Deletions in the Putative Cell Receptor-Binding Domain of Sindbis Virus Strain MRE16 E2 Glycoprotein Reduce Midgut Infectivity in
            <i>Aedes aegypti</i>

160

178

We recently demonstrated that a soluble protein, Gas6, can facilitate viral entry by bridging viral envelope phosphatidylserine to Axl, a receptor tyrosine kinase expressed on target cells. The interaction between phosphatidylserine, Gas6, and Axl was originally shown to be a molecular mechanism through which phagocytes recognize phosphatidylserine exposed on dead cells. Since our initial report, several groups have confirmed that Axl/Gas6, as well as other phosphatidylserine receptors, facilitate entry of dengue, West Nile, and Ebola viruses. Virus binding by viral envelope phosphatidylserine is now a viral entry mechanism generalized to many families of viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most of the known human phosphatidylserine-recognizing molecules, including MFG-E8, TIM-1, -3, and -4, CD300a, BAI1, and stabilin-1 and -2, for their abilities to facilitate virus binding and infection. Using pseudotyped lentiviral vectors, we found that a soluble phosphatidylserine-binding protein, MFG-E8, enhances transduction. Cell surface receptors TIM-1 and -4 also enhance virus binding/transduction. The extent of enhancement by these molecules varies, depending on the type of pseudotyping envelope proteins. Mutated MFG-E8, which binds viral envelope phosphatidylserine without bridging virus to cells, but, surprisingly, not annexin V, which has been used to block phagocytosis of dead cells by concealing phosphatidylserine, efficiently blocks these phosphatidylserine-dependent viral entry mechanisms. These results provide insight into understanding the role of viral envelope phosphatidylserine in viral infection. IMPORTANCEEnvelope phosphatidylserine has previously been shown to be important for replication of various envelope viruses, but details of this mechanism(s) were unclear. We were the first to report that a bifunctional serum protein, Gas6, bridges envelope phosphatidylserine to a cell surface receptor, Axl. Recent studies demonstrated that many envelope viruses, including vaccinia, dengue, West Nile, and Ebola viruses, utilize Axl/Gas6 to facilitate their entry, suggesting that the phosphatidylserine-mediated viral entry mechanism can be shared by various enveloped viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most human phosphatidylserine-recognizing molecules for their abilities to facilitate viral infection. The results provide insights into the role(s) of envelope phosphatidylserine in viral infection, which can be applicable to the development of novel antiviral reagents that block phosphatidylserine-mediated viral entry.

Section: Resultsmentioning

confidence: 99%

Role of Phosphatidylserine Receptors in Enveloped Virus Infection

Morizono

Chen

2014

160

178

“…To test this hypothesis, we utilized adult A. aegypti females of the Rexville D strain, which has been used extensively in studies with SINV (22)(23)(24). Adult female Rexville D mosquitoes were injected with 345 ng dsRNA corresponding to either Aeiap1, Aedronc, or a negative-control sequence not present in the A. aegypti genome (the bacterial cat gene) and monitored for survival.…”

Section: Resultsmentioning

confidence: 99%

Effects of Manipulating Apoptosis on Sindbis Virus Infection of Aedes aegypti Mosquitoes

Wang

Gort

Boyle

et al. 2012

Improved control of vector-borne diseases requires an understanding of the molecular factors that determine vector competence. Apoptosis has been shown to play a role in defense against viruses in insects and mammals. Although some observations suggest a correlation between apoptosis and resistance to arboviruses in mosquitoes, there is no direct evidence tying apoptosis to arbovirus vector competence. To determine whether apoptosis can influence arbovirus replication in mosquitoes, we manipulated apoptosis in Aedes aegypti mosquitoes by silencing the expression of genes that either positively or negatively regulate apoptosis. Silencing of the A. aegypti anti-apoptotic gene iap1 ( Aeiap1 ) caused apoptosis in midgut epithelium, alterations in midgut morphology, and 60 to 70% mosquito mortality. Mortality induced by Aeiap1 silencing was rescued by cosilencing the initiator caspase gene Aedronc , indicating that the mortality was due to apoptosis. When mosquitoes which had been injected with Aeiap1 double-stranded RNA (dsRNA) were orally infected with Sindbis virus (SINV), increased midgut infection and virus dissemination to other organs were observed. This increase in virus infection may have been due to the effects of widespread apoptosis on infection barriers or innate immunity. In contrast, silencing the expression of Aedronc , which would be expected to inhibit apoptosis, reduced SINV midgut infection and virus dissemination. Thus, our data suggest that some level of caspase activity and/or apoptosis may be necessary for efficient virus replication and dissemination in mosquitoes. This is the first study to directly test the roles of apoptosis and caspases in determining mosquito vector competence for arboviruses.

“…The results of a recent study indicate that C6/36 cells lack a functional RNA interference (RNAi) response, suggesting that these cells may not accurately model mosquito-arbovirus interactions at the molecular level (41). However, others have demonstrated that reduced Sindbis virus (an alphavirus) replication efficiency in C6/36 cells correlated with poor experimental infection of Aedes aegypti and sharply reduced midgut infectivity and dissemination (42). Future work should focus on elucidating the mechanistic basis underlying the geographically expansive genotype displacement by comparing GIII and GI-b isolates in terms of their in vitro fitness and temperature sensitivity phenotypes in C6/36 cells in addition to their phenotypes in C. tritaeniorhynchus.…”

Section: Gi-b Emerged Inmentioning

confidence: 99%

Dynamics of the Emergence and Establishment of a Newly Dominant Genotype of Japanese Encephalitis Virus throughout Asia

Schuh¹,

Ward

Brown

et al. 2014

125

140

In recent years, genotype I (GI) of Japanese encephalitis virus (JEV) has displaced genotype III (GIII) as the dominant virus genotype throughout Asia. In this study, the largest collection of GIII and GI envelope gene-derived viral sequences assembled to date was used to reconstruct the spatiotemporal chronology of genotype displacement throughout Asia and to determine the evolutionary and epidemiological dynamics underlying this significant event. GI consists of two clades, GI-a and GI-b, with the latter being associated with displacement of GIII as the dominant JEV genotype throughout Asia in the 1990s. Phylogeographic analysis indicated that GI-a diverged in Thailand or Cambodia and has remained confined to tropical Asia, whereas GI-b diverged in Vietnam and then dispersed northwards to China, where it was subsequently dispersed to Japan, Korea, and Taiwan. Molecular adaptation was detected by more than one method at one site (residue 15), and coevolution was detected at two pairs of sites (residues 89 to 360 and 129 to 141) within the GI E gene protein alignment. Viral multiplication and temperature sensitivity analyses in avian and mosquito cells revealed that the GI-b isolate JE-91 had significantly higher infectivity titers in mosquito cells from 24 to 48 h postinfection than did the GI-a and GIII isolates. If the JE-91 isolate is indeed representative of GI-b, an increased multiplicative ability of GI-b viruses compared to that of GIII viruses early in mosquito infection may have resulted in a shortened extrinsic incubation period that led to an increased number of GI enzootic transmission cycles and the subsequent displacement of GIII. IMPORTANCE Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, represents the most significant etiology of childhood viral neurological infection in Asia.Despite the existence of effective vaccines, JEV is responsible for an estimated 68,000 human cases and a reported 10,000 to 15,000 deaths annually. Phylogenetic studies divided JEV into five geographically and epidemiologically distinct genotypes (GI to GV). GIII has been the source of numerous JEV epidemics throughout history and was the most frequently isolated genotype throughout most of Asia from 1935 until the 1990s. In recent years, GI has displaced GIII as the most frequently isolated virus genotype. To date, the mechanism of this genotype replacement has remained unknown. In this study, we have identified genetic determinants underlying the genotype displacement as it unfolded across Asia. JEV provides a paradigm for other flaviviruses, including West Nile, yellow fever, and dengue viruses, and the critical role of the selective advantages in the mosquito vector.