Three cell-based models shed light on how herpes simplex virus type 1 (HSV-1) infection, which can spread to the fetal brain during pregnancy, may contribute to various neurodevelopmental disabilities and long-term neurological problems into adulthood, according to a study published October 22, 2020 in the open-access journal PLOS Pathogensby Pu Chen and Ying Wu of Wuhan University, and colleagues.
HSV-1 is a highly prevalent pathogen that can cause lifelong neurological problems such as cognitive dysfunction, learning disabilities, and dementia. But progress in understanding the role of HSV-1 in human fetal brain development has been hampered by restricted access to fetal human brain tissue as well as limitations of existing animal models.
To address this gap in knowledge, the researchers generated three different cell-based neurodevelopmental disorder models, including a 2D layer of cells and a 3D brain-like structure.
These models are based on human induced pluripotent stem cells (hiPSCs) – immature, embryonic stem cell-like cells that are generated by genetically reprogramming specialized adult cells.
HSV-1 infection in neural stem cells derived from hiPSCs resulted in activation of the caspase-3 apoptotic pathway, which initiates programmed cell death. HSV-1 infection also impaired the production of new neurons, and hindered the ability of hiPSC-derived neural stem cells to convert into mature neurons through a process called neuronal differentiation.
Moreover, the HSV-1-infected brain organoids mimicked the pathological features of neurodevelopmental disorders in the human fetal brain, including impaired neuronal differentiation and abnormalities in brain structure.
In addition, the 3D model showed that HSV-1 infection promotes the abnormal proliferation and activation of non-neuronal cells called microglia, accompanied by the activation of inflammatory molecules, such as TNF-α, IL-6, IL-10, and IL-4.
According to the authors, the findings open new therapeutic avenues for targeting viral reservoirs relevant to neurodevelopmental disorders.
The authors add, “This study provides novel evidence that HSV-1 infection impaired human brain development and contributed to the neurodevelopmental disorder pathogen hypothesis”.
Herpes Simplex Type 1
HSV-1 is a double-stranded DNA herpesvirus belonging to the Alphaherpesvirinae subfamily . It is an important neurotropic human pathogen that can infect also other species, especially non-human primates , as well as numerous cell types in vitro, although humans are the natural hosts .
HSV-1 is one of the most widely spread human viral pathogen, and around 67% of the global population have antibodies to this pathogen . Primary infection takes place in epithelial cells and the virus is transmitted to new hosts via saliva. In this stage, HSV-1 typically causes labial and oral lesions, and although it may also cause genital herpes, the most common sexually transmitted type is herpes simplex virus type 2 (HSV-2) [23,24,25].
In addition, HSV-1 can cause severe pathologies such as encephalitis or keratoconjunctivitis . HSE includes severe brain damage with hemorrhage, edema, and necrosis, and mostly affects the frontal and temporal lobes and the limbic system.
It is generally considered that HSV-1 primary infections utilize oral routes of entry, given the common presentation of oral lesions. However, it has been argued that the acute oral lesions of human HSV-1 infections do not necessarily reflect oral host entry, and that the routes used for primary infection and reactivation are not necessarily the same .
After infection of epithelial cells, HSV-1 spreads to the peripheral nervous system (PNS), entering sensory neurons by fusion with the plasma membranes of their sensory terminals. Then, HSV-1 travels retrogradely to the cell body and establishes a latent infection in the trigeminal ganglia (TG) .
However, latent virus may also locate to CNS structures such as the olfactory bulb (OB), brainstem, or temporal cortex. During latency, the virus persists in the cell nucleus as an episome; the expression of lytic genes is repressed, and conversely the expression of latency-associated transcripts (LAT) begins.
Periodically, HSV-1 may leave the latent state and reactivate, either spontaneously or in response to stimulation from immunosuppression, fever, ultraviolet light exposure, or injury to the tissues innervated by latently-infected neurons . This process may lead to a recurrent lesion, but it may also proceed asymptomatically.
Although reactivation is a complex process triggered by causes that are not fully understood, it has been demonstrated that the immune system plays a critical role, and in this regard host stress may lead to HSV-1 reactivation by increasing regulatory T cell (Treg) control of CD8+ T lymphocytes .
The roles of Tregs in the context of viral infections seem to be highly complex; Tregs may exert radically different roles depending on the infectious agent, the disease phase, or the genetic profile of the host, both suppressing antiviral immune responses and contributing to viral spread and establishment of latency, or conversely contributing to virus control . Latency is also an epigenetically controlled process  in which changes induced by different stressors may trigger viral reactivation [30,31].
During reactivation, the virus travels anterogradely along the axon, replicating in the tissue of the dermatome innervated by the sensory neuron in which the virus established latency.
HSV-1 Infection of the CNS
HSV-1 may enter the CNS by two main routes: peripheral neurons and the bloodstream. Two cellular barriers (the blood–brain barrier and the blood–cerebrospinal fluid barrier) protect the CNS, separating it from the circulatory system [5,32].
However, other pathways to the CNS are available to pathogens, such as the olfactory system and the trigeminal nerve, which bypass the cellular barriers and provide a direct portal into the brain. Therefore, the trigeminal and olfactory nerves constitute direct routes to the brain that can evade the barriers imposed by the circulatory system .
The neurotropic character of HSV-1 has been known for almost a century, since experimental corneal infection produced encephalitis in rabbits, suggesting that the virus propagated through axons and synapses by invasive proliferation . It is currently assumed that primary HSV-1 infection takes place in epithelial cells and subsequently reaches the PNS by direct cell-to-cell retrograde spread to the nerve endings of nearby sensory neurons (Figure 1A,1).
During reactivation, HSV-1 virions travel by anterograde transport; that is, from the cell soma of the sensory neuron to the epithelial cells where the primary infection arose (Figure 1A,2). Another pathway for viral spread is the trans-synaptic route, e.g., from one neuron to an adjacent one across the synaptic cleft (Figure 1A,3).
Unlike other neurotropic viruses that do not cross synapses, such as Moloney murine leukemia virus (MMLV), lentivirus, adeno-associated virus (AAV), or human adenovirus 5 (Ad5) , trans-synaptic spread is a major mode of HSV-1 propagation [35,36].
In fact, given their ability to spread bidirectionally along multi-synaptic pathways, herpesviruses, particularly HSV-1 and PRV, were the first to be widely used to trace neuronal circuits, providing information about connectivity between different areas of the brain [34,37,38,39].
Using HSV-1 as a trans-neuronal tracer, the virus has been observed to cross the synaptic space to label third- to fourth-order neurons .
After infection of sensory neurons, HSV-1 travels retrogradely to the cell bodies and establishes a latent infection in the TG (Figure 1B). All branches of the trigeminal nerve (ophthalmic, maxillary, and mandibular) may serve as portals for HSV-1 (Figure 1C), with entry from the oral and nasal epithelia or the cornea. Occasionally, the virus may infect the CNS if it spreads trans-synaptically from the TG to the trigeminal nucleus in the brainstem, from where it might disseminate to higher brain areas (Figure 1B).
In fact, there are polysynaptic pathways from the brainstem to the thalamus and somatosensory cortex (Figure 1C) that might be hypothetically utilized by the virus. However, in human HSV-1 infections, viral antigens are mostly found in the olfactory pathways, the temporal cortex, and the limbic system, but not in higher brain areas related to the trigeminal projection pathways, such as the somatosensory cortex [41,42,43,44].
Therefore, those findings support the hypothesis of olfactory spread to the CNS in humans, although spread from the trigeminal nerve to the orbitofrontal and medial temporal lobes has been also proposed. In this regard, the meninges of the middle and anterior fossae are innervated by nerves derived from the TG, and the trigeminal nerve projects to the dura mater via the tentorial nerve, which arises from its ophthalmic division .
Spread of the virus to the anterior and middle fossae via tentorial nerves has been proposed to explain viral dissemination to frontal and temporal lobes . However, if the trigeminal pathway was the main portal to the CNS, a higher rate of HSV-1 encephalitis affecting the brainstem would be expected .
Finally, the evidence is not sufficient to establish solid conclusions, and further studies are necessary to fully clarify this aspect regarding pathways of HSV-1 entry into the CNS.
Besides the trigeminal nerve, HSV-1 may enter the CNS via the sensory neurons of the olfactory neuroepithelium (Figure 2A). From there, the virus may access the OB and then spread through the olfactory tract to reach limbic structures such as the hippocampus, amygdala, or orbitofrontal cortex (Figure 2A).
In the olfactory neuroepithelium, the cilia of the olfactory sensory cells covering the upper nasal cavity provide a portal for viral entry (Figure 2B). The dendrites of these neurons are covered by a thin layer of mucus that protects them. The axons of these first-order olfactory sensory neurons gather in bundles that project through the cribriform plate of the ethmoid bone to reach the OB, forming the olfactory nerves.
Once in the OB, the olfactory sensory neurons synapse with mitral and tufted cells in the glomeruli; the virus may infect these cells trans-synaptically at the glomeruli and spread along the olfactory tract towards the olfactory projection pathways. In the olfactory tract, HSV-1 may access the ipsilateral olfactory projection areas via the lateral olfactory stria. However, virus circulating along the medial olfactory stria may reach the contralateral olfactory structures via the anterior commissure (Figure 2C).
HSV-1 and Demyelination
MS is an immune-mediated demyelinating and neurodegenerative disease of the CNS of unknown etiology, although several viruses are known to be involved in such demyelinating diseases [6,8,95,96].
The disease is multifactorial, influenced by genetic and environmental factors , and it is characterized by multifocal demyelinating lesions in both the white and gray matter  of the brain and spinal cord.
These lesions can be associated with axon degeneration and synaptic loss. MS is typically multifocal and multiphasic (relapsing), and lesions are thought to be caused by infiltration of immune cells into the CNS .
One hallmark of MS is the presence of oligoclonal IgG bands (OCBs) in the cerebrospinal fluid (CSF) of patients. These OCBs, which indicate an anomalous intrathecal B-cell response, are found in the CSF of more than 95% of MS patients and cannot be detected in serum.
OCBs are typically detected in inflammatory and infectious CNS disorders. In addition to MS, there are other pathologies with reported CSF OCBs, such as systemic lupus erythematosus, aseptic meningitis, HIV infection, and HSV-1 encephalitis .
It has been suggested that OCBs are directed against the infectious agent that causes the disease, and that MS might be triggered by an agent against which the antibody response in the brain and CSF was directed . In fact, OCBs from patients with infectious CNS diseases have been proven to recognize the relevant infectious agent .
Regarding MS, OCBs directed against EBV and HHV-6 have been identified in patients . OCBs directed against HSV-1 in the CSF of patients with MS has also been reported , although other studies did not find reactivity to HSV-1 antigens . Regardless, the antibody activity of most OCBs remains unknown to date.
It is known that human oligodendrocytic cells are susceptible to HSV-1 in vitro . In murine models, infected OLs were found in the mandibular division of the spinal trigeminal tract after infection of mice through cranial nerve XII . Early studies with animal models showed latent TG infections and demyelinating lesions in mice intranasally infected with HSV-1 .
Later research also reported that mice infected with HSV-1 can develop lethal encephalitis or virus-induced CNS multifocal demyelinating lesions, with outcome affected by several factors, including the route of infection and mouse strain [108,109,110,111,112].
Demyelinating lesions have also been associated with HSV-1-induced facial nerve paralysis . A recent study demonstrated a direct association between infection with HSV-1 and multifocal brain demyelination in a murine model . Moreover, in that study, demyelination was followed by remyelination, although it was incomplete and the presence of scars was observed.
As in studies with experimental animals, resistance to HSV-1 varies between primary cultures of human OLs and is donor-dependent . Susceptibility to HSV-1 encephalitis may be caused at least partly by mutations in Toll-like receptors that decrease the intrinsic resistance of CNS cells (neurons and OLs in particular) to HSV-1 infection [116,117].
An association between HSV-1 infection and demyelination has been also suggested from studies with human patients. HSV was detected early in the CNS of an MS patient , and later HSV-1 was also isolated from the CSF of a patient during the first attack of MS .
Postmortem brain samples from 37 cases of MS were screened for HSV-1 and HSV-2, finding higher prevalence of HSV in MS patients compared to controls and in more active plaques than inactive plaques [120,121]. A case of acute MS preceded by varicella-zoster virus (VZV) infection was reported at the same time as intrathecal reactivation of HSV-1 and HHV-6 , and a coincident onset of HSV-1 encephalitis and MS has been also described .
In another study, patients of MS treated with valacyclovir showed a reduction in the number of new active demyelinating lesions and a decrease in the number of scans free of new active lesions . HSV-1 may also play a role in triggering MS relapses during clinical acute attacks of MS, at least in the most frequent clinical presentation of the disease, the relapsing–remitting form .
HSV-1 DNA was detected by PCR in peripheral blood mononuclear cell (PBMC) samples from relapsing–remitting MS patients . However, the involvement of HSV-1 in MS etiology is far from confirmed, and other investigators have proposed other herpesviruses as more plausible etiological agents [127,128,129], or even doubt the role of herpesviruses in the etiology of demyelinating diseases .
Viruses, specifically HSV-1, might not operate as unique causative agents, but rather as risk factors, and indeed genetic susceptibility and the immune system are also crucial in demyelinating pathologies. For instance, when a recombinant HSV-1 constitutively expressing interleukin-2 (IL-2) was inoculated into mice, it provoked CNS demyelination and optic neuropathy, whereas infection with recombinant viruses expressing IL-4, gamma interferon, IL-12p35, IL-12p40, or IL-12p70 did not induce this effect . On the other hand, donor-dependent differences in resistance to infection with HSV-1 were established in primary cultures of human OLs .
HSV-1 and Endogenous Retroviruses
Endogenous retroviruses (ERVs) are vestiges of ancient retroviral infections that remain in the host genomes of all vertebrates. They make up around 8% of the human genome and their expression may be triggered by environmental factors, inducing pathogenesis under some circumstances.
Several human ERV (HERV) transcripts and proteins have been identified in the CNS, often associated with neuroinflammation , and there is a solid epidemiological association between MS and the expression of ERVs, which are upregulated in the brains of MS patients compared to healthy controls [12,133,134,135,136].
The MS-associated retrovirus (MSRV), a member of the HERV-W family, has been frequently isolated from MS patients , and its presence in the CSF of these patients has been associated with a greater rate of disability and progression of the disease .
Herpesviruses have been implicated in regulation of the HERV-W family , and a role for HSV-1 in HERV-W  and HERV-K  expression has been reported. HERV-W–MSRV expression may be enhanced by HSV-1 in leptomeningeal cells [142,143]. HSV-1 can activate HERV-W in cells involved in MS pathogenesis, such as B cells, macrophages, microglia, and astrocytes , and may induce ERV proteins .
Syncytins are Env glycoproteins encoded by ERV genes that are involved in mammalian placental morphogenesis [146,147]. These proteins stimulate cell–to–cell fusion in a process analogous to viral entry, promoting the formation of syncytia , and can activate pro-inflammatory and autoimmune processes .
Syncytin-1, an Env glycoprotein encoded by the HERV-W env gene, plays a crucial role in placental trophoblastic formation and has an immunosuppressive role that impedes rejection of the fetus by the maternal immune system. However, this protein has also been associated with different pathogenic processes, triggering neuroimmune activation and OL damage . I
n this regard, syncytin-1 may inhibit the differentiation of oligodendroglial precursors, thus hindering remyelination . In astrocytes, overexpression of syncytin-1 (which is upregulated in glial cells in demyelinating lesions of MS patients) triggered the release of redox reactants, inducing neuroinflammation and death of OLs .
In this context, HSV-1 has been demonstrated to upregulate syncytin-1 , and therefore deepening the understanding of this role may greatly increase knowledge of the demyelinating processes.
HSV-1 and Molecular Mimicry
Another mechanism that has been associated with HSV-1-related demyelination is molecular mimicry. In this process, the peptides of a pathogen are similar to those of the host organism, triggering activation of autoreactive immune cells in susceptible individuals.
Viruses may induce autoimmunity by molecular mimicry [151,152], and several viral peptides have been shown to activate autoreactive T cells . The triggering of autoimmunity by HSV-1 infection was demonstrated when an epitope expressed by the capsid protein was recognized by autoreactive T cells targeting corneal antigens in a murine model of autoimmune herpes stromal keratitis .
Mimicry between an epitope shared by the HSV-1 glycoprotein gB and a brain-specific factor has also been reported, supporting the hypothesis that viral infections may prompt the production of self-reactive CSF antibodies .
After a HSE episode, a complex immune cellular and humoral response starts. Cytotoxic T lymphocytes lyse cells infected by HSV-1 and the production of inflammatory cytokines (predominantly by Toll-like receptor [TLR] 2) commences .
The presence of cytokines such as IL-6, interferon gamma, or TNF alpha, which are detected in the serum and CSF of HSE patients, indicates a strong immune response and suggests that inflammation may contribute to the pathological impact of the viral infection .
However, autoimmunity may be another effect triggered by infection. In fact, HSV-1 infection may induce antibodies against neurotransmitter receptors.
After an episode of encephalitis caused by HSV-1, around 10–20% of patients may experience a relapse syndrome known as post–herpes simplex encephalitis (PHSE) .
This syndrome is immune-mediated, and many patients acquire antibodies against the ionotropic glutamate NMDA receptor (NMDAR). In addition to NMDAR, PHSE may trigger the release of antibodies against GABA A and AMPA receptors, or other still unidentified antigens [157,158,159].
However, although a link between HSV-1 and anti-NMDAR encephalitis has been found, it is not completely clear whether or not molecular mimicry is the responsible mechanism .
Regarding ocular HSV-1 infection, it has been proposed that virus infection might result in unmasking of corneal autoantigens, leading to chronic autoreactive T cell stimulation. Alternatively, autoimmunity might be explained by a process of molecular mimicry triggered by a viral peptide sharing reactivity to the unmasked corneal autoantigen. Thus, the initial antiviral response against the virus might subsequently become sustained by autoreactive Taggressor cells .
HSV-1: A Role in Remyelination Impairment?
Primary demyelination in the CNS is a process by which a direct injury to OLs harms the myelin sheath. On the contrary, in secondary demyelination (Wallerian degeneration), the myelin sheath degenerates as a result of primary axonal loss . After demyelinating processes, a mechanism of remyelination starts, which aims to repair the damaged myelin.
This process is critically regulated by numerous intracellular signaling pathways [162,163] and depends on generation of new mature OLs derived from a population of adult CNS precursor cells (adult OL precursor or progenitor cells (OPCs)).
Remyelinating OLs can derive from the adult subventricular zone (SVZ), a region which may be a source of remyelinating OLs during MS, although the contribution of cells derived from that region might be small compared to the local sources .
It has been suggested that infection of OPCs with HHV-6 might impair remyelination . In response to demyelinating damage, OPCs proliferate and migrate to the lesion site, where they differentiate into myelinating OLs and wrap damaged axons with new myelin sheaths.
A hypothetical viral infection of OPCs leading to impairment of differentiation or migration might affect remyelination in patients with demyelinating diseases . In this context, HSV-1 might exert a similar role, as it has been proven to infect OPCs in vitro, although the infection increases along with differentiation . Therefore, infection of a population of OPCs by HSV-1 during remyelinating processes might affect this process, resulting in impairment of remyelination.
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404202/