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Pseudorabies virus infections in explants of porcine nasal mucosa
Research in Veterinary Science 1991, 50, 45-53
Pseudorabies virus infections in explants of porcine nasal mucosa J. M. A. POL, Central Veterinary Institute, Virology Department, PO Box 365, 8200 A J Lelystad, The Netherlands, W. G. V. QUINT, Diagnostic Centre SSDZ, PO Box 5010 2500 GA Delft, The Netherlands, G. L. KOK, J. M. BROEKHUYSEN-DAVIES, Central Veterinary Institute,
Virology Department, PO Box 305, 8200 A J Lelystad, The Netherlands
The spread of infection and the morphogenesis of three psendorabies virus strains were studied in explants of porcine nasal mucosa. Virulent NIA-3 virus was compared with a deletion mutant 2.4N3A, and with a non-virulent Bartha virus. All three virus strains infected nasal epithelial cells. N|A-3 virus particles were enveloped mainly at the inner nuclear membrane; the virus rapidly invaded the stroma, causing widespread necrosis. In contrast, 2 . 4 N 3 A virus particles were enveloped mainly at the endoplasmic reticulum and the infection spread more slowly. Bartha virus particles were enveloped mainly at the endoplasmic reticulum; the infection spread slowly and remained restricted to the epithelial cells. In situ DNA hyhridisation showed an accumulation of Bartha virus DNA in the nucleus 24 hours after inoculation. In nasal mucosa viral virulence seemed directly related to the speed of replication and release of virus from infected cells.
vitro method of infecting porcine nasal mucosa was developed. Three PRY strains of different virulence were studied: the virulent Northern Ireland strain 3 (NIA-3) virus, the deletion mutant virus 2-4N3A, and the vaccine virus Bartha. The 2.4N3A strain is derived from NIA-3virus (Quint et al 1987). It is intermediate in virulence and lacks two proteins. The Bartha strain lacks four proteins and is defective in a gene involved in nucleocapsid assembly. At 24 and 48 hours after infection, the development of the viral infection in nasal epithelium and in stromal cells was examined immunohistochemically, morphometrically and ultrastructurally.
Materials and methods
Virus strains Virulent PRY NIA-3was kindly provided by Dr J. B. McFerran, Belfast, Northern Ireland. Strain 2-4N3A was derived from NIA-3 virus. The 2"4N3A strain has a deletion of 2.4 kilobase pairs (kb) in the Us region of the genome and was constructed as described elsewhere (Berns et al 1985, Quint et al 1987). Because of the deletion, the virus is unable to express glycoprotein I (gI), present in the parent strain. The 2.4N3A strain also lacks an 11 kilodalton (K) protein (Quint et a11987). The 11 K protein is probably a phosphoprotein of the integument (Frame et al 1986). Non-virulent Bartha virus was purchased as a commercial vaccine (Duphar). The Bartha virus genome has a 3.8 kb deletion in the same part of the U s region as 2.4N3A virus, it does not express gI and gp 63 (Mettenleiter et al 1985, Petrovskis et al 1986), and lacks the 11 K and 28 K proteins. In addition, glycoprotein gill is not efficiently glycosylated or matured (Robbins et al 1989); Bartha virus is also defective in a gene involved in nucleocapsid assembly (Lomniczi et al 1987). The viruses were passaged several times in
PSEUDORABIES virus (PRV) (syn herpesvirus suis, Aujeszky's disease virus) causes in pigs an infection similar to herpes simplex virus infection in humans (Gustafson t986). Many physical and chemical properties of PRY have been determined, and the biological activity of the virus has been studied in experimentally induced infections in many species, such as pigs, cattle, rabbits, chickens, rats and mice (Becker 1968, Baskerville 1972, McCracken et al 1973, Field and Hill 1974, Maes et a11979, Hagemoser et a11984). Several studies have demonstrated that the virulence of some PRY strains in rabbits and mice did not correspond with their virulence in pigs, the natural host ( P l a t t e t al 1980, Van Oirschot and Gielkens 1984). Because PRV infections in pigs cause great economic damage, much research has been done in pigs. Use of the intranasal infection route in pigs is preferred because it closely resembles the natural infection (Pol et al 1989). To compare the morphogenesis of PRV strains and to investigate whether differences in viral morphogenesis in nasal mucosa correspond with differences in virulence in vivo, an in 45
46
J. M. A. Pol, W. G. V. Quint, G. L. Kok, J. M. Broekhuysen-Davies
secondary pig kidney cells, as described elsewhere (De Leeuw et al 1982). All virus preparations were diluted to a final concentration of 107 plaque-forming units (PFU) ml -~ in Eagle's minimum essential medium (EMEM) with 10 per cent fetal calf serum. After inoculation, the remaining virus suspensions were titrated on secondary pig kidney cells to verify the virus titres.
P~ Dutch Landrace pigs from the specific pathogen free herd of the Central Veterinary Institute, Lelystad, were used. They were free of antibodies against PRV and were three to five weeks old.
and placed in pure methanol with 0.03 per cent hydrogen peroxide for 30 minutes to remove endogenous peroxidase activity. Sections were incubated with rabbit anti-PRY serum for 60 minutes. After washing in PBS, the slides were covered with sheep anti-rabbit serum conjugated to peroxidase (Institut Pasteur) in PBS for 60 minutes. The sections were washed again and stained with 0.5 mg 3,3 '-diaminobenzidine tetrahydrochloride (DAB; Sigma Chemical) m1-1 Tris-HC1 buffer (0.05 M, pH 7.6) containing 0.01 per cent hydrogen peroxide to detect peroxidase activity. Control sections were treated in the same way but the incubation step with rabbit anti-PRy serum was omitted.
DATA in situ hybridisation Nasal mucosa explants Pigs were killed by intravenous inoculation of barbiturates and were exsanguinated. The nose was sawed from the skull at the level of the eyes and split by cutting through the middle nasal bone and through the underlying nasal septum. The mucosal surface was washed with phosphate buffered saline (PBS) and rectangular (5 × 10 mm) pieces of nasal mucosa were cut and stripped from the surfaces of the turbinates and the nasal septum. Five pieces of mucosa with a total surface area of 2" 5 cm 2 were placed in sterile dry 35 mm plastic macroplates (Costar Europe). Since epithelial cells of nasal mucosa have an average diameter of 5 #m, 2.5 cm 2 of nasal mucosa contain about 107 epithelial cells.
Viral DNA was detected by hybridising tissue sections with biotinylated probes of PRY DNA, according to the method of Walboomers et al (1988). In short, sections were picked up on poly-L-lysinetreated glass slides, deparaffinised, treated with protease K, and fixed in paraformaldehyde. The sections were incubated at 100°C with a hybridisation mixture containing biotinylated denatured DNA probe, herring sperm DNA, formamide and dextran sulphate in a sodium citrate buffer supplemented with bovine serum albumin, Ficoll 400 and polyvinyl pyrrolidone. Hybridisation of the DNA probe to viral DNA in the sections was detected by using a peroxidase complex coupled to streptavidin (Detek 1-hrp; Enzo Biochem) and stained with DAB.
Morphometric examination Experimental procedure The nasal mucosa explants were infected by placing 1 ml of virus suspension (107 PFU m1-1) on top of 2.5 cm 2 of explant (multiplicity of infection: approximately 1 , v u cell-1) and incubating the explants at 37°C for one hour. Next, the explants were washed twice with PBS and transferred to a sterile 35 mm petri dish with 2 ml fresh medium (EMEMwith 10 per cent fetal calf serum and antibiotics). After 24 and 48 hours the explants were fixed for examination. Control mucosa explants were treated as described before, except that incubation with virus was replaced by incubation with sterile medium. The experiments were repeated six times under identical conditions.
Infected areas of nasal mucosa in immunohistochemically stained sections were measured with a cross-hair cursor on a magnetic tablet (MOPVideoplan; Kontron Image Analysis Systems) and a Leitz Orthoplan section was measured at an optical magnification of 25 times (one field = 7 mm). Infected areas were measured at a magnification of 250 times, the result was divided by 10 to correct for magnification and expressed as a percentage of the total length
TABLE 1 : Percentage of infected nasal mucosa 24 and 48 hours after inoculation of three pseudorabies virus (PRV) strains PRV strain
Immunohistochemistry Mucosa explants were fixed in 10 per cent neutral buffered formalin, dehydrated, and embedded in paraffin wax. Sections were cut 5 #m thick and picked up on glass slides. The sections were deparaffinised
NIA-3 2.4N3A Bartha
24 h
48 h
87"(7-1) 55"(7.0) 3 7 " (6"0)
86 (6"9) 84 (6.5) 70 ( 1 4 " 9 )
* Significantly different (P < 0 • 01 ), as calculated by Student's t test. Values are given as the mean score ± SD in a 100 mm length of mucosa
Morphogenesis and virulence of pseudorabies viruses
%
°
FIG 1 : Photomicrograph ( x 150) of porcine nasal mucosa explants infected with pseudorabies virus and stained by the immunoperoxidase method. NIA-3 virus infection: (A) 24 hours after inoculation, epithelial cells and few fibroblasts in the lamina propria contain viral antigens; (B) 48 hours, the number of infected fibroblasts has markedly increased. 2" 4 N 3 A virus infection; (C) 24 hours, only epithelial cells contain viral antigens; (D) 48 hours, a few fibroblasts have become infected. Bartha virus infection; (E) 24 hours, viral antigens are strictly confined to epithelial cells. Inset ( × 1000): viral DNA is detected by in situ hybridisation in the nucleus of infected cells; (F) 4 8 hours, viral antigens were still confined to epithelial cells. Inset ( × 1000): viral DNA is detected by in situ hybridisation in both. nucleus and cytoplasm
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48
J. M. A. Pol, W. G. V. Quint, G. L. Kok, J. M. Broekhuysen-Davies
FIG 2: Electron micrograph ( x 8000) of nasal epithelium cell 48 hours after inoculation with, NIA-3 virus. Viral nucleocapsids are detected in the nucleus (N), enveloped virus particles in the cytoplasm (arrows) and in the extracellular space. New nucleocapsids are budding through membranes of the smooth endoplasmic reticulum (arrowheads)
of the mucosa in that section. Each value represents the mean ( + so) of six experiments with a total of 600 mm of mucosal length.
Transmission electron microscopy The nasal mucosa was fixed in a mixture of 0.75 per cent glutaraldehyde and 0.8 per cent osmium tetroxide in a veronal acetate buffer, washed extensively and then stained in 1-0 per cent uranyl acetate (Hirsch and Fedorko 1968). Tissues were washed again and then dehydrated in alcohol and embedded in Epon resin. Ultra-thin sections were stained with 0"2 per cent lead citrate in 0.1 M sodium hydroxide and uranyl acetate (Venable and Coggeshall 1965), and examined in a Philips EM 300 microscope.
Spread o f PR V infections in nasal mucosa NIA-3 virus. At 24 hours after inoculation, viral antigens (Fig 1A) and DNA were detected in both nucleus and cytoplasm of epithelial and stromal cells. The staining patterns after DNA hybridisation and immunohistochemistry were identical. After 48 hours, large areas of epithelium had become necrotic and the infection had reached deep into the stroma, killing many cells (Fig 1B). 2"4N3A virus. At 24 hours after inoculation, viral antigens (Fig 1C) and DNA were detected in both nucleus and cytoplasm of epithelial and stromal cells. The staining patterns after DNA hybridisation and immunohistochemistry were identical. After 48 hours, 2.4N3A virus infection had invaded the stroma (Fig 1D), but had killed less cells than NIA-3 virus.
Results
PRV infections in nasal epithelium At 24 hours after inoculation, viral antigen was detected in most of the epithelial surface (87 per cent) after NIA-3 infection; in less than half after Bartha virus infection (37 per cent) and in an intermediate amount after 2.4N3A virus infection (55 per cent) (Table 1). At 48 hours after inoculation, the two less virulent strains had also infected most of the epithelial cells (84 to 70 per cent).
Bartha virus. At 24 hours after inoculation, viral antigens (Fig 1E) were mainly detected in the cytoplasm of epithelial ceils, whereas viral DNA was predominantly found in the nucleus of infected epithelial cells (inset in Fig 1E). Stromal cells under the epithelium were not infected. After 48 hours the infection was still strictly confined to epithelial cells (Fig 1F). Both nucleus and cytoplasm of infected cells contained viral antigens as well as viral DNA (inset in Fig IF).
Morphogenesis and virulence of pseudorabies viruses
49
FIG 3: Electron micrograph ( x 40,000) of nasal epithelial cell 48 hours after inoculation with NIA-3 virus. Nucleocapsids (arrows) are adjacent to the inner nuclear membrane. Enveloped virus particles (arrowheads) are detected in the perinuclear space between the inner and the outer nuclear membrane
The underside of the explants consisted of fibrous tissue which was readily infected by all three virus strains. The N1A-3 virus and 2-4N3A virus spread through several layers of fibroblasts, but Bartha virus infected only the outer layer.
Ultrastructural morphogenesis NIA-3 virus. At 24 and 48 hours after inoculation,
enveloped virus particles were detected in and around infected epithelial cells and fibroblasts. Few nucleocapsids were seen budding through membranes of the smooth endoplasmic reticulum (Fig 2). Most nucleocapsids were enveloped by budding through the inner nuclear membrane (Fig 3).
2.4N3A virus. At 24 and 48 hours after inoculation, naked nucleocapsids were detected in the
FIG 4: Electron micrograph ( x 8000) of nasal epithelial cell 48 hours after inoculation with 2.4N3A virus. Nucleocapsids (arrowheads) are detected in the nucleus (N) and in the cytoplasm. Nucleocapsids are enveloped by budding through membranes of the smooth endoplasmic reticulum (arrow). Enveloped virus particles are detected in the extracellular space
50
J. M. A. Pol, W. G. V. Quint, G. L. Kok, J. M. Broekhuysen-Davies
FIG 5: Electron micrograph (x 32,000) of nasal epithelium cell 48 hours after inoculation with 2 . 4 N 3 A virus. Nucleocapsids in the cytoplasm (C) are enveloped by budding through thickened membranes of the smooth endoplasmic reticulum (arrowheads). Enveloped virus particles are detected in cytoplasmic vacuoles and in the extracellular space
nucleus and cytoplasm of infected cells, and enveloped virus particles were detected outside the cells (Fig 4). Some nucleocapsids were enveloped by budding through the internal nuclear membrane as in the NIA-3 virus infected cells. More frequently, however, nucleocapsids accumulated in the cytoplasm and were enveloped by budding through membranes of the endoplasmic reticulum, which became thickened at the site of contact with the
nucleocapsids (Fig 5). The morphogenesis of 2' 4N3A virus in epithelial cells and stromal fibroblasts was identical. Bartha virus. At 24 and 48 hours after inoculation of Bartha virus, few virus particles were detected in infected epithelial cells and extracellular spaces (Fig 6). Few nucleocapsids were enveloped by budding through the inner nuclear membrane,
FIG 6: Electron micrograph ( x 8000) of nasal epithelium cell 48 hours after inoculation with 8artha virus. Few nucleocapsids are detected in the nucleus and cytoplasm (arrows). Few enveloped virus particles are detected in the extracellular space (arrowheads)
Morphogenesis and virulence of pseudorabies viruses
51
FIG 7: Electron micrograph ( x 32,000) of nasal epithelium cell 48 hours after inoculation with Bartha virus. Nucleocapsids (arrows) are detected in the nucleus (N) and in the cytoplasm (C). Membranes of the smooth endoplasmic reticulum are thickened at the site of viral envelopment. Few enveloped virus particles are detected in the cytopiasm (arrowhead)
smooth nucleocapsids were seen in the cytoplasm budding through thickened membranes of the smooth endoplasmic reticulum (Fig 7). Bartha virus did not penetrate the basal lamina under the epithelial cells, but the virus replicated in stromal fibroblasts on the underside of the explants with a similar morphogenesis as in epithelial cells. Discussion
Infection of nasal mucosa explants with three PRY strains of different virulence showed that nasal epithelial cells are target cells for the virus. N1A-3 virus quickly spread to large areas of the epithelium and invaded the subepitheliai stroma. In contrast, Bartha virus infection spread to fewer epithelial cells and did not penetrate through the basal lamina to the stromal fibroblasts. The deletion mutant virus 2.4N3A spread to epithelial cells as NIA-3 virus did but showed less invasion to stromal fibroblasts. These results agreed with those of in vivo studies of intranasal infections of pigs with the same three virus strains (Pol et al 1989). The stroma of oronasal epithelium contains many blood vessels and neural axons of the trigeminal, olfactory and vegetative nerves through which the virus can invade the central nervous system. Therefore, the ability to penetrate the basal lamina and infect the underlying fibroblasts is an important criterion for virulence. Staining for viral DNA and antigens revealed that the three strains replicate differently. In cells infected by NXA-3 virus or 2-4N3A virus the two methods
produced identical staining patterns, indicating that viral DNA was present in the nucleus and was transported from there to the cytoplasm. Moreover, NIA-3 virus spread rapidly to neighbouring cells, and its rapid release was confirmed by electron microscopy. In contrast, 2.4N3A virus spread slowly, which suggests that it was released more slowly; electron microscopy showed that many virus particles accumulated in the cytoplasm where they were enveloped before being released. At 24 hours after inoculation, the nuclei of Bartha virus infected cells contained both viral DNA and antigens, whereas the cytoplasm contained viral antigens but little DNA. Also, cells infected with Bartha virus contained fewer nucleocapsids than cells infected with the two other viruses. Lomniczi et al (1987) showed that in Bartha virus a U l gene involved in nucleocapsid assembly is defective. Bartha virus nucleocapsids, like those of 2.4N3A virus, were enveloped by budding through smooth endoplasmic reticulum membranes. 2.4N3A virus and Bartha virus both lack gI and an 11 K phosphoprotein of the nucleocapsid (Frame et al 1986). The exact role of these viral proteins in the morphogenesis is still unknown, but results suggest that the lack of a glycoprotein may account for the different site of envelopment compared to NIA-3 virus. Glycoproteins are produced on the rough endoplasmic reticulum and are glycosylated and processed in the smooth endoplasmic reticulum. Herpesvirus glycoproteins are incorporated in the inner nuclear
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J. M. A. Pol, W. G. V. Quint, G. L. Kok, J. M. Broekhuysen-Davies
membrane at the site of viral budding (Johnson and Spear 1982). It is proposed that, because gI is missing, both Bartha virus and 2.4N3A virus cannot bud through the inner nuclear membrane; they leave the nucleus unenveloped to bud through membranes of the smooth endoplasmic reticulum. Bartha virus also lacks a 28 K protein and gp 63 whereas glII is inefficiently incorporated in the virus (Schreurs et al 1988) which may further lower its virulence (Robbins et al i989). Because mucosal explants have no contact with the blood circulation, the only endogenous agents that can influence infection in vitro are lymphokines like interferons which can be produced locally by cells of the host. Conceivably, when virus replicates and spreads slowly, as Bartha virus does, epithelial cells have time to produce interferon which can protect the surrounding cells and underlying fibroblasts of the stroma. That a similar mechanism may be involved is also suggested by the fact that Bartha virus did not infect the stromal fibroblasts under the basal lamina although it infected one layer of fibroblasts on the underside of the explants. Viral budding through endoplasmic membranes was seldom observed in cells infected with NIA-3 but frequently in cells infected with Bartha virus or 2" 4N3A virus, indicating that the budding process is retarded. Interferons may play a role in the disruption of viral morphogenesis by changing membrane fluidity (Chatterjee et al 1982) and reducing the synthesis of certain herpesvirus glycoproteins (Chatterjee et al 1985). Nasal mucosa explants offer a natural combination of epithelial cells and stromal fibrobiasts that yields more information about the virulence of PRV than each type of cell on its own. Because conditions such as multiplicity of infection, contact between virus and cells, and temperature and pH can readily be controlled, the method is reliably reproducible. This method closely mimics the in vivo infection; the virulence of PRV strains and mutant viruses in mucosa can be studied and virus strains can be screened to select those that can be used in vaccines. This in vitro model is not suited for the study of neural virulence. Although the axons lying in the nasal mucosa explants can be infected, the possible consequences of such infections cannot be studied. Neurovirulence of PRV strains should preferably be studied in in vitro models that contain both neurons and glial cells to mimic the complex interactions of cells during PRY infection. Acknowledgements We thank F. Wagenaar and L. Juffermans for excellent technical assistance, V. Thatcher for editorial assistance and F. Propsma and P. de Haas for the photographs.
References BASKERVILLE, A. (1972) Aujeszky's disease encephalitis in pigs produced by diferent modes of infection. Research in Veterinary Science 13, 394-396 BECKER, CH. (1968) Die Mnltiplikation des Aujeszkyschen Virus in den Spinalganglien des Kaninchens. Archivfiir Experimentelle Veterinar Medizin 22, 363-381 BERNS, A., VAN DER OUWELAND, A., QUINT, W., VAN OIRSCHOT, J. & GIELKENS, A. (1985) Presence of markers for virulence in the unique shor t region or repeat region or both of pseudorabies hybrid viruses. Journal of Virology 58, 89-93 CHATTERJEE, S., CHEUNG, H. & HUNTER, E. (1982) Interferon inhibits Sendai virus induced cell fusion: an effect on cell membrane fluidity. Proceedings of the National Academy of Sciences, USA 79, 835-840 CHATTERJEE, S., HUNTER, E. & WHITLEY, R. (1985) Effect of cloned herpes simplex virus. Journal of Virology 56, 419-425 DE LEEUW, P. W., WlJSMULLER, J. M., ZANTINGA, J. W. & TIELEN, M. J. M. (1982) Intranasal vaccination of pigs against Aujeszky's disease. 1. Comparison of intranasal and parenteral vaccination with an attenuated vaccine in 12-week-old pigs from immunised dams. Veterinary Quarterly 4, 49-55 FIELD, H. J. & HILL, T. J. (1974) The pathogenesis of pseudorabies in mice following peripheral inoculation. Journal of General Virology 23, 145-157 FRAME, M. C., McGEOCH, D. J. & RIXON, F. J. (1986) The 10K virion phosphoprotein encoded by gene US9 from herpes simplex virus type 1. Virology 150, 320-322 GUSTAFSON, D. P. (I 986) Pseudorabies. In Diseases of Swine. Eds A. D. Leman, B. Straw, R. D. Glock, W. L. Mengeling, R. H. C. Penny and E. Scholl. Ames, Iowa State University Press. pp 274-288 HAGEMOSER, W. A., KLUGE, J. P. & HILL, H. T. (1984) Studies on the pathogenesis of pseudorabies in domestic cats following oral inoculation. Canadian Journal of Comparative Medicine 44, 192-202 HIRSCH, J. G. & FEDORKO, M. E. (1968) Ultrastructure of human leucocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and postfixation in uranyl acetate. Journal of Cellular Biology 38, 615-618 JOHNSON, D. C. & SPEAR, P. G. (1982) Monsensin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface, and the egress of virions from infected cells. Journal of Virology 43, 1102-1112 LOMNICZI, B., WATANABE, S., BEN-PORAT, T. & KAPLAN, A. S. (1987) Genome location and identification of functions defective in the Bartha vaccine strain of pseudorabies virus. Journal of Virology 61,796-801 MAES, R. K., KAN1TZ, C. L. & GUSTAFSON, D. P. (1979) Pseudorabies virus infections in wild and laboratory rats. American Journal of Veterinary Research 40, 393-396. McCRACKEN, R. M., McFERRAN, J. B. & DOW, C. (1973) The neural spread of pseudorabies virus in calves. Journal of General Virology 20, 17-28 METTENLEITER, T. C., LUKACS, N. & RZIHA, H. J. (1985) Pseudorabies virus avirulent strains fail to express a major glycoprotein. Journal of Virology 56, 307-311 PETROVSKIS, E., TIMMINS, J. G., GIERMAN, T. M. & POST, L. E. (1986) Deletions in vaccine strains and their effect on synthesis of glycoprotein gp63. Journal of Virology 60, 1166-1169 PLATT, K. B., MARE, C. J. & HINZ, P. N. (1980) Differentiation of vaccine strains and field isolates of pseudorabies (Aujeszky's disease) virus: trypsin sensitivity and mouse virulence markers. Archives of Virology 63, 107-114 POL, J. M., GIELKENS, A. J. & VAN OIRSCHOT, J. (1989) Comparative pathogenesis of three strains of pseudorabies virus in pigs. Microbial Pathogenesis 7, 361-371 QUINT, W., GIELKENS, A., VAN OIRSCHOT, J., BERNS, A. & CUYPERS, H. T. (1987) Construction and characterisation of
Morphogenesis and virulence of pseudorabies viruses deletion mutarrts of pseudorabies virus: A new generation of 'live' vaccines. Journal of General Virology 68, 523-524 ROBBINS, A. K., RYAN, J. P., WHEALY, M. E. & ENQUIST, L. W. (1989) The gene encoding the glII envelope protein of pseudorabies virus vaccine strain Bartha contains a mutation affecting protein localisation. Journal of Virology 63,250-258 SCHREURS, C., METTENLEITER, T. C., ZUCKERMANN, F., SUGG, N. & BEN-PORAT, T. (1988) Glycoprotein gill of pseudorabies virus is multifunctional. Journal of Virology 62,2251-2257 VAN OIRSCHOT, J. T. & GIELKENS, A. L. J. (1984) Some characteristics of four attenuated vaccine virus strains and a virulent strain of Aujeszky's disease virus. Veterinary Quarterly 6, 225-229
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VENABLE, J. H. & COGGESHALL, R. (1965) A simple lead citrate stain for use in electron microscopy. Journal of Cellular Biology 25,407-408 WALBOOMERS, J. M. M., MELCHERS, W. J. G., MULLINK, H., MEIJER, C. J. L. M., STRUYK, A., QUINT, W. G. J., VAN DER NOORDAA, J. & TER SCHEGGET, J. (1988) Sensitivity of in situ detection with biotinylated probes of human papilloma virus type 16 DNA in frozen tissue sections of squarnous cell carcinomas of the cervix. American Journal of Pathology 131,587-594
Received January 9, 1990 Accepted July 20, 1990
Pseudorabies virus infections in explants of porcine nasal mucosa J. M. A. POL, Central Veterinary Institute, Virology Department, PO Box 365, 8200 A J Lelystad, The Netherlands, W. G. V. QUINT, Diagnostic Centre SSDZ, PO Box 5010 2500 GA Delft, The Netherlands, G. L. KOK, J. M. BROEKHUYSEN-DAVIES, Central Veterinary Institute,
Virology Department, PO Box 305, 8200 A J Lelystad, The Netherlands
The spread of infection and the morphogenesis of three psendorabies virus strains were studied in explants of porcine nasal mucosa. Virulent NIA-3 virus was compared with a deletion mutant 2.4N3A, and with a non-virulent Bartha virus. All three virus strains infected nasal epithelial cells. N|A-3 virus particles were enveloped mainly at the inner nuclear membrane; the virus rapidly invaded the stroma, causing widespread necrosis. In contrast, 2 . 4 N 3 A virus particles were enveloped mainly at the endoplasmic reticulum and the infection spread more slowly. Bartha virus particles were enveloped mainly at the endoplasmic reticulum; the infection spread slowly and remained restricted to the epithelial cells. In situ DNA hyhridisation showed an accumulation of Bartha virus DNA in the nucleus 24 hours after inoculation. In nasal mucosa viral virulence seemed directly related to the speed of replication and release of virus from infected cells.
vitro method of infecting porcine nasal mucosa was developed. Three PRY strains of different virulence were studied: the virulent Northern Ireland strain 3 (NIA-3) virus, the deletion mutant virus 2-4N3A, and the vaccine virus Bartha. The 2.4N3A strain is derived from NIA-3virus (Quint et al 1987). It is intermediate in virulence and lacks two proteins. The Bartha strain lacks four proteins and is defective in a gene involved in nucleocapsid assembly. At 24 and 48 hours after infection, the development of the viral infection in nasal epithelium and in stromal cells was examined immunohistochemically, morphometrically and ultrastructurally.
Materials and methods
Virus strains Virulent PRY NIA-3was kindly provided by Dr J. B. McFerran, Belfast, Northern Ireland. Strain 2-4N3A was derived from NIA-3 virus. The 2"4N3A strain has a deletion of 2.4 kilobase pairs (kb) in the Us region of the genome and was constructed as described elsewhere (Berns et al 1985, Quint et al 1987). Because of the deletion, the virus is unable to express glycoprotein I (gI), present in the parent strain. The 2.4N3A strain also lacks an 11 kilodalton (K) protein (Quint et a11987). The 11 K protein is probably a phosphoprotein of the integument (Frame et al 1986). Non-virulent Bartha virus was purchased as a commercial vaccine (Duphar). The Bartha virus genome has a 3.8 kb deletion in the same part of the U s region as 2.4N3A virus, it does not express gI and gp 63 (Mettenleiter et al 1985, Petrovskis et al 1986), and lacks the 11 K and 28 K proteins. In addition, glycoprotein gill is not efficiently glycosylated or matured (Robbins et al 1989); Bartha virus is also defective in a gene involved in nucleocapsid assembly (Lomniczi et al 1987). The viruses were passaged several times in
PSEUDORABIES virus (PRV) (syn herpesvirus suis, Aujeszky's disease virus) causes in pigs an infection similar to herpes simplex virus infection in humans (Gustafson t986). Many physical and chemical properties of PRY have been determined, and the biological activity of the virus has been studied in experimentally induced infections in many species, such as pigs, cattle, rabbits, chickens, rats and mice (Becker 1968, Baskerville 1972, McCracken et al 1973, Field and Hill 1974, Maes et a11979, Hagemoser et a11984). Several studies have demonstrated that the virulence of some PRY strains in rabbits and mice did not correspond with their virulence in pigs, the natural host ( P l a t t e t al 1980, Van Oirschot and Gielkens 1984). Because PRV infections in pigs cause great economic damage, much research has been done in pigs. Use of the intranasal infection route in pigs is preferred because it closely resembles the natural infection (Pol et al 1989). To compare the morphogenesis of PRV strains and to investigate whether differences in viral morphogenesis in nasal mucosa correspond with differences in virulence in vivo, an in 45
46
J. M. A. Pol, W. G. V. Quint, G. L. Kok, J. M. Broekhuysen-Davies
secondary pig kidney cells, as described elsewhere (De Leeuw et al 1982). All virus preparations were diluted to a final concentration of 107 plaque-forming units (PFU) ml -~ in Eagle's minimum essential medium (EMEM) with 10 per cent fetal calf serum. After inoculation, the remaining virus suspensions were titrated on secondary pig kidney cells to verify the virus titres.
P~ Dutch Landrace pigs from the specific pathogen free herd of the Central Veterinary Institute, Lelystad, were used. They were free of antibodies against PRV and were three to five weeks old.
and placed in pure methanol with 0.03 per cent hydrogen peroxide for 30 minutes to remove endogenous peroxidase activity. Sections were incubated with rabbit anti-PRY serum for 60 minutes. After washing in PBS, the slides were covered with sheep anti-rabbit serum conjugated to peroxidase (Institut Pasteur) in PBS for 60 minutes. The sections were washed again and stained with 0.5 mg 3,3 '-diaminobenzidine tetrahydrochloride (DAB; Sigma Chemical) m1-1 Tris-HC1 buffer (0.05 M, pH 7.6) containing 0.01 per cent hydrogen peroxide to detect peroxidase activity. Control sections were treated in the same way but the incubation step with rabbit anti-PRy serum was omitted.
DATA in situ hybridisation Nasal mucosa explants Pigs were killed by intravenous inoculation of barbiturates and were exsanguinated. The nose was sawed from the skull at the level of the eyes and split by cutting through the middle nasal bone and through the underlying nasal septum. The mucosal surface was washed with phosphate buffered saline (PBS) and rectangular (5 × 10 mm) pieces of nasal mucosa were cut and stripped from the surfaces of the turbinates and the nasal septum. Five pieces of mucosa with a total surface area of 2" 5 cm 2 were placed in sterile dry 35 mm plastic macroplates (Costar Europe). Since epithelial cells of nasal mucosa have an average diameter of 5 #m, 2.5 cm 2 of nasal mucosa contain about 107 epithelial cells.
Viral DNA was detected by hybridising tissue sections with biotinylated probes of PRY DNA, according to the method of Walboomers et al (1988). In short, sections were picked up on poly-L-lysinetreated glass slides, deparaffinised, treated with protease K, and fixed in paraformaldehyde. The sections were incubated at 100°C with a hybridisation mixture containing biotinylated denatured DNA probe, herring sperm DNA, formamide and dextran sulphate in a sodium citrate buffer supplemented with bovine serum albumin, Ficoll 400 and polyvinyl pyrrolidone. Hybridisation of the DNA probe to viral DNA in the sections was detected by using a peroxidase complex coupled to streptavidin (Detek 1-hrp; Enzo Biochem) and stained with DAB.
Morphometric examination Experimental procedure The nasal mucosa explants were infected by placing 1 ml of virus suspension (107 PFU m1-1) on top of 2.5 cm 2 of explant (multiplicity of infection: approximately 1 , v u cell-1) and incubating the explants at 37°C for one hour. Next, the explants were washed twice with PBS and transferred to a sterile 35 mm petri dish with 2 ml fresh medium (EMEMwith 10 per cent fetal calf serum and antibiotics). After 24 and 48 hours the explants were fixed for examination. Control mucosa explants were treated as described before, except that incubation with virus was replaced by incubation with sterile medium. The experiments were repeated six times under identical conditions.
Infected areas of nasal mucosa in immunohistochemically stained sections were measured with a cross-hair cursor on a magnetic tablet (MOPVideoplan; Kontron Image Analysis Systems) and a Leitz Orthoplan section was measured at an optical magnification of 25 times (one field = 7 mm). Infected areas were measured at a magnification of 250 times, the result was divided by 10 to correct for magnification and expressed as a percentage of the total length
TABLE 1 : Percentage of infected nasal mucosa 24 and 48 hours after inoculation of three pseudorabies virus (PRV) strains PRV strain
Immunohistochemistry Mucosa explants were fixed in 10 per cent neutral buffered formalin, dehydrated, and embedded in paraffin wax. Sections were cut 5 #m thick and picked up on glass slides. The sections were deparaffinised
NIA-3 2.4N3A Bartha
24 h
48 h
87"(7-1) 55"(7.0) 3 7 " (6"0)
86 (6"9) 84 (6.5) 70 ( 1 4 " 9 )
* Significantly different (P < 0 • 01 ), as calculated by Student's t test. Values are given as the mean score ± SD in a 100 mm length of mucosa
Morphogenesis and virulence of pseudorabies viruses
%
°
FIG 1 : Photomicrograph ( x 150) of porcine nasal mucosa explants infected with pseudorabies virus and stained by the immunoperoxidase method. NIA-3 virus infection: (A) 24 hours after inoculation, epithelial cells and few fibroblasts in the lamina propria contain viral antigens; (B) 48 hours, the number of infected fibroblasts has markedly increased. 2" 4 N 3 A virus infection; (C) 24 hours, only epithelial cells contain viral antigens; (D) 48 hours, a few fibroblasts have become infected. Bartha virus infection; (E) 24 hours, viral antigens are strictly confined to epithelial cells. Inset ( × 1000): viral DNA is detected by in situ hybridisation in the nucleus of infected cells; (F) 4 8 hours, viral antigens were still confined to epithelial cells. Inset ( × 1000): viral DNA is detected by in situ hybridisation in both. nucleus and cytoplasm
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J. M. A. Pol, W. G. V. Quint, G. L. Kok, J. M. Broekhuysen-Davies
FIG 2: Electron micrograph ( x 8000) of nasal epithelium cell 48 hours after inoculation with, NIA-3 virus. Viral nucleocapsids are detected in the nucleus (N), enveloped virus particles in the cytoplasm (arrows) and in the extracellular space. New nucleocapsids are budding through membranes of the smooth endoplasmic reticulum (arrowheads)
of the mucosa in that section. Each value represents the mean ( + so) of six experiments with a total of 600 mm of mucosal length.
Transmission electron microscopy The nasal mucosa was fixed in a mixture of 0.75 per cent glutaraldehyde and 0.8 per cent osmium tetroxide in a veronal acetate buffer, washed extensively and then stained in 1-0 per cent uranyl acetate (Hirsch and Fedorko 1968). Tissues were washed again and then dehydrated in alcohol and embedded in Epon resin. Ultra-thin sections were stained with 0"2 per cent lead citrate in 0.1 M sodium hydroxide and uranyl acetate (Venable and Coggeshall 1965), and examined in a Philips EM 300 microscope.
Spread o f PR V infections in nasal mucosa NIA-3 virus. At 24 hours after inoculation, viral antigens (Fig 1A) and DNA were detected in both nucleus and cytoplasm of epithelial and stromal cells. The staining patterns after DNA hybridisation and immunohistochemistry were identical. After 48 hours, large areas of epithelium had become necrotic and the infection had reached deep into the stroma, killing many cells (Fig 1B). 2"4N3A virus. At 24 hours after inoculation, viral antigens (Fig 1C) and DNA were detected in both nucleus and cytoplasm of epithelial and stromal cells. The staining patterns after DNA hybridisation and immunohistochemistry were identical. After 48 hours, 2.4N3A virus infection had invaded the stroma (Fig 1D), but had killed less cells than NIA-3 virus.
Results
PRV infections in nasal epithelium At 24 hours after inoculation, viral antigen was detected in most of the epithelial surface (87 per cent) after NIA-3 infection; in less than half after Bartha virus infection (37 per cent) and in an intermediate amount after 2.4N3A virus infection (55 per cent) (Table 1). At 48 hours after inoculation, the two less virulent strains had also infected most of the epithelial cells (84 to 70 per cent).
Bartha virus. At 24 hours after inoculation, viral antigens (Fig 1E) were mainly detected in the cytoplasm of epithelial ceils, whereas viral DNA was predominantly found in the nucleus of infected epithelial cells (inset in Fig 1E). Stromal cells under the epithelium were not infected. After 48 hours the infection was still strictly confined to epithelial cells (Fig 1F). Both nucleus and cytoplasm of infected cells contained viral antigens as well as viral DNA (inset in Fig IF).
Morphogenesis and virulence of pseudorabies viruses
49
FIG 3: Electron micrograph ( x 40,000) of nasal epithelial cell 48 hours after inoculation with NIA-3 virus. Nucleocapsids (arrows) are adjacent to the inner nuclear membrane. Enveloped virus particles (arrowheads) are detected in the perinuclear space between the inner and the outer nuclear membrane
The underside of the explants consisted of fibrous tissue which was readily infected by all three virus strains. The N1A-3 virus and 2-4N3A virus spread through several layers of fibroblasts, but Bartha virus infected only the outer layer.
Ultrastructural morphogenesis NIA-3 virus. At 24 and 48 hours after inoculation,
enveloped virus particles were detected in and around infected epithelial cells and fibroblasts. Few nucleocapsids were seen budding through membranes of the smooth endoplasmic reticulum (Fig 2). Most nucleocapsids were enveloped by budding through the inner nuclear membrane (Fig 3).
2.4N3A virus. At 24 and 48 hours after inoculation, naked nucleocapsids were detected in the
FIG 4: Electron micrograph ( x 8000) of nasal epithelial cell 48 hours after inoculation with 2.4N3A virus. Nucleocapsids (arrowheads) are detected in the nucleus (N) and in the cytoplasm. Nucleocapsids are enveloped by budding through membranes of the smooth endoplasmic reticulum (arrow). Enveloped virus particles are detected in the extracellular space
50
J. M. A. Pol, W. G. V. Quint, G. L. Kok, J. M. Broekhuysen-Davies
FIG 5: Electron micrograph (x 32,000) of nasal epithelium cell 48 hours after inoculation with 2 . 4 N 3 A virus. Nucleocapsids in the cytoplasm (C) are enveloped by budding through thickened membranes of the smooth endoplasmic reticulum (arrowheads). Enveloped virus particles are detected in cytoplasmic vacuoles and in the extracellular space
nucleus and cytoplasm of infected cells, and enveloped virus particles were detected outside the cells (Fig 4). Some nucleocapsids were enveloped by budding through the internal nuclear membrane as in the NIA-3 virus infected cells. More frequently, however, nucleocapsids accumulated in the cytoplasm and were enveloped by budding through membranes of the endoplasmic reticulum, which became thickened at the site of contact with the
nucleocapsids (Fig 5). The morphogenesis of 2' 4N3A virus in epithelial cells and stromal fibroblasts was identical. Bartha virus. At 24 and 48 hours after inoculation of Bartha virus, few virus particles were detected in infected epithelial cells and extracellular spaces (Fig 6). Few nucleocapsids were enveloped by budding through the inner nuclear membrane,
FIG 6: Electron micrograph ( x 8000) of nasal epithelium cell 48 hours after inoculation with 8artha virus. Few nucleocapsids are detected in the nucleus and cytoplasm (arrows). Few enveloped virus particles are detected in the extracellular space (arrowheads)
Morphogenesis and virulence of pseudorabies viruses
51
FIG 7: Electron micrograph ( x 32,000) of nasal epithelium cell 48 hours after inoculation with Bartha virus. Nucleocapsids (arrows) are detected in the nucleus (N) and in the cytoplasm (C). Membranes of the smooth endoplasmic reticulum are thickened at the site of viral envelopment. Few enveloped virus particles are detected in the cytopiasm (arrowhead)
smooth nucleocapsids were seen in the cytoplasm budding through thickened membranes of the smooth endoplasmic reticulum (Fig 7). Bartha virus did not penetrate the basal lamina under the epithelial cells, but the virus replicated in stromal fibroblasts on the underside of the explants with a similar morphogenesis as in epithelial cells. Discussion
Infection of nasal mucosa explants with three PRY strains of different virulence showed that nasal epithelial cells are target cells for the virus. N1A-3 virus quickly spread to large areas of the epithelium and invaded the subepitheliai stroma. In contrast, Bartha virus infection spread to fewer epithelial cells and did not penetrate through the basal lamina to the stromal fibroblasts. The deletion mutant virus 2.4N3A spread to epithelial cells as NIA-3 virus did but showed less invasion to stromal fibroblasts. These results agreed with those of in vivo studies of intranasal infections of pigs with the same three virus strains (Pol et al 1989). The stroma of oronasal epithelium contains many blood vessels and neural axons of the trigeminal, olfactory and vegetative nerves through which the virus can invade the central nervous system. Therefore, the ability to penetrate the basal lamina and infect the underlying fibroblasts is an important criterion for virulence. Staining for viral DNA and antigens revealed that the three strains replicate differently. In cells infected by NXA-3 virus or 2-4N3A virus the two methods
produced identical staining patterns, indicating that viral DNA was present in the nucleus and was transported from there to the cytoplasm. Moreover, NIA-3 virus spread rapidly to neighbouring cells, and its rapid release was confirmed by electron microscopy. In contrast, 2.4N3A virus spread slowly, which suggests that it was released more slowly; electron microscopy showed that many virus particles accumulated in the cytoplasm where they were enveloped before being released. At 24 hours after inoculation, the nuclei of Bartha virus infected cells contained both viral DNA and antigens, whereas the cytoplasm contained viral antigens but little DNA. Also, cells infected with Bartha virus contained fewer nucleocapsids than cells infected with the two other viruses. Lomniczi et al (1987) showed that in Bartha virus a U l gene involved in nucleocapsid assembly is defective. Bartha virus nucleocapsids, like those of 2.4N3A virus, were enveloped by budding through smooth endoplasmic reticulum membranes. 2.4N3A virus and Bartha virus both lack gI and an 11 K phosphoprotein of the nucleocapsid (Frame et al 1986). The exact role of these viral proteins in the morphogenesis is still unknown, but results suggest that the lack of a glycoprotein may account for the different site of envelopment compared to NIA-3 virus. Glycoproteins are produced on the rough endoplasmic reticulum and are glycosylated and processed in the smooth endoplasmic reticulum. Herpesvirus glycoproteins are incorporated in the inner nuclear
52
J. M. A. Pol, W. G. V. Quint, G. L. Kok, J. M. Broekhuysen-Davies
membrane at the site of viral budding (Johnson and Spear 1982). It is proposed that, because gI is missing, both Bartha virus and 2.4N3A virus cannot bud through the inner nuclear membrane; they leave the nucleus unenveloped to bud through membranes of the smooth endoplasmic reticulum. Bartha virus also lacks a 28 K protein and gp 63 whereas glII is inefficiently incorporated in the virus (Schreurs et al 1988) which may further lower its virulence (Robbins et al i989). Because mucosal explants have no contact with the blood circulation, the only endogenous agents that can influence infection in vitro are lymphokines like interferons which can be produced locally by cells of the host. Conceivably, when virus replicates and spreads slowly, as Bartha virus does, epithelial cells have time to produce interferon which can protect the surrounding cells and underlying fibroblasts of the stroma. That a similar mechanism may be involved is also suggested by the fact that Bartha virus did not infect the stromal fibroblasts under the basal lamina although it infected one layer of fibroblasts on the underside of the explants. Viral budding through endoplasmic membranes was seldom observed in cells infected with NIA-3 but frequently in cells infected with Bartha virus or 2" 4N3A virus, indicating that the budding process is retarded. Interferons may play a role in the disruption of viral morphogenesis by changing membrane fluidity (Chatterjee et al 1982) and reducing the synthesis of certain herpesvirus glycoproteins (Chatterjee et al 1985). Nasal mucosa explants offer a natural combination of epithelial cells and stromal fibrobiasts that yields more information about the virulence of PRV than each type of cell on its own. Because conditions such as multiplicity of infection, contact between virus and cells, and temperature and pH can readily be controlled, the method is reliably reproducible. This method closely mimics the in vivo infection; the virulence of PRV strains and mutant viruses in mucosa can be studied and virus strains can be screened to select those that can be used in vaccines. This in vitro model is not suited for the study of neural virulence. Although the axons lying in the nasal mucosa explants can be infected, the possible consequences of such infections cannot be studied. Neurovirulence of PRV strains should preferably be studied in in vitro models that contain both neurons and glial cells to mimic the complex interactions of cells during PRY infection. Acknowledgements We thank F. Wagenaar and L. Juffermans for excellent technical assistance, V. Thatcher for editorial assistance and F. Propsma and P. de Haas for the photographs.
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Morphogenesis and virulence of pseudorabies viruses deletion mutarrts of pseudorabies virus: A new generation of 'live' vaccines. Journal of General Virology 68, 523-524 ROBBINS, A. K., RYAN, J. P., WHEALY, M. E. & ENQUIST, L. W. (1989) The gene encoding the glII envelope protein of pseudorabies virus vaccine strain Bartha contains a mutation affecting protein localisation. Journal of Virology 63,250-258 SCHREURS, C., METTENLEITER, T. C., ZUCKERMANN, F., SUGG, N. & BEN-PORAT, T. (1988) Glycoprotein gill of pseudorabies virus is multifunctional. Journal of Virology 62,2251-2257 VAN OIRSCHOT, J. T. & GIELKENS, A. L. J. (1984) Some characteristics of four attenuated vaccine virus strains and a virulent strain of Aujeszky's disease virus. Veterinary Quarterly 6, 225-229
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VENABLE, J. H. & COGGESHALL, R. (1965) A simple lead citrate stain for use in electron microscopy. Journal of Cellular Biology 25,407-408 WALBOOMERS, J. M. M., MELCHERS, W. J. G., MULLINK, H., MEIJER, C. J. L. M., STRUYK, A., QUINT, W. G. J., VAN DER NOORDAA, J. & TER SCHEGGET, J. (1988) Sensitivity of in situ detection with biotinylated probes of human papilloma virus type 16 DNA in frozen tissue sections of squarnous cell carcinomas of the cervix. American Journal of Pathology 131,587-594
Received January 9, 1990 Accepted July 20, 1990