key: cord-0004959-e6gdplxm
authors: Pearson, J.; Mims, C. A.
title: Selective vulnerability of neural cells and age-related susceptibility to OC43 virus in mice
date: 1983
journal: Arch Virol
DOI: 10.1007/bf01309260
sha: 1e66cb771ad693d5e0cd468c6701a8eef6a30638
doc_id: 4959
cord_uid: e6gdplxm

Suckling CD1 mice infected intracerebrally or extraneurally with OC43 virus developed a lethal neurotropic infection with high titres of virus in the brain. Examination of infected brain by routine H & E staining revealed no necrosis even in extensively infected tissue. Resistance to infection developed with increasing age, and by 20 days of age mice were completely insusceptible to i. c. inoculation. Virus replication was also demonstrable by FA staining, in spinal cord, dorsal root ganglia and retina. All other tissues were insusceptible and in particular, macrophages from both susceptible and resistant mice were found to be resistant to infection bothin vivo andin vitro. Immunosuppression rendered 15 day old mice more susceptible to infection but adult mice remained insusceptible. The transfer of immune or non immune spleen cells from resistant mice did not confer resistance to newborn mice. Treatment of resistant mice with anti interferon globulin (AIG) did not render them more susceptible. These results indicate that the immune response is partially responsible for the development of resistance to OC43 infection but that it is only partially protective and other factors must also be required. The basis for the unique susceptibility of neural tissues in suckling mice is being investigated.

An age-related resistance to infection has been described for various viruses in mice including members of the coronavirus family (5, 7, 1t) . Age-related resistance to eoronaviruses has been attributed to: i) Age-related changes in the ability of macrophages either to support infection or their ability to hmit virus spread (4, 7, 9) .

ii) Maturation of cell mediated immunity with increasing age (4) . iii) Age-related changes in interferon production (10) . iv) A combination of these mechanisms.

Macrophages have been shown to play an important role in the resistance of older mice, peritoneal macrophages taken from 4 week old (resistant) C 3 H mice infected in vitro with mouse hepatitis virus strains produced 1/10 as much virus as macrophages from 1 week old (susceptible) mice (9) . The same workers showed that peritoneal cells transferred from 4 week old mice to one week old mice rendered these mice resistant to subsequent infection. Also young SJL mice can be protected against infection with the JHM strain of MHV by transferring adherent peritoneal cells (macrophages) from older resistant mice (7) or by transferring spleen cells from immune adults (5) . The transfer of resistance to MI-IV 3 requires T lymphoeytes together with adherent ceils and older mice have been rendered susceptible to infection by treatment with immunosuppresive drugs (4) .

Interferon also appears to play a role in age-related resistance to MHV3. Strain A/J mice are normally resistant to MHV3 when older than 15 days but mice up to 6 weeks old can be rendered susceptible when treated with anti-interferon globulin one hour prior to inoculation of virus (9, 10) . Resistant 4 week old C3 H mice infected with MHV-S generate 4--8 times as much serum interferon as susceptible 1 week old mice (9) .

In different experiments therefore, age-related resistance of mice to infection with various types of MHV appears to be related to maerophages, with or without T cells, and also to interferon production.

All the above reports concern mouse eoronavirus. No studies on the pathogenesis or neurotropism of 0C43 or other human eoronaviruses, which are of eonceivable importance in diseases such as multiple sclerosis, (1) have been reported. Although these viruses are assumed to cause harmless upper respiratory tract infections the ability of other coronaviruses to invade the CNS make them worthy of study.

OC43 virus was received as the 8th passage of infected suckling mouse brain suspension and was obtained from Dr. D. A. J. Tyrrell. Clinical Research Centre, Northwiek Park, Middlesex. Stock virus was an infected suckling mouse brain suspension which had a titre of 109 SM ic LD 50/ml.

All CD 1 outbred and C57 BI6 mice were specific pathogen free, obtained from the Spaceway unit, Guy's Hospital Medical School. SJL/J inbred mice were received as a gift from Jay Wallace, Chester Beatty Institute, London.

Mouse embryo brain (MEB) cells were derived from 16 day old embryos of CD 1 mice and were plated on to plastic tissue culture flasks (Falcon) or "ring" eu]tures (2) on microscope slides.

Resident peritoneal cells were obtained from mice in Eagles Minimal Essential

(MEM) containing I0 U FIeparin/ml plus 15 per cent heat inactivated foetal calf serum (FCS). They were added directly into "ring" cultures, allowed to settle for zl hours, washed vigorously to remove non-adherent cells, and heparin-free medium replaced.

Homogenised tissues were obtained and dilutions added to ring cultures of primary MEB cells atlowed to absorb for 1 hour and then ruled with MEM containing 2 per cent heat inactivated FCS. Three days later the cell monolayers were fixed and stained by the fluorescent antibody (FA) technique (see below). Infected rings were then scored and the virus titre calculated as tissue culture infectious dose (TCID50)/ml. On occasions the virus titre was determined as the suckling mouse i. c. lethal dose 50 (SMie L D 50).

The indirect method was used for F A staining of both eryostat sections of tissues and celt cultures. Antiserum to OC 43 virus was raised in mice (a 1 in 2560 dilution of AS was capable of neutralizing 100 sm LD 50 as detected by ic inoculation into 1 day old mice) and FITC conjugated goat anti-mouse imrnunoglobutin was purchased from Nordic, Maidenhead, Berks. stained slides mounted in neutral glycerol-PBS were examined with a Leitz Orthoplan microscope equipped for UV observation, using water immersion objectives.

Spleens from each group of mice were pooled and a single cell suspension was made by passing the spleens through a metal sieve in ice cold Hanks medium supplemented with 5 per cent FCS. The cells were washed and resuspended to give 3 × 10 s cells/ml and 0.05 ml (15 × 106 cells) was inoculated i.p. into suckling mice. 80 per cent of cells were viable as indicated by trypan blue exclusion. The spleens and cell suspensions were maintained at 4 ° C to minimize the loss of adherent ceils.

Infection of suckling CD 1 mice with 0C43 virus b y either intraperitoneal or intracerebraI routes results in a lethal neurotropic infection and the mice die after 3 --7 days. 1 d a y old mice were infected either i.p. or i.c. and t a k e n when sick 2 --4 days post infection. The brains of these mice contained 1010 SMJc LDs0/G b u t neither viral antigen (FA) nor infectious virus could be detected in the heart, liver, spleen, lungs, kidney, intestine, muscle, t h y m u s or adrenals. I m m u n ofluoreseent examination of brains revealed occasional foei of infection at 16 hours post infection. B y 48 hours there was extensive and generalised inolvement of the cortical grey m a t t e r ( Fig. 1) with striking infection of large bands of hippocampal neurones. Basal ganglia and t h a l a m u s were sparsely infected. Infection of the cerebellum, however, was restricted to Purkinje cells, white m a t t e r a n d granule cells did not contain viral antigen (Fig. 2) . Infected cells were seen in spinal cord, dorsal root ganglia (Fig. 3 ) and in the retina (Fig. 4) . The experiment. was r e p e a t e d with 5 d a y old mice with identical results. E x a m i n a t i o n of infected brains b y H & E did not reveal cell damage even in tissue t a k e n from extensively infected brains.

CD1 mice infected with 0C43 virus display an age-related susceptibility becoming resistant to i. c. infection after 15 days old and resistant to i.p. infection after 10 days. The dose of virus r e q u i r e d to cause death increases w i t h the age of the animal (Table 1 ). S u b d e t h a l doses cause no detectable disease. Mice older t h a n 

Immunosuppression Immunosuppression of adult mice (12 weeks old) with cyelophosphamide (12) (200 mg/Kg 3 and 7 days p.i.) did not render them susceptible to very large amounts of OC43 virus inoculated i.c. ( Table 2 ). All the treated :mice remained well throughout the experiment and on FA examination at 7dpi all brains were negative for viral antigen. 40 per cent of the 15 day old mice, however, became susceptible to 102 SMie LDs0 following eyelophosphamide treatment and died within 7 days. The brains of 6 of these mice were examined by FA staining and 

Immune and non-immune spleen cells from 12 week old CD1 mice were transferred to 3 day old and also to 9 day old suckling mice by i.p. inoculation. The mice were challenged i.p. or i.c. with a lethal dose of virus 24 hours after transfer. Neither immune nor non immune spleen cells were capable of protecting the suckling mice ( Table 3) a The figure in brackets represents the virus dose in relation to the lethal dose for that route of inoculation in that age of mice,

Twelve 6 week old mice were treated with 0.1 mI anti-interferon globulin (10) (AIG) (a gift from Ion Gresser-Institute de Recherches Scientifiqnes sur le cancer, Villejuif) intravenously followed one hour later b y i.c. inoculation of 106 SMLDs0 0C43. A t 3 days post infection the animals were given a second dose of AIG. 15 control mice were t r e a t e d with virus onlyo 11 of the 12 mice t r e a t e d with AIG remained well and F A e x a m i n a t i o n of these brains at 7 d a y s p.i. did not reveal a n y viral antigen. One mouse died 3 days p.i. and extensive infection of the brain was seen by F A staining. Of the 15 mice treated with virus only, none became sick and none d e m o n s t r a t e d viral antigen in the brain.

W h e n 0C43 virus infects suckling mice it i n v a r i a b l y causes death following acute infection of the nervous system. High titres (1010 SMic LDs0/G) of virus can be recovered from the brain and extensive infection can be d e m o n s t r a t e d b y F A staining. Spinal cord, dorsal root ganglia and retina are also infected. There is, however, no growth of virus in non neural tissues as detectable either b y the presence of infectious virus or viral antigen. CD 1 mice become resistant to i.c. or i.p. infection by 20 d a y s of age a n d this resistance could reflect the inability of the virus to either re~ch or grow in its target organ. The failure to detect infected cells in the brain of adult mice following direct intracerebral injection of virus suggests t h a t t h e y are resistant because 0C43 virus does not grow in their brain cells. The timecourse of m a t u r a t i o n of the immune system (6) appears to parallel the development of resistance in mice. F o r this reason a d u l t (resistant) mice were immunosuppressed with cyclophosphamide (12) prior to infection with 0C43, but this did not render t h e m susceptible to infection. T r e a t m e n t of 15 d a y old mice with cyclophosphamide, however, rendered t h e m susceptible to a previously sub-lethal dose. This indicates t h a t the immune system, whilst being p a r t i a l l y protective in 15 d a y old mice, was not the sole factor in this resistance. Suckling mice could not be protected from infection b y the transfer of immune or non-immune spleen cells, in spite of the fact that large numbers of cells were transferred and care was taken to include adherent cells. The failure of transferred spleen cells to protect against infection could be due to their inability to localise in the site of viral growth i.e. the CNS as OC43 is exclusively neurotropie. I t has been demonstrated in MHV8 virus infections (3, 7) tha,t the maerophages of (susceptible) suckling mice support viral replication whilst those of adult (resistant) mice do not, and that this accounts for the differences in susceptibility. I n the above experiments the maerophages of CD 1 mice of all ages were uniformly insusceptible to OC43 virus infection, both in vitro and in vivo. The age-related resistance to 0C43 virus infection therefore does not depend on the inability of virus to grow in maerophages. This does not rule out a contribution of adult macrophages to control the infection by controlling virus replication in other cells (8) .

Treatment of adult (resistant) mice with AIG did not cause a significant increase in mortality following infection. This indicates that interferon is not involved in the resistance of adult mice to 0C43 virus, in contrast to the results reported for MIIV3 infection (10) . This difference in response to AIG could be due to the action of interferon on the extraneural growth of MHV3 whereas it was not capable of limiting the rapid growth of OC43 in the CNS. Resistance to i.p. inoculated virus appears earlier in life than resistance to i.c. inoculated virus ( Table 1) . I t is possible that the rate of clearance of virus from the blood is more efficient in older mice following i.p. infection, and this prevents virus from reaching the brain. Experiments to monitor the rate of clearance from the blood did indicate that inoculated virus could be detected in the blood of suckling mice at higher concentrations for slightly longer periods of time (data not shown). This could contribute to the development of resistance to i.p. infection. I t is concluded that age-related resistance of mice to 0C43 virus is likely to depend on changes in the ability of neural cells to support virus growth, possibly because of the absence of virus receptors. Experiments with cultured neural cells are being earried out to substantiate this. 

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

The initiation of vaceinia virus infection

Ontogeny of maerophage resistance to mouse hepatitis in rive and in vitro

Neonatal susceptibility to MHV~ infection in mice. I. Transfer of resistance

Analysis of age-dependent resistance to routine eoronavirus JHM infection in mice

Maturation of the humeral immune response in mice

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

Macrophage antiviral activity: Extrinsic versus instrinsic activity

Factors involved in the age-dependent l~sistanee of mice infected with low virulence mouse hepatitis virus

Role of interferon in the pathogenesis of viral diseases of mice as demonstrated by the use of anti-interferon serum. V. Protective role in mouse hepatitis virus type 3 infection of susceptible and resistant strains of mice

The biology and pathogenesis of coronaviruses

Effect of cyclophosphamide on the genetic resistance of C~H mice to mouse hepatitis virus

Received April 11, 1983