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1.
To analyze the contribution of neuraminidase (NA) toward protection against avian influenza virus (AIV) infection, three different recombinant Newcastle disease viruses (NDVs) expressing hemagglutinin (HA) or NA, or both, of highly pathogenic avian influenza virus (HPAIV) were generated. The lentogenic NDV Clone 30 was used as backbone for the insertion of HA of HPAIV strain A/chicken/Vietnam/P41/05 (H5N1) and NA of HPAIV strain A/duck/Vietnam/TG24-01/05 (H5N1). The HA was inserted between the genes encoding NDV phosphoprotein (P) and matrixprotein (M), and the NA was inserted between the fusion (F) and hemagglutinin-neuraminidase protein (HN) genes, resulting in NDVH5VmPMN1FHN. Two additional recombinants were constructed carrying the HA gene between the NDV P and M genes (NDVH5VmPM) or the NA between F and HN (NDVN1FHN). All recombinants replicated well and stably expressed the HA gene, the NA gene, or both. Chickens immunized with NDVH5VmPMN1FHN or NDVH5VmPM were protected against two different HPAIV H5N1 and also against HPAIV H5N2. In contrast, immunization of chickens with NDVN1FHN induced NDV- and AIV N1-specific antibodies but did not protect the animals against a lethal dose of HPAIV H5N1. Furthermore, expression of AIV N1, in addition to AIV H5 by NDV, did not increase protection against HPAIV H5N1.  相似文献   

2.
Inactivated influenza virus vaccine prepared from a non-pathogenic influenza virus strain A/duck/Hokkaido/Vac-1/2004 (H5N1) from the virus library conferred protective immunity to chickens against the challenge of antigenically drifted highly pathogenic avian influenza virus (HPAIV), A/whooper swan/Hokkaido/1/2008 (H5N1). The efficacy of the vaccine was comparable to that prepared from genetically modified HPAIV strain deltaRRRRK rg-A/ whooper swan/Mongolia/3/2005 (H5N1), which is more antigenically related to the challenge virus strain, in chickens.  相似文献   

3.
表达H5N1亚型禽流感病毒HA和NA基因的重组鸭瘟病毒的构建   总被引：1，自引：0，他引：1  
利用PCR技术扩增出鸭瘟病毒(DPV)生长非必需的TK基因(约1.1kb),将其克隆入pGEM-Teasy载体获得载体pGTK。根据已知的绿色荧光蛋白载体pEGFP-C1的序列设计了一对引物,PCR扩增出pEGFP-C1上含CMV启动子、EGFP及其多克隆位点的完整的基因表达盒插入pGTK的TK上,获得质粒pGTK-EGFP。根据Genbank已发表的H5N1亚型禽流感病毒的血凝素(HA)和神经氨酸酶(NA)基因序列,设计了两对引物,分别从pT-HA和pT-NA两个质粒上扩增出HA和NA基因,克隆到pGTK-EGFP的表达盒的多克隆位点Kpn2I与SmaI之间,构建含EGFP及HA和NA基因的转移质粒载体pGTK-EGFP-HA-NA。将这质粒载体与DPV34F2疫苗毒共转染鸡胚成纤维细胞(CEF),通过荧光方法筛选,获得了表达HA和NA基因的重组DPV(rDPV-HA-NA)。  相似文献   

4.
Duck virus enteritis (DVE) is an acute and contagious herpes virus infection of duck, geese and swans with high morbidity and mortality. The development of specific mucosal immune system against duck enteritis virus (DEV) infection for ducks has been hindered by a lack of knowledge concerning the purification of immunoglobulin A (IgA) of duck. In the present work, the method for purification of duck immunoglobulin A was developed, and the induction of intestinal mucosal immune responses against DEV was studied by orally infected ducklings with virulent DEV. The results showed that a continuous increased DEV DNA levels were observed in blood and various organs examined of orally infected ducklings throughout the infection, which was accompanied by the development of infection in ducklings from mild progressed to severe pathological lesions. Furthermore, a marked increased level of DEV-specific IgA and IgG antibodies in bile, serum and the intestinal tract, as well as the density of IgA+ cells in intestine were detected between 1 and 12 days p.i., followed by a drastic reduction of the antibody levels and the density of IgA+ cells at 15 days p.i. The results indicate that the DVE infection can stimulate both IgA-dominated antibody immune responses in the intestinal tract, and IgG-dominated antibody systemic immunity in the serum of ducklings orally inoculated with virulent DEV. The severe lesions of the villus epithelial cells and the lymphoid organs can suppress the intestinal mucosal immune responses.  相似文献   

5.
为研究鸭病毒性肠炎病毒(DEV)基因组异构体的存在形式,本研究在已建立的以pCC1FOS为载体的DEV疫苗株基因文库基础上对DEV全序列进行了测定,并进一步对DEV基因组的异构体进行了研究。对261个重组fosmid中的DEV基因组片段的末端序列进行测序及序列分析,初步证明了DEV基因组有3种异构体,即P型、IS型和ILS型,未发现IL型的存在。并且,以上3种异构体在基因组中所占比例分别为77.1%、8.6%和14.3%。通过对11个重组fosmid的全序列测定,进一步确认了以上3种异构体的存在。该结果为DEV的分类提供了重要实验依据。  相似文献   

6.
实时荧光定量PCR快速检测鸭病毒性肠炎标准方法的建立   总被引：1，自引：0，他引：1  
根据GenBank(登录号:EF417996)中鸭病毒性肠炎病毒U31基因的序列,设计了1对特异性引物及TaqMan探针,扩增片段长度为76 bp.以鸭病毒性肠炎弱毒疫苗株DNA为阳性标准品模板,建立了实时荧光定量PCR检测鸭病毒性肠炎病毒的方法.该方法能在鸭病毒性肠炎病毒DNA样本中检出荧光信号,定量范围为:2.1×109～2.1×100个拷贝数,最小检出量为2.1×100个拷贝;用该方法对人工感染试验的4只鸭组织器官、粪便、血液等样品重复测定3次,病毒模板DNA的检出率为100%,对鸭正常组织、巴氏杆菌、大肠杆菌、沙门菌和鸭病毒性肝炎、鹅源禽流感H5毒株、新城疫、小鹅瘟病毒等DNA检测不出现特异性荧光信号.经过重复性和实际临床样本检验证实,该方法真实可靠,而且从核酸提取到报告检测结果耗时不超过4 h,不仅实现了对鸭病毒性肠炎的快速诊断,也实现了对该病毒DNA由定性到定量的检测.  相似文献   

7.
Identification and characterization of duck enteritis virus dUTPase gene   总被引：3，自引：0，他引：3  
Zhao LC  Cheng AC  Wang MS  Yuan GP  Jia RY  Zhou DC  Qi XF  Ge H  Sun T 《Avian diseases》2008,52(2):324-331
Deoxyuridine triphosphatase (dUTPase) is a ubiquitous and important enzyme that hydrolyzes dUTP to dUMP. Many viruses encode virus-specific dUTPase, which plays an essential role in maintaining the integrity of the viral DNA both by reducing the dUTP levels and by providing the substrate for the thymidylate synthase. A 1344-bp gene of duck enteritis virus (DEV) homologous to herpesviral dUTPase was first reported in this paper. The gene encodes a protein of 477 amino acids, with a predicted molecular mass of 49.7 kDa. Multiple sequence alignment suggested that DEV dUTPase was quite similar to other identified herpesviral dUTPase and functioned as a homotrimer. The five conserved motifs of DEV dUTPase with 3-1-2-4-5 arrangement have been recognized, and the phylogenetic analysis showed that DEV dUTPase was genetically close to the avian herpesvirus. Furthermore, RNA dot blot, western blot, and immunofluorescence analysis indicated that the enzyme was expressed at early and late stages after infection. Immunofluorescence also confirmed that DEV dUTPase localized in the cytoplasm of DEV-infected duck embryo fibroblasts as early as 4 hr postinfection (hpi). Later, the enzyme transferred from cytoplasm to nucleus at 8 hpi, and then reached its expression peak at 12 hpi, both in the cytoplasm and nucleus. The results suggested that the DEV dUTPase gene might be an early viral gene in DEV vitro infection and contribute to ensuring the fidelity of genome replication.  相似文献   

8.
The UL49.5 gene of most herpesviruses is conserved and encodes glycoprotein N. However, the UL49.5 protein of duck enteritis virus (DEV) (pUL49.5) has not been reported. In the current study, the DEV pUL49.5 gene was first subjected to molecular characterization. To verify the predicted intracellular localization of gene expression, the recombinant plasmid pEGFP-C1/pUL49.5 was constructed and used to transfect duck embryo fibroblasts. Next, the recombinant plasmid pDsRed1-N1/glycoprotein M (gM) was produced and used for co-transfection with the pEGFP-C1/pUL49.5 plasmid to determine whether DEV pUL49.5 and gM (a conserved protein in herpesviruses) colocalize. DEV pUL49.5 was thought to be an envelope glycoprotein with a signal peptide and two transmembrane domains. This protein was also predicted to localize in the cytoplasm and endoplasmic reticulum with a probability of 66.7%. Images taken by a fluorescence microscope at different time points revealed that the DEV pUL49.5 and gM proteins were both expressed in the cytoplasm. Overlap of the two different fluorescence signals appeared 12 h after transfection and continued to persist until the end of the experiment. These data indicate a possible interaction between DEV pUL49.5 and gM.  相似文献   

9.
本实验应用了免疫组织化学的单克隆抗体间接酶标染色法，对人工感染鸭瘟病毒雏鸭的组织切片进行染色观察。旨在研究病毒在鸭体内分布，对其进行定位。研究结果显示，鸭的心脏、肝脏、脾、胸腺、肠、法氏囊、胰、肺、肾等组织的细胞浆内均出现了染色的特异阳性反应物。结果表明，鸭瘟病毒广泛分布于感染雏鸭的各种组织器官，并造成一定的组织病理变化。  相似文献   

10.
禽痘病毒感染对禽流感重组禽痘病毒疫苗免疫效力的影响   总被引：1，自引：0，他引：1  
表达禽流感病毒 (AIV)HA和NA基因的重组禽痘病毒rFPV_HA_NA能够诱导鸡体产生 10 0 %抵抗高致病性禽流感病毒 (HPAIV)H5N1的攻击。而当鸡群已进行禽痘疫苗免疫或者感染了禽痘病毒的情况下 ,此重组疫苗的免疫效力如何 ?首先用禽痘病毒S_FPV_0 17人工感染SPF试验鸡 ,既而在感染后的不同间隔时间接种重组疫苗 ,免疫后检测鸡群的HI抗体水平 ,同时用 10 0LD50 的HPAIVH5N1进行攻击。结果重组疫苗免疫与禽痘病毒人工感染时间间隔在 4周 (或以上 )时 ,预先感染禽痘病毒对重组疫苗的免疫效力不构成影响 ,对禽流感的保护力为 10 0 % ,而间隔时间在 1、2、3周时 ,重组疫苗的免疫保护效力则受到不同程度的影响。  相似文献   

11.
A chicken embryo-adapted duck enteritis virus (DEV) strain is the most widely used vaccine against duck virus enteritis (DVE) infection. The kinetics of attenuated DEV vaccine was examined in tissues of ducklings vaccinated by the mucosal or systemic route at 20 days of age and sampled regularly up to 60 days post-vaccination (p.v.). Significant numbers of virus genomes in the lymphoid and other parenchymatous organs were first detected at 60 min p.v., and subsequently rose to peak levels during 90 min to 1 day p.v. independent of the route of vaccine administration. The peak level of vaccine virus in the individual parenchymatous organs of subcutaneously immunized ducklings was significantly higher than that of orally or nasally immunized ducklings. The route of vaccine administration had significant effect on the initial tissue distribution of vaccine virus in respiratory and digestive tracts. Vaccine viruses spread to digestive tract and trachea tissues by mucosal route, i.e. oral and nasal administration, early than that by subcutaneous route. The rapid early increase of vaccine virus levels in all samples examined followed by a steady decline from 90 min to 6 days p.v. The real-time PCR analysis of a variety of tissues is significant for further investigation of the mechanism of vaccinal protection, and the optimization of vaccination regimes.  相似文献   

12.
In this paper,a 1,860 bp sequence in IRs region of duck enteritis virus(DEV) was amplified by single oligonucleotide nested PCR with a single primer designed according to partial sequence of US1 and then a pair of primers designed according to the 3' UTR of US8 gene and 5' end of the new getting sequence were used to amplify a 2,426 bp sequence toward the TRs region.Sequence analysis revealed that the both sequences contained an identical 990 bp open reading frame of DEV US1 gene.The two ORFs were in opposi...  相似文献   

13.
Qi X  Yang X  Cheng A  Wang M  Zhu D  Jia R 《Avian diseases》2008,52(2):338-344
To better understand the pathogenesis of duck virus enteritis (DVE), the levels of viral DNA in various tissues of ducklings during acute stage of virulent duck enteritis virus (DEV) infection were investigated by using quantitative real-time polymerase chain reaction. The results show that the viral levels of DEV in systemic organs have a close correlation with the progression of disease. The rapid dissemination and active replication of virulent DEV in multiple systemic organs at the early phase of acute infection accelerate the progression of disease. The levels of viral DNA increase sharply soon after developed clinical signs of disease, and the extent of increase and the magnitude of DEV DNA load in various tissues of ducklings after the exhibition of clinical signs may be a critical determinant of the outcome of DEV infection. The relatively high levels of DEV in bursa and small intestine tissues of dead ducklings most likely reflect the abundance of target epithelial and lymphoid cells in these tissues, which therefore play a key role in the pathogenesis of acute DVE and manifest as severe tissue lesions on the bursa and small intestine.  相似文献   

14.
Latency sites and reactivation of duck enteritis virus   总被引：16，自引：0，他引：16  
Shawky S  Schat KA 《Avian diseases》2002,46(2):308-313
Duck virus enteritis (DVE) is a contagious disease caused by herpesvirus in waterfowl populations. Recovered birds become carriers and shed the virus periodically. Reactivation of latent duck enteritis virus (DEV) has been implicated in outbreaks of DVE in domestic and migrating waterfowl populations. In this study, the sites for virus latency were determined in white Pekin ducks infected with the DEV-97 strain. At 3 wk postinfection, infectious virus was not detectable in tissues or cloacal swabs (CSs). At 7 and 9 weeks postinfection, the viral DNA was detected by polymerase chain reaction in the trigeminal ganglia (TG), suggesting that the virus is latent. Viral DNA was detected in the peripheral blood lymphocytes (PBL), spleen, thymus, bursa, and CSs only after in vitro cocultivation. In vivo virus reactivation was demonstrated when dexamethasone or a combination of dexamethasone and cyclophosphamide was inoculated in latently infected ducks. The reactivation of DEV occurred without any clinical evidence of the disease, but the virus was detected in PBL and CSs. We conclude from this study that DEV establishes latency in TG and lymphoid tissues including PBL.  相似文献   

15.
  相似文献   

16.
依据GenBank登录的鸭肠炎病毒(DEV)核苷酸序列设计引物,利用长片段PCR技术扩增了DEV基因组UL36与UL43基因之间的未知序列,扩增所得片段长度约为15 kb.经EcoRV单酶切,将其中的3.9 kb片段克隆到pUC18中.序列分析表明该3.9 kb EcoR V片段含有2个完整的转录方向相反的与单纯疱疹病毒(HSV)UL41和UL42基因同源的ORF,命名为DEV UL41和ULA2基因.通过氨基酸序列比对发现:DEV UL41基因含有5个高度保守位点,而UL42含有2个,进化树分析表明DEV与疱疹病毒科a疱疹病毒亚科的马立克病毒、火鸡疱疹病毒的进化关系非常相近,为DEV的分类提供了参考依据.  相似文献   

17.
Wu Y  Cheng A  Wang M  Zhang S  Zhu D  Jia AB  Luo BQ  Chen Z  Chen X 《Avian diseases》2011,55(4):626-632
A recombinant UL55 protein (pUL55) of duck enteritis virus (DEV), produced in Escherichia coli, was tested for diagnostic applicability in an indirect enzyme-linked immunosorbent assay (1-ELISA) as a coating antigen. Serum dilutions of 1:6400 (0.025microg) are the maximum detection limits of the pUL55-ELISA, according to the determined cut-off value of 0.330. Antigenic cross-reactivity investigation in heterologous sera of ducks failed to provide evidence that other viruses of ducks could hamper the serodiagnosis of DEV, and the inhibition assay revealed that the specific binding of antigen and antibody can be inhibited by pUL55, both of which demonstrated a good specificity of the established pUL55-ELISA. This assay was further validated by comparison with a commercial 1-ELISA based on DEV (DEV-ELISA) and a neutralization test (NT). The results suggested that the sensitivity of pUL55-ELISA was almost as good as DEV-ELISA but was much higher than NT. The established pUL55-ELISA is a rapid, simple, sensitive, specific, and inexpensive serodiagnosis for detecting antibodies against DEV and has a potential to complement the traditional assays for serodiagnosis of DEV; it can be used as a diagnosis alternative candidate for serologic surveillance of DEV infection.  相似文献   

18.
为研究鸭肠炎病毒(DEV)gL蛋白在感染鸡胚成纤维细胞(CEF)过程中的表达情况,本研究以DEVClone-03基因组为模板,应用PCR方法分别扩增得到截短的(gLt,181 bp～711 bp)和全长的gL(1 bp～711 bp)两个基因片段。将gLt基因片段克隆至pET-30a原核表达载体,转化E.coli BL21(DE3),经IPTG诱导表达并对表达产物进行纯化复性,免疫BALB/c小鼠,制备鼠抗gL蛋白多克隆抗体。同时将全长gL基因克隆至真核表达载体pcDNA3.1(+),构建真核表达重组质粒pcDNA-gL,转染293T细胞。采用获得的抗gL蛋白抗体检测DEV感染CEF后及真核表达质粒pcDNA-gL转染293T细胞后gL蛋白在不同时间点的表达情况。结果表明,在pcDNA-gL转染293T细胞后12 h应用western blot方法能够检测到gL蛋白的表达,其表达量随着转染时间增加而增加;在病毒感染CEF后24 h应用间接免疫荧光方法能够检测到gL蛋白少量的表达,western blot方法在病毒感染CEF48 h后检测到gL蛋白的表达,其表达量随着病毒感染时间增加而增加。上述结果提示,编码gL蛋白基因可能是病毒复制的晚期表达基因。  相似文献   

19.
为了建立重组鸭瘟病毒技术,构建了鸭瘟病毒转移质粒。在对鸭瘟强毒和弱毒株TK基因进行测序分析后,将鸭瘟病毒TK-UL24DNA片段克隆于pUC18载体中,构建了质粒pTK;将PCR扩增的GFP真核表达盒插入pTK质粒的TK基因内部,获得转移载体质粒pTK-GFP。鸭瘟病毒TK-UL24测序分析表明鸭瘟强、弱毒株TK基因序列完全相同;转移载体携带Pcmv-GFP-SV40pA表达盒,测序验证其序列与源序列一致。pTK-GFP在脂质体介导下,转染鸭胚成纤维细胞和鸭肾细胞,在荧光显微镜下观察绿色荧光蛋白表达情况。质粒转染细胞后,绿色荧光蛋白得到了有效的表达,为进一步开展重组鸭瘟病毒的研究和构建具有遗传标记的鸭瘟疫苗奠定了基础。  相似文献   

20.
Yu X  Jia R  Huang J  Shu B  Zhu D  Liu Q  Gao X  Lin M  Yin Z  Wang M  Chen S  Wang Y  Chen X  Cheng A 《Veterinary research》2012,43(1):56
Orally delivered DNA vaccines against duck enteritis virus (DEV) were developed using live attenuated Salmonella typhimurium (SL7207) as a carrier and Escherichia coli heat labile enterotoxin B subunit (LTB) as a mucosal adjuvant. DNA vaccine plasmids pVAX-UL24 and pVAX-LTB-UL24 were constructed and transformed into attenuated Salmonella typhimurium SL7207 resulting SL7207 (pVAX-UL24) and SL7207 (pVAX-LTB-UL24) respectively. After ducklings were orally inoculated with SL7207 (pVAX-UL24) or SL7207 (pVAX-LTB-UL24), the anti-DEV mucosal and systemic immune responses were recorded. To identify the optimum dose that confers maximum protection, we used different doses of the candidate vaccine SL7207 (pVAX-LTB-UL24) during oral immunization. The strongest mucosal and systemic immune responses developed in the SL7207 (pVAX-LTB-UL24) (1011 CFU) immunized group. Accordingly, oral immunization of ducklings with SL7207 (pVAX-LTB-UL24) showed superior efficacy of protection (60-80%) against a lethal DEV challenge (1000 LD50), compared with the limited survival rate (40%) of ducklings immunized with SL7207 (pVAX-UL24). Our study suggests that the SL7207 (pVAX-LTB-UL24) can be a candidate DEV vaccine.  相似文献   

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