Swine diseases could be caused by unrecognized or minor pathogens. In this study, two unknown cytopathogenic agents were isolated from swine, through cell culture. In order to identify these two cytopathogenic agent (designated CP129 and #2045-7), a particle associated nucleic acids PCR (PAN-PCR) from previous paper was used with simple modification. The cloning procedure was more specified in this study by adding cell control system. According to the modified PAN-PCR, two and four agents-specific DNA sequences were obtained from CP129 and #2045-7, respectively, and they were identified as Mycoplasma (M.) hyorhinis and Mammalian orthoreovirus by nucleotide BLAST. Since M. hyorhinis (CP129) was filterable and non-visible by microscope, this unusual virus-like nature of M. hyorhinis (CP129) was discussed. Especially, the reovirus (#2045-7) was a serotype 3 and a triple reassortant among three serotypes of reoviruses. It was grouped with recently reported reoviruses from disease cases (swine, human and feline), based on the genetic analysis of L1 and S1 partial sequences. In conclusion, two unknown cytopathogenic agents were successfully identified using modified PAN-PCR with cell control system and they were characterized in this study.
Base Sequence ; Cell Culture Techniques ; Clone Cells ; Cloning, Organism ; Humans ; Mammalian orthoreovirus 3 ; Mycoplasma ; Mycoplasma hyorhinis ; Nucleic Acids ; Orthoreovirus, Mammalian ; Polymerase Chain Reaction ; Swine ; Swine Diseases

Humans ; Persistent Infection ; Orthoreovirus/genetics* ; Reoviridae Infections/veterinary*

The virus strains were isolated from the liver and spleen of the dead young ducks characterized with symptoms of hemorrhagic-necrotic hepatitis. These isolates could cause the death of muscovy duck-embryo and chick-embryo. 1-day-old birds infected with these isolates had the same character with clinically dead birds and the virus could be isolated from artificially infected birds. These isolates could proliferate in MDEF and result in CPE. The virus could proliferate in the cytoplasm in order of crystals and arranged in the latlic-like. The viron was shown spherical, icosahedron, cubic symmetry, no-envelope, with double-layered capsid, about 70 nm in diameter by electron microscopy. The genome segments of the virus were consisted of L1-3, M1-3 and S1-4, which were similar to that of avian reovirus (ARV). Compared to 68.2%, 69.3% - 70.1%, respectively. The system evolution analysis showed that S3 gene coding sigmaB protein was placed in different branch of MDRV and ARV, indicating that S3 gene of the virus was different from ARV and MDRV. The main clinical symptoms and lesions of ducklings caused by the virus were different from the diseases caused by MDRV and ARV. It was concluded that the virus was a Novel duck reovirus belonging to Orthoreovirus genus of the Reoviridae family.
Animals ; Animals, Wild ; virology ; Bird Diseases ; pathology ; virology ; Chick Embryo ; China ; Ducks ; Molecular Sequence Data ; Orthoreovirus, Avian ; classification ; genetics ; isolation & purification ; Phylogeny ; Reoviridae Infections ; pathology ; veterinary ; virology ; Viral Proteins ; genetics

OBJECTIVETo study a newly isolated domestic mammalian reovirus, BYD1, its ability to induce apoptosis analyze the three-dimensional structure of its major membrane penetration protein to predict its function in inducing apoptosis.

METHODSHeLa cells infected with BYD1 reovirus were metered with flow cytometer (FCM) to quantify the ratio of apoptotic cells. The data were analyzed with Student's t-test to judge the ability of BYD1 strain to induce apoptosis. The primary sequence ranged from 582 to 675 per microliter protein of BYD1, T1L, T2J and T3D were aligned and compared. The three-dimensional comparative protein structure model of microliter protein was generated by homology-modeling pipeline SWISS MODEL was applied to annotate its secondary and tertiary structure.

RESULTSBYD1 strain was verified with the ability to induce the apoptosis of HeLa cells. The 643-675 segment composing an alpha-helix showed major difference compared with prototype T2J.

CONCLUSIONThe newly isolated reovirus BYD1 is an apoptosis inducing strain. The alpha-helix (residues 643 to 675) of microliter protein of BYD1 may play a key role to induce the proapoptotic activity of infected cells.

Apoptosis ; Apoptosis Regulatory Proteins ; chemistry ; genetics ; physiology ; Cell Differentiation ; Female ; Flow Cytometry ; HeLa Cells ; Host-Pathogen Interactions ; Humans ; Models, Molecular ; Orthoreovirus, Mammalian ; genetics ; metabolism ; physiology ; Protein Conformation ; Protein Structure, Tertiary ; Reoviridae ; genetics ; metabolism ; physiology ; Uterine Cervical Neoplasms ; pathology ; virology ; Viral Regulatory and Accessory Proteins ; chemistry ; genetics ; physiology

Genome, Viral ; Humans ; Malaysia/epidemiology* ; Orthoreovirus/isolation & purification* ; Outpatients ; Pharynx/virology* ; Reoviridae Infections/epidemiology* ; Seroepidemiologic Studies

Muscovy ducks reovirus (DRV) is an important pathogen with a high mortality rate in Muscovy ducks, the researches in the test and the immunity were useful for the prevention and control of DRV infection. In this study, the S3 genes of the three Fujian DRVs were cloned by RT-PCR and sequencing technology. It was found that DRV-YH and YJL were close to avian reovirus (ARV) in the genetic distance, with high identities ranged from 94. 6% to 98. 9%, however, the identities of DRV-YB strain and reference ARV strains in the S3 gene were only 60.6% - 61.7%. The expression vector pET-30a-S3 harboring DRV YB strain S3 gene was constructed and transformed into E. coli BL21, and then the fusion sigmaB protein expression was induced with IPTG. The SDS-PAGE of the expressed products indicated that the fusion protein of approximately 42ku in molecular weight was expressed highly in inclusion body, and made up 67. 7% of the total proteins. The most efficient concentration of IPTG and inducing time were 0. 1 mM and 5h respectively, while the best temperature for expression was 37 degrees C. After purification with the Ni2+ affinity chromatography, the fusion sigmaB protein was 93% of the total proteins, and the purified protein amounted to 0. 86g/L. The Western blot analysis showed that the fusion aB protein was recognized specifically by the antiserum against DRV, confirming that the recombinant fusion protein had good immunoreactivity.
Amino Acid Sequence ; Animals ; Capsid Proteins ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Molecular Sequence Data ; Orthoreovirus, Avian ; chemistry ; classification ; genetics ; isolation & purification ; Phylogeny ; Poultry Diseases ; virology ; RNA-Binding Proteins ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Reoviridae Infections ; veterinary ; virology ; Sequence Analysis ; Sequence Homology, Amino Acid

Reoviridae ; Cryoelectron Microscopy

OBJECTIVETo prepare purified and concentrated coltivirus high titer antigen in order to further detect antibodies against coltivirus in serum sample of patients.

METHODSThe coltivirus in C6/36 cells was cultured and harvested at different time, and the titer was titrated. The virus was purified and concentrated by polyethylene glycol (PEG), and stored at -20 degrees and 4 degrees, with and without glycerol, respectively, then the titer of coltivirus antigen was tested by indirect ELISA. By using the antigen, coltivirus antibodies in serum samples from both suspected Japanese encephalitis (JE) and viral encephalitis (VE) patients were detected.

RESULTSThe highest titer of coltivirus was found at 3-4 weeks of culturing. The antigen titer could be maintained at least for 6 months, especially antigen with glycerol either at 4 degrees or at -30 degrees even for two years. Totally 1141 serum samples from patients diagnosed clinically as JE and VE were tested. The results showed that 130 samples were coltivirus IgM antibody positive and the average positive rate was 11.4% (130/1141). Among 41 samples of paired-serum from patients in Guangzhou Children's Hospital, 9 samples were positive, the positive rate was 22.0% (9/41) in which 5 samples were diagnosed clinically as VE.

CONCLUSIONSStable and purified coltivirus antigen was obtained in order to test coltivirus antibodies as well as development of kits. Coltivirus probably can cause summer-autumn encephalitis in China.

Antibodies, Viral ; blood ; Antigens, Viral ; isolation & purification ; Cell Line ; Coltivirus ; immunology ; Cryopreservation ; methods ; Enzyme-Linked Immunosorbent Assay ; Humans ; Reoviridae Infections ; blood

OBJECTIVETo identify the virus isolated from a mosquito Culex modestus collected from Tongliao city of Inner Mongolia Autonomous Region.

METHODSA strain of virus isolated from mosquito in Tongliao city was identified by serological and molecular biological methods. The nucleotides of the virus isolate were amplified by RT-PCR, and the products were purified and sequenced. Multiple alignment, phylogenetic and amino acid (AA) analysis were carried out by software Clustal X, MEGA4 and MegAlign (DNAStar).

RESULTSThe new isolate was identified to be Banna virus by serological and molecular biological methods. Phylogenetic analysis showed that the Chinese isolates were distributed within one cluster. The homologue of nucleotide and amino acid of 12 segments between the new isolate and other strains isolated from China were 89.6%-98.4% and 90.4%-98.6%.

CONCLUSIONThe virus isolated from Culex modestus in Inner Mongolia belonged to Banna virus, and it is the first time that Banna virus was isolated in this region.

Animals ; Cell Line ; China ; Coltivirus ; classification ; genetics ; immunology ; isolation & purification ; Culicidae ; virology ; Insect Vectors ; virology ; Mice ; Molecular Sequence Data ; Phylogeny ; Reoviridae Infections ; immunology ; virology

Rotaviruses belong to Reoviridae causes diarrhea in human beings as well as domestic animals. This study was conducted to see what type of human rotaviruses are distributed in Seoul and Kyung-gi province. Twenty two of 81 patients showed rotavirus positive with diagnostic kit and RNA electropherosis. We isolated all of rotaviruses from the patients. Electropherotypes of 22 isolates showed 4:2:3 :2 patters whereas those migration patterns were long type. All of those isolates belonged to group 4. Twenty out of 22 isolates reacted with monoclonal antibodies specific to G1, P1A and subgroup II, whereas rest of them, 4-29 and K-30 reacted with subgroup I specific monoclonal antibody. The nucleotide sequence of an isolate K-21 showed 98~100% and 90~96% homologies with those of Wa and KU strain, respectively.
Animals, Domestic ; Antibodies, Monoclonal ; Base Sequence ; Diarrhea ; Gyeonggi-do ; Humans* ; Korea* ; Reoviridae ; RNA ; Rotavirus* ; Seoul