a-laser-capture-microdissection-protocol-that-yields-high-quality-rna

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

University of Veterinary Medicine Vienna

Using a PCR that amplifies a region of the thymidine kinase (TK) gene, an epidemic spread of koi herpesvirus (KHV) was determined in koi carps in Austria in 2007. A total of 15 virus samples from different locations in Austria were analyzed to determine their genetic relatedness following PCR and nucleic acid sequencing of the open reading frame 40 (ORF40) region of the KHV genome. ORF40-specific PCR amplification products that were obtained from tissue samples shared 100% nucleotide sequence identity with the published sequence of the Japanese strain of KHV. The ORF40 sequence of one isolate from the UK that was included in the present study was 100% identical with the published sequence of an Israeli strain of KHV. This is the first study that used a larger number of samples and a PCR method, which allowed distinguishing all 3 strains of KHV. The present investigation provides information on the epidemiology of KHV infections in Europe and describes a useful molecular tool for epidemiological studies.

Characterization of Austrian koi herpesvirus samples based on the ORF40 region.

A total of 44 fowl adenovirus (FAdV) samples from 6 European countries, Pakistan, India, Kuwait, Mexico, Peru and Ecuador were used in this study and the phylogenetic analyses based on the loop 1 (L1) region of hexon gene were performed. For comparison, available hexon sequences of representatives of different FAdV species were also used. At least 12 genotypes within the five FAdV species (A-E) were revealed and the existence of these genotypes was supported by high bootstrap values. Furthermore, three primer pairs binding to the conserved pedestal regions (HexL1s/HexL1as and HexA/HexB) and pedestal (P1) region and loop 2 (L2) region (HexF1/HexR1) of the FAdV hexon gene were used for high-resolution melting (HRM)-curve analysis and results were compared with those of phylogenetic analyses. HRM-curve analysis based on the HexL1s/HexL1as region grouped all tested field isolates and reference strains into 22 subgroups, consistently with phylogenetic analysis. This method is a rapid and cost-effective alternative to existing serotype identification methods and offers a possibility to classify FAdV isolates more precisely. However, it has limitations such as need for extensive interpretation of results and potential for indeterminate results. Gaining of hexon sequences of further field isolates offers the potential for novel and additional information in analysis of the molecular epidemiology of FAdV.

Classification of fowl adenoviruses by use of phylogenetic analysis and high-resolution melting-curve analysis of the hexon L1 gene region.

The complete genome or the genome region containing the two fiber genes of two reference strains and one field isolate representing both serotypes of Fowl adenovirus C were sequenced. Two fiber genes were revealed in the genomes of all three isolates. Fiber-1 and fiber-2 genes of several Fowl adenovirus C isolates were sequenced as well. Both serotypes 4 and 10 have two fiber genes. The genome region containing the fiber gene was also sequenced for the reference strain of Fowl adenovirus B. Just one fiber gene was revealed in this strain. Predicted amino acid sequences were compared to already published fiber sequences of different adenovirus isolates and one amino acid substitution within fiber-2 was detected in all of the Fowl adenovirus C isolates that were isolated from chickens with hepatitis-hydropericardium syndrome in comparison to apathogenic isolates. Phylogenetic analyses provided insights about the evolution of fiber genes in avian adenoviruses and their genetic relationships.

Two fiber genes of nearly equal lengths are a common and distinctive feature of Fowl adenovirus C members.

The present report documents an outbreak of adenoviral gizzard erosion in 22 broiler flocks in Germany. The clinical picture was characterized by uneven growth of affected broilers that resulted in considerably lower than average weight at slaughtering. Fowl adenovirus serotype 1 (FAdV-1) was isolated from gizzard lesions and histological examinations demonstrated FAdV-1-positive intranuclear inclusion bodies in gizzard epithelial cells of affected broilers by in-situ hybridization. Birds from all affected flocks originated from one broiler breeder farm. During production of affected birds, broiler breeders were between 27 and 32 weeks old. Enzyme-linked immunosorbent assay and specific virus neutralization assay of sera from parent birds demonstrated an acute FAdV-1 infection within the first 5 weeks of the production cycle. Clinically, broiler breeders exhibited a moderate fall in the hatchability of their chicks, while egg production remained normal. No further clinical signs could be observed. Genetically identical FAdV-1 strains were isolated from gizzards of embryos at the lowest point of hatchability and from affected broiler flocks raised on independent farms. For the first time, direct detection of viable FAdV-1 from gizzards of embryos and progenies of one FAdV-1-seropositive broiler breeder farm in the course of an outbreak of adenoviral gizzard erosion could be demonstrated, highlighting the importance of vertical transmission of this disease. Additionally, growth retardation and subsequent reduced average weight at the time of slaughter of broiler chickens underline the economic impact of adenoviral gizzard erosion for poultry production.

Vertical transmission and clinical signs in broiler breeders and broilers experiencing adenoviral gizzard erosion.

Fowl adenoviruses (FAdVs) are grouped into five different species: Fowl adenovirus A through Fowl adenovirus E (FAdV-A to FAdV-E), and so far, complete nucleotide sequences are only available for the genomes of FAdV-A, FAdV-C, FAdV-D and FAdV-E members. The aim of this study was to sequence and analyze the complete genome of the reference strain representing FAdV-B (FAdV-5, strain 340). By applying Next Generation Sequencing, the genome was found to be 45,781bp long with 56.5% G+C content, and being very similar to the other FAdV strains. Genome organization and phylogenetic analyses confirmed the present division of the genus Aviadenovirus into species and the closer genetic relationship between FAdV-D and FAdV-E. In the left end region of the genome, homologues to the first predicted genes (ORF0-ORF12) described for FAdV-C (strain KR5) were present in the genome of strain 340, but ORFs 14B and 14C were absent. The central part of the strain 340 genome (IVa2-pVIII) showed an organization identical to that of other adenoviruses (but lacking the gene of protein V, which occurs only in mastadenoviruses). Just one fiber gene was identified. The right end region of the genome showed more heterogeneity. The predicted gene content within this region varies among aviadenoviruses, while the gene order and orientation of shared ORFs are conserved between different aviadenoviruses. With the completion of full genomes from all fowl adenovirus species, additional insights into the evolution of genus Aviadenovirus were obtained.

Ana Marek

Department of Biomedical Research

University of Veterinary Medicine Vienna

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

Ana Marek

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Research

University of Veterinary Medicine Vienna

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

Department of Biomedical Research