Potential interactions among disease, pesticides, water quality and adjacent land cover in amphibian habitats in the United States.

To investigate interactions among disease, pesticides, water quality, and adjacent land cover, we collected samples of water, sediment, and frog tissue from 21 sites in 7 States in the United States (US) representing a variety of amphibian habitats. All samples were analyzed for >90 pesticides and pesticide degradates, and water and frogs were screened for the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) using molecular methods. Pesticides and pesticide degradates were detected frequently in frog breeding habitats (water and sediment) as well as in frog tissue. Fungicides occurred more frequently in water, sediment, and tissue than was expected based upon their limited use relative to herbicides or insecticides. Pesticide occurrence in water or sediment was not a strong predictor of occurrence in tissue, but pesticide concentrations in tissue were correlated positively to agricultural and urban land, and negatively to forested land in 2-km buffers around the sites. Bd was detected in water at 45% of sites, and on 34% of swabbed frogs. Bd detections in water were not associated with differences in land use around sites, but sites with detections had colder water. Frogs that tested positive for Bd were associated with sites that had higher total fungicide concentrations in water and sediment, but lower insecticide concentrations in sediments relative to frogs that were Bd negative. Bd concentrations on frog swabs were positively correlated to dissolved organic carbon, and total nitrogen and phosphorus, and negatively correlated to pH and water temperature. Data were collected from a range of locations and amphibian habitats and represent some of the first field-collected information aimed at understanding the interactions between pesticides, land use, and amphibian disease. These interactions are of particular interest to conservation efforts as many amphibians live in altered habitats and may depend on wetlands embedded in these landscapes to survive.

Heterogeneity of the woodchuck hepatitis virus genome in a chronically infected woodchuck.

The nucleotide sequence of an isolate of woodchuck hepatitis virus (WHV) from the serum of a woodchuck trapped in New York state (WHVNY) was compared with the sequences of previously published isolates. The nucleotide sequence of WHVNY was closest to that of an isolate originating from New Jersey: the two genomes shared a 15 nucleotide in-frame deletion in the region where the presurface and polymerase genes overlap (nucleotides 3260-3274) and differed by 54 point mutations (1.6% of genome). Amino acid differences ranged from 0.4% in the surface gene to 5.7% in the X gene. Three isolates from woodchucks that originated in Pennsylvania and Maryland did not contain the deletion and differed from WHVNY by 102 to 106 point mutations (3.0% to 3.2% of nucleotides). Amino acid changes ranged from 0.5% in the core gene to 5.7% in the X-gene. Thus, WHVNY differed little from previous isolates. Next, the genomes from 102 independent clones of WHVNY were compared to ascertain the extent of sequence variation among WHV genomes in a chronically infected animal. A total of 98 clones had genomes of unit length while 2 clones had genomes shorter than unit length and 2 clones had genomes longer than unit length. The clones not of unit length possessed deletions or inverted duplications of sequence. The rate of mutation in the viral genes was 2.65 mutations per 10,000 nucleotides in the precore domain, 1.27 per 10,000 in the X-gene, 0.98 per 10,000 in the presurface gene, and 3.77 per 10,000 at the 5' end of the core gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence against a requisite role for defective virus in the establishment of persistent hepadnavirus infections.

The factors involved in the establishment of persistent hepadnavirus infection are poorly understood. Recent findings demonstrate that the sequence of the genome of hepatitis B virus (HBV) is variable in infected individuals and that, in some cases, virus mutants predominate. Our objectives in the present study were to analyze the variability of woodchuck hepatitis virus (WHV) genomes in an infected animal and to determine whether sequence heterogeneity played a critical role in the ability of WHV to induce chronic infection. We cloned and determined the complete nucleotide sequence of three supercoiled genomes from an animal that became infected after inoculation with a standardized WHV serum pool (i.e., the WHV7 virus pool). We found that there were four nucleotide substitutions among the three genome sequences as well as a 73-nucleotide deletion in one of the recombinants. DNA transfection experiments revealed that only one of the three recombinants was capable of independent replication. These data suggest that a significant proportion of replicative templates in woodchucks that are infected with WHV are defective virus genomes. Next, we compared the outcome of acute infection after inoculation with a serum pool containing a uniform population of replication competent virus (i.e., the WHV7R pool) with a serum pool composed of WHV genomes of variable sequence. The WHV7R serum pool originated from a woodchuck that became a chronic carrier after in vivo transfection of the liver with the infectious WHV7 recombinant. Neonatal woodchucks were inoculated with 5 x 10(6) WHV genome equivalents of either the WHV7 pool or the WHV7R pool. All animals in the study became acutely infected with WHV. Of the animals infected with the WHV7 serum pool, 65% became chronic carriers, while 80% of the animals infected with the WHV7R serum pool developed chronic infection. Thus, infection of woodchucks with a serum pool containing defective virus resulted in a rate of chronic WHV infection that was similar to, or even lower than, a rate from a pool containing only wild-type virus. This suggests that the presence of defective virus in the inoculum is not a prerequisite for the establishment of persistent hepadnavirus infections.

The first-year costs of establishing an unrelated bone marrow donor registry.

The cost of therapies like bone marrow transplantation has been an important consideration for several decades. Bone marrow transplantation is becoming increasingly accepted as an effective treatment for hematologic disorders, including acute nonlymphocytic leukemia. To find suitable donors, bone marrow donor registries are being developed. The first-year costs of establishing an unrelated bone marrow donor registry are reported here. First-year costs are largely due to personnel costs and HLA typing charges. The cost per registrant decreases over time, but further decreases due to economies of scale are limited by the continued fixed requirement for HLA typing. Data are presented by separating costs into six unique categories, thereby allowing other blood centers to estimate start-up costs based on our experience.

Detection of hepatitis A virus by extraction of viral RNA and molecular hybridization.

Hepatitis A virus (HAV) RNA was extracted from cell culture, serum, liver, and feces and then detected by molecular hybridization with cloned HAV cDNA. Hybridization was approximately 10-fold more sensitive than immune electron microscopy or radioimmunoassay was and less sensitive than was assays of HAV infectivity in primates or in cell culture. As little as 10(3) 50% infective doses of HAV, or approximately 0.1 pg of viral RNA, was detected by this method. Analysis of fecal specimens from an experimentally infected marmoset and an epidemic of hepatitis A showed that HAV excretion could often be detected later in the illness by hybridization than by radioimmunoassay. This technique should be widely applicable for detection and analysis of HAV RNA.