key: cord-0921422-si960hzb
authors: Barthold, S. W.; Beck, Deborah S.; Smith, A. L.
title: Mouse hepatitis virus nasoencephalopathy is dependent upon virus strain and host genotype
date: 1986
journal: Arch Virol
DOI: 10.1007/bf01314284
sha: 4aadbb62eeea095e0b3849eda7089aeb19016c85
doc_id: 921422
cord_uid: si960hzb

Mouse hepatitis virus (MHV) S induced typical MHV spongiform lesions in brainstem 28 days following intranasal inoculation of adult A/J, BALB/cByJ, CBA/J, C3H/HeJ and C3H/RV, but not SJL mice. In all but SJL mice, brain lesions occurred at or near the infectious dose level, based on seroconversion by the indirect immunofluorescence assay. During the acute phase of infection (day 5), lesions were limited to the nose and brain in most genotypes. Exceptions were BALB mice, which had mild hepatitis and SJL mice, which had lesions restricted to the nose. No mortality occurred in any genotype. Following intranasal inoculation of adult mice, MHV-1, -3, -A 59,-JHM and -S all caused brain lesions at 28 days after inoculation. MHV-1 and-3 caused lesions that were usually restricted to the anterior olfactory tracts, while MHV-A 59, -S and -JHM also caused more generalized and pronounced lesions involving the midbrain and pons. These studies suggest that avirulent MHV-S given intranasally to most mouse genotypes is a good model for induction of brain infection in the absence of mortality. They also confirm observations made by others in which MHV-JHM, -S and -A 59 are relatively more neurotropic than other MHV strains, such as MHV-1 and -3.

Mouse hepatitis virus (MHV) is a coronavirus with several antigenically and biologically distinguishable strains (3) . MHV-JHM has been studied extensively as a model for virus-induced encephalitis and demyelination in mice and rats (1, 9, 10, 13, 14, 16, 17, 21, 22, 27, 31, 37) . The relevance of this model has recently been augmented by the association of coronaviruses with some eases of human multiple sclerosis (6, 8) . MHV-JHM is emphas~ed as a relatively unique neurotropie strain of MHV, but neurotropism may" be a common biological feature of several other MHV strains. It has been shown that three antigenically distinguishable MHV strains (MHV-JHM, -S and -A59) can induce demyelination (1, 4, 12, 19, 39) . At least 2 of these strains (MtlV-JHM and -S) can infect the brain by direct extension from the nasal mucosa to the olfactory tracts, apparently in the absence of other organ involvement (4, 9, 32, 33) . It remains unclear if other MHV strains can cause nasoencephalopathy in adult mice, but most MHV strains are at least encephMitogenic following oronasal inoculation of suckling mice (5) . Confhsion over the relative neurotropic properties of this group of viruses may emanate from differences in disease caused by factors such as virus dose, route of inoculation, passage history, host age and genotype (14) .

The purpose of this study was to determine if low-virulence, neurotropic MHV-S can induce brain lesions in adult mice of different genotypes following intranasal inoculation and if brain infection is dose-dependent by this route of exposure. We also investigated the ability of several different prototype MHV strains to infect brain following intranasal inoculation of adult mice.

Inbred A/J, BALB/cByJ, BALB/cJ, CBA/J, C 3 H/HeJ and SJL/J mice were purchased from The Jackson Laboratory, Bar Harbor, ME. Outbred Cr 1 : CD-1 (ICR) BR Swiss mice were purchased i?om Charles River Breeding Laboratories, Portage, MI. Inbred C 3 tt/RV mice were obtained from a pathogen-f?ee breeding colony at Yale. Mice from all sources were free of serum antibody to MHV as determined by indirect immunofluorescence (26) . Mice were transferred frora their source in filtered boxes at 6 weeks of age and placed immediately on arrival into autoclaved, filter-top Micro-Isolator cages (Lab Products, Maywood, NJ) with sterile food, water and bedding in a room separate from other laboratory rodents. Cages were handled aseptically as previously described (4) to preclude contamination by experimental or adventitious MHV. Uninoculated, sentinel mice in open cages were maintained in the experimental room and periodically tested for seroconversion to MHV. Mice were killed with carbon dioxide gas and exsanguinated by cardiac puncture.

MHV-S, -JHM, -1, -3 and -A 59 were obtained from the American ~l~gpe Culture Collection, I~ockville, MD and passaged in mycoplasma-free NCTC 1469 celt cultures. Inocula consisted of cell culture fluid containing approximately 10 a median tissue culture infectious doses (TCIDs0)/100 ~tl of each virus strain or dilutions thereof. Unanesthetized mice were inoculated intranasally with 10 IxI of virus stock in a Class IIB biological safety cabinet.

Sera were diluted 1 : l0 and tested for MHV antibody by an indirect immunofluorescence assay-, using a bivalent antigen consisting of cultured L cells infected with MHV-S mixed with L cells infected with MHV-JHM (26) . Median infectious dose based on seroconversion was calculated by the method of REED and MUENCH (23) .

Tissues were fixed in neutral buft~red formalin, pH 7.2 and processed by routine histological technique. Nose, olfactory bulb, brain, liver and intestine were examined on day 5 after inoculation. This interval has been determined to be best suited for examination of the peak acute phase of MHV infection (4, 5) . Sections of brain consisted of a coronal hemisection through the olfactory bulbs; anterior cerebrum; mid-cerebrum and brainstem; and cerebellum and pons. Spongiosis with demyelination is most apt to be detected in the mesencephalon and pons following intranasal inoculation ofMHV (1, 4, 9, 33) . Median infectious dose based on brain stem lesions was calculated by the method of ]~EED and MUENCH (23) . Statistical evaluations were performed using the Chi-square method (28) .

The susceptibility of several selected genot)Tes of mice to the effects of different doses of neurotropic MHV was tested using MHV-S. This virus strain was chosen because of its low virulence and proven ability to induce nasoencephalopathy in the absence of other organ involvement, thus minimizing mortality (4, 9, 32, 33) . Mouse genotypes were selected to represent a spectrum of strains with susceptibihty and resistance to various strains of MHV described by others (2, 14, 15, 17, 18, 20, 25, 29, 30, (34) (35) (36) 38) .

Groups of 5 mice of each genotype were inoculated with serial 100-fold dilutions of virus stock and the median infectious dose was established based on seroconversion and presence of brain stem spongiform lesions at 28 days. In addition, an extra 5 mice of each genotype were inoculated with the highest dose level (1 × 103 TCtDs0) of MttV-S and killed at 5 days for comparison of acute disease patterns among genotypes.

As expected, MHV-S induced only mild acute disease in the inoculated adult mice (Table 1) . No mortality was observed. Mice of all genotypes developed mild necrotizing rhinitis on day 5. Only BALB/cByJ mice had (Table 2) . Chi square anMysis rejected in@pen@nee of the 2 variables of seroconversion and spongiosis (P < 0.001). Furthermore, no differences in severity of spongiosis were noted between dose levels or genotypes. SJL mice were an exception, since they required a remarkably higher dose of MHV-S for seroconversion, and none developed brain lesions at any dose level. Five prototype MHV strains were evaluated for their ability to induce brain lesions in adult susceptible (BALB/cByJ) mice by the intranasal route. Because of the high virulence of MHV-3 and the high susceptibility of BALB mice, outbred CD-1 were inoculated with MHV-3. Strains JHM and S were used as positive controls, since both cause nasoencephalopathy with demyelination (4, 9, 21, 32, 33) . Strain A 59 has been reported to cause demyelination by intracerebral inoculation (12, 19, 39) , but its pathogenicity by the intranasal route has not been established. Strains 1 and 3 can infect brain following intracerebral or intraperitoneal inoculation (7, 36) but spongiosis or demyelination have not been observed with these strains.

Histological evidence of brain infection was present at 28 days after intranasal inoculation of mice with all MHV strains tested (Table 3 ). In some of the mice (5/14) inoculated with MHV-1 and most of the mice (8/9) inoculated with MHV-3, very mild nonsuppurative meningitis, gliosis and spongiosis selectively involved the tractus olfactorius of the olfactory bulb and of the anteroventral cerebral cortex (Fig. 1) , but not other areas of brain. Most of the mice inoculated with MHV-A 59, -JHM and -S and a single mouse inoculated with MHV-3 also developed prominent patches of spongiosis in the midbrain and pons (Fig. 2) . Chi square analysis of the prevalence of brainstem spongiosis among treatment groups revealed significant differences between virus strains (P < 0.001). Mortality varied markedly among treatment groups (Table 3) . Neither MHV-S nor MHV-1 caused mortality; MHV-A 59 caused a low prevalence of deaths; MHV-JHM caused death in half of the BALB mice inoculated between days 5-11. MHV-3 caused moderate mortality among the outbred Swiss mice. 

Although M H ¥ \ S has not been studied as extensively as MHV-JHM as a neurotropic MHV strain, MHV-S offers the distinct advantage of possessing equal neurotropism but low virulence. Both MHV-JHM and MHV-S appear to follow similar olfactory pathways into the brain and induce a similar distribution of spongiform brain lesions in intranasally-inoeulated mice (4, 9, 32, 33) . Results of this investigation indicate that following intranasal inoculation, MHV-S induces a reproducibly high prevalence of brainstem lesions with minimal involvement of other organs and no mortality in most adult mouse genotypes. Intranasal inoculation of MHV-S has been shown to be equally effective as intracerebral inoculation for inducing brain infection (32) , but this strain is remarkably avirulent, regardless of the route ofinoculation (11) . The spongiosis with minimal encephalitis and mortality seen in mice of the several genotypes tested parallel the effects of non-encephalitogenie, temperature-sensitive mutants of MHV-JHM following either intracerebral or intranasal inoculation. In contrast, as we noted in this study, wild-type MHV-JHM produces a high mortality dne to encephalitis (10, 11, 16, 17, 24) . Following intranasal inoculation of MHV-S, brain stem spongiosis occurred in most genotypes at or near the infectious dose level, based on comparison of brain lesions with seroconversion. The indirect immunofluorescenee assay" utilized in this study is very sensitive for detecting seroconversion to MHV following natural or experimental exposure of adult mice to MHV (26) . Our studies suggested that once an infectious intranasal dose of MHV-S is achieved, higher doses do not influence the subsequent severity of infection. This suggests that brain infection occurs after initial infection and replication in the nasal mueosa, which has been shown for both MHV-S and MHV-JHM (4, 9, 32, 33) . Thus, MHV-S given intranasally appears to be a good model for induction of nasoencephalopathy that does not require laboratory attenuation of virulence, has a wide effective dose range and can be used in many genotypes without excessive mortality.

Genotypic resistance to infection has been demonstrated with several MHV strains, usually using intraperitoneM or intracerebral inoculation. The genotypes examined for susceptibility to intranasal MHV-S were selected on this basis. BANG et al. noted resistance of C 3 H mice to MHV-2 (2) and later developed eongenie C 3 H mice which were susceptible to MHV-2 (38) ). The C 3 H/HeJ mice and C 3 H/RV mice utilized in this study are congenic for this resistance locus. Although C 3 H/HeJ mice are resistant to MHV-2, they have been found to be semi-susceptible to MHV-3 (36) . Resistance of A/J mice has been demonstrated to MHV-3 (20, 25) , but this genotype seems to be susceptible to MHV-JI-IM (15, 29) . A marked dichotomy of resistance and susceptibility to neurotropie JHM has been noted between BALB and SJL mice (14, 15, 17, 29, 30) . Mechanisms of resistance are multifaeterial and vary with host age and genotype, virus strain, dose and route of inoculation (14) . In our studies with low-virulence MHV-S given intranasally to adult mice a spectrum of resistance and susceptibility among genotypes was not particularly apparent. Strains A, BALB, CBA and C 3 H were all infected at, a similar virus dose level and all developed a similar degree of nasoencephalopathy. BALB/eByJ mice developed mild hepatitis, while other strains did not, suggesting slightly higher susceptibility. SJL mice were remarkably resistant to MHV-S and did not develop brain lesions, as noted by others with more ~rulent MHV-JHM (14, 15, 17, 29, 30) .

Most strains of MHV display neurotropism following oronasal inoculation of susceptible suckling mice (5) . On the other hand, brain stem spongiosis with demyelination has been observed with only a relatively few strains of MHV, including MHV-JHM (1), MHV-S (33) and MHV-A 59 (19, 39) . t~eports of histological studies in mice intracerebrally inoculated with MHV-1 and MHV-3 mention inflammatory changes but not spongiosis or demyelination (7, 36) . Results of our studies confirm the ability of MHV-JHM, -S and -A 59 to cause widespread brain lesions following intranasal inoculation. Our data also indicate that MHV-1 and -3 are also capable of infecting brain following intranasal inoculation, but lesions are generally restricted to the anterior olfactory tracts. The mechanism of this virus strain-related restriction requires further investigation. It does not appear to be related to virulence, since virulent MHV strains can cause both restricted (MHV-3) and generalized (MHV-JHM) patterns and avirulent MHV strains likewise cause both restricted (MHV:I) and generalized (MHV-S) patterns.

A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin. II. Pathology

Mouse macrophages as host cells for the mouse hepatitis virus and the genetic basis of their susceptibility

Complications of viral and mycoplasmal infections in rodents to toxicology research testing

Mouse hepatitis virus S in weanling Swiss mice following intranasal inoculation

Mouse hepatitis virus strain-related patterns of tissue tropism in suckling mice

Two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients

A virus related to that causing hepatitis in mice (MHV)

Coronavirus isolates SK and SD from multiple sclerosis patients are serologically related to routine coronaviruses A 59 and JHM and human coronavirus 0C 43, but not to human coronavirus 229 E

Nasoencephalopathy of mice infected intranasatly with a mouse hepatitis virus, JHM strain

Temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination

Comparison of mouse hepatitis virus strains for pathogenicity in weanling mice infected by various routes

Temperature-sensitive mutants of mouse hepatitis virus strain A 59: isolation, characterization and neuropathogenic properties

Selective localization of wild type and mutant mouse hepatitis virus (JtLM strain) antigens in CNS tissue by fluorescence, light and electron microscopy

Host and virus factors associated with CNS cellular tropism leading to encephalomyelitis or demyelination induced by the JHM strain of mouse hepatitis virus

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Selected mutants of mouse hepatitis virus type 4 (JHM strain) induce different CNS diseases

Host genetic control of mouse hepatitis virus type 4 (JHM strain) replication. I. Restriction of virus amplification and spread in macrophages from mice

Experimental demyelination produced by the A 59 strain of mouse hepatitis virus

Genetic study of mouse sensitivity to MHV-3 infection: influence of the H-2 complex

Pathology of infection with the JHM virus

01igodendrocytes and their myelin-plasma membrane connections in JHM mouse hepatitis virus encephalomyelitis

A simple method of estimating fifty percent endpoints

Pathogenic murine eoronaviruses. III. Biological and biochemical characterization of temperature-sensitive mutants of JHMV

Activation of natural killer cells and induction of interferon after injection of mouse hepatitis virus type 3 in mice

An immunofluorescenee test tbr deMction of serum antibody to rodent coronaviruses

In rive and in vitro models of demyelinating diseases

Principles and procedures of statistics

Resistance to fatal central nervous system disease by mouse hepatitis virus, strain

Macrophages and resistance to JtIM virus CNS infection

Chronic central nervous system demyelination in mice after JtIM virus infection

Age-dependent response of mice to a mouse hepatitis virus, MHRT-S

Pathogenesis of mouse hepatitis virus infection. The role of nasal epithelial cells as a primary target of low-virulence virus

Difference in response to mouse hepatitis virus among susceptible mouse strains

Correlation of persistent mouse hepatitis virus (MHV-3) infection with its effect on nlouse macrophages

NeuropathologicM effects of persisten~ infection of mice by mouse hepatitis virus

JHM infections in rats as a model for acute and subacute demyelination disease

Congenic strMns of mice susceptible and resistant to mouse hepatitis virus

Acute and snbacute demyelination induced by mouse hepatitis virus strain A59 in C3H mice

This work was supported by Public Health Service grants RR-020309 and R1%-00393 from the Division of I~esearch t~esourees.

l~eceived November 23, 1985