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

Schmallenberg virus (SBV), a novel orthobunyavirus, was first isolated in 2011. SBV preferentially infects the central nervous system of cattle and sheep and causes fever, diarrhea, a drop in milk yields, congenital malformations and stillbirths. Until June 2014, more than 200 scientific publications regarding SBV have been published. Although more than 20 articles on SVB were published in China, most of these articles provided only a brief introduction of the disease without fully discussing the associated disease characteristics. As a new disease, it has been made a focus of the National Research Center for Exotic Animal Diseases at the China Animal Health and Epidemiology Center. In this review, in order to provide a reference for research into SBV in China, we have reviewed the state of current research progress on the etiology, diagnosis and epidemiology of SBV, and vaccine development.
Animals ; Bunyaviridae Infections ; diagnosis ; epidemiology ; veterinary ; virology ; Cattle ; China ; epidemiology ; Goats ; Host Specificity ; Orthobunyavirus ; classification ; genetics ; isolation & purification ; physiology ; Sheep

OBJECTIVETo culture, isolate and identify new bunyavirus in Vero cell line.

METHODSSamples of 164 new bunyavirus positive by real time RT-PCR detection and well preserved serum specimens were selected from cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) in Xinyang, Henan province in 2009 - 2011. These sera were cultured in Vero cell line and new bunyavirus were detected by observing cytopathic effect (CPE), Real-time RT-PCR, indirect immunofluorescence assay (IFA) and thin-section electron microscopy observation. A total of 10 positive PCR products were selected randomly for sequencing and the results were compared with sequence in Genbank.

RESULTSAmong 164 FTLS serum specimens cultured in Vero cell line, no special CPE were observed and 67 strains (40.85%) were positive detected by Real-time RT-PCR. Nucleic acid similarity of 10 specimens were 97.8% - 100% and there's also a high similarity (> 99%) between specimens and new bunyavirus isolates (Accession No. HQ141600.1). Among 67 positive strains, 58 of them showed specific fluorescence particles by IFA. The viral particles were observed to be spheres with a diameter of 80 - 100 nm by electron microscopy.

CONCLUSIONVero cell line is suitable for culture, isolation and identification of new bunyavirus.

Animals ; Cercopithecus aethiops ; Humans ; Orthobunyavirus ; growth & development ; isolation & purification ; Serum ; virology ; Vero Cells ; virology ; Virus Cultivation ; methods

Animals ; Bunyaviridae Infections ; epidemiology ; virology ; China ; epidemiology ; Fever ; epidemiology ; virology ; Humans ; Orthobunyavirus ; genetics ; isolation & purification ; physiology ; Thrombocytopenia ; epidemiology ; virology

OBJECTIVETo investigate the distribution, species, seasonal fluctuation of ticks and detect new bunyavirus in some hematophagus in the endemic areas of fever thrombocytopenia and leukopenia syndrome (FTLS) in Henan province.

METHODSFrom March to December 2011, the free ticks were collected manually with white cloth from the grassland and the parasitic ticks were collected from the host skin by hand searching in Xinyang and Jiyuan. The density and seasonal fluctuation of ticks were analyzed after classification of the specimen. The hematophagus were collected including gadfly (38 in 16 groups), cattle lice (224 in 16 groups), mosquitoes (238 in 17 groups) and ticks (825 in 77 groups), then RNA of new bunyavirus were detected by RT-PCR.

RESULTSA total of 12 388 ticks were collected in Xinyang and Jiyuan, consisting of 2 families, 5 geniuses and 6 species. In Xinyang city, 622 ticks were identified, consisting of 2 families, 3 geniuses and 3 species, including 2 (0.32%) Ornithodoros lahorensis, 451 (72.51%) Haemaphysalis longicornis and 117 (18.81%) Boophilus microplus. In Jiyuan city, 11 766 ticks were identified, consisting of 1 family, 4 geniuses and 5 species, including 7718 (65.60%) Haemaphysalis longicornis, 164 (1.39%) H.anatolicum anatolicum and 710 (6.03%) other ticks such as H. detritum, Boophilus microplus and Rhipicephalus sanguineus. Haemaphysalis longicornis were found in both districts as the predominant species in Henan province. Ticks were active from March to October. The average density was 160 per person hour and the peak was from May to July with density 278, 209 and 542 per person hour respectively. The results was positive in RNA detection of new bunyavirus in 11 groups of tick and 3 groups of gadfly by RT-PCR. The results were negative in all other hematophagus.

CONCLUSIONOrnithodoros lahorensis, Haemaphysalis longicornis, Boophilus microplus, H.anatolicum anatolicum, Rhipicephalus sanguineus and H. detritum were found in Henan province. Haemaphysalis longicornis was the predominant species. The density of ticks varied with the seasons. The detection of new bunyavirus by PCR was positive in some ticks and gadflies.

Animals ; China ; epidemiology ; Fever ; complications ; epidemiology ; Humans ; Insecta ; virology ; Leukopenia ; complications ; epidemiology ; Orthobunyavirus ; isolation & purification ; Ticks ; classification ; physiology ; virology

Recent global warming trends may have a significant impact on vector-borne viral diseases, possibly affecting vector population dynamics and disease transmission. This study measured levels of hemagglutination-inhibition (HI) antibodies against Japanese encephalitis virus (JEV) and neutralizing antibodies against Akabane virus (AKAV) and Aino virus (AINV) for Thoroughbred horses in Korea. Blood samples were collected from 989 racehorses in several provinces, between October 2005 and March 2007. Sera were tested using either an HI assay or a virus neutralization test. Approximately half (49.7%; 492/989) of the horses tested were antibody-positive for JEV. The HI titer against JEV was significantly correlated with racehorse age (p < 0.05). Horses with an HI antibody titer of 1: 160 or higher accounted for 3.9% of the animals tested, indicating that vectors transmitting arthropod- borne viruses bit relatively few horses. In contrast, 3.8% (19/497) and 19.5% (97/497) of horse sera collected in March 2007 were positive against AKAV and AINV, respectively. The presence of antibodies against AKAV and AINV may indicate the multiplication of AKAV and AINV in these horses.
Aging ; Animals ; Encephalitis Virus, Japanese/*isolation & purification ; Hemagglutination Inhibition Tests/veterinary ; Horse Diseases/blood/*epidemiology ; Horses ; Korea/epidemiology ; Orthobunyavirus/*isolation & purification ; Seroepidemiologic Studies

To evaluate the prevalence of mosquito-borne viruses in Manshi and Ruili (Yunnan Province, China), we collected 2 149 mosquitoes (17 species) in August 2010. Virus isolation was undertaken by the cul- ture of baby hamster kidney cells (BHK-21 cells). Two virus-like isolates were obtained: DHL10M117 was isolated from collected in Mangshi; DHL10M110 was obtained from Anopheles vagus collected in Rui- li. Both isolates caused cytopathic effects,illness and death in suckling mice inoculated with these isolates via the intracerebral route. Two positive amplicons, 702-bp from the S segment and 456-bp from the M segment,were obtained using reverse transcription-polymerase chain reaction using primers specific for the Akabane virus (AKV). Phylogenetic analysis suggested that these two virus stains had a distant relation- ship with AKVs from Kenya and Australia,but were genetically close to those from Japan,South Korea, and Taiwan. However,they were separate from other Asian strains and grouped into a small branch. The highest nucleotide and amino-acid sequence identity of the S segment was found with the CY-77 strain from Taiwan (96.6% and 99.6% for DHL10M117 and 96.7% and 100% for DHL10M110,respectively). Com- parison of the M segment showed they shared the highest amino acid identity with CY-77 (99.6% and 100%, respectively), whereas the highest nucleotide identity was found with the Iriki strain from Japan (99.6% and 100%, respectively). Compared with the MP496 strain from Kenya,they displayed lower lev- els of sequence homology, at 69.7% and 70.0% for nucleotide sequences of the two loci,and 91. 0% for a- mino acids. Our results identified that DHL10M117 and DHL10M110 were strains of AKV,and provided molecular biological evidence for the existence of AKV in Yunnan Province. These AKV strains that are circulating in Yunnan Province share a close genetic relationship with strains from the rest of Asia. Culex tritaeniorhynchus and Anopheles vagus may serve as transmission vectors.
Amino Acid Sequence ; Animals ; Anopheles ; virology ; Base Sequence ; Bunyaviridae Infections ; virology ; China ; Cricetinae ; Female ; Humans ; Insect Vectors ; virology ; Male ; Mice ; Orthobunyavirus ; classification ; genetics ; isolation & purification ; physiology ; Phylogeny ; Sequence Homology ; Viral Proteins ; chemistry ; genetics

We wished to sequence the full-length genomes of the DHL10M110 strain of the Akabane virus (AKV) isolated from mosquitoes in Yunnan Province, China, in 2010. We also wished to analyze the characteristics of these complete nucleotide sequences. The complete genomic sequence of the DHL10M110 strain from Yunnan Province was obtained by reverse transcription-polymerase chain reaction and direct sequencing. We found that the length of the L, M and S gene nucleotide sequences of the DHL10M110 strain were 6 869-bp, 4 309-bp and 856-bp, respectively, including the open reading frame (ORF) nucleotide sequences of 6 756-bp (L), 4 206-bp (M) and 702-bp (S), encoding 2252, 1402 and 234 amino-acid polyproteins, respectively. Phylogenetic analyses based on L-fragment ORF showed that the DHL10M110 strain had a close relationship with the OBE-1 strain of the AKV from Japan and AKVS-7/SKR/2010 strain of the AKV from South Korea. Phylogenetic analyses based on M- and S-fragment ORF showed that the DHL10M110 strain had a close relationship with the epidemic strains of the AKV from Japan, South Korea and Taiwan, but that the DHL10M110 strain had a lone evolutionary branch. In terms of nucleotide (amino acid) homology, the similarity of L-, M- and S-fragment ORFs of the DHL10M110 strain to the OBE-1 strain from Japan was 92.6% (98%), 88.5% (94%) and 96.4% (99.1%), respectively. When comparing the DHL10M110 strain with the OBE-1 strain, we noted 45, 84, and 2 different sites in the amino acids of L, M and S fragments, respectively. Homology and phylogenetic analyses also suggested that the DHL10M110 strain had a distant relationship with the epidemic strains of the AKV from Kenya and Australia. Also, we confirmed by complete genomic sequence analyses that the DHL10M110 strain was clade-Asia of the AKV. However, differences between the DHL10M110 strain compared with strains from Japan and South Korea were also noted. These results suggest that the DHL10M110 strain harbored relatively stable genetic characteristics and distinct regional features. This is the first time that full-length genomic sequences of the DHL10M110 strain of the AKV in mainland China have been obtained.
Amino Acid Sequence ; Animals ; Base Sequence ; Bunyaviridae Infections ; transmission ; virology ; China ; Culicidae ; virology ; Female ; Genome, Viral ; Humans ; Insect Vectors ; virology ; Male ; Molecular Sequence Data ; Open Reading Frames ; Orthobunyavirus ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics

OBJECTIVETo develop an indirect immunofluorescence assay (IFA) for detection of IgG antibodies against new bunyavirus.

METHODSThe antigen slides were prepared with 5 new bunyavirus strains isolated using Africa green monkey kidney (Vero) cells. Specificity and sensitivity evaluation of IFA were carried out by optimizing working conditions of IFA. Using established IFA, serum samples from both acute and recovery phases were tested for 126 cases with fever thrombocytopenia and leukopenia syndrome in Xinyang, Henan province in 2007 - 2011. The results were compared with detections by RT-PCR.

RESULTSThe new bunyavirus stable immunofluorescence specific WZ69 strain was selected to prepare antigen slides of IFA. The optimum conditions of IFA were: optimum dilution for primary antibody (serum) and secondary antibody (isosulfocyanic acid fluorescence marked goat anti-human IgG antibody) was 1:40 and 1:150 respectively. The optimum dilution for Evans blue in secondary antibody was 1:20 000. Among the 126 patients, 96 paired serum specimens were tested positive to the new bunyavirus and 30 patients were tested negative to the virus. The positive rate of antibodies was 76.19%. There was no significant difference in results between IFA and RT-PCR (72.22% (91/126)) (P > 0.05).

CONCLUSIONThe IFA has high sensitivity and specificity with easy operation. It can be used in detecting the new bunyavirus infection in patients with fever, thrombocytopenia and leukopenia syndrome.

Animals ; Antibodies, Viral ; analysis ; immunology ; Bunyaviridae Infections ; diagnosis ; immunology ; Cercopithecus aethiops ; Female ; Fluorescent Antibody Technique, Indirect ; Humans ; Immunoglobulin G ; analysis ; immunology ; Male ; Middle Aged ; Orthobunyavirus ; immunology ; isolation & purification ; Sensitivity and Specificity ; Vero Cells

OBJECTIVETo analyze the epidemiological characteristics of fever thrombocytopenia and leukopenia syndrome (FTLS) in Henan province, China in 2007 - 2011.

METHODSData from specific surveillance system for FTLS in Henan and Information Management System of Chinese Center for Disease Control and Prevention were used to collect the information of the cases.Descriptive epidemiological methods were used to analyze the surveillance data during 2007 - 2011. Patients' sera were collected to detect new bunyavirus using fluorescent RT-PCR and virus isolation.

RESULTSDuring 2007 - 2011, 1021 FTLS cases were reported in Henan province. The fatality rate was 2.25%with 23 deaths. The cases reported in Xinyang city were 1007, accounting for 98.75%.Cases were mainly occurred between April and October, accounting for 96.47% (985/1021). Epidemic peak was May to July, accounting for 59.16% (604/1021). The second peak occurred in September, accounting for 12.05% (123/1021). The age of the cases ranged from 1 to 88 years old with the median age of 59. Sex ratio (male:female) was 1:1.50 (408:613). In all cases, 93.73% (957/1021) were farmers. In 465 patients' sera, the positive rate of new bunyavirus was 69.25% (322/465) using fluorescent RT-PCR. In 164 patients' sera, 67 strains of new bunyavirus were isolated with isolation rate of 40.85% (67/164).

CONCLUSIONFTLS in Henan province is caused mainly by the new bunyavirus and has certain regional and seasonal characteristics. Most cases are female older farmers.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bunyaviridae Infections ; epidemiology ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Fever ; epidemiology ; virology ; Humans ; Infant ; Male ; Middle Aged ; Orthobunyavirus ; isolation & purification ; Sex Ratio ; Thrombocytopenia ; epidemiology ; virology ; Young Adult