Varicella zoster virus (VZV) is a ubiquitous, human alphaherpesvirus that produces varicella on primary infection then becomes latent in ganglionic neurons along the entire neuraxis. In elderly and immunocompromised individuals, VZV reactivates and travels along nerve fibers peripherally resulting in zoster. However, VZV can also spread centrally and infect cerebral and extracranial arteries (VZV vasculopathy) to produce transient ischemic attacks, stroke, aneurysm, sinus thrombosis and giant cell arteritis, as well as granulomatous aortitis. The mechanisms of virus-induced pathological vascular remodeling are not fully elucidated; however, recent studies suggest that inflammation and dysregulation of programmed death ligand-1 play a significant role.

In varicella-zoster virus (VZV)-infected primary human brain vascular adventitial fibroblasts (BRAFs), levels of beta interferon (IFN-␤V aricella-zoster virus (VZV) vasculopathy is often protracted, perhaps due in part to persistent infection of arterial adventitial fibroblasts (1). A potential mechanism of virus persistence is evasion of the antiviral type I interferon (interferon alpha [IFN-␣] and IFN-␤) response (2) which induces antiviral interferon-inducible genes such as Mx1. Mx1 belongs to the dynamin superfamily of large GTPases (3,4), is present in all vertebrates in one to three copies, and is strictly controlled by type I and type III interferons. Mx1 encodes the human MxA protein, which accumulates in the cytoplasm, recognizes viral nucleocapsids, and blocks viral replication. In human lung embryonic fibroblasts, VZV immediateearly protein (IE) 62 blocks phosphorylation of interferon regulatory factor 3 (IRF3) and subsequent induction of IFN-␤ (5). Similarly, in HEK 293T and MeWo cells, VZV IE61 degrades activated IRF3 (6), and in VZV-infected epidermal cells, IFN-␣ is downregulated and IFN-␣-induced phosphorylated STAT1 (pSTAT1) is absent (7). Thus, we examined interference with type 1 IFN signaling in VZVinfected cerebrovascular adventitial fibroblasts as a potential mechanism for virus persistence.Primary human brain vascular adventitial fibroblasts (BRAFs) (ScienCell, Carlsbad, CA) were seeded at 5,000 cells/cm 2 in basal fibroblast medium supplemented with 2% fetal bovine serum (FBS), 1% fibroblast growth serum, and 1% 100ϫ penicillinstreptomycin (ScienCell). After 24 h, medium was changed to basal fibroblast medium supplemented with 0.1% FBS and 1% 100ϫ penicillin-streptomycin and replenished every 48 h for 1 week to establish quiescence. Quiescent BRAFs were cocultivated with VZV-infected or uninfected BRAFs or treated for 24 h with 1,000 U of IFN-␣ (PBL Interferon Source, Piscataway, NJ). VZVinfected BRAFs were analyzed at the height of the cytopathic effect (CPE) 3 days later and positive controls 24 h after treatment in 4 independent experiments. mRNA was analyzed by reverse-transcription PCR (RT-PCR) using SYBR green (8) and primers for IFN-␣, IFN-␤, STAT1, STAT2, Mx1, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Fig. 1A). Primer efficiencies were 104%, 93%, 92%, 89%, 107%, and 102%, respectively. Data were normalized to GAPDH and analyzed using the delta delta threshold cycle (C T ) method (9).BRAFS were propagated on coverslips, fixed, and permeabilized (10). Mouse anti-VZV gE (Santa Cruz Biotechnology, Santa Cruz, CA) (1:500) was added in addition to another primary antibody: rabbit anti-STAT1 (Epitomics, Burlingame, CA) (1:200); rabbit anti-pSTAT1 (Epitomics) (1:300); or rabbit anti-Mx1 (Abcam, Cambridge, MA) (1:100). Secondary antibodies were 1:1,000 dilutions of both Alexa Fluor 594 donkey anti-mouse IgG and Alexa Fluor 488 donkey anti-rabbit IgG 488 (Life Technologies, Grand Island, NY). Coverslips were mounted with Vectashield containing DAPI (4=,6-]diamidino-2-phenylindo...

Virological confirmation of varicella zoster virus (VZV) vasculopathy is provided by presence of virus in cerebral arteries, frequently associated with inflammation. Yet cerebral arteries from normal subjects have never been studied for VZV DNA or antigen. We analyzed 63 human cerebral arteries from 45 subjects for VZV DNA and antigen, control herpes simplex virus (HSV)-1 DNA and antigen and leukocyte-specific CD45 antigen. No cerebral arteries contained VZV or HSV-1 DNA or antigen; 8 arteries from 7 subjects contained leukocytes expressing CD45. Thus, the presence of VZV antigen in cerebral arteries of patients with stroke is likely to be clinically significant.

Upon reactivation, varicella zoster virus (VZV) spreads transaxonally, infects cerebral arteries and causes ischemic or hemorrhagic stroke, as well as aneurysms. The mechanism(s) of VZV-induced aneurysm formation is unknown. However, matrix metalloproteinases (MMPs), which digest extracellular structural proteins in the artery wall, play a role in cerebral and aortic artery aneurysm formation and rupture. Here, we examined the effect of VZV infection on expression of MMP-1, -2, -3, and -9 in primary human brain vascular adventitial fibroblasts (BRAFS). At 6 days post-infection, VZV- and mock-infected BRAFs were analyzed for mRNA levels of MMP-1, -2, -3 and -9 by RT-PCR and for corresponding total intra- and extracellular protein levels by multiplex ELISA. The activity of MMP-1 was also measured in a substrate cleavage assay. Compared to mock-infected BRAFs, MMP-1, MMP-3 and MMP-9 transcripts, cell lysate protein and conditioned supernatant protein were all increased in VZV-infected BRAFs, whereas MMP-2 transcripts, cell lysate protein and conditioned supernatant protein were decreased. MMP-1 from the conditioned supernatant of VZV-infected BRAFs showed increased cleavage activity on an MMP-1-specific substrate compared to mock-infected BRAFs. Differential regulation of MMPs in VZV-infected BRAFs may contribute to aneurysm formation in VZV vasculopathy.

Varicella zoster virus (VZV) is the only human virus known to replicate in cerebral arteries and produce stroke. After reactivation, virus travels transaxonally from ganglia and initially infects the arterial adventitia. To analyze the virus-host relationship, we infected primary human cerebrovascular adventitial fibroblasts with VZV and studied the interferon-α initiated STAT pathway, which is commonly activated after virus infection. Quantitative PCR showed that expression of STAT1, STAT2 and Mx1 mRNA were significantly decreased in VZV-infected, but not uninfected adventitial fibroblasts (p<0.05). Western blotting confirmed decreased STAT1 and Mx1 expression in VZV-infected fibroblasts compared to uninfected cells. Although Western blotting did not reveal phosphorylated STAT1 (pSTAT1) in VZV-infected cells, interferon-α is known to trigger STAT1 phosphorylation; thus, we treated uninfected adventitial fibroblasts with interferon-α, after which pSTAT1 expression was readily seen on Western blots. Furthermore, immunocytochemical staining detected pSTAT1 exclusively in the cytoplasm of VZV-infected cells. In interferon-α -treated uninfected cells, pSTAT1 was found exclusively in the nucleus, suggesting that VZV infection blocks egress of pSTAT1 from the cytoplasm to the nucleus in cerebral adventitial fibroblasts.