Zika virus (ZIKV) causes mostly asymptomatic infection or mild febrile illness. However, with an increasing number of patients, various clinical features such as microcephaly, Guillain-Barré syndrome and thrombocytopenia have also been reported. To determine which host factors are related to pathogenesis, the E protein of ZIKV was analyzed with the Informational Spectrum Method, which identifies common information encoded by primary structures of the virus and the respective host protein. The data showed that the ZIKV E protein and the complement component C1q cross-spectra are characterized by a single dominant peak at the frequency F = 0.338, suggesting similar biological properties. Indeed, C1q-specific antibodies were detected in sera obtained from mice and monkeys infected with ZIKV. As C1q has been known to be involved not only in immunity, but also in synaptic organization and different autoimmune diseases, a ZIKV-induced anti-C1q antibody response may contribute to the neurological complications. These findings might also be exploited for the design of safe and efficacious vaccines in the future.

Although live attenuated vaccines (LAVs) have been effective in the control of flavivirus infections, to date they have been excluded from Zika virus (ZIKV) vaccine trials due to safety concerns. We have previously reported two ZIKV mutants, each of which has a single substitution in either envelope (E) glycosylation or nonstructural (NS) 4B P36 and displays a modest reduction in mouse neurovirulence and neuroinvasiveness, respectively. Here, we generated a ZIKV mutant, ZE4B-36, which combines mutations in both E glycosylation and NS4B P36. The ZE4B-36 mutant is stable and attenuated in viral replication. Next-generation sequence analysis showed that the attenuating mutations in the E and NS4B proteins are retained during serial cell culture passages. The mutant exhibits a significant reduction in neuroinvasiveness and neurovirulence and low infectivity in mosquitoes. It induces robust ZIKV-specific memory B cell, antibody, and T cell-mediated immune responses in type I interferon receptor (IFNR) deficient mice. ZIKV-specific T cell immunity remains strong months post-vaccination in wild-type C57BL/6 (B6) mice. Vaccination with ZE4B-36 protects mice from ZIKV-induced diseases and vertical transmission. Our results suggest that combination mutations in E glycosylation and NS4B P36 contribute to a candidate LAV with significantly increased safety but retain strong immunogenicity for prevention and control of ZIKV infection.

Currently, the laboratory diagnosis of Zika virus (ZIKV) infection is primarily through the detection of ZIKV RNA or antibodies against ZIKV proteins. The detection of viral RNA is highly sensitive and specific, but periods of viremia and viruria are brief, limiting the utility of ZIKV RNA assays. Instead, most ZIKV infections are diagnosed serologically, using an IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for screening, followed by a confirmatory plaque reduction neutralization test (PRNT). Typical turnaround times vary, due to assay incubation periods and a lack of clinical laboratories performing these tests. Recently, a novel luciferase-ZIKV- and -dengue virus (DENV)-based serological assay, which considerably improves the turnaround times and throughput for ZIKV diagnosis, was described. Using the traditional PRNT as a reference method, we evaluated the performance characteristics of the reporter virus neutralization test (RVNT) with 258 clinical serum specimens. The ZIKV RVNT produced primary ZIKV screening and secondary confirmation results in 4 days, with 100% reproducibility. As a screening assay, the ZIKV RVNT displayed excellent diagnostic accuracy, sensitivity, and specificity of 98.2%, 100%, and 98.1%, respectively. As a confirmatory assay, the ZIKV RVNT titers displayed 93.1% agreement with the traditional ZIKV PRNT titers. Overall, the RVNT accurately and reliably detects neutralizing antibodies in patient serum specimens, with improved turnaround times, and can be used for the serological detection of ZIKV infections. Due to the homogeneous 96-well format, the RVNT has also significantly improved the assay throughput to allow testing of a large number of specimens in a single run.

The explosive emergence of Zika virus has inspired a global effort to develop vaccines. Zika virus, which is a flavivirus primarily transmitted by mosquitoes, can cause devastating congenital syndrome in fetuses of pregnant women, including microcephaly, craniofacial disproportion, spasticity, ocular abnormalities, and miscarriage. In adults, Zika infection has been linked to the autoimmune disorder Guillain-Barré syndrome. Thus, despite the current waning in newly reported Zika infections, an efficacious vaccine is urgently needed to help limit the emergence of another detrimental epidemic. Here we summarize the current status of the Zika vaccine pipeline and highlight the challenges for clinical efficacy trials.

The current epidemic of Zika virus (ZIKV) has underscored the urgency to establish experimental systems for studying viral replication and pathogenesis, and countermeasure development. Here we report two ZIKV replicon systems: a luciferase replicon that can differentiate between viral translation and RNA synthesis; and a stable luciferase replicon carrying cell line that can be used to screen and characterize inhibitors of viral replication. The transient replicon was used to evaluate the effect of an NS5 polymerase mutation on viral RNA synthesis and to analyze a known ZIKV inhibitor. The replicon cell line was developed into a 96-well format for antiviral testing. Compare with virus infection-based assay, the replicon cell line allows antiviral screening without using infectious virus. Collectively, the replicon systems have provided critical tools for both basic and translational research.

Zika virus (ZIKV) infection of the mother during pregnancy causes devastating Zika congenital syndrome in the offspring. A ZIKV vaccine with optimal safety and immunogenicity for use in pregnant women is critically needed. Toward this goal, we have developed a single-dose live-attenuated vaccine candidate that infects cells with controlled, limited infection rounds. The vaccine contains a 9-amino-acid deletion in the viral capsid protein and replicates to titers of > 106 focus-forming units (FFU)/mL in cells expressing the full-length capsid protein. Immunization of A129 mice with one dose (105 FFU) did not produce viremia, but elicited protective immunity that completely prevented viremia, morbidity, and mortality after challenge with an epidemic ZIKV strain (106 PFU). A single-dose vaccination also fully prevented infection of pregnant mice and maternal-to-fetal transmission. Intracranial injection of the vaccine (104 FFU) to 1-day-old mice did not cause any disease or death, underscoring the safety of this vaccine candidate.

Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts.

Current efforts to develop Zika virus (ZIKV) subunit vaccines have been focused on pre-membrane (prM) and envelope (E) proteins, but the role of NS1 in ZIKV-specific immune response and protection is poorly understood. Here, we develop an attenuated recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing ZIKV prM-E-NS1 as a polyprotein. This vectored vaccine candidate is attenuated in mice, where a single immunization induces ZIKV-specific antibody and T cell immune responses that provide protection against ZIKV challenge. Co-expression of prM, E, and NS1 induces significantly higher levels of Th2 and Th17 cytokine responses than prM-E. In addition, NS1 alone is capable of conferring partial protection against ZIKV infection in mice even though it does not induce neutralizing antibodies. These results demonstrate that attenuated rVSV co-expressing prM, E, and NS1 is a promising vaccine candidate for protection against ZIKV infection and highlights an important role for NS1 in ZIKV-specific cellular immune responses.

An important goal of the Zika virus (ZIKV) vaccine is to prevent a congenital syndrome in fetuses of pregnant women, but studies directly evaluating maternal vaccination for ZIKV are lacking. Here we report maternal vaccination using a live-attenuated ZIKV vaccine (3'UTR-∆10-LAV) in a pregnant mouse model. Maternal immunization with 3'UTR-∆10-LAV does not cause any adverse effects on pregnancy, fetal development, or offspring behavior. One maternal immunization fully protects dams against ZIKV infection and in utero transmission. Although neutralizing antibody alone is sufficient to prevent in utero transmission, a higher neutralizing titer is required to protect pregnant mice against in utero transmission than that required to protect non-pregnant mice against viral infection. The immunized dams transfer maternal antibodies to pups, which protect neonates against ZIKV infection. Notably, pregnancy weakens maternal T cell response to 3'UTR-∆10-LAV vaccination. Our results suggest that, besides vaccinating non-pregnant individuals, 3'UTR-∆10-LAV may also be considered for maternal vaccination.

Zika virus (ZIKV) remained obscure until the recent explosive outbreaks in French Polynesia (2013-2014) and South America (2015-2016). Phylogenetic studies have shown that ZIKV has evolved into African and Asian lineages. The Asian lineage of ZIKV was responsible for the recent epidemics in the Americas. However, the underlying mechanisms through which ZIKV rapidly and explosively spread from Asia to the Americas are unclear. Non-structural protein 1 (NS1) facilitates flavivirus acquisition by mosquitoes from an infected mammalian host and subsequently enhances viral prevalence in mosquitoes. Here we show that NS1 antigenaemia determines ZIKV infectivity in its mosquito vector Aedes aegypti, which acquires ZIKV via a blood meal. Clinical isolates from the most recent outbreak in the Americas were much more infectious in mosquitoes than the FSS13025 strain, which was isolated in Cambodia in 2010. Further analyses showed that these epidemic strains have higher NS1 antigenaemia than the FSS13025 strain because of an alanine-to-valine amino acid substitution at residue 188 in NS1. ZIKV infectivity was enhanced by this amino acid substitution in the ZIKV FSS13025 strain in mosquitoes that acquired ZIKV from a viraemic C57BL/6 mouse deficient in type I and II interferon (IFN) receptors (AG6 mouse). Our results reveal that ZIKV evolved to acquire a spontaneous mutation in its NS1 protein, resulting in increased NS1 antigenaemia. Enhancement of NS1 antigenaemia in infected hosts promotes ZIKV infectivity and prevalence in mosquitoes, which could have facilitated transmission during recent ZIKV epidemics.

Reporter virus neutralization test (RVNT) has been used as an alternative to the more laborious and time-demanding conventional PRNT assay for both DENV and ZIKV. However, few studies have investigated how these techniques would perform in epidemic areas with the circulation of multiple flavivirus. Here, we evaluate the performance of ZIKV and DENV Rluc RVNT and ZIKV mCh RVNT assays in comparison to the conventional PRNT assay against patient sera collected before and during ZIKV outbreak in Brazil. These samples were categorized into groups based on (1) acute and convalescent samples according to the time of disease, and (2) laboratorial diagnostic results (DENV and ZIKV RT-PCR and IgM-capture ELISA). Our results showed that DENV Rluc assay presented 100% and 78.3% sensitivity and specificity, respectively, with 93.3% accuracy, a similar performance to the traditional PRNT. ZIKV RVNT90, on the other hand, showed much better ZIKV antibody detection performance (around nine-fold higher) when compared to PRNT, with 88% clinical sensitivity. Specificity values were on average 76.8%. Even with these results, however, ZIKV RVNT90 alone was not able to reach a final diagnostic conclusion for secondary infection in human samples due to flavivirus cross reaction. As such, in regions where the flavivirus differential diagnosis represents a challenge, we suggest the establishment of a RVNT panel including other flaviviruses circulating in the region, associated with the other serological techniques such as IgM ELISA and the investigation of seroconversion, in order to help define an accurate diagnostic conclusion using serology.

Zika virus (ZIKV), a mosquito-borne flavivirus, can cause severe eye disease and even blindness in newborns. However, ZIKV-induced retinal lesions have not been studied in a comprehensive way, mechanisms of ZIKV-induced retinal abnormalities are unknown, and no therapeutic intervention is available to treat or minimize the degree of vision loss in patients. Here, we developed a novel mouse model of ZIKV infection to evaluate its impact on retinal structure. ZIKV (20 plaque-forming units) was inoculated into neonatal wild type C57BL/6J mice at postnatal day (P) 0 subcutaneously. Retinas of infected mice and age-matched controls were collected at various ages, and retinal structural alterations were analyzed. We found that ZIKV induced progressive neuronal and vascular damage and retinal inflammation starting from P8. ZIKV-infected retina exhibited dramatically decreased thickness with loss of neurons, initial neovascular tufts followed by vessel dilation and degeneration, increased microglia and leukocyte recruitment and activation, degeneration of astrocyte network and gliosis. The above changes may involve inflammation and endoplasmic reticulum stress-mediated cell apoptosis and necroptosis. Moreover, we evaluated the efficacy of preclinical drugs and the safety of ZIKV vaccine candidate in this mouse model. We found that ZIKV-induced retinal abnormalities could be blocked by a selective flavivirus inhibitor NITD008 and a live-attenuated ZIKV vaccine candidate could potentially induce retinal abnormalities. Overall, we established a novel mouse model and provide a direct causative link between ZIKV and retinal lesion in vivo, which warrants further investigation of the underlying mechanisms of ZIKV-induced retinopathy and the development of effective therapeutics.

Virus-host interactions determine an infection outcome. The Asian lineage of Zika virus (ZIKV), responsible for the recent epidemics, has fixed a mutation in the NS1 gene after 2012 that enhances mosquito infection. Here we report that the same mutation confers NS1 to inhibit interferon-β induction. This mutation enables NS1 binding to TBK1 and reduces TBK1 phosphorylation. Engineering the mutation into a pre-epidemic ZIKV strain debilitates the virus for interferon-β induction; reversing the mutation in an epidemic ZIKV strain invigorates the virus for interferon-β induction; these mutational effects are lost in IRF3-knockout cells. Additionally, ZIKV NS2A, NS2B, NS4A, NS4B, and NS5 can also suppress interferon-β production through targeting distinct components of the RIG-I pathway; however, for these proteins, no antagonistic difference is observed among various ZIKV strains. Our results support the mechanism that ZIKV has accumulated mutation(s) that increases the ability to evade immune response and potentiates infection and epidemics.

Compared with other flaviviruses, Zika virus (ZIKV) is uniquely associated with congenital diseases in pregnant women. One recent study reported that (i) ZIKV has higher thermostability than dengue virus (DENV [a flavivirus closely related to ZIKV]), which might contribute to the disease outcome; (ii) the higher thermostability of ZIKV could arise from an extended loop structure in domain III of the viral envelope (E) protein and an extra hydrogen-bond interaction between E molecules (V. A. Kostyuchenko, E. X. Y. Lim, S. Zhang, G. Fibriansah, T.-S. Ng, J. S. G. Ooi, J. Shi, and S.-M. Lok, Nature 533:425-428, 2016, https://doi.org/10.1038/nature17994). Here we report the functional analysis of the structural information in the context of complete ZIKV and DENV-2 virions. Swapping the prM-E genes between ZIKV and DENV-2 switched the thermostability of the chimeric viruses, identifying the prM-E proteins as the major determinants for virion thermostability. Shortening the extended loop of the E protein by 1 amino acid was lethal for ZIKV assembly/release. Mutations (Q350I and T351V) that abolished the extra hydrogen-bond interaction between the E proteins did not reduce ZIKV thermostability, indicating that the extra interaction does not increase the thermostability. Interestingly, mutant T351V was attenuated in A129 mice defective in type I interferon receptors, even though the virus retained the wild-type thermostability. Furthermore, we found that a chimeric ZIKV with the DENV-2 prM-E and a chimeric DENV-2 with the ZIKV prM-E were highly attenuated in A129 mice; these chimeric viruses were highly immunogenic and protective against DENV-2 and ZIKV challenge, respectively. These results indicate the potential of these chimeric viruses for vaccine development.

Glioblastoma (GBM) is the deadliest type of brain tumor, and glioma stem cells (GSCs) contribute to tumor recurrence and therapeutic resistance. Thus, an oncolytic virus targeting GSCs may be useful for improving GBM treatment. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we investigated the safety and efficacy of a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a GSC-derived orthotopic model. Intracerebral injection of ZIKV-LAV into mice caused no neurological symptoms or behavioral abnormalities. The neurovirulence of ZIKV-LAV was more attenuated than that of the licensed Japanese encephalitis virus LAV 14-14-2, underlining the superior safety of ZIKV-LAV for potential GBM treatment. Importantly, ZIKV-LAV significantly reduced intracerebral tumor growth and prolonged animal survival by selectively killing GSCs within the tumor. Mechanistically, ZIKV infection elicited antiviral immunity, inflammation, and GSC apoptosis. Together, these results further support the clinical development of ZIKV-LAV for GBM therapy.IMPORTANCE Glioblastoma (GBM), the deadliest type of brain tumor, is currently incurable because of its high recurrence rate after traditional treatments, including surgery to remove the main part of the tumor and radiation and chemotherapy to target residual tumor cells. These treatments fail mainly due to the presence of a cell subpopulation called glioma stem cells (GSCs), which are resistant to radiation and chemotherapy and capable of self-renewal and tumorigenicity. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we tested a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a human GSC-derived orthotopic model. Our results showed that ZIKV-LAV retained good efficacy against glioblastoma by selectively killing GSCs within the tumor. In addition, ZIKV-LAV exhibited an excellent safety profile upon intracerebral injection into the treated animals. The good balance between the safety of ZIKV-LAV and its efficacy against human GSCs suggests that it is a potential candidate for combination with the current treatment regimen for GBM therapy.

Zika virus infection during pregnancy can cause congenital abnormities or fetal demise. The persistence of Zika virus in the male reproductive system poses a risk of sexual transmission. Here we demonstrate that live-attenuated Zika virus vaccine candidates containing deletions in the 3' untranslated region of the Zika virus genome (ZIKV-3'UTR-LAV) prevent viral transmission during pregnancy and testis damage in mice, as well as infection of nonhuman primates. After a single-dose vaccination, pregnant mice challenged with Zika virus at embryonic day 6 and evaluated at embryonic day 13 show markedly diminished levels of viral RNA in maternal, placental, and fetal tissues. Vaccinated male mice challenged with Zika virus were protected against testis infection, injury, and oligospermia. A single immunization of rhesus macaques elicited a rapid and robust antibody response, conferring complete protection upon challenge. Furthermore, the ZIKV-3'UTR-LAV vaccine candidates have a desirable safety profile. These results suggest that further development of ZIKV-3'UTR-LAV is warranted for humans.Zika virus infection can result in congenital disorders and cause disease in adults, and there is currently no approved vaccine. Here Shan et al. show that a single dose of a live-attenuated Zika vaccine prevents infection, testis damage and transmission to the fetus during pregnancy in different animal models.

Zika virus (ZIKV) and Japanese encephalitis virus (JEV) are mosquito-borne flaviviruses with sequence homology. ZIKV circulates in some regions where JEV also circulates, or where JE vaccination is used. Cross-immunity between flaviviruses exists, but the precise mechanisms remain unclear. We previously demonstrated that T cell immunity induced by the live-attenuated Japanese encephalitis (JE) SA14-14-2 vaccine conferred protective immunity against ZIKV infection in mice, which could even bypass antibody-dependent enhancement. However, the role of T cell immune, especially memory T cell subsets, in cross-reactive immune responses between JE vaccine and ZIKV in humans has not been reported.

Global Zika virus (ZIKV) outbreaks and their link to microcephaly have raised major public health concerns. However, the mechanism of maternal-fetal transmission remains largely unknown. In this study, we determined the role of yolk sac (YS) microglial progenitors in a mouse model of ZIKV vertical transmission. We found that embryonic (E) days 6.5-E8.5 were a critical window for ZIKV infection that resulted in fetal demise and microcephaly, and YS microglial progenitors were susceptible to ZIKV infection. Ablation of YS microglial progenitors significantly reduced the viral load in both the YS and the embryonic brain. Taken together, these results support the hypothesis that YS microglial progenitors serve as "Trojan horses," contributing to ZIKV fetal brain dissemination and congenital brain defects.

Global Zika virus (ZIKV) outbreaks and their strong link to microcephaly have raised major public health concerns. ZIKV has been reported to affect the innate immune responses in neural stem/progenitor cells (NS/PCs). However, it is unclear how these immune factors affect neurogenesis. In this study, we used Asian-American lineage ZIKV strain PRVABC59 to infect primary human NS/PCs originally derived from fetal brains. We found that ZIKV overactivated key molecules in the innate immune pathways to impair neurogenesis in a cell stage-dependent manner. Inhibiting the overactivated innate immune responses ameliorated ZIKV-induced neurogenesis reduction. This study thus suggests that orchestrating the host innate immune responses in NS/PCs after ZIKV infection could be promising therapeutic approach to attenuate ZIKV-associated neuropathology.

The Asian lineage of Zika virus (ZIKV) has recently caused epidemics and severe disease. Unraveling the mechanisms causing increased viral transmissibility and disease severity requires experimental systems. We report an infectious cDNA clone of ZIKV that was generated using a clinical isolate of the Asian lineage. The cDNA clone-derived RNA is infectious in cells, generating recombinant ZIKV. The recombinant virus is virulent in established ZIKV mouse models, leading to neurological signs relevant to human disease. Additionally, recombinant ZIKV is infectious for Aedes aegypti and thus provides a means to examine virus transmission. The infectious cDNA clone was further used to generate a luciferase ZIKV that exhibited sensitivity to a panflavivirus inhibitor, highlighting its potential utility for antiviral screening. This ZIKV reverse genetic system, together with mouse and mosquito infection models, may help identify viral determinants of human virulence and mosquito transmission as well as inform vaccine and therapeutic strategies.