Mutational Analysis of the Avian Adenovirus CELO, Which Provides a Basis for Gene Delivery Vectors

The avian adenovirus CELO is being developed as a gene transfer tool. Using homologous recombination in Escherichia coli, the CELO genome was screened for regions that could be deleted and would tolerate the insertion of a marker gene (luciferase or enhanced green fluorescent protein). For each mutant genome, the production of viable virus able to deliver the transgene to target cells was monitored. A series of mutants in the genome identified a set of open reading frames that could be deleted but which must be supplied in trans for virus replication. A region of the genome which is dispensable for viral replication and allows the insertion of an expression cassette was identified and a vector based on this mutation was evaluated as a gene delivery reagent. Transduction of avian cells occurs at 10- to 100-fold greater efficiency (per virus particle) than with an adenovirus type 5 (Ad5)-based vector carrying the same expression cassette. Most important for gene transfer applications, the CELO vector transduced mammalian cells as efficiently as an Ad5 vector. The CELO vector is exceptionally stable, can be grown inexpensively in chicken embryos, and provides a useful alternative to Ad5-based vectors.     

Temporal Organization in Avian Reproduction

Recent studies have demonstrated that biological rhythms haveimportant roles in avian reproduction. In the photoperiodicstimulation of the reproductive complex, there are two systemsinvolved in the interpretation of day length. One system isentrained by the photoperiod, probably by dawn. This entrainedsystem in turn entrains two light-sensitive phases which occurlater in the day. If the photoperi od is long enough, it ispresent during the sensitive phases when it can induce the productionof luteinzing hormone and follicle stimulating hormone. Theinterrenal gland appears to be a part of the entraining systeminasmuch as injections of corticosterone can set a sensitivephase when light can induce gonadal development. The annual cycle of photosensitivity and photorefractorinessappeals to be controlled by the temporal relations between thedaily rhythms of corticosterone and prolactin which change seasonally.When the hormonal relations of photosensitive and photorefractorybirds are simulated by injections of the hormones, the appropriateconditions (photosensitivity or photorefractonness) can be produced.Seasonal changes in the hormonal relations are not direct reflectionsof changes in the photoperiod; they depend on more complex physiologicalexperiences. The systems involved in egg laying and parental behavior mayalso have a temporal basis of biological rhythms. For example,the intensity of the pigeon cropsac response depends on thetime of daily injections of prolactin relative to those of corticosterone.It is apparent that the daily rhythm constitutes the basic structuialunit in the temporal organizationtion of avian reproduction.     

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 29.3·10⁶.     

Complementary strands of CELO virus DNA.

When alkali-denatured DNA from CELO virus (an avian adenovirus) was annealed for 15 min at 37 C in 0.1 M NaCl, 70% of the molecules formed single-stranded circles. This is probably due to base pairing of complementary sequences not more than 110 nucleotides long at the ends of the single strands and implies an inverted terminal repetition in the duplex DNA similar to that reported for the DNA from human adenoviruses. The circular molecules had a uniform length that was approximately the same as that of linear single-stranded molecules. The complementary strands of CELO virus DNA were separated on a preparative scale, and at least 40% of the heavy strands and 56% of the light strands were found to be intact as judged by the formation of single-stranded circles.     

Construction of avian adenovirus CELO recombinants in cosmids

The avian adenovirus CELO is a promising vector for gene transfer applications. In order to study this potentiality, we developed an improved method for construction of adenovirus vectors in cosmids that was used to engineer the CELO genome. For all the recombinant viruses constructed by this method, the ability to produce infectious particles and the stability of the genome were evaluated in a chicken hepatocarcinoma cell line (LMH cell line). Our aim was to develop a replication-competent vector for vaccination of chickens, so we first generated knockout point mutations into 16 of the 22 unassigned CELO open reading frames (ORFs) to determine if they were essential for virus replication. As the 16 independent mutant viruses replicated in our cellular system, we constructed CELO genomes with various deletions in the regions of these nonessential ORFs. An expression cassette coding for the enhanced green fluorescent protein (eGFP) was inserted in place of these deletions to easily follow expression of the transgene and propagation of the vector in cell monolayers. Height-distinct GFP-expressing CELO vectors were produced that were all replication competent in our system. We then retained the vector backbone with the largest deletion (i.e., 3.6 kb) for the construction of vectors carrying cDNA encoding infectious bursal disease virus proteins. These CELO vectors could be useful for vaccination in the chicken species.     

H5N1禽流感病毒HA和NA基因重组腺病毒共表达载体的构建

利用口蹄疫病毒(FMDV)2A蛋白具有自我裂解的功能,将其作为连接肽构建携带有H5N1亚型AIVHA和NA基因的重组腺病毒表达载体,进而为AIV基因工程疫苗的开发以及相关诊断试剂的开发提供依据。采用融合PCR的方法扩增出含有H5N1 AIV HA-2A-NA的基因,定向插入pAdtrack-CMV腺病毒穿梭质粒中,含有目的基因的腺病毒穿梭质粒pAdtrack-HA-2A-NA与腺病毒骨架质粒pAdeasy-1在基因工程菌BJ5183中进行同源重组,获得腺病毒质粒pAdeasy-HA-2A-NA,将pAdeasyd-H5经PacI线性化后转染HEK293细胞株包装出含有HA-2A-NA基因的腺病毒pAd-HA-2A-NA。结果表明,构建的含有目的基因的腺病毒穿梭质粒pAdtrack-HA-2A-NA和含有目的基因的腺病毒质粒pAdeasy-HA-2A-NA经PCR、双酶切及核苷酸测序测定无误。线性化后的pAdeasy-HA-2A-NA转染HEK293细胞包装成功获得腺病毒pAd-HA-2A-NA载体,经绿色荧光蛋白和RT-PCR分析证实,目的基因在该细胞中成功表达。本试验构建的含有AIV H5N1亚型HA-2A-NA基因的重组腺病毒表达载体,将为进一步研究开发基因工程疫苗提供病毒模型。     

表达H5N1亚型禽流感病毒HA基因重组腺病毒的遗传稳定性分析及滴度测定

研究表达H5N1亚型禽流感病毒HA基因重组腺病毒pAd-H5的遗传稳定性及重组病毒的滴度测定。将重组腺病毒pAd-H5在293细胞上连续传代20次,取第5、10、15和20代的重组病毒采用PCR 方法扩增禽流感病毒HA基因,并进行基因序列测定分析;用标记为GFP的快速测定法计算出20代次时重组病毒的滴度。从各代重组病毒DNA 中均扩增出了约1 700bp的目的条带,与HA基因片段长度一致,基因序列分析表明：第5 、10 、15 代重组病毒中的HA基因序列与原始转移载体序列完全一致,第20代重组病毒插入基因有1处发生了点突变(即HA基因417位A→G),但其编码的氨基酸未发生变化(即同义突变),表明表达的目的蛋白抗原表位未发生变化;计算出的重组病毒滴度为108.875pfu/0.1ml。重组腺病毒pAd-H5在293细胞上连续传代20次,具有良好的遗传稳定性,重组腺病毒的病毒滴度相对较高。     

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.