2005 Vol.20(6)
Using hydrodynamic injection. pUC18-HBV1. 3 containing a replication-competent, HBV1.3 supergenomic DNA, was injected into BALB/cJ mice through tail vain. HBV viremia was measured by real-time PCR. Serum concentrations of HBsAg were detected by sandwich ELISA HBeAg was detected and quantified by RIA using HBcAg standards to generate. Antibodies specific for HBsAg, and HBcAg were assayed by endpoint titration ELISA. HBV core protein expressed in the liver was visualized by immunohistochemical staining. Viral RNA and replicative DNA intermediates in liver were detected by Northern and Southern blot analyses, respectively. Hydrodynamic injection of pUC18-HBV1. 3 lead to viral gene expression and replication in vivo . The immune response to HBV. which was comparable with the characteristic response during acute HBV infection in man, was elicited in mice. A mouse model of acute hepatitis B virus infection can be developed by transfection of hepatocytes in vivo .
Hepadnavirus surface, core antigen and c genes and their products which may exist in liver tissues of Marmots himalayana were detected by immunohistochemical staining and in situ hybridization, respectively. The data suggested that hepadnavirus surface antigens were detected in the liver of 13 out of 50 animals (26%) and the core antigen in 18 out of 50 animals (36%). Some of the samples were proven positive by dejection of the c gene by in situ hybridization. The core antigen was located in the cytoplasm and/or nucleus of liver cells and had multiple appearance such as scatterd, clustered or diffused. The C antigen was located in a scattered manner in the nucleus of liver cells. Mild inflammation was observes in 5 liver sections and did not correlate to antigen expression. Using histological detection methods of Woodchuck hepatitis virus (WHV), we demonstrated that a hepadnavirus similar to WHV infection may exist in Marmota himalayana in Qinhai, China. Marmota himalayana may be a potential animal model to s
A recombinant plasmid named pCI-HBs145 containing a fragment of G145R mutate S gene of Hepatitis B virus was constructed. Seven HeLa cell lines stablely expressing the protein of G145R HBsAg were developed by G418 selection after transfecting with the PCI-HBs145. Biological characteristics of one of the 7 cell lines, namely, 2A8 were studied. The immunoreactivity of G145R HBsAg expressed by 2A8 was different from wild type HBsAg. The genetic stability and purity of the cell population were confirmed. The kinetics and optimal conditions for mutation HBsAg secretion from 2A8 cells were studied.
To investigate the effect of chemokine gene immunization on immune responses induced by HIV-1 envelope protein gene vaccine and to explore new strategies against HIV, Balb/c mice were immunized with pVAX1GP120 alone or co-administered with the DNA encoding for RANTES、and MIP-1α. Their sera were collected for analyzing anti-HIV antibody and IFN-γ by ELISA, and splenocytes of Balb/c mice were isolated for detecting antigen-specific lymphoprolif-erative responses and specific CTL response by MTT and LDH assays, respectively. Our results showed that the anti-HIV antibody tilers of mice co-immunized with pVAX1GP120 and the DNA encoding for RANTES and MIP-1α were higher than that of mice immunized with pVAX1GP120 alone (P0. 01). The IFN-γ level of mice co-immunized with pVAX1GP120 and the DNA encoding for RANTES and MIP-1α was higher than that of mice immunized with pVAX1GP120 alone (P0. 01). In comparison with mice injected with pVAX1GP120 alone, the specific CTL cyto-toxity activity and antigen-specific lymphop
The gene encoding the N protein of SARS virus was cloned and expressed in E. coli. The re-combinant protein was identified by anti-SARS positive sera obtained from recovered SARS patients. The recombinant N protein (55 kDa) reacted specifically with anti-SARS positive sera. The recombinant N protein was purified and used to immunize horses, and the antibody was detected. The recombinant protein of SARS virus displayed specific reaction with SARS antibody. The results showed that the horses immunized with this recombinant protein produced higher antibody titers (1:2560). Our results provide information on the immunization against the SARS virus.
The etiology of respiratory tract infection (RTI) pathogens, e. g. Human respiratory syncytial virus A and B (RSVA, RSVB), Severe acute respiratory syndrome coronavirus (SARS-Cov), Influenza A virus and Influenza B virus (INFa, INFb), Influenza A virPara-influenza virus 1 and 3 (PIV1、PIV3), M. pneumoniae (MPN) and C. pneumoniae (CPN), were investigated using Multi-Analyte Suspension Array (MASA) technology. Samples were collected from throat swabs of 140 children symptomatic of typical respiratory tract infection. Ninety-five samples (67. 85%) were positive for at least one of the above pathogens. The infection rate was 35. 71% for RSVB, 4. 29% for PIV3, 28. 57% for INFa, 2. 14% for INFb, 3. 57% for MPN and 17. 86% for CPN. The ratio of mixed infection was 17. 14%. RSVA、PIV1、SARS were not detected. The infection rate of RSVB in children younger than 3 years was higher than that for children older than 3 years, while, IFNa in children younger than 3 years was lower than that of children older than 3 years. The
Genes encoding nucleocapsid (N) and envelope (E) proteins of SARS coronavirus (SARS-CoV) were obtained by RT-PCR and were cloned into the expression vector pGEX-KG. The genes were expressed in E. coli and the recombinant proteins were in a soluble form. The proteins were purified by affinity chromatography. The recombinant N protein reacted specifically with anti-SARS antibody. The results may provide a good tool for further research of immune responses and early diagnosis of SARS virus.
Host genetic factors, such as human leukocyte antigen (HLA) alleles, are important in Human immunodeficiency virus ( HIV) infection and its progression to AIDS. HLA class I genes, especially highly polymorphic HLA-B genes, are involved in the activation of HLA-restricted cytotoxic T lymphocytes (CTLs) against HIV, and thus control susceptibility to or protect against this virus. The present study was aimed to determine the distribution of HLA-B alleles in the Chinese Uygur ethnic group and its association with HIV infection. One hundred ten healthy control (HIV negative) and 128 HIV positive Chinese Xinjiang Uygur ethnic individuals were used in this study. HLA typing for B allele was performed by polymerase chain reaction (PCR) with sequence-specific primers (SSP). Hardy-Weinberg equilibrium was calculated using POPGENE software for the healthy control group. The HLA-B frequency of each alleic was compared between the patients and the controls using the chi-square test. In HIV-1-pos-itive group, gene frequen
The objective of this study was to determine the relationship between the nucleic acid and antibody of HIV/HCV in a high-risk population. Plasma samples (320) were collected from drug users in Xinjiang and the nucleic acid and antibody of HIV/HCV were detected. A total of 38% of the subjects were positive for both HIV and HCV. The positive rate of HIV antibody was 41. 9%, and the relative coincident rate of HIV RNA and antibody was 98. 9%. HIV RNA was detected in 2 cases out of 186 HIV antibody negative cases. The positive rate of HCV antibody was 80. 3% , and the positive accordant rate of HCV RNA and antibody is 92. 6%, the general accordant rate of those is 90. 0%. These results indicated that HIV/HCV infection during the so-called window period can be found by the nucleic acid assay in high-risk areas, however, 8% of HCV antibody positive cases with negative HCV RNA requires further investigations.
A multiplex PCR(mPCR) assay was developed and evaluated for its effectiveness as a means to simultaneously detect multiple viral infection of swine . Specific primers for each of the three common DNA viruses, Pseudorabies virus(PRV) , Porcine parvovirus ( PPV) and Porcine circovirus type 2(PCV2) were used to test the procedure, Four specific bands of 269bp(PCV2), 583 bp(PPV) 372 bp(PRV gB)and147 bp(PRV gE)were amplified. The assay proved to be sensitive when a composite of all three viruses were amplified, including both field and gene-deleted permutations of PRV. No specific band was amplified from other pathogenic viruses and bacteria. As little as 10 pg PCV2, 10-6.2 TCID50 PPV,10-3.8 TCID50\PRV gB and 10-5.8 TCID50 PRV gE were detected in this mPCR. This method could effectively detect infection of PCV2, PPV, field and gene-deleted permutations of PRV from clinical samples.
Three recombinant fowlpox viruses: rFPV-H5HA-IL18、rFPV-H5HA-H7HA-IL18 and rFPV-H5HA were administered to one-day-old specific pathogen free (SPF) chicken and seven-day-old commercial Leghorn egg laying chicken by wing web route. At 7, 14 and 21 days post infect, hemagglutination inhibition (HI) antibody titer and nonspecific cellular immunity level were quantified. The results showed all rFPV-vaccinated groups produced HI antibody, and cellular immunity levels induced by rFPV-H5HA-IL18 and rFPV-H5HA-H7HA-IL18 strains were significantly higher than that induced by rFPV-H5HA. At 21days post-inoculation, immunized SPF chicken and commercial Leghorn egg laying chicken were challenged with H5N1 HPAIV. rFPV-H5HA-IL18 and rFPV-H5HA-H7HA-IL18 strains could induce 10/10 protection against challenge with HPAIV. rFPV-H5HA strain induced 90% protection. For immunized egg laying chicken groups, cloacal swabbing samples were collected at 7 days post challenge, and no shedding was found in groups vaccinated with rFPV-H5HA-I
Inhibitory effects of sodium selenite, DL-selenomethionine and Kappa-selenocarrag-eenan on Porcine parvovirus (PPV) infection of the PK-15 cells were studied. The influences of reduced glutathione (GSH) and D-mannitol on antiviral effects of selenium were also carried out in PK-15 cells. The results showed that all of the three selenium sources have an inhibitory effect on the PPV replication in vitro. DL-selenomethionine had the highest inhibitory effect while Kap-pa-selenocarrageenan had the lowest, and the inhibition was concentration-dependent. The GSH and D-mannitol have the ability to increase the antiviral effects of selenium. The antiviral effect was enhanced with the addition of both GSH and D-mannitol.
To screen out vaccine candidate against PRRSV, adenovirus vector was used to express the M protein of Porcine reproductive and respiratory syndrome virus (PRRSV). A recombinant adenovirus was constructed and the expression of the M protein was identified by RT-PCR and indirect immunofluorescence assay (IFA). The purified recombinant adenovirus M (rAd-M) was passaged 25 times in 293A cells. The titer of stocks of rAd-M was stably 107.8\CID50/mL. Furthermore, the recombinant M protein adenovirus could induce PRRSV specific humoral immunity and cell mediated immunity in mice. It indicated that the recombinant adenovirus expressing the main structural proteins of PRRSV is a potentially viable candidate vaccine against PRRSV.
Sandwich ELISA with neutralizing McAbs against rabies virus glycoprotein can be used as a substitutive method to the NIH test. Kunming mice (12-14g) were inoculated ip with different dilution of rabies vaccines two times for NIH test (M-NIH), then challenged with CVS strain of rabies virus. At the same time, glycoprotein of rabies vaccines were tested with sandwich ELISA. A reference vaccine whose efficacy had been known was also tested with sandwich ELISA. Analysis of the data indicated that there was a significant positive correlation (0. 98) between E-NIHs and relevant M-NIHs of rabies vaccines. The range of E-NIHs resulted from one batch of rabies vaccine was between 2. 41 and 5. 85 while the range of M-NIH was between 5. 11 and 10. 19. There was a significant linear relation between E-NIHs and M-NIHs of rabies vaccines. E-NIHs showed a better reproducibility, lower cost and faster than M-NIHs. ELISA is a potential substitute of rabies vaccine NIH test.
Two earlier Chinese isolates of bovine viral diarrhea virus , bovine CC-184 and pig ZM-95 were phylogenetically analyzed. The E2 gene encoding a major antigenic was obtained by reverse transcription polymerase chain reaction (RT-PCR) and nest-PCR. Sequencing results showed that the E2 genes of CC-184 and ZM-95 are 1122 and 1125 nucleotides in length, encoding 374 and 375 amino acids, respectively. Sequence comparison and phylogenetic analyses demonstrated that both isolates fall into the BVDV-1 group and distant from other Pestiviruses such as BVDV-2, Border disease virus (BDV) and Classical swine fever virus (CSFV). The E2 gene of CC-184 had the highest identity (91.8%) with its homologue in the Osloss strain. However, the E2 gene of ZM-95 is significantly divergent from other known Pestiviruses, with the highest sequence identity of 72. 4% to strain Oregon c24v. Furthermore, isolate ZM-95 addressed significant evolution distance compared with the other established subgroups of BVDV-1, as indicated by phylog
A virus isolate from domestic chicken agglutinated chicken erythrocytes and was found as globular enveloped virion of 90nm-100nm diameters under TEM. The isolate was identified as H7N2 Avian influenza virus(AIV) by HI and NI assays and designated as A/Chicken/Hebei/1/ 2002(H7N2,or briefly as CK/HB/1/02. After inoculating to SPF chicken, the virus was recovered from cloacal swabs and the antibody to H7 was detected at 7 days post-infection (DPI). The IVPI was 0. 00 and postmortem examination showed hemorrhages in several tissues and organs indicating that the virus was LPAIV. HA gene of the isolate exhibited 99. 4% nucleotide sequence identity to A/Afri. Star. /Eng-Q/79(H7N1) virus, 96. 8%-98. 2% to H7N2 virus isolated from Italy and Israel, and only about 81. 0% to American H7N2 Strain. The amino acid at the cleavage site of HA is -KGR-GLF-, which implies the isolate should be of low pathogenicity.
In order to construct a recombinant canine adenovirus type 2 (CAV-2) of the VP2 of Feline distemper virus or Feline panleukopenia virus (FPV), the vp2 gene fragment of FPV GT-2 strain was amplified by PCR and cloned into pVAX1 vector. The complete VP2 expression cassette was subcloned into the shuttle vector pVAXΔE3 and then inserted into the pPoly2-CAV-2 backbone vector that contains the complete genome of CAV-2. Recombinant viral genome was linearized by ClaI/AscI and transfected into MDCK cell. The recombinant CAV-2-VP2 was a-chieved through 4 passages in MDCK, which showed typical cytopathogenic effect (CPE). The expressed FPV VP-2 was detected in the infected MDCK cells of the recombinant CAV-2-VP-2 by using FPV polyclonal antibodies. The specific antibodies of VP-2 and CAV-2 were induced in cats by the recombinant CAV-2-VP-2. The results indicated that the recombinant CAV-2-VP-2 may have the potential to be used as a FPV vaccine strain.
A novel flow cytometric assay for evaluating Equine infectious anemia virus (EIAV) antigen-specific CTL responses was established and by using PKH-26 and CFSE as cell colouration dyes. Compared with the traditional 51Cr -release assay, the new method is more sensitive, has a decrease background and avoids the risk of radioactive exposure to workers. The cellular immuno-response against EIAV infection was detected using this method. The maturation of EIAV antigen-specific CTL response was characterized. The result showed that the CTL response reached a maximum at three months and remained relatively high. The novel method is directly applicable to research on the immune mechanism of the EIAV attenuated vaccine.
Eight Spring Viraemia Viruses (SVCV) have been isolated from cultured carp and koi in China between 2002 to 2004. Based on the complete genome of SVCV reference strain, primers were designed and the glycoprotein genes from the 8 isolates were amplified, cloned and se-quenced. These genes were compared with other SVCVs in GenBank and showed nucleotide identities of more than 92%. There was more than 97. 7% nucleotide identities within the eight isolates. The homology was more than 94. 5% among the deduced amino acid sequences of the 8 isolates and 92. 0-94. 0% with other SVCV isolates. Phylogenetic analysis showed that the isolates shared evolutionary direction with USA isolates, and were different from other SVCV isolates from European countries. Nineteen common enzyme sites were found in the 8 isolates. Ten hy-drophobic regions, 10 possible antigenic sites and 10 transmembrane domains were also searched by the software and showed almost the same patterns. The functional sites include MYRISTYL, RGD, CK2-PHOSP
Tumor necrosis factor receptor (TNFR) plays an important role in the evasion of immune response by large DNA viruses. TNFR is a homologue of cellular receptors. Lymphocystis disease virus, an iriduvirus, isolated in China (LCDV-C) is a large DNA virus. Primers were designed based on conserved TNFR nucleotide sequences in iridoviruses and used to amplify the homologue in LCDV-C. A DNA fragment of 834 bp was generated and sequenced. The recombi-nant prokaryotic expression plasmid containing this fragment was constructed and expressed in E. coli. DE3. An expected 45kDa fusion protein was isolated by SDS-PAGE. Computer analysis indicated that LCDV-C TNFR homologue encodes a 278aa putative protein, which contains a typical cysteine-rich domain. It shares 34% identity with flounder TNFRII.
The activity of two isolates of Spotoptera exigua nucleopolyhedrovirus , SeMNPV-M and SeMNPV-Z, was compared by bioassays. The LD50 values of SeMNPV-M and SeMNPV-Z in 3rd instar host larvae were 195. 8 PIBs/ gram diet and 242. 4 PIBs/gram diet, respectively. The LT50 values infected with 6000PIBs/gram diet were 3. 50 and 3. 68 days, respectively. The viral suspension formulation pesticide with SeMNPV-Z was produced and in field trials, the SeMNPV pesticide proved to be an effective control agent.
Primers based on published sequences of granulin genes were synthesized and used to amplify the granulin gene from Clostera anachoreta granulovirus (ClanGV). The generated product was se-quenced. Sequence analysis indicated the entire ORF of ClanGV granulin gene was 747bp, encoding a protein of 248 aa with an estimated molecular weight of 29. 3kDa. A bacluloviral late promoter was present at 24 bp upstream of the ATG condon. Two TATA boxes were at 26 and 65bp upstream of the ATG codon, respectively. Phylogenetic analysis indicated that ClanGV was closely related to Adoxophyes ora-na granulovirus (AoGV)、Cydia pomonella granulovirus (CpGV)、Choristoneura fumiferana granulovirus (CfGV)、and Phthorimaea operculella granulovirus (PoGV). Antibodies prepared against the protein were used in Western blot analysis and indicated that ClanGV granulin reacted positively with occlusion body proten from HaSNPV and CaLGV and reacted weakly with polyhedrin of SeMNPV.
The major pathological changes of Pieris rapae infected by a new nodavirus (PrNV) and a mixture of PrNV and Pieris rapae Granulosis virus (PiraGV) were studied by electron microscopy and histochemisty. The results showed that PrNV replicated only in the cytoplasm of larval midgut cells. After infection, prominent changes took place in the organelles of permissive cell, such as mitochondrion, endoplasmic recticulum and ribosome. The PrNV assembly was also studied.
根据GenBank中VT1、VT2毒素的基因序列设计合成2对引物,以大肠杆菌O157:H7菌株DNA为模板,扩 增vt1、vt2。诱导只扩增出vt2的菌株释放噬菌体,利用多种指示菌经双层琼脂平板法来分离纯化VT2噬菌体,观 察噬菌斑的特征,提纯病毒粒子进行电镜观察,并对噬菌体中vt2基因检测、克隆和序列分析。结果显示VT2噬菌 体感染MC1061在双层琼脂平板上形成的噬菌斑小而混浊,多呈磨玻璃样;而首次感染大肠杆菌CC118(λpir),此 后用MC1061分离的噬菌体,再以MC1061为指示菌,在双层琼脂平板上形成小而清晰透明的噬菌斑。电镜下噬 菌体头部呈六边形外廓,尾部细长无尾鞘结构。以噬菌体DNA为模板进行PCR扩增,检测到vt2特异性DNA 带,克隆的vt2基因序列与GenBank中编码VT2毒素的核苷酸序列(X07865,NC_002655,BA000007,AF291819) 的同源性分别达到99％,确定编码VT2毒素的基因位于噬菌体上,并获得VT2噬菌体(?)HY。
The development of effective strategies against cervical cancer require knowledge on the prevalence and molecular biology of the Human papillomavirus (HPV). We have investigated HPV infection in 60 women suffering from cervical cancer. HPV was typed by PCR of DNA extracts. The prevalence of cervical HPV 59 infection was 5% (3/60). We also characterized E6 region nucleotide variation by PCR-directed sequencing. 1 of 3 cases showed 4 sites variations (T102C, C306T, A363C and T402C) in E6 region.
