Arthropod Cell Cultures and Their Application to the Study of Viruses


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Methods ARTICLE

Insect parvoviruses of the genus Densovirus also are potential gene transfer vectors for mosquitoes 2. Extrachromosomal paratransgenic expression of exogenous genes in arthropods by bacterial symbionts is another possible means of genetic alteration. Scott O'Neill's group showed that the obligate intracellular bacteria Wolbachia will infect a variety of insect tissues and suggested that genetically modified Wolbachia could be used to transduce mosquitoes with transmission-blocking genes Ben Beard's group has demonstrated that expression of the antimicrobial peptide cecropin A in the midgut of triatomine bugs by the nutritional mutualist bacterium Rhodococcus rhodnii renders a large proportion of the bugs refractory to infection by Trypanosoma cruzi 9.

Despite the development of several promising mosquito transformation systems, the construction of transgenic mosquitoes is still very labor-intensive and inefficient, and we have thus used transient gene expression systems to test the effect of introducing heterologous genetic material into mosquitoes and mosquito cell cultures. SIN is an arbovirus and the prototype of the genus Alphavirus in the family Togaviridae.

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The natural cycle of SIN involves birds and Culex mosquitoes SIN also will infect Aedes and Anopheles mosquitoes 43 and has a broad host range in vertebrate animal cells. Of particular importance for our work, SIN replicates efficiently in many cells and tissues of Aedes mosquitoes 74 , causing a persistent, noncytocidal infection.

The molecular biology of SIN is well studied A complete cDNA copy of the genome, from which infectious viral RNA can be transcribed in vitro an infectious clone , was produced by Rice et al. Two types of expression vectors that have been derived from the infectious clone have been of particular use in identifying successful viral interference strategies Recombinant virus will express the inserted gene in a broad range of host cells and spread to surrounding cells and tissues A second expression system, the SIN replicon, consists of the SIN infectious clone with the structural protein genes deleted and replaced by up to 6, bp of heterologous genetic material that is expressed from the native internal promoter The replicon is capable of intracellular self-amplification but can be packaged into infectious virions only in cells that are coinfected with a helper virus expressing the viral structural genes Packaged replicons efficiently infect cells but do not spread from the initially infected cell in the absence of helper.

To test the level, duration, and fidelity of expression of heterologous genes from the dsSIN vector in mosquitoes, a recombinant dsSIN virus was constructed to express bacterial chloramphenicol acetyltransferase CAT from the second subgenomic promoter. When cultured A. An assay of the enzyme in cell lysates showed that 8. After intrathoracic inoculation of A. These results demonstrated several properties of the SIN expression systems that made them ideal for testing the concept of PDR in mosquitoes.

The last two points suggested that any DNA-based system that ultimately deploys antiviral genes identified by expression with the SIN systems may require a similar high level of expression in the cytoplasm for efficacy. Plant researchers have used both transgenesis and transient expression by RNA virus transduction systems to demonstrate that interference to virus replication can be mediated by expression of the viral coat protein 68 , its mRNA 52 , or its antisense complement 8 , Therefore, Powers, Kamrud, et al.

LAC is a member of the genus Bunyavirus in the family Bunyaviridae. Its principal vector is A. Humans may be tangential hosts for LAC infection when they are bitten by infected A.

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Portions of the S segment that were inserted for expression by recombinant viruses included the entire S cDNA, the NS S coding sequence, and the conserved complementary terminal sequences 69 , An enzyme-linked immunosorbent assay was used to measure progeny LAC released from the cells. A high degree of sequence relatedness also was shown to be necessary to effect RNA-mediated interference with heterologous viruses in transgenic plants Interference was tested in adult A.

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After 12 to 18 days, the mosquitoes were assayed for infectious LAC or viral envelope antigens. Inoculation with the recombinant virus resulted in a to fold reduction in LAC titers compared to those in mosquitoes inoculated with nonrecombinant SIN and challenged with LAC These studies showed that expression in mosquito cells and adult mosquitoes of certain regions of LAC RNA, particularly those from the S RNA segment with antisense polarity, rendered the cells resistant to infection by the homologous and closely related viruses.

The resistance was RNA mediated and was dependent on a high degree of sequence identity between the effector RNA and its target. Our initial attempts to inhibit DEN replication also used the expression of genes encoding structural proteins. Flaviviruses have a nonsegmented, positive-sense RNA genome about 11, nt in length.

The genome acts as the only mRNA, encoding a single polyprotein that is co- and posttranslationally cleaved to give three structural and seven nonstructural polypeptides. Use of the indirect-immunofluorescence assay to detect the inhibition of DEN replication in mosquito cells and tissues. DEN-2 replication was detected 5 days postchallenge by the indirect-immunofluorescence assay. Adult female A. Salivary glands were removed and subjected to IFA with the use of anti-DEN E monoclonal primary antibodies and a biotinylated sheep anti-mouse secondary antibody.

Fluorescence produced by bound fluorescein-streptavidin Amersham, Arlington Heights, Ill.

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Cell cultures were counterstained with Evans blue. Interestingly, expression of the full-length C protein was toxic to mosquito cells A truncated C protein, lacking the 24 carboxy-terminal hydrophobic amino acids that serve as a signal sequence for the prM protein 77 , could be expressed at high levels without killing the mosquito cells D2prMa virus was used to transduce mosquitoes in experiments in which both recombinant dsSIN and challenge DEN-2 were simultaneously inoculated into the thoraxes of female A. After 11 days, the midguts and salivary glands were removed by dissection and examined by immunofluorescence for presence of SIN envelope and DEN-2 envelope proteins.

Surprisingly, abundant DEN antigen was observed in midgut epithelial cells; however, DEN-2 replication was dramatically inhibited in all tissues of the salivary glands Fig. SIN envelope antigen was seen only in the nerve and muscle tissues of the midgut but in all tissues of the salivary gland.

The ultimate test of effective interference is interruption of DEN transmission in A. Thus, the expression of antisense DEN RNA in epidemiologically relevant mosquito tissues can block homologous virus transmission. In related experiments, A.

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In an attempt to identify a more broadly based interference strategy that could be targeted to a heterologous group of viruses with a common vector mosquito, we explored the possibility of interference mediated by conserved, nonstructural proteins. In plant virus interference studies, expression of certain domains or truncated versions of viral RDRP proteins conferred resistance to both homologous virus and a variety of related viruses 27 , For both truncated and full-length protein expression, interference with the replication of YFV but not DEN-2 was observed in cultured mosquito cells Furthermore, the level of interference directly correlated with the degree of sequence identity, and antisense RNA to the GDD-encompassing coding region was more effective than protein-expressing sense RNA, suggesting RNA-mediated interference Most reports of RDRP-mediated plant virus interference involved the expression of a truncated, GDD-containing domain of the enzyme 32 or a protein with site-directed mutations in the putative RNA-binding site 5 , 15 , As with interference studies involving structural genes and proteins, the RDRP-mediated flavivirus interference appeared to be due to expression of RNA rather than of protein.

Since RNA-mediated interference is highly sequence specific, it did not broaden the range of target viruses. Other approaches such as expression of RDRP protein with site-directed mutations should perhaps be pursued because they have the potential to inhibit a broader range of viruses. Any DNA-based system must be engineered to achieve initial high expression levels, possibly by transformation with an entire cDNA copy of a recombinant SIN replicon genome which would then self-amplify its subgenomic RNA in the cytoplasm.

With the continuing goal of identifying an effector that will confer interference to several distinct viruses when expressed in transgenic mosquitoes, we embarked on a series of experiments to further define the parameters of RNA-mediated interference by asking the following questions. Are all regions of the genome equally vulnerable targets for antisense RNA effectors? What is the minimum size requirement of an antisense effector? By what mechanism does antisense RNA interfere? Recombinant viruses that elicited a high level of interference in cell culture were also tested in mosquitoes.

Although the reason s for the variability in interference effectiveness is unknown, it could be due to the position on the genome e. Adelman, K. Olson, S. Higgs, B. Beaty, and C. Blair, unpublished data. Similarly, truncation of the insert size reduced its ability to interfere in mosquitoes Adelman et al.


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Subsequently, other dsSIN viruses with chimeric inserts have exhibited interference with replication of two DEN serotypes, although interference results were unpredictable with regard to the origin of the effector sequence or its position in the chimera Adelman et al. All our results suggest that the induction of RNA-mediated interference requires the presence of sequence-specific double-stranded RNA.

The reduction in interference effectiveness observed on either decreasing the length or arraying antisense effectors in tandem could be the result of a reduction in the ability to form RNA-RNA duplexes. Although it has been shown that antisense RNAs as small as 60 nt can bind stably to their targets 79 , the heterologous RNA transcribed from dsSIN virus templates is embedded within a larger RNA expressed from the second subgenomic promoter. It is difficult to estimate the effect of these extraneous sequences on the formation of double-stranded RNA Adelman et al.

An alternative explanation for the lack of interference by the shorter effector RNA sequences is the proliferation of escape mutants. Bull et al. Since RNA viruses such as DEN exhibit sequence variation, even in a single isolate 40 , it is possible that nucleotide substitutions in the genome were sufficient to disrupt stable RNA duplex formation with shorter effectors.

The generation of escape mutants does not explain the decrease in interference sometimes seen when individually effective antisense sequences are tandemly arrayed, however. Several mechanisms have been proposed by which the expression of sense or antisense RNA or the presence of double-stranded RNA in eukaryotic cells results in interference with superinfecting homologous virus.

Hybrid formation may also block viral RDRP binding or movement for transcription. The presence of double-stranded RNA in cells that have been exposed to interferon leads to a cascade of events resulting in the inhibition of translation by phosphorylation of an initiation factor and degradation of mRNA by induced RNase L Furthermore, double-stranded RNA itself can elicit antiviral effects, even in the absence of interferon Interferon-related double-stranded RNA-mediated interference has not been reported to occur in invertebrate animals.

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Nevertheless, double-stranded RNA appears to play a role in complex regulatory mechanisms in a number of nonvertebrate systems. Evidence has been presented for degradation, in plant cells, of mRNA that is part of an RNA-RNA hybrid by a cellular RNase that, along with other induced enzymes, is involved in posttranscriptional regulation of gene expression and recovery from plant virus infection 8 , Fire et al. The interference was due to a decrease or elimination of the endogenous mRNA.

Double-stranded RNA has recently been shown to trigger sequence-specific gene silencing in a number of other organisms 28 , including Trypanosoma brucei 63 and D. Further pursuit of the mechanism of RNA-mediated virus interference should lead to new insights into mosquito cell gene regulation. Preliminary studies in our laboratory indicate that antisense RNA-mediated interference with DEN replication in mosquito cells occurs within the first 8 h after infection Adelman et al.

It is possible that more than one of these mechanisms is operating with different effector RNAs or even with a single effector. Further studies are required to determine the possible role of double-stranded RNA, the level of RNA expression required, and the best way to target the cellular compartment to most effectively interrupt infection.

Although we do not fully understand the mechanism of RNA-mediated interference, we have shown that expression of effector RNA in mosquito cells is an effective molecular strategy for interrupting specific Bunyavirus and Flavivirus replication in mosquitoes. The SIN expression systems have the potential not only to test PDR as a strategy for interrupting arbovirus transmission but also to lead to a better understanding of the regulation of mosquito and viral gene expression and virus-vector interactions 45 , However, two additional objectives stated earlier in this review must be accomplished in order to apply RNA-mediated interference in the field: i DNA-based, heritable expression will be necessary, and ii a means of driving transgenes into mosquito populations must be found.

Although the SIN expression systems have been invaluable in establishing these principles, direct use of SIN is not an option for long-term expression of an antiviral agent in mosquito field populations. As mentioned above, several methods to introduce stable, DNA-based exogenous genetic material into mosquitoes are under active development. The introduced DNA could contain a single gene or gene segment or an entire cDNA copy of a recombinant SIN replicon genome, preferably under the control of an inducible promoter.

The ideal transgene should be able to spread unaided and become fixed in natural mosquito populations. One possible mechanism for such spread is introduction in a transposon. Kidwell has presented evidence that the transposable P-element, despite a possible fitness reduction in carriers, has recently and rapidly spread worldwide in D. Since a transposable element introduced by one parental genome during mating may integrate at many sites in the genome of the other wild-type parent, the element can eventually spread to all chromosomes.

Arthropod Cell Cultures and Their Application to the Study of Viruses Arthropod Cell Cultures and Their Application to the Study of Viruses
Arthropod Cell Cultures and Their Application to the Study of Viruses Arthropod Cell Cultures and Their Application to the Study of Viruses
Arthropod Cell Cultures and Their Application to the Study of Viruses Arthropod Cell Cultures and Their Application to the Study of Viruses
Arthropod Cell Cultures and Their Application to the Study of Viruses Arthropod Cell Cultures and Their Application to the Study of Viruses
Arthropod Cell Cultures and Their Application to the Study of Viruses Arthropod Cell Cultures and Their Application to the Study of Viruses
Arthropod Cell Cultures and Their Application to the Study of Viruses Arthropod Cell Cultures and Their Application to the Study of Viruses
Arthropod Cell Cultures and Their Application to the Study of Viruses Arthropod Cell Cultures and Their Application to the Study of Viruses
Arthropod Cell Cultures and Their Application to the Study of Viruses Arthropod Cell Cultures and Their Application to the Study of Viruses

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