isolation-quantification-zika-virus-from-multiple-organs

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Saint Louis University

Zika virus (ZIKV) is a re-emerging virus that has recently spread into dengue virus (DENV) endemic regions and cross-reactive antibodies (Abs) could potentially affect ZIKV pathogenesis. Using DENV-immune serum, it has been shown in vitro that antibody-dependent enhancement (ADE) of ZIKV infection can occur. Here we study the effects of pre-existing DENV immunity on ZIKV infection in vivo. We infect two cohorts of rhesus macaques with ZIKV; one cohort has been exposed to DENV 2.8 years earlier and a second control cohort is naïve to flaviviral infection. Our results, while confirming ADE in vitro, suggest that pre-existing DENV immunity does not result in more severe ZIKV disease. Rather our results show a reduction in the number of days of ZIKV viremia compared to naïve macaques and that the previous exposure to DENV may result in modulation of the immune response without resulting in enhancement of ZIKV pathogenesis.

Zika virus pathogenesis in rhesus macaques is unaffected by pre-existing immunity to dengue virus.

Approximately 10% of all bone fractures do not heal, resulting in patient morbidity and healthcare costs. However, no pharmacological treatments are currently available to promote efficient bone healing. Inhibition of Ca /calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) reverses age-associated loss of trabecular and cortical bone volume and strength in mice. In the current study, we investigated the role of CaMKK2 in bone fracture healing and show that its pharmacological inhibition using STO-609 accelerates early cellular and molecular events associated with endochondral ossification, resulting in a more rapid and efficient healing of the fracture. Within 7 days postfracture, treatment with STO-609 resulted in enhanced Indian hedgehog signaling, paired-related homeobox (PRX1)-positive mesenchymal stem cell (MSC) recruitment, and chondrocyte differentiation and hypertrophy, along with elevated expression of osterix, vascular endothelial growth factor, and type 1 collagen at the fracture callus. Early deposition of primary bone by osteoblasts resulted in STO-609-treated mice possessing significantly higher callus bone volume by 14 days following fracture. Subsequent rapid maturation of the bone matrix bestowed fractured bones in STO-609-treated animals with significantly higher torsional strength and stiffness by 28 days postinjury, indicating accelerated healing of the fracture. Previous studies indicate that fixed and closed femoral fractures in the mice take 35 days to fully heal without treatment. Therefore, our data suggest that STO-609 potentiates a 20% acceleration of the bone healing process. Moreover, inhibiting CaMKK2 also imparted higher mechanical strength and stiffness at the contralateral cortical bone within 4 weeks of treatment. Taken together, the data presented here underscore the therapeutic potential of targeting CaMKK2 to promote efficacious and rapid healing of bone fractures and as a mechanism to strengthen normal bones. © 2018 American Society for Bone and Mineral Research.

Inhibition of CaMKK2 Enhances Fracture Healing by Stimulating Indian Hedgehog Signaling and Accelerating Endochondral Ossification.

Zika virus (ZIKV) has gained worldwide attention since it emerged, and a global effort is underway to understand the correlates of protection and develop diagnostics to identify rates of infection. As new therapeutics and vaccine approaches are evaluated in clinical trials, additional effort is focused on identifying the adaptive immune correlates of protection against ZIKV disease. To aid in this endeavor we have begun to dissect the role of CD4+T cells in the protection against neuroinvasive ZIKV disease. We have identified an important role for CD4+T cells in protection, demonstrating that in the absence of CD4+T cells mice have more severe neurological sequela and significant increases in viral titers in the central nervous system (CNS). The transfer of CD4+T cells from ZIKV immune mice protect type I interferon receptor deficient animals from a lethal challenge; showing that the CD4+T cell response is necessary and sufficient for control of ZIKV disease. Using a peptide library spanning the complete ZIKV polyprotein, we identified both ZIKV-encoded CD4+T cell epitopes that initiate immune responses, and ZIKV specific CD4+T cell receptors that recognize these epitopes. Within the ZIKV antigen-specific TCRβ repertoire, we uncovered a high degree of diversity both in response to a single epitope and among different mice responding to a CD4+T cell epitope. Overall this study identifies a novel role for polyfunctional and polyclonal CD4+T cells in providing protection against ZIKV infection and highlights the need for vaccines to develop robust CD4+T cell responses to prevent ZIKV neuroinvasion and limit replication within the CNS.

CD4+T cells mediate protection against Zika associated severe disease in a mouse model of infection.

Most of the world is at risk of being infected with a flavivirus such as dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and Zika virus, significantly impacting millions of lives. Importantly, many of these genetically similar viruses co-circulate within the same geographic regions, making it likely for individuals living in areas of high flavivirus endemicity to be infected with multiple flaviviruses during their lifetime. Following a flavivirus infection, a robust  T cell response is generated and the memory recall of this response has been demonstrated to provide long-lasting immunity, protecting against reinfection with the same pathogen. However, multiple studies have shown that this flavivirus specific T cell response can be cross-reactive and active during heterologous flavivirus infection, leading to the question:  It has been proposed that in some cases unfavorable disease outcomes may be caused by lower avidity cross-reactive memory T cells generated during a primary flavivirus infection that preferentially expand during a secondary heterologous infection and function sub optimally against the new pathogen. While in other cases, these cross-reactive cells still have the potential to facilitate cross-protection. In this review, we focus on cross-reactive T cell responses to flaviviruses and the concepts and consequences of T cell cross-reactivity, with particular emphasis linking data generated using murine models to our new understanding of disease outcomes following heterologous flavivirus infection.

Mouse Models of Heterologous Flavivirus Immunity: A Role for Cross-Reactive T Cells.

Many flaviviruses including dengue (DENV), and Zika (ZIKV) have attracted significant attention in the past few years. As many flaviviruses are spread by arthropods, most of the world's population is at risk of encountering a flavivirus, and infection with these viruses has created a significant disease burden worldwide. Vaccination against flaviviruses is thought to be one of the most promising avenues for reducing the disease burden associated with these viruses. The optimism surrounding a vaccine approach is supported by the highly successful vaccines for yellow fever and Japanese encephalitis. Central to the development of new successful vaccines is the understanding of the correlates of protection that will be necessary to engineer into new vaccines. To aid in this endeavor we have directed our efforts to identify correlates of protection that will reduce the disease burden associated with ZIKV and DENV. Within this study we have identified a novel murine ZIKV specific CD8 T cell epitope, and shown that the ZIKV epitope specific CD8 T cell response has a distinct immunodominance hierarchy present during acute infection and is detectible as part of the memory T cell responses. Our studies confirm that ZIKV-specific CD8 T cells are an important correlate of protection for ZIKV and demonstrate that both naïve and ZIKV immune CD8 T cells are sufficient for protection against a lethal ZIKV infection. Overall this study adds to the body of literature demonstrating a role for CD8 T cells in controlling flavivirus infection.

Article	Year
Zika virus pathogenesis in rhesus macaques is unaffected by pre-existing immunity to dengue virus. Nature communications\| PubMed ID: 28643775	2017
Inhibition of CaMKK2 Enhances Fracture Healing by Stimulating Indian Hedgehog Signaling and Accelerating Endochondral Ossification. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research\| PubMed ID: 29314250	2018
CD4+T cells mediate protection against Zika associated severe disease in a mouse model of infection. PLoS pathogens\| PubMed ID: 30212537	2018
Mouse Models of Heterologous Flavivirus Immunity: A Role for Cross-Reactive T Cells. Frontiers in immunology\| PubMed ID: 31143185	2019
Identification of Protective CD8 T Cell Responses in a Mouse Model of Zika Virus Infection. Frontiers in immunology\| PubMed ID: 31379867	2019

Mariah Hassert

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