Development of Novel Flow Cytometry Technique to Study Yellow Fever Virus

BY DEVORAH SAFFERN

Researchers in the Ploss lab of the Molecular Biology department at Princeton University have utilized a new technology to analyze the pathogenesis of the yellow fever virus (YFV), which can be applied to other viruses and lead to significant progress in understanding disease mechanisms. Published in Nature Communications on March 14, their study discusses the ways in which viruses interact with host cells, in an effort to discover more effective vaccines. Currently, there is a lack of scientific knowledge on how viruses interact with the host cells and cause illness or proper immune response, and their research begins to uncover these behaviors.

Yellow fever and other similar viruses affect many people – there are approximately 30,000 deaths due to yellow fever per year, and 200,000 infected people. YFV is a positively-stranded, prototypic flavivirus, meaning it is similar to other disease-causing viruses, such as Dengue and Zika. Analysis of YFV however could reveal the inner mechanisms not only of viruses in the Flaviridae genus family, but also of other viruses that similarly interact with host cells. This includes the many positively-stranded infectious viruses like Hepatitis C, West Nile, and the common cold. YFV is therefore a prime virus to study, and results can be broadly applicable.

The experiments consisted of tracking the positive and negative sense RNA in YFV viruses found in the immune systems of mice. Typically, researchers track viruses by targeting the viral proteins with fluorophore-labeled antibodies and analyzing the pathways of the proteins using flow cytometry. Cellular behaviors can be analyzed at high resolution through these labeled antibodies. This process is limited, however, in that highly specific antibodies must be used to attach to proteins for the vast virus types that are prevalent. In addition, it is important to understand not only viral protein behavior during translation, but also other antibody behaviors in the host cells of the immune system. It is also difficult to track antibodies in vivo. The viral RNA might affect the immune system in a number of other ways, which calls for a more broad analysis of the virus in action.

A new and advanced flow cytometry method called Prime RNA Flow, first presented in a Nature Communications paper published in 2014, enables improved tracking of viral RNA. The technology involves antibodies that can track multiple RNA strands at once, through amplifications. In this particular study, researchers used the technique to track YFV RNA replication in mice immune cells found in the blood stream. They built a new device that consists of two probes that analyze a specific set of base pairs on the RNA strand – one probe to sense positive RNA strands, and one to sense negative. Positive and negative YFV-17D RNA strands, which are strains of YFV used in the yellow fever vaccine, were then injected into mice and tested with the Prime Flow technique. Fluorescence signals as well as amplifiers were built into the probes to detect and track the RNA and obtain resolution on the single cellular level.

In experimenting with these new tools, the researchers found that removal of STAT1 on the RNA segment, a transcription factor important in the antiviral process, increased YFV-17D replication. YFV-17D normally does not replicate in mice but does in human cells. That result shows that signals within STAT1 are what normally shield mice from infection by YFV-17D, as opposed to humans who are susceptible. The researchers also found that YFV-17D is able to replicate in human B cells, a type of white blood cell, and NK cells, which are blood cells in mice in the immune system, facts unknown prior to this study. During infection, different cells in the blood and spleen are infected at various points through the course of the virus. These new understandings were possible using the RNA Prime Flow technique.

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Six tissue sections from three biological replicates (three animals) were examined for each experimental condition (non-infected or infected) and tissue (Liver and Spleen). The histopathological manifestations observed in the infected animal tissues were absent in all non-infected animals.

 

More research will be necessary to further elucidate and demonstrate more specific viral mechanisms. Future work may include verifying the YFV replication process in immune cells of sick and vaccinated patients, analyzing more of the range of host immune cells involved, and studying other viral mutations, as well as their varying symptoms. Outbreaks are commonly found in tropical areas of South America and Africa and are transmitted through mosquitoes. They occur relatively frequently, and in order to control outbreaks and produce effective vaccines, we need to understand the mechanisms of the virus. Analyzing these host cell interactions is significant progress toward understanding and targeting viruses of many diseases.

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