1997 Vol.12(2)
In the present study, with a new foreign epitope - presenting system, FHV - RNA2 carrier system, on the precursor capsid protein of an insect virus, flock house virus (FHV) as a carrier, in recombinant baculovirus system and in pET system three epitopes derived from the amino acid residues surrounding the trypsin cleavage site(s) of SAll Vp4 (epitope A, aa 223 to 242;epitope'B, aa 243 to 262; and epitope C, aa 234 to 251 ) was constructed and expressed. The ability of the three epitopes inducing neutralizing antibodies in animals was studies. The results showed that these three epitopes induced production of seral antibodies and neutralizing anbodies in guinea pigs. These findings suggested that the amino acid sequences surrounding the trypsin cleavage site (s) on SAll Vp4 have great significance in the development of epitope - based recombinant ro-tavirus subunit vaccines.Keywoeds: Rotavirus Vp4, Trypsin cleavage site, Epitopes, Neutralizing antibodies, FHV RNA2 carrier system
Highly reiterated Sequence AGMr (Hind Ⅲ)-l was isolated from genome of Vero cell line derived from African green monkey and cloned into plasmid pUC18. Sequence analysis confirmed that the insert Sequence is right but 6 out of 172 bp had mutated in the 165th generation of Vero cell. Southern Blot and Dot Blot analysis of rabies vaccine prepared by Vero cell showed that residual cell DNA(RC DNA) displayed heterogeneous size from 400 ～ 5000 bp, and that the amount of RCDNA can be determined specifically and sensitively (minimun 4 pg). The fact that AGMr (Hind Ⅲ )-l Sequence represents about 20% of the genome DNA and repeats 6.8 × 106times and is rare in Primates except in members of the same genus also suggested that the recombinant plasmid could be applied as probe for detecting RC DNA existed in vaccine prepared by Vero cell and maybe used for identifying test of Vero cell.
In order to observe further dynamic changes on proliferation of HFRSV in Leptotrombidium Scutellare, the raised Leptotrombidium scutellare were ground and sterilized with an interval of 20-40 days, the filterate of each batch was inoculated separately into Vero - E6 cells, titres of HFRSV and HFRSV-RNA were detected. The results confirmed that HFRSV were positive in most batches of larva. The above results indicated that the Leptotrombidlum sutellare is a transmitting vector of HFRSV.
Hantavirus (HV) has a tripartite, single - stranded, negative - sense RNA genome. Two pairs of oligonucleotides flanking S segment were chosen as primers for reverse transcription Polymerase chain reaction(RT - nested PCR). The method of RT - nested PCR was used for detection of virus RNA in HV infected Vero E6 cells and clinical serum specimens from 35 hemorrhagic fever with renal syndrome(HFRS) patients, with HV gene S segment plasmid cDNA as postive control,and virus RNA was extracted by Acid Guanidinium Thiocyanate - Phenol - Chloroform Single- Step Method. The results showed that all the patients' serum samples and HV infected Vero E6 cells were positive, on the other hand, all the twenty normal serum samples and Vero E6 cells were negative. It suggested that RT - nested PCR was sensitive, specific, rapid and simple, and can be applied for study on the pathogenesis of HFRS in molecular level and early clinical diagnosis
The plasmid p CMVCVSRG containing the full- length cDNA of G protein of rabies virus CVS strain was constructed and transiently transfected into NIH 3T3 cell. The expression of Gprotein in transfected NIH 3T3 was confirmed by indirect immunfluorescent(IIF) and alkaline phosphatase anti-alkaline phosphatase (APAAP ) methods. The mice were inoculated withpCMVCVSRG, pSV2X3CVSRG, pCMV, pSV2X3, respectively. Only mice immuned with the G gene-containing vectors developed neutralizing antibody to rabies virus. After rabies virus challenge, the survival rates of DNA vaccine inoculated group have very significant difference comparing with negative control group (p < 0.01 ). The immune responses to the DNA vaccines containing different promoters(from CMV or SV40) and different G protein genes(from CVS strain or ERA strain) were compared and it was found to be similar in magnitude. The specific glycoprotein gene was detectable by PCR at least 120 days postinjection.
A hammerhead structure ribozyme was designed and synthesized to potato leafroll virus Chinese isolate (PLRV - Ch) genome within 356 to 358 nt "GUC" in coat protein (CP) gene. DNA sequence encoding the ribozyme was inserted into the position downstream from SP6 promoter of in vitro transcription vector pSPT19. The target PLRV CP gene cDNA was subcloned in vector pSPT18. The RNA molecules of riboZyme and target Sequence were generated by transcription with SP6 RNA polymerase in vitro. After cleavage reaction at 41℃, two.expected short RNA fragments were observed.
A near full - length cDNA library of Wheat Rosette Stunt Virus (WRSV) was constructed.Purified viral RNA was polyadenylated by E. coli poly(A) polymerase. The poly(A) - tailed RNA was used as a template complementary to oligo dT(12 - 18) primer for cDNA synthesis.The cDNA was separately digested with restriction enzymes Pst I and EcoR I/Sma I. The pUC19 plasmid was digested with the same enzymes to poduce the both cohesive - ends, or one blunt and the other cohesive - ends respectively. Ten inserts with different sizes were obtained and the total size of them reached approximately 14 kb. One insert of near 6 kb length was identified as the insert containing the N protein gene and covering the Ns, M, G, genes as well as a part of L gene.The cDNA inserts with orientated - direction were sequenced and the sequence was compared to 5' and 3' ends of the genomic RNAs of two other plant rhabdoviruses, SYNV and LNYV using computer assisted searching method with the Software Sequence Analysis Version 3. 00.
With electron microscope, a kind of spherovirus was observed in plasma of penaeid shrimp Puns Chinensis at different stages, such as eggs, larvae and juvenile shrimps. The artificial infection experiment verified that. the spheroviral particle has infectivity. The offsprings coming from carrying virus parental shrimp were infected generally in the separated breeding condition. A kind of spherovirus - like particle was found out in just spawned eggs. The result showed that the spherovirus probablly can be transmitted vertically.
Three viruses were isolated from the kidney cell culture of Tupaia belangeri (tree shrew).They would like to replicate in mitosis of TL cells, to produce CPE and hemagglutinin and to hemaggulate the erythrocytes of guinea pig and mouse. The viruses belong to one serum type by hemagglutination inhibition test. The virus antigen appeared first at the nuclei by immunoenzymatic stain. The morphology of the virus was similar to round and hexagonal without capsule, diameter 30 nm by negative stain under electron microscope. Most number of sera in tree shrews appeared the virus antibody by serological assay. It was proved that the virus was a latent virus in tree shrews and identified preliminarily as parvo- like virus.
With electron microscopy techniques the major virus pathogens of artificially infected Penaeus chinensis were studied. It showed that there are two kinds of virus pathogens in diseased shrimps: spherical virus and baculovirus. Sometimes they were co - existing in the same tissue or even in the same cell. It suggested that a mixed infection maybe occurred. Using immunogold labelling method to label the spherical viruses appearing in the cytoplasm of diseased shrimp, preliminary results showed that the purified spherical viruses were the same as those observed in the infected cells. During the infection, prominent changes took place in main cell organelles of the host cell, such as mitochondrias, endoplasmic reticulum, and ribosome. Especially in late infection, a lot of lysosomes and various multilayered membrane structures were found, some nuclei were wrapped up by some microtubule structures.
Titrating infective virus by plaque assay is one of the most accurate methods which could determind the numbers of infective virus. The result shows that a considerable number of vaccinia viruses in inoculum were still unadsorbed to cv - 1 cell monolayer after a period of 4 hours adsorption time. A reciprocal and unlinear relationship between the titer of virus and the volume of inoculum, the volume of liquid medium was detected. According to this relationship, several new plaque assay methods, which had the advantage over routine method, were designed. By means of electronic computer equipped with mathematical software package, the curve fitting model and the surface fitting model of titer with the volume of virus inoculum and the volume of medium had been established. By curve fitting model, the extrapolated value of vaccinia virus titers was obtained five fold higher than the titers by routine method.
Hierarchical clustering analysis have been done on 48 strains of the representative insects inclusion body viruses (26 strains of nuclear polyhedrosls viruses, 13 strains of granulosis viruses and 9 strains of cytoplasmic polyhedrosis viruses) by capillary gas chromatography, using the eight strategies of hierarchical clustering of Euclidean distance coefficient. A comparison between the dendrograms obtained by these strategies has been made. The results showed that there are definite discriminations between baculovirus group (genus) and cytoplasmic polyhedrosis virus group (genus). The relationships among the genera, groups, subgroups and strains could clearly be unfolded. The similarities and differences among strains also be distinguished. The average method, flexible method （β=0.00）, and group average method in eight strategies of hierarchical clustering had the advantage of other strategies.
After infection with Trichoplusia ni nuclear polyhedrosis viruses, the esterases in the hemolymph of the fifth instar larvae of the Argyrogramma agnata were examined by the polyacrylamide gel electrophoresis (PAGE) and thin - layer scanning. The results showed that TnNPV can inhibite the metabolism of the esterases at the early stages and the effects of heterologous NPV were different from those of homlogous NPV.
