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1998 Vol.13(3)



Research Advances Oil Structures and Functions of Baculovirus pl0 Genes and Proteins

CHEN Chang-Wu, WEI Yong-Jie, LONG Qi-Xin

1998, 13(3): 185

Random RNA pool selection:developments and prospects

WANG Bing, JIANG Hong, KE Li-Hua

1998, 13(3): 192

Effects of long terminal repeat (LTR) of human immunodeficiency virus type 1 on the expression of gag gene in recombinant vaccinia viruses

MAO Chun-Sheng, JIN Ning-Yi, TONG Ming-Hua, GUO Qin-Jun, WANG Xiu-Qing, GUO Zhi-Ru, YAN Zhen

1998, 13(3): 207

To investigate the function of LTR in vaccinia virus, 6 recombinant viruses were constructed by inserting intact or serially truncated LTR together with their downstream of gag ORF of human immunodeficiency virus type 1 (HIV 1) into the vaccinia virus genome. Indirect fluorescence assay, Western blot and immunoenzyme assay showed that all the 6 recombinant vaccinia viruses expressed Gag protein, but the efficiency were significantly different. These results suggested that the function of each function domain in HIV 1 LTR exhibited distinct characteristics on gag gene expression in vaccinia virus: (1) The function of intact LTR on gag gene expression varied with the upstream poxvirus promoter; (2) NR sequence neither decreased nor increased the Gag protein exprssion level; (3) Enhancer sequence might not be recognized by recombinant vaccinia virus expression system; (4) TAR sequence enhanced Gag protein expression effectively; (5) U5 region and its downstream non translated sequence had little effect on

Large-scale purification of inactivated Japanese encephalitis vaccine from Veto cells by zonal centrifugation

DAN Hui-Ying, DING Zhi-Fen, DIAO Min, LONG Cheng-Hua, YANG Kang-Kang, LI Jing

1998, 13(3): 213

Japanese Encephalitis (JE) Vaccine in Vero cell can be easily purified by zonal centrifugation at non continuous sucrose gradients (36% and 60%), 32 600g for 4h. The calf serum protein and other nonviral proteins in the vaccine were almost separated from the JE virus. The residual calf serum protein was less than 0.5g/mL, and the total protein was less than 30 g/mL. The residual Vero cell DNA in the vaccine was less than 100 pg/0.5mL. The titer of purified Japanese Encephalitis vaccine is six times higher than China control vaccine. This method is recommended as an available method to purify JE vaccine from Vero cell in large-scale because it is simple, rapid and inexpensive.

Genetic variation characteristics of hepatitis C virus hypervariable region

PAN Wen-Sheng, WANG Hai-Tao, WANG Tao, GUO Hua-Zhang, LUO Gang-Xian

1998, 13(3): 221

At three time points duing the period of five years, the hypervaviable region gene of HCV from two patients with HCV persistent infection was amplified, sequenced and analyzed. The results demonstrated that there were five significant characteristics in genetic variation of HCV HVR. 1. Wide mutation degree and range. HVR is consistent of 81 nt and 27 aa, 89%nt (72/81) and 96%aa (26/27) mutated in 168 clones; 2.Different mutation types. Except the common substitution mutation, deletion was 22.5%; 3. Apparently dominant clones. Dominant clones always existed, although genetic variation was significant among clones at one time point;4. Phenomenon of quasispecies is very severe. In conclusion, this study demonstrated that the genetic variation of HCV HVR showed high complexity and diversity.

Susceptibility of GC32 cell to three strains of arbovirus

ZHANG Yu-Zhen, HUANG Wen-Li, HOU Zong-Liu, YUAN Qiang-Hong

1998, 13(3): 225

GC32 cell line established by authors from a carcinoma patient is susceptible to JE and Chikungunya. The susceptibility of GC32 cell to JE and Chikungunya is similar to that of BHK21 regarded as control. BHK21 and GC32 cells infected by JE,Chikungunya and Langat viruses make +++~++++ results by IFAT. Both BHK21 and GC32 cells to JE and Chikungunya form laques.The PFU of BHK21 cell to JE and Chikungunya are 5.36 and 5.65 and GC32 cell to JE and Chikungunya are 5.61 and 6.48, respectively. The results showed that GC32 cell line is a suitable cell strain for studying virus.

Comparison of Amino Acid Consensus sequences of M embrane Protein V3 Region between International and Local Epidem ic HIV-1 Strains from Yunnan During 1992 1994

WANG Bin, LU Xiao-Qing, SHAO Ji-Jun, SHAO Yi-Ming, CENG Yi

1998, 13(3): 230

The amino acid consensus sequences of membrane protein V3 region of HIV 1 Yunnan epidemic strain named YNV3 and two groups of amino acid consensus sequence YNV3A and YNV3B\ were deduced from DNA sequences of env gene V3 region of 16 local strains collected during 1992-1994,and analysed by DNASIS and PROSIS software. The conservaty of each amino acid in the YNV3 was calculated. The amino acid sequences of YNV3A and YNV3B were compared respectively with that of epidemic strains from other parts of the world for the homology analysis. The results showed that average variation of amino acid was 7.66% in the 35 amino acids which composed the consensus sequences of YNV3. The amino acid consensus sequences of YNV3A and YNV3B showed higher homology with that of HIV 1 American European strain and B subtype of Thailand strain, respectively. The results revealed that there is a closed relation ship between Yunnan Ruili HIV 1 epidemic strains in evolution. The domain epidemic strains in the area were mainly from HIV

Studies on some immunological properties of HHV-6 infected cells

ZHAO Bei, JI Xiao-Hui, ZHAO Wei-Hong, DING Ru-Yu, ZHOU Yao-Xi

1998, 13(3): 236

The local strain of human herpesvirus 6 (HHV 6),named CN5, was used to study some immunological properties of infected cells.By the methods of indirect immunofluorescence test, APAAP and MTT, the morphological and kinetic character of viral antigen expression, the changes of CD antigens expressions and the cellular proliferation reaction induced by PHA were detected. The viral antigens were appeared in cord blood mononuclear cells (CBMCs) 8-12 hours after infection. By 48 hours positive cells reached 36%. After CN5 infection, in both cases of CBMCs and adult peripheral blood mononuclear cells (PBMCs), the CD3 positive cells decreased while the CD4 positive cells increased. No significant changes were found in the percentages of CD2, CD8 and CD45 RA positive cells. CN5 infected cell lysates inhibited the proliferation of PBMCs induced by PHA in a protein concentration dependent pattern, and this inhibition could be partially eradicated by the antisera to HHV 6.

Microcarrier cultivation of silkworm BmN cell and high-level expression of HBeAg gene

DENG Xiao-Zhao, DIAO Zhen-Yu, ZHU Ying, HE Liang, JIAO Ren-Liang, ZHOU Zong-An, ZHANG Lin-Yuan

1998, 13(3): 237

Distribution of silkworm Bm N cell attachment to Cytodex 3 microcarrier was observed and the influence of cell inoculation concentration and microcarrier concentration on cell growth was studied. The minimal cell inoculation concentration was 1.010 5 cells/mL when Cytodex 3 microcarrier concentration was 3g/L. Cultured under the proper conditions (inoculation concentration: 3.610 5 cells/mL; microcarrier cytodex 3 concentration: 5g/L) for 5 days, the final cell concentration of BmN was 2.810 6 cells/mL. The cell growth index was 7.9. At 48-60 hours after cell passage, the Bm N cells was inoculated with rBmHBe (multiplicity of infection Mol=0.4 PFU/cell). Five days later the HBeAg titer in the medium was 1∶96000 and in the control group it was 1∶32000.

Gene expression of gold fish growth hormone Il in baculovirus

LIN Guang-Yun, LONG Qi-Xin, HUANG An-Lin, PANG Yi, YU Qi-Li

1998, 13(3): 250

Using a transfer vector plasmid pSXIVVI +X3 without an initiation codon, the occluded recombinant Trichoplusia ni nuclear polyhedrosis virus as an expressing vector carrying the cDNA encoding gold fish growth hormone Ⅱ(gf GHⅡ) under the control of the SynXIV promoter has been constructed. Immunoblot analysis revealed that the virus mediated gfGH Ⅱ can be detected as early as 24 hr pi and the expression level reached to the highest in 96 hr pi in the Sf cells and culture medium or larvae and haemolymph, the molecular weight of the expressed protein is 22.5 kDa, which is equivalent to the value calculated from the predicted amino acid sequence. The expression level in vivo and in vitro was quantified using RIA. Average 10 5 Sf9 cells may secret gfGH Ⅱ into medium reaching level of 86.74 ng. The expression level of gfGHⅡ in larvae may reach to 214g per gram of dry larvae.

An antiapoptosis p35 gene from Heliothis armigera polyhedrosis virus rescues Acp352 replication in non-permissible cell line

WANG Ye-Fu, JI Xi-Peng, ZHU Ying, LI Xiao-Feng, LI Zhi-Da

1998, 13(3): 256

p35 gene from baculovirus is an antiapoptosis gene which can sustain virus multiplication when infected by baculovirus. Both p35 deleted and inserting inactivated AcNPV can not replicate in Sf cell lines and Ha cell line.It is almost the same in BmNPV.Acp35Z which generated from AcNPV inserted LacZ gene whthin p35 gene causes apoptosis in Ha cell line ,but the abortion replication can be rescued by transient expression of HaNPV p35 gene drived by AcNPV IE1 promotor.The result was reconfirmed with X-gal manifest and Dot-ELISA.

Genetic transformation of flue-cured tobacco cultivars G28 and K326 protecting to cucumber mosaic virus

CHEN Yuan-Sheng, XIAO Huo-Gen, ZHANG Shu-Guang, FAN Fu-Zhong

1998, 13(3): 262

Two flue cured tobacco cultivars, G28 and K326, from Guangdong,were transformed by CMV BS CP gene from diseased banana via Agrobacterium tumefaciens , and 313 transgenic tobacco plants were obtained. Calli were observed on the small square sections of leaves of the transgenic plants in culture medium containing 100 g/mL Km,indicating the Km resistant marker genes (NPTⅡ) has been successfully introduced into the plants. It was confirmed with PCR analysis and DAS ELISA assay that the CMV BS CP genes were stably integrated into the genomes of the transgenic tobacco plants, and were subsequently found to be expressed in these plants. The transgenic tobacco plants were inoculated mechanically or by aphids with the hallenging CMV strains BS,27 and 37 from diseased banana,tobacco and cucumber in Guangdong,respectively. The transgenic tobacco plants expressing coat protein of BS were demonstrated protecting effect to the infection of these CMV strains significantly, delaying the disease expression and reducing

Partial cloning of the genome of non-occluded baculovirus from Penaeus chinensis and preparing the probe for detection

SHI Zheng-Li, HUANG Can-Hua, CHEN Dai-Hua

1998, 13(3): 267

Viral particles similar to non occluded baculoviruses were isolated from penaeid shrimp Penaeus chinensis in China. The size of enveloped virion is 250-300110nm.After extraction,the viral DNA was digested by EcoRⅠ and cloned into the pUC 18. Two clones, L46 and M13,containing inserts of 2.14kb and 3.58kb in size respectively, were obtained.The inserts were labeled by Digoxingenin. Diseased shrimp samples issued from different regions of China were investigated by dot blot hybridization with the previously preparing probes L46, M13 and with A26 probe. All the probes reacted with the tested samples showing baculovirus infection. It indicated that baculovirus from P.chinensis is very close to the WSSV.

Sequence determination of the specific DNA fragment from the genome of strong virulent GA strain of the Marek's disease virus

LU Chun, ZHU Hong-Fei, ZHANG Xun-Hai, CAI Bao-Xiang

1998, 13(3): 270

Differentiation of strong virulent and vaccinal Marek's disease virus (MDV) was carried out by using DNA molecular hybridization. DNA probe was made of BamHI L fragment from MDV gene library (strain GA) by labelled with digoxigenin using random priming. And the complete nucleotide sequence of BamHI L fragment was further determined from subcloned DNAs by using double stranded sequencing method. The results of computer analysis showed that, (1) the entire DNA fragment is 2893 bp long and has a base composition of 55.91% G+C;(2) it has no any open reading frames (ORFs); (3) this DNA fragment is possibly involved in transcription of meq gene of MDV.

Identification and characterization of an isolate of peanut stunt virus from black locust mosaic tree in China

ZHANG Zong-Xi, CHEN Kun-Rong, XU Ze-Yong, CHEN Jin-Xiang, FANG Xiao-Ping

1998, 13(3): 274

A virus isolated from a diseased black locust tree (Robinia pseudo accacia) in the suburb of Beijing was identified as peanut stunt virus (PSV),designated PSV Robinia isolate (PSV-R). PSV-R infected 14 of 24 species of plants in four families. The thermal inactivation point was 55-60℃. The dilution end point was 10 -4 -10 -5 and the longevity in vitro was 10 days.Aphid (Aphis craccivora Koch) transmitted the virus in a nonpersistent mamner. Virus particles were spherical with 30nm in diameter. An antiserum against PSV-R was produced with titers of 1∶1024 and 1∶256 in microprecipition and gel diffusion tests, respectively. Serologically PSV-R was closely related to PSV-Mi, distantly related to PSV-E. The molecular weight of PSV-R coat protein is about 26000D. PSV-R has four main components of RNAs with a small RNAs.

Detection of group B rotavirus by RT-PCR

HAN Xin-Bing, ZHANG Fu-Ping, WANG Zheng-Dang, HUANG Jia-Si

1998, 13(3): 279

A diagnostic assay was presented to detect group B rotavirus (GBRV) in fecal specimens with reverse transcription polymerase chain reaction (RT PCR). GBRV double strand RNAs (dsRNA) isolated from stool samples were reverse transcribed and amplified by PCR using two oligonucleotide primers which were derived from genomic segment 3 of the IDIR (intestinal disease of infants rat) strain of GBRV. This RT PCR assay permitted the sensitive and specific detection of a variety of GBRV in fecal specimens. Wild GBRV strains of lamb, kid were detected with these primer pairs by RT PCR. Moreover, RT PCR also permitted the detection of genomic RNA of lamb GBRV KB 63 strain which has been adapted to serial passage in cattle. The specific PCR products of 290 bp suggested that GBRV from lamb, kid and calf had identical sequences in genomic RNA;