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At least 919 records · Page 51Linked to original sources
PubMed2006

Efficacy of inactivated vaccines against H5N1 avian influenza infection in ducks.

The current Asian H5N1 highly pathogenic avian influenza virus has spread over much of Asia and into Europe and Africa. As well as affecting village and commercial chicken operations in many South East Asian countries, it differs from past H5 avian influenza viruses in that it causes morbidity and mortalities in other domesticated birds, such as ducks and turkeys and in wild water birds. Effective vaccines that can prevent infection, as well as disease, and be used in a variety of avian species are needed for field use. In this report, a bivalent H5N9+H7N1 oil emulsion vaccine is compared, in ducks, to a monovalent H5N3 oil emulsion vaccine that has been derived by reverse genetics with an H5 from A/chicken/Vietnam/C58/04. While both vaccines protected against morbidity, the monovalent vaccine provided effective protection, with no evidence of shedding of the challenge virus and no serological response to the H5N1 challenge virus.

Animals↗
PubMed2006

Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation.

The measles virus (MV) P gene encodes three proteins: P, an essential polymerase cofactor, and C and V, which have multiple functions including immune evasion. We show here that the MV P protein also contributes to immune evasion, and that tyrosine 110 is required to block nuclear translocation of the signal transducer and activator of transcription factors (STAT) after interferon type I treatment. In particular, MV P inhibits STAT1 phosphorylation. This is shown not only by transient expression but also by reverse genetic analyses based on a new functional infectious cDNA derived from a MV vaccine vial (Moraten strain). Our study also identifies a conserved sequence around P protein tyrosine 110 as a candidate interaction site with a cellular protein.

Animals↗
PubMed2004

Packaging, replication and recombination of the segmented genome of bacteriophage Phi6 and its relatives.

The genomes of bacteriophage Phi6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus strand transcripts of the segmented dsRNA genomes into preformed polyhedral structures called procapsids or inner cores. The packaging requires hydrolysis of NTPs and takes place in the order S:M:L. Minus strand synthesis begins after the completion of the plus strand packaging. The packaging and replication reactions can be studied in vitro with purified components. A model has been presented that proposes that the program of serially dependent packaging is determined by the conformational changes at the surface of the procapsid due to the amount of RNA packaged at each step. The in vitro packaging and replication system has facilitated the application of reverse genetics and the study of recombination in the family of Cystoviridae.

Bacteriophage phi 6↗
PubMed2004

Recovery of a persistent Canine distemper virus expressing the enhanced green fluorescent protein from cloned cDNA.

Wild-type A75/17-Canine distemper virus (CDV) is a highly virulent strain, which induces a persistent infection in the central nervous system (CNS) with demyelinating disease. Wild-type A75/17-CDV, which is unable to replicate in cell lines to detectable levels, was adapted to grow in Vero cells and was designated A75/17-V. Sequence comparison between the two genomes revealed seven nucleotide differences located in the phosphoprotein (P), the matrix (M) and the large (L) genes. The P gene is polycistronic and encodes two auxiliary proteins, V and C, besides the P protein. The mutations resulted in amino acid changes in the P and V, but not in the C protein, as well as in the M and L proteins. Here, a rescue system was developed for the A75/17-V strain, which was shown to be attenuated in vivo, but retains a persistent infection phenotype in Vero cells. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein (eGFP) was inserted at the 3' proximal position in the A75/17-V cDNA clone. Reverse genetics technology will allow us to characterize the genetic determinants of A75/17-V CDV persistent infection in cell culture.

Adaptation, Physiological↗
PubMed2004

Generation of an shRNAi expression library against the whole human transcripts.

RNA interference is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA, homologous to the gene, and is receiving attention as a powerful tool for reverse genetics in the post-genome era. Throughout our current research to generate an siRNA expression library for the whole human genome, we face many technical difficulties. We present here the strategies for overcoming some of the difficulties, including the development of genetically stable and highly active siRNA expression vectors, the selection procedure of the favorable target sites, and the efficient and low cost procedure for constructing an siRNA expression library. Furthermore, we demonstrate that the screening using the constructed siRNA-expression library indeed works, by evaluating siRNA-expression library against apoptosis-related genes.

Algorithms↗
PubMed2004

Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments.

A reverse genetics system for the generation of influenza virus A/PR/8/34 (NIBSC vaccine strain) from plasmid DNA was developed. Upon transfection of eight bidirectional transcription plasmids encoding the gene segments of A/PR/8/34 into 293T cells, virus titers in the supernatant were about 10(4) TCID50/ml. The production of A/PR/8/34 in 293T cells was compared to that of A/WSN/33, for which virus titers in the supernatant were 10(7)-10(8) TCID50/ml. Time-course analysis of virus production indicated that the differences in virus titers were due to reinfection of 293T cells by A/WSN/33 but not A/PR/8/34. Indeed, virus titers of A/PR/8/34 comparable to those of A/WSN/33 were achieved upon addition of trypsin to the culture medium of transfected cells. The production of chimeric viruses revealed that the difference in virus titers between A/PR/8/34 and A/WSN/33 are determined primarily by differences in the surface glycoproteins hemagglutinin and neuraminidase and the polymerase protein PB1. In conclusion, high-titer virus stocks of recombinant influenza A/PR/8/34 virus can be produced as well as virus stocks with much lower titers, but without the requirement of virus amplification through replication.

Animals↗
PubMed2004

Generation of serotype 1/serotype 2 reassortant viruses of the infectious bursal disease virus and their investigation in vitro and in vivo.

Infectious bursal disease virus (IBDV) is the causative agent of acute or immunosuppressive disease in chickens. Serotype 1 strains are pathogenic whereas serotype 2 strains neither cause disease nor protect against infection with the serotype 1 strains. The target organ of serotype 1 strains is the bursa Fabricii (BF). The molecular determinants of this tropism, and therefore pathogenicity, are poorly understood. IBDV is a non-enveloped icosahedral virus particle of 60 nm in diameter, which contains two genome segments of double-stranded RNA. Here, the generation of interserotypic reassortants using the reverse genetics approach is reported. The results of in vitro and in vivo investigations show that genome segment A determines the bursa tropism of IBDV, whereas segment B is involved in the efficiency of viral replication; they further indicate the significance of the interaction of the polymerase (segment B) with the structural protein VP3 (segment A) or the viral genome for efficient virus formation and replication.

Animals↗
PubMed2005

Full genome sequence of peste des petits ruminants virus, a member of the Morbillivirus genus.

Peste des petits ruminants virus (PPRV) causes an acute febrile illness in small ruminant species, mostly sheep and goats. PPRV is a member of the Morbillivirus genus which includes measles, rinderpest (cattle plague), canine distemper, phocine distemper and the morbilliviruses found in whales, porpoises and dolphins. Full length genome sequences for these morbilliviruses are available and reverse genetic rescue systems have been developed for the viruses of terrestrial mammals, with the exception of PPRV. This paper presents the first published full length genome sequence for PPRV. The genome was found to be consistent with the rule-of-six and open reading frames (ORFs) were identified that encoded the eight proteins characteristic of morbilliviruses. At the nucleotide (nt) level, the full length genome of PPRV was most similar to that of rinderpest, the other ruminant morbillivirus. However, at the protein level five of the six structural proteins and the V protein showed a greater similarity to the dolphin morbillivirus (DMV) while only the C and L proteins showed a high relationship to rinderpest.

Base Sequence↗
PubMed2005

Characterization of P gene-deficient rabies virus: propagation, pathogenicity and antigenicity.

The RNA polymerase of rabies virus (RV) is a two-protein complex composed of L (a large catalytic component) and P (a non-catalytic phosphoprotein cofactor) proteins. We generated a gene-deficient RV lacking the entire P gene from HEP-Flury (HEP) strain, one of the most attenuated RV strains, by the method of reverse genetics. This P gene-deficient (def-P) virus could replicate and produce progeny viruses with a slightly retarded rate in the cell lines that constitutively express the P protein. The def-P virus could perform the primary RNA transcription by the virion-associated polymerase even in the infected host without de novo P protein synthesis. However, the def-P virus required the newly synthesized P protein for the secondary RNA transcription and genome RNA replication of virus. No progeny virus was produced in the infected host that did not express P protein. The def-P virus was apathogenic in adult and suckling mice even when inoculated intracranially. On the other hand, inoculation of the def-P virus into mice induced a high titer of virus-neutralizing antibody and protected mice from lethal challenge with the CVS strain. These results demonstrated that the def-P virus could induce strong protective immunity against rabies virus without the production of progeny virus and the severe host damage. The def-P virus would be a potential resource of safe live-attenuated rabies vaccine.

Animals↗
PubMed2005

Oral vaccination of dogs with recombinant rabies virus vaccines.

Oral rabies virus (RV) vaccines are used to immunize a diversity of mammalian carnivores, but no single biological is effective for all major species. Recently, advances in reverse genetics have allowed the design of recombinant RV for consideration as new vaccines. The objective of this experiment was to examine the safety, immunogenicity and efficacy of recombinant RV vaccines administered to captive dogs by the oral route, compared to a commercial vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine. Animals consisted of naive purpose-bred beagles of both sexes, and were 6 months of age or older. Dogs were randomly assigned to one of six groups, and received either diluent or vaccine (PBS; V-RG; RV SN10-333; RV SPBN-Cyto c; RV SPBNGA; RV SPBNGAGA), with at least six animals per group. On day 0, 1 ml of each vaccine (or PBS) was administered to the oral cavity of each dog, at an approximate concentration of 10(8) to 10(9) TCID50. After vaccination, dogs were observed daily and bled weekly, for 5 weeks, prior to RV challenge. No signs of illness related to vaccination were detected during the observation period. Excluding the controls, RV neutralizing antibodies were detected in the majority of animals within 1-2 weeks of primary vaccination. Thereafter, all dogs were inoculated in the masseter muscle with a street virus of canine origin. All control animals developed rabies, but no vaccinates succumbed, with the exception of a single dog in the V-RG group. Review of these preliminary data demonstrates the non-inferiority of recombinant RV products, as concerns both safety and efficacy, and supports the suggestion that these vaccines may hold promise for future development as oral immunogens for important carnivore species, such as dogs.

Administration, Oral↗
PubMed2005

Replication strategies of rabies virus.

Rabies virus (RV) is a prototype neurotropic virus that causes fatal disease in human and animals. RV infects hosts at the periphery, enters motoneurons or sensory nerves and moves to the central nervous system (CNS) via retrograde axonal transport. At later stages, there is also centrifugal spread to major exit portals, such as the salivary glands. Transmission to other hosts is facilitated by behavioral changes related to the CNS infection. Successful accomplishment of the RV infectious cycle depends on multiple functions of the virus, and of individual virus proteins, all together defining the typical pathogenicity and virulence, i.e. the biological fitness of this virus. In particular, it appears important for RV to sneak into the host without causing pronounced host responses and to preserve, at least for some time, the integrity of infected cells and of the neuronal network. The availability of reverse genetics systems that allow generation of engineered recombinant RV has provided tools for a more detailed analysis of viral functions relevant to the typical RV pathogenesis. Novel developments such as tracking of live fluorescent RV are further increasing the opportunities to decipher RV pathogenicity factors. In this review, we describe different aspects of the molecular biology of RV that are relevant to pathogenesis, with a particular emphasis on the accurate control of RV transcription, gene expression, and replication. In addition, the role of individual virus proteins in maintaining host cell integrity and supporting retrograde transport is discussed. The potential of recombinant RVs with single or multiple pathogenicity factors eliminated is being discussed in terms of vaccine and virus vector development.

Amino Acid Sequence↗
PubMed2005

Molecular mechanisms of measles virus persistence.

As measles virus causes subacute sclerosing panencephalitis and measles inclusion body encephalitis due to its ability to establish human persistent infection, without symptoms for the time between the acute infection and the onset of clinical symptoms, it has been the paradigm for a long term persistent as opposed to chronic infection by an RNA virus. We have reviewed the mechanisms of persistence of the virus and discuss specific mutations associated with CNS infection affecting the matrix and fusion protein genes. These are placed in the context of our current understanding of the viral replication cycle. We also consider the proposed mechanisms of persistence of the virus in replicating cell cultures and conclude that no general mechanistic model can be derived from our current state of knowledge. Finally, we indicate how reverse genetics approaches and the use of mouse models with specific knock-out and knock-in modifications can further our understanding of measles virus persistence.

Animals↗
PubMed2006

Efficient expression of the 15-kDa form of infectious pancreatic necrosis virus VP5 by suppression of a UGA codon.

Infectious pancreatic necrosis virus (IPNV), a member of the Birnaviridae family, encodes a nonstructural VP5 protein from a small open reading frame (ORF), which overlaps with a major ORF encoding pVP2, VP4 and VP3 proteins. In majority of the Sp strains of IPNV sequenced to date, VP5 gene codes for a 15-kDa protein. However, we have shown that in highly virulent strains, there is a premature in-frame stop codon (UGA) at nucleotide (nt) position 427, (preceding the 15-kDa stop codon at nt position 511) which could encode a 12-kDa protein. Using reverse genetics, we recovered recombinant rNVI15, rNVI15-15K and rNVI15-DeltaVP5 viruses (which could encode 12 or 15-kDa VP5 or lack the expression of VP5, respectively) and demonstrated that VP5 is dispensable for viral replication in vivo but is not involved in virulence (Santi, N., Song, H., Vakharia, V. N., Evensen, Ø., 2005a. Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence. J. Virol. 79, 9206-9216). Here, we utilized these viruses to investigate the gene expression of VP5 in vitro. Our results indicate that a 15-kDa VP5 is produced in rNVI15-infected cells, albeit at lower levels than in rNVI15-15K-infected cells, suggesting that the opal stop codon at nt 427 is suppressed. Furthermore, to examine translational suppression of the opal stop codon in VP5 gene, we constructed plasmids containing VP5-specific sequence and employed a yeast-based bicistronic dual-luciferase reporter system (Harger, J.W., Dinman, J.D., 2003. An in vivo dual-luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae. RNA 9, 1019-1024). Our results demonstrate that the VP5 sequence (with or without a stop codon) yielded approximately 13% termination suppression and the efficiency is directly related to the base immediately 3' of the termination codon, C>A>U>G.

Animals↗
PubMed2006

Pollen-specific pectin methylesterase involved in pollen tube growth.

Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation.

Arabidopsis↗
PubMed2004

Target-selected mutagenesis of the rat.

The rat is one of the most extensively studied model organisms, and with its genome being sequenced, tools to manipulate gene function in vivo have become increasingly important. We here report proof of principle for target-selected mutagenesis as a reverse genetic or knockout approach for the rat.

Animals↗
PubMed2005

Conditional knockout mice to study alternative splicing in vivo.

Analysis of genomes has revealed that the total number of human genes is comparable to those of simpler organisms, and thus, the number of genes does not correlate with the complexity and functional diversity of different organisms. Multiple mechanisms, including alternative splicing, are believed to contribute to the molecular complexity in higher eukaryotes. Given the fact that more than half of human genes undergo alternative splicing, however, little is known about the biological relevance of most alternative splicing events and their regulatory mechanisms. Recent work has highlighted the power of reverse genetic approaches in addressing regulated splicing in animal models. Here, we focus on the conditional knockout approach adapted for splicing research with the intention to provide a general guide to the generation of mouse models to study regulated splicing in development and disease.

Alternative Splicing↗
PubMed2006

High-throughput zebrafish histology.

The morphological effects of mutation and disease are often critical to our understanding of normal and abnormal function. The power and popularity of zebrafish as a forward and reverse genetic vertebrate model system, combined with its small size, have made it an ideal model in which to study the genetics of histologically scorable phenotypes. The presence of multiple tissue types in this organism's small larvae also makes it a potentially important model for toxicological analysis. Studying histological phenotypes is greatly enhanced by high-throughput methods of histology. Here, we describe details of high-throughput histology of the zebrafish using larval arrays, along with recent advances in mold design and discussion of work in progress that will lead to easier ways for people in the field to more rapidly score phenotypes in arrays. These detailed descriptions, together with the troubleshooting guide, should enable any laboratory with ties to a histology facility to perform high-throughput histology of zebrafish.

Animals↗
PubMedDate not supplied

RNA interference and human disease.

The completion of the human genome project has left researchers searching for an efficient method to study gene function in mammalian cells. RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing (PTGS) mechanism mediated by double-stranded RNA (dsRNA). The dsRNA is processed into small duplex RNA molecules of approximately 21-22 nucleotides (nts) termed small interfering RNAs (siRNAs) by a RNase III enzyme called Dicer. Interaction of siRNAs with a multi-protein complex, termed the RNA-induced silencing complex (RISC), results in sequence specific association of the activated RISC complex with the cognate RNA transcript. This interaction leads to sequence-specific cleavage of the target transcript. Originally discovered in Caenorhabditis elegans, the study of RNAi in mammalian cells has blossomed in the last couple of years with the discovery that introduction of siRNA molecules directly into somatic mammalian cells circumvents the non-specific response vertebrate cells have against larger dsRNA molecules. Emerging as a powerful tool for reverse genetic analysis, RNAi is rapidly being applied to study the function of many genes associated with human disease, in particular those associated with oncogenesis and infectious disease. This review summarizes the mechanism of RNAi and provides an overview of its current applications in medicine.

Animals↗