SUMMARY: Evolution of RNA viruses occurs through disequilibria of collections of closely related mutant spectra or mutant clouds termed viral quasispecies. Here we review the origin of the quasispecies concept and some biological implications of quasispecies dynamics. Two main aspects are addressed: (i) mutant clouds as reservoirs of phenotypic variants for virus adaptability and (ii) the internal interactions that are established within mutant spectra that render a virus ensemble the unit of selection. The understanding of viruses as quasispecies has led to new antiviral designs, such as lethal mutagenesis, whose aim is to drive viruses toward low fitness values with limited chances of fitness recovery. The impact of quasispecies for three salient human pathogens, human immunodeficiency virus and the hepatitis B and C viruses, is reviewed, with emphasis on antiviral treatment strategies. Finally, extensions of quasispecies to nonviral systems are briefly mentioned to emphasize the broad applicability of quasispecies theory.

Background: Tenofovir (TDF) is an adenosine nucleotide analogue that has been approved for the treatment of HIV-1 infection. It also shows activity against hepatitis B virus (HBV) in patients with or without lamivudine (LAM)-associated mutations. Development of clinical or virological HBV breakthrough during TDF therapy has not been reported so far. The aim of this study was to analyse the HBV polymerase (pol) from HIV/HBV-coinfected patients with detectable serum levels of HBV DNA during treatment with TDF for longer than 6 months. Methods: The HBV pol was sequenced from 43 patient's serum before and during TDF therapy. Phenotypic analyses were performed using HBV replication-competent plasmids carrying unique mutations selected under TDF therapy. Results: Mean exposure to LAM was 35.3 ±27.5 months and to TDF 11.2 ±6.7 months. Genotypic analyses from 21 of the patients revealed LAM-associated mutations, and a further two patients developed a novel mutation, rtA194T, along with LAM-resistance-associated mutations. Phenotypic analyses revealed that constructs harbouring rtA194T combined with rtL180M and rtM204V displayed an over 10-fold increase in the IC ₅₀ for TDF compared with the wild type. Conclusion: The selection of HBV mutations in HBV/HIV-coinfected patients failing TDF therapy is an unlikely event within the first 12 months of treatment. However, HBV from two of the 43 patients treated with TDF for more than 12 months was found to contain one novel mutation located distal to the catalytic site of the HBV pol. In vitro, rtA194T conferred a reduced susceptibility to TDF in the presence of LAM-associated mutations.

To investigate transmission of human herpesvirus (HHV)-8, 2546 mother-child pairs were recruited from rural clinics in South Africa and were tested f

Latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) mediates the episomal replication of the KSHV genome, as well as transcriptional regulation, in latently infected cells. Interaction of LANA-1 with cellular chromatin is required for both these functions. An N-terminal heterochromatin-binding site in LANA-1 is essential for the replication and maintenance of latent episomes, as well as transcriptional regulation. We have recently described a C-terminal domain in LANA-1 that modulates the interaction with cellular interphase chromatin or elements of the nuclear matrix. Here, we used a series of LANA-1 deletion mutants to investigate the relationship between the different functions of LANA-1 and its interaction with the host chromatin-binding protein Brd2/RING3. Our findings suggest that the C-terminal chromatin-binding domain in LANA-1 is required for multiple LANA-1 functions, including the ability to bind to and replicate viral episomal DNA, to modulate transcription, and to interact with Brd2/RING3. Similar to the recently described tethering of bovine papillomavirus E2 protein to host chromatin via Brd4/MCAP, Brd2/RING3, another member of the Brd family of chromatin-binding proteins, therefore interacts with a chromatin-binding region of another viral latent nuclear protein and could play a role in its multiple functions.

Tenofovir is a new effective treatment option for patients with chronic hepatitis B, but could be potentially hampered by mutations in the hepatitis B virus (HBV) polymerase conferring drug resistance. Drug resistance may occur preferentially if long-term administration is required, for example, in patients with hepatitis B e antigen (HBeAg)-negative HBV infection bearing precore (PC) and basal core promoter (BCP) mutations. The rtA194T polymerase mutation has been found in HBV/HIV coinfected patients during tenofovir treatment and may be associated with tenofovir resistance. We generated replication-competent HBV constructs harboring rtA194T alone or in addition to lamivudine (LAM) resistance (rt180M + rtM204V), PC mutations, and BCP mutations and assessed their replicative capacity after transient transfection in human hepatoma cells. The rtA194T polymerase mutation alone or in conjunction with LAM resistance reduced the replication efficiency as compared with wild-type (WT) HBV. In contrast, combination of rtA194T (± LAM resistance) with HBeAg-negative PC or BCP mutants increased the replication capacity of the drug-resistant polymerase mutants, thereby restoring the viral replication to similar levels as WT clones. Clones harboring rtA194T showed partial resistance to tenofovir in vitro and also to LAM but remained susceptible to telbivudine and entecavir. Conclusion: The rtA194T polymerase mutation is associated with partial tenofovir drug resistance and negatively impacts replication competence of HBV constructs. Viral replication, however, can be restored to WT levels, if these polymerase mutations occur together with precore or basic core promoter substitutions as found in HBeAg-negative hepatitis B. Patients with HBeAg-negative chronic HBV infection may therefore be at particular risk when developing drug resistance to tenofovir. Telbivudine or entecavir should be considered as effective alternative treatment options for these patients. (Hepatology 2009.)

To look for a virus related to Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) in chimpanzees and to investigate phylogenetic and biological similarities to KSHV. Peripheral blood mononuclear cell (PBMC) DNA samples from chimpanzees (Pan troglodytes troglodytes) were screened with newly designed consensus oligonucleotide primers for the DNA polymerase gene of KSHV-related γ2-herpesviruses (rhadinoviruses). Samples from HIV-1-infected and -uninfected chimpanzees were screened with virus-specific primers. Antibodies to KSHV structural and latent antigens were measured by immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and Western blot. We identified 972 base pairs (bp) of a new viral DNA polymerase sequence with 81.6% (nucleotides) and 93.2% (protein) identity to that of KSHV/HHV8. It was detected in 15/37 (41%) animals experimentally infected with HIV-1, but only in one out of 30 uninfected animals (P < 0.001). Antibodies were found by immunofluorescence to structural, but not latent, KSHV antigens in nearly all HIV-1-infected and uninfected animals. Like man and two other Old World primate species, chimpanzees harbour a virus closely related to KSHV/HHV8, termed Pan troglodytes rhadinovirus-1 (PtRV-1). Like KSHV, PtRV-1 is more easily detected by polymerase chain reaction (PCR) in the PBMC of HIV-1-infected than of HIV-1-uninfected individuals, suggesting increased viral load. Despite the close phylogenetic relationship and biological similarities between KSHV and PtRV-1, Kaposi's sarcoma (KS) has not been reported in HIV-1-infected chimpanzees. PtRV-1 may lack some of the pathogenic determinants of KSHV, or humans and chimpanzees may differ in how they control the infection with their respective rhadinoviruses.