Comparative Characteristics of Two Coxsackie Viruses in Tissue Culture
Authors: Stulberg Cyril S. , Schapira Ruth , Richard Eidam C. 
Published: January 2023

Discussion and Summary
The results of an investigation of the ability of two immunologically distinct Coxsackie viruses to propagate in cells derived from different tissues of the newborn mouse have been presented. The Conn. 5 strain (Group B, type 1) was previously shown to multiply and produce fibroblastic degeneration in cells derived from mouse fat tissue (5). The present data indicate that this strain also could be propagated in primary cultures of skeletal muscle and in fibroblast-like cells derived from muscle of the newborn mouse. In contrast, the Easton-2 strain of Coxsackie virus (Group A, type 1) exhibited questionable growth in primary cultures of skeletal muscle and failed to grow in fibroblastic cells derived from muscle, in primary cultures of interscapular fat tissues, and in cultures of fibroblast-like cells derived from fat tissue.
The heterogeneous properties of the members of the Coxsackie group of viruses are well known. In tissue culture studies, Riordan, et al. (2) found that of 15 antigenically distinct Coxsackie viruses, only a few produced evidence of fibroblastic degeneration in cultures of monkey testicular tissues. Weller, et al. (3) demonstrated that a Group B strain grew in cultures of certain human tissues with no evidence of cytopathogenicity while a Group A strain multiplied in the same tissues and produced cytopathogenic effects. In this regard, the differences observed with the Conn. 5 and Easton-2 strains in cultures of mouse tissue, as well as in cells of human origin (HeLa), may be further indication that these viruses, and perhaps others included in the Coxsackie group, are entirely different viruses rather than members of a related group.
The question as to whether persistence of Easton-2 virus in primary muscle cultures actually represented growth was studied in two ways. Consideration was given to the effect of horse serum, which is known to be inhibitory for Coxsackie-like agents in tissue culture (11). In an experiment in which horse serum was eliminated from the medium, the evidence for multiplication of Easton-2 virus did appear to be more suggestive. Secondly, since virus titers in the culture fluids are a reflection of multiplication in the tissues, greater concentrations of virus in the tissue cells as compared to the culture fluids should be more easily detected. The data revealed that the virus titers in primary muscle cultures were higher in the tissues by factors of 10 to 1000 times with the Conn. 5 virus and 10 to 100 times with the Easton-2 virus. However, while the Conn. 5 virus titers increased daily in the tissues paralleling the increase in the fluids, the Easton-2 virus, although higher in titer in the tissues than in fluids, produced a picture of virus persistence rather than rapid multiplication.
The observations of Godman, et al. (4) have pointed out the relative pathogenicity of certain Group A and Group B strains for various tissues of the infant mouse. Their findings that Conn. 5 virus exhibited severe pathogenicity for fat tissues, whereas the Easton-2 virus did not affect that tissue at all, corresponds to the results obtained in the present and previous (5) studies in tissues culture. Similarly, the growth and fibroblastic degeneration produced by the Conn. 5 virus in cultures of skeletal muscle also is analogous to results obtained in the intact animal. On the other hand, the surprising finding that the Easton-2 virus did not unequivocally propagate in primary muscle cultures is more difficult to interpret in view of the widespread lesions reported to occur (4) in skeletal muscle in vivo. However, it is appreciated that the histotropisms of viruses in vitro do not necessarily reflect those that occur in vivo. For example, Lansing poliomyelitis virus is easily propagated in the brains of mice but does not multiply in that tissue in vitro (12).
The second aspect of this investigation concerned the viral susceptibility of fibroblast-like cells derived from tissues shown to be susceptible in primary passage. Previous studies (5) (13) have indicated that when tissues are susceptible in vitro to herpes simplex and Conn. 5 viruses, fibroblasts derived from those tissues in serial transfer were also found to be susceptible. This did not appear to be true for influenza A virus (13) which failed to propagate in fibroblastic cells derived from susceptible tissues.
The present data indicate that the Conn. 5 virus multiplied in the third passage of fibroblast-like cells from skeletal muscle but that such multiplication was variable. These findings could be accounted for by several possibilities. Either sufficient “muscle” cells from the explanted tissue were carried through three tissue culture passages to provide susceptible host cells for the virus, or different types of fibroblasts with diverse viral susceptibilities were derived from the explants. Since many cell types are able to assume a fibroblastic morphology in vitro, it would be difficult to determine the origin of such cells. Thus one might postulate that if the fibroblasts were derived from muscle cells, the Conn. 5 virus multiplied, but that if cultural conditions favored the predominance of fibroblasts from a contaminating but unsusceptible cell type, the virus failed to propagate. It should be recalled however, that fibroblast-like cells derived from interscapular fat pad tissue of newborn mice were consistently susceptible to Conn. 5 virus (5).