key: cord-0004849-1xglujqk
authors: Chasey, D.; Alexander, D. J.
title: Morphogenesis of avian infectious bronchitis virus in primary chick kidney cells
date: 1976
journal: Arch Virol
DOI: 10.1007/bf01317869
sha: 95514ee6cf44283a6c830a26d28b54cffa2e37dc
doc_id: 4849
cord_uid: 1xglujqk

Primary chick kidney cells were infected with avian infectious bronchitis virus (IBV) and examined by electron microscopy. Virus particles entered the cells by viropexis and distinction could be made between engulfment by cell processes (phagocytosis) and entry by micropinocytosis in coated transport vesicles. Virus maturation occurred by budding into either the cisternae of the endoplasmic reticulum or cytoplasmic vacuoles, and evidence was obtained to suggest that the viral surface projections could be attached during the budding process. Late in infection large numbers of virus particles were present, mainly in cytoplasmic vacuoles, and the majority were released by cell lysis. Release by fusion of vacuoles with the plasma membrane was also observed, and individual virions could be transported from the endoplasmic reticulum to the surface within coated vesicles.

Studies on the morphogenesis of coronaviruses have sho~ that replication takes place in the cytoplasm of the host cell by a budding process from either the membranes of the endoplasmie retieulum or cytoplasmic vacuoles (11). This method of development has been demonstrated for human coronaviruses in WI-38 cells (3, 8) and human embryo lung cells (7) , and avian infectious bronchitis virus (IBV) in ehorioatlantoie membrane cells (3) , chick embryo fibroblasts (10) , VERO cells (6) and the trachea of infected fowls (15) . The budding mechanisms of virus particles in infected cells have been recorded in detail but little is known of the modes of entry and release although it has been generally reported that infection and release occur by viropexis and cell lysis respectively.

In the present study we have examined the morphogenesis of two IBV strains in primary chick kidney (CK) cells with particular emphasis on aspects of entry and release.

T h e B e a u d e t t e strain of I B V was used as described previously (1) . Tissue culture a d a p t e d 'T' s t r a i n of I B V was o b t a i n e d from C. D. Braeewell, Central V e t e r i n a r y L a b o r a t o r y , W e y b r i d g e , U.K. a n d h a d been passaged six times in CK cells. A stock was g r o w n a n d this virus was used for infecting m o n o t a y e r tissue cultures.

Virus infectivity was e s t i m a t e d as plaque forming units (PFU) as described (4) .

P r i m a r y C K cells were p r e p a r e d from 4-week-old chicks as described (4) a n d t h e m e d i u m was m a i n t a i n e d a t pI-I 7.0--7.2 w i t h 15 rn51 I t E P E S buffer.

Cells were grown on 7.5 cm~ surfaces in 30 ml plastic culture bottles a n d inoculated a t conflueney w i t h ' 2 --5 P F U of virus per cell. Cultures that. were to be e x a m i n e d at, times up to one h o u r after infection were inoculated with a p p r o x i m a t e l y 500 P F U of virus per cell. A f t e r a n a d s o r p t i o n period of one hour, t h e inoculum was r e m o v e d a n d t h e monolayers were w a s h e d prior to overlay a n d i n c u b a t i o n a t 37 ° C. A t specified times after i n o c u l a t i o n cell cultures, including u n i n f e c t e d controls, were p r e p a r e d for electron microscopy.

T h e m e d i u m was r e m o v e d from t h e cells a n d t h e monolayers were rinsed briefly in 1 per cent g l u t a r a l d e h y d e in 0.05 N sodium p h o s p h a t e buffer p H 7.2 (SPB). F u r t h e r g l u t a r a l d e h y d e was a d d e d a n d t h e ceils fixed for 3 0 --6 0 m i n u t e s a t room t e m p e r a t u r e before washing in S P B a n d post-fixation in 1 per cent o s m i u m tetroxide for 3 0 6 0 minutes. A f t e r d e h y d r a t i o n in g r a d e d e t h a n o l sohations, t h e monolayers were covered w i t h araldite a n d left for a p p r o x i m a t e l y one hour. The m i x t u r e of resin a n d residual alcohol was replaced w i t h fresh araldite which was allowed to h a r d e n . T h e araldite blocks c o n t a i n i n g t h e m o n o l a y e r s were finally prised from t h e plastic s u b s t r a t e a n d small chips were r e -e m b e d d e d in capsules. T h i n sections of t h e s a n d w i c h e d monolayers were cut on a n L K B u l t r a t o m e , using glass knives, a n d s t a i n e d in 25 p e r cent m e t h a n o l i e u r a n y l a c e t a t e for 

CK cells were inoculated with approximately 500 PFU of IBV strain Beaudette per cell and examined 10, 20 and 60 minutes later. The inoculum, which was concentrated by centrifugation, contained large amounts of cell debris visible with virus particles outside the cells. One hour after inoculation particles and cell debris were also seen within the cells in cytoplasmic vacuoles (Fig. la) .

Virus particles appeared to enter CK cells by one of two me~hods, phagocytosis or micropinocytosis, both of which may be termed viropexis. Phagocytosis occurred with the engulfment of virus particles and cell debris by surface processes to produce phagocy.tic vacuoles (Fig. 1a--d) . Entry by micropinocytosis occurred with the formation of small invaginations, each containing a single virion, which were distinctive in that the membrane enveloping the virus appeared thickened (Fig. 2 a) . One hour after inoculation small virus-containing vesicles with thickened membranes could be seen within the cells (]Pig. 2b).

The uncoating of ingested virions and subsequent core replication was not evident in our preparations. Six to eight hours after infection with strain Beaudette, virus was visible within the cisternae of the endoplasmic reticulum. Virus particles could not be seen in large numbers until about ten hours after inoculation by which time extracellular particles were also observed. Evidence was obtained which showed that virus particles were formed by budding from the membranes of the smooth endoplasmic reticulum into the cisternae (Fig. 3) .

Electron microscopy of cells infected with strain Beaudette indicated that virus release occurred by at least two distinct mechanisms. A small number of virus particles were enveloped individually by thickened portions of the endoptasmic reticutum which formed rounded vesicles (Fig. 4a, b) . These vesicles, with a diameter in the range 90--180 nm, resembled those associated with incoming particles and often exhibited small exterior spicules. Vesicles with thickened membranes were seen in infected cells throughout the cytoplasm both empty and containing virus particles, and empty vesicles were also present in uninfected cells. The observations of vesicles at cell boundaries suggested that, after formation in the endoplasmic reticulum, the vesicles moved to the cell surface where they could be seen fused with the plasma membrane (Fig. 4c, d) . Since this mechanism is the reverse of micropinocytosis it was important to establish in which direction the vesicles at the cell surface were moving in order to distinguish outgoing particles from already released progeny virus re-entering the monolayer in a secondary cycle of infection. That infection or re-infection of cells occurred for a considerable time (at least ten hours) after inoculation was evident from the continued phagocytosis of virus particles (Fig. 5) . As a rule it was not possible to judge the direction of movement for vesicles opening onto the exposed cell surface (i. e. away from the substrate) since the large amount of extracellul~r virus seen close to the surface prevented unambiguous interpretation. However, virus release by reverse micropinocytosis could be demonstrated for thickened vesicles opening into regions of the monolayer in which different cells were in close contact. In this ease virus particles could be seen to have been effectively trapped in the narrow (Fig. 6a, b) . The majority of virus particles remained within the endoplasmic retieulum and were seen later in infection in large cytoplasmic vacuoles, many of which were apparently formed by dilation of the eisternae. The size of the vacuoles was not related to the number of enclosed virus particles and many vacuoles were empty (Fig. 7) ; control cells often contained large numbers of empty vacuoles. There was evidence to suggest that the -¢acuoles fused with each other (Fig. 7) but with strain Beaudette there was no observation of fusion with the plasma membranes.

The overall density of the cytoplasm increased during the last stages of infection, cells fused, and individual organelles tended to lose their identity Fig. 6 . l~elease of virus, strain Beaudette, by reverse micropinocytosis a) A thickened vesicle (arrowed) enclosing a virus particle close to the cell surface. I0 hours after inoculation. The confined extraeellular space already contains released particles. Note the 'spicules' on the vesicle membrane × 41,000. b) Virus particle released (arrowed) from a vesicle into a confined extracellular space, 14 hours after inoculation; × 41,000 in a granular mass; the nuclei were fragmented and appeared as small axeas of densely stained material, while regions of endoplasmic reticulum remained electron transparent. The cell masses rounded-up and detached from the substrate prior to lysis and, from the beginning of the stage at which cells were seen dying, large numbers of extracellular virus particles were present in the sections. Disintegrating cells were observed and progeny virus within the cyCoplasmic vacuoles was seen to be released b y m e m b r a n e rupture. Virus particles released into confined extracellular spaces tended ~o lie in dose-packed arrays with hexagonal configuration. Virus particles were seen in the process of budding from internal membranes but, unlike strain Beaudette, appeared to form predominantly within pre-existing cytoplasmic vacuoles (Fig. 8) . However, because of the difficulty in tracing membrane continuity in thin sections it was not always possible to distinguish between vacuoles and endoptasmic retieulum. The majority of progeny virus particles appeared to be released by cell lysis. Virus release by reverse micropinocytosis was not observed with 'T' strain but there was evidence that the large virus-containing cytoplasmic vacuoles could fuse with the plasma membranes and release virus particles before gross degenerative changes had taken place in the cytoplasm (Fig. 9) . 

Virus particles of both strains were circular in cross-section with diameters of 60--100 nm although some ]urger particles were also observed. IndividuM virions consisted of an outer envelope, approximately 20 nm thick, surrounding a pale central region. Areas of more densely stained material were often seen at the periphery of the central region close to the envelope and apparently 'empty' particles were also present.

Many virus particles, especially those seen outside the cells after release, possessed well-defined surface projections or spikes. These could be seen clearly on both strains of virus, and were particularly distinctive on 'T' strain particles (Fig. 10a, b) , but the number of spikes visible varied considerably from one particle to another. Each spike was 15--20 n m in length and approximately 5 n m across with a small thickened knob at the distal end.

Although projections were most numerous and clearly visible on released virions they could also be identified on particles still in the process of budding; generally no more than one or two spikes were seen attached to the incomplete envelope (Figs. 10% 3) . Fig. 10 . Morphology of IBV particles a) Extracellular T strain particles 46 hours after inoculation. Note the well-defined surface projections × 120,000. b) Extracellular sSrain Beaudette virus 21 hours after inoculation also exhibiting surface spikes x 75,000. e) A strain T virus particle budding into a cytoplasmic vacuole 46 hours after infection. A surface spike can be seen (arrowed) on the immature particle; × 120,000

The general features of I B V development in primary CK cells observed in this study are consistent with those reported in previous investigations of eoronavirus morphogenesis (11). I n common with other I B V studies the intraeellular inclusions seen in ceils infected ~dth mouse hepatitis or human respiratory viruses (7, 12) were not observed in our material.

The I B V particles seen in this investigation showed morphological characteristics similar to those already described for the eoronaviruses (11). A distinctive feature, however, was the presence on m a n y particles of well-defined surface projections which have not previously been clearly observed in sections of cells infected with I B V (2) or other eoronaviruses. The generaI shape of the individual spikes was similar to that seen in negatively-stained preparations (3, 9) . The presence of spikes on budding particles was also established and this observation demonstrates the early attachment of at least some of these subunits during virus formation. The apparent absence of surface projections on m a n y particles, particularly those within large vacuoles, could represent fnndamental differences due to different methods of maturation or, alternatively, be merely an artifact of specimen preparation.

Further information was obtained concerning the mode of virus entry and release. The electron micrographs indicate that IBV was able to enter CK cells by two distinct mechanisms both of which may be classified as viropexis. Although we did not observe any morphological evidence of uncoating, no other means of entry was detected and we assume that viropexis initiates infection. The mechanism by which the majority of particles entered the cells involved engulfment of the virus, and cell debris present in the inoculum, by surface processes or pseudopodia to form phagocytic vacuoles. The second method involved the uptake of individual particles by micropinocytosis. The thickened membrane forming the enveloping invagination can be identified with the modified or 'coated' merebrane characteristic of the transport vesicles present in many cell types (13, 7) . Virus entry by micropinocytosis and subsequent association with coated vesicles has not been described previously for coronaviruses but has been observed in L cells infected with a rhabdovirus, vesicular stomatitis virus (14) , and in HeLa cells infected with type 5 adcnovirus (5).

The budding of particles from the endoptasmie reticuIum and the release of progeny virus took place by processes which may be general for all eoronaviruses (11). However, the envelopment of virus particles by coated vesicles and their subsequent release from the cells by reverse mieropinocytosis are phenomena not previously described for any virus. This mechanism may be peculiar to those members of the coronaviru s group which bud into the cisternae of the endopl~smic reticulum since the particles are formed within the membranes from which the coated transport vesicles are produced (t4). In this respect our failure to observe any association of 'T' strain virus particles with coated vesicles may be significant since this strain was seen to bud predominantly into cytoplasmic vacuoles. The method of release of virus particles by fusion of vacuoles with the cell plasma membranes seen in 'T' strain infected cells appears similar to the process described for coronaviruscs from human respiratory infections (12) .

One of the more conspicuous aspects of the morphogenesis of IBV in CK cells was the large number of virus particles seen in the cytoplasmic vacuoles and outside the cells late in infection. Although infectious particle release was not measured in the present study, ALEXANDER and COLLI~S (i) have demonstrated release of infectious virus, measured as PFU, from CK cells infected with approximately 5 PFU of IBV strain Beaudette per cell. The maximum titre of released virus was reached 12--14 hours after inoculation, at a time when light microscopy of the infected cells revealed cytopathie effect in the form of small syncytia, but no obvious cell death or lysis had occurred. In their study the maximum titre represented a total infectious particle production of 10--20 PFU per cell (ALs~XA~DER, unpublished). This tow total and the failure to detect an increase in the rate of infectious particle release late in infection would suggest that the majority of particles seen in our thin sections were non-infectious. Similarly, observations on human coronaviruses grown in tissue culture have shown a lack of correlation between the infectivity titres and the large numbers of particles seen by electron microscopy (12) . While it is tempting, in the ease of strain Beaudette, to correlate the number of released infectious particles with the frequency of virus release from coated vesicles in our work, clearly further study is required to show more exactly the relationships between the different methods of particle release and the production of infectious virus.

Effect of pt-I on the growth and eytopathogenicity of avian infectious bronchitis virus in chick kidney cells

The biology of large I~NA viruses

Morphogensis of avian infectious bronchitis virus and a related human virus (strain 229E)

Antigenic relationships between strains of infectious bronchitis virus as shown by the plaque reduction test in chicken kidney cells

Early events in the interaction of adenoviruses with HeLa eelIs. I. Penetration of type 5 and intraeellular release of the DNA genome

l%eplication of avian infectious bronchitis virus in African green monkey kidney cell line VERO

An electron microscope study of the development of a mouse hepatitis virus in tissue culture cells

Growth and intracellular development of a new respiratory virus

Isolation from man of "avian infect.ious bronchitis virus-like" viruses (coronaviruses) sit~filar to 229 E virus, with some epiderniological observations

Morphogenesis of avian infectious bronchitis in chicken embryo fibroblasts

UItrastructure of animal viruses and bacteriophages: An atlas

Electron microscopic studies of coronavirus

Specialized sites on the cell surface for protein uptake

Viropexis of vesicular stomatitis virus by L ceils

An electron-microscope study of the trachea of the fowl infected with avian infectious bronchitis virus

A simplified lead citrate stain for use in electron microscopy

V~;e thank Judy Luddingto~l, Mike Collins, and Nick Chettle for their excellent assistance.

t~eceived March 8, 1976