In vitro permissivity of bovine cells for wild-type and vaccinal myxoma virus strains
© Pignolet et al; licensee BioMed Central Ltd. 2007
Received: 07 August 2007
Accepted: 27 September 2007
Published: 27 September 2007
Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe, non-replicative vaccine vector for non-host species. However, there is very little information concerning infection of non-laboratory animals species cells with MYXV. In this study, we investigated interactions between bovine cells and respectively a wild type strain (T1) and a vaccinal strain (SG33) of MYXV. We showed that bovine KOP-R, BT and MDBK cell lines do not support MYXV production. Electron microscopy observations of BT-infected cells revealed the low efficiency of viral entry and the production of defective virions. In addition, infection of bovine peripheral blood mononuclear cells (PBMC) occurred at a very low level, even following non-specific activation, and was always abortive. We did not observe significant differences between the wild type strain and the vaccinal strain of MYXV, indicating that SG33 could be used for new bovine vaccination strategies.
Until now, most of the ruminant vaccines use attenuated strains of pathogens, and for that reason, naturally infected and vaccinated animals cannot easily be differentiated. Development of recombinant vaccines for ruminant species would help to implement vaccine policies. The development of poxviruses as vectors for producing recombinant vaccines is well documented [1–6]. Although vaccinia virus was the first and most extensively developed poxvirus vector, concerns over its use in immunocompromised persons and its broad host-range specificity  had led to search for alternative poxviruses which might prove more suitable vectors. Myxoma virus (MYXV), a leporipoxvirus causing myxomatosis, a highly lethal disease of European rabbit, could be an interesting tool for animals vectored vaccination. MYXV attenuated strains were shown to be efficient vaccine vector to vaccinate its natural host against both myxomatosis and rabbit viral hemorrhagic disease [8, 9]. Recently, MYXV was successfully developed as a non replicative vector to vaccinate cats against feline calicivirus [10, 11]. However, for each target species, evaluation of host restriction is of importance for the development of safe and potent vaccine vectors. MYXV is reported to be restricted to rabbits in vivo  and to replicate in vitro in some non natural host cell lines such as simian BGMK and some cancer cells . No information concerning interactions between MYXV and bovine cells is available yet. In this study, we characterized the infection of bovine cell lines and bovine peripheral blood mononuclear cells (PBMC) with MYXV. By comparing two different MYXV strains (a wild-type strain (T1) and a cell-cultured attenuated vaccinal strain (SG33) ) we verified the stability of the viral tropism in vitro.
Three bovine cell lines were tested for MYXV permissivity: KOP-R cells (RIE 244, CCLV Federal Research Centre for Virus Diseases of Animals, Island Riems), BT cells (ATCC CRL-1390) and MDBK cells (ATCC CCL-22). Each cell line was infected at a multiplicity of infection (m.o.i.) of 1 and cultured for 72 h. Then, infected cells were lysed by three freeze/thaw cycles. One fifth of each cell lysate was inoculated to new cells and further incubated for 72 h. Virus productions were determined by serial dilution-titration of each cell lysate on permissive rabbit RK13 cells (ATCC CCL-37) (Figure 1A).
In RK13 cells, used as positive control, we observed a high virus titer maintained over the three passages for both MYXV strains (Figure 1A). In contrast, in the bovine cell lines, viral titers decreased during the three passages for both T1 and SG33 (Figure 1A). After three passages we measured a low virus titer for KOP-R, BT and MDBK indicating that both strains are not able to spread over serial passages.
To evaluate MYXV infection in blood primary bovine cells, peripheral blood mononuclear cells (PBMC) were infected. Bovine blood was collected in EDTA tubes, diluted (1:2), loaded on a density gradient (FicollPaque Plus, Amersham) and centrifugated at 900 g for 20 minutes. PBMC were then harvested, washed in PBS, recovered by centrifugation at 870 g for 10 minutes and cultured. To detect infected cells by flow cytometry, we used a recombinant SG33 virus expressing the enhanced green fluorescent protein (GFP) under the control of strong early/late vaccinia virus P7.5 promoter. The GFP encoded gene was inserted into the M11L/MGF locus. We also used the T1-Serp2-GFP recombinant virus which expresses the fused protein Serp2-GFP.
Resting PBMC were infected with T1-Serp2-GFP or SG33-GFP at a m.o.i. of 1 (Figure 3A). Cells were collected 16 h p.i., and infection levels were determined by counting living GFP-positive cells (Figure 3A). We observed that only a small fraction of bovine PBMC was susceptible to MYXV infection. An average of 1.2 % and 0.8 % of GFP-positive resting cells was detected for T1-Serp2-GFP and SG33-GFP respectively (Figure 3B). As activation may be required to allow infection by poxviruses , chemically-activated bovine PBMC were also infected at the same m.o.i.. The percentage of GFP-positive cells remained low following activation, as only 2.4 % and 5.1 % for T1-Serp2-GFP and SG33-GFP of positive cells were detected respectively (Figure 3B). The infection level remained below 5 % with an m.o.i. up to 10 (data not shown). In contrast to the infection level in activated rabbit PBMC (about 50 % of infected cells) (data not shown), activation have very low effect on bovine leukocytes infection with MYXV.
In this study, we investigated the interactions between bovine cells (cell lines and PBMC) and MYXV wild type (T1) strain or vaccinal (SG33) strain. In bovine cell lines, serial viral passages analysis and infection with both T1 and SG33 expressing LacZ gene showed that these cells failed to support spread of either MYXV strain. Electron microscopy study of BT-infected cells enabled us to identify at least two blocking events, the first one involving virus entry. Indeed, we observed many viral particles adsorbed on the cell surface throughout the experiment but very few infected cells. This result indicates that MYXV can bind to the cell surface, but enters the cells with low efficiency. The second blocking event involves the final steps of virus maturation, as numerous electron dense particles, similar to those already described in non-permissive cells infected with MVA [17–20] were present. In addition, very few IEV particles and no mature virions could be observed. As already suggested, these dense particles are more likely the products of defective virions morphogenesis . The very low level and abortive infection of bovine PBMC make it impossible for MYXV to disseminate via leukocytes in these animal species. Taken together, these results are compatible with the potential use of the SG33 MYXV strain as a safe non replicative vector for bovine vaccination.
BP was supported by a grant from the Institut National de la Recherche Agronomique (INRA), the Agence Française de la Sécurité Sanitaire Alimentaire (AFSSA) and ANR Génanimal 2006 "VacGenDC project".
The authors are especially grateful to Martine Deplanche, Martine Moulignié, Brigitte Peralta and Josyane Loupias for excellent technical assistance, Jean-Philippe Nougareyde for critical reading of the manuscript.
- Kieny MP, Lathe P, Drillien R, Spehner D, Skory S, Schmitt D, Wiktor T, koprowski H, Lecocq JP: Expression of rabies virus glycoprotein from a recombinant vaccinia virus. Nature 1984, 312: 163-166. 10.1038/312163a0PubMedView Article
- Taylor J, Paoletti E: Fowlpox virus as a vector in non-avian species. Vaccine 1988, 6: 466-8. 10.1016/0264-410X(88)90091-6PubMedView Article
- Taylor J, Weinberg R, Languet B, Desmettre P, Paoletti E: Recombinant fowlpox virus inducing protective immunity in non-avian species. Vaccine 1988, 6: 497-503. 10.1016/0264-410X(88)90100-4PubMedView Article
- Tartaglia J, Jarrett O, Neil JC, Desmettre P, Paoletti E: Protection of cats against feline leukemia virus by vaccination with a canarypox virus recombinant, ALVAC-FL. J Virol 1993, 67: 2370-2375.PubMedPubMed Central
- Moss B, Carroll MW, Wyatt LS, Bennink JR, Hirsch VM, Goldstein S, Elkins WR, Fuerst TR, Lifson JD, Piatak M, Restifo NP, Overwijk W, Chamberlain R, Rosenberg SA, Sutter G: Host range restricted, non replicating vaccinia virus vectors as vaccine candidates. Adv Exp Med Biol 1996, 397: 7-13.PubMedPubMed CentralView Article
- Aspen K, Passmore J, Tiedt F, Williamson A: Evaluation of lumpy skin disease virus, a capripoxvirus, as a replication-defecient vaccine vector. J Gen Virol 2003, 84: 1985-1996. 10.1099/vir.0.19116-0View Article
- Redfield RR, Wright DC, James WD, Jones TS, Brown C, Burke DC: Disseminated vaccinia in military recruit with human immunodeficiency virus (HIV) disease. N Engl J Med 1987, 316: 673-676.PubMedView Article
- Bertagnoli S, Gelfi J, Le Gall G, Boilletot E, Vautherot J, Rasschaert D, Laurent S, Petit F, Boucraut-Baralon C, Milon A: Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein. J Virol 1996, 70: 5061-5066.PubMedPubMed Central
- Barcena J, Morales M, Vazquez B, Boga JA, Parra F, Lucientes J, Pages-Mante A, Sanchez-Vizcaino JM, Blasco R, Torres JM: Horizontal transmissible protection against myxomatosis and rabbit hemorrhagic disease by using a recombinant myxoma virus. J Virol 2000,74(3):1114-23. 10.1128/JVI.74.3.1114-1123.2000PubMedPubMed CentralView Article
- McCabe VJ, Tarpey I, Spibey N: Vaccination of cats with an attenuated myxoma virus expressing feline calicivirus capsid protein. Vaccine 2002, 20: 2454-2462. 10.1016/S0264-410X(02)00186-XPubMedView Article
- McCabe VJ, Spibey N: Potential for broad-spectrum protection against feline calicivirus using an attenuated myxoma virus expressing a chimeric FCV capsid protein. Vaccine 2005,23(46–47):5380-5388. 10.1016/j.vaccine.2005.05.038PubMedView Article
- Fenner F, Ross J: Myxomatosis. In The European Rabbit, the History and Biology of a Successful Colonizer. Edited by: Thompson GV, king CM. Oxford, New York, Tokyo: Oxford University Press; 1994:205-239.
- Sypula J, Wang F, Ma Y, Bell J, Mcfadden G: Myxoma virus tropism in human tumor cells. Gene Ther Mol Biol 2004, 8: 103-114.
- Saurat P, Gilbert Y, Ganière JP: Etude d'une souche de virus myxomateux modifié. Rev Med Vet 1978, 129: 415-451. (In French)
- Duteyrat JL, Gelfi J, Bertagnoli S: Ultrastructure study of myxoma virus morphogenesis. Arch Virol 2006,151(11):2161-2180. 10.1007/s00705-006-0791-2PubMedView Article
- Chahroudi A, Chavan R, Koyz N, Waller EK, Silvestri G, Feinberg NB: Vaccinia virus tropism for hematolymphoid cells is determined by restricted expression of a unique virus receptor. J Virol 2005,79(16):10397-10407. 10.1128/JVI.79.16.10397-10407.2005PubMedPubMed CentralView Article
- Carroll MW, Moss B: Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line. Virology 1997, 238: 198-211. 10.1006/viro.1997.8845PubMedView Article
- Gallego-Gomez JC, Risco C, Rodriguez D, Cabezas P, Guerra S, Carrascosa JL, Esteban M: Differences in virus-induced cell morphology and in virus maturation between MVA and other strains (WR, Ankara, and NYCBH) of vaccinia virus in infected cells. J Virol 2003,77(19):10606-10622. 10.1128/JVI.77.19.10606-10622.2003PubMedPubMed CentralView Article
- Meiser A, Sancho C, Krijnse-Locker J: Plasma membrane budding as an alternative release mechanism of the extracellular enveloped form of vaccinia virus from Hela cells. J Virol 2003, 77: 9931-9942. 10.1128/JVI.77.18.9931-9942.2003PubMedPubMed CentralView Article
- Okeke MI, Nilssen O, Traavik T: Modified vaccinia virus-Ankara multiplies in rat IEC-6 cell and limited production of mature virions occurs in other mammalian cell lines. J Gen Virol 2006, 87: 21-27. 10.1099/vir.0.81479-0PubMedView Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.