Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF) is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV) also does this,but the mecha...Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF) is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV) also does this,but the mechanisms and particular receptors remain unclear.Virus overlay protein blot assays were carried out on CEF membrane proteins,which were extracted respectively with a Mem-PER TM kit,a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method,and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells,indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.展开更多
[Objective] This study aimed to construct the recombinant expression plasmids containing H and F protein genes of Canine distemper virus isolated from a mink and to express these two genes in prokaryotic cells as well...[Objective] This study aimed to construct the recombinant expression plasmids containing H and F protein genes of Canine distemper virus isolated from a mink and to express these two genes in prokaryotic cells as well as to study the reactogenicity of the expressed products. [Method] RT-PCR amplification was used to obtain H and F protein genes; TA cloning and subcloning techniques were used to construct the cloning plasmids( pMD-18T-H and pMD-18T-F) and recombinant expression plasmids( pET28a-H and pET28a-F) ; SDS-PAGE and Western-blotting were adopted to verify whether the target proteins were successfully expressed. [Result] The recombinant expression plasmids pET28a-H and pET28a-F containing H and F protein genes of Canine distemper virus isolated from a mink were successfully constructed,and both the expressed H and F proteins with respectively relative molecular mass of 31 400 and 38 200 produced positive reaction with the CDV standard positive serum. [Conclusion] The H and F proteins expressed in prokaryotic cells were the same with the natural ones in terms of reactogenicity,which can be utilized for diagnosis of a CDV's infection or for an epidemiological investigation. Meanwhile,they also provide a basis for developing genetically engineered subunit vaccines.展开更多
Canine distemper virus(CDV)has recently been identified in populations of wild tigers in Russia and India.Tiger populations are generally too small to maintain CDV for long periods,but are at risk of infections arisin...Canine distemper virus(CDV)has recently been identified in populations of wild tigers in Russia and India.Tiger populations are generally too small to maintain CDV for long periods,but are at risk of infections arising from more abundant susceptible hosts that constitute a reservoir of infection.Because CDV is an additive mortality factor,it could represent a significant threat to small,isolated tiger populations.In Russia,CDV was associated with the deaths of tigers in 2004 and 2010,and was coincident with a localized decline of tigers in Sikhote-Alin Biosphere Zapovednik(from 25 tigers in 2008 to 9 in 2012).Habitat continuity with surrounding areas likely played an important role in promoting an ongoing recovery.We recommend steps be taken to assess the presence and the impact of CDV in all tiger range states,but should not detract focus away from the primary threats to tigers,which include habitat loss and fragmentation,poaching and retaliatory killing.Research priorities include:(i)recognition and diagnosis of clinical cases of CDV in tigers when they occur;and(ii)collection of baseline data on the health of wild tigers.CDV infection of individual tigers need not imply a conservation threat,and modeling should complement disease surveillance and targeted research to assess the potential impact to tiger populations across the range of ecosystems,population densities and climate extremes occupied by tigers.Describing the role of domestic and wild carnivores as contributors to a local CDV reservoir is an important precursor to considering control measures.展开更多
A polymerase chain reaction was performed using re-ported primers for detection of Canine Parvo virus (CPV) in the stool sample obtained from repository. The PCR primers were specific to VP1/VP2 gene of CPV. Sensi-tiv...A polymerase chain reaction was performed using re-ported primers for detection of Canine Parvo virus (CPV) in the stool sample obtained from repository. The PCR primers were specific to VP1/VP2 gene of CPV. Sensi-tivity assay of PCR detection was performed by making dilutions of CPV positive DNA extracted from fecal sample, carrying out PCR for each dilution and visualiz-ing amplicons in ethidium bromide stained agarose gel under UV radiation. Study was valuable in determining the efficiency of PCR. The sensitivity of PCR in present study was determined to be equivalent to detection of .00 2pg/μl of CPV DNA. The study was conducted to analyze the variation, sensitivity and repeatability.展开更多
So far, the pathogenesis of demyelination caused by canine distemper virus (CDV) in the central nervous system has remained unclear, although a lot of studies have been done extensively. To further investigate the rel...So far, the pathogenesis of demyelination caused by canine distemper virus (CDV) in the central nervous system has remained unclear, although a lot of studies have been done extensively. To further investigate the relation of variety cells in brain to demyelination, this study was performed on 15 dogs with spontaneous acute canine distemper and 2 controls. According to anatomical relation, the brain was divided into cerebrum, cerebral stem and cerebellum. The sections with no, mild, moderate, or severe demyelinating lesions were selected respectively and stained by HE and immunohistochemistry. Immuno-localisation of CDV antigen was used to confirm CDV infection. The brain was examined for co-localisation of the CDV antigen with either an astrocyte-specific marker, glial fibrillary acidic protein (GFAP), or an oligodendrocyte-specific marker, galactocerebroside (GalC). Apoptotic cell was detected by TdT-mediated nick end-labeling assay (TUNEL). The results demonstrated that the local disturbance of blood circulation mainly included congestion, edema, thrombosis, and disseminated intravascular coagulation (DIC). The CDV neucleocapsid protein positive reaction, metabolic disorder and apoptosis of oligodendrocytes were observed in demyelinating areas. Lots of astrocytes displayed CDV antigen-positive, especially in their process. Some of them became apoptotic cell confirmed by TUNEL staining. Fibrous astrocytes showed more intense GFAP-positive in mild and moderate demyelinating area. Some of nervous cells located in pyramidal cell layers and nucleus nervi were in degeneration, necrosis. Satellitosis, neuronophagia and apoptotic neurons were examined by hematoxylin and eosin (HE) and TUNEL staining. The results suggested that the demyelinating changes in brain tissues infected with CDV mainly related to the metabolic disorder and apoptosis of ogliodendrocytes and astrocytes; also involved with the local disturbance of blood circulation and some neuron lost.展开更多
[Objective]The aim was to survey relationship between acute canine distemper and parasitic enteritis from pathology. [Method]Twelve cases of acute canine distemper with diarrhea were researched as per immunohistochemi...[Objective]The aim was to survey relationship between acute canine distemper and parasitic enteritis from pathology. [Method]Twelve cases of acute canine distemper with diarrhea were researched as per immunohistochemistry,Haematoxylin Eosin,and PAS staining kit. [Result] Of the twelve diseased dogs ( with diarrhea) ,six were detected caused by coccidium and two were detected by cryptosporidium. Coccidian protozoa is mainly in epithelial cells of jejunum and ileum,and some can be found in cut-off intestinal epithelial cells and in mucus formed by destroyed intesti- nal villus. The most common shapes of coccidian protozoa are trophozoite and schizont. The former is mainly within or among epithelial cells; nucle- us is in center and stained by hematoxylin; protoplasm is in " fined mesh" shape. The latter,round or oval,contains much glycogenosome in de- generated intestinal epithelial cells. On the other hand,cryptosporidium is mainly in striated borders of intestinal epithelial cells and intestinal gland cells,leading to destruction of villus and cut-off of cells. Through detection on monoclonal antibody of nucleocapsid proteins of anti-canine distemper virus,it was found that epithelial cells in intestinal mucosa,glandular cells in recesses,lymphocytes and macrophage infittrated in lamina propria and dendritic cells in aggregated nodule were all with positive reactions. [Conclusion]Parasitic diarrhea caused by acute canine distemper occurs when resistance of intestinal mucosa caused by canine distemper virus begins to decline.展开更多
犬细小病毒病是由犬细小病毒(canine parvovirus,CPV)引起犬的一种急性、高度传染性和致死性的病毒性疾病。该病主要通过接种疫苗预防,没有特效治疗方法。为了制备犬细小病毒样颗粒(virus like particles,VLPs),本研究通过杆状病毒表达...犬细小病毒病是由犬细小病毒(canine parvovirus,CPV)引起犬的一种急性、高度传染性和致死性的病毒性疾病。该病主要通过接种疫苗预防,没有特效治疗方法。为了制备犬细小病毒样颗粒(virus like particles,VLPs),本研究通过杆状病毒表达系统表达CPV VP2蛋白。将表达后的蛋白经过蔗糖密度梯度离心法纯化后,负染色处理置于透射电子显微镜下观察。结果显示VP2蛋白组装成直径25 nm左右的六边形或圆形样VLPs。血凝试验表明,该VLPs可以凝集猪红细胞,血凝价为27。进一步将该VLPs经弗氏佐剂乳化后免疫小鼠,血凝抑制试验结果显示VLPs可诱导小鼠产生211的血凝抑制抗体。因此,通过杆状病毒表达系统表达VP2蛋白,VP2蛋白可在细胞内组装成与病毒粒子类似的VLPs,该VLPs可以诱导小鼠产生血凝抑制抗体,为CPV VLPs疫苗的制备奠定了基础。展开更多
旨在研究犬腺病毒-2型(canine adenovirus type 2,CAV-2)作为溶瘤病毒的潜力并进一步探索精子黏附因子(SPAM1)协同CAV-2重塑肿瘤微环境在肿瘤免疫治疗中的应用价值。本研究以P15A-CAV-2反向遗传操作平台为基础,使用RED/ET同源重组系统...旨在研究犬腺病毒-2型(canine adenovirus type 2,CAV-2)作为溶瘤病毒的潜力并进一步探索精子黏附因子(SPAM1)协同CAV-2重塑肿瘤微环境在肿瘤免疫治疗中的应用价值。本研究以P15A-CAV-2反向遗传操作平台为基础,使用RED/ET同源重组系统构建携带SPAM1外源基因的中间载体,使用中间载体将CAV-2骨架载体E3区域缺失并替换SPAM1外源基因表达盒。再利用合成引物敲除kmccdB反向筛选表达盒构建P15A-CAV-2-mCMV-SPAM1-SV40 polyA感染性克隆质粒并在MDCK-E1A细胞系中拯救重组溶瘤病毒,对重组病毒体外溶瘤效应进行验证。根据测序与酶切验证结果证明,本研究成功构建并拯救出稳定表达外源基因SPAM1的重组溶瘤病毒1株;IFA试验证明,重组毒株能够高水平稳定表达外源基因且外源蛋白具有可弥散分布于细胞间的特点。缺失E3区域表达外源基因SPAM1的重组溶瘤病毒通过光镜观察和CCK8检测发现具有对犬癌细胞系A72强烈的杀伤效应。综上,本研究成功构建1株具有良好溶瘤效应的重组CAV-2,为后续应用于宠物肿瘤治疗奠定了基础。展开更多
The number of human rabies cases acquired from dog bites constitutes a high proportion of the total rabies cases in China, although the number of human rabies cases has gradually decreased in recent years. The pivotal...The number of human rabies cases acquired from dog bites constitutes a high proportion of the total rabies cases in China, although the number of human rabies cases has gradually decreased in recent years. The pivotal role of dogs in the spread of rabies indicates that controlling and preventing canine rabies could be a key step in eradicating human rabies in China. The primary aims of this review are to discuss the properties and pathogenesis of the rabies virus, the clinical signs and diagnosis of canine rabies, threshold host density and vaccination of dogs, and the prevention and control of canine rabies in China.展开更多
[Objective] The study aims to determine the optimal concentration of trypsin for the proliferation of avian influenza virus(AIV) H9N2 subtype in Madin-Darby canine kidney(MDCK) cells.[Method] Three AIV H9 subtype isol...[Objective] The study aims to determine the optimal concentration of trypsin for the proliferation of avian influenza virus(AIV) H9N2 subtype in Madin-Darby canine kidney(MDCK) cells.[Method] Three AIV H9 subtype isolates were inoculated on MDCK cells respectively.Then,DMEM containing different concentrations of trypsin as maintenance media were added to MDCK monolayer cells.The cytopathic effect(CPE) was observed once every 24 h,and the HA titer of the supernatant was measured by HA assay.[Result] When the trypsin concentration was 10-20 μg/ml in DMEM,the HA titer of virus culture reached 7 log2(1:128).Almost all cells were cytopathic after 96 h post inoculation with 1:1 000 or 1:10 000 dilution of AIV culture,and the virus titer reached a peak after 72-96 h.[Conclusion] The optimal concentration of trypsin is 10-20 μg/ml for proliferation of AIV H9N2 subtype in MDCK cells.展开更多
狂犬病、犬瘟热和犬细小病毒病是当前危害养犬业的三种重要传染病。为探讨狂犬病毒(RABV)、犬瘟热病毒(CDV)和犬细小病毒(CPV)灭活制备三联灭活疫苗的可行性,将该3种病毒HEP-Flury株、LN(10)1株和JL(18)1-Beagle株分别在BHK-21、Vero/Do...狂犬病、犬瘟热和犬细小病毒病是当前危害养犬业的三种重要传染病。为探讨狂犬病毒(RABV)、犬瘟热病毒(CDV)和犬细小病毒(CPV)灭活制备三联灭活疫苗的可行性,将该3种病毒HEP-Flury株、LN(10)1株和JL(18)1-Beagle株分别在BHK-21、Vero/DogSLAM(VDS)和F81细胞培养,经β-丙内酯灭活剂灭活后,分别与三种不同类型免疫佐剂(MONTANIDE GEL 02、ADJ-801(W)、Al(OH)3)配制成三联灭活疫苗。疫苗经三次免疫比格犬后,通过测定动物血清抗体水平和攻毒保护情况评价其免疫效果。实验结果显示,不同佐剂的三联灭活疫苗对增强比格犬RABV、CDV和CPV血清抗体应答反应效果不同。其中ADJ-801(W)佐剂能显著提高针对RABV、CDV和CPV三种病毒的抗体水平,疫苗三次免疫比格犬后血清抗体效价分别为7.4 EU/mL、1∶50和1∶1024;而Al(OH)3佐剂效果较差,三免后抗体效价分别为4.01 EU/mL、1∶6和1∶256。疫苗三次免疫后,分别应用强毒CDV SD(14)7株和CPVJL(18)1-Beagle株对比格犬进行攻毒实验,实验结果显示,ADJ-801(W)组对SD(14)7和JL(18)1-Beagle攻毒保护率均为100%,MONTANIDE GEL 02组攻毒保护率均为60%,而Al(OH)3组攻毒保护率均为0,表明ADJ-801(W)佐剂制备的狂犬病、犬瘟热和犬细小病毒病三联灭活疫苗对比格犬提供较好的免疫保护作用。本研究为犬狂犬病、犬瘟热和犬细小病毒病三联灭活疫苗的研制奠定了基础。展开更多
基金supported by a grant from Yunnan Provincial Education Board(08C0070)a grant from Yunnan Provincial Program for Introducing High-level Scientists (2009CI125)
文摘Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF) is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV) also does this,but the mechanisms and particular receptors remain unclear.Virus overlay protein blot assays were carried out on CEF membrane proteins,which were extracted respectively with a Mem-PER TM kit,a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method,and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells,indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.
基金Supported by the Natural Science Foundation of Jilin Province(201115194)Education Department of Jilin Province(2009.No.66)
文摘[Objective] This study aimed to construct the recombinant expression plasmids containing H and F protein genes of Canine distemper virus isolated from a mink and to express these two genes in prokaryotic cells as well as to study the reactogenicity of the expressed products. [Method] RT-PCR amplification was used to obtain H and F protein genes; TA cloning and subcloning techniques were used to construct the cloning plasmids( pMD-18T-H and pMD-18T-F) and recombinant expression plasmids( pET28a-H and pET28a-F) ; SDS-PAGE and Western-blotting were adopted to verify whether the target proteins were successfully expressed. [Result] The recombinant expression plasmids pET28a-H and pET28a-F containing H and F protein genes of Canine distemper virus isolated from a mink were successfully constructed,and both the expressed H and F proteins with respectively relative molecular mass of 31 400 and 38 200 produced positive reaction with the CDV standard positive serum. [Conclusion] The H and F proteins expressed in prokaryotic cells were the same with the natural ones in terms of reactogenicity,which can be utilized for diagnosis of a CDV's infection or for an epidemiological investigation. Meanwhile,they also provide a basis for developing genetically engineered subunit vaccines.
基金We would like to thank the Morris Animal Foundation,Zoo Boise,and the Biotechnology and Biological Sciences Research Council for their generous support of the project.In addition,none of this work would have been possible without the continued partnership of the Sikhote-Alin Biosphere Zapovednik(Director D.Yu.Gorskhov),Lazovskii Zapovednik(Director A.A.Laptev)and the Russian Ministry of Natural Resources.Thanks also to V.Keahey(In-Sync Exotics)for insights into the epidemiology of CDV.
文摘Canine distemper virus(CDV)has recently been identified in populations of wild tigers in Russia and India.Tiger populations are generally too small to maintain CDV for long periods,but are at risk of infections arising from more abundant susceptible hosts that constitute a reservoir of infection.Because CDV is an additive mortality factor,it could represent a significant threat to small,isolated tiger populations.In Russia,CDV was associated with the deaths of tigers in 2004 and 2010,and was coincident with a localized decline of tigers in Sikhote-Alin Biosphere Zapovednik(from 25 tigers in 2008 to 9 in 2012).Habitat continuity with surrounding areas likely played an important role in promoting an ongoing recovery.We recommend steps be taken to assess the presence and the impact of CDV in all tiger range states,but should not detract focus away from the primary threats to tigers,which include habitat loss and fragmentation,poaching and retaliatory killing.Research priorities include:(i)recognition and diagnosis of clinical cases of CDV in tigers when they occur;and(ii)collection of baseline data on the health of wild tigers.CDV infection of individual tigers need not imply a conservation threat,and modeling should complement disease surveillance and targeted research to assess the potential impact to tiger populations across the range of ecosystems,population densities and climate extremes occupied by tigers.Describing the role of domestic and wild carnivores as contributors to a local CDV reservoir is an important precursor to considering control measures.
文摘A polymerase chain reaction was performed using re-ported primers for detection of Canine Parvo virus (CPV) in the stool sample obtained from repository. The PCR primers were specific to VP1/VP2 gene of CPV. Sensi-tivity assay of PCR detection was performed by making dilutions of CPV positive DNA extracted from fecal sample, carrying out PCR for each dilution and visualiz-ing amplicons in ethidium bromide stained agarose gel under UV radiation. Study was valuable in determining the efficiency of PCR. The sensitivity of PCR in present study was determined to be equivalent to detection of .00 2pg/μl of CPV DNA. The study was conducted to analyze the variation, sensitivity and repeatability.
基金Supported by the National Natural Science Foundation of China(30771601)
文摘So far, the pathogenesis of demyelination caused by canine distemper virus (CDV) in the central nervous system has remained unclear, although a lot of studies have been done extensively. To further investigate the relation of variety cells in brain to demyelination, this study was performed on 15 dogs with spontaneous acute canine distemper and 2 controls. According to anatomical relation, the brain was divided into cerebrum, cerebral stem and cerebellum. The sections with no, mild, moderate, or severe demyelinating lesions were selected respectively and stained by HE and immunohistochemistry. Immuno-localisation of CDV antigen was used to confirm CDV infection. The brain was examined for co-localisation of the CDV antigen with either an astrocyte-specific marker, glial fibrillary acidic protein (GFAP), or an oligodendrocyte-specific marker, galactocerebroside (GalC). Apoptotic cell was detected by TdT-mediated nick end-labeling assay (TUNEL). The results demonstrated that the local disturbance of blood circulation mainly included congestion, edema, thrombosis, and disseminated intravascular coagulation (DIC). The CDV neucleocapsid protein positive reaction, metabolic disorder and apoptosis of oligodendrocytes were observed in demyelinating areas. Lots of astrocytes displayed CDV antigen-positive, especially in their process. Some of them became apoptotic cell confirmed by TUNEL staining. Fibrous astrocytes showed more intense GFAP-positive in mild and moderate demyelinating area. Some of nervous cells located in pyramidal cell layers and nucleus nervi were in degeneration, necrosis. Satellitosis, neuronophagia and apoptotic neurons were examined by hematoxylin and eosin (HE) and TUNEL staining. The results suggested that the demyelinating changes in brain tissues infected with CDV mainly related to the metabolic disorder and apoptosis of ogliodendrocytes and astrocytes; also involved with the local disturbance of blood circulation and some neuron lost.
基金funded by Scientific Research Staring Foundation for the Returned Overseas Chinese Scholars,Ministry of Education of ChinaFoundation of Talents and Science Researching,Henan Institute of Scince and Technology
文摘[Objective]The aim was to survey relationship between acute canine distemper and parasitic enteritis from pathology. [Method]Twelve cases of acute canine distemper with diarrhea were researched as per immunohistochemistry,Haematoxylin Eosin,and PAS staining kit. [Result] Of the twelve diseased dogs ( with diarrhea) ,six were detected caused by coccidium and two were detected by cryptosporidium. Coccidian protozoa is mainly in epithelial cells of jejunum and ileum,and some can be found in cut-off intestinal epithelial cells and in mucus formed by destroyed intesti- nal villus. The most common shapes of coccidian protozoa are trophozoite and schizont. The former is mainly within or among epithelial cells; nucle- us is in center and stained by hematoxylin; protoplasm is in " fined mesh" shape. The latter,round or oval,contains much glycogenosome in de- generated intestinal epithelial cells. On the other hand,cryptosporidium is mainly in striated borders of intestinal epithelial cells and intestinal gland cells,leading to destruction of villus and cut-off of cells. Through detection on monoclonal antibody of nucleocapsid proteins of anti-canine distemper virus,it was found that epithelial cells in intestinal mucosa,glandular cells in recesses,lymphocytes and macrophage infittrated in lamina propria and dendritic cells in aggregated nodule were all with positive reactions. [Conclusion]Parasitic diarrhea caused by acute canine distemper occurs when resistance of intestinal mucosa caused by canine distemper virus begins to decline.
文摘旨在研究犬腺病毒-2型(canine adenovirus type 2,CAV-2)作为溶瘤病毒的潜力并进一步探索精子黏附因子(SPAM1)协同CAV-2重塑肿瘤微环境在肿瘤免疫治疗中的应用价值。本研究以P15A-CAV-2反向遗传操作平台为基础,使用RED/ET同源重组系统构建携带SPAM1外源基因的中间载体,使用中间载体将CAV-2骨架载体E3区域缺失并替换SPAM1外源基因表达盒。再利用合成引物敲除kmccdB反向筛选表达盒构建P15A-CAV-2-mCMV-SPAM1-SV40 polyA感染性克隆质粒并在MDCK-E1A细胞系中拯救重组溶瘤病毒,对重组病毒体外溶瘤效应进行验证。根据测序与酶切验证结果证明,本研究成功构建并拯救出稳定表达外源基因SPAM1的重组溶瘤病毒1株;IFA试验证明,重组毒株能够高水平稳定表达外源基因且外源蛋白具有可弥散分布于细胞间的特点。缺失E3区域表达外源基因SPAM1的重组溶瘤病毒通过光镜观察和CCK8检测发现具有对犬癌细胞系A72强烈的杀伤效应。综上,本研究成功构建1株具有良好溶瘤效应的重组CAV-2,为后续应用于宠物肿瘤治疗奠定了基础。
基金supported by grants from the National Natural Science Foundation of China(No.3097016081160353)+1 种基金China Mega-Project for Infectious Disease(2011ZX10004-001)the Development Grant of the State Key Laboratory for Infectious Disease Prevention and Control(2011SKLID705)
文摘The number of human rabies cases acquired from dog bites constitutes a high proportion of the total rabies cases in China, although the number of human rabies cases has gradually decreased in recent years. The pivotal role of dogs in the spread of rabies indicates that controlling and preventing canine rabies could be a key step in eradicating human rabies in China. The primary aims of this review are to discuss the properties and pathogenesis of the rabies virus, the clinical signs and diagnosis of canine rabies, threshold host density and vaccination of dogs, and the prevention and control of canine rabies in China.
基金funded by the Beijing Academy of Agriculture and Forestry Sciences (2010A007)
文摘[Objective] The study aims to determine the optimal concentration of trypsin for the proliferation of avian influenza virus(AIV) H9N2 subtype in Madin-Darby canine kidney(MDCK) cells.[Method] Three AIV H9 subtype isolates were inoculated on MDCK cells respectively.Then,DMEM containing different concentrations of trypsin as maintenance media were added to MDCK monolayer cells.The cytopathic effect(CPE) was observed once every 24 h,and the HA titer of the supernatant was measured by HA assay.[Result] When the trypsin concentration was 10-20 μg/ml in DMEM,the HA titer of virus culture reached 7 log2(1:128).Almost all cells were cytopathic after 96 h post inoculation with 1:1 000 or 1:10 000 dilution of AIV culture,and the virus titer reached a peak after 72-96 h.[Conclusion] The optimal concentration of trypsin is 10-20 μg/ml for proliferation of AIV H9N2 subtype in MDCK cells.
文摘狂犬病、犬瘟热和犬细小病毒病是当前危害养犬业的三种重要传染病。为探讨狂犬病毒(RABV)、犬瘟热病毒(CDV)和犬细小病毒(CPV)灭活制备三联灭活疫苗的可行性,将该3种病毒HEP-Flury株、LN(10)1株和JL(18)1-Beagle株分别在BHK-21、Vero/DogSLAM(VDS)和F81细胞培养,经β-丙内酯灭活剂灭活后,分别与三种不同类型免疫佐剂(MONTANIDE GEL 02、ADJ-801(W)、Al(OH)3)配制成三联灭活疫苗。疫苗经三次免疫比格犬后,通过测定动物血清抗体水平和攻毒保护情况评价其免疫效果。实验结果显示,不同佐剂的三联灭活疫苗对增强比格犬RABV、CDV和CPV血清抗体应答反应效果不同。其中ADJ-801(W)佐剂能显著提高针对RABV、CDV和CPV三种病毒的抗体水平,疫苗三次免疫比格犬后血清抗体效价分别为7.4 EU/mL、1∶50和1∶1024;而Al(OH)3佐剂效果较差,三免后抗体效价分别为4.01 EU/mL、1∶6和1∶256。疫苗三次免疫后,分别应用强毒CDV SD(14)7株和CPVJL(18)1-Beagle株对比格犬进行攻毒实验,实验结果显示,ADJ-801(W)组对SD(14)7和JL(18)1-Beagle攻毒保护率均为100%,MONTANIDE GEL 02组攻毒保护率均为60%,而Al(OH)3组攻毒保护率均为0,表明ADJ-801(W)佐剂制备的狂犬病、犬瘟热和犬细小病毒病三联灭活疫苗对比格犬提供较好的免疫保护作用。本研究为犬狂犬病、犬瘟热和犬细小病毒病三联灭活疫苗的研制奠定了基础。
