Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
J. gen. Virol. (1986), 67, 2515-2520. Printed in Great Britain 2515 Key words: BHV-1/ latent infection/in situ hybrMization/transcription Detection of Bovine Herpesvirus Type 1 RNA in Trigeminal Ganglia of Latently Infected Rabbits by in situ Hybridization By D. L. R O C K , * ~ W. A. H A G E M O S E R , F. A. OSORIO:~ AND D. E. R E E D § Veterinary Medical Research Institute and Department o f Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011, U.S.A. (Accepted 16 July 1986) SUMMARY At times after conjunctival inoculation with bovine herpesvirus type 1 (BHV-1), representing the acute and latent phases of infection, rabbit trigeminal ganglia were examined for the presence of BHV-1 nucleic acids by in situ hybridization using a 3Hlabelled BHV-1 DNA probe. During the acute phase of virus infection, both BHV-1 DNA and RNA were detected in ganglionic neurons and occasionally in adjacent satellite cells. However, during the latent phase of infection only viral RNA was detectable in involved neurons. Viral RNA appeared restricted to the nucleus of latently infected cells and was present in varying amounts in individual cells. These results indicate that the BHV-1 genome is transcriptionally active in ganglionic neurons during latent infection. Bovine herpesvirus type 1 (BHV-1), a member of the alphaherpesvirus group, is responsible for a variety of disease conditions in cattle including rhinotracheitis, conjunctivitis, vulvovaginitis, balanoposthitis, meningoencephalitis and fatal systemic infection (Kahrs, 1981). Like other herpesviruses, BHV-1 establishes latent infections. In all likelihood, it is this virus property alone that is responsible for perpetuation and transmission of infection in cattle (Snowdon, 1965; Sheffy & Davies, 1972). Latent infection of cattle with BHV-1 is characterized by: (i) most, if not all, animals are latently infected after experimental infection (Sheffy & Davies, 1972; Davies & Carmichael, 1973); (ii) spontaneous and sporadic shedding of virus occurs in latently infected animals (Snowdon, 1965; Bitsch, 1973; Huck et al., 1973); (iii) virus can be reactivated predictably from latently infected animals following the administration of glucocorticoids (Kubin, 1969; Sheffy & Davies, 1972; Davies & Carmichael, 1973); (iv) virus persists in sensory and autonomic nerve ganglia during viral latency (Narita et al., 1978; Homan & Easterday, 1980, 1983; Ackermann et al., 1982). The specific virus-cell interactions underlying establishment, maintenance and reactivation of latent BHV-1 infection are unknown. We have described a rabbit model for latent BHV-1 infection that is consistent with the above-mentioned observations made for latently infected cattle (Rock & Reed, 1982). In this report, we make use of this model system together with in situ nucleic acid hybridization to examine latent BHV-1 infection at the molecular/cellular level. Our results indicate that the BHV-1 genome is transcriptionally active in ganglionic neurons during the latent phase of infection. t Present address: Department of Veterinary Science, North Dakota State University, P.O. Box 5406, Fargo, North Dakota 58105, U.S.A. $ Present address: Veterinary Diagnostic Center, University of Nebraska-Lincoln, Lincoln, Nebraska 685830907, U.S.A. § Present address: Molecular Genetics Inc., 10320 Bren Road East, Minnetonka, Minnesota 55343, U.S.A. 0000-7241 © 1986 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 16:04:22 2516 Short communication New Zealand white rabbits (2.5 to 3.0 kg) were lightly anaesthetized with methoxyflurane and inoculated in both the right and left conjunctival sac with 1.0 × 1 0 7 p.f.u, of the Cooper strain of BHV-1 (obtained from Dr A. Strating, National Veterinary Services Laboratory, Ames, Iowa, U.S.A.) as previously described (Rock & Reed, 1982). Ganglionic homogenates (10~ in MEM), prepared using Ten Broeck tissue grinders, were assayed on bovine lung (BL) cells. Ganglia for explant culture were washed in Eagle's MEM, cut into 1 to 5 mm pieces, and cultured in MEM supplemented with 20~ foetal bovine serum, penicillin (100 U/ml) and streptomycin (100 ~tg/ml). Cultures were maintained for 30 to 45 days before being discarded as negative. Extracellular virions from BHV-1 (Cooper) and pseudorabies virus (PRV) (strain BE12, obtained from K. B. Platt, Iowa State University) infected BL cells were purified using the procedure of Talens & Zee (1976). DNA was prepared from purified virions by Pronase-SDS treatment followed by phenol-chloroform extraction and two cycles of sodium iodide/ethidium bromide gradient centrifugation (Walboomers & Schegget, 1976; PeUicer et al., 1978). Herpes simplex virus type 1 (HSV-1) (strain F) and plasmids containing cloned B a m H I fragments of this virus genome were obtained from B. Roizman, University of Chicago (Post et al., 1980). Plasmid DNAs were prepared using standard techniques (Maniatis et al., 1982). Blot hybridizations of rabbit genomic DNA were performed as described by Rock & Fraser (1983). Blots were hybridized with a nick-translated 3Zp-labelled BHV-1 probe (2 × 107 to 4 × 107 c.p.m./blot) for 48 h at 50 °C in 50~ formamide, 10~ dextran sulphate, 4X SSC (IX SSC is 0.15 M-NaC1, 0.015 M-sodium citrate), 0"1 M-EDTA, 0.1 ~ SDS and 250 p.g/ml sonicated denatured salmon sperm DNA. Filters were washed once for 30 min under each of the following conditions: 1X SSC, 0.1~ SDS at 37 °C; 1X SSC, 0.1~ SDS at 65 °C; 0-5X SSC, 0.1~ SDS at 65 °C; 0-25X SSC, 0.1 ~ SDS at 65 °C. Autoradiographic exposure was 1 to 3 days. Sensitivities approaching 10 pg BHV-I DNA were routinely obtained. In situ hybridization was performed essentially as described previously (Brahic & Haase, 1978; Stroop et al., 1984). At various times post-infection animals were anaesthetized and killed by perfusing with periodate-lysine-paraformaldehyde fixative (PLP) (McLean & Nolsane, 1974). Trigeminal ganglia were dissected, immersion-fixed in PLP for 24 h and embedded in paraffin for sectioning. Paraffin was removed from tissue sections with xylene, the sections were rehydrated in graded ethanol solutions and then sequentially pretreated with 0.2 M-HC1 and proteinase K as described by Brahic & Haase (1978). Slides probed for viral RNA were dehydrated in graded ethanol solutions and stored desiccated at room temperature. Slides probed for viral DNA were rinsed in 2X SSC and further treated with DNase-free RNase A (100 ~tg/ml in 2X SSC) for 30 rain at 37 °C, refixed in 5 ~ paraformaldehyde for 30 min, dehydrated in graded ethanol solutions and stored desiccated at room temperature. DNA within tissue sections probed for viral DNA was denatured by heating to 65 °C in deionized formamide in 0-1X SSC for 15 min, quenched in ice-cold 0.1X SSC and dehydrated in graded ethanol solutions prior to hybridization. Probe DNAs used in these experiments were radiolabelled by nick translation with 3H-labelled nucleotides as described previously (Rigby et al., 1977; Stroop et al., 1984). Sections were hybridized with 1 ng (approx. 105 c.p.m.) of 3H-labelled probe DNA per slide for 72 h at 45 °C in 2X SSC, 45 ~ formamide, 10~ dextran sulphate, 10 mM-Tris-HC1 pH 7.4, 1 mMEDTA, 1X Denhardt's solution (0"02~o bovine serum albumin, 0-02~ polyvinylpyrrolidone, 0.02~o Ficoll) and 1.0 mg/ml rabbit brain total nucleic acids. Following hybridization, slides were washed in 2X SSC, 45~ formamide, 10 mM-Tris-HC1 pH 7.4, 1 mM-EDTA for 3 to 4 days at room temperature. Following dehydration in graded ethanol solutions containing 0.3 Mammonium acetate, the slides were coated with NTB-2 emulsion, exposed for 3 weeks at 4 °C, developed and stained with haematoxylin and eosin as described by Stroop et al. (1984). All sections were examined and scored in a masked fashion. In general, backgrounds were relatively free of non-specific autoradiographic grains; positive ceils were considered to be those cells exhibiting grains too numerous to count easily. Tissue culture cells lytically infected with the various herpesviruses were included as positive controls in all in situ hybridization experiments. Cells were infected at a multiplicity of 5 and fixed (20 min at room temperature in 75 ~ ethanol25~ glacial acetic acid) at 18 h post-infection. All viral probes used in these experiments Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 16:04:22 Short communication 2517 Table 1. Detection of BHV-1 nucleic acids in rabbit trigeminal ganglia during acute and latent infection by in situ hybridization* Hybridization in no. of rabbits/total no. examined r Probed for: BHV-1 DNA BHV-1 RNA PRV RNA HSV-1 RNA pRB 112 (BamHI fragment B) pRB113 (BamHI fragment Y) Uninfected 0/5 0/5 0/4 BHV-1 acute phase 8/9 8/9 0/6 BHV-1 latent phase 0/12 10/11 0/10 ND~ 0/3 0/9 ND 0/3 0/5 * Hybridization results were from two independent experiments and were recorded at 4 to 10 days postinoculation for BHV-1 acute phase and 2 to 15 months post-inoculation for BHV-1 latent phase. t ~o, Not done. hybridized to tissue culture cells infected with the homologous virus under the in situ hybridization assay conditions detailed above. The specificity of the BHV-1 in situ hybridization described was demonstrated in the following ways: (i) under the given hybridization conditions the BHV-1 probe failed to show appreciable hybridization to uninfected BL cells, PRV-infected BL cells and to Southern blots of rabbit genomic DNA (data not shown); (ii) BHV-l-specific hybridization was not detected in sections of trigeminal ganglion taken from uninfected rabbits (Table 1, Fig. 1e); (iii) heterologous herpesvirus probes high in 700G + C content (PRV, HSV-1) failed to hybridize to BHV-l-infected ganglionic tissue sections (Table 1) under conditions that allowed hybridization in an homologous system. The acute phase of BHV-1 infection was as previously described (Rock & Reed, 1982). Virus was present in ocular swabs taken from inoculated rabbits on day 1 post-inoculation and was detectable for an additional 9 to 15 days. Infectious BHV-1 was present in trigeminal ganglionic homogenates from two of three animals at 5 days post-inoculation. At this time, viral DNA and RNA were observed in neurons and occasionally other small supporting cells of the trigeminal ganglion. Localization of viral DNA in involved neurons was predominantly intranuclear (Fig. 1a) whereas viral RNA was routinely evident in both the nucleus and cytoplasm (Fig. 1 b). On histological examination, many BHV-l-hybridizing cells showed evidence of early necrotic changes including pyknotic nuclei, pale eosinophilic staining cytoplasm and irregular cell membrane borders (Fig. I a). During the latent phase of infection (2 to 15 months post-inoculation), BHV- 1 was detected in explant cultures of trigeminal ganglia from 19 of 24 animals, but was not isolated from ganglionic homogenates (N = 5). BHV-1 RNA, but not DNA, was detected in ganglionic neurons from 10 of 11 latently infected animals (Table 1). Involved cells, making up approximately 0-3 ~o of all neurons, were morphologically normal in appearance. In contrast to the RNA hybridization pattern seen in acutely infected cells, latently infected neurons contained autoradiographic grains almost exclusively over the nucleus of the cell, indicating an apparent restriction of viral RNA to the cell nucleus (Fig. 1 c, d). In addition, it was noted that individual latently infected neurons appeared to express varying levels of BHV-I RNA. The above results indicate that the BHV-1 genome is transcriptionally active in latently infected ganglionic neurons. Latency-related viral transcription has also been observed with HSV-1 and HSV-2 in ganglia from humans and experimental animals (Tenser et al., 1981; Galloway et al., 1982; Stroop et al., 1984). Thus, transcription from latent viral genomes appears to be a common feature of the alphaherpesviruses. The apparent restriction of viral RNA to the nucleus of latently infected neurons described here with BHV-1 has also been observed with HSV-1 (Stroop et al., 1984). It seems unlikely that Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 16:04:22 2518 S h o r t communication Fig. 1. Detection of BHV-1 nucleic acids in trigeminal ganglia of acutely and latently infected rabbits by in situ hybridization. (a) BHV-l-specific DNA in a neuron of the trigeminal ganglion of a rabbit 5 days post-infection. Note that autoradiographic grains are localized in the nucleus of the cell and that the cell shows evidence of early necrotic changes. (b) BHV-1 RNA in a ganglionic neuron during acute phase of infection (5 days post-inoculation). Viral RNA is evident in both the nucleus and cytoplasm of the involved cell. (c,d) Detection of BHV-l-specific RNA in ganglionic neurons from latently infected rabbits (120 days post-inoculation). Viral RNA is localized in the nucleus of both hybridizing cells. (e) Section of trigeminal ganglion taken from an uninfected control animal and hybridized with a BHV-Ispecific probe. Typical background levels of non-specific grains can be seen. Autoradiographic exposure for all slides was 3 weeks. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 16:04:22 Short communication 2519 this restriction is absolute because available evidence from HSV-1 latency studies suggests that at least some viral proteins are present and most likely synthesized, at least intermittently, throughout the latent phase of infection (Yamamoto et al., 1977; Green et al., 1981). However, this relative restriction and apparent accumulation of viral R N A in the nucleus of latently infected neurons possibly could represent a post-transcriptional regulatory event involved in the maintenance of this virus-cell interaction. Post-transcriptional regulation of viral gene expression involving preferential retention of specific viral transcripts in the nucleus of cells during the course of productive infection has been described with several of the herpesviruses, including HSV-1 (Stringer et al., 1977; Jacquemont et al., 1980; King et al., 1980; Wathen & Stinski, 1982). B HV-1 was recovered from explant cultures of trigeminal ganglia taken from latently infected rabbits, thus demonstrating the presence of the latent viral genome in this tissue. The failure to detect viral D N A in latently infected ganglia with the in situ hybridization procedure described here most likely represents a lack of the necessary sensitivity to detect a single or a small number of viral genomes in a single cell. Under conditions of active R N A transcription from a single or a small number of viral genomes it would be more likely to detect R N A than the D N A template because of the amplification effect caused by the high R N A to template ratio present within the cell (Moar & Jones, 1975; Copple & McDougall, 1976). Interestingly, BHV-1 D N A was easily detected (Fig. 1 a) during the acute phase of the infection when viral D N A replication was occurring and infectious virus was present. It would appear that amounts of viral D N A comparable to levels seen in productively infected cells are not present in neurons during the latent phase of the infection. This would suggest that significant replication of the viral genome had not occurred prior to establishment of latency in these neurons. The fact that D N A ( - ) temperature-sensitive mutants of HSV- 1 appear to be capable of establishing latent infections at the non-permissive temperature would support this contention (Gerdes et al., 1979; Watson et al., 1980). An alternative, although less likely, explanation for this observation could involve a qualitative difference in the viral D N A present in acutely and latently infected cells (physical structure, degree of association with protein, etc.) that would affect the ability of the latter to hybridize under the assay conditions used here. The significance of viral transcription during latent BHV-1 infection is unknown. It remains to be determined what role, if any, it plays in establishment and/or maintenance of the latent state. We wish to thank Dr M. S. Hofstad for his encouragement and support, Dr W. G. Stroop for helpful advice and Ms J. Wheeler for excellent technical assistance. REFERENCES ACKERMANN,M., PETERHANS,~. 8, WYLER,R. (1982). DNA of bovine herpesvirus type 1 in the trigeminal ganglia of latently infected calves. American Journal of Veterinary Research 43, 36-40. BITSeH,V. (1973). Infectious bovine rhinotracheitis virus infection in bulls, with special reference to preputial infection. Applied Microbiology 26, 337-343. BRAtlIC,M.&EIA~E,A. T. (1978). Detection of viral sequences of low reiteration frequency by in situ hybridization. Proceedings of the National Academy of Sciences, U.S.A. 75, 6125-6129. COPPLE,C. D. &MeDOUOALL,J. K. (1976). Clonal derivatives of a herpes type 2 transformed hamster cell line (333-89): cytogenic analysis, tumorigenicity and virus sequence detection. International Journal of Cancer 17, 501510. DAVIES,D. ~. &CARMICHArL,L. E. (1973). Role of cell mediated immunity in the recoveryof cattle from primary and recurrent infections with infectious bovine rhinotracheitis virus. Infection and Immunity 8, 510-518. GALLOWAY, D. A., FENOGLIA, C. M. & McDOUGALL,J. K. (1982). Limited transcription of the herpes simplex virus genome when latent in human sensory ganglia. Journal of Virology 41, 686-691. GERDES, J. C., MARSDEN,H. S., COOK, M. L. & STEVENS,J. G. (1979). Acute infection of differentiated neuroblastoma cells by latency-negative herpes simplex virus ts mutants. Virology 94, 430-441. GREEN, M. T., COURTNEY,R. J. & DUNKEL,E. C. (1981). Detection of an immediate early herpes simplex virus type 1 polypeptide in trigeminal ganglia from latently infected animals. Infection and Immunity 34, 987-992. HOMAY, E. J. & EASTERDAY, a. C. (1980). Isolation of bovine herpesvirus-1 from trigeminal ganglia o f clinically normal cattle. American Journal of Veterinary Research 41, 1212-1213. HOMAN,E. J. &EASTERDAY,B. C. (1983). Experimental latent and recrudescent bovine herpesvirus-1 infections in calves. American Journal of Veterinary Research 44, 309-313. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 16:04:22 2520 Short communication BucK, R. A., MILLER,P. G. & WOODS, D. G. (1973). Experimental infection of maiden heifers by the vagina with infectious bovine rhinotracheitis/infectious pustular vulvovaginitis virus. Journal of Comparative Pathology 83, 21-27. JACQUEMONT,B., GARCIA,A. & HUPPERT,J. (1980). Nuclear processing of viral high-molecular-weight RNA in cells infected with herpes simplex virus type 1. Journal of Virology 35, 382-389. K~T-IRS,R. F. (1981). Infectious bovine rhinotracheitis. In Viral Diseases of Cattle, pp. 135-156. Ames: Iowa State University Press. KINC, w., THOMAS-POWELL,A. L, R~B-TRAUB, N., rt~WKE, M. a KIEFr, E. (1980). Epstein-Barr virus RNA. V. viral RNA in a restringently infected, growth transformed cell line. Journal of Virology 36, 506-518. KUBIN, G. (1969). Intermittent recovery of IPV virus from a naturally infected bull. Wiener tier~rzttiche MonatsschriJt 56, 336-337. McLEAN, I. W. & NOLSANE, P. K. (1974). Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy. Journal of Histoehemistry and Cytoehemistry 22, 1077-1083. MANIATIS, T., FRITSCH, E. F. & SAMBROOK,J. (1982). Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory. MOAR,M. 1t. & JONES,K. W. (1975). Detection of virus-specific DNA and RNA base-sequences in individual cells transformed or infected by adenovirus type 2. International Journal of Cancer 16, 998 1007. NARITA, M., INUI, S., NAMBA, K. & SHIMIZU, Y. (1978). Neural changes in calves after intraconjunctival inoculation with infectious bovine rhinotracheitis virus in calves treated with dexamethasone. American Journal of Veterinary Research 39, 1399-1403. PELLIeER, A., WIGLER, M., AXEL,R. a SILVERSTEIN,S. (1978). Biochemical transfer of single-copy eukaryotic genes using total cellular DNA as a donor. Cell 14, 725-731. POST, L. E., CONLEY, A. J., MORCARSKI, E. S. & ROIZMAN, B. (1980). Cloning of reiterated and nonreiterated herpes simplex virus 1 sequences as Barn H1 fragments. Proceedings of the National Academy of Sciences, U.S.A. 77, 4201-4205. RIGBY, P. W. J., DIECKMANN, M., RHODES, C. & BERG, P. (1977). Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. Journal of Molecular Biology 113, 237-251. ROCK,D. L. & FRASER,N. W. (1983). Detection of HSV-I genome in central nervous system of latently infected mice. Nature, London 302, 523-525. ROCK, D. L. & REED, D. E. (1982). Persistent infection with bovine herpesvirus type 1 : rabbit model. Infection and Immunity 35, 371-373. SHEFFY, B. E. & DAVIES, D. H. (1972). Reactivation of a bovine herpesvirus after corticosteroid treatment. Proceedings of the Society for Experimental Biology and Medicine 140, 974-976. SNOWDON, W. A. (1965). The IBR-IPV: reaction to infection and intermittent recovery of virus from experimentally infected cattle. Australian Veterinary Journal 41, 135-142. STRINGER, J. R., HOLLAND, L. E., SWANSTROM, R. I., PIVO, K. & WAGNER, E. K. (1977). Quantitation of herpes simplex virus type 1 RNA in infected HeLa cells. Journal of Virology 21,889-901. STROOP, W. G., ROCK, D. L. & FRASER, N. W. (1984). Localization of herpes simplex virus in the trigeminal and olfactory systems of the mouse central nervous system during acute and latent infections by in situ hybridization. Laboratory' Investigation 51, 27-38. TALENS, L. T. & ZEE, Y. C. (1976). Purification and buoyant density of infectious bovine rhinotracheitis virus. Proceedings of the Society for Experimental Biology and Medicine 151, 132-135. TENSER, R. B., DAWSON, M., RESSEL, S. J. & DUNSTAN, M. E. (1981). Detection of herpes simplex virus m-RNA in latently infected trigeminal ganglion neurons by in situ hybridization. Annals of Neurology I1, 285-291. WALBOOMERS,J. M. M. & SCHEGGET,J. T. (1976). A new method for isolation of herpes simplex virus type 2 DNA. Virology 74, 256-258. WATHEN, M. W. & STINSKI, M. F. (1982). Temporal patterns of human cytomegalovirus transcription: mapping the viral RNAs synthesized at immediate early, early, and late times after infection. Journal of Virology 41,462477. WATSON, K., STEVENS, J. G., COOK, M. L. & SUBAK-SHARPE, J. H. (1980). Latency competence of thirteen HSV-1 temperature-sensitive mutants. Journal of General Virology 49, 149-159. YAMAMOTO,H., WALZ, M. A. & NOTKINS,A. L. (1977). Viral-specific thymidine kinase in sensory ganglia of mice infected with herpes simplex virus. Virology 76, 866-869. (Received 7 M a y 1986) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 16:04:22