We demonstrate here for the first time infection of hESC-derived neurons by the human-specific neurotropic virus VZV. The only previous report, to our knowledge, using hESC-derived neural cells for study of viral infection was one using hESC-derived oligodendrocyte precursors as a model for studying neural infection by the polyomavirus JC virus (23
). In contrast to previous in vitro
studies using human fetal ganglia or dissociated neurons (8
), our use of GFP-expressing VZV allowed immediate, live observation of infection of neurons. In preliminary experiments we have also observed infection of these hESC-derived neurons by two additional GFP-labeled herpesviruses, pseudorabies virus (PRV) and HSV-1 (data not shown), suggesting that this model is useful for study of other members of Herpesviridae
Mitotic inhibition of MeWo cells permitted efficient cell-cell infection with four different GFP-expressing VZVs without the input cells overwhelming the quiescent neural cultures. Infection occurred rapidly, resulting in GFP expression by neurons within 2 days of adding VZV, with about half of the neurons being infected by 4 days, consistent with observations of VZV-infected human DRG neurons (9
). At 7 to 9 days, we observed that a majority of neurons were infected. As expected, increasing the number of input cells resulted in more rapid infection of the cultures.
The use of free GFP-expressing VZV and VZV expressing GFP protein fusions each conferred its own experimental advantage. The use of GFP-tagged proteins allows the distribution of viral proteins during various stages of neuronal infection to be observed, and in the case of the capsid protein ORF23, the movements of nucleocapsids/virions can be followed. On the other hand, infection with the VZVBAC virus allowed the tracing individual neurons and their entire arborizations because of the diffuse filling of infected cells with GFP.
It was difficult to assess the ratio of input MeWo cells to neurons in these experiments for two reasons. First, most of the neurons were present in large clumps, which prevented the input cells from coming in direct contact the large majority of neurons in the plate upon initial seeding since many neurons are below the surface of the clumps. In addition, the remaining neurons were scattered at low density on the plate, and therefore many did not come into direct contact with the seeded MeWo cells. As expected, however, increasing the number of input cells increased the pace of infection of the cultures since more neurons were contacted by the MeWo cells.
Neuronal infection producing cell-free infectious virus has been demonstrated by using supernatant from VZV-infected cultured human DRG to infect fibroblasts (8
). The presence of intact extracellular virions was also observed using TEM in that study. Using both these techniques, we have confirmed the ability of VZV-infected neurons to package and release infectious virus. GFP-labeled VZV greatly simplified the assay for infectious virus, allowing infections to be observed in real time.
Long-term, persistent VZV infection is characteristic of the SCID-hu DRG model, with neurons apparently transitioning from a productive to a latency-like state (reviewed in reference 31
). We attempted to follow some of our cultures for 3 to 8 weeks. From the point that most of the neurons were infected at 7 to 9 days, neurons were observed to start to die and detach from the substrate. It was unfortunately difficult to follow the time course of the death of specific neurons because of the constant death, migration of neurons, and the presence of many of the neurons in 3D clumps. We have observed, though, some GFP-expressing neurons at extended culture periods (3 to 8 weeks), suggesting the possibility of persistent infection. However, since we were unable to determine at what point the GFP-expressing neurons had been infected and whether the GFP-expressing neurons had been infected at one time and ceased to make GFP, we do not yet know if the model is a valid model for persistent and/or latent infection. This will require development of experimental conditions that allow long-term observation of individual neurons after VZV infection.
This new model for the study of VZV, like all in vitro
systems, has advantages and disadvantages compared to in vivo
studies. Compared to the SCID-hu DRG system, this new model has the advantages of rapid setup, simple infection, and the ability to follow the time course of infection in real-time studies. Another advantage of using hESC-derived neurons is the availability of virtually unlimited amounts of human neural cells, compared to the difficulty in obtaining human fetal material for SCID mouse xenografts or cell/ganglion culture. This combination of ready availability of neural cells and rapid infection makes the system ideal for testing of newly engineered viruses that have proved so invaluable for understanding the function of VZV gene products. For example, the roles of specific VZV gene products in axonal transport, viral entry into neural cells from the distal portion of the axons, and viral assembly can be quickly and rapidly approached using genetically modified virus and live imaging or electron microscopy. In addition, this system can easily be scaled up and automated, which would allow the high-throughput testing of libraries of antiviral agents. However, a disadvantage of this system is the lack of the appropriate histological organization of the DRG and the possibility that not all types of the neurons found in the DRG are represented. Somewhat mitigating this is the fact that hESC cells can be differentiated into many neural phenotypes, potentially allowing the study of infection of specific classes of neurons and glia (see below). A further disadvantage of this model is that the interactions of the neurons with their in vivo
neighbors cannot yet be achieved. The SCID-hu DRG model has allowed the description, for example, of the interaction between satellite cells and DRG neurons during VZV pathogenesis (22
The use of hESC-derived neurons for studying VZV permits two additional types of studies hitherto impossible. First, specific neural phenotypes can be derived from hESC, including spinal motoneurons (24
), DRG neurons (19
), retinal neurons (14
), and central nervous system (CNS) (11
) and peripheral nervous system (PNS) (19
) glia. This would enable the comparison of VZV infection/life cycles in each type of neuron and the type of infection. For example, work with HSV-1 and HSV-2 has suggested that latency of each virus is preferentially established in specific neuronal subtypes (16
). A second, perhaps more important, aspect of this model is the ability to genetically manipulate the hESC before making them into neurons (5
), which will allow the study of the effects of specific host genes (receptors, axonal transport machinery proteins, etc.) on the VZV life cycle in neural cells. The feasibility of such experiments has already been shown in a study of the myelin-associated glycoprotein (MAG) proteins in VZV infection of an oligodendrocyte cell line (25
Growing hESC-derived neurons in compartmented microfluidic chambers combined with the use of GFP-labeled viruses allowed the demonstration for the first time of experimental axonal infection and retrograde transport of VZV using two different viruses. The amount of time until cell bodies were retrogradely infected was variable for reasons that we do not understand. Interestingly, we have not yet observed anterograde infection of MeWo cells by axons following VZV infection of cell bodies of neurons using microfluidic chambers to date. The reasons for this are not yet clear, but it may be due to insufficient copies of the virus made in the cell body to allow viral transport to the axon terminals for infection of targets. A relatively low quantity of virus produced and/or transported in VZV-infected neurons is consistent with clinical observations that reactivation and replication of VZV in the DRG do not always lead to zoster lesions. Study of the conditions required for anterograde VZV transport is currently under active investigation.
The microfluidic chamber experiments confirmed neuronal productive infection since individual infected neurons went on to infect its neighbors. Use of VZV GFP fusion protein constructs of viral structural proteins allowed observation of viruses in axons. Live-imaging techniques are now being used for study of VZV axonal transport kinetics, as has been performed for HSV-1 and pseudorabies viruses (15
). This system should also allow testing of the roles of specific VZV proteins in retrograde transport, as well as of screening drugs that block trafficking and neuronal transport to the site of VZV latency.