scholarly journals Trehalose improves PPR vaccine virus stability in diluent

2020 ◽  
Vol 17 (2) ◽  
Author(s):  
N. Mohanto ◽  
A. Khatun ◽  
J. A. Begum ◽  
M. M. Parvin ◽  
M. S. I. Siddiqui ◽  
...  
Keyword(s):  
Ambient Temperature ◽  
Vero Cells ◽  
Vaccine Virus ◽  
Virus Infectivity ◽  
Different Temperatures ◽  
Ppr Virus ◽  
Pcr Targeting ◽  
Infectivity Titer

Background: Specialized freeze-drying process is being used in the field for different thermostable vaccine preparation worldwide. The thermostability remains only in undiluted conditions. If dilution is made at the morning and used for the whole day, the vaccine efficacy is compromised at high ambient temperature. In this study, trehalose based specialized vaccine diluent was used to improve the stability of Peste des Petits Ruminants (PPR) vaccine in diluted condition. Methods: The available PPR vaccine was reconstituted with conventional diluent and with trehalose based test diluent. The diluted vaccine was kept at ambient temperature without maintaining any cool chain. Stability of diluted vaccine virus was further assessed in vivo and in vitro at different temperatures. Goats were vaccinated and Vero cells were infected with reconstituted vaccines and were assessed at 0, 3, 6, 9 and 24 hours post dilution. Antibody titer was measured and virus infectivity titer was determined in both cultured cell lysate and supernatant. The presence of the virus particles in Vero cell was confirmed by standard RT-PCR targeting Fusion (F) gene of PPR virus. Results: In vivo results revealed that the number of goats possessed antibodies to PPR virus was higher in trehalose based vaccine formulation than the conventional PBS based diluent. Reconstituted vaccine virus (using PBS and trehalose diluent) infected Vero cells produced 70-80% cytopathic effect (CPE) in 5th days of post infection. Both diluents produced and maintained infectivity titer from log10 TCID50 5.5 to log10 TCID50 3.6, until the use of vaccines incubated for 9 hours after dilution. On the other hand, at 24 hours of post dilution only trehalose formulated vaccine produced log10 TCID502.5 whereas no infectivity titer was observed at the same time using conventional one. Conclusion: The present study suggests that trehalose preserves the quality of reconstituted vaccine in terms of infectivity titers. Trehalose can be a diluent of choice for reconstitution of PPR vaccine in field.

scholarly journals Construction of an Excisable Bacterial Artificial Chromosome Containing a Full-Length Infectious Clone of Herpes Simplex Virus Type 1: Viruses Reconstituted from the Clone Exhibit Wild-Type Properties In Vitro and In Vivo

2003 ◽  
Vol 77 (2) ◽  
pp. 1382-1391 ◽  
Author(s):  
Michiko Tanaka ◽  
Hiroyuki Kagawa ◽  
Yuji Yamanashi ◽  
Tetsutaro Sata ◽  
Yasushi Kawaguchi
Keyword(s):  
Vero Cells ◽  
Infectious Molecular Clone ◽  
Wild Type ◽  
Molecular Clone ◽  
Viral Genes ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT In recent years, several laboratories have reported on the cloning of herpes simplex virus type 1 (HSV-1) genomes as bacterial artificial chromosomes (BACs) in Escherichia coli and on procedures to manipulate these genomes by using the bacterial recombination machinery. However, the HSV-BACs reported so far are either replication incompetent or infectious, with a deletion of one or more viral genes due to the BAC vector insertion. For use as a multipurpose clone in research on HSV-1, we attempted to generate infectious HSV-BACs containing the full genome of HSV-1 without any loss of viral genes. Our results were as follows. (i) E. coli (YEbac102) harboring the full-length HSV-1 genome (pYEbac102) in which a BAC flanked by loxP sites was inserted into the intergenic region between UL3 and UL4 was constructed. (ii) pYEbac102 was an infectious molecular clone, given that its transfection into rabbit skin cells resulted in production of infectious virus (YK304). (iii) The BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus YK304 by coinfection of Vero cells with YK304 and a recombinant adenovirus, AxCANCre, expressing Cre recombinase. (iv) As far as was examined, the reconstituted viruses from pYEbac102 could not be phenotypically differentiated from wild-type viruses in vitro and in vivo. Thus, the viruses grew as well in Vero cells as did the wild-type virus and exhibited wild-type virulence in mice on intracerebral inoculation. (v) The infectious molecular clone pYEbac102 is in fact useful for mutagenesis of the HSV-1 genome by bacterial genetics, and a recombinant virus carrying amino acid substitutions in both copies of the α0 gene was generated. pYEbac102 will have multiple applications to the rapid generation of genetically engineered HSV-1 recombinants in basic research into HSV-1 and in the development of HSV vectors in human therapy.

Elucidation of the full genetic information of Japanese rubella vaccines and the genetic changes associated with in vitro and in vivo vaccine virus phenotypes

Vaccine ◽  
2011 ◽  
Vol 29 (10) ◽  
pp. 1863-1873 ◽  
Author(s):  
Noriyuki Otsuki ◽  
Hitoshi Abo ◽  
Toru Kubota ◽  
Yoshio Mori ◽  
Yukiko Umino ◽  
...  
Keyword(s):  
Genetic Information ◽  
Vaccine Virus ◽  
Genetic Changes

scholarly journals Antiplasmodial and Cytotoxic Cytochalasins from an Endophytic Fungus, Nemania sp. UM10M, Isolated from a Diseased Torreya taxifolia Leaf

Molecules ◽  
2019 ◽  
Vol 24 (4) ◽  
pp. 777 ◽  
Author(s):  
Mallika Kumarihamy ◽  
Daneel Ferreira ◽  
Edward Croom ◽  
Rajnish Sahu ◽  
Babu Tekwani ◽  
...  
Keyword(s):  
Mouse Model ◽  
Solid Tumor ◽  
Endophytic Fungus ◽  
Antimalarial Activity ◽  
Vero Cells ◽  
Antiplasmodial Activity ◽  
Etoac Extract ◽  
Bioassay Guided Fractionation

Bioassay-guided fractionation of an EtOAc extract of the broth of the endophytic fungus Nemania sp. UM10M (Xylariaceae) isolated from a diseased Torreya taxifolia leaf afforded three known cytochalasins, 19,20-epoxycytochalasins C (1) and D (2), and 18-deoxy-19,20-epoxy-cytochalasin C (3). All three compounds showed potent in vitro antiplasmodial activity and phytotoxicity with no cytotoxicity to Vero cells. These compounds exhibited moderate to weak cytotoxicity to some of the cell lines of a panel of solid tumor (SK-MEL, KB, BT-549, and SK-OV-3) and kidney epithelial cells (LLC-PK11). Evaluation of in vivo antimalarial activity of 19,20-epoxycytochalasin C (1) in a mouse model at 100 mg/kg dose showed that this compound had weak suppressive antiplasmodial activity and was toxic to animals.

scholarly journals Differential Expression of the p44 Gene Family in the Agent of Human Granulocytic Ehrlichiosis

2002 ◽  
Vol 70 (9) ◽  
pp. 5295-5298 ◽  
Author(s):  
Jacob W. IJdo ◽  
Caiyun Wu ◽  
Sam R. Telford ◽  
Erol Fikrig
Keyword(s):  
Differential Expression ◽  
Antigenic Variation ◽  
Hypervariable Region ◽  
Differentially Expressed ◽  
Granulocytic Ehrlichiosis ◽  
Reverse Transcription Pcr ◽  
Human Granulocytic Ehrlichiosis ◽  
Pcr Targeting

ABSTRACT Using reverse transcription-PCR targeting of the p44 genes of the agent of human granulocytic ehrlichiosis (HGE) with primers flanking the hypervariable region, we show differential expression in a murine model of HGE infection and during tick transmission. The p44 genes were differentially expressed in salivary glands of infected nymphal ticks removed during transmission feeding but not in nonfeeding infected ticks. Similarly, the p44 genes were differentially expressed in infected C3H mice, in SCID mice, and in cultured HGE bacteria. Thus, differential p44 expression exists in vivo and in vitro and could provide a basis for antigenic variation.

Europium-Doped Hydroxyapatite: Influence of Excitation Wavelength on the Eu3+ Luminescence in the Hydroxyapatite

2015 ◽  
Vol 820 ◽  
pp. 335-340 ◽  
Author(s):  
Flávia R.O. Silva ◽  
Nelson B. de Lima ◽  
Deiby S. Gouveia ◽  
Nildemar A.M. Ferreira ◽  
Valter Ussui ◽  
...  
Keyword(s):  
Precipitation Method ◽  
Excitation Wavelength ◽  
Careful Evaluation ◽  
Lanthanide Ion ◽  
Site Occupation ◽  
Different Temperatures ◽  
Co Precipitation ◽  
The Eu

Hydroxyapatite (HA) doped with europium (HAEu) offers the advantage of making the hydroxyapatite a fluorescent biomarker, allowing their imaging through emissionin vivoandin vitrotests. Several authors had been based their studies about europium site occupation (CaI and CaII) in hydroxyapatite by the lanthanide ion luminescence, verifying the influence of the method of synthesis and concentration of the dopant ion. In this study HA nanoparticles doped with 1.4 mol% of trivalent europium were synthesized by co-precipitation method and thermal treated at different temperatures (600°C and 1200°C). A careful evaluation of the influence of the excitation wavelength of europium luminescence in the HAEu was performed and it has been verified that both the characteristics transitions of europium, at CaI and CaII sites, and the luminescent intensity are dependent on the excitation wavelength. The non-observance of this fact can lead to erroneous conclusions about the site occupation of europium in hydroxyapatites.

scholarly journals Live-Attenuated Measles Virus Vaccine Targets Dendritic Cells and Macrophages in Muscle of Nonhuman Primates

2014 ◽  
Vol 89 (4) ◽  
pp. 2192-2200 ◽  
Author(s):  
Linda J. Rennick ◽  
Rory D. de Vries ◽  
Thomas J. Carsillo ◽  
Ken Lemon ◽  
Geert van Amerongen ◽  
...  
Keyword(s):  
Measles Virus ◽  
Nonhuman Primates ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Vaccine Virus ◽  
Target Cells ◽  
Recombinant Viruses ◽  
Green Fluorescent

ABSTRACTAlthough live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replicationin vivoare still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb (EZ), allowing recovery of recombinant (r)MVEZ. Three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMVEZEGFP(1), rMVEZEGFP(3), and rMVEZEGFP(6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were comparedin vitroby growth curves, which indicated that rMVEZEGFP(1) was overattenuated. Intratracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMVEZEGFP(1) and rMVEZEGFP(6) did not induce satisfactory serum antibody responses, whereas bothin vitroandin vivorMVEZEGFP(3) was functionally equivalent to the commercial MVEZ-containing vaccine. Intramuscular vaccination of macaques with rMVEZEGFP(3) resulted in the identification of EGFP+cells in the muscle at days 3, 5, and 7 postvaccination. Phenotypic characterization of these cells demonstrated that muscle cells were not infected and that dendritic cells and macrophages were the predominant target cells of live-attenuated MV.IMPORTANCEEven though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the primary cells in which the virus replicatesin vivo. This is vital information given the push to move toward needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the nonrecombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in nonhuman primates. The virus primarily replicated in professional antigen-presenting cells, which may explain why this LAV is so immunogenic and efficacious.

scholarly journals A Novel Methyltransferase Methylates Cucumber Mosaic Virus 1a Protein and Promotes Systemic Spread

2008 ◽  
Vol 82 (10) ◽  
pp. 4823-4833 ◽  
Author(s):  
Min Jung Kim ◽  
Sung Un Huh ◽  
Byung-Kook Ham ◽  
Kyung-Hee Paek
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Protein Interactions ◽  
Protein Localization ◽  
Binding Activity ◽  
Virus Infectivity ◽  
Gene Encoding ◽  
Systemic Spread

ABSTRACT In mammalian and yeast systems, methyltransferases have been implicated in the regulation of diverse processes, such as protein-protein interactions, protein localization, signal transduction, RNA processing, and transcription. The Cucumber mosaic virus (CMV) 1a protein is essential not only for virus replication but also for movement. Using a yeast two-hybrid system with tobacco plants, we have identified a novel gene encoding a methyltransferase that interacts with the CMV 1a protein and have designated this gene Tcoi1 (tobacco CMV 1a-interacting protein 1). Tcoi1 specifically interacted with the methyltransferase domain of CMV 1a, and the expression of Tcoi1 was increased by CMV inoculation. Biochemical studies revealed that the interaction of Tcoi1 with CMV 1a protein was direct and that Tcoi1 methylated CMV 1a protein both in vitro and in vivo. The CMV 1a binding activity of Tcoi1 is in the C-terminal domain, which shows the methyltransferase activity. The overexpression of Tcoi1 enhanced the CMV infection, while the reduced expression of Tcoi1 decreased virus infectivity. These results suggest that Tcoi1 controls the propagation of CMV through an interaction with the CMV 1a protein.

scholarly journals The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death

2009 ◽  
Vol 84 (2) ◽  
pp. 1097-1109 ◽  
Author(s):  
Eric C. Freundt ◽  
Li Yu ◽  
Cynthia S. Goldsmith ◽  
Sarah Welsh ◽  
Aaron Cheng ◽  
...  
Keyword(s):  
Cell Death ◽  
Severe Acute Respiratory Syndrome ◽  
Vero Cells ◽  
Small Gtpase ◽  
Open Reading Frames ◽  
Accessory Proteins ◽  
Reading Frame ◽  
Golgi Fragmentation

ABSTRACT The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.

scholarly journals In vitro and in vivo fertilization potential of cryopreserved spermatozoa from bull epididymides stored for up to 30 hours at ambient temperature (18 °C–20 °C)

2016 ◽  
Vol 86 (4) ◽  
pp. 1014-1021 ◽  
Author(s):  
Melina Andrea Formighieri Bertol ◽  
Romildo Romualdo Weiss ◽  
Luiz Ernandes Kozicki ◽  
Ana Claudia Machinski Rangel de Abreu ◽  
João Filipi Scheffer Pereira ◽  
...  
Keyword(s):  
Ambient Temperature

Levamisole and bovine immunity: in vitro and in vivo effects on immune responses to herpesvirus immunization

1981 ◽  
Vol 27 (12) ◽  
pp. 1312-1319 ◽  
Author(s):  
L. A. Babiuk ◽  
V. Misra
Keyword(s):  
Vaccine Virus ◽  
Pokeweed Mitogen ◽  
Infectious Bovine Rhinotracheitis ◽  
Purified Protein Derivative ◽  
Macrophage Activity ◽  
Bovine Lymphocytes ◽  
Humoral Responses ◽  
In Vivo Effects

Levamisole was shown to enhance in vitro blastogenic responses of bovine lymphocytes to nonspecific mitogens (phytohemagglutinin and pokeweed mitogen) as well as to infectious bovine rhinotracheitis virus and purified protein derivative. Greatest enhancement was observed at suboptimal concentrations of viral antigen. In addition to enhancing lymphocyte reactivity levamisole also affected macrophage activity as determined by increased Fc receptor activity and [3H]glucosamine incorporation. Levamisole (5–50 μg/mL) enhanced type II immune (or γ) interferon production by macrophage–lymphocyte cultures. Administration of levamisole and attenuated infectious bovine rhinotracheitis vaccine virus in vivo did not elevate cellular or humoral responses.

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