key: cord-0958256-6id8rb35
authors: Chaloner-Larsson, Gillian; Johnson-Lussenburg, C. Margaret
title: Establishment and maintenance of a persistent infection of L132 cells by human coronavirus strain 229E
date: 1981
journal: Arch Virol
DOI: 10.1007/bf01315155
sha: e733d7270f79a64af5fda173dcc78d1d7485037e
doc_id: 958256
cord_uid: 6id8rb35

A persistent infection by human coronavirus 229E (HCV/229E) was established in a human continuous cell line (L132). Following the initial infection with stock HCV/229E, several cultures were established of which two (HV1 and HV4) have been maintained by continuous passage for two years. These cultures have shed high titres of infectious virus continuously into the supernatant fluid since their initiation. The persistently infected cells were resistant to homologous super-infection but supported polio virus replication to normal titres. Preliminary tests indicated that 50–100 percent of the cells contain virus. Neither interferon nor reverse transcriptase could be detected in these cultures and the presence of defective interfering particles could not be demonstrated. VH1 and VH4 coronaviruses, isolated from these persistently infected cultures (HV) and identified by 229E antiserum neutralization, were more cytocidal than the parent virus as judged by plaque characteristics and CPE however, they were indistinguishable on the basis of density, EM morphology, and genome size. Present evidence indicates that temperature plays an important but as yet undetermined role in the establishment and maintenance of stable 229E persistently infected cell cultures.

Coronaviruses are important pathogens of humans and animals causing a variety of respiratory and enteric infections of worldwide distribution (17) . In addition to the well known acute infections of man, swine, dogs, eats, calves and chickens, long term eoronaviral infections have been described in chickens, mice and swine. Also, it has been reported that human eoronavirus (HCV), which causes a mild upper respiratory infection, can be isolated from nasal washings of humans for up to 18 days (11) and human enteric coronavirus (HECV) has been isolated GILLIAN CIIALONER-LABssoN and C. MARGARET JOHNSON-LUSSENBURG:

from faeces for several months (12) . Thus it seems evident t h a t closer a t t e n t i o n should be given to the existence of chronic i n a p p a r e n t eoronaviral infections of humans. Increasingly, interest is being focussed on h u m a n coronavirus because of its suggested i n v o l v e m e n t in persistent infection (Balkan endemic nephropathy) (1) and its possible etiological role in multiple sclerosis (MS) (3, 20) . But, the inability to temporally assay h u m a n tissues for HCV or HECV after acute infection has made the investigation of persistent h u m a n coronavirus infection very difficult. This difficulty is further compounded because coronaviruses are notoriously fastidious, their host range is usually restricted to the n a t u r a l host, and there are no suitable h u m a n coronavirus models of in vitro persistent infection reported to date.

On the other hand, in vivo a n d in vitro, persistent or chronic infection of mouse cells by strains of mouse hepatitis virus, the m a r i n e eoronavirus, are well documented (5, 10, 19 ). I n the absence of a suitable h u m a n host eoronavirns model, these murine systems have been proposed as suitable models for the s t u d y of h u m a n disease (10).

This paper describes the suecesful establishment of an in vitro persistent infection of a h u m a n cell line (L132) with h u m a n eoronavirus, strain 229E (HCV/229E), which has to date been m a i n t a i n e d for more t h a n 300 passages over a period of 24 months and continues to shed high titres of infectious virus. This system has great potential for further studies on virus replication, pathogenesis of eoronavims disease and eoronavirus genetics.

Cells and Cell Culture I-Iuman fetal lung cells L 132, a continuous cell line which supports human coronavirus replication were used throughout. They were routinely propagated as described previously (8) , using Eagle's minimal essential medium (Flow Laboratories, Inc., Mississauga, Ontario, Canada) supplemented with 10 percent fetal bovine serum (FBS, Flow Labs., Inc.), sodium bicarbonate (20 m~r), penicillin (100 U/ml), streptomycin (100 ,ug/ml), neomycin (50 p.g/ml) and glutamine (2 m~f) at 37 ° C. The ceils were passaged every two days at a split ratio of 1 : 3.

I-Iuman eoronavirus strain 229E used in these experiments was stock virus grown in L 132 cells and maintained in this laboratory (8) . Poliovirus Type I, Sabin strain, used for the superinfeetion study was obtained from Dr. S. A. Sattar of this department.

Normal cells --L132 (I-I); Standard virus 229E (V); Persistently infected cells --L132/229E (I-IV); Virus derived from persistently infected cells -229E/ L 132 (V~).

Growth o/ Virus L 132 monolayers were infected with 229E at a multiplicity of 3---5 PFU/cell and, following an adsorption period of 1 hour at room temperature, were incubated ar 33°C for 24 to 40 hours in medium 199 (2¢i199, Flow Labs. Inc.) without serum, supplemented with glutamine, antibiotics and I per cent sodium bicarbonate. The infected cultures were subjee~ed to three freeze-thaw cycles at --20°C and then stored in aliquots at --70 ° C for use as virus inoculum as required. The titre of stock virus was 0.7--1.0 × 10 s PFU/ml. F o r t h e p r e p a r a t i o n of radioisotope labelled virus, t h e cells were infected as above. A t t h e end of t h e a d s o r p t i o n period, either -uridine (10 ~Ci/ml: specific a c t i v i t y 28 Ci/rnmol) or 14C-amino acid m i x t u r e (1 ~Ci/ml) were included in the m a i n t e n a n c e m e d i u m (M 199). R a d i o c h e m i c a l s were from N e w E n g l a n d Nuclear.

All steps were carried o u t a t 0 ° to 4 ° C. Following t h r e e freeze-thaw cycles to release virus, 229 E d n f e c t e d cell lysates were clarified b y two cycles of centrifugation, first at low speed ( I E C / P R J , 1800 r p m × 15 minutes) a n d t h e n a t m e d i m n speed ( I E C / B -2 0 A , t 5 , 0 0 0 × g × 30 minutes). T h e virus was t h e n cushioned onto 7 ml of 65 p e r c e n t (w/v) sucrose in p h o s p h a t e buffer (0.001 •, p t I 7.2) in a B e c k m a n S~725.2 rotor at 20,000 r p m (48,000 × g) for 60 minutes. The c o n c e n t r a t e d virus was collected from t h e interface a n d was pelleted in a B e c k m a n F A 5 0 rotor a t 40,000 r p m (97,000 × g) for 2 hours. T h e pellet, was resuspended in 0.001 ~i s o d i u m p h o s p h a t e buffer (pI-I 7.2), overlaid onto a linear 2 5 --6 5 p e r c e n t (w/w) sucrose g r a d i e n t in 0.001 M sodium p h o s p h a t e a n d centrifuged to e q u i l i b r i u m in a B e c k m a n SW41 rotor at 22,000 r p m (63,000×g) for 16 to 24 hours. :Fractions were collected a n d samples up to 200 ~zl were c o u n t e d in a cocktail composed of BBS-3,60 m l ; b u t y l -P B D , 15 g (both from B e c k m a n I n s t r u m e t l t s ) ; water, 1 2 0 m l ; a n d toluene to 3liters, using a B e c k m a n LS-250 liquid scintillation counter. T h e p e a k fractions were pooled a n d t h e virus pelleted for two h o u r s in a B e c k m a n F A 5 0 rotor a t 40,000 r p m (97,000×g). T h e virus pellets were r c s u s p e n d e d in a few drops of 0.001 M sodium p h o s p h a t e buffer, pooled a n d either used i m m e d i a t e l y or stored at --70 ° C.

All virus t i t r a t i o n s were p e r f o r m e d b y t h e s t a n d a r d plaque assay in monolayers of L132 cells in 75 cm 2 disposable culture flasks (Lux Scientific Corporation) as described previously (8) . F o r p l a q u e d e v e l o p m e n t , 2 2 9 E i n c u b a t i o n was a t 33°C for 6 days a n d polio at 37°C for two days. Virus titres are expressed as plaqueforming units per ml ( P F U / m l ) .

To d e t e r m i n e t h e p e r c e n t a g e of p e r s i s t e n t l y infected cells releasing infectious virus, m o n o l a y e r s of L 132/229E ceils (HV) were washed, ~rypsinized a n d a p p r o p r i a t e dilutions of t h e cells in 5 to I0 m l of m a i n t e n a n c e m e d i u m were a d d e d to confluent u n i n f e c t e d L 132 monolayers, w i t h or w i t h o u t p r e t r e a t m e n t w i t h a n t i -2 2 9 E g~uinea pig serum. After two to t h r e e hours a t 37 ° C to allow t h e cells to settle a n d a t t a c h to t h e layer, t h e m o n o l a y e r s were covered w i t h overlay m e d i u m (8) a n d plaques allowed to develop for 5---6 days a t 33 ° C.

A n t i s e r u m a g a i n s t purified a n d c o n c e n t r a t e d 2 2 9 E virus a n t i g e n was p r e p a r e d in male g u i n e a pigs (I-Iartley/Albino o u t b r e e d weighing 4 0 0 --5 0 0 g) according to t h e following schedule: 1st day, 0.2 ml a n t i g e n in t h e f o o t p a d ; 4th day, 1 rnl of a n t i g e n w i t h complete F r e u n d ' s a d j u v a n t (GIBCO) t : 1 s u b c u t a n e o u s l y ; 19th a n d 32nd day, t ml a n t i g e n / a d j u v a n t 1 : 1 i n t r a m u s c u l a r l y . T h e a n i m a l s were bled b y h e a r t p u n c t u r e prior to i m m u n i z a t i o n (control) a n d weekly t h r o u g h o u t the procedure. Virus neut r a l i z a t i o n titres of 10 -3.7 were o b t a i n e d b y s t a n d a r d plaque r e d u c t i o n assay.

Ten-fold serial dilutions of guinea pig a n t i -2 2 9 E serum were p r e p a r e d in saline. 0.5 m l of each were m i x e d in equal a m o u n t s w i t h suspensions of 2 2 9 E or 2 2 9 E / L 132 (VII) viruses, diluted a p p r o p r i a t e l y to give b e t w e e n 20 a n d 50 P F U / m l . 0.5 m l saline w i t h o u t a n t i s e r u m was a d d e d in parallel to serve as virus controls. After one h o u r at room t e m p e r a t u r e , 0.33 m l of each sample was seeded in duplicate onto L 132 monolayers, allowed to a d s o r b for one hour, covered w i t h overlay m e d i u m a n d t h e plaques allowed to develop for 5 --6 days at 33 ° C. E n d p o i n t s were calculated on t h e basis of 50 p e r cent r e d u c t i o n in p l a q u e formation.

All preparations were negatively stained with sodium or potassium phosphotungstate (2 per cent) following standard procedures as described previously (8) . Grids were made directly from gradient fractions in sucrose or, more commonly, the fractions were diluted in 0.001 5I sodium phosphate buffer and centrifuged at 97,000 × g for I to 2 hours. I~esulting pellets were resuspended in distilled water and grids prepared immediately. All grids were examined in a Philips EM300 electron microscope.

The assay for reverse transeriptase (RT) activity was carried out through the courtesy of Dr. A. Greig (Animal Disease I%eseareh Institute, Agriculture Canada, Ottawa) using the Kit for Mammalian Viral Reverse Transcriptase from Collaborative Research Inc., Waltham, Mass. and following the recommended procedures. This kit is designed to distinguish the I~T enzyme from other DNA polymerase activities, and E. coli poIymerase I, active on the provided templat.es, is included as an internal control for test performance. Supernatant fluid from uninfected and persistently infected L 132 Cells was pelleted, resuspended in 25 p.1 and tested for viral RT activity using the primer template provided (Oligo dT. Poly rA). Positive and negative controls were supernatant fluid from Bovine Leukemia Virus infected fetal lamb kidney cells and uninfected fetal lamb spleen cells respectively. The cation requirements (Mg++ or Mn ++) of the enzyme were assessed in the tests. 

Monolayers of L 132 cells were infected with stock 229E virus at multiplicities (MOI) of 0.03, or 3.0 in 75 cm 2 tissue culture flasks. After adsorption at room temperature for one hour, m a i n t e n a n c e m e d i u m (M 199) or growth medium (MEM) was added and the flasks incubated at either 33 ° or 37 ° C. After 30 to 36 hours the medium was decanted, the layers rinsed with phosphate buffered saline (PBS) and, depending on the integrity of the monolayer, the cells were either trypsinized a n d reseeded into fresh flasks or overlaid with fresh growth medium. The cell layers were monitored daily, the growth medium was changed daily and the cells were passaged when the layer approached confluence. Once it became clear t h a t a monotuyer was growing satisfactorily (approximately 2 weeks), atiquots of the m e d i u m were collected ~nd assayed for infectious virus by plaque titration.

The successful m a i n t e n a n c e of the persistently infected cell cultures was directly related to the i n c u b a t i o n temperature of the cultures subsequent to the initial infection with 229E virus. No distinct visual difference in the cell monolayers could be correlated with the virus i n p u t multiplicity b u t the infected 121.

cultures kept at 33 ° C did not fare as well as those at 37 ° C. Therefore, the effect of temperature on maintenance was examined, the temperatures chosen being 33 ° C which is optimal for 229E replication and 37 ° C the optimum for L 132 cell growth. Only those cultures kept at 37 ° C (supraoptimal for 229E virus replication) during the initial incubation with virus or changed to 37 ° C shortly after the 30 to 36 hour incubation at 33 ° C survived the infection and formed a stable population of cells which appeared normal and grew at rates characterist~ie of L 132 cell cultures. In contrast, those cells infected and maintained at 33 ° C throughout or changed from 37 ° to 33°C during the course of our experiments grew slowly and irregularly, never reaching a confluent monolayer. During 6--8 weeks of maintenance at 33 ° C these cells continued to shed virus into the medium but in progressively decreasing amounts (up to 3 logs less before termination). Furthermore, after severM weeks, these cells did not recover when returned to an incubation temperature of 37 ° C. By comparison, control uninfected L 132 cultures grew more slowly at 33 ° C than at 37 ° C, but they appeared normal and resumed normal growth rates when returned to their optimal incubation temperature (37 ° C).

The stable persistently infected cells maintained at 37 ° C have been stored at --80 ° C for up to 15 months and have been revived with no adverse effects--still growing characteristically and shedding virus. To distinguish these cells and their virus from the standard system, the cells were termed I-IV (L132/229E) and the virus derived from these cultures VH (229E/L 132).

Several subsequent attempts have been made to establish new 229 E persistently infected cell lines. New long term stable virus-shedding populations have been obtained, but, the successful outcome of each attempt could not be predicted. Further work aimed at defining the critical procedure(s) for consistent production of such persistent infections is in progress.

Since the time of the initial infection with 229E virus, strict precautions have been taken to ensure that virus has not been reintroduced. At the time of writing, the persistently infected cells have been passaged over 300 times on the same splitting schedule as uninfected L 132 cells (1 : 3 every two days) for over 24 months. They have continued to shed high titres of virus (105 to 106 PFU/ml representing 5--10 virions/cell) and show no apparent deleterious effects.

A visual difference between L 132 and HV cell layers was apparent on the first day after passage before a confluent monolayer was reached. Consistently, the IiV cultures showed a pattern of cell growth which was distinguishable from L 132 cell cultures. During the initial growth, the individual cells of the persistently infected HV cultures did not show the characteristic elongation and spreading seen in the uninfected L 132 cells (Figs. 1 A and 1 B) . Once confluent however, the monolayers were visually identical (Figs. 1 C and t D) . Since the number of cells in 48 hour confluent monolayers of both uninfected and persistently infected cells were equivalent (Fig. 2) , this characteristic pattern probably reflects the presence of replicating virus. Infectious center assays to determine the number of infected cells in the HV cultures tentatively indicate that 50--100 percent were producing virus. When HV cells were cloned, all resulting cultures (15/15) were virus produe-ing and/or resistant to snperinfection by 229E virus. These results need to be confirmed by immunofluoreseent methods.

Throughout this study, the quantity of infectious virus shed into the culture medium (25 mt per 75 em 2 flask) was routinely determined at weekly intervals by At intervals the amount of cell associated virus was also measured and consistently revealed five to ten times greater amounts of virus. On the basis of the total yield of virus/cell during a two day growth period, i.e. one cell passage, the production of virus correlated directly with the increase in the number of cells ( Fig. 2A and  2B ). Both showed a 3 to 4 fold increase during the 48 hour period. When the cells

for virus production i n this persistent system. 

Both of these monolayers retained their integrity under agar at 37 ° C and at 33 ° C for up to seven days. 

The results of neutralization tests performed with anti-229 E serum indicated t h a t the persistent virus was in fact a coronavirus. The serum was equally effective in neutralizing both V H and 229E viruses, the 50 percent plaque reduction endpoints being between 10 -3.4 and 10 -3-s. The kinetics of neutralization were not evaluated.

The morphology of both types of coronaviruses was compared by examining negatively stained preparations of each in the electron microscope. No relevant difference could be seen between the persistently shed virions and the 229E virions.

Despite the lack of eytopathic effects in the persistently infected culture, the isolated V I I virus seemed to be more eytocidat t h a n stock 229 E virus. W h e n acute V I I virus infections in L 132 cells were carried out under liquid medium, generalized In addition to tile 25 txl sample, the reaction mixtures contained: 50 mM Tris. ttCI cell deterioration (CPE) was evident 12--15 hours earlier than with standard 229 E virus infections under similar conditions. Furthermore, a one-step growth curve experiment with both of these viruses showed that replication of V H virus was consistently earlier and reached higher titres than the standard 229E virus (Fig. 3) . This increase in replication efficiency of the V H virus was further indicated by the earlier development (by 1--2 days) of clearer and slightly larger plaques as compared to those of 229E virus (Fig. 4) . Samples were titrated in duplicate by plaque assay. Titres are expressed as log PFU/ml VII virus, labelled in 8itu with [5-3H] uridine or 14C-amino acids, and VH virus isolated and labelled also with [5-3H] uridine or 14C-amino acids during an acute infection of L 132 cells, were harvested, purified and compared with the standard 229 E virus similarly prepared. The density of the virus as determined by isopyenic sucrose gradient analysis was the same for 229 E and V H virus (1.18--1.19 gm/ce) (Fig. 5) . lZIXZA isolated from these viruses in isokinetie sucrose gradients gave the same profile of a single large molecular weight species of R N A (data not shown.). Viral yields from lyric infections carried out at 33 ° C were always ~--1 log higher for the V H than 229E viruses (Table 3) .

The mechanism of HCV/229E persistence in L132 cells does not appear to involve either interferon production, integration or defective interfering particles. Assays for reverse transeriptase in L / 3 2 cells and in persistently infected cells measured in the presence of Mg++ or Mn++ were negative (Table 2) . Interferon, as assayed by the plaque reduction method, was not detected in normal, acutely infected, or persistently infected cells (data not shown). Interference by defective interfering particles (DI) could not be completely ruled out b u t all efforts to detect such particles have been unsuccessful to date. The VH virus could not be distinguished from standard stock 229E virus by a n y of the methods tFied i.e. density (Fig. 5) , (Table 3) . Also, the fa.et t h a t the virus titre of the H V cells has not varied over the course of 2 years suggests t h a t D I particles are unlikely to be involved. T e m p e r a t u r e sensitivity was therefore, the more promising avenue for investigation, since the persistent state showed some t e m p e r a t u r e dependence. The persistently infected cell cultures had been successfully m a i n t a i n e d at 37 ° C, but not at 33 ° C which is the optimal t e m p e r a t u r e for 229 E virus replication. A t 33 ° C, the initial persistent infection had aborted after several weeks a n d several a t t e m p t s to change H V cultures from 37 ° to 33 ° C met with a gradual deterioration and eventual halt in growth and loss of integrity of the layer. This was however not accompanied b y an increase in virus shedding. F u r t h e r m o r e plaquing efficiencies of each of these viruses was 90 percent lower at 37 ° C as compared to 33 ° C. These facts do not indicate,the emergence of t e m p e r a t u r e sensitive m u t a n t s per se, b u t r a t h e r imply a contribution by the host cell which is t e m p e r a t u r e dependent,

The in vitro virus/host system described here appears to be the first reported instance of h u m a n coronavirus HCV 229 E giving rise to a persistent infection in cell culture. After infection with 229 E and an initial a d j u s t m e n t period, persistently infected L132 cells (HV cells) have been continuously subcultured and have shed virus into the m e d i u m at high titres for more t h a n two years. Preliminary experiments suggest t h a t the m a j o r i t y of these H V cells are infected.

I n other in vitro persistent infections, several different mechanisms have been proposed. These include suppression of virus production b y interferon (4), interterence b y defective interfering (DI) particles (6, 7, 13, t6) , integration of the viral genome (23) , and genetic m u t a t i o n s of the virus (15).

In the L132/229E system reported here, the first three mechanisms do not seem to play a role. Neither interferon nor reverse transcriptase activity has been detected. DI particles did not seem to be involved since there was no significant change in the virus shed over 300 passages of the persistently infected cell cultures and there was no biochemical or morphologicM evidence for two t~pes of particles. Also, no interference could be demonstrated in mixed infections of 229E and VH virus. However, further experiments are necessary to provide sufficient evidence to exclude the involvement of DI particles.

A common factor in severM other in vitro persistent virus/host systems is the production of temperature sensitive virus progeny. For example, such mutants have been isolated in persistent infections of measles (2), Newcastle disease virus (14) , vesicular stematitis virus (22) , and mumps virus (21) . Among the Coronaviruses, only mouse hepatitis virus has been reported to dev-elop a persistent infection in vitro (17) . Both temperature sensitive mutants of MHV (K. HOLMES, abstract, Int'l Virol. IV: 453, 1978) and cold sensitive mutants of JHM (a neurotropic strain of MttV) (18) have been isolated. LvcAs et al. (9, 10) have described a temperature association with in vitro persistence of mouse hepatitis virus. They found that viral replication was thermosensitive in cell cultures persistently infected with strain JHM, although the virus progeny from the persistent infection were not thermolabile. In our HV system, some temperature effect is associated with both the establishment and maintenance of persistence, however, further experiments are required to determine the role of temperature in this virus/host system. Since the virus isolated from the persistently infected cells (HV) caused more pronounced CPE than 229 E virus in L 132 cells, it seems probable that changes have occurred in both the virus and the host cells during the early stages of the establishment of persistence. Experiments are in progress to elucidate tile mechanism or mechanisms involved in the establishment and maintenance of persistence of human eoronavirus in vitro.

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