Downregulation of Cell Surface Major Histocompatibility Complex Class I Expression Is Mediated by the Left-End Transcription Unit of Fowl Adenovirus 9.

Major histocompatibility complex class I (MHC-I) molecules play a critical role in the host's antiviral response by presenting virus-derived antigenic peptides to cytotoxic T lymphocytes (CTLs), enabling the clearance of virus-infected cells. Human adenoviruses evade CTL-mediated cell lysis, in part, by interfering directly with the MHC-I antigen presentation pathway through the expression of E3-19K, which binds both MHC-I and the transporter associated with antigen processing protein and sequestering MHC-I within the endoplasmic reticulum. Fowl adenoviruses have no homologues of E3-19K. Here, we show that representative virus isolates of the species Fowl aviadenovirus C, Fowl aviadenovirus D, and Fowl aviadenovirus E downregulate the cell surface expression of MHC-I in chicken hepatoma cells, resulting in 71%, 11%, and 14% of the baseline expression level, respectively, at 12 h post-infection. Furthermore, this work reports that FAdV-9 downregulates cell surface MHC-I through a minimum of two separate mechanisms-a lysosomal-independent mechanism that requires the presence of the fowl adenovirus early 1 (FE1) transcription unit located within the left terminal genomic region between nts 1 and 6131 and a lysosomal-dependent mechanism that does not require the presence of FE1. These results establish a new functional role for the FE1 transcription unit in immune evasion. These studies provide important new information about the immune evasion of FAdVs and will enhance our understanding of the pathogenesis of inclusion body hepatitis and advance the progress made in next-generation FAdV-based vectors.

Antibody response and virus shedding of chickens inoculated with left end deleted fowl adenovirus 9-based recombinant viruses.

The nonpathogenic fowl adenoviruses (FAdVs) are suitable recombinant virus vectors. Two different replication-competent FAdV-9-based recombinant viruses carrying the enhanced green fluorescent protein (EGFP) gene within a nonessential DNA sequence at the left end genomic region were tested in chickens to study the antibody response by enzyme-linked immunosorbent assay to both the foreign proteins, EGFP and FAdV-9, and virus shedding through the feces. All inoculations were done intramuscularly: groups 1 and 2 with the recombinant viruses and group 3 with the wild-type FAdV-9 virus. Group 4 was mock inoculated. Sentinel birds also were included in groups 1-3 to study virus transmission. Boosting inoculations were done in all groups at 2, 3, and 4 wk after the first inoculation. Antibodies to EGFP were detected at 3-7 wk postinoculation in groups 1 and 2 only. Antibody response to FAdV-9 in groups 1-3 did not differ significantly (P > 0.06). Virus was not detected in the feces of chickens in groups 1 and 2, including the sentinel birds, but virus was present in the feces of chickens in group 3, including the sentinel birds. These results further supported our previous findings regarding the suitability of the nonessential region at the left end of the viral genome as an insertion site for foreign genes and its importance in in vivo replication. In this work, we demonstrated the potential of FAdV-9-based recombinant viruses as vaccines for poultry.

Oncolytic viruses as experimental treatments for malignant gliomas: using a scourge to treat a devil.

The concept of oncolytic viral therapy has a century-old history, but only within the last 20 years have oncolytic viruses been considered for the treatment of brain cancers. Viruses such as herpes, measles, and vaccinia have all been known to cause devastating cases of neurological disease in humans, yet these 'scourges' are now being harnessed in such a way that they prove very useful as cancer therapeutics. There have been 8 formal clinical trials and 3 case studies using oncolytic viruses to treat malignant glioma patients. Although some success has been reached with oncolytic therapy, overall it has fallen short of expectations. In this review we analyze the results of these trials and bring to light some of the limitations and pitfalls of this therapy, as well as present some promising preclinical work that has been proposed to circumvent such problems.

The non-essential left end region of the fowl adenovirus 9 genome is suitable for foreign gene insertion/replacement.

The goals of this study were to demonstrate that a non-essential region at the left end of the fowl adenovirus 9 (FAdV-9) genome could be used to generate recombinant viruses, examine their in vitro growth characteristics and determine their ability to transduce non-avian cells. Three FAdV-9 vectors (rFAdV-9s) were generated carrying the enhanced-green fluorescent protein (EGFP) gene: FAdV-9inEGFP, FAdV-9 Delta 1-EGFP and FAdV-9 Delta 4-EGFP. FAdV-9inEGFP carried the EGFP cassette inserted into the non-essential region without deletion resulting in an increase of the genome size to 103.7% of the wild-type. FAdV-9 Delta 1-EGFP and FAdV-9 Delta 4-EGFP (rFAdV-9 Delta s) carried the EGFP cassette replacing the non-essential sequences at nucleotides 1194-2342 and 491-2782, respectively. All rFAdV-9s had wild-type growth kinetics and plaque morphology. The rFAdV-9 Delta s replicated in CH-SAH cells with the same titers as the wild-type virus. The FAdV-9inEGFP titers were approximately 1 log lower than those of rFAdV-9 Delta s and wt FAdV-9 at 36 and 48 h post-infection (h.p.i.). EGFP was expressed in avian and mammalian cells infected with rFAdV-9s. EGFP expression, based on spectrofluorometry, was significantly higher in chicken hepatoma cells infected with FAdV-9inEGFP than in those with rFAdV-9 Delta s at 18 and 24h.p.i, suggesting a functional role of some or all non-essential ORFs on foreign gene expression. This study demonstrated the suitability of the non-essential region as an insertion/replacement site for foreign genes to generate FAdV-9-based vectors that can be applied as recombinant vaccines for poultry or gene delivery vehicles for mammalian systems.

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.

Detection and quantitation of fowl adenovirus genome by a real-time PCR assay.

The purpose of the study was to develop a highly sensitive real-time polymerase chain reaction (PCR) assay to detect and quantitate fowl adenovirus (FAdV) DNA in chicken tissues, using FAdV-9 as a model. The assay had a dynamic range of 7 logs and minimum detection limit of 9.4 viral genome copies. It was shown to be highly specific, as tissues from uninfected chickens and other viral genomes, such as those of Marek's disease virus, fowlpox virus and infectious laryngotracheitis virus did not produce positive signal. The sensitivity of the real-time PCR was comparable with nested PCR and it was 100 times more sensitive than the conventional PCR. The assay was validated by testing DNA from tissues of chickens infected with FAdV-9 collected at different days post-infection. FAdV-9 DNA was detected in liver, bursa of Fabricius and cecal tonsil tissues in a range of 10(2)-10(7) copies per 100 ng of total DNA. High amounts of viral DNA were present in the cecal tonsils for a week after inoculation making this tissue an ideal sample source for the diagnosis of FAdV infection. This assay is an excellent research and diagnostic tool that provides high sensitivity, specificity and rapid post-PCR analyses.

Sequence comparison of the right end of fowl adenovirus genomes.

The nucleotide sequence at the right end of the genomes of fowl adenoviruses (FAdVs) representing species groups C (FAdV-4 CA and FAdV-10 C-2B), D (FAdV-2 CA), and E (FAdV-8 CA) was determined and analyzed and compared to FAdV-1 (species group A virus) and FAdV-9 A-2A (species group B virus). High nucleotide sequence identities and amino acid identities (74-94%) were found among viruses in the same species group. Homologues to known open reading frames (ORFs) of the right end of the reported FAdV genomes, such as lipase and Gam-1, were also found in all analyzed viral genomes. Homologues to a few FAdV ORFs were found in viruses other than FAdVs as well. Several novel FAdV ORFs were also found. Most identified ORFs have unknown function and thus further studies are necessary to establish their importance in virus replication. ORF content within the right end of the FAdV genomes varied among FAdVs, while the gene order and orientation of shared ORFs are conserved between different FAdVs.

Sequence analysis of the left end of fowl adenovirus genomes.

Nucleotide sequence analysis of the left end of the genome of fowl adenoviruses (FAdV) representing species group C (FAdV-4 and -10), D (FAdV-2) and E (FAdV-8) were carried out, and the sequence data was compared to those of FAdV-1 (FAdV-A) and FAdV-9 (FAdV-D). The viruses were propagated in chicken hepatoma cell line for viral DNA isolation. Restriction endonuclease analysis was performed followed by hybridization with two DNA probes representing the left end of FAdV-9. The identified fragments were sequenced, and the generated data were compared with the GenBank database. Nucleotide sequence homology and amino acid sequence identities were high between members of the same species group, FAdV-2 and -9, and FAdV-4 and -10, whereas different degrees of variations were observed among all FAdVs. Gene arrangement and position of ORFs at the left end of FAdV genomes were largely conserved suggesting similar gene functions. All previously characterized left end ORFs in CELO virus and FAdV-9 were found in all analyzed FAdVs. However, ORF 1C was absent in FAdV-4 and -10, but additional ORFs, most likely corresponding to duplicates of ORF 14, were observed in these viruses.