The aims of this study were to test the hypothesis of a role of HCV quasispecies and to estimate the degree of HCV heterogeneity in an extrahepatic systemic HCV-related disease such as MC.
The large number of examined clones showed a clear trend toward less HVR1 heterogeneity in the MC patients, whose median nonsynonymous substitution rate was 3.3-fold lower than that of the controls, whereas the median synonymous substitution rates in both groups were similar. Nevertheless, the tested patients were probably insufficient to reach statistical significance for all of the analyzed parameters. Interestingly, we observed that the subjects infected by HCV genotype 1 (both MC and CH patients) had significantly fewer divergent quasispecies than those infected with genotype 2, mainly due to an eightfold-lower rate of nonsynonymous substitutions.
After the results were stratified on the basis of the infecting genotype, the MC patients, among the subjects infected by genotype 1, were found to have significantly less quasispecies complexity than did the controls. In contrast, both MC and CH patients infected with HCV-2 had similar diversity and complexity values but, although among the controls the higher variability of HCV-2 than HCV-1 was due to a 11 times increase of nonsynonymous substitutions, among MC patients it was largely due to an increase of synonymous mutations, thus suggesting less positive selection in MC subjects than in CH subjects. Since selection affects only a few amino acid sites in a protein, the dN
ratios averaged over codon sites is frequently not significantly higher than 1, but new models have been recently developed to allow for heterogeneous dN
ratios among sites (58
) and can be used to identify the residues of a protein under positive selection. In a recent report, Sheridan et al. (52
) showed the importance of this high-resolution analysis when studying the interaction between HCV and the host immune response. Analysis of our data with these models yielded two interesting results. The first was that there was a significant reduction in positive selection in both MC and control subjects infected by genotype 1 compared to those infected with HCV genotype 2. This is the first study that has applied high-resolution analysis to the association between the infecting genotype and positive selective pressure on E1/E2 sites. It has recently been suggested that HCV type 1b is less complex than other HCV genotypes (5
), and this may correlate with relatively weak humoral and cell-mediated immune responses against HCV 1b (34
). Our findings of the significantly greater diversity of genotype 2 HCV, together with the higher selective pressure at HVR1 level in this genotype, clearly confirm the association between host immune pressure and the HCV genotype.
The second finding was the relatively low level of selection pressure in our study population: only two (20%) of the MC patients (and none of those infected by HCV-1) showed an averaged ω higher than 1 in the E1/E2 region versus four (50%) of the controls (one with HCV-1). The great majority of the sites under positive selection were in HVR1 (both in MC patients and in controls), thus confirming the presence of an immunodominant B-cell epitope in this region (52
). A number of studies have suggested that the heterogeneity of HVR1 is related to the host immune response and postulated that viral diversity is driven by immune pressure (15
); in particular, it has been observed that immunocompromised hosts (such as agammaglobulinemic or human immunodeficiency virus type 1-infected patients) have a less heterogeneous mutant population (8
). Our finding of less quasispecies heterogeneity in MC patients may therefore be due to an impaired immune response in patients with autoimmune disorders, as is also suggested by the low dN
values constantly observed among the MC patients.
The results of a phylogenetic study of HCV mutants in donor-recipient pairs led Allain et al. to suggest that the evolutionary rate of the quasispecies is inversely proportional to the duration of the infection (4
), whereas other authors (32
) have found that E1/E2 genetic drift seems to be largely unrelated to immune pressure, thus suggesting the existence of possible alternative mechanisms. Mixed cryoglobulinemia mainly affects patients aged 40 to 60 years, almost all of whom have no history of acute hepatitis infection. Our case file did not include the period of time in which the subjects were infected. However, except for one CH patient infected by HCV genotype 2e/f, which has been already described in Italy (6
), all of our MC patients and controls were infected by HCV genotypes 1b and 2a/c (which have been spreading in Western countries for decades) (46
), and we can argue that they have probably been infected for many years. The lower rate of viral replication and evolution in MC subjects with an “old” infection (4
) may partially explain the low level of viral diversity and selective pressure observed by us.
Finally, although it has been reported that viral heterogeneity directly correlates with serum ALT levels (5
), there was no difference in the mean serum ALT levels of our MC patients and controls. One possible confounding factor in the present study is that the group of MC patients included three subjects with cirrhosis, whereas only one of the controls had cirrhosis. However, stratification of the results by the stage of liver disease did not reveal any significantly different quasispecies heterogeneity in the subjects with or without cirrhosis (data not shown).
Taken together, these observations suggest that HCV quasispecies in MC patients are the product of a number of virus-related (genotype, replication activity, and evolutionary rate) and host-related (age, immune response, and period of infection) factors, but longitudinal observations will be required to define the role of each.
The second main objective of the present study was to assess the compartmentalization of HCV in plasma, cryoprecipitate, and PBMC. Although HCV-RNA has been found in the mononuclear blood cells (particularly B lymphocytes) of infected patients (28
), including MC patients (60
), the ability of the virus to replicate inside these cells is still questioned. We found that the HCV quasispecies in PBMC were significantly less complex and divergent than those in plasma and that both synonymous and nonsynonymous substitution rates were significantly lower in PBMC than in plasma. It can be hypothesized that the reduced complexity and diversity of PBMC quasispecies is due to the slow replication of selected quasispecies “inside” the cells and/or the selective absorption of some variants at the cell surface (1
The compartmentalization of the HCV quasispecies in PBMC was evaluated by applying phenetic and phylogenetic methods, using Mantel's test, and bootstrap and maximum-likelihood analyses.
Our data confirm the observations of others (1
) showing that significant compartmentalization in PBMC is possible, but it was only found in four of nine subjects. Although compartmentalization seems to be a frequent finding in MC, its role in the pathogenesis of the syndrome has not yet been defined.
Analysis of the cryoprecipitates showed that, with the exception of two patients with highly homogeneous quasispecies (a phenomenon previously described by others) (26
), all of the other MC patients had at least partially different quasispecies in their plasma and cryoprecipitate samples, and the master sequences were always different. These observations are in agreement with other studies showing the compartmentalization of HCV quasispecies in immunocomplexed and free plasma virus (the last representing the escape mutants) (3
Aiyama et al. (3
) have reported a highly homogeneous quasispecies population complexed with anti-HVR1 antibodies in cryoprecipitates. In our study, the degree of diversity and complexity of the HCV clones in cryoglobulins and plasma significantly correlated. It is probable that antibodies directed against viral antigens other than HVR1 participate in cryoprecipitate formation, thus justifying the HVR1 heterogeneity observed by us and others in cryoglobulin or and immunocomplexes (26
One of our MC patients had an insertion of five amino acids in HVR1 codons 385 to 389, which was observed with only minimal variations in all of the clones obtained from the patient's plasma, cryoprecipitate, and lymphocyte samples. A number of studies have suggested that HVR1 induces antibodies that can neutralize E1/E2 binding to susceptible cells and, more recently, it has been reported that E1/E2 glycoprotein binds CD81, a tetraspanin largely expressed by human cells (47
), and E1/E2 peptide motifs (also included in HVR1) have been identified that can bind the surface of different cell types such as hepatocytes or B cells (22
). Sequence variations in this region may affect binding to cell receptors or neutralizing antibodies, as suggested by the observation in our isolate of amino acid sites under positive selection in the insertion. In particular, four of the five newly inserted residues were polar and therefore capable of affecting the protein-protein interactions in the region and its antigenic and binding properties. Gerotto et al. recently found a one-residue insertion/deletion at codons 384 to 385 in about one-third of their MC patients (23
), but we did not observe any other case of one-residue insertion or deletion. One reason for the different results of the two studies (which were both performed in Northern Italy) may be the different distribution of HCV genotypes. Further studies are needed to demonstrate the existence of specific mutations characteristic of MC.
In conclusion, the heterogeneity of HCV quasispecies and the positive selective pressure tend to be less in MC patients than in noncryoglobulinemic controls, possibly because of factors related to the impaired immune response, the HCV genotype, and the duration of infection. The mechanisms regulating HCV quasispecies in the PBMC and cryoprecipitates of many MC patients, as well as their pathogenic significance, remain to be defined.