key: cord-0007689-eyk015n3
authors: Powell, H.C.; Lehrich, J.R.; Arnason, B.G.
title: Electron-microscopic appearance of the DA virus, a demyelinating murine virus()
date: 2003-03-06
journal: J Neurol Sci
DOI: 10.1016/0022-510x(77)90087-9
sha: a327ca577be60740ff8562f671bd0d7bd67385ba
doc_id: 7689
cord_uid: eyk015n3

The DA virus is a neurotropic murine virus which can induce acute encephalomyelitis in suckling mice and a chronic myelopathy in weanlings. The agent has been attenuated by serial passage in baby hamster kidney (BHK-21) cells. When attenuated virus is inoculated in 8-week-old C(3)HeJ mice a myelopathy of delayed onset with prominent demyelination of lateral and anterior columns occurs. The DA virus is believed to be related to the Theiler murine encephalomyelitis (TME) viruses because of the similar clinical and pathological conditions which it causes, and because neutralization tests indicate shared antigens between it and GD7, a TME virus. This paper reports electron-microscopic studies of BHK-21 cells infected with DA virus. The cells were prepared 24 and 48 hr after inoculation. Cytopathic effects were observed and infected cells contained plaques consisting of numerous 25 nm virus particles in crystalline array. The virions were exclusively intracytoplasmic and were morphologically indistinguishable from human poliomyelitis virus. These observations appear to establish DA as a picorna virus, related to the TME virus group. The chronic myelopathy caused by DA may prove relevant to chronic demyelinative myelopathies in man, such as multiple sclerosis, and also to amyotrophic lateral sclerosis.

tlOUS agents which have common antigenic properties and can cause pollomyehtJs ill mice The first of these viruses, Theller's original virus (TO), was isolated from "l weanling unmoculated mouse which had developed flaccid paralysis of the hind limbs (Theller 1937) The disease could be reproduced by injecting infected CNS material into the brains of healthy mice TO virus is a neurotroplc agent capable of destroying neurons in the brain and anterior horn cells of the spinal cord Although TO virus can be Isolated from the intestines of almost all weanhng mice less than 6 weeks old, it ts rarely found in mouse CNS and occasional CNS infection can, like human pol,omyelitis, be described as a "biological accident" (Cahsher and Rowe 1966) The DA virus was recovered in 1948 from a mouse in the Harvard colony which had shown spontaneous paralysis over a period of 2 months (Daniels, Pappenheimer and Richardson 1952) The agent was propagated by serial passage in mouse brain and induced an acute encephalomyehtls in suckling mice Danlels et al (1952) also observed a more chronic myelopathy which developed m weanling animals The virus size was determined to be less than 100 nm by ultra-filtration techmques and DA virus was assumed to be a member of the plcorna viruses of lower animals, a group which includes TMEV (Darnels et al 1952) The DA virus was classified as a plcorna virus on the basis of its chmcal behavior and the pathologic lesions it caused Recently antlgemc relationships have been demonstrated between DA and GDVll (Lehrich, Arnason and Hochberg 1976), a plcorna virus which is a member of the TM EV group (Sturman and Tamm 1966) While serologic studies and its biologic acttvlty supported the original claim that DA virus is a plcorna virus, virus particles have not been visualized ultrastructurally Failure to see them in electron microscopic examination of white matter undergoing demyehnation was attributed to a very low content of infectious agent in the CNS of affected mice (Dal Canto and Lipton 1975) However, the DA virus has been tissue culture adapted (Lehrlch et al 1976) and the purpose of this paper is to show the ultrastructural appearance of DA virus grown m BHK-21 hamster fibroblasts

The DA virus, in mouse brain suspension (fifth passage, prepared in 1952) was kindly supplied by Mrs Joan B Danlels (Commonwealth of Massachusetts Virus Laboratories) The virus was adapted to BHK-21 hamster fibroblasts by means of serial passage Cells were disrupted by three cycles of freezing and thawing on the fourteenth day after inoculation of virus material, and the tissue homogenates were then inoculated on to monolayers of BHK-21 cells Cytopathic effects first appeared on the second passage in BHK-21 cells Thtrd passage virus was used to inoculate BHK-21 cell monolayers for electron-microscopic study

Specimens were harvested for electron microscopy at 24 and 48 hr after inoculation with DA virus Uninoculated BHK-21 fibroblasts were used as controls The cells were removed from the surface of the tissue culture vessel by trypslnlzatlon and were recovered from the tissue culture medium by centrlfugatlon in an International centrifuge operating at 150 × g The cell pellets were allowed to fix in 2 5 ~ phosphatebuffered glutaraldehyde for 4 hr at 4 °C The glutaraldehyde was then decanted and the pellets retained in phosphate buffer prior to processing for electron microscopy The cell pellets were postfixed for 45 mln in Dalton's chrome-osmium solution, dehydrated in a graded alcohol series, infiltrated with a propylene-oxlde Epon mixture and embedded in Epon One micron sections cut from Epon blocks were stained with methylene blue and examined by light microscopy Ultrathln sections were cut from selected blocks, stained with uranyl acetate and lead citrate, and examined on a Siemens Elm~skop 101 operating at 80 kV Viral size was determined from electron mlcrographs using the method described by Hatta 0976)

Cytopathlc effects were evident 24-48 hr after Inoculation of BHK-21 cells Llght-m~croscoplc examination of I #m plastic sections showed that many of the cells became rounded and that nucleoll became more prominent than in control BHK-21 cells Many degenerating cells were seen at 24 hr but destruction of cells was much more prevalent in the samples taken at 48 hr Electron microscopy Pathologic alterations in the infected tissue cultures were present both in cells which showed viral replication and in other cells without evident viral particles Cytoplasmic changes consisted of prohferation of membranous structures and vesicles in the centrosphere region of the cell (Fig 1) , dilated endoplasmlc retlculum and numerous cytoplasmic filaments Nuclear shape was often irregular, with marglnatlon of chromatin to the periphery and very prominent nucleoh Viruses were identified in sections from every block examined, both m specimens collected 24 hr after inoculation and m those taken at 48 hr Viruses were more frequently identified and degenerative changes more pronounced m the latter group of cells Vzrus particles aggregated to form lntracytoplasmlc crystals which were generally large (Fig 2) and exclusively mtracytoplasmlc The viral crystals were composed of orthogonal arrays of spherical electron-dense particles, the average dmmeter of which was 25 nm In many instances organelles such as mltochondrm and other membrane-bounded structures were encompassed by the virus crystal (Fig 3) Individual VlnOns were separated from each other by a 2-4 nm electron-lucent space Virus pamcles were present m apparently viable cells and also in degenerating cells which had markedly swollen endoplasmlc retlculum and ruptured nuclear membranes and lacked recogmzable mltochondrla In sections from tissue culture preparations harvested 48 hr after inoculation most of the cells had undergone necrosis, but wral crystals were prominent Within these crystals individual virus particles showed varying electron density and wral arrays were irregular (Fig 4) , as &stmct from the homogeneously electron-dense viral umts m crystals from cells processed 24 hr after inoculation

Electron-microscopic studies of tissue culture cells inoculated w~th DA virus show that the agent is a 25 nm sphencal electron-dense particle morphologically re-sembhng human polio virus as well as certain murlne plcorna viruses The particles are arranged orthogonally in the cytoplasm forming crystalline lattices (Fig 2) and can thus be readily distinguished from other 25 nm electron-dense structures such as polyrlbosomes and glycogen granules The International Committee on Enterovlruses (Melnlck et al 1963) has ruled that plcorna virus size ranges from 15 to 30 nm The estimated diameter of pohovlrus type I cultured in HeLa cells is 26-28 nm (Dales, Eggers, Tamm and Palade 1965) Other enterovlruses which lie within the 15-30 nm size range are FA virus, also a member of the TMEV group, encephalomyocarditlS (EMC) virus, mengovlrus, and Coxsackle virus These agents grow within the cytoplasmic matrix forming orthogonal crystalline arrays as does human poliomyelitis virus (Dales et al 1965) The changes associated with DA virus growth in BHK-21 cells included marked proliferation of smooth walled vesicles in the centrosphere region of the cytoplasm, lntracytoplasmlc filamentous accumulation, flattening and displacement of the nuclei and nucleolar changes In the cells containing viruses the particles showed hexagonal packing, and viral crystals frequently formed around mltochondrla and other cytoplasmic organelles Each of these changes has been observed during growth of EMC virus and mengovirus in tissue cultured L cells Both EMC and mengovlrus are plcorna viruses which measure 27-28 nm (Dales and Franklin 1962) and which cause encephalomyehtls in mice Similar cytopathlc changes have been observed in tissue cultured mouse liver cells infected with murlne hepatitis virus (MHV) (Davld-Ferreira and Manaker 1965), a member of the larger coronavirus group

In DA virus-infected cells harvested 48 hr after inoculation, viral particles showed varying electron density and the crystalline arrays became Irregular This change was described as "melting" by Mattern and Daniel (1965) , who observed it in HeLa cell cultures infected with human pohovlrus Dal Canto and Lipton (1975) have described an electron-microscopic study of the sequence of events leading to demyehnatlon in DA virus-infected mice, but the virus was not seen in the infected central nervous system tissue They attributed this to the low tlters of virus within infected spinal cord (maximum tlters never exceeded 105LDso/g tissue) It is also conceivable that the virus may exert a cytocldal effect without new virus production In studies of mengovirus, a plcorna virus which shares some biologic properties with TMEV (Andrewes and Perrelra 1972) tissue cultured macrophages were killed without virus production, whereas infected murlne fibroblasts produced large quantities of virus (Buck, Granger, Taylor and Holland 1967) It is possible that the DA virus like the mengovlrus has varying tropisms for cells of different tissues

The long persistence of infective DA virus in mouse CNS (Lipton 1975 , Lehrlch et al 1976 raises the possiblhty that demyehnatlon may be due to a cytocldal effect of the DA virus on the ohgodendrocyte Virus replication within ohgodendrocytes has been described in JHM virus encephalomyelitis (Lampert, Sims and Kniazeff 1973) and abnormalities of the myelin-plasma membrane connections of infected ohgodendrocytes have been illustrated (Powell and Lampert 1975) Lipton and Dal Canto (1976) present evidence suggesting an immune basis for the late appearing demyellnat-mg lesions m DA varus infected mace They report that ammunosuppres~on pre~el~t~ the onset of demyelmatlon In thear wew local persastence of antigen for a long period of time may be a crat~cal factor m reducing an ~mmune reaction Striking inflammatory changes have been seen m mouse spinal cords 9 months after DA wrus moculataon (Salomon 1976 ) and further ultrastructural stu&es are reqmred to determine whether the delayed lesions m white matter are due solely to an immune mechamsm or to a &rect cytocldal effect Tissue culture adapted DA wrus ~s a slow wrus capable of causing a chromc demyehnatmg myelopathy Its study may assist investigation of such human diseases as multiple sclerosis, amyotrophlc lateral sclerosis (ALS) and the pohomyelms-ALS syndrome

Viruses of Vertebrates

Efficient, inefficient and abortive mfectlon of different mammahan cells by small RNA viruses

Mouse hepatitis, reo-3 and Theder viruses In Viruses ot Laboratory Rodents

A comparison of the changes m fine structure of cells during smgte cycles of viral multlphcatlon following their mfect~on with the viruses of mengo and encephalomyocardltls

Electron microscopic study of the formation of pohovlrus

Primary demyehnatlon m Theder's virus refection

Observations on encephalomyelms of mice (DA strata)

An electron m~croscop~c study of the development of a mouse hepatitis wrus m tissue culture cells

Recogmtlon and measurement of small isometric wrus particles m thin sections

Mechamsm of demyehnatlon m JHM virus encephalomyehtls --Electron microscopic studies

Demyehnatwe myelopathy m mice reduced by the DA wrus

Theder's virus injection in mice --An unusual blphaslc d~sease leading to demyehnatlon

Theder's wrus reduced demyehnatlon --Prevention by ~m-munosuppresslon

Rephcatlon of pohowrus m HeLa cells --Electron microscopic observations, Vtrology

ODgodendrocytes and their myehn-plasma membrane con-nectJons m JHM mouse hepatitis virus encephalomyehtm

Host dependence of GD7 virus --Complete or abortive multlphcation m various cell types

Spontaneous encephalomyehtls of mice, a new virus disease

The authors would hke to thank Mrs Claire Bao and Mr Theodore Llsczcac for their excellent techmcal assistance