised against RGSV particles.
The gene encoding the core and NS3 proteins of hepatitis C virus was amplified by PCR, respectirely. Two genes were fused and formed the recombinant plamid pHCV CN (Core+NS3) and pHCV NC (NS3+Core). The fused plasmid were expressed in E.coli 06. The pHC CN plasmid expressed fussion protein was shown by a major band with a expected molecular weight about 68 kD on SDS PAGE. However, the pHCV NC plasmid expressed fussion protein was 66 kD in molecular weight. The results indicate that the pHCV NC fussion protein differs from the pHCV CN fussion protein in mulecular weight. It suggests that the fusion way is important for the structure of recombinant proteins.
