[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstep...[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstepoort was produced by RT-PCR,inserted into pcDNA3.1（＋）vector to construct a expression cassette,which was then subcloned into transfer vector p8AA,prior to the insertion of LacZ expression cassette.The resulting new transfer vector was named as p8AAZH.Subsequently,p8AAZH was co-transfected with the genome of pseudorabies virus（PRV）Bartha-K61 into BHK-21 cells to enable gene recombination and virus package,and the virus solution was collected as cytopathic effect occurring.A series of procedures including blue plaque purification,PCR identification,observation under electron microscope and Western blot were carried out to screen the recombinant pseudorabies virus and identify the protein expression of target gene.Meanwhile,growth curve of the recombinant virus was determined in BHK-21 cells.[Result] The H gene had been inserted into the genome of Bartha-K61 strain,and RPRV-H was the same as Bartha-K61 in the one-step growth curve and cytopathic effect in BHK-21 cells.[Conclusion] The recombinant pseudorabies virus was constructed,and the insertion of H gene did not influence proliferation of recombinant virus,which laid a foundation for development of recombinant canine distemper virus vaccine.展开更多
[Objective] The aim of the present study was to develop a direct immunofluorescence method for the diagnosis of canine distemper （CD） with FITC-conjugated monoclonal antibodies （FITC-McAb）.[ Metbod] The McAb again...[Objective] The aim of the present study was to develop a direct immunofluorescence method for the diagnosis of canine distemper （CD） with FITC-conjugated monoclonal antibodies （FITC-McAb）.[ Metbod] The McAb against CDV, designated as CE3, was purified with protein G and labeled with FITC through agitation method. After purification and identification, the optimal working concentration of FITC-labeled CE3 was determined. Then 61 clinical samples of suspected canine distemper were detected by direct immunofluorescence assay. [ Result] The absorption test, blocking test and specificity test showed that the labeled antibody had high specificity and sensitivity, but didn＇t have cross reaction with canine parvovirus （CPV）, canine parainfluenza virus （CPIV）, canine adenovirus （CAV） and rabies virus （RV）. The optimal working concentration was 1：80. The positive rate of clinical suspected samples was 48%. [ Conclusion] The direct immunofluorescence assay developed in this study was rapid, specific and convenient, and had great significance for the early diagnosis of canine distemper.展开更多
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 prekaryotic 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 prekaryotic cells as well as to study the reactogenieity of the expressed products. [ Method ] RT-PCR amplification was used to obtain H and F protein genes; TA cloning and subclonlng 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 reac- tion 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 reac- togenicity, which can be utilized for diagnosis of a CDV＇s infection or for an epidemiological investigation. Meanwhile, they also provide a basis for developing ge- netically 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
文摘[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstepoort was produced by RT-PCR,inserted into pcDNA3.1（＋）vector to construct a expression cassette,which was then subcloned into transfer vector p8AA,prior to the insertion of LacZ expression cassette.The resulting new transfer vector was named as p8AAZH.Subsequently,p8AAZH was co-transfected with the genome of pseudorabies virus（PRV）Bartha-K61 into BHK-21 cells to enable gene recombination and virus package,and the virus solution was collected as cytopathic effect occurring.A series of procedures including blue plaque purification,PCR identification,observation under electron microscope and Western blot were carried out to screen the recombinant pseudorabies virus and identify the protein expression of target gene.Meanwhile,growth curve of the recombinant virus was determined in BHK-21 cells.[Result] The H gene had been inserted into the genome of Bartha-K61 strain,and RPRV-H was the same as Bartha-K61 in the one-step growth curve and cytopathic effect in BHK-21 cells.[Conclusion] The recombinant pseudorabies virus was constructed,and the insertion of H gene did not influence proliferation of recombinant virus,which laid a foundation for development of recombinant canine distemper virus vaccine.
基金Supported by Science and Technology Foundation of PLA General Lo-gistics Department(06G138)~~
文摘[Objective] The aim of the present study was to develop a direct immunofluorescence method for the diagnosis of canine distemper （CD） with FITC-conjugated monoclonal antibodies （FITC-McAb）.[ Metbod] The McAb against CDV, designated as CE3, was purified with protein G and labeled with FITC through agitation method. After purification and identification, the optimal working concentration of FITC-labeled CE3 was determined. Then 61 clinical samples of suspected canine distemper were detected by direct immunofluorescence assay. [ Result] The absorption test, blocking test and specificity test showed that the labeled antibody had high specificity and sensitivity, but didn＇t have cross reaction with canine parvovirus （CPV）, canine parainfluenza virus （CPIV）, canine adenovirus （CAV） and rabies virus （RV）. The optimal working concentration was 1：80. The positive rate of clinical suspected samples was 48%. [ Conclusion] The direct immunofluorescence assay developed in this study was rapid, specific and convenient, and had great significance for the early diagnosis of canine distemper.
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 prekaryotic cells as well as to study the reactogenieity of the expressed products. [ Method ] RT-PCR amplification was used to obtain H and F protein genes; TA cloning and subclonlng 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 reac- tion 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 reac- togenicity, which can be utilized for diagnosis of a CDV＇s infection or for an epidemiological investigation. Meanwhile, they also provide a basis for developing ge- netically 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.
