Mesocyclops were collected from 631 water areas and fresh water containers over 31 provinces and city between 1998 - 1999. It was found 10 species that involved in Mesocyclops gender. Messocyclops distribute widely and are available in fresh water containers in crowded regions. They can reproduce and develop easily under natural conditions. They have high potential in killing Aedes aegypti larvae. Mesocyclops can become an effective biological agent in preventing actively the Dengue fever/Dengue hemorrhage vector in Viet Nam
Copepoda ; Densovirinae

Background: Vietnam is one of eight countries with dengue fever / dengue hemorrhagic fever circulating seriously in the region. Some recent studies showed Mesocyclops dropping is one of effectively dengue fever/dengue hemorrhagic fever preventive approaches.\r\n', u'Objectives: To survey the distribution and role of mesocyclops in preventing dengue fever/dengue hemorrhagic fever in three communes in Thanh Hoa province.\r\n', u'Subjects and methods: The study was conducted in Dong Hai, Thanh Hoa, Hai Chau communes of Tinh Gia district, Nga Lien commune of Nga Son district. Research subjects are Mesocyclops collected in the artificial water containers and Adesaegypti mosquito larvae in water containers.\r\n', u'Results: Two species found are M.woutersi mesocyclops and M.thermo. M.woutersi exits in all of three local studied Mesocyclops rate in the water containers is 8.2%. Mesocyclops rate in water containers in Nga Son district is highest (13.26%) and in city is lower (13.26%). Of the experimental five species dropped on field, there are four species survived after a long-term period. Among them, M. Woutersi is highest (65.51%), M.aspericornis (15:52%). They breed and grow very fast, easy to adapt to natural conditions in Thanh Hoa.\r\n', u'Conclusion: The results show that Mesocyclops can be cultured in Thanh Hoa province.\r\n', u'\r\n', u'\r\n', u'
Dengue ; Dengue Hemorrhagic Fever ; Copepoda ; Densovirinae ;

OBJECTIVETo probe the primary characteristic of 0507JS11 virus isolated from Culex sp. and determine the classification of 0507JS11 virus in taxonomy.

METHODS0507JS11 virus was cultured in Aedes albopictus C6/36 cells and cytopathic effects (CPEs) were recorded. Electro-microscopic morphology of 0507JS11 virus was observed. Total DNA extract of 0507JS11 virus was detected by 1% Agarose Gel Electrophoresis. Complete genomic sequence of 0507JS11 virus was sequenced and then made phylogenetic analysis.

RESULTS0507JS11 virus could cause CPEs in Aedes albopictus C6/36 cells. Viral particles have no envelope and appear icosahedron symmetry with diameter of 20 nm. The genome of 0507JS11 virus was positive single strand DNA (ssDNA) with full length of 3977 nt. However, a DNA band about 4 kbp was observed in the electrophoresis of total DNA extract of 0507JS11 virus. The coding region of the genome included three ORFs, ORF1 and ORF2 code NSP1 and NSP2, ORF3 codes VP. Phylogenetic analysis of the complete genomic sequence of 0507JS11 virus indicated an independent linear in Brevidensovirus.

CONCLUSION0507JS11 virus is a new member in Brevidensovirus.

Animals ; Culex ; virology ; DNA, Viral ; genetics ; Densovirinae ; classification ; genetics ; isolation & purification ; Genome, Viral ; Sequence Analysis, DNA

Loop-mediated isothermal amplification (LAMP) assay is a novel method of gene amplification with high specificity, sensitivity and rapidity, which can be applied for disease diagnosis in shrimp aquaculture. The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. In the present study, according to the conservative regions of non-structural protein gene NS1, a set of four specific primers were designed, and a rapid detection of IHHNV was established by LAMP assay. The parameters of reaction time and temperature were optimized, and its specificity and sensitivity were assessed. The reactions were carried out at 60 degrees C, 62 degrees C, 63 degrees C, 64 degrees C, 65 degrees C, 66 degrees C, 67 degrees C, 68 degrees C for different time (0 min; 15 min; 30 min; 45 min; 60 min; 75 min). A plasmid pMDIHHNV carrying target sequence of LAMP detection was constructed. Ten-fold serially diluted pMDIHHNV (10(7)-10(0)copies/microL) was used as template for LAMP assay to investigate the detection limit. To determine the specificity, LAMP assays were carried out with DNA templates from other pathogens (White spot syndrome virus; WSSV, Taura Syndrome Virus; TSV, Aeromonas. hydrophila, V. alginolyticus, Vibrio. parahaemolytious, Escherichia. coli). The results showed the optimized LAMP assay for the rapid detection of IHHNV was performed at 65 degrees C for 60 min. The LAMP assay had an unequivocal detection limit of 100 copies/microL, and it was 1,000 times lower than that of PCR. The nucleic acids of other pathogens were not amplified by this LAMP system with the specific primers, which showed a good specificity. The resulting amplicons were detected using visual observation after the addition of SYBR Green I and gel electrophoresis. We investigated the efficacy of UNG (uracil-N-glycosylase) and dUTP in avoiding carry-over contamination in the LAMP assay procedure and explored its effect on the amplification efficiency. Products of LAMP with dUTP adding could be lysed by UNG to avoid LAMP products carry-over contamination effectively. The LAMP assay could be finished within an hour, requiring only a regular laboratory water bath or heat block for reaction and the result could easily be detected using visual observation. Clinically suspected IHHNV-infected shrimp samples were detected by both LAMP and PCR assay, and the result indicated that IHHNV was detected rapidly by LAMP instead of by PCR.
Animals ; DNA Primers ; genetics ; Densovirinae ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; methods ; Penaeidae ; virology ; Viral Proteins ; genetics

Several virus families have been shown to encode microRNAs (miRNAs), which have roles in the infection and replication of viruses in host cells. These virus-encoded miRNAs are identified in double-stranded DNA virus (dsDNA virus) and in several RNA virus families, but not in single-stranded DNA virus (ssDNA virus). We used a bioinformatics approach based on VMir, miRNAFold and MaturePred software to predict virus-encoded miRNA-like small RNAs from the genome of a ssDNA virus: Aedes aegypti densovirus (AaeDV). Northern blotting and stem-loop reverse transcription-polymerase chain reaction (RT-PCR) were used to detect predicted small RNAs. A miRNA-like small RNA termed "AaeDVMD" was identified by stem-loop RT-PCR from predicted candidates. This is the first report demonstrating that a ssDNA virus can encode miRNA-like small RNAs. These data will aid further exploration of the interaction between the AaeDV and its mosquito host.
Aedes ; virology ; Animals ; Base Sequence ; Computational Biology ; Densovirinae ; chemistry ; genetics ; metabolism ; MicroRNAs ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; RNA, Viral ; chemistry ; genetics ; metabolism

Bombyx mori bidensovirus (BmBDV) has been identified as causing chronic densonucleosis in Bombyx mori specifically. The replication mechanism of BmBDV remains unknown. Its genome comprises two single stands DNA (VD1 and VD2). In order to rescue infectious virions in vitro, we obtained the total viral DNA extracted from the BmBDV-infected larvae midguts, subsequently cloned the full-length sequence of BmBDV genome fragments by PCR and constructed recombinant plasmids pMD18T-VD1 and pUC-VD2. The linear genome fragments were obtained by digesting recombinant plasmids with corresponding restriction enzymes, and then collectively transfected BmN cells by the method of liposome-embedding. We determined the replication of the virus gene by PCR with the template of demethylated total DNA extracted from the post-transfect BmN cells. Meanwhile, we collected the total proteins from the post-transfect BmN cells and the larvae midgut of feeding the post-transfect BmN cells to perform Western blotting analysis, and detected the expression of viral genes. Here we firstly confirm that infectious virions can be rescued in BmN cells by linear co-transfect method.
Animals ; Bombyx ; DNA, Viral ; Densovirus ; growth & development ; Larva ; Transfection ; Virion ; Virus Cultivation

OBJECTIVETo investigate arboviruses in Wenshan and Hekou county which are the Sino-Vietnam frontier regions of Wenshan, Yunnan province, China.

METHODSIn September 2007, 6091 Culicines, 1334 Anophelines, 848 Aedes vexans and 53 Armigeres obturbans were collected from 5 field sites. Mosquitoes were collected and stored in liquid nitrogen after classification. The mosquito pools were homogenized, and centrifuged, then the supematant was inoculated onto C6/36 and BHK-21 cells, and the viral isolates were identified by serological and molecular biological methods.Sequence alignment and phylogenetic analysis on the viral isolates were carried out using Clustal X 1.85, GENEDOC and MEGA4 software.

RESULTSA total of 4 pairs of virus isolated with C6/36 cells cytopathic effect were observed, and other mosquito species have not cytopathic effect.Strain WS0704-2 was Banna virus which identified by antibody response and PCR. Strain WS0704-1, WS0708-1, WS0708-2 were Culex pipens pallens densovirus (CppDNV) which identified by PCR. The phylogenetic analysis the 12th segment showed significant difference between the new Banna virus and other strains isolated in China.

CONCLUSIONThere are many mosquito vectors in frontier regions (China and Vietnam) of Wenshan in Yunnan province of China, and mosquito-borne arbovirus, such as BAV were isolated here.

Animals ; Arboviruses ; classification ; genetics ; isolation & purification ; China ; Culicidae ; virology ; Densovirus ; genetics ; isolation & purification ; Phylogeny ; Sequence Alignment ; Sequence Analysis, DNA

An artificial intron consisting of the 5'-donor site (from the first intron of the human beta-globin gene) and the 3'-acceptor site (from the intron of an immunoglobulin gene heavy chain variable region) was obtained with a splice overlap extension PCR and was then inserted in frame into the coding sequence of nostructural protein NS1 gene fused to GFP gene in a recombinant mosquito densovirus plasmid p7NS1-GFP. The constructed plasmid was named as p7NS1-Intron-GFP. The plasmid p7NS1-Intron-GFP was co transfected with the helper plasmid pUCA into C6/36 cells, then the packaged recombinant and wild type viruses were purified and recovered. The second-instars of Aedes albopictus larvae were exposed to recombinant and wild type virus mixed stock. The high level GFP expression in C6/36 cells and larvae was observed under fluorescence microscope, indicating that the inserted artificial intron exerted its normal function in self-splicing both in vitro and in vivo. This study laid a foundation for application of an artificial intron in insect cells and development of new strategy for genetic engineering technology of mosqtuito and its pathogens.
Animals ; Cell Line ; Culicidae ; genetics ; virology ; Densovirus ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Introns ; genetics ; Polymerase Chain Reaction

To better understand the genomic structure and function of Bombyx mori bidensovirus (China isolate) VD1, the VD1 was purified and cloned into the pUC119 vector, and the complete nucleotide sequence of VD1 was determined. Sequence analysis showed that VD1 genome consisted of 6543 nts including inverted terminal repeats (ITRs) of 224 nts. In the viral genome, three major open reading frames (ORF1, ORF2 and ORF3) in the plus strand and one major ORF (ORF4) in the complementary strand were identified. Comparison of the complete genome sequence between Bombyx mori bidensovirus (China isolate) and BmDNV-2 (Yamanashi isolate) showed an identity of 98.4% in VD1, with a total number of 104 bp substitutions and 1 bp insertions found in Bombyx mori bidensovirus (China isolate), the highly variable regions were mainly located in VD1 ORF3 and VD1 ORF4. Northern blotting revealed that VD1 contained 1.1 kb and 1.5 kb transcript in the left-half 'plus' strand, and one transcript about 3.3 kb of 'minus' strand in the right-half. Sequencing of 3' and 5' ends of transcript products showed the 1.1 kb transcript started at nt 290 and ended at nt 1437, the 1.5 kb transcript was found to start nt 1423 and ended at 2931, and the 3.3 kb transcript was found to start nt 6287 and ended at nt 2922. Therefore, the 1.5 kb transcript in the left-half plus' strand and 3.3 kb transcripts of minus' strand in the right-half overlapped for 10 nts at the 3' ends. These results indicate that this virus employs a transcription strategy that is radically different from that of the other reported DNVs.
Amino Acid Sequence ; Animals ; Base Sequence ; Bombyx ; virology ; Densovirus ; genetics ; Genome, Viral ; Molecular Sequence Data ; Transcription, Genetic

The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3(Zhenjiang isolate), named BmDNV-3, is a kind of bidensovirus. The most obvious characteristic in the genome of BmDNV-3 is that it has 2 sets of DNA molecular (VD1, VD2),and each of them is encapsidated respectively in the form of single-stranded liner DNA ( + VD1, - VD1, + VD2, - VD2) in equal percentage. So the BmDNV-3 has 4 kinds of virions. Furthermore the sequence of BmDNV-3 is able to encode DNA polymerase itself. Some strains of silkworm revealed complete resistance to BmDNV-3, so they didn' t fall sick. To investigate the difference in the process of infection and replication between the 2 virions ( VD1, VD2) of this bidensovirus, and the difference of the increment in the resistant or susceptible host, the 5th instar larvae of the susceptible silkworm strain (HUABA 35) and the resistant silkworm strain(QIUFENG d) were inoculated determinate dose of BmDNV-3 by oral ingestion. Then the midgut were collected at 9 timepoints. The silkworm cytoplasm actin A3 was used to be normalized gene, so the number of cells in collected tissue could be determined. The result shows that whatever in the susceptible silkworm strain or in the resistant one, the copies of VD1 and VD2 in the genome of BmDNV-3 collected at the different timepoint were almost at the equal level respectively, so that the VD1 and VD2 were replicated with synchronization. The process of infection in the susceptible silkworm strain was devided into 3 partitions, latent period( 2 - 12 hours post inoculation), exponential phase (12 - 36 hours post inoculation)and stationary phase (36- 96 hours post inoculation and there are about 2 x 10(5) copies per cell) . In the resistant silkworm strain, the virus were replicated at a very low level, that was from 6 - 10 copies 2 hours post inoculation to 150 - 200 copies 96 hours post inoculation (about 20 times) . So we predict that the resistance in some of the silkworm strains from BmDNV-3 was a kind of chronic representation that the host carried virus without being caused flacherie.
Animals ; Bombyx ; genetics ; virology ; DNA, Viral ; genetics ; Densovirus ; genetics ; physiology ; Female ; Genome, Viral ; genetics ; Host-Pathogen Interactions ; Male ; Polymerase Chain Reaction ; methods ; Time Factors ; Virion ; genetics ; physiology ; Virus Replication ; genetics