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At least 955 records · Page 53Linked to original sources
PubMed2003

Human papillomavirus type 45 propagation, infection, and neutralization.

The organotypic (raft) culture system has allowed the study of the entire differentiation-dependent life cycle of human papillomaviruses (HPVs), including virion morphogenesis. We introduced linearized HPV45 genomic DNA into primary keratinocytes, where it recircularized and maintained episomally at a range of 10-50 copies of HPV genomic DNA. Following epithelial stratification and differentiation in organotypic culture, virion morphogenesis occurred. HPV45 virions were purified from raft cultures and were able to infect keratinocytes in vitro. By testing a panel of HPV VLP antisera, we were able to demonstrate that the infection was neutralized not only with human HPV45 VLP-specific antiserum, but also with human HPV18 VLP-specific antiserum, demonstrating serological cross-reactivity between HPV18 and HPV45.

Antibodies, Viral↗
PubMed2003

Construction and characterisation of a full-length infectious molecular clone from a fast replicating, X4-tropic HIV-1 CRF02.AG primary isolate.

Based on our previous analysis of HIV-1 isolates from Cameroon, we constructed a full-length infectious molecular clone from a primary isolate belonging to the CRF02.AG group of recombinant viruses which dominate the HIV-epidemic in West and Central Africa. The virus derived by transfection of the proviral clone pBD6-15 replicated with similar efficiency compared to its parental isolate and used CXCR4 as coreceptor as well. Furthermore, HIV-1 BD6-15 exhibited similar replication properties and virus yield as the reference B-type HIV-1 strain NL4-3. Sequence analysis revealed open reading frames for all structural and accessory genes apart from vpr. Phylogenetic and bootscanning analyses confirmed that BD6-15 clusters with CRF02.AG recombinant strains from West and Central Africa with similar cross-over points as described for the CRF02.AG prototype strain lbNG. Thus, pBD6-15 represents the first non-subtype B infectious molecular clone of a fast replicating, high producer, X4-tropic primary HIV-1 isolate, which had only been briefly passaged in primary cells.

Adult↗
PubMed1994

SV40 virus expression vectors.

SV40 late-replacement vectors provide an excellent means for expressing large amounts of proteins from cDNAs of less than 2400 bp. The amount of protein made 28 hr after infection is sufficient for extremely brief pulse-chase protocols. At periods postinfection when cells are still healthy, sufficient protein has been made for techniques in which only a small fraction of the protein is detected, including immunocytochemistry on cryosections. Because of the ease of subcloning DNA fragments into them, the ease of making virus stocks, and the ability to achieve comparable amounts of protein expression reproducibly with different infections, these vectors are superb tools for comparing series of mutants made by site-directed mutagenesis, especially if a normal cellular environment is important for the measurements to be made.

Animals↗
PubMed1982

Isolation of virus from the spinal fluid of three patients with multiple sclerosis and one with amyotrophic lateral sclerosis.

A transmissible agent with the properties of a thermolabile filterable virus was isolated from the cerebrospinal fluid (CSF) of 4 Houston patients with chronic central nervous system (CNS) disease. 3 had multiple sclerosis and I had amyotrophic lateral sclerosis. No isolations were made from 27 patients whose CSF was taken for diagnosis of illness unrelated to degenerative CNS disease. Neutralising antibodies to the 4 isolates were present in the serum of each of the 4 patients with virus (14 positive results among 15 tests) but in hardly any of the sera from 8 patients without virus (3 antibody-positive results among 32 tests). The agent is antigenically related to the virus isolated in Japan from cases of subacute myelo-optico-neuropathy.

Adult↗
PubMed2001

Cucurbit protoplast isolation for the study of plant virus replication.

A cucurbit protoplast isolation protocol was established for the study of plant virus replication in vivo. This protocol is applicable to both cucumber and squash leaf tissue with significant increases in yields of viable protoplasts suitable for electroporation, compared to other published methods. A combination of Cellulase RS, Macerozyme R10 and mannitol was used as digestion enzymes and osmoticum. An average of 1.7x10(7) protoplasts per gram of fresh leaf tissue were obtained from cucumber cultivar Bet-alpha. Both cucumber cultivar Shimson and squash cultivar First Taste produced an average yield of 6.0x10(6) protoplasts per g of fresh leaf tissue. Electroporation of 10 microg of Zucchini yellow mosaic potyvirus (ZYMV-S) RNA into the protoplasts resulted in virus replication and synthesis of coat protein (CP). SDS-PAGE and immunoblotting were used to detect the CP 48 h post-electroporation. This protocol is highly reproducible and will assist researchers who require cucurbit protoplasts to study virus replication.

Cell Separation↗
PubMed2001

Optimization of recombinant adeno-associated virus production using an herpes simplex virus amplicon system.

A major limitation of adeno-associated virus (AAV) based vectors for clinical applications to date is the production of high-titer recombinant AAV vector stocks. Despite recent improvements, the amount of recombinant adeno-associated virus vectors (rAAV) particles produced per cell continues to be significantly lower than that of wild-type AAV. In this study, an HSV-based system for rAAV production was used to examine the influence of different parameters including transfection conditions (vector-to-packaging plasmid ratio, amount of total transfected DNA, cell confluency) and multiplicity of infection of herpes helper virus on the resulting titre of rAAV stocks. For herpes helper virus, time-course experiments were carried out to analyse the effect on rAAV yields up to 72 h postinfection and to determine the ideal harvesting time. Taken together, the optimized production scheme consistently yields more than 3x10(3) transducing units per producer cell.

Animals↗