Laser-Capture Microdissection: Refining Estimates of the Quantity and Distribution of Latent Herpes Simplex Virus 1 and Varicella-Zoster Virus DNA in Human Trigeminal Ganglia at the Single-Cell Level

Sobel

Lai

et al. 2012

Analyses of varicella-zoster virus (VZV) protein expression during latency have been discordant, with rare to many positive neurons detected. We show that ascites-derived murine and rabbit antibodies specific for VZV proteins in vitro contain endogenous antibodies that react with human blood type A antigens in neurons. Apparent VZV neuronal staining and blood type A were strongly associated (by a 2 test, ␣ ‫؍‬ 0.0003). Adsorption of ascites-derived monoclonal antibodies or antiserum with type A erythrocytes or the use of in vitro-derived VZV monoclonal antibodies eliminated apparent VZV staining. Animal-derived antibodies must be screened for anti-blood type A reactivity to avoid misidentification of viral proteins in the neurons of the 30 to 40% of individuals who are blood type A. V aricella-zoster virus (VZV) is a human alphaherpesvirus that establishes latency in ganglionic sensory neurons. In autopsied human ganglia, the frequency of individual VZV-positive neurons was 1.0 to 6.9% by single-cell laser capture microdissection (LCMD) and PCR (29). However, immunostaining analyses of VZV protein expression in latently infected neurons have differed significantly. Mahalingam et al. reported infrequent detection of VZV proteins in cadaver ganglia, and in positive tissues VZV protein expression was restricted to very few neurons, a finding confirmed by Kennedy et al. and our recent study (13,16,32). In contrast, Lungu et al. detected VZV proteins in 9 to 24% of neurons in ganglia from three of three subjects (15). Theil et al. found immediate-early 62 (IE62) proteins in ganglia from 38% of individuals and in 3 to 7% of neurons (25). Discrepancies among reports describing the frequencies of VZV protein-positive neurons in autopsied ganglia require further investigation because of their implications for understanding mechanisms of VZV latency.Using a high-potency rabbit anti-IE63 antibody and preimmune serum control, we detected IE63 proteins in ganglia from only 1 of 18 individuals and in Ͻ2.8% of neurons (32). Subsequently, we observed cytoplasmic immunoreactivity in many neurons in ganglia from several individuals when the neurons were stained with monoclonal antibodies (MAbs) to IE62, glycoprotein E (gE), and the capsid protein ORF40 (MAB8616, MAB8612, and MAB8614, respectively; EMD Millipore, Billerica, MA); different lots had similar reactivities. These commercial reagents against IE62 and gE, known to be specific for viral proteins in cultured cells, have been used in studies that report cytoplasmic localization of VZV proteins in neurons (6,9,10,25,26,28). The failure to confirm IE62 and gE detection with other VZV antibodies, the detection of the ORF40 capsid protein detection in latency, and the Golgi zone-like localization of all three VZV proteins suggested a possible staining artifact.The mouse ascites Golgi (MAG) reaction results from endogenous anti-human blood type A antibodies in MAbs derived from mouse ascites (2, 5, 14, 21-23, 30, 31). Importantly, in blood type A individuals, sensory neurons exp...

Section: Demonstration Of Mag Reactivity In Human Neuronal Golgi Zonescontrasting

confidence: 51%

“…In autopsied human ganglia, the frequency of individual VZV-positive neurons was 1.0 to 6.9% by single-cell laser capture microdissection (LCMD) and PCR (29). However, immunostaining analyses of VZV protein expression in latently infected neurons have differed significantly.…”

mentioning

confidence: 99%

Apparent Expression of Varicella-Zoster Virus Proteins in Latency Resulting from Reactivity of Murine and Rabbit Antibodies with Human Blood Group A Determinants in Sensory Neurons

Sobel

Lai

et al. 2012

“…In the ocular model, mice that survive have no apparent replicating virus in DRG or trigeminal ganglia four to six weeks after infection (10,36,40,41). However, the frequency of infected ganglion cells in murine models is much lower than the estimate of 2 to 10.5% from human autopsy studies (42). The different outcomes of infection of human and mouse ganglia in vivo are likely to reflect adaptations of HSV-1 to its natural host.…”

Section: Discussionmentioning

confidence: 69%

Herpes Simplex Virus 1 Tropism for Human Sensory Ganglion Neurons in the Severe Combined Immunodeficiency Mouse Model of Neuropathogenesis

Che

Reichelt

et al. 2013

bThe tropism of herpes simplex virus (HSV-1) for human sensory neurons infected in vivo was examined using dorsal root ganglion (DRG) xenografts maintained in mice with severe combined immunodeficiency (SCID). In contrast to the HSV-1 lytic infectious cycle in vitro, replication of the HSV-1 F strain was restricted in human DRG neurons despite the absence of adaptive immune responses in SCID mice, allowing the establishment of neuronal latency. At 12 days after DRG inoculation, 26.2% of human neurons expressed HSV-1 protein and 13.1% expressed latency-associated transcripts (LAT). Some infected neurons showed cytopathic changes, but HSV-1, unlike varicella-zoster virus (VZV), only rarely infected satellite cells and did not induce fusion of neuronal and satellite cell plasma membranes. Cell-free enveloped HSV-1 virions were observed, indicating productive infection. A recombinant HSV-1-expressing luciferase exhibited less virulence than HSV-1 F in the SCID mouse host, enabling analysis of infection in human DRG xenografts for a 61-day interval. At 12 days after inoculation, 4.2% of neurons expressed HSV-1 proteins; frequencies increased to 32.1% at 33 days but declined to 20.8% by 61 days. Frequencies of LAT-positive neurons were 1.2% at 12 days and increased to 40.2% at 33 days. LAT expression remained at 37% at 61 days, in contrast to the decline in neurons expressing viral proteins. These observations show that the progression of HSV-1 infection is highly restricted in human DRG, and HSV-1 genome silencing occurs in human neurons infected in vivo as a consequence of virus-host cell interactions and does not require adaptive immune control.T he simplex viruses, herpes simplex virus types 1 and 2 (HSV-1 and -2), are ubiquitous human viral pathogens that may cause mild to severe mucocutaneous infections (1). In rare instances, these viruses cause meningoencephalitis. The prevalence of HSV-1 infection among adults ranges from 50 to 90%. Primary HSV infection is acquired by inoculation of mucosal epithelium and abraded skin. Local replication facilitates retrograde axonal transport of HSV virions within innervating cutaneous sensory nerve fibers to the corresponding neuronal cell bodies in the peripheral dorsal root ganglia (DRG) or trigeminal ganglia, where latency is established. Although most primary infections are asymptomatic, HSV-1 and HSV-2 have a dynamic relationship with the human host, and transmission to other susceptible individuals is ensured by frequent episodes of reactivation; these episodes are also usually asymptomatic but release infectious virus into oral and genital tract secretions. Notably, the many hundreds of HSV reactivations from latency that occur in sensory ganglia over the lifetime of the infected individual seldom result in ganglionitis, peripheral neuropathy, or spread to the central nervous system (2-4).Lytic HSV-1 infection, which produces infectious progeny virions, involves transcription of more than 80 viral genes in a highly ordered process governed by sequential derepression ...

“…By in situ hybridization and PCR methods, VZV DNA has been reported in as few as 1.5% of neurons exclusively (none in satellite cells) to as many as 30% of ganglion cells (neurons as well as satellite cells) (24,26). Most recently, Wang et al used a laser-capture microdissection/PCR method to provide more precise information at the single-cell level by examining 1,722 neurons in trigeminal ganglia (TGG) from seven individuals (49). These experiments identified VZV DNA in 4.1% of neurons (range, 1.0 to 6.9%) (49).…”

mentioning

confidence: 99%

Expression of Varicella-Zoster Virus Immediate-Early Regulatory Protein IE63 in Neurons of Latently Infected Human Sensory Ganglia

Sobel

Ramachandran

et al. 2010

Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory nerve ganglia, but the characteristics of VZV latency are not well defined. Immunohistochemical detection of the VZV immediateearly 63 (IE63) protein in ganglion neurons has been described, but there are significant discrepancies in estimates of the frequency of IE63-positive neurons, varying from a rare event to abundant expression. We examined IE63 expression in cadaver ganglia using a high-potency rabbit anti-IE63 antibody and corresponding preimmune serum. Using standard immunohistochemical techniques, we evaluated 10 ganglia that contained VZV DNA from seven individuals. These experiments showed that neuronal pigments were a confounding variable; however, by examining sections coded to prevent investigator bias and applying statistical analysis, we determined that IE63 protein, if present, is in a very small proportion of neurons (<2.8%). To refine estimates of IE63 protein abundance, we modified our protocol by incorporating a biological stain to exclude the pigment signal and evaluated 27 ganglia from 18 individuals. We identified IE63 protein in neurons within only one ganglion, in which VZV glycoprotein E and an immune cell infiltrate were also demonstrated. Antigen preservation was shown by detection of neuronal synaptophysin. These data provide evidence that the expression of IE63 protein, which has been referred to as a latency-associated protein, is rare. Refining estimates of VZV protein expression in neurons is important for developing a hypothesis about the mechanisms by which VZV latency may be maintained.Varicella-zoster virus (VZV), the human alphaherpesvirus that causes varicella during primary infection, establishes a lifelong latency in neurons of the sensory ganglia along the cerebrospinal axis (7). Sensory ganglia are composed of heterogeneous populations of neurons surrounded by satellite cells and other nonneuronal cells; axons extend from these neurons to innervate the skin and mucous membranes (50). Herpes zoster (shingles) results from reactivation of latent virus within ganglion cells and transfer of newly synthesized infectious particles to the skin via axonal transport (7).The number and type of neural cells that harbor latent virus are fundamental to the question of VZV latency. Studies to address this question have used methods to detect viral DNA, RNA, and proteins in cadaver ganglia. Although RNA is especially vulnerable to RNA-degrading enzymes during the postmortem interval between death and fixation of histological specimens at autopsy, DNA and proteins are reasonably stable (16). Nevertheless, reports using either VZV DNA or protein detection to assess the numbers of cells that contain VZV in human cadaver ganglia have yielded estimates that are extremely variable. By in situ hybridization and PCR methods, VZV DNA has been reported in as few as 1.5% of neurons exclusively (none in satellite cells) to as many as 30% of ganglion cells (neurons as well as satellite cells) (24, 26). Most recently,...