scholarly journals Lack of involvement of haemocytes in the establishment and spread of infection in Spodoptera frugiperda larvae infected with the baculovirus Autographa californica M nucleopolyhedrovirus by intrahaemocoelic injection

2002 ◽  
Vol 83 (7) ◽  
pp. 1565-1572 ◽  
Author(s):  
Thomas E. Clarke ◽  
Rollie J. Clem
Keyword(s):  
Spodoptera Frugiperda ◽  
Trichoplusia Ni ◽  
Fluorescent Protein ◽  
Fat Body ◽  
Autographa Californica ◽  
Green Fluorescent Protein Gene ◽  
Primary Role ◽  
Late Instar ◽  
Noctuid Species ◽  
Green Fluorescent

It is thought that insect haemocytes, or blood cells, play an important role in baculovirus pathogenesis by amplifying and helping to spread the infection within the insect. Here, infection is described of the lepidopteran noctuid species Spodoptera frugiperda with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV). Late instar S. frugiperda larvae were infected by intrahaemocoelic injection using a recombinant of AcMNPV expressing the enhanced green fluorescent protein gene to visualize infected cells. Approximately 1000-fold higher doses of injected virus were required to initiate infection in S. frugiperda larvae than in another permissive noctuid species, Trichoplusia ni. Infected S. frugiperda larvae survived twice as long as T. ni larvae and exhibited a slower build-up of virus in the haemolymph. In S. frugiperda, infection of fat body and epithelium was observed prior to significant infection of haemocytes, even though the virus was delivered by intrahaemocoelic injection. Expression of eGFP was first detected 12–18 h post-injection within the fat body and, by 24 h, infection had spread to the tracheal and body wall epithelium. In contrast, only 5% of haemocytes were infected at 24 h and the proportion of infected haemocytes increased slowly to only around 50% at 5 days post-infection, when most larval death occurred. Thus, in S. frugiperda, haemocytes do not appear to have a primary role in AcMNPV pathogenesis. This relative lack of infection of haemocytes may in part explain why S. frugiperda larvae are more resistant to AcMNPV infection than T. ni larvae.

Expression of EGFP-Spider Dragline Recombination Gene in BmN Cells Using piggyBac Transposon-Derived Vector

2011 ◽  
Vol 175-176 ◽  
pp. 116-120
Author(s):  
Jian Nan Wang ◽  
Hong Gen Yi
Keyword(s):  
Bombyx Mori ◽  
Reporter Gene ◽  
Trichoplusia Ni ◽  
Fluorescent Protein ◽  
Pcr Amplification ◽  
Green Fluorescent Protein Gene ◽  
Transformation System ◽  
Helper Plasmid ◽  
Bombyx Mori L ◽  
Green Fluorescent

A transformation system was developed for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni, and a helper plasmid. The transposon consists of the piggyBac inverted terminal repeats, the enhanced green fluorescent protein gene as the reporter gene and the spider dragline gene. A nonautonomous helper plasmid encodes the piggyBac transposase. The transformation system was cotransfected into BmN (Bombyx mori L. Nucleopolyhedrovirus) cells using lipofection. PCR amplification on cellular genomic DNA using specific primers showed that a fragment of reporter gene, the spider dragline derived gene and A3 promoter were successfully amplified respectively. Plasmids without being transpositioned were not assayed. Green fluorescence cells were observed at 48 hours after transfection and the fluorescence intensity increased at 72 hours after transfection.

scholarly journals Avidin expression does not increase speed of kill of Spodoptera litura by baculovirus

2003 ◽  
Vol 56 ◽  
pp. 194-200 ◽  
Author(s):  
L.N. Gatehouse ◽  
N.P. Markwick ◽  
J. Poulton ◽  
J.T. Christeller
Keyword(s):  
Spodoptera Frugiperda ◽  
Spodoptera Litura ◽  
Culture Medium ◽  
Fluorescent Protein ◽  
Insect Cell Culture ◽  
Autographa Californica ◽  
Green Fluorescence ◽  
Strong Fluorescence ◽  
Nuclear Polyhedrosis ◽  
Green Fluorescent

A disarmed (polhminus) Autographa californica nuclear polyhedrosis virusbased baculovirus expressing green fluorescent protein (GFP) on the strong late promotor p10 was constructed A second virus was made which also expressed the biotinbinding protein avidin from the strong late promotor polh Both viruses caused strong fluorescence in Spodoptera frugiperda cells in insect cell culture Sevendayold larvae of Spodoptera litura were inoculated by microinjection with GFP virus GFPavidin virus or tissue culture medium (control) Mortality growth rate and the development of green fluorescence were followed and after death the larvae were analysed for GFP and avidin content All virusinjected larvae developed strong fluorescence However no significant differences in growth or mortality were seen between the larvae injected with virus expressing and not expressing avidin Both GFP and avidin accumulated to high levels in the larvae The expression of avidin did not change the level of expression of GFP

Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs

2014 ◽  
Vol 16 (6) ◽  
pp. 674-683 ◽  
Author(s):  
Chao Qiu ◽  
Bin Cheng ◽  
Yunsheng Zhang ◽  
Rong Huang ◽  
Lanjie Liao ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Green Fluorescent

scholarly journals Hierarchy among Viral RNA (vRNA) Segments in Their Role in vRNA Incorporation into Influenza A Virions

2006 ◽  
Vol 80 (5) ◽  
pp. 2318-2325 ◽  
Author(s):  
Yukiko Muramoto ◽  
Ayato Takada ◽  
Ken Fujii ◽  
Takeshi Noda ◽  
Kiyoko Iwatsuki-Horimoto ◽  
...  
Keyword(s):  
Influenza A ◽  
Fluorescent Protein ◽  
Virus Assembly ◽  
Viral Rna ◽  
Green Fluorescent Protein Gene ◽  
Influenza A Viruses ◽  
Coding Regions ◽  
Virion Incorporation ◽  
Green Fluorescent ◽  
Efficient Viral Replication

ABSTRACT The genome of influenza A viruses comprises eight negative-strand RNA segments. Although all eight segments must be present in cells for efficient viral replication, the mechanism(s) by which these viral RNA (vRNA) segments are incorporated into virions is not fully understood. We recently found that sequences at both ends of the coding regions of the HA, NA, and NS vRNA segments of A/WSN/33 play important roles in the incorporation of these vRNAs into virions. In order to similarly identify the regions of the PB2, PB1, and PA vRNAs of this strain that are critical for their incorporation, we generated a series of mutant vRNAs that possessed the green fluorescent protein gene flanked by portions of the coding and noncoding regions of the respective segments. For all three polymerase segments, deletions at the ends of their coding regions decreased their virion incorporation efficiencies. More importantly, these regions not only affected the incorporation of the segment in which they reside, but were also important for the incorporation of other segments. This effect was most prominent with the PB2 vRNA. These findings suggest a hierarchy among vRNA segments for virion incorporation and may imply intersegment association of vRNAs during virus assembly.

scholarly journals Response of immunocompetent and immunosuppressed Spodoptera littoralis larvae to baculovirus infection

2006 ◽  
Vol 87 (8) ◽  
pp. 2217-2225 ◽  
Author(s):  
Hadassah Rivkin ◽  
Jeremy A. Kroemer ◽  
Alexander Bronshtein ◽  
Eduard Belausov ◽  
Bruce A. Webb ◽  
...  
Keyword(s):  
Immune System ◽  
Immune Responses ◽  
Spodoptera Littoralis ◽  
Fluorescent Protein ◽  
Oral Route ◽  
Green Fluorescent Protein Gene ◽  
Successful Infection ◽  
Baculovirus Infection ◽  
Budded Virus ◽  
Green Fluorescent

The Mediterranean lepidopteran pest Spodoptera littoralis is highly resistant to infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via the oral route, but highly sensitive to infection with budded virus (BV) via the intrahaemocoelic route. To study the fate of AcMNPV infection in S. littoralis, vHSGFP, an AcMNPV recombinant that expresses the reporter green fluorescent protein gene under the control of the Drosophila heat-shock promoter, and high-resolution fluorescence microscopy were utilized. S. littoralis fourth-instar larvae infected orally with vHSGFP showed melanization and encapsulation of virus-infected tracheoblast cells serving the midgut columnar cells. At 72 h post-infection, the viral foci were removed during the moult clearing the infection. Thus, oral infection was restricted by immune responses to the midgut and midgut-associated tracheal cells. By contrast, injection of BV into the haemocoel resulted in successful infection of tracheoblasts, followed by spread of the virus through the tracheal epidermis to other tissues. However, in contrast to fully permissive infections where tracheoblasts and haemocytes are equally susceptible to infection, a severe limitation to vHSGFP infection of haemocytes was observed. To investigate the resistance of S. littoralis haemocytes to BV infection with AcMNPV, the larval immune system was suppressed with the Chelonus inanitus polydnavirus or a putatively immunosuppressive polydnavirus gene, P-vank-1. Both treatments increased the susceptibility of S. littoralis larvae to AcMNPV. It is concluded that the resistance of S. littoralis to AcMNPV infection involves both humoral and cellular immune responses that act at the gut and haemocyte levels. The results also support the hypothesis that tracheolar cells mediate establishment of systemic baculovirus infections in lepidopteran larvae. The finding that polydnaviruses and their encoded genes synergize baculovirus infection also provides an approach to dissecting the responses of the lepidopteran immune system to viruses by using specific polydnavirus immunosuppressive genes.

Laser-induced gene expression in specific cells of transgenic zebrafish

Development ◽  
2000 ◽  
Vol 127 (9) ◽  
pp. 1953-1960 ◽  
Author(s):  
M.C. Halloran ◽  
M. Sato-Maeda ◽  
J.T. Warren ◽  
F. Su ◽  
Z. Lele ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Transgenic Zebrafish ◽  
Hsp70 Promoter ◽  
Expression Of Genes ◽  
Transgenic Lines ◽  
Transgenic Embryos ◽  
Green Fluorescent

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

A Novel Approach To Investigate the Uptake and Internalization of Escherichia coli O157:H7 in Spinach Cultivated in Soil and Hydroponic Medium†

2009 ◽  
Vol 72 (7) ◽  
pp. 1513-1520 ◽  
Author(s):  
MANAN SHARMA ◽  
DAVID T. INGRAM ◽  
JITENDRA R. PATEL ◽  
PATRICIA D. MILLNER ◽  
XIAOLIN WANG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Green Fluorescent Protein Gene ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Novel Approach ◽  
Efficient Uptake ◽  
Green Fluorescent

Internalization of Escherichia coli O157:H7 into spinach plants through root uptake is a potential route of contamination. ATn7-based plasmid vector was used to insert a green fluorescent protein gene into the attTn7 site in the E. coli chromosome. Three green fluorescent protein–labeled E. coli inocula were used: produce outbreak O157:H7 strains RM4407 and RM5279 (inoculum 1), ground beef outbreak O157:H7 strain 86-24h11 (inoculum 2), and commensal strain HS (inoculum 3). These strains were cultivated in fecal slurries and applied at ca. 103 or 107 CFU/g to pasteurized soils in which baby spinach seedlings were planted. No E. coli was recovered by spiral plating from surface-sanitized internal tissues of spinach plants on days 0, 7, 14, 21, and 28. Inoculum 1 survived at significantly higher populations (P < 0.05) in the soil than did inoculum 3 after 14, 21, and 28 days, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than are nonpathogenic E. coli isolates. Inoculum 2 applied at ca. 107 CFU/ml to hydroponic medium was consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 days (3.73 log CFU per shoot) and 21 days (4.35 log CFU per shoot). Fluorescent E. coli cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils. No internalized E. coli was microscopically observed in shoot tissue of plants grown in inoculated soil. These studies do not provide evidence for efficient uptake of E. coli O157:H7 from soil to internal plant tissue.

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