Multiplication of Boston Strains of Coxsackievirus A9 in the Adult Mouse Heart.

Strains of coxsackievirus group A type 9 were isolated in Boston from 1959 through 1961 from patients with rash, aseptic meningitis, or pneumonia (Table 1). One of these strains (no. 13) multiplied in the myocardium of 7to 12-monthold mice, producing focal myocarditis (Lerner et al., New Engl. J. Med. 269:678, 736, 1963; Lerner, Levin, and Finland, J. Exptl. Med. 115: 745, 1962; Lerner, Progr. Med. Virol., in press). Since this property, to our knowledge, had not been noted in other strains of coxsackievirus A9 recovered elsewhere, or in the other serotypes of the group A coxsackieviruses, the other strains of coxsackie A9 virus isolated in this laboratory during the same period were examined for virus replication in the myocardium, and for resultant myocarditis. Stock viruses prepared from rhesus kidney cell cultures which had been frozen (-20 C) for several years were thawed, and 0.1 ml of a 10-1 dilution of each of them was inoculated into three monkey-kidney tissue-culture tubes per specimen. The cultures were incubated in stationary racks (37 C), and the fluids were harvested 24 to 72 hr later when cytopathic effects were maximal. These new virus pools were two to six tissue culture passages beyond their primary isolation. Freshly prepared viruses titered 106 5 to 107 5 TCD5o per milliliter. Amounts of 0.03 to 0.05 ml of each virus strain containing approximately 105 TCDSo were inoculated intraperitoneally into 7 to 15 white male mice (8 to 12 months old; supplied by Charles River Breeding Laboratories, Inc., Boston, Mass.). The animals (which all appeared well) were killed 5 days later by exsanguination, and the hearts were examined simultaneously for the presence of virus and for focal myocarditis by 1 Formerly Postdoctoral Research Fellow; presently Trainee in Infectious Diseases, National Institutes of Health. 2 Fellow in Medicine and Infectious Diseases. light microscopy of tissue sections stained with hematoxylin and eosin. Four of these same mice were studied for simultaneous viremia, and the amount of coxsackievirus A9 in their hearts at the time was determined. The methods used were standard, and have been described previously (Lerner and Shaka, Proc. Soc. Exptl. Biol. Med. 111:804, 1962). Hearts of mice which were killed on day 5, and which had been inoculated with strains 13, 186, 711, and 713, contained 1046, 102.5, 104 , and 103. TCDWo per gram of coxsackievirus A9. Although the sera of the inoculated mice tested were free from virus on the 5th day after infection, 6 of 15, 3 of 3, 3 of 3, and 3 of 3 mouse hearts which had been inoculated with these respective virus strains contained virus. Although virus replication was evident in these hearts 5 days after infection, all of the myocardiums appeared normal under light microscopy. At 9 days after inoculation, however, strain 711 (the only strain tested) induced a focal myocarditis (Fig. 1). With this same strain, none of 10, 4 of 10, 2 of 10, and none of 10 hearts of mice showed focal myocarditis at 5, 9, 20, and 30 days after infection. These data are similar to results obtained previously with coxsackievirus A9 strain 13. Another of these strains (186) was passaged 20 times in monkey-kidney tissue culture. At the 3rd and the 20th passages, crude virus suspensions of tissue-culture fluids were inoculated intraperitoneally into 10 mice each. The animals were killed 5 days later, and the hearts were processed for the presence of virus. Of 10 hearts, 8 contained virus at the third passage, but only 1 of them was positive 17 rhesus-kidney passages later. Of the 15 new strains of coxsackievirus A9, 11 multiplied in the myocardium of adult mice, and, in the one strain tested, a focal myocarditis was seen on the 9th day of infection. It would be