A Tale of 20 Alphaviruses; Inter-species Diversity and Conserved Interactions Between Viral Non-structural Protein 3 and Stress Granule Proteins.

Alphaviruses infect a diverse range of host organisms including mosquitoes, mammals, and birds. The enigmatic alphavirus non-structural protein 3 (nsP3) has an intrinsically disordered, C-terminal hypervariable domain (HVD) that can interact with a variety of host proteins associated with stress granules (SGs). The HVD displays the highest variability across the more than 30 known alphaviruses, yet it also contains several motifs that are conserved amongst different subgroups of alphaviruses. For some alphaviruses, specific nsP3-SG protein interactions are essential for virus replication. However, it remains difficult to attribute general roles to these virus-host interactions, as multiple amino acid motifs in the HDV display a degree of redundancy and previous studies were performed with a limited number of alphaviruses. To better understand nsP3-host protein interactions we conducted comprehensive co-localization experiments with the nsP3s of 20 diverse alphaviruses: chikungunya, Semliki Forest, Sindbis, Bebaru, Barmah Forest, Getah, Mayaro, Middelburg, O'nyong-nyong, Ross River QML and T48, Una, Whataroa, Southern Elephant Seal, Eilat, Tai Forest (TAFV), Venezuelan/Eastern/Western equine encephalitis (V/E/WEEV) and the aquatic Salmonid alphavirus (SAV), with three different SG proteins (G3BP and its insect homolog Rasputin, FMRP) and BIN1 in mammalian and mosquito cell lines. Despite that all terrestrial alphavirus nsP3s contained at least one BIN1-binding motif (PxPxPR), not all nsP3s co-localized with BIN1. Further, all alphaviruses except SAV, TAFV and VEEV displayed co-localization with G3BP. Although viruses lacking FGxF-like motifs contained Agenet-like domain binding motifs to facilitate interaction with FMRP, cytoplasmic nsP3 granules of all tested alphaviruses co-localized with FMRP. Crispr-Cas9 knockout of G3BP in mammalian cells abolished nsP3-FMRP co-localization for all alphaviruses except V/E/WEEV nsP3s that bind FMRP directly. G3BP knockout also changed nsP3 subcellular localization of Bebaru, Barmah Forest, Getah, and Sindbis viruses. Taken together this study paints a more detailed picture of the diverse interactions between alphavirus nsP3 and SG-associated host proteins. The interaction between nsP3 and G3BP clearly plays a central role and results in recruitment of additional host proteins such as FMRP. However, direct binding of FMRP can make the interaction with G3BP redundant which exemplifies the alternate evolutionary paths of alphavirus subgroups.

Experimental adaptation of dengue virus 1 to Aedes albopictus mosquitoes by in vivo selection.

In most of the world, Dengue virus (DENV) is mainly transmitted by the mosquito Aedes aegypti while in Europe, Aedes albopictus is responsible for human DENV cases since 2010. Identifying mutations that make DENV more competent for transmission by Ae. albopictus will help to predict emergence of epidemic strains. Ten serial passages in vivo in Ae. albopictus led to select DENV-1 strains with greater infectivity for this vector in vivo and in cultured mosquito cells. These changes were mediated by multiple adaptive mutations in the virus genome, including a mutation at position 10,418 in the DENV 3'UTR within an RNA stem-loop structure involved in subgenomic flavivirus RNA production. Using reverse genetics, we showed that the 10,418 mutation alone does not confer a detectable increase in transmission efficiency in vivo. These results reveal the complex adaptive landscape of DENV transmission by mosquitoes and emphasize the role of epistasis in shaping evolutionary trajectories of DENV variants.

Impact of Gut Bacteria on the Infection and Transmission of Pathogenic Arboviruses by Biting Midges and Mosquitoes.

Tripartite interactions among insect vectors, midgut bacteria, and viruses may determine the ability of insects to transmit pathogenic arboviruses. Here, we investigated the impact of gut bacteria on the susceptibility of Culicoides nubeculosus and Culicoides sonorensis biting midges for Schmallenberg virus, and of Aedes aegypti mosquitoes for Zika and chikungunya viruses. Gut bacteria were manipulated by treating the adult insects with antibiotics. The gut bacterial communities were investigated using Illumina MiSeq sequencing of 16S rRNA, and susceptibility to arbovirus infection was tested by feeding insects with an infectious blood meal. Antibiotic treatment led to changes in gut bacteria for all insects. Interestingly, the gut bacterial composition of untreated Ae. aegypti and C. nubeculosus showed Asaia as the dominant genus, which was drastically reduced after antibiotic treatment. Furthermore, antibiotic treatment resulted in relatively more Delftia bacteria in both biting midge species, but not in mosquitoes. Antibiotic treatment and subsequent changes in gut bacterial communities were associated with a significant, 1.8-fold increased infection rate of C. nubeculosus with Schmallenberg virus, but not for C. sonorensis. We did not find any changes in infection rates for Ae. aegypti mosquitoes with Zika or chikungunya virus. We conclude that resident gut bacteria may dampen arbovirus transmission in biting midges, but not so in mosquitoes. Use of antimicrobial compounds at livestock farms might therefore have an unexpected contradictory effect on the health of animals, by increasing the transmission of viral pathogens by biting midges.

The invasive Asian bush mosquito Aedes japonicus found in the Netherlands can experimentally transmit Zika virus and Usutu virus.

BACKGROUND: The Asian bush mosquito Aedes japonicus is invading Europe and was first discovered in Lelystad, the Netherlands in 2013, where it has established a permanent population. In this study, we investigated the vector competence of Ae. japonicus from the Netherlands for the emerging Zika virus (ZIKV) and zoonotic Usutu virus (USUV). ZIKV causes severe congenital microcephaly and Guillain-Barré syndrome in humans. USUV is closely related to West Nile virus, has recently spread throughout Europe and is causing mass mortality of birds. USUV infection in humans can result in clinical manifestations ranging from mild disease to severe neurological impairments. METHODOLOGY/PRINCIPAL FINDINGS: In our study, field-collected Ae. japonicus females received an infectious blood meal with ZIKV or USUV by droplet feeding. After 14 days at 28°C, 3% of the ZIKV-blood fed mosquitoes and 13% of the USUV-blood fed mosquitoes showed virus-positive saliva, indicating that Ae. japonicus can transmit both viruses. To investigate the effect of the mosquito midgut barrier on virus transmission, female mosquitoes were intrathoracically injected with ZIKV or USUV. Of the injected mosquitoes, 96% (ZIKV) and 88% (USUV) showed virus-positive saliva after 14 days at 28°C. This indicates that ZIKV and USUV can efficiently replicate in Ae. japonicus but that a strong midgut barrier is normally restricting virus dissemination. Small RNA deep sequencing of orally infected mosquitoes confirmed active replication of ZIKV and USUV, as demonstrated by potent small interfering RNA responses against both viruses. Additionally, de novo small RNA assembly revealed the presence of a novel narnavirus in Ae. japonicus. CONCLUSIONS/SIGNIFICANCE: Given that Ae. japonicus can experimentally transmit arthropod-borne viruses (arboviruses) like ZIKV and USUV and is currently expanding its territories, we should consider this mosquito as a potential vector for arboviral diseases in Europe.

Subgenomic flavivirus RNA binds the mosquito DEAD/H-box helicase ME31B and determines Zika virus transmission by Aedes aegypti.

Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

Mosquito Small RNA Responses to West Nile and Insect-Specific Virus Infections in Aedes and Culex Mosquito Cells.

Small RNA mediated responses are essential for antiviral defence in mosquitoes, however, they appear to differ per virus-vector combination. To further investigate the diversity of small RNA responses against viruses in mosquitoes, we applied a small RNA deep sequencing approach on five mosquito cell lines: Culex tarsalis CT cells, Aedes albopictus U4.4 and C6/36 cells, Ae. aegypti Aag2 cells (cleared from cell fusing agent virus and Culex Y virus (CYV) by repetitive dsRNA transfections) and Ae. pseudoscutellaris AP-61 cells. De novo assembly of small RNAs revealed the presence of Phasi Charoen-like virus (PCLV), Calbertado virus, Flock House virus and a novel narnavirus in CT cells, CYV in U4.4 cells, and PCLV in Aag2 cells, whereas no insect-specific viruses (ISVs) were detected in C6/36 and AP-61 cells. Next, we investigated the small RNA responses to the identified ISVs and to acute infection with the arthropod-borne West Nile virus (WNV). We demonstrate that AP-61 and C6/36 cells do not produce siRNAs to WNV infection, suggesting that AP-61, like C6/36, are Dicer-2 deficient. CT cells produced a strong siRNA response to the persistent ISVs and acute WNV infection. Interestingly, CT cells also produced viral PIWI-interacting (pi)RNAs to PCLV, but not to WNV or any of the other ISVs. In contrast, in U4.4 and Aag2 cells, WNV siRNAs, and pi-like RNAs without typical ping-pong piRNA signature were observed, while this signature was present in PCLV piRNAs in Aag2 cells. Together, our results demonstrate that mosquito small RNA responses are strongly dependent on both the mosquito cell type and/or the mosquito species and family of the infecting virus.

Conserved motifs in the hypervariable domain of chikungunya virus nsP3 required for transmission by Aedes aegypti mosquitoes.

BACKGROUND: Chikungunya virus (CHIKV) is a re-emerging arthropod-borne (arbo)virus that causes chikungunya fever in humans and is predominantly transmitted by Aedes aegypti mosquitoes. The CHIKV replication machinery consists of four non-structural proteins (nsP1-4) that additionally require the presence of a number of host proteins for replication of the viral RNA. NsP3 is essential for CHIKV replication and has a conserved macro, central and C-terminal hypervariable domain (HVD). The HVD is intrinsically disordered and interacts with various host proteins via conserved short peptide motifs: A proline-rich (P-rich) motif that has affinity for SH3-domain containing proteins and duplicate FGDF motifs with affinity for G3BP and its mosquito homologue Rasputin. The importance of these motifs for infection of mammalian cells has previously been implicated. However, their role during CHIKV infection of mosquito cells and transmission by mosquitoes remains unclear. METHODOLOGY / PRINCIPAL FINDINGS: Here, we show that in-frame deletion of the P-rich motif is lethal for CHIKV replication in both mosquito and mammalian cells. However, while mutagenesis of the P-rich motif negatively affects replication both in mammalian and mosquito cells, it did not compromise the infection and transmission of CHIKV by Ae. aegypti mosquitoes. Mutagenesis of both FGDF motifs together completely inactivated CHIKV replication in both mammalian and mosquito cells. Importantly, mutation of a single FGDF motif attenuated CHIKV replication in mammalian cells, while replication in mosquito cells was similar to wild type. Surprisingly, CHIKV mutants containing only a single FGDF motif were efficiently transmitted by Ae. aegypti. CONCLUSIONS / SIGNIFICANCE: The P-rich motif in CHIKV nsP3 is dispensable for transmission by mosquitoes. A single FGDF motif is sufficient for infection and dissemination in mosquitoes, but duplicate FGDF motifs are required for the efficient infection from the mosquito saliva to a vertebrate host. These results contribute to understanding the dynamics of the alphavirus transmission cycle and may help the development of arboviral intervention strategies.

The Methyltransferase-Like Domain of Chikungunya Virus nsP2 Inhibits the Interferon Response by Promoting the Nuclear Export of STAT1.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has evolved effective mechanisms to counteract the type I interferon (IFN) response. Upon recognition of the virus, cells secrete IFNs, which signal through transmembrane receptors (IFNAR) to phosphorylate STAT proteins (pSTAT). pSTAT dimers are transported into the nucleus by importin-α5 and activate the transcription of IFN-stimulated genes (ISGs), increasing cellular resistance to infection. Subsequently, STAT proteins are shuttled back into the cytoplasm by the exportin CRM1. CHIKV nonstructural protein 2 (nsP2) reduces ISG expression by inhibiting general host cell transcription and by specifically reducing the levels of nuclear pSTAT1 via an unknown mechanism. To systematically examine where nsP2 acts within the JAK/STAT signaling cascade, we used two well-characterized mutants of nsP2, P718S and KR649AA. Both mutations abrogate nsP2's ability to shut off host transcription, but only the KR649AA mutant localizes exclusively to the cytoplasm and no longer specifically inhibits JAK/STAT signaling. These mutant nsP2 proteins did not differentially affect IFNAR expression levels or STAT1 phosphorylation in response to IFNs. Coimmunoprecipitation experiments showed that in the presence of nsP2, STAT1 still effectively bound importin-α5. Chemically blocking CRM1-mediated nuclear export in the presence of nsP2 additionally showed that nuclear translocation of STAT1 is not affected by nsP2. nsP2 putatively has five domains. Redirecting the nsP2 KR649AA mutant or just nsP2's C-terminal methyltransferase-like domain into the nucleus strongly reduced nuclear pSTAT in response to IFN stimulation. This demonstrates that the C-terminal domain of nuclear nsP2 specifically inhibits the IFN response by promoting the nuclear export of STAT1.IMPORTANCE Chikungunya virus is an emerging pathogen associated with large outbreaks on the African, Asian, European, and both American continents. In most patients, infection results in high fever, rash, and incapacitating (chronic) arthralgia. CHIKV effectively inhibits the first line of defense, the innate immune response. As a result, stimulation of the innate immune response with interferons (IFNs) is ineffective as a treatment for CHIKV disease. The IFN response requires an intact downstream signaling cascade called the JAK/STAT signaling pathway, which is effectively inhibited by CHIKV nonstructural protein 2 (nsP2) via an unknown mechanism. The research described here specifies where in the JAK/STAT signaling cascade the IFN response is inhibited and which protein domain of nsP2 is responsible for IFN inhibition. The results illuminate new aspects of antiviral defense and CHIKV counterdefense strategies and will direct the search for novel antiviral compounds.

Functional RNA during Zika virus infection.

Zika virus (ZIKV; family Flaviviridae; genus Flavivirus) is a pathogenic mosquito-borne RNA virus that currently threatens human health in the Americas, large parts of Asia and occasionally elsewhere in the world. ZIKV infection is often asymptomatic but can cause severe symptoms including congenital microcephaly and Guillain-Barré syndrome. The positive single-stranded RNA genome of the mosquito-borne ZIKV requires effective replication in two evolutionary distinct hosts - mosquitoes and primates. In addition to some of the viral proteins, the ZIKV genomic RNA and functional RNAs produced thereof aid in the establishment of productive infection and the evasion of host cell antiviral responses. ZIKV has evolved to contain a nucleotide composition and RNA modifications, such as methylation and the formation of G-quadruplexes that allow effective replication in both hosts. Furthermore, a number of host factors interact with the viral genome to modulate RNA replication. Importantly, the ZIKV genome produces non-coding subgenomic flavivirus RNA (sfRNA) due to stalling of host 5'- 3' ribonucleases on viral RNA structures in the 3' untranslated region (UTR). This sfRNA (sfRNA) exerts important proviral functions such as antagonizing the innate interferon response and RNA interference. Here, we discuss the ZIKV genomic RNA and functional RNAs thereof to assess their significance during ZIKV infection. Understanding the details of the ZIKV infection cycle will aid in the development of effective antiviral strategies and safe vaccines.

Vector competence of European mosquitoes for West Nile virus.

West Nile virus (WNV) is an arthropod-borne flavivirus of high medical and veterinary importance. The main vectors for WNV are mosquito species of the Culex genus that transmit WNV among birds, and occasionally to humans and horses, which are 'dead-end' hosts. Recently, several studies have been published that aimed to identify the mosquito species that serve as vectors for WNV in Europe. These studies provide insight in factors that can influence vector competence of European mosquito species for WNV. Here, we review the current knowledge on vector competence of European mosquitoes for WNV, and the molecular knowledge on physical barriers, anti-viral pathways and microbes that influence vector competence based on studies with other flaviviruses. By comparing the 12 available WNV vector competence studies with European mosquitoes we evaluate the effect of factors such as temperature, mosquito origin and mosquito biotype on vector competence. In addition, we propose a standardised methodology to allow for comparative studies across Europe. Finally, we identify knowledge gaps regarding vector competence that, once addressed, will provide important insights into WNV transmission and ultimately contribute to effective strategies to control WNV.

Mosquito co-infection with Zika and chikungunya virus allows simultaneous transmission without affecting vector competence of Aedes aegypti.

BACKGROUND: Zika virus (ZIKV) and chikungunya virus (CHIKV) are highly pathogenic arthropod-borne viruses that are currently a serious health burden in the Americas, and elsewhere in the world. ZIKV and CHIKV co-circulate in the same geographical regions and are mainly transmitted by Aedes aegypti mosquitoes. There is a growing number of case reports of ZIKV and CHIKV co-infections in humans, but it is uncertain whether co-infection occurs via single or multiple mosquito bites. Here we investigate the potential of Ae. aegypti mosquitoes to transmit both ZIKV and CHIKV in one bite, and we assess the consequences of co-infection on vector competence. METHODOLOGY/PRINCIPAL FINDINGS: First, growth curves indicated that co-infection with CHIKV negatively affects ZIKV production in mammalian, but not in mosquito cells. Next, Ae. aegypti mosquitoes were infected with ZIKV, CHIKV, or co-infected via an infectious blood meal or intrathoracic injections. Infection and transmission rates, as well as viral titers of positive mosquitoes, were determined at 14 days after blood meal or 7 days after injection. Saliva and bodies of (co-)infected mosquitoes were scored concurrently for the presence of ZIKV and/or CHIKV using a dual-colour immunofluorescence assay. The results show that orally exposed Ae. aegypti mosquitoes are highly competent, with transmission rates of up to 73% for ZIKV, 21% for CHIKV, and 12% of mosquitoes transmitting both viruses in one bite. However, simultaneous oral exposure to both viruses did not change infection and transmission rates compared to exposure to a single virus. Intrathoracic injections indicate that the selected strain of Ae. aegypti has a strong salivary gland barrier for CHIKV, but a less profound barrier for ZIKV. CONCLUSIONS/SIGNIFICANCE: This study shows that Ae. aegypti can transmit both ZIKV and CHIKV via a single bite. Furthermore, co-infection of ZIKV and CHIKV does not influence the vector competence of Ae. aegypti.

Vector competence of northern European Culex pipiens biotypes and hybrids for West Nile virus is differentially affected by temperature.

BACKGROUND: Outbreaks of West Nile virus (WNV) have not occurred in northern Europe despite nearby circulation of WNV in the southern part of the continent. The main vector for WNV, the mosquito Culex (Cx.) pipiens, consists of two behaviorally distinct biotypes, pipiens and molestus, which can form hybrids. Although temperature has been shown to influence vector competence of Cx. pipiens for WNV and biotypes are differentially susceptible towards infection, the interaction between the two has not been elucidated. METHODS: We determined vector competence of the Cx. pipiens biotypes and hybrids, after 14 days of incubation at 18, 23 and 28 °C. Mosquitoes were orally infected by providing an infectious blood meal or by injecting WNV directly in the thorax. Infection and transmission rates were determined by testing the bodies and saliva for WNV presence. In addition, titers of mosquitoes with WNV-positive bodies and saliva samples were determined. RESULTS: Orally infected biotype pipiens and hybrids showed significantly increased transmission rates with higher temperatures, up to 32 and 14 %, respectively. In contrast, the molestus biotype had an overall transmission rate of 10 %, which did not increase with temperature. All mosquitoes that were infected via WNV injections had (close to) 100 % infection and transmission rates, suggesting an important role of the mosquito midgut barrier. We found no effect of increasing temperature on viral titers. CONCLUSIONS: Temperature differentially affected vector competence of the Cx. pipiens biotypes. This shows the importance of accounting for biotype-by-temperature interactions, which influence the outcomes of vector competence studies. Vector competence studies with Cx. pipiens mosquitoes differentiated to the biotype level are essential for proper WNV risk assessments.

Dengue Non-coding RNA: TRIMmed for Transmission.

Dengue virus RNA is trimmed by the 5'→3' exoribonuclease XRN1 to produce an abundant, non-coding subgenomic flavivirus RNA (sfRNA) in infected cells. In a recent paper in Science, Manokaran et al. (2015) report that sfRNA binds TRIM25 to evade innate immune sensing of viral RNA by RIG-I.