Department of Microbiology and Immunology, The Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan1
Department of Pathology, National Institute of Infectious Diseases, 1-23-1 Toyama-cho, Shinjuku-ku, Tokyo 162-8540, Japan2
Department of Laboratory Animal Science, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan3
Author for correspondence: Satoshi Koike. Fax +81 42 321 8678. e-mail koike{at}tmin.ac.jp
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Abstract |
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Introduction |
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The poliovirus receptor (PVR) has been considered as a major determinant of tissue tropism and host-range specificity (Holland et al., 1959 ; Holland, 1961
). PVR is an integral membrane protein of the immunoglobulin (Ig) superfamily (Mendelsohn et al., 1989
). PVR proteins, PVR
and PVR
, which are alternative splicing isoforms, consist of a signal peptide, three Ig-like domains, a transmembrane domain and either one of the two cytoplasmic tails (Koike et al., 1990
). Rodents lack the PVR molecule and are generally non-permissive to PV infection. Mouse cells in culture and transgenic (tg) mice expressing PVR acquire PV susceptibility, indicating that PVR is a determinant of host-range specificity in mice (Mendelsohn et al., 1989
; Ren et al., 1990
; Koike et al., 1991
). With regard to the tissue tropism, PVR also plays an important role. In the tg mice, PVR mRNA expression was detected in the neurons in the CNS, and these neurons were infected by PV (Ren & Racaniello, 1992
; Koike et al., 1994
). Although PVR expression is necessary to acquire susceptibility to PV, it does not seem to be sufficient because PVR expression was observed in extraneural non-target tissues (Kunin & Jordan, 1961
; Mendelsohn et al., 1989
; Nomoto et al., 1994
).
If the neuropathogenicity of PV is largely dependent on cell tropism, and if cell tropism is mainly determined by PVR expression in the CNS, PV would exhibit quite a different pathogenicity in tg mice expressing PVR in different patterns. In order to examine the influence of PVR expression on the neuropathogenicity of PV, we produced tg mice with different PVR distributions. Tg mice carrying the human genomic DNA for PVR (designated hgPVR mice) have already been produced: ICRPVRTg1 and ICRPVRTg21 by our group (Koike et al., 1991 ) and TgPVR117 by others (Ren et al., 1990
). In most cases, PV antigens were detected in the motor neurons in the spinal cord and in neurons in the brainstem. Thus, this is a mouse model for an acute CNS disease that resembles human poliomyelitis. In this study, we have produced a new tg mouse in which PVR is expressed under the transcriptional control of the CAG promoter (Niwa et al., 1991
) (designated CAGPVR mice). In these CAGPVR mice, PVR is also expressed in both glial and ependymal cells. We focused on the difference in histopathology of the CNS of these two tg mouse strains with different transgenes. Here, we describe the marked changes in the neuropathogenicity of PV, including the site of virus replication and progression of virus infection caused by alteration of PVR expression. Interestingly, the pathological changes induced by PV in the CAGPVR mice are similar to those observed in humans infected with CVB (Price et al., 1970
) or those in rodents infected with mumps virus (Wolinskey, 1996
), rather than those in humans with PV infection (Bodian, 1955
; Sabin, 1956
). The relationship between neuropathogenicity and cell tropism is discussed.
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Methods |
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In situ hybridization.
An XhoIXhoI human PVR cDNA fragment (nt 11461687) was inserted into the SalI site of pBluescript SK in both orientations. Plasmid DNA was digested with HindIII and transcribed in vitro using T7 RNA polymerase and the NTP mixture containing digoxigenin (DIG)UTP (Roche) as substrates. Mice were fixed with 4% paraformaldehyde/PBS (PFA/PBS) by perfusion and overnight immersion at 4 °C. Tissues were embedded in paraffin and sections at 7 µm thickness were prepared. In situ hybridization was carried out as described previously (Koike et al., 1994 ). The hybridized DIG-labelled RNA probe was detected and visualized using a DIG Nucleic Acid Detection kit (Roche). Sections were stained with methyl green to visualize the nucleus.
PV binding assay.
The mouse brain, spinal cord, heart, lung, liver, kidney, spleen and skeletal muscle were homogenized in 10 vols of ice-cold Dulbecos modified minimum essential medium (DMEM) using a Polytron homogenizer. PV (1000 p.f.u.) was incubated with 1 ml of the tissue homogenates at 37 °C for 30 min. The virus titre of the virushomogenate mixture was determined by plaque assay on an African green monkey kidney (AGMK) cell line, JVK-03 (Koike et al., 1992 ).
PV infection in mice.
The PV type 1 Mahoney strain was obtained by transfection of in vitro-transcribed RNA from an infectious cDNA clone, pOM (Shiroki et al., 1995 ). The RNA was isolated from the recovered virus and its sequence was verified. No nucleotide change was observed in the recovered virus compared with the sequence of the infectious cDNA clone. The virus was propagated in AGMK cells. The titre of stock virus was measured by plaque assay on the AGMK cells. Five-week-old mice were used in the infection experiments. The PV solution was inoculated into mice by intracerebral, intraperitoneal and intravenous routes (Koike et al., 1991
). PV solution (25 µl) was also administered orally by dropping solution into the mouth.
Measurement of neutralizing antibody titre.
Two weeks after inoculation, the antibody titre of the serum of the surviving mice was measured by a microneutralizing test (World Health Organization, 1997 ). We considered that the mice elicited neutralizing antibody when virus was neutralized with the serum diluted more than fourfold.
Immunohistochemistry.
Mice that died following the virus infection were fixed in PFA/PBS. Mice were sacrificed by deeply anaesthetizing with ether and then perfused with PBS followed by PFA/PBS. The brains and the spinal cords were embedded in paraffin and 3 µm thick sections were prepared. PV antigens were detected with a rabbit anti-PV polyclonal antibody using an immunoperoxidase method (Koike et al., 1991 ).
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Results |
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Southern blot hybridization was carried out to confirm the presence and the integrity of the transgene (Fig. 1B). Bands with the expected size were found after restriction map analysis of the three strains, while no band was detected in DNA digests from non-tg mice. The copy numbers of the transgene in the three strains were roughly estimated to be between two and four.
PVR expression in tg mice
We determined PVR expression by four different methods. First, RTPCR was carried out on total RNA isolated from various tissues of the mice using PVR51 and PVR31 as primers. A human PVR-specific band was observed in all tissues of tg mice tested but not in non-tg mice (data not shown). The specific band was observed only in the presence of reverse transcriptase in the reaction. Secondly, we performed Northern blot hybridization to investigate the expression of human PVR mRNA. The PVR transcript (approximately 2000 nt) was detected in all tissues of tg mice tested, but the expression level varied both among tissues and among tg strains (Fig. 1C). The same amount of RNA from non-tg mice did not contain any human PVR transcript (data not shown). The expression level of the PVR mRNA was less than 10% of the endogenous
-actin mRNA in three tg strains. Thirdly, we tested the neutralization activity of PV using tissue homogenates of mice. The functional PVR protein is able to bind PV and it induces conformational changes of the virion, which initiates its uncoating. The neutralization activity of the tissue homogenate therefore indicates the presence of functional PVR protein. As shown in Fig. 2
, all tissue homogenates had virus titres reduced to less than 10% of the levels of the inoculated virus, while no reduction in the virus titre was observed when tissue homogenates from non-tg mice were used. Fourthly, in situ hybridization was performed to determine the expression pattern of PVR mRNA in each cell type. In sections of the brain and spinal cord, signals from the PVR transcript were detected in the cytoplasm of almost all neurons, such as those in the cerebral cortex (data not shown), hippocampus (Fig. 3E
), cerebellum (data not shown) and spinal cord (Fig. 3I
). The PVR transcript was also detected in some glial cells (Fig. 3C
, I
) and ependymal cells (Fig. 3G
). The hybridization signals from some glial cells were evident but some were not. No signal was observed in controls using sense PVR RNA as the probe in these cells (Fig. 3D
, F
, H
, J
, L
). It is clear that PVR is expressed not only in neurons but also in ependymal cells and at least in some glial cells in CAGPVR mice, although we could not conclude that the transgene expression is ubiquitous. The results were the same for the three CAGPVR mouse strains.
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Different mortality rates between the two tg strains are due to the difference in transgene expression
There are at least three possibilities to explain the difference in mortality rates between the two tg strains. It may be due to the expression of PVR both in neuronal and non-neuronal cells in the CAGPVR mice, the difference in genetic background among strains, or the lack of PVR
, PVR
and PVR
isoforms in CAGPVR mice. To examine these possibilities, we mated homozygotes of ICRPVRTg21 with hemizygotes of B6CAGPVRTg41. All of their F1 progenies have the same genetic background because both strains are inbred. Half of these had both the hgPVR and the CAGPVR transgenes (hgPVR+/-, CAGPVR+/-). The other half carried only the hgPVR transgene (hgPVR+/-, CAGPVR-/-). All four of the splicing isoforms of PVR are supposed to be expressed in neurons since all the mice carry the hgPVR transgene. PVR
is additionally expressed in many cells in the former tg littermates (hgPVR+/-, CAGPVR+/-), but only in the neurons in the latter (hgPVR+/-, CAGPVR-/-). Both genotypes of littermates were challenged by intracerebral inoculation of PV. The former were resistant to 106 p.f.u. of PV (no mice died in 23 inoculated mice). All of the latter (20 inoculated mice) died at the same dose of virus. We therefore concluded that the difference in mortality rate is due to the PVR expression in non-neuronal cells in CAGPVR mice, but not due to the difference in genetic background nor to the lack of different PVR isoforms.
Time courses of PV levels in the CNS of tg mice
We next recovered PV from the infected mice to examine whether the virus replicated in the CNS of the ICRPVRTg21 and B6CAGPVRTg41 mice (Fig. 4). The time courses of recovered virus in the two tg strains were quite different. In hgPVR mice, the amount of recovered virus increased and reached maximum level in the spinal cord on day 3 p.i. when most of the inoculated tg mice were paralysed and died. This indicated that the virus had spread from the inoculation site as a result of multiple cycles of infection.
In CAGPVR mice, 106 p.f.u. of the virus was recovered from the brain on day 1 p.i. It is clear that PV multiplication occurred in the brain of CAGPVR mice since the amount of virus recovered was greater than that of inoculated virus. After 2 days p.i., the virus titre began to decrease in the brain. This suggests that the virus multiplication may occur only in an early period and not spread efficiently from the initial multiplication site, or that virus clearance may begin as early as day 2 p.i. In the spinal cord, only a small amount of virus was recovered on day 2 p.i. The inoculated virus did not appear to spread to the spinal cord. These results explain the difference in the clinical symptoms and mortality rates between the two tg mouse strains.
Pathological changes in tg mice
The clinical symptoms and mortality rates greatly differed between hgPVR and CAGPVR mice after intracerebral inoculation of PV. We therefore examined the sites of virus multiplication and pathological changes of these mice. We have previously described the histopathological changes in hgPVR mice (Koike et al., 1991 ; Horie et al., 1994
). PV infection in the hgPVR mice is restricted to neurons. This is in good agreement with the detection of PVR expression only in the neurons (Koike et al., 1994
). Neurons in the ventral horn of the spinal cord, medulla oblongata, pons, thalamus and the midbrain were the major targets of PV. Infection of hippocampal neurons was sometimes observed but at a low frequency. Compared with the natural PV infection in humans and experimental infection in monkeys, cellular infiltration was minimal in hgPVR mice, particularly in the case of intracerebral inoculation of virulent strains.
In contrast, major sites of PV replication in CAGPVR mice were quite different from those in hgPVR mice. In the brains of mice that died within 3 days p.i., ependymal cells degenerated with granulocyte infiltration. PV antigens were detected in the ependymal cells (Fig. 5B, C
). In the corpus callosum and in the white matter between the lateral ventricle and the cerebral cortex, where no neurons exist, PV antigens were detected in glial cells (Fig. 5B
, E
). A preliminary experiment of double-labelling with PV antigen and an oligodendrocyte marker (glutathione S-transferase) suggested that the infected glial cells were mostly oligodendrocytes. PV antigens were also detected in pyramidal neurons in the hippocampus (Fig. 5B
, D
, E
). PV antigens were not clearly detected in vascular endothelial cells and meningeal cells. All of the infected cells were localized within and around the ventricles, suggesting that the virus spread from this area. Neurons in the brainstem, which are often infected by PV in humans, monkeys and the hgPVR mice (Bodian, 1955
; Koike et al., 1991
; Horie et al., 1994
), were seldom infected. The lateral ventricle became narrowed or almost closed in all of the mice that died within 3 days p.i. (Fig. 5C
). In the spinal cord, PV antigens were detected in the motor neurons in the ventral horn, ependymal cells, neurons and glial cells near the central canal, and glial cells in the white matter of dorsal funiculi (Fig. 5G
, H
, I
). These results indicate that many cell types, including neurons, glial cells and ependymal cells, became susceptible to PV when they expressed PVR. The ependymal cells seem to be highly susceptible. This indicates that mouse glial and ependymal cells possess all host factors sufficient for PV replication except for PVR. No difference was observed in the distribution of PV antigens among the three CAGPVR tg strains.
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Ventricular dilation caused by PV infection
Most of the CAGPVR mice survived until the end of the observation period. We observed for as long as 8 weeks p.i. We examined the time-course of pathological changes. B6CAGPVRTg41 mice were inoculated with 106·7 p.f.u. of PV and sacrificed on days 2, 3, 5, 14, 28, 42 and 56 p.i. On day 14 p.i., ventricular dilation became evident (Fig. 6). Mild ventricular dilation without cortical thinning was observed in mice in the control experiments, using non-tg mice inoculated with PV and tg mice inoculated only with DMEM without virus. The ventricular dilation observed in tg mice inoculated with PV is greater and is associated with cortical thinning. Table 2
shows the incidence of severe ventricular dilation associated with cortical thinning on day 14 p.i. Ventricular dilation occurred more frequently as the inoculated virus titre increased. Dose dependency was most clearly seen in B6CAGPVRTg55 mice. Dilation proceeded in a time-dependent manner in B6CAGPVRTg41 mice (Fig. 6
).
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Discussion |
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There is an example in which a change of cell tropism alters the pathogenicity of a virus. Neurovirulent strains of mouse hepatitis virus (MHV) produce an acute and often fatal encephalitis with extensive neuronal involvement, whereas neuroattenuated variant strains spread only slowly in neurons and generally infect glia, with attendant chronic white matter disease (Fleming et al., 1986 ). These pathological changes induced by MHV strains are similar to those in hgPVR mice and those in CAGPVR mice induced by PV, respectively. This observation is consistent with the idea that the acquisition of susceptibility in the non-neuronal cells alters the virus phenotype into an attenuated one.
Among members of the Picornaviridae, CVs and echoviruses invade the CNS, cause aseptic meningitis and terminate with good recovery in many cases (Johnson, 1998 ). The clinical and histopathological changes in CAGPVR mice after PV infection were mainly on the ependymal surface. The parenchyma of the brain and the spinal cord were not severely damaged in most cases. This resembles the pathological changes observed in a patient (Price et al., 1970
) who died of myocarditis accompanied by meningitis due to CVB5 infection, rather than those observed in PV infection in human and hgPVR mice. The cellular receptor for CVBs, the coxsackieadenovirus receptor (CAR), has been identified (Bergelson et al., 1997
, 1998
; Tomko et al., 1997
). The distribution of CAR in the human CNS is not known. However, CAR is expressed in ependymal cells in rats (Johansson et al., 1999
). It is possible that CAR is expressed in the ependymal and glial cells in the human CNS and that CVBs do not frequently cause fatal encephalitis because of these non-neuronal cells.
The most interesting observation is that the viral invasion of the CNS did not spread and did not result in the death or paralysis of infected CAGPVR mice, although the neurons are susceptible to the virus, similar to those of hgPVR mice. This was most evident in the motor neurons in the spinal cord of the CAGPVR mice. A small amount of virus was recovered and a few antigen-positive cells were detected in the spinal cord of CAGPVR mice on day 2 p.i., but infection did not spread to larger areas to cause paralysis. Virus clearance may have occurred as a result of adsorption and neutralization of infectious particles by PVR protein ectopically expressed. This prevents secondary infection near the initial infection sites. Alternatively, immunological responses may have begun from the early stage of infection only in CAGPVR mice. Inflammatory cells are observed as early as day 2 p.i. in CAGPVR mice, whereas cell infiltration was not observed at such an early period of infection in hgPVR mice. This suggests that the immune response was induced to clear the virus in the CAGPVR mice before the virus infected neurons that are essential for the maintenance of life. Non-neuronal cells may have a much greater capacity to induce immunological response than neurons in the CNS. The CNS shows a generalized lack of constitutive major histocompatibility complex (MHC) expression. Microglias, oligodendrocytes and endothelial cells, but not neurons, were induced to express MHC molecules in the CNS infected with viruses or treated with interferon- (Joli et al., 1991
; Horwitz et al., 1999
). In the CAGPVR mice, infected non-neuronal cells would likely be MHC class I-positive and therefore could be a good target for an anti-viral response. In contrast, neurons would not express sufficient levels of the MHC class I molecule in hgPVR mice. The infecting PV is therefore able to escape immunological surveillance. In our preliminary experiments, the CAGPVR mice became resistant to infection by Japanese encephalitis virus (JEV), when JEV was inoculated intracerebrally 1 day after PV inoculation (data not shown). This suggested that an anti-virus state was established by PV infection in CAGPVR mice. We are now investigating immune responses in CAGPVR mice.
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Acknowledgments |
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References |
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Received 12 September 2001;
accepted 18 January 2002.