Quantitation of Latent Varicella-Zoster Virus and Herpes Simplex Virus Genomes in Human Trigeminal Ganglia

Quantitation of Latent Varicella-Zoster Virus and Herpes Simplex Virus Genomes in Human Trigeminal Ganglia
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    1999, 73(12):10514. J. Virol. StrausMcChesney, Erik K. Mont, John E. Smialek and Stephen E. Stephanie R. Pevenstein, Richard K. Williams, Daniel  Human Trigeminal Gangliaand Herpes Simplex Virus Genomes in Quantitation of Latent Varicella-Zoster Virus information and services can be found at: These include:  REFERENCES This article cites 34 articles, 20 of which can be accessed free CONTENT ALERTS  more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new Information about commercial reprint orders: To subscribe to to another ASM Journal go to:  onM ar  c h 1 2  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  onM ar  c h 1 2  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   J OURNAL OF  V IROLOGY ,0022-538X/99/$04.00  0Dec. 1999, p. 10514–10518 Vol. 73, No. 12 Quantitation of Latent Varicella-Zoster Virus and HerpesSimplex Virus Genomes in Human Trigeminal Ganglia STEPHANIE R. PEVENSTEIN, 1 RICHARD K. WILLIAMS, 1 DANIEL M C CHESNEY, 1 ERIK K. MONT, 2 JOHN E. SMIALEK, 3  AND  STEPHEN E. STRAUS 1 *  Medical Virology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, 1  and Anatomical Pathology Service, Laboratory of Pathology, National Cancer Institute, 2  National Institutes of Health, Bethesda, and Department of Pathology, University of Maryland School of Medicine, Baltimore, 3  Maryland Received 10 May 1999/Accepted 31 August 1999 Using real-time fluorescence PCR, we quantitated the numbers of copies of latent varicella-zoster virus(VZV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) genomes in 15 human trigeminal ganglia.Eight (53%) and 1 (7%) of 15 ganglia were PCR positive for HSV-1 or -2 glycoprotein G genes, with means of 2,902  1,082 (standard error of the mean) or 109 genomes/10 5 cells, respectively. Eleven of 14 (79%) to 13 of 15 (87%) of the ganglia were PCR positive for VZV gene 29, 31, or 62. Pooling of the results for the three VZV genes yielded a mean of 258  38 genomes/10 5 ganglion cells. These levels of latent viral genome loads haveimplications for virus distribution in and reactivation from human sensory ganglia. Herpes simplex virus types 1 and 2 (HSV-1 and -2) and varicella-zoster virus (VZV) are alphaherpesviruses that infect,establish latency in, and subsequently reactivate from humansensory nerve ganglia (1, 36). Following reactivation from la-tent ganglia reservoirs, each of these herpesviruses may causesignificant clinical disease in the individual and may spread touninfected persons. Symptomatic VZV reactivation is an in-frequent, usually once-in-a-lifetime event that results in zoster(shingles), while HSV-1 and -2 reactivation occurs frequentlyand results in numerous symptomatic and asymptomatic recur-rences of oral and genital herpes.Little is known regarding the mechanism underlying theparticular patterns of latency and reactivation that distinguishHSV-1 and -2 infection from that with VZV. Abundant datafrom both human studies and animal models confirm thatHSV-1 and -2 persist in sensory neurons but that satellite glialcells are spared from harboring latent HSV (7, 29, 30). Dataregarding the site of VZV latency have been controversial, with various reports indicating it to be neurons, nonneuronalcells, or both (7, 9, 17, 22, 26). Moreover, estimates of theproportions of cells harboring HSV and VZV and the quantityof latent viral DNA in ganglia have varied widely (7, 17, 22, 25,30). Recent animal studies show that latent viral genome levelsin sensory ganglia influence the reactivation frequency of HSV-1 and -2, suggesting that the quantity of latent viral ge-nome copies per ganglion—the latent viral load—may be asignificant determinant of herpesvirus reactivation from thenervous system (21, 31, 32). To further clarify the nature anddistribution of latent HSV and VZV genomes in human gan-glia, we developed and used several sensitive and specific PCRassays. Human tissue samples.  Human trigeminal ganglia were har- vested within 24 h postmortem, frozen in dry ice, and stored at  70°C until DNA extraction. The general clinical histories andcauses of death are summarized in Table 1.DNA was extracted by the protocol described in the instruc-tions for a Puregene DNA isolation kit (D-5000A; GentraSystems, Minneapolis, Minn.) with a few modifications. Gan-glia were pulverized to a fine powder on dry ice and incubatedin cell lysis buffer and 10 mg of proteinase K per ml for 3 daysto ensure complete homogenization. Following protein precip-itation, DNA was ethanol precipitated and resuspended in water. DNA concentration was estimated by spectrophotome-try, and purity was determined from the ratios of the opticaldensity at 260 nm to that at 280 nm. On average, the ganglia yielded 478  46 (mean  standard error of the mean [SEM])  g of DNA. QF-PCR assays.  Quantitative fluorescent (QF) PCR wasperformed with a Prism 7700 sequence detector (PE AppliedBiosystems, Foster City, Calif.) according to supplied guide-lines for real-time DNA amplification. Real-time PCR relieson a quantitative increase in fluorescence due to cleavage of a5  reporter dye from a dually labeled fluorogenic probe oligo-nucleotide by the 5  3  3   nuclease activity of   Taq  DNA poly-merase.The genes encoding VZV glycoprotein B (gB; open readingframe [ORF] 31), ORF 62 (which encodes the major immedi-ate early transactivator), ORF 29 (which encodes a putativeearly major DNA-binding protein), HSV-1 glycoprotein G(gG1), and HSV-2 glycoprotein G (gG2) were selected forquantification. The forward and reverse primers and probe forthe VZV gB gene were described by Kimura et al. (18) (Table2). The forward and reverse primers and probes for VZV ORF29 and ORF 62 and for the gG genes of HSV-1 and HSV-2 were designed with the Primer Express program (PE AppliedBiosystems) (Table 2) and synthesized by Bioserve Biotechnol-ogies (Laurel, Md.). QF PCR was also performed with theprimers and the probe (Table 2) provided with the Taqman  -actin reagent kit (PE Applied Biosystems) to normalize eachof the ganglion extracts for amplifiable human DNA. All PCRs were performed in triplicate on two separate oc-casions with a Taqman PCR kit (PE Applied Biosystems) inthe absence of reverse transcriptase, so that only DNA wasamplified. Each 50-  l PCR mixture contained 500 ng of humantrigeminal ganglion DNA with final concentrations of eachprimer of 1,000 nM and of each probe sequence of 200 nM.Multiple human trigeminal DNA samples were run togetheron a plate in parallel with duplicate sets of DNA standards.Each primer set mixture was run on a separate plate. Standard * Corresponding author. Mailing address: LCI, NIAID, NIH, Build-ing 10, 11N228, 10 Center Dr., Bethesda, MD 20892. Phone: (301)496-5807. Fax: (301) 496-7383. E-mail:   onM ar  c h 1 2  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   curves for each targeted viral gene were generated by mixingserial 10-fold dilutions of plasmids containing 10 0 to 10 6 copiesof the desired genes. Additional dilutions within that range were analyzed for plasmids bearing the genes for gB, ORF 62,and gG1 to better delineate the sensitivity limits at the lowends of the standard curves. Uninfected BALB/c mouse tri-geminal DNA (500 ng) was added to all plasmid standards tocompensate for the potential inhibition of amplification reac-tions by added DNA. For VZV gB, the gB gene contained inthe pUC19 vector, a gift of Liyanage Perera, was used. ForORF 29 and ORF 62, their respective  Eco RI-B and  Eco RI-A fragments from the VZV strain Ellen contained in the pGEM vector were used. Plasmids containing the HSV-1 and HSV-2gG genes were obtained from Mark Challberg and PhilipKrause, respectively. In addition, no template control reactionmixtures containing the appropriate probe and primer system without DNA were run on all plates in triplicate. PCR mixtures were subjected to 2 min at 50°C (reaction of AmpErase uracil-  N  -glycosylase), 10 min at 95°C (activation of AmpliTaq Gold),and 55 cycles of 15 s at 95°C and 1 min at 60°C. For   -actinamplification, 40 cycles were performed.For each reaction, real-time fluorescence values were mea-sured as a function of the quantity of a reporter dye (6-carboxy-fluorescein [FAM]) released during amplification. A thresholdcycle ( C t ) value for each sample was determined as the numberof the first cycle at which the measured fluorescence exceededthe threshold limit (10 times the standard deviation of thebaseline).  C t  values observed for human trigeminal DNA sam-ples were used to calculate the viral genome copy number foreach sequence amplified based on the standard curves forplasmids containing test sequence.Standard curves for three VZV-containing plasmids areshown in Fig. 1 as examples. No cross-reactivity was observedbetween any of the viral DNA assays. The limits of sensitivityof the QF PCRs for all of the viral genes were estimated as thelowest plasmid dilutions that yielded comparable  C t  values inreplicate samples: they were all   10 copies/500 ng of inputDNA. The human  -actin standards were not evaluated below100 copies/500 ng of DNA. All standard curves were fit bylinear regression, with correlation coefficients of   0.92. C t  values for plasmid dilutions in these standard curves werecompared with  C t  values of human trigeminal ganglia samplesto estimate the number of copies of each viral gene present inthe approximately 500 ng of extracted DNA analyzed for eachreaction. To more accurately estimate the amount of humangenomic DNA in each trigeminal ganglion extract, we quanti-tated the number of copies of    -actin genes in that sample volume and normalized the number of DNA copies observedto the number of copies per 2  10 5  -actin copies, i.e., per 10 5 cells. Because the mean number of copies of   -actin quantifiedin 500 ng of DNA was 6.4  10 4  0.8  10 4 , we calculated thateach cell contained 15.6 pg of DNA.  VZV and HSV latent DNA load.  The numbers of copies of the various VZV and HSV genes per 10 5 cells are displayed inFig. 2 along with tabulations of their mean numbers (  SEM)for ganglia that yielded positive results (  10 copies/500 ng of DNA). By these criteria, the proportions (and percentages) of ganglia positive for each viral gene were as follows: 11 of 14 were positive (79%) for the ORF 29 gene, 12 of 15 werepositive (80%) for the gB gene, 13 of 15 were positive (87%)for the ORF 62 gene, 8 of 15 were positive (53%) for the gG1gene, and 1 of 14 were (7%) positive for the gG2 gene.By the Spearman rank test, the numbers of copies of allthree VZV genes correlated highly significantly with eachother (  P     0.001). There were no statistical associations be-tween HSV gene copy numbers and VZV gene numbers.Moreover, the mean number of VZV ORF 62 genes, 2.7  0.4times the numbers of the gB and ORF 29 genes, was notdissimilar from the expected ratio of 2.0 for this diploid viralgene. With the numbers obtained for gB and ORF 29 andone-half of the number obtained for the diploid ORF 62 genebeing pooled, the QF-PCR assay revealed that PCR-positiveganglia contained a mean of 258  38 VZV genome copies/10 5 cells. This value was significantly less than the 2,902    1,082HSV-1 genomes per 10 5 cells in positive ganglia (  P     0.02;Wilcoxon rank sum test) but comparable to the copy numberof genomes in the one ganglion containing detectable HSV-2DNA, 109 genomes/10 5 cells. Latent viral DNA loads were notgrossly different for ganglia obtained from individuals knownto be immunocompromised and those presumed to be immu-nocompetent. Implications of the data.  The detection of VZV DNA in 79to 87% of ganglia is in concordance with the very high pro-portion of American adults who are seropositive for VZV and with data obtained from prior standard nonquantitative PCRassays and in situ hybridization (1, 13, 27). Slightly more thanhalf of the ganglia were HSV-1 DNA positive, also commen-surate with the seroprevalence of 50 to 70% reported for this TABLE 1. Demographic data on subjects from whom trigeminal ganglia were recovered Subject Gender  a  Age(yr) Immediate cause of death Underlying and/or contributing disease 669 M 65 Alcohol abuse None known671 M 27 Motor vehicle accident None known672 M 25 Drug overdose HIV infected673 M 34 Gunshot wound None known674 F 35 Unknown None known675 M 40 Gunshot wounds None known676 M 73 Drug overdose Systemic lupus erythmatosus677 M 45 Unknown None known678 M 18 Drug overdose None known679 F 43 Drug overdose None known680 F 62 Drug overdose None known681 F 30 Respiratory failure Aplastic anemia682 M 41 Sepsis Large granular lymphocytic leukemia683 M 23 Pneumonia Multiple myeloma, bone marrow transplant684 M 18 Diffuse alveolar damage Graft vs host disease, organ transplant recipient  a M, male; F, female. V OL  . 73, 1999 NOTES 10515   onM ar  c h 1 2  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om    virus for young American adults and with prior molecularstudies of human ganglia (7, 11, 36). HSV-2 DNA was detectedin only 1 of 15 ganglia, reflecting the relatively low rate of facialinfection with this virus (36).Extrapolations of our data permit estimates of the total copynumbers and distributions of these three viruses in humantrigeminal ganglia. We recovered a mean of 478   g of DNA per ganglion. Based on our estimate of 15.6 pg of DNA/cell,the average trigeminal ganglion contained upward of 3  10 7 cells. Thus, we estimate that, on average, each ganglion latentlyinfected with VSV, HSV-1, or HSV-2 contained 7.7    10 4 ,8.7  10 5 , or 3.2  10 4 genomes, respectively. Our estimate forlatent HSV-1 load in human ganglia (Fig. 2) is similar to thatreported by Efstathiou et al. (11) (1,000 to 10,000 copies), butthe estimate for VZV load is higher than the 6 to 31 copies/10 5 cells reported by Mahalingam et al. using the less precise PCRand Southern hybridization methods (26).Ball et al. reported that human trigeminal ganglia contain anaverage of 8.1  10 4 neurons (2). Since HSV-1 and -2 persistexclusively in neurons (5, 7, 8, 29, 30, 34, 35), we can projectthat each latently infected neuron contains at least 11 copies of HSV-1 DNA, assuming all neurons are infected. While in situhybridization studies for HSV-1 latency-associated transcriptssuggested that only 1 to 4% of neurons are positive (7, 8, 10,34, 35), the more recent PCR and in situ-PCR analyses dem- FIG. 1. Standard curves for QF-PCR assays for VZV genes encoding ORFs29 and 62 and gB. Shown are the  C t  values at which a given input of a VZVDNA-containing plasmid was detected by PCR. Plasmid concentrations testedranged from 10 0 to 10 6 copies per reaction mixture. The Spearman rank coeffi-cient of correlation (  r  ) for lines fitting each standard curve are given.     T    A    B    L    E    2 .    P   r   o    b   e   a   n    d   p   r    i   m   e   r   s   e   q   u   e   n   c   e   s    f   o   r   g   e   n   e   s   a   m   p    l    i    fi   e    d    b   y   q   u   a   n    t    i    t   a    t    i   v   e    P    C    R     V    i   r   a    l   o   r   c   e    l    l   u    l   a   r   g   e   n   e    P   r   o    b   e      a     F   o   r   w   a   r    d   p   r    i   m   e   r    R   e   v   e   r   s   e   p   r    i   m   e   r     V    Z    V   g    B    5            -    (    F    A    M    )    A    T    T    A    C    T    G    G    A    A    C    C    T    G    C    A    G    C    G    C    G    G    A    (    T    A    M    R    A    )  -    3              5            -    G    A    T    G    G    T    G    C    A    T    A    C    A    G    A    G    A    A    C    A    T    T    C    C  -    3              5            -    C    C    G    T    T    A    A    A    T    G    A    G    G    C    G    T    G    A    C    T    A    A  -    3              O    R    F    2    9    5            -    (    F    A    M    )    C    C    C    G    T    G    G    A    G    C    G    C    G    T    C    G    A    A    A    (    T    A    M    R    A    )  -    3              5            -    C    G    T    A    C    A    C    G    T    A    T    T    T    T    C    A    G    T    C    C    T    C    T    T    C  -    3              5            -    G    G    C    T    T    A    G    A    C    G    T    G    G    A    G    T    T    G    A    C    A  -    3              O    R    F    6    2    5            -    (    F    A    M    )    T    C    T    C    G    A    C    T    G    G    C    T    G    G    G    A    C    T    T    G    C    G    (    T    A    M    R    A    )  -    3              5            -    T    C    T    T    G    T    C    G    A    G    G    A    G    G    C    T    T    C    T    G  -    3              5            -    T    G    T    G    T    G    T    C    C    A    C    C    G    G    A    T    G    A    T  -    3              H    S    V  -    1   g    G    1    5            -    (    F    A    M    )    C    C    C    T    G    G    A    C    A    C    C    C    T    C    T    T    C    G    T    C    G    T    C    A    G    (    T    A    M    R    A    )  -    3              5            -    C    T    G    T    T    C    T    C    G    T    T    C    C    T    C    A    C    T    G    C    C    T  -    3              5            -    C    A    A    A    A    A    C    G    A    T    A    A    G    G    T    G    T    G    G    A    T    G    A    C  -    3              H    S    V  -    2   g    G    2    5            -    (    F    A    M    )    A    C    A    C    A    T    C    C    C    C    C    T    G    T    T    C    T    G    G    T    T    C    C    T    A    A    C    G    (    T    A    M    R    A    )  -    3              5            -    C    A    A    G    C    T    C    C    C    G    C    T    A    A    G    G    A    C    A    T  -    3              5            -    G    G    T    G    C    T    G    A    T    G    A    T    A    A    A    G    A    G    G    A    T    A    T    C    T    A    G    A  -    3              H   u   m   a   n   g   e   n   o   m    i   c            -   a   c    t    i   n    5            -    (    F    A    M    )    A    T    G    C    C    C    X    (    T    A    M    R    A    )    C    C    C    C    C    A    T    G    C    C    A    T    C    C    T    G    C    G    T   p  -    3              5            -    T    C    A    C    C    C    A    C    A    C    T    G    T    G    C    C    C    A    T    C    T    A    C    G    A  -    3              5            -    C    A    G    C    G    G    A    A    C    C    G    C    T    C    A    T    T    G    C    C    C    A    A    T    G    G  -    3               a     T    A    M    R    A ,    6  -   c   a   r    b   o   x   y  -    t   e    t   r   a   m   e    t    h   y    l  -   r    h   o    d   a   m    i   n   e .    X    i   n    t    h   e            -   a   c    t    i   n   p   r   o    b   e   s   e   q   u   e   n   c   e    i   s   a    l    i   n    k   e   r   a   r   m   n   u   c    l   e   o    t    i    d   e ,   a   n    d   p    i   s   a   p    h   o   s   p    h   o   r   y    l   a    t    i   o   n   s    i    t   e . 10516 NOTES J. V IROL  .   onM ar  c h 1 2  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   onstrated that 3 to 10 times that percentage are positive (6–8,29–32, 34, 35). These data indicate that latently infected tri-geminal neurons contain an average of 28 or more HSV-1genomes, a copy number similar to those of Epstein-Barr virusepisomes carried in B cell lines (12, 28) and papillomavirusepisomes in human cervical cancer cells (3, 4, 15, 16).The low copy number of detectable HSV-2 DNA in trigem-inal ganglia may parallel the very low frequency with which this virus recurs following initial infection in the mouth (20). Sincethe latent viral load correlates well with recurrence rates ininfected animals, if further studies confirm the low HSV-2 copynumber in human ganglia, these data suggest that the same isalso true for humans (21, 24, 31, 32). If so, some obstacle mustimpede infection and establishment of latency of HSV-2 inhuman trigeminal ganglia. Although our initial data suggested that VZV persists onlyin nonneuronal cells (7), recent data compel us to concludethat both neurons and nonneuronal cells are infected by VZVboth during productive infection (7, 22) and during latency (7,9, 14, 17, 22, 25). Were VZV to persist exclusively in neurons,the average of 7.7  10 4 genome copies we detected could bedistributed among nearly all of the estimated 8.1    10 4 neu-rons; however, they are not distributed in this way (17, 22).While we found no published estimates of the numbers of satellite and other nonneuronal cell populations in humanganglia, sensory neurons are very large (50 to 100   m in di-ameter) and are encircled and contacted by about 10 nucleatedsatellite cells each in most thin (6 to 8-  m) histologic sections(unpublished data and references 6, 10, and 19), which impliesthat there are at least 100 satellite cells surrounding everyneuron or at least 8  10 6 satellite cells per human trigeminalganglion.If we were to assume that VZV persisted in the same pro-portions of neurons and nonneuronal cells and at roughly thesame copy number per cell as that of HSV-1 (  28), fewer than1 in 1,000 total cells would prove positive. Lungu et al. (22, 23)estimated that 5 to 30% of both neurons and satellite cells arepositive for VZV DNA or VZV proteins, an estimate that isfar higher than that permitted by these data. Kennedy et al.(17), however, estimated by in situ PCR that 2% of neurons(  1,600 cells according to the estimate of Ball et al. [2]) and0.1% of satellite cells (  8,000 cells according to our estimate)are VZV DNA positive. If the estimate of Kennedy et al. iscorrect, the 7.7    10 4 VZV genomes could persist in thisnumber of cells at a density of eight copies/cell, not dissimilarfrom that for HSV-1. We are currently testing the validity of these extrapolations from our QF-PCR data by analyzing sec-tions microdissected from human ganglia (33).The present data provide refined estimates of the proportionof human trigeminal ganglia containing VZV, HSV-1, andHSV-2 DNAs and the latent DNA loads for each of theseneurotropic viruses. Moreover, they have implications regard-ing the distribution of these three viruses in human ganglia andthe pathogenesis of reactivation infections associated withthem. We thank Jeffrey Cohen, Philip Krause, and Nancy Tresser foradvice and assistance in this project, Peter Kennedy for additionalcomments on the manuscript, and Uri Lopatin for help with statisticalanalyses. REFERENCES 1.  Arvin, A. M.  1996. Varicella-zoster virus, p. 2547–2585.  In  B. N. Fields, D. M.Knipe, and P. M. Howley (ed.), Fields virology, 3rd ed., vol. 2. Lippincott-Raven, Philadelphia, Pa.2.  Ball, M. J., K. Nuttall, and K. G. Warren.  1982. Neuronal and lymphocyticpopulations in human trigeminal ganglia: implications for aging and forlatent virus. Neuropathol. Appl. Neurobiol.  8: 177–187.3.  Berumen, J., L. Casas, E. Segura, J. L. Amezcua, and A. Garcia-Carranca. 1994. Genome amplification of human papillomavirus types 16 and 18 incervical carcinomas is related to the retention of E1/E2 genes. Int. J. Cancer 56: 640–645.4.  Brown, D. R., J. T. Bryan, H. Cramer, B. P. Katz, V. Handy, and K. H. Fife. 1994. Detection of multiple human papillomavirus types in condylomataacuminata from immunosuppressed patients. J. Infect. Dis.  170: 759–765.5.  Croen, K. D., J. M. Ostrove, L. Dragovic, and S. E. Straus.  1991. Charac-terization of herpes simplex virus type 2 latency-associated transcription inhuman sacral ganglia and in cell culture. J. Infect. Dis.  163: 23–28.6.  Croen, K. D., J. M. Ostrove, L. J. Dragovic, J. E. Smialek, and S. E. Straus. 1987. Latent herpes simplex virus in human trigeminal ganglia. Detection of an immediate early gene “anti-sense” transcript by in situ hybridization.N. Engl. J. Med.  317: 1427–1432.7.  Croen, K. D., J. M. Ostrove, L. J. Dragovic, and S. E. Straus.  1988. Patternsof gene expression and sites of latency in human nerve ganglia are differentfor varicella-zoster and herpes simplex viruses. Proc. Natl. Acad. Sci. USA  85: 9773–9777.8.  Deatly, A., and A. Lumsden.  1984. RNA from an immediate early region of the type 1 herpes simplex virus genome is present in the trigeminal gangliaof latently infected mice. Proc. Natl. Acad. Sci. USA   84: 3204–3208.9.  Dueland, A. N., T. Ranneberg-Nilsen, and M. Degre.  1995. Detection of latent varicella zoster virus DNA and human gene sequences in humantrigeminal ganglia by in situ amplification combined with in situ hybridiza-tion. Arch. Virol.  140: 2055–2066.10.  Ecob-Prince, M. S., C. M. Preston, F. J. Rixon, K. Hassan, and P. G. E.Kennedy.  1993. Neurons containing latency-associated transcripts are nu-merous and widespread in dorsal root ganglia following footpad inoculationof mice with herpes simplex virus type 1 mutant in 1814. J. Gen. Virol. 74: 985–994.11.  Efstathiou, S., A. C. Minson, H. J. Field, J. R. Anderson, and P. Wildy.  1986.Detection of herpes simplex virus-specific DNA sequences in latently in-fected mice and in humans. J. Virol.  57: 446–455.12.  Ernberg, I., M. Andersson-Anvret, and G. Klein.  1977. Relationship betweenamount of Epstein-Barr virus-determined nuclear antigen per cell and num-ber of EBV-DNA copies per cell. Nature  226: 269–271.13.  Furuta, Y., T. Takasu, S. Fukuda, K. C. Sato-Matsumura, Y. Inuyama, R.Hondo, and K. Nagashima.  1992. Detection of varicella-zoster virus DNA inhuman geniculate ganglia by polymerase chain reaction. J. Infect. Dis.  166: 1157–1159.FIG. 2. Copies of VZV, HSV-1, and HSV-2 genes in human trigeminalganglia. The mean  SEM number of copies per 10 5 cells for each gene in thePCR-positive ganglia are tabulated below the figure. The filled circles show thegene copy numbers for samples that exceeded the assay limit of detection (  10copies/500 ng of total DNA). Open circles represent values for samples for whichgene copy numbers may have been extrapolated from the QF-PCR results but were below the threshold of reliable DNA detection (  10 copies/500 ng of DNA). The inability to define a precise threshold for positive ganglia on theper-10 5 -cell basis used here reflects the variability in   -actin gene number per500 ng of total DNA from the individual ganglia. V OL  . 73, 1999 NOTES 10517   onM ar  c h 1 2  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om 
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