Simian Varicella Virus Expresses a Latency-Associated Transcript That Is Antisense to Open Reading Frame 61 (ICP0) mRNA in Neural Ganglia of Latently Infected Monkeys

SVV infection of nonhuman primates.Six St. Kitts vervet monkeys (Chlorocebus aethiops) (3 to 4 years old), designated ET66, ET68, ET69, ET70, FV91, and FV93, were confirmed to be seronegative for SVV by serum neutralization assay prior to experimental infection. The animals were infected with 5 × 10⁴ PFU of SVV strain Delta by intratracheal inoculation. One animal (FV91) was part of a study to investigate the immunogenicity of recombinant SVV expressing SIV Env and Gag antigens (40). The clinical and virological parameters of simian varicella were evaluated as previously described (20). Viremia was monitored by harvesting peripheral blood mononuclear cells (PBMCs) from Ficoll-Hypaque gradients and determining infectious SVV titers by coculture of PBMCs on Vero cells. Skin rash was scored on an established scale of 0 to 4, with 0 for no rash and 4 for severe rash. Hepatitis was assessed by aspartate transaminase assays of blood specimens. Serum neutralization assays were used to assess antibody responses to SVV, with the neutralization titer determined to be the serum dilution that reduced the viral plaque count by 50%. One animal (FV93) developed severe simian varicella, and tissues were collected at necropsy on day 10 postinfection (p.i.). Monkeys ET66, ET68, ET69, and ET70 were given booster immunizations on days 28 and 56 p.i. by intratracheal inoculation with 5 × 10⁴ PFU of SVV and were sacrificed by ketamine anesthesia on day 119 p.i. Monkey FV91 was given a booster immunization on day 42 by intratracheal and subcutaneous inoculation with 5 × 10⁴ PFU of SVV and was sacrificed on day 108 p.i. Liver, lung, and neural ganglia (trigeminal, cervical, and lumbar) tissues were harvested at necropsy and quick-frozen for subsequent analysis. Studies involving animals were conducted in compliance with the Public Health Service policy on humane care and use of laboratory animals.

RNA isolation and detection of transcripts in tissues.Total cellular RNA was isolated from liver, lung, and ganglion tissues by the TRI Reagent protocol (Molecular Research Center Inc., Cincinnati, OH). Briefly, 1 ml of TRI Reagent was added to 50 to 100 mg of tissue and immediately homogenized in a Polytron homogenizer. After addition of a phase separation reagent and a 15-min incubation, the mixture was centrifuged at 10,000 rpm for 15 min at 4°C. RNA in the upper aqueous phase was precipitated by the addition of isopropanol and centrifugation at 10,000 rpm for 8 min at 4°C. The RNA pellet was washed with 75% ethanol, dissolved in FORMAzol (300 to 500 μl/50 to 100 mg tissue), and stored at −20°C. RNA was precipitated by centrifugation at 14,000 rpm for 5 min, resuspended in H₂O, and DNase I treated with a commercial kit (Ambion Inc., Austin, TX).

Standard one-step reverse transcriptase PCR (RT-PCR) was conducted with the Access RT-PCR system (Promega Corp.). The RT reaction mixture, including RNA (0.5 μg) and gene-specific primers (1 μM), was incubated at 45°C for 45 min to generate cDNA and was followed by PCR amplification conducted under the following conditions: initial denaturation, 94°C for 2 min; 31 cycles of 94°C for 30 s, 60°C for 30 s, and 68°C for 1 min; and a final 5-min extension at 68°C. Control reactions without RT were conducted to confirm the absence of contaminating SVV DNA. Amplified cDNA products were fractionated by 1.0% agarose gel electrophoresis and visualized by ethidium bromide staining and UV illumination. The oligonucleotide primers used for RT-PCR are indicated in Table 1.

The specificity of RT-PCR products was confirmed by Southern blot hybridization. Briefly, the amplified cDNA was fractionated by agarose gel (1.0%) electrophoresis and then transferred to a nylon membrane by capillary transfer and UV cross-linking (Roche Applied Science). SVV DNA probes were labeled with digoxigenin (DIG)-dUTP by random priming, denatured, and then hybridized to the immobilized cDNA on the filters at 42°C for 16 h in Easy Hyb hybridization buffer (Roche Applied Science). After extensive washing, hybridized products were incubated with anti-DIG antibody conjugated with alkaline phosphatase, followed by washing and incubation with chemiluminescent substrate for alkaline phosphatase. The membranes were exposed to X-ray film, and the hybridized DNA products were detected by chemiluminescence.

In some cases, RT-PCR products were confirmed by nested PCR. Briefly, 2 μl of cDNA amplified in the initial PCR was used as the template for a second PCR using nested primers internal to the original PCR primers. Amplified DNA was analyzed by 1.0% agarose gel electrophoresis, ethidium bromide staining, and UV illumination.

Determination of SVV transcript orientation.A two-step RT-PCR procedure to determine the orientation of the SVV LAT was performed with the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen Corp.). Briefly, individual RT reaction mixtures, including either sense or antisense primers (0.1 μM) and DNase I-treated total cellular RNA template (0.5 μg), were incubated for 15 min at 50°C and then terminated at 85°C, after which the RNA template was degraded with RNase H. The cDNA product was amplified by PCR using gene-specific primers and the PCR conditions described above. The identity of the amplified products was confirmed by agarose gel electrophoresis and Southern blot hybridization analysis using DIG-labeled SVV ORF 61 DNA probes, as described above.

SVV infection of nonhuman primates.Five vervet monkeys (ET66, ET68, ET69, ET70, and FV93) were infected with wild-type SVV by intratracheal inoculation. An additional monkey (FV91) was infected with a recombinant SVV expressing the SIV Env and Gag antigens (40). The clinical and virological parameters of infection for each animal are given in Table 2. A transient viremia occurred between days 3 and 6 p.i., as indicated by detection of infectious SVV in PBMCs. Each animal exhibited clinical signs of simian varicella, including vesicular skin rash and hepatitis during the first 2 weeks of infection. One monkey (FV93) developed severe disease, and acutely infected tissues were harvested from this animal on day 10 p.i. The remaining five animals generated immune responses to SVV, including neutralizing antibody, and resolved the acute disease by 20 days p.i. Monkeys ET66, ET68, ET69, and ET70 were administered booster immunizations with SVV on days 28 and 56 p.i., and monkey FV91 was given a booster immunization with the recombinant SVV on day 42. Neither viremia nor clinical symptoms (rash, hepatitis) were detected following these booster immunizations. The animals were sacrificed 9 weeks after the final immunization, and liver, lung, and neural ganglion tissues were collected and analyzed as indicated below. These animals were initially determined to be latently infected, based on the lack of clinical symptoms of simian varicella at the time of sacrifice, an inability to detect infectious SVV in tissues by coculture on CV-1 cell monolayers, and the ability to detect SVV DNA in the neural ganglia (data not shown).

Detection of SVV transcripts in tissues of acutely and latently infected monkeys.SVV transcripts in acutely and latently infected tissues were detected by RT-PCR and confirmed by nested PCR and/or Southern blot hybridization. Table 3 summarizes the analysis leading to the detection of SVV transcripts in liver, lung, and various neural ganglion tissues in each of the infected monkeys. Figure 2 shows representative RT-PCR/DNA hybridization results for detection of SVV transcripts in tissues derived on day 10 p.i. from an acutely infected monkey (FV93) (Fig. 2A) and in tissues derived on day 119 p.i. from a latently infected monkey (ET68) (Fig. 2B).

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FIG. 2.

Detection of viral transcripts in tissues of an acutely infected monkey (FV93) (A) and a latently infected monkey (ET68) (B). SVV transcripts were detected by RT-PCR using RT (+) and gene-specific primer sets, followed by Southern blot hybridization with DIG-labeled gene-specific DNA probes. Control reactions without RT (−) were conducted to confirm the absence of contaminating DNA. Based on molecular size standards (100-bp ladder), the sizes of the RT-PCR products corresponded to the expected size for each primer set: 345 bp for ORF 61, 431 bp for ORF 29, 420 bp for ORF 31, 502 bp for ORF 62, 551 for ORF 63, and 539 bp for ORF 21.

SVV IE (ORFs 61, 62, and 63), early (ORF 29), and late (ORF 31) genes were expressed in acutely infected liver and lung tissues and trigeminal, cervical, and lumbar ganglia derived from monkey FV93 on day 10 p.i., as indicated by detection of viral transcripts by RT-PCR and Southern blot hybridization (Fig. 2A; Table 3). The results confirm active viral replication in these tissues during the acute stage of simian varicella.

SVV transcripts mapping to SVV 61 ORF were consistently detected in neural ganglia (trigeminal, cervical, and lumbar) of each of the five animals (ET66, ET68, ET69, ET70, and FV91) that were latently infected with SVV (Fig. 2B; Table 3). Transcripts mapping to SVV ORF 21 were detected in 5 of 12 of the ganglia analyzed and in four of the five latently infected monkeys, confirming a previous report that ORF 21 is expressed in SVV latently infected ganglia (6). The ORF 61 and ORF 21 transcripts were detected exclusively in the neural ganglia; they were not detected in liver or lung tissues. In contrast, SVV ORF 28, 29, 31, 62, and 63 transcripts were not detected in the neural ganglia or liver or lung tissues, indicating a lack of viral replication in these tissues and confirming latent infection.

In order to confirm ORF 61 transcription in latent neural ganglia, five different ORF 61 primer sets (61-1, 61-2, 61-3, 61-4, and 61-5 [Fig. 1]) were used to detect transcripts in latent ganglia by RT-PCR. Figure 3 demonstrates that RT-PCR primer sets 61-1, 61-2, 61-3, and 61-4 each amplified cDNA from RNA template isolated from the trigeminal and lumbar ganglia, but not lung tissue, of a latently infected monkey (ET69). Primer set 61-5 did not detect ORF 61 RNA, indicating that the left-most terminus of the ORF 61 transcript maps within a 132-bp region between SVV DNA nt 102,392 and 102,261. Neither SVV ORF 61 transcripts nor cellular transcripts homologous to ORF 61 were detected in neural ganglia derived from an uninfected monkey, as confirmed in a previous study (15).

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FIG. 3.

Detection of the SVV ORF 61 LAT in the trigeminal and lumbar ganglia of a latently infected monkey (ET69) by RT-PCR employing multiple ORF 61 primer sets. Total cellular RNA isolated from ganglia or lung tissue was used as a template for RT-PCRs with RT (+) and without RT (−) and five different primer sets. Amplified LAT was detected by Southern blot hybridization using an ORF 61-specific DIG-labeled DNA probe. The map location of each RT-PCR primer set is shown in Fig. 1. Based on molecular size standards (100-bp ladder), the sizes of the RT-PCR products corresponded to the expected size for each primer set: 345 bp for 61-1, 345 for 61-2, 322 for 61-3, and 330 for 61-4.

Orientation of the SVV ORF 61 LAT.In order to determine the orientation of the SVV ORF 61 transcript, oligonucleotide sense or antisense primers, relative to the ORF 61 mRNA, were used in RT reactions, and the cDNA products were amplified by PCR using ORF 61-specific primer sets. The size and specificity of the amplified products were confirmed by Southern blot hybridization using ORF 61 DIG-labeled DNA probes. Figure 4A demonstrates that RT reactions including the ORF 61 sense primers generated a cDNA product of the expected size from an RNA template derived from the neural ganglia of latently infected monkeys (ET66, ET70, and FV91), but RT reactions including antisense primers were less efficient at generating such a cDNA product. Because the sense primer has the same orientation as gene 61 mRNA, this primer must bind to a transcript that is antisense to ORF 61 mRNA. In contrast, when RNA template derived from liver tissue or neural ganglia of an SVV acutely infected monkey (FV93) was used, RT reactions including ORF 61 antisense primers, which bind to gene 61 mRNA, efficiently generated the expected cDNA product (Fig. 4B). Although the ORF 61 sense primers did detect antisense transcripts in acutely infected liver and cervical ganglia tissues, the sense primers were not as efficient as the antisense primers for generating a cDNA product from RNA template derived from acutely infected tissues. These results indicate that SVV gene 61 mRNA is predominantly expressed in acutely infected neural ganglia, while an antisense LAT is primarily expressed in latently infected ganglia (Fig. 1). Both ORF 61 mRNA and the LAT could be detected in some ganglia, but gene 61 mRNA was detected most efficiently in acutely infected ganglia and the LAT was detected most efficiently in latently infected ganglia.

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FIG. 4.

Determination of SVV ORF 61 LAT orientation by strand-specific RT-PCR. Total cellular RNA isolated from ganglia derived from the latently infected monkeys ET66, ET70, and FV91 and the acutely infected monkey FV93 was used as a template for RT reactions conducted with ORF 61 sense (S) and antisense (AS) primers. Reactions were conducted with (+) and without (−) RT. The cDNA product was amplified by PCR and detected by Southern blot hybridization using an ORF 61-specific DIG-labeled DNA probe. The map location of each RT-PCR primer set is shown in Fig. 1. Based on molecular size standards (100-bp ladder), the sizes of the amplified products corresponded to the expected size for each primer set, as indicated.