A small haemagglutinating porcine DNA virus

J. COW. PATH.

196%

A SMALL

VOL.

79.

371

HAEMAGGLUTINATING DNA VIRUS I. ISOLATION

PORCINE

AND PRO~PERTIES BY

SHEILA

F. CARTWRIGHT,

MARGARET

LUCAS and R. A. HUCK

Central VeterinaryLaboratory Minis~y of Agkdture, Wqbridge, Suwq

INTRODUCTION

An investigation carried out at the Central Veterinary Laboratory over the past 5 years into pig breeding herds with histories of abortions, stillbirths and neonatal losses,resulted in 111 isolations of viruses. Thirteen per cent. of these were enteroviruses, but the remaining 87 per cent. were small haemagglutinating viruses which appeared to be serologically identical (Car&right and Huck, 1967). One isolation, 59e/63, was selected as a prototype. This paper reports the results of investigations into the physical and chemical properties of this vii-us, together with clinical histories of the relevant herds and data from serological surveys. MATERIALS

AND

METEIODS

Preparation of t&e cultures. Primary pig kidney (PK) monolayer cultures in 1 oz. medical flat (MF) bottles, 20 oz. MF bottles or Leighton tubes, were prepared as previously described (Huck, Cartwright and Paterson, 1962). Primary pig testis (PT), calf kidney (CK), calf testis (CT) and lamb testis (LT) cultures were prepared in a similar manner. Monkey kidney (MK), dog kidney (DK), HEP 2 and HeLa tissue cultures were obtained from a commercial source.+. Collection and preparation of specimens. Tissues from field and experimental cases were prepared as 1/ 10 suspensions by grinding with sand and Earle’s saline solution plus 05 per cent. la&albumin hydrolysate and 0.01 per cent. yeast extract (EYL) containing 10 times the normal concentration of antibiotics i.e. 1,000 i.u. penicillin, 1,000 mg streptomycin and 200 i.u. mycostatin per ml. These suspensions were allowed to stand for 15 minutes at room temperature and then centrifuged at 223 g for 30 minutes. The supematant fluids were removed with a pipette and stored at -2OOC. until required for inoculation onto tissue cultures. Rectal and vaginal swabs were rotated in EYL plus antibiotics and fluid extracts clarified by centrifugation. Vaginal mucus samples were treated as tissues. Inoculation and examination of cultures. All samples were inoculated in 02 ml. amounts onto washed 1 ox. MF bottles of PK cultures, which were held at 37%. for 30 minutes for absorption of virus and then overlaid with EYL plus 1 per cent. calf or lamb serum and returned to 37OC. These cultures were examined daily for cytopathic effects. After seven days the supematant fluids were harvested and passaged onto further cultures, the original cultures being refreshed with media and examined daily for a further 7 days. Three blind passages were carried out. All supernatants were tested against chick red cells for the presence of haemagglutinins. Stocks of virus were prepared by the inoculation of 20 oz. M!F bottles of PK monolayers with l Wellcome Laboratories,Beckenbam,Kent.

372

PORCINE

DNA

VIRUS:

ISOLATION

AND

PROPERTIES

1 ml. of seed virus. Cultures were frozen and thawed on the 5th day after inoculation, the suspensions were pooled and centrifuged, the supernatants collected, dispensed in 1 ml. amounts and stored at -2OOC. Haemugglutination test (HAT) and titration of virus. Chick, quail, guinea-pig, sheep, calf, human ‘O’, rhesus and patas monkey red blood cells were tested for agglutination by the virus. Chick red cells were selected as those most easily obtained and as giving the clearest end point. Two-fold dilutions of virus were made in 0.2 ml. volumes of Verona1 buffer in perspex trays. To each was added 0.2 ml. of 0.7 per cent. red cells. The trays were kept either at room temperature for 30 minutes or at 4’c. overnight. Whenever possible estimations were made of both the infectivity titre and HA titre of the virus. Infectivity titrations were made by inoculation of dilutions of the virus suspension into Leighton tube cultures, the presence of virus being determined both by testing for the production of haemagglutinin and demonstration of intranuclear inclusions in coverslip cultures stained with haematoxylin and eosin. Haemagglutination titres were expressed directly as the reciprocal of the highest dilution to give a positive reaction. Infectivity titres were expressed as log. TC.IDBO/ml. of virus suspension. Huemugglutinution inhibition test (HIT). Test sera were treated by one of 2 methods to remove non-specific inhibitors : (a) b heating at 37OC. with 0.02 per cent. crystalline trypsin followed by heating for 40 minutes at 6OOC. according to the method of Sampaio and Isaacs (1953); (b) by inactivation at 56OC. for 30 minutes and allowing to cool. To remove non-specific red cell agglutinins O-2 ml. of 10 per cent. washed chick red cells were added to 0.02 ml. of serum. The sera were then left at 37OC. for 1 hour and 0.6 ml. of Verona1 buffer was added to give a l/10 dilution. The red cells were then centrifuged off and the supernatants stored at -2OOC. Except in the initial tests all sera were treated with heat and 10 per cent. chick red cells as described above. A variation was found between birds with regard to the sensitivity of their red cells towards the virus. To overcome this 6 stock birds were maintained and bled on alternative weeks in groups of three. Two-fold dilutions of serum were tested against 4 HA units of virus. Pre- and post-infection pig sera of known titre were included as controls. The serum titres were expressed as the reciprocal of the highest dilution of serum inhibiting haemagglutination of 4 HA units of virus. Huemudsorfition test (HAD). Tests using the methods of Vogel and Shelokov (1957) were carried out at room temperature, 37OC. and 4OC. with chick and guinea-pig red blood cells. Inoculation of embryonuted hens’ eggs. Six-day-old embryonated hens’ eggs were inoculated with virus suspensions by the yolk sac (YS) route. Three blind passages were carried out at weekly intervals. Inoculation of mice. Two-day-old and 7-day-old suckling mice were inoculated by the intraperitoneal (i/p) and intracerebral (i/c) routes. A further passage in mice was carried out using ground mouse tissues from the first passage. Production of antiserum. Rabbits were given 3 weekly i/p injections of tissue culture virus and were bled out 10 days after the last injection. Fluorescent antibody technique. A conjugate was prepared according to the method of Mengeling, Gutekunst, Fernelius and Pirtle (1963) using a pig serum with an HI titre of 1 / 1280. PK cover-slips were infected with strain 59e/63 (PK 10) and stained at 1, 2, 3 and 6 days. RESULTS

Zncidence Details of the herds and the samples from which these viruses were isolated are recorded in Table 1. Ninety-six isolations of virus were made from tissues,

SHEILA

F. CARTWRIGHT

373

et al.

swabs and semen from 13 of the 141 herds examined. These isolates were serologically identical. Strain 59e/63, isolated from the colon of a stillborn piglet (Herd l), was selected as a prototype for e.xami+ion in detail. In addition to the isolations recorded in Table 1, the virus was rec~ered from monolayer cultures prepared from the kidneys of a 3-week-old pig. TABLE DETAILS

Herd

OF ISOLATIONS

1

Stillbirths

2

Infertility in sows, heavy losses in piglets within 12 hours of birth

OF

5%/63

AititZkZlS examined

Case hisby

NO.

1

and neonatal losses

Samfibssyielding

virus

2 sows,3 stillborn piglets, 3 neonatal dead piglets

Colon, liver, lung, 2 spleens, 4 spinal cords

23 sows, 2 foetuses

2 placentae; foetal

SdOg9 2 sow sera positive titres

14 vaginal swabs, NT

colon, lung, spleen 25/ 150 piglets died at 2-3 weeks. Followed by a high percentage of stillbirths in litters

3 stillborn piglets, 1 live piglet,

Virus recovered throughout piglet tissues, 2 vaginal swabs

NT

Neonatal losses at 2-3 days, Sows vaginal discharge

2 sows, 4 piglets

Pi let cord, spf eat, rectum and cerebellum

NT

5

Abortions

4sowa

1 vaginal swab

NT

6

Stillbirths

2-2

1 piglet

piglet spinal cord

2 sows positive titres

3

6~0~s

_4

7

Boars of low fertility

6 boars

4 semen

6 positive titres

8

Loss of condition in sows, coughing, vaginal discharge, stillborn piglets

S’sows, 3 stillborn piglets

@le;tlez:iver, ,

‘,:I

9

Neonatal death, weakness of piglets. Myofibrillar hypoplasia diagnosed

2 sows, 2 dead piglets, 2 live piglets

Spleen, intestine and lung oflive piglets

2 sows had positive titres

Stillbii

1 stillborn piglet

liver, kidney,

10

positive

NT

StOllliSCh

11

Stillbirths

1 sow, 2 stilllxJm piglets

12

Abortions

Saborted foemses

13

Stillbirtha Full term

3slnallintestines,

NT

2 liven, kidney, spleen P~klate~

2sowIhad positive titres

kidney and 1~; NT = not tested

Four hundred and thirty sexa collected in 1966 from pigs of all ages from abattoits and farms in England apd Scotland w&e examined for antibodies to strain 59e/63. Thirty-three per cent. had titrcs of a l/320. There was a definite age incidence with a sharp decline in the titres of pigs about 4 months of age,

374

PORCINE

DNA

VIRUS:

ISOLATION

AND

PROPERTIES

presumably due to waning maternal immunity. There was a sharp rise in titres in stock of breeding age, probably caused by active infection. In 1967, 70 per cent. of 40 sera examined from herds with breeding problems were positive, but this sample would obviously be heavily weighted in age incidence. Rising antibody levels were detected in herds with developing “breeding” problems. Antibodies to the virus were detected in sera from Canada, United States, Holland and Northern Ireland. Characterisation

of Strain 59e/63

Growth properties. The cytopathic effect (cpe) of strain 59e/63 on pig kidney monolayer cultures varied according to the state of the culture. It was most obvious in young, actively growing cultures inoculated about 3 or 4 days after preparation, and was practically non-existent in overgrown granular cultures. In the young culture vacuolation of the cytoplasm was seen 2 to 3 days after inoculation. This was sometimes followed by a gradual necrosis of the cell sheet by the 7th or 8th day. Observation of cpe was found not to be a satisfactory method for virus detection and titration. Coverslip PK cultures stained with haematoxylin and eosin showed the development of intranuclear inclusions as early as 16 hours after inoculation. These inclusions were eosinophilic and granular in the early stages, but later became denser and more darkly stained. Eosinophilic inclusions then appeared in the cytoplasm followed by vacuolation and necrosis of the cell (Figs. 1 to 4). There was evidence of a slight multiplication in PT and Hep-2 cells, and intranuclear inclusions were observed in stained preparations of PT. Other tissue culture systems tried were HeLa, MK, CK and CT; in none of these was growth of the virus detected. The virus agglutinated chick, rat, guinea-pig, human ‘O’, rhesus and patas monkey red cells, but did not agglutinuate the red cells of quail, sheep and calf. Chick red cells gave the clearest end point, but those of the rhesus monkey and human ‘0’ were the most sensitive. Haemagglutination was first detected in the supernatant fluid on the 2nd day after inoculation, although with primary isolates it was often not detectable until the 2nd or even 3rd passage. With an adapted strain the peak HA was observed at 3 or 4 days with titres as high as l/51 2. Infectivity reached a peak about the 3rd day after inoculation with a titre of about 10”’ TCIDao/ml. Ultrasonication of the deposited cell debris resulted in HA titres up to l/4,096 and infectivity titres as high as 107’0 TCIDao/ml. Slight haemadsorption of chick red cells occurred at 4OC. on infected monolayem, but it was not always repeatable. This effect was never observed in control cultures. There was no evidence of infection following the inoculation of 5 or 6-day-old embryonated eggs by the YS route or of suckling mice by i/p and i/c mutes. Physical and chemical properties. Filtration studies using both HA and infectivity determinations showed that the virus passed a 42 rnp A.P.D. and not a 21 rnp A.P.D. filter. Applying the factor 0.64 (Black, 1958) to the average of these two, gave an approximate size of 20 rnp. Electron microscopical examination of concentrated tissue culture virus suspension showed a particle of size approximately 28 rnp with cubical symmetery (Cruickshank, personal communication).

SHEILA

F. CARTWRIGHT

375

#t d.

Treatment of the virus with 20 per cent. ethyl’ ether for 18 hours at 4OC. resulted in no loss of infectivity or of HA titrc compared with an untreated control. There were no significant di%rences in infectivity or HA titms between aliquots of virus held at pH 7-O and pH 3-O for 3 hours at 37OC. There was little or no loss in HA titres after storage at -20°C. and -7OOC. for 6 months. Virus held at 70°C. for 2 hours, 56OC. for 48 hours and 37OC. for 7 days was still infective. At 80°C. infectivity appeared to be lost after 5 minutes. Tests were carried out to determine the nucleic acid type using 5-iodo-2deoxyuridine (IUDR) as a known inhibitor of DNA synthesis (Hamparian, Hilleman and Kelter, 1963 ; Lam and Atherton, 1963). IUDR was incorporated into the overlay of infected cultures at a concentration of 10m4 M. Cultures inoculated with Talfan and Aujeszky viruses were treated in, an identical fashion as known RNA and DNA viruses respectively. Results showed no difference in titre between Talfan and Talfan plus IUDR, but Aujeszky plus IUDR resulted in a definite drop in titre. Addition of IUDR to strain 59e/63 infected cultures resulted in a statistically significant decrease both in HA titre and intranuclear inclusion count. The intranuclear inclusions were shown to be Feulgen-positive. Serological properties. Hyperimmune serum to 59e/63 prepared in rabbits gave a titre of l/2560 against 4 HAU of virus. Table 2 shows that cross inhibition was TABLE sEROL~OICAL

-

Vit7d.Y (4 HAU) F;/”

RELATIONVUP,

B/63 (ram) 2560 2560

BETWREN

2

59~163

PRP,

WAVRE

AND

PPV STRAIN

PRP (rabbit)

Wavrc (rabbif)

25120 25120

640 NT

do/l

GIOIl MA?)

-

isi

NT 3 Not tested. Wak7e antiserum GlO/l antiserum

ologour titre of l/1024.

demonstrated between,59e/63 virus and antisera ‘to PRP virus (Darbyshire and Roberts, 1968), “Wavre” virus (Huygelen and Peetermans, 1967) and PPV virus strain GlO/l (Mayr, Bachmann, Siegl, Mahnel and Sheffy, 1968). The use of the fluorescent antibody technique demonstrated strong nuclear fluorcscense by 24 hours in infected PK monolayers coverslip cultures. By 48 hours the number of fluorescing nuclei had increased and a few fluorescent spots appeared in the cytoplasm of these cells. At 3 days the fluorescence in the cytoplasm was more pronounced, some cells appearing to fluoresce overall. At 6 days there was still marked fluorescence in the cytoplasm, but the nuclei were no longer very obvious. DISCUSSION

During the past few years several small DNA animal viruses have been described. They are the rat virus (RV) (Kilham and~olivier, 1959) and the related hamster osteolytic ‘H’ viruses (Chandra and Toolan, 1961), feline panleucopaenia

376

PORCINE

DNA VIRUS:

ISOLATION

AND PROPERTIES

virus (FPL) (Johnson, 1965), mink enteritis virus (Johnson, 1967b) and a group of small DNA virus-like particles which are associated with both simian (Atchison, Cast0 and Hammon, 1965) and human (Melnick, Mayor, Smith and Rapp, 1965) adenoviruses referred to as adeno-associated virus (AAV) or adeno-satellite virus (ASV). It was suggested by Mayor and Melnick (1966) that this group should be referred to as the picodnavirus group by analogy to the picornavirus group. However this group has now been retermed the parvovirus group, by the International Nomenclature Committee for Virus Classification. The results obtained in this study indicate that strain 59e/63 virus is a DNA haemagglutinating virus 20 to 28 rnp in size, both ether and acid resistant and extremely heat resistant, all properties consistent with those of the above group. Serological relationships have been established between strain 59e/63 and the “Wavre” virus (Huygelen and Peetermans, 1967) and the pig parvoviruses isolated by Mayr et al. (1968) and Darbyshire and Roberts (1968). “Wavre” virus was, however, described as a haemagglutinating picornavirus but, apart from its more obvious cpe, the properties described are similar to those of strain 59e/63 virus. It is of interest that “Wavre” virus was isolated as a spontaneous contaminant of pig kidney monolayer cultures. All isolations of 59e/63 have, with only one exception, been made from herds with breeding problems. The exception was the isolation of the virus from primary cultures prepared in the normal routine manner from kidneys of a 3-week-old piglet. Antibodies to the virus were demonstrated in animals on the farm from which the piglet had been obtained. The rather vague cytopathic effect of this virus could easily allow contamination of stock viruses grown on pig tissue monolayer cultures to pass undetected. Because of this danger all batches of pig tissue cultures prepared at this laboratory are now routinely checked for haemagglutinating activity and stained preparations are examined for the presence of the typical intranuclear inclusions. Experience has also shown that despite normal aseptic precautions bench contamination of cultures with this virus can occur presumably because of its extreme resistance. All primary isolations of virus are therefore carefully checked by re-isolation from the original material. The necessity to USA very young actively growing cultures to obtain successful growth of strain 59e/63 virus is in agreement with the apparent predilection of this virus for foetal tissues in vivo and also with the re@ements of other viruses of this group. Toolan (1968) points out that both RV and H viruses require rapidly proliferating tissues for growth and Johnson (1967a) has reported similar findings for FPL virus. Serological survey results show antibodies to 59e/63 to be widespread throughout the pig population of this country, particularly in breeding herds. The high percentage of positive titres in adult pigs might be due to subclinical infection. The occurrence of clinical manifestations might depend on such variables as the physiological state of the individual, the mute of infection and the concentration of virus available. The repeated isolation of this virus from aborted foetuses and stillborn piglets suggests that it may be one of the causes of embryonic and neonatal loss in the pig industry. Isolation from semen and vaginal mucus indicates one probable method of transmiss ion. Further studies of this virus including the experimental infection of pigs and their antibody responses will be described later.

SHEILA

PK primary Fig. Fig.

1. 2.

monolayer

Intranuclear Intranuclear

cultures, inclusions inclusions,

infected (arrowed), 24 hours

F. CART-WRIGHT

with

strain

et Ul.

59e/63.

24 hours after after infection.

H. & E.

infection. x 1750.

x 460.

PORCINE

Fig. Fig.

3. 4.

Intracytoplasmic Vacuolation

DNA

inclusions, of cytoplasm,

VIRUS:

ISOLATION

72 hours after infection. 72 hours after infection.

AND

PROPERTIES

x 670. x 460.

SHEILA

F. CARTTqHT

Gt rd.

377

SUMMARY

Ninety-six isolations of a small haemagglutinating virus were made from cases of infertility, abortions, stillbirths and neonatal losses in pig breeding herds. Isolation 59e/63, selected as a prototype, multiplied in young, actively growing PK monolayer cultures producing intranuclear inclusions. It haemagglutinated the red blood cells of chick, rat, guinea-pig, human ‘0’, and rhesus and patas monkeys. The virus particle appeared to be 20 to 28 ma in size, was ether and acid resistant and extremely heat resistant. Inhibition of multiplication by IUDR together with positive Feulgen staining indicated a DNA virus. These properties suggest that 59e/63 is a member of the picodna or parve virus group. ACKNOWLEDCMENTS

We are indebted to Dr. J. G. Cruikshank of the University of Birmingham for carrying out the electronmicroscopy and acknowledge with pleasure the technical assistance of Miss V. Pusey, and Messrs. P. Napthine, A. S. Scott, D. gtagg and G. Wibberley. REFERENCES

Atchison, R. W., Cast?, B. C., and Hammon, W. McD. (1965). Science, 149, 754. Black, F. L. (1958). vzrology, 5, 391. Cartwright, S. F., and Huck, R. A. (1967). vet. Rec., 81, 196. Chandra, S., and Toolan, H. W. (1961). 1. Nat. Cancer Inst., 27, 1405. Darbyshne, J. H., and Roberts, D. H. (1968). 1. clin, Path., 21, suppl. No. 2, 61. Harnparian, V. V., Hilleman, M. R., and Kelter, A. (1963). PTOC. Sot. exp. Biof. Med.,

112,104O.

Huck, R. A., Cartwright, S. F., and Paterson, A. B. (1962). Res. vet. Sci., 3, 429. Huygelen, C., and Peetermans, J. (1967). Arch. ges. Virusforsch., 20, 260. Johnson, R. H. (1965). Nature, 2Q5, 107; (1967a). Res. vet. Sci., 8, 256; (1967b). J. small anim. Pratt., S, 319. Kilham, L., and Olivier, L. J. (1959). Virology, 7, 428. Lam, K. S. K., and Atherton, J. G. (1963). Nature, 197, 820. Mayor, H. D., and Melnick, J. L. (1966). Ibid., 210, 331. Mayr, A., Bachmann, P. A., Siegl, G., Mahnel, H., and Sheffy, B. E. (1968). Arch. fiir. Virusforsch.,

25, 38.

Melnick, J. L., Mayor, H. D., Smith, K. O., and Rapp, F. (1965). /. Bacterial., !I@, 271. Mengeling, W. L., Gutekunst, B. E., Fernelius, A. L., and Pirtle, E. C. (1963). Can. J. camp. Med. vet. Sci., 27, 162. Sampaio, A. A. de C., and Isaacs, A. (1953). Brit. J. exp. Path., 34, 152. Toolan, H. W. (1968). Znf. Rev. exp. Path., 6, 135. Vogel, J., and Shelokov, A. (1957). Science, 126, 358. [Received

for publication,

January

17th, 19691