scholarly journals Mechanistic Aspects of Adenovirus Serotype 2 Inactivation with Free Chlorine

2010 ◽  
Vol 76 (9) ◽  
pp. 2946-2954 ◽  
Author(s):  
Martin A. Page ◽  
Joanna L. Shisler ◽  
Benito J. Mariñas
Keyword(s):  
Life Cycle ◽  
Cell Attachment ◽  
Adenovirus Type ◽  
Free Chlorine ◽  
Host Cells ◽  
Coxsackie Adenovirus Receptor ◽  
Genome Damage ◽  
Fiber Proteins ◽  
Molecular Components

ABSTRACT Free chlorine is an effective disinfectant for controlling adenoviruses in drinking water, but little is known about the underlying inactivation mechanisms. The objective of this study was to elucidate the molecular components of adenovirus type 2 (Ad2) targeted by free chlorine during the inactivation process. The effects of free chlorine treatment on several Ad2 molecular components and associated life cycle events were compared to its effect on the ability of adenovirus to complete its life cycle, i.e., viability. Free chlorine treatment of Ad2 virions did not impair their ability to interact with monoclonal antibodies specific for hexon and fiber proteins of the Ad2 capsid, as measured by enzyme-linked immunosorbent assays, nor did it impair their interaction with recombinant, purified Coxsackie-adenovirus receptor (CAR) proteins in vitro. Free chlorine-treated Ad2 virions also retained their ability to bind to CAR receptors on A549 cell monolayers, despite being unable to form plaques, suggesting that free chlorine inactivates Ad2 by inhibiting a postbinding event of the Ad2 life cycle. DNA isolated from Ad2 virions that had been inactivated by free chlorine was able to be amplified by PCR, indicating that genome damage was not the cause of inactivation. However, inactivated Ad2 virions were unable to express E1A viral proteins during infection of A549 host cells, as measured by using immunoblotting. Collectively, these results indicate that free chlorine inactivates adenovirus by damaging proteins that govern life cycle processes occurring after host cell attachment, such as endocytosis, endosomal lysis, or nuclear delivery.

scholarly journals Adenovirus Type 11 Uses CD46 as a Cellular Receptor

2003 ◽  
Vol 77 (17) ◽  
pp. 9183-9191 ◽  
Author(s):  
Anna Segerman ◽  
John P. Atkinson ◽  
Marko Marttila ◽  
Veronica Dennerquist ◽  
Göran Wadell ◽  
...  
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Host Cells ◽  
Cellular Receptor ◽  
Transfected Cells ◽  
Coxsackie Adenovirus Receptor ◽  
Adenovirus Receptor

ABSTRACT The 51 human adenovirus serotypes are divided into six species (A to F). Many adenoviruses use the coxsackie-adenovirus receptor (CAR) for attachment to host cells in vitro. Species B adenoviruses do not compete with CAR-binding serotypes for binding to host cells, and it has been suggested that species B adenoviruses use a receptor other than CAR. Species B adenoviruses mainly cause disease in the respiratory tract, the eyes, and in the urinary tract. Here we demonstrate that adenovirus type 11 (Ad11; of species B) binds to Chinese hamster ovary (CHO) cells transfected with CD46 (membrane cofactor protein)-cDNA at least 10 times more strongly than to CHO cells transfected with cDNAs encoding CAR or CD55 (decay accelerating factor). Nonpermissive CHO cells were rendered permissive to Ad11 infection upon transfection with CD46-cDNA. Soluble Ad11 fiber knob but not Ad7 or Ad5 knob inhibited binding of Ad11 virions to CD46-transfected cells, and anti-CD46 antibodies inhibited both binding of and infection by Ad11. From these results we conclude that CD46 is a cellular receptor for Ad11.

scholarly journals Organoids and Bioengineered Intestinal Models: Potential Solutions to the Cryptosporidium Culturing Dilemma

2020 ◽  
Vol 8 (5) ◽  
pp. 715 ◽  
Author(s):  
Samantha Gunasekera ◽  
Alireza Zahedi ◽  
Mark O’Dea ◽  
Brendon King ◽  
Paul Monis ◽  
...  
Keyword(s):  
Life Cycle ◽  
Host Cells ◽  
Protozoan Parasites ◽  
Future Directions ◽  
In Vivo Models ◽  
Severe Diarrhea ◽  
Transformed Cell Lines ◽  
Gnotobiotic Piglets

Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes.

scholarly journals Complementation of the fadA Mutation in Fusobacterium nucleatum Demonstrates that the Surface-Exposed Adhesin Promotes Cellular Invasion and Placental Colonization

2009 ◽  
Vol 77 (7) ◽  
pp. 3075-3079 ◽  
Author(s):  
Akihiko Ikegami ◽  
Peter Chung ◽  
Yiping W. Han
Keyword(s):  
Host Cell ◽  
Cell Attachment ◽  
Adverse Pregnancy Outcome ◽  
Fusobacterium Nucleatum ◽  
Host Cells ◽  
Mouse Placenta ◽  
Host Cell Attachment ◽  
Adverse Pregnancy

ABSTRACT Fusobacterium nucleatum is a gram-negative oral anaerobe implicated in periodontal disease and adverse pregnancy outcome. The organism colonizes the mouse placenta, causing localized infection and inflammation. The mechanism of placental colonization has not been elucidated. Previous studies identified a novel adhesin from F. nucleatum, FadA, as being involved in the attachment and invasion of host cells. The fadA deletion mutant F. nucleatum 12230 US1 was defective in host cell attachment and invasion in vitro, but it also exhibited pleiotropic effects with altered cell morphology and growth rate. In this study, a fadA-complementing clone, F. nucleatum 12230 USF81, was constructed. The expression of FadA on USF81 was confirmed by Western blotting and immunofluorescent labeling. USF81 restored host cell attachment and invasion activities. The ability of F. nucleatum 12230, US1, and USF81 to colonize the mouse placenta was examined. US1 was severely defective in placental colonization compared to the wild type and USF81. Thus, FadA plays an important role in F. nucleatum colonization in vivo. These results also represent the first complementation studies for F. nucleatum. FadA may be a therapeutic target for preventing F. nucleatum colonization of the host.

scholarly journals Complete In Vitro Assembly of the Reovirus Outer Capsid Produces Highly Infectious Particles Suitable for Genetic Studies of the Receptor-Binding Protein

2001 ◽  
Vol 75 (11) ◽  
pp. 5335-5342 ◽  
Author(s):  
Kartik Chandran ◽  
Xing Zhang ◽  
Norman H. Olson ◽  
Stephen B. Walker ◽  
James D. Chappell ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Cell Attachment ◽  
Molecular Genetic ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
Dependent Manner ◽  
Viral Attachment ◽  
Structure Assembly

ABSTRACT Mammalian reoviruses, prototype members of theReoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins—ς1, μ1, and ς3—to enter host cells. ς1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of ς1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by “recoating” genome-containing core particles that lacked ς1, μ1, and ς3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to ς1. The recoated particles bound to and infected cultured cells in a ς1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant ς1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the ς1 protein. Additional experiments showed that recoated particles containing ς1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound ς1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of ς1 with respect to its structure, assembly into particles, and roles in entry.

scholarly journals Flexibility of the Adenovirus Fiber Is Required for Efficient Receptor Interaction

2003 ◽  
Vol 77 (13) ◽  
pp. 7225-7235 ◽  
Author(s):  
Eugene Wu ◽  
Lars Pache ◽  
Dan J. Von Seggern ◽  
Tina-Marie Mullen ◽  
Yeshi Mikyas ◽  
...  
Keyword(s):  
Virus Infection ◽  
Cell Surface ◽  
Cell Attachment ◽  
Molecular Model ◽  
Host Cells ◽  
Receptor Interaction ◽  
Particle Analysis ◽  
Fiber Protein ◽  
Fiber Proteins ◽  
Fiber Flexibility

ABSTRACT The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.

scholarly journals The Arg279Glu Substitution in the Adenovirus Type 11p (Ad11p) Fiber Knob Abolishes EDTA-Resistant Binding to A549 and CHO-CD46 Cells, Converting the Phenotype to That of Ad7p

2006 ◽  
Vol 80 (4) ◽  
pp. 1897-1905 ◽  
Author(s):  
Dan J. Gustafsson ◽  
Anna Segerman ◽  
Kristina Lindman ◽  
Ya-Fang Mei ◽  
Göran Wadell
Keyword(s):  
Amino Acid ◽  
Adenovirus Type ◽  
Cell Interaction ◽  
Amino Acid Identity ◽  
Host Cells ◽  
Single Amino Acid ◽  
Receptor Interaction ◽  
Coxsackie Adenovirus Receptor ◽  
Cellular Attachment ◽  
Adenovirus Receptor

ABSTRACT The major determinant of adenovirus (Ad) attachment to host cells is the C-terminal knob domain of the trimeric fiber protein. Ad type 11p (Ad11p; species B2) in contrast to Ad7p (species B1) utilizes at least two different cellular attachment receptors, designated sBAR (species B adenovirus receptor) and sB2AR (species B2 adenovirus receptor). CD46 has recently been identified as one of the Ad11p attachment receptors. However, CD46 did not seem to constitute a functional receptor for Ad7p. Although Ad7p shares high knob amino acid identity with Ad11p, Ad7p is deficient in binding to both sB2AR and CD46. To determine what regions of the Ad11p fiber knob are necessary for sB2AR-CD46 interaction, we constructed recombinant fiber knobs (rFK) with Ad11p/Ad7p chimeras and Ad11p sequences having a single amino acid substitution from Ad7p. Binding of the constructs to A549 and CHO-CD46 BC1 isoform-expressing cells was analyzed by flow cytometry. Our results indicate that an Arg279Glu substitution is sufficient to convert the Ad11p receptor-interaction phenotype to that of Ad7p and abolish sB2AR and CD46 interaction. Also a Glu279Arg substitution in Ad7p rFKs increases CD46 binding. Thus, the lateral HI loop of the Ad11p fiber knob seems to be the key determinant for Ad11p sB2AR-CD46 interaction. This result is comparable to another non-coxsackie-adenovirus receptor binding Ad (Ad37p), where substitution of one amino acid abolishes virus-cell interaction. In conjunction with previous results, our findings also strongly suggest that sB2AR is equivalent to CD46.

scholarly journals Cryptosporidium parvum Subtilisin-Like Serine Protease (SUB1) Is Crucial for Parasite Egress from Host Cells

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Samantha Nava ◽  
A. Clinton White ◽  
Alejandro Castellanos-González
Keyword(s):  
Life Cycle ◽  
Serine Protease ◽  
Host Cells ◽  
Effective Vaccine ◽  
Infection Model ◽  
Target Cells ◽  
Calcium Dependent ◽  
Argonaute 2 ◽  
Parasite Egress

ABSTRACT Despite the severity and global burden of Cryptosporidium infection, treatments are less than optimal, and there is no effective vaccine. Egress from host cells is a key process for the completion of the life cycle of apicomplexan parasites. For Plasmodium species, subtilisin-like serine protease (SUB1) is a key mediator of egress. For Toxoplasma species, calcium-dependent protein kinases (CDPKs) are critical. In this study, we characterized Cryptosporidium SUB1 expression and evaluated its effect using an infection model. We found increased expression between 12 and 20 h after in vitro infection, prior to egress. We induced silencing of SUB1 (ΔSUB1) mRNA using SUB1 single-stranded antisense RNA coupled with human Argonaute 2. Silencing of SUB1 mRNA expression did not affect parasite viability, excystation, or invasion of target cells. However, knockdown led to a 95% decrease in the proportion of released merozoites in vitro (P < 0.0001). In contrast, silencing of CDPK5 had no effect on egress. Overall, our results indicate that SUB1 is a key mediator of Cryptosporidium egress and suggest that interruption of the life cycle at this stage may effectively inhibit the propagation of infection.

Morphological characterization ofCryptosporidium parvumlife-cycle stages in anin vitromodel system

Parasitology ◽  
2009 ◽  
Vol 137 (1) ◽  
pp. 13-26 ◽  
Author(s):  
H. BOROWSKI ◽  
R. C. A. THOMPSON ◽  
T. ARMSTRONG ◽  
P. L. CLODE
Keyword(s):  
Electron Microscopy ◽  
Life Cycle ◽  
Protozoan Parasite ◽  
Morphological Characterization ◽  
Host Cells ◽  
Epithelial Cell Line ◽  
Life Cycle Stages ◽  
Complete Life Cycle ◽  
Behavioural Characteristics

SUMMARYCryptosporidium parvumis a zoonotic protozoan parasite that mainly affects the ileum of humans and livestock, with the potential to cause severe enteric disease. We describe the complete life cycle ofC. parvumin anin vitrosystem. Infected cultures of the human ileocecal epithelial cell line (HCT-8) were observed over time using electron microscopy. Additional data are presented on the morphology, development and behavioural characteristics of the different life-cycle stages as well as determining their time of occurrence after inoculation. Numerous stages ofC. parvumand their behaviour have been visualized and morphologically characterized for the first time using scanning electron microscopy. Further, parasite-host interactions and the effect ofC. parvumon host cells were also visualized. An improved understanding of the parasite's biology, proliferation and interactions with host cells will aid in the development of treatments for the disease.

scholarly journals Hepatitis E Virus Assembly and Release

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 539 ◽  
Author(s):  
Xiaohui Ju ◽  
Qiang Ding
Keyword(s):  
Life Cycle ◽  
Hepatitis E Virus ◽  
Current Knowledge ◽  
Virus Assembly ◽  
Antiviral Treatment ◽  
Hepatitis E ◽  
Host Cells ◽  
Direct Acting Antiviral ◽  
Direct Acting

Hepatitis E is an underestimated threat to public health, caused by the hepatitis E virus (HEV). HEV is the most common cause of acute viral hepatitis in the world, with no available direct-acting antiviral treatment. According to a recent WHO report, 20 million people become infected with HEV annually, resulting in 44,000 deaths. However, due to the scarcity of efficient in vitro cell culture systems for HEV, our knowledge of the life cycle of HEV is incomplete. Recently, significant progress has been made towards gaining a more comprehensive view of the HEV life cycle, as several in vitro culturing systems have been developed in recent years. Here, we review current knowledge and recent advances with regard to the HEV life cycle, with a particular focus on the assembly and release of viral particles. We also discuss the knowledge gaps in HEV assembly and release. Meanwhile, we highlight experimental platforms that could potentially be utilized to fill these gaps. Lastly, we offer perspectives on the future of research into HEV virology and its interaction with host cells.

Synthetic Polymer Matrices for Neural Cell Transplantation

1993 ◽  
Vol 2 (3) ◽  
pp. 229-239 ◽  
Author(s):  
S. Woerly ◽  
K. Ulbrich ◽  
V. Chytrý ◽  
K. Smetana ◽  
P. Petrovický ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Cell Attachment ◽  
Neuronal Cell ◽  
Polymer Matrices ◽  
Host Cells ◽  
Donor Cells ◽  
Neural Transplant ◽  
Neuritic Growth ◽  
Neural Graft

This study proposes a strategy to promote the integration of a neural graft into the host brain tissue. It involves the attachment of donor cells to a polymeric matrix, and the implantation of this cell-polymer matrix. We have synthesized hydrogels based on N-(2-hydroxypropyl)-methacrylamide (HPMA) to produce highly porous matrices. As preliminary steps, we have examined: 1) The response of the brain tissue to the implantation of PHPMA/collagen hydrogels; 2) adhesion, growth, differentiation, and viability of embryonic neuronal cells, and embryonal carcinoma-derived neurons seeded onto PHPMA substrates containing hexosamine residues (glucosamine and N-acetylglucosamine), and after entrapment of cells within the hydrogels. Histological analysis seven wk after implantation showed the tolerance of PHPMA hydrogels, and the penetration of host cells into the pore structures. However, cellular ingrowth requires the presence of collagen, and is dependent upon porosity. In vitro data showed that PHPMA substrates supported neuronal cell attachment and neuritic growth, but the biocompatibility of the substrate was enhanced after incorporation of N-acetylglucosamine into the hydrogel. The data also showed the feasibility of entrapping cells into the polymer matrices, and that these “cellular” hydrogel matrices could be maintained in vitro with preservation of cell viability and differentiation. These findings suggest that PHPMA-based hydrogels can serve as carriers for neural transplant, and as a support to guide tissue ingrowth and organization.

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