Department of Infection, Guys, Kings & St Thomas School of Medicine, Kings College London, St Thomas Campus, Lambeth Palace Road, London SE1 7EH, UK1
Author for correspondence: Peter Muir. Fax +44 20 7922 8387. e-mail peter.muir{at}kcl.ac.uk
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Abstract |
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Introduction |
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A number of investigators have developed murine models of picornavirus-induced disease, such as myocarditis (Grodums & Dempster, 1959 ; Woodruff & Kilbourne, 1970
), IDDM (Webb et al., 1976
; Yoon et al., 1978
), poliomyelitis (Jubelt et al., 1980
; Ren et al., 1991
), polymyositis (Strongwater et al., 1984
) or chronic demyelinating disease (Lipton, 1975
). These models have been used to study the natural course of infection and immunity, and the mechanisms of pathogenesis. Numerous studies have documented virus persistence beyond the acute phase of infection in mice. These persistent infections can be classified as either productive, where infectious virus is detectable continuously or intermittently during the chronic phase of infection (Lipton & Dal Canto, 1976
; Miller, 1981
), or non-productive, where infectious virus cannot be isolated beyond the acute phase of infection, but viral RNA can nonetheless be detected using molecular hybridization or amplification techniques (Destombes et al., 1997
; Klingel et al., 1992
; Kyu et al., 1992
; Tam et al., 1991
). Persistent productive enterovirus has been observed in patients with gammaglobulin deficiency (reviewed by McKinney et al., 1987
). However, the second pattern of non-productive enterovirus persistence appears similar to that reported in immunocompetent humans with chronic enterovirus-associated disease. The molecular mechanism of enterovirus persistence in the absence of productive infection, the extent of viral genome expression, and its possible consequences for the infected cell or organism are not fully understood.
The duration for the persistence of the virus genome is likely to be determined by the rates of residual viral RNA replication, catabolic degradation of viral RNA and virus- or immune-mediated cytolysis of infected cells. We considered that quantitative analysis of viral RNA kinetics would shed light on the net effect of these mechanisms on the fate of viral RNA in a model of persistent picornavirus infection. Therefore, we studied viral RNA kinetics in acute and chronic phases of CVB3-induced murine myocarditis using quantitative-competitive PCR (Reetoo et al., 1999 ). Viral RNA levels in the myocardium were quantified over a 90 day period following initiation of infection. Viral RNA persistence was also investigated in other target organs of CVB3 infection. For these studies we used the SWR (H-2q) mouse strain, which is highly susceptible to both acute and chronic CVB3-induced myocarditis but shows little or no morbidity or mortality (Klingel et al., 1992
; Zhang et al., 1994
) and is, therefore, suitable for studying persistent infection. This approach yielded valuable insight into the rate of viral RNA clearance during acute and persistent phases of CVB3 infection in different target organs and its temporal relationship to inflammatory responses.
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Methods |
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Murine model of CVB3 myocarditis.
Inbred male four-week-old SWR mice (Harlan) were inoculated intraperitoneally with 106 TCID50 CVB3 in 100 µl PBS. Mice inoculated with 100 µl PBS served as uninfected controls. Internal organs, whole EDTA-anticoagulated blood and serum were aseptically collected from four uninfected mice at day 0 post-infection (p.i.), and from infected mice (four or five) and uninfected mice (two or three) at time-points from day 3 to day 90 p.i. Organs were washed in sterile PBS and a portion was snap-frozen in liquid nitrogen and stored at -70 °C for virus isolation and PCR analysis. The remainder was fixed in formal-buffered saline for histological studies. Serum was also stored at -70 °C, but whole blood was processed immediately for virus infectivity and PCR analysis as described below.
Titration of virus infectivity in murine myocardium.
Pre-weighed portions of approximately 10 mg cryopreserved tissue were ground to a fine powder in liquid nitrogen, homogenized in 200 µl sterile PBS, filtered through a 0·45 µm pore size and serially diluted in PBS. Replicate aliquots (100 µl) of serial tenfold dilutions were inoculated onto Vero cell monolayers in microwell cultures. After 7 days incubation at 37 °C in a 5% CO2 atmosphere, infectivity titres were determined and expressed as TCID50/mg tissue. Whole blood samples and filtered serum samples were cultured undiluted; the inoculum was aspirated after 24 h and replaced with 100 µl maintenance medium.
Qualitative enterovirus PCR.
Total RNA was extracted from whole blood and serum using RNAzol B (Biogenesis) and from cryopreserved tissue after grinding in liquid nitrogen and homogenization in RNAzol B. The presence of enterovirus RNA was tested by qualitative nested RTPCR (EV nPCR) as described previously (Nicholson et al., 1994 ). In titration experiments using synthetic RNA transcripts corresponding in sequence to the 5' nontranslated region of the CVB3 genome (Reetoo et al., 1999
), this assay was found to have a lower detection level of 10 to 100 genome equivalents.
Quantitative enterovirus PCR (EV qPCR).
CVB3 RNA in murine tissues was quantified by EV qPCR as described previously (Reetoo et al., 1999 ). Briefly, total RNA in 100 µl whole blood or serum, or pre-weighed portions of approximately 10 mg cryopreserved tissue was co-extracted and co-amplified with 104 copies of an internal standard synthetic RNA transcript (IS RNA). The CVB3 target sequence of the EV qPCR assay, with a modified internal probe recognition sequence to allow differentiation of IS RNA-derived PCR product from CVB3 RNA-derived product, was contained within the IS RNA sequence. The ratio of CVB3-derived and IS RNA-derived PCR product was then determined by hybridization with specific ruthenium-labelled probes and electrochemiluminescent quantification using QPCR 5000 (Perkin Elmer/Applied Biosystems). From this ratio, the CVB3 RNA copy number in the original sample was determined by reference to a standard curve generated from EV qPCR analysis of increasing copy numbers of a synthetic transcript corresponding to nucleotides 1 to 645 of the CVB3 genome. The use of this standard curve to determine viral RNA copy number accounts for any difference in the amplification efficiencies of the IS RNA and CVB3 RNA templates. Where high levels of viral RNA in the test sample resulted in complete inhibition of IS RNA amplification, samples were retested in the presence of higher IS RNA copy numbers to allow accurate quantification. For samples in which the amplification of either CVB3 RNA or IS RNA failed, RNA was reprecipitated in ethanol and washed three times with 70% ethanol, prior to repeating EV qPCR in an effort to remove possible enzymatic inhibitors. The EV qPCR assay has been previously shown to be able to quantify as little as 100 copies of viral RNA/mg tissue, or per 10 µl whole blood or serum (Reetoo et al., 1999
). All samples were tested in duplicate and the mean values obtained were used in subsequent analyses. Blank RNA extractions, in which sterile H2O was processed in place of tissue, and PCR reagent blanks, in which sterile H2O was added to EV qPCR reagents in place of test RNA extract, were included in each test batch to exclude the possibility of PCR contamination.
Histology.
Tissue portions were fixed in formal-buffered saline and embedded in paraffin. Sections were stained with haematoxylin and eosin and observed by light microscopy. Inflammatory lesions were enumerated blind with respect to the infection status of individual animals or to the time after infection at which tissues were collected.
Data analysis.
Viral RNA levels and virus infectivity in murine tissues were calculated as described previously (Reed & Muench, 1938 ; Reetoo et al., 1999
) and assembled using Microsoft Excel (1997). Mean logarithmic values and 95% confidence intervals were determined for each time-point. Trendlines obtained by linear regression were employed to determine mean rates of viral RNA clearance during different phases of infection and to estimate the time required for complete clearance of viral RNA (i.e. <1 viral genome/mg tissue).
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Results |
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Discussion |
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Several innate and acquired immune effectors have been shown to be important in virus clearance, including NK cell responses (Godeny & Gauntt, 1987 ), macrophage-derived inducible nitric oxide synthase (Lowenstein et al., 1996
), B cell responses (Mena et al., 1999
) and CD4+ and CD8+ T cell responses (Henke et al., 1995
). The reduced clearance rate of viral RNA during the chronic phase of infection suggests that these responses are minimally effective against persistent CVB3 infection. Persisting viral RNA is focally restricted in infected tissues and the level of genomic positive-sense viral RNA is reduced such that equal amounts of genomic- and template-sense RNA are present (Cunningham et al., 1990
; Klingel et al., 1992
, 1996
). Recently Tam & Messner (1999
) showed that viral RNA persists in a double-stranded form in a murine model of CVB1 infection of skeletal muscle. Double-stranded RNA may be resistant to ribonuclease activity and may be associated with reduced viral antigen expression, which would in turn minimize immune-mediated killing of persistently infected cells. Thus, if CVB3 also persists in a double-stranded form, this may account for the long half-life of viral RNA during the chronic phase of infection.
We found considerable variation in the duration of viral RNA persistence in different organs and the time to clearance bore little relation to the initial level of viral RNA. This is consistent with our hypothesis that different mechanisms of virus clearance operate during acute and persistent phases of infection. Previous studies have documented persistence of viral RNA for at least 42 days p.i. in CVB3-induced murine myocarditis (Klingel et al., 1996 ). In other non-productive picornavirus infections in mice, viral RNA was found to persist from 80 days to 12 months (Destombes et al., 1997
; Kyu et al., 1992
; See & Tilles, 1995
; Tam et al., 1994
). It therefore seems probable that such long-term persistence of viral RNA is not uncommon in picornavirus infections.
The consequences of such low-grade virus persistence for the infected cell or organ are uncertain. Picornavirus replication and gene expression in cultured cells has a profound effect on cellular metabolism and cytoskeletal organization in cultured cells and in the murine heart during acute viral myocarditis (Badorff et al., 1999 ). The extent of virus replication and genome expression during the persistent enterovirus infection has proved difficult to assess. However, Li et al. (2000
) recently identified VP1, the major enterovirus capsid protein, in myocardial tissue from 54·5% of patients with myocarditis and 37·5% with dilated cardiomyopathy by employing improved immunohistochemical detection methods. It will be interesting to apply these methods to the study of murine models of persistent enterovirus infection. There is currently some debate as to whether immune responses in chronic murine myocarditis are directed against viral antigens or host myocardial determinants. The persistence of both viral RNA and viral antigen raises the possibility that immune responses may target viral or virus-induced antigens.
Since enterovirus persistence may be established without the selection of replication-defective mutants (Tam & Messner, 1999 ), it is theoretically possible that productive infection might resume under appropriate conditions. The persistence of non-productive infection beyond the resolution of myocardial inflammation in the present study indicates that the inflammatory response is not required to contain virus persistence. However, Shioi et al. (1996
) found increased cytokine expression and localization in the myocardium even during the post-inflammatory phase of encephalomyocarditis virus-induced murine myocarditis. It will clearly be important to determine the role of these chronic immune responses in both virus persistence and ongoing cardiac pathology.
We previously suggested that virus persistence in non-cardiac tissues might act as a reservoir of antigenic stimulation, which could explain the persistence of enterovirus-specific IgM and IgA responses in patients with chronic heart disease (Muir et al., 1989 ). Klingel et al. (1996
) found that the spleen and the lymph nodes were major sites of extracardiac persistence in murine CVB3-induced myocarditis at 42 days p.i. and suggested that this may play a role in the maintenance of chronic disease. In the present study, clearance of viral RNA from spleen was observed at about day 77 p.i. It is interesting to note that clearance of virus from the spleen coincided with resolution of myocardial inflammation. Anderson et al. (1996
) showed that localization of viral RNA in splenic germinal centres only occurred in mouse strains that are susceptible to myocarditis. Taken together, these observations suggest that virus replication or retention in follicular germinal centres of the spleen drives the immune response that gives rise to inflammation of the myocardium and other virus-infected organs.
Although the concept that enteroviruses may persist in the immunocompetent host challenges current understanding of enterovirus biology, the findings presented here, together with other recent data, indicate that the enterovirus RNA persistence is a true biological phenomenon characterized by a reduced rate of clearance of the viral genome and is not an artefact caused by the greater sensitivity of genome detection methods relative to infectivity assays. Future studies should aim to define further the molecular basis and biochemical consequences of enterovirus persistence, to identify the antigenic stimulus that drives the inflammatory response in enterovirus-induced myocardial disease and to clarify further the role of immune effectors in virus clearance, persistence and immunopathology.
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Acknowledgments |
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This study was funded by the CFS Research Foundation. K.N.R. is the recipient of a scholarship from the Association of Commonwealth Universities. S.J.I. is supported by the Philip Fleming Trust. We thank the staff of the Biological Research Services Facility, Rayne Institute, St Thomas Campus, Kings College London, for invaluable technical assistance and Dr J. Cason for useful discussions during preparation of this manuscript.
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References |
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Badorff, C., Lee, G.-H., Lamphear, B. J., Martone, M. E., Campbell, K. P., Rhoads, R. E. & Knowlton, K. U.(1999). Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy.Nature Medicine5, 320-326.[Medline]
Cunningham, L., Bowles, N. E., Lane, R. J. M., Dubowitz, V. & Archard, L. C.(1990). Persistence of enteroviral RNA in chronic fatigue syndrome is associated with the abnormal production of equal amounts of positive and negative strands of enteroviral RNA.Journal of General Virology71, 1399-1402.[Abstract]
Destombes, J., Couderc, T., Thiesson, D., Girard, S., Wilt, S. G. & Blondel, B.(1997). Persistent poliovirus infection in mouse motorneurons.Journal of Virology71, 1621-1628.[Abstract]
Godeny, E. K. & Gauntt, C. J.(1987). Murine natural killer cells limit coxsackievirus B3 replication.Journal of Immunology139, 913-918.
Grodums, E. I. & Dempster, G.(1959). Myocarditis in experimental coxsackie B3 infection. CanadianJournal of Microbiology5, 605-615.
Henke, A., Huber, S., Stelzner, A. & Whitton, J. L.(1995). The role of CD8+ T lymphocytes in coxsackievirus B3-induced myocarditis.Journal of Virology69, 6720-6728.[Abstract]
Jubelt, B., Gallez-Hawkins, G., Narayan, O. & Johnson, R. T.(1980). Pathogenesis of human poliovirus infection in mice. I. Clinical and pathological studies.Journal of Neuropathology & Experimental Neurology39, 138-148.
Kandolf, R. & Hofschneider, P. H.(1985). Molecular cloning of the genome of a cardiotropic coxsackie B3 virus: full length reverse-transcribed recombinant cDNA generates infectious virus in mammalian cells.Proceedings of the National Academy of Sciences, USA82, 4818-4822.[Abstract]
Klingel, K., Hoenadl, C., Canu, A., Albrecht, M., Seemann, M., Mall, G. & Kandolf, R.(1992). Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: quantitative analysis of virus replication, tissue damage and inflammation.Proceedings of the National Academy of Sciences, USA89, 314-318.[Abstract]
Klingel, K., Stephan, S., Sauter, M., Zell, R., McManus, B. M., Bultmann, B. & Kandolf, R.(1996). Pathogenesis of murine enterovirus myocarditis: virus dissemination and immune cell targets.Journal of Virology70, 8888-8895.[Abstract]
Kyu, B., Matsumori, A., Sato, Y., Okada, Y., Chapman, N. & Tracy, S.(1992). Cardiac persistence of cardioviral RNA detected by polymerase chain reaction in a murine model of dilated cardiomyopathy.Circulation86, 522-530.[Abstract]
Li, Y., Bourlet, T., Andreoletti, L., Mosnier, J.-F., Peng, T., Yang, Y., Archard, L. C., Pozzetto, B. & Zhang, H.(2000). Enteroviral capsid protein VP1 is present in myocardial tissues from some patients with myocarditis or dilated cardiomyopathy.Circulation101, 231-234.
Lipton, H. L.(1975). Theilers virus infection in mice: an unusual biphasic disease process leading to demyelination.Infection and Immunity11, 1147-1155.[Medline]
Lipton, H. L. & Dal Canto, M. C.(1976). Chronic neurological disease in Theilers virus infection of SJL/J mice.Journal of Neurological Sciences34, 201-207.
Lowenstein, C. J., Hill, S. L., Lafond-Walker, A., Wu, J., Allen, G., Landavere, M., Rose, N. R. & Herskowitz, A.(1996). Nitric oxide inhibits viral replication in murine myocarditis.Journal of Clinical Investigation97, 1837-1843.
McKinney, R. E., Katz, S. L. & Wilfert, C. M.(1987). Chronic enteroviral meningoencephalitis in agammaglobulinemic patients.Reviews of Infectious Diseases9, 334-356.[Medline]
Melchers, W., Zoll, J., van Kuppeveld, F., Swanink, C. & Galama, J.(1994). There is no evidence for persistent enterovirus infections in chronic medical conditions in humans.Reviews of Medical Virology4, 235-243.
Mena, I., Perry, C. M., Harkin, S., Rodriguez, F., Gebhard, J. & Whitton, J. L.(1999). The role of B lymphocytes in coxsackievirus B3 infection.American Journal of Pathology155, 1205-1215.
Miller, J. R.(1981). Prolonged intracerebral infection with poliovirus in asymptomatic mice.Annals of Neurology9, 590-596.[Medline]
Modlin, J. F.(2000). Coxsackieviruses, echoviruses, and newer enteroviruses. In Mandell, Douglas & Bennetts Principles & Practice of Infectious Diseases, pp. 1904-1919. Edited by G. L. Mandell, J. E. R. Bennett & R. Dolin. Philadelphia:Churchill Livingstone.
Muir, P. & Archard, L. C.(1994). There is evidence for persistent enterovirus infections in chronic medical conditions in humans.Reviews in Medical Virology4, 245-250.
Muir, P., Nicholson, F., Tilzey, A. J., Signy, M., English, T. A. H. & Banatvala, J. E. (1989). Chronic relapsing pericarditis and dilated cardiomyopathy: serological evidence of persistent enterovirus infection. Lancet i, 804807.
Nicholson, F., Meetoo, G., Aiyar, S., Banatvala, J. E. & Muir, P.(1994). Detection of enterovirus RNA in clinical samples by nested polymerase chain reaction for rapid diagnosis of enterovirus infection.Journal of Virological Methods48, 155-166.[Medline]
Reed, L. J. & Muench, H.(1938). A simple method of estimating fifty per cent endpoints.American Journal of Hygiene27, 493-497.
Reetoo, K. N., Osman, S. A., Illavia, S. J., Banatvala, J. E. & Muir, P.(1999). Development and evaluation of quantitative-competitive PCR for quantitation of coxsackievirus B3 RNA in experimentally infected murine tissues.Journal of Virological Methods82, 145-156.[Medline]
Ren, R. B., Costantini, F., Gorgacz, E. J., Lee, J. J. & Racaniello, V. R.(1991). Transgenic mice expressing a human poliovirus receptor: a new model for poliomyelitis.Cell63, 353-362.
See, D. M. & Tilles, J. G.(1995). Pathogenesis of virus-induced diabetes in mice.Journal of Infectious Diseases171, 1131-1138.[Medline]
Shioi, T., Matsumori, A. & Sasayama, S.(1996). Persistent expression of cytokine in the chronic stage of viral myocarditis in mice.Circulation94, 2930-2937.
Strongwater, S. L., Dorivini-Zis, K., Ball, R. D. & Schnitzer, T. J.(1984). A murine model of polymyositis-induced by coxsackievirus B1 (Tucson strain).Arthritis and Rheumatism27, 422-433.[Medline]
Tam, P. E. & Messner, R. P.(1999). Molecular mechanisms of coxsackievirus persistence in chronic inflammatory myopathy: viral RNA persists through formation of a double-stranded complex without associated genomic mutations or evolution.Journal of Virology73, 10113-10121.
Tam, P. E., Schmidt, A. M., Ytterberg, S. R. & Messner, R. P.(1991). Viral persistence during the developmental phase of coxsackie B1-induced murine polymyositis.Journal of Virology65, 6654-6660.[Medline]
Tam, P. E., Schmidt, A. M., Ytterberg, S. R. & Messner, R. P.(1994). Duration of virus persistence and its relationship to inflammation in the chronic phase of coxsackievirus B1-induced murine polymyositis.Journal of Laboratory and Clinical Medicine123, 346-356.[Medline]
Webb, S. R., Loria, R. M., Madge, G. E. & Kibrick, S.(1976). Susceptibility of mice to group B coxsackievirus is influenced by the diabetic gene.Journal of Experimental Medicine143, 1239-1248.[Abstract]
Woodruff, J. F. & Kilbourne, E. D.(1970). The influence of quantitated post-weaning undernutrition on coxsackievirus B3 infection of adult mice. I. Viral persistence and increased severity of lesions.Journal of Infectious Diseases121, 137-163.[Medline]
Yoon, J. W., Onodera, T. & Notkins, A. L.(1978). Virus-induced diabetes mellitus. XV. Beta cell damage and insulin-dependent hyperglycaemia in mice infected with coxsackie virus B4.Journal of Experimental Medicine148, 1068-1080.[Abstract]
Zhang, H., Yousef, G. E., Ouyang, X. & Archard, L. C.(1994). Characterization of a murine model of myocarditis induced by a reactivated coxsackievirus B3.International Journal of Experimental Pathology75, 99-110.[Medline]
Received 16 June 2000;
accepted 3 August 2000.