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Biomedical subjects

B J Beaty

Publications and source records attributed to B J Beaty.

At least 55 records · Page 3Linked to original sources

Mosquito feeding-induced enhancement of Cache Valley Virus (Bunyaviridae) infection in mice.

Cache-Valley (CV) virus, an arthropod-borne bunyavirus, recently has emerged as a significant veterinary pathogen causing infertility and congenital malformations in North American ruminants. To investigate the role of vector feeding on CV infection, adult mice were injected subcutaneously with CV, CV and vector thorax extract (a source of vector saliva), or CV into the site of intense, noninfected-mosquito feeding. Mice did not become infected after injection of CV or CV and vector saliva, nor did they produce antibodies to CV. However, injection of CV into sites of mosquito feeding resulted in viremia and production of anti-CV antibody by 2 wk after infection. This enhancement of CV infection resulted after feeding by Aedes triseriatus (Say), Ae. aegypti (L.), or Culex pipiens (L.). Enhancement occurred when injection was delayed up to 4 h after mosquito feeding, but it was not observed when virus injection was performed at a site distant from mosquito feeding. These results indicate that arbovirus infection may be enhanced by mosquito-vertebrate host interactions and that replication of arboviruses in arthropod vectors may not be responsible for increased virulence of infections mediated by infected arthropods. Enhanced CV infection in pregnant mice did not result in infertility or malformed pups, indicating that the mouse is not a suitable model to study CV-induced malformations.

Aedes↗

La Crosse virus infection of Aedes triseriatus (Diptera: Culicidae) ovaries before dissemination of virus from the midgut.

La Crosse (LAC) virus is efficiently transmitted transovarially by the mosquito Aedes triseriatus (Say). To determine the time course and tropisms of LAC virus infection of ovaries, immunofluorescent antibody staining, in situ hybridization, and reverse transcription-polymerase chain reaction techniques were used to detect viral antigen and RNA in the ovaries. LAC virus was detected in the ovaries (presumably in calyx tissues) by all 3 assays at day 2 after infection and before dissemination from the midgut on day 6. Apparently, ovaries can become infected by mechanisms other than by dissemination of virus from a midgut infection. By days 8-14 after infection, virus analytes became detectable in many tissues within the ovary including follicular epithelium, oocytes, nurse cells, and calyx, reflecting the remarkable host parasite relationship between LAC virus and its mosquito vector.

Aedes↗

Characterization of La Crosse virus RNA in autopsied central nervous system tissues.

A reverse transcription-PCR (RT-PCR) technique was used to detect La Crosse (LAC) virus RNA in the central nervous system (CNS) tissues of two patients who died of LAC encephalitis in 1960 and 1978. Viral RNA was readily detected by RT-PCR although the tissues had been stored frozen for up to 37 years. LAC virus was detected in the cerebral cortex but not in other CNS tissues. RT-PCR allowed detection of replicative forms of the virus, indicating that the virus was actively replicating in the specific CNS tissues. The small (S) RNA segments of the viruses from the CNS samples were demonstrated to be genetically similar by single-strand conformation polymorphism analyses. These S RNA segments were then sequenced; only two base changes were demonstrated between the 1960 and the 1978 samples, suggesting that LAC virus is genetically stable in areas of endemicity. The RT-PCR analyses of analyte directly from CNS tissues allows study of the virus without passage in cell culture.

Animals↗

Gene expression and regulation from the p7 promoter of Aedes densonucleosis virus.

The nonstructural proteins NS1 and NS2 are thought to be expressed from the p7 promoter of Aedes densonucleosis virus (AeDNV). To study gene expression from the p7 promoter, eight different plasmids were constructed by fusing beta-galactosidase or beta-glucuronidase into the genome so that the reporter gene was in different open reading frames and under the transcriptional control of the p7 promoter. After transfection into C6/36 Aedes albopictus cells, constructs generated comparable amounts of RNA, but only the NS1 and NS2 fusion constructs produced appreciable levels of active enzyme. NS1 and NS2 fusion constructs contained wild-type AeDNV sequences from the p7 promoter downstream to nucleotide 458. The remaining constructs, with the exception of p7GUS.rf3, lacked some or all of these necessary sequences and inefficiently produced protein. These data suggest that sequences downstream of the p7 promoter play a role in translational regulation of gene expression from the p7 promoter of AeDNV.

Aedes↗

Molecular and genetic ecotoxicologic approaches to aquatic environmental bioreporting.

Molecular and population genetic ecotoxicologic approaches are being developed for the utilization of arthropods as bioreporters of heavy metal mixtures in the environment. The explosion of knowledge in molecular biology, molecular genetics, and biotechnology provides an unparalleled opportunity to use arthropods as bioreporter organisms. Interspecific differences in aquatic arthropod populations have been previously demonstrated in response to heavy metal insult in the Arkansas River (AR) California Gulch Superfund site (CGSS). Population genetic analyses were conducted on the mayfly Baetis tricaudatus. Genetic polymorphisms were detected in polymerase chain reaction amplified 16S mitochondrial rDNA (a selectively neutral gene) of B tricaudatus using single-strand conformation polymorphism analysis. Genetic differences may have resulted from impediments to gene flow in the population caused by mortality arising from exposure to heavy metal mixture pollution. In laboratory studies a candidate metal-responsive mucinlike gene, which is metal and dose specific, has been identified in Chironomus tentans and other potential AR-CGSS bioreporter species. Population genetic analyses using the mucinlike gene may provide insight into the role of this selectable gene in determining the breeding structure of B. tricaudatus in the AR-CGSS and may provide mechanistic insight into determinants of aquatic arthropod response to heavy metal insult. Metal-responsive (MR) genes and regulatory sequences are being isolated, characterized, and assayed for differential gene expression in response to heavy metal mixture pollution in the AR-CGSS. Identified promoter sequences can then be engineered into previously developed MR constructs to provide sensitive in vitro assays for environmental bioreporting of heavy metal mixtures. The results of the population genetic studies are being entered into an AR geographic information system that contains substantial biological, chemical, and geophysical information. Integrated spatial, structural, and temporal analyses of these parameters will provide invaluable information concerning environmental determinants that restrict or promote gene flow in bioreporter populations.

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Effect of La Crosse virus infection on overwintering of Aedes triseriatus.

The effect of La Crosse (LAC) virus infection on Aedes triseriatus overwintering success was determined. Eggs from LAC virus transovarially infected (LAC TOT+) and uninfected (LAC TOT-) Ae. triseriatus colonies were induced into diapause, held in natural conditions, and returned to the laboratory at predetermined times for assay of diapause, mortality, and filial infection rates, and to examine viral transcription and replication during diapause. Embryos from the LAC TOT+ colony exhibited greater cumulative mortality (16.7%) than the LAC TOT- eggs (7.3%) throughout the overwintering periods. The increased mortality rate in LAC TOT+ eggs corresponded with a decrease in filial infection rates. Eggs from the LAC TOT+ colony terminated diapause more readily than the LAC TOT- colony. An RNA strand-specific reverse transcriptase-polymerase chain reaction technique was used to monitor viral transcription and replication in mosquito eggs during overwintering, and to compare viral replication in diapausing and nondiapausing embryos. Viral messenger and replicative form RNA were present in eggs in all sample periods, suggesting that some virus replication occurred during diapause.

Aedes↗

Short report: Diapause, transovarial transmission, and filial infection rates in geographic strains of La Crosse virus-infected Aedes triseriatus.

La Crosse (LAC) virus is transmitted horizontally to vertebrates and vertically to progeny by Aedes triseriatus mosquitoes, and in northern midwestern states, this virus overwinters in diapausing eggs of the vector. In Florida, the vector remains active throughout the year and does not diapause. To determine if there is an association between diapause and vertical transmission efficiency of LAC virus, transovarial transmission (TOT), and filial infection (FI) rates were determined for geographic strains of Ae. triseriatus. The TOT rates were not significantly different for Ae. triseriatus originating from Florida (78%) and those from Wisconsin (85%). The FI rates did differ significantly between the two groups (33% and 45%, respectively, for the Florida and Wisconsin mosquitoes). Furthermore, a line of mosquitoes was selected from a Wisconsin colony that had a reduced diapause phenotype (the AD- strain). While this strain displayed TOT rates that were the same as the other Wisconsin mosquitoes (85%), the FI rates were significantly lower (34%), indicating a reduction in TOT efficiency. The role of vertical transmission capacity in LAC virus endemicity remains to be determined.

Aedes↗

Engineered resistance in Aedes aegypti to a West African and a South American strain of yellow fever virus.

Double subgenomic Sindbis (dsSIN) viruses were engineered to transduce mosquito cells with antisense RNA derived either from the premembrane (prM) or polymerase (NS5) coding regions of the 17D vaccine strain of yellow fever virus (YFV). Aedes albopictus C6/36 cells were infected at high multiplicities of infection (MOI) with each dsSIN virus. Forty-eight hours later, the transduced cells were challenged with an MOI of 0.1 of the Asibi strain of YFV. At 72-hr postchallenge, the cells were assayed by immunofluorescence for the presence of YFV antigen. Cells transduced with prM or NS5 antisense RNAs derived from the YFV genome displayed no YFV-specific antigens. In contrast, cells infected with control dsSIN viruses that expressed no antisense RNA or dengue virus-derived antisense RNAs were permissive for the challenge virus. To analyze resistance in the mosquito, five log10 50% tissue culture infective doses (TCID50) of each dsSIN virus and three log10TCID50 of either a West African (BA-55) or South American (1899/81) strain of wild-type YFV were coinoculated into Ae. aegypti. Mosquitoes transduced with effector RNAs targeting the prM or NS5 gene regions did not transmit West African YFV and poorly transmitted the South American strain of YFV.

Aedes↗

Quantitative genetics of vector competence for dengue-2 virus in Aedes aegypti.

A quantitative genetic study of the ability of Aedes aegypti to propagate dengue-2 (DEN-2) virus in the midgut and in a disseminated infection in the head was conducted with a standard half-sib breeding design. Aedes aegypti aegypti and A. aegypti formosus differ markedly in oral susceptibility to DEN-2 virus. Mosquitoes were orally infected and, after an extrinsic incubation period of 14 days, virus titer (by tissue culture infectious dose, 50% endpoint) was determined in the midgut (MT) and head (HT). Body size as measured by wing length was not significantly different between infected and uninfected mosquitoes and was not correlated with MT or HT The heritability for MT in both subspecies was 0.41 and was 0.39 for HT in A. aegypti formosus. In A. aegypti aegypti, HT appeared to be controlled by dominant alleles. The MT was not correlated with HT nor did MT determine whether virus disseminated out of the midgut. These results suggest that it is the barriers to infection and dissemination, independent of virus titer, that determine vector competence for DEN-2 virus.

Aedes↗

Detection of expressed chloramphenicol acetyltransferase in the saliva of Culex pipiens mosquitoes.

Mosquito salivary glands play an important role in the transmission of arthropod-borne pathogens. The ability to express genes in mosquitoes would be a powerful approach to characterize salivary gland genes, and to reveal important vector determinants of pathogen transmission. Here we report the use of a double subgenomic Sindbis (dsSIN) virus, designated TE/3'2J/CAT, and a packaged Sindbis replicon virus, designated rep5/CAT/26S, to express chloramphenicol acetyltransferase (CAT) protein in the salivary glands and saliva of transduced female Culex pipiens pipiens. Indirect immunofluorescence analysis revealed that salivary glands of these mosquitoes infected with either TE/3'2J/CAT or rep5/CAT/26S virus (4 or 6 days post-infection (p.i.)) were positive for both SIN E1 antigen and CAT protein. Saliva collected from mosquitoes transduced with TE/3'2J/CAT virus contained a unique 25 kDa protein that corresponded to the size of CAT protein. Additionally, CAT activity assays revealed that saliva collected from mosquitoes transduced with either TE/3'2J/CAT or rep5/CAT/26S virus could contain greater than 5.0 x 10(-5) units of CAT enzyme (3.0 x 10(6) CAT trimers).

Animals↗

Analysis of LaCrosse virus S mRNA 5' termini in infected mosquito cells and Aedes triseriatus mosquitoes.

Nucleotide sequences were determined for the 5' termini of La Crosse virus (LAC) S segment mRNA from persistently infected mosquito cell cultures (C6/36 from Aedes albopictus) and embryos (Aedes triseriatus). LAC primes transcription of its mRNA with "scavenged" 5' caps and adjacent oligonucleotides from host mRNAs, and these non-virus-encoded 5'-terminal extensions are heterogeneous in infected mammalian cells. The nature of mosquito host-derived primers has not been previously investigated. During early C6/36 cell infection, LAC mRNA 5'-terminal sequences were heterogeneous, but variability decreased as infection persisted. One predominant sequence, 5' CCACTCGCCACT (sequence 1), was observed throughout C6/36 cell infection but was more prevalent after 15 days postinfection. This LAC mRNA 5'-terminal sequence comprised 81% of the scavenged host oligonucleotides from vertically infected A. triseriatus eggs during embryogenesis. As these embryos progressed in the dormant overwintering stage (diapause), the predominant scavenged sequence became 5' AGGAAAAGATGGT (sequence 2), and sequence 1 became less prevalent. As the eggs emerged from diapause, the LAC mRNA 5' termini were more variable; 33% had sequence 1, and the remainder were heterogeneous. In post-diapausing eggs, 100% of viral mRNAs had sequence 1 at their 5' termini. Molecular analyses thus revealed continuous but selective LAC cap scavenging during persistent C6/36 cell infection and during embryogenesis and diapause in A. triseriatus eggs. The variety of host-derived sequences was limited in both biosynthetically active (embryonating) and dormant (diapausing) eggs.

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Rapid characterization of genetic diversity among twelve dengue-2 virus isolates by single-strand conformation polymorphism analysis.

Single-strand conformation polymorphism (SSCP) analysis was used to characterize genetic polymorphisms among 12 isolates of dengue-2 virus, which were previously genetically characterized by RNase T1 oligonucleotide mapping and by sequencing the viral envelope (E) gene. Specific cDNA fragments from the dengue-2 isolates were amplified by the reverse transcriptase-polymerase chain reaction. The viral E, premembrane (prM), and nonstructural 5 (NS5) gene cDNAs of 291 basepairs (bp), 291 bp, and 201 bp, respectively, were denatured, rapidly chilled to promote intrastrand reassociation, electrophoretically separated on nondenaturing polyacrylamide gels, and SSCP patterns were observed by silver staining. The SSCP analysis revealed polymorphisms among a number of dengue-2 isolates from the same topotype, and these were markedly different between isolates of different topotype (distinct genetic group). Comparison of nucleotide sequence and SSCP analyses of the 291-bp E cDNA demonstrated that virus isolates that produced identical SSCP patterns contained 0-7 nucleotide substitutions, whereas isolates that showed different SSCP patterns contained 4-25 nucleotide substitutions. Positive predictive value and negative predictive value as measures of certainty for predicting identical and different sequences were 26% and 100%, respectively. The SSCP patterns of the 12 dengue-2 isolates suggested greater genetic variation in the prM gene region than in either the E or NS5 gene regions. The SSCP analyses should allow easy, sensitive, and rapid screening of dengue viruses isolates and the assessment of variation at a number of sites in the virus genome. Additionally, SSCP screening of dengue-2 virus for genetic variability may reveal the introduction of new viral genotypes in a given geographic area. These genetic variants of the virus could serve as markers of the epidemic potential of the virus strain.

DNA, Complementary↗

Genetically engineered resistance to dengue-2 virus transmission in mosquitoes.

The control of arthropod-borne virus diseases such as dengue may ultimately require the genetic manipulation of mosquito vectors to disrupt virus transmission to human populations. To reduce the ability of mosquitoes to transmit dengue viruses, a recombinant Sindbis virus was used to transduce female Aedes aegypti with a 567-base antisense RNA targeted to the premembrane coding region of dengue type 2 (DEN-2) virus. The transduced mosquitoes were unable to support replication of DEN-2 virus in their salivary glands and therefore were not able to transmit the virus.

Aedes↗

Molecularly engineered resistance to California serogroup virus replication in mosquito cells and mosquitoes.

Introduction of genetic elements derived from a viral pathogen's genome may be used to reduce the vectorial capacity of mosquitoes for that virus. A double subgenomic Sindbis virus expression system was utilized to transcribe sequences of LaCrosse (LAC) virus small (S) or medium (M) segment RNA in sense or antisense orientation; wild-type Sindbis and LaCrosse viruses have single-stranded RNA genomes, the former being positive sense and the latter being negative sense. Recombinant viruses were generated and used to infect Aedes albopictus (C6/36) mosquito cells, which were challenged with wild-type LAC virus and then assayed for LAC virus replication. Several recombinant viruses containing portions of the LAC S segment were capable of inducing varying degrees of interference to the challenge virus. Cells infected with TE/3'2J/ANTI-S virus, expressing full-length negative-sense S RNA of LAC virus, yielded 3-6 log10TCID50 (tissue culture 50% infective dose) less LAC virus per ml than did cells infected with a double subgenomic sindbis virus containing no LAC insert. When C6/36 cells infected with TE/3'2J/ANTI-S were challenged with closely related heterologous bunyaviruses, a similar inhibitory effect was seen. Adult Ae. triseriatus mosquitoes infected with TE/3'2J/ANTI-S were also resistant to challenge by LAC virus. Organs that were productively infected by the double subgenomic Sindbis virus expressing the LAC anti-S sequences demonstrated little LAC virus or antigen. These studies indicate that expression of carefully selected antiviral sequences derived from the pathogen's genome may result in efficacious molecular viral interference in mosquito cells and, more importantly, in mosquitoes.

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Analysis of La Crosse virus S-segment RNA and its positive-sense transcripts in persistently infected mosquito tissues.

La Crosse (LAC) virus is an important cause of pediatric arboviral encephalitis in the United States. LAC virus is biologically transmitted by the mosquito Aedes triseriatus, and, like other arthropod-borne viruses, it establishes a persistent, nonpathogenic infection in its vector following oral infection. To investigate LAC virus persistent infection of mosquitoes, a reverse transcription-PCR assay was developed for the amplification of LAC virus negative-sense small (S) genome RNA segment, its full-length complement, and its mRNA transcript for qualitative analysis of transcription and replication in persistently infected mosquito tissues. RNAs were assayed from midguts removed at predetermined times after infection with a LAC virus-containing blood meal. LAC virus genome was detected almost uniformly in midguts at days 3 to 28 postinfection (p.i.) and, as the time p.i. progressed, in more of the samples than either mRNA or viral cRNA (vcRNA). Thus, persistent LAC virus infection of A. triseriatus midguts was correlated with a reduction in detectable viral mRNA and vcRNA. The assay was also used for analysis of virus-specified RNA in both quiescent and biosynthetically active mosquito ovaries. Viral replication decreased, as indicated by the absence of viral mRNA and vcRNA, in the ovaries of mosquitoes that did not receive further blood meals after their original oral infection. Viral replication increased in ovaries of mosquitoes that took an additional blood meal 30 days p.i. and was continuous in mosquitoes that took multiple meals to stimulate oogenesis. Thus, virus replication in persistently infected mosquito ovaries was dependent on host cell biosynthetic status.

Aedes↗

Pathogen-derived resistance to dengue type 2 virus in mosquito cells by expression of the premembrane coding region of the viral genome.

The full-length premembrane (prM) coding region of the dengue virus type 2 (DEN-2; Jamaica) genome was expressed in C6/36 (Aedes albopictus) cells in either the sense or the antisense orientation from a double subgenomic Sindbis (dsSIN) virus. Northern (RNA) blot analysis confirmed the expression of sense or antisense DEN-2 prM RNA in infected C6/36 cells. PrM protein was demonstrated in cells infected with dsSIN virus expressing DEN-2 sense RNAs by an immunofluorescence assay. C6/36 cells were infected with each dsSIN virus at a multiplicity of infection (MOI) of 50 and challenged 48 h later with DEN-2 virus at an MOI of 0.1. Whereas C6/36 cells infected with a control of dsSIN virus supported high levels of DEN-2 replication, C6/36 cells infected with the dsSIN virus expressing prM antisense RNA were completely resistant to DEN-2 challenge. Cells expressing prM protein or untranslatable prM sense RNA also were resistant to DEN-2 challenge. Cells expressing prM protein demonstrated some breakthrough of DEN-2 virus when challenged at an MOI of 10. However, expressed untranslatable sense prM RNA conferred complete protection to challenge at the high MOI.

Aedes↗

Green fluorescent protein expressed in living mosquitoes--without the requirement of transformation.

Mosquitoes transmit viruses, protozoa and nematodes that are major causes of morbidity and mortality in humans. Details of arthropod anatomy and development, and the replication and development of pathogens in the arthropod vector, have relied upon examination of dissected or histologically processed material. We constructed a double-subgenomic Sindbis (dsSIN) virus expressing green fluorescent protein to demonstrate the potential of this protein for studying pathogen development in living arthropods. We were able to observe dissemination of virus, and furthermore, it was possible to observe components of the nervous system of mosquito larvae in extraordinary detail and record this on video tape. Although green fluorescent protein has been used as a reporter gene in a number of organisms, expression has relied upon transformation of cells or embryos. Transformation technology has limited applicability, thus we have described an alternative system that, due to the broad host range and viral tropisms of dsSIN viruses, may be useful to scientists in a range of disciplines. Green fluorescent protein may also provide a non-lethal selection method for use in transgenic arthropod research.

Animals↗

The expression of chloramphenicol acetyltransferase in mosquitoes and mosquito cells using a packaged Sindbis replicon system.

Sindbis (SIN) replicon virus was used to express chloramphenicol acetyltransferase (CAT) in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes. RNA transcribed in vitro from a SIN replicon plasmid (pSINrep5/CAT) and from SIN virus helper constructs (pDH-EB or pDH(26S)5'SIN) was coelectroporated into BHK-21 cells to generate replicon viruses, designated rep5/CAT/EB and rep5/CAT/26S. C6/36 cells infected with rep5/CAT/EB or rep5/CAT/26S virus at a multiplicity of infection of 3, expressed 3.8 x 10(6) and 6.0 x 10(6) CAT trimers per cell, respectively, at 2 days postinfection (pi). Both viruses attained peak titers by Day 2 pi. Adult female A. triseriatus mosquitoes were intrathoracically inoculated with 7 x 10(4) IFU rep5/CAT/EB or 1 x 10(5) IFU rep5/CAT/26S virus. Virus titers remained at approximately 10(5) IFU/ml through Day 2 pi and decreased roughly 1 log by Day 10 pi. CAT enzyme activity was detected 2 days pi (rep5/CAT/EB, 1.49 x 10(-4) units CAT/10 micrograms protein; rep5/CAT/26S, 2.03 x 10(-5) units CAT/10 micrograms protein) and remained near these levels through Day 10 pi. CAT was detected in the head, salivary glands, midgut, and ovaries of inoculated mosquitoes by indirect immunofluorescence or CAT activity assays. These results suggest that packaged replicon viruses can be useful for expression of heterologous genes in mosquito cells and whole mosquitoes.

Aedes↗