An In Vitro Model of Latency and Reactivation of Varicella Zoster Virus in Human Stem Cell-Derived Neurons

An In Vitro Model of Latency and Reactivation of Varicella Zoster Virus in Human Stem Cell-Derived Neurons

Ingenol mebutate (Picato®) gel may cause severe skin reactions, eye injury, or herpes zoster (shingles) reactivation. The attenuated vaccine virus can reactivate and cause herpes zoster; Later in life, the virus can reactivate, causing shingles, which is a very different illness than chickenpox. All of us who had chickenpox can later have herpes zoster. Quiescent infections were established by exposing neurons to low titer cell-free VZV either by using acyclovir or by infection of axons in compartmented microfluidic chambers without acyclovir. Severe eye injuries identified include severe pain, chemical conjunctivitis, corneal burn, eyelid edema, eyelid ptosis, and periorbital edema. What is the varicella-zoster virus and how does it cause shingles? To date it has not been fully clarified which factors lead to reactivation of the virus from its dormant state.

Comparison of VZV genome transcription in quiescently-infected to productively-infected neurons using RNASeq revealed preferential transcription from specific genome regions, especially the duplicated regions. It should not be applied in or near the mouth, lips, or eye area and not to an area larger than 25 cm2. If you are having treatment for cancer, for example, you are more likely to get shingles. These symptoms can be present one to three days before the redness on the skin. Study of VZV latency and reactivation has been severely hampered by the inability to reproduce a persistent state in vitro or in vivo that can be experimentally reactivated. Our study establishes a system using human neurons derived from embryonic stem cells where multiple stimuli can induce reactivation from long term experimental latency. While the disease is not life-threatening, it can cause a painful rash anywhere on your body and usually appears as a single stripe of blisters that wraps around one side of the torso.

Very rarely, herpes zoster can manifest it self only in pain and without blisters, or with blisters but with no sense of pain. Herpes Zoster, which results from reactivation of latent varicella zoster virus (VZV) is a common and debilitating disease that is frequently complicated by acute pain, diverse neurological sequelae, vision problems and difficult-to-treat chronic pain known as post-herpetic neuralgia. The VZV latent state is established in human sensory neurons of ganglia along the entire neuraxis during primary infection and disease, chickenpox. Usually the virus does not cause any further problems; however, the virus may re-emerge years later, causing shingles. Persons who have herpes zoster can transmit the virus if the blisters are not intact. [3],[4]) in latently-infected ganglia. However, the recent recognition that latent VZV genomes undergo viral transcription in ganglia following post mortem removal raised doubt as to what transcriptional events occur in the latent state [2].

The same virus also causes herpes zoster, or shingles, in adults. In more severe cases and when the eye is caught in the infection, the dermatologist prescribes a combination of systemic corticosteroids and antiviral medication. The most commonly reported transcript in human ganglia is that for ORF63 [7] (which has also been observed in rodent neurons in a model for VZV latency, i.e. [8], that encodes a transcriptional regulatory protein during lytic infection that may influence apoptosis and host cell survival [9],[10]. Shingles is due to a reactivation of varicella zoster virus (VZV) within a person’s body. In contrast to HSV, for which there are both small animal and in vitro models for latent infection that can be reactivated, there is no widely-used in vivo small animal model of latency or any in vitro system of persistent infection in which reactivation can be experimentally induced. Indeed, VZV has proven to be difficult to induce to reactivate, even from post-mortem human ganglia harboring latent VZV genomes.

An In Vitro Model of Latency and Reactivation of Varicella Zoster Virus in Human Stem Cell-Derived Neurons
The strict human specificity of VZV has precluded the use of most rodents as models of latency because no animal model reproduces human disease and most rodents do not even support VZV replication. Anyone 60 years of age or older should get the shingles vaccine, regardless of whether they recall having had chickenpox, which is caused by the same virus as shingles. However, it is possible that data obtained from it may not extend to human ganglionic latency due to species differences. Human dorsal root ganglia tissue transplanted to SCID mice have been pioneered for study of VZV neuronal infection by Arvin and colleagues (reviewed in [13], [14]). Human DRG obtained from 2nd trimester fetuses can be infected with VZV administered either directly into the fetal DRG graft, or following venous administration of VZV infected human T-cells. A slight weakening of the immune system in older people may account for the virus reactivating and multiplying to cause shingles. Low levels of transcripts from the ORF63 genomic region were detected in this model system, but reactivation of VZV in the model has not yet been documented.

As a closed system of a complex tissue, it is not amenable to real time evaluation of VZV infection and reactivation. It is also hampered by the limited supply of fetal tissues, expense and being a technically demanding system. If you are having treatment for cancer, for example, you are more likely to get shingles. Several in vitro models of using cells of human origin have therefore been proposed for studying for VZV neuronal replication and persistence. These include the use of SH-SY5Y neuroblastoma derived neuron-like cells [15], and human neurons obtained from fetal DRG or differentiated from stem cells (hESC, iPSC and neural stem cells, i.e. [16],[17],[18],[19]. If you present with the symptoms of the virus and only have a rash that presents with papulae around your trunk, he may just say you have the Zoster Virus.

We recently presented evidence that the different outcomes of VZV infection is partly influenced by the quantity of virus used to infect the cultures [21], although lack of spread of infection observed in some models complicates the interpretation of what kind of infection the persistence actually reflects. One report in which persistent non-productive VZV infection of iPSC-derived neurons occurred, presented evidence for widespread transcription suggestive of an abortive type of infection, rather than a persistent quiescent state [6]. We argue that a compelling experimental model of neuronal VZV latency requires the ability of the cells to be able to fully support VZV infection and spread upon experimental reactivation. Also disseminated herpes zoster is more likely to occur in such people. We present here an extension of our previously described hESC-derived neuron model system, for which we have established conditions that lead to a prolonged, non-productive neuronal VZV infection that can be experimentally reactivated. Persistent VZV infections can be established either using acyclovir to block lytic infections, or by infection of axons without use of antiviral drugs. Persistently-infected human neuronal cultures maintain viral genomes for up to two months, with minimal transcription and undetectable translation of several VZV proteins.

If the deeper layers of the cornea are involved, this is more serious and is called stromal keratits. We further show that stimulation combined with incubation at a reduced temperature (34°C) results in a productive, spreading infection. Comparison of the transcriptomes of quiescently vs productively-infection human neurons by RNAseq analysis reveals a preferential RNA transcription of select genomic regions during latency. In particular, the short repeated regions of the VZV genome encoding the regulatory proteins IE62 and IE63 are enriched for transcription in persistently-infected neurons. Herpes zoster (shingles) is a painful rash caused by the same virus that causes chickenpox. We have previously reported that hESC-derived neurons exposed to high MOI cell-free recombinant pOKA-derived VZV with fluorescent protein reporters of viral protein expression, results in a spreading, productive infection [21]. In order to obtain non-productive persistent VZV infections in hESC-derived neurons, we exposed them to low PFU (0.001 MOI) of cell-free VZV in the presence of acyclovir (ACV) for 6 days.

To detect productive infection, we used VZV66GFP [22] where GFP is fused to the N terminus of ORF66. This virus initially causes chickenpox but then remains dormant in nerve cells. Using this approach (shown schematically in S1 Fig), in approximately half of neuron-containing wells exposed to VZV individual ORF66GFP+ neurons appeared in 1–5 small clusters, while the remainder did not contain any GFP+ cells (Fig 1). By comparison, infection of parallel cultures with higher levels of cell free VZV (MOI 0.02) resulted in GFP expression in many neurons by 3 dpi and an obvious productive, spreading infections. After 6 days we removed the ACV from the low MOI exposed cultures, and maintained them in its absence up to 7 weeks. The herpes zoster virus can cause numerous eye problems, including:. We conjecture that the combination of ACV treatment and low MOI conditions are at the threshold for generating a productive infection, resulting in ½ the wells containing GFP+ cells that overrode the ACV inhibition, while the other half were effectively prevented from expressing ORF66.

No visible cytopathic effect was observed in these GFP- wells throughout the experimental period. S1A Table indicates the numbers of GFP+ and GFP- negative wells we observed in our initial 7 experiments. The duration of protection afforded by the vaccine remains a matter of investigation, and vaccinated individuals may require additional booster doses later in life.

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