通过使用血清3型鸭甲型肝炎疫苗毒免疫SPF鸡,制备血清3型鸭甲型肝炎诊断用标准阳性血清,对制备血清进行无菌、支原体和特异性检验以及效价测定,并用该抗血清进行鸭病毒性肝炎治疗试验.结果表明,该血清无菌检验合格,无支原体污染,特异性良好,无禽类常见13种外源病毒抗体,血清中和效价为10-2.8.发病雏鸭紧急治疗保护率为91.67％,而与血清1型鸭甲型肝炎病毒(DHAV-1)无交叉保护.

In order to develop a positive serum for the differential diagnosis of duck hepatitis A virus, SPF chickens were inoculated with serum type 3 duck hepatitis vaccine, and a series of tests have been done for the serum acquired form those immune SPF chickens, including sterility test, mycoplasma test, specific test, and neutralization titer test. The results showed that the antiserum was specific for the serum type 3 duck hepatitis A virus, without 13 types of avian common exogenous virus antibody, and free of bacterial and mycoplasma contamination, the neutralization titer was as high as 10-2.8 . The research provided material for serological identification and related research of duck hepatitis A virus.

In order to develop a real-time quantitative PCR to detect duck hepatitis A virus serotype 1 ( DHAV-1) , a pair of primers and one TaqMan probe were designed and synthesized, according to the 5 ’ untranslated sequences of DHAV-1 published on GenBank, The recombinant plasmid was built as a standard control for the method, the specificity, sensitivity, and repeatability of the method were determined, and also preliminarily applicated in the detection of clinical samples. The results showed that the detection assay was specific for DHAV-1, and there was no cross reaction between duck hepatitis A virus serotype 3( DHAV-3) and the other viruses including duck plague virus, Newcastle disease virus, avian influenza virus, duck reovirus, avian infectious bronchitis virus, etc. The method also showed high sensitivity, as low as 10 copies virus could be detected for each reaction, and good reproducibility, there was a coefficient of variations less than 3% for both inter-assay and intra-assay. The detection results of clinical samples were consistent with the sequencing. These results indicated that the developed TaqMan fluorescence quantitative PCR assay had the advantages of good specificity, sensitivity and repeatability;it was useful for the rapid diagnosis and quantification analysis of DHAV-1.

通过对GenBank公布的鸭甲型肝炎病毒全序列比对,在3型鸭甲肝炎病毒（DHVA-3）5’非编码区的保守区,设计了一对检测引物和一条特异性TaqMan探针,以构建的阳性重组质粒经体外转录合成的RNA作为标准品绘制标准曲线,建立了一种快速检测DHAV-3的实时荧光定量RT-PCR方法。该方法能特异性地检测出DHAV-3,而与血清1型鸭肝炎病毒（DHAV-1）、鸭瘟病毒、新城疫病毒、禽流感病毒（H9）、呼肠孤病毒、传染性支气管炎病毒无交叉反应。在1.8×103～1.8×108copies/μL的检测范围内标准曲线线性关系较好,R2为0.992。敏感性试验表明,最低检测限为36拷贝数RNA。用该方法和胚半数致死量法对尿囊液中病毒含量进行检测,表明二种方法检测结果呈正相关。本研究所建立的检测方法特异性好,灵敏度高,且操作方便,可为该病毒的快速诊断和流行病学调查提供技术手段。

The duck viral hepatitis( DVH) live vaccines and inactivated vaccines are the most widely used vaccines for the DVH at present,and there are also some developments in the research of duck viral hepatitis genetic engineering vaccines. In this paper we briefly reviewed the progress on DVH vaccine in order to offer some guides for the further research of DVH vaccine.

In this study,the M gene of Porcine reproductive and respiratory syndrome virus(PRRSV) CH-1a strain was amplified from the PRRSV genome by RT-PCR and cloned into the plasmid pSC11.The recombinant plasmid pSC11-PRRSV-M was transfected into TK-143 cells infected with vaccinia virus WR strain.Recombinants vaccinia virus of rWR-PRRSV-M was obtained by selecting blue plaques on the TK-143 cells overlaid with agar containing X-gal,and confirmed by PCR.The M gene expressed in TK-143 cells were identified by SDS-PAGE and confirmed by immunofluorescence assay(IFA).The rWR-PRRSV-M recombinant virus could induce specific humoral immune responses against PRRSV in the immunized mice.

OBJECTIVE To evaluate the effects of the fusion gene of ubiquitin (Ub) and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) M gene on the immune response in inoculated mice. METHODS Mouse Ub gene and PRRSV M gene were amplified by RT-PCR from BALB/c mice spleen cells and PRRSV Ch-1a strain, respectively, and the M and Ub gene (U-M) was fused by SOE PCR. Therefore, pVAX1-U-M and pVAX1-M recombinant plasmid were constructed for eukaryotic expression. RESULTS The fusion U-M and M protein expressions were verified in transfected BHK-21 cells by indirect fluorescence assay. Furthermore, both pVAX1-M and pVAX1-U-M induced specific humoral and cellar immune responses against PRRSV in the recombinant plasmid injected mice. However, pVAX1-U-M was able to induce higher level of T cell response then that of pVAX1-M (P<0.05), but lower level of antibody (P<0.05). CONCLUSION Expression of U-M fusion gene had ability to enhance specific T cell response against PRRSV, but no effect on stimulation of humoral response in inoculated mice.

Objective To determine the gene-chip sensitivity on detecting zoonotic viruses,and found a basis for large scale screening and identifying zoonotic viruses by gene-chip technique.Methods Japanese encephalitis virus(JEV) of genus Flavivirus was chosen as the model to determine the sensitivity of the gene-chip.The virus genome was amplified by random PCR,and the PCR products were then labeled with fluorescence dye and hybridized with zoonotic viruses gene-chip.Two methods were employed to determine the sensitivity of the gene-chip.One was to determine the minimal quantity of random PCR products,and the other was to determine the minimal TCID50.Finally,the sensitivity of zoonotic virus gene-chip was also compared with that of specific RT-PCR.Results The minimal quantity of random PCR products detected by the zoonotic virus gene-chip was 300ng,and the detective limitation of virus titer was 10 folds of TCID50 dilution,i.e.105 TCID50/200μl.The sensitivity of the gene-chip was equivalent to that of specific RT-PCR.Conclusion The zoonotic virus gene-chip achieves a high sensitivity,indicating that to establish a large scale screening for identifying zoonitic viruses by gene-chip-based detection is feasible and practical.

Some highly pathogenic viruses, such as Chikungunya virus, Japanese encephalitis virus, Yellow fever virus, Dengue virus, Hanta virus, SARS‐CoV, and H5N1 avian influenza virus can cause severe infectious diseases. However, the consensus method for detecting these viruses has not been well established. A rapid and sensitive microarray approach for detection of these viruses and a panel of specific probes covering nine genera and 16 virus species were designed. 70‐mer oligonucleotides were used at the genus level and 50‐mer oligonucleotides were at the species level, respectively. To decrease the interference of the host genome in hybridization, the consensus genus primers were designed and used to reverse transcribe only virus genome. The synthesis of the second strand was carried out with a random primer sequence (5′‐GTTTCCCAGTAGGTCTCNNNNNNNN‐3′). The amplified products were labeled and processed for microarray analyses. This microarray‐based method used the highly conserved consensus primers to synthesize specifically the virus cDNA and could identify effectively Chikungunya virus, Japanese encephalitis virus, Yellow fever virus, Dengue virus, Tick borne encephalitis virus, and H5N1 avian influenza virus. Using this method, one unknown virus isolated from pig brain in Shanxi Province, China was identified. This method may have an important potential application for the diagnosis of virus infection. J. Med. Virol. 81:1945–1950, 2009. © 2009 Wiley‐Liss, Inc.

To screen and identify 25 zoonotic viruses,300 viral oligonucleotide microarray probes were designed and validated by bioinformatic analyses.Random amplification of viral sequences were explored and the microarray hybridization conditions were optimized.The specificity and sensitivity of the customed microarray were evaluated with cell-cultivated viruses and simulated clinical samples.Before hybridization,the microarray was blocked with 0.25% NaBH_4.51 ℃,2 hour incubation and 50% formamide were applied for the optimal hybridization.All the 25 important zoonotic viruses were successfully identified by our screening in given samples.The results indicated that the anchored random PCR amplification of viral oligonucleotide was suitable for the microarray assays to detect zoonotic viruses.

Objective: To label equine IFN-γ monoclonal antibody (McAb) with fluorescein isothiocyanate (FITC) and provide an experimental material for intracellular cytokine staining (ICS) assay of IFN-γ in equine lymphocytes. Methods: Purified equine IFN-γ McAb SB10 was labeled with FITC, then diluted serially and used for ICS assay of IFN-γ in equine PBMCs by flow cytometry. The result was compared with that of flow cytometry by using commercial FITC-labeled bovine IFN-γ McAb CC302. Results: When 106 PBMCs were stained with 1 μl of FITC-CC302, the frequency of PBMCs secreting IFN-γ was 9. 35%. However, when 106 PBMCs were stained with 0. 25 μl of FITC-SB10, the frequency was 9. 90%. Conclusion: The prepared FITC-labeled equine IFN-γ McAb laid a material foundation of developing the methods for immunological study on equine cells and provided a technical platform for monitoring the immune state and investigating the immunological mechanism.

To develop a multiplex RT-PCR method for simultaneous detection of RVFV,HEV,RV,VSV and FMDV,a set of specific primers were designed based on the published sequences.PCR products of 499 bp,137 bp,274 bp,224 bp and 389 bp were obtained for the respective viruses.The specificity of this method was evaluated by testing representative viruses in the same family and related zoonosis viruses and no PCR products were detected for NDV,AKAV,BEFV and JBEV.This method has a sensitivity of detecting as low as 2.91 ×104 (RVFV),3.14 ×103 (HEV),1.25 ×103 (RV),6.65 ×103 (VSV) and 2.38 ×104 (FMDV) copies of template RNA.Test on 11 RV samples and 7 HEV samples all produced positive results.

To examine the bioactivity of recombinant equine interleukin-18 (rEIL-18) obtained from Escherichia coli, rEIL-18 was evaluated to see if it showed a synergistic effect with recombinant human interleukin-12 (rhIL-12) for the induction of equine interfer-on-γ (EIFN-γ ) gene expression from equine peripheral blood mononuclear cells (PBMCs) by Real-time RT-PCR. REIL-18 could in- duce EIFN-γ mRNA expression from equine PBMCs about 20-fold higher than that of negative control in the presence of rhIL-12. And the effect to induce EIFN-γ gene expression was dose-dependent with rEIL-18 in a fixed concentration of rhIL-12. Nine an-ti-rEIL-18 monoclonal antibodies (mAbs) were also evaluated if they could neutralize the bioactivity of rEIL-18. And one of the 9 mAbs could neutralize rEIL-18 bioactivity and its effect was dose-dependent.

To obtain a recombinant glycoprotein G1 of Akabane virus(AKAV)for diagnostic purpose,the gene fragment coding high antigenic domain of AKAV G1 protein was amplified from the AKAV genome by RT-PCR after analyzing glycoprotein G1 with bio-software.The amplified product was cloned into pMD1S-T vector and sequenced.Then the gene fragment was subcloned into pET-28a(+)vector directionally.The recombinant plasmid was transformed into Escherichia coli BL21(DE3).SDS-PAGE and Western blotting analysis indicated that the fusion protein was insoluble and approximately 44 kD in molecular weight and had immunological activity.After purified by affinity chromatography,the concentration of purified protein was 2 mg/mL and the purity was 92.6%.An indirect enzyme linked immunosorbent assay(ELISA)was developed using the purified recombinant protein.The optimal reaction conditions of ELISA were determined:the coating concentration of the purified recombinant protein was 20μg/mL; the dilution fold of the serum samples was 1:200.There was no cross reaction with positive sera of other six infectious diseases. Bovine serum samples from Yunnan(77)and Inner Mongolia(70)had been detected by serum neutralizing(SN)test before detected by the ELISA.In accordance with SN test,the positive threshold of the ELISA was determined as 0.493 and 0.488 in two districts respectively;the speciality of the ELISA was 73% and 86.9% respectively.And the agreement ratio between the two methods was 80%(52/65)and 85.9%(55/64)respectively.

To establish a rapid and sensitive microarray-base method for detecting multiple virulent viruses,we constructed a panel of specific 50-mer oligonucleotide probes covering 21 viral at 5 probes for each virus.Appropriate methods for the purification and amplification of viral genes were developed from cell culture producing Chikungunya virus or Yellow fever virus.The virus RNA was extracted was treated with DNase I for the digestion of host cell genomic DNAs,then conserved genus primers to special reverse transcript from the virus,which was least hybridized with cellular genes,were designed and utilized for RT reactions.The PCR products amplified following random PCRs of the viral genomes,were labeled with fluorescent dye and hybridized to the microarray.4 Chikungunya virus probes and 5 Yellow fever virus probes gave positive signals,thus demonstrated a satisfied specificity and certain capability for viral detections.Our strategy based on microarray technologies may have a substantial potential in the diagnosis of viral infections.

Equine interferon-gamma (eIFN-gamma) expressed both in E. coli and baculovirus were evaluated for antiviral activity against recombinant Vesicular Stomatits Virus expressing green fluorescence protein (rVSV-GFP) in EFK-78 cells. The assays were conducted in 96-well plate. Virus infectivity was measured by quantifying GFP-positive cells, instead of quantifying the CPE reduction. Prior to infection of EFK-78 cells with rVSV-GFP, the cells were incubated with eIFN-gamma. The GFP expression in the EFK-78 cells dramatically decreased in the cells treated with eIFN-gamma in a dose-dependent manner, comparing with the mock-treated cells. The titers of antiviral activity were 1 x 10(3) AU/mL and 1 x 10(5) AU/mL of eIFN-gamma expressed from E. coli and baculovirus, respectively. The antiviral activities of the recombinant eIFN-gamma were highly efficient and specific, as it was blocked by mAbs against eIFN-gamma.

The G1 gene of Akabane virus OBE-1 strain was amplified from the AKAV genome by RT-PCR.The PCR product was cloned into pFastBacHT A donor plasmid of Bac-to-Bac baculovirus expression system.Recombinant DNA containing the G1 gene was obtained through homologous recombination of the donor plasmid with Bacmid DNA at the site of Tn7.Recombinant virus BAC-G1P1 was generated by transfecting recombinant DNA into Sf9 cells.SDS-PAGE analysis indicated that the G1 protein expressed in Sf9 cells was approximately 120 ku in size.Western blot and indirect immunofluorescence assay(IFA) showed that the recombinant G1 protein had good antigenicity.