ABSTRACT Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins. IMPORTANCE Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.
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A Review on Ultrasonic Neuromodulation of the Peripheral Nervous System: Enhanced or Suppressed Activities?
Ultrasonic (US) neuromodulation has emerged as a promising therapeutic means by delivering focused energy deep into the nervous tissue. Low-intensity ultrasound (US) directly activates and/or inhibits neurons in the central nervous system (CNS). US neuromodulation of the peripheral nervous system (PNS) is less developed and rarely used clinically. The literature on the neuromodulatory effects of US on the PNS is controversial, with some studies documenting enhanced neural activities, some showing suppressed activities, and others reporting mixed effects. US, with different ranges of intensity and strength, is likely to generate distinct physical effects in the stimulated neuronal tissues, which underlies different experimental outcomes in the literature. In this review, we summarize all the major reports that document the effects of US on peripheral nerve endings, axons, and/or somata in the dorsal root ganglion. In particular, we thoroughly discuss the potential impacts of the following key parameters on the study outcomes of PNS neuromodulation by US: frequency, pulse repetition frequency, duty cycle, intensity, metrics for peripheral neural activities, and type of biological preparations used in the studies. Potential mechanisms of peripheral US neuromodulation are summarized to provide a plausible interpretation of the seemly contradictory effects of enhanced and suppressed neural activities of US neuromodulation.
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- Award ID(s):
- 1727185
- NSF-PAR ID:
- 10173985
- Date Published:
- Journal Name:
- Applied Sciences
- Volume:
- 9
- Issue:
- 8
- ISSN:
- 2076-3417
- Page Range / eLocation ID:
- 1637
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/app9081637
