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 capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-µm-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

Genetic diversity of species Fowl aviadenovirus D and Fowl aviadenovirus E.

Complete genomes of eight reference strains representing different serotypes within the species Fowl aviadenovirus D (FAdV-D) and Fowl aviadenovirus E (FAdV-E) were sequenced. The sequenced genomes of FAdV-D and FAdV-E members comprise 43 287 to 44 336 bp, and have a gene organization identical to that of an earlier sequenced FAdV-D member (strain A-2A). Highest diversity was noticed in the hexon and fiber genes and ORF19. All genomes sequenced in this study contain one fiber gene. Phylogenetic analyses and G+C content support the division of the genus Aviadenovirus into the currently recognized species. Our data also suggest that strain SR48 should be considered as FAdV-11 instead of FAdV-2 and similarly strain HG as FAdV-8b. The present results complete the list of genome sequences of reference strains representing all serotypes in species FAdV-D and FAdV-E.

C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells.

The infection of chickens with avian Hepatitis E virus (avian HEV) can be asymptomatic or induces clinical signs characterized by increased mortality and decreased egg production in adult birds. Due to the lack of an efficient cell culture system for avian HEV, the interaction between virus and host cells is still barely understood. In this study, four truncated avian HEV capsid proteins (ORF2-1 - ORF2-4) with an identical 338aa deletion at the N-terminus and gradual deletions from 0, 42, 99 and 136aa at the C-terminus, respectively, were expressed and used to map the possible binding site within avian HEV capsid protein. Results from the binding assay showed that three truncated capsid proteins attached to avian LMH cells, but did not penetrate into cells. However, the shortest construct, ORF2-4, lost the capability of binding to cells suggesting that the presence of amino acids 471 to 507 of the capsid protein is crucial for the attachment. The construct ORF2-3 (aa339-507) was used to study the potential binding of avian HEV capsid protein to human and other avian species. It could be demonstrated that ORF2-3 was capable of binding to QT-35 cells from Japanese quail and human HepG2 cells but failed to bind to P815 cells. Additionally, chicken serum raised against ORF2-3 successfully blocked the binding to LMH cells. Treatment with heparin sodium salt or sodium chlorate significantly reduced binding of ORF2-3 to LMH cells. However, heparinase II treatment of LMH cells had no effect on binding of the ORF2-3 construct, suggesting a possible distinct attachment mechanism of avian as compared to human HEV. For the first time, interactions between avian HEV capsid protein and host cells were investigated demonstrating that aa471 to 507 of the capsid protein are needed to facilitate interaction with different kind of cells from different species.

Detailed molecular analyses of the hexon loop-1 and fibers of fowl aviadenoviruses reveal new insights into the antigenic relationship and confirm that specific genotypes are involved in field outbreaks of inclusion body hepatitis.

Forty-eight fowl aviadenoviruses (FAdVs) isolated from recent IBH outbreaks across Europe were investigated, by utilizing for the first time the two major adenoviral antigenic domains, hexon loop-1 and fiber, for compound molecular characterization of IBH-associated FAdVs. Successful target gene amplification, following virus isolation in cell culture or from FTA-card samples, demonstrated presence of FAdVs in all cases indicative for IBH. Based on hexon loop-1 analysis, 31 European field isolates exhibited highest nucleotide identity (>97.2%) to reference strains FAdV-2 or -11 representing FAdV-D, while 16 and one European isolates shared >96.0% nucleotide identity with FAdV-8a and -8b, or FAdV-7, the prototype strains representing FAdV-E. These results extend recognition of specific FAdV-D and FAdV-E affiliate genotypes as causative agents of IBH to the European continent. In all isolates, species specificity determined by fiber gene analysis correlated with hexon-based typing. A threshold of 72.0% intraspecies nucleotide identity between fibers from investigated prototype and field strains corresponded with demarcation criteria proposed for hexon, suggesting fiber-based analysis as a complementary tool for molecular FAdV typing. A limited number of strains exhibited inconsistencies between hexon and fiber subclustering, indicating potential constraints for single-gene based typing of those FAdVs. Within FAdV-D, field isolate fibers shared a high degree of nucleotide (>96.7%) and aa (>95.8%) identity, while FAdV-E field isolate fibers displayed greater nucleotide divergence of up to 22.6%, resulting in lower aa identities of >81.7%. Furthermore, comparison with FAdVs from IBH outbreaks outside Europe revealed close genetic relationship in the fiber, independent of the strains' geographic origin.

Complete genome sequences of pigeon adenovirus 1 and duck adenovirus 2 extend the number of species within the genus Aviadenovirus.

Complete genomes of the first isolates of pigeon adenovirus 1 (PiAdV-1) and Muscovy duck adenovirus (duck adenovirus 2, DAdV-2) were sequenced. The PiAdV-1 genome is 45,480bp long, and has a gene organization most similar to turkey adenovirus 1. Near the left end of the genome, it lacks ORF0, ORF1A, ORF1B and ORF1C, and possesses ORF52, whereas six novel genes were found near the right end. The DAdV-2 genome is 43,734bp long, and has a gene organization similar to that of goose adenovirus 4 (GoAdV-4). It lacks ORF51, ORF1C and ORF54, and possesses ORF55A and five other novel genes. PiAdV-1 and DAdV-2 genomes contain two and one fiber genes, respectively. Genome organization, G+C content, molecular phylogeny and host type confirm the need to establish two novel species (Pigeon aviadenovirus A and Duck aviadenovirus B) within the genus Aviadenovirus. Phylogenetic data show that DAdV-2 is most closely related to GoAdV-4.

Recombinant FAdV-4 fiber-2 protein protects chickens against hepatitis-hydropericardium syndrome (HHS).

Virulent fowl adenovirus (FAdV) serotype 4 strains are the etiological agents of hepatitis-hydropericardium syndrome (HHS), a highly infectious disease in chickens with severe economic impact. In the present study, three different FAdV-4 derived capsid proteins, fiber-1, fiber-2, and hexon loop-1, were expressed in a baculovirus system and tested for their capacity to induce protection in chickens. Purified recombinant proteins were administered to day-old specific pathogen-free (SPF) chickens allocated in three separate groups and challenged with virulent FAdV-4 at 21 days of life. Two additional groups served as controls, a challenge control group with mock-vaccinated but infected birds and a negative control group with PBS injection substituting both vaccination and challenge. The fiber-2 vaccinated group displayed high resistance against the adverse effects of the challenge with only one dead bird out of 28, as compared to the challenge control group where the infection caused 78% mortality. A moderate protective effect resulting in 38% mortality was observed for fiber-1, whereas the hexon loop-1 vaccinated group was not effectively protected as manifested by 73% mortality. While a fiber-2 specific ELISA showed a gradual antibody increase after immunization of birds with the homologous protein, a commercial ELISA did not detect vaccination-induced antibodies in any of the groups but displayed a difference in challenge virus-directed response in protected and non-protected birds. Although immunoblotting confirmed the presence of specific antibodies in all vaccinated groups, the anti-protein sera did not exhibit neutralizing activity. Fecal excretion of challenge virus DNA was detected with a real-time PCR in the majority of tested birds until termination of the study independent of protection, indicating the prevention of clinical symptoms, but not infection, by vaccination. In conclusion, recombinant fiber-2 was identified as a protective immunogen and is proposed as an attractive candidate for a subunit vaccine to prevent hepatitis-hydropericardium syndrome in chickens.

Whole-genome sequences of two turkey adenovirus types reveal the existence of two unknown lineages that merit the establishment of novel species within the genus Aviadenovirus.

There are eight species established for aviadenoviruses: Fowl adenovirus A-E, Goose adenovirus A, Falcon adenovirus A and Turkey adenovirus B. The aim of this study was to sequence and analyse the complete genomes of turkey adenovirus 4 (TAdV-4) and TAdV-5 (strain 1277BT) in addition to almost two-thirds of the genome of another TAdV-5 strain (strain D1648). By applying next-generation sequencing, the full genomes were found to be 42 940 and 43 686 bp and the G+C content was 48.5 and 51.6 mol% for TAdV-4 and TAdV-5, respectively. One fiber gene was identified in TAdV-4, whereas two fiber genes were found in TAdV-5. The genome organization of TAdV-4 resembled that of fowl adenovirus 5 (FAdV-5), but it had ORF1C near the left end of the genome. TAdV-4 also had five 123 bp tandem repeats followed by five 33 bp tandem repeats, but they occurred before and not after ORF8, as in several fowl adenoviruses. The genome organization of TAdV-5 was almost the same as that of FAdV-1 but with a possible difference in the splicing pattern of ORF11 and ORF26. Phylogenetic analyses and G+C content showed differences that seem to merit the establishment of two new species within the genus Aviadenovirus: Turkey adenovirus C (for TAdV-4) and Turkey adenovirus D (for TAdV-5). Our analyses suggest a common evolutionary origin of TAdV-5 and FAdV-1.

The first whole genome sequence of a Fowl adenovirus B strain enables interspecies comparisons within the genus Aviadenovirus.

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 45,781 bp 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.

Species determination of fowl adenoviruses based on the 52K gene region.

In the present study, the classification of fowl adenoviruses (FAdVs) based on a part of the 52K gene region was described. A total of 44 FAdV field samples from different countries and sources were detected using a recently developed SYBR Green-based real-time PCR. Amplified products were sequenced, and phylogenetic analyses were conducted on the basis of the 116-bp region. For comparison, the already published sequences of the 52K gene region of aviadenoviruses were used in the analyses. The phylogenetic analysis allowed the grouping of the FAdVs into the established five different FAdV species: Fowl adenovirus A to Fowl adenovirus E. The existence of the species was supported by high bootstrap values (> 70%). This method provides the advantages of quantitation and high sensitivity for FAdV detection in combination with species assignment.

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

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.

Real-time PCR assay for universal detection and quantitation of all five species of fowl adenoviruses (FAdV-A to FAdV-E).

The present study describes the development of a SYBR Green based real-time polymerase chain reaction (real-time PCR) for detection and quantitation of all fowl adenovirus (FAdV) species. Primers were designed based on conserved nucleotide sequences within the 52K gene. Ten-fold serial dilutions of a vector DNA were used as standard for quantitation. The real-time PCR had an efficiency of 98%, a regression squared value of 0.999 and showed a range of 6.73-6.73×10(8) copies of FAdV DNA per reaction. The assay was highly specific for FAdVs and an exact quantitation of all 5 FAdV species (FAdV-A to FAdV-E) could be demonstrated. It was shown, that twelve FAdV serotypes (FAdV-1 to 8a, and 8b to 11) were detectable and quantifiable. Other viral genomes as well as uninfected chicken embryo liver (CEL) cells did not produce positive signal. Cloacal swabs were taken during the animal experiment, which was performed with all FAdV species. Shedding of FAdVs was investigated in cell culture, by conventional PCR and by the developed real-time PCR. The real-time PCR was found more sensitive than cell culture and conventional PCR. Detection and quantitation of FAdVs in different type of samples was possible by the new real-time PCR.

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

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.

TaqMan real-time reverse transcription-PCR assay for universal detection and quantification of avian hepatitis E virus from clinical samples in the presence of a heterologous internal control RNA.

Avian hepatitis E virus (HEV) isolates could be separated into at least three genotypes. In this study, the development of the first duplex TaqMan real-time reverse transcription-PCR (RT-PCR) assay for detection and quantification of avian HEV is presented. Primers and probes binding within relatively conserved open reading frame 3 (ORF3) were designed. Tenfold dilution series of in vitro-transcribed avian HEV RNA were used as the standard for quantification. A 712-bp region of the green fluorescent protein gene was transcribed in vitro and used as a heterologous internal control for both RNA isolation and real-time RT-PCR. The duplex real-time RT-PCR for avian HEV had an efficiency of 1.04, a regression squared value of 0.996, and a sensitivity of approximately 3.6 × 10(3) copies per reaction mixture when in vitro-transcribed RNA was used as the template. The presence of in vitro-transcribed heterologous internal control RNA did not affect amplification of avian HEV RNA compared to that achieved by the single assay. The sensitivity of the real-time RT-PCR assay was comparable to that of conventional RT-PCR, and it was shown to be highly specific, as tissues from uninfected chickens, mammalian HEVs, and other viral genomes did not produce positive signals. All tested field samples with virus belonging to different avian HEV genotypes were successfully detected with this new duplex TaqMan real-time RT-PCR assay.

Comparison of the fibers of Fowl adenovirus A serotype 1 isolates from chickens with gizzard erosions in Europe and apathogenic reference strains.

A total of 18 samples from 4 outbreaks of gizzard erosions in broiler chickens in Europe were used in the current study. Fowl adenoviruses were found in samples from all 4 outbreaks, and isolates were identified as Fowl adenovirus A (FAdV-A) serotype 1. As described earlier, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the long fiber gene was conducted. However, all 18 samples showed the same pattern as apathogenic FAdV-1 strains: Ote and chicken embryo lethal orphan (CELO) viruses. Nucleotide and amino acid sequences of the long and short fiber of several isolates from broiler chickens with gizzard erosions were analyzed, and 100% identity between the field isolates on the protein level was revealed. Only 1 nonsynonymous mutation (T→A) was present in the long fiber of studied isolates compared to the CELO strain. The same mutation was also present in the Ote strain. Four nonsynonymous mutations were present in the long fiber of studied isolates compared to Ote strain. In the short fiber, 6 nonsynonymous mutations were found in the studied isolates compared to the CELO strain. However, the short fiber of pathogenic isolates was 100% identical to apathogenic Ote strain. In conclusion, the usefulness of PCR-RFLP analysis of the long fiber gene of FAdV-1 isolates in distinguishing between those that induce gizzard erosions and those that do not remains questionable for the isolates obtained from European poultry flocks. The role of certain FAdV-1 strains with their long and short fiber in pathogenicity regarding gizzard erosions is still not clear.

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