Naturally Acquired Simian Varicella Virus Infection in African Green Monkeys

Simian varicella virus (SVV) infection of primates shares clinical,pathological, immunological, and virological features with varicella-zostervirus infection of humans. Natural varicella infection was simulatedby exposing four SVV-seronegative monkeys to monkeys inoculatedintratracheally with SVV, in which viral DNA and RNA persistin multiple tissues for more than 1 year (T. M. White, R. Mahalingam,V. Traina-Dorge, and D. H. Gilden, J. Neurovirol. 8:191-205,2002). The four naturally exposed monkeys developed mild varicella10 to 14 days later, and skin scrapings taken at the time ofthe rash contained SVV DNA. Analysis of multiple ganglia, liver,and lung tissues from the four naturally exposed monkeys sacrificed6 to 8 weeks after resolution of the rash revealed SVV DNA inganglia at multiple levels of the neuraxis but not in the lungor liver tissue of any of the four monkeys. This animal modelprovides an experimental system to gain information about varicellalatency with direct relevance to the human disease.

INTRODUCTION

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Introduction

Primary varicella-zoster virus (VZV) infection causes chickenpox (varicella). The virus then becomes latent in ganglia andreactivates decades later to produce shingles (zoster) and multipleother neurological complications (3). VZV causes disease onlyin humans. Attempts to produce the disease by experimental infectionof animals with VZV have led to seroconversion but not to disease(14). However, clinical, pathological, immunological, and virologicalevidence suggests that simian varicella virus (SVV) infectionof nonhuman primates is the counterpart of human VZV infection(10, 12).

Both the 1968 and 1974 outbreaks of varicella in Erythrocebuspatas monkeys at the Tulane National Primate Research Centerwere attributed to reactivated SVV (11). Further, SVV is antigenicallyrelated to human VZV (2), and sequence analysis of the completegenome of SVV has revealed a high degree of homology betweenthe two viruses at the nucleic acid and protein levels (5).

Studies with nonhuman primates inoculated intratracheally withSVV showed that multiple tissues, including blood mononuclearcells (MNCs), contain viral DNA and RNA for months after experimentalinfection (13). These findings differ from those for humanslatently infected with VZV, in which case virus is present onlyin ganglia and viral transcription is limited (1). To developa model of SVV latency that mimics VZV latency in humans, wesimulated natural infection by exposing SVV-seronegative monkeysto monkeys that had been inoculated intratracheally with SVV,as primary infection is thought to occur through aerosol ordroplet exposure of respiratory secretions from actively infectedindividuals. We observed the monkeys clinically for the developmentof varicella. Skin, blood MNCs, ganglia, and lung and livertissue were analyzed for virus DNA.

MATERIALS AND METHODS

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Materials and Methods

SVV and inoculation. The deltaherpesvirus strain of SVV, isolated from a naturallyinfected monkey (E. patas), was propagated in Vero cells, anda virus stock was prepared as described previously (6). TwoSVV-seronegative African green monkeys were inoculated intratracheallywith 10³ PFU of SVV as described previously (7). After 0 to3 days, each of these monkeys was placed in a cage with twoSVV-seronegative African green monkeys (monkeys 165 and 166and monkeys 190 and 191). All monkeys were examined daily. Bloodwas obtained once a week from all monkeys until they were sacrificed2 months later. Total body varicella developed 10 to 12 dayslater in the two monkeys that received the virus intratracheally.In the four monkeys that were naturally exposed to SVV, a mildrash occurred 10 to 14 days later, at which time the monkeyswere anesthetized and the skin area containing the rash wascleaned and scraped with a sterile swab into sterile medium.

DNA isolation. DNA was extracted by using a QIAamp blood mini kit (Qiagen,Valencia, Calif.) from blood MNCs, from the African green monkeykidney line BSC-1 infected with SVV (4), and from medium containingthe skin rash scrapings. All monkeys were euthanized 6 to 8weeks after intratracheal inoculation or natural exposure, andtotal DNA was extracted from either individual or pooled trigeminal,cervical, thoracic, lumbar, and sacral ganglia as well as fromliver and lung tissue by using a DNeasy tissue kit (Qiagen)according to the manufacturer's instructions.

PCR. Because both transcriptional and translational products of itsVZV homologue have been identified in human ganglia during latency(1, 8), we used oligonucleotide primers (Sigma-Genosys, St.Louis, Mo.) and probes specific for SVV open reading frame (ORF)63 to amplify and detect SVV DNA as described previously (13).DNA (1 µg) extracted from tissue or MNCs was scored aspositive if it was detectable in at least one of the triplicatePCRs carried out. As a control, DNA was omitted in one reactiontube.

RESULTS AND DISCUSSION

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Results and Discussion

The two monkeys that had been inoculated intratracheally withSVV developed diffuse varicella 10 to 12 days later. The fourmonkeys that were caged with each of the intratracheally infectedmonkeys developed a mild rash 2 weeks later. Analysis of theDNA extracted from skin scrapings of one intratracheally inoculatedmonkey and monkeys 190 and 191, which were caged with the inoculatedmonkey, revealed SVV ORF 63 sequences in the skin of all threemonkeys (Fig. 1), indicating that the rash in the simulatednatural infection was caused by SVV.

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FIG. 1. Detection of SVV DNA in skin scrapings of rash areas from monkeys infected naturally or intratracheally with SVV. DNA samples extracted from scrapings of rash from a monkey intratracheally infected with SVV and from two monkeys (no. 190 and 191) exposed to the experimentally infected monkey were analyzed by nested PCR with primers and probes specific for SVV ORF 63 to amplify a 219-bp fragment. No DNA was included in one of the reactions (No DNA). DNA samples from uninfected (BSC-1 DNA) and SVV-infected (SVV-BSC-1 DNA) BSC-1 cells in culture were used as negative and positive controls, respectively.

In the four monkeys that developed varicella after natural exposure,blood MNCs were obtained once a week for the next 2 months.SVV DNA was detected in one monkey (no. 190) 10 days later (datanot shown), consistent with the peak viremic phase seen daysbefore and at the time of varicella infection (9). SVV DNA wasnot detected in blood MNCs at any other times or at necropsyfrom any of the four naturally infected monkeys (data not shown).It is unclear why the three other naturally infected monkeysthat developed varicella were not viremic and why MNCs of onlyone monkey revealed SVV DNA. Perhaps the milder disease, indicatedby minimal rash in the naturally infected monkeys compared withextensive rash in the intratracheally inoculated monkeys, accountsfor this.

Six to 8 weeks after resolution of the rash, the four naturallyinfected monkeys were sacrificed and ganglionic and nonganglionictissues were harvested. Pools of ganglia were prepared frommultiple dermatomes (cervical, thoracic, lumbar, and sacral)of two naturally infected monkeys (no. 165 and 166) and fromone intratracheally inoculated monkey. PCR analysis of DNA extractedfrom these pools of ganglia and from the lungs and livers ofall three monkeys revealed sequences specific for SVV ORF 63in all of the samples from the intratracheally inoculated monkey,consistent with earlier observations that SVV is found in multipletissues months after experimental infection (13). SVV DNA wasdetected in pooled sacral ganglia of one naturally infectedmonkey (no. 165) and in pooled thoracic and pooled sacral gangliaof another naturally infected money (no. 166) but not in thelung or liver tissue of either monkey (Fig. 2 and Table 1).

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FIG. 2. Detection of SVV DNA in ganglia from monkeys naturally infected with SVV. DNA samples extracted from pooled cervical (C), thoracic (T), lumbar (L), and sacral (S) ganglia and from the lungs and livers of two monkeys (no. 165 and 166) exposed to an intratracheally infected monkey (no. 164) were analyzed by nested PCR with primers and probes specific for SVV ORF 63 to amplify a 219-bp fragment. No DNA was included in one of the reactions (No DNA). DNA samples from uninfected (BSC-1 DNA) and SVV-infected (SVV-BSC-1 DNA) BSC-1 cells in culture were used as negative and positive controls, respectively.

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TABLE 1. PCR detection of SVV DNA in ganglia of naturally infected monkeys

Because pooling multiple ganglia for DNA extraction might havediluted any low-abundance SVV DNA present in individual gangliafrom each monkey, we examined DNA extracted from both individualand pooled ganglia of two naturally infected monkeys (no. 190and 191) (Table 1). When analyzed individually, one of the trigeminalganglia from monkey no. 190, but neither of the two trigeminalganglia from monkey no. 191, revealed SVV DNA. Five of eightcervical ganglia from monkey no. 190, compared to none of eightcervical ganglia from monkey no. 191, contained SVV DNA. Becauseof their small size, individual thoracic ganglia were not tested.In both monkeys, SVV DNA was found in two of four lumbar ganglia,but not in sacral ganglia. Overall, SVV DNA was detected in8 of 17 ganglia in monkey no. 190 and in 2 of 20 ganglia ofmonkey no. 191, both of which were naturally infected.

In pools of two to four ganglia from monkeys 190 and 191, SVVDNA was detected in cervical ganglia in one of two pools frommonkey no. 190 but not in either of two pools from monkey no.191 (Table 1). In thoracic ganglia, SVV DNA was present in fourof four pools from monkey no. 190 and in three of six poolsfrom monkey no. 191. None of the three pools of lumbar gangliafrom either monkey revealed SVV DNA. Together, the results obtainedfrom PCR analysis of individual and pooled ganglia indicatethat SVV DNA was distributed along the trigeminal, cervical,thoracic, and lumbar regions in monkey no. 190 and only in thethoracic and lumbar regions in monkey no. 191. Overall, thedetection of SVV DNA in ganglia but not in the lung or livertissue from any of four naturally infected monkeys suggeststhe presence of latent infection in the ganglia.

It was previously shown that intratracheal inoculation of SVVresults in persistence of viral DNA in multiple tissues formany months (13). In contrast, in the naturally infected monkeysdescribed herein, virus DNA was restricted to the ganglia. Thevirological differences could reflect the large amount of virusadministered intratracheally compared to a presumed smalleraerosol inoculum in the naturally exposed monkeys. Taken together,SVV infection of monkeys provides a unique animal model thatclosely resembles human VZV infection. Our model will allowanalysis of the physical state of varicella nucleic acid inlatently infected ganglia (e.g., cell type and extent of transcriptionand translation) and experiments to reactivate the virus fromlatency.

ACKNOWLEDGMENTS

This work was supported in part by NIH grants NS 32623 and AG06127.

We thank Marina Hoffman for editorial review and Cathy Allenfor preparing the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Department of Neurology, Mail Stop B182, University of Colorado Health Sciences Center, 4200 East 9th Ave., Denver, CO 80262. Phone: (303) 315-8281. Fax: (303) 315-8720. E-mail: don.gilden{at}uchsc.edu.

REFERENCES

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