Detection and clearance of a mosquito densovirus contaminant from laboratory stocks of Zika virus

BACKGROUND The Zika virus (ZIKV) epidemics that affected South America in 2016 raised several research questions and prompted an increase in studies in the field. The transient and low viraemia observed in the course of ZIKV infection is a challenge for viral isolation from patient serum, which leads to many laboratories around the world sharing viral strains for their studies. C6/36 cells derived from Aedes albopictus larvae are commonly used for arbovirus isolation from clinical samples and for the preparation of viral stocks. OBJECTIVES Here, we report the contamination of two widely used ZIKV strains by Brevidensovirus, here designated as mosquito densovirus (MDV). METHODS Molecular and immunological techniques were used to analyse the MDV contamination of ZIKV stocks. Also, virus passages in mammalian cell line and infecting susceptible mice were used to MDV clearance from ZIKV stocks. FINDINGS MDV contamination was confirmed by molecular and immunological techniques and likely originated from C6/36 cultures commonly used to grow viral stocks. We applied two protocols that successfully eliminated MDV contamination from ZIKV stocks, and these protocols can be widely applied in the field. As MDV does not infect vertebrate cells, we performed serial passages of contaminated stocks using a mammalian cell line and infecting susceptible mice prior to re-isolating ZIKV from the animals' blood serum. MDV elimination was confirmed with immunostaining, polymerase chain reaction (PCR), and analysis of the mosquitoes that were allowed to feed on the infected mice. MAIN CONCLUSIONS Since the putative impact of viral contaminants in ZIKV strains generally used for research purposes is unknown, researchers working in the field must be aware of potential contaminants and test viral stocks to certify sample purity.

Rescuing Bombyx mori bidensovirus in BmN cells in vitro / 生物工程学报

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.

Genetic and biological characterization of a densovirus isolate that affects dengue virus infection

Brevidensoviruses have an encapsidated, single-stranded DNA genome that predominantly has a negative polarity. In recent years, they have received particular attention due to their potential role in the biological control of pathogenic arboviruses and to their unnoticed presence in cell cultures as contaminants. In addition, brevidensoviruses may also be useful as viral vectors. This study describes the first genetic and biological characterization of a mosquito densovirus that was isolated in Brazil; moreover, we examined the phylogenetic relationship between this isolate and the other brevidensoviruses. We further demonstrate that this densovirus has the potential to be used to biologically control dengue virus (DENV) infection with in vitro co-infection experiments. The present study provides evidence that this densovirus isolate is a fast-spreading virus that affects cell growth and DENV infection.

Preliminary application of a mosquito densovirus-mediated artificial intron in vitro and in vive of mosquito / 病毒学报

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.

Bioinformatics analysis of mosquito densovirus nostructure protein NS1 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze and predict the structure and function of mosquito densovirus (MDV) nostructual protein1 (NS1).</p><p><b>METHODS</b>Using different bioinformatics software, the EXPASY pmtparam tool, ClustalX1.83, Bioedit, MEGA3.1, ScanProsite, and Motifscan, respectively to comparatively analyze and predict the physic-chemical parameters, homology, evolutionary relation, secondary structure and main functional motifs of NS1.</p><p><b>RESULTS</b>MDV NS1 protein was a unstable hydrophilic protein and the amino acid sequence was highly conserved which had a relatively closer evolutionary distance with infectious hypodermal and hematopoietic necrosis virus (IHHNV). MDV NS1 has a specific domain of superfamily 3 helicase of small DNA viruses. This domain contains the NTP-binding region with a metal ion-dependent ATPase activity. A virus replication roller rolling-circle replication(RCR) initiation domain was found near the N terminal of this protein. This protien has the biological function of single stranded incision enzyme.</p><p><b>CONCLUSION</b>The bioinformatics prediction results suggest that MDV NS1 protein plays a key role in viral replication, packaging, and the other stages of viral life.</p>

Investigation on arboviruses at Sino-Vietnam border areas in Wenshan of Yunnan province / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate arboviruses in Wenshan and Hekou county which are the Sino-Vietnam frontier regions of Wenshan, Yunnan province, China.</p><p><b>METHODS</b>In 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.</p><p><b>RESULTS</b>A 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.</p><p><b>CONCLUSION</b>There 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.</p>

A new densovirus isolated from the mosquito Toxorhynchites splendens (Wiedemann) (Diptera: Culicidae).

A new densovirus was isolated and characterized in laboratory strains of Toxorhynchites splendens. The virus was detected by polymerase chain reaction (PCR) from mosquitoes reared in our laboratory. PCR fragments from each mosquito were compared by single strand conformation polymorphism (SSCP) assay and found to be indistinguishable. Thus, it is likely the densoviruses from these mosquitoes contain homologous nucleotide sequences. The PCR fragment corresponding to a 451 bp densovirus structural gene segment from each of 5 mosquitoes had 100% identical nucleotide sequences. Phylogenetic analysis of the structural gene sequence suggests the newly isolated densovirus is more closely related to Aedes aegypti densovirus (AaeDNV) than to Aedes albopictus densovirus (AalDNV). Analysis of offspring and predated larvae suggests that vertical and horizontal transmission are responsible for chronic infections in this laboratory strain of Toxorhynchites splendens. The virion DNA is 4.2 kb in size, is closely related to, but distinct from, known densoviruses in the genera Brevidensovirus and Contravirus. Thevirus is tentatively named Toxorhynchites splendens densovirus (TsDNV).

Replication of Bombyx mori Densonucleosis Virus (Zhenjiang isolate) in different silkworm strains / 生物工程学报

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.

Organization and transcription strategy of genome of Bombyx mori bidensovirus (China isolate) VD1 / 生物工程学报

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.

Pathogenicity and infectivity of the Thai-strain densovirus (AThDNV) in Anopheles minimus S.L.

We report the first experimental infection of an anopheline, An. minimus s.l., with the Thai-strain densovirus, AThDNV. Two hundred first-instar larvae were raised in 2 virus concentrations; mortality in the low virus concentration was no different than that in controls (9.5% vs 7.5%, respectively). Mortality in the high virus concentration was 17.5%. Among surviving adults, infection rates were 33.3% (low concentration) and 15.5% (high concentration), as judged by PCR-screening. Infection rates did not differ between males and females. All orally-infected females transmitted densovirus to at least some of their offspring. Vertical transmission rates ranged from 25.0-53.8%. Densovirus infection did not appear to affect fecundity, either in the number of eggs laid or the number of eggs hatched.

Seasonal and spatial variation in natural densovirus infection in Anopheles minimus S.L. in Thailand.

We report the first detection of a mosquito densovirus in anophelines, An. minimus species A and species C, and describe temporal and spatial variation in natural densovirus infection. A total of 814 (136 species A; 678 species C) adult mosquitos, obtained over a one year period from human biting catches at three locations in a village in western Thailand, were PCR tested for densovirus infection. Overall infection prevalence did not differ between species (15.4% species A; 14.5% species C). Infection prevalence showed significant seasonal variation. Some spatial heterogeneity in infection was also noted, with timing of peak infection prevalence varying between sites. PCR-screening of An. minimus s.l. larvae found an overall infection prevalence of 18.8%. Larval infection showed a significant positive association with rainfall recorded two months previous to larval collection. Infection in adult mosquitos showed a moderate relationship to environmental variables, but a significant negative correlation with larval infection in the previous month.