Simian varicella virus infection and reactivation in rhesus macaques trigger cytokine and Aß40/42 alterations in serum and cerebrospinal fluid.

Simian varicella virus (SVV) produces peripheral inflammatory responses during varicella (primary infection) and zoster (reactivation) in rhesus macaques (RM). However, it is unclear if peripheral measures are accurate proxies for central nervous system (CNS) responses. Thus, we analyzed cytokine and Aß42/Aß40 changes in paired serum and cerebrospinal fluid (CSF) during the course of infection. During varicella and zoster, every RM had variable changes in serum and CSF cytokine and Aß42/Aß40 levels compared to pre-inoculation levels. Overall, peripheral infection appears to affect CNS cytokine and Aß42/Aß40 levels independent of serum responses, suggesting that peripheral disease may contribute to CNS disease.

Simian Varicella Virus Infection and Reactivation in Rhesus Macaques Trigger Cytokine and Aß40/42 Alterations in Serum and Cerebrospinal Fluid.

Simian varicella virus (SVV) produces peripheral inflammatory responses during varicella (primary infection) and zoster (reactivation) in rhesus macaques (RM). However, it is unclear if peripheral measures are accurate proxies for central nervous system (CNS) responses. Thus, we analyzed cytokine and Aß42/Aß40 changes in paired serum and cerebrospinal fluid (CSF) during the course of infection. During varicella and zoster, every RM had variable changes in serum and CSF cytokine and Aß42/Aß40 levels compared to pre-inoculation levels. Overall, peripheral infection appears to affect CNS cytokine and Aß42/Aß40 levels independent of serum responses, suggesting that peripheral disease may contribute to CNS disease.

Zoster-Associated Prothrombotic Plasma Exosomes and Increased Stroke Risk.

Herpes zoster (HZ; shingles) caused by varicella zoster virus reactivation increases stroke risk for up to 1 year after HZ. The underlying mechanisms are unclear, however, the development of stroke distant from the site of zoster (eg, thoracic, lumbar, sacral) that can occur months after resolution of rash points to a long-lasting, virus-induced soluble factor (or factors) that can trigger thrombosis and/or vasculitis. Herein, we investigated the content and contributions of circulating plasma exosomes from HZ and non-HZ patient samples. Compared with non-HZ exosomes, HZ exosomes (1) contained proteins conferring a prothrombotic state to recipient cells and (2) activated platelets leading to the formation of platelet-leukocyte aggregates. Exosomes 3 months after HZ yielded similar results and also triggered cerebrovascular cells to secrete the proinflammatory cytokines, interleukin 6 and 8. These results can potentially change clinical practice through addition of antiplatelet agents for HZ and initiatives to increase HZ vaccine uptake to decrease stroke risk.

Simian Varicella Virus Pathogenesis in Skin during Varicella and Zoster.

Primary simian varicella virus (SVV) infection and reactivation in nonhuman primates is a valuable animal model in the study of varicella zoster virus disease [varicella (chickenpox) and herpes zoster (shingles)]. To understand SVV pathogenesis in skin, we inoculated 10 rhesus macaques with SVV, resulting in varicella rash. After the establishment of latency, eight of the monkeys were immunosuppressed using tacrolimus with or without irradiation and prednisone and two monkeys were not immunosuppressed. Zoster rash developed in all immunosuppressed monkeys and in one non-immunosuppressed monkey. Five monkeys had recurrent zoster. During varicella and zoster, SVV DNA in skin scrapings ranged from 50 to 107 copies/100 ng of total DNA and 2-127 copies/100 ng of total DNA, respectively. Detection of SVV DNA in blood during varicella was more frequent and abundant compared to that of zoster. During varicella and zoster, SVV antigens colocalized with neurons expressing ß-III tubulin in epidermis, hair follicles, and sweat glands, suggesting axonal transport of the virus. Together, we have demonstrated that both SVV DNA and antigens can be detected in skin lesions during varicella and zoster, providing the basis for further studies on SVV skin pathogenesis, including immune responses and mechanisms of peripheral spread.

The Enduring Legacy of Randall Cohrs: A Meeting of the Minds in the Rocky Mountains.

Randall Cohrs established the Colorado Alphaherpesvirus Latency Society (CALS) in 2011 [...].

Azadirachta indica A. Juss bark extract and its Nimbin isomers restrict ß-coronaviral infection and replication.

Emerging mutations in the SARS-CoV-2 genome pose a challenge for vaccine development and antiviral therapy. The antiviral efficacy of Azadirachta indica bark extract (NBE) was assessed against SARS-CoV-2 and m-CoV-RSA59 infection. Effects of in vivo intranasal or oral NBE administration on viral load, inflammatory response, and histopathological changes were assessed in m-CoV-RSA59-infection. NBE administered inhibits SARS-CoV-2 and m-CoV-RSA59 infection and replication in vitro, reducing Envelope and Nucleocapsid gene expression. NBE ameliorates neuroinflammation and hepatitis in vivo by restricting viral replication and spread. Isolated fractions of NBE enriched in Nimbin isomers shows potent inhibition of m-CoV-RSA59 infection in vitro. In silico studies revealed that NBE could target Spike and RdRp of m-CoV and SARS-CoV-2 with high affinity. NBE has a triterpenoids origin that may allow them to competitively target panoply of viral proteins to inhibit mouse and different strains of human coronavirus infections, suggesting its potential as an antiviral against pan-ß-Coronaviruses.

Targeted RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Temporal Arteries From Giant Cell Arteritis Cases Reveals Viral Signatures.

BACKGROUND AND OBJECTIVES: Varicella zoster virus (VZV) antigen has been detected in temporal arteries (TAs) of individuals with giant cell arteritis (GCA), the most common systemic vasculitis in older adults. Thus, we explored the contribution of VZV to GCA pathogenesis. METHODS: Formalin-fixed, paraffin-embedded TA sections from biopsy-positive GCA participants with VZV antigen (GCA/VZV-positive; n = 20) and without (GCA/VZV-negative, n = 20) and from normal participants with VZV antigen (control/VZV-positive, n = 11) and without (control/VZV-negative, n = 20) were analyzed by targeted RNA sequencing of the whole human transcriptome (BioSpyder TempO-Seq). Ingenuity pathway analysis and R-computational program were used to identify differentially expressed genes and pathways between groups. RESULTS: Compared with control/VZV-negative TAs, GCA/VZV-negative and GCA/VZV-positive TAs were significantly enriched for human transcripts specific for pathways involved in viral infections, including viral entry, nuclear factor kappa B activation by viruses, and other pathogen-related immune activation pathways. Similarly, human gene sets supporting viral infection were found in control/VZV-positive TAs that showed no morphological signs of inflammation, suggesting that the enriched pathways were not nonspecific signatures of infiltrating immune cells. All GCA TAs and control/VZV-positive TAs showed enrichment of transcripts involved in vascular remodeling, including smooth muscle cell migration. DISCUSSION: The detection of viral and immune activation pathways in GCA TAs supports a role for virus infection in GCA pathogenesis. In addition, the detection of viral pathways in control/VZV-positive TAs, along with vascular remodeling pathways, suggests that these samples may represent early infection with progression to clinical disease, depending on host and other environmental factors.

Histopathological Analysis of Adrenal Glands after Simian Varicella Virus Infection.

Latent varicella zoster virus (VZV) has been detected in human adrenal glands, raising the possibility of virus-induced adrenal damage and dysfunction during primary infection or reactivation. Rare cases of bilateral adrenal hemorrhage and insufficiency associated with VZV reactivation have been reported. Since there is no animal model for VZV infection of adrenal glands, we obtained adrenal glands from two non-human primates (NHPs) that spontaneously developed varicella from primary simian varicella virus (SVV) infection, the NHP VZV homolog. Histological and immunohistochemical analysis revealed SVV antigen and DNA in the adrenal medulla and cortex of both animals. Adrenal glands were observed to have Cowdry A inclusion bodies, cellular necrosis, multiple areas of hemorrhage, and varying amounts of polymorphonuclear cells. No specific association of SVV antigen with ßIII-tubulin-positive nerve fibers was found. Overall, we found that SVV can productively infect NHP adrenal glands, and is associated with inflammation, hemorrhage, and cell death. These findings suggest that further studies are warranted to examine the contribution of VZV infection to human adrenal disease. This study also suggests that VZV infection may present itself as acute adrenal dysfunction with "long-hauler" symptoms of fatigue, weakness, myalgias/arthralgias, and hypotension.

Amylin, Aß42, and Amyloid in Varicella Zoster Virus Vasculopathy Cerebrospinal Fluid and Infected Vascular Cells.

BACKGROUND: Varicella zoster virus (VZV) vasculopathy is characterized by persistent arterial inflammation leading to stroke. Studies show that VZV induces amyloid formation that may aggravate vasculitis. Thus, we determined if VZV central nervous system infection produces amyloid. METHODS: Aß peptides, amylin, and amyloid were measured in cerebrospinal fluid (CSF) from 16 VZV vasculopathy subjects and 36 stroke controls. To determine if infection induced amyloid deposition, mock- and VZV-infected quiescent primary human perineurial cells (qHPNCs), present in vasculature, were analyzed for intracellular amyloidogenic transcripts/proteins and amyloid. Supernatants were assayed for amyloidogenic peptides and ability to induce amyloid formation. To determine amylin's function during infection, amylin was knocked down with small interfering RNA and viral complementary DNA (cDNA) was quantitated. RESULTS: Compared to controls, VZV vasculopathy CSF had increased amyloid that positively correlated with amylin and anti-VZV antibody levels; Aß40 was reduced and Aß42 unchanged. Intracellular amylin, Aß42, and amyloid were seen only in VZV-infected qHPNCs. VZV-infected supernatant formed amyloid fibrils following addition of amyloidogenic peptides. Amylin knockdown decreased viral cDNA. CONCLUSIONS: VZV infection increased levels of amyloidogenic peptides and amyloid in CSF and qHPNCs, indicating that VZV-induced amyloid deposition may contribute to persistent arterial inflammation in VZV vasculopathy. In addition, we identified a novel proviral function of amylin.

Elevated serum substance P during simian varicella virus infection in rhesus macaques: implications for chronic inflammation and adverse cerebrovascular events.

Varicella and zoster, produced by varicella-zoster virus (VZV), are associated with an increased risk of stroke that may be due to persistent inflammation and hypercoagulability. Because substance P is associated with inflammation, hypercoagulability, and atherosclerotic plaque rupture that may contribute to increased stroke risk after VZV infection, we measured serum substance P in simian varicella virus-infected rhesus macaques. We found significantly increased and persistent serum substance P concentrations during varicella and zoster compared with pre-inoculation, supporting the hypothesis that VZV-induced increases in serum substance P may contribute to increased stroke risk associated with VZV infection.

Design and preparation of nanostructures based on Krafft point of nonionic amphiphiles for delivery of poorly water-soluble compounds.

Micellar solubilization can effectively dissolve low water-soluble compounds in aqueous environment, however, the micellar systems are not able to withstand dilution and maintain solubilization of poorly water-soluble drugs below critical micelle concentration. To overcome the drawbacks of conventional micellar solubilization, nonionic polyoxyethylated surfactants with Krafft points at or higher than body temperature were chosen to create novel micelle-based nanostructures as a delivery vehicle for poorly water-soluble compounds. A technique "thermo-spray process" was developed for the preparation of the nanostructures-containing formulation, in which the drug-containing micelle solution was first prepared and maintained at the elevated temperature above the Krafft point of the surfactant, then spray dried to solidify the obtained micelle-like nanostructure at room temperature. Lactose was used as an excipient to embed the nanostructures in the thermo-spray products. Water insoluble spherical nanoparticles with size range from 80 to 250 nm were obtained after reconstitution of the product at the temperature lower than Krafft point. When paclitaxel was used as model drug, the micelle-like nanostructures exhibited similar drug entrapment efficiency, solubility enhancement and drug release facilitation as conventional micelles, but provided lower critical micellar concentration at body temperature, and good encapsulation stability upon storage and dilution. These findings indicated that the developed thermo-spray product can serve as a promising delivery platform for drugs with low aqueous solubility.

Varicella-Zoster Virus Infection of Primary Human Spinal Astrocytes Produces Intracellular Amylin, Amyloid-ß, and an Amyloidogenic Extracellular Environment.

BACKGROUND: Herpes zoster is linked to amyloid-associated diseases, including dementia, macular degeneration, and diabetes mellitus, in epidemiological studies. Thus, we examined whether varicella-zoster virus (VZV)-infected cells produce amyloid. METHODS: Production of intracellular amyloidogenic proteins (amylin, amyloid precursor protein [APP], and amyloid-ß [Aß]) and amyloid, as well as extracellular amylin, Aß, and amyloid, was compared between mock- and VZV-infected quiescent primary human spinal astrocytes (qHA-sps). The ability of supernatant from infected cells to induce amylin or Aß42 aggregation was quantitated. Finally, the amyloidogenic activity of viral peptides was examined. RESULTS: VZV-infected qHA-sps, but not mock-infected qHA-sps, contained intracellular amylin, APP, and/or Aß, and amyloid. No differences in extracellular amylin, Aß40, or Aß42 were detected, yet only supernatant from VZV-infected cells induced amylin aggregation and, to a lesser extent, Aß42 aggregation into amyloid fibrils. VZV glycoprotein B (gB) peptides assembled into fibrils and catalyzed amylin and Aß42 aggregation. CONCLUSIONS: VZV-infected qHA-sps produced intracellular amyloid and their extracellular environment promoted aggregation of cellular peptides into amyloid fibrils that may be due, in part, to VZV gB peptides. These findings suggest that together with host and other environmental factors, VZV infection may increase the toxic amyloid burden and contribute to amyloid-associated disease progression.

Current In Vivo Models of Varicella-Zoster Virus Neurotropism.

Varicella-zoster virus (VZV), an exclusively human herpesvirus, causes chickenpox and establishes a latent infection in ganglia, reactivating decades later to produce zoster and associated neurological complications. An understanding of VZV neurotropism in humans has long been hampered by the lack of an adequate animal model. For example, experimental inoculation of VZV in small animals including guinea pigs and cotton rats results in the infection of ganglia but not a rash. The severe combined immune deficient human (SCID-hu) model allows the study of VZV neurotropism for human neural sub-populations. Simian varicella virus (SVV) infection of rhesus macaques (RM) closely resembles both human primary VZV infection and reactivation, with analyses at early times after infection providing valuable information about the extent of viral replication and the host immune responses. Indeed, a critical role for CD4 T-cell immunity during acute SVV infection as well as reactivation has emerged based on studies using RM. Herein we discuss the results of efforts from different groups to establish an animal model of VZV neurotropism.

Simian Varicella Virus DNA in Saliva and Buccal Cells After Experimental Acute Infection in Rhesus Macaques.

Simian varicella virus (SVV) infection of non-human primates is the counterpart of varicella zoster virus (VZV) infection in humans. To develop non-invasive methods of assessing SVV infection, we tested for the presence of SVV DNA in saliva, as has been documented in human VZV infection, and in buccal cells to determine whether epithelial cells might provide a more reliable source of material for analysis. Five rhesus macaques intratracheally inoculated with SVV all developed varicella with viremia and macular-papular skin rash in 1-2 weeks, which resolved followed by establishment of latency. DNA extracted from longitudinal blood peripheral blood mononuclear cells (PBMCs), saliva and buccal samples collected during acute infection and establishment of latency were analyzed by real-time qPCR. After intratracheal inoculation, viremia was observed, with peak levels of 101-102 copies of SVV DNA in 100 ng of PBMC DNA at 4 and 7 days post inoculation (dpi), which then decreased at 9-56 dpi. In saliva and buccal cells at 7 dpi, 101-104 copies and 101-105 copies of SVV DNA in 100 ng of cellular DNA, respectively, were detected in all the five monkeys. At 9 dpi, saliva samples from only two of the five monkeys contained SVV DNA at 102-103 copies/100 ng of saliva DNA, while buccal cells from all five monkeys showed 100-103 copies of SVV DNA/100 ng of buccal cell DNA. Similar to viremia, SVV DNA in saliva and buccal cells at 11-56 dpi was lower, suggesting clearance of viral shedding. SVV DNA levels were generally higher in buccal cells than in saliva. Our findings indicate that SVV shedding into the oral cavity parallels acute SVV infection and underscore the relevance of both saliva and buccal cell samples to monitor acute varicella virus infection.

Reactivation of Simian Varicella Virus in Rhesus Macaques after CD4 T Cell Depletion.

Rhesus macaques intrabronchially inoculated with simian varicella virus (SVV), the counterpart of human varicella-zoster virus (VZV), developed primary infection with viremia and rash, which resolved upon clearance of viremia, followed by the establishment of latency. To assess the role of CD4 T cell immunity in reactivation, monkeys were treated with a single 50-mg/kg dose of a humanized monoclonal anti-CD4 antibody; within 1 week, circulating CD4 T cells were reduced from 40 to 60% to 5 to 30% of the total T cell population and remained low for 2 months. Very low viremia was seen only in some of the treated monkeys. Zoster rash developed after 7 days in the monkey with the most extensive CD4 T cell depletion (5%) and in all other monkeys at 10 to 49 days posttreatment, with recurrent zoster in one treated monkey. SVV DNA was detected in the lung from two of five monkeys, in bronchial lymph nodes from one of the five monkeys, and in ganglia from at least two dermatomes in three of five monkeys. Immunofluorescence analysis of skin rash, lungs, lymph nodes, and ganglia revealed SVV ORF63 protein at the following sites: sweat glands in skin; type II cells in lung alveoli, macrophages, and dendritic cells in lymph nodes; and the neuronal cytoplasm of ganglia. Detection of SVV antigen in multiple tissues upon CD4 T cell depletion and virus reactivation suggests a critical role for CD4 T cell immunity in controlling varicella virus latency.IMPORTANCE Reactivation of latent VZV in humans can result in serious neurological complications. VZV-specific cell-mediated immunity is critical for the maintenance of latency. Similar to VZV in humans, SVV causes varicella in monkeys, establishes latency in ganglia, and reactivates to produce shingles. Here, we show that depletion of CD4 T cells in rhesus macaques results in SVV reactivation, with virus antigens found in zoster rash and SVV DNA and antigens found in lungs, lymph nodes, and ganglia. These results suggest the critical role of CD4 T cell immunity in controlling varicella virus latency.

Simian Varicella Virus Infects Enteric Neurons and α4ß7 Integrin-Expressing Gut-Tropic T-Cells in Nonhuman Primates.

The pathogenesis of enteric zoster, a rare debilitating complication of reactivation of latent varicella-zoster virus (VZV) in the enteric nervous system (ENS), is largely unknown. Infection of monkeys with the closely related Varicellovirus simian varicella virus (SVV) mimics VZV disease in humans. In this study, we determined the applicability of the SVV nonhuman primate model to study Varicellovirus infection of the ENS. We confirmed VZV infection of the gut in latently infected adults and demonstrated that SVV DNA was similarly present in gut of monkeys latently infected with SVV using quantitative real-time PCR. In situ analyses showed that enteric neurons expressed SVV open reading frame (ORF) 63 RNA, but not viral nucleocapsid proteins, suggestive of latent ENS infection. During primary infection, SVV-infected T-cells were detected in gut-draining mesenteric lymph nodes and located in close vicinity to enteric nerves in the gut. Furthermore, flow cytometric analysis of blood from acutely SVV-infected monkeys demonstrated that virus-infected T-cells expressed the gut-homing receptor α4ß7 integrin. Collectively, the data demonstrate that SVV infects ENS neurons during primary infection and supports the role of T-cells in virus dissemination to the gut. Because SVV reactivation can be experimentally induced, the SVV nonhuman primate model holds great potential to study the pathogenesis of enteric zoster.

Attenuation of Simian Varicella Virus Infection by Enhanced Green Fluorescent Protein in Rhesus Macaques.

Simian varicella virus (SVV), the primate counterpart of varicella-zoster virus, causes varicella (chickenpox), establishes latency in ganglia, and reactivates to produce zoster. We previously demonstrated that a recombinant SVV expressing enhanced green fluorescent protein (rSVV.eGFP) is slightly attenuated both in culture and in infected monkeys. Here, we generated two additional recombinant SVVs to visualize infected cells in vitro and in vivo One harbors eGFP fused to the N terminus of open reading frame 9 (ORF9) (rSVV.eGFP-2a-ORF9), and another harbors eGFP fused to the C terminus of ORF66 (rSVV.eGFP-ORF66). Both recombinant viruses efficiently expressed eGFP in cultured cells. Both recombinant SVV infections in culture were comparable to that of wild-type SVV (SVV.wt). Unlike SVV.wt, eGFP-tagged SVV did not replicate in rhesus cells in culture. Intratracheal (i.t.) or i.t. plus intravenous (i.v.) inoculation of rhesus macaques with these new eGFP-tagged viruses resulted in low viremia without varicella rash, although SVV DNA was abundant in bronchoalveolar lavage (BAL) fluid at 10 days postinoculation (dpi). SVV DNA was also found in trigeminal ganglia of one monkey inoculated with rSVV.eGFP-ORF66. Intriguingly, a humoral response to both SVV and eGFP was observed. In addition, monkeys inoculated with the eGFP-expressing viruses were protected from superinfection with SVV.wt, suggesting that the monkeys had mounted an efficient immune response. Together, our results show that eGFP expression could be responsible for their reduced pathogenesis.IMPORTANCE SVV infection in nonhuman primates has served as an extremely useful animal model to study varicella-zoster virus (VZV) pathogenesis. eGFP-tagged viruses are a great tool to investigate their pathogenesis. We constructed and tested two new recombinant SVVs with eGFP inserted into two different locations in the SVV genome. Both recombinant SVVs showed robust replication in culture but reduced viremia compared to that with SVV.wt during primary infection in rhesus macaques. Our results indicate that conclusions on eGFP-tagged viruses based on in vitro results should be handled with care, since eGFP expression could result in attenuation of the virus.

Simian varicella virus inhibits the interferon gamma signalling pathway.

The alphaherpesvirus simian varicella virus (SVV) causes varicella and zoster in nonhuman primates. Herpesviruses evolved elaborate mechanisms to escape host immunity, but the immune evasion strategies employed by SVV remain ill-defined. We analysed whether SVV impairs the cellular response to key antiviral cytokine interferon-γ (IFNγ). SVV infection inhibited the expression of IFNγ-induced genes like C-X-C motif chemokine 10 and interferon regulatory factor 1. Phosphorylation and nuclear translocation of the signal transducer and activator of transcription 1 (STAT1) was blocked in SVV-infected cells, which did not involve cellular and viral phosphatases. SVV infection did not downregulate IFNγ receptor α and ß chain expression on the cell surface. Instead, STAT1, Janus tyrosine kinases 1 (JAK1) and JAK2 protein levels were significantly decreased in SVV-infected cells. Collectively, these results demonstrate that SVV targets three proteins in the IFNγ signal transduction pathway to escape the antiviral effects of IFNγ.

Characterization of the immune response in ganglia after primary simian varicella virus infection.

Primary simian varicella virus (SVV) infection in non-human primates causes varicella, after which the virus becomes latent in ganglionic neurons and reactivates to cause zoster. The host response in ganglia during establishment of latency is ill-defined. Ganglia from five African green monkeys (AGMs) obtained at 9, 13, and 20 days post-intratracheal SVV inoculation (dpi) were analyzed by ex vivo flow cytometry, immunohistochemistry, and in situ hybridization. Ganglia at 13 and 20 dpi exhibited mild inflammation. Immune infiltrates consisted mostly of CD8(dim) and CD8(bright) memory T cells, some of which expressed granzyme B, and fewer CD11c(+) and CD68(+) cells. Chemoattractant CXCL10 transcripts were expressed in neurons and infiltrating inflammatory cells but did not co-localize with SVV open reading frame 63 (ORF63) RNA expression. Satellite glial cells expressed increased levels of activation markers CD68 and MHC class II at 13 and 20 dpi compared to those at 9 dpi. Overall, local immune responses emerged as viral DNA load in ganglia declined, suggesting that intra-ganglionic immunity contributes to restricting SVV replication.