Biomedical subjects

B J Beaty

Publications and source records attributed to B J Beaty.

At least 19 recordsLinked to original sources

PubMed1992

Stable transformation of a mosquito cell line results in extraordinarily high copy numbers of the plasmid.

Stable incorporation of high copy numbers (greater than 10,000 per cell) of a plasmid vector containing a gene conferring resistance to the antibiotic hygromycin was achieved in a cell line derived from the Aedes albopictus mosquito. Plasmid sequences were readily observed by ethidium bromide staining of cellular DNA after restriction endonuclease digestion and agarose gel electrophoresis. The plasmid was demonstrated by in situ hybridization to be present in large arrays integrated in metaphase chromosomes and in minute and double-minute replicating elements. In one subclone, approximately 60,000 copies of the plasmid were organized in a large array that resembles a chromosome, morphologically and in the segregation of its chromatids during anaphase. The original as well as modified versions of the plasmid were rescued by transformation of Escherichia coli using total cellular DNA. Southern blot analyses of recovered plasmids indicate the presence of mosquito-derived sequences.

Aedes↗

PubMed1992

Specificity of molecular hybridization techniques for the detection of bluetongue virus serotypes in Culicoides variipennis.

Direct blot hybridization (DBH) and sandwich hybridization (SH) were evaluated for their ability to detect bluetongue virus (BTV) RNA in the biting midge Culicoides variipennis (Coquillett). Probes were derived from the L3 RNA segment of BTV, serotype 17. RNA of the five BTV serotypes occurring in the USA (BTV-2, BTV-10, BTV-11, BTV-13, and BTV-17) was extracted from pools of varying numbers of infected and uninfected biting midges and assayed by direct blot and sandwich hybridization tests. Direct blot hybridization using an RNA transcript probe or cDNA probe was a fast, efficient and sensitive technique, detecting as few as one midge infected with any BTV serotype in a pool of 50 or 100. Sandwich hybridization was able to detect the homologous serotype, BTV-17, in pools containing a single infected midge in a total of 50 or 100. However, detection of the heterologous serotypes, BTV-10, BTV-11, and BTV-13, was limited to pools containing 5 or more infected midges in a total of 50, and BTV-2 was undetectable by SH. Hybridization techniques provide an alternative to the conventional detection methods of inoculation of cell culture or embryonated chicken eggs for detection of BTV.

Animals↗

PubMed1992

A Drosophila metallothionein promoter is inducible in mosquito cells.

Expression from a Drosophila metallothionein promoter (Mtn) was investigated in mosquito cells. Recombinant plasmids carrying a transcription unit comprised of the Escherichia coli beta-galactosidase gene (lacZ) fused to the metallothionein promoter were stably introduced into Aedes albopictus C6/36 cells. A low copy transformant containing approximately 60 copies of plasmid per cell, and a high copy transformant (1-2 x 10(4) copies/cell) were characterized. The expression of beta-galactosidase from the metallothionein promoter could be induced and controlled in this heterologous system, even when the copy number of introduced plasmid was several thousand.

Aedes↗

PubMed1992

Expression of the chloramphenicol acetyltransferase gene in Aedes albopictus (C6/36) cells using a non-infectious Sindbis virus expression vector.

Genomic RNA was transcribed in vitro from the non-infectious Sindbis (SIN) virus expression vector (pTRCAT) and introduced into C6/36 (Aedes albopictus) cells by liposome-mediated transfections. The chloramphenicol acetyltransferase (CAT) polypeptide expressed within cells was detected by an indirect immunofluorescent assay directly into 24-well polystyrene tissue culture plates. Approximately 1 in 1000 C6/36 cells showed fluorescence when the transfection was optimized. CAT enzyme activity was also assayed; in C6/36 cells CAT expression was detected as early as 8 h after transfection, peaked at 24 h, and could still be detected at 7 days. At 24 h posttransfection each transfected C6/36 cell was calculated to express 1.3 x 10(7) CAT polypeptides.

Aedes↗

PubMed1992

Enhancement of the antibody response to flavivirus B-cell epitopes by using homologous or heterologous T-cell epitopes.

We have been investigating the T-helper (Th)-cell response to the flavivirus envelope (E) glycoprotein. In our studies with Murray Valley encephalitis (MVE) virus, we previously identified synthetic peptides capable of priming Th lymphocytes for an in vitro antivirus proliferative response (J. H. Mathews, J. E. Allan, J. T. Roehrig, J. R. Brubaker, and A. R. Hunt, J. Virol. 65:5141-5148, 1991). We have now characterized in vivo Th-cell priming activity of one of these peptides (MVE 17, amino acids 356 to 376) and an analogous peptide derived from the E-glycoprotein sequence of the dengue (DEN) 2, Jamaica strain (DEN 17, amino acids 352 to 368). This DEN peptide also primed the Th-cell compartment in BALB/c mice, as measured by in vitro proliferation and interleukin production. The failure of some MVE and DEN virus synthetic peptides to elicit an antibody response in BALB/c mice could be overcome if a Th-cell epitope-containing peptide was included in the immunization mixture. A more detailed analysis of the structural interactions between Th-cell and B-cell epitope donor peptides revealed that the peptides must be linked to observe the enhanced antibody response. Blockage or deletion of the free cysteine residue on either peptide abrogated the antibody response. The most efficient T-B-cell epitope interaction occurred when the peptides were colinearly synthesized. These Th-cell-stimulating peptides were also functional with the heterologous B-cell epitope-containing peptides. The Th-cell epitope on DEN 17 was more potent than the Th-cell epitope on MVE 17.

Amino Acid Sequence↗

PubMed1992

Genomic and biologic analyses of snowshoe hare virus field and laboratory strains.

Low-passage field strains of snowshoe hare (SSH) virus (Bunyaviridae), the prototype SSH virus (originally isolated in Montana), and La Crosse (LAC) virus were compared serologically by plaque-reduction neutralization (PRNT) and molecularly by oligonucleotide fingerprinting (ONF). The PRNT and ONF results confirmed the identity of the field strains, although some differences in the fingerprints were observed. We have examined the RNA genome variability in the two field and three laboratory strains of SSH virus, using direct sequence analysis of selected RNase T1 oligonucleotides. Few changes were observed among three Montana prototype-derived laboratory isolates, although they have different passage histories. In contrast, the field isolates differed greatly from the laboratory strains. In addition, we have located several of the larger T1 oligonucleotides within the known sequence of the small and large RNA genome segments. We then compared the viruses for their ability to replicate in and be transmitted by Aedes triseriatus mosquitoes. The oral infection rates for LAC, the field isolates, and the SSH prototype, as determined by immunofluorescent examination of midgut tissues, were 100%, 82%, and 47%, respectively. All viruses were also transmissible from mosquitoes to mice.

Aedes↗

PubMed1991

Reassortment of La Crosse and Tahyna bunyaviruses in Aedes triseriatus mosquitoes.

Experiments were conducted to determine if La Crosse (LAC) and Tahyna (TAH) viruses reassort in Aedes triseriatus mosquitoes and to determine the genotypic frequencies of viruses selected by in vivo vector interactions. A molecular hybridization technique was used to analyze progeny viruses. Probes specific for the La Crosse L, M and S segments (pLAC4.16: LAC L RNA; pLAC4.27: LAC M RNA; pLAC4C-26: LAC S RNA) were used to determine the parental origin of the progeny RNA segments. Following infection with a mixture of LAC and TAH viruses, mosquitoes were held for 23 days extrinsic incubation, then assayed for reassortment. Individual progeny viruses were isolated by plaque assay and propagated in BHK-21 cells. Cytoplasmic RNA was extracted from the cells, blotted in triplicate to Nytran, and each blot was hybridized with 32P-labelled pLAC4.16, pLAC4.27 or pLAC4C-26 to determine the parental origin of each RNA segment. High frequency reassortment occurred in these mosquitoes. All of the expected genotypes resulting from a cross of LAC and TAH were obtained from these mosquitoes. Genotypic frequencies of 708 virus isolates from 39 mosquitoes were: LLL, 150 (21%); LLT, 71 (10%); LTL, 39 (5.5%); LTT, 109 (15%); TTT, 259 (36%); TTL, 16 (2.2%); TLT, 55 (7.8%); TLL, 9 (1.2%).

Aedes↗

PubMed1991

A single amino acid change in the E2 glycoprotein of Venezuelan equine encephalitis virus affects replication and dissemination in Aedes aegypti mosquitoes.

Four monoclonal antibody-resistant variants (MARVs) of Venezuelan equine encephalitis (VEE) virus were used to study mosquito-virus interactions. In vitro experiments using an Aedes albopictus cell line, C6/36, demonstrated that an amino acid change in the glycoprotein E2h epitope (MARV 1A3B-7) decreased virus growth when compared with the wild-type, Trinidad donkey virus, and its vaccine derivative, TC-83. The MARVs replicated as efficiently as the parent virus when inoculated into Aedes aegypti mosquitoes, but MARV 1A3B-7 was restricted in its ability to infect and disseminate from the midgut following oral infection. These results demonstrate that a single amino acid change in the E2 glycoprotein can affect the ability of VEE virus to replicate and disseminate in Ae. aegypti mosquitoes.

Aedes↗

PubMed1991

Detection of bluetongue virus RNA by in situ hybridization: comparison with virus isolation and antigen detection.

An in situ nucleic acid hybridization (ISH) technique was developed to detect bluetongue virus (BTV) RNA in cell culture. The sensitivity of the ISH technique was compared with virus isolation (VI) and antigen detection, using an indirect fluorescent-antibody (IFA) or an enzyme immunocytoassay (EICA) technique, for detection of 5 BTV serotypes indigenous to the United States. The VI was the most sensitive technique, detecting BTV early after infection of the cells. The IFA and EICA were of similar sensitivity; BTV antigen could be detected shortly after demonstration of virus by isolation. The sensitivity of ISH for detection of BTV-17 was equivalent to that of antigen detection. The ISH was not as sensitive as VI or antigen detection when assaying for the other BTV serotypes.

Animals↗

PubMed1991

Infection of Aedes albopictus and Aedes aegypti mosquitoes with dengue parent and progeny candidate vaccine viruses: a possible marker of human attenuation.

Dengue (DEN-1) and DEN-4 parent (P) and progeny candidate vaccine (CV) viruses were compared in their abilities to infect and to replicate in Aedes aegypti and Aedes albopictus mosquitoes. The DEN CV clones were temperature sensitive (ts) and had small plaque morphology. The DEN-1 and DEN-4 CV viruses differed in their ability to infect, to replicate in, and to be transmitted by mosquitoes. The DEN-1 CV virus was not attenuated for the vector mosquitoes; oral infection rates with the CV virus were as high as or higher than the P virus, and the CV virus replicated efficiently in mosquitoes after oral infection. The DEN-4 CV virus was attenuated; it was less efficient than its P virus in infection and replication in mosquitoes. Thus, the ts phenotype and small plaque morphology are not reliable biological markers for prediction of vector attenuation. Similar results were reported by others for attenuation in man and monkeys. These studies with DEN-1 and DEN-4 viruses, and previously reported studies with DEN-2 virus and with DEN-3 virus suggest that vector and vertebrate host attenuation are genetically linked. Thus, vector attenuation may be a biological marker for human attenuation.

Aedes↗

PubMed1991

Bunyaviridae--natural history.

Obviously, the family Bunyaviridae is comprised of a large number of epidemiologically diverse viruses. They vary dramatically in their vector and vertebrate host relationships, geographic distributions, and epidemic potential in humans and animals. Public health practitioners, veterinarians, virologists, entomologists, biologists, ecologists, molecular biologists, and other scientists will all benefit from increased study and knowledge of this fascinating group of viruses.

Animals↗

PubMed1990

Detection of dengue viral RNA in mosquito vectors by mixed phase and solution hybridization.

A mixed phase hybridization technique was developed to detect dengue virus type 2 (DEN-2) RNA in pools of infected Aedes albopictus mosquitoes using radiolabelled RNA probes. This technique used a guanidine thiocyanate extraction procedure to simplify analyte preparation. The probes contained sequences complementary to portions of the NS-1 or NS-5 genes of the DEN-2 viral genome. One infected mosquito in a pool of 25 could be detected in approximately 48 h. RNAs from DEN serotypes 1-4 were extracted from cultured mosquito (C6/36) cells. The NS-1 RNA probe was highly specific for DEN-2 RNA. The NS-5 RNA probe detected both DEN-2 and DEN-4 RNA. DEN-2 RNA was also detected by molecular hybridization in concentrated solutions of guanidine thiocyanate using the NS-1 probe. Solution hybridization was 10-fold more sensitive when detecting RNA from purified DEN-2 virus than the mixed phase assay and could detect one infected mosquito in a pool of 25 within 6-8 h. Solution hybridizations were performed in 2-3 h vs 16-20 h for mixed phase hybridizations, and solution hybridizations required 5-10 times less mosquito RNA than mixed phase hybridizations to attain comparable sensitivities. However, solution hybridizations did result in a broader probe specificity than mixed phase hybridizations.

Aedes↗

PubMed1990

Heterologous reassortment of bunyaviruses in Aedes triseriatus mosquitoes and transovarial and oral transmission of newly evolved genotypes.

Aedes triseriatus mosquitoes were orally infected with two different California serogroup bunyaviruses (La Crosse and snowshoe hare viruses) and high frequency reassortment occurred in these mosquitoes. Increased viral replication and subsequent gene segment reassortment was noted in the ovaries of mosquitoes that had ingested multiple blood-meals. To determine whether newly generated reassortant viruses could be transmitted transovarially to progeny mosquitoes, adult female mosquitoes were inoculated with the two temperature-sensitive (ts) parental viruses, and allowed to blood-feed and oviposit. Of 58 infected progeny mosquitoes assayed, six (10%) contained non-ts viruses, and three of these transmitted non-ts viruses to a susceptible host. Selected viruses of the non-ts phenotype, which were isolated from mosquitoes and from mice fed upon by the mosquitoes, were demonstrated to be reassortant viruses by oligonucleotide fingerprinting.

Aedes↗

PubMed1990

Detection of bluetongue virus serotype 17 in Culicoides variipennis by nucleic acid blot and sandwich hybridization techniques.

Molecular hybridization techniques were developed for the detection and surveillance of bluetongue virus (BTV) serotype 17 in the insect vector Culicoides variipennis, a biting midge. Radiolabeled RNA and cDNA probes were generated from sequences of the L3 segment of BTV serotype 17. These probes were used to detect BTV RNA in pools of infected C. variipennis by hybridizing the probes directly to analyte immobilized on nylon membranes or by using a nucleic acid sandwich hybridization test. Hybridization procedures were able to detect 1 infected C. variipennis in a pool of 50 and as little as 3.55 log10 50% tissue culture infective doses per ml of virus. These hybridization techniques provide an alternative to virus isolation for the surveillance of BTV in vector populations.

Animals↗

PubMed1990

Dengue 3 virus infection of Aedes albopictus and Aedes aegypti: comparison of parent and progeny candidate vaccine viruses.

DEN-3 parent (strain CH53489) and progeny candidate vaccine (clone 24/28) viruses were compared in their abilities to interact with Aedes aegypti and Ae. albopictus. The parent and progeny virus were equivalent in their ability to infect, to replicate in, and to be transmitted by both species of mosquitoes. The candidate vaccine DEN-3 clone was temperature sensitive and had small plaque morphology. These phenotypic markers remained stable during mosquito passage. Thus, temperature sensitivity and small plaque morphology are not reliable biological markers for attenuation.

Aedes↗

PubMed1989

Genomic stability of La Crosse virus during vertical and horizontal transmission.

We have used ribonuclease T1 oligonucleotide fingerprint analysis to study genomic stability of La Crosse virus (Bunyaviridae) during vertical and horizontal transmission in the laboratory. No RNA genomic changes were detected in vertebrate cell culture-propagated virus isolated (following ingestion and replication) from the natural host, Aedes triseriatus. Genomic changes were not detected during transovarial passage of the virus through two generations of mosquitoes, nor were changes detected in the genomes of virus isolated from suckling mice that had been fed upon by second generation transovarially-infected mosquitoes. These results demonstrate that despite the well-documented phenomena of rapid nucleotide change in RNA virus genomes under various conditions, the La Crosse virus genome can remain stable during transovarial transmission in the insect host and during transfer between the insect and vertebrate hosts. The evolutionary implications of these results are discussed.

Aedes↗

PubMed1989

Asynchronous mixed infection of Culicoides variipennis with bluetongue virus serotypes 10 and 17.

Culicoides variipennis (Diptera: Ceratopogonidae) the primary vector of bluetongue virus (BTV) in the U.S.A. were asynchronously mixedly infected with two BTV serotypes (BTV-10 and BTV-17); flies first ingested a blood meal that contained BTV-17 and 1, 3, 5, 7, and 9 days later selected flies ingested a second blood meal that contained BTV-10. Control flies ingested each parental virus separately, or both viruses simultaneously, in a single blood meal. Electrophoretic analysis of progeny virus clones indicated that superinfection with BTV-10 occurred when the flies ingested the second virus 1, 3 and 5 days post-initial infection. Parental BTV-17 and reassortant virus clones were isolated from these flies, but parental BTV-10 virus was not isolated from any flies. Reassortant clone frequencies were 67%, 71% and 17% when superinfection occurred on days 1, 3 and 5 after initial infection, respectively, as compared to 48% for simultaneously infected flies. Only parental BTV-17 clones were isolated from flies that ingested the second virus on days 7 and 9 after initial BTV-17 infection. The results indicated that interference to superinfection occurred in C. variipennis by 5 days and flies were refractory to superinfection by 7 days post-initial infection. Analysis of segregation of the parental origin of genome segments in the reassortant clones indicated selection against most segments of BTV-10 parental origin. This occurred both in individual flies and in individual groups. The fact that C. variipennis readily fed on a second blood meal and their ability to produce new viral genotypes suggested that these vectors are highly permissive hosts for evolution of BTV by genome reassortment.

Animals↗

PubMed1989

Detection of bluetongue virus antigens in Culicoides variipennis by enzyme immunoassay.

A solid-phase enzyme immunoassay (EIA) was developed to detect bluetongue (BT) virus antigens in infected cell cultures and in suspensions of infected Culicoides variipennis midges. The technique was equally sensitive for detecting the five U.S. BT virus serotypes (2, 10, 11, 13, and 17) in cell cultures. EIA reliably detected about 3.8 log10 median tissue culture infective doses per ml of BT virus in infected cell culture lysates. The EIA readily detected virus antigens in pools of midges infected with BT serotypes 2, 10, 11, 13, and 17 and contained 2.3 to 4.8 log10 median tissue culture infective doses per ml of BT virus. The technique was sensitive enough to detect a single infected midge in a pool with 99 noninfected midges. The EIA may be a sensitive and rapid alternative to virus isolation for surveillance of BT viruses in vector populations.

Animals↗