Chick Embryo Lethal Orphan Virus Research Articles

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.

Structure of the C-terminal head domain of the fowl adenovirus type 1 short fibre

There are more than 100 known adenovirus serotypes, including 50 human serotypes. They can infect all 5 major vertebrate classes but only Aviadenovirus infecting birds and Mastadenovirus infecting mammals have been well studied. CELO (chicken embryo lethal orphan) adenovirus is responsible for mild respiratory pathologies in birds. Most studies on CELO virus have focussed on its genome sequence and organisation whereas the structural work on CELO proteins has only recently started. Contrary to most adenoviruses, the vertices of CELO virus reveal pentons with two fibres of different lengths. The distal parts (or head) of those fibres are involved in cellular receptor binding. Here we have determined the atomic structure of the short-fibre head of CELO (amino acids 201–410) at 2.0 Å resolution. Despite low sequence identity, this structure is conserved compared to the other adenovirus fibre heads. We have used the existing CELO long-fibre head structure and the one we show here for a structure-based alignment of 11 known adenovirus fibre heads which was subsequently used for the construction of an evolutionary tree. Both the fibre head sequence and structural alignments suggest that enteric human group F adenovirus 41 (short fibre) is closer to the CELO fibre heads than the canine CAdV-2 fibre head, that lies closer to the human virus fibre heads.

The Helper Activities of Different Avian Viruses for Propagation of Recombinant Avian Adeno-Associated Virus

To compare the helper activities of different avian viruses for propagation of recombinant avian adeno-associated virus (rAAAV), AAV-293 cells were cotransfected with the AAAV vector pAITR-GFP containing green fluorescent protein (GFP) gene, the AAAV helper vector pcDNA-ARC expressing the rep and cap genes, and the adenovirus helper vector pHelper expressing Ad5 E2A,E4, and VA-RNA genes. Chicken embryonic fibroblast (CEF) or chicken embryonic liver (CEL) cells were cotransfected with the AAAV vector and the AAAV helper vector, followed by infection with Marek's disease virus (MDV), avian adenovirus, chicken embryo lethal orphan (CELO) virus or infectious bursal disease virus (IBDV). Infectious rAAAV particles generated by the two strategies were harvested and titrated on CEF and CEL cells. A significantly higher viral titer was obtained with the helper activity provided by the pHelper vector than by MDV or CELO virus. Further experiments showed that rAAAV-mediated green fluorescent protein (gfp) expression was overtly enhanced by MDV or CELO virus super infection or treatment with sodium butyric acid, but not by IBDV super infection. These data demonstrated that MDV and CELO viruses could provide weak helper activity for propagation of rAAAV, and rAAAV-mediated transgene expression could be enhanced by super infection with the helper viruses.

Structure of the C-terminal head domain of the fowl adenovirus type 1 long fiber

Avian adenovirus CELO (chicken embryo lethal orphan virus, fowl adenovirus type 1) incorporates two different homotrimeric fiber proteins extending from the same penton base: a long fiber (designated fiber 1) and a short fiber (designated fiber 2). The short fibers extend straight outwards from the viral vertices, whilst the long fibers emerge at an angle. In contrast to the short fiber, which binds an unknown avian receptor and has been shown to be essential to the invasiveness of this virus, the long fiber appears to be unnecessary for infection in birds. Both fibers contain a short N-terminal virus-binding peptide, a slender shaft domain and a globular C-terminal head domain; the head domain, by analogy with human adenoviruses, is likely to be involved mainly in receptor binding. This study reports the high-resolution crystal structure of the head domain of the long fiber, solved using single isomorphous replacement (using anomalous signal) and refined against data at 1.6 A (0.16 nm) resolution. The C-terminal globular head domain had an anti-parallel beta-sandwich fold formed by two four-stranded beta-sheets with the same overall topology as human adenovirus fiber heads. The presence in the sequence of characteristic repeats N-terminal to the head domain suggests that the shaft domain contains a triple beta-spiral structure. Implications of the structure for the function and stability of the avian adenovirus long fiber protein are discussed; notably, the structure suggests a different mode of binding to the coxsackievirus and adenovirus receptor from that proposed for the human adenovirus fiber heads.

Nanoporous crystals of chicken embryo lethal orphan (CELO) adenovirus major coat protein, hexon

CELO (chicken embryo lethal orphan) virus is an avian adenovirus that is being developed as a gene transfer vector. Its trimeric major coat protein (942 residues, 106,709 Da) has 42% sequence identity to human adenovirus type 2 (AdH2) hexon and 45% to AdH5 hexon. For structural studies, the growth of CELO virus has been optimized, and its hexon purified and crystallized. The hexon crystals, the first non-human example, diffract to 3.9 Å resolution. Molecular replacement using the AdH5 model was used to identify the location of the CELO hexon within the unit cell. There is one hexon monomer in the asymmetric unit of the trigonal space group P321 ( a = b = 157.8 Å, c = 114.2 Å, γ = 120°) and the solvent content is 67.8%. The hexons pack in a hexagonal honeycomb so that large ∼100 Å diameter channels run through the entire crystal. This remarkable property of the crystals lends itself to their exploitation as a nanomaterial. Structural studies on CELO will elucidate the differences between avian and human adenoviruses and contribute to a better understanding of adenoviruses with non-human hosts.

Chick embryo lethal orphan virus can be polymer-coated and retargeted to infect mammalian cells

Non-human adenovirus vectors have attractive immunological properties for gene therapy but are frequently restricted by inefficient transduction of human target cells. Using chicken embryo lethal orphan (CELO) virus, we employed a nongenetic mechanism of polymer coating and retargeting with basic fibroblast growth factor (bFGF-pc-CELOluc), a strategy that permits efficient tropism modification of human adenovirus. bFGF-pc-CELOluc showed efficient uptake and transgene expression in chick embryo fibroblasts (CEF), and increased levels of binding and internalization in a variety of human cell lines. Transgene expression was also greater than unmodified CELOluc in PC-3 human prostate cells, although the specific activity (RLU per internalized viral genome) was decreased. In CEF, the specific activity of bFGF-pc-CELOluc was considerably higher than in the human prostate cell line PC-3. Retargeted virus was fully resistant to inhibition by human serum with known adenovirus-neutralizing activity in vitro, while in mice CELOluc was cleared less rapidly from the blood than Adluc following i.v. administration in the presence of adenovirus neutralizing serum. Polymer coating and retargeting with bFGF further reduced rates of clearance for both viruses, suggesting protection against both neutralizing and opsonizing factors. The data indicate that CELO virus may be retargeted to infect human cells via alternative, potentially disease-specific, receptors and resist the effects of pre-existing humoral immunity.

Increased adiposity in animals due to a human virus

Four animal models of virus-induced obesity including adiposity induced by an avian adenovirus have been described previously. This is the first report of adiposity induced in animals by a human virus. We investigated the adiposity promoting effect of a human adenovirus (Ad-36) in two different animal models. Due to the novel nature of the findings we replicated the experiments using a chicken model three times and a mammal model once. In four separate experiments, chickens and mice were inoculated with human adenovirus Ad-36. Weight matched groups inoculated with tissue culture media were used as non-infected controls in each experiment. Ad-36 inoculated and uninfected control groups were housed in separate rooms under biosafety level 2 or better containment. The first experiment included an additional weight matched group of chickens that was inoculated with CELO (chick embryo lethal orphan virus), an avian adenovirus. Food intakes and body weights were measured weekly. At the time of sacrifice blood was drawn and visceral fat was carefully separated and weighed. Total body fat was determined by chemical extraction of carcass fat. Animals inoculated with Ad-36 developed a syndrome of increased adipose tissue and paradoxically low levels of serum cholesterol and triglycerides. This syndrome was not seen in chickens inoculated with CELO virus. Sections of the brain and hypothalamus of Ad-36 inoculated animals did not show any overt histopathological changes. Ad-36 DNA could be detected in adipose tissue, but not skeletal muscles of randomly selected animals for as long as 16 weeks after Ad-36 inoculation. Data from these animal models suggest that the role of viral disease in the etiology of human obesity must be considered.

The complete DNA sequence and genomic organization of the avian adenovirus CELO

The complete DNA sequence of the avian adenovirus chicken embryo lethal orphan (CELO) virus (FAV-1) is reported here. The genome was found to be 43,804 bp in length, approximately 8 kb longer than those of the human subgenus C adenoviruses (Ad2 and Ad5). This length is supported by pulsed-field gel electrophoresis analysis of genomes isolated from several related FAV-1 isolates (Indiana C and OTE). The genes for major viral structural proteins (Illa, penton base, hexon, pVI, and pVIII), as well as the 52,000-molecular-weight (52K) and 100K proteins and the early-region 2 genes and IVa2, are present in the expected locations in the genome. CELO virus encodes two fiber proteins and a different set of the DNA-packaging core proteins, which may be important in condensing the longer CELO virus genome. No pV or pIX genes are present. Most surprisingly, CELO virus possesses no identifiable E1, E3, and E4 regions. There is 5 kb at the left end of the CELO virus genome and 15 kb at the right end with no homology to Ad2. The sequences are rich in open reading frames, and it is likely that these encode functions that replace the missing El, E3, and E4 functions.

Increase in DNA polymerase alpha in nuclei of chicken kidney cells after avian adenovirus infection.

DNA polymerase activity which sedimented at about 7s in the presence of 0.5M KC1 increased in cultured kidney cells after infection with a chicken embryo lethal orphan virus, an avian adenovirus. Elevated levels of antigenic material to a monoclonal antibody specific for the large subunit of chicken DNA polymerase alpha were also detected in nuclei of virus-infected cells by an immunofluorescence method. Since the elevations of DNA polymerase activity and of nuclear immunofluorescence was not inhibited with 1-beta-D-arabinofuranosylcytosine, the early viral gene(s) seems to be responsible for the induction of the synthesis and nuclear accumulation of DNA polymerase alpha.

DNA-binding proteins of chick embryo lethal orphan virus: lack of complementation between early proteins of avian and human adenoviruses.

Chick cells infected by chick embryo lethal orphan (CELO) virus (fowl adenovirus type 1) contained four prominent virus-specific, structurally related DNA-binding proteins with mol. wt. of 74K, 64K, 56K, 52K, and two minor forms. The CELO virus DNA-binding proteins were phosphorylated, delayed-early nuclear proteins. CELO virus early proteins were expressed in BHK cells, but did not complement human adenovirus type 5 mutants with lesions E1A, E2A or E2B. Moreover, CELO virus DNA-binding proteins were not produced in 293 cells, which express human adenovirus E1 genes. These results suggest that activation of transcription by adenovirus E1A genes involves specific interactions between the E1A gene products and viral early promoters.

The structural proteins of chick embryo lethal orphan virus (fowl adenovirus type 1).

Chick embryo lethal orphan (CELO) virus (fowl adenovirus type 1) contains at least 14 structural proteins with polypeptide molecular weights ranging from 100K to about 6K. A nomenclature of the CELO virion polypeptides is presented and the molar proportion of each polypeptide has been estimated. The CELO virus pentons were specifically released from the virion by dialysis against borate-based calcium-magnesium saline. The penton base (polypeptide III, mol. wt. 92K) and two fibres were separated, characterized and their polypeptides were correlated with their morphological positions in the virion. Peptide mapping suggested that the long fibre (polypeptide IV, mol. wt. 65K), and the short fibre (polypeptide VII, mol. wt. 44.5K) were not related in their primary sequences and are therefore probably encoded by separate genes. The time course of synthesis of the CELO virion polypeptides indicated that, like their mammalian adenovirus counterparts, they are synthesized late (after viral DNA replication).

Restriction map of CELO virus DNA

A restriction map of chicken embryo lethal orphan (CELO) virus DNA was reported with ten restriction endonucleases ( XbaI, XhoI, SalI, HindIII, EcoRI, BglI, KpnI, BamHI, PstI and SstI). CELO virus DNA was estimated by comparing CELO virus DNA fragments with marker DNA fragments to have a molecular weight of 29.3·10 6.

A common sequence in the inverted terminal repetitions of human and avian adenovimses

The termini of the avian chick embryo lethal orphan (CELO) virus DNA have been sequenced. The results revealed a 63-bp-long inverted terminal repetition (ITR) which shared the sequence ATAATA with all adenovirus termini, thus far analyzed. The CELO virus ITR differed from those of the mammalian adenoviruses in two major aspects: (i) it is not a perfect duplication; (ii) it begins with a 5′-guanylic acid residue instead of the cytidylic acid normally observed in adenoviruses.

Sequence homology between avian and human adenoviruses.

Studies of hybridization between fowl adenovirus type 1 (chicken embryo lethal orphan virus) DNA and human adenovirus type 2 DNA revealed two short but distinct regions which cross-hybridized under stringent conditions. One of the homologous regions was located between map positions 18.1 and 19.3 and did not correspond to any gene recognized so far. The second region mapped in the hexon gene between position 57 and 58.

Studies on egg drop syndrome (EDS) and chick embryo lethal orphan (CELO) avian adenovirus DNAs by restriction endonucleases.

Restriction endonucleases EcoRI, BamHI, HindIII, BglI, BglII, HpaI and PstI recognized 48 cleavage sites in EDS adenovirus strain B8/78 DNA, whereas CELO virus DNA was cut into 61 fragments by the same enzymes. No similarity could be detected in the restriction patterns obtained from the two avian adenoviruses. The calculated mol. wt. of B8/78 DNA was 22.6 X 10(6) [about 34.2 kilobases (kb)] and that of CELO virus DNA was 28.9 X 10(6) (about 43.7 kb). The fragments generated from B8/78 DNA by EcoRI and BamHI were physically mapped.

Virus-Specific Markers and Virus-Like Particles in Cell Lines of Tumors Produced by CELO Virus in Syrian Golden Hamsters2

Cell lines were established from 2 primary hepatocellular carcinomas (HC's) and 3 sarcomas produced in Syrian golden hamsters inoculated as newborns with chicken embryo lethal orphan (CELO) virus. Cell lines from 2 sarcomas (COT, CMT) and 1 HC (CEHEP) produced CELO virus-specific T-antigen. The antigen was not detected in cells of the third sarcoma line (RCT) until they had undergone more than 34 passages in vitro. Although 5-10% of cells in the second HC line (CILT/2) contained T-antigen during early passages, it was not demonstrable after the fifth subculture. Nevertheless, cells of both HC lines possessed CELO virus tumor-specific transplantation antigen. All 5 cell lines also contained hamster type R particles, and both HC lines had type C and intracytoplasmic type A particles. The percentage of carcinoma cells producing type R particles increased during cultivation in vitro, whereas the number of cells with type A particles decreased. Treatment with dibutyryl cyclic AMP and theophylline enhanced the number of cells producing type C particles in 1 HC line and type R particles in 2 sarcoma lines.

Some adenovirus DNA is associated with the DNA of permissive cells during productive or restricted growth

We have investigated the association of viral DNA with cell DNA in chicken embryo kidney (CEK) cells productively infected with chicken embryo lethal orphan (CELO) virus and in human (HEK) cells infected with mutants ts36 and ts125 of human adenovirus type 5 under permissive and restrictive conditions. Cell and viral DNA molecules were separated after CELO virus infection of CEK cells by alkaline sucrose gradient centrifugation, network formation, and CsCl density gradient centrifugation, methods that rely on different properties of the DNA. The cell DNA was then tested for viral sequences by DNA reannealing kinetics. Between 500 and 1,000 viral genome equivalents per cell were found at 36 h postinfection associated with cell DNA purified by each method. These values greatly exceeded the amount of free viral DNA found contaminating cell DNA prepared by the same methods from uninfected cells to which CELO virus DNA had been added. Quantitative agreement in the amounts of viral DNA found associated with cell DNA purified by these different methods suggests that CELO virus DNA is integrated into chick cell DNA during lytic infection. Similar experiments in HEK cells using mutants ts36 and ts125 of adenovirus type 5 at both restrictive and permissive temperatures showed that the same proportion of viral DNA is associated with cell DNA in the absence of viral DNA replication, and this suggests that the difference in the frequency with which cells are transformed by these mutants is not due to a difference in the frequency integration.

Chicken Embryo Lethal Orphan (CELO) Virus DNA Present in Hamster Cell Lines Derived from CELO-Induced Hepatomas

The kinetics of renaturation of the isolated DNA fragments of chicken embryo lethal orphan (CELO) virus generated by the restriction enzyme EcoR1 was measured in the presence of DNA extracted from two lines of CELO virus-induced hamster hepatoma cells and from control hamster cells. One hepatoma cell line (CILT-2), which did not produce the CELO virus tumor (T) antigen, lacked sequences from about one-half the virus genome while the other hepatoma cell line (CEHEP), which did produce the CELO virus T antigen, lacked sequences corresponding to one-fifth of the viral genome.

Chick embryo lethal orphan (CELO) virus-induced early and late polypeptides

Polypeptides of chick embryo lethal orphan virus (an avian adenovirus) were analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Virions ( p = 1.338 g/cm 3) and virion-like particles having lower density (light particles, p = 1.289 g/cm 3) were composed of at least 11 and 14 types of polypeptides, respectively. At least 23 virus-induced polypeptides in infected cells (primary monolayer culture of chicken kidney cells) were detected; eight polypeptides (early polypeptides) were synthesized even in the presence of 1-β- d-arabinofuranosyl cytosine, but the others (late polypeptides) were not. On the basis of migration in SDS-PAGE, 5 polypeptides were found to be common to virions and infected cells; 5, to light particles and infected cells; 10, to virions and light particles; and 4, to all three. Scarcely any of the early polypeptides were found in the soluble fraction when extracted with the low salt buffer. Some of the early polypeptides were solubilized with the high salt buffer. Other early polypeptides were solubilized by sodium deoxycholate; three of these were found in the so-called “M-band,” as are early polypeptides of human adenoviruses. An experiment with radioactive glucosamine indicated that one of early polypeptides found in the M-band was a glycoprotein.

A study of the sequences of chicken embryo lethal orphan (CELO) virus DNA present in a transformed hamster cell line with use of specific fragments of the virus genome

Chicken embryo lethal orphan (CELO) virus DNA (molecular weight 28.3 × 10 6) was cleaved by the restriction enzyme EcoR1 into seven fragments whose molecular weights ranged from 1.8 × 10 6 to 10.2 × 10 6 as measured by gel electrophoresis and electron microscopy. 32P-labeled CELO virus DNA was treated with EcoR1 and the resulting fragments were separated by gel electrophoresis. The kinetics of renaturation of the isolated fragments was measured in the presence of DNA extracted from a line of CELO virus-transformed hamster skin (THS) cells and from control hamster cells. THS cell DNA contained sequences homologous to only six of the seven EcoR1-produced CELO virus DNA fragments. No sequence homologous to a 2.0 × 10 6 molecular weight fragment was detected indicating that the DNA of THS cells did not contain a complete copy of the virus genome.