OBJECTIVE: To investigate the mechanisms underlying ozone-induced inactivation of poliovirus type 1 (PV1). METHODS: We used cell culture, long-overlapping RT-PCR, and spot hybridization assays to verify and accurately locate the sites of action of ozone that cause PV1 inactivation. We also employed recombinant viral genome RNA infection models to confirm our observations. RESULTS: Our results indicated that ozone inactivated PV1 primarily by disrupting the 5'-non-coding region (5'-NCR) of the PV1 genome. Further study revealed that ozone specifically damaged the 80-124 nucleotide (nt) region in the 5'-NCR. Recombinant viral genome RNA infection models confirmed that PV1 lacking this region was non-infectious. CONCLUSION In this study, we not only elucidated the mechanisms by which ozone induces PV1 inactivation but also determined that the 80-124 nt region in the 5'-NCR is targeted by ozone to achieve this inactivation.
5' Untranslated Regions ; Animals ; Cercopithecus aethiops ; Genome, Viral ; drug effects ; Oxidants, Photochemical ; pharmacology ; Ozone ; pharmacology ; Poliovirus ; drug effects ; Vero Cells ; Virus Inactivation

Sec61β, a subunit of the Sec61 translocon complex, is not essential in yeast and commonly used as a marker of endoplasmic reticulum (ER). In higher eukaryotes, such as Drosophila, deletion of Sec61β causes lethality, but its physiological role is unclear. Here, we show that Sec61β interacts directly with microtubules. Overexpression of Sec61β containing small epitope tags, but not a RFP tag, induces dramatic bundling of the ER and microtubule. A basic region in the cytosolic domain of Sec61β is critical for microtubule association. Depletion of Sec61β induces ER stress in both mammalian cells and Caenorhabditis elegans, and subsequent restoration of ER homeostasis correlates with the microtubule binding ability of Sec61β. Loss of Sec61β causes increased mobility of translocon complexes and reduced level of membrane-bound ribosomes. These results suggest that Sec61β may stabilize protein translocation by linking translocon complex to microtubule and provide insight into the physiological function of ER-microtubule interaction.
Animals ; COS Cells ; Caenorhabditis elegans Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cercopithecus aethiops ; Endoplasmic Reticulum ; metabolism ; Homeostasis ; Humans ; Microtubules ; metabolism ; SEC Translocation Channels ; deficiency ; genetics ; metabolism

Animals ; Antibodies, Monoclonal ; immunology ; Cell Line ; Cercopithecus aethiops ; Cricetinae ; Culicidae ; Dengue Virus ; immunology ; Encephalitis Virus, Japanese ; immunology ; Encephalitis, Japanese ; immunology ; Mice ; Mice, Inbred Strains ; Vero Cells ; Viral Vaccines ; immunology

Animals ; Apoptosis ; drug effects ; genetics ; COS Cells ; Cercopithecus aethiops ; Cytoskeletal Proteins ; genetics ; metabolism ; Eye Proteins ; genetics ; metabolism ; Glaucoma, Open-Angle ; genetics ; metabolism ; Glycoproteins ; genetics ; metabolism ; Humans ; Mutation ; Ubiquinone ; analogs & derivatives ; pharmacology

In order to develop a recombinant attenuated Salmonella typhimurium as oral live vaccine vector, we constructed recombinant plasmid pYA-sopENt100 by replacing the trc promoter with the sopE promoter and secretion signal sequence sopENt100 of Salmonella typhimurium on the basis of plasmid pYA3493. Then, the complementary plasmid pYA-sopENt100 was transformed into ΔcrpΔasdSL1344 by electroporation to generate attenuated Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100). We further characterized ΔcrpΔasdSL1344 (pYA-sopENt100). We also constructed a recombinant strain ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) that harbored the reporter gene-enhanced green fluorescent protein (egfp) gene. Vero cells were infected with ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) and the ability of delivery foreign antigens was tested via Western blotting analysis. The results of PCR, enzyme digestion and sequencing showed that the ΔcrpΔasdSL1344 (pYA-sopENt100) type III secretion system was constructed successfully. The serotype of ΔcrpΔasdSL1344 (pYA-sopENt100) was identical to ΔcrpΔasdSL1344 and SL1344. Compared with wild strain SL1344, the biochemical characteristics of ΔcrpΔasdSL1344 (pYA-sopENt100) had obvious change, but it was basically the same with ΔcrpΔasdSL1344. The growth speed was much slower than that of the wild strain SL1344. The chicken virulence test (LD₅₀) showed that the virulence of ΔcrpΔasdSL1344 (pYA-sopENt100) was 7×10⁴ times lower than SL1344. In addition, we observed the 37 kDa SopENt100-egfp protein in the cultured supernatant of ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) strain by Western blotting analysis. However, both the 37 kDa SopENt100-egfp protein and 27 kDa EGFP protein were detected in ΔcrpΔasdSL1344 (pYA-sopENt100-egfp)-infected Vero cells. These results demonstrated that the recombinant Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100) was successfully constructed, and it should be used as a live vaccine vector for expressing foreign genes.
Animals ; Bacterial Proteins ; genetics ; Cercopithecus aethiops ; Mice ; Plasmids ; Promoter Regions, Genetic ; Salmonella typhimurium ; genetics ; Type III Secretion Systems ; genetics ; Vaccines, Attenuated ; genetics ; Vero Cells ; Virulence

The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.
Animals ; Antibodies, Viral/blood ; Cercopithecus aethiops ; Chickens ; *HN Protein/genetics/immunology ; Immunogenicity, Vaccine/*immunology ; Newcastle Disease/immunology ; Newcastle disease virus/enzymology/*genetics/immunology ; Specific Pathogen-Free Organisms ; Vaccines, DNA/genetics/*immunology ; Vaccines, Inactivated/immunology ; Vero Cells ; *Viral Fusion Proteins/genetics/immunology ; Viral Vaccines/genetics/*immunology/*standards

During the 2015 outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) in Korea, 186 persons were infected, resulting in 38 fatalities. We isolated MERS-CoV from the oropharyngeal sample obtained from a patient of the outbreak. Cytopathic effects showing detachment and rounding of cells were observed in Vero cell cultures 3 days after inoculation of the sample. Spherical virus particles were observed by transmission electron microscopy. Full-length genome sequence of the virus isolate was obtained and phylogenetic analyses showed that it clustered with clade B of MERS-CoV.
Animals ; Cercopithecus aethiops ; Coronavirus Infections/*diagnosis/epidemiology/*virology ; Disease Outbreaks ; Humans ; Microscopy, Electron ; Middle East Respiratory Syndrome Coronavirus/classification/genetics/*isolation & purification/ultrastructure ; Phylogeny ; Polymerase Chain Reaction ; RNA, Viral/analysis/chemistry/metabolism ; Republic of Korea/epidemiology ; Sequence Analysis, RNA ; Vero Cells

To construct an HSV-1 vector vaccine carrying HIV-1 antigens, HIV-1 gp160, gag, protease and the expression elements were chained together, and then inserted into the internal inverted repeat sequence region of HSV-1 by bacterial artificial chromosome technology. Firstly, HIV-1 gp160 (including type B and C), gag and protease genes were cloned into pcDNA3 in series to generate the pcDNA/gBgp and pcDNA/gCgp, then the recombinant plasmids were transfected into 293FT cells, and HIV-1 antigen was detected from transfected cells by Western blotting. Then the expression cassettes from pcDNA/gBgp and pcDNA/gCgp, comprising HIV-1 antigen genes and expression elements, were cloned into pKO5/BN to generate the shuttle plasmids pKO5/BN/gBgp and pKO5/BN/gCgp. The shuttle plasmids were electroporated into E. coli cells that harbor an HSV-BAC, the recombinant bacteria were screened, and the recombinant DNA was extracted and transfected into Vero cells. The recombinant virus was purified through picking plaques, the virus' DNAs were identified by Southern blotting; HIV-1 antigen was detected from the recombinant HSV-1 infected cells by Western blotting, and the virus' replication competent was analyzed. As the results, gp160 and gag proteins were detected from 293FT cells transfected with pcDNA/gBgp and pcDNA/gCgp by Western blotting. The recombinant bacteria were generated from the E. coli electroporated with pKO5/BN/gBgp or pKO5/BN/gCgp. The recombinant HSV was purified from the Vero cells transfected with the recombinant DNA, the unique DNA fragment was detected from the genome of recombination HSV by Southern blotting; gp120 and gp41 were detected from the infected cells by Western blotting, and the recombinant HSV retained replication competent in mammalian cells. The results indicate that the recombinant HSV carrying HIV-1 gp160, gag and protease genes was generated, the virus retains replication competent in mammalian cells, and could be used as a replicated viral vector vaccine.
Animals ; Cercopithecus aethiops ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; genetics ; DNA, Viral ; genetics ; Escherichia coli ; HIV Antigens ; genetics ; immunology ; HIV Envelope Protein gp160 ; genetics ; immunology ; HIV Protease ; genetics ; immunology ; Herpes Simplex Virus Vaccines ; immunology ; Herpesvirus 1, Human ; physiology ; Plasmids ; Transfection ; Vero Cells ; Virus Replication ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; immunology

OBJECTIVETo diagnose imported dengue fever case from Henan province, and to sequence and analyze the characteristics of whole genome sequence, and to explore the possible viral origin source.

METHODSA suspected dengue fever case was reported in Yuzhou city, Henan province. The patient returned from foshan, Guangdong province on September 19, 2014, after the epidemiological investigation and serum specimen collected, which dengue fever case was diagnosed in the laboratory, then it was inoculated on Vero cells. Whole genome sequence was amplified by several pairs primers and characterized using biologic software.

RESULTSThe imported case was diagnosed as dengue virus 1 serotype infection. Dengue 1 strain was isolated using Vero cells successfully. Whole genome was 10,670 nt, which belonged to dengue virus 1 serotype V genotype and didn't found any recombination event. The phylogenetic analysis demonstrated that the strain was closed to Indian starins isolated in 2008-2011, and the homology of nucleotide sequence was between 98.2%-99.4%.

CONCLUSIONIt was the first time to discover imported dengue 1 serotype case in Henan province. However, according to the patient has been to Guangdong province before onset, it inferred that the Indian strain had been imported to Guangdong province before this case in Henan province.

Animals ; Cercopithecus aethiops ; China ; Dengue ; Dengue Virus ; Genes, Viral ; Genotype ; Humans ; India ; Molecular Epidemiology ; Serogroup ; Vero Cells

To investigate the phenotypic characteristics of the strain of the rabies virus CTNCEC25, the strain of the China rabies virus CTN-1 adapted to primary chicken embryo cells (CECs), Vero cells, and mouse neuroblastoma N2a cells was inoculated with CTNCEC25 and parental CTN-1 strains to explore the cytopathic effect (CPE) and growth kinetics of CTNCEC25 on cultured cells. To determine the pathogenicity of CTNCEC25, suckling mice, adult mice, guinea pigs and rabbits were inoculated with CTNCEC25 via the intracerebral route and their survival monitored every day. Furthermore, the CTNCEC25 strain was passed serially in CECs for 20 passages and then 3 passages in the brains of suckling mice to determine phenotypic stability. CTNCEC25 achieved similar growth kinetics in Vero cells and N2a cells compared with parental CTN-1, but CTNCEC25 replicated more efficiently in CECs than the CTN-1 strain with a titer 72 h after infection reaching 10(7.5-7.6) FFU/mL, which was significantly higher than the 10(5.8) FFU/mL achieved by its parental strain, CTN-1. Moreover, CTNCEC25 induced apparent CPE in Vero cells, CECs and N2a cells. Analyses of intracerebral inoculation demonstrated that CTNCEC25 was attenuated profoundly in adult mice and was completely apathogenic to guinea pigs and rabbits, though it caused death in suckling mice. The CTNCEC25 strain proliferated steadily after serial passage in CECs and the brains of suckling mice, and remained avirulent in adult mice. These results suggest that CTNCEC25 is a highly attenuated and genetically stable strain of the rabies virus. CTNCEC25 replicated stably and efficiently in cultured cells and achieved high titers, so it could be a promising and safe vaccine strain for rabies prevention in China.
Animals ; Cell Line ; Cercopithecus aethiops ; China ; Guinea Pigs ; Humans ; Mice ; Rabbits ; Rabies ; virology ; Rabies virus ; genetics ; growth & development ; pathogenicity ; Serial Passage ; Viral Vaccines ; adverse effects ; genetics ; Virulence