Effect of Newcastle disease virus-like particles on dendritic cell maturation / 中国兽医学报

Newcastle disease virus-like particles (NDV VLPs) are composed of matrix protein (M) as the skeleton,with the insertion of hemagglutinin-neuraminidase and/or fusion protein.NDV VLPs are reported to be immunogenic and can induce specific humoral and cellular immune responses.However,its relationship with innate immunity remains elusive.Dendritic cells (DCs) are a group of specialized antigen presenting cells,which are crucial in connecting innate immunity and adaptive immunity.In this study,NDV VLPs and murine DCs were used to investigate the connection between NDV VLPs and innate immunity.The DC maturation induced by NDV VLPs (M+ HN) was evaluated.The results showed that NDV VLPs could be effectively taken up by DC and presented to naive T cells.NDV VLPs-induced DC significantly up-regulated the expression of MHC Ⅱ and costimulatory molecules on DC surface,and subsequently promoted the secretion of proinflammatory cytokines.This experiments also showed that different assembled NDV VLPs induced significant stimulating ability in cytokine levels.In summary,NDV VLPs can induce DC maturation,which gives insights to better understanding of VLPs-mediated innate immunity and provide information in selecting preferred NDV VLPs candidate.

Study on immunogenicity of recombinant lentivirus expressing trimeric HA of swine H1 N1 influenza virus / 中国免疫学杂志

Objective:To evaluate the protective immunity by vaccination of BALB/c mice with rLV-HA-GCN4,a recombinant lentivirus expressing the trimeric HA of swine H1N1 influenza virus. Methods:The female mice were randomly divided into rLV-HA-GCN4,rLV-HA,LV and PBS groups. Mice were primed with plasmid and boosted with lentivirus by the administration of intramuscular thigh injections at an interval of two weeks. At day 28 post-prime immunization,mice were inoculated intranasally with 100TCID50 of swine H1N1 influenza virus in a 50 μl volume. The immune levels were assessed by the T lymphocyte transformation test, flow cytometry,indirect ELISA and the indexes of spleen and lung. Results:The spleen lymphocyte transformation rate was 0. 3±0. 11 in the rLV-HA-GCN4 group at day 14 post-boost immunization, showing a statistical significance ( P<0. 01 ) compared to the PBS group. Meanwhile,rLV-HA-GCN4 could cause T lymphocyte response mainly based on the Th1-type CD4+ T cells. The IgG antibody titer reached to 1:8 000 at day 14 post-boost immunization and approximately 1:7 000 at day 14 post challenge. After challenge,the spleen and lung indexes of rLV-HA-GCN4 group were significantly lower than those of PBS group (P<0. 05). The body weight of rLV-HA-GCN4 group demonstrated a slight decrease before 3 days post challenge and then a gradual increase compared to the LV and PBS groups (P<0. 05). Conclusion:rLV-HA-GCN4 can effectively induce cellular and humoral immune response in BALB/c mice against swine H1N1 influenza virus.

Induction of S phase arrest and apoptosis by ethyl acetate extract from Tetrastigma hemsleyanum in human hepatoma HepG2 cells.

Tetrastigma hemsleyanum, a rare and endangered medicinal plant, has attracted much attention due to antitumor and immunomodulatory activities. In this study, the effect and mechanism of ethyl acetate extract from T. hemsleyanum (EET) on cell cycle and apoptosis in human hepatoma HepG2 cells were investigated. Twenty-five to 200 µg/mL of EET were found to have the antiproliferation effect toward HepG2 cells determined by MTT assay. The morphology of EET-treated HepG2 cells showed evidence of apoptosis that included blebbing and chromatin condensation, nucleic fragmentation, and so on. The DNA laddering assay confirmed that DNA fragmentation had occurred during late apoptosis. The cell-cycle analysis indicated that EET was able to induce S phase arrest and typical subdiploid peak in a dose- and time-dependent manner. The apoptosis rate of 200 µg/mL treatment for 24 h was 42.24 ± 4.90%. The protein expression of Bax and P53 was increased after treatment, while that of Bcl2 was significantly decreased in a dose-dependent manner, which suggested that a high Bax/Bcl2 ratio and an upregulated P53 might contribute to the pro-apoptotic activity of EET via the mitochondria-dependent pathway. The protein expression of cyclin-dependent kinase 1 (CDK1) was decreased in EET-treated HepG2 cells, suggesting that EET evoked S phase arrest possibly through the downregulation of cyclin A-CDK1 complex. In conclusion, the cytotoxicity on HepG2 cells induced by EET is a result of both cell-cycle arrest and apoptosis. Thus, it may have therapeutic potential for the treatment of liver cancer.

Proteomic analysis of chicken peripheral blood mononuclear cells after infection by Newcastle disease virus

Characteristic clinical manifestations of Newcastle disease include leukopenia and immunosuppression. Peripheral blood mononuclear cells (PBMCs) are the main targets of Newcastle disease virus (NDV) infection. To survey changes in proteomic expression in chicken PBMCs following NDV infection, PBMC proteins from 30 chickens were separated using two-dimensional electrophoresis (2-DE) and subjected to mass spectrometry analysis. Quantitative intensity analysis showed that the expression of 78 proteins increased more than two-fold. Thirty-five proteins exhibited consistent changes in expression and 13 were identified as unique proteins by matrix assisted laser desorption ionization-time of flight mass spectrometer/mass spectrometer including three that were down-regulated and 10 that were up-regulated. These proteins were sorted into five groups based on function: macromolecular biosynthesis, cytoskeleton organization, metabolism, stress responses, and signal transduction. Furthermore, Western blot analysis confirmed the down-regulation of integrin-linked kinase expression and up-regulation of lamin A production. These data provide insight into the in vivo response of target cells to NDV infection at the molecular level. Additionally, results from this study have helped elucidate the molecular pathogenesis of NDV and may facilitate the development of new antiviral therapies as well as innovative diagnostic methods.

Comparison of plaque-forming characteristics and morphogenetic changes of Newcastle disease virus isolated from chickens and geese on host cells / 病毒学报

The plaque-forming characteristics of Newcastle disease viruses of chickens and geese source were compared on various cells. The result showed that there were obvious differences of plaque formation between F48E9 and NA-1 on Vero cells, chicken embryo fibroblast cells (CEF) and goose embryo fibroblast cells (GEF). The plaque-forming ability of NA-1 was higher than F48E9 on GEF, but lower than F48E9 on CEF. On Vero cells, the plaque-forming ability of NA-1 was slightly stronger than F48E9. It demonstrated that the plaque-forming characteristics were consistent with host tropism of virus. The morphogenesis of F48E9 and NA-1 on Vero cells was observed with transmission electron microscope. There were different replication processes between F48E9 and NA-1 on cells at different stages. NA-1 had stronger adaptability to host than F48E9 according to budding processes and envelope integrity.

Isolation,identification and sequence analysis of the strain JL/07/SW with deletion mutations of PRRSV / 中国兽医学报

One strain of PRRSV was isolated from tissue of piglets died of obvious respiratory syndrome and high fever.It was identified as a strain of American type PRRSV by serological test and gene sequencing.According to American type PRRSV VR-2332,we designed 3 pairs of primers to aim directly at N gene,GP5 gene and variation sequence of NSP2.The results of sequence analysis indicated a discontinuous deletion of 30 amino acids in nonstructural protein 2 (NSP2).N and GP5 gene is conservative relatively.

Inhibition of Newcastle disease virus (NDV) replication by RNA interference targeting phosphoprotein (P) gene in CEF / 中国兽医学报

A plasmids of continuous expressing shRNAs targeting the NDV NA-1 Phosphoprotein (P) gene was designed.Virus titration,Real Time RT-PCR,CPE indicated that P-specific siRNA could inhibit virus replication at 36 h post-virus infection.In future studies,a combination of siRNAs targeting the NP and L gene may be used as a tool to study NDV replication and antiviral therapy.

Rhodococcus equi distribution in the soil environment of horses in Inner Mongolia,China——An investigation report / 中国兽医学报

Little is known about the distribution of Rhodococcus equi in the soil environment of native horses in China. One hundred and eight soil samples were collected from native-horse farms in the Hulun Beier grasslands of eastern Mongolia, the Xilin Goler grasslands of southern Mongolia, and Tongliao city in Inner Mongolia of China for investigating the distribution of R. equi in these regions. The isolation rates of R. equi from soil samples from the Hulun Beier and Xilin Goler grasslands ranged from 25.9% to 30.0%. In contrast, isolation rates from soil samples from Tongliao city was as high as 82.3% and the mean number of R. equi in soil samples from Tongliao city was 10 times more than those of samples from the grasslands. The 488 isolates were examined using PCR for the presence of genes that encode virulence-associated 15 000-17 000 antigen protein (VapA) and the 20 000 antigen protein (VapB). All isolates were negative for virulence-associated proteins. Plasmid profiles of these avirulent isolates showed that cryptic plasmids of various sizes were present with an incidence of 13.3% to 21.5%. The results of the present study contrast with those of our recent study, in which we reported that R. equi was absent from Mongolian horses in Ulaanbaatar, Mongolia. It is suggested that the difference between the results of these two studies is due to the mobile pasturing system in Mongolia and nonmobile pasturing system in Inner Mongolia.

Study on the histopathology of cats inoculated with H5N1 subtype high pathogenic avian influenza virus originated from tigers / 病毒学报

In this study, the HPAIV A/Tiger/Harbin/01/2002 (H5N1) used was originated from tigers and propagated in SPF embryonated hen eggs. TCID5, of the virus was 10(-7.36)/0. 05mL on MDCK cell. The cats were inoculated through bronchus route and then, the cats of dead and control were collected for histopathological and immunohistochemistry examination. Meanwhile, the emulsion supernatant fluid of organs and the pharyngeal swab samples of the dead cats were collected for RT-PCR, survived cats and the control cats were tested for the presence of HI antibody by standard method. The results indicated that the damage of lungs from the dead cats were most obvious, the wide range of red consolidation focus emerged on the lobus pulmonis, the fused focus of infection caused injury of lungs. Histology under the microscope revealed diffuse alveolar damage, confluence phlegmasia pathology, infiltration of lymphomonocytes, sackful of infiltration of macrophages and manipulus protein-like effusion in the alveolar. By immunohistochemistry, the positively stained virus particles were found on the epithelial cells of bronchus and alveolus, and also in the endochylema of lymphomonocytes. The specific electophoretic band of 464bp amplified by RT-PCR from samples of pharyngeal swabs, lungs, kidneys, hearts and brains was as same as the theory value. HI antibody titers of the survived cat were 1:32.

Rhodococcus equi in the soil environment of horses in Inner Mongolia, China.

Little is known about the distribution of Rhodococcus equi in the soil environment of native horses in China. One hundred and eight soil samples were collected from native-horse farms in the Hulun Beier grasslands of eastern Mongolia, the Xilin Goler grasslands of southern Mongolia, and Tongliao City in Inner Mongolia, China. The isolation rates of R. equi from soil samples from the Hulun Beier and Xilin Goler grasslands ranged from 25.9% to 30.0%. In contrast, isolation rates from soil samples from Tongliao City were as high as 82.3% and the mean number of R. equi in soil samples from Tongliao City was 10 times more than those of samples from the grasslands. The 488 isolates were examined using PCR for the presence of genes that encode virulence-associated 15-17 kDa antigen protein (VapA) and the 20 kDa antigen protein (VapB). All isolates were negative for virulence-associated proteins. Plasmid profiles of these avirulent isolates showed that cryptic plasmids of various sizes were present with an incidence of 13.3% to 21.5%. The results of the present study contrast with those of our recent study (J. Vet. Med. Sci. 67:611-613, 2005), in which we reported that R. equi was absent from Mongolian horses in Ulaanbaatar, Mongolia. It is suggested that the difference between the results of these two studies is due to the mobile pasturing system in Mongolia and nonmobile pasturing system in Inner Mongolia.

Study on the coinfection status of Borrelia burgdorferi sensu lato and spotted fever group Rickettsia in ticks from Hunchun, Jilin province / 中华流行病学杂志

<p><b>OBJECTIVE</b>To understand the coinfection status of Borrelia burgdorferi sensu lato (B.b.s.l) and spotted fever group Rickettsia (SFGR) in Hunchun of Jilin province, China.</p><p><b>METHODS</b>Polymerase chain reaction (PCR) was used to detect the 5S-23S rRNA intergenic spacer of B. b. s. l and ompA of SFGR in ticks was collected in Hunchun,Jilin province. The amplification products of positive ticks were sequenced, and phylogenetic analysis was conducted by PHYLIP software package.</p><p><b>RESULTS</b>The infection rate of B. b. s. l was 36.0% in Ixodes persulcatus ticks and the SFGR was discovered in I. persulcatus ticks,with an infection rate of 2.0%. The coinfection rate of both agents was 2.0%. In 327 Dermacentor siltarum ticks, the positive rates of B. b. s. l and SFGR were 30.9% and 29.1% respectively. 55 ticks (16.8%) were coinfected with the two pathogens. The sequence analysis of B. b. s. l showed that the B. b. s. l in Jilin area, which were highly homologous, all belonged to B. garinii genotypes. The sequence analysis of SFGR positive products showed that the DNA secquence of the newly detected agent (JL-95) was close to the two previously described rickettsiae which were detected in I. ricinus from Slovakia (called IRS3 and IRS4). Phylogenetic relationships inferred from the comparison of these sequences with those of other genus Rickettsiae indicated that JL-95, IRS3 and IRS4 constituted a new rickettsial genotype and formed a separate cluster among the spotted fever group Rickettsiae.</p><p><b>CONCLUSION</b>Coinfection of B. b. s. l and SFGR existed in Hunchun, Jilin province. The sequencing of specific fragment confirmed a new SFGR which was different from other rickettsiae known in China.</p>

Polymerase Chain Reaction Detection for Porcine Endogenous Retrovirus in 4 Pig Cell Lines / 中国兽医学报

Porcine endogenous retroviruses (PERV) can infect human cell in vitro, which raised widely concerns re-garding the transmission of PERV to xenograft recipients. It's essential to establish a method for detection of PERV.3 pairs of primers were synthesized according to the sequence of gag, pol and env gene of PERV. Polymerase chainreaction (PCR) and reverse transcription PCR (RT-PCR) assays were performed for detection of PERV provirusDNA and PERV specific mRNA. The results showed that provirus DNA and mRNA of PERV existed and expressedin all 4 tested cell lines. The sizes of amplified fragments are identical with the predicted. These methods may be suit-able for monitoring PERV in other cells or tissue.

A pilot study of functional magnetic resonance imaging of the brain during manual and electroacupuncture stimulation of acupuncture point (LI-4 Hegu) in normal subjects reveals differential brain activation between methods.

OBJECTIVES: To characterize the brain activation patterns evoked by manual and electroacupuncture on normal human subjects. DESIGN: We used functional magnetic resonance imaging (fMRI) to investigate the brain regions involved in electroacupuncture and manual acupuncture needle stimulation. A block design was adopted for the study. Each functional run consists of 5 minutes, starting with 1-minute baseline and two 1-minute stimulation, the interval between the two stimuli was 1 minute. Four functional runs were performed on each subject, two runs for electroacupuncture and two runs for manual acupuncture. The order of the two modalities was randomized among subjects. During the experiment, acupuncture needle manipulation was performed at Large Intestine 4 (LI4, Hegu) on the left hand. For each subject, before scanning started, the needle was inserted perpendicular to the skin surface to a depth of approximately 1.0 cm. Electroacupuncture stimulation was delivered using a continuous rectangular wave form (pulse width 30 ms) at a frequency of 3 Hz. For manual acupuncture, the needle was rotated manually clockwise and counterclockwise at a rate of about 180 times per minute (3 Hz). SUBJECTS: Eleven right-handed, normal, healthy volunteer adults, 6 male and 5 female, ages 21-64 participated in the experiment. RESULTS: Results showed that electroacupuncture mainly produced fMRI signal increases in precentral gyrus, postcentral gyrus/inferior parietal lobule, and putamen/insula; in contrast, manual needle manipulation produced prominent decreases of fMRI signals in posterior cingulate, superior temporal gyrus, putamen/insula. CONCLUSION: These results indicate that different brain networks are involved during manual and electroacupuncture stimulation. It suggests that different brain mechanisms may be recruited during manual and electroacupuncture.