Deciphering human immunodeficiency virus type 1 transmission and early envelope diversification by single-genome amplification and sequencing

Accurate identification of the transmitted virus and sequences evolving from it could be instrumental in elucidating the transmission of human immunodeficiency virus type 1 (HIV-1) and in developing vaccines, drugs, or microbicides to prevent infection. Here we describe an experimental approach to analyze HIV-1 env genes as intact genetic units amplified from plasma virion RNA by single-genome amplification (SGA), followed by direct sequencing of uncloned DNA amplicons. We show that this strategy precludes in vitro artifacts caused by Taq-induced nucleotide substitutions and template switching, provides an accurate representation of the env quasispecies in vivo, and has an overall error rate (including nucleotide misincorporation, insertion, and deletion) of less than 8 x 10(-5). Applying this method to the analysis of virus in plasma from 12 Zambian subjects from whom samples were obtained within 3 months of seroconversion, we show that transmitted or early founder viruses can be identified and that molecular pathways and rates of early env diversification can be defined. Specifically, we show that 8 of the 12 subjects were each infected by a single virus, while 4 others acquired more than one virus; that the rate of virus evolution in one subject during an 80-day period spanning seroconversion was 1.7 x 10(-5) substitutions per site per day; and that evidence of strong immunologic selection can be seen in Env and overlapping Rev sequences based on nonrandom accumulation of nonsynonymous mutations. We also compared the results of the SGA approach with those of more-conventional bulk PCR amplification methods performed on the same patient samples and found that the latter is associated with excessive rates of Taq-induced recombination, nucleotide misincorporation, template resampling, and cloning bias. These findings indicate that HIV-1 env genes, other viral genes, and even full-length viral genomes responsible for productive clinical infection can be identified by SGA analysis of plasma virus sampled at intervals typical in large-scale vaccine trials and that pathways of viral diversification and immune escape can be determined accurately.     

Bovine leukemia virus, an animal model for the study of intrastrain variability.

Intradermal injection of a cloned bovine leukemia virus (BLV) provirus (pV344) into sheep allowed direct evaluation of intrastrain variability. A sheep was injected with pV344 DNA mixed with DEAE-dextran and became persistently infected with BLV strain 344. After 18 months, DNA was extracted from peripheral blood leukocytes from a single 0.5-ml blood sample. The long terminal repeat (LTR) and the env gene were amplified by using the polymerase chain reaction, cloned, and sequenced. Nineteen independent LTR clones (0.6-kb inserts) and 16 env clones (1-kb inserts) were analyzed. The in vivo rate of nucleotide change was 0.009%/year (two mutations out of 14,464 bp in 1.5 years), corresponding to only one amino acid change in the env gene. Five point mutations (all transitions), corresponding to a modification rate of 0.034%/year (five mutations out of 9,709 bp in 1.5 years), were identified in the LTR. As a control for Taq DNA polymerase errors, the same procedure using pV344 plasmid DNA was carried out. Out of 9,944 bp sequenced, three point mutations were found (i.e., one misincorporation in 3,315 nucleotides). These data demonstrate the extremely low level (or absence) of intrastrain variability of BLV in vivo. Consequently, BLV persistence in the infected host does not seem to result from an escape mutant strategy, in sharp contrast with the high mutation rates observed in the lentivirus family. The lack of genetic variation supports the possibility of successful vaccine against BLV and probably against the related human T-cell leukemia viruses.     

Evolutionary strategies of human T-cell lymphotropic virus type II

Human T-cell lymphotropic virus type II (HTLV-II) primarily infects two different populations in which the virus is transmitted in very diverse ways. In endemically infected populations, the virus is propagated through sexual contact, and by mother to child transmission via breast-feeding, among intravenous drug users (IDUs), spread is mainly due to blood-borne transmission via needle sharing. The phylogeny of HTLV-II strains isolated from American Indian and Pygmy tribes and strains from IDUs, reveal that the virus originated on the African continent as a result of a simian to human transmission at least 400,000 years ago. HTLV-II was very likely introduced into the American continent during one or more migrations of HTLV-II infected Asian populations over the Bering land bridge, some 15,000-35,000 years ago. During the last few decades, HTLV-II has been transmitted from native American Indians to IDUs at least twice, followed by a rapid spread of the virus in the drug users population world-wide due to the practice of needle sharing. Molecular clock analysis showed that HTLV-II has two different evolutionary rates, with the molecular clock for the virus in IDUs ticking 150-350 times faster than the one in endemically infected tribes: 2.7x10(-4) compared to 1.7/7.3x10(-7) nucleotide substitutions per site per year in the LTR region. Although many of the HTLV-II infected drug users are co-infected with HIV, the dramatic acceleration of the evolutionary rate seems to be mainly related to the different modes of transmission in the two populations. These contrasting evolutionary rates correlate with an endemic spread of HTLV-II in infected tribes compared to an epidemic spread in IDUs.     

Molecular epidemiology of 58 new African human T-cell leukemia virus type 1 (HTLV-1) strains: identification of a new and distinct HTLV-1 molecular subtype in Central Africa and in Pygmies.

To gain new insights on the origin, evolution, and modes of dissemination of human T-cell leukemia virus type I (HTLV-1), we performed a molecular analysis of 58 new African HTLV-1 strains (18 from West Africa, 36 from Central Africa, and 4 from South Africa) originating from 13 countries. Of particular interest were eight strains from Pygmies of remote areas of Cameroon and the Central African Republic (CAR), considered to be the oldest inhabitants of these regions. Eight long-term activated T-cell lines producing HTLV-1 gag and env antigens were established from peripheral blood mononuclear cell cultures of HTLV-1 seropositive individuals, including three from Pygmies. A fragment of the env gene encompassing most of the gp21 transmembrane region was sequenced for the 58 new strains, while the complete long terminal repeat (LTR) region was sequenced for 9 strains, including 4 from Pygmies. Comparative sequence analyses and phylogenetic studies performed on both the env and LTR regions by the neighbor-joining and DNA parsimony methods demonstrated that all 22 strains from West and South Africa belong to the widespread cosmopolitan subtype (also called HTLV-1 subtype A). Within or alongside the previously described Zairian cluster (HTLV-1 subtype B), we discovered a number of new HTLV-1 variants forming different subgroups corresponding mainly to the geographical origins of the infected persons, Cameroon, Gabon, and Zaire. Six of the eight Pygmy strains clustered together within this Central African subtype, suggesting a common origin. Furthermore, three new strains (two originating from Pygmies from Cameroon and the CAR, respectively, and one from a Gabonese individual) were particularly divergent and formed a distinct new phylogenetic cluster, characterized by specific mutations and occupying in most analyses a unique phylogenetic position between the large Central African genotype (HTLV-1 subtype B) and the Melanesian subtype (HTLV-1 subtype C). We have tentatively named this new HTLV-1 genotype HTLV-1 subtype D. While the HTLV-1 subtype D strains were not closely related to any known African strain of simian T-cell leukemia virus type 1 (STLV-1), other Pygmy strains and some of the new Cameroonian and Gabonese HTLV-1 strains were very similar (>98% nucleotide identity) to chimpanzee STLV-1 strains, reinforcing the hypothesis of interspecies transmission between humans and monkeys in Central Africa.     

5'-long terminal repeat-selective CpG methylation of latent human T-cell leukemia virus type 1 provirus in vitro and in vivo

CpG methylation of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) has been implicated in proviral latency, but there is presently little information available regarding the pattern of LTR methylation and its effect on viral gene expression. To gain insight into the mechanisms of HTLV-1 latency, we have studied methylation of individual CpG sites in the U3-R region of the integrated proviral LTR by using bisulfite genomic sequencing methods. Surprisingly, our results reveal selective hypermethylation of the 5' LTR and accompanying hypomethylation of the 3' LTR in both latently infected cell lines and adult T-cell leukemia (ATL) cells having a complete provirus. Moreover, we observed a lack of CpG methylation in the LTRs of 5'-defective proviruses recovered from ATL samples, which is consistent with the selective hypomethylation of the 3' LTR. Thus, the integrated HTLV-1 provirus in these carriers appears to be hypermethylated in the 5' LTR and hypomethylated in the 3' LTR. These results, together with the observation that proviral gene expression is reactivated by 5-azacytidine in latently infected cell lines, indicate that selective hypermethylation of the HTLV-1 5' LTR is common both in vivo and in vitro. Thus, hypermethylation of the 5' LTR appears to be an important mechanism by which HTLV-1 gene expression is repressed during viral latency.     

Simian T-Cell Lymphotropic Virus Type 1 from Mandrillus sphinx as a Simian Counterpart of Human T-Cell Lymphotropic Virus Type 1 Subtype D

A recent serological and molecular survey of a semifree-ranging colony of mandrills (Mandrillus sphinx) living in Gabon, central Africa, indicated that 6 of 102 animals, all males, were infected with simian T-cell lymphotropic virus type 1 (STLV-1). These animals naturally live in the same forest area as do human inhabitants (mostly Pygmies) who are infected by the recently described human T-cell lymphotropic virus type 1 (HTLV-1) subtype D. We therefore investigated whether these mandrills were infected with an STLV-1 related to HTLV-1 subtype D. Nucleotide and/or amino acid sequence analyses of complete or partial long terminal repeat (LTR), env, and rex regions showed that HTLV-1 subtype D-specific mutations were found in three of four STLV-1-infected mandrills, while the remaining monkey was infected by a different STLV-1 subtype. Phylogenetic studies conducted on the LTR as well as on the env gp21 region showed that these three new STLV-1 strains from mandrills fall in the same monophyletic clade, supported by high bootstrap values, as do the sequences of HTLV-1 subtype D. These data show, for the first time, the presence of the same subtype of primate T-cell lymphotropic virus type 1 in humans and wild-caught monkeys originating from the same geographical area. This strongly supports the hypothesis that mandrills are the natural reservoir of HTLV-1 subtype D, although the possibility that another monkey species living in the same area could be the original reservoir of both human and mandrill viruses cannot be excluded. Due to the quasi-identity of both human and monkey viruses, interspecies transmission episodes leading to such a clade may have occurred recently.     

Identification of human T-cell lymphotropic virus infection in a semi-isolated Afro-Brazilian quilombo located in the Marajó Island (Pará, Brazil)

Antibodies to human T-cell lymphotropic virus-1 and 2 (HTLV-1 and 2) were tested in 259 inhabitants (98 males and 161 females) of four villages of the Marajó Island (Pará, Brazil) using enzyme immunoassays (ELISA and Western blot). Types and subtypes of HTLV were determined by nested polymerase chain reaction (PCR) targeting the pX, env and 5 LTR regions. HTLV-1 infection was detected in Santana do Arari (2.06%) and Ponta de Pedras (1%). HTLV-2 was detected only in Santana do Arari (1.06%). Sequencing of the 5 LTR region of HTLV-1 and the phylogenetic analysis identified the virus as a member of the Cosmopolitan Group, subgroup Transcontinental. Santana do Arari is an Afro-Brazilian community and the current results represent the first report of HTLV-1 infection in a mocambo located in the Brazilian Amazon region.     

Human T-cell leukemia virus type 1 (HTLV-1) infection of mice: proliferation of cell clones with integrated HTLV-1 provirus in lymphoid organs

Human T-cell leukemia virus type 1 (HTLV-1) is suggested to cause adult T-cell leukemia after 40 to 50 years of latency in a small percentage of carriers. However, little is known about the pathophysiology of the latent period and the reservoir organs where polyclonal proliferation of cells harboring integrated provirus occurs. The availability of animal models would be useful to analyze the latent period of HTLV-1 infection. At 18 months after HTLV-1 infection of C3H/HeJ mice inoculated with the MT-2 cell line, which is an HTLV-1-producing human T-cell line, HTLV-1 provirus was detected in spleen DNA from eight of nine mice. No more than around 100 proviruses were found per 10(5) spleen cells. Cellular sequences flanking the 3' long terminal repeat (LTR) and the clonalities of the cells which harbor integrated HTLV-1 provirus were analyzed by linker-mediated PCR. The results showed that the flanking sequences are of mouse genome origin and that polyclonal proliferation of the spleen cells harboring integrated HTLV-1 provirus had occurred in three mice. A sequence flanking the 5' LTR was isolated from one of the mice and revealed the presence of a 6-nucleotide duplication of cellular sequences, consistent with typical retroviral integration. Moreover, PCR was performed on DNA from infected tissues, with LTR primers and primers derived from seven novel flanking sequences of the three mice. Data revealed that the expected PCR products were found from lymphatic tissues of the same mouse, suggesting that the lymphatic tissues were the reservoir organs for the infected and proliferating cell clones. The mouse model described here should be useful for analysis of the carrier state of HTLV-1 infection in humans.     

Genomic quasispecies associated with the initiation of infection and disease in ponies experimentally infected with equine infectious anemia virus.

Equine infectious anemia virus (EIAV) provides a uniquely dynamic system in which to study the mechanism and role of genomic variation in lentiviral persistence and pathogenesis. We have used a Shetland pony model of infection to investigate the association of specific long terminal repeat (LTR) and env gene genomic sequences with the initiation of infection and the onset of disease. We analyzed viral RNA isolated from a pathogenic stock of virus (EIAV PV) and from plasma taken during the first disease episode from two ponies infected with EIAV PV. Overall sequence variation within gp90 was low in EIAV PV and only slightly higher in plasma virus samples isolated from ponies during the first disease episode. However, a high proportion of mutations were localized to the principal neutralizing domain in EIAV PV and to the principal neutralizing domain and the gp90 hypervariable region in the two pony-derived samples. The rate of fixation of mutations was analyzed and determined to be approximately 4 x 10(-2) mutations per site per year. Sequence diversity within the U3 region of the LTR was extremely low, which suggested that the previously reported hypervariability of this region may be a consequence of selection for replication of EIAV in different host cells. The predominant EIAV PV env and LTR sequences were used to construct chimeric viruses so that the contribution of these sequences to viral pathogenicity could be examined. The chimeras replicated in cultured equine monocytes to the same extent as the parental nonpathogenic virus and did not cause disease in Shetland ponies by 120 days postinfection, suggesting that the EIAV genomic determinants of pathogenesis are complex.     

A genome scanning approach to assess the genetic effects of radiation in mice and humans

We used Restriction Landmark Genome Scanning (RLGS) to assess, on a genome-wide basis, the mutation induction rate in mouse germ cells after radiation exposure. Analyses of 1,115 autosomal NotI DNA fragments per mouse for reduced spot intensity, indicative of loss of one copy, in 506 progeny derived from X-irradiated spermatogonia (190, 237 and 79 mice in 0-, 3-, and 5-Gy groups, respectively), permitted us to identify 16 mutations affecting 23 fragments in 20 mice. The 16 mutations were composed of eight small changes (1-9 bp) at microsatellite sequences, five large deletions (more than 25 kb), and three insertions of SINE B2 or LINE1 transposable elements. The maximum induction rate of deletion mutations was estimated as (0.17 +/- 0.09) x 10(-5)/locus Gy(-1). The estimate is considerably lower than 1 x 10(-5)/locus Gy(-1), the mean induction rate of deletion mutations at Russell's 7 loci, which assumed that deletion mutations comprise 50% of all mutations. We interpret the results as indicating that the mean induction rate of mutations in the whole genome may be substantially lower than that at the 7 loci. We also demonstrate the applicability of RLGS for detection of human mutations, which allows direct comparisons between the two species.     

Evaluation of accumulation of hepatitis C virus mutations in a chronically infected chimpanzee: comparison of the core, E1, HVR1, and NS5b regions

Four hepatitis C virus genome regions (the core, E1, HVR1, and NS5b) were amplified and sequenced from yearly samples obtained from a chronically infected chimpanzee over a 12-year span. Nucleotide substitutions were found to accumulate in the core, E1, and HVR1 regions during the course of chronic infection; substitutions within the NS5b region were not detected for the first 8 years and were found to be minimal during the last 4 years. The rate of accumulation of mutations in the core and E1 regions, based on a direct comparison between the first 1979 sequence and the last 1990 sequence, was 1.120 x 10(-3), while phylogenetic ancestral comparison using the 12 yearly sequences showed a rate of 0.816 x 10(-3) bases per site per year. Temporal evaluation of the sequences revealed that there appeared to be periods in which substitutions accumulated and became fixed, followed by periods with relative stasis or random substitutions that did not persist. Synonymous and nonsynonymous substitutions within the core, E1, and HVR1 regions were also analyzed. In the core and E1 regions, synonymous substitutions predominated and gradually increased over time. However, within the HVR1 region, nonsynonymous substitutions predominated but gradually decreased over time.     

Molecular epidemiology of endemic human T-lymphotropic virus type 1 in a rural community in Guinea-Bissau

Background

Human T-Lymphotropic Virus Type 1 (HTLV-1) infection causes lethal adult T-cell leukemia (ATL) and severely debilitating HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in up to 5% of infected adults. HTLV-1 is endemic in parts of Africa and the highest prevalence in West Africa (5%) has been reported in Caio, a rural area in the North-West of Guinea-Bissau. It is not known which HTLV-1 variants are present in this community. Sequence data can provide insights in the molecular epidemiology and help to understand the origin and spread of HTLV-1.

Objective

To gain insight into the molecular diversity of HTLV-1 in West Africa.

Methods

HTLV-1 infected individuals were identified in community surveys between 1990–2007. The complete Long Terminal Repeat (LTR) and p24 coding region of HTLV-1 was sequenced from infected subjects. Socio-demographic data were obtained from community census and from interviews performed by fieldworkers. Phylogenetic analyses were performed to characterize the relationship between the Caio HTLV-1 and HTLV-1 from other parts of the world.

Results

LTR and p24 sequences were obtained from 72 individuals (36 LTR, 24 p24 only and 12 both). Consistent with the low evolutionary change of HTLV-1, many of the sequences from unrelated individuals showed 100% nucleotide identity. Most (45 of 46) of the LTR sequences clustered with the Cosmopolitan HTLV-1 subtype 1a, subgroup D (1aD). LTR and p24 sequences from two subjects were divergent and formed a significant cluster with HTLV-1 subtype 1g, and with the most divergent African Simian T-cell Lymphotropic Virus, Tan90.

Conclusions

The Cosmopolitan HTLV-1 1aD predominates in this rural West African community. However, HTLV-1 subtype 1g is also present. This subtype has not been described before in West Africa and may be more widespread than previously thought. These data are in line with the hypothesis that multiple monkey-to-man zoonotic events are contributing to HTLV-1 diversity.     

The visna virus genome: evidence for a hypervariable site in the env gene and sequence homology among lentivirus envelope proteins.

The complete nucleotide sequence of the visna virus 1514 genome was determined. Our sequence confirms the relationship of visna virus and other lentiviruses to human immunodeficiency virus (HIV) both at the level of sequence homology and of genomic organization. Sequence homology is shown to extend to the transmembrane proteins of lentivirus env genes; this homology is strongest in the extracellular domain, suggesting that close structural and functional similarities may also exist among these envelope proteins. Comparison of our data with the sequence of visna virus LV1-1, an antigenic variant derived from strain 1514, demonstrates that the rate of divergence has been about 1.7 x 10(-3) substitutions per nucleotide per year in vivo. This rate is orders of magnitude higher than that for most DNA genomes, but agrees well with estimates of the rate for HIV. A statistically significant cluster of mutations in the env gene appears to represent a hypervariable site and may correspond to the epitope responsible for the antigenic differences between 1514 and LV1-1. Analysis of the potential RNA folding pattern of the visna virus env gene shows that this hypervariable site falls within a region with little potential for intramolecular base pairing. This correlation of hypervariability with lack of RNA secondary structure is strengthened by the fact that it also holds for a hypervariable site in the env gene of HIV.     

Human glyceraldehyde-3-phosphate dehydrogenase pseudogenes: Molecular evolution and a possible mechanism for amplification

We screened two human genomic libraries and isolated 14 different clones, designated λG1 and EG1-EG13, homologous to human glyceraldehyde-3-phosphate dehydrogenase (GAPD) cDNA. Subcloning and sequencing these recombinant phages led us to classify them as five different pseudogenes (ψG1–ψG5). All these sequences show such features typical of processed pseudogenes as numerous mutations, insertions, and deletions. The identity of numerous mutated sites among these pseudogenes and the presence of two Alu sequences flanking both ends of ψG1 suggest that GAPD pseudogenes originated from a unique reverse transcribed mRNA followed by gene duplication. The rate of nucleotide substitutions per site per year for known GAPD functional genes is low both for the synonymous substitutions (1.87×10⁻⁹) and for the nonsynonymous substitutions (0.12￠10⁻⁹) and indicates that the GAPD cDNA sequence is well conserved not only at the amino acid level, but also at the nucleotide level. The rate of nucleotide substitutions per site per year for GAPD pseudogenes shows a higher value (5.9×10⁻⁹) and suggests that these pseudogenes do not have any functional role. This work was supported by grants from the Consiglio Nazionale delle Ricerche and the Ministero Pubblica Istruzione (Rome, Italy). Special acknowledgment is given to the “Progetto Finalizzato Ingegneria Genetica e Basi Molecolari delle Malattie Ereditarie.”     

Simian T-cell leukemia virus (STLV) infection in wild primate populations in Cameroon: evidence for dual STLV type 1 and type 3 infection in agile mangabeys (Cercocebus agilis)

Three types of human T-cell leukemia virus (HTLV)-simian T-cell leukemia virus (STLV) (collectively called primate T-cell leukemia viruses [PTLVs]) have been characterized, with evidence for zoonotic origin from primates for HTLV type 1 (HTLV-1) and HTLV-2 in Africa. To assess human exposure to STLVs in western Central Africa, we screened for STLV infection in primates hunted in the rain forests of Cameroon. Blood was obtained from 524 animals representing 18 different species. All the animals were wild caught between 1999 and 2002; 328 animals were sampled as bush meat and 196 were pets. Overall, 59 (11.2%) of the primates had antibodies cross-reacting with HTLV-1 and/or HTLV-2 antigens; HTLV-1 infection was confirmed in 37 animals, HTLV-2 infection was confirmed in 9, dual HTLV-1 and HTLV-2 infection was confirmed in 10, and results for 3 animals were indeterminate. Prevalences of infection were significantly lower in pets than in bush meat, 1.5 versus 17.0%, respectively. Discriminatory PCRs identified STLV-1, STLV-3, and STLV-1 and STLV-3 in HTLV-1-, HTLV-2-, and HTLV-1- and HTLV-2-cross-reactive samples, respectively. We identified for the first time STLV-1 sequences in mustached monkeys (Cercopithecus cephus), talapoins (Miopithecus ogouensis), and gorillas (Gorilla gorilla) and confirmed STLV-1 infection in mandrills, African green monkeys, agile mangabeys, and crested mona and greater spot-nosed monkeys. STLV-1 long terminal repeat (LTR) and env sequences revealed that the strains belonged to different PTLV-1 subtypes. A high prevalence of PTLV infection was observed among agile mangabeys (Cercocebus agilis); 89% of bush meat was infected with STLV. Cocirculation of STLV-1 and STLV-3 and STLV-1-STLV-3 coinfections were identified among the agile mangabeys. Phylogenetic analyses of partial LTR sequences indicated that the agile mangabey STLV-3 strains were more related to the STLV-3 CTO604 strain isolated from a red-capped mangabey (Cercocebus torquatus) from Cameroon than to the STLV-3 PH969 strain from an Eritrean baboon or the PPA-F3 strain from a baboon in Senegal. Our study documents for the first time that (i) a substantial proportion of wild-living monkeys in Cameroon is STLV infected, (ii) STLV-1 and STLV-3 cocirculate in the same primate species, (iii) coinfection with STLV-1 and STLV-3 occurs in agile mangabeys, and (iv) humans are exposed to different STLV-1 and STLV-3 subtypes through handling primates as bush meat.     

PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines.

Polymerase chain reaction (PCR) has been used to amplify the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from Western Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from highly conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have amplified by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from 1-2.3 kb from gag pol and env genes have been successfully amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a highly conserved stretch of the viral genome.     

Estimation of mutation induction rates in AT-rich sequences using a genome scanning approach after X irradiation of mouse spermatogonia

We have previously used NotI as the marker enzyme (recognizing GCGGCCGC) in a genome scanning approach for detection of mutations induced in mouse spermatogonia and estimated the mutation induction rate as about 0.7 x 10(-5) per locus per Gy. To see whether different parts of the genome have different sensitivities for mutation induction, we used AflII (recognizing CTTAAG) as the marker enzyme in the present study. After the screening of 1,120 spots in each mouse offspring, we found five mutations among 92,655 spots from the unirradiated paternal genome, five mutations among 218,411 spots from the unirradiated maternal genome, and 13 mutations among 92,789 spots from 5 Gy-exposed paternal genome. Among the 23 mutations, 11 involved mouse satellite DNA sequences (AT-rich), and the remaining 12 mutations also involved AT-rich but non-satellite sequences. Both types of sequences were found as multiple, similar-sequence blocks in the genome. Counting each member of cluster mutations separately and excluding results on one hypermutable spot, the spontaneous mutation rates were estimated as 3.2 (+/- 1.9) x 10(-5) and 2.3 (+/- 1.0) x 10(-5) per locus per generation in the male and female genomes, respectively, and the mutation induction rate as 1.1 (+/- 1.2) x 10(-5) per locus per Gy. The induction rate would be reduced to 0.9 x 10(-5) per locus per Gy if satellite sequence mutations were excluded from this analysis. The results indicate that mutation induction rates do not largely differ between GC-rich and AT-rich regions: 1 x 10(-5) per locus per Gy or less, which is close to 1.08 x 10(-5) per locus per Gy, the current estimate for the mean mutation induction rate in mice.     

Molecular evolution of hepatitis B virus over 25 years

Determining the longitudinal molecular evolution of hepatitis B virus (HBV) is difficult due to HBV's genomic complexity and the need to study paired samples collected over long periods of time. In this study, serial samples were collected from eight hepatitis B virus e antigen-negative asymptomatic carriers of HBV genotype B in 1979 and 2004, thus providing a 25-year period to document the long-term molecular evolution of HBV. The rate, nature, and distribution of mutations that emerged over 25 years were determined by phylogenetic and linear regression analysis of full-length HBV genome sequences. Nucleotide hypervariability was observed within the polymerase and pre-S/S overlap region and within the core gene. The calculated mean number of nucleotide substitutions/site/year (7.9 x 10(-5)) was slightly higher than published estimates (1.5 x 10(-5) to 5 x 10(-5)). Nucleotide changes in the quasispecies population did not significantly alter the molecular evolutionary rate, based on linear regression analysis of evolutionary distances among serial clone pre-S region sequences. Therefore, the directly amplified or dominant sequence was sufficient to estimate the putative molecular evolutionary rate for these long-term serial samples. On average, the ratio of synonymous (dS) to nonsynonymous (dN) substitutions was highest for the polymerase-coding region and lowest for the core-coding region. The low dS/dN ratios observed within the core suggest that selection occurs within this gene region, possibly as an immune evasion strategy. The results of this study suggest that HBV sequence divergence may occur more rapidly than previously estimated, in a host immune phase-dependent manner.