In the present study, we have shown that after exposure of HCV-positive recipients to one or two blood donors, the original recipients' strains became undetectable while all donor strains established infection in the recipient. However, one donor strain was clearly predominant, while the other was detected only occasionally. These observations documenting the predominance of a single strain after superinfection are compatible with the results of our recent study on the outcome of liver transplantation in HCV-infected patients who received HCV-infected grafts (19
). In that study, analysis of sequential follow-up sera revealed that half of the patients retained their original HCV infecting strain while in the other half the donor strain took over. However, as the study was conducted with severely immunosuppressed liver transplant recipients and the viral load transplanted with the graft was exceedingly large, it is unclear whether this observation would be pertinent to other clinical situations.
The observed predominance of one strain could perhaps be explained from the evolutionary point of view. The major hypothesis of classical population biology, the competitive exclusion principle, states that in the absence of niche differentiation, one competing species will eventually eliminate or exclude the other (11
). HCV is a rapidly replicating virus (37
), and even small fitness differences, understood as the overall replication and survival ability, could result in overgrowth of one strain by another. The applicability of the competitive exclusion principle to RNA virus populations in cell culture is highly likely, as it has been demonstrated that even virus populations of approximately equal fitness are inherently unstable, since stochastic changes in the balance do eventually occur due to the sudden evolution of higher-fitness variants (5
). Presumably, such a displacement of one virus population by another could also occur in infected animals and humans; however, in the case of an infected host, the equilibrium is infinitely more complex, and any shift in the virus population(s) might be mitigated by changes in the adaptive environment.
One theoretical way to prevent competitive exclusion is the development of a parasitic relationship, where one virus population adapts to use the resources of another virus population and in this way compensates for fitness disadvantages; the end result is the development of an equilibrium. Well-known example of such a relationship are “defective interfering particles,” described for many human and animal viruses (13
) and recently also for HCV (24
). An intriguing possibility is that the minor strain was more adept at infecting certain cells within the liver or even in extrahepatic sites. For example, it has been demonstrated for lymphocytic choriomeningitis virus that strains differing by a single amino acid substitution, when inoculated together into a mouse, are competitively selected either by the liver and spleen or by neurons (6
). In light of recent findings (17
), the presence of extrahepatic HCV replication is highly likely. Nevertheless, regardless of the mechanisms that allowed the minor strain to survive, their effectiveness was probably limited, as it was detectable only transiently and constituted a small fraction of the circulating virus population.
Whether the immune system has played any role in the selection of infecting strains is unclear. It can be argued that the superinfecting strain is immunologically favored, as it represents, to a certain extent, an “escape mutant.” However, neither in our previous study of liver transplant recipients (19
) nor in published chimpanzee studies (9
) did the donor strains seem to be privileged in any way, which suggests the likely role of viral factors, such as the replication fitness of individual strains, in establishing dominance in superinfection and coinfection.
Patients 1 and 2 received blood from two infected donors. While both donor strains established infection in the recipient, one was clearly predominant. What determined this strain predominance is unclear, although one possibility is the size of the inoculum. Interestingly, in case 1, the predominant strain was the one with the higher viral load in the donor. However, in both donors in case 2, the levels of viremia were below the sensitivity limit of the quantification assay. Whether the amount of virus introduced during transfusion determines the outcome of superinfection is unclear, as in all three analyzed cases the donor strain overtook the recipient strain. In any case, the overall amount of virus in the recipient, taking into account the amount of virus in body fluids and the liver, must have been much larger than the amount transfused in 1 U of blood.
The E2 region quasispecies analysis provided the opportunity to study the evolution of quasispecies of two different HCV strains in the same host. Interestingly, two different pictures emerged: in patient 1, the number of viral variants within quasispecies was reduced and the quasispecies composition remained largely unchanged during the follow up, while in patient 3 the number of variants increased after the superinfection and quasispecies showed constant evolution thereafter. These observations suggest that the interplay between different HCV strains and hosts may result in different, perhaps unique, quasispecies compositions. This is compatible with the assumption that at any given moment during the natural history of the infection, the quasispecies distribution represents the best-fitting population that has established a status of equilibrium with a particular host (7
Studies of superinfection and coinfection phenomena have important practical implications, as repeated exposure to HCV is common in high-risk groups, such as drug addicts, but is also likely in many other epidemiological settings. Furthermore, detailed analysis of virological outcome in situations where more than one viral strain is involved, and particularly the evidence for eradication of one strain by another, could have implications for novel treatment options or for the development of a live, attenuated vaccine. The feasibility of exploiting viral interference for antiviral therapy has been demonstrated for influenza A virus infections. An attenuated cold-adapted influenza virus was shown to block the growth of wild-type virus in vitro (34
) and, when administered to infected animals or humans, would prevent or reduce the disease symptoms (35
In summary, we have described three cases of blood transfusion from HCV-positive donors into HCV-positive recipients. In each case, the recipient's pretransfusion strain was apparently displaced by a predominant strain from the transfused blood. In both patients exposed to two HCV RNA-positive donors, the second donor's strain also produced infection. These observations are compatible with the presence of direct competition between infecting strains, which could result in the dominance of a single strain and the competitive exclusion or suppression of other strains.