Intervirology Editor-in-Chief. MJ. Buchmeier, Lalblla, Calif: CR, Howard, London Reprint Publisher: §.Karger AG, Basel Original Paper a Intervirology 1993,6:153-140 Ricando M, Gémez* Differential Behaviour in Pancreas Xiaodai Cui* * Gina Cement and Heart of Two Coxsackievirus B3 Marfa f. Berria® Variants Department of Microbiology, Faculty of Medicine, Universi Bucnos Aires, Angenti * Department of Biochemistry and Immunol; Capital Iistitute of Pediatrics, Beijing, People's Republic of Chi Key Words Summary Coxsackicvirus B3 vari: In order to charactet experimental infection of two intratypic variants of coxsackievirus B type 3, Balb/ Intratypic variants, mice were intraperitoneally inoculated and serial samples hy coxsackievirus vested from days 2 to 28. Although both CB3o (amyocardi Viral myocardi and CB3m (myocarditic) variants induced similar early infecti Viral pancreatitis ity titres in pancreas, only the latter led to severe acinur necrosis, and subsequent focal I kinetics and pathogenic properties followed in turn by patent viraemi myocarditis. Nevertheless, when both variants were inoculated in cultured cardiac cells, neither infectivity nor cell death rate differed noticeable. Therefore, our findings indicate that overt myocarditis is not attributable to contrasting cardiomyocyte susceptibility tothe tested variants but rather to prior viral events in pancreatic tissues. Introduction mouse has been employed for nearly 40 years, singe il reproduces selective multiorgan lesions Types I-6 of coxsackievirus B (CBI-6) are [2]. human enteroviruses liable to induce a wide Both cell injury mechanisms and histopa- spectrum of clinical manifestations, including — thological pattems in infected mice have been myocarditis, myositis, pancreatitis, hepatitis, shown to depend on viral strain [3, 4) and and CNS involvement[] Amongexperimental mouse strain[5, 6], among other factors. To gain models used for the study of such diseases, the deeper insight into viral effects om a given cell Sperber by 10)type. as a useful tool cardiac cell cultures derived from diverse species have been devel- oped [7, 8 From both in vivo and in vitro systems, the existence of CB intratypical vari- ants has been demonstrated [9-1l]. The con- ‘trasting behaviour recorded for CB3 variants, so patent in the whole animal and on occasion in cell culture, still lacks explanation at the genomic level [12, 13]. Likewise, the variant-de- pendent induction of selective cell damage re- mains to be elucidated. For a better understanding of CB ex- Perimental infection, here we resorted to the Balb/c mouse, a host for which cardiomyocyte injury has been recently attributed to a direct viral action [6, 14, 15] rather than to an immune= mediated mechanism [16-18]. Therefore, the purpose of the present study was to charac- terize viral kinetics and pathogenic features of two CB3 intratypic variants a3 well as to com= pare their behaviour in cardiac cell culture of murine origin. Materials and Methods Virws ‘Two intratypic variants of coxsackievirus B3 were used. One(C1B30) was generously provided by Dr. M.H. Hatch (Center for Disease Control, Atlanta, Ga, USA) in 1979, and the other (CB3m) was kindly sent by Dr. ‘Charles J. Gauntt (University of Texas Health Scence ‘Center, San Antonio. Tex., USA) who hadicharacterized itas myocarditic (9. In both cases, after passage through Vero cells, supematants were titrated by PFU assay as. previously repaarted [19], with final infectivity stock titres ranging from 10* to 10? PFU /mal in bath cases, Animale Twocolonies of inbred Balb/c mice were emplayed: one from the Depariment of Micrabislogy, Faculty of Medicine, University of Buenos Aires, and the other from the Chinese Academy of Sciences, Beijing. The former was used for in vivo studies and the latter to ‘obtain cardiac cells for cultures Isa Giicnea/CuiCantapsina’ Bervia Jn vivo Snadies Weanling mice were intraperitoneally inoculated ‘wih 0.2 mil of virus dilution in phosphatesbuffered saline (PIES) containing W PFU, ml of CB3o of CB3m. 2, 4, 6, 8, 10, 12, 21, and 28 days after inoculation, 3 animals were bled todeath underether anaesthesia cach day, and specimens of Blood, heart, pancreas, liver, brain, spleen, and skelctal muscle collected: the organs ‘were washed with PBS and divided into two parts. For Viral infectivity assay, tissues were pooled by type for each day of collection, homogenized in a vortex in 20Ps PBS (w/v), clarified by centrifugation at 2,008 rpen far 15 min, and maintained at -70° until use. For histalogi= cal studies, organ tissues were fixed in 8% parafarmal- dehyde, embedded in paraffin, and rautinely processed ‘Sections (5 jum) were oblained and stained with hagmane toxplia and eosin Coll Cilteres Hears from newborn mice were processed acconi- ing to a procedure previously described [20]. Briefly, tissue was finely minced and washed in PES and the resultant fragments subjected to three successive Memin treatments with 0.129% trypsin at 37°. Aer discarding the firstsupernatant gontaining blood and debris. disso- ciated cells from the subsequent trypsinization Ot = Benth rae Viral tire, toss PFU Fa oS8 88 BS iin Meh sme Time since infection, h Fig. 5. Myocardial tissue harvested from CB3m-in. oculated mice, & 6 days after inoculation, widespread freerotic foci exhibiting basophilic properties are shown, Note the scarcity of cell exudate, 2 20M), b 28 days after inoculation, an incipient fibrotic patch made Up by interstitial tissue and necrotic myocyte debris, 200, ‘ © 50 0 5 my 20 5 wg @ ASNT wer ae ara oF b Time since inlection. h Fig. 6. Supernatant infectivity titres and rate of cell death in cardiac cell cultures inoculated with CB3o (a) and CB3m (b).Discussion Although both CB3o- and CB3m-inecu- lated mice showed similar infectivity titres in pancreas, CB3o failed to induce the severe aci- nar cell damage caused by CB3m; besides, viraemia proved undetectable in samples har- vested from CB30-infected animals where liver and heart presented threshold infectivity levels. In contrast, CB3m-inoculated mice ex- hibited patent viraemia followed by heart in- volvement. Although pioneer work relating CB replica- tion in pancreas with pathological changes [23, 24] s00n found support (25, 26], our former [19, 27Jand present results highlight the pancreas as, the earliest organ where viral colonization takes place. Likewise, infectivity was recorded earlier in the pancreas than in heart and liver, as re- ported for CBI by Landau et al. (28) and found forCB2\Gémez, unpubl, data}. As faras mouse strain dependence is concemed, aur prelimi- nary studies in C3H/Hel mice inoculated with ‘CB3m also supported the pancreas as the pri- mary teplication site. Therefore, it may be speculated that, regardless of CB strain and mouse strain, the pancreas seems to behave as a highly permissive tissue fostering virus coloni- zation. Itis worthwhile stressing that when viral replication in the pancreas induces severe local damage, viraemia may reach high enough lev. els to involvewther tissues, particularly myocar- dial. Indeed, it is only in the presence of overt acinar necrosis, as the kind induced by CR3m [27], that zymogen granules could release pan= creatic enzymes which, once activated, have been described to increase dramatically the in- fectivity of certain enteric viruses [29]. How- ever, it remains to be seen whether such en: hancement is operative in the case of coxsac- Kieviruses, Since a number of CB3 variants have been reported to infect cultured endothelial cells from liver, lung, and heart in an organ-depend- ‘ent fashion, such cells may well be capable of channeling both viral tropism and cell injury (30), [tis not unlikely that CB3 variants follow ‘the same course until they replicate in pancreas, ‘the endothelial cells of which would behave as a selective barrier, enabling or disabling subse- quent viraemia. Differing in relation to other amyocarditic varianis [9, 31], the one employed herein failed to replicate significantly in the heart, a finding that is not uncommon in certain prototype Strains [Gauntt, personal commun}. The in- ability io multiply in myocardial tissues has been attributed by Gaunt et al. [32] to a rapid clearance not operative for other intratypic variants ortoa lack of initial replication in cells: readily permissive to other myocarditic viral strains, Asuitable tool to check whether a given cell is susceptible and/or permissive to a given virus involves a tissue culture that allows the characterization of a direct viral effect but avoids the immune reaction at work in the whole organism, In this contest, when murine cardiac cells were cultured and infected as de- seribed, they proved permissive to both vari« ants, even tothe extent of sustaining productive viral replication and causing a similar CPE. However, such findings cannot be readily extrapolated to whole organs, since cultured cells commonly exhibit greater permissiveness than their in vivo counterparts, as well recog- nized for poliovirus in non-neural cells (33. Furthermore, poliovirus has been shown to bind to kidney tissue homogenates from trans- genic host mice expressing the gene from its receptor, although subsequent viral replication was only feasible in culture [34]. Bearing in mind that the viral receptor plays a leading role in recognition, binding, and uncoating of the enteraviruses[34], it may well happen that such receptor functions only become fully operative in vitro to enable the completion of a produc- tive cycle. 158 (Giiener Ci Castagnino ‘Bereia Pancreas ard Heart Infecice by (CHE VariantsTo sum up, our comparison of two CB3 variants indicates that early viral colonization in the pancreas, in the comtest af widespread necrosis, regularly leads ta viraemia high enough to induce overt myocarditis. In an ef- fort to elucidate whether pancreatic enzyme release is involved in modifying virus tropism and pathogenicity, further work is already Acknowledgements This work was partly supported by grants from ‘Conices (Consejo Nacional de Investigaciones Cientifi- as y Técnicas) and Melvietle and Peruilh Fellowship, Faculty of Medicine, University of Buenos Aires, Ar- gentina, and from The Thitd World Academy af Scien: wes, Trieste, aly. under way. References | Grist NR Reid 1D: Greeral pathoge- nicity and epidemiology; in Hendi- felli M, Friedman H (eds): Consac- Rievinas: A General Update. New ‘York, Plenum Press, 1988, pp 22 28. 2 Woodruff JF: Viral enyocarditis: A eviews Ami J Pathe 1980:101:425 483 3) Melnick J: Enteroniruses: Pobiowi- ruses, eonsacicviruses, echovinuses and newer enteroviruses: in Field B (edi: Virology. 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