Adaptation of the viral haemorrhagic disease virus of rabbits to the

Rev. sci. tech. Off. int. Epiz.,
1991, 10 (2), 337-345
Adaptation of the viral haemorrhagic
disease virus of rabbits to the
DJRK cell strain
CHUAN-YI JI, NIAN-XING D U and WEI-YAN X U *
Summary: Liver emulsion of rabbits which had died of viral haemorrhagic
disease (VHD) was inoculated onto DJRK cell culture. After two passages,
specific cytopathic effect was observed. Immunofluorescence was found in the
nucleus at the early stage of infection and later also in the cytoplasm. The virus
propagated in cell culture at the fifth, tenth and sixteenth passages was found
to cause typical VHD. Electron microscopy showed that there were numerous
virions in the infected cells. The cultured virus, inactivated with formaldehyde,
could elicit haemagglutination inhibition antibodies in the inoculated rabbits
and protect them against the challenge of virulent VHD virus.
KEYWORDS: Cell culture - DJRK cell strain - Viral haemorrhagic disease virus
of rabbits.
INTRODUCTION
Viral haemorrhagic disease (VHD) of rabbits is a new, acute and highly
communicable disease (7). The aetiological agent, V H D virus (VHDV), has many
unique characteristics ( 1 , 6, 10, 12) and is very difficult to grow in cell culture. Since
1984, various cell cultures, including primary rabbit cells (kidney, liver, lung and testis),
as well as cell lines (PK-15, BHK-21, MA-104, IBRS-2, H e L a and V E R O ) have been
used to cultivate V H D V . Despite the addition of trypsin to the media, the substitution
of rabbit serum for calf serum, the use of culture fluid together with cells in passage
and inoculation just after adsorption of cells on a glass surface (2, 3, 9), n o attempt
to cultivate V H D V has succeeded. A cell strain of epithelial type was established in
this laboratory from primary rabbit kidney cells. The purified m u t a n t cell obtained
was named Du and Ji Rabbit Kidney (DJRK) after its originators. There is evidence
that V H D V has been adapted to D J R K .
ESTABLISHMENT
OF T H E
DJRK
CELL
STRAIN
The kidney of an unweaned rabbit is digested with 0 . 2 5 % trypsin and cultured
in medium " R P M I 1640" (containing 10% calf serum). The primary culture is treated
with trypsin and diluted 1:2 for further passages. After culturing the fourth passage
* Division of Veterinary Microbiology, Nanjing Agricultural University, Nanjing 210014, People's
Republic of China.
338
for four days, foci of epithelioid cells appear in the cell monolayers. The foci
three-dimensional and are visible to the naked eye after another ten days of
The other cells fail to divide and undergo gradual degeneration. Confluent
the transformed focus may be washed off without the need for dispersal by
In this way, a single focus can be purified.
become
culture.
cells of
trypsin.
DJRK cells are passaged in dilutions of 1:4 in " R P M I 1640" or McCoy's 5A media
at a seeding efficiency close to 100%. The culture becomes confluent in 2-3 days and
then superconfluent, losing the property of contact inhibition. It m a y be subcloned
at high plating efficiency without the need for accessory cells. The epithelioid cells
have a high nucleus to cytoplasm ratio. Each nucleus contains several large nucleoli.
Mitochondria, endoplasmic reticula and polyribosomes are abundant. The cell surface
bears m a n y long microvilli. T h e chromosomes are identified as heteroploid.
Multinucleated giant cells are present. These features indicate that the D J R K cell line
is a highly viable mutant with active metabolism and rapid growth. The characteristics
have been maintained after more t h a n 50 passages.
VIRUS CULTIVATION
A N D
CYTOPATHIC
EFFECT
Liver emulsion, extracted with chloroform from rabbits which had died of V H D ,
was inoculated onto D J R K cell culture and adsorbed at 37°C for 45 min. Having
been washed with H a n k s ' solution and added to McCoy's 5A medium, the cell culture
was cultivated at 37°C for 24 to 72 h. Haemagglutination (HA) was not detected
in the culture supernatant, but reached titres of approximately 2 after freezing and
thawing with the cells. This suspension was used for passage. Specific cytopathic effect
(CPE), characterised by rounding, aggregation and detachment of the cells, appeared
at the third passage. Some cells shrunk and formed thread-like intercellular bridges
(Fig. 1). The colour of the indicator showed that the p H of the infected culture was
lower t h a n that of the uninfected culture.
3
IMMUNOFLUORESCENCE
O n the tenth passage, the V H D V antigens in the infected cells were detected
by the indirect immunofluorescence test (IIFT). Fluorescence was first localised
in the nucleus 2-4 h post infection (p.i.). At the 8th h p . i . , fluorescence appeared
intensely a r o u n d the nuclear membrane; later, it could also be observed in the
cytoplasm.
ELECTRON
MICROSCOPY
Numerous virions were observed by electron microscopy in thin sections of infected
cells of the tenth passage. The nuclear membranes were intact and the accumulated
granular materials were observed in the nuclei 4-8 h p . i . (Fig. 2). Virus-like particles
339
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FIG. 2
Thin section of the VHDV-infected DJRK cell
Granular materials accumulated in the nucleus
at the early stage of infection
with a diameter of about 32 nm were seen after 12 h p.i. These had primarily
accumulated outside the nuclei and near the disrupted nuclear membranes or enlarged
nuclear pores; some were scattered in the nuclei (Fig. 3).
PATHOGENICITY
Eight susceptible rabbits which had been inoculated with the cell culture of the
fifth passage died of typical V H D within five to eleven days. The titres of the virus
H A reached 2 - 2 from the first to third day following inoculation and quickly rose
2
6
341
FIG. 3
Thin section of the VHDV-infected DJRK cell
Numerous virions have accumulated around the nucleus and
some are scattered in the nucleus (single arrow)
The nuclear membrane is partially disrupted and the nuclear pores
are enlarged (double arrow)
6
1 2
1
to 2 - 2 ; this lasted two to eight days. H A titres increased to approximately 2 in
most of the dying rabbits (Fig. 4), all of which experienced pathological changes typical
of V H D ; the kidney lesions, however, were more serious than those associated with
natural disease. The viruses of the tenth and sixteenth passages were proved to have
similar pathogenicity.
342
FIG.
4
The serum haemagglutination kinetic curves of two rabbits
inoculated with cultured (•
•
)
and virulent ( X
X
) VHDV
IMMUNOGENICITY
Six susceptible rabbits were inoculated with the cell culture of the eleventh passage,
inactivated with formaldehyde. The titres of induced haemagglutination inhibition
antibodies were approximately 2 on the 7th day and 2 on the 14th day post
inoculation. The rabbits were able to resist the challenge of virulent V H D V on the
fifteenth day.
5
1 0
DISCUSSION
Establishment of the D J R K cell line is important for the propagation of V H D
virus in cell culture. The localisation of virus-specific antigens and virions in vivo
has shown that kidney epithelial cells are target cells for the virus (11). T h e virus
343
replicates mainly in cells which are metabolically active and capable of regeneration,
such as the endothelial cells of blood vessels, liver cells and lymphocytes within
germinal centres of the spleen. The D J R K cell line is a m u t a n t of the rabbit kidney
epithelial cell which is highly viable, metabolically active and rapidly dividing, suitable
for the replication of V H D virus.
Owing to the results of such classical methods as high performance liquid
c h r o m a t o g r a p h y and synthesis of double-stranded D N A in vitro, it is suggested that
the genome of V H D V is linear, single-stranded D N A ( 1 , 13, 14). V H D V is assumed
to be a parvo-like virus (12). Contradictory results have also been reported on the
nature of the genome as single-stranded R N A . The morphology and the major
structural protein of the virus have led some research workers to conclude that V H D V
is a calicivirus (8). The results of the morphogenesis of V H D V obtained by the authors
and the localisation and development of the viral antigens in cell culture indicate that
the early stages of viral replication take place in the nucleus. This would support the
idea that the virus is a parvovirus. In addition, although the virions display cup-shaped
depressions on the surface, the morphological pattern is different from that of
caliciviruses (6).
Although V H D V has been adapted to D J R K cell strain, there are still many
unsolved problems. A fraction of the population of infected cells may remain alive
and, by dividing, form a new monolayer. Because V H D V antigens are still detectable
in the cytoplasm by IIFT after five passages of the infected cell culture, it is suggested
that a persistent infection in cell culture may exist, and that this infection possibly
correlates with defective interfering virus particles (4, 5).
*
ADAPTATION DU VIRUS DE LA MALADIE HÉMORRAGIQUE VIRALE DU LAPIN
EN CULTURE CELLULAIRE DJRK. - Chuan-Yi Ji, Nian-Xing Du et Wei-Yan Xu.
Résumé: Les auteurs ont inoculé un broyat de foie de lapins ayant succombé
à la maladie hémorragique virale du lapin (VHD), en cultures de cellules DJRK
(souches obtenues à partir de broyats de reins de lapereaux ; les cellules traitées
à la trypsine 0,25 % sont cultivées en milieu RPMI 1640). Après deux passages,
ils ont observé un effet cytopathogène spécifique. Ils ont constaté peu de temps
après l'infection un phénomène d'immunofluorescence dans le noyau, puis plus
tard dans le cytoplasme. Le virus obtenu aux cinquième, dixième et seizième
passages en culture cellulaire pourrait provoquer une maladie typique. La
présence de nombreux virions dans les cellules auxquelles a été inoculé le virus
a été mise en évidence par la microscopie électronique. Le virus adapté en culture,
et inactivé par le formol, a pu provoquer la formation d'anticorps inhibant
l'hémagglutination chez les lapins auxquels il a été administré et qui ont résisté
à l'épreuve virulente.
MOTS-CLÉS : Culture cellulaire - Souche cellulaire DJRK - Virus de la maladie
hémorragique virale du lapin.
*
* *
344
ADAPTACIÓN CELULAR DEL VIRUS DE LA ENFERMEDAD HEMORRÁGICA
VIRAL DEL CONEJO EN CEPAS CELULARES DJRK. - Chuan-Yi Ji, Nian-Xing Du
y Wei-Yan Xu.
Resumen: Los autores han inoculado trituraciones de hígado de conejos muertos
de la enfermedad hemorrágica viral (VHD), en cultivos celulares DJRK (cepas
obtenidas a partir de trituraciones de riñon de gazapos; las células tratadas por
tripsina a 0,25% son cultivadas en medio RPMI 1640). Al cabo de dos pasajes,
se observó un efecto citopatogénico específico. Los autores constataron, poco
tiempo después de la infección, un fenómeno de inmuno-fluorescencia en el
núcleo, y luego a nivel del citoplasma. El virus obtenido en el cultivo celular
durante el quinto, décimo y decimoquinto pasajes provocó una forma típica
de la enfermedad. La presencia de numerosos viriones en las células en las cuales
se había inoculado el virus fue puesta en evidencia por microscopía elecrónica.
El virus de cultivo, inactivado por formaldehído, ha podido provocar la
formación de anticuerpos inhibidores de la hemaglutinación en los conejos
inoculados, que resistieron a la prueba virulenta.
PALABRAS CLAVE: Cepa celular DJRK - Cultivo celular - Virus de la
enfermedad hemorrágica viral del conejo.
*
* *
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