key: cord-1014594-zu5ns4yq
authors: Fennestad, K. L.; MacNaughton, M. R.
title: Pleural effusion disease in rabbits. Properties of the aetiological agent
date: 1983
journal: Arch Virol
DOI: 10.1007/bf01311102
sha: c5693c87fa1c8b99d18d2ee0f40e55430d53a253
doc_id: 1014594
cord_uid: zu5ns4yq

The size and heat sensitivity of Pleural effusion disease (PED) agent or virus (PEDV) propagated in rabbits were examined. The infectious particles were estimated to be between 25 and 50 nm by filtration. Residual infectivity of infectious serum was 0.1 per cent after heating at 56° C for 4 hours. PEDV and the Stockholm agent appeared identical concerning pathogenic and immunogenic properties by infection experiments and protection tests in rabbits. Two of the three PEDV isolates were less pathogenic but appeared immunogenically identical to PEDV. The third isolate, obtained from the laboratory, which several years previously had supplied material for demonstration of the Stockholm agent, differed from PEDV in pathogenic and immunogenic properties. Serological examinations of paired rabbit sera did not indicate any antigenic relationship between PEDV and representative members of the two mammalian coronavirus antigenic groups. It is concluded that the aetiological agent of PED is a virus not belonging to the coronaviridae.

Pleural effusion disease (PED) agent is found as a passenger in rabbit testieular suspensions of Treponema pallidum and causes subclinical to fatal infections in rabbits (1, 6, 17, 22) . PED has been reported as a new disease (1) and another name proposed for it is rabbit infectious cardiomyopathia (19) . The disease has been known since the 1960s (4, 9) , but as yet the agent or ~Arus (PEDV) has not been convincingly demonstrated by tissue culture or electron microscopy. A K . L . PENNESTAD and M. 1~. MACNAUGHTON: remarkable feature of P E D is the long persisting viraemia (1, 3) ; nevertheless, the infection induces clinical protection a n d there is also evidence indicating t h a t protective IgG antibodies are produced as a result of the infection (3) .

During a survey in 1978/79, isolates were obtained from several laboratories. All these isolates induced clinical protection to challenge with PEDV, except one (SBL) from Stockholm which induced only partial protection (2) .

S~ALL et at. (19) have suggested t h a t the Stockholm agent, which was brought to the J o h n s Hopkins University School of Medicine, Baltimore in 1970 (5) , might be a coronavirus antigenically related to h u m a n coronaviruses (HCVs) 229 E and OC43. This concept was supported by OSTER~AUS et al. (18) who observed coronavirus-like particles in rabbits infected with P E D V . However, in this s t u d y we show t h a t P E D V appear to be smaller and more heat resistant t h a n eoronaviruses, and t h a t P E D V and related isolates are antigenieMly unrelated to representative m a m m a l i a n coronaviruses by enzyme-linked i m m u n o s o r b e n t assay (ELISA).

Stock PEDV consisted of pooled rabbit sera obtained 48 hours after subcutaneous inoculation with pleural fluid or infectious serum. A low virulent variant of PEDV from the blood of a rabbit, six months after infection was also used (3). Stock of this variant was infectious serum h'om an l tth serial rabbit passage, obtained 72 hours after inoculation. The Stockholm agent was kindly supplied in 1980 by Dr. J. D. Small, NIH, Bethesda, U.S.A. as freeze dried infectious serum 73 015. Three other isolates used were from different laboratories and designated SILL, Paris I and Minneapolis (Table 1) . Stocks of the Stockholm agent and the three isolates were from the first or second rabbit passage, prepared in the same manner as for PEDV. All stocks were stored at ~-70 ° C. 

The number of rabbit-infectious doses per ml of the virus stocks was determined by making 10-fold dilutions in PBS (pH 7.0) using one to four rabbits per dilution. The term rabbit-infectious dose refers to a dose capable of producing typical clinical responses, i.e. fever together with uveitis or death with characteristic necropsy findings and/or clinical protection to challenge with PEDV 30 days after inoculation. An exception to this procedure was the titration of SBL, where the homologous stock virus was used for challenge.

lZepresentative m e m b e r s of t h e two m a m m M i a n eoronavirus antigenic groups were used. These were HCVs 229E (7) a n d CV Paris (20) , t h e l a t t e r being closely r e l a t e d to I-ICV OC 43 (8) . All m a m m a l i a n coronaviruses, apart, from one or two possible exceptions t h a t h a v e n o t b e e n fully charaeterised, are antigenicMly r e l a t e d b y E L I S A to one or t h e o t h e r of these strains (8, 13, 21, 23) .

H C V 2 2 9 E was g r o w n in M R C c o n t i n u o u s cells (14) a n d CV Paris was g r o w n in I t R T 18 cells (8) . T h e cells were frozen a n d t h a w e d once a n d t h e resulting" suspensions were clarified a t 2000 × g for 30 m i n u t e s . T h e p r e p a r a t i o n s of H C V 2 2 9 E a n d CV P a r i s c o n t a i n e d b e t w e e n 107 a n d 10 s particles p e r ml, as d e t e r m i n e d b y electron microscopy.

T h e E L I S A was b a s e d on m e t h o d s described previously for d e t e c t i n g t t C V antibodies in r a b b i t (10) a n d h u m a n (11, 15) sera.

Samples of P E D V stock were d i l u t e d 1 : 10 in P B S (pH %0) a n d passed serially t h r o u g h Millipore filter m e m b r a n e s g r a d u a t e d in porosity from 800 to 25 n m . Samples of filtrates were t a k e n for t i t r a t i o n in r a b b i t s a f t e r passage t h r o u g h 220, 100, 50 a n d 25 n m filters. Similar e x p e r i m e n t s were done w i t h I-ICV229E a n d polio virus, t y p e 3, s t r a i n S a u k e t t . T h e i n f e c t i v i t y of t h e I-ICV 229 E samples were d e t e r m i n e d b y fluorescent focus assay (t 6). T h e S a u k e t t s t r a i n was grown a n d t i t r a t e d in p r i m a r y m o n k e y k i d n e y cells. T h e culture was c o n c e n t r a t e d a n d purified, b u t was diluted 1 : 100. (The E n t e r ovirus D e p a r t m e n t a t S t a t e n s S e r u m i n s t i t u t supplied ~/he polio virus a n d p e r f o r m e d t h e i n f e c t i v i t y titrations).

Two ml c r y o t u b e s c o n t a i n i n g P E D V stock, u n d i l u t e d a n d d i l u t e d t : 1 0 0 in P B S (pH 7.4), were s u b m e r g e d in a stirred w a t e r b a t h at 37 o a n d 56 o C. A t selected i n t e r v a l s t h e r e a f t e r t h e t u b e s were r e m o v e d a n d t h e c o n t e n t s i m m e d i a t e l y t i t r a t e d in r a b b i t s using chilled P B S diluent.

Albino rabbits, aged 3 --5 m o n t h s , from t h e closed colony at S t a t e n s S e r u m i n s t i t u t (Ssc : CPtI), were used. T h e m a j o r i t y of these animals h a d b e e n used once for p y r o g e n t e s t i n g of p r o t e i n fractions of h u m a n blood. All inoculations were m a d e s u b c u t a n e o u s l y .

F o r serological e x a m i n a t i o n paired sera were o b t a i n e d before a n d 30 days after p r o v e n infections of groups of fore" r a b b i t s w i t h P E D V , S t o c k h o l m agent, SBL, Paris I, or Minneapolis. T h e sera were stored a t --2 0 ° C until examined.

U n i f o r m groups of r a b b i t s were infected w i t h t h e same dose of t h e various isolates. T h e S t o c k h o l m a g e n t was passaged serially in r a b b i t s a t i n t e r v a l s of 3 --1 0 days a n d 30 days as described for P E D V (1). T h e results of t h e P E D V passages are s h o w n in Table 3 for comparison. Passages of t h e t h r e e P E D V isolates were done in t h e same w a y b u t only a t i n t e r v a l s of 3~1 0 days. T h e i n o c u l u m for t h e first passage was t h e original m a t e r i a l while 1 ml of blood or pleural fluid was used for t h e s u b s e q u e n t passages. T h e low v i r u l e n t v a r i a n t of P E D V was also passed serially in rabbits, w i t h a n i n t e r v a l of 7 days b e t w e e n passages. 

Infectious serum diluted 1 : 10 and with a titre of 105 passed the 100 nm filter without loss in infectivity. The virus also passed through 50 nm membrane, but with a 10 to 100-fold loss in titre. No detectable infectivity passed 25 nm filter. Infectious ttCV229E particles did not pass the 100 nm filter, and polio virus particles passed the 50 and 25 nm filters with a 100-fold loss in infectivity. These findings indicate that P E D V particles are between 25 and 50 nm, smaller than coronavirus particles and larger than polio virus particles.

The rate of inactivation of undiluted and diluted infectious serum at 56 ° C was about the same and the calculated half life of infectivity of the two preparations was 0.12 hours (Fig. 1 ). There was a 1 to 10-fold loss in infectivity of the diluted preparation after 2 days at 37 ° C, but after 5 days residual infectivity was 0.01 per cent. Stock virus kept at 4--5 ° C for 5 months showed no loss of infectivity. 

The pathogenic properties of the isolates were compared directly (Table 2 ) and by passage in rabbits to simulate the "natural" infection, i.e. a concomitant infection of rabbits used for propagation of pathogenic treponemes (Table 3) .

Both the Stockholm agent and PEDV produced high mortality with almost the same incidence of fever and uveitis among the survivors. When passed at an Number of rabbit-infectious doses b All survivors failing to show fever or uveitis were protected when challenged with PEDV, except the SBL-infeeted animals i n t e r v a l of 30 days, there was no m o r t a l i t y a n d the clinical signs of P E D were reduced. This indicates t h a t the two isolates are similar in their pathogenesis and persistence in blood. Minneapolis a n d the low virulent v a r i a n t of P E D V produced a febrile response, b u t rarely detectable uveitis. The clinical response by the SBL or Paris I was intermediate between these responses. A distinguishing feature of the SBLinfection was the delayed onset of uveitis, i.e. 8 t h --l l t h day post-inoculation as compared with 3 r d --6 t h days for the other isolates.

As measured by clinical response only P E D V appeared to increase in virulence during passage. When rabbits were challenged with PEDV 30 days after infection, they were nearly always chnically protected, the only exception being the SBL-infected animals.

PEDV and the Stockholm agent, which both caused high mortality in rabbits, were not used for active immunization. Minneapolis was used for challenge in only one experiment since this isolate does not cause typical P E D ( Table 4 ).

The immunizing infections of the various groups of rabbits produced the same clinical syndromes as observed in the infection experiments. When challenged all the rabbits were protected except for the SBL-infected rabbits challenged with PEDV or the Stockholm agent. The majority of animals in these two groups developed F E D symptoms, although none died. The SBL-infected rabbits challenged with Paris I did not produce PED symptoms, but several showed a transient ephemera] fever. Challenge with SBL did not cause clinical signs of PED.

These results together with those of the infection experiments demonstrate a similarity in immunogenicity between PEDV and the Stockholm agent, and between Paris I and Minneapolis. The immunogenicity of SBL is evidently different from PEDV and the Stockholm agent. 

a Inoculum expressed as number of rabbit-infectious doses, given subcutaneously b Numerator equals number of protected rabbits and denominator number of challenged rabbits c Not done

HCV229E and CV Paris were used as antigens in E L I S A to detect antibody rises to coronaviruses in paired sere from 20 rabbits infected with the 5 isolates. A ratio of 2 or more in the absorbance values obtained with postinocnlation serum compared to preinocnlation serum at the same serum dilution was considered to represent a significant antibody rise. Using this criterium no antibody rises to HCV229E were detected in any of the paired sere, whereas only one paired sere from an SBL-infected rabbit showed an antibody rise to CV Paris, i.e. it had a ratio of 2.2. The same results were obtained when the same or other pairs of sera were examined by a neutralisation test with HCV229E (kindly performed by Dr. Sylvia E. Reed, Central Public Health Laboratory, London, England) and in a CF test with HCV 0C43 antigen (kindly performed by Dr. T. Hovi, University of Helsinki, Finland).

I t has been suggested that the Stockholm agent and P E D V are similar on the basis of history and pathology (1, 5, 22) , but they have never been compared directly. This study shows that they are very similar if not identical. The results also indicate that the less pathogenic Paris I and Minneapolis are similar to PEDV, and that SBL is probably closely related to PEDV, but differs in its pathogenic and immunogenic properties. This agrees with the observations at isolation, although Paris I did not cause mortality (2) .

SBL was isolated in 1978 from treponema-infected rabbits from the laboratory, that had supplied the rabbit material from which the Stockholm agent was isolated in 1970 (2) . Attempts in this laboratory to remove the treponemes from the Stockholm agent were made on several occasions. This was done by passaging contaminated treponemal suspensions through hamsters, which are not susceptible to PEDV, but which allow the multiplication of treponemes (2, 6) . These efforts appeared to be successful since intercurrent rabbit mortality fell to less than one per cent after 1976. However, the isolation of SBL two years later suggests this was a failure and that the reduction in intercurrent mortality was due to a mutational change in the Stockholm agent.

The apparent stability in the virulence of the isolates during the 20 to 54 rabbit passages may well be due to the number of passages. PEDV had 170 rabbit passages over a 3-year period and during this time the annual mortality increased from 39 to 68 per cent.

Sh~ALL et al. (19) and OSTER~A~-S et al. (18) suggested a role for coronaviruses in PED, while the latter and LAPIERRE et al. (12) also reported the association of coronavinls-like agents with intestinal infections in rabbits. Both S5~ALI~ et al. and OSTEI~I~AVS st al. detected coronavirus-like particles in infectious serum. However, in our hands no coronavirus-like particles were detected by electron microscopy in rabbits infected with P E D V (FE~NESTAD et al., unpublished results), and by filtration infectious PEDV particles were clearly smaller than infectious HCV 229E particles. The heat stability of PEDV, which is in agreement with S~AI~L et al. (19) , was greater than that reported for coronaviruses (21, 23) . SMALL et al. used a CF assay to show that antigen(s) :in infectious serum displayed a cross-reactivity with HCVs 229E and 0C43 and that hyperimmune sera from convalescent rabbits contained antibodies to HCV229E. These results are surprising as these HCVs have been shown to be antigcnically unrelated (13, t5, 23) . Our ELISAs show that P E D V does not generally produce antibodies related to HCV 229E and CV Paris. One of 20 rabbits showed a positive reaction to CV Paris, but this is probably not due to infection with PEDV, as normal rabbit sera contains antibodies to both CV Paris and HCV 0C43, but not HCV229E, indicating the existence of a possible rabbit coronavirus antigenically related to CV Paris and HCV OC43 (MAoNAuGHTO~¢, unpublished results). K . L . FENNESTAD and M. R. I~¢IAcNAUGHTON: B o t h S~L L et al. (19) a n d 0STERI~AUS et al. (18) left open t h e possibility t h a t t h e cause of P E D m i g h t be a virus different f r o m coronavirus. Our findings agree w i t h this i n t e r p r e t a t i o n .

Pleural effusion disease in rabbits. Clinical and post morton observations

Pleural effusion disease agent as passenger of Treponema pallidum suspensions from rabbits

Pleural effusion disease in rabbits. Observations on viraemia, i m m u n i t y and transmissibility

F e v e r after inoculation of rabbits with Treponema pallidum

D e m o n s t r a t i o n of a virus-like agent contaminating material containing the Stockholm substrain of the Nichols pathogenic Treponema pallidum

Screening out a virus-like agent from the testicular suspension of the Nichols pathogenic T. pallidum. W i t h observations on certain characteristics of the agent

A new virus isolated from the h u m a n respiratory tract

Prevalence of h u m a n coronavirus a n t i b o d y in the population of Southern Iraq

Spontaneous deaths a m o n g rabbits inoculated with Treponema pallidum less t h a n 2 weeks before

1~.: E n z y m e -l i n k e d i m m u n o s o r b e n t assay for coronaviruses IcICV229E and M H V 3

E n z y m e -l i n k e d immunosorbent assay for detection of antibody in volunteers experimentally infected with h u m a n eoronavirus 229E group viruses

Preliminary report on the observation of a eoronavirus in the intestine of the laboratory rabbit

Structural and antigenic relationships between human

The genome of h u m a n eoronavirus strain 229E

Two antigenic groups of h u m a n coronavirus d e t e e t e d by using enzyme-linked i m m u n o s o r b e n t assay. Infect. I m m u n

IK~-I

I n t e r c u r r e n t death of rabbits after inoculation with Treponema pallidum in the Netherlands. 7th 1CLAS Syrup. U t r e c h t 1979

Coronavirus-like particles in laboratory rabbits with different syndromes in The Netherlands

Rabbit cardiomyopathy associated with a virus antigenically related to h u m a n coronavirus strain 229E

Une dpid6mie d'ent6rocolites ulc6ron6crosantes en maternit6

Coronaviridae : second report

The biology and pathogenesis of coronaviruses

Received September 20, 1982