scholarly journals Temperature-sensitive mutants of enterovirus 71 show attenuation in cynomolgus monkeys

2005 ◽  
Vol 86 (5) ◽  
pp. 1391-1401 ◽  
Author(s):  
Minetaro Arita ◽  
Hiroyuki Shimizu ◽  
Noriyo Nagata ◽  
Yasushi Ami ◽  
Yuriko Suzaki ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Virulent Strain ◽  
Infectious Clone ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Infection Model ◽  
Temperature Sensitive ◽  
Cynomolgus Monkeys

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is sometimes associated with serious neurological disorders. In this study, an attempt was made to identify molecular determinants of EV71 attenuation of neurovirulence in a monkey infection model. An infectious cDNA clone of the virulent strain of EV71 prototype BrCr was constructed; temperature-sensitive (ts) mutations of an attenuated strain of EV71 or of poliovirus (PV) Sabin vaccine strains were then introduced into the infectious clone. In vitro and in vivo phenotypes of the parental and mutant viruses were analysed in cultured cells and in cynomolgus monkeys, respectively. Mutations in 3D polymerase (3Dpol) and in the 3′ non-translated region (NTR), corresponding to ts determinants of Sabin 1, conferred distinct temperature sensitivity to EV71. An EV71 mutant [EV71(S1-3′)] carrying mutations in the 5′ NTR, 3Dpol and in the 3′ NTR showed attenuated neurovirulence, resulting in limited spread of virus in the central nervous system of monkeys. These results indicate that EV71 and PV1 share common genetic determinants of neurovirulence in monkeys, despite the distinct properties in their original pathogenesis.

scholarly journals An Attenuated Strain of Enterovirus 71 Belonging to Genotype A Showed a Broad Spectrum of Antigenicity with Attenuated Neurovirulence in Cynomolgus Monkeys

2007 ◽  
Vol 81 (17) ◽  
pp. 9386-9395 ◽  
Author(s):  
Minetaro Arita ◽  
Noriyo Nagata ◽  
Naoko Iwata ◽  
Yasushi Ami ◽  
Yuriko Suzaki ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Tissue Specificity ◽  
Virulent Strain ◽  
Enterovirus 71 ◽  
Infection Model ◽  
Intravenous Route ◽  
Lethal Challenge ◽  
Neutralization Activity ◽  
Cynomolgus Monkeys ◽  
Genotype A

ABSTRACT Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and is also sometimes associated with serious neurological disorders. In this study, we characterized the antigenicity and tissue specificity of an attenuated strain of EV71 [EV71(S1-3′)], which belongs to genotype A, in a monkey infection model. Three cynomolgus monkeys were inoculated with EV71(S1-3′), followed by lethal challenge with the parental virulent strain EV71(BrCr-TR) via an intravenous route on day 45 postinoculation of EV71(S1-3′). Monkeys inoculated with EV71(S1-3′) showed a mild neurological symptom (tremor) but survived lethal challenge by virulent EV71(BrCr-TR) without exacerbation of the symptom. The immunized monkey sera showed a broad spectrum of neutralizing activity against different genotypes of EV71, including genotypes A, B1, B4, C2, and C4. For the strains examined, the sera showed the highest neutralization activity against the homotype (genotype A) and the lowest neutralization activity against genotype C2. The order of decreasing neutralization activity of sera was as follows: A > B1 > C4 > B4 > C2. To examine the tissue specificity of EV71(S1-3′), two monkeys were intravenously inoculated with EV71(S1-3′), followed by examination of virus distribution in the central nervous system (CNS) and extraneural tissues. In the CNS, EV71(S1-3′) was isolated only from the spinal cord. These results indicate that EV71(S1-3′) acts as an effective antigen, although this attenuated strain was still neurotropic when inoculated via the intravenous route.

scholarly journals Candida albicans PEP12 Is Required for Biofilm Integrity and In Vivo Virulence

2009 ◽  
Vol 9 (2) ◽  
pp. 266-277 ◽  
Author(s):  
Suresh K. A. Palanisamy ◽  
Melissa A. Ramirez ◽  
Michael Lorenz ◽  
Samuel A. Lee
Keyword(s):  
Candida Albicans ◽  
Lipolytic Activity ◽  
Infection Model ◽  
Temperature Sensitive ◽  
Null Mutant ◽  
Macrophage Infection ◽  
Content Type ◽  
Vacuolar Acidification

ABSTRACT To investigate the role of the prevacuolar secretion pathway in biofilm formation and virulence in Candida albicans, we cloned and analyzed the C. albicans homolog of the Saccharomyces cerevisiae prevacuolar trafficking gene PEP12. C. albicans PEP12 encodes a deduced t-SNARE that is 28% identical to S. cerevisiae Pep12p, and plasmids bearing C. albicans PEP12 complemented the abnormal vacuolar morphology and temperature-sensitive growth of an S. cerevisiae pep12 null mutant. The C. albicans pep12 Δ null mutant was defective in endocytosis and vacuolar acidification and accumulated 40- to 60-nm cytoplasmic vesicles near the plasma membrane. Secretory defects included increased extracellular proteolytic activity and absent lipolytic activity. The pep12Δ null mutant was more sensitive to cell wall stresses and antifungal agents than the isogenic complemented strain or the control strain DAY185. Notably, the biofilm formed by the pep12Δ mutant was reduced in overall mass and fragmented completely upon the slightest disturbance. The pep12Δ mutant was markedly reduced in virulence in an in vitro macrophage infection model and an in vivo mouse model of disseminated candidiasis. These results suggest that C. albicans PEP12 plays a key role in biofilm integrity and in vivo virulence.

scholarly journals Antiviral Efficacy of Flavonoids against Enterovirus 71 Infection in Vitro and in Newborn Mice

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 625 ◽  
Author(s):  
Wenwen Dai ◽  
Jinpeng Bi ◽  
Fang Li ◽  
Shuai Wang ◽  
Xinyu Huang ◽  
...  
Keyword(s):  
Protective Efficacy ◽  
Lethal Dose ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Neurological Complications ◽  
Ev71 Infection ◽  
Newborn Mice ◽  
Reported Data

Enterovirus 71 (EV71) infection is known to cause hand, foot, and mouth disease (HFMD), which is associated with neurological complications; however, there is currently no effective treatment for this infection. Flavonoids are a large group of naturally occurring compounds with multiple bioactivities, and the inhibitory effects of several flavonoids against EV71 have been studied in cell cultures; however, to date, there are no reported data on their effects in animal models. In this study, we confirmed the in vitro activities of eight flavonoids against EV71 infection, based on the inhibition of cytopathic effects. Moreover, these flavonoids were found to reduce viral genomic RNA replication and protein synthesis. We further demonstrated the protective efficacy of these flavonoids in newborn mice challenged with a lethal dose of EV71. Apigenin, luteolin, kaempferol, formononetin, and penduletin conferred survival protection of 88.89%, 91.67%, 88.89%, 75%, and 66.67%, respectively, from the lethal EV71 challenge. In addition, isorhamnetin provided the highest mice survival protection of 100% at a dose of 10 mg/kg. This study, to the best of our knowledge, is the first to evaluate the in vivo anti-EV7l activities of multiple flavonoids, and we accordingly identified flavonoids as potential leading compounds for anti-EV71 drug development.

scholarly journals Site-Directed Mutagenesis of the 19-Kilodalton Lipoprotein Antigen Reveals No Essential Role for the Protein in the Growth and Virulence of Mycobacterium intracellulare

1998 ◽  
Vol 66 (8) ◽  
pp. 3626-3634 ◽  
Author(s):  
Eshwar Mahenthiralingam ◽  
Britt-Inger Marklund ◽  
Lucy A. Brooks ◽  
Debbie A. Smith ◽  
Gregory J. Bancroft ◽  
...  
Keyword(s):  
Essential Role ◽  
Genetic Manipulation ◽  
Virulent Strain ◽  
Site Directed Mutagenesis ◽  
Infection Model ◽  
Directed Mutagenesis ◽  
Exact Nature ◽  
Mycobacterium Intracellulare

ABSTRACT The mycobacterial 19-kilodalton antigen (19Ag) is a highly expressed, surface-associated glycolipoprotein which is immunodominant in infected patients and has little homology with other known proteins. To investigate the pathogenic significance of the 19Ag, site-directed mutagenesis of the Mycobacterium intracellulare 19Ag gene was carried out by using a suicide vector-based strategy. Allelic replacement of the 19Ag gene of a mouse-avirulent M. intracellulare strain, 1403, was achieved by double-crossover homologous recombination with a gentamicin resistance gene-mutated allele. Unfortunately, an isogenic 19Ag was not achievable in the mouse-virulent strain, D673. However, a 19Ag mutant was successfully constructed in M. intracellulare FM1, a chemically mutagenized derivative of strain D673. FM1 was more amenable to genetic manipulation and susceptible to site-directed mutagenesis of the 19Ag gene yet retained the virulent phenotype of the parental strain. No deleterious effects of 19Ag gene mutation were observed during in vitro growth ofM. intracellulare. Virulence assessment of the isogenic 19Ag mutants in a mouse infection model demonstrated that the antigen plays no essential role in the growth of M. intracellularein vivo. Site-directed mutagenesis of the 19Ag gene demonstrated that it plays no essential role in growth and pathogenicity of M. intracellulare; however, the exact nature of its biological function remains unknown.

scholarly journals Immunity to Avirulent Enterovirus 71 and Coxsackie A16 Virus Protects against Enterovirus 71 Infection in Mice

2007 ◽  
Vol 81 (19) ◽  
pp. 10310-10315 ◽  
Author(s):  
Te-Chia Wu ◽  
Ya-Fang Wang ◽  
Yi-Ping Lee ◽  
Jen-Ren Wang ◽  
Ching-Chuan Liu ◽  
...  
Keyword(s):  
Passive Immunization ◽  
Enterovirus 71 ◽  
Clinical Score ◽  
Cross Reactivity ◽  
Immune Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infection Model ◽  
Ev71 Strain

ABSTRACT In this study, we sought to determine whether intratypic and intertypic cross-reactivity protected against enterovirus 71 (EV71) infection in a murine infection model. We demonstrate that active immunization of 1-day-old mice with avirulent EV71 strain or coxsackie A16 virus (CA16) by the oral route developed anti-EV71 antibodies with neutralizing activity (1:16 and 1:2, respectively). Splenocytes from both EV71- and CA16-immunized mice proliferated upon EV71 or CA16, but not coxsackie B3 virus (CB3), antigen stimulation. Immunized mice became more resistant to virulent EV71 strain challenge than nonimmunized mice. There was an increase in the percentage of activated splenic T cells and B cells in the immunized mice 2 days after EV71 challenge. The CA16 immune serum reacted with EV71 antigens in an enzyme-linked immunosorbent assay and neutralized EV71 but not CB3 or poliovirus at a titer of 1:4. Passive immunization with the CA16 immune serum reduced the clinical score, diminished the organ viral load, and increased the survival rate of mice upon EV71 challenge. CB3 neither shared in vitro cross-reactivity with EV71 nor provided in vivo protection after both active and passive immunization. These results illustrated that live vaccine is feasible for EV71 and that intertypic cross-reactivity of enteroviruses may provide a way to determine the prevalence of EV71.

scholarly journals In vitro and in vivo characterization of an infectious clone of a European strain of porcine circovirus type 2

2004 ◽  
Vol 85 (5) ◽  
pp. 1259-1266 ◽  
Author(s):  
M. Roca ◽  
M. Balasch ◽  
J. Segalés ◽  
M. Calsamiglia ◽  
E. Viaplana ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Infectious Clone ◽  
Experimental Period ◽  
Porcine Circovirus Type ◽  
Virus Genome ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus

The aim of this study was to describe the generation of a PCV2 (porcine circovirus type 2) infectious clone (pIC-PCV2) and its infectivity under in vitro and in vivo conditions. The constructed pIC-PCV2 contained the whole PCV2 genome from a German isolate together with a partial duplication of 467 bp. PK-15 cells were transfected with pIC-PCV2 and an indirect immune fluorescence assay (IFA) was performed 7 days post-transfection. The PCV2 Cap gene was expressed in approximately 20 % of the cultured cells, and only the recombination product, and not pIC-PCV2, was subsequently detected by PCR and Southern blot. This result indicated that infection by pIC-PCV2 delivered genomic PCV2 DNA specifically into susceptible cells and led to the expression of a functional virus genome. Eighteen 30- to 40-day-old conventional pigs were distributed into three groups. Group 1 pigs (n=6) were inoculated intranasally (i.n.) with a Spanish isolate of PCV2 propagated in cell culture; pigs from group 2 (n=6) were inoculated with pIC-PCV2 intramuscularly (i.m.), and the last group of pigs (n=6) was inoculated with pIC-PCV2 intraperitoneally (i.p.). All pigs remained clinically healthy during the whole experimental period (35 days). Pigs that received pIC-PCV2 i.p. and i.m., as well as those PCV2 i.n. inoculated, became infected based on an in situ hybridization (ISH), PCR, TaqMan PCR and serological results. The results of this study confirm that cloned PCV2 genomic DNA is infectious both in vitro and in vivo, and is able to cause PMWS-like lesions in i.p. and i.m. experimentally inoculated pigs.

scholarly journals A novel strategy to express different antigens from one modified vaccinia Ankara vaccine vector

2017 ◽  
Author(s):  
K. B. Lauer ◽  
E. A. McKenzie ◽  
Y. Hall ◽  
C.P.C. Gowda ◽  
P. Klapper ◽  
...  
Keyword(s):  
Enterovirus 71 ◽  
Infection Model ◽  
Antigen Delivery ◽  
Serum Antibodies ◽  
T7 Promoter ◽  
B Virus ◽  
Novel Strategy ◽  
Immunogenicity Study

AbstractTo enhance global control of encephalitis and hepatitis caused by rabies-(RABV), Japanese encephalitis-(JEV), hepatitis B-virus (HBV), and enterovirus 71 (EV71) novel immunisation strategies are needed. Therefore, a multipathogen modified Vaccinia Ankara vector, expressing antigens from the above pathogens, was constructed. Two recombinants, one carrying the EV71 and JEV pathogen sequence and one the RABV-HBV pathogen sequence were generated. To ensure similar expression of the antigens, a T7-promoter was linked to the expression cassettes of all pathogen sequences. Direct regulation of this promoter was achieved through co-infection with a second T7-polymerase expressing MVA. Protein expression using this co-infection model of expression was demonstrated in vitro. To investigate the co-infection model of antigen delivery in vivo, a murine immunogenicity study was performed using the MVA-RABV-HBV recombinant. Although, serum antibodies against MVA were induced in all mice, no serum antibodies against RABV or HBV could be detected.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

In Vitro, In Silico and Ex Vivo Studies of Dihydroartemisinin Derivatives as Antitubercular Agents

2019 ◽  
Vol 19 (8) ◽  
pp. 633-644 ◽  
Author(s):  
Komal Kalani ◽  
Sarfaraz Alam ◽  
Vinita Chaturvedi ◽  
Shyam Singh ◽  
Feroz Khan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Silico ◽  
Ex Vivo ◽  
Virulent Strain ◽  
Infection Model ◽  
Mouse Bone Marrow ◽  
Significant Activity ◽  
Macrophage Infection ◽  
In Silico Studies

Introduction: As a part of our drug discovery program for anti-tubercular agents, dihydroartemisinin (DHA-1) was screened against Mtb H37Rv, which showed moderate anti-tubercular activity (>25.0 µg/mL). These results prompted us to carry out the chemical transformation of DHA-1 into various derivatives and study their antitubercular potential. Materials and Methods: DHA-1 was semi-synthetically converted into four new acyl derivatives (DHA-1A – DHA-1D) and in-vitro evaluated for their anti-tubercular potential against Mycobacterium tuberculosis H37Rv virulent strain. The derivatives, DHA-1C (12-O-(4-nitro) benzoyl; MIC 12.5 µg/mL) and DHA-1D (12-O-chloro acetyl; MIC 3.12µg/mL) showed significant activity against the pathogen. Results: In silico studies of the most active derivative (DHA-1D) showed interaction with ARG448 inhibiting the mycobacterium enzymes. Additionally, it showed no cytotoxicity towards the Vero C1008 cells and Mouse bone marrow derived macrophages. Conclusion: DHA-1D killed 62% intracellular M. tuberculosis in Mouse bone marrow macrophage infection model. To the best of our knowledge, this is the first-ever report on the antitubercular potential of dihydroartemisinin and its derivatives. Since dihydroartemisinin is widely used as an antimalarial drug; these results may be of great help in anti-tubercular drug development from a very common, inexpensive, and non-toxic natural product.

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