Sequencing of multiple recombinant clones generated from polymerase chain reaction-amplified products demonstrated that the degree of heterogeneity of two well-conserved regions of the hepatitis C virus (HCV) genome within individual plasma samples from a single patient was consistent with a quasispecies structure of HCV genomic RNA. About half of circulating RNA molecules were identical, while the remaining consisted of a spectrum of mutants differing from each other in one to four nucleotides. Mutant sequence diversity ranged from silent mutations to appearance of in-frame stop codons and included both conservative and nonconservative amino acid substitutions. From the relative proportion of essentially defective sequences, we estimated that most circulating particles should contain defective genomes. These observations might have important implications in the physiopathology of HCV infection and underline the need for a population-based approach when one is analyzing HCV genomes.

Introduction There are numerous popular misconceptions about sign languages. One of these misconceptions has it that sign languages are “languages on the hands,” that is, that sign language lexemes as well as morphologically and syntactically complex structures are articulated entirely by the hands (and possibly the lower arm). In the course of this chapter, we will show that this statement is far from appropriate. Surely, the hands play an important role in the articulation of signed utterances, but other articulators – the body, the head and (parts of) the face – are just as important. All linguistically significant elements that are not expressed by the hands are referred to as “nonmanual markers” or just “nonmanuals.” Actually, it has been shown that signers, while communicating, do not focus their attention on each other's hands but rather on the face, where essential grammatical information is encoded nonmanually (Siple 1978, Swisher, Christie & Miller 1989). Linguistically significant nonmanuals have to be distinguished from purely affective nonmanual markers such as facial expressions or head movements expressing disgust, disbelief or surprise, which are used by signers just as they are used by speakers. Differentiating between the two types of markers is not always straightforward, but certain distinguishing criteria have been suggested. The scope and the timing of linguistic nonmanual behaviors, for instance, is linguistically constrained relative to the manual sign(s) they accompany in a way that affective markers are not (e.g., Baker-Shenk 1983, Reilly & Anderson 2002).

In the course of a study conducted from 1992 through 1994 of the efficacy of screening blood donors for antibodies to hepatitis C virus (HCV), we found that two patients had acquired hepatitis C after cardiac surgery, with the transmission apparently unrelated to blood transfusions. Because their surgeon had chronic hepatitis C, we sought to determine whether he was transmitting the virus to his patients. Of 222 of the surgeon's patients who participated in studies of post-transfusion hepatitis between 1988 and 1994, 6 contracted postoperative hepatitis C, despite the use of only seronegative blood for transfusions. All six patients had undergone valve-replacement surgery. Analyses were performed to compare nucleotide sequences encompassing the hypervariable region at the junction between the coding regions for envelope glycoproteins E1 and E2 in the surgeon, the patients, and 10 controls infected with the same HCV genotype. The surgeon and five of the six patients with hepatitis C unrelated to transfusion were infected with HCV genotype 3; the sixth patient had genotype 1 and was considered to have been infected from another source. Thirteen other patients of the surgeon had transfusion-associated hepatitis C and were also infected with genotype 1. The average net genetic distance between the sequences from the five patients with HCV genotype 3 and those from the surgeon was 2.1 percent (range, 1.1 to 2.5 percent; P<0.001), as compared with an average distance of 7.6 percent (range, 6.1 to 8.3 percent) between the sequences from the patients and those from the controls. The results of a phylogenetic-tree analysis indicated a common epidemiologic origin of the viruses from the surgeon and the five patients. Our findings provide evidence that a cardiac surgeon with chronic hepatitis C may have transmitted HCV to five of his patients during open-heart surgery.

The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses.