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Journal of Virology, January 2003, p. 150-158, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.150-158.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Extent of Measles Virus Spread and Immune Suppression Differentiates between Wild-Type and Vaccine Strains in the Cotton Rat Model (Sigmodon hispidus)

Joanna Pfeuffer, Karen Püschel, Volker ter Meulen, Jürgen Schneider-Schaulies, and Stefan Niewiesk^*

Institute of Virology and Immunobiology, University of Wuerzburg, 97078 Wuerzburg, Germany

Received 10 June 2002/ Accepted 18 September 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Infection of humans with wild-type measles virus leads to strong immune suppression and secondary infections, whereas immunization with an attenuated vaccine strain does not. Using the cotton rat model (Sigmodon hispidus), we investigated whether vaccine and wild-type viruses differ in viral spread and whether this is correlated with inhibition of of proliferation of spleen cells ex vivo after mitogen stimulation. After intranasal infection of cotton rats with wild-type and vaccine strains, it was found that wild-type virus replicates better in lung tissue, spreads to the mediastinal lymph nodes, and induces a more pronounced and longer-lasting inhibition of proliferation of spleen cells ex vivo after mitogen stimulation than does vaccine virus. To induce the same degree of proliferation inhibition, 1,000-fold less wild-type virus was required than vaccine virus. With this system, the virulence of various measles virus isolates and recombinant viruses was tested. Four (in humans and/or monkeys) highly pathogenic virus strains were immunosuppressive, whereas viruses of vaccine virus genotype A were not. Using virus pairs which, due to passage on fibroblasts versus lymphoid cells or due to a point mutation in the hemagglutinin (N481 Y), differed in their usage of the two receptor molecules CD46 and CD150 on human cells, it was found that viruses using exclusively CD150 in vitro spread to mediastinal lymph nodes and induced strong immune suppression. These data demonstrate that important parameters of virulence seen in humans, such as viral spread and immune suppression, are reflected in the cotton rat model.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Infection with wild-type measles virus (MV) induces a severe immune suppression in humans, which is thought to be responsible for the occurence of secondary infections (11). In contrast, live attenuated vaccine virus induces no overt clinical disease. By laboratory diagnosis, an ex vivo inhibiton of mitogen-stimulated proliferation of peripheral blood lymphocytes (PBL) is seen for 2 to 4 weeks (8). An in vitro correlate for the immunosuppressive capacity of MV is the inhibition of mitogen-stimulated proliferation of PBL by MV-infected cells. In this system it was found that the fusion and hemagglutinin proteins are necessary and sufficient to induce proliferation inhibition (24) by disturbing the Akt kinase pathway in T cells (4). However, no difference in the inhibitory capacity was found between wild-type and vaccine virus in tissue culture. In investigations of MV-induced immune suppression in vivo, the cotton rat (Sigmodon hispidus) has proven to be a useful animal model (16). After infection with a high titer of vaccine virus, inhibition of mitogen-induced proliferation of spleen cells was seen ex vivo. As in tissue culture, the fusion (F) and hemagglutinin (H) protein were necessary and sufficient to induce proliferation inhibition (16) and disturb the Akt kinase pathway (4) in cotton rats. However, the ability of vaccine versus wild-type virus to inhibit the proliferation of spleen cells ex vivo was not investigated. Using the lymphoid B95-8 cell line as indicator cells (12), Wyde et al. were able to recover clinical isolates of MV efficiently from a lung tissue homogenate of infected cotton rats (33). One clinical isolate (MO2) was tested for viral spread and was found (apart from the lungs) sometimes in mediastinal lymph node cells (9 of 16 animals) and spleen (2 of 16 animals) but not in other organs. Using a different wild-type virus (WTFb) and molecularly cloned viruses, we found that the wild-type H determines viral spread to mediastinal lymph nodes (17). Although it has not been proven formally, this difference in viral spread might be related to receptor usage. The two known receptors for MV are the human CD46 (5, 14) and CD150 (7, 9, 30) molecules. Wild-type viruses bind to CD150 and retain this receptor usage after passage on lymphoid cells (18). If passaged on fibroblasts (like Vero cells), they acquire the capacity to bind to both CD46 and CD150. This appears to be part of an attenuation process since the currently known vaccine strains were produced this way. Although no structural or functional homologues for human CD46 or CD150 have yet been described in cotton rats, it is reasonable, in light of the above data to assume the presence of such homologues. In this study we focused on the question whether viral spread and proliferation inhibition are related in vivo and may differ between wild-type and vaccine MV viruses.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals. Cotton rats (inbred strain COTTON/NIco) were obtained from Iffa Credo (Lyon, France). Female animals 6 to 10 weeks of age were used. The animals were specific pathogen free according to the breeder’s specifications and were maintained in a barrier system. They were kept under controlled environmental conditions of 22 ± 1°C, 55 to 60% humidity, and a 12-h light cycle.

Cells and viruses. Vero (African green monkey) cells were grown in minimal essential medium with 5% fetal calf serum (FCS), and BJAB and B95a cells were grown in RPMI 1640 containing 10% FCS, 1% nonessential amino acids, 1% sodium pyruvate, 2 mmol of glutamine per ml, 50 IU of penicillin, and 50 µg of streptomycin per liter (referred to as RPMI/10). To obtain CR-T2 cells, cotton rat spleen cells were stimulated for 3 days with concanavalin A (2.5 µg/ml) and subsequently fused with the mouse thymoma BW5147 using polyethyleneglycol 1500 (Boehringer). The resulting hybridoma cells were cloned in RPMI/10 supplemented with 100 IU of interleukin-2 per ml and 1% hypoxanthine-aminopterin-thymidine (HAT). One of the stable clones designated CR-T2 was shown to be infectable with MV (data not shown).

MV strains used in this study, their designations, and the cell line in which they were grown are listed in Table 1. The Bilthoven and ICB strains were isolated from patients with measles and induce measles in rhesus and cynomolgus macaques, respectively (3, 13). ICB was kindly provided by H. Okada and M. Tashiro, Tokyo, Japan, and was passaged once through cotton rats and subsequently on BJAB cells. The HU2 strain was isolated from a child with measles-induced encephalitis after vaccination with Schwarz vaccine (31). Wü5404 is a clinical isolate from a recent outbreak (6). A low-passage stock of the Edmonston strain [Edm(wt)] was obtained from Lee Martin, Zurich, Switzerland. This stock had been passaged seven times in primary human kidney (HK) cells and six times in Vero cells after the original isolation by J. F. Enders in 1954 (23). Cotton rats were infected from the original aliquot. Vaccine-like viruses were grown on Vero cells, and wild-type viruses were grown on BJAB cells. Both were subjected to titer determination on B95 cells, with the exception of a previously published Edm (16) stock, for which Vero cells were used. The titers of Edm and Ed-NSE, in a plaque formation assay on Vero cells and a 50% tissue culture infective dose (TCID₅₀) assay on B95 cells, differed by not more than 0.5 log10. Generally, Ed-NSE was used as vaccine virus since it is the basis for the recombinant viruses used in this study. No difference in the induction of proliferation inhibition was found between Ed-NSE and Edm (data not shown).

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TABLE 1. Measles virus strains used in this study

Infection of cotton rats and virus titer determination. For intranasal (i.n.) infection, MV was given in phosphate-buffered saline to ether anesthezised cotton rats. i.n. inoculations of virus were administered in a volume of not more then 100 µl. At different times after infection, the animals were asphyxiated using CO₂ and their lungs were removed and weighed. Lung tissue was minced with scissors and dounced with a glass homogenizer. Serial 10-fold dilutions of virus containing supernatant were assessed for the presence and levels of infectious virus in a 48-well microassay using Vero cells and B95a cells with cytopathic effect (CPE) as an end point. Plates were scored for CPE microscopically after 7 days. The amount of virus in inocula was expressed as the quantity of virus that could infect 50% of inoculated tissue culture monolayers (TCID₅₀). The TCID₅₀ was calculated by the method of Reed and Muench (20). Lymph nodes from infected animals were pooled from a group of animals, nicked with a scapel blade, and passaged through a steel sieve. In a 96-well plate, 10⁵ B95a cells were plated per well and coincubated with lymph node cells starting at 10⁶ cells per well in 10-fold dilutions. Scoring for CPE after 7 days and titer calculations were done as above.

Proliferation assay. Spleen cells from infected and mock-infected animals were plated in triplicate at 5 x 10⁵ cells/well in a 96-well plate containing RPMI 1640 with 10% FCS and were left untreated (medium) or treated with concanavalin A (2.5 µg/ml). After 40 h, 0.5 µCi of [³H] thymidine/well was added, and 16 to 20 h later the cells were harvested onto glass filters and counted with a Betaplate counter (Wallac). Variation between wells usually did not exceed 10%. The stimulation index was calculated as the mean of proliferation of mitogen-stimulated cells (cpm)/proliferation of cells in medium (cpm). The percent proliferation inhibition was expressed by comparing the stimulation indices of a group of infected cells to to those of mock-infected animals. If proliferation of spleen cells from infected animals was identical to or higher than that of proliferation of spleen cells from mock-infected animals (100%), proliferation was given as 100%.

For the proliferation assay with infected CR-T2 cells, CR-T2 cells were infected at a multiplicity of infection of 0.01 for 3 days with MV. The expression of both MV glycoproteins (H and F proteins) was tested with antibody L77 (for H) or A504 (for F) by flow cytometry. CR-T2 cells were UV- irradiated at 1 J/cm² and plated at different ratios with spleen cells (5 x 10⁵/well) from naive cotton rats and concanavalin A (2.5 µg/ml). Otherwise, the proliferation assay was performed as above.

Top Abstract Introduction Materials and Methods Results Discussion References

 
No difference in proliferation inhibition between MV wild-type (WTFb) and vaccine (Edm) strains in vitro. In vitro, mitogen-stimulated proliferation of human PBL is inhibited after contact or infection with MV. To test whether the same is true for cotton rat spleen cells, an in vitro assay for proliferation inhibition was developed. Concanavalin A-stimulated spleen cells from cotton rats were fused with mouse thymoma BW 5147 cells, and hybridomas were selected with HAT medium. Growing T-cell hybridomas were cloned, and a line designated CR-T2 was used for these assays. CR-T2 cells were infected with either wild-type virus (WTFb) or molecularly cloned vaccine virus (Ed-NSE). After 2 days, CR-T2 cells infected with either virus demonstrated equivalent levels of glcoproteins at the cell surface as determined by flow cytometry (data not shown). After UV irradiation, CR-T2 cells were mixed with spleen cells from naive animals at different ratios and stimulated with concanavalin A. Contact with non-infected CR-T2 cells did not inhibit the proliferation of spleen cells (Fig. 1). In contrast, contact with MV-infected CR-T2 cells inhibited mitogen-stimulated proliferation of spleen cells in a dose-dependent manner. However, no difference in the inhibitory capacity between CR-T2 cells infected with wild-type virus (WTFb) or vaccine (Edm) virus was observed. Thus, as reported for human cells, in vitro proliferation of cotton rat spleen cells is inhibited by wild-type and vaccine MV strains to the same degree.

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FIG. 1. In vitro inhibition of mitogen-stimulated proliferation by MV-infected CR-T2 cells. Spleen cells from naive cotton rats (5 x 105/well) were cocultured at different ratios with uninfected CR-T2 cells () or CR-T2 cells infected with Ed-NSE () or WTFb () and stimulated with 2.5 µg of concanavalin A per ml. One experiment representative of four is shown.

Differences between WTFb and Edm infection in vivo. To test whether differences in proliferation inhibition after infection with wild-type and vaccine virus might be found in vivo, cotton rats were infected i.n. with 10⁵ TCID₅₀ of WTFb or Ed-NSE. Infection with WTFb induced a pronounced ex vivo proliferation inhibition of spleen cells from days 2 to 20 (Fig. 2). In contrast, infection with 10⁵ TCID₅₀ of Ed-NSE did not result in proliferation inhibition. However, it has been shown previously (16) that a high-dose inoculum (2 x 10⁶ PFU of Edm) resulted in inhibition of proliferation of of spleen cells after mitogen stimulation ex vivo. In comparison to proliferation inhibition induced by a high-dose inoculum of Edm, proliferation inhibition after infection with 10⁵ TCID₅₀ of WTFb was noted 1 day earlier and lasted at least 4 days longer (Figure 2).

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FIG. 2. Differences in inhibition of of proliferation of spleen cells ex vivo after infection of cotton rats with Ed-NSE or WTFb. After i.n. infection of groups of four cotton rats with 105 TCID50 of Ed-NSE or WTFb, spleen cells were stimulated with concanavalin A, on various days postinfection. Proliferation was expressed as the percentage of proliferation in naive animals. Previously reported results (16) after infection with 2 x 106 to 4 x 106 PFU of Edm are included for comparison. Error bars indicate standard deviations.

One possible explanation for the differences beteen wild-type and vaccine virus strains is a difference in viral burden and spread in cotton rats. To test this possibility, cotton rats were tested for the presence of virus in the lungs, the lung-draining mediastinal lymph nodes (MDLN), PBL, or spleen after i.n. inoculation with 10⁵ TCID₅₀ of WTFb or Ed-NSE or 4 x 10⁶ PFU of Edm. From PBL or spleen, the virus could not be reisolated irrespective of the virus strain used. WTFb was found in MDLN on days 2, 3, and 4 after i.n. inoculation (Fig. 3A). After inculation of 4 x 10⁶ PFU, of Edm, virus was found in MDLN on day 2 only; no virus was detected after inoculation with 10⁵ TCID50 of Ed-NSE (Fig. 3A). The presence of virus in MDLN correlated with the virus titer in lung tissue on day 4 (Fig. 3B) and proliferation inhibition ex vivo on day 4 (Fig. 3C).

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FIG. 3. Comparison of viral burden and spread with proliferation inhibition. (A and B) At 4 days after i.n. infection of groups of four cotton rats with 105 TCID50 of WTFb or Ed-NSE or 4 x 106 PFU of Edm, the titer of virus from MDLN cells (A) and lung tissue (B) was determined on days 2 and 4. (C) After stimulation of spleen cells with concanavalin A, proliferation was expressed as a percentage of proliferation in naive animals. Error bars indicate standard deviations.

To investigate the inhibitory capacity of WTFb in more detail, cotton rats were infected with 10⁵, 10⁴, and 10³ TCID₅₀ and tested for the presence of virus in MDLN and inhibition of proliferation of spleen cells ex vivo. The extent of proliferation inhibition (Fig. 4A) and the presence of virus in MDLN (Fig. 4B) correlated with the titer of the inoculum. WTFb still had some inhibitory effect at a titer of 10³ TCID₅₀ and was therefore approximately 1,000-fold more effective than Edm in inducing proliferation inhibition in spleen cells.

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FIG. 4. Proliferation inhibition induced by WTFb is dependent on the virus dose. Inbition of proliferation of mitogen-stimulated spleen cells (A) and titers in MDLN (B) were tested 4 days after i.n. infection of groups of four cotton rats with 103, 104, and 105 TCID50 of WTFb. Error bars indicate standard deviations.

Comparison of wild-type and vaccine MV strains in cotton rats. To determine whether the results obtained with WTFb were representative, cotton rats were inoculated with other MV strains such as BIL, ICB, and Wü5404, which were isolated from patients with clinical measles. In addition, BIL and ICB induce measles in rhesus (3) and cynomologus (13) macaques. Groups of cotton rats were infected with 10⁵ TCID₅₀ of each virus, and on day 4 the virus was isolated from lung tissue and MDLN and proliferation was assessed in spleen cells. Titers in lung tissue of animals infected with vaccine viruses (Edm and Ed-NSE) were <10³ TCID₅₀/g of lung, whereas titers in lung tissue of animals infected with wild-type viruses were >10⁴ TCID₅₀/g of lung (data not shown). Edm and Ed-NSE did not induce proliferation inhibition (Fig. 5A) and no or little virus was found in draining lymph nodes (Fig. 5B). In contrast, the wild-type strains BIL and ICB (like WTFb) induced proliferation inhibition and virus was present in draining lymph nodes. The same was true for Wü5405, which could be tested only at an inoculum of 10⁴ TCID₅₀ (Fig. 5). These data indicate that vaccine-like and wild-type viruses can be differentiated in cotton rats by virus titer in lung tissue, spread to MDLN, and proliferation inhibition in spleen cells.

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FIG. 5. Comparison of proliferation inhibition and presence of virus in MDLN with different wild-type MV strains. Groups of four cotton rats were infected i.n. with vaccine viruses (Edm and Ed-NSE) or wild-type viruses (BIL, ICB, WTFb, and Wü5404). Then spleen cells were tested for proliferation inhibition (A) and MDNL cells were tested for the presence of virus (B). Because Wü5405 did not reach the same titers as the other wild-type strains, it was tested at an inoculum of 104 TCID50. Error bars indicate standard deviations.

Testing of MV strains with uncertain pathogenic potential. With this system, we tried to investigate viruses of more uncertain pathogenic potential. Edm(wt) was derived from a patient with measles and has been passaged for 13 passages on human and monkey fibroblasts (summarized in reference 19). By serial passage, all vaccine strains of the Edmonston lineage were derived from this isolate. Edm(wt) did not induce acute measles in rhesus macaques (3), but at high titers induced encephalitis in marmosets (1). Chi-1 was derived from a patient with measles and passaged afterwards on Vero cells. It did not induce acute measles in rhesus macaques (3). The HU2 strain was isolated from a child with MV encephalitis after MV immunization and belongs to genotype A of the vaccine strains (31). After infection of cotton rats with Edm(wt) and HU2, no or little virus was found in MDLN and no proliferation inhibition was observed (Fig. 6). With Chi-1, a slight proliferation inhibition was observed, although no virus was found in MDLN. After infection with a 10-fold-higher dose, virus was found in MDLN (100 TCID₅₀/10⁷ lymph node cells), correlating with 50% proliferation inhibition (data not shown). It appears that the passage on Vero cells correlates with a loss of virulence in cotton rats when proliferation inhibition and spread of virus to MDLN are used as parameters.

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FIG. 6. Evaluating MV strains with uncertain pathogenic potential. Groups of four cotton rats were infected i.n. with Edm, Edm (wt), HU2, Chi-1, and WTFb. Then spleen cells were tested for proliferation inhibition (A), and mediastinal lymph node cells were for tested the presence of virus (B). The titer of the inoculum was 105 TCID50. Error bars indicate standard deviations.

Comparison of MV pairs with CD150 versus CD46/CD150 receptor usage. After passage on Vero cells, wild-type viruses acquire the ability to use CD46 as receptor molecule, and this was supposed to be crucial for attenuation (15, 21, 25-27). To investigate whether the in vitro receptor usage reflects virulence in vivo, cotton rats were infected with two MV pairs with different receptor usage and minimal amino acid exchanges. WTFb was propagated on BJAB lymphoid cells, whereas WTFv was propagated on Vero cells (21). This propagation resulted in defined changes in the H protein (at positions 192 and 546), and whereas WTFb uses CD150 as the receptor, WTFv is able to interact with both (6). After i.n. infection with 10⁵ TCID₅₀ of WTFb, mitogen-stimulated proliferation was inhibited whereas infection with WTFv had nearly no effect (Fig. 7A). This correlated well with the presence or absence of virus in draining lymph nodes (Fig. 7B). The second virus pair investigated was MV (WTF-H)Ed and (WTF-H481N-Y)Ed. In the molecularly cloned (WTF-H)Ed, the H gene (Edm-H) is replaced by WTF-H, resulting in the exclusive utilization of CD150 (6). In (WTF-H481N-Y)Ed, a single point mutation at amino acid 481 is introduced into WTF-H which changes receptor usage to both CD46 and CD150 (6). After i.n. infection, (WTF-H)Ed induced proliferation inhibition and virus was found in MDLN (Fig. 7). In contrast, after infection with (WTF-H481N-Y)Ed, proliferation was not impaired and no virus was found in MDLN (Fig. 7).

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FIG. 7. Comparison of MV pairs with different receptor usage. Groups of four cotton rats were infected i.n. with Ed-NSE, (WTF-H)Ed, (WTF-H481N-Y)Ed, WTFb, and WTFv. Then spleen cells were tested for proliferation inhibition (A), and MDLN cells were tested for the presence of virus (B). The titer of the inoculum was 105 TCID50. Error bars indicate standard deviations.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In humans, wild-type MV infection causes immune suppression, which often leads to secondary infections. In contrast, vaccine virus does not induce a clinically relevant immune suppression but causes only a slight reduction in the proliferation of PBL after stimulation with mitogen ex vivo. The difference in virulence between wild-type and vaccine strains seen in humans is not reflected in vitro in a contact- inhibition assay of mitogen-stimulated proliferation of human cells (24). When a similar assay was applied to cotton rat cells in vitro, no difference in proliferation inhibition was found between vaccine and wild-type strains. A likely explanation for the in vivo differences is a difference in viral replication and spread. In monkeys, wild-type strains disseminate more widely than vaccine strains, leading to higher titers (32) and acute measles (3, 13). Viral spread in humans has not been studied comparatively between wild-type and vaccine strains in detail. However, the appearance of rash after a wild-type infection that is not seen after vaccination also indicates the growth of wild-type virus to higher titers and enhanced virus spread after infection. In cotton rats, both viral titers in lung tissue and the presence of virus in MDLN after wild-type virus infection correlate well with proliferation inhibition ex vivo. Using the presence of virus in draining lymph nodes and proliferation inhibition as parameters, it is possible to distinguish between vaccine and wild-type virus strains in cotton rats.

All vaccine-related viruses (genotype A) did not spread well and did not induce proliferation inhibition. This includes also HU2, a virus derived from a vaccine-induced encephalitis case (31). This indicates that the aforementioned parameters do not correlate with neurovirulence of a virus. As in monkeys (3, 13), the Bilthoven and ICB strains were virulent, as were the clinical isolates WTFb and Wü5404 grown on lymphoid cells (BJAB). In contrast, clinical isolates grown on Vero cells were attenuated in cotton rats [Edm(wt), Chi-1, and WTFv]. This correlates well with data from rhesus macaques, in which both Edm (wt) and Chi-1 did not induce acute measles (3). It is known that MV strains grown on fibroblasts adapt to the usage of CD46 although they retain the ability to bind to CD150 (7, 30). The change in receptor usage is correlated with mutations in the H protein (6, 9, 27). Our data using the two virus pairs of closely related (WTFv and WTFb) or the molecularly cloned viruses [(WTF-H)Ed and (WTF-H481Y-N)Ed] differing in one amino acid indicate the importance of receptor usage in vivo. Viruses which use human CD46 and CD150 in vitro [WTFv and (WTF-H481Y-N)Ed] (6) did not spread well in cotton rats and induced no proliferation inhibition. In contrast, viruses using only human CD150 in vitro [WTFb and (WTF-H)Ed] spread well and induced proliferation inhibition. The results using virus pair MV (WTF-H)Ed and (WTF-H481Y-N)Ed confirm in vitro data that amino acid position 481 is important for receptor usage and biological activity. They also indicate that although both F and H are required for the induction of proliferation inhibition, H is the more important determinant in vivo. Not only do mutations in the H protein seem to be important for attenuation of MV strains, but also virulence is determined by mutations in other genes. After passage of ICB on Vero cells, a mutation resulting in a loss of expression of the C protein occurred (29). Although no effect was seen in vitro, this mutant was not able to induce acute measles in cynomolgus macaques. Genotypic analysis has suggested a number of changes important to the virulence of MV strains, and due to the reverse genetic system available, many mutants can be generated. Because in cotton rats vaccine and wild-type strains can be differentiated on the basis of viral spread and proliferation inhibition, it appears to be an excellent in vivo model to further investigate parameters of MV virulence.

 
Measles virus strain ICB was kindly provided by H. Okada and M. Tashiro, Tokyo, Japan; Edm(wt) was obtained from L. Martin, Zurich, Switzerland.

This work was supported by Deutsche Forschungsgemeinschaft.

 
* Corresponding author. Mailing address: Institut für Virologie and Immunbiologie, Versbacher Str. 7, 97078 Würzburg, Germany. Phone: 49 931 201 49896. Fax: 49 931 201 49553. E-mail: niewiesk{at}vim.uni-wuerzburg.de.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, January 2003, p. 150-158, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.150-158.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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