Construction of Recombinant Pseudorabies Virus Expressing Canine Distemper Virus H Gene and Analysis on Its Biological Characters

[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.

Preparation and Preliminary Identification of Fluorescein Labeled Monoclonal Antibody against Canine Distemper Virus

[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.

ELISA Detection of Antibody against Canine Distemper Virus in Fox

Canine distemper is one of the most important common viral epidemics in farmed foxes. In this study, the whole blood samples of foxes that had been immunized with canine distemper vaccine were collected from a farming area in Changli County. The antibody against canine distemper virus in the 85 prepared serum samples were detected with a double-antibody sandwich ELtSA method. The results showed among the 85 samples from the 4 farms, 81 samples were antibody-positive, and only 4 samples had no antibody against canine distemper. The overall antibody level was higher in that farming area. It is indicated canine distemper vaccination and vaccination program have gotten attention from most of the local farmers, and the immune effect of canine distemper vaccine is better.

Histopathological Observation of Canine Distemper in Raccoon Dogs

In this study, histopathological changes in the heart, liver, spleen, lung, kidney and other organs of raccoon dogs died of canine distemper were observed. According to the results, the lung of infected raccoon dogs exhibited severe hemor- rhage and emphysema; the liver was congested and degenerated; the spleen was enlarged; the exhibited severe hemorrhage and hyaline degeneration. Multiple organs of infected raccoon dogs were congested and hemorrhaged with tissue damage, in- flammatory cell infiltration and a series of pathological changes. This study laid a solid foundation for clinical diagnosis and treatment of canine distemper in raccoon dogs.

Canine Distemper Virus Utilizes Different Receptors to Infect Chicken Embryo Fibroblasts and Vero cells

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.

Pathogenesis of Demyelinating Encephalopathy in Dogs with Spontaneous Acute Canine Distemper

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 conftrm 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.

Pathomorphologic Study of Parasitic Enteritis Caused by Acute Canine Distemper

[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.

Construction of Recombinant Expression Plasmids Containing H and F Protein Genes of Canine Distemper Virus Isolated from a Mink and Their Expression in Prokaryotic Cells

[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.

Addressing the impact of canine distemper spreading on an isolated tiger population in northeast Asia

The continuation of the isolated Amur tiger(Panthera tigris altaica)population living along the China-Russia border is facing serious challenges due to factors such as its small size(including 38 individuals)and canine distemper virus(CDV).We use a population viability analysis metamodel,which consists of a traditional individual-based demographic model linked to an epidemiological model,to assess options for controlling the impact of negative factors through domestic dog management in protected areas,increasing connectivity to the neighboring large population(including more than 400 individuals),and habitat expansion.Without intervention,under inbreeding depression of 3.14,6.29,and 12.26 lethal equivalents,our metamodel predicted the extinction within 100 years is 64.4%,90.6%,and 99.8%,respectively.In addition,the simulation results showed that dog management or habitat expansion independently will not ensure tiger population viability for the next 100 years,and connectivity to the neighboring population would only keep the population size from rapidly declining.However,when the above three conservation scenarios are combined,even at the highest level of 12.26 lethal equivalents inbreeding depression,population size will not decline and the probability of extinction will be<5.8%.Our findings highlight that protecting the Amur tiger necessitates a multifaceted synergistic effort.Our key management recommendations for this population underline the importance of reducing CDV threats and expanding tiger occupancy to its former range in China,but re-establishing habitat connectivity to the neighboring population is an important long-term objective.

Canine distemper virus as a threat to wild tigers in Russia and across their range

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.

Mortality of Amur tigers:The more things change,the more they stay the same

Poaching as well as loss of habitat and prey are identified as causes of tiger population declines.Although some studies have examined habitat requirements and prey availability,few studies have quantified cause-specific mortality of tigers.We used cumulative incidence functions(CIFs)to quantify cause-specific mortality rates of tigers,expanding and refining earlier studies to assess the potential impact of a newly emerging disease.To quantify changes in tiger mortality over time,we re-examined data first collected by Goodrich et al.(2008;study period 1:1992–2004)as well as new telemetry data collected since January 2005(study period 2:2005–2012)using a total of 57 tigers(27 males and 30 females)monitored for an average of 747 days(range 26–4718 days).Across the entire study period(1992 to 2012)we found an estimated average annual survival rate of 0.75 for all tigers combined.Poaching was the primary cause of mortality during both study periods,followed by suspected poaching,distemper and natural/unknown causes.Since 2005,poaching mortality has remained relatively constant and,if combined with suspected poaching,may account for a loss of 17–19%of the population each year.Canine distemper virus(CDV)may be an additive form of mortality to the population,currently accounting for an additional 5%.Despite this relatively new source of mortality,poaching remains the main threat to Amur tiger survival and,therefore,population growth.