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Detection of Varicella-Zoster Virus DNA in Human Geniculate Ganglia by Polymerase Chain Reaction Yasushi Furuta, Tsuyoshi Takasu, Satoshi Fukuda, Kazuko C. Sato-Matsumura, Yukio Inuyama, Ryo Hondo, and Kazuo Nagashima

Departments of Pathology and Otolaryngology. Hokkadio University School ofMedicine. Sapporo; Department ofMicrobiology. Institute of Public Health. Tokyo. Japan

It is well known that Ramsay Hunt syndrome (facial paralysis, eighth cranial nerve symptoms, and herpes zoster in the head and neck) is caused by reactivation of varicella-zoster virus (VZV). A significantly elevated antibody titer for VZV has been demonstrated in patients with Ramsay Hunt syndrome [1]. After primary infection in children, VZV becomes latent in the sensory ganglia and is reactivated many years later to cause zoster in adults. Hunt concluded that the geniculate ganglion was affected in the syndrome in a manner analogous to the involvement of the spinal ganglion [2]. According to this hypothesis, latent VZV in the geniculate ganglion causes zoster of the external ear after reactivation and migration of the virus along the sensory fibers; ganglionitis and neuritis in the narrow fallopian canal result in facial nerve palsy. Furthermore, the inflammation may spread to the eighth cranial nerve and cause auditory and vestibular disorders. The human geniculate ganglia during VZV latency have not been examined because they are located in the geniculate part of the facial nerve and are surrounded by the bony wall of the fallopian canal. We previously reported the detection of herpes simplex virus (HSV) type 1 nucleic acid in human geniculate ganglia by the in situ hybridization

Received 20 March 1992; revised 12 June 1992. Financial support: Special Coordination Funds for Promoting Science and Technology. Science and Technology Agency (to K.N.); grant-in-aid for Scientific Research. Ministry of Education. Science. and Culture (C 03670803 to S.F.). Reprints or correspondence: Dr. Yasushi Furuta. Department of Pathology. Hokkaido University School of Medicine. Kita 15. Nishi 7. Kita-ku, Sapporo 060. Japan. The Journal of Infectious Diseases 1992;166:1157-9 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6605-0030$01.00

method [3]. In the present study, we looked for VZV DNA in human geniculate ganglia obtained from autopsy eases.

Materials and Methods Ganglia from autopsied cases. The geniculate areas of the facial nerves were obtained from 7 adults autopsied 1-9 h after death. At autopsy, the temporal bones were dissected from the base of the cranium, and the facial nerves were exposed from the internal auditory canal to the horizontal portion of the nerve by use of an electric drill and a microscope as described previously [3]. The geniculate portions of the facial nerves were removed from the fallopian canals and were stored at -70°C. The trigeminal ganglia were also removed. We used ganglia obtained from an autopsy of a newborn as negative controls. DNA extraction. Confluent monolayers of Vero cells grown in Eagle MEM supplemented with 5% fetal calf serum at 37°C with 5% CO2 were infected with VZV strains H-N3 and ONI, HSV-I strain Kl92, HSV-2 strain 20, and human cytomegalovirus (CMV) strain KH, respectively. Viral DNA was extracted by the method ofHirt [4]. We also extracted DNA from the ganglia of the 7 adults and the neonate according to the method of Sambrook et al. [5]. Polymerase chain reaction (peR). .To detect VZV DNA, we used a pair of oligonucleotide primers that are specific for the major DNA binding protein ofVZV (VZV I, 5'-TACGGGTCTTGCCGGAGCTGGTAT-3'; VZV 2, 5'-AATGCCGTGACCACCAAGTATAAT-3'), as described previously [6, 7]. We also used a pair of human o-tubulin primers (TUB I, 5'-GACAGAATTCCAGACCAACC-3'; TUB 2, 5'-GCACCAATCCACAAACGTGA-3') [8] to show that each DNA sample contained an amplifiable cellular gene. These primer pairs were synthesized on a DNA synthesizer (model 380; Applied Biosysterns, Warrington, UK). In each sample, 1 J.Lg of tissue DNA or 1 ng of viral DNA was amplified 30 times on a DNA thermal cycler (thermal cyclic reactor model TC-100; Hoei Science, Tokyo) with lOX PCR buffer (Perkin-Elmer Cetus, Norwalk, CT), primer pairs (each 100 pmol), deoxynucleotide triphosphate

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Latent varicella-zoster virus (VZV) has been demonstrated in the human trigeminal and thoracic ganglia by means of nucleic acid hybridization. However, the human geniculate ganglia in VZV latency have not been examined. Tissue DNA extracted from the trigeminal and geniculate ganglia of a newborn and 7 adults was examined by polymerase chain reaction with a pair of VZV-specific primers. None had symptoms of recent infection with VZV (chickenpox or shingles). VZV DNA was detected in 11 (79%) of 14 trigeminal ganglia and in 9 (69%) of 13 geniculate ganglia of the adults. VZV DNA was not detected in either type of ganglion from the newborn or from 1 adult who was seronegative for VZV antibodies. These findings indicate that VZV becomeslatent in human geniculate ganglia after primary infection and suggest the possibility that reactivation of the virus from the geniculate ganglia may cause Ramsay Hunt syndrome.

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Concise Communications

Results Sensitivity and specificity of Pc'R. We could detect as little as 0.1 fg ofVZV DNA (about equal to I copy ofVZV) by PCR with the VZV primers and Southern blot hybridization with a nonisotopic probe (data not shown). Thus, our method was highly sensitive for detecting VZV DNA. When we used DNA extracts from VZV strains H-N3 and ONI, a 273-bp product was visible in the ethidium bromidestained gel and was hybridized with the digoxigenin-labeled VZV probe. However, no amplified fragments were seen in the ethidium bromide-stained gel or by Southern blot hybridization when we used DNA extracted from HSV-l, HSV-2, and human CMV strains (data not shown). Thus, the 273-bp product was specific to VZV DNA. VZV DNA in human ganglia. By PCR with the tubulin primers, a 286-bp product was not detected in one sample (the right geniculate ganglion of case I) and we omitted this sample. Other DNA samples contained amplifiable cellular genes. By PCR with the VZV primers, we detected a 273-bp product ofVZV DNA in II (79%) of 14 trigeminal ganglia and in 9 (69%) of 13 geniculate ganglia of the 7 adults (table I; figure I). VZV DNA was not detected in the geniculate and trigeminal ganglia from the newborn (case 8). Serum was obtained from 2 cases. Case I was positive for VZV antibody, and we detected VZV DNA in the left trigeminal ganglion in this case. However, we did not detect VZV DNA in either type of ganglion from I seronegative case (case 7).

Table 1. Results of polymerase chain reaction for detection of VZV DNA. Case no .. age (years)

I. 63 2.77

3. 48 4. 69 5. 50 6.68 7.63 8.0

Clinical diagnosis Brain hemorrhage Lung cancer Malignant lymphoma Rectal cancer Lung cancer Cardiomyopathy Esophageal cancer Immature infant

Trigeminal ganglia

Geniculate ganglia

Right

Left

Right

+ +

_t

+ + + + +

+

+ + +

+ + + + +

Left

VZV antibody index"

1.06

+ + + +

NA NA NA NA NA

0.66 NA

NA

NA

NOTE. Cases 3 and 5 were female; all others were male. -, negative; +. positive; NA. not available. * YZY antibody index = EIA titer of patient/minimal EIA titer ofpositive controls: ~ 1.00. positive; < 1.00. negative. t Case had no amplification by PCR with o-tubulin primer pairs.

Discussion VZV latency in human sensory ganglia has been detected by means of nucleic acid hybridization. Gilden et al. I I0] reported the detection of VZV DNA in normal trigeminal ganglia from autopsy cases by Southern blot hybridization. However, they failed to detect VZV in normal thoracic ganglia by this method because of the small size of this ganglion and the limited quantity of DNA available. The cellular localization of the VZV genome in latently infected ganglia has been studied by in situ hybridization, but there were discrepancies among the reports. Hyman et al. [II] and Gilden et al. [12J found VZV nucleic acid in neurons of the human trigeminal and thoracic ganglia and not in satellite cells, whereas Croen et al. [13J detected VZV RNA in both neuronal and nonneuronal cells of the human trigeminal and dorsal root ganglia. i>CR is a highly sensitive and simple method for detection of viral genomes, and only a small amount oftissue is needed for analysis. Mahalingam et al. [6], who used PCR, reported that VZV becomes latent in the trigeminal ganglia more often than in any thoracic ganglion of man. In the present study, we used PCR for detecting VZV DNA in the human geniculate ganglia because these are very small ganglia located in the geniculate portion of the facial nerves and because we can extract only a small amount of DNA. We detected VZV DNA in most of the trigeminal and geniculate ganglia from the adults. However, VZV was negative in both types ofganglia from I seronegative adult and in those from a newborn. These results indicate that VZV becomes latent not only in the trigeminal ganglia but also in the geniculate ganglia after primary infection, and we suggest that reactivation of the virus from the geniculate ganglia may

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mixture (dATP, dGTP, dCTP, and dTTP at a final concentration of200 J,LM), and 2.5 units of Thermus aquaticus DNA polymerase (Perkin-Elmer Cetus). Detection of amplified DNA. The EcoRI-B restriction fragment ofVZV strain H-S I DNA, which contains the major DNA binding protein domain, was amplified by PCR with the VZV primer pairs, and 50 ng of the amplified DNA fragment was labeled with digoxigenin-dUTP (Boehringer, Mannheim, Germany) according to the method of Lanzillo [9]. One-fourth of the final reaction product from tissue DNA was electrophoresed in agarose gels composed of 1% Seakem and 3% NuSieve (FMC BioProducts, Rockland, ME) and was transferred to nylon filters (Hybond N; Amersham, Amersham, UK). Southern blot hybridization was done with the VZV probe labeled by PCR, and the hybridization was detected by the use of an antidigoxigenin Fab fragment conjugated to alkaline phosphatase (Boehringer) and a chemiluminescent substrate, 3-(2'-spiroadamantane )-4-methoxy-4~(3"-phosphoryloxy)phenyl-1 ,2-dioxetane disodium salt (AMPPD; Tropix, Bedford, MA), as described by Lanzillo [9]. After the immunologic reaction, the filter was exposed to radiographic film (X-Omat; Eastman Kodak, Rochester, NY) at room temperature for 7 h. VZVantibody. Serum was taken by cardiac puncture in 2 cases (cases I and 7). VZV IgG antibody index was measured by EIA (SMI Bristol, Sagamihara, Japan).

Concise Communications

JID 1992 ;166 (November)

Case 6 TrL Gen.

p

Case 7 TrL Gen.

R L R L R L

References

R L

vzv

... 273 bp

Tubulin

. - -

-

-

- -

- -

-

-

1159

... 286bp

I

OX174 /Haelll diges t marker

Figure 1. Detection ofVZV DNA in human trigeminal and geniculate ganglia. DNA (I ~g) was extracted from human ganglia and amplified with VZV primers; 25%of final reaction product was analyzed by Southern blot hybridization. 273-bp products were detected in left and right trigeminal ganglia and in right geniculate ganglion of case 6. Hybridized bands below 273-bp products were not specific to VZV primer pairs. P. VZV strain H-N3 DNA (10 fg), used as positive control; tubulin, I ~g of DNA from same samples amplified with tubulin primers, and 10% of product was electrophoresed. Amplified products (286 bp) were seen in all ganglia.

cause peripheral facial nerve palsy. zoster in the auricles, and eighth cranial nerve disorders. The auditory and vestibular symptoms of Ramsay Hunt syndrome are thought to be caused by the spread of inflammation or of virus to the eighth cranial nerve and the labyrinth. However, the possibility exists that simultaneous reactivation of the virus from the spiral or vestibular ganglion causes the auditory or vestibular dysfunction. We are now planning to analyze the VZV latency in these ganglia by peR.

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•

I. Peitersen E. Caunt AE. The incidence of herpes zoster antibodies in patients with peripheral facial palsy. J Laryngol Otol 1970;84 :6570. 2. Hunt JR . On herpetic inflammation of the geniculate ganglion. a new syndrome and its complications. J Nerv Ment Dis 1907 ;34:73-96. 3. Furuta Y, Takasu T. Sato K. Fukuda S. Inuyama Y. Nagashima K. Latent herpes simplex virus type I in human geniculate ganglia. Acta Neuropathol (Berl) 1992 ;84:39-44. 4. Hirt B. Replication molecules of polyoma virus DNA. J Mol Bioi 1969:40: 141-4. 5. Sambrook J. Fritsch EF. Maniatis T. Isolation of DNA from mammalian cells: protocol I. In: Sambrook J. Fritsch EF. Maniatis T. eds. Molecular cloning . 2nd ed. Vol 2. Cold Spring Harbor. NY: Cold Spring Harhor Laboratory, 1989 ;9. 16-9.19. 6. Mahalingam R. Wellish M. Wolf W, et al. Latent varicella-zoster viral DNA in human trigeminal and thoracic ganglia. N Engl J Med 1990:323:627-31. 7. Davison AJ. Scott JE . The complete DNA sequence of varicella-zoster virus. J Gen ViroI1986;67: I759-816. 8. Cowan NJ. Dobner PRoFuchs EV. Cleveland DW. Expression ofhuman a-tubulin genes: interspecies conservation of 3' untranslated regions. Mol Cell Bioi 1983 ;3:1738-45. 9. Lanzillo JJ . Chemiluminescent nucleic acid detection with digoxigenin-labeled probes: a model system with probes for angiotensin converting enzyme which detect less than one attomole of target DNA. Anal Biochem 1991:194:45-53. 10. Gilden DH. Vafai A. Shtram Y. Becker Y. Devlin M. Wellish M. Varicella-zoster virus DNA in human sensory ganglia. Nature 1983;306:478-80. II. Hyman RW. Ecker JR. Tenser RB. Varicella-zoster virus RNA in human trigeminal ganglia. Lancet 1983 ;2:814-6. 12. Gilden DH. Rozenman Y. Murray R. Devlin M. Vafai A. Detection of varicella-zoster virus nucleic acid in neurons of normal human thoracic ganglia. Ann Neurol 1987;22:377-80. 13. Croen KD. Ostrove JM. Dragovic U. Straus SE. Patterns of gene expression and sites of latency in hum an nerve ganglia are different for varicella-zoster and herpes simplex viruses. Proc Natl Acad Sci USA 1988;85:9773-7.