Transcriptome analysis of fowl adenovirus serotype 4 infection in chickens.

Fowl adenovirus serotype 4 (FAdV-4) is a causative agent of inclusion body hepatitis and hydropericardium-hepatitis syndrome. These diseases cause considerable economic losses in the global poultry industry and are significant stressors for infected chickens. However, the molecular mechanisms of FAdV-4 pathogenesis are poorly understood. In the present study, we identified differentially expressed genes from the livers of FAdV-4-infected chickens using RNA-seq at 7, 14 and 21 days after FAdV-4 infection. We identified 2395 differentially expressed genes at the three time points. These genes were enriched in variety of biological processes and pathways including PPAR and Notch signaling, cytokine-cytokine receptor interactions and Toll-like receptor signaling pathways. The transcriptional data were validated by quantitative real-time PCR. Our results will assist in the understanding of the molecular pathogenesis of FAdV-4 infection and for developing novel antiviral therapies.

A region at the left end of the fowl adenovirus 9 genome that is non-essential in vitro has consequences in vivo.

The regions at the left and right ends of fowl adenovirus (FAdV) genomes are not well-characterized in comparison to those of human adenoviruses. Using a series of deletion mutants, we analysed a 2.4 kb region near the left end of the FAdV-9 genome (nt 400-2782) that contains packaging-signal motifs VI and VII and open reading frames (ORFs) 0, 1, 1A, 1B, 1C and 2. Viable viruses with specific deletions in this region had wild-type characteristics in vitro, as measured by cytopathic effect, plaque morphology, virus titres and growth kinetics. However, one mutant (FAdV-9Delta4), which lacked these ORFs and retained the packaging motifs, did not replicate at wild-type levels in vivo, as judged in infected eggs by virus titres in allantoic fluid and in infected chickens by antibody responses, virus titres in faeces and virus genome copy numbers in tissues. These findings indicate that some of the ORFs in this region, although dispensable in vitro, are important for in vivo replication of FAdV-9.

☆DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus.

Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics.

Protein profile of FAdV-4 based on SDS-PAGE and Western blot isolated from chickens in India.

Fowl adenovirus-4 (FAdV-4) causes hydropericardium syndrome (HPS) in poultry worldwide. An understanding of viral structural protein composition is important for developing novel immunodiagnostics and immunoprophylactics. Here we report isolation, culture, molecular and protein profile of FAdV-4 isolates recovered from HPS outbreaks in chicken in the states of Himachal Pradesh and Tamil Nadu in India. We performed a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting-based protein profiling of FAdV-4 isolates against a reference FAdV-1 or Chicken Embryo Lethal Orphan (CELO) virus. SDS-PAGE analysis showed that seven protein bands in FAdV-4 isolates were similar to CELO expect an additional band of 110 kDa in CELO virus. On Western blotting, two protein fractions of 43 kDa and 78 kDa size were observed in FAdV-4 isolates. Overall, results show that FAdV-4 isolates recovered from different regions of the country had similar protein profile and possibly a common source of origin.

Replication cycle and associated biosynthetic reactions for a non-oncogenic avian adenovirus.

The replication cycle of the non-oncogenic fowl adenovirus serotype 10 (FAV-10) has been examined. The onset of viral DNA synthesis was shown to commence at about 10 h postinfection (hpi) defining the early period of viral replication as prior to this time and the late phase as that time following the initiation of DNA replication. Virus titre rapidly increased between 18 and 24 hpi with maximum virus yield between 28 and 30 hpi. The late phase transcription profiles of the FAV-10 genome from 10 hpi to 24 hpi were determined. Late translation of virus protein began about 14 hpi increasing rapidly between 18 and 30 hpi.

Growth characteristics of fowl adenovirus type 8 in a chicken hepatoma cell line.

Fowl adenoviruses, many of which appear to be non-pathogenic, are ubiquitous in birds. In addition, the genome of these viruses is large, making them ideal candidates for construction as vectors for foreign genes. Current methods to cultivate fowl adenoviruses use primary cell cultures derived from embryonated chicken eggs. In order to provide a more suitable culture method, the growth of fowl adenovirus type 8 (FAdV-8) was investigated in CH-SAH, a continuous hepatoma cell line. A one step growth curve demonstrated release of extracellular virus beginning by 18 h p.i. and with a final yield about 100 fold higher than that in chicken embryo liver cells. Viral DNA synthesis was first detected 8 h prior to this. The CH-SAH cell line supported the production of progeny viruses similar to the wild-type virus after being transfected with purified FAdV-8 DNA. This study demonstrated that the continuous hepatoma cell line is an appropriate in vitro host for FAdV-8.

Growth of avian adeno-associated virus in chicken cells transfected with fowl adenovirus serotype 1 DNA.

Using the chloroquine-modified calcium phosphate coprecipitation technique, fowl adenovirus serotype 1 (FAV 1) DNA transfects efficiently chicken cell cultures. Infection of FAV 1 DNA transfected cells with helper dependent avian adeno-associated virus (AAAV) results in the production of AAAV progeny, being detected by an indirect immunofluorescence assay. These findings indicate that FAV 1 DNA introduced into the host cell promotes actively the growth of AAAV.

Chicken embryo propagation of type I avian adenoviruses.

Forty-two clone-purified, cell-culture-propagated type I avian adenoviruses (AAV) representing 11 serotypes and two intermediate strains were evaluated for virus replication (evidenced by embryo death and lesions) resulting from the inoculation of specific-pathogen-free chicken embryos via the chorioallantoic sac or yolk sac. Commonly observed embryonic changes were death, stunting and curling, hepatitis, splenomegaly, congestion and hemorrhage of body parts, and urate formation in the kidneys. Basophilic or eosinophilic intranuclear inclusion bodies characteristic of fowl adenoviruses were observed in hepatocytes. The magnitude and relative uniformity of intra- and interserotypic embryo mortality, gross lesions, and virus titers was greater in embryos inoculated via the yolk sac. This work identifies the yolk sac as a practical and sensitive chicken embryo inoculation route for poultry diagnosticians to employ. It is suggested that the yolk sac may be a reliable alternative to cell culture for the successful isolation of all type I avian adenoviruses.

Effect of composting poultry carcasses on survival of exotic avian viruses: highly pathogenic avian influenza (HPAI) virus and adenovirus of egg drop syndrome-76.

Eight-week-old chickens were inoculated with one of two exotic viruses to determine the effect of composting on virus survival. Group 1 chickens were inoculated with highly pathogenic avian influenza (HPAI) virus via the caudal thoracic air sac. Group 2 chickens were inoculated with the adenovirus that causes egg drop syndrome-76 (EDS-76) by the oral route. Five days after inoculation, lung, trachea, and air sacs for HPAI and spleen, cecal tonsils, and bursa of Fabricius for EDS-76 were collected and composted with poultry carcasses. At the end of the first 10 days of composting, virus-isolation efforts showed that the HPAI virus had been inactivated, and only 1 of 20 tissue samples yielded the adenovirus of EDS-76. The viruses of HPAI and EDS-76 were completely inactivated at the end of the second 10-day period of the two-stage composting process. Control tissues collected at necropsy and frozen at -70 C for virus isolation were all positive for virus.

Physicochemical characterization and pathogenicity studies of two turkey adenovirus isolants.

Two turkey adenoviruses were isolated from poults with respiratory disease, and their physicochemical properties were studied. The virus particles were unenveloped. contained DNA genome, replicated within the nuclei of infected cells, and were icosahedral in shape. Pathogenicity studies in poults indicated that North Carolina Kennedy isolant was a pathogen of relatively low virulence whereas the North Carolina Jackson isolant was nonpathogenic in commercial turkey poults.

Antigenic analysis of several turkey respiratory adenoviruses by reciprocal-neutralization kinetics.

Five turkey adenoviruses were classified into four serologic groups by reciprocal-neutralization kinetics. Two viruses were found to be serologically homologous, while three others were found to be heterologous and placed in separate serologic groups.

Experimental infection of bobwhite quail with Indiana C adenovirus.

Bobwhite quails (Colinus virginianus) were inoculated intratracheally, intraperitoneally, or subcutaneously with Indiana C adenovirus at 1, 3, 6, or 9 weeks of age. Mortality rates were 33-100% in quails inoculated at 1 or 3 weeks of age and 0-10% in quails inoculated at 6 or 9 weeks of age. Gross and histologic lesions included necrotizing tracheitis and bronchitis with pneumonia, necrotizing hepatitis and splenitis, and lymphoid depletion of the bursa of Fabricius; these were consistent with quail bronchitis. Indiana C is highly pathogenic in bobwhite quails and cannot be recommended as a vaccine to prevent quail bronchitis.

Further studies on in vitro and in vivo assays of hemorrhagic enteritis virus (HEV).

Isolation of hemorrhagic enteritis virus (HEV) from spleens of infected turkeys in the MDTC-RP19 lymphoblastoid cell line was compared with detection of HEV antigen in the same spleens using the agar gel precipitation (AGP) test. A concordance of 80% was found between the two assays. Virus isolation had a sensitivity of 84% and a specificity of 88% compared with the AGP test. RP19 cells were also susceptible to infection with several other avian adenoviruses, but such infection was easily differentiated from that of HEV by a fluorescent-antibody (FA) test. Turkeys required 10(2) tissue-culture-infectious doses (TCID) to develop HE-specific lesions and 10(5) TCID to be killed. On the other hand, as little as 10 TCID of apathogenic HEV protected the poults against challenge with virulent HEV. The enzyme-linked immunosorbent assay (ELISA) for detection of HEV antibody was improved by using virus-infected RP19 cells as antigen. The ELISA appears to be more sensitive than the serum-neutralization test.

Variations in response of four lines of ring-necked pheasants to infection with marble spleen disease virus.

This study evaluated the response and susceptibility of four lines of ring-necked pheasants to infection with marble spleen disease (MSD) virus. Fifteen birds of the following lines were used: the standard ring-necked gamebird, the highly inbred jumbo white, the Sichuan, and the first-generation cross of the Sichuan and the standard gamebird. Baseline immune responses were assessed at 6 weeks of age by lymphoblastogenesis assays. All birds were orally challenged at 7 weeks of age with cell -culture-propagated MSD virus. Eight birds of each group were necropsied at 6-7 days post-inoculation (PI), and gross splenic lesions were recorded. Appropriate tissue sections were collected, fixed in 10% buffered formalin, and routinely processed for microscopic evaluation. The remaining birds were bled weekly from week 2 through week 8 Pl. Serum was tested for antibodies to MSD virus using a commercial enzyme-linked immunosorbent assay kit. Results of the lymphoblastogenesis assays and the gross and microscopic lesion incidences indicated that variations did exist between several lines evaluated. The jumbo white pheasant had significantly higher lymphoblastogenesis stimulation index and was more resistant to developing gross and histologic lesions than was the standard ring-necked gamebird.

A technique for isolating turkey respiratory adenoviruses.

Several turkey respiratory adenoviruses were isolated in turkey embryonic liver cells from nasal turbinate filtrate collected from clinically ill turkey poults during postmortem examination. Suspect adenovirus inoculum had to remain in cell culture for 5 to 7 days, and 9 blind passages were required before a sample was declared negative. Once isolated, the turkey adenoviruses adapted rapidly to turkey kidney cells.

Some properties of avian adenoviruses isolated from chickens with inclusion body hepatitis in Japan.

Outbreaks of inclusion body hepatitis were observed in broiler chickens on a poultry farm during 3 years. Avian adenovirus-like agents were isolated during these years from livers of diseased chickens. Round-cell-type cytopathogenic effect and intranuclear inclusion bodies were produced in chicken kidney cell cultures inoculated with these agents. Properties of the agents were as follows: resistant to ether, chloroform, socium deoxycholate, trypsin, heating at 50 C, and pH 3.0; sensitive to 5-iodo-deoxyuridine; and pathogenic to chicken embryos. From these properties and ultrastructural findings of the agents, these were identified as avian adenovirus. Day-old commercial chicks were insusceptible to these viruses. Maternal antibody levels in commercial chicks were considerable. Surveys for neutralizing index to the virus were performed on chickens in the field, and all sera tested were positive. Electron-microscope examination showed that these viruses contained avian-adenovirus-associated virus.

Avian cell cultures grown in an open system (dishes) without CO2 incubator, and their use for virus cultivation.

An organic buffer, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), in a culture medium made it possible to culture chick embryo fibroblast (CEF), duck embryo fibroblast (DEF), and chick kidney (CK) cells in an open system (dishes) under incubation in a bacteriological incubator, requiring no CO2 incubation. CEF, DEF, and CK cells cultured with this system were suitable for cultivation of avain viruses such as Newcastle disease virus, CELO virus, Marek's disease virus, and turkey herpesvirus.

Characterization of two strains of fowl adenovirus type 1.

An adenovirus isolated from the trachea of a chicken with respiratory disease was compared with an isolate obtained from the trachea of a clinically normal chicken. The isolates caused similar respiratory disease experimentally but differed in plaque morphology and fluorescent-antibody reactions in infected chick embryos.

An adenovirus from a turkey pathogenic to both chicks and turkey poults.

An adenovirus, designated T-75 isolant, was isolated from the cloacal swab of a clinically normal turkey, 13 weeks old. The T-75 isolant was identified as an adenovirus on the basis of physicochemical properties, cytopathology, and agar-gel precipitin test. Producing two-way cross-neutralization reactions with CELO virus, the isolant was classified as an avian adenovirus of serotype 1. The T-75 isolant was pathogenic to both chicks and turkey poults, causing hepatitis, respiratory disease, atrophy of the bursa of Fabricius, and/or growth depression of experimentally inoculated birds, varying with the host and route of inoculation.

Biological characterisation of an Indian isolate of egg drop syndrome-76 virus.

An isolate of egg drop syndrome-76 virus replicated best in primary chicken embryo liver cells and less well in duck embryo liver cells, duck embryo fibroblast cells and chicken embryo kidney cells. The cytopathic effect in chicken embryo liver cells was marked by the presence of round and refractile cells and detachment of cells from the glass surface. The intranuclear eosinophilic inclusion bodies were observed by 24 to 48 hours after infection. No virus multiplication was observed in primary quail embryo fibroblast cells, chicken embryo fibroblast cells or mammalian cells like Vero, BHK-21 and MDBK. Duck embryos supported the maximum growth of the virus, with allantoic fluid having the highest haemagglutinin titre, followed in order by chorioallantoic membrane, skin and internal organs. Chicken and quail embryos did not support the growth of the virus.