Identification, Isolation, and Characterization of an Ectromelia Virus New Strain from an Experimental Mouse.
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ABSTRACT:
SUBMITTER: Wang J
PROVIDER: S-EPMC7973332 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND: Bovine viral diarrhea virus (BVDV) is one of the most important pathogens in cattle. Previously, BVDV sub-genotypes of 1b, 1c, 1d, and 1 m were detected in China. However, isolation of BVDV type 1a from cattle has not been reported in China. In 2010, twenty nasal swabs and blood samples were collected from the cattle suspected BVDV infection in Henan province, China. A BVDV isolate was isolated using cell culture, and the pathogenesis of the virus isolate was studied. METHODS: Virus isolation was performed on MDBK cells. The virus identification was conducted by RT-PCR, neutralization test and immunofluorescence assay. In order to determine the genotype of the newly isolated virus, the 5' un-translated region (5'UTR) of the virus isolate was cloned, sequenced and phylogenetically analyzed. To evaluate the virulence of the virus isolate, four BVDV sero-negative calves were intranasally inoculated with the virus suspension. Rectal temperatures and clinical signs were recorded daily. Blood samples were analyzed for changes in white blood cell counts, and tissue samples were taken for histopathology analysis. RESULTS: A new isolate of bovine viral diarrhea virus (BVDV), named HN01, was isolated from the nasal swabs using MDBK cell culture. The HN01 strain caused cytopathic effect (CPE) in MDBK cell cultures after two passages. The virus specifically reacted to BVDV1-specific monoclonal antibody in an immunofluorescence assay. A fragment of 288 bp of genome from this isolate was amplified by the RT-PCR. Phylogenetic analysis of 5'UTR indicated that the virus was BVDV 1a. In the pathogenesis study, four calves experimentally infected with the BVDV strain developed depression, cough and other clinical signs. Calves showed high temperature over 40°C, and white blood cell counts dropped more than 40%. CONCLUSIONS: A new subgenotype 1a strain of BVDV was firstly isolated from dairy cattle in China. The experimental infection showed that the virus was moderate pathogenic to cattle and can be used as a BVDV challenge virus to evaluate the efficacy of BVDV vaccines in the target animals.
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Project description:Erythromelagia is a condition characterized by attacks of burning pain and inflammation in the extremeties. An epidemic form of this syndrome occurs in secondary students in rural China and a virus referred to as erythromelalgia-associated poxvirus (ERPV) was reported to have been recovered from throat swabs in 1987. Studies performed at the time suggested that ERPV belongs to the orthopoxvirus genus and has similarities with ectromelia virus, the causative agent of mousepox. We have determined the complete genome sequence of ERPV and demonstrated that it has 99.8% identity to the Naval strain of ectromelia virus and a slighly lower identity to the Moscow strain. Small DNA deletions in the Naval genome that are absent from ERPV may suggest that the sequenced strain of Naval was not the immediate progenitor of ERPV.
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Project description:T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A*02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8+ as well as CD4+ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma.
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Project description:In the present study, a Lactobacillus plantarum FPL strain exhibiting fructophilic behavior has been isolated for the first time from honeydew. It is a probably syntrophic bacterium inhabiting the gastrointestinal tract of Coccus hesperidum L. and taking part in sugar metabolism. The promising growth characteristics and biochemical properties of Lb. plantarum FPL indicate that this may be a facultatively fructophilic species, whose properties are not associated with the loss of the alcohol/acetaldehyde dehydrogenase gene. The article attempts to classify the peculiar behavior of this strain by means of tests that are characteristic for FLAB as well as through a classic identification approach. In this study, we used a reference strain Lb. plantarum NRRL B-4496, which showed no fructophilic properties. With the FLAB group, the new strain shares the habit, such as a fructose-rich environment, the preference of this sugar for growth, and similar growth curves. However, it exceeds FLAB in terms of osmotolerance to high sugar content. The fructophilic Lb. plantarum FPL strain can proliferate and grow on a medium wherein the sugar concentration is 45 and 50% (w/v). Our findings indicate that honeydew can be a promising source of new fructophilic lactic acid bacteria.
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Project description:BackgroundTrypanosoma equiperdum causes dourine via sexual transmission in Equidae. T. equiperdum is classified under the subgenus Trypanozoon along with the T. brucei sspp. and T. evansi; however, the species classification of Trypanozoon remains a controversial topic due to the limited number of T. equiperdum reference strains. In addition, it is possible that some were misclassified T. evansi strains. Thus, there is a strong need for a new T. equiperdum strain directly isolated from the genital mucosa of a horse with a clinically- and parasitologically-confirmed dourine infection.MethodsTrypanosomes isolated from the urethral tract of a stallion with suspected dourine, were directly cultivated using soft agarose media at 37 °C in 5 % CO2. For molecular characterization, 18S ribosomal RNA (rRNA) gene, the internal transcribed spacer (ITS) and 8 maxicircle DNA regions were amplified by a PCR and their sequences were determined. To analyze the ratio of the kinetoplastic/akinetoplastic population, the kinetoplasts and the nuclei of trypanosomes were subjected to Hoechst staining and observed by fluorescence microscopy.ResultsIn addition to the clinical symptoms and the molecular diagnosis, this stallion was definitively diagnosed with dourine by the detection of trypanosomes in the urethral mucosa. These results strongly suggested that the isolated trypanosome was true T. equiperdum. T. equiperdum isolated from the urethral tract was adapted in vitro using soft agarose media. Based on the results of a phylogenetic analysis of 18S rRNA and ITS, this T. equiperdum isolate was classified into the Trypanozoon clade. In a PCR of the maxicircle DNA region, only NADH-dehydrogenase subunits 4 and 5 was amplified. Clear kinetoplasts were observed in most of the T. equiperdum isolates. In contrast, most culture-adapted T. equiperdum were of the akinetoplastic form.ConclusionWe concluded that our isolated trypanosome was the first confirmed case of T. equiperdum in Mongolia and named it "T. equiperdum IVM-t1". T. equiperdum IVM-t1 was well adapted and propagated in soft agarose media, which indicates that this culture method is useful for isolation of T. equiperdum from horses with dourine.
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Project description:Severe outbreaks of porcine epidemic diarrhea virus (PEDV) have re-emerged in Korea and rapidly swept across the country, causing tremendous economic losses to producers and customers. Despite the availability of PEDV vaccines in the domestic market, the disease continues to plague the Korean pork industry, raising issues regarding their protective efficacy and new vaccine development. Therefore, PEDV isolation in cell culture is urgently needed to develop efficacious vaccines and diagnostic assays and to conduct further studies on the virus biology. In the present study, one Korean PEDV strain, KOR/KNU-141112/2014, was successfully isolated and serially propagated in Vero cells for over 30 passages. The in vitro and in vivo characteristics of the Korean PEDV isolate were investigated. Virus production in cell culture was confirmed by cytopathology, immunofluorescence, and real-time RT-PCR. The infectious virus titers of the viruses during the first 30 passages ranged from 10(5.1) to 10(8.2) TCID50 per ml. The inactivated KNU-141112 virus was found to mediate potent neutralizing antibody responses in immunized guinea pigs. Animal studies showed that KNU-141112 virus causes severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis, indicating that strain KNU-141112 is highly enteropathogenic in the natural host. In addition, the entire genomes or complete S genes of KNU-141112 viruses at selected cell culture passages were sequenced to assess the genetic stability and relatedness. Our genomic analyses indicated that the Korean isolate KNU-141112 is genetically stable during the first 30 passages in cell culture and is grouped within subgroup G2b together with the recent re-emergent Korean strains.
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Project description:Borna disease virus (BDV), the causative agent of severe meningoencephalitis in a wide variety of animal species, has been considered to be genetically invariable and to form a single type within the genus Bornavirus of the family Bornaviridae. BDV infections are of particular interest, because for the first time a virus infection appears to be linked to human psychiatric disorders. We now describe a new subtype of BDV isolated from a horse which was euthanatized due to severe, incurable neurological disease. The nucleotide sequence of this new strain, named No/98, differs from the reference strains by more than 15%, and the subtype is difficult to detect by standard reverse transcriptase PCR protocols. The nucleotide exchanges of the novel BDV isolate have surprisingly little effect on the primary structures of most viral proteins, with the notable exception of the X protein (p10), which is only 81% identical to its counterpart in reference strains. Our data indicate that the genome of BDV is far more variable than previously assumed and that naturally occurring subtypes may escape detection by currently used diagnostic assays.
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Project description:In 2013, porcine epidemic diarrhea (PED) was reported in Japan for the first time in 7years and caused significant economic losses. In the present study, we isolated PED virus (PEDV) circulating in Japan using Vero cell cultures and analyzed sequences of S1 genes of these PEDV isolates. Sequence analysis revealed that one of these strains contained distinct insertion and deletions in the S gene (i.e., S INDEL). Furthermore, inoculation of PEDV into 1-week-old pigs demonstrated that the S INDEL strain had a lower pathogenicity than the North American (NA) prototype strain. This is the first report comparing pathogenicity of an S INDEL strain with the NA prototype strain following experimental inoculation. Excretion of PEDV in the feces of S INDEL strain-inoculated pigs occurred later than in NA prototype strain-inoculated pigs. Thus, our findings suggested that the S INDEL strain had different viral dynamics than the NA prototype strain.
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