Akabane, Aino and Chuzan virus are arthropod-borne (arbo)viruses mainly associated with reproductive failures in cattle. We investigated apoptosis in Vero cells (C-1586) infected with Akabane, Aino and Chuzan virus. The fragmentation of chromosomal DNA was simultaneously detected with the progress of cytopathic effect from 48 hr to 72 hr post infection, depending on viruses. Although the treatment of cycloheximide blocked apoptosis in Vero cells infected with three viruses, actinomycin D did not prevent DNA oligomerization, thus indicating that de novo viral protein synthesis is critical for viral apoptosis. In addition, the activation of caspase-3 was also detected in Vero cells by indirect fluorescent assay. From the present results, it is of future interest whether apoptotic characteristics of these viruses are related to pathogenecity in vivo.
Animals ; Apoptosis/*physiology ; Bunyaviridae/*physiology ; Caspase 3 ; Caspases/metabolism ; Cercopithecus aethiops ; Cytopathogenic Effect, Viral/*physiology ; DNA Fragmentation/physiology ; Dactinomycin ; Enzyme Activation ; Orbivirus/*physiology ; Vero Cells

OBJECTIVETo investigate the epidemic state of arboviruses in the downstream area of Lancang river in Yunnan province.

METHODSMosquitoes were collected from Lancang river downstream area (including Lancang county and Simao city) during summer in 1998 and stored in liquid nitrogen after classification. The mosquito pools were homogenized and centrifuged, then the supernatant was inoculated into C6/36 cells for virus isolation. New isolates were identified by neutralization test(NT), ELISA, immunofluorescence assay(IFA) and polyacrylamid gel electrophoresis(PAGE).

RESULTSTotally 22 isolates of arbovirus were obtained from 233 mosquito pools by inoculation of C6/36 cells and positive rate of the isolation was 9.4%. Ten strains were resistant to both ether and 5 prime-IDU. So they were non-enveloped double-stranded (ds) RNA virus. Twelve segmented RNAs were shown by PAGE and PAGE profiles from the ten strains were 6-6 with minor variation. The isolates can be neutralized by immunized mouse ascites fluid of BJ95-75 strains of coltivirus by NT, and reacted with monoclonal antibody against BJ95-75 by ELISA. These viruses were identified as coltivirus. Nine isolates were sensitive to ether and resistant to 5 prime-IDU. So they were non-enveloped RNA viruses. PAGE showed 10 segmented RNA, and they were identified to be orbiviruses. Three isolations were sensitive to ether. One of them can be neutralized with JEV A2 strain antibody by NT and was positive to the homologous antibody by IFA. It was identified being strain of JE virus. One strain(YN92-4) can be reacted with anti-bunyavirus group specific immune ascites fluid by both IFA and ElISA, but reacted neither with anti-alpha virus group, nor with anti-flavivirus group JE virus ascites fluid. The virions are spherical and about 87 nm in diameter with surface projections by negative staining. Conclusion Twenty-two isolates have been obtained from wild caught-mosquitoes of Lancang river down-stream area in Yunnan province. Among them ten, nine, one and one were identified as coltivirus, orbivirus, JE virus and bunyavirus, respectively. One is under identification. This is the first report on bunyavirus isolated from mosquitoes in China.

Animals ; Arboviruses ; classification ; isolation & purification ; China ; Coltivirus ; isolation & purification ; Culicidae ; virology ; Encephalitis Virus, Japanese ; isolation & purification ; Insect Vectors ; virology ; Orbivirus ; isolation & purification ; Orthobunyavirus ; isolation & purification

Bluetongue virus (BTV) causes Bluetongue (BT) of ruminants vectored by culicoides midges. It is also a classic model for studying the release mechanism of non-enveloped virus. This review begins with the infection and assembly of BTV, then summarizes the advances of different ways of releasing BTV. This includes BTV-induced autophagy and the release as extracellular vesicles via multivesicular bodies, BTV-induced apoptosis and the lytic release, as well as different pathways of release through budding via plasma membrane. The regulatory mechanisms of NS3 which is a key non-structural protein during the release of BTV are also discussed, providing a basis for further understanding the molecular mechanisms underpinning the infection, proliferation and release of BTV.
Animals ; Bluetongue ; Bluetongue virus ; Ceratopogonidae ; Sheep ; Viral Nonstructural Proteins

There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping.
African horse sickness virus/*isolation & purification ; Animals ; Antibodies, Immobilized ; Antibodies, Viral/*immunology ; Cercopithecus aethiops ; Chickens ; Enzyme-Linked Immunosorbent Assay/methods/*veterinary ; Immunoglobulin G ; *Peptide Library ; Serologic Tests/methods/veterinary ; Serotyping ; Single-Chain Antibodies/*immunology ; Vero Cells

OBJECTIVETo isolate viruses from mosquitoes in the south of Xinjiang and identify these viruses primarily.

METHODSA total of 13 491 mosquitoes were collected in the south of Xinjiang from Jul to Aug, 2005. These mosquitoes were divided into 130 groups and grinded respectively. The supernates were inoculated in C6/36 and Vero cells. Viruses isolated were detected, the genomic nucleic types by electrophoresis of viral genomes and the morphologies observed under electronmicroscope.

RESULTSAll 42 viruses were isolated, which caused CPEs on C6/36 but not on Vero cells. 27 viruses showed similar genomic profiles with 12 dsRNA segments. 1 virus displayed genomic profile with 10 dsRNA segments. 5 viruses took on similar genomic profiles with about 4 kbp DNA band. 9 viruses did not get any taxonomy information. Electromicroscopic pictures of these viruses revealed that above four types of viruses had distinguished morphologies indicating different virus species.

CONCLUSIONThere should be several virus species in the mosquitoes in the south of Xinjiang. dsRNA virus with 12 genomic segments should play analysis a predominant role in the south of Xinjiang.

Animals ; Bluetongue virus ; classification ; genetics ; isolation & purification ; Cercopithecus aethiops ; China ; Culicidae ; virology ; Dengue Virus ; classification ; genetics ; isolation & purification ; Genome, Viral ; Insect Viruses ; classification ; genetics ; isolation & purification ; RNA, Double-Stranded ; genetics ; RNA, Viral ; genetics ; Reassortant Viruses ; genetics ; Sequence Analysis, DNA ; Vero Cells

OBJECTIVETo study the death mode of human hepatocellular carcinoma Hep-3B cells and human lung adenocarcinoma A549 cells induced by bluetongue virus strain HbC3 (BTV-HbC3) and the mechanism of its action.

METHODSBTV-HbC3 was used to infect the tumor cells, and the cytopathic effects (CPE) was observed. TUNEL staining was used to detect the apoptosis of tumor cells induced by BTV-HbC3. The changes of endoplasmic reticulum and nuclei treated with BTV-HbC3 were further examined by laser scanning confocal microscopy. The activities of caspase-3/7, caspase-8 and caspase-9 were determined by fluorescence analysis.

RESULTSHep-3B cells were sensitive to BTV-HbC3. Lots of early apoptotic cells were found by TUNEL staining. The laser scanning confocal microscopic examination showed characteristics of apoptosis, such as pyknotic nuclei, margination of nuclear chromatin and vacuolization of endoplasmic reticulumin in Hep-3B cells exposed to BTV-HbC3. The activity of caspase-3/7 was increased, but the activity changes of caspase-8 and caspase-9 were not found. A549 cells were sensitive to BTV-HbC3 too. But no apoptotic cells were observed by TUNEL staining. The results of laser scanning confocal microscopy showed marked vacuolization of endoplasmic reticulum, but chromatin margination was not found after A549 cells was exposed to BTV-HbC3. The activity of caspase-3/7 and caspase-9 was increased, but the activity of caspase-8 was not changed.

CONCLUSIONBTV-HbC3 induces apoptosis of Hep-3B tumor cells mainly through endoplasmic reticulum signal transduction pathway, and the features of cell death in A549 cells could be described as paraptosis.

Adenocarcinoma ; metabolism ; pathology ; virology ; Apoptosis ; Bluetongue virus ; pathogenicity ; physiology ; Carcinoma, Hepatocellular ; metabolism ; pathology ; virology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cell Nucleus ; pathology ; Endoplasmic Reticulum ; pathology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; virology ; Lung Neoplasms ; metabolism ; pathology ; virology ; Oncolytic Viruses ; pathogenicity ; physiology ; Signal Transduction

Arthropod-borne viruses (Arboviruses) are transmitted by arthropods such as Culicoides biting midges and cause abortion, stillbirth, and congenital malformation in ruminants, apparently leading to economic losses to farmers. To monitor the distribution of Culicoides and to determine their relationship with different environmental conditions (temperature, humidity, wind speed, and altitude of the farms) on 5 cattle farms, Culicoides were collected during summer season (May-September) in 2016 and 2017, and analyzed for identification of species and detection of arboviruses. About 35% of the Culicoides were collected in July and the collection rate increased with increase in temperature and humidity. The higher altitude where the farms were located, the more Culicoides were collected on inside than outside. In antigen test of Culicoides against 5 arboviruses, only Chuzan virus (CHUV) (2.63%) was detected in 2016. The Akabane virus (AKAV), CHUV, Ibaraki virus and Bovine ephemeral fever virus (BEFV) had a positive rate of less than 1.8% in 2017. In antigen test of bovine whole blood, AKAV (12.96%) and BEFV (0.96%) were positive in only one of the farms. As a result of serum neutralization test, antibodies against AKAV were generally measured in all the farms. These results suggest that vaccination before the season in which the Culicoides are active is probably best to prevent arbovirus infections.
Agriculture ; Altitude ; Animals ; Antibodies ; Arbovirus Infections ; Arboviruses ; Arthropods ; Cattle ; Ceratopogonidae ; Ephemeral Fever Virus, Bovine ; Farmers ; Humidity ; Jeollabuk-do ; Korea ; Neutralization Tests ; Palyam Virus ; Ruminants ; Seasons ; Stillbirth ; Vaccination ; Wind

A survey was performed in Korea to monitor the prevalence of five bovine arboviruses [Akabane virus, Aino virus, Chuzan virus, bovine ephemeral fever (BEF) virus, and Ibaraki virus] in arthropod vectors, such as Culicoides species. To determine the possible applications of survey data in annual monitoring and warning systems in Korea, we examined the prevalence of bovine arboviruses in arthropod vectors using RT-PCR. To compare the sensitivity and specificity of virus detection, nested PCR was also performed in parallel for all five viruses. Using the RT-PCR, the detection limits were at least up to 10(1.5), 10(2.8), 10(2.0), 10(1.8), and 10(4.0) TCID50/ml for Akabane virus, Aino virus, Chuzan virus, BEF virus, and Ibaraki virus, respectively. When nested PCR was performed using 1 micronl of PCR product, the detection limits were increased, to 10(0.05), 10(1.8), 10(1.0), 10(0.008), and 10(2.0) TCID50/ml for Akabane virus, Aino virus, Chuzan virus, BEF virus, and Ibaraki virus, respectively. Thus, nested PCR increased the sensitivity of the virus detection limit by 1~2 log. We pooled 30~40 mosquitoes in one sample. We collected 113 samples in 2006, 135 samples in 2007, and 100 samples in 2008. Among these samples, Chuzan virus and BEF virus genes were detected at a range between 0.82% and 1.19%, and Akabane virus, Aino virus, and Ibaraki virus genes were detected at less than 0.20%. These data may provide some insight into future epidemiological studies of bovine arboviral diseases in Korea.
Animals ; Arboviruses ; Arthropod Vectors ; Arthropods ; Cattle ; Ceratopogonidae ; Culicidae ; Ephemeral Fever ; Epidemiologic Studies ; Korea ; Limit of Detection ; Organothiophosphorus Compounds ; Palyam Virus ; Polymerase Chain Reaction ; Prevalence ; Sensitivity and Specificity ; Viruses