A Tn7-transposition approach was utilized for site-specific insertion of foreign genes into the genome of simian varicella virus (SVV), the causative agent of simian varicella in nonhuman primates. The severe acute respiratory syndrome coronavirus (SARS-CoV-2) nucleocapsid (N) gene and receptor binding domain (RBD) of the spike gene were inserted into the ORF 14 region of the SVV genome cloned into a bacterial artificial chromosome and then transfected into Vero cells to generate the infectious recombinant SVV (rSVV). The rSVV replicated efficiently in infected Vero cells and expressed the N and RBD antigens as indicated by immunoblot and immunofluorescence assays. Tn7-mediated transposition provides a rapid and efficient method for constructing rSVVs which may be evaluated as live-attenuated vaccines.

Varicella zoster virus (VZV) and simian varicella virus (SVV) cause varicella (chickenpox) in children and nonhuman primates, respectively. After resolution of acute disease, the viruses establish latent infection in neural ganglia, after which they may reactivate to cause a secondary disease, such as herpes zoster. SVV infection of nonhuman primates provides a model to investigate VZV pathogenesis and antiviral strategies. The VZV and SVV genomes are similar in size and structure and share 70–75% DNA homology. SVV and VZV DNAs are co-linear in gene arrangement with the exception of the left end of the viral genomes. Viral gene expression is regulated into immediate early, early, and late transcription during in vitro and in vivo infection. During viral latency, VZV and SVV gene expression is limited to transcription of a viral latency-associated transcript (VLT). VZV and SVV are closely related alphaherpesviruses that likely arose from an ancestral varicella virus that evolved through cospeciation into species-specific viruses.

HIV vaccine mediated efficacy, using an expanded live attenuated recombinant varicella virus-vectored SIV rSVV-SIVgag/env vaccine prime with adjuvanted SIV-Env and SIV-Gag protein boosts, was evaluated in a female rhesus macaques (RM) model against repeated intravaginal SIV challenges. Vaccination induced anti-SIV IgG responses and neutralizing antibodies were found in all vaccinated RMs. Three of the eight vaccinated RM remained uninfected (vaccinated and protected, VP) after 13 repeated challenges with the pathogenic SIVmac251-CX-1. The remaining five vaccinated and infected (VI) macaques had significantly reduced plasma viral loads compared with the infected controls (IC). A significant increase in systemic central memory CD4+ T cells and mucosal CD8+ effector memory T-cell responses was detected in vaccinated RMs compared to controls. Variability in lymph node SIV-Gag and Env specific CD4+ and CD8+ T cell cytokine responses were detected in the VI RMs while all three VP RMs had more durable cytokine responses following vaccination and prior to challenge. VI RMs demonstrated predominately SIV-specific monofunctional cytokine responses while the VP RMs generated polyfunctional cytokine responses. This study demonstrates that varicella virus-vectored SIV vaccination with protein boosts induces a 37.5% efficacy rate against pathogenic SIV challenge by generating mucosal memory, virus specific neutralizing antibodies, binding antibodies, and polyfunctional T-cell responses.

BACKGROUND:Simian varicella virus (SVV) is a primate herpesvirus that causes a natural varicella-like disease in Old World monkeys and may cause epizootics in facilities housing nonhuman primates. SVV infection of nonhuman primates is used as an experimental model to investigate varicella pathogenesis and to develop antiviral strategies. METHODS:An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect SVV antibodies in infected rhesus macaque monkeys. RESULTS:An ELISA determined SVV antibody titers following experimental infection. SVV IgG was detected by day 14 post-infection and remained elevated for at least 84 days. CONCLUSIONS:The SVV ELISA is a safe and rapid approach to confirm SVV seropositivity and to determine SVV antibody titers in naturally and experimentally SVV-infected monkeys. In addition to being a useful diagnostic assay to rapidly confirm acute disease or past SVV infection, the SVV ELISA is a valuable epidemiological tool to determine the incidence of SVV in non-human primate facilities.

Background: Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus that infects humans and results in chickenpox and herpes zoster. A number of VZV genes remain functionally uncharacterized and since VZV is an obligate human pathogen, rigorous evaluation of VZV mutants in vivo remains challenging. Simian varicella virus (SVV) is homologous to VZV and SVV infection of rhesus macaques (RM) closely mimics VZV infection of humans. Recently the SVV genome was cloned as a bacterial artificial chromosome (BAC) and BAC-derived SVV displayed similar replication kinetics as wild-type (WT) SVV in vitro.Methods: RMs were infected with BAC-derived SVV or WT SVV at 4x10(5) PFU intrabronchially (N=8, 4 per group, sex and age matched). We collected whole blood (PBMC) and bronchoalveolar lavage (BAL) at various days post-infection (dpi) and sensory ganglia during latent infection (>84 dpi) at necropsy and compared disease progression, viral replication, immune response and the establishment of latency.Results: Viral replication kinetics and magnitude in bronchoalveolar lavage cells and whole blood as well as rash severity and duration were similar in RMs infected with SVV BAC or WT SVV. Moreover, SVV-specific B and T cell responses were comparable between BAC and WT-infected animals. Lastly, we measured viral DNA in sensory ganglia from both cohorts of infected RMs during latent infection.Conclusions: SVV BAC is as pathogenic and immunogenic as WT SVV in vivo. Thus, the SVV BAC genetic system combined with the rhesus macaque animal model can further our understanding of viral ORFs important for VZV pathogenesis and the development of second-generation vaccines.

Varicella-zoster virus (VZV) vaccines induce immunity against childhood chickenpox and against shingles in older adults. The safety, efficacy, and widespread use of VZV vaccines suggest that they may also be effective as recombinant vaccines against other infectious diseases that affect the young and the elderly. The generation of recombinant VZV vaccines and their evaluation in animal models are reviewed. The potential advantages and limitations of recombinant VZV vaccines are addressed.

ABSTRACTVaricella zoster virus (VZV) is a neurotropic alphaherpesvirus that causes chickenpox during primary infection and establishes latency in sensory ganglia. Infection of rhesus macaques (RM) with the homologous simian varicella virus (SVV) recapitulates hallmarks of VZV infection. We have shown that an antisense transcript of SVV open reading frame 61 (ORF61), a viral transactivator, was detected most frequently in latently infected RM sensory ganglia. In this study, we compared disease progression, viral replication, immune response, and the establishment of latency following intrabronchial infection with a recombinant SVV lacking ORF61 (SVVΔORF61) to those following infection with wild-type (WT) SVV. Varicella severity and viral latency within sensory ganglia were comparable in RMs infected with SVVΔORF61 and WT SVV. In contrast, viral loads, B and T cell responses, and plasma inflammatory cytokine levels were decreased in RMs infected with SVVΔORF61. To investigate the mechanisms underlying the reduced adaptive immune response, we compared acute SVV gene expression, frequency and proliferation of dendritic cell (DC) subsets, and the expression of innate antiviral genes in bronchoalveolar lavage (BAL) samples. The abundance of SVV transcripts in all kinetic classes was significantly decreased in RMs infected with SVVΔORF61. In addition, we detected a higher frequency and proliferation of plasmacytoid dendritic cells in BAL fluid at 3 days postinfection in RMs infected with SVVΔORF61, which was accompanied by a slight increase in type I interferon gene expression. Taken together, our data suggest that ORF61 plays an important role in orchestrating viral gene expressionin vivoand interferes with the host antiviral interferon response.

The simian varicella virus (SVV) genome encodes ORF A, a truncated homolog of SVV ORF 4. The SVV ORF A was expressed as a 1.0-kb transcript in SVV-infected Vero cells. The ORF A promoter was active in infected Vero cells and was stimulated by the SVV immediate-early gene ORF 62 product (IE62), a viral transactivator of SVV genes. The SVV ORF A did not transactivate SVV IE, early, or late gene promoters in transfected Vero cells and was unable to augment IE62-mediated transactivation of SVV promoters. A SVV mutant lacking ORF A replicated as efficiently as wild-type SVV in infected Vero cells, indicating that ORF A expression is not essential for in vitro replication.

Background An effective AIDS vaccine remains one of the highest priorities in HIV-research. Our recent study showed that vaccination of rhesus macaques with recombinant simian varicella virus (rSVV) vector – simian immunodeficiency virus (SIV) envelope and gag genes, induced neutralizing antibodies and cellular immune responses to SIV and also significantly reduced plasma viral loads following intravenous pathogenic challenge with SIV MAC251 /CX1. Findings The purpose of this study was to define cellular immunological correlates of protection in rSVV-SIV vaccinated and SIV challenged animals. Immunofluorescent staining and multifunctional assessment of SIV-specific T-cell responses were evaluated in both Experimental and Control vaccinated animal groups. Significant increases in the proliferating CD4+ T-cell population and polyfunctional T-cell responses were observed in all Experimental-vaccinated animals compared with the Control-vaccinated animals. Conclusions Increased CD4+ T-cell proliferation was significantly and inversely correlated with plasma viral load. Increased SIV-specific polyfunctional cytokine responses and increased proliferation of CD4+ T-cell may be crucial to control plasma viral loads in vaccinated and SIV MAC251 /CX1 challenged macaques.

Simian varicella virus (SVV) is closely related to human varicella-zoster virus and causes varicella and zoster-like disease in nonhuman primates. In this study, a mini-F replicon was inserted into a SVV cosmid, and infectious SVV was generated by co-transfection of Vero cells with overlapping SVV cosmids. The entire SVV genome, cloned as a bacterial artificial chromosome (BAC), was stably propagated upon serial passage in E. coli. Transfection of pSVV-BAC DNA into Vero cells yielded infectious SVV (rSVV-BAC). The mini-F vector sequences flanked by loxP sites were removed by co-infection of Vero cells with rSVV-BAC and adenovirus expressing Cre-recombinase. Recombinant SVV generated using the SVV-BAC genetic system has similar molecular and in vitro replication properties as wild-type SVV. To demonstrate the utility of this approach, a SVV ORF 10 deletion mutant was created using two-step Red-mediated recombination. The results indicate that SVV ORF 10, which encodes a homolog of the HSV-1 virion VP-16 transactivator protein, is not essential for in vitro replication but is required for optimal replication in cell culture.

Simian varicella virus (SVV) open reading frame (ORF) 63, duplicated in the virus genome as ORF 70, is homologous to varicella zoster virus ORF 63/70. Transfection of bacterial artificial chromosome clones containing the wild-type SVV genome and mutants with stop codons in ORF 70, in both ORFs 63 and 70 and the repaired virus DNA sequences into Vero cells produced a cytopathic effect (CPE). The onset of CPE was much slower with the double-mutant transfectants (10 days vs. 3 days) and plaques were smaller. While SVV ORF 63 is not required for replication in culture, its expression leads to robust virus replication.

Simian varicella virus (SVV) is a primate herpesvirus that is closely related to varicella-zoster virus (VZV), the causative agent of varicella (chickenpox) and herpes zoster (shingles). Epizootics of simian varicella occur sporadically in facilities housing Old World monkeys. This review summarizes the molecular properties of SVV. The SVV and VZV genomes are similar in size, structure, and gene arrangement. The 124.5 kilobase pair (kbp) SVV genome includes a 104.7 kbp long component covalently linked to a short component, which includes a 4.9 kbp unique short segment flanked by 7.5 kbp inverted repeat sequences. SVV DNA encodes 69 distinct open reading frames, three of which are duplicated within the viral inverted repeats. The viral genome is coordinately expressed, and immediate early (IE), early, and late genes have been characterized. Genetic approaches have been developed to create SVV mutants, which will be used to study the role of SVV genes in viral pathogenesis, latency, and reactivation. In addition, SVV expressing foreign genes are being investigated as potential recombinant varicella vaccines.

The development of an effective AIDS vaccine remains one of the highest priorities in HIV research. The live, attenuated varicella-zoster virus (VZV) Oka vaccine, safe and effective for prevention of chickenpox and zoster, also has potential as a recombinant vaccine against other pathogens, including human immunodeficiency virus (HIV). The simian varicella model, utilizing simian varicella virus (SVV), offers an approach to evaluate recombinant varicella vaccine candidates. Recombinant SVV (rSVV) vaccine viruses expressing simian immunodeficiency virus (SIV) env and gag antigens were constructed. The hypothesis tested was that a live, attenuated rSVV-SIV vaccine will induce immune responses against SIV in the rhesus macaques and provide protection against SIV challenge. The results demonstrated that rSVV-SIV vaccination induced low levels of neutralizing antibodies and cellular immune responses to SIV in immunized rhesus macaques and significantly reduced viral loads following intravenous challenge with pathogenic SIVmac251-CX-1.

Neurotropic herpesviruses express viral deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and uracil DNA glycosylase (UDG) enzymes which may reduce uracil misincorporation into viral DNA, particularly in neurons of infected ganglia. The simian varicella virus (SVV) dUTPase (ORF 8) and UDG (ORF 59) share 37.7% and 53.9% amino acid identity, respectively, with varicella-zoster virus (VZV) homologs. Infectious SVV mutants defective in either dUTPase (SVV-dUTPase−) or UDG (SVV-UDG−) activity or both (SVV-dUTPase−/UDG−) were constructed using recA assisted restriction endonuclease cleavage (RARE) and a cosmid recombination system. Loss of viral dUTPase and UDG enzymatic activity was confirmed in CV-1 cells infected with the SVV mutants. The SVV-dUTPase−, SVV-UDG−, and SVV-dUTPase−/UDG− mutants replicated as efficiently as wild-type SVV in cell culture. SVV dUTPase and UDG expression was detected in tissues derived from acutely infected animals, but not in tissues derived from latently infected animals. Further studies will evaluate the pathogenesis of SVV dUTPase and UDG mutants and their potential as varicella vaccines.

Recombinant simian varicella viruses (rSVVs) were engineered to express respiratory syncytial virus (RSV) antigens. The RSV surface glycoprotein G and second matrix protein M2 (22k) genes were cloned into the SVV genome, and recombinant viruses were characterized in vitro and in vivo. rSVVs were also engineered to express the membrane-anchored or secreted forms of the RSV-G protein as well as an RSV G lacking its chemokine mimicry motif (CX3C), which may have different effects on priming the host immune response. The RSV genes were efficiently expressed in rSVV/RSV-infected Vero cells as RSV-G and -M2 transcripts were detected by RT-PCR, and RSV antigens were detected by immunofluorescence and immunoblot assays. The rSVVs replicated efficiently in Vero cell culture. Rhesus macaques immunized with rSVV/RSV-G and rSVV/RSV-M2 vaccines produced antibody responses to SVV and RSV antigens. The results demonstrate that recombinant varicella viruses are suitable vectors for the expression of RSV antigens and may represent a novel vaccine strategy for immunization against both pathogens.

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.

Simian varicella virus (SVV) and varicella-zoster virus (VZV) are closely related alphaherpesviruses that cause varicella (chickenpox) in nonhuman primates and humans, respectively. After resolution of the primary disease, SVV and VZV establish latent infection of neural ganglia and may later reactivate to cause a secondary disease (herpes zoster). This study investigated SVV gene expression in neural ganglia derived from latently infected vervet monkeys. SVV transcripts were detected in neural ganglia, but not in liver or lung tissues, of latently infected animals. A transcript mapping to open reading frame (ORF) 61 (herpes simplex virus type 1 [HSV-1] ICP0 homolog) was consistently detected in latently infected trigeminal, cervical, and lumbar ganglia by reverse transcriptase PCR. Further analysis confirmed that this SVV latency-associated transcript (LAT) was oriented antisense to the gene 61 mRNA. SVV ORF 21 transcripts were also detected in 42% of neural ganglia during latency. In contrast, SVV ORF 28, 29, 31, 62, and 63 transcripts were not detected in ganglia, liver, or lung tissues of latently infected animals. The results demonstrate that viral gene expression is limited during SVV latency and that a LAT antisense to an ICP0 homolog is expressed. In this regard, SVV gene expression during latency is similar to that of HSV-1 and other neurotropic animal alphaherpesviruses but differs from that reported for VZV.

Simian varicella virus (SVV) is closely related to varicella-zoster virus (VZV), the causative agent of chickenpox and shingles. The SVV and VZV gene 61 polypeptides are homologs of the HSV-1 ICP0, a viral transactivator which appears to play a role in viral latency and reactivation. In this study, the molecular properties of the SVV 61 were characterized. The SVV open reading frame (ORF) 61 encodes a 54.1-kDa polypeptide with 37% amino acid identity to the VZV 61. Homology to the HSV-1 ICP-0 is limited to a conserved RING finger motif at the amino terminus of the protein. A nuclear localization sequence (nls) at the carboxy-terminus directs the SVV 61 to the cell nucleus, while a SVV 61nls(-) mutant is confined to the cell cytoplasm. The SVV 61 transactivates its own promoter as well as SVV immediate early (IE, ORF 62), early (ORFs 28 and 29), and late (ORF 68) gene promoters in transfected Vero cells. The RING finger and nls motifs are required for efficient SVV 61 transactivation. The SVV 61 has no effect on the ability of the major SVV transactivator (IE62) to induce SVV promoters. Generation and propagation of a SVV gene 61 deletion mutant demonstrated that the SVV 61 is non-essential for in vitro replication. SVV gene 61 is expressed in liver, lung, and neural ganglia of infected monkeys during acute simian varicella.