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M K Bechtel

Publications and source records attributed to M K Bechtel.

9 recordsLinked to original sources
PubMed1999

Recombinant feline leukemia virus (FeLV) variants establish a limited infection with altered cell tropism in specific-pathogen-free cats in the absence of FeLV subgroup A helper virus.

Feline leukemia virus subgroup B (FeLV-B) is commonly associated with feline lymphosarcoma and arises through recombination between endogenous retroviral elements inherited in the cat genome and corresponding regions of the envelope (env) gene from FeLV subgroup A (FeLV-A). In vivo infectivity for FeLV-B is thought to be inefficient in the absence of FeLV-A. Proposed FeLV-A helper functions include enhanced replication efficiency, immune evasion, and replication rescue for defective FeLV-B virions. In vitro analysis of the recombinant FeLV-B-like viruses (rFeLVs) employed in this study confirmed these viruses were replication competent prior to their use in an in vivo study without FeLV-A helper virus. Eight specific-pathogen-free kittens were inoculated with the rFeLVs alone. Subsequent hematology and histology results were within normal limits, however, in the absence of detectable viremia, virus expression, or significant seroconversion, rFeLV proviral DNA was detected in bone marrow tissue of 4/4 (100%) cats at 45 weeks postinoculation (pi), indicating these rFeLVs established a limited but persistent infection in the absence of FeLV-A. Altered cell tropism was also noted. Focal infection was seen in T-cell areas of the splenic follicles in 3/4 (75%) rFeLV-infected cats analyzed, while an FeLV-A-infected cat showed focal infection in B-cell areas of the splenic follicles. Nucleotide sequence analysis of the surface glycoprotein portion of the rFeLV env gene amplified from bone marrow tissue collected at 45 weeks pi showed no sequence alterations from the original rFeLV inocula.

Animals↗
PubMed1998

In vivo evolution and selection of recombinant feline leukemia virus species.

Ecotropic feline leukemia viruses subgroup A (FeLV-A) is known to recombine with endogenous FeLV (enFeLV) env elements yielding polytropic FeLV-B viruses. However, scattered nucleotide differences exist between enFeLV env elements and corresponding sequences of exogenous FeLV-B isolates. To address this disparity, we examined recombinant FeLV (rFeLV) viruses obtained from three experimentally-induced feline thymic tumors, along with rFeLVs derived from one naturally-occurring thymic tumor. Two of the three experimental cats were challenged with a FeLV-A/Rickard preparation, while one cat received this FeLV-A along with a mixture of in vitro-generated rFeLVs. The FeLV-A/Rickard preparation employed in this study was shown to be free of detectable rFeLVs since no recombinant products were observed in this preparation following nested PCR analyses. For each of the four tumor DNAs, nucleotide sequence analysis was performed on multiple clones of rFeLV-specific PCR products derived from the surface glycoprotein (SU) portion of the recombinant proviral env gene. Relative to the parental enFeLV sequence used to generate the rFeLVs, a total of 19 nucleotide differences were found scattered within the SU region of the env gene in these in vivo-derived rFeLV clones. Most interestingly, this set of 19 differences led to complete sequence identity with natural FeLV-B isolates. Our results indicate these differences are present early in the in vivo evolution of recombinant viruses, suggesting that rFeLVs harboring these differences are strongly selected. We also present evidence indicating an in vivo selection pattern exists for specific recombinant species containing relatively greater amounts of enFeLV-derived SU sequence. This in vivo selection process appears to be gradual, occurring over the infection timecourse, yielding rFeLV species which have recombination structural motifs similar to those seen in natural FeLV-B isolates.

Amino Acid Sequence↗
PubMed1998

Pathogenicity induced by feline leukemia virus, Rickard strain, subgroup A plasmid DNA (pFRA).

A new provirus clone of feline leukemia virus (FeLV), which we named FeLV-A (Rickard) or FRA, was characterized with respect to viral interference group, host range, complete genome sequence, and in vivo pathogenicity in specific-pathogen-free newborn cats. The in vitro studies indicated the virus to be an ecotropic subgroup A FeLV with 98% nucleotide sequence homology to another FeLV-A clone (F6A/61E), which had also been fully sequenced previously. Since subgroup B polytropic FeLVs (FeLV-B) are known to arise via recombination between ecotropic FeLV-A and endogenous FeLV (enFeLV) env elements, the in vivo studies were conducted by direct intradermal inoculation of the FRA plasmid DNA so as to eliminate the possibility of coinoculation of any FeLV-B which may be present in the inoculum prepared by propagating FeLV-A in feline cell cultures. The following observations were made from the in vivo experiments: (i) subgroup conversion from FeLV-A to FeLV-A and FeLV-B, as determined by the interference assay, appeared to occur in plasma between 10 and 16 weeks postinoculation (p.i.); (ii) FeLV-B-like recombinants (rFeLVs), however, could be detected in DNA isolated from buffy coats and bone marrow by PCR as early as 1 to 2 weeks p.i.; (iii) while a mixture of rFeLV species containing various amounts of N-terminal substitution of the endogenous FeLV-derived env sequences were detected at 8 weeks p.i., rFeLV species harboring relatively greater amounts of such substitution appeared to predominate at later infection time points; (iv) the deduced amino acid sequence of rFeLV clones manifested striking similarity to natural FeLV-B isolates, within the mid-SU region of the env sequenced in this work; and (v) four of the five cats, which were kept for determination of tumor incidence, developed thymic lymphosarcomas within 28 to 55 weeks p.i., with all tumor DNAs harboring both FeLV-A and rFeLV proviruses. These results provide direct evidence for how FeLV-B species evolve in vivo from FeLV-A and present a new experimental approach for efficient induction of thymic tumors in cats, which should be useful for the study of retroviral lymphomagenesis in this outbred species.

3T3 Cells↗
PubMed1995

Feline leukemia virus variants in experimentally induced thymic lymphosarcomas.

This study was initiated to evaluate the in vivo infectivity and pathogenicity of a group of recombinant feline leukemia viruses (rFeLVs) previously generated by in vitro forced recombination between a FeLV subgroup A virus (FeLV-A) and an endogenous FeLV (enFeLV) envelope (env) element (Sheets et al., 1992, Virology 190, 849-855). To determine infectivity of rFeLVs, neonatal cats were inoculated with rFeLVs alone or in combination with FeLV-A. The recombinant viruses were able to replicate efficiently in vivo only when administered along with FeLV-A. Of six co-infected cats, three developed thymic lymphosarcomas, one severe aplastic anemia, and two cachexia and depression; all were viremic and seroconverted shortly after inoculation. While both virus types were detected in virtually all tissues examined from these tumor-bearing cats, there was a particularly noteworthy sequence reversion in the rFeLVs. It is known that exogenous FeLV isolates carry a conserved neutralizing MGPNL epitope in the middle of the surface glycoprotein domain of the env gene. In contrast, the parental recombinant viruses used to inoculate these cats harbored the enFeLV-derived MGPNP sequence at this position. However, all in vivo-propagated recombinants displayed the MGPNL sequence, while the env-encoded backbone flanking the MGPNL sequence was that of the parental recombinant virus. These results suggest that viruses with the MGPNL epitope have an in vivo proliferative advantage. The data also provide an explanation for the conservation of this epitope in exogenous FeLVs despite the existence of variant forms in enFeLV proviral elements with which they can recombine.

Amino Acid Sequence↗
PubMed1994

Abnormal processing of a recombinant feline leukemia virus envelope polyprotein and its interference with subgroup C virus infection.

Processing of the env polyprotein of a noninfectious feline leukemia virus (FeLV) recombinant, named r6gp, was examined in human-transfected cells. The r6gp provirus was previously generated in the frame of FeLV, subgroup B, GA clone with substitution of all but 40 C-terminal amino acid sequences of the surface glycoprotein (SU) from an endogenous FeLV provirus element (CFE-6). Although r6gp produced a normal size (85 kDa) env glycoprotein precursor, the product, unlike the precursor of the parental virus, was neither additionally glycosylated nor further processed into mature env proteins. Biochemical observations were consistent with the idea that the chimeric env polyprotein was trapped in the endoplasmic reticulum (ER) and were directly supported by immunofluorescence microscopy analyses. Interestingly, the residence of the chimeric protein in the ER specifically interfered with FeLV, subgroup C (Sarma) virus infection but not the parental FeLV-B virus infection. Since FeLV-C provirus sequences could be readily detected in the infected cells, it appeared that r6gp env expression did not block entry of the challenge virus. While FeLV-B and CFE-6 env genes share an extensive overall sequence homology, a variable region (region VI) of CFE-6 near the C-terminus of SU, which was retained in the r6gp construct, exhibits a considerably higher degree of homology to FeLV-C than FeLV-B. Thus, we propose that region VI is involved in conferring specificity for the env polyprotein oligomerization in the ER, and that co-oligomerization of the trapped r6gp env with FeLV-C is the reason for specific interference with FeLV-C infection. The results also demonstrate for the first time a functional abnormality of a recombinant FeLV env gene which is structurally similar to those commonly detected in FeLV-induced feline lymphosarcomas.

Amino Acid Sequence↗
PubMed1992

Circulating cytomegalovirus (CMV) neutralizing activity in bone marrow transplant recipients: comparison of passive immunity in a randomized study of four intravenous IgG products administered to CMV-seronegative patients.

Forty-two cytomegalovirus (CMV)-seronegative bone marrow transplant (BMT) recipients were randomized in a double-blind fashion to receive one of four commercially available intravenous Ig (IVIgG) products (Gamimmune N, Immune Globulin Intravenous, Gammagard, or Sandoglobulin) at a dose of 500 mg/kg every other week. The four treatment groups were similar in distribution of patient ages, weights, autologous versus allogeneic donor type, and underlying diseases. Every other week administration of IVIgG provided total serum IgG levels within the physiologic range for age. CMV titers by latex agglutination were stable (average geometric mean titer of 18.4 after the second IVIgG dose), with no statistically significant differences among the four product groups. CMV neutralizing activity (CMVNA) and CMV enzyme-linked immunosorbent assay (ELISA) titers were determined on a subset of sera from 27 study patients representing the four product groups. Patient serum samples obtained before IVIgG infusions and 2 weeks after the second IVIgG dose (ie, 3 weeks post-BMT) were assayed for CMVNA and CMV ELISA titers. Geometric mean titers of CMVNA and CMV ELISA varied among the product groups. The highest mean 50% CMVNA was 1:43 for product B, whereas the lowest mean 50% CMVNA was 1:14 for product A; two of the IVIgG product groups showed intermediate 50% mean titers of 1:27 (product C) and 1:26 (product D) for an overall P = .02. CMV ELISA titers (expressed as Paul Ehrlich International units [PEI U]) also showed the highest mean of 2.95 PEI U/mL for product B and the lowest mean of 1.34 PEI U/mL for product A. Intermediate mean values of 2.27 PEI U/mL and 2.03 PEI U/mL were obtained with products C and D, respectively (overall P = .003). The CMV ELISA titers show a minimal correlation (r = .566) to the observed CMVNA titers. We conclude that commercially available IVIgG products provide passive CMVNA, and that the level of circulating CMVNA is affected by the IVIgG product used.

Adolescent↗
PubMed1988

Virus removal or inactivation in hemoglobin solutions by ultrafiltration or detergent/solvent treatment.

Two procedures to eliminate virus infectivity from hemoglobin solutions at ambient temperature were evaluated. In the first, virus removal was assessed during the ultrafiltration of hemoglobin solutions through a membrane with a nominal molecular weight cut-off of 100,000 Daltons. The results of this study demonstrated that less than 0.1% of any virus originally spiked into the solution was detectable in the ultrafiltrate. In the second procedure the inactivation of viruses in hemoglobin solutions incubated with tri(n-butyl)phosphate mixed with sodium cholate was studied. Greater than 99% of each of the enveloped viruses tested was inactivated during the first 15 minutes of incubation with greater than 10(5) plaque forming units/ml of each being inactivated after one to six hours. No inactivation of the non-enveloped poliovirus was effected by this treatment. The data imply that both ultrafiltration and detergent/solvent incubation may reduce virus infectivity in hemoglobin solutions, but neither method yields a completely virus free product.

Cholic Acid↗
PubMed1988

Virus inactivation in hemoglobin solutions by heat.

To determine the feasibility of heat treating hemoglobin solutions to inactivate viruses, we performed experiments on the thermal stability of this protein and found that the structure and function of deoxyhemoglobin are well preserved during incubation at 60 degrees C for 10 hours at a pH of 7.5. Model viruses and the human immunodeficiency virus were rapidly inactivated under these conditions. The results imply that incubation at 60 degrees C is a practical method for inactivating viruses in hemoglobin solutions.

Dithionite↗