Ultrastructural study of the renal tubular system in acute experimental African swine fever: virus...

Arch Virol (1995) 140:581-589

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Vifrology © Springer-Verlag 1995 Printed in Austria

Ultrastructural study of the renal tubular system in acute experimental African swine fever: virus replication in glomernlar mesangial cells

and in the collecting ducts

Brief Repor t

J. C. G6mez-Villamandos 1, J. Hervfis 1, A. M6ndez 1, L. Carrasco 1, C. J. Villeda 2, P. J. Wilkinson 3, and M. A. Sierra 1

1Dpto Anatomia y Anatomia Patol6gica, Facultad de Veterinaria, Universidad de C6rdoba, C6rdoba, 2Centro de Biologia Molecular (CSIC-UAM), Facultad

de Ciencias, Universidad Aut6nomad de Madrid, Madrid, Spain 3 A.F.R.C., Institute for Animal Health, Pirbright Laboratory, Pirbright, U.K.

Accepted November 12, 1994

Summary. Despite the considerable attention given to kidney lesions in African swine fever (ASF), a number of questions remain to be answered. Structural and ultrastructural examination showed that a highly virulent isolate of ASF virus (Malawi 83) replicated in glomerular mesangial cells and renal collecting duct epithelial cells, with hyperplasia of the latter in infected pigs. Replication in mesangial cells may be due to their contact with the bloodstream, as well as to their phagocytic capacity and high metabolism rate. Virus replication in macrophages and endothelial cells of interstitial capillaries, and the necrosis of these infected cells gave rise to a large number of free virus in interstitial tissue. This, together with the lesser thickness of the basal membrane of collecting ducts in comparison to the rest of the tubular system, probably facilitates ASFV infection of tubular epithelial cells. Virus replication in these cells may account for the presence of virus in the urine of pigs with acute ASF where haematuria is not observed.

African swine fever (ASF) is an infectious infectious disease caused by a large DNA virus that has been classified by the International Committee of Taxonomy of Viruses as the only member of an unnamed floating genus [10]. In the acute form of the disease, the mononuclear phagocyte system (MPS) is destroyed by ASF virus (ASFV) replication [15]; however, ASFV also replicates in other cell populations in infected animals, such as hepatocytes [19] and endothelial

582 J.C. G6mez-Villamandos et al.

cells [20]. For that reason, the disease provides an ideal model for the study of virus action on populations of cells other than the usual target cells. ASF has also been used as a model for the study of immunopathological mechanisms [18] and of circulatory abnormalities [22].

Kidney involvement in ASF has been widely studied ever since the disease was first described, because gross lesions observed in the kidney have been considered of great value in the diagnosis of the disease [6]. Ultrastructural studies of the kidney in pigs infected with virulent isolates of ASFV have identified virus particles or replication sites in interstitial macrophages and interstitial capillary endothelial cells only [20].

Heuschele [7] and Plowright et al. [16] reported low virus titres in the kidney following inoculation of the highly virulent Tengani strain. Greig and Plowright [4] showed that ASFV is eliminated in the urine of affected swine, even though viral titers are only high in the presence of haematuria [11]. The pathogenic mechanism leading to the presence of virus in the urine of animals with no haematuria is unknown.

This paper reports ultrastructural lesions observed in the renal tubular system, including the nephron and the collecting ducts, of swine infected with the virulent Malawi'83 ASFV isolate, and presents morphological evidence of ASFV replication in the glomerular mesangial (GM) cells and collecting duct epithelial cells, with hyperplasia in the latter.

Nine Large White x Landrace cross-bred pigs of both sexes were used for this study; pigs weighed approximately 30kg at the beginning of the experiment, and were free from infectious and parasitic diseases; they were fed with Prime Grover (Scats Ltd., Winchester, U.K.) and housed in an environ- mentally controlled isolation room. One pig was used as an uninfected control. Eight pigs were inoculated by the intramuscular route with 105 50~ haemadsor- bing doses (HADso) of the Malawi'83 ASFV isolate [-5]. The animals showed no changes in behaviour caused by experimental conditions. The clinical signs observed in these animals were characteristic of ASF [6]. Animals were sacrificed in pairs at 1, 3, 5 and 7 days postinoculation (dpi). This experiment was performed in the Institute of Animal Health, Pirbright (U.K.) in accordance with the Code of Practice for the Housing and Care of Animals Used in Scientific Procedures.

Tisssues of all pigs were fixed by vascular perfusion with 2.5~o glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at a pressure of 120mm Hg. Prior to perfusion, animals were sedated with azaperone (Stresnil) (Jannsen Animal Health, Belgium) and anaesthetised with thiopental-sodium (Thiovet) (Vet Limited, England). Samples from perfused animals were embedded in Epon 812 (Fluka, Buchs, Switzerland). Sections were cut to 1 gm and stained with toluidine blue (1~o aqueous solution). For ultrastructurat study, 50 nm sections of kidneys were contrasted with uranyl acetate and lead citrate and observed with a Philips CM-10 transmission electron microscope.

All animals were asymptomatic on day 2 post inoculation. From 3 to 4 days only two pigs developed a slight diarrhoea and suffered varying degrees

ASFV replication in renal cells 583

Fig. 1. A Monocyte in glomerular capillary with ASFV replication site (arrow). Bar: 400 nm. B Cell debris and networks of fibrin (arrows) in the glomerular capsular space. The glomerular capillaries also showed cell debris (,). Bar: 3gin. Insert: virus in capsular

space. Bar: 500 nm

of loss of appetite but were otherwise normal. No sign of haemorrhage was seen. From day 5 onwards the onset of illness was extremely sudden, with rectal temperatures of over 42 °C, markedly decreased activity, difficulty in breathing with coughing, swollen and haemorrahagic conjunctival membranes. Episodes of nasal bleeding and diarrhoea with blood were seen. Animals progressively became drowsy.

Structural and ultrastructural examination of the kidney revealed no visible lesions in the pigs sacrificed at 1 and 3 dpi, with structural and ultrastructural features resembling the structures of the kidneys of control pigs. However, at 5 dpi glomerular capillaries contained an increased number of monocytes, lymphocytes and neutrophils. A few monocytes showed an evident viral cytopathic effect, with peripheral margination of chromatin, cytoplasmatic vacuolisation and visible intracytoplasmic ASFV replication sites, which appeared as areas of organelle-free cytoplasm, containing icosahedral virus with electrodense nucleoids (mature particles) or without nucleoids immature particles); membranous structures were also observed (Fig. 1A).

At 7 dpi, glomerular capillary endothelial cells showed light evidence of activation and phagocytosis, with long pseudopodia enveloping cell debris. Capillary lumina contained monocytes, many of which showed cytopathic effect


and visible intracytoplasmic ASFV replication sites. Lymphocytes and neutrophils with phagosomes were observed in these vessels. A few of these circulating cells were necrosed. Cell debris and viral particles, with and without envelope, were also observed in lumina, some times enmeshed in fibrin to form micro- thrombi (Fig. 1B).

Structural study of the mesangium revealed evidence of hypercellularity, and a small number of cells showed peripheral margination of chromatin and inclusion bodies, which appeared as areas of more lightly-staining cytoplasm close to the nucleus. Ultrastructurally, GM cells were characterised by their location between glomerular capillaries and had cytoplasmic processes containing fibrils connected to the basal membrane; some showed clear signs of activation with an evident increased number of lysosomes. Ultrastructurally, the inclusion bodies observed in GM cells by light microscopy were also found to be ASFV replication sites, similar to those described for monocytes (Fig. 2). Necrosis was not commonly observed in these cells.

Bowman's capsule epithelial cells and podocytes showed evidence of swelling; there were also signs of podocyte pedicel fusion, and moderately electrodense globular material was visible in the capsular space. Congestion and necrosis were observed in a few glomeruli, which contained cell debris and a few virus in the capsular space; these were enmeshed in fibrin (Fig. 1B). Proximal and

Fig. 2. Mesangial cells (M) with peripheral margination ofchromatin and intracytoplasmic virus replication site (arrow). Bar: 1 ~m


distal tubular epithelial cells showed evidence of apical ballooning and desquamation. Some proximal epithelial cells contained numerous highly electrodense absorption granules, which varied in size. Condensation of nuclear chromatin was evident in a small number of tubular epithelial cells.

Collecting ducts contained a mild intraepithelial infiltrate consisting of lymphocytes and macrophages. Mature virus, virus replication sites and virus budding were observed in a small number of these macrophages. Collecting duct epithelial cells were characterised by intercellular junctions and close contact with the basal membrane, Mature virus particles were found in vesicles and free within the cytoplasm of these cells. Some epithelial cells showed peripheral margination of nuclear chromatin, together with organelle-free areas of cytoplasm in the vicinity of the nucleus; structures observed in these areas were similar in size and shape to ASFV particles, although they appeared to be incomplete.

A moderate number of epithelial cells of these ducts showed ASFV cytopathic effect and viral replication sites, with virus at varying stages of maturity. Mature virus were observed close to the cytoplasmic membrane of these cells; some were being released by budding from the cell membrane (Fig. 3). In some collecting ducts, the virus replication in epithelial cells was associated with hyperplasia phenomena (Fig. 4A, 4B). Tubular lumina contained a few epithelial cells with cytopathic effect and replication sites or virus particles.

Fig. 3. Collecting duct epithelial cell with intracytoplasmic virus replication site (,) and budding virus (arrows). Bar: 1 p.m

586 J.C. Gdmez-Villamandos et al.

Fig. 4. A Focal epithelial hyperplasia (arrowheads) in collecting duct. The cells of this focus show severe cytopathic effect by ASFV replication. In the tubular lumina there are two desquamated epithelial cells with peripheral margination of chromatin (arrows). Bar: 15 gm. B Electron micrograph of a focus of epithelial hyperplasia associated with ASFV replication. Note the severe cytopathic effect and ASFV replications sites with virus particles (arrows). Bar: 3 lam. C Interstitial virus particle in close contact with the basal membrane of a tubule.

Bar: 200 nm

In the renal interstitium, a small number of macrophages were observed at 5 dpi with ASFV replication. However, the renal interstitium of animals sacrificed at 7 dpi showed an intense mononuclear infiltrate; it was formed chiefly by macrophages, most of which contained ASFV replication sites and necrosis. Cytopathic effect and virus replication were observed in a large number of interstitial capillary endothelial cells. Interstitial areas contained abundant ASFV particles with and without envelopes, some of which were in close contact with the tubule basal membrane (Fig. 4C).

This work presents evidence, and the first morphological description, of ASFV replication in glomerular mesangial cells and collecting duct epithelial cells in the kidney of pigs infected with a highly virulent isolate of ASFV. Moreover, ASFV replication in collecting duct epithelial cells is shown to be associated to hyperplasia; in this respect, ASFV clearly resembles Poxvirus [8]. Interaction of ASFV with a specific receptor is necessary not only virus penetration, but also for activation of a component in the virus particle required for virus replication, as has been described in picornavirus [1]. This suggests


that GM cells, collecting duct epithelial cells and endothelial cells, must all express in the cytoplasmic membrane the specific receptor for ASFV, in order for virus replication to take place. This in turn raises a question which may contribute to the identification of the as yet unknown specific receptor for ASFV. What membrane structure/s, able to act as receptors, are common to these cell populations and to MPS cells, the target cells of ASFV? Further research is required to answer this question.

The function of mesangial cells is to renew the mesangial matrix in which they are immersed [13]. Although they are not considered to be MPS cells [17], they can phagocyte blood-borne substances or structures with which they come into contact by fenestration of the glomerular capillaries or the projection of pseudopodia towards the capillary lumen [9]. The location and function of these cells, coupled with the presence of ASFV particles both free in the blood and bound to monocytes and erythrocytes, may determine virus-specific receptor interaction, ASFV penetration and replication in GM cells. The virus replication in these cells at 7 dpi, coinciding with evident hyperplasia of mesangial cells and the fusion ofpodocytes, gives rise to haemodynamic changes and filtration [12].

Virus replication in collecting duct intraepithelial macrophages and epithelial cells was abundant. In contrast, Fernandez et al. [-2] detected virus antigen in a small number of cells present in the collecting ducts of pigs experimentally infected with a virulent isolate. They maintained that the presence of virus antigens in these unidentified cells did not constitute proof of virus infection, pointing out that the presence of ASFV antigens in those may be due to reabsorption. Our results showed that this could not be the case. The data obtained here show that ASF virus replicates in collecting duct epithelial cells, suggesting that the infection of these may be caused by virus from the interstice because there were a large number of virus particles free in the interstice, and virus particles were also seen in close contact with tubule basal membranes. Given that the amount of free virus in the interstice remains constant throughout the nephron tubular system, the lesser thickness of the basal membrane of collecting ducts in comparison to the rest of the tubular system [23] might facilitate infection of duct epithelial cells by ASFV.

Excretion of virus in the urine may be the result of various pathogenic processes: Garcia e Costa and Alves de Matos [3] attribute it to the changes in the glomerular filtration barrier which occur during the course of the disease. Such alterations were observed here in some glomeruli following inoculation of the Malawi'83 isolate, and reached a maximum with the necrosis of glomerular elements, which might facilitate the presence of virus particles in the urinary space of the glomeruli. Necrosis of the glomeruli is associated with alteration of the glomerular capillary wall, a phenomenon not related here to virus replication in capillary endothelial cells and which may be caused by the action of enzymes released by neutrophils and monocytes, which were found to be activated from 3 dpi and necrotic at 7 dpi. Another possible origin of ASFV particles present in the urine as a result of tubular necrosis and severe kidney


and urinary tract bleeding [14]. The combination of these phenomena would explain the high virus titers in urine associated with haematuria, reported by McVicar [11].

Replication in intraepithelial macrophages and collecting duct epithelial cells from which mature virus are released by budding, and the hyperplasia, desquamation and necrosis of these epithelial cells, may account for the presence of the virus in urine when there is no haematuria.

To conclude, collecting duct epithelial cells may be infected by ASFV from infected macrophages and interstitial endothelial cells, a process probably favoured by histological predisposition (lesser thickness of basal membrane). Moreover, ASFV replication in these epithelial cells is associated to hyperptasia. On the other hand, the small number of MPS cells present in the kidney, in which ASFV may replicate, the fact that a highly virulent strain was employed in this study may have led to the infection of other cell populations, such as glomerular mesangial cells, collecting duct epithelial cells and interstitial capillary endothelial cells.

Acknowledgements We wish to think to Prof. Dr. A. Jover and Dr. E. Vifiuela for their support in this experiment; Dr. J. Martin de las Mulas for reviewing the manuscripts C. Romero, G. H. Hutchings and S.M. Williams for technical laboratory assistance; T. Kinsella and L. Fitzpatrick for assistance in the animal unit. This work was supported by grants from CAICYT, Junta de Andalucia and Junta de Extremadura (Spain) and the Ministry of Agriculture, Food and Fisheries (U.K.).

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Authors' address: Dr. J. C. Gomez-Villamandos, Dpto Anatomia Patologica, Facultad de Veterinaria, 14005 C6rdoba, Spain.

Received July 14, 1994

Ultrastructural study of the renal tubular system in acute experimental African swine fever: virus...

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