Project Title:

Definition of the ability of zika virus (ZIKV) to infect the placenta, brain and eye that will impact on our understanding of debilitating microcephaly and macular scarring in ZIKV exposed newborns.

Discipline:

Infectious Diseases

Chief Investigator:

Dr Jillian Carr

Funding Amount:

$74,027

Recipient:

Flinders University

Overview:

The world was shocked by the threat of Ebola. Now we face the more insidious ZIKV. ZIKV is transmitted by a mosquito that spreads dengue, which with 390 million infections pa (Bhatt et al., 2013. Nature), paints a grim picture for future ZIKV. Epidemiology links ZIKV infection of pregnant women to microcephaly and macular scars in newborns with intellectual and visual impairment. A WHO alert suggests travel restrictions in pregnancy and presents ZIKV as an emerging threat to the health of newborns with permanent sequela for the next generation. ZIKV is thus a clear priority for child health.

Research Outcomes:

Researchers:

Jillian Carr, Justine Smith

Research Completed:

2017

Research Findings:

This study looked at the basic biology of zika virus (ZIKV) infection in the developing brain and eye, at a time when ZIKV was a new and significant health concern, in particular for the catastrophic developmental defects in the brain and eyes of babies born to mothers infected with ZIKV. Using cells from the human eye, our findings have established unique responses of different cell types to ZIKV in terms of protective anti-viral and damaging inflammatory responses. Additionally, our team has established a newborn mouse model of ZIKV infection that has allowed us to define the infection in the brain and eye during development. Results from both these systems are currently in preparation for publication. Excitingly, the newborn mouse model will allow our researchers in Adelaide to be at the forefront of future studies of infections with ZIKV as well as other important viruses in the brain and eye during development, which can significantly impact on child health and development..

Key Outcomes:

Our original study AIMS were to investigate the ability of ZIKV to infect different tissues and cells of the eye and brain. The study aimed to compare findings with dengue virus (DENV), a well described and closely related flavivirus with ZIKV, a new virus associated with developmental defects in the brain and eye of newborns. Specifically, we aimed to

  • Infect human cells in vitro, reflecting potential tissue targets associated with ZIKV or DENV disease.
  • Assess ZIKV tropism in vivo, in particular the brain and eye, by infection of pregnant and newborn mice with ZIKV and DENV.

We have successfully achieved both of these aims with the key outcomes of:

  1. Definition of ZIKV infection and the antiviral and inflammatory responses induced in different cell types of the human eye in vitro. We utilised human eye cell lines and primary cells isolated from human donor tissue, with cell types representing parts of the retina – retinal pigment epithelium, retinal endothelial cells and retinal Mueller cells. These cell types were chosen due to their clinical links to defects seen in ZIKV affected babies, and their known functions in forming physical and immunological barriers to infection in the eye. All of these eye cell types were readily infected by ZIKV and to a greater extent than our comparative control, DENV – which has no described developmental effects on the eye or brain. Each cell type showed unique immunological responses to infection suggesting cell-type specific roles in controlling ZIKV infection in the eye. The profile of ZIKV infection in the different cells was not related to the presence of the TAM receptors, proposed to influence ZIKV entry in some cell and tissue types, suggesting different viral entry mechanisms in these cells. Overall, this data is a solid foundation for our understanding of the factors that influence ZIKV infection in the eye and provides knowledge to assist the field moving towards strategies to restrict infection and damage in this important tissue type.
  2. Establishment of a newborn mouse model of ZIKV infection in the developing brain and eye. We have established the logistics of injecting PC2 pathogens (ZIKV and DENV) into a live 1-day-old mouse pup and the dose and timing for these viruses to move from the periphery into the developing brain and eye. This model has already yielded outcomes, as below but additionally, is an exciting new tool that will allow us to investigate the process and influences of movement of a virus and infection in the developing brain and eye.
  3. Definition of ZIKV infection and the antiviral and inflammatory responses induced in the developing mouse eye in vivo. Using the newborn suckling mouse model established above, our results have shown that ZIKV moves quickly to both the brain and eye (within 3 days following infection in the periphery). In contrast DENV is not detected in these tissues until 6 days after infection and is not detected in all animals challenged. We have also defined the tissues’ antiviral and inflammatory responses to infection and are in the process of comparing these with individual human cell-type specific responses to determine which are the dominant cell-type specific responses seen in the context of a natural ZIKV or DENV infection in a whole tissue. Together these findings are novel and will be of significant interest to general virology, those with a focus on viral infections in relation to child development and those with a specific interest of ZIKV in the eye.
  4. Publication of data. Selected parts of the above were presented as a poster by Dr Gustavo Bracho at the premier Australian Virology meeting – The Australasian Society for Virology (AVS) meeting, Dec, 2018. Additionally, these results are currently in process of final compilation into a manuscript that we anticipate will be ready for submission to the Journal of Virology, a Schimago Q1 journal and among the top ranking discipline specific journals in the virology field.

E. Positive impact on further funding and research. CI’s Smith and Carr have obtained NHMRC funding, 2018-20 for a project largely on Ebola virus infection in the eye, but the data here will also form a comparative model of benefit. Additionally, the data on infection in Mueller cells will be extended by PhD student Genevieve Oliver, a retinal surgeon, to investigate the role of this cell type specifically. Lastly and excitingly, this study has allowed us to form a new collaboration with A/Prof Alice Pebay, and we have performed a ZIKV infection in ‘optic cups’ representing retinal stem cell progenitors, as a future model to study the effects of viral infection on development of the retina.

Research Papers:

Manuscript in preparation:

Gustavo Bracho, Julie Calvert, Liam Ashander, Sheila Cabezas-Falcon, Yufang Ma, Justine Smith and Jillian M Carr. Zika virus (ZIKV) infects human eye cells in vitro and the eye and brain of newborn mice with characteristics that contrast to dengue virus (DENV). Journal of Virology (manuscript in preparation, expected submission April 2018)

Poster presentation:

Gustavo Bracho, Julie Calvert, Yuefang Ma, Sheila Cabezas, Justine Smith, Jillian Carr. Dengue and zika virus infect retinal cells and induce differential host defence gene expression. 9th Australasian Society for Virology meeting, Dec 4-6, Stamford Grand Hotel, Adelaide, SA

Future Outcomes:

Remarks:

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