key: cord-0010025-o6wj8z6e authors: Wroblewska, Zofia; Wellish, Mary; Rorke, Lucy B.; Gilden, Donald H. title: Rat tracheal organ culture supports replication of parainfluenza 1 (6/94) virus and promotes 6/94 virus rescue from latently infected human brain cells date: 2005-12-06 journal: J Med Virol DOI: 10.1002/jmv.1890030204 sha: 59680a284c53491683fcb9ae1fa6ab5faa070c02 doc_id: 10025 cord_uid: o6wj8z6e Rat tracheal organ culture (TOC) supported replication of parainfluenza 1 (6/94) virus. Cell‐associated and cell‐free viruses were found after primary infection of TOC. In contrast to other mammalian systems, rat TOC was capable of maintaining 6/94 virus infectivity after primary infection. Rat TOC may be considered a potential indicator system that could be used to detect virus latent in human tissue. In the past ten years, much evidence has been accumulated t o indicate that chronic neurologic disease may be caused by persistence of viruses that normally produce acute disease [ter Meulen and Katz, 19771 . Exactly how virus persists despite a normal and often enhanced host immune response is still unclear. Even less well understood is the mechanism by which viruses establish latency in the human nervous system [Fenner and White, 19761 and periodically become activated. alitis (SSPE), and that herpes simplex virus is latent in the human nervous system stimulated a search for a viral etiology in relatively common, chronic neurologic diseases such as multiple sclerosis (MS). Although a parainfluenza 1(6/94) virus has been isolated from the brains of two patients with MS [ter Meulen et al, 19721 , subsequent attempts at isolation have failed. Furthermore, failure t o isolate cell-associated measles virus from SSPE brain heavlly laden with viral antigen is still a common occurrence [Katz and Koprowski, 19731. The finding that measles virus is the causative agent of subacute sclerosing panenceph-Expansion of the indicator systems and techniques used increases the chances of virus rescue. For example, Cytochalasin treatment of indicator cells has been shown to enhance the rescue of 6/94 virus from latently infected human brain cells [Wroblewska et al, 19781 after cocultivation. Since 6/94 is a parainfluenza virus that infects man most frequently through the respiratory tract, it became of interest to see if this favored entry site would be useful for an in vitro study of virus replication. It has already been shown that for some fastidious coronaviruses, tracheal organ culture (TOC) is the only host system useful for virus propagation in vitro [Tyrell and Hoorn, 1965; Tyrell and Bynoe, 1965; McIntosh et al, 1970; Thomas et al, 1976; Tyrrell et al, 19681 . For these reasons, we decided to study the replication of 6/94 virus in rat TOC and to determine its applicability for rescue of 6/94 virus from latently infected human brain cell cultures. Weanling Lewis rats were obtained from Microbiological Associates, Bethesda, Maryland. Virus 6/94 virus (kindly provided by Dr. David Waters of The Wistar Institute) was originally isolated from multiple sclerosis brain tissue [ter Meulen et al, 19721 and characterized as a temperature-sensitive variant of parainfluenza type 1 virus, replicating more efficiently at 33°C than at 37°C [Lief et al, 19751 . 6/94 virus was propagated in embryonated hens eggs, and the titer of stock 6/94 virus was 10' egg infectious doses (EID)So/ml. [Collier et al, 19711 . Lewis rats were exsanguinated by cardiac puncture. Under aseptic conditions, the chest and anterior neck were opened and the trachea was exposed. The peritracheal tissue was trimmed, and the trachea was washed with Eagle's minimum essential medium (MEM). The proximal trachea was cut, the free end was grasped with a fine forceps, and the tracheal rings were cut separately to the level of the thyroid cartilage with a sterile scalpel. Each tracheal ring was placed on the scratched surface of a 30-mm plastic petri dish, Two to three rings were placed in each dish and incubated at 37°C in MEM supplemented with glutamine and 10% fetal bovine serum (E + 10). The beating motion of cilia was readily seen in healthy cultures for at least three weeks. TOC was obtained with slight niodifications of previously described techniques In each experiment, 10 petri dishes each containing two or three tracheal rings were infected with virus 24 hours after explantation. The medium was removed, and the rings were washed in MEM and incubated for one hour at 33°C with 1 .O ml of MEM containing I08EIDS0 of 6/94 virus. Control TOC were treated with MEM only. The inoculum was then removed, and the TOC were rinsed twice with MEM, refed with 3 ml of E + 10, and incubated at 33°C. infected and three control tracheal organ cultures were removed and stored at -80°C. At various intervals after inoculation, samples of tissue culture medium from three Tracheal rings were rinsed in sterile phosphate-buffered saline (PBS). One ring was cut frozen on a cryostat 4 microns thin, fixed for five minutes in cold acetone, and stored at -20°C for immunofluorescence (IF) studies. Another ring was fixed in 10% neutral buffered formalin for histopathologic examination, and the third ring was transferred to a 30-mm petri dish containing CV, cells (see Virus Detection). five days after infection with 6/94 virus tracheal rings were cocultivated with uninfected CV, cells. Cocultivated cultures were incubated for five days. The tracheal rings were removed, the CV, cells were washed three times in MEM, and then cocultivated with a fresh, uninfected TOC. Three to five days after the second cocultivation, these tracheal rings were transferred to uninfected CV, cells (see Virus Detection). Human brain cells latently infected with 6/94 virus have been described previously [Wroblewska et al, 19761. Briefly, virus is cell-associated and no antigen is detectable by hemadsorption, immunofluorescence, or immunoprecipitation. Infectious virus can be recovered only after prolonged cocultivation or fusion of latently infected human brain cells with indicator cells. To determine whether 6/94 virus maintains its infectivity after passage through TOC, of latently infected human brain cell cultures, and two T75 flasks of normal brain cell cultures. Two "control" flasks of latently infected cells and normal brain cells were not cocultivated with TOC. All cultures were fed with E + 10 and incubated at 33OC in an atmosphere of 5% COz. At various intervals after cocultivation, samples of medium from all experimental and control cultures were removed and stored at -80°C. In addition, two to three rings from all cocultivated cultures were transfered to monolayers of uninfected CV, cells and incubated for five days. The tracheal rings were then removed, and the washed monolayer of CV, cells was checked for virus presence (see Virus Detection). Six rings of uninfected TOC were placed on to each of two T75 plastic Falcon flasks Serial 10-fold dilutions of medium harvested from experimental and control cutlures were inoculated intra-allantoically into ten-day-old embryonated hens eggs. Inoculated eggs were incubated for five days at 33"-35"C, and the harvested allantoic fluid was tested for its ability to hemagglutinate chicken red blood cells as previously described [Lennette and Schmidt, 19691 . The titer of infectious virus was calculated according to the method of Reed and Muench. In addition, CV1 cell cultures that had been cocultivated with 6/94 virus-infected TOC and uninfected CVI cells were tested by hemadsorption with 0.5% guinea pig red blood cells at 4°C [Lennette and Schmidt, 19691 . The results of the tests were expressed as an average of the percent of cells that were hemadsorption-positive in the culture. In addition, 6/94 virus in TOC was detected by indirect immunofluorescence [Gilden et al, 19761 using a 1: 10 dilution of immune serum prepared in rabbits against 6/94 virus and a 1 : 10 dilution of fluorescein-conjugated goat anti-rabbit IgG [Cappel Laboratories, Downingtown, Pennsylvania] , Controls were provided by the substitution of normal rabbit serum for the immune iabbit serum. Observations were made with a Leitz Orthoplan fluorescent microscope illuminated with a mercury HBO 200 bulb, a Schott BG12 excitor filter, and a K510 barrier filter. was identified by indirect iinmunofluorescence [Gilden et al, 19761 and also by hemaglutination inhibition (HAI) using rabbit immune serum against 6/94 virus [Lennette and Schmidt, 19691 . HA1 serum was treated with receptor-destroying enzymes before HAI testing [Lennette and Schmidt, 19691 . At various intervals after infection, sections of paraffin-embedded, formalin-fixed TOC were cut at 6 p and stained with hematoxylin and eosin. Uninfected tracheal organ cultures were examined at identical intervals. Rat TOG infected with 6/94 virus appeared normal until six days post-infection (p.i.), when ciliary activity slowed. By day 10 ciliary beating was markedly decreased, and by day 12 it ceased entirely. Pathological changes were characterized by flattening of epithelial cells and loss of cilia (Figs, LA and 1B) . The kinetics of the release of infectious virus from cultures is illustrated in Figure 2 . Cell-free virus peaked on day 5, and then decreased rapidly, although a small amount of infectious virus was released until day 24 (the total observation time). Cell-associated 6/94 viius, as determined by the ability of 6/94-infected TOC to produce infection in CV, cells (measured by hemadsorption), was detected by day 3, gradually increased until day 13, and was maintained at that level until day 24 p.i. Infectious 6/94 virus could always be obtained from these CV1 cells by cocultivation with a fresh uninfected TOC and subsequent transfer of the TOC to uninfected CV1 cells. Uninfected TOC failed to produce hemadsorption in CV1 cells. In addition, 6/94 viral antigen was seen by indirect immunofluorescence in the cytoplasm of epithelial cells and in fibroblasts in the external layer of the tracheal rings from day 8 to day 24 p.i. (Figs. 3A and 3B ). The intensity of the immunofluorescence was greatest at day 14; by day 24 only traces of viral antigen were detectable. The ability of rat TOC to promote 6/94 virus rescue from latently infected human brain cells is shown in Table 1 . When the brain cells were cocultivated with rat TOC, and the rings were then transferred to uninfected CV, cells, 6/94 virus was detected as soon as day 1 p.i. and also on day 8 after the latter cocultivation. Transfer of rat TOC that had been cocultivated with normal human brain cells to CV, cells did not result in 6/94 virus detection. In addition, virus was not detected in any of the following controls: untreated latently infected or normal human brain cells, or untreated CV, cells. Furthermore, direct cocultivation of latently infected human brain cells with CV, cells did not result in virus rescue (Table I>. was identified as 6/94 virus by both HA1 and immunofluoresence. Virus that was recovered after cocultivation of latently infected brain cells with TOC These studies demonstrate that Lewis rat TOC is a favorable host system for replication of 6/94 virus. Infection of rat TOC with 6/94 virus led to the cessation of ciliary activity after 12 days and to the production of infectious virus. This finding is consistent with the observation of Tyrrell and Hoorn that parainfluenza types 1 , 2 , 3 , and 4 are capable of growing in human and ferret TOC [Tyrrell and Hoorn, 19651 . Of interest is the fact that the only parainfluenza virus capable of damaging ciliary epithelium was the Sendai strain of type 1 parainfluenza virus. The close relationship between the Sendai and 6/94 strains of parainfluenza 1 virus has already been demonstrated [Lief e t al, 19751 . Other RNA viruses, including coronaviruses and influenza virus, will also grow in TOC [McIntosh et al, 1970; Tyrrell and Hoorn, 1965) . Characteristic findings after primary infection include impairment of ciliary function followed by degeneration of cells 4-12 days after infection. Thus, TOC appears to be not only a susceptible host system for replication of a wide variety of viruses but also a convenient indicator system for monitoring virus presence. Of considerable importance is the ability of rat TOC to support replication of fully infectious 6/94 virus. As has been reported previously, the infectivity of both Sendai and After cocultivation with LIHB or normal (uninfected) human brain cells, TOCs were transferred to uninfected CV1 cells, and 6/94 virus was assayed for b y the ability of medium removcd from the CV1 cells to infect einbryonated hen eggs. 6/94 virus was further identified by liemagglutination-inhibition and indirect immunofluorescence using rabbit anti-6/94 virus immune serum. aNormal human brain. p.i. = post-infection. Homma, 1961; Lief et al, 19751. This was associated with the development of a virus-induced specific glycoprotein in infected mammalian cell cultures [Choppin and Compans, 1975. Virus infectivity could be restored by treatment with trypsin, which produced cleavage of this glycoprotein [Homma, 19711. It is possible that organotypic cultures of trachea, as opposed to dissociated cell cultures, may produce a trypsin-like substance or other proteolytic enzymes that could result in maintenance of virus infectivity. The usefulness of rat TOC as an intermediary for the rescue of virus from latently infected cells cannot be overstated. TOC provides a favorable growth environment for a large number of viruses as well as a system that is capable of rescuing virus from latently infected human brain cells. TOC may be a useful indicator system that could be applied to human tissue suspected of harboring latent virus. Reproduction of pardmyxoviruses Mycoplasma pneumoniae in hamster tracheal organ culture Persistent infections Experi-Immunofluorescent and electron microscopic studies mental panencephalitis induced in suckling mice b y parainfluenza typc 1 (6/94) virus. 11. Virologic studies Trypsin action o n the growth of Sendai virus in tissue culture cells. 1. Restoration of the infectivity for L cells by direct action of trypsin on L cell-borne Sendai virus The significance of failure to isolate infectious viruses in cases of subacute sclerosing panencephalitis Diagnostic procedures for viral and rickettsia1 infections Antigenic variation among parainfluenza type 1 (Sendai) viruses: analyqis of 6/94 virus Seroepidemiologic studies of coronavirus infcction in adults and childrcn 977): Slow virus infections of the central nervous system Fusion of cultured multiple sclerosis brain cells with indicator cells: presence of nucleocapsids and virions and isolation of parainfluenza-type virus. Lancet 2:l-5. cultures of bovine foetal trachea 18. latently infected hurnan brain cell cultures by treatment with Cytochalasin D and dimethyl sulfoxide type 1 (6/94) infection of brain cells in tissue culture The growth of respiratory syncytial virus in organ Cultivation of a novel type of common-cold virus in organ cultures Cultivation of difficult viruses from patients with common 'Iyrrell DAJ This study was supported by Public Health Service research grant NS 11036, National Institute of Neurological Diseases and Stroke, and by a grant from the National Multiple Sclerosis Society.The authors wish to thank Mary Devlin, Elsa Aglow, and Anita Jackson for their excellent technical assistance, and Susan Thomas for her help in the editing and preparation of this manuscript. The immunofluorescence photographs were printed by Arthur Siegel.