In vivo and in vitro models of demyelinating diseases: Tropism of the JHM strain of murine hepatitis virus for cells of glial origin

Infection of mice with the neurotropic JHM strain of murine hepatitis virus causes demyelinating lesions resulting from an infection of the oligodendroglia. This was most evident in mice inoculated intraperitoneally with JHM. Such CNS lesions were not observed in mice inoculated intraperitoneally with the MHV3 strain. An in vitro system is described in which the rat glial RN2 cell line functions as a discriminating host for the JHM virus. Shortly after inoculation, this virus establishes a persistent infection in which there is a cyclical rise and fall in titer with an accompanying cytopathology. Furthermore, this host cell confers a thermal lability which the virus does not demonstrate in the fully permissive host cell, L-2. By comparison, infection of RN2 cells with the prototype MHV3 is aborted immediately. In the persistent infection of RN2 cells with measles virus, Hallé strain, the cell again confers a temperature sensitivity which the virus does not possess when replicating in Vero cells. This appears to be the first instance in which a cloned cell line of glial origin determines the outcome of the infectious process, discriminating in favor of a neurotropic variant which possesses a tropism for the glia in vivo. Systems such as the one described here may now offer a specific screening procedure for selecting, identifying and characterizing the nature of neurotropic viruses.


Infection
of mice with the neurotropic JHM strain of murine hepatitis virus causes demyelinating lesions resulting from an infection of the oligodendroglia.
This was most evident in mice inoculated intraperitoneally with JHM. Such CNS lesions were not observed in mice inoculated intraperitoneally with the MHV, strain. An in vitro system is described in which the rat glial RN2 cell line functions as a discriminating host for the JHM virus. Shortly after inoculation, this virus establishes a persistent infection in which there is a cyclical rise and fall in titer with an accompanying cytopathology.
Furthermore, this host cell confers a thermal lability which the virus does not demonstrate in the fully permissive host cell, L-2. By comparison, infection of RN2 cells with the prototype MHV, is aborted immediately.
In the persistent infection of RN2 cells with measles virus, Hal& strain, the cell again confers a temperature sensitivity which the virus does not possess when replicating in Vero cells. This appears to be the first instance in which a cloned cell line of glial origin determines the outcome of the infectious process, discriminating in favor of a neurotropic variant which possesses a tropism for the glia in vivo. Systems such as the one described here may now offer a specific screening procedure for selecting, identifying and characterizing the nature of neurotropic viruses.

Introduction
One of the most attractive systems available for studying phenomena pertinent to human demyelinating disease such as multiple sclerosis (MS) involves infection of the central nervous system (CNS) by the JHM variant of the murine coronavirus, mouse hepatitis virus, MHV (Baileyet al., 1949;Cheever et al., 1949;Weiner, 1973). This agent causes pathological effects, including zones of demyelination, similar in some aspects to the plaques of human MS. While the JHM virus was initially isolated from a mouse showing paralytic symptoms (Cheever et al., 1949), another variant producing a similar CNS pathology was subsequently derived from mice injected with murine hepatitis virus, MHV3 (Dick, Niven and Gledhill, 1956). These two independent isolates have maintained their neurotropsim and, after numerous passages, continue to produce CNS demyelination in both mice (Bailey et al., 1949;Weiner, 1973;Herndon et al., 1975) and rats (Bailey et al., 1949). As a further analogy to the human disease, demyelination with JHM may occur intermittently, particularly when older mice are inoculated with JHM (Herndon et al., 1975). Examination of the CNS by immunofluorescence (Weiner, 1973) and electron microscopy (Lambert, Sims and Kniazeff, 1973) has revealed that JHMspecified products are in cells of the white matter, specifically in the cytoplasm of oligodendrocytes. Since immunosuppression of JHM-infected mice does not alter either the intensity or pattern of CNS lesions (Weiner, 1973), the mechanism of demyelination is presumed to involve a more direct effect of the virus on its target cells, the oligodendroglia.
This murine system acquires an even greater significance as a model for MS because JHM is serologically related to human coronavirus 229E, associated with upper respiratory disease especially in children (McIntosh, 1974). In this regard, it is worth mentioning that electron microscopic examination of a single biopsy from a terminal MS patient by Tanaka, lwasaki and Koprowski (1976) revealed the presence of numerous intracisternal particles resembling, morphologically, a coronavirus. In undertaking further studies using the murine model with a view to advancing our understanding of JHM tropism for glial cells, we deemed it logical and desirable to develop an in vitro system that might be a counterpart to the in vivo events. We now report the chance discovery of a selection process demonstrated by a rat glial tumor cell line which discriminates between the parental and neurotropic variants of JHM. In this cell-virus interaction, the host becomes persistently infected, causing cyclical manifestations of cytopathology and virus production.

Results
Growth in Mouse L-2 Cells Since we wished to compare the growth of the MHV, and JHM virus strains in various cell lines, it was necessary to use a cell type that was fully permissive for both agents. The cell selected for this purpose was the mouse L-2, a subline of mouse L-929 (Rothfels et al., 1959).
Following infection of L-2 monolayers with either virus at 32.5"C, using a multiplicity of infection (moi) of 0.01, multinucleate giant cells first became apparent at 16 hr. Maximum titers of IO6 PFU/ml of cell-free virus in the medium were recorded 30 hr postinfection, at the time monolayers had become virtually one continuous syncytium. The capability of MHV, and JHM to grow to high titer in L-2 cells allowed the use of these cells as host for routine virus propagation and assay by the plaque technique (see Experimental Procedures).

Growth in Rat Glial Cells
The presence of JHM antigen and virus in the oligodendrocytes of infected mice (Weiner, 1973;Lambert et al., 1973)  involving the entire monolayer, with RN2-2 cells, the syncytia developed only to a limited extent and remained discrete in sizeat least during the initial period of 2 weeks after infection -even though virus was being continually shed into the medium (indicated in Figure 1). It thus became apparent that JHM virus could readily establish a persistent infection in RN2-2 glial cells. Judging by PFU/ml in the medium, virus was produced in a cyclical manner over a period of at least 5 weeks and five cell passages.
independently inoculated RN2-2 cultures conformed to the same pattern and have undergone a number of crises and recovery cycles.
Although to perpetuate each of the above cultures, we passaged them at approximately weekly intervals by releasing the attached cells with trypsin, this manipulation did not affect either the nature of the carrier state or the amount of infectious virus released within 24 hr after subculture.

Properties
of Virus Released from the Carrier Culture To determine whether the JHM virus had been genetically altered in any obvious manner as a consequence of being propagated in the glial cells, virus released from carrier cells was tested for its ability to reestablish a persistent infection in RN2-2 cells, and for its capacity to form plaques on L-2 cells at 32.5 and 395°C.
JHM released from the carrier culture could once again readily establish a persistent infection in the rat glial cells in the same way as the L-2 cell-grown virus. JHM derived from the carrier culture was not temperature-sensitive for growth on L-2 cells, since the number and size of plaques formed at 39.5 and 325°C were the same. Furthermore, the appearance of plaques formed on L-2 cell monolayers by JHM grown on RN2-2 cells is indistinguishable from that of JHM propagated in L-2 cells. It may be of significance, however, that JHM virus, when propagated in RN2-2 cells, is highly temperature-sensitive.
This became evident upon shift of RN2-2 carrier cultures from 32.5 to 39.5"C, which resulted in a complete suppression of CPE and the release of PFU into the medium. The fact that freshly inoculated JHM virus failed to replicate but became eclipsed in RN2-2 cells at 39.5% is consistent with this observation. It is interesting that measles virus, HalIe strain, similarly acquires thermal lability when propagated persistently in RN2-2 cells (manuscript in preparation). These results imply that expression of temperature restriction on JHM and measles virus was somehow conferred by a property of the host cell rather than by the virus itself.

Heterologous
Viral Challenge To determine whether the JHM persistently infected RN2-2 cultures were resistant to superinfection by an unrelated virus, vesicular stomatis virus (VSV) was inoculated onto either uninfected or persistently infected RN2-2 cells (passage 5 or 6) at an moi of 0.1. Following incubation for 24 hr at 32.5"C, the yield of PFU of VSV in the supernatant of each culture was assayed on monolayers of both L-2 and RN2-2 cells. In two separate experiments, we found that yields of VSV from the JHM persistently infected RN2-2 culture, whether determined on L-2 cells or RN2-2 monolayers, were only about 10% of those from uninfected RN2-2 cells. in appearance or location from those found in the CNS of mice inoculated with the JHM strain. In the liver, disseminated foci of hepatic necrosis were frequent, emphasizing the viscerotropic properties of MHV,. A comparison of the histological findings of the MHV,-and JHM-inoculated mice reveals the following general differences.
First, those mice with CNS lesions due to MHV, inoculation had a more striking necrotizing meningitis than animals inoculated with the JHM strain. Second, the involvement of white matter and histological evidence of demyelination were more striking in JHM-infected mice than in MHV,-inoculated mice. The destructive lesions of the white matter were characterized by vacuolation, disruption of the normal architecture  Infection of the glial cells by the prototype virus MHV,, on the other hand, was neither productive nor persistent, since there was no detectable virus replication, and the fraction of cells scoring as infectious centers was at best very small indeed. Infection with either JHM or MHV, in the glial cells is clearly quite different from that associated with L-2 cells. In the latter case, both viruses undergo fully productive replication cycles, culminating in a complete destruction of the culture.
The tropism of the JHM strain for the rat RN2-2 glial cells uncovered in the present study indicates that a host cell can exert a selective pressure on a virus and determine the outcome of an infection. The ability to discriminate between the JHM and MHV, viruses, however, is not a general property of all cultured rodent glial cells examined by us to date. As indicated in Table 2, the C6 line, an astrocytoma-derived line of rat cells (Benda et al., 1968), failed to become infected altogether by JHM or MHV,. By contrast, inoculation of glial lines G26-20 and G26-24 of murine origin (Sundarraj, Schachner and Pfeiffer, 1975) resulted in the establishment of persistent infections with both JHM and MHV,. In these latter cases, the carrier state is associated with restricted CPE and cyclical production of low quantities of infectious virus. To date, the ability to discriminate between JHM and MHV, therefore appears to be a rather unique property of the RN2-2 cells.
As indicated in The molecular events by which JHM establishes and maintains its persistent infection and by which MHV, is suppressed in the RN2-2 cells remain to be elucidated.
In other similar carrier systems, several mechanisms have been proposed. These include competition at the synthetic level by defective interfering (DI) particles (Holland et al., 1976;Kawai and Matsumoto, 1977), cyclical suppression of virus production by interferon (Wiktor and Clark, 1972) and genotypic alterations of the virus (Thacore and Younger, 1969;Kawai, Matsumoto, and Tanabe, 1975;Younger et al., 1976;Truant and Hallum, 1977).
In the JHM persistent infection, the role of DI particles may be improbable since the initial moi used was low, and by changing the medium daily, and DI particles that may have accumulated would have been removed unless they somehow had remained cell-associated. Nevertheless, this argument is based on circumstantial evidence, so that proof against the presence of DI particles must await direct determination.
Partial resistance of RN2-2 cells, persistently infected with JHM, to superinfection with VSV suggests that interferon or an interferon-like mechanism may be functioning.
Clearly, additional work is also required to ascertain whether interferon may be a factor in the observed persistence. By analogy to the present system under study, reference should be made to a persistent infection of BHK-21 cells by rabies virus (Wiktor and Clark, 1972). In this system, the virus yield and interferon levels are produced in a cyclical sequence whereby the times when high levels of interferon are present coincide with periods of low yields of virus, and vice versa.
A common feature of other viral carrier systems is the outgrowth of, or selection for, a variant progeny virus that is temperature-sensitive for growth. Thus temperature-sensitive mutants have been isolated in persistent infections of measles virus (Haspel et al., 1973;Gould and Linton, 1975;Armen et al., 1977) and Hallum, 1977) and Sendai virus (Kimura et al., 1975). The JHM restriction at elevated temperature is not associated with growth in the fully permissive L-2 cells, however, but only in RN2-2 cells. The JHM strain fails to establish persistence when inoculation is carried out at 395°C and is not replicated when carrier cultures are shifted from 32.5 to 39.5%.
Similarly, temperature sensitivity is con-RN2-2 When taken together, these results imply that the determination of the outcome of the infection, whether fully productive, persistent or aborted, is controlled at least in some measure by the host cell.
Nevertheless, the biological properties of either MHV, grown on L-2 cells or JHM grown on L-2 cells or RN2-2 are not host-modified as a result of passaging through the different hosts. Thus the pathological manifestations produced by MHV, grown in mice or on L-2 cells are like those previously described for MHV, (Piazza, 1969), while the CNS lesions resulting from inoculation by JHM, whether derived from brain passage, L-2 cells or RN2-2 cells, are similar to those previously reported by Bailey et al. (1949), Pappenheimer (1958, Waksman andAdams (1962) andWeiner (1973 Plaque Assay Virus production was monitored by a plaque assay on L-2 cell monolayers. L-2 cells were grown in 6-well multidishes (Costar) at 37°C in a humidified atmosphere of 5% CO*. Confluent monolayers were inoculated with 0.2 ml of virus dilution in phosphatebuffered saline (PBS). After adsorption at 32.5"C for 1 hr, the infected cells were overlaid with 3 ml of MEM containing 10% FBS and 0.5% methyl cellulose, 4000 cps. The plates were incubated at 32.5"C for 2 days in the humidified atmosphere, after which the medium was removed and the cells were fixed with neutral buffered formalin. The plates were then stained with 0.1% crystal violet. The virus titer is represented as the plaque-forming units per ml (PFU/ml).
There is no apparent difference in the morphology or the size of the plaques formed by the two virus strains on L-2 cells.

Infectious
Center Assay To determine the percentage of glial cells releasing infectious virus, infected monolayers of RN2-2 cells were washed and trypsinized.
After trypsinization, various dilutions of the cells were plated on L-2 cell monolayers.
After 3-5 hr at 32.5X, the plates were overlaid with the methyl cellulose-MEM mixture, as described for the plaque assay, and processed in a similar manner.