Sf9 Cell Metabolism Throughout the Recombinant Baculovirus and Rabies Virus-Like Particles Production in Two Culture Systems.

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10-12 mmol cell/h) in SF and for glutamine (30.8 × 10-12 mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10-10 mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.

Production of Recombinant Zika Virus Envelope Protein by Airlift Bioreactor as a New Subunit Vaccine Platform.

The Zika Virus (ZIKV) is an emerging arbovirus of great public health concern, particularly in the Americas after its last outbreak in 2015. There are still major challenges regarding disease control, and there is no ZIKV vaccine currently approved for human use. Among many different vaccine platforms currently under study, the recombinant envelope protein from Zika Virus (rEZIKV) constitutes an alternative option for vaccine development and has great potential for monitoring ZIKV infection and antibody response. This study describes a method to obtain a bioactive and functional rEZIKV using an E. coli expression system, with the aid of a 5-L airlift bioreactor and following an automated fast protein liquid chromatography (FPLC) protocol, capable of obtaining high yields of approximately 20 mg of recombinant protein per liter of bacterium cultures. The purified rEZIKV presented preserved antigenicity and immunogenicity. Our results show that the use of an airlift bioreactor for the production of rEZIKV is ideal for establishing protocols and further research on ZIKV vaccines bioprocess, representing a promising system for the production of a ZIKV envelope recombinant protein-based vaccine candidate.

Zika virus-like particles (VLPs) produced in insect cells.

Introdutcion: The Zika virus (ZIKV) infections are a healthcare concern mostly in the Americas, Africa, and Asia but have increased its endemicity area beyond these geographical regions. Due to the advances in infections by Zika virus, it is imperative to develop diagnostic and preventive tools against this viral agent. Virus-like particles (VLPs) appear as a suitable approach for use as antiviral vaccines. Methods: In this work, a methodology was established to produce virus-like particles containing the structural proteins, C, prM, and E of Zika virus produced in insect cells using the gene expression system derived from baculovirus. The vector pFast- CprME -ZIKV was constructed containing the gene sequences of Zika virus structural proteins and it was used to generate the recombinant bacmids (Bac- CprME -ZIKV) through transformation into DH10BacTM cells. The Bac- CprME -ZIKV was transfected in Spodoptera frugiperda (Sf9) insect cells and batches of BV- CprME -ZIKV were obtained by infection assays using a multiplicity of infection of 2. The Sf9 cells were infected, and the supernatant was collected 96 h post-infection. The expression of the CprME -ZIKV protein on the cell surface could be observed by immunochemical assays. To concentrate and purify virus-like particles, the sucrose and iodixanol gradients were evaluated, and the correct CprME -ZIKV proteins' conformation was evaluated by the Western blot assay. The virus-like particles were also analyzed and characterized by transmission electron microscopy. Results and discussion: Spherical structures like the native Zika virus from 50 to 65 nm containing the CprME -ZIKV proteins on their surface were observed in micrographs. The results obtained can be useful in the development path for a vaccine candidate against Zika virus.

RBD and Spike DNA-Based Immunization in Rabbits Elicited IgG Avidity Maturation and High Neutralizing Antibody Responses against SARS-CoV-2.

Neutralizing antibodies (nAbs) are a critical part of coronavirus disease 2019 (COVID-19) research as they are used to gain insight into the immune response to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections. Among the technologies available for generating nAbs, DNA-based immunization methods are an alternative to conventional protocols. In this pilot study, we investigated whether DNA-based immunization by needle injection in rabbits was a viable approach to produce a functional antibody response. We demonstrated that three doses of DNA plasmid carrying the gene encoding the full-length spike protein (S) or the receptor binding domain (RBD) of SARS-CoV-2 induced a time-dependent increase in IgG antibody avidity maturation. Moreover, the IgG antibodies displayed high cross neutralization by live SARS-CoV-2 and pseudoviruses neutralization assays. Thus, we established a simple, low cost and feasible DNA-based immunization protocol in rabbits that elicited high IgG avidity maturation and nAbs production against SARS-CoV-2, highlighting the importance of DNA-based platforms for developing new immunization strategies against SARS-CoV-2 and future emerging epidemics.

Biochemical monitoring throughout all stages of rabies virus-like particles production by Raman spectroscopy using global models.

This work aimed to quantify growth and biochemical parameters (viable cell density, Xv; cell viability, CV; glucose, lactate, glutamine, glutamate, ammonium, and potassium concentrations) in upstream stages to obtain rabies virus-like particles (rabies VLP) from insect cell-baculovirus system using on-line and off-line Raman spectra to calibrate global models with minimal experimental data. Five cultivations in bioreactor were performed. The first one comprised the growth of uninfected Spodoptera frugiperda (Sf9) cells, the second and third runs to obtain recombinant baculovirus (rBV) bearing Rabies G glycoprotein and matrix protein, respectively. The fourth one involved the generation of rabies VLP from rBVs and the last one was a repetition of the third one with cell inoculum infected by rBV. The spectra were acquired through a Raman spectrometer with a 785-nm laser source. The fitted Partial Least Square models for nutrients and metabolites were comparable with those previously reported for mammalian cell lines (Relative error < 15 %). However, the use of this chemometrics approach for Xv and CV was not as accurate as it was for other parameters. The findings from this work established the basis for bioprocess Raman spectroscopical monitoring using insect cells for VLP manufacturing, which are gaining ground in the pharmaceutical industry.

Sf9 Cells Metabolism and Viability When Coinfected with Two Monocistronic Baculoviruses to Produce Rabies Virus-like Particles.

This work aimed to describe the dynamics of the Sf9 insect cells death and primary metabolism when this host is infected simultaneously by two recombinant baculoviruses (BV) expressing rabies glycoprotein (BVG) and matrix protein (BVM) genes to produce rabies virus-like particles (VLP) at different multiplicities of infection (MOI). Schott flasks essays covering a wide range of MOI for both BV were performed. Viable cell density, cell viability, glucose, glutamine, glutamate, lactate, ammonium, and rabies proteins concentrations were monitored over the infection phase. The expression of both recombinant proteins was not limited by glucose, glutamine, and glutamate in a broad MOI (pfu/cell) range of BVG (0.15-12.5) and BVM (0.1-5.0) using SF900 III serum free culture medium. Death phase initiation and the specific death rate depend on BV MOI. The wave pattern of nutrient/metabolite profiles throughout the viral infection phase is related to the baculovirus lytic cycle. The optimal MOIs ratio between BVG (2.5-4.5) and BVM (1.0-3.0) for maximum protein expression was defined. The produced rabies VLP sizes are close to 78 nm. In general, these work outputs bring a better understanding of the metabolic performance of Sf9 cells when infected by BV for producing VLP, and specifically, for progressing in a rabies VLP vaccine development.

Production of recombinant Zika virus envelope protein by airlift bioreactor as a new subunit vaccine platform

The Zika Virus (ZIKV) is an emerging arbovirus of great public health concern, particularly in the Americas after its last outbreak in 2015. There are still major challenges regarding disease control, and there is no ZIKV vaccine currently approved for human use. Among many different vaccine platforms currently under study, the recombinant envelope protein from Zika Virus (rEZIKV) constitutes an alternative option for vaccine development and has great potential for monitoring ZIKV infection and antibody response. This study describes a method to obtain a bioactive and functional rEZIKV using an E. coli expression system, with the aid of a 5-L airlift bioreactor and following an automated fast protein liquid chromatography (FPLC) protocol, capable of obtaining high yields of approximately 20 mg of recombinant protein per liter of bacterium cultures. The purified rEZIKV presented preserved antigenicity and immunogenicity. Our results show that the use of an airlift bioreactor for the production of rEZIKV is ideal for establishing protocols and further research on ZIKV vaccines bioprocess, representing a promising system for the production of a ZIKV envelope recombinant protein-based vaccine candidate.

Zika virus-like particles (VLPs) produced in insect cells

Introdutcion: The Zika virus (ZIKV) infections are a healthcare concern mostly in the Americas, Africa, and Asia but have increased its endemicity area beyond these geographical regions. Due to the advances in infections by Zika virus, it is imperative to develop diagnostic and preventive tools against this viral agent. Virus-like particles (VLPs) appear as a suitable approach for use as antiviral vaccines. Methods: In this work, a methodology was established to produce virus-like particles containing the structural proteins, C, prM, and E of Zika virus produced in insect cells using the gene expression system derived from baculovirus. The vector pFast- CprME -ZIKV was constructed containing the gene sequences of Zika virus structural proteins and it was used to generate the recombinant bacmids (Bac- CprME -ZIKV) through transformation into DH10BacTM cells. The Bac- CprME -ZIKV was transfected in Spodoptera frugiperda (Sf9) insect cells and batches of BV- CprME -ZIKV were obtained by infection assays using a multiplicity of infection of 2. The Sf9 cells were infected, and the supernatant was collected 96 h post-infection. The expression of the CprME -ZIKV protein on the cell surface could be observed by immunochemical assays. To concentrate and purify virus-like particles, the sucrose and iodixanol gradients were evaluated, and the correct CprME -ZIKV proteins’ conformation was evaluated by the Western blot assay. The virus-like particles were also analyzed and characterized by transmission electron microscopy. Results and discussion: Spherical structures like the native Zika virus from 50 to 65 nm containing the CprME -ZIKV proteins on their surface were observed in micrographs. The results obtained can be useful in the development path for a vaccine candidate against Zika virus.

Sf9 cell metabolism throughout the recombinant Baculovirus and rabies virus-like particles production in two culture systems

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to fnally obtain rabies VLP in two culture systems: Schott fask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specifc rates were quantifed over the exponential growth phase and infection stage. The highest uptake specifc rate was observed for glucose (42.5× 10–12 mmol cell/h) in SF and for glutamine (30.8× 10–12 mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10–10 mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The fndings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.

RBD and spike DNA-based immunization in rabbits elicited IgG avidity maturation and high neutralizing antibody responses against SARS-CoV-2

Neutralizing antibodies (nAbs) are a critical part of coronavirus disease 2019 (COVID-19) research as they are used to gain insight into the immune response to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections. Among the technologies available for generating nAbs, DNA-based immunization methods are an alternative to conventional protocols. In this pilot study, we investigated whether DNA-based immunization by needle injection in rabbits was a viable approach to produce a functional antibody response. We demonstrated that three doses of DNA plasmid carrying the gene encoding the full-length spike protein (S) or the receptor binding domain (RBD) of SARS-CoV-2 induced a time-dependent increase in IgG antibody avidity maturation. Moreover, the IgG antibodies displayed high cross neutralization by live SARS-CoV-2 and pseudoviruses neutralization assays. Thus, we established a simple, low cost and feasible DNA-based immunization protocol in rabbits that elicited high IgG avidity maturation and nAbs production against SARS-CoV-2, highlighting the importance of DNA-based platforms for developing new immunization strategies against SARS-CoV-2 and future emerging epidemics.

Biochemical monitoring throughout all stages of rabies virus-like particles production by Raman spectroscopy using global models

This work aimed to quantify growth and biochemical parameters (viable cell density, Xv; cell viability, CV; glucose, lactate, glutamine, glutamate, ammonium, and potassium concentrations) in upstream stages to obtain rabies virus-like particles (rabies VLP) from insect cell-baculovirus system using on-line and off-line Raman spectra to calibrate global models with minimal experimental data. Five cultivations in bioreactor were performed. The first one comprised the growth of uninfected Spodoptera frugiperda (Sf9) cells, the second and third runs to obtain recombinant baculovirus (rBV) bearing Rabies G glycoprotein and matrix protein, respectively. The fourth one involved the generation of rabies VLP from rBVs and the last one was a repetition of the third one with cell inoculum infected by rBV. The spectra were acquired through a Raman spectrometer with a 785-nm laser source. The fitted Partial Least Square models for nutrients and metabolites were comparable with those previously reported for mammalian cell lines (Relative error < 15 %). However, the use of this chemometrics approach for Xv and CV was not as accurate as it was for other parameters. The findings from this work established the basis for bioprocess Raman spectroscopical monitoring using insect cells for VLP manufacturing, which are gaining ground in the pharmaceutical industry.

Performance comparison of recombinant Baculovirus and rabies virus-like particles production using two culture platforms

This work aimed to assess, following upstream optimization in Schott flasks, the scalability from this culture platform to a stirred-tank bioreactor in order to yield rabies-recombinant baculovirus, bearing genes of G (BVG) and M (BVM) proteins, and to obtain rabies virus-like particles (VLP) from them, using Sf9 insect cells as a host. Equivalent assays in Schott flasks and a bioreactor were performed to compare both systems and a multivariate statistical approach was also carried out to maximize VLP production as a function of BVG and BVM’s multiplicity of infection (MOI) and harvest time (HT). Viable cell density, cell viability, virus titer, BVG and BVM quantification by dot-blot, and BVG quantification by Enzyme-Linked Immunosorbent Assay (ELISA) were monitored throughout the assays. Furthermore, transmission electron microscopy was used to characterize rabies VLP. The optimal combination for maximum VLP expression was BVG and BVM MOI of 2.3 pfu/cell and 5.1 pfu/cell, respectively, and 108 h of harvest time. The current study confirmed that the utilization of Schott flasks and a benchtop bioreactor under the conditions applied herein are equivalent regarding the cell death kinetics corresponding to the recombinant baculovirus infection process in Sf9 cells. According to the results, the hydrodynamic and chemical differences in both systems seem to greatly affect the virus and VLP integrity after release.

Oxygen uptake and transfer rates throughout production of recombinant baculovirus and rabies virus-like particles

The technologies used in rabies vaccines manufacturing for human use are based on the inactivated virus platform. An alternative to traditional vaccines is virus-like particles (VLPs). This work aimed to characterize the oxygen uptake and transfer rate parameters throughout recombinant baculovirus (rBV) and rabies VLPs production using Sf9 cells in stirred tank bioreactor (STB) for a better bioprocess understanding and scalability. Four runs in a bench STB were performed: cell culture without infection; cells infected singly with rBV bearing rabies virus glycoprotein (rBVG, multiplicity of infection, MOI=0.1 pfu/cell) and matrix protein (rBVM, MOI=0.1 pfu/cell), and coinfected with BVG and BVM at MOI of 3 and 2 pfu/cell, respectively. The specific oxygen uptake rate () and volumetric oxygen transfer coefficient () were monitored throughout the reactions, as well as viable cell concentration, viability, rBV titers, and protein concentration. According to the results herein, the aeration and agitation systems in a bioreactor at a higher scale could be designed using the criterium for scale-up of constant , without oxygen facilities. Besides, rabies VLPs volumetric yield of 2.8 mg/L with a typical size (55–68 nm) was obtained. These findings suggest a promising bioprocess for rabies VLPs at a commercial scale.

ZIKV-envelope proteins induce specific humoral and cellular immunity in distinct mice strains.

Recent outbreaks of Zika virus (ZIKV) infection have highlighted the need for a better understanding of ZIKV-specific immune responses. The ZIKV envelope glycoprotein (EZIKV) is the most abundant protein on the virus surface and it is the main target of the protective immune response. EZIKV protein contains the central domain (EDI), a dimerization domain containing the fusion peptide (EDII), and a domain that binds to the cell surface receptor (EDIII). In this study, we performed a systematic comparison of the specific immune response induced by different EZIKV recombinant proteins (EZIKV, EDI/IIZIKV or EDIIIZIKV) in two mice strains. Immunization induced high titers of E-specific antibodies which recognized ZIKV-infected cells and neutralized the virus. Furthermore, immunization with EZIKV, EDI/IIZIKV and EDIIIZIKV proteins induced specific IFNγ-producing cells and polyfunctional CD4+ and CD8+ T cells. Finally, we identified 4 peptides present in the envelope protein (E1-20, E51-70, E351-370 and E361-380), capable of inducing a cellular immune response to the H-2Kd and H-2Kb haplotypes. In summary, our work provides a detailed assessment of the immune responses induced after immunization with different regions of the ZIKV envelope protein.

Recapitulation of Retinal Damage in Zebrafish Larvae Infected with Zika Virus.

Zebrafish are increasingly being utilized as a model to investigate infectious diseases and to advance the understanding of pathogen-host interactions. Here, we take advantage of the zebrafish to recapitulate congenital ZIKV infection and, for the first time, demonstrate that it can be used to model infection and reinfection and monitor anti-viral and inflammatory immune responses, as well as brain growth and eye abnormalities during embryonic development. By injecting a Brazilian strain of ZIKV into the yolk sac of one-cell stage embryos, we confirmed that, after 72 h, ZIKV successfully infected larvae, and the physical condition of the virus-infected hosts included gross morphological changes in surviving embryos (84%), with a reduction in larval head size and retinal damage characterized by increased thickness of the lens and inner nuclear layer. Changes in locomotor activity and the inability to perceive visual stimuli are a result of changes in retinal morphology caused by ZIKV. Furthermore, we demonstrated the ability of ZIKV to replicate in zebrafish larvae and infect new healthy larvae, impairing their visual and neurological functions. These data reinforce the deleterious activity of ZIKV in the brain and visual structures and establish the zebrafish as a model to study the molecular mechanisms involved in the pathology of the virus.

Anti-SARS-CoV-2 equine F (Ab')2 immunoglobulin as a possible therapy for COVID-19.

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab')2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab')2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Performance Comparison of Recombinant Baculovirus and Rabies Virus-like Particles production Using Two Culture Platforms.

This work aimed to assess, following upstream optimization in Schott flasks, the scalability from this culture platform to a stirred-tank bioreactor in order to yield rabies-recombinant baculovirus, bearing genes of G (BVG) and M (BVM) proteins, and to obtain rabies virus-like particles (VLP) from them, using Sf9 insect cells as a host. Equivalent assays in Schott flasks and a bioreactor were performed to compare both systems and a multivariate statistical approach was also carried out to maximize VLP production as a function of BVG and BVM's multiplicity of infection (MOI) and harvest time (HT). Viable cell density, cell viability, virus titer, BVG and BVM quantification by dot-blot, and BVG quantification by Enzyme-Linked Immunosorbent Assay (ELISA) were monitored throughout the assays. Furthermore, transmission electron microscopy was used to characterize rabies VLP. The optimal combination for maximum VLP expression was BVG and BVM MOI of 2.3 pfu/cell and 5.1 pfu/cell, respectively, and 108 h of harvest time. The current study confirmed that the utilization of Schott flasks and a benchtop bioreactor under the conditions applied herein are equivalent regarding the cell death kinetics corresponding to the recombinant baculovirus infection process in Sf9 cells. According to the results, the hydrodynamic and chemical differences in both systems seem to greatly affect the virus and VLP integrity after release.

Sf9 Cells metabolism and viability when coinfected with two monocistronic baculoviruses to produce rabies virus‑like particles

This work aimed to describe the dynamics of the Sf9 insect cells death and primary metabolism when this host is infected simultaneously by two recombinant baculoviruses (BV) expressing rabies glycoprotein (BVG) and matrix protein (BVM) genes to produce rabies virus-like particles (VLP) at diferent multiplicities of infection (MOI). Schott fasks essays covering a wide range of MOI for both BV were performed. Viable cell density, cell viability, glucose, glutamine, glutamate, lactate, ammonium, and rabies proteins concentrations were monitored over the infection phase. The expression of both recombinant proteins was not limited by glucose, glutamine, and lutamate in a broad MOI (pfu/cell) range of BVG (0.15–12.5) and BVM (0.1–5.0) using SF900 III serum free culture medium. Death phase initiation and the specifc death rate depend on BV MOI. The wave pattern of nutrient/metabolite profles throughout the viral infection phase is related to the baculovirus lytic cycle. The optimal MOIs ratio between BVG (2.5–4.5) and BVM (1.0–3.0) for maximum protein expression was defned. The produced rabies VLP sizes are close to 78 nm. In general, these work outputs bring a better understanding of the metabolic performance of Sf9 cells when infected by BV for producing VLP, and specifcally, for progressing in a rabies VLP vaccine development.

ZIKV-envelope proteins induce specific humoral and cellular immunity in distinct mice strains

Recent outbreaks of Zika virus (ZIKV) infection have highlighted the need for a better understanding of ZIKV-specific immune responses. The ZIKV envelope glycoprotein (EZIKV) is the most abundant protein on the virus surface and it is the main target of the protective immune response. EZIKV protein contains the central domain (EDI), a dimerization domain containing the fusion peptide (EDII), and a domain that binds to the cell surface receptor (EDIII). In this study, we performed a systematic comparison of the specific immune response induced by different EZIKV recombinant proteins (EZIKV, EDI/IIZIKV or EDIIIZIKV) in two mice strains. Immunization induced high titers of E-specific antibodies which recognized ZIKV-infected cells and neutralized the virus. Furthermore, immunization with EZIKV, EDI/IIZIKV and EDIIIZIKV proteins induced specific IFNγ-producing cells and polyfunctional CD4+ and CD8+ T cells. Finally, we identified 4 peptides present in the envelope protein (E1–20, E51–70, E351–370 and E361–380), capable of inducing a cellular immune response to the H-2Kd and H-2Kb haplotypes. In summary, our work provides a detailed assessment of the immune responses induced after immunization with different regions of the ZIKV envelope protein.

Recapitulation of retinal damage in zebrafish larvae infected with Zika virus

Zebrafish are increasingly being utilized as a model to investigate infectious diseases and to advance the understanding of pathogen–host interactions. Here, we take advantage of the zebrafish to recapitulate congenital ZIKV infection and, for the first time, demonstrate that it can be used to model infection and reinfection and monitor anti-viral and inflammatory immune responses, as well as brain growth and eye abnormalities during embryonic development. By injecting a Brazilian strain of ZIKV into the yolk sac of one-cell stage embryos, we confirmed that, after 72 h, ZIKV successfully infected larvae, and the physical condition of the virus-infected hosts included gross morphological changes in surviving embryos (84%), with a reduction in larval head size and retinal damage characterized by increased thickness of the lens and inner nuclear layer. Changes in locomotor activity and the inability to perceive visual stimuli are a result of changes in retinal morphology caused by ZIKV. Furthermore, we demonstrated the ability of ZIKV to replicate in zebrafish larvae and infect new healthy larvae, impairing their visual and neurological functions. These data reinforce the deleterious activity of ZIKV in the brain and visual structures and establish the zebrafish as a model to study the molecular mechanisms involved in the pathology of the virus.