Sindbis virus induces transport processes and alters expression of innate immunity pathway genes in the midgut of the disease vector, Aedes aegypti

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Abstract

The midgut of hematophagous insects is the initial site of infection by arthropod-borne viruses (arboviruses) and plays a crucial role in vector competence. To further understand processes that occur in the midgut in response to infection by an arbovirus, DNA microarrays were used to analyze gene expression changes following infection by the alphavirus, Sindbis (MRE16 Malaysian strain). Midgut transcription profiles from mosquitoes fed blood containing 108 pfu/ml of virus were compared with those from mosquitoes ingesting blood meals having no virus. Transcription profiles from both experimental groups were analyzed at 1, 4, and 8 days post-feeding. Among the many transcription changes observed by microarray analysis, the most dramatic involved three genes that had 25–40-fold increases in transcript levels in virus infected mosquitoes at 4 days post-infection. These genes were synaptic vesicle protein-2 (SV2), potassium-dependent sodium/calcium exchanger (NCKX), and a homologue of Caenorhabditis elegans Unc-93, a putative component of a two-pore potassium channel. We speculate that these changes represent changes in vesicle transport processes. In addition to these observations, transcript changes were observed in infected mosquitoes that suggested involvement of Toll and c-jun amino terminal kinase immune cascades as a response to viral infection

Introduction

Arthropod-borne viruses (arboviruses) remain a major threat to human and animal health throughout much of the world. The arbovirus transmission cycle involves replication in hematophagous insects, such as mosquitoes, and a vertebrate host. When a mosquito ingests a viremic blood meal from a veterbrate host, the virus initially infects the midgut epithelial cells, replicates, and then exits the midgut to infect secondary tissues such as fat body and salivary glands (Woodring et al., 1996). Productive infection of the midgut is critical for a mosquito species or population to be competent to transmit an arbovirus (Black et al., 2002). Despite the central role of the midgut in vector competence, our understanding of how the vector responds to arbovirus infections is very limited.

Arboviruses within Alphavirus genus (Family, Togaviridae) consist of at least 25 mosquito-borne viruses, some of which cause severe morbidity and mortality in mammalian hosts (Schlesinger and Schlesinger, 1996). Alphaviruses such as Sindbis virus (SINV), have proven to be very useful in examining gene expression and RNA interference mediated gene silencing in insect hosts (Adelman et al., 2001; Attardo et al., 2003; Sanchez-Vargas et al., 2004; Uhlirova et al., 2003). MRE16 SINV is an enveloped RNA virus with a positive sense, single-stranded genome that readily infects Aedes aegypti by the per os route. MRE16 SINV and Ae. aegypti are an excellent model system to identify virus–vector interactions and may also reveal fundamental virus–vector interactions between medically important arboviruses within the Flaviviridae family such as dengue and yellow fever viruses and their natural vectors.

Much of what is known about replication of arthropod-borne viruses is based in studies performed in mammalian cell lines with less information from insect cell lines. While these studies are quite useful, studies performed in insect cell lines do not give us a complete picture of the actual infection process in intact insects. In fact, upon analysis of SINV morphogenesis in three Ae. albopictus subclones, Miller and colleagues showed variability between the different cell line subclones and concluded that subclone u4.4 and not C6/36, the most commonly used Ae. albopictus cell line, most closely resembled infectivity in the adult mosquito (Miller and Brown, 1992). Such data suggest that our basic understanding of viral infection may be biased by the cell line chosen to study virus–vector interactions. It is clear that more effort is required to understand viral replication not only in insect cell lines but also in vivo in insects.

Moreover, there is very limited knowledge of insect immune responses to viral infection. Much is known of involvement of insect innate immunity in bacterial and fungal infections, but few studies have analyzed the role of these pathways in arthropod-borne viral infection. Lack of adaptive immunity in insects demands powerful defenses mediated by innate immunity against invading microorganisms. Indeed, insects are able to mount effective innate immune responses against bacterial and fungal infections that lead to killing of these microorganisms by signaling proteolytic cascades and activating Toll or immunodeficiency (IMD) pathways that lead to induction of antimicrobial peptides such as defensins and cecropins (reviewed in Imler and Zheng, 2004).

A recent study analyzing viral-induced genes from a subtractive cDNA library in the Western Flower Thrip, Frankliniella occidentalis, revealed that the innate immune system is in fact activated by Tomato Spotted Wilt Tospovirus (TSWV; Bunyaviridae) infection (Medeiros et al., 2004). They identified 2–3-fold induction of genes involved in the Toll and c-jun amino terminal kinase (JNK) pathway, as well as a number of antimicrobial peptides and proteases, and the gene encoding Relish of the IMD pathway. Although TSWV has a segmented, negative sense ssRNA genome, SINV and TSWV may elicit similar immune responses in the infected insect.

Involvement of innate immunity in antiviral defense is becoming well established in mammalian systems. For example, recent work has demonstrated activation of Toll receptors (TLRs) (Alexopoulou et al., 2001; Krug et al., 2004; Lund et al., 2003, Lund et al., 2004), JAK/STAT pathways, and JNK signaling in response to viral infection (for review see Williams, 2001). Viruses interfere with these pathways in order to bypass host defenses. Examples of this include the following: (1) inhibition of STAT phosphorylation in C6/36 cells by Japanese encephalitis virus (JEV) (Lin et al., 2004); (2) inhibition of mammalian (Bour et al., 2001) and insect (Leulier et al., 2003) Toll-dependent immune responses by the HIV-1 accessory protein, viral protein U (Vpu); and (3) Inhibition of RNA silencing-based antiviral response (RSAR) in Drosophila cells by interferon antagonist proteins of mammalian viruses (Li et al., 2004). Clearly adaptation to vertebrate immune responses would also allow pathogens to evade insect immune responses due to the striking similarity of innate immunity pathways between insects and mammals. While insects are not killed by these types of arboviruses, the virus is able to maintain high levels of infection and disseminate to tissues such as the salivary glands. Thus, the ability of arboviruses to replicate successfully without causing mortality in their insect hosts must involve some level of evasion of innate immune responses in order to preserve viability of the pathogen and its host.

Much of the recent focus on prevention of arthropod-borne diseases has centered on creating disease refractory vectors by genetic manipulation (Ito et al., 2002; Olson et al., 2002). However, to more efficiently block viral transmission, we should thoroughly understand the processes of viral entry, replication, release, and dissemination to secondary tissues, as well as host defense responses in the insect system. Because of their extensive use in analyzing gene function in insects as well as a need to further understand insect–virus interactions, DNA microarray analysis of viral-induced gene expression patterns using SINV as a model for ssRNA viral infections is performed here. The results presented suggest involvement of vesicle transport processes as well as activation of innate immunity pathways in response to viral infection.

Section snippets

EST library

Poly(A)+ RNA was isolated from isolated midgut and Malpighian tubules of adult female unfed Ae. aegypti. The mRNA was then used for the construction of cDNA library as previously described (Jin et al., 2003). An additional cDNA library was constructed from the midgut and Malpighian tubules of fourth instar larvae using similar methods. Individual colonies from this library were isolated and the 5′ sequences were determined and compared to NCBI Genbank sequences to determine homologies.

Gene chip construction

Plasmid

Results

Global changes in gene transcription within the Ae. aegypti midgut were detected following oral infection with SINV (MRE16 Malaysian strain). cDNAs representing 2170 unique Ae. aegypti genes were printed on a gene chip and hybridized with labeled probes synthesized using RNA of dissected midguts at 1, 4, and 8 days post-infection (DPI). Midgut transcription profiles from infected mosquitoes were compared with those from mosquitoes receiving non-infectious blood meals at the same time points.

Discussion

These microarray data revealed a large number of genes that had altered transcription levels in response to SINV infection. Based on these transcript changes, pathways that appear to be altered include induction of vesicle transport and fusion at 4 DPI, induced and repressed innate immunity pathways, changes in ion and solute transport, induced protein translation at 8 DPI (Table 7), and changes in metabolic processes. In addition, 29 genes with unknown functions demonstrated significant

Acknowledgments

We thank Cindy Merideth for technical assistance in raising mosquitoes. This research was funded by NIH grants AI 32572 and AI 48049 at UCR and AI 25489, AI 46435 and AI 060960 at CSU.

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