Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Sirokanedai, Minato-ku, Tokyo 108-8639, Japan1
Institute for Animal Health, Pirbright, Woking, Surrey GU24 0NF, UK2
Author for correspondence: Chieko Kai. Fax +81 3 5449 5379. e-mail ckai{at}ims.u-tokyo.ac.jp
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Abstract |
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Introduction |
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The most commonly used vaccine for rinderpest is the tissue culture attenuated RBOK strain, which does not cause any disease in rabbits. This vaccine was derived by repeated passage of the wild-type parent virus (Kabete O strain) in primary bovine kidney cells. Recently, the RPV-RBOK strain was rescued from a full-length DNA copy of its genome (Baron & Barrett, 1997 ). The recovery of infectious RPV from recombinant cDNAs of RPV-RBOK with exchanged components from the RPV-L strain should allow us to directly address questions concerning the molecular determinants of virulence of RPV using the rabbit experimental model. In paramyxoviruses, the surface glycoproteins, the haemagglutinin (H) and fusion (F) proteins, mediate virus attachment to, and penetration of, host cells (Scheid et al., 1972
), and so these surface proteins play an important role in allowing virus entry into the host. Here we describe experiments using a virus, designated rRPV-lapH, which was rescued from a modified form of the RBOK DNA clone where the H protein gene was replaced by the corresponding gene from RPV-L. We show that the H protein plays a key role in allowing infection of rabbits by RPV, but that it does not determine pathogenicity in this species.
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Methods |
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Viruses and rescue of recombinant virus from cDNA.
The L strain of RPV, adapted to B95a cells, was used in this study. RPV-L grown in B95a cells maintains its virulence for rabbits (Ishii et al., 1986 ). rRPV-RBOK was rescued from a full-length DNA copy of the genome of the RBOK vaccine strain (Baron & Barrett, 1997
) which had two new restriction enzyme sites engineered into the less conserved, untranslated regions flanking the H gene to allow its replacement with the H gene from other virus strains. For this an AscI site was introduced just before the FH intergenic region and a PmlI site just before the HL intergenic sequence (Das et al., 2000
). The H gene from the lapinized strain was amplified by RTPCR from total RNA of RPV-L-infected B95a cells using oligonucleotide primers with the appropriate restriction enzyme sites and then inserted in place of the normal H gene into the RBOK cDNA. rRPV-lapH was rescued from this cDNA according to the procedure previously described (Baron & Barrett, 1997
). The rescued viruses were passaged once in tissue culture, in B95a cells, and stored at -70 °C as the stock viruses for tissue culture infectivity studies and animal experiments.
Rabbits, virus inocula and samples.
Three-month-old female albino rabbits (JW-NIBS strain) with an average body weight of 2·0 kg, which are highly sensitive to RPV-L (Okita et al., 1993 ), were obtained from the Nippon Institute for Biological Science (Ome, Tokyo). One ml each of rRPV-RBOK, rRPV-lapH and RPV-L, diluted to 104 TCID50/ml with maintenance medium, was intravenously inoculated into two rabbits per experiment. One control rabbit was inoculated with 1 ml of maintenance medium. The rabbits were euthanized at 3 or 6 days post-inoculation (p.i.), using Dormitor (Orion) and Dormicum (Roche), and selected tissues collected for further investigation. Other rabbits, two per virus strain, were each inoculated with 1 ml of 102 TCID50/ml of the three strains of RPV for analysis of the humoral antibody responses. These rabbits were euthanized at 21 days p.i. and the sera collected for antibody titration.
Clinical investigations.
Inoculated cattle were examined daily for clinical signs of RPV infection and rectal temperatures recorded. Inoculated rabbits were also examined daily and rectal temperatures and body weights were recorded. Total white blood cell (WBC) counts in the peripheral blood of the rabbits were determined with a commercial kit (Unopette Test 58.56; Becton Dickinson).
Virological investigations.
The rabbits were euthanized at 3 days p.i. and the lymphoid tissues (spleen, Peyers patch, mesenteric lymph nodes and appendix) were collected and weighed. Virus infectivity titres in 10% (w/v) homogenates of these tissues were determined in B95a cells and expressed as TCID50/ml.
RTPCR.
Total RNA was extracted from mesenteric lymph nodes of virus-infected rabbits using a commercial reagent (Isogen; Nippon Gene). The cDNA of a part of the P gene was amplified by RTPCR using primers PF3 and PR6 (nt 17631784 and 26562677 of the RPV-RBOK strain).
Histopathological examination.
The various lymphoid tissues and other organs removed at autopsy were fixed in 10% formalin, dehydrated and embedded in paraffin using routine techniques. Thin sections were stained using haematoxylin and eosin.
Virus growth.
B95a cells were infected with rRPV-RBOK, rRPV-lapH or RPV-L at an m.o.i. of 0·1 for 1 h. The virus inoculum was then removed, the cells washed once with PBS and 1·5 ml of maintenance medium was added to each well of 12-well-plate. Samples (200 µl) for virus titration were collected immediately and at various times thereafter and stored at 80 °C. The TCID50/ml of released virus was quantified by standard methods.
Measurement of antibody titres.
The anti-RPV antibody titres in the rabbit sera were determined using an indirect ELISA. Ninety-six well microtitre plates (Nunc) were coated with 50 µl of RPV-L-infected B95a cell lysate diluted in coating buffer (0·1 M carbonatehydrogen carbonate buffer, pH 9·6) at 4 °C overnight. The wells were blocked with 100 µl of 0·8% Block Ace (Dainihonseiyaku) in PBS at room temperature for 1 h, washed with PBS containing 0·05% Tween 20 (PBS-T) and 100 µl of diluted (from 10-fold to 10000-fold) rabbit serum was added in duplicate wells. After 2 h incubation at 4 °C, the wells were washed with PBS-T and incubated for 1 h with 100 µl of 1000-fold diluted HRP-conjugated goat anti-rabbit IgG (Tago). Following a final wash with PBS-T, 100 µl of peroxidase substrate (Bio-Rad) was added to each well and the absorbance at 655 nm measured 30 min later.
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Results |
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Discussion |
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Histopathological examination of lymphoid tissues from rabbits infected with rRPV-lapH did not reveal the severe lesions, consisting of giant cell formation and necrosis of lymphoid follicles, observed in all rabbits infected with RPV-L. However, immunoreactive degeneration (starry sky effect) was observed in the lymphoid tissues of rabbits infected with rRPV-lapH and this result suggested that multiplication of rRPV-lapH might have taken place in these tissues, which would explain the generation of humoral antibody responses following infection with this virus. Paradoxically, rRPV-lapH could not be detected in homogenates of the different lymphoid organs from infected rabbits. It may be that growth of the virus was below detectable levels for the tests employed. The results of RTPCR on the tissues of the infected rabbits at 3 days p.i. indicated that rRPV-lapH could grow in lymphoid tissues although the growth rate was much lower than for RPV-L. In addition, rRPV-lapH was quickly eliminated from the tissues (by 6 days p.i.), which may be due to the immune reactions implied by histopathological observation.
In the case of Measles virus (MV), the H gene of a rodent-adapted neurovirulent strain was shown to confer the ability to replicate in brain tissue on the vaccine strain of the virus in vivo. Mice infected with a recombinant vaccine whose H protein was replaced with that of a neurovirulent MV strain became clinically ill. However, the authors concluded that replacement of H gene alone is not sufficient to cause the full pathology of the neurovirulent strain since the neuropathology induced by infection with the recombinant MV was not as severe (Duprex et al., 1999 ). Similarly, transgenic mice expressing the CD46 receptor for MV allowed replication of the vaccine strain of MV in neuronal tissue culture and in neonatal, but not adult, mice intracerebrally inoculated with the virus (Rall et al., 1997
). In contrast, transgenic rats expressing CD46 are not susceptible to MV infection when inoculated by the natural respiratory route, while rat fibroblasts expressing the CD46 receptor could internalize the virus but replication could not be demonstrated (Niewiesk et al., 1997
). In other virus systems it appears that the proteins responsible for virus entry into the host cell are the predominant molecular determinant of species specificity. Transgenic mice expressing the human poliovirus receptor are susceptible to infection with poliovirus strains, although mice are normally resistant to infection with this virus, and these mice show clinical symptoms similar to those observed in humans and monkeys (Koike et al., 1993
). Therefore, virus entry appears to be the most crucial event for initiating pathogenic poliovirus infection.
While the RPV H protein proved to play an important role in allowing the RBOK virus entry into some cells of the rabbit host, it was not capable of replicating to high levels in that species. Viral proteins other than the H protein are required after virus entry to enable replication and morphogenesis of new virus particles. It is likely that the extensive passaging of the lapinized virus in rabbits selected variants with the potential for faster growth, and also greater pathogenic potential, in the rabbit. Using reverse genetics and the rabbit model to test other recombinant RPVs, the molecular determinants of pathogenicity and species specificity can now be analysed in a relatively tractable animal model system. Construction of recombinant viruses with other proteins replaced and further studies on their pathogenicity in rabbits are under investigation.
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Acknowledgments |
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Footnotes |
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References |
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Received 5 October 2001;
accepted 4 January 2002.