Simian Varicella Virus in Pigtailed Macaques (Macaca nemestrina): Clinical, Pathologic, and Virologic Features

Simian varicella virus (SVV; Cercopithecine herpesvirus 9) is a naturally occurring herpesvirus of nonhuman primates. Here we present the clinical, pathologic, and virologic findings from 2 cases of SVV in adult female pigtailed macaques (Macaca nemestrina). The initial case presented with hyperthermia and a diffuse inguinal rash which spread centripetally, progressing to vesiculoulcerative dermatitis of the trunk, face, and extremities. At 96 h after presentation, the animal was anorexic and lethargic and had oral and glossal ulcerations. Euthanasia was elected in light of the macaque''s failure to respond to clinical treatment. Seven days after the first case was identified, a second macaque presented with a vesicular rash and was euthanized. Gross necropsy lesions for both cases included vesicular, ulcerative dermatitis with mucocutaneous extension and hepatic necrosis; the initial case also demonstrated necrohemorrhagic gastroenterocolitis and multifocal splenic necrosis. Histology confirmed herpetic viral infection with abundant intranuclear inclusion bodies. Immunofluorescence assays detected antibodies specific for SVV. PCR assays of vesicular fluid, tissue, and blood confirmed SVV and excluded varicella–zoster virus (Human herpesvirus 3). Serology for Macacine herpesvirus 1 (formerly Cercopithecine herpesvirus 1), poxvirus (monkeypox), and rubella was negative. Banked serum samples confirmed SVV exposure and seroconversion. Investigation into the epidemiology of the seroconversion demonstrated a SVV colony prevalence of 20%. The described cases occurred in animals with reconstituted immune systems (after total-body irradiation) and demonstrate the clinical effects of infection with an endemic infectious agent in animals with a questionable immune status.Abbreviations: IFA, immunofluorescence assay; SVV, simian varicella virus; TBI, total body irradiation; WaNPRC, Washington National Primate Research Center; VZV, varicella–zoster virus; McHv1, Macacine herpesvuris 1; SRV-2, Simian retrovirus 2 (type D)Simian varicella virus (SVV; Cercopithecine herpesvirus 9) is a naturally occurring herpesvirus of Old World primates responsible for sporadic epizootics in biomedical research facilities.² Signs of infection include fever, vesicular skin lesions, hemorrhagic ulceration throughout the gastrointestinal tract, and multifocal hemorrhagic necrosis of the liver, spleen, lymph nodes, and endocrine organs.^6,7,8 Other names for SVV include Delta herpesvirus, Liverpool vervet virus, patas herpesvirus, and Medical Lake macaque virus.^{16, 20-23} Like many other herpesviruses, SVV establishes persistent lifelong infections, with viral DNA detectable in neural ganglia.¹² Infection with SVV does not necessarily lead to lifelong latency, and periodic reactivation of SVV may occur.³ SVV is genetically and antigenically similar to Human herpesvirus 3,² commonly known as varicela–zoster virus (VZV), the etiologic agent of chickenpox and shingles in humans. SVV in macaques and VZV in man present with similar clinical signs; SVV has been proposed as an animal model of VZV disease in man.²⁴ Rarely, VZV may occur in higher primates (Gorilla).¹⁸ The 2 viruses must be distinguished from one another through molecular techniques.^1,410,11 Both viral infections are usually mild and self-limiting in immunocompetent hosts,^4,8 reactivation and viral shedding may occur during times of stress or immunosupression.^80,21,22A recent review of SVV in Old World Monkeys⁸ focused on SVV as a disease of nonhuman primates. This case report expands on the 2 most recent cases of SVV mentioned in that review.⁸ The animals described were housed in accordance with the regulations of the Animal Welfare Act and the recommendations of the Guide for the Care and Use of Laboratory Animals¹¹ at the Washington National Primate Research Center (WaNPRC) facility in Seattle. The Institutional Animal Care and Use Committee of the University of Washington approved all aspects of the study to which the animals were assigned. The 2 clinical cases described in this report originated at the WaNPR–Seattle facility; contact animals described originated at the WaNPR–Tulane facility. When animals are relocated between the 2 facilities, they are processed through a domestic quarantine consisting of isolation for 30 d, during which time 3 tuberculin skin tests, 2 physical examinations, and 1 complete blood count and serological panel are performed. The WaNPRC–Tulane facility houses a breeding colony founded by animals relocated to Louisiana from the WaNPRC–Medical Lake facility in 1996.     

Simian varicella virus antibody response in experimental infection of African green monkeys

Abstract: The humoral immune response to simian varicella virus (SVV) was investigated following primary and secondary experimental infection of African green monkeys. Neutralization and immunoprecipitation assays were used to determine antibody titers to SVV throughout the course of infection. The immune response to specific viral polypeptides was analyzed by immunoprecipitation analysis. The results demonstrate that the simian varicella model offers a useful approach to investigate immune mechanisms in human varicella zoster virus (VZV) infections.     

Simian Varicella Virus Induces Apoptosis in Monkey Kidney Cells by the Intrinsic Pathway and Involves Downregulation of Bcl-2 Expression

Simian varicella virus (SVV) causes varicella in primates, becomes latent in ganglionic neurons, and reactivates to produce zoster. SVV produces a cytopathic effect in monkey kidney cells in tissue culture. To study the mechanism by which SVV-infected cells die, we examined markers of apoptosis 24 to 64 h postinfection (hpi). Western blot analysis of virus-infected cell lysates revealed a significant increase in the levels of the cleaved active form of caspase-3, accompanied by a parallel increase in caspase-3 activity at 40 to 64 hpi. Caspase-9, a marker for the intrinsic pathway, was activated significantly in SVV-infected cells at all time points, whereas trace levels of the active form of caspase-8, an extrinsic pathway marker, was detected only at 64 hpi. Bcl-2 expression at the mRNA and protein levels was decreased by 50 to 70% throughout the course of virus infection. Release of cytochrome c, an activator of caspase-9, from mitochondria into the cytoplasm was increased by 200% at 64 hpi. Analysis of Vero cells infected with SVV expressing green fluorescent protein (SVV-GFP) at 64 hpi revealed colocalization of the active forms of caspase-3 and caspase-9 and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining with GFP. A significant decrease in the bcl-2 mRNA levels along with an abundance of mRNA specific for SVV genes 63, 40, and 21 was seen in the fraction of Vero cells that were infected with SVV-GFP. Together, these findings indicate that SVV induces apoptosis in cultured Vero cells through the intrinsic pathway in which Bcl-2 is downregulated.Apoptosis, a regulated form of cell death, plays a critical role in the homeostasis of multicellular organisms. Key features include membrane blebbing, chromatin condensation, and cell shrinkage. UV irradiation, deprivation of growth factors, and viral infection all cause apoptosis in cultured cells. Apoptosis is triggered by sequential activation of a group of cysteine proteases known as caspases. Apoptosis proceeds primarily through two pathways. The extrinsic pathway involves activation of caspase-8 and is initiated by ligand interaction with Fas or death receptors, while the intrinsic pathway is activated by an imbalance between proapoptotic (e.g., Bad and Bax) and antiapoptotic (e.g., Bcl-2 and Bcl-xL) proteins in mitochondria (21), resulting in release of cytochrome c from mitochondria, which in turn activates caspase-9. Bcl-2 plays an important role in cell survival (22, 32). Both caspase-8 and caspase-9 activate caspase-3, which along with other effector caspases, cleave critical cellular proteins, resulting in apoptosis.Simian varicella virus (SVV), the primate counterpart of human varicella zoster virus (VZV), produces a naturally occurring exanthematous disease that mimics human varicella (9, 18). Clinical and pathological changes produced by SVV infection of primates are similar to those produced by human varicella, and both VZV and SVV reactivate from latently infected ganglionic neurons (4, 13, 23, 33). The SVV and VZV genomes share a high degree of nucleotide homology (3, 10), and SVV-specific antibodies cross-react with human VZV in serum neutralization and complement fixation tests (5, 6, 30). Both viruses produce a cytopathic effect in monkey kidney cells in tissue culture (2, 29, 31). VZV has been shown to cause apoptosis in cultured Vero cells, human foreskin fibroblasts, and peripheral blood mononuclear cells isolated from healthy donors but not in primary human dorsal root ganglionic neurons (12, 13, 16, 28). Apoptosis is also seen in peripheral blood mononuclear cells of children infected with VZV in vivo (25). Thus, VZV-induced apoptosis may be cell type specific. The main objectives of this study were to determine if SVV induces apoptosis in cultured Vero cells, a monkey kidney cell line, and to identify the specific pathways.     

Cercopithecine Herpesvirus 9 (Simian Varicella Virus) Infection after Total-Body Irradiation in a Rhesus Macaque (Macaca mulatta)

This case report describes a rhesus macaque (Macaca mulatta; male; age, 5 y; weight, 6.7 kg) with anorexia, dehydration, lethargy, ataxia, and generalized skin rashes that occurred 30 d after total-body irradiation at 6.5 Gy (⁶⁰Co γ-rays). Physical examination revealed pale mucus membranes, a capillary refill time of 4 s, heart rate of 180 bpm. and respirations at 50 breaths per minute. Diffuse multifocal maculopapulovesicular rashes were present on the body, including mucocutaneous junctions. The CBC analysis revealed a Hct of 48%, RBC count of 6.2 × 10⁶/µL, platelet count of 44 × 10³/µL, and WBC count of 25 × 10³/µL of WBC. The macaque was euthanized in light of a grave prognosis. Gross examination revealed white foci on the liver, multifocal generalized petechiation on serosal and mucosal surfaces of the gastrointestinal tract, hemorrhagic lymph nodes, and hemorrhagic fluid in the thoracic cavity. Microscopic examination revealed cutaneous vesicular lesions with intranuclear eosinophilic viral inclusions within the epithelial cells, consistent with herpesvirus. Immunohistochemistry was positive for herpesvirus. The serum sample was negative for antibodies against Macacine herpesvirus 1 and Cercopithecine herpesvirus 9 (simian varicella virus, SVV). Samples submitted for PCR-based identification of the etiologic agent confirmed the presence of SVV DNA. PCR analysis, immunohistochemistry, and histology confirmed that lesions were attributed to an active SVV infection in this macaque. This case illustrates the importance of screening for SVV in rhesus macaques, especially those used in studies that involve immunosuppressive procedures.Abbreviations: SVV, simian varicella virus; TBI, total-body irradiation     

Comprehensive Analysis of Varicella-Zoster Virus Proteins Using a New Monoclonal Antibody Collection

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus''s restricted host and cell type tropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251 monoclonal antibodies (MAbs) against 59 of the 71 (83%) currently known unique VZV proteins to characterize VZV protein expression in vitro and in situ. Using this new set of MAbs, 44 viral proteins were detected by Western blotting (WB) and indirect immunofluorescence (IF); 13 were detected by WB only, and 2 were detected by IF only. A large proportion of viral proteins was analyzed for the first time in the context of virus infection. Our study revealed the subcellular localization of 46 proteins, 14 of which were analyzed in detail by confocal microscopy. Seven viral proteins were analyzed in time course experiments and showed a cascade-like temporal gene expression pattern similar to those of other herpesviruses. Furthermore, selected MAbs tested positive on human skin lesions by using immunohistochemistry, demonstrating the wide applicability of the MAb collection. Finally, a significant portion of the VZV-specific antibodies reacted with orthologs of simian varicella virus (SVV), thus enabling the systematic analysis of varicella in a nonhuman primate model system. In summary, this study provides insight into the potential function of numerous VZV proteins and novel tools to systematically study VZV and SVV pathogenesis.     

T-Cell Infiltration Correlates with CXCL10 Expression in Ganglia of Cynomolgus Macaques with Reactivated Simian Varicella Virus

Ganglia of monkeys with reactivated simian varicella virus (SVV) contained more CD8 than CD4 T cells around neurons. The abundance of CD8 T cells was greater less than 2 months after reactivation than that at later times and correlated with that of CXCL10 RNA but not with those of SVV protein or open reading frame 61 (ORF61) antisense RNA. CXCL10 RNA colocalized with T-cell clusters. After SVV reactivation, transient T-cell infiltration, possibly mediated by CXCL10, parallels varicella zoster virus (VZV) reactivation in humans.     

Perspectives on optimal control of varicella and herpes zoster by mass routine varicella vaccination

Herpes zoster arises from reactivation of the varicella–zoster virus (VZV), causing varicella in children. As reactivation occurs when cell-mediated immunity (CMI) declines, and there is evidence that re-exposure to VZV boosts CMI, mass varicella immunization might increase the zoster burden, at least for some decades. Fear of this natural zoster boom is the main reason for the paralysis of varicella immunization in Europe. We apply optimal control to a realistically parametrized age-structured model for VZV transmission and reactivation to investigate whether feasible varicella immunization paths that are optimal in controlling both varicella and zoster exist. We analyse the optimality system numerically focusing on the role of the cost functional, of the relative zoster–varicella cost and of the planning horizon length. We show that optimal programmes will mostly be unfeasible for public health owing to their complex temporal profiles. This complexity is the consequence of the intrinsically antagonistic nature of varicella immunization programmes when aiming to control both varicella and zoster. However, we show that gradually increasing—hence feasible—vaccination schedules can perform better than routine programmes with constant vaccine uptake. Finally, we show the optimal profiles of feasible programmes targeting mitigation of the post-immunization natural zoster boom with priority.     

Genome-Wide Analysis of T Cell Responses during Acute and Latent Simian Varicella Virus Infections in Rhesus Macaques

Varicella zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (HZ [shingles]). Clinical observations suggest that VZV-specific T cell immunity plays a more critical role than humoral immunity in the prevention of VZV reactivation and development of herpes zoster. Although numerous studies have characterized T cell responses directed against select VZV open reading frames (ORFs), a comprehensive analysis of the T cell response to the entire VZV genome has not yet been conducted. We have recently shown that intrabronchial inoculation of young rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we characterized the specificity of T cell responses during acute and latent SVV infection. Animals generated a robust and broad T cell response directed against both structural and nonstructural viral proteins during acute infection in bronchoalveolar lavage (BAL) fluid and peripheral blood. During latency, T cell responses were detected only in the BAL fluid and were lower and more restricted than those observed during acute infection. Interestingly, we identified a small set of ORFs that were immunogenic during both acute and latent infection in the BAL fluid. Given the close genome relatedness of SVV and VZV, our studies highlight immunogenic ORFs that may be further investigated as potential components of novel VZV vaccines that specifically boost T cell immunity.     

Experimental simian varicella virus infection of St. Kitts vervet monkeys

Experimental simian varicella virus (SVV) infection of St. Kitts vervet monkeys was evaluated as an animal model to investigate human varicella-zoster virus (VZV) infections. During the incubation period, viremia disseminated infectious virus throughout the body via infected peripheral blood lymphocytes (PBLs). A vesicular skin rash in the inguinal area, and on the abdomen, extremities, and face appeared on day 7–10 postinfection. Necrosis and hemorrhage in lung and liver tissues from acutely infected monkeys were evident upon histologic analysis. Recovery from simian varicella was accompanied by a rise in the serum neutralizing antibody response to the virus. SVV latency was established in trigeminal ganglia of monkeys which resolved the acute infection. This study indicates that experimental SVV infection of St. Kitts vervets is a useful animal model to investigate SVV and VZV pathogenesis and to evaluate potential antiviral agents and vaccines.     

Evaluation of Chosen Cytokine Levels among Patients with Herpes Zoster as Ability to Provide Immune Response

Aim and Background

Herpes zoster is a viral disease caused by the reactivation of varicella–zoster virus (VZV) which remained latent in the cranial nerve or dorsal root ganglia. Cell-mediated immunity is known to decline with age as part of immunosenescence and can lead to the reactivation of VZV. Whereas herpes zoster is usually mild in healthy young persons, older patients are at increased risk for complications. In the present study we investigated the serum cytokine profile (IL-17, IL-23, IL-21, IL-4, IL-12), representing cellular and humoral immunity and assessed the level of VZV IgG antibodies in patients with herpes zoster.

Methods

We investigated the serum concentrations of IL-17, IL-23, IL-21, IL-4, IL-12 and the level of VZV IgG antibodies in 23 patients with herpes zoster who did not develop superinfection. The control group was represented by 21 individuals in similar age with no inflammatory and infectious diseases. Cytokine and antibodies levels were measured by ELISA method. Statistical analysis was performed using the ROC curve (receiver operating characteristic), t-test, Welch’s t-test, and nonparametric tests with STATISTICA 10 software.

Results

In patients with herpes zoster, the serum level of IL-17, IL-23, IL-21, IL-4 and IL-12 as well as VZV IgG antibodies titer were statistically significantly increased compared to control group.

Conclusion

Our results confirm the broad activation of the immune system involving humoral and cell-mediated immunity.     

Naturally acquired simian varicella virus infection in African green monkeys

Simian varicella virus (SVV) infection of primates shares clinical, pathological, immunological, and virological features with varicella-zoster virus infection of humans. Natural varicella infection was simulated by exposing four SVV-seronegative monkeys to monkeys inoculated intratracheally with SVV, in which viral DNA and RNA persist in multiple tissues for more than 1 year (T. M. White, R. Mahalingam, V. Traina-Dorge, and D. H. Gilden, J. Neurovirol. 8:191-205, 2002). The four naturally exposed monkeys developed mild varicella 10 to 14 days later, and skin scrapings taken at the time of the rash contained SVV DNA. Analysis of multiple ganglia, liver, and lung tissues from the four naturally exposed monkeys sacrificed 6 to 8 weeks after resolution of the rash revealed SVV DNA in ganglia at multiple levels of the neuraxis but not in the lung or liver tissue of any of the four monkeys. This animal model provides an experimental system to gain information about varicella latency with direct relevance to the human disease.     

Inactivity of the bicyclic pyrimidine nucleoside analogues against simian varicella virus (SVV) does not correlate with their substrate activity for SVV-encoded thymidine kinase

Simian varicella virus (SVV) and human varicella-zoster virus (VZV) are closely related viruses that share many structural and functional properties. 5-Substituted 2'-deoxyuridine derivatives (e.g., BVDU, BVaraU) and acyclic guanine nucleoside derivatives (i.e., ACV and GCV) show comparable antiviral efficacy against VZV and SVV in cell culture. In contrast, the novel bicyclic nucleoside analogues (BCNAs) are exquisitely inhibitory to VZV (EC50 in the lower nanomolar range) but completely inactive against SVV. The VZV-encoded thymidine kinase (TK) appeared to be essential for BCNA activation (phosphorylation) and anti-VZV activity. Also SVV TK is able to recognize the BCNAs as substrate, although with a different structure-affinity relationship. Thus, viral TK-catalyzed phosphorylation is necessary but not sufficient for the BCNAs to display antiviral activity. Our data suggest that the eventual target of the BCNAs against VZV is either absent in SVV or, alternatively, is insensitive for the (phosphorylated) BCNAs.     

A qualitative analysis of a model for the transmission of varicella-zoster virus

Varicella-zoster virus (VZV) is a herpesvirus which is the known agent for causing varicella (chickenpox) in its initial manifestation and zoster (shingles) in a reactivated state. The standard SEIR compartmental model is modified to include the cycle of shingles acquisition, recovery, and possible reacquisition. The basic reproduction number R(0) shows the influence of the zoster cycle and how shingles can be important in maintaining VZV in populations. The model has the typical threshold behavior in the sense that when R(0)1, the virus persists over time and so chickenpox and shingles remain endemic.