Annual cycle in LH and testosterone release in response to GnRH challenge in male woodchucks (Marmota monax).
Testosterone and LH concentrations were determined in serum samples obtained before and 15 min after injections of GnRH (1 microgram kg-1) administered at 4-7 week intervals over 20 months to groups of male woodchucks (n = 6-7) born and maintained in Northern Hemisphere (boreal) versus Southern Hemisphere (austral) simulated natural photoperiods, beginning at 18-24 months of age. Nadir and peak unstimulated testosterone (0.1 +/- 0.01 and 7.0 +/- 0.1 ng ml-1, respectively) and LH (0.8 +/- 0.2 and 8.1 +/- 1.1 ng ml-1, respectively) concentrations did not differ in boreal versus austral males. In the five boreal and five austral males that were confirmed to be photoentrained, basal (pre-GnRH) concentrations of LH and testosterone were lowest in summer, increased simultaneously in late autumn or early winter, and declined in the spring. GnRH stimulated some LH release throughout the year except for a 1-4 month period in the summer. The initial annual increase in the LH response to GnRH occurred in early autumn, and in 17 of 20 cycles it occurred 1-2 months before the initial increase in basal LH was detected. In the three free-running males not entrained to the photoperiod, the endocrine patterns were similar but were advanced by several months. The results demonstrate that in woodchucks there is a late autumn increase in LH secretion associated with the onset of testicular recrudescence, and an early autumn increase in pituitary response to GnRH before a detectable increase in serum testosterone. (+info)
Hepadnavirus evolution and molecular strategy of adaptation in a new host.
In order to elucidate the mechanisms of hepadnavirus evolution in vivo and to trace the fate of known quasispecies in a single animal during the acute phase of infection, a woodchuck (Marmota monax) was infected with the hepadnavirus woodchuck hepatitis B virus (WHV). Woodchuck 197 (W197) was injected intravenously with pooled sera collected from a chronic carrier that had been infected originally with a molecular clone of known genome sequence (WHV7). Viral genome variants from both the inoculum and the follow-up sera from W197 were characterized for the presence of quasispecies related to the WHV7 sequence. Interestingly, WHV7-related genomes were predominant 6 weeks post-infection (p.i.), whereas a highly heterogeneous virus population was present in the first viraemic serum (4 weeks p.i.). Using WHV7 as the prototype, the variability of the Pol and PreS/S regions in the first 11 weeks p.i. has been calculated. The sequence population in serum collected 6 weeks p.i. was highly homogeneous, with a mean variability of 0.36% in the region analysed. Mean variability values ranging from 0.82% to 1.61% were found in quasispecies from the other sera. The presence of possible selective pressure was analysed by means of the non-synonymous versus synonymous variation ratio (dn/d5). We found that the dn/d5 values were stable for the S ORF (ranging from 2.6 to 3.0), whereas a wider range was observed for the Pol ORF (from 1.4 to 3.0). Furthermore, from the analysis of the variability of the codon positions for the two overlapping ORFs it was found that, in most cases, non-synonymous mutations at position 1 of the Pol ORF (position 3 of the S ORF) corresponded to synonymous variation in the S (Pol) ORF, indicating independent evolution of the encoded proteins. (+info)
Activation of the N-myc2 oncogene by woodchuck hepatitis virus integration in the linked downstream b3n locus in woodchuck hepatocellular carcinoma.
In the woodchuck hepatitis virus (WHV)/woodchuck model for hepatitis B virus-induced hepatocellular carcinoma, frequent activation of N-myc oncogenes by WHV integration has been firmly established. N-myc2, the most frequently affected gene, was reported to be activated by WHV insertion either in the proximity of the gene or in a distant uncoding locus, win. We previously reported that a WHV integration cloned from a liver tumor was located in a chromosomal locus already described by others as the site of WHV integration in another hepatocellular carcinoma. On this basis, the locus, named b3n, was defined as a recurrent site of WHV integration. A scaffold or matrix attachment region (S/MAR) element was subsequently shown to be located in this locus approximately 1 kb from the WHV insertion sites. S/MARs are genetic elements involved both in structural and functional organization of chromosomal DNA and in stimulation of gene expression. In the present work, we investigated the possibility that an N-myc gene might be affected by integration in b3n. Analysis of a liver tumor harboring WHV integration in this locus showed N-myc2 overexpression. By restriction analysis, the b3n locus was shown to be located downstream of N-myc2, so the known sites of viral insertion in b3n were approximately 11 kb downstream of the N-myc2 promoter. Although these data support that WHV insertion in b3n activates N-myc2, the mechanisms previously described to be involved in N-myc2 activation do not appear to properly account for activation in this subset of WHV integrations. Available data suggest that activation of N-myc2 by WHV integration in b3n might be mediated by the S/MAR located near the WHV insertion. (+info)
Characterization of the 5' ends for polyadenylated RNAs synthesized during the replication of hepatitis delta virus.
The genome of hepatitis delta virus (HDV) is a 1,679-nucleotide (nt) single-stranded circular RNA that is predicted to fold into an unbranched rodlike structure. During replication, two complementary RNAs are also detected: an exact complement, referred to as the antigenome, and an 800-nt polyadenylated RNA that could act as the mRNA for the delta antigen. We used a 5' rapid amplification of cDNA ends procedure, followed by cloning and sequencing, to determine the 5' ends of the polyadenylated RNAs produced during HDV genome replication following initiation under different experimental conditions. The analyzed RNAs were from the liver of an infected woodchuck and from a liver cell line at 6 days after transfection with either an HDV cDNA or ribonucleoprotein (RNP) complexes assembled in vitro with HDV genomic RNA and purified recombinant small delta protein. In all three situations the 5' ends mapped specifically to nt 1630. In relationship to what is called the top end of the unbranched rodlike structure predicted for the genomic RNA template, this site is located 10 nt from the top, and in the middle of a 3-nt external bulge. Following transfection with RNP, such specific 5' ends could be detected as early as 24 h. We next constructed a series of mutants of this predicted bulge region and of an adjacent 6-bp stem and the top 5-nt loop. Some of these mutations decreased the ability of the genome to undergo antigenomic RNA synthesis and accumulation and/or altered the location of the detected 5' ends. The observed end located at nt 1630, and most of the novel 5' ends, were consistent with transcription initiation events that preferentially used a purine. The present studies do not prove that the detected 5' ends correspond to initiation sites and do not establish the hypothesis that there is a promoter element in the vicinity, but they do show that the location of the observed 5' ends could be controlled by nucleotide sequences at and around nt 1630. (+info)
Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis.
Mother-to-child transmission is an important route for hepatitis B virus (HBV) dissemination. It has been established that HBV traces persist for years after complete clinical recovery from hepatitis B. Similarly, resolution of hepatitis caused by HBV-related woodchuck hepatitis virus (WHV) is followed by occult lifelong carriage of pathogenic virus. In this study, we documented that WHV persisting after termination of acute hepatitis is transmittable to newborns as an asymptomatic long-term infection. All 11 offspring from 4 dams studied carried transcriptionally active WHV genomes for 3.5 years after birth without immunovirological markers of infection. WHV genomes and mRNA were detected both in the liver and lymphoid tissue in the majority of offspring; WHV covalently closed circular DNA was detected in some samples. In 4 offspring, however, the virus was restricted to the lymphatic system. In the circulation, WHV DNA-reactive particles were DNase resistant and of comparable size and density to complete virions. Importantly, the virus in offspring with or without hepatic WHV DNA expression was infectious to WHV-naive woodchucks. Finally, offspring challenged with WHV were not protected against reinfection. These findings show that mothers with occult hepadnaviral carriage transmit pathogenic virus to their offspring, inducing a persistent infection invariably within the lymphatic system but not always in the liver. (+info)
Emergence of drug-resistant populations of woodchuck hepatitis virus in woodchucks treated with the antiviral nucleoside lamivudine.
Lamivudine [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine] reduces woodchuck hepatitis virus (WHV) titers in the sera of chronically infected woodchucks by inhibiting viral DNA synthesis. However, after 6 to 12 months, WHV titers begin to increase toward pretreatment levels. Three WHV variants with mutations in the active site of the DNA polymerase gene are present at this time (W. S. Mason et al., Virology 245:18-32, 1998). We have asked if these mutant viruses were responsible for the lamivudine resistance and if their emergence caused an immediate rise in virus titers. Cell cultures studies implied that the mutants were resistant to lamivudine. Emergence of mutant WHV was not always associated, however, with an immediate rise in virus titers in the serum. One of the three types of mutant viruses became prominent in serum up to 7 months before titers in serum actually began to increase, at a time when wild-type virus was still predominant in the liver. The two other mutants did not show this behavior but were detected in serum and liver later, just at the time that virus titers began to rise. A factor linking all three mutants was that a similar duration of drug administration preceded the rise in titers, irrespective of which mutant ultimately prevailed. A simple explanation for these results is that the increase in virus titers following emergence of drug-resistant mutants can occur only as the preexisting wild-type virus is cleared from the hepatocyte population, allowing spread of the mutants. Thus, prolonged suppression of virus titers in the serum may sometimes be a measure of the stability of hepatocyte infection rather than of a successful therapeutic outcome. (+info)
Perforin and Fas/Fas ligand-mediated cytotoxicity in acute and chronic woodchuck viral hepatitis.
The Fas ligand (FasL)/Fas and the perforin-granzyme cytotoxic pathways presumably play a central role in the development of hepatocellular injury in viral hepatitis. To recognize the potential contribution of FasL and perforin-based cell killing in hepadnaviral infection, we adopted a cytotoxic assay using murine Fas+ P815 and human Fas- K562 cells as targets. Freshly isolated peripheral blood mononuclear cells (PBMC) from woodchucks with newly acquired woodchuck hepatitis virus (WHV) infection (n = 6), with chronic WHV hepatitis (n = 9), and from healthy animals (n = 11) were used as effector cells. We have found that woodchuck lymphoid cells kill cell targets via both the FasL/Fas and the perforin death pathways. The contribution of Fas-dependent cytolysis was ascertained in blocking experiments with anti-Fas antibody and by incubation of PBMC with cyclohexamide to prevent de novo synthesis of FasL. The involvement of the perforin pathway was confirmed by treatment of K562 cells with colchicine to inhibit the microtubule-dependent perforin release. Comparative analysis showed that peripheral lymphoid cells from acute WHV hepatitis, but not those from chronic WHV infection, are more cytotoxic and that this increase seems to be entirely due to activation of perforin-mediated killing. The data indicate that acute infection in woodchucks is associated with the augmented capacity of lymphoid cells to elicit perforin-dependent killing, but in chronic infection, independent of the severity of liver disease and duration of chronicity, these cells have the same or lower cytotoxic potential as PBMC from healthy controls. These findings suggest a role for non-specific cellular immunity, presumably natural killer (NK) cells, in the control of early WHV infection and in the progression of chronic hepatitis. (+info)
Circannual changes in free thyroxine, prolactin, testes, and relative food intake in woodchucks, Marmota monax.
Woodchucks (n = 12-14/group) with circannual cycles entrained to northern versus southern hemisphere photoperiods were assessed monthly for 16 mo. Changes in serum total triiodothyronine (TT(3)), free thyroxine (T(4)), total thyroxine (TT(4)), and prolactin were determined in a subset of five animals per group. Metabolic hormone results were examined in relation to changes in body weight, food intake, and serum testosterone (n = 12-14/group). Seasonal changes in each parameter were similar in both groups as were nadir and peak TT(3) (162 +/- 6 and 392 +/- 12 ng/ml, respectively), free T(4) (19 +/- 2 and 86 +/- 7 ng/ml, repectively), TT(4) (3.2 +/- 0.2 and 8.0 +/- 0.5 ng/ml, respectively), and prolactin (0.6 +/- 0.1 and 14 +/- 2 ng/ml, respectively). In late winter and early spring, simultaneous increases in both free T(4) and prolactin were associated with 1) a large increase in food intake, 2) a decline in body weight to nadir values, 3) a corresponding negative energy balance, 4) a peak and decline in serum testosterone, and 5) a modest increase in TT(4) and large decline in serum TT(3). Low levels of free T(4) and prolactin were observed in summer when energy balance was very positive. The results demonstrate that, in woodchucks, serum T(4) and prolactin undergo seasonal changes during annual cycles entrained by photoperiod. The results suggest that changes in free T(4), acting as a calorigenic hormone, and changes in both T(4) and prolactin, potentially acting as lipolytic, antilipogenic, and/or orectic hormones, are likely involved in the mechanisms underlying the corresponding seasonal changes in food intake, fat metabolism, and energy balance in this species. Their potential roles in gonadal regression and recrudescence are less clear. (+info)