Serologic response against fowl poxvirus and reticuloendotheliosis virus after experimental and natural infections of chickens with fowl poxvirus.

Two infection studies in chickens were done to investigate the humoral immune response against fowl poxvirus (FPV) and reticuloendotheliosis virus (REV) after intradermal infection with different passages of a field isolate and with the vaccine strain HP B. The field isolate in a low passage carried the near-full-length REV provirus and induced antibodies to REV, but not to FPV. The vaccine strain carried only remnants of the long terminal repeat and induced antibodies against FPV, but not against REV. The field isolate lost the provirus after 36 passages in vitro, and it induced few antibodies against FPV and no antibodies against REV. Intravenous challenge with the low passage field isolate caused some antibody development against FPV in the birds that had previously been infected with the field isolate, but it caused no antibodies against REV in the previously vaccinated birds. REV proviral DNA was found in peripheral blood mononuclear cells of most birds that had been infected with the low passage field isolate. However, FPV DNA was found only once. The findings showed that the integrated REV provirus had an effect on the pathogenesis of fowlpox and that the tested vaccine strain is effective against FPV strains carrying REV provirus. Investigation of sera from FPV diseased flocks and flocks vaccinated against FPV showed a similar proportion of sera with antibodies against FPV. Sera from all diseased flocks but only from two of 10 vaccinated flocks had antibodies against REV. This indicated that the integrated REV provirus is common in FPV field strains.

Quantitative PCR as a tool to determine the reticuloendotheliosis virus-proviral load of fowl poxvirus.

Sequences of the reticuloendotheliosis virus (REV), an avian retrovirus, are integrated into the genome of fowl poxvirus (FPV). We developed and evaluated a quantitative multiplex real-time PCR (multiplex qPCR) assay to determine the REV-proviral load of FPV strains. Amplification efficiencies were 98.7% for the amplification of the FPV DNA and 88.7% for the amplification of REV-proviral DNA. The ratio between FPV DNA and REV-proviral DNA was calculated from the PCR efficiencies and the threshold cycle deviation of the unknown samples vs. a standard. The intraassay variation was determined by investigating triplets of different dilutions of the standard. The coefficient of variation between the threshold cycles was below 0.05 in all tested dilutions. The ratios of the triplet had a coefficient of variation of 0.201. Generally, the method overestimated the relative amount of REV-proviral DNA. Skin lesions from fowlpox outbreaks were investigated with the multiplex qPCR. The FPV:REV ratio was between 1:0.803 and 1:1.411 in samples with sufficient DNA to allow a conclusion. In addition, the investigation of cell culture material of several passages of a FPV field isolate showed a complete loss of the REV provirus after 36 passages. The loss rate of the REV provirus was approximately 50% per passage. In conclusion, we established the multiplex qPCR assay as a convenient and reliable method to determine the REV-proviral load of FPV. The first results we obtained with it show that it is of value for further investigations about the significance of the integration of the REV provirus into the genome of FPV.

Adenovirus of psittacine birds: investigations on isolation and development of a real-time polymerase chain reaction for specific detection.

Liver samples of psittacine birds with a histological suspicion of an adenovirus infection, confirmed by electron microscopy examination, were subjected to virus isolation attempts using a heterologous cell culture system and a homologous cell culture system in the form of chicken embryo liver cells and psittacine embryo fibroblasts, respectively. Whereas isolation in chicken embryo liver cells failed, virus was isolated successfully in the psittacine embryo fibroblasts cell culture system. Molecular investigations identified the virus as a specific psittacine adenovirus (PsAdV). Additionally, on the basis of the hexon gene sequence data obtained, a real-time polymerase chain reaction (PCR) for specific detection of PsAdV was developed. To ensure an exclusive hybridization with PsAdV, selected primers were located within the variable L1 region of the hexon gene. Furthermore, the specificity of the real-time PCR was confirmed by investigation of a panel of different avian adenoviruses and unrelated DNA viruses. Using this PCR, the threshold cycle values obtained support the propagation of PsAdV in the homologous cell culture system in comparison with the chicken cell culture system. Moreover, the developed PCR represents a reliable method for specific and sensitive detection of PsAdV in clinical samples.

Presence of avian pneumovirus type A in continental Europe during the 1980s.

Three isolates of avian pneumovirus (APV) were isolated in Germany during 1987 and 1988 from turkeys with clinical signs of turkey rhinotracheitis and one was isolated during 1990 from a broiler breeder flock with typical signs of swollen head syndrome. The isolates were typed using type-specific reverse transcriptase-polymerase chain reactions. The three isolates from turkeys were identified as type A, while the isolate from the broiler breeders was type B. During the late 1980s no APV live-virus vaccine was used in poultry flocks in Germany, which is indicative of the presence of both types at that time. Previous isolates detected from elsewhere in Europe during the 1980s had been only of type B.

[Epizootiology of fowls adenoviruses]. / Epizootiologische Untersuchungen über Hühneradenoviren.

Fowl adenoviruses of the serotype 4 from Germany were characterised by restriction enzyme analysis in comparison to isolates from Asia, South America and the FAV4 reference strain KR5. Only strain Da60 which was isolated from a psittacine aviary was identical with the reference strain KR5. None of the isolates was identical with the highly pathogenic strains from India and Ecuador. One-day-old chicks were infected orally and intramuscularly with the reference strain KR5, the psittacine isolate Da60 and isolate K1013 from Ecuador. Whereas no mortality was seen with the two strains KR5 and Da60, the mortality with K1013 was 100%. The main pathological signs were a swollen liver with necrosis and a lymphocyte depletion with a loss of the follicle structure. To investigate a second subject of avian adenovirus epizootiology several FAVs were characterized serologically and with PCR which was combined with the digestion of the PCR products. Including the reference strains, both methods were compared. It was shown that the digestion of the PCR products allows a clear attribution to a specific serotype, which underlines the usefulness of this method for diagnostic purposes.

Characterization of fowl adenoviruses from outbreaks of inclusion body hepatitis/hydropericardium syndrome in Chile.

Three fowl adenovirus (FAV) isolates (341, 344, and 215) obtained during 1996-97 from field outbreaks of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS) affecting broilers and broiler breeders in Chile were characterized by virus neutralization tests (VNTs) and restriction enzyme analysis of a DNA fragment. Furthermore, the pathologic characteristics of one of these FAV isolates (FAV 341) was studied in experimentally infected chickens. The VNTs conducted with isolates 341 and 344 against reference strains and antisera belonging to each of 12 FAV serotypes demonstrated a close antigenic relationship with strain KR5 of the FAV serotype 4. Polymerase chain reaction using the primers H3/H4 and subsequent HpaII digestion was used for serotype identification of isolates 341 and 215. The length of the PCR products and the restriction profiles of isolates 341, 215, and the reference strain KR5 (FAV4) were identical. The present results confirmed the classification of all three isolates as FAV4. The pathogenicity test with 1000 mean tissue infectious dose of isolate 341 inoculated intramuscularly in 20-day-old specific-pathogen-free chickens resulted in the death of 9% (two birds) six days postinoculation (PI). Both birds showed characteristic IBH/HPS gross and microscopic lesions; the remaining birds, sacrificed at day 10 PI, showed less severe lesions. On the basis of epidemiologic and experimental data of the virulence of Chilean FAV isolates, and the pathogenicity results with isolate 341, we speculate that Chilean FAV strains may require an association with other agents (immunosuppressive agents) to induce IBH/HPS outbreaks in the field.

Epidemiological studies on fowl adenoviruses isolated from cases of infectious hydropericardium.

Serology, restriction enzyme analysis and polymerase chain reactions were used to classify a total of 12 fowl adenoviruses (FAV) isolated from clinical cases of infectious hydropericardium from field outbreaks in seven countries in Asia and America. All isolates belonged to FAV serotype 4. Two isolates were contaminated with avian adeno-associated virus and one of them also contained FAV1. Minor differences were observed in the BamHI restriction profiles. More variability was seen with SmaI, BglII and PstI restriction profiles. However, more than 80% of the fragments were identical in size in the five different PstI profiles, indicating the close genomic relationship between the isolates. Polymerase chain reaction assays supported the classification of the isolates as FAV4 strains. All isolates could be detected using H1/H2 or H3/H4 primer pairs. Restriction enzyme analysis of the H1/H2 polymerase chain reaction (PCR) products allowed no differentiation between the isolates, whereas the three isolates from India and Pakistan could be separated from all others after HpaII digestion of the H3/H4 PCR products. Although strain variation was demonstrated, it could be shown that all adenoviruses isolated from various field cases of infectious hydropericardium (Angara Disease) in several countries belong to fowl adenovirus serotype 4.

Isolation of fowl adenoviruses serotype 4 from pigeons with hepatic necrosis.

Nine homogenized livers were taken to isolate the causative agent of adenovirus type I and type II infections in pigeons. The samples were passaged up to four times on primary chicken embryo hepatocytes. Adenoviruses were isolated from all of the six type II but none of the three type I infections. Serologically the isolated adenoviruses were classified as fowl adenovirus (FAV) serotype 4. Restriction enzyme analysis of two isolates in comparison with FAV4 reference strain KR5 confirmed the serological results and classification of the pigeon isolates as FAV4 strains.

[Adenovirus infections in psittacines]. / Adenovirusinfektion bei Psittaziden.

Diagnostic investigations were carried out in a fatal disease of several African Grey Parrots (Psittacus erithacus) and Cape Parrots (Poicephalus robustus) in a large breeding plant. Electron microscopically examination of liver and intestine, the organs with the most prominent pathomorphological changes, regularly revealed adenoviruses. Necrotizing hepatitis and catarrhal to haemorrhagic enteritis dominated on histopathological examination. Numerous basophilic intranuclear inclusion bodies in hepatocytes and enterocytes and their ultrastructural characteristics underline the etiological role of the detected adenoviruses. Adenoviruses were isolated from livers of three different birds and once from the intestine. Serologically the isolates were classified as fowl adenovirus serotype 4 (FAV4). Restriction enzyme analysis of two isolates showed the identity with the FAV4 reference strain KR5.

Growth analysis of adenoviruses isolated from pigeons in chicken cells and serological characterization of the isolates.

The susceptibility of chicken embryo liver (CEL) and chicken kidney (CK) cells to eight adenoviruses isolated from different pigeons were investigated. Isolation and propagation was most successful in CEL cells. The cytopathic effect, i.e. cell rounding and detachment of the cells was typical for adenoviruses. Titres of up to 10(6.6) plaque-forming units/ml could be reached on CEL cells. Transferring CEL-grown virus onto CK cells also resulted in a cytopathic effect but with much lower titres, whereas the isolation of pigeon adenoviruses on these cells was not successful. Antibodies against fowl adenovirus reference strains (FAV1-12) were used to serotype the isolates in neutralisation tests. Six were identified as FAV serotypes 2,5,6,10 and 12. Two isolates could not be typed. An antiserum produced in chickens against one of these untypable strains was able to neutralize both. The neutralization indexes of these strains were very similar, indicating that they are probably the same serotype. A cross neutralization test confirmed that this serotype is different from known fowl adenoviruses.

Some strains of serotype 4 fowl adenoviruses cause inclusion body hepatitis and hydropericardium syndrome in chickens.

Fowl adenoviruses were isolated and characterized from severe cases of hydropericardium syndrome in Ecuador and Pakistan. All were neutralized by antibodies against serotypes 4 and 11. Cross-neutralization tests and restriction enzyme analysis strengthen the classification as serotype 4 strains. The restriction endonucleases used allowed the differentiation among field isolates and reference strains. All field isolates tested induced high embryo mortality. One-day-old specific pathogen free (SPF) chicks were infected with 10(5) plaque-forming units by natural routes to reproduce the disease with plaque purified virus. Whereas no mortality was seen with the reference strain, the mortality with the field isolates was 100%. A reduced mortality occurred with a lower infectious dose. Field isolate K1013 from Ecuador was also highly pathogenic for 1- to 3-week-old SPF chicks after intramuscular inoculation. The main pathological signs were swollen livers, severe hydropericardium and nephritis, reflecting the field situation and underlining the role of FAV4 strains in the aetiology of infectious hydropericardium.

Identification and characterization of penton base and pVIII protein of egg drop syndrome virus.

The nucleotide sequence and location of the penton base and pVIII genes of the egg drop syndrome (EDS) virus an avian adenovirus, were determined. The penton base gene is located at 34.8-38.8 map units. The coding sequence has a length of 1359 nt and encodes a polypeptide of 452 amino acids (aa) with a molecular weight of 51 105 Da. The amino acid sequence shows a homology of 57.3 and 55.3% to the structural protein IH of human adenovirus serotype 2 (HAd2) and fowl adenovirus serotype 1 (FAV1). The penton base protein lacks the integrin binding motifs RGD (Arg-Gly-Asp) and LDV (Leu-Asp-Val). At 65.1-67.3 map units an open reading frame of 753 nucleotides was identified which encodes the structural protein pVIH. It forms a protein of 250 aa with an expected molecular weight of 28 501 Da. Possible protease cleavage sites were identified. Amino acid homologies of 30.8 and 27.7% were found to the HAd2 and FAV1 pVIII genes. Remarkably, the overall amino acid identity with ovine adenovirus pVIII is 52%. The start codons of both genes are shifted leftwards compared with the respective structural elements on the HAd2 or FAV1 genomes which indicates a different genomic organization of the EDS virus.