Recombinant Mosquito Densovirus with Bti Toxins Significantly Improves Pathogenicity against Aedes albopictus.

Mosquito densoviruses (MDVs) are mosquito-specific viruses that are recommended as mosquito bio-control agents. The MDV Aedes aegypti densovirus (AeDNV) is a good candidate for controlling mosquitoes. However, the slow activity restricts their widespread use for vector control. In this study, we introduced the Bacillus thuringiensis (Bti) toxin Cry11Aa domain II loop α8 and Cyt1Aa loop ß6-αE peptides into the AeDNV genome to improve its mosquitocidal efficiency; protein expression was confirmed using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). Recombinant plasmids were transfected into mosquito C6/36 cell lines, and the expression of specific peptides was detected through RT-PCR. A toxicity bioassay against the first instar Aedes albopictus larvae revealed that the pathogenic activity of recombinant AeDNV was significantly higher and faster than the wild-type (wt) viruses, and mortality increased in a dose-dependent manner. The recombinant viruses were genetically stable and displayed growth phenotype and virus proliferation ability, similar to wild-type AeDNV. Our novel results offer further insights by combining two mosquitocidal pathogens to improve viral toxicity for mosquito control.

The Effect of Phenoloxidase Activity on Survival Is Host Plant Dependent in Virus-Infected Caterpillars.

An important goal of disease ecology is to understand trophic interactions influencing the host-pathogen relationship. This study focused on the effects of diet and immunity on the outcome of viral infection for the polyphagous butterfly, Vanessa cardui Linnaeus (Lepidoptera: Nymphalidae) (painted lady). Specifically, we aimed to understand the role that larval host plants play when fighting a viral pathogen. Larvae were orally inoculated with the entomopathogenic virus, Junonia coenia densovirus (JcDV) (Parvovirididae: Densovirinae, Lepidopteran Potoambidensovirus 1) and reared on two different host plants (Lupinus albifrons Bentham (Fabales: Fabaceae) or Plantago lanceolata Linnaeus (Lamiales: Plantaginaceae)). Following viral infection, the immune response (i.e., phenoloxidase [PO] activity), survival to adulthood, and viral load were measured for individuals on each host plant. We found that the interaction between the immune response and survival of the viral infection was host plant dependent. The likelihood of survival was lowest for infected larvae exhibiting suppressed PO activity and feeding on P. lanceolata, providing some evidence that PO activity may be an important defense against viral infection. However, for individuals reared on L. albifrons, the viral infection had a negligible effect on the immune response, and these individuals also had higher survival and lower viral load when infected with the pathogen compared to the controls. Therefore, we suggest that host plant modifies the effects of JcDV infection and influences caterpillars' response when infected with the virus. Overall, we conclude that the outcome of viral infection is highly dependent upon diet, and that certain host plants can provide protection from pathogens regardless of immunity.

Mass mortality in freshwater mussels (Actinonaias pectorosa) in the Clinch River, USA, linked to a novel densovirus.

Freshwater mussels (order Unionida) are among the world's most biodiverse but imperiled taxa. Recent unionid mass mortality events around the world threaten ecosystem services such as water filtration, nutrient cycling, habitat stabilization, and food web enhancement, but causes have remained elusive. To examine potential infectious causes of these declines, we studied mussels in Clinch River, Virginia and Tennessee, USA, where the endemic and once-predominant pheasantshell (Actinonaias pectorosa) has suffered precipitous declines since approximately 2016. Using metagenomics, we identified 17 novel viruses in Clinch River pheasantshells. However, only one virus, a novel densovirus (Parvoviridae; Densovirinae), was epidemiologically linked to morbidity. Clinch densovirus 1 was 11.2 times more likely to be found in cases (moribund mussels) than controls (apparently healthy mussels from the same or matched sites), and cases had 2.7 (log10) times higher viral loads than controls. Densoviruses cause lethal epidemic disease in invertebrates, including shrimp, cockroaches, crickets, moths, crayfish, and sea stars. Viral infection warrants consideration as a factor in unionid mass mortality events either as a direct cause, an indirect consequence of physiological compromise, or a factor interacting with other biological and ecological stressors to precipitate mortality.

Sea Star Wasting Disease in Asterias forbesi along the Atlantic Coast of North America.

As keystone species, sea stars serve to maintain biodiversity and species distribution through trophic level interactions in marine ecosystems. Recently, Sea Star Wasting Disease (SSWD) has caused widespread mass mortality in several sea star species from the Pacific Coast of the United States of America (USA) and Asterias forbesi on the Atlantic Coast. A densovirus, named Sea Star associated Densovirus (SSaDV), has been associated with the wasting disease in Pacific Coast sea stars, and limited samples of A. forbesi. The goal of this research is to examine the pathogenesis of SSWD in A. forbesi on the Atlantic Coast of the USA and to determine if SSaDV is associated with the wasting disease in this species. Histological examination of A. forbesi tissues affected with SSWD showed cuticle loss, vacuolation and necrosis of epidermal cells, and oedema of the dermis, but no consistent evidence indicating the cause of the lesions. Challenge experiments by cohabitation and immersion in infected water suggest that the cause of SSWD is viral in nature, as filtration (0.22 µm) of water from tanks with sea stars exhibiting SSWD did not prevent the transmission and progression of the disease. Death of challenged sea stars occurred 7-10 d after exposure to infected water or sea stars, and the infectivity crossed species (A. forbesi and Pateria miniata) with equal penetrance. Of the 48 stars tested by quantitative real time PCR, 29 (60%) were positive for the SSaDV VP1 gene. These stars represent field-collected sea stars from all geographical regions (South Carolina to Maine) in 2012-2015, as well as stars exposed to infected stars or water from affected tanks. However, a clear association between the presence of SSaDV and SSWD signs in experimental and field-collected A. forbesi was not found in this study.

Sea Star Wasting Disease in the Keystone Predator Pisaster ochraceus in Oregon: Insights into Differential Population Impacts, Recovery, Predation Rate, and Temperature Effects from Long-Term Research.

Sea star wasting disease (SSWD) first appeared in Oregon in April 2014, and by June had spread to most of the coast. Although delayed compared to areas to the north and south, SSWD was initially most intense in north and central Oregon and spread southward. Up to 90% of individuals showed signs of disease from June-August 2014. In rocky intertidal habitats, populations of the dominant sea star Pisaster ochraceus were rapidly depleted, with magnitudes of decline in density among sites ranging from -2x to -9x (59 to 84%) and of biomass from -2.6x to -15.8x (60 to 90%) by September 2014. The frequency of symptomatic individuals declined over winter and persisted at a low rate through the spring and summer 2015 (~5-15%, at most sites) and into fall 2015. Disease expression included six symptoms: initially with twisting arms, then deflation and/or lesions, lost arms, losing grip on substrate, and final disintegration. SSWD was disproportionally higher in orange individuals, and higher in tidepools. Although historically P. ochraceus recruitment has been low, from fall 2014 to spring 2015 an unprecedented surge of sea star recruitment occurred at all sites, ranging from ~7x to 300x greater than in 2014. The loss of adult and juvenile individuals in 2014 led to a dramatic decline in predation rate on mussels compared to the previous two decades. A proximate cause of wasting was likely the "Sea Star associated Densovirus" (SSaDV), but the ultimate factors triggering the epidemic, if any, remain unclear. Although warm temperature has been proposed as a possible trigger, SSWD in Oregon populations increased with cool temperatures. Since P. ochraceus is a keystone predator that can strongly influence the biodiversity and community structure of the intertidal community, major community-level responses to the disease are expected. However, predicting the specific impacts and time course of change across west coast meta-communities is difficult, suggesting the need for detailed coast-wide investigation of the effects of this outbreak.

Gene expression and localization analysis of Bombyx mori bidensovirus and its putative receptor in B. mori midgut.

Bombyx mori bidensovirus (BmBDV), which causes fatal flacherie disease in the silkworm, replicates only in midgut columnar cells. The viral resistance expressed by some silkworm strains, which is characterized as non-susceptibility irrespective of the viral dose, is determined by a single gene, nsd-2. We previously identified nsd-2 by positional cloning and found that this gene encodes a putative amino acid transporter that might function as a receptor for BmBDV. In this study, we investigated the relationship between the part of the midgut expressing nsd-2 (resistance gene), +(nsd-2) (susceptibility gene) and BmBDV propagation. Quantitative RT-PCR (qRT-PCR) analysis using total RNA isolated from the anterior, middle, and posterior parts of the midgut showed that nsd-2 and +(nsd-2) were strongly expressed in the posterior part of the midgut. The expression levels of both genes were very low in the anterior and middle parts. The qRT-PCR analysis showed that the expression levels of BmBDV-derived transcripts were correlated with the levels of +(nsd-2) expression. However, BmBDV-derived transcripts were clearly detected in all parts of the midgut. These results suggest that the infectivity of BmBDV depends mainly on the expression level of +(nsd-2) in the midgut and that viral infection is supported even by very faint expression of +(nsd-2). By contrast, the expression levels of +(nsd-2) were exceedingly low or undetectable in the middle part of the midgut, indicating that BmBDV infection might occur via another mechanism, independent of +(nsd-2), in the middle part of the midgut.

Up in Arms: Immune and Nervous System Response to Sea Star Wasting Disease.

Echinoderms, positioned taxonomically at the base of deuterostomes, provide an important system for the study of the evolution of the immune system. However, there is little known about the cellular components and genes associated with echinoderm immunity. The 2013-2014 sea star wasting disease outbreak is an emergent, rapidly spreading disease, which has led to large population declines of asteroids in the North American Pacific. While evidence suggests that the signs of this disease, twisting arms and lesions, may be attributed to a viral infection, the host response to infection is still poorly understood. In order to examine transcriptional responses of the sea star Pycnopodia helianthoides to sea star wasting disease, we injected a viral sized fraction (0.2 µm) homogenate prepared from symptomatic P. helianthoides into apparently healthy stars. Nine days following injection, when all stars were displaying signs of the disease, specimens were sacrificed and coelomocytes were extracted for RNA-seq analyses. A number of immune genes, including those involved in Toll signaling pathways, complement cascade, melanization response, and arachidonic acid metabolism, were differentially expressed. Furthermore, genes involved in nervous system processes and tissue remodeling were also differentially expressed, pointing to transcriptional changes underlying the signs of sea star wasting disease. The genomic resources presented here not only increase understanding of host response to sea star wasting disease, but also provide greater insight into the mechanisms underlying immune function in echinoderms.

Densovirus crosses the insect midgut by transcytosis and disturbs the epithelial barrier function.

Densoviruses are parvoviruses that can be lethal for insects of different orders at larval stages. Although the horizontal transmission mechanisms are poorly known, densoviral pathogenesis usually starts with the ingestion of contaminated food by the host. Depending on the virus, this leads to replication restricted to the midgut or excluding it. In both cases the success of infection depends on the virus capacity to enter the intestinal epithelium. Using the Junonia coenia densovirus (JcDNV) as the prototype virus and the lepidopteran host Spodoptera frugiperda as an interaction model, we focused on the early mechanisms of infection during which JcDNV crosses the intestinal epithelium to reach and replicate in underlying target tissues. We studied the kinetics of interaction of JcDNV with the midgut epithelium and the transport mechanisms involved. Using several approaches, in vivo, ex vivo, and in vitro, at molecular and cellular levels, we show that JcDNV is specifically internalized by endocytosis in absorptive cells and then crosses the epithelium by transcytosis. As a consequence, viral entry disturbs the midgut function. Finally, we showed that four mutations on the capsid of JcDNV affect specific recognition by the epithelial cells but not their binding.

Detailed investigation of the sequential pathological changes in silkworm larvae infected with Bombyx densovirus type 1.

Bombyx mori densovirus type 1 (BmDNV-1) is a pathogen causing flacherie disease in silkworms. BmDNV-1 multiplies only in the nuclei of the columnar cells of larval midgut epithelium. Although several immunohistochemical studies using anti-BmDNV-1 antibody have been reported to date, sequential pathological changes in BmDNV-1-infected larvae have not been completely elucidated. In this paper, sequential investigations were performed on the pathological features of BmDNV-1-infected larvae and BmDNV-1 propagation. Oral infection experiments using newly ecdysed 4th instar larvae revealed that the larvae began to die 9 days post infection (dpi), and the remaining died 10 dpi. Histological observations revealed phenotypic alterations in the midgut cells from 4 dpi, and complete disruption of the midgut structure at 9 dpi. Quantitative RT-PCR of two BmDNV-1 genes indicated that BmDNV-1 began to propagate from 4 dpi, and gradually increased until the larvae died. These expression patterns revealed marked correlation with the histological changes observed in the virus-infected midgut cells. Moreover, bioassays using larvae at various developmental stages clearly indicated that the pathogenicity of this virus is not dependent on the larval stage or the molting process.

The interaction between viral protein and host actin facilitates the virus infection to host.

Although the virus-host interaction has attracted extensive studies, the host proteins essential for virus infection remain largely unknown. To address this issue, the shrimp Penaeus stylirostris densovirus (PstDNV), belonging to the family Parvoviridae, was characterized. PstDNV, a single-stranded DNA virus with a 3.9-kb genome, encoded only three open reading frames (ORFs). Among the three viral proteins, the PstDNV ORF2-encoded protein was discovered to interact with the shrimp actin, suggesting that the host actin played a very important role in virus infection. The RNAi assays revealed that the ORF2-encoded protein was required for the PstDNV infection. The confocal evidence demonstrated that the interaction between the ORF2-encoded protein and actin was essential for the virus infection. Therefore our study indicated that the manipulation of the host actin cytoskeleton was a necessary strategy for viral pathogens to invade host cells.

Four amino acids of an insect densovirus capsid determine midgut tropism and virulence.

Densoviruses are insect parvoviruses that are orally infectious for Lepidoptera. To assess the mechanisms underlying their specificity and their virulence, we investigated the role of eight candidate residues in the densovirus capsid. We showed that the substitutions of four amino acids were associated with decreased virulence due to a decreased ability to cross the host midgut epithelium, without an effect on viral replication in other tissues.

Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics.

The European house cricket, Acheta domesticus L., is highly susceptible to A. domesticus densovirus (AdDNV). Commercial rearings of crickets in Europe are frequently decimated by this pathogen. Mortality was predominant in the last larval stage and young adults. Infected A. domesticus were smaller, less active, did not jump as high, and the adult females seldom lived more than 10-14 days. The most obvious pathological change was the completely empty digestive caecae. Infected tissues included adipose tissue, midgut, epidermis, and Malpighian tubules. Sudden AdDNV epizootics have decimated commercial mass rearings in widely separated parts of North America since the autumn of 2009. Facilities that are producing disease-free crickets have avoided the importation of crickets and other non-cricket species (or nonliving material). Five isolates from different areas in North America contained identical sequences as did AdDNV present in non-cricket species collected from these facilities. The North American AdDNVs differed slightly from sequences of European AdDNV isolates obtained in 1977, 2004, 2006, 2007 and 2009 and an American isolate from 1988. The substitution rate of the 1977 AdDNV 5kb genome was about two nucleotides per year, about half of the substitutions being synonymous. The American and European AdDNV strains are estimated to have diverged in 2006. The lepidopterans Spodoptera littoralis and Galleria mellonella could not be infected with AdDNV. The Jamaican cricket, Gryllus assimilis, and the European field cricket, Gryllus bimaculatus, were also found to be resistant to AdDNV.

Pathogenesis of Junonia coenia densovirus in Spodoptera frugiperda: a route of infection that leads to hypoxia.

To evaluate densovirus potential against lepidopteran pests and their capacity to invade new hosts, we have characterised in vivo the infection and pathogenesis of the Junonia coenia densovirus (JcDNV) in the noctuid pest Spodoptera frugiperda. Here we show that infection starts with the ingestion of viral particles that cross the midgut epithelium without replicating. By quantitative PCR we established the kinetic and the route of infection, from virus ingestion to replication in visceral tracheae and hemocytes. JcDNV has a high particle-to-infection ratio mostly due to the barrier function of the midgut. Pathology and cytopathology suggested that infection of tracheal cells impairs oxygen delivery to demanding tissues leading to cytopathic effects in all the tissues. Finally, larval death results from several physiological shocks, including molting arrest and anoxia.

Persistent, triple-virus co-infections in mosquito cells.

BACKGROUND: It is known that insects and crustaceans can carry simultaneous, active infections of two or more viruses without showing signs of disease, but it was not clear whether co-infecting viruses occupied the same cells or different cells in common target tissues. Our previous work showed that successive challenge of mosquito cell cultures followed by serial, split-passage resulted in stabilized cultures with 100% of the cells co-infected with Dengue virus (DEN) and an insect parvovirus (densovirus) (DNV). By addition of Japanese encephalitis virus (JE), we tested our hypothesis that stable, persistent, triple-virus co-infections could be obtained by the same process. RESULTS: Using immunocytochemistry by confocal microscopy, we found that JE super-challenge of cells dually infected with DEN and DNV resulted in stable cultures without signs of cytopathology, and with 99% of the cells producing antigens of the 3 viruses. Location of antigens for all 3 viruses in the triple co-infections was dominant in the cell nuclei. Except for DNV, this differed from the distribution in cells persistently infected with the individual viruses or co-infected with DNV and DEN. The dependence of viral antigen distribution on single infection or co-infection status suggested that host cells underwent an adaptive process to accommodate 2 or more viruses. CONCLUSIONS: Individual mosquito cells can accommodate at least 3 viruses simultaneously in an adaptive manner. The phenomenon provides an opportunity for genetic exchange between diverse viruses and it may have important medical and veterinary implications for arboviruses.

Non-susceptibility genes to Bombyx densovirus type 1, Nid-1 and nsd-1, affect distinct steps of the viral infection pathway.

In the silkworm Bombyx mori, densovirus type 1 (BmDNV-1) replicates only in the midgut and causes fatal disease. Resistance to BmDNV-1 is determined by two genes, nsd-1 and Nid-1, respectively. Neither of them has been identified yet. We investigated the viral transcript by RT-PCR in inoculated silkworms carrying different sets of nsd-1 and Nid-1 genotype. BmDNV-1 transcript was not detected in nsd-1-carrying strains irrespective of existence of Nid-1 but clearly detected in strains carrying Nid-1 without nsd-1. The result suggests that nsd-1 blocks early step of infection. On the other hand, Nid-1 does not block cell and nucleus entry and viral transcription in nuclei but blocks later step in the viral infection cycle.

Non-structural proteins of Periplaneta fuliginosa densovirus inhibit cellular gene expression and induce necrosis in Sf9 cell cultures.

The non-structural protein NS1 of Periplaneta fuliginosa densovirus (PfDNV) is a multifunctional protein that has previously been shown to possess ATP-binding, ATPase, site-specific DNA-binding, helicase, and transcription activation activities. We report here an investigation of the cytopathogenicity of this viral non-structural (NS) protein, as well as other two NSs, NS2, and NS3, in cultured insect cells. The expression of NS1 alone potently inhibited cellular gene expression, whereas NS2 and NS3 did not produce a similar effect. The inhibition of gene expression by NS1 was confirmed to be specific and not a simple manifestation of toxicity. For example, NS1 inhibited expression of several reporter genes under the control of different RNA polymerase II promoters, whereas it did not inhibit expression from a T7 RNA polymerase promoter construct. Mapping analysis identified the carboxy-terminal peptide of this protein as the region important for the inhibition of cellular gene expression, suggesting that this inhibition is independent of its DNA-binding activity. Next, the mutagenesis assay showed that ATP-binding was essential for the unique function of this protein. Furthermore, we found that NS2 and NS3 cooperatively enhanced the NS1-induced transcription inhibition. Co-expression of all the three NS proteins in Sf9 cells also led to necrotic cell death by ATP depletion.

Viral paratransgenesis in the malaria vector Anopheles gambiae.

Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

The use of insects as a bioassay for Penaeus merguiensis densovirus (PmergDNV).

The lack of available cell lines has hampered the study of viral diseases in crustaceans. This is particularly important for aquaculture which has been plagued by viral diseases since its rapid expansion to meet with the growing demand for seafood products. This study was designed to find an alternative bioassay to cell lines by investigating the use of insects as potential animal models for Penaeus merguiensis densovirus (PmergDNV). Acheta domesticus (house cricket) and Tenebrio molitor (mealworms) were challenged with approximately 1x10(6) virions of PmergDNV by inoculation. PmergDNV was detected in 20% of Tenebrio molitor and 86.6% of Acheta domesticus challenged with PmergDNV. During a subsequent time course experiment, there was a non significant increase in PmergDNV titres (10(4-5) virions), reaching a maximum peak at day 5 (10(6) copies). A threshold of PmergDNV DNA level equal to or greater than 10(3) virions was necessary for mortality in Acheta domesticus. As the inoculum increased from 10(3) DNA copies to 10(4), 10(5), 10(6), mortality increased from 20% to 60%, 80% and 100%, respectively. This is the first evidence that insects may be directly used to study viruses from crustaceans and concludes Acheta domesticus may be used as a potential model to study Penaeus merguiensis densovirus.

The pathogenicity of mosquito densovirus (C6/36DNV) and its interaction with dengue virus type II in Aedes albopictus.

When Aedes albopictus larvae were infected with C6/36 densovirus (C6/36DNV), the mortality reached 97.46% within 21 days for those larvae infected at the first day after hatching, and 14.17% for control. A real-time quantitative polymerase chain reaction (qPCR) was used to trace the dynamic change of the quantity of C6/36DNV genomes in the larvae and the adults, and to study the interaction of C6/36DNV with dengue virus type II(DEN-II) in mosquitoes. It showed that C6/36DNV could persist in the adults that could transmit C6/36DNV vertically to the next generation. The quantity of C6/36DNV after DEN-II infection increased by 10(2)~10(3) times in the C6/36DNV-positive mosquitoes, and the quantity of DEN-II in the C6/36DNV-positive mosquitoes was about 100 times lower than that in the C6/36DNV-negative mosquitoes, which suggested that DEN-II could remarkably stimulate the reproduction of C6/36DNV, while C6/36DNV persisted in mosquitoes could inhibit the reproduction of DEN-II. The study throws light on C6/36DNV as a possible biologic control agent against dengue virus and Ae. albopictus.

Densoviruses for control and genetic manipulation of mosquitoes.

Mosquito densoviruses (MDV) are parvoviruses that replicate in the nuclei of mosquito cells and cause the characteristic nuclear hypertrophy (densonucleosis) that gives them their name. Several MDV that differ in pathogenicity both in vitro and in vivo have been isolated. MDV have a number of features that make them potentially attractive as biological control agents for mosquito-borne disease. They are nonenveloped and relatively stable in the environment. They are highly specific for mosquitoes and they infect and kill larvae in a dose dependent manner in the aqueous larval habitat. Infected larvae that survive to become adult mosquitoes exhibit a dose-dependent shortening of lifespan and many do not survive longer than the extrinsic incubation period for arboviruses. Thus they may have a significant impact on transmission of pathogens. Infected females can transmit the virus vertically by laying infected eggs in new oviposition sites. Studies on how MDV affect populations are relatively limited. Population cage studies suggest that they will persist and spread in populations and limited field studies have shown similar preimaginal mortality in wild populations to that seen in laboratory studies. The availability of infectious clones of MDV genomes allows the development of densovirus vectors for expressing genes of interest in mosquito cells and mosquitoes. Recently short hairpin RNA expression cassettes that induce RNA interference have been inserted into densovirus genomes. These expression cassettes should be useful for both research and disease-control applications.