Nucleotide and Amino Acid Complexity of Hepatitis C Virus Quasispecies in Serum and Liver

Abstract

The quasispecies nature of the hepatitis C virus (HCV) is thought to play a central role in maintaining and modulating viral replication. Several studies have tried to unravel, through the parameters that characterize HCV circulating quasispecies, prognostic markers of the disease. In a previous work we demonstrated that the parameters of circulating viral quasispecies do not always reflect those of the intrahepatic virus. Here, we have analyzed paired serum and liver quasispecies from 39 genotype 1b-infected patients with different degrees of liver damage, ranging from minimal changes to cirrhosis. Viral level was quantified by real-time reverse transcription-PCR, and viral heterogeneity was characterized through the cloning and sequencing of 540 HCV variants of a genomic fragment encompassing the E2-NS2 junction. Although in 95% of patients, serum and liver consensus HCV amino acid sequences were identical, quasispecies complexity varied considerably between the viruses isolated from each compartment. Patients with HCV quasispecies in serum more complex (26%) than, less complex (28%) than, or similarly complex (41%) to those in liver were found. Among the last, a significant correlation between fibrosis and all the parameters that measure the viral amino acid complexity was found. Correlation between fibrosis and serum viral load was found as well (R ‫؍‬ 0.7). With regard to the origin of the differences in quasispecies complexity between serum and liver populations, sequence analysis argued against extrahepatic replication as a quantitatively important contributing factor and supported the idea of a differential effect or different selective forces on the virus depending on whether it is circulating in serum or replicating in the liver. Flaviviridae (7,29,45). Its genome consists of a single-stranded RNA, with plus polarity, of 9,600 nucleotides, which does not integrate in the host genome, yet persistence is the rule. The damage caused during infection ranges from minimal changes to cirrhosis of the liver and hepatocarcinoma, but little is known about the mechanism of hepatocyte injury due to chronic infection. It seems very likely that the pathogenesis of HCV infection is directly related to a strong interplay between the host defense mechanisms and the virus's ability to evade them efficiently. Moreover, in order to persist, HCV must regulate its lytic potential and avoid elimination by the host immune system. Due to the quasispecies structure of the HCV viral population infecting single patients (39), the virus may use a variety of strategies to fulfill both requirements (11,17). Hepatitis C virus (HCV) is an enveloped virus classified in the familyThe dynamic component of the quasispecies structure is responsible for the rapid virus evolution (12). It works through a complex mixture of genomic sequences (quasispecies) which behaves as a single unit when facing changes in the environment. The genetic interaction within the viral population allows the system to distinguish the best possibility at any given...

Hepatitis C virus dynamics and pathology: the role of CTL and antibody responses

“…As severe pathology develops, the number of susceptible host cells is reduced and this can prevent invasion of further antigenic variants. The hypothesis that virus evolution can drive disease progression is supported further by the observation that the population diversity, as well as the ratio of amino acid changing to silent substitutions was higher in individuals with severe liver disease (Cabot et al, 2000;Curran et al, 2002). Similar patterns have been observed in studies of virus evolution following liver transplants in patients with mild and severe disease (Lyra et al, 2002).…”

“…This concept is very difficult to test with data. Studies have quantified sequence diversity and the rate of virus evolution in patients who differ in the severity of liver disease (Cabot et al, 2000;Curran et al, 2002;Farci, 2001;Lyra et al, 2002;Major et al, 1999;Thomson et al, 2001). Even if virus evolution does drive disease progression -as suggested here -this does not mean that one would expect higher virus diversity or faster evolution in patients with severe compared to mild disease.…”

“…Therefore, if pathology develops as a result of virus evolution towards antigenic escape, it is possible that patients with severe disease show reduced levels of virus diversification and evolution compared to patients with milder disease. Data on virus diversity have given rise to different and contradictory results (Cabot et al, 2000;Curran et al, 2002;Farci, 2001;Lyra et al, 2002;Major et al, 1999;Thomson et al, 2001). Studies with HCV-infected patients report the emergence of antibody escape mutants early after infection (Farci et al, 2000), while data from the chimpanzee model argue against the frequent occurrence of antibody escape mutants during the early stages of the infection (Major et al, 1999).…”

CD81 is an entry coreceptor for hepatitis C virus

“…RT-PCR-and electron microscopy-based approaches were relied on to demonstrate the presence of HCV RNA and proteins in primary hepatocytes and certain hepatoma cell lines (3)(4)(5)(6)(7)(8). Furthermore, the existence of extrahepatic HCV reservoirs was suggested by the detection of viral RNA in serum and peripheral blood mononuclear cells (PBMC) (9)(10)(11). Recently, a major technical advance in the field has been the discovery that unmodified HCV envelope glycoproteins can pseudotype retroviral particles and mediate entry into target cells (12)(13)(14)(15).…”

Characterization of the structure and variability of an internal region of hepatitis C virus RNA for M1 RNA guide sequence ribozyme targeting

“…We took advantage of the 540 nucleotide sequences obtained from virus samples (serum and liver) from 39 patients chronically infected with HCV (genotype 1b); these sequences had been obtained previously in our laboratory (Cabot et al, 2000). On average, seven sequences (4-12) were aligned for each sample, serum and liver, and the 'quasispecies consensus sequence' (i.e.…”