RESUMO
Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or ß1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and ß1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvß1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV.
Assuntos
Fusão Celular , Metapneumovirus/fisiologia , Infecções por Paramyxoviridae/fisiopatologia , Receptores de Vitronectina/fisiologia , Proteínas Virais de Fusão/fisiologia , Animais , Linhagem Celular , Infecções por Paramyxoviridae/virologia , Replicação ViralRESUMO
A bacterial artificial chromosome clone, designated LCY, was constructed from a Gallid herpesvirus 2 (GaHV-2) isolate from a GaHV-2 and reticuloendotheliosis virus co-infected clinical sample. The LCY GaHV-2 insert was sequenced and found to consist of 175,319 nucleotides. LCY GaHV-2 open reading frames (ORFs) had a high sequence identity to those of reference strains. The major difference was that two REV long terminal repeats (LTRs), in the same direction, were inserted at the internal repeat short (IRs)/unique short (Us) and Us/terminal repeat short (TRs) junctions. In addition, the a-like sequence and UL36 were different from other strains. Phylogenetic analysis revealed that LCY was closely related to pandemic strains in China. A pathogenicity study and a vaccination-challenge test were performed on LCY and the reference strain, GA. The results showed that LCY induced gross Marek's disease (MD) lesions and mortality in 71.4 and 7.1% of chickens, respectively, which are lower rates than those observed for the reference strain GA (85.7 and 35.7%). The commercially available CVI988 vaccine provided complete protection against LCY and GA (100%). These results showed that the isolate exhibited lower pathogenicity in SPF chickens. This study revealed that a novel pattern of LTR inserts was found in the strain LCY and that the strain was of low virulence. The present work expands the available genetic information for GaHV-2 and will be useful for the control of MD in China.
Assuntos
Galinhas/virologia , Herpesvirus Galináceo 2/genética , Doença de Marek/virologia , Mutagênese Insercional , Vírus da Reticuloendoteliose/genética , Sequências Repetidas Terminais/genética , Animais , China , Coinfecção/virologia , Modelos Animais de Doenças , Escherichia coli/genética , Genoma Viral , Herpesvirus Galináceo 2/efeitos dos fármacos , Herpesvirus Galináceo 2/imunologia , Doença de Marek/imunologia , Doença de Marek/mortalidade , Doença de Marek/prevenção & controle , Proteínas Oncogênicas Virais/genética , Fases de Leitura Aberta , Filogenia , Doenças das Aves Domésticas/virologia , Análise de Sequência de DNA , Vacinação , Vacinas Virais , Virulência , Sequenciamento Completo do GenomaRESUMO
During the course of our continuous surveillance of Gallid herpesvirus 2 (GaHV-2), 44 isolates were obtained from GaHV-2-positive chickens of different flocks in China from 2009 to 2013. The meq gene, considered as a major GaHV-2 oncogene, was sequenced and was found to contain an open reading frame of 1020 nucleotides encoding a 339 amino acid (aa) polypeptide in all isolates. Compared with the GaHV-2 GA strain, the meq genes in 15.9 % (7/44) of the isolates analyzed in this study contained an aa substitution mutation at position 88 (A to T) of which is the first report. The main characteristics of Chinese GaHV-2 isolates meq genes included the substitutions K77E, D80Y, V115A, T139A, P176R, and P217A, and the aa substitution frequency at positions 139 and 176 showed an increase. To test the pathogenicity of the isolates, a pathogenicity study and a vaccination-challenge test were performed on three selected isolates (ZY/1203, WC/1203, and WC/1110) and reference strain GA. The results showed that the three isolates induced gross Marek's disease (MD) lesions in 95.0-100 % cases, which was a higher rate than that obtained for strain GA (82.4 %). Three isolates induced mortality in 10-21.1 % of specific-pathogen-free chickens, which was similar to results with strain GA (23.5 %). The commercially available CVI988 vaccine induced lower protective indices (PIs) against ZY/1203 (82.4) and WC/1110 (83.3) as compared to those against WC/1203 (100) and GA (100). These results showed an evolving trend in the meq genes of the isolates; three isolates exhibited higher morbidity as compared to the reference strain and the vaccine induced lower PIs against two isolates as compared to that against the reference strain.
Assuntos
Galinhas/virologia , Herpesvirus Galináceo 2/patogenicidade , Animais , China/epidemiologia , Herpesvirus Galináceo 2/classificação , Herpesvirus Galináceo 2/genética , Doença de Marek/epidemiologia , Doença de Marek/virologia , Proteínas Oncogênicas Virais/genética , Filogenia , VirulênciaRESUMO
Reticuloendotheliosis virus (REV), classified as a gammaretrovirus, has a variety of hosts, including chickens, ducks, geese, turkeys, and wild birds. REV causes a series of pathological syndromes, especially the immunosuppression of the host, which may lead to an increased susceptibility to other pathogens, thus greatly damaging the poultry industry. Mixed infections of REV and Marek's disease virus (MDV) have been reported in many countries, including China. Previous reports revealed that MDV vaccines were not efficacious, and even less-virulent MDV strains would cause some losses due to mixed infections with REV. Additionally, contaminants in the MDV vaccine might be the main source of REV. In this study, two clinical samples were collected from two flocks of chickens that were diagnosed with MDV. Subsequently, two REV isolates were obtained from the clinical samples. The isolates, named CY1111 and SY1209, were further confirmed through an indirect immunofluorescence assay and electron microscopy. Complete genome sequences of the two REV strains were determined to test the relationship between them and other REV strains. Phylogenetic trees showed that the two REV strains were closely related to most REV strains that were isolated from a variety of hosts. Therefore, REVs might spread freely among these hosts under natural conditions. Additionally, most REV strains in China were in the same clade. The present work offers some information regarding REV in China.
Assuntos
Coinfecção/veterinária , Coinfecção/virologia , Genoma Viral , Doenças das Aves Domésticas/virologia , Vírus da Reticuloendoteliose/genética , Vírus da Reticuloendoteliose/isolamento & purificação , Infecções por Retroviridae/veterinária , Animais , Galinhas , China , Análise por Conglomerados , Herpesvirus Galináceo 2/isolamento & purificação , Doença de Marek/complicações , Microscopia , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Infecções por Retroviridae/complicações , Infecções por Retroviridae/virologia , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
Reproductive toxicity research takes an important place in traditional Chinese medicine pre-clinical safety evaluation. Modern reproductive toxicity experiment includes drug-related miscarriage, fetal death, teratism, and adverse effects on fertility, genital system, embryonic development and fetus, which is different from contraindicated in pregnancy in traditional Chinese medicine theory. Now the three-phases reproductive toxicity study is the method mainly applied in traditional Chinese medicine reproductive toxicity evaluation. Besides that, alternative methods of whole embryos culture and embryonic stem cell test are also used in traditional Chinese medicine embryo toxicity evaluation. This article reviews research progress and pre-clinical evaluation on reproductive toxicity of traditional Chinese medicine.
Assuntos
Medicamentos de Ervas Chinesas/toxicidade , Medicina Tradicional Chinesa , Reprodução/efeitos dos fármacos , Animais , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Desenvolvimento Embrionário/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Humanos , Gravidez , Testes de ToxicidadeRESUMO
The purpose of this study is to evaluate the embryotoxicity of alkaloids in Senecionis Scandentis Hebra on in vitro cultured mouse embryos. Mouse whole embryo culture (WEC) was applied in this study. Post-implantation (8.5 d) mouse embryos were isolated from their mothers, and cultured in medium of immediately centrifuged serum (ICS) with different concentrations of seneciphylline (target concentrations were 100, 50, 25 and 12.5 µg x mL(-1)) or senkirkine (target concentrations were 50, 25 and 12.5 µg x mL(-1)) for 48 h. After culturing completed, the development and organic morphodifferentiation of the cultured embryos were evaluated microscopically. Treatment with seneciphylline and senkirkine had adverse effects on the development and organic morphodifferentiation of embryos. The effect also had clear dose-response. Alkaloidals in Senecionis Scandentis Hebra had embryotoxicity on cultured embryos, which indicated that pregnant people exposed to Senecionis Scandentis Hebra may get potential risk on fetus.
Assuntos
Embrião de Mamíferos/efeitos dos fármacos , Alcaloides de Pirrolizidina/toxicidade , Senécio/química , Teratogênicos/toxicidade , Animais , Técnicas de Cultura Embrionária , Feminino , Camundongos , GravidezRESUMO
Reviewing the literatures about adverse reactions induced by traditional Chinese medicine injections (TCMI) reported on CNKI from 1983 to 2013. Analyzing the causes of adverse reactions induced by TCMI from its quality standards. Provide ideas for improving security of TCMI and completing its quality standards. This review indicates that TCMI-induced adverse reactions have little relationship with the number of compositions, but have tight connection with chemical ingredients and solvents. Adverse reactions can be decreased by perfecting the quality standards of TCMI.
Assuntos
Tratamento Farmacológico/normas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Medicamentos de Ervas Chinesas/efeitos adversos , Sistemas de Notificação de Reações Adversas a Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/normas , Humanos , Injeções , Controle de QualidadeRESUMO
Pseudoallergic reactions of Qingkailing injection (QKLI) was assessed by vascular hyperpermeability which were indicated by ear blue staining in ICR mice after single intravenous injection of QKLI mixed with Evans blue (EB) and skin blue spot formation in SD rats after intradermal injection of QKLI and intravenous injection of EB. In addition, QKLI-induced histamine, VEGF, TNF-alpha release was measured after ICR mice received the single dosing of QKLI iv. The mild vascular hyperpermeability characterized by ear blue staining could be observed in mice after intravenous injection of QKLI and EB. Intracutaneous injection of 50 micro L of test solution containing QKLI (25,50 microL) in rat back skin caused obvious local vascular hyperpermeability at the injection sites so as to result the larger diameters of blue spots than that in negative control group (P <0. 01). QKLI induced a significant increase of VEGF and a slight elevation of histamine in mice after intravenous administration, while TNF-alpha showed no change after QKLI iv. The results in this study indicated that both intravenous injection and intracutanous injection of QKLI could induce vascular hyperpemeability so as to cause pseudoallergic reaction in mice and rats. QKLI-induced pseudoallergic reaction may be associated with the release of histamine and VEGF.
Assuntos
Hipersensibilidade a Drogas/etiologia , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/efeitos adversos , Animais , Hipersensibilidade a Drogas/sangue , Hipersensibilidade a Drogas/imunologia , Histamina/sangue , Injeções , Masculino , Camundongos , Ratos , Pele/efeitos dos fármacos , Pele/imunologia , Fator de Necrose Tumoral alfa/sangue , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
As an important part of die steels, hot-work die steels are mainly used to manufacture molds made of solid metal or high-temperature liquid metal from heating to recrystallization temperature. In view of the requirements for mechanical properties and service life for hot-work die steel, it is conducive to improve the thermal fatigue resistance, wear resistance, and oxidation resistance of hot work die steel. In this review, the main failure modes of hot-work die steel were analyzed. Four traditional methods of strengthening and toughening die steel were summarized, including optimizing alloying elements, electroslag remelting, increasing the forging ratio, and heat treatment process enhancement. A new nano-strengthening method was introduced that aimed to refine the microstructure of hot-work abrasive steel and improve its service performance by adding nanoparticles into molten steel to achieve uniform dispersion. This review provides an overview to improve the service performance and service life of hot work die steel.
RESUMO
Despite highly effective vaccines, Marek's disease (MD) causes great economic loss to the poultry industry annually, largely due to the continuous emergence of new MD virus (MDV) strains. To explore the pathogenic characteristics of newly emerged MDV strains, we selected two strains (AH/1807 and DH/18) with clinically different pathotypes. We studied each strain's infection process and pathogenicity and observed differences in immunosuppression and vaccine resistance. Specific pathogen-free chickens, unvaccinated or vaccinated with CVI988, were challenged with AH/1807 or DH/18. Both infections induced MD damage; however, differences were observed in terms of mortality (AH/1807: 77.8%, DH/18: 50%) and tumor rates (AH/1807: 50%, DH/18: 33.3%). The immune protection indices of the vaccine also differed (AH/1807: 94.1, DH/18: 61.1). Additionally, while both strains caused interferon-ß and interferon-γ expression to decline, DH/18 infection caused stronger immunosuppression than AH/1807. This inhibition persisted even after vaccination, leading to increased replication of DH/18 that ultimately broke through vaccine immune protection. These results indicate that both strains have different characteristics, and that strains such as DH/18, which cause weaker pathogenic damage but can break through vaccine immune protection, require further attention. Our findings increase the understanding of the differences between epidemic strains and factors underlying MD vaccination failure in China.
Assuntos
Herpesvirus Galináceo 2 , Vacinas contra Doença de Marek , Doença de Marek , Doenças das Aves Domésticas , Vacinas , Animais , Doença de Marek/epidemiologia , Doença de Marek/prevenção & controle , Galinhas , Virulência , China/epidemiologia , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/prevenção & controleRESUMO
Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphoma in poultry. The complete genome of the avirulent vaccine strain MDV-814 was cloned as an infectious bacterial artificial chromosome (BAC) using an 8.8-kb fragment containing the self-designed selective marker guanosine phosphoriboxyl transferase. The recombinant virus MDV-814-BAC was generated by co-transfection of a BAC transfer vector and MDV-814 total DNA, and was purified by eight rounds of selective passaging. The infectivity of the BAC DNA clone was validated by MDV reconstitution from chicken embryo fibroblasts transfected with MDV-BAC DNA, which was extracted from electroporated Escherichia coli DH10B cells. In vitro, the BAC-derived virus had similar biological characteristics and growth kinetics as the wild-type parental and recombinant viruses, and chickens immunized with BAC derivatives by various delivery mechanisms acquired protection against virulent MDV challenge. Construction of this MDV-BAC may aid the development of recombinant vaccines-containing multiple antigens.
Assuntos
Galinhas/virologia , Cromossomos Artificiais Bacterianos/genética , Mardivirus/genética , Vacinas contra Doença de Marek/genética , Doença de Marek/prevenção & controle , Animais , Galinhas/imunologia , Clonagem Molecular , DNA Viral/genética , Vetores Genéticos , Genoma Viral , Mardivirus/crescimento & desenvolvimento , Mardivirus/imunologia , Doença de Marek/imunologia , Doença de Marek/virologia , Vacinas contra Doença de Marek/imunologia , Vacinas de DNA/genéticaRESUMO
The prevalence of Marek's disease (MD) caused by Gallid herpesvirus-2 (GaHV-2) has been increasing in chickens in China despite universal vaccination. Among the possible reasons for this trend, of Reticuloendotheliosis virus (REV) contamination in vaccines could lead to co-infection and reduce the vaccine efficacy. Here, we report the epidemiological findings of our continuous surveillance of MD, and an examination of the effects of REV and/or GaHV-2 co-infection. A total of 1230 samples were collected between 2011 and 2015 from 305 flocks covering many of the chicken-raising regions of China. Among these, 606 samples were determined to be GaHV-2-positive, 13.0% of which were found to be co-infected with REV from 18.8% of the flocks. One GaHV-2 strain (HS/1412), a REV strain (HS/1412R), and a GaHV-2 and REV-co-infected strain (HS/1412â¯GR) were isolated from different chickens of a GaHV-2 and REV co-infected flock. Pathogenicity tests showed that HS/1412 and HS/1412â¯GR caused disease in all chickens and that HS/1412R induced morbidity in 84.6% of the infected chickens. HS/1412â¯GR induced 100% mortality and 76.9% tumor formation, which were significantly higher frequencies than those observed with strain HS/1412 (38.5% and 15.4%, respectively) and HS/1412R (0% and 0%). These results indicate that co-infection with GaHV-2 and REV might explain the persistent, sporadic outbreaks of neoplastic disease in some commercial flocks, resulting in a significant economic burden to the poultry industry of China.
Assuntos
Galinhas , Coinfecção/veterinária , Doença de Marek/complicações , Neoplasias/veterinária , Doenças das Aves Domésticas/virologia , Infecções por Retroviridae/veterinária , Animais , China/epidemiologia , Coinfecção/epidemiologia , Coinfecção/virologia , Herpesvirus Galináceo 2/isolamento & purificação , Doença de Marek/epidemiologia , Neoplasias/epidemiologia , Neoplasias/virologia , Vírus da Reticuloendoteliose/isolamento & purificação , Infecções por Retroviridae/complicações , Infecções por Retroviridae/epidemiologia , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/complicações , Infecções Tumorais por Vírus/epidemiologia , Infecções Tumorais por Vírus/veterinária , Infecções Tumorais por Vírus/virologiaRESUMO
Marek's disease virus (MDV), an α-herpesvirus targeting avian species, causes fatal Marek's disease (MD) in chickens. The host interferon (IFN) responses play a key role in resisting viral infection. However, host IFN responses following MDV infection in the chicken central immune organs (thymus and bursa of Fabricius), which contain numerous MDV target cells, is poorly understood. In this study, we performed animal experiments in specific pathogen-free chickens infected with two virulent MDV strains (BS/15 and Md5) or without infection as negative controls. Specifically, the type I IFN (IFN-α and IFN-ß) transcriptional and proteomic expression levels at 7, 10, 14, 17, and 21 days post infection (dpi) were detected and analyzed. Our results indicated that the mRNA and protein expression levels of IFN-α and IFN-ß in the thymus and bursa of Fabricius were mainly downregulated in cytolytic infection (such as 10 dpi) and reactivation (such as 17 dpi) stages, but not the latent (such as 14 dpi) stage of MDV infection, which was determined by comprehensively analyzing the MDV viral load and immune organ damage caused by MDV infection. These data suggest that MDV could inhibit the expression of host type I IFNs, which may be involved in the MDV-induced host immunosuppression and contribute to the immune escape of MDV from host immunity. Furthermore, we found that the downregulated expression of the host type I IFNs induced by BS/15 and Md5 infection was significantly different, which we speculated may be related to the diverse virulence and pathogenicity of MDV strains. In conclusion, our study demonstrated that MDV mostly inhibited the expression of type I IFNs in infected hosts, which may be associated to its pathogenesis.
Assuntos
Interferon Tipo I/imunologia , Doença de Marek/imunologia , Doenças das Aves Domésticas/imunologia , Animais , Bolsa de Fabricius/imunologia , Galinhas , Expressão Gênica , Herpesvirus Galináceo 2 , Interferon Tipo I/genética , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Doenças das Aves Domésticas/virologia , Proteômica , RNA Mensageiro/genética , Organismos Livres de Patógenos Específicos , Timo/imunologia , Carga Viral , VirulênciaRESUMO
Marek's disease (MD) virus (MDV) has been evolving continuously, leading to increasing vaccination failure. Here, the MDV field strain BS/15 was isolated from a severely diseased Chinese chicken flock previously vaccinated with CVI988. To explore the causes of vaccination failure, specific-pathogen free (SPF) chickens vaccinated with CVI988 or 814 and unvaccinated controls were challenged with either BS/15 or the reference strain Md5. Both strains induced MD lesions in unvaccinated chickens with similar mortality rates of 85.7% and 80.0% during the experimental period, respectively. However, unvaccinated chickens inoculated with BS/15 exhibited a higher tumor development rate (64.3% vs. 40.0%), but prolonged survival and diminished immune defects compared to Md5-challenged counterparts. These results suggest that BS/15 and Md5 show a similar virulence but manifest with different pathogenic characteristics. Moreover, the protective indices of CVI988 and 814 were 33.3 and 66.7 for BS/15, and 92.9 and 100 for Md5, respectively, indicating that neither vaccine could provide efficient protection against BS/15. Taken together, these data suggest that MD vaccination failure is probably due to the existence of variant MDV strains with known virulence and unexpected vaccine resistance. Our findings should be helpful for understanding the pathogenicity and evolution of MDV strains prevalent in China.
Assuntos
Mardivirus/imunologia , Mardivirus/isolamento & purificação , Doença de Marek/imunologia , Doença de Marek/virologia , Vacinas Virais/imunologia , Animais , Galinhas , China , Mardivirus/genética , Mardivirus/patogenicidade , Doença de Marek/prevenção & controle , Falha de Tratamento , VirulênciaRESUMO
Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) cause Marek's disease (MD) and reticuloendotheliosis (RE), respectively. Co-infection with MDV and REV is common in chickens, causing serious losses to the poultry industry. However, experimental studies of such co-infection are lacking. In this study, Chinese field strains of MDV (ZW/15) and REV (JLR1501) were used as challenge viruses to evaluate the pathogenicity of co-infection and the influence of MD vaccination in chickens. Compared to the MDV-challenged group, the mortality and tumor rates increased significantly by 20.0% (76.7 to 96.7%) and 26.7% (53.3 to 80.0%), in the co-challenged group, respectively. The protective index of the MD vaccines CVI988 and 814 decreased by 33.3 (80.0 to 47.7) and 13.3 (90.0 to 76.7), respectively. These results indicated that MDV and REV co-infection significantly increased disease severity and reduced the vaccine efficacy. The MDV genome load showed no difference in the feather pulps and spleen, and pathogenicity-related MDV gene expression (meq, pp38, vIL-8, and ICP4) in the spleen significantly increased at some time points in the co-challenged group. Clearly, synergistic pathogenicity occurred between MDV and REV, and the protective efficacy of existing MD vaccines was attenuated by co-infection with Chinese field MDV and REV strains.
Assuntos
Coinfecção/veterinária , Vacinas contra Doença de Marek/imunologia , Doença de Marek/patologia , Doenças das Aves Domésticas/patologia , Infecções por Retroviridae/veterinária , Infecções Tumorais por Vírus/veterinária , Animais , Galinhas , Coinfecção/imunologia , Coinfecção/patologia , Herpesvirus Galináceo 2/imunologia , Herpesvirus Galináceo 2/patogenicidade , Doença de Marek/imunologia , Doenças das Aves Domésticas/imunologia , Vírus da Reticuloendoteliose Aviária/patogenicidade , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/patologia , Análise de Sobrevida , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/patologia , Carga ViralRESUMO
Bacterial artificial chromosomes (BACs) are powerful tools for the manipulation of the large genomes of DNA viruses, such as herpesviruses. However, the methods currently used to construct the recombinant viruses, an important intermediate link in the generation of BACs, involve the laborious process of multiple plaque purifications. Moreover, some fastidious viruses may be lost or damaged during these processes, making it impossible to generate BACs from these large-genome DNA viruses. Here, we introduce the CRISPR/Cas9 as a site-specific gene knock-in instrument that promotes the homologs recombination of a linearized transfer vector and the Pseudorabies virus genome through double incisions. The efficiency of recombination is as high as 86%. To our knowledge, this is the highest efficiency ever reported for Pseudorabies virus recombination. We also demonstrate that the positions and distances of the CRISPR/Cas9 single guide RNAs from the homology arms correlate with the efficiency of homologous recombination. Our work show a simple and fast cloning method of BACs with large genome inserted by greatly enhancing the HR efficiencies through CRISPR/Cas9-mediated homology-directed repair mechanism, and this method could be of helpful for manipulating large DNA viruses, and will provide a successful model for insertion of large DNA fragments into other viruses.
RESUMO
The virulence of Marek's disease virus (MDV) is continuously evolving, and more virulent MDV pathotypes are emerging, thereby reducing the effectiveness of the existing vaccines. In this study, feather pulps were collected from diseased chickens in commercial chicken flocks in China that presented significant MD visceral tumors in 2011 and were inoculated into a monolayer of duck embryo fibroblasts (DEFs). Three field isolates of MDV were obtained by plaque cloning and identified as MDV via PCR and designated strains LCC, LLY, and LTS. Unvaccinated and CVI988 vaccine-vaccinated specific pathogen-free chickens were challenged at 7 days post vaccination (dpv) with 1000 plaque forming units of each of the respective MDV isolates. These strains induced gross MD lesions in all (100%) of the unvaccinated chickens, and the mortality rates of the unvaccinated chickens were 42.9%, 46.7%, and 23.1% by 60 days post challenge (dpc), respectively. The CVI988 vaccine induced protective indices (PIs) of 85.7, 92.3, and 66.7, respectively. These results showed that the pathogenic characteristics of the Chinese isolates were diverse and that vaccine CVI988 provided different levels of protection against them. These data indicated that the existence of variant MDV strains was a possible reason of immunity failure in China.
Assuntos
Herpesvirus Galináceo 2/genética , Vacinas contra Doença de Marek/imunologia , Doença de Marek/virologia , Animais , Galinhas/imunologia , China/epidemiologia , Plumas/virologia , Doença de Marek/epidemiologia , Doença de Marek/prevenção & controle , Reação em Cadeia da Polimerase , Prevalência , Organismos Livres de Patógenos Específicos , Virulência/efeitos dos fármacosRESUMO
PURPOSE: To study the association of intraoperative intraocular pressure (IOP) at the conclusion of primary trabeculectomy with postoperative IOP on days 1, 7, and 30 and report the ability of intraoperative IOP to predict early postoperative IOP. PATIENTS AND METHODS: Prospective, observational, case series. Ninety-seven consecutive patients with primary open-angle or angle-closure glaucoma underwent primary trabeculectomy. The IOP was measured 3 minutes after the closure of scleral flap with a formed anterior chamber. Clinical details and complications were recorded over a 30-day follow-up. The patients were grouped as A (intraoperative IOP≤10.0 mm Hg), B (>10, ≤15.0 mm Hg), and C (>15 mm Hg). RESULTS: Mean age was 60.8±9.9 years. Forty (41.3%) patients were males. Eighty-two (84.5%) had primary angle-closure glaucoma. Mitomycin-c (MMC) was used in 75 (77.3%) eyes. Mean IOP in group C was significantly higher than group A at all visits (P=0.005, 0.002, and 0.05). Postoperative IOP was associated with intraoperative IOP, age, duration, and dose of MMC in univariable regressions. On multivariable analysis, the intraoperative IOP was associated with postoperative IOP's on day 1 (regression coefficient b=0.24, P=0.039, R(2)=0.24) and day 7 (b=0.47, P<0.001, R=0.42), but not on day 30 (b=0.22, P=0.065, R(2)=0.12). IOP on day 7 was predicted by -8.6+0.47×(intraoperative IOP)+0.27×age-11.7×(dose of MMC in mg/mL). Prediction for day 30=9.8+0.27×(intraoperative IOP). CONCLUSIONS: In patients undergoing uncomplicated primary trabeculectomy, intraoperative IOP is associated with and may predict early postoperative IOP. Adjusting the IOP during the operation may optimize postoperative IOP.
Assuntos
Glaucoma de Ângulo Fechado/cirurgia , Glaucoma de Ângulo Aberto/cirurgia , Pressão Intraocular/fisiologia , Trabeculectomia , Adulto , Idoso , Feminino , Glaucoma de Ângulo Fechado/fisiopatologia , Glaucoma de Ângulo Aberto/fisiopatologia , Humanos , Período Intraoperatório , Masculino , Pessoa de Meia-Idade , Mitomicina/uso terapêutico , Período Pós-Operatório , Estudos Prospectivos , Esclera/cirurgia , Retalhos Cirúrgicos , Tonometria Ocular , Resultado do TratamentoRESUMO
A recombinant fowlpox virus (rFPV-IFNγS1) that co-expressed the infectious bronchitis virus (IBV) S1 gene and the chicken interferon-γ gene has been constructed. To evaluate the efficacy of the recombinant fowlpox virus vaccine against heterotypic IBV strains, 60 4-week-old Specific-Pathogen-Free (SPF) chickens were inoculated with this vaccine and 3 weeks post inoculation challenged with the homotypic IBV strain LX4 and the heterotypic IBV strains LHB, LHLJ04XI, LTJ95I and LSC99I. Antibodies against IBV were detected in vaccinated chickens 1-week post inoculation. The number of CD4(+) and CD8(+) T-lymphocytes in the peripheral blood increased rapidly in the vaccinated groups challenged with strains LX4, LHB and LHLJ04XI. There were significant differences in the number of CD4(+) and CD8(+) T-lymphocytes between the vaccinated groups challenged with strains LTJ95I and LSC99I and all the control groups. The morbidity was below 30% in vaccinated groups challenge with strains LX4, LHB and LHLJ04XI, but was 40% greater than that in the other groups. In addition, the lesions and the amount of virus shedding were less severe in the vaccinated groups challenged by strains LX4, LHB and LHLJ04XI when compared with the other groups, but there was no significant difference in the average body weight of the chickens in all groups (all p>0.05). These results indicate that the rFPV-IFNγS1 protected chickens against challenge with homotypic IBV strain LX4 and heterotypic strains LHLJ04XI and LHB.