Department of Pathology and Laboratory Medicine, University of British Columbia, #318, BCRICWH, 950 West 28th Avenue, Vancouver, British Columbia, CanadaV5Z 4H41
Author for correspondence: Janet Chantler. e-mail chantler{at}interchange.ubc.ca
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Abstract |
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Introduction |
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Heart disease associated with CVB infection usually occurs in adulthood although serological studies indicate widespread exposure of the population to this group of viruses during childhood. Moreover, patients presenting with acute myocarditis have been reported to have antibody against a greater number of coxsackieviruses than age-matched controls, indicating that they are not undergoing a primary CVB infection at the time of development of clinical cardiomyopathy (Horr et al., 1981 ). The possibility that secondary infection with a different strain of CVB might have a role in pathogenesis was investigated by Beck et al. (1990)
in a murine model of coxsackievirus myocarditis. These investigators showed that tandem infection of C3H/HeJ mice with CVB2 followed 1 month later by CVB3 resulted in more intense myocarditis than found in animals injected with either CVB2 or CVB3 alone. They concluded that the effect was due to cell-mediated immune responses to a conserved enterovirus epitope, although the mechanism of this was not further investigated. Another explanation for the phenomenon they observed is antibody enhancement of the secondary CVB3 infection by cross-reactive antibody from the antecedent inoculation with CVB2. Such exacerbation of virus infections by pre-existing antibody which reacts with virus particles but is non-neutralizing has been observed with a number of viruses, notably members of the Flaviviridae such as dengue (Halstead, 1982
; Halstead & ORourke, 1977
) and yellow fever virus (Porterfield, 1986
) as well as respiratory syncytial virus (Gimenez et al., 1996
) and recently human immunodeficiency virus (Montefiori et al., 1996
). Antibody enhancement of infectivity (AEI) of disease is believed to be due to uptake of virusantibody complexes via Fc receptors on cells (usually macrophages) with subsequent replication and release of virus resulting in higher levels of viraemia and thus virus reaching the target tissues. AEI is believed to be responsible for the haemorrhagic fever accompanying secondary dengue virus infections (dengue shock syndrome) and has been suggested to have a role in AIDS pathogenesis (Homsy et al., 1989
).
The study reported here examines the possible role of cross-reactive antibody in mediating the enhancement of myocarditis seen in secondary CVB infections in mice. The effect of polyclonal anti-CVB2 antibody (IgG fraction) on CVB3 infection of macrophages in vitro is reported and also the result of injecting susceptible strains of mice with CVB3 together with anti-CVB2 IgG.
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Methods |
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Virus.
The RK (Reinhardt Kandolf) strain of coxsackievirus B3 was derived from an infectious clone, pCVB3/T7, that was transcribed with T7 polymerase and the RNA subsequently transfected into HeLa cells. The progeny virus was passaged in mice and the virus obtained from heart tissue is the strain that we received from R. Kandolfs laboratory and designated CVB3(RK). Stock virus was prepared by infection of Vero cells at 0·1 p.f.u. per cell. When 100% cytopathology was evident at 2448 h post-infection, the cultures were freezethawed twice to release intracellular virus into the medium which was then centrifuged at 800 g for 10 min at 4 °C to remove cell debris. The supernatant was stored in aliquots at -70 °C, and was titred in Vero cells by plaque assay.
Production of anti-CVB2 antibody.
Polyclonal anti-CVB2 IgG was prepared in BALB/c mice against the Ohio strain of CVB2, obtained from the ATCC. The mice were injected intra-peritoneally with 105 p.f.u. of CVB2 and were sacrificed after 4 weeks. Approximately 0·8 ml of intraventricular blood was obtained from each mouse and allowed to clot at 4 °C. The serum obtained was pooled prior to immunoglobulin purification. This was carried out by ammonium sulphate precipitation and fractionation by protein ASepharose chromatography.
Virus infection of J774.1 cells and peritoneal macrophages.
Cells were infected at 520 p.f.u. per cell for 1 h. After removal of the inoculum the cells were washed three times and then incubated in growth medium containing 5% heat-inactivated FBS and 1% gentamycin. At 2 h intervals the cultures were harvested and freezethawed twice to release virus from the cells. Virus was titred by plaque assay.
Infectious focus assay.
Cells were infected and incubated as before. At 2 h intervals, cultures were harvested, the cells were washed three times in DMEM and then plated at known numbers in 35 mm dishes containing a monolayer of Vero cells. After gently centrifuging the cells onto the monolayer, the supernatant was removed and the cells were overlaid with agar. Plaques were stained and counted after 2 days, and the numbers were correlated with the number of cells plated.
Infection of mice.
Male A/J mice (45 weeks old) were obtained from Jackson Laboratories. The animals were allowed to acclimatize for 1 week before inoculation with 0·2 ml PBS containing 104 p.f.u. of CVB3 alone or together with 10 µl of anti-CVB2 antibody. On days 3 or 5 post-infection five animals of each group were anaesthetized with sodium pentobarbital (60 mg per kg) and heart, spleen, kidney, pancreas, liver and lung were removed aseptically. The tissues were sectioned transversely into two pieces, one section was fixed with 4% paraformaldehyde solution for histopathology and in situ hybridization and the other was snap-frozen in liquid nitrogen for plaque titration.
Plaque titration of virus in organs.
Snap-frozen sections were homogenized in 1 ml of DMEM and the homogenate was then centrifuged to remove the cell debris at 12000 g. The supernatant was stored at -70 °C for plaque assay.
Histology.
Sections (3 µm thick) were prepared from organs fixed in 4% paraformaldehyde solution using a Leica microtome. The sections were stained with haemotoxylin and eosin and Massons trichrome to assess histopathological damage.
In situ hybridization.
Tissues were fixed with 4% paraformaldehyde solution overnight, rinsed with PBS, embedded in paraffin blocks, cut into 4 µm sections and placed on silanated glass slides. The sections were baked overnight at 60 °C, deparaffinized using xylene and rehydrated in graded alcohols. The tissues were permeabilized using 0·2 M HCl, 2x SSC, 20 mM Tris/2 mM CaCl2 containing 1 µg/ml of proteinase K, and 0·25% acetic anhydride containing 0·1 M triethanolamine. The slides were then dehydrated using graded alcohols. Once the tissues were completely dried, 25 µl of the hybridization solution containing 100 ng/ml of the DIG-labelled sense or anti-sense probe was added to each section. The sections were then covered with glass coverslips and placed in a sealed humidified dish at 42 °C overnight. Post-hybridization washes were performed overnight using 50% formamide, 10 mM Tris/1mM EDTA and 600 mM NaCl in a 56 °C rocking water bath, followed by several washes in 2x SSC. The slides were equilibrated in buffer 1 containing 0·15 M NaCl and 0·1 M TrisHCl and blocked with 2% lamb serum. Anti-DIGalkaline phosphatase (100 µl; diluted 1:500 in buffer 1 containing 1% lamb serum and 0·1% Tween-20) was added per section and incubated for 45 min at room temperature in a humidified chamber. The slides were washed with buffer 1 and were equilibrated to pH 9·5 in buffer 3. The alkaline phosphatase-linked anti-DIG antibody was detected by incubation with NBT/BCIP substrate (Sigma fast tablets) for 24 h at room temperature. The slides were counterstained with eosin and were examined for a positive reaction indicated by a blueblack colour.
The ISH score is an arbitrary scale of 05, where 0 represents no viral genome detected and 5 represents massive detection of bound probe at high levels over 2530% of the heart section. It is used to record a composite value for the amount of viral genome detected in all the mice in one set.
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Results |
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Determination of the percentage of infected cells in each culture
Infectious focus assays were performed to determine the proportion of infected cells in each of the cultures. The results showed that at 6 h post-infection, essentially all (>95%) Vero cells were infected when high multiplicities of infection (520 p.f.u. per cell) were used. In contrast only 36% of J774.1 cells were infected and an even smaller proportion of peritoneal macrophages (0·51·5%), explaining at least in part the low yields of virus with these cells.
Effect of anti-CVB2 antibody on CVB3 infection of J774.1 cells
Aliquots of CVB3 virus (2x107 p.f.u.) were mixed with 1:500 and 1:1000 dilutions of anti-CVB2 IgG, or with an equivalent volume of PBS, and were incubated with 1x106 J774.1 cells for 1 h. The inoculum was then removed and the cells were washed three times with PBS and then incubated in RPMI medium. Supernatant medium was harvested and replaced at 2, 4, 6, 8 and 24 h post-infection and was titred on Vero cells. The result is shown in Fig. 2.
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Infectious focus assays were also performed on J774.1 cells infected in the presence and absence of different dilutions of anti-CVB2 IgG and harvested at 6 h post-infection. The results of two separate experiments are shown in Fig. 3. The proportion of infected cells increased 1020-fold in the presence of 1:500 or 1:1000 dilutions of anti-CVB2 but the enhancement was lost with the 1:5000 dilution of antibody (Fig. 3
).
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Using equivalent amounts of F(ab')2 to original IgG, the previous experiment to assess the proportion of infected J774.1 cells was repeated and the result is shown in Fig. 4.
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Effect of anti-CVB2 IgG on the pathogenesis of CVB3 infection in A/J mice
Groups of five mice (5 weeks old) were injected intraperitoneally with 105 p.f.u. of CVB3 alone or together with anti-CVB2 IgG. Control mice received an equivalent volume of PBS. The animals were sacrificed on days 3 or 5 post-injection and samples of serum, pancreas, heart and spleen were removed for plaque assay, histopathology and in situ hybridization.
Virus titres
The levels of infectious virus detected in the tissues are shown in Fig. 5. In serum, 1000-fold higher titres were detected in animals injected with CVB3 plus anti-CVB2 than in those injected with CVB3 alone. Similarly, much higher levels of virus (about 100-fold) were found in pancreas, heart and spleen on day 5 post-inoculation.
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Pancreas.
Massive necrosis of exocrine pancreas can be seen in Fig. 6(c
, e
, g
) in animals injected with virus alone, or with virus plus antibody, in comparison with the control tissue in Fig. 6(a)
. Interestingly, CVB3 does not infect pancreatic islet tissue and intact islets can be seen, despite the widespread destruction of the surrounding tissue.
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Heart.
The degree of damage to heart tissue in animals injected with CVB3 in the presence or absence of anti-CVB2 IgG is shown in photomicrographs of transverse heart sections in Fig. 7. Tissue pathology seen in animals injected with CVB3 alone, on day 5 post-infection, was assessed as minor, while widespread necrosis was apparent in animals injected with virus plus antibody. A similar result was seen by in situ hybridization (Fig. 8
), where the size of lesions containing CVB3 genome and the amount of genome detected can be seen to be much greater in mice injected with virus plus antibody (ISH score of 3·5) than in mice injected with virus alone (ISH score of 1).
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Discussion |
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AEI was also demonstrated in vivo when CVB3 was injected together with anti-CVB2 IgG into 5-week-old A/J mice (intra-peritoneally). Animals injected with virus plus antibody showed 1000-fold increases in circulating virus and 50100-fold increases in virus titre in several organs including pancreas and heart. We interpret this as follows. In animals with pre-existing antibody to a different CVB strain, virus titres may be augmented by enhanced replication in local macrophages at the site of entry. In the case of the enteroviruses, this would occur in Peyers patches in the mucosal layer of the small intestine and in nearby mesenteric lymph nodes (Melnick, 1996 ). This in turn would raise the level of viraemia and therefore the amount of virus reaching the target tissues. In tissues that are susceptible to CVB3, the larger infectious load would cause more cells to be infected and therefore increase the amount of tissue pathology induced before the immune system was able to bring the infection under control.
These findings have considerable implications for understanding CVB pathogenesis and are in agreement with the results of Beck et al. (1990) who showed that tandem infections of mice with CVB2 followed 1 month later by CVB3 also resulted in much more serious disease. While their interpretation of the results related to T-cell reactivity to a conserved CVB epitope, it would seem likely from our study that enhancement by antibody was at least partly the explanation. The relevance of these results to humans relates to the fact that there are six strains of CVB and the majority of individuals become infected by multiple strains during their lifetime. The possibility therefore exists that a CVB infection may be exacerbated by pre-existing antibody to a different strain, resulting in greater tissue damage in heart and other organs. This correlates with the known increase in acute viral myocarditis with age (McManus et al., 1986
) and exposure to multiple CVB strains (Horr et al., 1981
).
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Acknowledgments |
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References |
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Received 27 October 2000;
accepted 25 September 2001.