Varicella zoster virus vasculopathy: analysis of virus-infected arteries.

OBJECTIVE: Varicella zoster virus (VZV) is an under-recognized yet treatable cause of stroke. No animal model exists for stroke caused by VZV infection of cerebral arteries. Thus, we analyzed cerebral and temporal arteries from 3 patients with VZV vasculopathy to identify features that will help in diagnosis and lead to a better understanding of VZV-induced vascular remodeling. METHODS: Normal and VZV-infected cerebral and temporal arteries were examined histologically and by immunohistochemistry using antibodies directed against VZV, endothelium, and smooth muscle actin and myosin. RESULTS: All VZV-infected arteries contained 1) a disrupted internal elastic lamina; 2) a hyperplastic intima composed of cells expressing α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-myosin) but not endothelial cells expressing CD31; and 3) decreased medial smooth muscle cells. The location of VZV antigen, degree of neointimal thickening, and disruption of the media were related to the duration of disease. CONCLUSIONS: The presence of VZV primarily in the adventitia early in infection and in the media and intima later supports the notion that after reactivation from ganglia, VZV spreads transaxonally to the arterial adventitia followed by transmural spread of virus. Disruption of the internal elastic lamina, progressive intimal thickening with cells expressing α-SMA and SM-MHC, and decreased smooth muscle cells in the media are characteristic features of VZV vasculopathy. Stroke in VZV vasculopathy may result from changes in arterial caliber and contractility produced in part by abnormal accumulation of smooth muscle cells and myofibroblasts in thickened neointima and disruption of the media.

The varicella zoster virus vasculopathies: clinical, CSF, imaging, and virologic features.

BACKGROUND: Varicella zoster virus (VZV) vasculopathy produces stroke secondary to viral infection of cerebral arteries. Not all patients have rash before cerebral ischemia or stroke. Furthermore, other vasculitides produce similar clinical features and comparable imaging, angiographic, and CSF abnormalities. METHODS: We review our 23 published cases and 7 unpublished cases of VZV vasculopathy. All CSFs were tested for VZV DNA by PCR and anti-VZV IgG antibody and were positive for either or both. RESULTS: Among 30 patients, rash occurred in 19 (63%), CSF pleocytosis in 20 (67%), and imaging abnormalities in 29 (97%). Angiography in 23 patients revealed abnormalities in 16 (70%). Large and small arteries were involved in 15 (50%), small arteries in 11 (37%), and large arteries in only 4 (13%) of 30 patients. Average time from rash to neurologic symptoms and signs was 4.1 months, and from neurologic symptoms and signs to CSF virologic analysis was 4.2 months. CSF of 9 (30%) patients contained VZV DNA while 28 (93%) had anti-VZV IgG antibody in CSF; in each of these patients, reduced serum/CSF ratio of VZV IgG confirmed intrathecal synthesis. CONCLUSIONS: Rash or CSF pleocytosis is not required to diagnose varicella zoster virus (VZV) vasculopathy, whereas MRI/CT abnormalities are seen in almost all patients. Most patients had mixed large and small artery involvement. Detection of anti-VZV IgG antibody in CSF was a more sensitive indicator of VZV vasculopathy than detection of VZV DNA (p < 0.001). Determination of optimal antiviral treatment and benefit of concurrent steroid therapy awaits studies with larger case numbers.

The value of detecting anti-VZV IgG antibody in CSF to diagnose VZV vasculopathy.

BACKGROUND: Factors that may obscure the diagnosis of varicella zoster virus (VZV) vasculopathy include the absence of rash before TIAs or stroke as well as similar clinical features and imaging, angiographic, and CSF abnormalities to those of other vasculopathies. Diagnosis relies on virologic confirmation that detects VZV DNA, anti-VZV IgG antibody, or both in the CSF. METHODS: We reviewed our current 14 cases of patients diagnosed with VZV vasculopathy based on combined clinical, imaging, angiographic, or CSF abnormalities. All CSFs must have been tested for VZV DNA by PCR and for anti-VZV IgG antibody by enzyme immunoassay and found to be positive for either or both. Of the 14 subjects, 8 had a history of recent zoster, whereas 6 had no history of zoster rash before developing vasculopathy. RESULTS: All 14 subjects (100%) had anti-VZV IgG antibody in their CSF, whereas only 4 (28%) had VZV DNA. The detection of anti-VZV IgG antibody in CSF was a more sensitive indicator of VZV vasculopathy than detection of VZV DNA (p < 0.001). CONCLUSIONS: In varicella zoster virus (VZV) vasculopathy, the diagnostic value of detecting anti-VZV IgG antibody in CSF is greater than that of detecting VZV DNA. Although a positive PCR for VZV DNA in CSF is helpful, a negative PCR does not exclude the diagnosis of VZV vasculopathy. Only when the CSF is negative for both VZV DNA and anti-VZV IgG antibody can the diagnosis of VZV vasculopathy be excluded.

Presence of VZV and HSV-1 DNA in human nodose and celiac ganglia.

Polymerase chain reaction (PCR) revealed herpes simplex virus (HSV) and varicella zoster virus (VZV) DNA in human nodose and celiac ganglia. This is the first detection of VZV DNA in ganglia of the human autonomic nervous system. The ability of reactivated VZV to produce serious, sometimes fatal neurological disease in the absence of rash, raises the possibility that VZV reactivation from autonomic ganglia might be involved in visceral disease.

Acute, chronic, and recurrent varicella zoster virus neuropathy without zoster rash.

The authors report three patients with acute, chronic, and recurrent neuropathy associated with varicella zoster virus (VZV) infection but without zoster rash. CSF of all three patients contained VZV immunoglobulin G antibody, but not herpes simplex virus. In two patients, serum/CSF ratios of VZV immunoglobulin G were reduced compared to normal ratios for immunoglobulin G and albumin, and one patient also had VZV immunoglobulin M in CSF. All three patients received antiviral therapy and improved. The diagnosis of nervous system infection by VZV may be confirmed by the presence of antibody to VZV in CSF even without detectable VZV DNA.

Simian varicella virus infects ganglia before rash in experimentally infected monkeys.

Monkeys experimentally infected with simian varicella virus (SVV) develop rash 10-14 days later. However, the route and the time of ganglionic infection are unknown. Using PCR, we analyzed DNA extracted from tissues of 13 monkeys 5 to 60 days after either intratracheal or intravenous inoculation with SVV. SVV DNA was detected in ganglia from four of five monkeys sacrificed 6 to 7 days after intratracheal inoculation. Further, analysis of ganglia from monkeys sacrificed at 10 days revealed that intravenous inoculation produced a higher proportion of SVV DNA-positive ganglia (63%) than that after intratracheal inoculation (13%), pointing to the role of hematogenous spread in ganglionic infection. Like other organs, monkey ganglia become infected with SVV before the appearance of rash.

Sequence analysis of the leftward end of simian varicella virus (EcoRI-I fragment) reveals the presence of an 8-bp repeat flanking the unique long segment and an 881-bp open-reading frame that is absent in the varicella zoster virus genome.

Simian varicella virus (SVV) causes varicella (chickenpox) in nonhuman primates, becomes latent in cranial and dorsal root ganglia, and reactivates to produce zoster (shingles). Because the clinical and molecular features of SVV closely resemble those of varicella zoster virus (VZV) infection of humans, SVV infection of primates has served as an experimental model of VZV pathogenesis and latency. The SVV genome has been completely mapped, but attempts to clone the 3600-bp EcoRI fragment located at the leftward end of the virus genome have hitherto been unsuccessful. Herein, we report the cloning and the complete nucleotide sequence of this region. Comparison of the SVV and VZV sequences in this region revealed an 8-bp inverted repeat sequence flanking the unique long segment of the SVV genome; an 879-bp open-reading frame (ORF) A in SVV that is absent in VZV but has 42% amino acid identity to SVV ORF 4 and 49% to VZV ORF 4; a 342-bp ORF B in SVV with 35% amino acid identity to a 387-bp ORF located to the left of ORF 1 on the VZV genome; and a 303-bp ORF in SVV with 27% amino acid identity to VZV ORF 1. No homologue of VZV ORF 2 was detected. Transcripts specific for ORFs A and B were present in SVV-infected cells in culture and in acutely infected monkey ganglia. Overall, there are more than 2000 bp of DNA in the SVV genome that are absent in the VZV genome.

Identification of simian varicella virus gene 21 promoter region using green fluorescent protein.

Clinical, pathological, immunological and virological features of simian varicella virus (SVV) infection in primates closely resemble those of varicella zoster virus (VZV) infection in humans. In ganglia infected latently of humans and monkeys, gene 21 of VZV and SVV is transcribed, respectively. We determined the nucleotide sequence of the intragenic region between SVV genes 20 and 21 to identify the putative promoter region for SVV gene 21. A recombinant clone was prepared in which the gene encoding green fluorescent protein (GFP) was inserted ten base pairs upstream of the predicted translational start site for SVV gene 21. SVV-infected monkey kidney cells transfected with the recombinant clone showed the presence of green fluorescence, whereas transfection of these cells with a construct containing the GFP gene in the opposite orientation, produced no fluorescence. The recombinant clone containing GFP flanked by SVV sequences can be used to prepare a SVV mutant in which the virus gene 21 promoter drives GFP. Such a mutant will be useful in analyzing varicella pathogenesis and latency in experimentally infected animals, studies not possible in humans.

Attempts to identify herpes simplex virus DNA in normal and diseased human peripheral nerve.

PCR analysis of DNA extracted from 31 sections of formalin-fixed sural nerve biopsies did not reveal herpes simplex virus (HSV) DNA. Unlike the presence of HSV DNA sequences in normal human brain, spinal cord, and ganglia, HSV DNA is not present in normal or diseased human distal peripheral nerve.

Search for varicella zoster virus in giant cell arteritis.

Polymerase chain reaction and immunohistochemical analyses of formalin-fixed temporal arteries from 10 pathologically verified cases of giant cell arteritis did not reveal varicella zoster virus antigen or DNA.

Profound cerebrospinal fluid pleocytosis and Froin's Syndrome secondary to widespread necrotizing vasculitis in an HIV-positive patient with varicella zoster virus encephalomyelitis.

Demonstration of the direct involvement of cranial blood vessels by varicella zoster virus (VZV) is facilitated by immunohistochemistry (IHC), in situ hybridization (ISH) and polymerase chain reaction (PCR) techniques. The extent to which an inflammatory vasculitis serves as the pathogenic mechanism for VZV encephalomyelitis (VZVE) is still, however, debated. Most VZVE patients are immunocompromised and show little inflammation, either pre-mortem in cerebrospinal fluid (CSF) or at autopsy. We describe an HIV-positive patient with a moderately depressed CD4 count (304) who presented with massively elevated CSF protein (1800 mg/dl), bloody CSF and pleocytosis (1300 white blood cells (WBC)/mm3). His CSF was positive for VZV DNA by PCR. He was treated with acyclovir and foscarnet, but died. At autopsy, an unusually widespread, inflammatory, transmural vasculitis caused by VZV affected meningeal vessels at virtually all brain stem and spinal cord levels, causing multiple subpial hemorrhages and necrosis. Virus DNA in multiple areas of brain, brainstem and spinal cord was readily revealed by PCR, but not by the presence of viral inclusions, IHC or ISH. This case, with a clinically confusing presentation for VZVE, illustrates the extensive, albeit infrequent, degree of necrotizing vasculitis and CSF abnormalities that VZV is capable of producing. Antiviral therapy may have inhibited VZV genome replication and subsequent antigen production, resulting in negative ISH and IHC studies, but generated increased VZV genomic fragments that were detectable by the more sensitive PCR technique.

Infectious simian varicella virus expressing the green fluorescent protein.

Clinical, pathologic, immunologic and virologic features of simian varicella virus (SVV) infection in primates closely resemble varicella-zoster virus (VZV) infection in humans. Such similarities provide a rationale to analyze SVV infection in primates as a model of varicella pathogenesis and latency. Thus, we constructed an SVV-expressing green fluorescent protein (SVV-GFP) by inserting the GFP gene into the unique short segment of the virus genome by homologous recombination. Analysis of recombinant viral DNA and the expressed proteins of plaque-purified SVV-GFP confirmed the location of the GFP insert and that the recombinant SVV expressed the 27 kDa GFP. Infection of monkey kidney cells in tissue culture with SVV-GFP revealed bright green fluorescence associated with the characteristic focal cytopathic effect produced by SVV infection. Microscopic examination of lung from a 3-month-old African green monkey 10 days after infection with SVV-GFP revealed bright green fluorescence in areas of acute necrotizing pneumonitis. SVV-GFP allows ready identification of cells infected with SVV both in vitro and in vivo, and will be useful for further analysis of varicella pathogenesis and latency in experimentally infected animals--studies not possible in humans.

Localization of varicella-zoster virus gene 21 protein in virus-infected cells in culture.

Although four varicella-zoster virus (VZV) genes have been shown to be transcribed in latently infected human ganglia, their role in the development and maintenance of latency is unknown. To study these VZV transcripts, we decided first to localize their expression products in productively infected cells. We began with VZV gene 21, whose open reading frame (ORF) is 3,113 bp. We cloned the 5' and 3' ends and the predicted antigenic segments of the ORF as 1292-, 1280-, and 880-bp DNA fragments, respectively, into the prokaryotic expression vector pGEX-2T. The three VZV 21 ORFs were expressed as approximately 75-, 73-, and 59-kDa glutathione S-transferase fusion proteins in Escherichia coli. To prepare polyclonal antibodies that would recognize all potential epitopes on the VZV gene 21 protein, rabbits were inoculated with a mixture of the three fusion proteins, and antisera were obtained and affinity purified. Immunohistochemical and immunoelectron microscopic analyses using these antibodies revealed VZV ORF 21 protein in both the nucleus and cytoplasm of VZV-infected cells. When these antibodies were applied to purified VZV nucleocapsids, intense staining was seen in their central cores.

Varicella zoster virus, a cause of waxing and waning vasculitis: the New England Journal of Medicine case 5-1995 revisited.

A 73-year-old man developed an ill-defined fatal vasculitis involving the central nervous system. The case report was published as a clinicopathologic exercise in February 1995 in The New England Journal of Medicine. We restudied the pathologic material and found both varicella zoster virus (VZV) DNA and VZV-specific antigen, but not herpes simplex virus (HSV) or cytomegalovirus (CMV) DNA or HSV- or CMV-specific antigen, in three of the five cerebral arteries examined. The inflammatory response, disruption of the internal elastic lamina, and detection of viral antigen were patchy from one artery to another, as well as within a given artery. A search for VZV should be conducted in cases of vasculitis when both the central and peripheral nervous systems are involved, when focal narrowing is present in large arteries, when brain imaging reveals infarction in gray and white matter, both deep and superficial, and when white matter is disproportionally involved. Zosteriform rash is not required for diagnosis.

Epstein-Barr virus--associated acute autonomic neuropathy.

A 44-year-old man developed blurry vision, photosensitivity, orthostasis, constipation, and acrodysesthesias after a febrile illness. The neurologic examination and ancillary studies were consistent with a dysautonomic small fiber neuropathy. The cerebrospinal fluid (CSF) contained both Epstein-Barr virus (EBV) DNA and antibody to EBV. This is the first report of an acute autonomic neuropathy with documented EBV infection in CSF.

Expression of protein encoded by varicella-zoster virus open reading frame 63 in latently infected human ganglionic neurons.

The ganglionic cell type in which varicella-zoster virus (VZV) is latent in humans was analyzed by using antibodies raised against in vitro-expressed VZV open reading frame 63 protein. VZV open reading frame 63 protein was detected exclusively in the cytoplasm of neurons of latently infected human trigeminal and thoracic ganglia. This is, to our knowledge, the first identification of a herpesvirus protein expressed during latency in the human nervous system.

Varicella-zoster virus (VZV) transcription during latency in human ganglia: prevalence of VZV gene 21 transcripts in latently infected human ganglia.

Reverse transcriptase-linked PCR was used to determine the prevalence of varicella-zoster virus (VZV) gene 21 transcription in latently infected human ganglia. Under conditions wherein reverse transcriptase-linked PCR detected > or = 1,000 transcripts, VZV gene 21 RNA, but not VZV gene 40 RNA, was found in ganglia but not other tissues from five of seven humans.

In situ polymerase chain reaction detection of varicella zoster virus in infected cells in culture.

Polymerase chain reaction (PCR) technology provides a means of identifying genes and studying their expression with specificity and precision. Combined with in situ hybridization (ISH), the amplified gene can be localized within cells. Cell gene sequences (human alpha-tubulin) were identified by PCR-ISH first in uninfected cells in culture, then in human blood mononuclear cells, and finally in sections of human liver. Varicella zoster virus (VZV) DNA was detected for the first time in virus-infected cells in culture by PCR-ISH, and compared to ISH alone. Efficient PCR-ISH was achieved by fixation of cells or tissue sections with 2% paraformaldehyde, by amplification under conditions of complete exposure of the samples to the reagents, and by detection of amplified products using non-radioactive digoxigenin-labeled oligonucleotide probes internal to the amplified DNA segment. PCR-ISH increased the detection of VZV DNA in infected cells 5-fold compared to ISH alone. Compared to ISH alone, PCR-ISH enhances significantly the detection of virus DNA in infected cells.

Persistence of varicella-zoster virus DNA in elderly patients with postherpetic neuralgia.

The most common complication of zoster in the elderly is postherpetic neuralgia, operationally defined as pain persisting longer than 1-2 months after rash. The cause of postherpetic neuralgia is unknown. Using polymerase chain reaction, we detected varicella zoster virus DNA in blood mononuclear cells from 11 of 51 postherpetic neuralgia patients, but not in any of 19 zoster patients without postherpetic neuralgia, or in any of 11 elderly individuals without a history of zoster. Blood mononuclear cells from nine of 27 serially-bled postherpetic neuralgia patients were positive for varicella zoster virus DNA; six were positive once, and three patients were positive more than once. Our results indicated that postherpetic neuralgia may be related to persistence of varicella zoster virus.

Varicella-zoster virus (VZV) transcription during latency in human ganglia: construction of a cDNA library from latently infected human trigeminal ganglia and detection of a VZV transcript.

The entire varicella-zoster virus (VZV) genome appears to be present in latently infected human ganglia, but the extent of virus DNA transcription is unknown. Conventional methods to study virus gene transcripts by Northern (RNA) blotting are not feasible, since ganglia are small and VZV DNA is not abundant. To circumvent this problem, we prepared radiolabeled cDNA from ganglionic RNA, hybridized it to Southern blots containing VZV DNA, and demonstrated the presence of a transcript within the SalI C fragment of the virus genome (R. Cohrs, R. Mahalingam, A. N. Dueland, W. Wolf, M. Wellish, and D. H. Gilden, J. Infect. Dis. 166:S24-S29, 1992). To further map VZV transcripts, in the work described here we constructed a cDNA library from poly(A)+ RNA obtained from latently infected human ganglia. Phage DNA isolated from the library was used in PCR amplifications to detect VZV-specific inserts. The specificity of the PCRs was provided by selection of a primer specific for VZV gene 17, 18, 19, 20, or 21 and a second vector-specific primer. VZV gene 21-specific sequences were identified by PCR amplification. The PCR product contained the XhoI cloning site and poly(A)+ sequences between vector and VZV gene 21 sequences. The sequence motif at the 3' end of VZV gene 21, determined by cloning and sequencing of the PCR product, consisted of 49 to 51 nucleotide bases of 3'-untranslated DNA, the termination codon for the VZV gene 21 open reading frame, and DNA sequences reading into the VZV gene 21 open reading frame.