Reserve genetic approaches for the development of Zika vaccines and therapeutics

SUMMARY

In 2015–2016, the little known Zika virus (ZIKV) caused an epidemic in which it became recognized as a unique human pathogen associated with a range of devastating congenital abnormalities collectively categorized as congenital Zika syndrome (CZS). In adults, the virus can trigger the autoimmune disorder Guillain-Barré syndrome (GBS), characterized by ascending paralysis. In February 2016, the World Health Organization (WHO) declared ZIKV to be a Public Health Emergency of International Concern. The global public health problem prompted academia, industry, and governments worldwide to initiate development of an effective vaccine to prevent another ZIKV epidemic that would put millions at risk. The development of reverse genetic systems for the study and manipulation of RNA viral genomes has revolutionized the field of virology, providing platforms for vaccine and antiviral development. In this review, we discuss the impact of reverse genetic systems on the rapid progress of ZIKV vaccines and antiviral therapeutics.

INTRODUCTION

The Flaviviridae family, genus Flavivirus, includes several medically important pathogens that cause human disease, such as Zika virus (ZIKV), dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne-encephalitis virus (TBEV) (1). ZIKV emerged at a global level after being linked to microcephaly during the 2015–2016 epidemic in South America (2). Flaviviruses have a single-strand, positive-sense RNA genome that encodes three structural proteins (capsid [C], precursor membrane [prM], and envelope [E]) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The structural proteins and viral genomic RNA form viral particles, while the nonstructural proteins are involved in replication, assembly, and immune evasion (3).

ZIKV was initially isolated in 1947 in the Zika Forest of Uganda from a sentinel rhesus monkey (2). Prior to a 2007 outbreak, ZIKV circulation was restricted to Africa and Asia, causing a limited number of human infections, with symptoms described as asymptomatic or mild (4). The first major outbreak of ZIKV occurred in Micronesia in 2007. By 2013, the virus had spread to French Polynesia, where neurological complications like Guillain-Barré syndrome were first noted. Subsequently, ZIKV disseminated to the Americas, where it was initially identified in the northeast of Brazil (5). Startling reports showed an increase of children born with microcephaly in the areas affected by ZIKV. Soon thereafter, the virus was discovered in the amniotic fluid of pregnant women whose fetuses presented with ultrasound-detected microcephaly (6). From 2015 to 2018, ZIKV spread rapidly; more than 800,000 human cases were reported in the Americas (7). ZIKV is mainly transmitted through the bite of infected Aedes aegypti mosquitoes (8); however, the virus can also be acquired through sexual contact, vertical transmission, blood transfusion, and organ transplantation (9). During the 2015–2016 epidemic ZIKV exhibited an enhanced potential to infect millions of people, and despite the high rate of asymptomatic infections (~80%) (10), the devastating congenital syndrome associated with ZIKV infection has underscored the importance of countermeasure development (11**). Therefore, the creation of a safe and efficacious vaccine is urgently needed. Since the epidemic, a number of platforms have been rapidly used for ZIKV vaccine development, including DNA, RNA, viral vector, chimeric flavivirus, and live-attenuated vaccine platforms (12). Among these technologies, advances like reverse genetic systems have led to a rapid research response to the ZIKV epidemic including the swift initiation of vaccine and therapeutic development (13*, 14**).

Reverse genetics describes a technique for manipulating the RNA genome of a virus. It begins with the reverse transcription of the viral RNA to cDNA. The cDNA of the complete viral genome is then cloned into a single plasmid. Such a plasmid allows for mutations to be introduced at any location of the genome (15). The engineered DNA is then in vitro transcribed to generate an RNA transcript, which is then transfected into susceptible cells. Engineering a 5’ eukaryotic promoter in place of a bacterial promoter is also common (16). Since the flavivirus genome is positive sense, the transfected RNA is directly translated to make viral proteins, which initiate viral replication, and generate infectious virus in cells (17). Reverse genetic systems are routinely used to make infectious viral particles and to manipulate the viral genome by the introduction of mutations, insertions, and deletions. Shan, et al. developed the first infectious cDNA clone of ZIKV as well as the first luciferase reporter ZIKV (14**). A low-copy plasmid strategy was used to overcome the problem of bacterial instability. Other reverse genetic strategies involve segmenting the genome into smaller plasmids and then combining them by either self-splicing ribozyme (18), Gibson assembly (19), or type IIS restriction enzyme sites (20). Other successful approaches involve in silico prediction of, and removal of, cryptic promoters (21) and the bacterial free approaches of circular polymerase extension reaction (22) and infectious subgenomic amplicons (23). A non-infectious ZIKV replicon with deletion of viral structural genes has also been developed (13*). These genetic systems of ZIKV have provided a tractable platform to develop vaccines, define genetic changes, characterize structural and nonstructural genes, and map determinants for virulence, vector competence, and therapeutic targets (24–27).

PREVENTIVE VACCINATION

Vaccination has proven to be the most effective strategy for the prevention of infectious diseases, like ZIKV (28). Reverse genetics technology has played a role in the development of five types of ZIKV vaccine candidates: (I) conventional live-attenuated vaccines (LAV), (II) DNA-launched LAV, (III) chimeric flavivirus vaccines, (IV) virus-like particles (VLP), (V) and other virus-based vectors (Fig 1). LAVs are based on the engineering of mutations that confer attenuated viral replication, but retain the ability to induce durable protective immunity after a single dose vaccination (12). DNA vaccines expressing single or multiple antigens have the advantage of rapid and cost-effective production by eliminating cell culture and cold chain requirements. However, multiple doses are typically required due to poor immunogenicity in non-human primates (NHP) and humans. As detailed below, a DNA-launched LAV was developed to combine the strengths of conventional LAV and DNA vaccination (29**). ZIKV chimeric vaccine approaches have employed the use of a licensed flavivirus LAV vaccine as a backbone to express ZIKV structural genes (30). VLPs are non-infectious particles that morphologically resemble the live virus. VLPs have been shown to elicit a protective immune response against flavivirus infection (31). The rapid progress of multiple vaccine candidates has been enabled by previous expertise in flavivirus vaccinology (28).

Figure 1. Zika virus reverse genetics and vaccine platforms.

Zika vaccine categories based on a full-length cDNA clone of ZIKV fall into five main categories: live-attenuated virus (LAV) vaccines, DNA vaccines, chimeric virus vaccines, virus-like particle (VLP) vaccines, and vaccines based on other viral vectors. LAV vaccines are live, replicating viruses that are attenuated through mutations in the genome (denoted by an asterisk). DNA vaccines take two forms: eukaryotic promoter-driven LAVs or eukaryotic promoter-driven viral proteins. Chimeric virus vaccines use previously licensed flavivirus LAVs to express ZIKV prME. VLPs are prME proteins that self-assemble into empty virions. Other viruses can also be used to express ZIKV prME, including VSV, as is represented in cartoon.

(I). Conventional Live-Attenuated Vaccine (LAV).

LAVs are designed to mimic the natural viral infection and, as such, often require only a single dose immunization and induce rapid and durable immunity, which is of practical importance since ZIKV is endemic in developing countries (32*). The most devastating manifestations of ZIKV are the array of fetal abnormalities, jointly termed Congenital Zika Syndrome, found in babies born to infected pregnant women. Furthermore, ZIKV infection can persist in the male reproductive tract inducing damage to the testes. The optimal vaccine will need to prevent infection in reproductive tissues and establish protection in the context of pregnancy (33). In addition to the regular vaccine safety requirements, due to the infectious nature of LAV, these candidates should bear no risk of transmission via mosquitoes (34).

An infectious full-length cDNA clone was used to develop three types of ZIKV LAV candidates (14**): ZIKV-NS1-LAV, C-deletion LAV, and ZIKV-3’UTR-Δ10. The Cambodian strain FSS13025 was selected for LAV development because it is a pre-epidemic strain without replication- and virulence-enhancing mutations (24–27). For the ZIKV-NS1-LAV, mutations were engineered in the NS1 gene to abolish its N-glycosylation (11**). NS1 glycosylation was previously shown to play an important role in viral replication (35) and this knowledge guided the rational design of this vaccine. After a single dose vaccination, the ZIKV-NS1-LAV showed efficacy in A129 mice with a strong antibody response (neutralizing titer of 1:7000) and no detectable viremia after challenge. The glycosylation mutant LAV was shown to protect both pregnant C57BL/6 mice and their fetuses from ZIKV infection (11**). The ZIKV-NS1-LAV virus was also not infectious in mosquitoes. Surprisingly, non-human primates (NHPs) vaccinated with ZIKV-NS1-LAV did not develop protective, neutralizing titers. It is not currently known what factors contribute to the discrepancy between the mouse and NHP studies.

The C-deletion LAV was made by introducing a nine-amino acid deletion in the viral capsid (C) protein, resulting in a LAV that can infect cells with controlled, limited infection rounds. These viruses carry functional capsid proteins as part of the virion, but encode a defective C protein in their genome, allowing only one round of productive infection. Virions released following this first infection are infection competent but fail to form more infectious viruses. This restricted infection strategy shows improved safety compared with the conventional LAV, which is particularly important when vaccinating pregnant women and immunocompromised individuals. The C-deletion LAV was protective and immunogenic in both non-pregnant and pregnant A129 mice, the latter model showing full protection against maternal-to-fetal transmission (32*).

The ZIKV-3’UTR-Δ10 vaccine strain was engineered by deleting a 10-nucleotide stretch in the 3’ untranslated region (3’UTR) of the ZIKV genome. The ZIKV-3’UTR-Δ10 virus is highly attenuated, immunogenic, and protective in mice and nonhuman primates (34, 36**, 37). It prevented both transmission during pregnancy and damage of the testes in A129 mice, and is not competent in a mosquito host (34, 37). Furthermore, ZIKV-3’UTR-Δ10 showed single-dose efficacy when used in maternal vaccination (i.e., vaccination during pregnancy), providing protection during pregnancy while not causing fetal abnormalities. Importantly, the three LAVs discussed above exhibited excellent safety profiles, showing less neurovirulence than licensed flavivirus LAVs such as YF17D and JEV SA14–14-2 (11**, 12, 32*, 34, 36**, 38).

(II). DNA-launched LAV.

Standard DNA vaccines have the advantage of chemical stability, simple manufacturing, no cold chain requirement, and low cost. These candidates typically consist of a plasmid encoding one or more viral protein(s) under control of a constitutive, eukaryotic promoter. However, DNA vaccines (expressing single or multiple antigens) usually require multiple doses and/or periodic boosting (12). Since ZIKV is endemic in developing countries, immunizing populations in remote areas with multiple doses renders the DNA vaccination approach less practical and more challenging. In contrast, as described in (I), LAVs, which lack the shipping and shelf stability of DNA vaccines, have the advantage of a single dose eliciting rapid, durable protection. Zou, et al. developed a DNA-launched LAV that combines the strengths of both LAV and DNA vaccine platforms by engineering a eukaryotic promoter upstream of the full-length cDNA copy of an attenuated ZIKV encoded on a plasmid. The DNA-launched LAV candidate contains a 20-nucleotide deletion within the 3’UTR of the ZIKV genome (ZIKV-3’UTR-Δ20) and is administered by injection and electroporation of the DNA plasmid. A previous study showed that a single dose of ZIKV-3’UTR-Δ20 in the conventional format (i.e. attenuated live virus) conferred sterilizing immunity and had an excellent safety profile in both A129 mouse (36**) and NHP models (37). The balance between efficacy and safety made the ZIKV-3’UTR-Δ20 an ideal candidate to be converted into a plasmid DNA-launched LAV, Pzikv-3’UTR-Δ20. Remarkably, a single-dose immunization of 0.5 μg DNA-LAV plasmid by intramuscular injection with electroporation (IM & EP) conferred 100% protective immunity with high neutralizing antibody titers (about 1:10,000) in A129 mice. After challenge with WT ZIKV, no increase in neutralizing antibody titers was observed in the vaccinated animals, suggesting that the vaccination had elicited sterilizing immunity. Additionally, immunization prevented maternal-to-fetal transmission and testis damage in A129 mouse models (29**). The single dose of 0.5 μg DNA required for 100% seroconversion for the pZIKV-3’UTR-Δ20 is much more potent than the 50 μg dose required for the DNA subunit vaccine (expressing ZIKV prM-E genes) in BALB/c mice (39**, 40). The DNA subunit vaccine recently completed a Phase II trial, with results still pending (NCT03110770).

(III). Chimeric Virus Vaccines.

The high safety and efficacy in humans of the licensed YFV vaccine strain 17D (YF17D) and JE vaccine virus SA14–14-2 (SA14–14-2) make them an ideal genetic backbone for chimeric constructs (41). The prM and E genes of these vaccine viruses, the most antigenic flavivirus proteins, can be interchanged with other flaviviruses and still give viable virus. The YF17D backbone-based chimeric approach has been successfully used to develop the licensed dengue vaccine Dengvaxia (42, 43) and Japanese encephalitis vaccine Imojev (44) as well as a WNV vaccine candidate (44, 45). Using reverse genetic techniques, chimeric ZIKV vaccines were generated using YF17D and SA14–14-2 to express ZIKV prM-E genes. A single immunization with either chimeric SA14–14-2 or YF17D harboring ZIKV prM-E elicited a strong antibody response. The immunized immunodeficient and immunocompetent mice were both protected from ZIKV infection (41, 46, 47*) or maternal-to-fetal transmission in mice (41, 47*). The SA14–14-2 chimeric ZIKV vaccine also showed efficacy in NHPs (41). As chimeric viruses are ordinarily more attenuated than their parental viruses, it is not surprising that both SA14–14-2 and YF17D chimeric ZIKV vaccine candidates exhibited lower neurovirulence than SA14–14-2 and YF17D, respectively (41, 46–48).

Along with previously licensed vaccine viruses as backbones, Hobson-Peters, et al. isolated an insect-specific flavivirus, Binjari virus (BinJV), and used reverse genetics to create BinJV backbone WNV, ZIKV, DENV1,2, and 4, and YFV prME chimeric viruses. These viruses are incapable of replicating in mammalian cells and as such are considered very safe. An immunization with 20 μg of BinJ/ZIKV-prME protected A129 mice from challenge with a contemporary ZIKV strain (49). Despite being a live virus, as a vaccine strategy it resembles more closely an inactivated vaccine because of its inability to infect mammalian cells. This could explain the large amount of material (20 μg) needed for efficient vaccination.

(IV). Virus-like particles (VLP).

VLPs are produced in cells engineered to express the viral prM-E proteins. The viral proteins self-assemble into particles that resemble noninfectious empty virus produced during viral infection (50). The use of VLPs recently emerged as a powerful technology for vaccines due to their excellent safety profile (because they’re noninfectious), ability to generate an effective immune response, and cost-effective scale-up production (13*). Garg, et al. developed two approaches to create prM-E and C-prM-E-based VLP vaccines. The first approach used a stable cell line that constitutively expressed ZIKV prM-E protein, leading to secretion of extracellular VLPs. The second approach co-transfected cells with two DNA plasmids: one plasmid expressing ZIKV C-prM-E and another plasmid expressing viral NS2B-3 protease to mediate cleavage between C and prM. The second approach seemed to generate a higher yield of VLPs than the first approach. Nevertheless, the VLPs produced from both approaches exhibited efficacy as vaccines, with neutralizing antibody titers of >1:1,000 (reporter virus particle-based microneutralization assay) in BALB/c mice (51).

(V). Other Viral-vectored Vaccines.

Combinations of reverse genetics for ZIKV and other viruses have yielded vaccine candidates using diverse viruses that carry and express ZIKV genes and proteins. These platforms have the advantage of presenting whole viral particles to the immune system, which can help stimulate a more robust immune response when compared to subunit vaccines. Multiple adenovirus-vectored vaccines, from rhesus macaque, chimpanzee, and human lineages, have been developed for ZIKV. Recombinant rhesus adenovirus serotype 52 (RhAd52) expressing prME was completely protective in NHPs after immunization with a single 10¹¹ dose and showed a better cellular immune response than a DNA-launched prME vaccine (52). A chimpanzee adenovirus type 7 carrying ZIKV M and E genes (AdC7-M/E) elicited a robust antibody and T cell response after single immunization in IFNAR1^−/− mice and protected them from viremia and testis damage after challenge (53). Human adenovirus type 26, carrying ZIKV M and E genes, has also been tested as a vaccine candidate due to its ability to grow to high titers and protect immunocompetent mice and monkeys from viremia after challenge (54).

Other viral vectors include vesicular stomatitis virus (VSV) and measles virus. VSV-based vaccines have been engineered to express prME and prME-NS1 polyproteins. The first of these constructs elicited both humoral and cellular immunity and protected ~50% of the offspring born to previously vaccinated female C57BL/6 mice after challenge (55). VSV-prM-E-NS1 vaccine viruses induced protective levels of ZIKV-specific antibodies and cellular responses in BALB/c mice. Notably, the VSV-prM-E-NS1 virus stimulated larger Th2 and Th17 responses than VSV-prM-E virus (56). The Schwarz measles virus vaccine strain was engineered to express ZIKV prME and a prime-boost immunization strategy with this virus led to reduced viremia and protection of offspring in vaccinated IFNAR^−/− female mice (57). Together, these diverse viral platforms reflect the utility of reverse genetics in developing vaccine candidates.

ZIKV AS A THERAPEUTIC AGENT FOR GLIOBLASTOMA TREATMENT

Oncolytic viruses are emerging as promising cancer therapeutic options (58). Glioblastoma (GBM) is the most common and malignant form of primary brain tumor and despite aggressive treatment with surgery, radiation, and chemotherapy, GBM remains the most lethal of all human cancers (59*, 60). Thus, novel treatment options are urgently needed. ZIKV’s ability to cause microcephaly by killing neural progenitor cells (NPCs) in the fetus raised the possibility of its utility as an oncolytic virus to target glioblastoma stem cells (GSCs) (61). GSCs have self-renewal, tumorigenic and differentiation potential, and thus are similar to NPCs. Zhu, et al. first demonstrated that ZIKV selectively kills human GSCs in vitro (59*). This was followed by Chen, et al. testing ZIKV-3’UTR-Δ10’s oncolytic activity against human GSCs (60). These results showed that ZIKV-3’UTR-Δ10, a safer strain than WT ZIKV, retained the oncolytic activity against patient-derived GSCs in vivo despite being significantly attenuated when compared to WT ZIKV, paving the way for clinical development of this vaccine candidate for GBM therapy (60).

ZIKA THERAPEUTICS

Although significant progress has been made towards ZIKV vaccine development, there is no licensed vaccine yet. This gap highlights the importance of developing therapeutics for ZIKV. Antiviral therapy could provide an important countermeasure to treat infected individuals, particularly in the case of pregnant women with fetuses at risk of congenital Zika syndrome (62). As detailed below, the reserve genetics system of ZIKV has been used to generate important tools for antiviral drug discovery, including reporter ZIKV and replicon-containing cell lines for compound library screening as well as inhibitor mode-of-action studies (Fig 2).

Figure 2. ZIKV reverse genetics and reporter viruses.

ZIKV based reporter constructs come in three main types: replicon, full-length reporter virus, and chimeric reporter virus. Replicon constructs have the reporter gene inserted in place of the structural genes and so form no infectious virions. Reporter genes for full-length reporter ZIKV are typically engineered before the polyprotein. Replicon systems can be combined with ZIKV structural proteins to make a chimeric, reporter, single round infectious particle.

Reporter viruses and replicons constructed from cDNA clones can facilitate the discovery of antiviral compounds against ZIKV (63). Luminescent and fluorescent protein-expressing viruses can significantly increase the throughput and quantification of samples, while concomitantly reducing the workload and turnaround time. The level of expression of the reporter gene is a surrogate measure of viral replication and can be used to quantify the inhibitory activities of drugs or antibodies (64). Reporter viruses and replicons have been used for high-throughput drug repurposing screening, compound library screening, diagnostic assays, and studies of viral entry, translation and replication (13*, 14**, 65, 66). Flavivirus replicons lack the viral structural C-prM-E genes, hence they are non-infectious and have a safety advantage over infectious reporter viruses. Infectious reporter viruses have the benefit of completing the whole viral life cycle and thus are more robust tools when a range of viral proteins are being targeted. Engineering a reporter gene to be stable after multiple rounds of infection has been a long-standing challenge for reporter flaviviruses. Recently, Volkova, et al. have elegantly developed a method to stabilize a reporter ZIKV. Reporter genes in flaviviruses are typically placed after the 5’ UTR, at the beginning of the viral ORF; however, RNA signals for translation and replication are continuous from the 5’UTR into the capsid gene. This requires duplication of part of the capsid gene. Volkova, et al. systematically tested different lengths of this duplicated capsid and its effect on viral replication, determining that 50 amino acids are sufficient for WT levels of viral replication. To block recombination between the two regions of duplicated capsid, they optimized the codons and added a frameshift mutation in the duplicated capsid portion. Thus, if recombination occurs, the capsid gene will be mistranslated, producing non-viable virus. Using this clever method, they established a reporter virus with stability greater than ten passages in cell culture. (67*) This method, translated to diagnostics and antiviral assays, can simplify large scale experiments and instill greater confidence in results.

Shan, et al. engineered a luciferase ZIKV when first publishing the ZIKV reverse genetic system (14**). When tested head-to-head against wild-type (without reporter) virus, comparable EC₅₀ values were obtained with a known pan-flavivirus inhibitor (nucleoside analog NITD008) (68). Additionally, this luciferase reporter virus led to the development of a high-throughput neutralization assay to replace the traditional plaque-reduction neutralization test (PRNT) to measure neutralization antibody levels (69, 70). Framunce, et al. developed an alternative to the conventional PRNT, a flow cytometry neutralization test (FNT), using a GFP reporter ZIKV. PRNT₅₀ and FNT₅₀ values revealed a linear relationship (R² = 0.90, p-value <0.0001), indicating a concordance between both methods based on a single infection round (71).

The risk of laboratory-acquired viral infection is eliminated when employing a replicon system as infectious particles are not generated. Replicons are useful tools to study ZIKV replication and to develop antiviral therapies (13*, 14**). Xie, et al. developed both a transient ZIKV replicon and a stable cell line carrying a ZIKV replicon. This system has been demonstrated to be useful for identifying ZIKV inhibitors, as shown by testing with a known inhibitor NITD008 (13*). Li, et al. developed and optimized a robust HTS assay using a stable ZIKV replicon cell line with a calculated Z’ factor of > 0.5 in all tested conditions (cell density, NITD008 concentration, and incubation time), indicating a good quality HTS replicon assay (72).

Additional, chimeric-based approaches have also been developed. Similar to vaccination, these have the advantage over VLPs in more authentic antigen presentation. In 2005, Pierson, et al. described the production of pseudo-infectious WNV reporter virus particles (RVPs) that allow rapid quantification of virus infection and its inhibition by neutralizing antibodies (73*). This approach has been modified to produce ZIKV RVPs by complementation of a GFP-expressing WNV replicon with a plasmid encoding the C-prM-E structural genes of ZIKV (51, 74). As an alternative to the traditional PRNT (70), the RVP-based neutralization assay reduces the biosafety concern as it does not require the use of live virus and offers a rapid and quantitative approach to measuring antibody titers (51, 73*, 74). The ZIKV RVP neutralization assay developed by Dowd, et al. has been used in the preclinical and clinical evaluation of several vaccine candidates (11**, 75). Additionally, RVPs offers an approach for measuring virus entry and could be used to screen for inhibitors of ZIKV entry (13*). Similarly, Matsuda, et al. made single round infectious DENV1 chimeric particles by trans-complementing various prME-containing plasmids with DENV1 C and a luciferase-containing DENV1 replicon. These were then used as a safer, more high-throughput alternative to neutralization tests (76). Together, these reporter virus strategies all represent improved methods for antiviral therapeutic discovery or, as is needed with vaccine trials or disease diagnosis, serum antibody quantification.

CONCLUSION

In the recent epidemics in Asia and the Americas, ZIKV infection has caused devastating disease, most notably Guillain-Barre syndrome in adults and congenital malformation in fetuses. Since 2016, ZIKV vaccine remains a global health priority as shown by the continued progress in ZIKV vaccine development. Several vaccine candidates have advanced to clinical trials: DNA vaccines, modified RNA vaccines, purified formalin inactivated virus (PIV), and vectored vaccines (75, 77–79). However, an approved vaccine or antiviral is still not available to prevent and/or treat ZIKV infection. Therefore, the urgency remains to develop safe, efficacious, and cost-effective vaccines and therapeutics to meet the unmet medical need. Reverse genetics technology has allowed for a rapid response to the ZIKV epidemic, assisting in areas like basic science research to understand ZIKV factors and their role in the viral infection cycle, as well as to advance the development of therapeutics and vaccines (13*). The generation of cDNA infectious clones has enabled rational engineering of ZIKV to develop various vaccine approaches (11**, 28-30, 32*, 38, 51). Additionally, the reporter, replicon, and RVP systems have provided critical tools for basic and translational research to aid in the development of these vaccines, as well as therapeutics and diagnostics.