How rabies virus moves through nerve cells, and how it might be stopped

Cells in red against black background

To successfully infect its host, the rabies virus must move from the nerve ending to the nerve cell body where it can replicate. In a study published July 20 in the journal PLoS Pathogens, researchers from Princeton University reveal that the rabies virus moves differently compared to other neuron-invading viruses and that its journey can be blocked by a drug commonly used to treat amoebic dysentery.

Most viruses only infect the nervous system accidentally when the immune system is compromised. But some “neurotropic” viruses have evolved to target neurons as part of their normal infectious cycle. The rabies virus, for example, is transmitted when an infected animal bites into a host’s muscle. It then spreads into the end terminals of motor neurons innervating the muscle and travels along the neurons’ long axon fibers to the neuronal cell bodies. From there, the virus can spread throughout the central nervous system and into the salivary glands, where it can be readily transmitted to other hosts. Though rabies infections in humans are rare in the United States, the virus kills nearly 60,000 people annually.

Alpha herpesviruses, such as herpes simplex viruses, also enter peripheral nerve terminals and move along axons to the neuronal cell body, where they can lie dormant for the life of the host.

“Transport to the neuronal cell body is not a passive process, but an active one relying on the neuron’s own motor proteins and microtubule tracks,” said Lynn Enquist, Princeton’s Henry L. Hillman Professor in Molecular Biology, a professor of molecular biology and the Princeton Neuroscience Institute, and the study’s senior author. “Virus particles must engage this machinery for efficient transport in axons, otherwise infection cannot start.”

Viruses moving in a narrow nerve cell
Twenty-four hours after infecting nerve terminals, the rabies virus (red) reaches the cell bodies (green) of control neurons (left) but not neurons treated with emetine (right). Image credit: MacGibeny et al., 2018.

Enquist and colleagues previously found that alpha herpesviruses engage the neuronal transport machinery by stimulating protein synthesis at infected nerve terminals. Viral transport to the cell body can therefore be blocked by drugs that inhibit protein synthesis, as well as by cellular antiviral proteins called interferons.

In the current study, Enquist and colleagues investigated how the rabies virus engages the neuronal transport machinery. The researchers infected neurons with a virulent strain of the virus tagged with a red fluorescent protein, allowing the researchers to observe viral transport in real time by live-cell fluorescence microscopy.

The study was led by Margaret MacGibeny, who earned her Ph.D. in 2018, and associate research scholar Orkide Koyuncu, at Princeton, with contributions from research associate Christoph Wirblich and Matthias Schnell, professor and chair of microbiology and immunology at Thomas Jefferson University.

In contrast to alpha herpesvirus infections, the team found that interferons had no effect on rabies virus transport, perhaps because, until it reaches the neuronal cell body, the rabies virus hides out inside cellular structures called endosomes.

“We also couldn’t detect increased protein synthesis in axons upon rabies virus infection,” MacGibeny said. “But, to our surprise, we saw that a protein synthesis inhibitor called emetine efficiently blocked rabies virus transport to the cell body.”

Emetine had no effect on the transport of endosomes devoid of the rabies virus. But endosomes carrying the virus were either completely immobilized, or were only able to move short distances at slower-than-normal speeds.

Other protein synthesis inhibitors did not block rabies virus transport, however, suggesting that emetine works by inhibiting a different process in infected neurons.

“Emetine has been used to treat amoebic dysentery,” Koyuncu said. “In the laboratory it is widely used to inhibit protein synthesis but there are recent reports indicating that emetine has anti-viral effects that are independent of protein synthesis inhibition. Our study shows that this drug can inhibit rabies virus invasion of the nervous system through a novel mechanism that hasn’t been reported before.”

“The manuscript by MacGibeny et al. both advances and complicates our understanding of how neurotropic viruses make their way from the axon terminus to the cell body,” said Professor Glenn Rall, an expert in neurotropic virus infections at Fox Chase Cancer Center, who was not involved in the study. “Revealing variations in the axonal transport of neurotropic viruses, coupled with intriguing insights into new roles for well-known drugs, has both mechanistic and clinical implications for these life-threatening infections.

“Our next step is to figure out how emetine disrupts rabies virus transport in axons,” Enquist says. “Does it inhibit cell signaling pathways after rabies virus entry, or does it directly block the recruitment of motor proteins to virus-carrying endosomes?”

This study was funded by the US National Institutes of Health (grants P40 OD010996, RO1 NS33506, and F30 NS090640).

The study, Retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons, by Margaret A. MacGibeny, Orkide O. Koyuncu, Christoph Wirblich, Matthias J. Schnell, and Lynn W. Enquist was published in PLoS Pathogens. 2018. DOI: 10.1371/journal.ppat.1007188

Text courtesy of the Department of Molecular Biology

An immune signaling pathway for control of Yellow Fever Virus infection

By the Department of Molecular Biology

Princeton University researchers have uncovered a critical role for a new immune signaling pathway in controlling infection by the flavivirus Yellow Fever Virus (YFV).  The paper describing this discovery was published today in the journal mBio.

Infection with YFV causes a devastating illness with a mortality rate of up to 50%.  Like other members of its viral family—which includes West Nile Virus, Dengue Virus and Zika Virus—YFV is transmitted to humans by mosquitos that are expanding into new areas across the globe, exposing more people to these dangerous viruses. Fortunately, there is an effective vaccine for YFV: a live-attenuated strain of the virus, called YFV-17D, which differs by only a few amino acids from the virulent viral strain YFV-Asibi, but nonetheless provokes a potent and durable protective immune response in humans.

“An improved understanding of the complex mechanisms regulating YFV-17D attenuation will provide insights into key viral-host interactions that regulate host immune responses and infection outcomes, [and] open novel avenues for the development of innovative vaccine strategies,” said Alexander Ploss, assistant professor at Princeton’s Department of Molecular Biology, who led the study with first author Florian Douam, a postdoctoral research associate. However, research efforts have been hampered due to the fact that mice, which are used in the study of viral infections, are resistant to YFV infection. Nonetheless, recent mouse experiments have pointed to an important role for cytokines called interferons (IFN) in controlling the virus.

Mice, like humans, possess three types of interferons, molecules produced by the immune system during infection: type I interferons, which signal through the widely distributed IFN-α/β receptor; type II interferons that act on IFN-γ receptors present in most tissues; and type III interferons, which activate signaling by IFN-λ receptors found on epithelial cells. Mice lacking type I receptors die after infection by YFV-Asibi, but survive YFV-17D infection despite extensive viral replication at early stage of infection.Type II IFN signaling has also been shown to be important for clearing up late stage YFV-Asibi and YFV-17D infection when type I IFN signaling is defective. By contrast, the contribution of type III IFN signaling to control of YFV infection was unknown.

To address this question, Douam and colleagues studied YFV-17D infection in mice lacking the type III receptor. Initial experiments showed that these mice were able to control viral replication and rapidly cleared YFV-17D, indicating that type III signaling alone wasn’t necessary for resistance to YFV-17D. However, mice lacking both type I and type III receptors succumbed after YFV-17D infection, suggesting type III signaling does contribute to the antiviral immune response.

To find out more, the authors examined YFV-17D levels in various tissues. Early in the infection, the virus was present in every tissue of each mouse model examined. However, although viral loads were low in wild-type mice and type III receptor-deficient mice, they were much higher in type I and type I/III receptor-deficient mice. Surprisingly, the viral loads in brains of type I/III receptor-deficient mice increased over time in comparison to type I receptor-deficient mice, showing that loss of type III IFN signaling enhances the susceptibility of type I-receptor deficient animals to brain infection. This is significant because the presence of viruses in the brain can cause brain damage such as spongiosis or encephalitis. The low level of YFV-17FD brain invasion in wild-type mice caused mild spongiosis, whereas type I/III receptor-deficient mice had severe spongiosis—potentially explaining YFV-17D lethality in those animals.  However, this raised the question of why YFV-17 was present at such high levels in the animals’ brains.

Brain tissues
Left panel: Loss of Type I IFN signaling leads to active replication of the attenuated YFV strain (YFV-17D), which is accompanied by viral invasion of the brain and damage to brain tissues (spongiosis). Right panel: The additional loss of Type III IFN signaling in Type I IFN-deficient mice impairs the integrity of the blood-brain-barrier and alters immune cell function, which aggravates spongiosis and is ultimately lethal. Image Credit: Florian Douam and Alexander Ploss

Another study recently showed that type III IFN signaling affects the epithelial cells that make up the blood brain barrier (BBB), and modulates BBB integrity during infection by another flavivirus, West Nile Virus. Consistent with this, Ploss’s group observed that the BBB of type I/III receptor-deficient mice was especially leaky to a blue dye. But this wasn’t the only way that loss of type III IFN signaling impaired the body’s response to YFV; the researchers also found evidence that type III receptor deficiency provokes strong imbalances in several different kinds of immune cells during YFV-17D infection. In particular, type I/III receptor-deficient mice were defective in the activation of T cells, critical immune cells that control YFV-17D infection.

“We uncovered a critical role of type III IFN-mediated signaling in preserving the integrity of the blood brain barrier and preventing viral brain invasion,” Ploss said. More work is needed to explore how type III IFN signaling affects YFV infection in primates, but this study already provides important new insights about a poorly understood immune signaling pathway.

The study was supported by grants from National Institutes of Health (R01 AI107301, R21AI117213 to Alexander Ploss and R01 AI104669 to Sergei Kotenko). Additional funding included a Research Scholar Award from the American Cancer Society (RSG-15-048-01-MPC), the Princeton Environmental Institute‘s Grand Health Challenge program from Princeton University, and an Investigator in Pathogenesis Award by the Burroughs Wellcome Fund (all to Alexander Ploss).

The study, “Type III Interferon-mediated signaling is critical for controlling live attenuated Yellow Fever Virus infection in vivo.,” by Florian Douam, Yentil E. Soto-Albrecht, Gabriela Hrebikova, Evita Sadimin, Christian Davidson, Sergei V. Kotenko and Alexander Ploss was published in the journal mBio.  (2017). doi:10.1128/mBio.00819-17

Researchers propose surveillance system for Zika virus and other infectious diseases (The Lancet)

Test tube image courtesy of the NIH
Image courtesy of the National Institutes of Health

By Catherine Zandonella, Office of the Dean for Research

A group of prominent researchers from seven institutions including Princeton University are calling for the establishment of a worldwide program to collect and test blood and other human bodily fluids to aid in the study and prevention of emerging infectious diseases such as the mosquito-borne Zika fever, which is caused by the Zika virus and has spread throughout Latin America and the Caribbean since early 2015.

In an article published in The Lancet April 5, the authors call for the creation of a World Serology Bank that would include storage repositories located around the world that use a common set of best practices to collect and evaluate blood and other bodily fluids from people infected by various pathogens. The resulting data would help scientists better understand the susceptibility of humans to emerging diseases such as Zika fever. The information could be shared widely among scientists who track disease.

The authors include Princeton’s C. Jessica Metcalf, an assistant professor of Ecology and Evolutionary Biology and Public Affairs in the Woodrow Wilson School for Public and International Affairs, and Bryan Grenfell, the Kathryn Briger and Sarah Fenton Professor of Ecology and Evolutionary Biology and Public Affairs. In the interview below, Metcalf explains more about the World Serology Bank proposal.

Why is it important to create a World Serology Bank?

Serology is the study of bodily fluids including serum, the part of the blood that contains antibodies, with the aim of detecting the body’s immune response to pathogens. Serology provides us with the clearest window we have onto the landscape of susceptibility to pathogens across human populations, and the consequent risk of outbreaks. A World Serology Bank would shed light on the global risk of infectious disease outbreaks, and would be of tremendous public health benefit.

Why do you feel it is important to address this issue now?

With the emergence of pathogens like Zika virus, it becomes ever more important to understand what enhances or limits the global spread of these pathogens, and what the consequences of such spread may be across our pathogen communities. A World Serology Bank would provide a powerful mechanism toward such a global perspective.

What are the challenges involved in creating the Bank?

Challenges range from developing systems for collecting fluids, which can be done on a regular schedule or during specific disease events, to methods for sera storage and sera testing. Other challenges include defining who will administer the World Serology Bank, and what global data-sharing agreements will be put in place. Finally, we will need to develop new methods to translate what we learn from the evaluation of sera, such as patterns of susceptibility to specific pathogens, or protection from those pathogens. These methods will be driven by the underlying biology, and are likely to require an array of analytical innovations.

Read more

The article, “Use of serological surveys to generate key insights into the changing global landscape of infectious disease,” by C. Jessica E Metcalf, Jeremy Farrar, Felicity T. Cutts, Nicole E. Basta, Andrea L. Graham, Justin Lessler, Neil M. Ferguson, Donald S. Burke and Bryan T. Grenfell was published online in the journal The Lancet on April 5, 2016.