Zika virus recruits karyopherin \u03b16 for efficient replication via NS2B

Febrile or Exanthematous Illness Associated with Zika, Dengue, and Chikungunya Viruses, Panama.

Zika virus (ZIKV) is a single-stranded, positive-sense RNA virus that is mainly transmitted by mosquito biting.ZIKV belongs to the family Flaviviridae, genus Flavivirus, which includes other important human pathogens including dengue virus, yellow fever virus, West Nile virus, and Japanese encephalitis virus.ZIKV was first isolated from a sentinel rhesus monkey in the Zika forest of Uganda in 1947.Following the outbreak of ZIKV in Yap Island of Micronesia in 2007 and the largest Zika pandemic in Brazil in 2015, ZIKV has rapidly spread through the Pacific islands and the Americas, resulting in an estimated two million infections per year.Due to its unexpected causal link to microcephaly in fetus and Guillain-Barre syndrome in adults, the current ongoing ZIKV epidemic was declared as a public health emergency of international concern by the World Health Organization in February, 2016.Accumulated evidence has demonstrated that ZIKV preferentially targets human neuronal progenitor cells, disrupts proliferation and differentiation, and triggers a strong immune response, resulting in significant neuronal cell death and microcephaly phenotype.Currently, no vaccines or specific antiviral drugs are available to prevent or treat ZIKV infections.Upon ZIKV infection, host cells immediately initiate several lines of antiviral innate immune responses, including the production of type I interferon (IFN) and its downstream signaling pathways.Type I IFN response is well evidenced to play a critical role during ZIKV infection, and mice deficient in type I IFN receptor are highly susceptible to ZIKV infection upon various injection routes.Type I IFN and its downstream signaling molecules (e.g.IFITM1 and IFITM3) show significant anti-ZIKV effects in both cell culture and animal models.Meanwhile, ZIKV has evolved diverse strategies to antagonize the host defense system through blocking the production of type I IFN and its downstream signaling, or escaping from antiviral effects of individual ISGs.Like other mosquito-borne flaviviruses, the viral proteins NS2B, NS3, NS4B and NS5, as well as the subgenomic flavivirus RNA (sfRNA), have been identified as potent suppressors of type I IFN induction and its effector pathways.Especially, ZIKV NS5 directly binds to STAT2, and leads to the proteasomal degradation of STAT2, resulting in the silencing of type I IFN signaling.These remarkable achievements in recent two years not only lead to the better understanding of ZIKV infection and pathogenesis, but also provide useful clues for the development of antiviral drugs and vaccine candidates.Several ISGs and their enzymatic products, including 25-Hydroxycholesterol, have been well characterized with potent anti-ZIKV activity.A few recombinant live-attenuated ZIKV vaccine candidates have been rationally constructed via reverse genetics with increased IFN sensitivity, which attributed to the attenuation phenotype in immunocompetent animals.Here, we summarize recent advances in the interplay between ZIKV and host type I IFN-mediated immune response.

To the Editor: Isolated in 1947 from a rhesus monkey in Zika forest, Uganda, Zika virus (ZIKV) is a mosquito-borne flavivirus (1).For half a century, ZIKV was described only as causing sporadic human infections in Africa and Asia, which was mostly confirmed by serologic methods (2).In 2007, the first ZIKV outbreak reported outside Africa and Asia was retrospectively documented from biological samples of patients on Yap Island, Federated States of Micronesia, North Pacific, who had received an incorrect diagnosis of dengue virus (DENV) (3,4).We report here the early investigations that led to identification of ZIKV as the causative agent of an outbreak that started in October 2013 in French Polynesia.French Polynesia is a French overseas territory located in the South Pacific.The ≈270,000 inhabitants live on 67 islands distributed into 5 archipelagoes (Society, Marquesas, Tuamotu, Gambier, and Austral Islands).Surveillance for acute febrile illnesses is coordinated by the Department of Health with the contribution of a sentinel network of public and private practitioners, the main public hospital (Centre Hospitalier

Zika virus (ZIKV) is a member of the Flaviviridae family that received public attention and scientific interest after the outbreak in French Polynesia (2013-2014) and the epidemic in the Americas (2015-2016). Even though only 20% of infected people exhibit clinical manifestations and they are predominantly flu-like symptoms, these events unveiled neurological complications associated with ZIKV infection, such as the Guillain-Barré syndrome in adults and microcephaly in newborns. Lacking a preventive vaccine and a specific antiviral therapy against ZIKV allied to the fact that this pathogen is a re-emerging virus, uncovering and comprehending novel virus-host interactions is crucial to the identification of new antiviral targets and the development of innovative antiviral approaches. Previous research work uncovered that the Chinese hamster ovary (CHO) cells do not support ZIKV infection.459 As this cell line does not express endogenous epidermal growth factor receptor (EGFR), this study aimed to investigate whether EGFR and EGFR-dependent signaling are relevant for the ZIKV life cycle in vitro. In the first part of the study, viral infection was investigated in CHO cells and compared to A549 cells, a highly ZIKV permissive cell line. After performing binding and entry assays, ZIKV entry, but not the attachment, was significantly decreased in CHO cells in comparison to A549 cells. Additionally, in A549-EGFR KO cells, ZIKV entry was diminished relatively to the off-target control. These results show the clear impact that the absence of EGFR has on viral entry, implicating EGFR during this process. Even though EGFR overexpression in CHO cells could not render these cells permissive to ZIKV infection, as demonstrated by the lack of viral infection after electroporation with in vitro transcribed capped ZIKV-Renilla luciferase RNA, it was possible to rescue ZIKV entry. These findings suggest that there are additional elements, which are not expressed in CHO cells, required for viral replication. Furthermore, the impact of ZIKV infection on EGFR mRNA and protein levels as well as on the EGFR subcellular localization and distribution was evaluated. The relative number of EGFR specific transcripts continuously increased with ZIKV infection, whereas the EGFR protein level diminished at later times of infection. Moreover, changes in the subcellular localization of EGFR and its colocalization with the early endosomal marker EEA1 in ZIKV-infected cells revealed that ZIKV triggers EGFR internalization. The relevance of EGFR in the ZIKV entry process was further corroborated by the observation of EGFR internalization at 30 min post-infection (mpi) and to less extent at 60 mpi, which concurs with the expected time of ZIKV entry into the host cells. In the remaining part of the study, the influence of ZIKV infection in EGFR-dependent signaling as well as the contribution of EGFR and EGFR signaling for viral infection were studied. Activation of EGFR and the MAPK/ERK signaling cascade was detected as early as 5 mpi and ceased within 30 mpi in ZIKV-infected cells. Taking into account that EGFR internalization was observed at 30 mpi in infected cells, the activation of EGFR and ERK and subsequent dephosphorylation within this period go along with this previous observation. Vice-versa, inhibition of the activation of EGFR and the MAPK/ERK pathway declines ZIKV infection. On the one hand, inhibition of EGFR activation by Erlotinib affected ZIKV entry, as a consequence of impaired EGFR internalization. On the other hand, Raf and MEK inhibitors reduced ZIKV infection without disturbing viral replication or viral entry. These data suggest that the activation of the MAPK/ERK signaling cascade is necessary for a step of the viral life cycle before the onset of genome replication and morphogenesis and after viral entry. The importance of EGFR signaling was additionally investigated by the determination of EGFR half-life in ZIKV-infected cells upon EGF stimulation. While the EGFR half-life was similar in uninfected and Uganda-infected cells, a delay in EGFR degradation was observed in French Polynesia-infected cells. This observation might indicate an extended usurpation of the EGFR signaling since EGFR seems to still be active in the endosomes. Moreover, disruption of lipid rafts by MβCD, a cholesterol-depleting agent, hampered ZIKV entry. In uninfected cells, MβCD treatment led to the activation of EGFR, but at the same time prevented EGFR internalization, indicating that EGFR activation exclusively is not sufficient for an efficient ZIKV entry and further supporting the importance of EGFR internalization during the ZIKV entry process. Taken together, this study uncovers EGFR as a relevant host factor in the early stages of ZIKV infection, providing novel insights into the ZIKV entry process. Since numerous monoclonal antibodies and substances that target EGFR are licensed, repurposing these compounds might be a helpful tool for the establishment of an antiviral therapy in case of ZIKV re-emergence.

mammalian adaptation. Various analyses are ongoing to answer questions about the route of transmission among seals and possible transmissibility to humans.

Zika virus in the dock

Zika virus is a single-stranded RNA virus in the genus Flavivirus and is closely related to dengue, West Nile, Japanese encephalitis, and yellow fever viruses (1,2). Among flaviviruses, Zika and dengue virus share similar symptoms of infection, transmission cycles, and geographic distribution. Diagnostic testing for Zika virus infection can be accomplished using both molecular and serologic methods. For persons with suspected Zika virus disease, a positive real-time reverse transcription-polymerase chain reaction (rRT-PCR) result confirms Zika virus infection, but a negative rRT-PCR result does not exclude infection (3-7). In these cases, immunoglobulin (Ig) M and neutralizing antibody testing can identify additional recent Zika virus infections (6,7). However, Zika virus antibody test results can be difficult to interpret because of cross-reactivity with other flaviviruses, which can preclude identification of the specific infecting virus, especially when the person previously was infected with or vaccinated against a related flavivirus (8). This is important because the results of Zika and dengue virus testing will guide clinical management. Pregnant women with laboratory evidence of Zika virus infection should be evaluated and managed for possible adverse pregnancy outcomes and be reported to the U.S. Zika Pregnancy Registry or the Puerto Rico Zika Active Pregnancy Surveillance System for clinical follow-up (9,10). All patients with clinically suspected dengue should have proper management to reduce the risk for hemorrhage and shock (11). If serologic testing indicates recent flavivirus infection that could be caused by either Zika or dengue virus, patients should be clinically managed for both infections because they might have been infected with either virus.

Zika virus (ZIKV) infection has emerged as a significant public health concern due to its association with fetal microcephaly and Guillain-Barre syndrome (GBS). Unfortunately, its detailed pathogenesis remains unclear. To better understand how ZIKV evades host antiviral immunity, we analyzed the microarray dataset (GSE98889) of ZIKV-infected primary human brain microvascular endothelial cells (hBMECs) retrieved from the gene expression omnibus (GEO). 160, 1423, 969, 829, and 600 differentially expressed genes (DEGs) were identified at 12, 24, 48, 72, and 216hours post-ZIKV infection in hBMECs, respectively. Subsequently, 31 common DEGs across all time-points were selected for further analysis. Gene ontology (GO) functional analysis showed these 31 DEGs were mainly involved in the host antiviral innate immune responses. Protein-protein interaction (PPI) network analysis identified 10 hub genes (MX1, OAS1, OAS2, IFI44, IFI44L, IFIT1, IFIT2, IFIT3, IFIH1, and XAF1), which were all interferon-stimulated genes (ISGs) and upregulated. qRT-PCR was used to validate the expression patterns of these 10 hub genes in different ZIKV-infected cell lines. Finally, miRNA-mRNA regulatory network analysis revealed that hsa-miR-129-2-3p, hsa-miR-138-5p, hsa-miR-21-3p, hsa-miR-27a-5p, hsa-miR-449a, and hsa-miR449b-5p were key miRNAs regulating these hub genes. Our study showed that ZIKV infection activated the host innate immune response to restrict ZIKV infection. The common pathways, hub genes, and their regulatory miRNA network offer new insights into virus-host interactions, enhancing our understanding of ZIKV pathogenesis.

ABSTRACT.Serological cross-reactivity has proved to be a challenge to diagnose Zika virus (ZIKV) infections in dengue virus (DENV) endemic countries. Confirmatory testing of ZIKV IgM positive results by plaque reduction neutralization tests (PRNTs) provides clarification in only a minority of cases because most individuals infected with ZIKV were previously exposed to DENV. The goal of this study was to evaluate the performance of a ZIKV/DENV DUO IgM antibody capture ELISA (MAC-ELISA) for discriminating between DENV and ZIKV infections in endemic regions. Our performance evaluation included acute and convalescent specimens from patients with real-time reverse transcription polymerase chain reaction (RT-PCR)-confirmed DENV or ZIKV from the Sentinel Enhanced Dengue Surveillance System in Ponce, Puerto Rico. The ZIKV/DENV DUO MAC-ELISA specificity was 100% for DENV (N = 127) and 98.4% for ZIKV (N = 275) when specimens were tested during the optimal testing window (days post-onset of illness [DPO] 6–120). The ZIKV/DENV DUO MAC-ELISA sensitivity of RT-PCR confirmed specimens reached 100% for DENV by DPO 6 and for ZIKV by DPO 9. Our new ZIKV/DENV DUO MAC-ELISA was also able to distinguish ZIKV and DENV regardless of previous DENV exposure. We conclude this novel serologic diagnostic assay can accurately discriminate ZIKV and DENV infections. This can potentially be useful considering that the more labor-intensive and expensive PRNT assay may not be an option for confirmatory diagnosis in areas that lack PRNT capacity, but experience circulation of both DENV and ZIKV.

Background: Zika virus (ZIKV) infection is a public health concern. The first ZIKV outside Africa was detected in mosquito in Malaysia. More than six decades ago, serological surveys indicated the presence of human infection with ZIKV in the Malaysian Borneo state of Sabah. It has also been demonstrated that orangutans in Sabah have antibodies against ZIKV. Several years ago, a case of human ZIKV infection was reported in a traveler who visited Sabah. Therefore, it is thought that ZIKV is endogenous to Sabah and is widely distributed. During the recent global epidemic of ZIKV, the first autochthonous case and two subsequent autochthonous cases were detected in Sabah. Because ZIKV infection is mainly asymptomatic or mildly symptomatic, the extent of ZIKV infection in the population of Sabah is not known. Furthermore, the presence of ZIKV in vector mosquitoes and animals has not been investigated. Therefore, the present study was performed to analyze the outbreak cases of ZIKV infection and to determine their relationship with the burden of ZIKV infection in the local population, mosquitoes, and wild nonhuman primates in Sabah. Methods: Serum and urine samples were collected from two local patients with ZIKV infection, their household members, and those who resided within 400m of the patients’ residences. Serum samples were also collected from four wild Maca fascicularis. Mosquito samples, mostly female Aedes albopictus, were collected from 30 sites in Kota Kinabalu. The presence of ZIKV was assessed by RT-qPCR and RT-PCR. Phylogenetic analysis was performed using the neighbor-joining method. Results: Two cases of ZIKV infection were identified by reverse-transcription quantitative PCR (RT-qPCR) in residents of Kota Kinabalu, and the Taiwanese health authorities reported one case in an individual who visited Kota Kinabalu during the study period. All household members of both local patients and people living within a 400 m radius of the patients were negative for ZIKV. Furthermore, mosquitoes collected from the surroundings of the residences and places visited by the patients and four serum samples from M. fascicularis were also negative for ZIKV. A phylogenetic tree constructed using the nucleotide sequences of the envelope genes of ZIKV showed that the strains from Sabah formed a cluster with strains from Thailand and Cambodia, and belong to the Asian lineage. Conclusions: Our study revealed that ZIKVs in Sabah is of Asian lineage and are not related to the recent outbreak strains in the Americas and Singapore. ZIKV infection in Sabah is sporadic, possibly because of limited transmission of the virus. Further studies are needed to characterize the evolutionary history of ZIKV in Sabah to understand the epidemiology of this infection in Borneo.

To the Editor: Zika virus (ZIKV), a mosquito-borne flavivirus, causes Zika fever, a self-limiting febrile and exanthematic arthralgia syndrome closely resembling dengue fever. Most often, signs and symptoms are maculopapular rash, fever, arthralgia, myalgia, headache, and conjunctivitis; edema, sore throat, cough, and vomiting occur less frequently (1). The virus, which was initially isolated from a rhesus monkey (Macaca mulatta) in 1947 in Uganda, has come to attention recently after a large outbreak occurred in the western Pacific region, including French Polynesia, New Caledonia, Easter Island, and the Cook Islands (2). Travel-related imported infections have thus been increasingly reported from the western Pacific and sporadically also in travelers to other regions of the world, including Thailand, Indonesia, and Senegal (2,3). ZIKV is transmitted by different Aedes mosquito species, and nonhuman primates play a role as reservoirs (1). After the beginning of the ZIKV epidemic in late 2013, a 20-fold increase of Guillain-Barre syndrome incidence was noted in French Polynesia; 1 patient was infected a week before neurologic symptoms started (4). We report an acute ZIKV infection in a traveler returning from Malaysian Borneo who experienced bilateral hearing difficulties during the course of illness. On September 1, 2014, a 45-year-old woman was seen in an outpatient clinic in Heidelberg, Germany for fever of up to 39°C and maculopapular rash covering her trunk, arms, and legs. Fever had started on August 30, which was 6 days after she had returned from a 3-week vacation to peninsular Malaysia and Sabah, Malaysian Borneo. Laboratory analyses showed a slightly elevated C-reactive protein level of 5.2 mg/L (reference range <5.0), but liver function test and complete blood count results were within reference range. During the next 3 days, the fever subsided, but the patient experienced a sore throat, bilateral conjunctivitis, and a burning sensation of the palms and soles. These symptoms were accompanied by swelling of the hands and increasing arthralgia of the wrists, palms, and fingers. There was no lymphadenopathy. An indirect immunofluorescence assay for ZIKV (3) demonstrated an IgM titer of 1:640 and an IgG titer of 1:320 (cutoff <1:20) on day 6 of illness (Figure). An indirect immunofluorescence assay for dengue virus demonstrated an IgG titer of 1:80 and no IgM (cutoff <1:20). Figure Clinical course and laboratory results (reverse transcription-PCR [RT-PCR]) for a patient with Zika virus (ZIKV) infection acquired from Malaysian Borneo. Two days later, the patient experienced sudden bilateral dull and metallic hearing; in her left ear, she experienced a very short delay between a sound and her perception of the sound. Follow-up ZIKV serologic testing on day 11 of illness showed a decreased IgM titer of 1:160 and an increased IgG titer of 1:2,560 (Figure). Viral neutralization testing (3) of the same sample demonstrated the presence of ZIKV-specific neutralizing antibodies. Chikungunya virus serology results were negative. An archived serum sample from day 3 of illness studied by ZIKV serology and a ZIKV-specific real-time reverse transcription PCR (3) was negative (Figure). Hearing difficulties lasted for 10 days and resolved gradually (Figure). During her journey to several cities and villages in Sabah, Malaysian Borneo, the patient had noticed several mosquito bites even though she had used repellents. She had stayed in hotels, private homes, and remote church homes under various conditions (Technical Appendix). In Asia, Zika fever has been described sporadically in Cambodia, Thailand, and Indonesia (Java and Lombok) (1,3,5,6). On the basis of the incubation time of ≈6 days in returning travelers (2,3), we assumed that the patient became infected in Keningau or surrounding villages, in northern Borneo. Although ZIKV was detected in Ae. aegypti mosquitoes in peninsular Malaysia in 1969 (7) and antibodies against ZIKV were demonstrated in serum samples from 15 of 79 patients on peninsular Malaysia and 9 of 50 patients in Borneo in 1953 (8), Zika fever in peninsular Malaysia or Borneo has not been reported. In 2001, ZIKV seropositivity was demonstrated in a native Bornean, 2 migrants to Borneo, and 2 Bornean orangutans (Pongo pygmaeus) (9). A later study found an additional 8 Bornean orangutans to be seropositive for antibodies against ZIKV (10). Thus, in Borneo, either the virus only rarely infects humans or the disease is mistaken for dengue fever. Neurologic complications of ZIKV infections had previously been reported only as Guillain-Barre syndrome, and hearing difficulties in Zika fever patients have not been reported. Because this symptom resolved spontaneously, no audiometry or auditory brainstem response testing was performed, and the cause of the disorder remains unclear. Because of increasing travel and migration and heightened clinical and laboratory awareness, more ZIKV infections are likely to be diagnosed outside of epidemic events. Technical Appendix: Travel itinerary of a patient with Zika fever. Click here to view.(217K, pdf)

Association between Guillain-Barré syndrome and Zika virus infection

Zika virus (ZIKV) is a mosquito-borne flavivirus causing a major epidemic in the Americas in 2015. Dendritic cells (DCs) are leukocytes with key antiviral functions, but their role in ZIKV infection remains under investigation. While most studies have focused on the monocyte-derived subtype of DCs, less is known about conventional dendritic cells (cDCs), essential for the orchestration of antiviral adaptive immunity. This study investigates the mechanisms by which cDCs respond to ZIKV for antiviral cytokine production. Here, using murine cultures, we demonstrate that ZIKV infection and not detection of ZIKV-infected dead cells activates cDCs by inducing type I interferons (IFN-I) and proinflammatory cytokines. Furthermore, ZIKV-infected cDCs markedly activated the IRE1/XBP1s axis of the unfolded protein response (UPR). Flow cytometry analysis indicates that among cDCs, type 1 cDCs (cDC1s) are responsible for ZIKV detection. Functionally, genetic loss of XBP1s curtailed expression of the costimulatory molecule CD86 and the production of IFN-I and proinflammatory cytokines by cDCs, without exhibiting increased susceptibility to ZIKV infection. These effects are attributable to perturbations in the IRE1/XBP1s axis and not due to overcompensation of PERK or IRE1 kinase signaling. Finally, tissue resident cDCs also exhibit susceptibility to infection, potentially establishing these cells as ZIKV targets invivo. These findings underscore a critical role for the IRE1/XBP1s pathway in fine-tuning cDC activation to ZIKV, linking viral recognition to cDC functional maturation and opening new avenues for exploring UPR pathways targeting cDCs in the context of flavivirus infections.

Three pathogenic mosquito-borne viruses that have been on the rise in recent years are Zika virus (ZIKV), Usutu virus (USUV) and Mayaro virus (MAYV). ZIKV unexpectedly caused a large-scale epidemic of human illness in Central and South America in 2015 and 2016. The zoonotic USUV has recently spread throughout Europe, causing massive bird die-off and rare but severe neuroinvasive disease in humans. The tropical MAYV is currently emerging in Central and South America, and infection in humans can result in long-lasting, debilitating arthralgia. The rapid emergence of these three arthropod-borne (arbo)viruses urged for an in-depth analysis of the mosquito vectors capable of transmitting these viruses, as well as for the development of effective strategies to confine and prevent epidemics of these arboviral diseases.Although the number of ZIKV cases has declined after the outbreak in the Americas, the virus is still present in tropical regions and therefore remains a threat to public health. Especially in areas with human populations naive to the virus, ZIKV may suddenly emerge, which could result in new, major disease outbreaks. Since arboviruses replicate in both their vertebrate host and invertebrate vector, the risk of ZIKV outbreaks in a particular region is also determined by the presence of competent mosquito vectors. In this thesis, it was investigated how effectively indigenous and invasive mosquito species present in the Netherlands transmit ZIKV in the laboratory. The invasive Asian bush mosquito Aedes japonicus, permanently established in Flevoland, the Netherlands, was found capable of experimentally transmitting ZIKV, hence suggesting that this mosquito species could be a vector for ZIKV. The indigenous common house mosquito Culex pipiens, however, was unable to transmit ZIKV after an infectious blood meal. Nevertheless, bypassing the mosquito midgut by intrathoracic injection of ZIKV resulted in limited virus accumulation in Cx. pipiens saliva. This indicates that the mosquito midgut normally restricts ZIKV dissemination in Cx. pipiens after oral exposure. Additionally, a general replication deficiency of ZIKV in Culex mosquito cells was identified, which occurred post-entry. These results indicate that Cx. pipiens should be considered a highly inefficient vector for ZIKV. Cx. pipiens mosquitoes can, however, effectively transmit USUV. This virus is maintained in an enzootic transmission cycle between birds and mosquitoes. Humans and other mammals can also become infected via mosquito bites but are thought to be dead-end hosts due to low levels of viraemia. Thus, when local mosquitoes are evaluated for their ability to transmit USUV under experimental conditions, the use of avian blood for the infectious blood meal would be preferable. Nonetheless, the origin of blood used to study vector competence generally varies between studies, while it is unknown to what extent the blood source affects the experimental outcomes. In this thesis, it was found that the use of chicken or human blood resulted in comparable vector competence of Cx. pipiens for USUV. Interestingly, this study also revealed that the USUV titers in the saliva of the two biotypes of Cx. pipiens (pipiens and molestus) markedly differed. Biotype molestus accumulated much lower USUV titers in the saliva as compared to biotype pipiens, regardless of which blood type was offered. This may indicate that biotype molestus is a less efficient vector for USUV than biotype pipiens, which is especially interesting considering that biotype pipiens preferentially feeds on birds, whereas biotype molestus is more attracted to mammals including humans. Importantly, it was also found that the opportunistic feeder Ae. japonicus is capable of experimentally transmitting USUV, thus making this mosquito species a potential bridge vector between birds and humans. Besides arboviruses, mosquitoes can also carry insect-specific viruses (ISVs). Recently, ISVs have received increasing attention due to their ability to influence arbovirus transmission, and it is therefore important to characterize the collection of viruses (i.e., the virome) present in mosquito vectors. ISV replication in mosquito cells activates RNA interference (RNAi)-based immune responses, resulting in the production of virus-derived small RNAs. Sequencing and de novo assembly of these small RNAs provides an overview of the ISVs present in mosquito populations. In this thesis, novel virus species were discovered in Ae. japonicus populations in the Netherlands and France using a small RNA-based metagenomic approach. The newly discovered Ae. japonicus narnavirus 1 (AejapNV1) showed the strongest RNAi response. Narnaviruses have been described as positive-sense RNA viruses with only a forward open reading frame (ORF) coding for the RNA-dependent RNA polymerase (RdRp). Interestingly, AejapNV1 showed an ambigrammatic coding strategy with a forward ORF encoding the RdRp on the positive strand and a reverse ORF with unknown function on the negative strand. This was remarkable, as positive-sense RNA viruses usually code for proteins only on the positive strand.The arboviruses ZIKV and MAYV have the potential to invade new geographical areas, whilst no licenced antivirals or vaccines are available to treat or prevent disease. Here, virus-like particle (VLP) vaccines against both these viruses were developed using the scalable baculovirus-insect cell expression system. Vaccination of mice with MAYV VLPs induced high levels of neutralising antibodies, and completely protected the animals from viraemia and arthritic disease after challenge with wild-type MAYV, allowing this vaccine to be further developed for human use. Immunisation of mice with two developed ZIKV vaccine candidates, VLPs and subviral particles (SVPs), however, only induced limited levels of ZIKV-neutralising antibodies and did not protect against wild-type ZIKV infection, although the viraemic period became shorter. Epitope analysis showed that the ZIKV VLPs and SVPs do not display quaternary structure epitopes normally present on envelope protein homodimers found on the ZIKV virion. These epitopes induce potent neutralising antibodies following natural ZIKV infection in humans. To improve the efficacy of the ZIKV SVP vaccine, novel variants were developed with the specific aim to stabilise the envelope protein homodimers. The improved vaccine candidates now await further testing in mouse models of ZIKV disease.In conclusion, the results of this thesis enhance our understanding of the mosquito vectors involved in ZIKV and USUV transmission. Also, the developed VLP vaccines against ZIKV and MAYV will hopefully help to control outbreaks of these arboviral diseases in the future.

Zika virus is a flavivirus transmitted primarily by Aedes aegypti and Aedes albopictus mosquitoes, and infection can be asymptomatic or result in an acute febrile illness with rash (1). Zika virus infection during pregnancy is a cause of microcephaly and other severe birth defects (2). Infection has also been associated with Guillain-Barré syndrome (GBS) (3) and severe thrombocytopenia (4,5). In December 2015, the Puerto Rico Department of Health (PRDH) reported the first locally acquired case of Zika virus infection. This report provides an update to the epidemiology of and public health response to ongoing Zika virus transmission in Puerto Rico (6,7). A confirmed case of Zika virus infection is defined as a positive result for Zika virus testing by reverse transcription-polymerase chain reaction (RT-PCR) for Zika virus in a blood or urine specimen. A presumptive case is defined as a positive result by Zika virus immunoglobulin M (IgM) enzyme-linked immunosorbent assay (MAC-ELISA)* and a negative result by dengue virus IgM ELISA, or a positive test result by Zika IgM MAC-ELISA in a pregnant woman. An unspecified flavivirus case is defined as positive or equivocal results for both Zika and dengue virus by IgM ELISA. During November 1, 2015-July 7, 2016, a total of 23,487 persons were evaluated by PRDH and CDC Dengue Branch for Zika virus infection, including asymptomatic pregnant women and persons with signs or symptoms consistent with Zika virus disease or suspected GBS; 5,582 (24%) confirmed and presumptive Zika virus cases were identified. Persons with Zika virus infection were residents of 77 (99%) of Puerto Rico's 78 municipalities. During 2016, the percentage of positive Zika virus infection cases among symptomatic males and nonpregnant females who were tested increased from 14% in February to 64% in June. Among 9,343 pregnant women tested, 672 had confirmed or presumptive Zika virus infection, including 441 (66%) symptomatic women and 231 (34%) asymptomatic women. One patient died after developing severe thrombocytopenia (4). Evidence of Zika virus infection or recent unspecified flavivirus infection was detected in 21 patients with confirmed GBS. The widespread outbreak and accelerating increase in the number of cases in Puerto Rico warrants intensified vector control and personal protective behaviors to prevent new infections, particularly among pregnant women.