Browse
Submit Data
Databases
API
Help

Dataset Information

17 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Application of cross-priming amplification (CPA) for detection of fowl adenovirus (FAdV) strains.

ABSTRACT: Fowl adenoviruses (FAdVs) are widely distributed among chickens. Detection of FAdVs is mainly accomplished by virus isolation, serological assays, various polymerase chain reaction (PCR) assays, and loop-mediated isothermal amplification (LAMP). To increase the diagnostic capacity of currently applied techniques, cross-priming amplification (CPA) for the detection of the FAdV hexon gene was developed. The single CPA assay was optimised to detect all serotypes 1-8a-8b-11 representing the species Fowl aviadenovirus A-E. The optimal temperature and incubation time were determined to be 68 °C for 2 h. Using different incubation temperatures, it was possible to differentiate some FAdV serotypes. The results were recorded after addition of SYBR Green I(®) dye, which produced a greenish fluorescence under UV light. The CPA products separated by gel electrophoresis showed different "ladder-like" patterns for the different serotypes. The assay was specific for all serotypes of FAdV, and no cross-reactivity was observed with members of the genus Atadenovirus, duck atadenovirus A (egg drop syndrome virus EDS-76 [EDSV]) or control samples containing Marek's disease virus (MDV), infectious laryngotracheitis virus (ILTV) or chicken anaemia virus (CAV). The results of the newly developed FAdV-CPA were compared with those of real-time PCR. The sensitivity of CPA was equal to that of real-time PCR and reached 10(-2.0) TCID50, but the CPA method was more rapid and cheaper than the PCR systems. CPA is a highly specific, sensitive, efficient, and rapid tool for detection of all FAdV serotypes. This is the first report on the application of CPA for detection of FAdV strains.

SUBMITTER: Niczyporuk JS

PROVIDER: S-EPMC4369288 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Fowl Adenovirus (FAdV) Recombination with Intertypic Crossovers in Genomes of FAdV-D and FAdV-E, Displaying Hybrid Serological Phenotypes.

Project description:After analyzing 27 new genomes from fowl adenovirus (FAdV) field isolates and so-far unsequenced prototypes, we report the first evidence for recombination in FAdVs. Recombination was confined to species FAdV-D and FAdV-E, accommodating the largest number of, and the intraspecies-wise most differentiated, types. The majority of detected events occurred in FAdV-E, involving segments with parental origin of all constitutive types. Together with the diversity of breakpoints, this suggests widespread recombination in this species. With possible constraints through species-specific genes and diversification patterns, the recombinogenic potential of FAdVs attains particular interest for inclusion body hepatitis (IBH), an important disease in chickens, caused by types from the recombination-prone species. Autonomously evolving, recombinant segments were associated with major sites under positive selection, among them the capsid protein hexon and fiber genes, the right-terminal ORFs 19, 25, and the ORF20/20A family. The observed mosaicism in genes indicated as targets of adaptive pressures points toward an immune evasion strategy. Intertypic hexon/fiber-recombinants demonstrated hybrid neutralization profiles, retrospectively explaining reported controversies on reference strains B3-A, T8-A, and X11-A. Furthermore, cross-neutralization supported sequence-based evidence for interdomain recombination in fiber and contributed to a tentatively new type. Overall, our findings challenge the purported uniformity of types responsible for IBH, urging more complete identification strategies for FAdVs. Finally, important consequences arise for in vivo studies investigating cross-protection against IBH.

| S-EPMC6950264 | biostudies-literature

Detection of fowl adenovirus D strains in wild birds in Poland by Loop-Mediated Isothermal Amplification (LAMP).

Project description:BackgroundThe present study on the role of strains of adenovirus in wildlife reservoirs, and their prevalence is under exploration. In several previous studies, the presence of adenovirus strains in wild birds has been investigated. Worldwide distribution and outbreaks of adenovirus infections have been reported by many authors. The present study investigated the prevalence of FAdVs in 317 samples of different bird species from the northwestern region of Poland. An applied specific, sensitive, and efficient, without cross-reactivity loop-mediated isothermal amplification (LAMP) method to gauge the prevalence of fowl adenovirus strains in wild birds was developed and used.ResultsThe method was based on the sequence of the loop L1 HVR1-4 region of the hexon gene of the FAdV genome reference strains FAdV-2 KT862805 (ANJ02325), FAdV-3 KT862807 (ANJ02399) and FAdV-11 KC750784 (AGK29904). The results obtained by LAMP were confirmed by real-time PCR. Among 317 samples obtained from wild birds, eight FAdV isolates (2.52%) were identified and produced a cytopathic effect (CPE) in chicken embryo kidney cells (CEK). Three FAdV types belonging to species Fowl adenovirus D were detected, which were isolated from three adenovirus types 2/3/11, and have been confirmed in three mute swans (Cygnus olor), three wild ducks (Anas platyrhynchos), one owl (Strigiformes), and one common wood pigeon (Columba palumbus).ConclusionsThis study provides the first accurate quantitative data for the replication of fowl adenovirus strains in wild birds in Poland, indicating adenovirus interspecies transmission, and demonstrating the circulation of FAdVs in wild birds.

| S-EPMC7023798 | biostudies-literature

Establishment and application of cross-priming isothermal amplification coupled with lateral flow dipstick (CPA-LFD) for rapid and specific detection of red-spotted grouper nervous necrosis virus.

Project description:Red-spotted grouper nervous necrosis virus (RGNNV) is an important pathogen that causes diseases in many species of fish in marine aquaculture. The larvae and juveniles are more easily infected by RGNNV and the cumulative mortality is as high as 100 % after being infected with RGNNV. This virus imposes a serious threat to aquaculture of grouper fry. This study aimed to establish a simple, accurate and highly sensitive method for rapid detection of RGNNV on the spot.In this study, the primers specifically targeting RGNNV were designed and cross-priming isothermal amplification (CPA) system was established. The product amplified by CPA was detected through visualization with lateral flow dipstick (LFD). Three important parameters, including the amplification temperature, the concentration of dNTPs and the concentration of Mg(2+) for the CPA system, were optimized. The sensitivity and specificity of this method for RGNNV were tested and compared with those of the conventional RT-PCR and real-time quantitative RT-PCR (qRT-PCR).The optimized conditions for the CPA amplification system were determined as follows: the optimal amplification temperature, the optimized concentration of dNTPs and the concentration for Mg(2+) were 69 °C, 1.2 mmol/L and 5 mmol/L, respectively. The lowest limit of detection (LLOD) of this method for RGNNV was 10(1) copies/?L of RNA sample, which was 10 times lower than that of conventional RT-PCR and comparable to that of RT-qPCR. This method was specific for RGNNV in combination with SJNNV and had no cross-reactions with 8 types of virus and bacterial strains tested. This method was successfully applied to detect RGNNV in fish samples.This study established a CPA-LFD method for detection of RGNNV. This method is simple and rapid with high sensitivity and good specificity and can be widely applied for rapid detection of this virus on the spot.

| S-EPMC4583742 | biostudies-literature

A novel recombinase polymerase amplification assay for rapid detection of epidemic fowl adenovirus.

Project description:Fowl adenovirus (FAdV) has posed a grave threat to the health of poultry, and the sudden outbreak highlights the importance of the new rapid diagnostic method for the control and prevention of transmission. Hence, in the present study, a novel recombinase polymerase amplification (RPA) assay, which was suitable for all 12 serotypes (FAdV-1 to 8a and 8b to 11) had been successfully launched to detect FAdV. Also, the entire amplification process could be completed in the isothermal condition when temperature ranged from 26 to 42°C within no more than 14 min, which was remarkably superior to endpoint polymerase chain reaction (98 min) with the same detecting sensitivity (as low as 0.1 fg viral DNA), avoiding sophisticated thermal cyclers with simple operation. Additionally, the same primers did not produce positive reactions with other viruses tested, demonstrating that the specificity of the RPA assay was acceptable. Moreover, this developed method could be efficiently used in the diagnosis of FAdV references and epidemic strains from different avian origins, thus making it a rapid, reliable, and point-of-care FAdV diagnostics tool, as well as an alternative to endpoint PCR.

| S-EPMC7449135 | biostudies-literature

Serological and Pathogenic Analyses of Fowl Adenovirus Serotype 4 (FAdV-4) Strain in Muscovy Ducks.

Project description:Hydropericardium hepatitis syndrome (HHS) is a lethal disease caused by Fowl adenovirus serotype 4(FAdV-4) that mainly infects 3- to 6-week-old broiler chicks. In 2015, an infectious disease characterized similar symptom to HHS in broilers outbroke in commercial duck flocks in Shandong province. FAdV-4 was isolated from naturally infected ducks and determined by polymerase chain reaction (PCR) amplification and DNA sequence analysis. In order to investigate the effect of FAdV-4 infection on muscovy ducks, we determined and characterized the FAdV-4 Isolate, and assessed its pathogenicity. In this study, HHS was respectively reproduced in 5-week-old muscovy duck by intramuscular injection and intranasal inoculation of allantoic fluid containing FAdV-4, ducks in the negative control group were inoculated with allantoic fluids of healthy duck embryos in the same manner. Clinical symptoms, gross and microscopic lesions, cytokines and antibodies, blood biochemical indices were detected and recorded for 12 days after infection. Typical hydropericardium and hepatitis was observed in experimental muscovy duck in the 3rd day post-inoculation (dpi). FAdV-4 can be replicated in tissues and cause pathological damage, especially in the liver and immune organs. Most of the immune-related cytokines and antibodies levels are up-regulated and then decreased, which may be caused by the initial infection and the normal immune response, later the virus caused the immunosuppression and led to the decrease of levels. To the best of our knowledge, this is the first systematic trial of the pathogenicity of FAdV-4 in muscovy ducks mainly based on the serological test, which will provide new insights into the study of the disease.

| S-EPMC5996943 | biostudies-literature

Insights into LMH cells response to fowl adenovirus serotype 4 (FAdV-4) infection by transcriptome analysis

Project description:To better understand the interactions between host and FAdV-4 infection at transcription level, and to explore the pathogenesis of this virus, a high-throughput RNA-seq technology was utilized in white leghorn male hepatocellular (LMH) cells at 12, 24, and 48 hours after FAdV-4 infection.

2017-11-14 | GSE106839 | GEO

Fowl Adenovirus 4 (FAdV-4)-Based Infectious Clone for Vaccine Vector Development and Viral Gene Function Studies.

Project description:Fowl adenovirus 4 (FAdV-4) is associated with economically important poultry diseases. Recent studies of fully sequenced genomes of FAdV-4 isolates suggest potential genomic regions associated with virulence and amenable for manipulation and vector development. Direct manipulation of viral genomes is cumbersome, as opposed to that of infectious clones-viral genomes cloned into plasmid or cosmid vectors. In this work, we generated an infectious clone, pFAdV-4 ON1, containing the entire viral genome of a nonpathogenic FAdV-4 (ON1 isolate). pFAdV-4 ON1 was used for targeted deletion of open reading frames (ORFs) 16 and 17 and replacement with the enhanced green fluorescence protein (EGFP) expression cassette to generate recombinant viruses. These viruses were viable, and EGFP was expressed in infected cells. Their replication, however, was significantly reduced with respect to that of the wild-type virus. These observations suggest the potential utility of FAdV-4 as a vaccine vector and the importance of ORFs 16 and 17 for virus replication at wild-type levels. To our knowledge, this is the first report of an infectious clone based on the FAdV-4 genome, and our results demonstrate its utility for studies of virulence determinants and as a platform for either vaccine or gene delivery vectors.

| S-EPMC5850404 | biostudies-literature