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In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors. Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described. This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1(st) instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

The Aedes aegypti densovirus (AeDNV) has previously shown potential in mosquito control. To improve its efficacy as a biopesticide, the gene for an excitatory insect-specific toxin from Buthus martensii Karsch (BmK IT1) was inserted into the AeDNV genome and cloned into pUCA plasmid. The coding sequence for green fluorescent protein was ligated to the C-terminus of the BmK IT1 gene as a screening marker. Recombinant and helper plasmids were cotransfected into C6/36 cells; wild-type viruses were the controls. The recombinant viruses were identified and quantified by real-time polymerase chain reaction and exposed to Ae. albopictus larvae for the evaluation of its bioinsecticidal activity. LT(50) and LD(50) bioassays showed that the recombinant AeDNV had stronger and faster pathogenic effects on Ae. albopictus than the wild-type virus. This is the first report on the recombinant AeDNA containing the insect-specific toxin, BmK IT1, which may be used to develop a novel type of insecticide.

We previously reported that mosquito densoviruses (MDVs) are potential vectors for delivering foreign nucleic acids into mosquito cells. However, considering existing expression strategies, recombinant viruses would inevitably become replication-defective viruses and lose their ability for secondary transmission. The packaging limitations of the virion represent a barrier for the development of MDVs for viral paratransgenesis or as high-efficiency bioinsecticides. Herein, we report the development of a non-defective recombinant Aedes aegypti densovirus (AaeDV) miRNA expression system, mediated by an artificial intron, using an intronic miRNA expression strategy. We demonstrated that this recombinant vector could be used to overexpress endogenous miRNAs or to decrease endogenous miRNAs by generating antisense sponges to explore the biological functions of miRNAs. In addition, the vector could express antisense-miRNAs to induce efficient gene silencing in vivo and in vitro. The recombinant virus effectively self-replicated and retained its secondary transmission ability, similar to the wild-type virus. The recombinant virus was also genetically stable. This study demonstrated the first construction of a non-defective recombinant MDV miRNA expression system, which represents a tool for the functional analysis of mosquito genes and lays the foundation for the application of viral paratransgenesis for dengue virus control.

The DNA and putative amino acid sequences of representative insect and shrimp parvoviruses (subfamily Densovirinae) were analyzed using computer programs. Shrimp viruses included hepatopancreatic parvovirus (HPV) of Penaeus monodon (HPVmon) and P. chinensis (HPVchin), spawner-isolated mortality virus from P. monodon (SMVmon) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) from P. vannamei. Insect viruses included Aedes aegypti densovirus (AaeDNV), Aedes albopictus densovirus (AalDNV), Junonia coenia densovirus (JcDNV), Galleria mellonella densovirus (GmDNV), Bombyx mori densovirus 5 (BmDNV), Diatraea saccharalis densovirus (DsDNV) and Periplaneta fuliginosa densovirus (PfDNV). Virion size for all these viruses ranged between 18 and 30 nm diameter and ssDNA genome length was between 4 and 6 kb. Using BLAST or Clustal W with the sequence fragments available, no significant DNA homology was found except for 77% DNA identity between HPVmon and HPVchin. However, phylogenetic trees constructed by comparing DNA genome sequences for putative viral polypeptides, capsid proteins and nonstructural proteins placed the parvoviruses into two Clades: Clade 1 with SMVmon, PfDNV, DsDNV, GmDNV, JcDNV, and BmDNV; and Clade 2 with HPVmon, HPVchin, IHHNV, AalDNV and AaeDNV. The four shrimp parvoviruses fell into two different clades that grouped with different insect parvoviruses.

Aedes aegypti densovirus (AeDNV) is a small DNA virus that has been developed into an expression and transducing vector for mosquitoes [Afanasiev et al. (1994) Exp Parasitol 79: 322–339; Afanasiev et al. (1999) Virology 257: 62–72; Carlson et al. (2000) Insect Transgenesis: Methods and Applications (Handler, A.M. & James, A.A., eds), pp. 139–159. CRC Press, Boca Raton]. Virions carrying a recombinant genome expressing the GFP gene were used to characterize the pathogenesis of the virus in 255 individual Aedes aegypti larvae. The anal papillae of the larvae were the primary site of infection confirming previous observations (Afanasiev et al., 1999; Allen‐Muira et al. (1999) Virology 257: 54–61). GFP expression was observed in most cases to spread from the anal papillae to cells of the fat body, and subsequently to many other tissues including muscle fibers and nerves. Infected anal papillae were also observed to shrink, or melanize and subsequently fall off in a virus dependent manner. Three to four day‐old larvae were less susceptible to viral infection and, if infected, were more likely to survive into adulthood, with 14% of them still expressing GFP as adults. Higher salt concentrations of 0.10–0.15 m inhibited viral infection. Anopheles gambiae larvae also showed infection of the anal papillae (17%) but subsequent viral dissemination did not occur. The persistence of the reporter gene expression into adulthood of Aedes aegypti indicates that transduction of mosquito larvae with recombinant AeDNV may be a means of introducing a gene of interest into a mosquito population for transient expression.

During an investigation of arboviruses in China, a novel densovirus (DNV) was isolated from the adult female Culex pipiens pallens. The virus, designated Culex pipiens pallens densovirus (CppDNV), caused cytopathic effect in C6/36 cells. The virus particles were icosahedral, non-enveloped and had a mean diameter of 24 nm. The complete coding region of CppDNV was found to be 3335 nt and it contained three open reading frames (ORFs). CppDNV shares 82–93 % identical nucleotides with isolates of the Aedes albopictus densovirus [isolates AalDNV-1, AalDNV-2 (C6/36 DNV) and AalDNV-3], Aedes aegypti densovirus (AaeDNV) and Haemagogus equines densovirus (HeDNV). The nucleotide sequence identity among CppDNV isolates exceeds 98 %. Phylogenetic trees based on non-structural (NS1 and NS2) and capsid (VP) genes show that CppDNV clustered with the species AaeDNV and represents a novel variant of this species within the genus Brevidensovirus.

We have constructed an infectious DNA clone containing the genome of Aedes aegypti densovirus (AeDNV) in a bacterial plasmid. When this clone was transfected into Aedes albopictus C6/36 mosquito cells, the AeDNV genome rescued from the plasmid and replicated as the wild-type virus. To investigate the cloned virus as an expression vector, the reporter gene encoding beta-galactosidase (beta-gal) was inserted into four large open reading frames (ORF) observed in the AeDNV genome. When these recombinant constructs were transfected into Aedes albopictus C6/36 cells, the beta-gal was expressed efficiently from the right ORF (encoding capsid proteins, Vps) and the mid ORF (encoding putative nonstructural protein 2). A low level of expression was found from the left ORF (encoding nonstructural protein 1, NS1), and no expression was detected from the ORF observed on the minus strand of the AeDNV genome. The expression from the right, mid, and left ORFs can be trans-activated with NS1. A putative nuclear targeting sequence observed in the N-terminus of the AeDNV Vps is presumed to be responsible for transport of the chimeric beta-gal into nucleus. The recombinant AeDNV genomes (carrying the beta-gal gene) supplied with the AeDNV capsid proteins can be packaged into infectious transducing particles. Our results indicate that the genome of AeDNV can serve as a vector for delivery and expression of foreign genes in mosquito cells with subsequent targeting of the product to the desired cell compartment.

The Aedes aegypti densovirus (AeDNV) has potential as a delivery vector for foreign nucleic acids into mosquito cells. In this study, we investigated the ability of plasmids containing recombinant viral transducing genome to induce RNA interference (RNAi) effects in C6/C36 cells. We then evaluated the efficiency of a recombinant AeDNV vector to induce RNAi in Aedes albopictus larvae. We found that the expression of V-ATPase was inhibited by up to 90% at 96 h post-transfection in transfected C6/C36 cells. In addition, the bioinsecticidal activities of various RNAi-expressing AeDNV vectors used to infect Ae. albopictus larvae were also tested. We found that when Ae. albopictus larvae were infected with recombinant AeDNV, expression of V-ATPase was downregulated by nearly 70% compared to controls. Furthermore, the median survival time bioassays demonstrated that recombinant AeDNV caused more serious pathogenic effects than the wild type virus. This is the first report showing that recombinant virus plasmid and corresponding recombinant AeDNV can be used as an effective in vitro and in vivo RNAi delivery system, respectively.

We report the isolation, sequencing, biochemical, and structural characterization of a previously undescribed virus in a chronically infected Aedes albopictus C6/36 cell line. This virus is identified as a new densovirus under the Densovirinae subfamily of the Parvoviridae based on its biological and morphologic properties as well as sequence homologies, and is tentatively designated A. albopictus C6/36 cell densovirus (C6/36 DNV). Analysis of the 4094 nt of the C6/36 DNV genome revealed that the plus strand had three large open reading frames (ORFs): a left ORF, a right ORF, and a mid-ORF (within the left ORF), whose potential coding capacities are 91.0, 40.8, and 41.2 kDa, respectively. The left ORF likely encodes the nonstructural protein NS-1, which contains NTP-binding and helicase domains. The right ORF likely encodes structural proteins, VP1 and VP2. Our analyses revealed that C6/36 DNV has a similar genomic organization and shares very high homology in nucleotide sequence and amino acid sequences with Aedes aegypti densovirus (AaeDNV) and A. albopictus densovirus (AalDNV), members of the genus Brevidensovirus of the Densovirinae. Similar to other densoviruses, C6/36 DNV has a different genomic organization and no recognizable sequence homology with viruses in the Parvovirinae. The three-dimensional (3D) reconstruction of the C6/36 DNV at 15.6-Å resolution by electron cryomicroscopy (cryoEM) revealed distinctive outer surface features not previously seen in other parvoviruses, indicating structural divergence of densoviruses, in addition to its genomic differences, while the inner surface of the C6/36 DNV capsid exhibits features that are conserved among parvoviruses.