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.     

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.     

Simian Varicella in Old World Monkeys

Simian varicella virus (SVV) causes a natural erythematous disease in Old World monkeys and is responsible for simian varicella epizootics that occur sporadically in facilities housing nonhuman primates. This review summarizes the biology of SVV and simian varicella as a veterinary disease of nonhuman primates. SVV is closely related to varicella–zoster virus, the causative agent of human varicella and herpes zoster. Clinical signs of simian varicella include fever, vesicular skin rash, and hepatitis. Simian varicella may range from a mild infection to a severe and life-threatening disease, and epizootics may have high morbidity and mortality rates. SVV establishes a lifelong latent infection in neural ganglia of animals in which the primary disease resolves, and the virus may reactivate later in life to cause a secondary disease corresponding to herpes zoster. Prompt diagnosis is important for control and prevention of epizootics. Antiviral treatment for simian varicella may be effective if administered early in the course of infection.Abbreviations: FEAU, 1-(2′-deoxy-2′-flouro-β-D-arabinofuranosyl)-5-iodouracil, IE, immediate early, ORF, open reading frame, PBL, peripheral blood lymphocyte, SVV, simian varicella virus, VZV, varicella–zoster virusSimian varicella is a natural erythematous disease of Old World primates (Superfamily Cercopithecoidea, Subfamily Cercopithecinae), involving particularly patas (Erythrocebus patas), African green or vervet (Chlorocebus aethiops), and various species of macaque (Macaca spp.) monkeys. Epizootics of simian varicella occur sporadically in facilities housing nonhuman primates. These outbreaks are sometimes associated with high morbidity and mortality and the loss of valuable research animals. Simian varicella virus (SVV; Cercopithecine herpesvirus 9), a primate herpesvirus, is the etiologic agent of the disease. SVV is antigenically and genetically related to varicella–zoster virus (VZV; Human herpesvirus 3), the cause of human varicella (chickenpox) and herpes zoster (shingles). The clinical similarities between simian and human varicella and the relatedness of SVV and VZV, indicate that SVV infection of nonhuman primates is a useful model for study of varicella pathogenesis and development of antiviral therapies. A previous comprehensive review emphasized simian varicella as an experimental model for VZV infections.²² This review focuses on simian varicella as a veterinary disease of nonhuman primates. Simian varicella outbreaks and their epidemiology are considered, and the etiologic agent, clinical manifestations, pathogenesis, diagnosis, treatment, and control of the disease are discussed.     

Acyclovir treatment prevents varicella-zoster virus replication in PBMC during viremia.

The most effective antiviral therapy of varicella and zoster has become acyclovir. Using polymerase chain reaction specific for VZV ORF 14, ORF 29, ORF 63 as well as nucleic acid sequence-based amplification (ORF 63, ORF 68) we tested PBMC of patients with VZV-associated diseases for the presence of viral DNA and RNA, respectively. In PBMC of patients treated with acyclovir neither DNA nor RNA was detectable already one day after the onset of therapy. In three blood sample pairs from zoster patients we were able to detect viral nucleic acid before but not after acyclovir treatment. These results confirm clinical and epidemiological data. It can be concluded that treatment with acyclovir prevents VZV replication in peripheral blood mononuclear cells.     

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.     

A self-excisable infectious bacterial artificial chromosome clone of varicella-zoster virus allows analysis of the essential tegument protein encoded by ORF9

In order to facilitate the generation of mutant viruses of varicella-zoster virus (VZV), the agent causing varicella (chicken pox) and herpes zoster (shingles), we generated a full-length infectious bacterial artificial chromosome (BAC) clone of the P-Oka strain. First, mini-F sequences were inserted into a preexisting VZV cosmid, and the SuperCos replicon was removed. Subsequently, mini-F-containing recombinant virus was generated from overlapping cosmid clones, and full-length VZV DNA recovered from the recombinant virus was established in Escherichia coli as an infectious BAC. An inverted duplication of VZV genomic sequences within the mini-F replicon resulted in markerless excision of vector sequences upon virus reconstitution in eukaryotic cells. Using the novel tool, the role in VZV replication of the major tegument protein encoded by ORF9 was investigated. A markerless point mutation introduced in the start codon by two-step en passant Red mutagenesis abrogated ORF9 expression and resulted in a dramatic growth defect that was not observed in a revertant virus. The essential nature of ORF9 for VZV replication was ultimately confirmed by restoration of the growth of the ORF9-deficient mutant virus using trans-complementation via baculovirus-mediated gene transfer.     

The varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase is dispensable for viral replication and is not required for phosphorylation of ORF63 protein, the VZV homolog of herpes simplex virus ICP22.

To investigate the role of varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase during infection, a VZV mutant was generated in which two contiguous stop codons were introduced into ORF47, thus eliminating expression of the ORF47 kinase. ORF47 kinase was not essential for the growth of VZV in cultured cells, and the growth rate of the VZV mutant lacking ORF47 protein was indistinguishable from that of parental VZV. Nuclear extracts from cells infected with parental VZV contained several phosphorylated proteins which were not detected in extracts from cells infected with the ORF47 mutant. The herpes simplex virus type 1 (HSV-1) UL13 protein (the homolog of VZV ORF47 protein) is responsible for the posttranslational processing associated with phosphorylation of HSV-1 ICP22 (the homolog of VZV ORF63 protein). Immunoprecipitation of 32P-labeled proteins from cells infected with parental virus and those infected with ORF47 mutant virus yielded similar amounts of the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 (VZV gE), and the electrophoretic migration of these proteins was not affected by the lack of ORF47 kinase. Therefore, while the VZV ORF47 protein is capable of phosphorylating several cellular or viral proteins, it is not required for phosphorylation of the ORF63 protein in virus-infected cells.     

Characterization of Varicella-Zoster virus glycoprotein K (open reading frame 5) and its role in virus growth

Varicella-zoster virus (VZV) is an alphaherpesvirus that is the causative agent of chickenpox and herpes zoster. VZV open reading frame 5 (ORF5) encodes glycoprotein K (gK), which is conserved among alphaherpesviruses. While VZV gK has not been characterized, and its role in viral replication is unknown, homologs of VZV gK in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) have been well studied. To identify the VZV ORF5 gene product, we raised a polyclonal antibody against a fusion protein of ORF5 codons 25 to 122 with glutathione S-transferase and used it to study the protein in infected cells. A 40,000-molecular-weight protein was detected in cell-free virus by Western blotting. In immunogold electron microscopic studies, VZV gK was in enveloped virions and was evenly distributed in the cytoplasm in infected cells. To determine the function of VZV gK in virus growth, a series of gK deletion mutants were constructed with VZV cosmid DNA derived from the Oka strain. Full and partial deletions in gK prevented viral replication when the gK mutant cosmids were transfected into melanoma cells. Insertion of the HSV-1 (KOS) gK gene into the endogenous VZV gK site did not compensate for the deletion of VZV gK. The replacement of VZV gK at a nonnative AvrII site in the VZV genome restored the phenotypic characteristics of intact recombinant Oka (rOka) virus. Moreover, gK complementing cells transfected with a full gK deletion mutant exhibited viral plaques indistinguishable from those of rOka. Our results are consistent with the studies of gK proteins of HSV-1 and PRV showing that gK is indispensable for viral replication.     

Neuronal Subtype and Satellite Cell Tropism Are Determinants of Varicella-Zoster Virus Virulence in Human Dorsal Root Ganglia Xenografts In Vivo

Varicella zoster virus (VZV), a human alphaherpesvirus, causes varicella during primary infection. VZV reactivation from neuronal latency may cause herpes zoster, post herpetic neuralgia (PHN) and other neurologic syndromes. To investigate VZV neuropathogenesis, we developed a model using human dorsal root ganglia (DRG) xenografts in immunodeficient (SCID) mice. The SCID DRG model provides an opportunity to examine characteristics of VZV infection that occur in the context of the specialized architecture of DRG, in which nerve cell bodies are ensheathed by satellite glial cells (SGC) which support neuronal homeostasis. We hypothesized that VZV exhibits neuron-subtype specific tropism and that VZV tropism for SGC contributes to VZV-related ganglionopathy. Based on quantitative analyses of viral and cell protein expression in DRG tissue sections, we demonstrated that, whereas DRG neurons had an immature neuronal phenotype prior to implantation, subtype heterogeneity was observed within 20 weeks and SGC retained the capacity to maintain neuronal homeostasis longterm. Profiling VZV protein expression in DRG neurons showed that VZV enters peripherin+ nociceptive and RT97+ mechanoreceptive neurons by both axonal transport and contiguous spread from SGC, but replication in RT97+ neurons is blocked. Restriction occurs even when the SGC surrounding the neuronal cell body were infected and after entry and ORF61 expression, but before IE62 or IE63 protein expression. Notably, although contiguous VZV spread with loss of SGC support would be predicted to affect survival of both nociceptive and mechanoreceptive neurons, RT97+ neurons showed selective loss relative to peripherin+ neurons at later times in DRG infection. Profiling cell factors that were upregulated in VZV-infected DRG indicated that VZV infection induced marked pro-inflammatory responses, as well as proteins of the interferon pathway and neuroprotective responses. These neuropathologic changes observed in sensory ganglia infected with VZV may help to explain the neurologic sequelae often associated with zoster and PHN.     

Requirement of varicella-zoster virus immediate-early 4 protein for viral replication

Varicella-zoster virus (VZV) is an alphaherpesvirus that causes two diseases, chickenpox and zoster. VZV open reading frame 4 (ORF4) encodes the immediate-early 4 (IE4) protein, which is conserved among alphaherpesvirus and has transactivation activity in transient transfections. To determine whether the ORF4 gene product is essential for viral replication, we used VZV cosmids to remove ORF4 from the VZV genome. Deleting ORF4 was incompatible with recovery of infectious virus, whereas transfections done by using repaired cosmids with ORF4 inserted at a nonnative site yielded virus. To analyze the functional domain of IE4, we introduced a mutation altering the C-terminal amino acids, KYFKC (K443S), which was designed to disrupt the dimerization of IE4 protein. Transfections with these mutant cosmids yielded no virus, indicating that this KYFKC motif was essential for IE4 function.     

Varicella‐Zoster virus ORF9 is an antagonist of the DNA sensor cGAS

Varicella‐Zoster virus (VZV) causes chickenpox and shingles. Although the infection is associated with severe morbidity in some individuals, molecular mechanisms that determine innate immune responses remain poorly defined. We found that the cGAS/STING DNA sensing pathway was required for type I interferon (IFN) induction during VZV infection and that recognition of VZV by cGAS restricted its replication. Screening of a VZV ORF expression library identified the essential VZV tegument protein ORF9 as a cGAS antagonist. Ectopically or virally expressed ORF9 bound to endogenous cGAS leading to reduced type I IFN responses to transfected DNA. Confocal microscopy revealed co‐localisation of cGAS and ORF9. ORF9 and cGAS also interacted directly in a cell‐free system and phase‐separated together with DNA. Furthermore, ORF9 inhibited cGAMP production by cGAS. Taken together, these results reveal the importance of the cGAS/STING DNA sensing pathway for VZV recognition and identify a VZV immune antagonist that partially but directly interferes with DNA sensing via cGAS.