Simian Varicella Virus Research Articles

Identification of simian varicella virus homologues of varicella zoster virus genes.

Clinical, pathologic, immunologic and virologic features of simian varicella virus (SVV) infection in primates resemble human varicella-zoster virus (VZV) infection. Further, the SVV and VZV genomes are similar in size and structure, show striking homology in their configuration and DNA sequences, and encode antigenically related polypeptides. Although the entire VZV genome is present during latency in human ganglia, transcription is limited. VZV genes 21, 29, 62 and 63 are transcribed during latency, while genes 4, 10, 40, 51 and 61 are not transcribed. The entire VZV genome has been sequenced, but the SVV genome has not. Thus, to analyze SVV genes transcribed during latency, we have begun to identify SVV homologues of the above VZV genes. We used nick-translated [32p]-labeled-VZV open reading frame (ORF)-specific probes to screen Southern blots containing EcoRI-digested SVV genomic DNA and recombinant clones of SVV EcoRI and BamHI DNA fragments spanning approximately 97% of the virus genome. We showed that SVV homologues of VZV ORFs 4, 10, 29, 40, 51 and 61 mapped to SVV DNA fragments EcoRI I, A, N, BamHI E, EcoRI D and E, respectively. We also confirmed earlier reports that SVV homologues of VZV genes 21 and 63 mapped to SVV EcoRI DNA fragments H and C, respectively. Viral genes on the SVV and VZV genomes seem to be collinear.

Viral Isolates Derived from Simian Varicella Epizootics Are Genetically Related but Are Distinct from Other Primate Herpesviruses

Epizootics of a natural varicella-like disease occur in populations of nonhuman primates. Several primate herpesviruses have been isolated from these epizootics, but the relatedness of these isolates to each other is not well-defined. In this study, we demonstrated that the restriction endonuclease (REn) profiles of four epidemiologically distinct isolates were similar, although not identical, indicating that simian varicella epizootics are caused by various strains of simian varicella virus (SVV). The genetic variation among the isolates did not map to a specific region of the SVV genome and REn differences were detected within the SVV DNA long component and the inverted repeat region. Southern blot hybridization demonstrated that SVV is more closely related to varicella-zoster virus than to other primate herpesviruses. The study indicates that the current herpesvirus classification scheme should be changed to include SVV as a single taxonomic group within theVaricellovirusgenus of alphaherpesviruses. In addition, REn profiles of SVV isolates, derived from primary and secondary episodes of simian varicella in the same monkey, were identical, providing evidence for SVV reactivation in a latently infected monkey.

A simian varicella virus (SVV) homolog to varicella-zoster virus gene 21 is expressed in monkey ganglia latently infected with SVV.

We have sequenced a simian varicella virus (SVV) open reading frame (ORF), 3,123 bp in length, whose product has 51% amino acid homology with the sequence encoded by the ORF of varicella-zoster virus gene 21. Several regions are highly conserved between the two ORFs, with homologies of approximately 80%. The SVV gene is transcribed in tissue culture cells productively infected with SVV and in monkey ganglia latently infected with SVV.

Identification and analysis of the simian varicella virus thymidine kinase gene

The thymidine kinase (TK) of herpesviruses, in contrast to cellular TKs, phosphorylates a variety of substrates including antiherpetic nucleoside analogues. This study reports the identification and DNA sequence of the simian varicella virus (SVV) TK gene. A 32P-labeled varicella zoster virus (VZV) TK DNA probe hybridized to the HindIII B subclone of the SVV BamHI B restriction endonuclease (RE) fragment, indicating the presence of a SVV DNA sequence homologous to the VZV TK gene. DNA sequence analysis of the SVV HindIII B subclone revealed a 1014 base pair (bp) open reading frame (ORF) encoding a 337 amino acid polypeptide homologous to herpesvirus TKs. The predicted SVV and VZV TK polypeptides share 51.3% identity, and alignment of the putative protein sequence of several TK homologues suggests the position of a conserved nucleotide binding site and a nucleoside (substrate) binding site in the SVV TK. Identification of the 5' end of the SVV TK transcript by primer extension analysis allowed a comparison of the SVV and VZV TK promoter regions indicating extensive conservation of the DNA sequence and transcription factor binding sites. Plaque reduction assays demonstrate that the SVV TK is active based on the susceptibility of SVV to acyclovir treatment and that SVV is less sensitive to acyclovir than VZV and herpes simplex virus (HSV-1) in infected Vero cells. Identification of the SVV TK ORF will facilitate studies that examine the role of viral TKs in pathogenesis and antiviral sensitivity and provides a potential insertion site for the expression of foreign genes.

The inverted repeat regions of the simian varicella virus and varicella-zoster virus genomes have a similar genetic organization

Simian varicella virus (SVV) causes a varicella-like disease in nonhuman primates. The DNA sequence and genetic organization of the inverted repeat region (RS) of the SVV genome was determined. The SVV RS is 7559 bp in size with 56% guanine + cytosine (G+C) content and includes 3 open reading frames (ORFs). The SVV RS1 ORF encodes a 1279 amino acid (aa) protein with 58 and 39% identity to the varicella-zoster virus (VZV) gene 62 and herpes simplex virus type 1 (HSV-1) ICP4 homologs, respectively. The predicted 261 aa SVV RS2 polypeptide possesses 52% identity with the VZV gene 63 homolog and 23% identity with the HSV-1 ICP22. The SVV RS3 encodes a 187 aa polypeptide with 56% and 28% identity to the VZV gene 64 and the HSV-1 US10 homologs, respectively, and includes an atypical zinc finger motif. A G+C-rich 16 base-pair (bp) sequence which is repeated 7 times and a putative SVV origin of replication were identified between the RS1 and RS2 ORFs. Comparison with the VZV RS indicates the SVV and VZV RS regions are similar in size and genetic organization.

DNA sequence of the simian varicella virus (SVV) gH gene and analysis of the SVV and varicella zoster virus gH transcripts

The varicella zoster virus (VZV) glycoprotein H (gH) stimulates VZV-specific immune responses and may be involved in virus penetration. This study reports the genomic map position and the DNA sequence of a simian varicella virus (SVV) homologue of the VZV gH gene. A 32P-labeled VZV gH-specific DNA probe hybridized to the HindIII B subclone of the SVV BamHI B restriction endonuclease (RE) fragment. The DNA sequence of the SVV HindIII B subclone was determined and analysis indicated a SVV open reading frame (ORF) homologous to several herpesvirus gH genes. The SVV gH ORF is 2559 base pairs in size and encodes a 852-amino acid protein. The SVV gH contains characteristics of a transmembrane glycoprotein including: 9 consensus N-linked glycosylation sites, a potential amino terminal signal sequence, and a predicted transmembrane segment located near the carboxyl terminus. The SVV and VZV gH genes exhibit 60.0% identity and the predicted polypeptides exhibit 54.5% identity. The SVV and VZV gH transcripts were analyzed and the promoter regions were compared. 32P-labeled SVV and VZV gH-specific DNA probes each hybridized to a single 2.9 kilobase transcript. The mRNA start sites of the SVV and VZV gH genes were determined by primer extension analysis, and alignment of the promoter regions indicated similar content and arrangement. The extensive conservation of SVV and VZV genes and predicted polypeptides further supports the use of SVV infection of non-human primates as a model of VZV infection of humans.

Configuration and Terminal Sequences of the Simian Varicella Virus Genome

The simian varicella virus (SVV) genome is a linear DNA molecule consisting of a unique short (U S) and a unique long (U L) region. The U S is bounded by internal (IR) and terminal inverted repeats and inverts such that virion DNA contains equimolar amounts of two genome isomers. We have sequenced the right and leftward termini and the U L-IR junction region of the SVV genome. A sequence motif common to other herpesviruses, consisting of A and T residues surrounded by G+C-rich regions, was found near the rightward terminus of the SVV genome. Sequence analysis showed no repeats surrounding the U L region of the genome. Nucleic acid hybridization and polymerase chain reaction (PCR) amplification using primers from the right end leftward ends of the SVV genome indicated that the U L region inverts. PCR amplification also showed that, compared with virion DNA, SVV genomes with connected termini are increased in infected cell DNA, suggesting the presence of circular or concatemeric genomic molecules.

Transcriptional Analysis of Two Simian Varicella Virus Glycoprotein Genes Which Are Homologous to Varicella-Zoster Virus gpI (gE) and gpIV (gI)

Simian varicella virus (SVV) causes a natural, varicella-like disease in nonhuman primates. The unique short region of the SVV genome contains four open reading frames (ORFs), two of which encode glycoproteins that exhibit extensive homology with varicella-zoster virus (VZV) gpIV (gI) and gpI (gE). Northern hybridization, primer extension, and RNase protection analyses were employed to define precisely the transcripts mapping to the SVV gpIV and gpI genes. A total of five transcripts composing two coterminal families of RNAs were mapped to the SVV gpIV end gpI ORF region. Based on transcriptional mapping and previous DNA sequence analysis, two transcripts 1.3 and 2.2 kb in size were assigned to the SVV gpIV and gpI genes, respectively. The transcriptional patterns described in this study for the SVV gpIV and gpl ORFs are analogous to those previously reported for the homologous glycoproteins genes encoding the herpes simplex virus type 1 Us7 (gI) and Us8 (gE) and VZV gpIV and gpI genes. In addition, the transcriptional start site for the VZV gpl RNA was determined. DNA alignments of the promoter regions for the SVV and VZV gpIV and gpl genes revealed a number of cis-acting elements which are conserved between the two viruses. The characterization of SVV glycoprotein genes will facilitate future studies to define their role in SVV pathogenesis and immunity and assist in the construction of recombinant vaccines which could be evaluated in the simian varicella model.

Pharmacokinetics and antiviral activity of a novel isonucleoside, BMS-181165, against simian varicella virus infection in African green monkeys

A novel nucleoside analog BMS-181165 with potent activity against varicella-zoster virus was tested for efficacy in a simian varicella virus infection in African green monkeys. BMS-181165 was effective in preventing the development of a rash, decreasing the development of viremia and preventing death in infected monkeys when administered orally at 4, 16 or 64 mg/kg/day. The compound is well orally absorbed in monkeys, between 44 to 50% oral bioavailability, and may prove of value in therapy of varicella-zoster infections in humans.

117 GR95168X (chiral carbocyclic BVdU) is highly effective against simian varicella virus infections of African green monkeys: RE Boehme 1, A Beresford 1, GJ Hart 1, SJ Angier 1, G Thompson 1, C Van Wely 1, J-L Huang 2, P Mack 2 and KF Soike 21Glaxo Group Research Ltd, Greenford, Middlesex, UK; 2Tulane Regional Primate Research Center, Covington, LA, USA

117 GR95168X (chiral carbocyclic BVdU) is highly effective against simian varicella virus infections of African green monkeys: RE Boehme 1, A Beresford 1, GJ Hart 1, SJ Angier 1, G Thompson 1, C Van Wely 1, J-L Huang 2, P Mack 2 and KF Soike 21Glaxo Group Research Ltd, Greenford, Middlesex, UK; 2Tulane Regional Primate Research Center, Covington, LA, USA

DNA Sequence of a Simian Varicella Virus Gene That Encodes a Homologue of Varicella Zoster Virus IE62 and Herpes Simplex Virus ICP4

Clinical and biological studies indicate that simian varicella virus (SVV) infection of primates is the counterpart of human varicella zoster virus (VZV) infection. We have identified an SVV open reading frame (ORF) that is homologous to the VZV protein IE62 and herpes simplex virus (HSV) ICP4. Like the genes encoding the VZV and HSV proteins, the SVV gene is located in the repeat region of the virus genome. Its expression in SVV-infected cells yields a 4.2-kb transcript. DNA sequencing of the SVV gene reveals a coding region 3834 nucleotides in length and a G + C content of 60.3%. Like ICP4 homologues in other herpesviruses, the SVV protein has five regions, two of which (regions 2 and 4) are highly conserved. The SVV protein also contains a run of serine residues in its amino terminus that is characteristic of herpesvirus ICP4 homologues. The SVV protein shows an overall homology of 24% to HSV ICP4 and 53% to VZV IE62.

Characterization and mapping of simian varicella virus transcripts

The size and genomic location of viral transcripts expressed in simian varicella virus (SVV)-infected Vero cells were determined. Total cellular RNA and polyadenylated RNA were isolated from SVV-infected and mock-infected Vero cells. Viral transcripts were detected by Northern blot hybridization analysis using overlapping SVV DNA probes representative of the entire SVV genome. The results indicated that all regions of the SVV genome are transcribed during SVV infection in vitro. At least 53 distinct viral RNA species ranging in size from 9.2 to 0.8 kb were detected. DNA probes derived from the SVV DNA long (L) and short (S) components hybridized to 44 RNAs (9.2 to 0.8 kb) and nine RNAs (4.9 to 0.8 kb), respectively. A transcript map of the SVV genome was constructed. The comparison made between the transcript maps of SVV and varicella-zoster virus (VZV) provides further support that the SVV and VZV genomes have an analogous gene organization.

Efficacy of (-)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine in African green monkeys infected with simian varicella virus

(-)-9-[4-Hydroxy-2-(hydroxymethyl)butyl]guanine was evaluated for its efficacy in African green monkeys infected with simian varicella virus. Treatment by intramuscular injection was initiated 48 h after virus inoculation and was continued for 10 days; the treatment showed therapeutic effects on rash and viremia at dosages down to 1 mg/kg of body weight per day.

DNA Sequence and Genetic Organization of the Unique Short (U s) Region of the Simian Varicella Virus Genome

Simian varicella virus (SVV) infection of nonhuman primates is a model for the study of human varicella zoster virus (VZV) infections. The DNA sequence of the entire SVV unique short (U s) region and adjacent flanking sequences of the inverted repeats were determined. The U s region is 4904 bp in size and has a 60.9% A + T base composition. Four potential open reading frames (ORFs), designated SVUS 1, SVUS 2, SVUS 3, and SVUS 4, were identified and found to be remarkably similar in size, genetic content, and transcriptional orientation to their respective VZV U s counterparts; ORF 65, ORF 66 (U s PK), ORF 67 (gpIV), and ORF 68 (gpl). The SVUS 1 ORF encodes a putative 9 kDa homolog of the herpes simplex virus type-1 (HSV-1) U s9 tegument phosphoprotein. The SVUS 2 ORF encodes a predicted 345 amino acid polypeptide that contains a number of sequence domains conserved in cellular and viral serine/threonine (S/T) protein kinases and exhibits extensive homology with previously reported alphaherpesviral U s S/T PKs, including VZV ORF 66, HSV-1 U s3, pseudorabies virus (PRV) PK, and equine herpesvirus (EHV-1) ORF 69. The SVUS 3 and SVUS 4 ORFs exhibit features characteristic of membrane glycoproteins: an amino terminal signal sequence, potential N-linked glycosylation sites, and a large hydrophobia transmembrane domain. The predicted 353 amine acid protein encoded by SVUS 3 ORF is homologous to the VZV gpIV (ORF 67), HSV-1 gl (U s7), PRV gp63, and EHV-1 gl (ORF 73) gene products. The SVUS 4 ORF encodes a putative 604 amino acid polypeptide which exhibits extensive homology with VZV gpl and more limited homology with HSV-1 gE (U s8), PRV gpl, and EHV gE (ORF 74). This report describes the initial characterization of individual SVV genes and further defines the evolutionary relationships between SVV, VZV, and other alphaherpesviruses.

150 Efficacy of (-)-9-[4-Hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) in African green monkeys infected with simian varicella virus: K. F. Soike 1, R. Bohm 1, J.-L. Huang 1 and B. Öberg 2. 1Tulane Regional Primate Research Center, Covington, LA 70433 USA and 2Medivir AB, Huddinge, Sweden

150 Efficacy of (-)-9-[4-Hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) in African green monkeys infected with simian varicella virus: K. F. Soike 1, R. Bohm 1, J.-L. Huang 1 and B. Öberg 2. 1Tulane Regional Primate Research Center, Covington, LA 70433 USA and 2Medivir AB, Huddinge, Sweden

The genomes of simian varicella virus and varicella zoster virus are colinear

Simian varicella virus (SVV) causes an exanthematous disease in non-human primates which is clinically similar to varicella zoster virus (VZV) infection of humans. In this study, the genetic relatedness of SVV and VZV was confirmed and the location of SVV DNA sequences homologous to VZV restriction endonuclease (RE) fragments and viral genes was determined. VZV DNA RE fragments representing 98.3% of the VZV genome were 32P-labeled and hybridized to RE digested, immobilized SVV DNA. Homologous sequences were located throughout the viral DNAs in similar map positions, indicating a colinear relationship between the VZV and SVV genomes. 32P-labeled VZV glycoprotein (gp I, II, III, and IV) and gene 62 DNA probes also hybridized to SVV DNA in a colinear manner. The results suggest that the location of specific SVV genes may be predicted from the known map positions of homologous VZV genes. This study provides further support for SVV infection of non-human primates as a model for VZV infection of humans.

Molecular analysis of simian varicella virus DNA

Clinical and pathological studies indicate that simian varicella virus (SVV) infection in primates is the counterpart of human varicella zoster virus (VZV) infection. The SVV and VZV genomes are also similar in size and structure. To extend studies of SVV DNA, we analyzed virus DNA from African green monkey kidney cells infected with the Delta-herpesvirus strain of SVV. The infectivity of SVV DNA was 88 PFU/μg. The buoyant density of SVV DNA, determined by isopycnic banding in CsCl gradients, was 1.700 ± 0.002 g/ml, corresponding to a G + C molar ratio of 40.8%. The size of SVV DNA, estimated by analysis of restriction endonuclease digestion products and pulsed-field gel electrophoresis was 125.1 and 124.9 kbp, respectively. Electron microscopy of SVV DNA revealed a long region of 110.0 kbp, a unique short (Us) region of 5.1 kbp, and inverted repeat regions of 7.5 kbp flanking the Us. An EcoRI map of SVV DNA revealed two fragments not previously reported; our complete PstI map also shows some differences. Mapping of SVV DNA with an additional restriction enzyme, measurement of full-length SVV DNA molecules, and the first use of pulsed-field electrophoresis to size SVV DNA, confirm and extend Gray's recent finding that SVV DNA has the same size and molecular configuration as VZV. We also show for the first time that the density of SVV DNA is similar to that of VZV DNA and that SVV DNA is infectious.

Simian varicella virus infection in African and Asian monkeys. The potential for development of antivirals for animal diseases.

Simian varicella virus infection in African and Asian monkeys. The potential for development of antivirals for animal diseases.

Prevalence and distribution of latent simian varicella virus DNA in monkey ganglia

We used polymerase chain reaction to analyze the prevalence and distribution of latent simian varicella virus (SVV) in ganglionic and nonganglionic tissues from nine African green monkeys experimentally infected with SVV. Primers specific for three different regions of the SVV genome were used for amplification. SVV DNA sequences were detected in trigeminal ganglia from seven of nine monkeys and in thoracic ganglia from seven of nine monkeys. Analysis of DNA from nonneuronal tissues of three monkeys and from adrenal glands of nine monkeys revealed the presence of SVV-specific sequences in the adrenal gland of one monkey. The results indicate that, like human varicella, SVV becomes latent primarily in ganglia at multiple levels of the neuraxis, and more than one region of the SVV genome is present in latently infected ganglia. SVV latency in primates maybe a useful model for varicella latency in humans.

Simian varicella virus: characterization of virion and infected cell polypeptides and the antigenic cross-reactivity with varicella-zoster virus.

Simian varicella virus (SVV) causes a varicella-like disease in non-human primates. In this study, SVV virions were purified from SVV-infected BSC-1 cells by zonal and differential gradient centrifugation and the virion polypeptide composition was analysed by SDS-PAGE. SVV virions had a buoyant density of 1.21 g/ml, identical to the value obtained for varicella-zoster virus (VZV) virions purified by the same method. Electron microscopy of the concentrated SVV virions revealed characteristic herpesvirus morphology. SVV virions consisted of at least 30 polypeptide species ranging from 16K to greater than 200K. The electrophoretic profiles of radiolabelled SVV and VZV virion polypeptides were very similar. Immunoprecipitations of solubilized SVV-infected cell preparations using SVV immune sera revealed at least 18 viral polypeptides with an Mr range of 12K to 142K and six glycoproteins ranging from 46K to 115K. In addition, extensive cross-reactivity between SVV and VZV proteins and glycoproteins was demonstrated by immunoprecipitation with heterologous immune sera. The high degree of antigenic relatedness between SVV and VZV provides further support for simian varicella as a model for VZV infections.