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PubMed1966

Replication of adenovirus type 7 in monkey cells: a new determinant and its transfer to adenovirus type 2.

A strain of human adenovirus type 7, adapted to replication in green-monkey kidney cells, requires the interaction of two particles to initiate plaque formation in the simian cells. One particle is a true adenovirion. The second, apparently defective, consists of a genome carrying amonkey-adapting component in an adenovirus capsid; this genome does not express known SV40 determinants. The addition of human adenovirus type 7 that is not adapted enhances the titer and changesconditions for plaque formation by the adapted virus to a one-particle requirement. Addition of nonadapted human adenovirus type 2 as helper virus results in the transfer of the monkey-adaptingcomponent from adenovirus type 7 to adenovirus type 2. The population containing the adenovirus 2 transcapsidant then has the ability to replicate in simian cells.

Adenoviridae↗
PubMed1975

DNA-minus temperature-sensitive mutants of adenovirus type 5 help adenovirus-associated virus replication.

Efficient potentiation of adenovirus-associated viruses (AAV) replication occurs in coinfections with either of two DNA-minus temperature-sensitive mutants of adenovirus type 5 (Ad5), ts125 and ts149. The helper activity of these mutants does not result from leakiness. At the nonpermissive termperature (39.5 C) there was little or no detectable adenovirus DNA synthesis, and only a relatively low level of adenovirus transcription was observed. However, the synthesis of AAV DNA and RNA and the yield of infectious AAV were comparable in amounts to those found when wild-type Ad5 was the helper. Furthermore, an apparent lag in the initiation of AAV transcription after the onset of AAV DNA synthesis was seen in coinfections with both wild type or ts125. These findings strongly suggest that the adenovirus factor(s) required for AAV multiplication is produced early in the adenovirus DNA replication, this requirement does not include all factors directly needed for adenovirus DNA synthesis.

Adenoviridae↗
PubMed1988

Antigenic structure of mammalian adenoviruses studied by monoclonal antibodies against bovine adenovirus type 2.

The cross-reactivity pattern of eight monoclonal antibodies (MAbs) raised against bovine adenovirus type 2 (BAV2) hexon was studied by indirect ELISA using seven different adenoviruses. All of the eight MAbs reacted with the two BAV2 subtypes (A and B), while the other adenoviruses, human adenovirus type 1 (HAV1), ovine adenovirus type 3 (OAV3), BAV1, BAV4 and BAV8 gave different combinations of positive reactions. Based on the results of cross-reactivity there is a close antigenic relationship between the two different BAV2 subtypes. There is an antigenic relationship among the adenovirus types studied, including viruses of human and animal origin. One of the MAbs (IV. F5) seems to be type specific. Another MAb (BH5) detects a common epitope among adenoviruses of animal origin, and reacts even with BAV4 and BAV8.

Adenoviridae↗
PubMed1989

Cloning of the herpes simplex virus ICP4 gene in an adenovirus vector: effects on adenovirus gene expression and replication.

To assess the ability of the herpes simplex virus ICP4 protein to complement adenovirus E1a mutants we have constructed an adenovirus type 5 vector containing a temperature-sensitive ICP4 gene, under control of its own promoter, within the E1 region of the genome. The recombinant virus expresses ICP4 in cells which are permissive (293) or nonpermissive (KB and R970-5) for viral replication, and at levels which approximate those obtained in herpes simplex infection. The adenovirus-encoded protein is functional in that it complements an ICP4 deletion mutant of herpes simplex virus; however, it is incapable of complementing adenovirus E1a mutants for viral growth or DNA replication. At the level of activation of gene expression, ICP4 stimulates the expression of the adenovirus E2a gene but not that of other early genes. Our results indicate that ICP4 does not possess all of the functions of the E1a proteins and, furthermore, that adenovirus early genes differ in their susceptibility to heterologous trans-activators.

Adenovirus Early Proteins↗
PubMed2000

Adult adenovirus infections: loss of orphaned vaccines precipitates military respiratory disease epidemics. For the Adenovirus Surveillance Group.

Adenovirus vaccines have greatly reduced military respiratory disease morbidity since the 1970s. However, in 1995, for economic reasons, the sole manufacturer of these vaccines ceased production. A population-based adenovirus surveillance was established among trainees with acute respiratory illness at 4 US military training centers as the last stores of vaccines were depleted. From October 1996 to June 1998, 1814 (53.1%) of 3413 throat cultures for symptomatic trainees (78% men) yielded adenovirus. Adenovirus types 4, 7, 3, and 21 accounted for 57%, 25%, 9%, and 7% of the isolates, respectively. Unvaccinated trainees were much more likely than vaccinated trainees to be positive for types 4 or 7 (odds ratio [OR] = 28.1; 95% CI, 20.2-39.2). Two training centers experienced epidemics of respiratory disease affecting thousands of trainees when vaccines were not available. Until a new manufacturer is identified, the loss of orphaned adenovirus vaccines will result in thousands of additional preventable adenovirus infections.

Adenovirus Infections, Human↗
PubMed1985

E1a regions of the human adenoviruses and of the highly oncogenic simian adenovirus 7 are closely related.

Simian adenovirus 7 (SA7) is a highly oncogenic virus, capable of causing tumors in hamsters upon the direct injection of viral DNA. We determined the transcriptional organization of the transforming region and compared it with that of the human adenoviruses. This analysis demonstrated that there are two independently promoted transcription units similar to the E1a and E1b regions of the human adenoviruses. The nucleotide sequence of the SA7 E1a region demonstrated considerable homology with the human adenoviruses, both in the sequences that regulate E1a expression and in the encoded polypeptides. The amino acid homology was reflected in the ability of SA7 to complement the growth of human adenoviruses mutant in the E1a region. Furthermore, we found two regions of amino acid homology unique to SA7 and the highly oncogenic human adenovirus 12.

Adenoviridae↗
PubMed2001

Selective effects of E1B-defective adenoviruses and adenovirus E1A mutants in deficient mouse primary embryonic fibroblasts.

E1B-defective adenoviruses have been described as exerting selective cytopathic effects on transformed cells. Previously, we showed that adenovirus dl118, lacking both E1B proteins, very efficiently kills most human malignant cell lines. In order to study whether these selective effects were due to selective replication of dl118 in cells harboring specific genetic alterations, we compared the viability of various deficient mouse primary fibroblasts. We studied mouse embryonic fibroblasts (MEFs) derived from p16, p21, p27 and p53 knockout mice, as well as wild-type MEFs. We infected them with 100 p.f.u. of adenoviruses adl118, adwt300, and adenoviruses carrying the E1A mutant 922 (the E1a product only binds to the p300 and related proteins) and Ad646 (the E1A product binds to the pRb and related proteins). The percentage of infectivity was evaluated with an adenovirus carrying the green fluorescent protein (AdGFP). With AdGFP, clear green fluorescent signals were detected in more than 70% of the cells after 3 days of infection. After infection with several adenoviruses, we observed that E1A mutant 922 killed all the MEFs. Conversely, the E1a mutant Ad646 exerted its major effects on control wild-type MEFs. Moreover, Adl118 killed the wtMEFs and other MEFs slightly more efficiently than did wtAd, but less than Ad922. No viral replication was detected by adding the obtained supernatants to HEK293 cells. Due to the absence of significant viral replication on these cells, the results could be interpreted as direct effects of E1A and E1A mutant proteins on the different mouse cells carrying diverse genetic alterations.

Adenoviridae↗
PubMed1999

Coxsackie and adenovirus receptor (CAR)-dependent and major histocompatibility complex (MHC) class I-independent uptake of recombinant adenoviruses into human tumour cells.

The role of two receptors, previously proposed to mediate the entry of adenoviruses into human cells, the coxsackie and adenovirus receptor (CAR) and the major histocompatibility complex (MHC) class I heavy chain has been investigated. The expression of MHC class I in many tumours is reduced or absent, therefore if this were a means by which adenoviruses gained entry into cells, it would have important implications for their application in cancer treatment. In order to determine if MHC class I heavy chain is involved in adenovirus type 5 (Ad5) uptake, the binding of recombinant Ad5 fibre knob domain (which mediates viral attachment) to human cell lines that had greatly different levels of surface MHC class I was studied. We also created derivatives of a non-permissive Chinese hamster ovary (CHO) cell line that expressed human class I (HLA-A2) and found that these cells did not bind fibre or take up virus. In addition, the extracellular domain of CAR was expressed in E. coli and used to generate a polyclonal anti-CAR antibody. This antibody blocked both 125I labelled fibre knob binding and virus uptake. Thus CAR, and not MHC class I, is a receptor for human adenoviruses in cultured tumour cells. Tissue CAR levels may therefore be an important factor in the efficiency of adenovirus-mediated gene therapy.

Adenoviridae↗
PubMed1999

Expression of the primary coxsackie and adenovirus receptor is downregulated during skeletal muscle maturation and limits the efficacy of adenovirus-mediated gene delivery to muscle cells.

Skeletal muscle fibers are infected efficiently by adenoviral vectors only in neonatal animals. This lack of tropism for mature skeletal muscle may be partly due to inefficient binding of adenoviral particles to the cell surface. We evaluated in developing mouse muscle the expression levels of two high-affinity receptors for adenovirus, MHC class I and the coxsackie and adenovirus receptor (CAR). The moderate levels of MHC class I transcripts that were detected in quadriceps, gastrocnemius, and heart muscle did not vary between postnatal day 3 and day 60 adult tissue. A low level of CAR expression was detected on postnatal day 3 in quadriceps and gastrocnemius muscles, but CAR expression was barely detectable in adult skeletal muscle even by reverse transcriptase-polymerase chain reaction. In contrast, CAR transcripts were moderately abundant at all stages of heart muscle development. Ectopic expression of CAR in C2C12 mouse myoblast cells increased their transducibility by adenovirus at all multiplicities of infection (MOIs) tested as measured by lacZ reporter gene activity following AVCMVlacZ infection, with an 80-fold difference between CAR-expressing cells and control C2C12 cells at an MOI of 50. Primary myoblasts ectopically expressing CAR were injected into muscles of syngeneic hosts; following incorporation of the exogenous myoblasts into host myofibers, an increased transducibility of adult muscle fibers by AVCMVlacZ was observed in the host. Expression of the lacZ reporter gene in host myofibers coincided with CAR immunoreactivity. Furthermore, sarcolemmal CAR expression was markedly increased in regenerating muscle fibers of the dystrophic mdx mouse, fibers that are susceptible to adenovirus transduction. These analyses show that CAR expression by skeletal muscle correlates with its susceptibility to adenovirus transduction, and that forced CAR expression in mature myofibers dramatically increases their susceptibility to adenovirus transduction.

Adenoviridae↗
PubMed2004

Analysis of the first complete genome sequence of an Old World monkey adenovirus reveals a lineage distinct from the six human adenovirus species.

Simian adenovirus 3 (SAdV-3) is one of several adenoviruses that were isolated decades ago from Old World monkeys. Determination of the complete DNA sequence of SAdV-3 permitted the first full genomic comparison of a monkey adenovirus with adenoviruses of humans (HAdVs) and chimpanzees, which are recognized formally as constituting six of the species (HAdV-A to HAdV-F) within the genus Mastadenovirus. The SAdV-3 genome is 34 246 bp in size and has a G+C content of 55.3 mol%. It contains all the genes that are characteristic of the genus Mastadenovirus and has a single VA-RNA gene and six genes in each of the E3 and E4 regions. The genetic organization is the same as that of HAdV-12, a member of the HAdV-A species. Phylogenetic analyses showed that although SAdV-3 is related marginally more closely to HAdV-A and HAdV-F than to other species, it represents a unique lineage that branched at an early stage of primate adenovirus divergence. The results imply that the genetic layout in SAdV-3 and HAdV-12 may also have characterized the common ancestor of all sequenced primate adenoviruses.

Adenoviruses, Simian↗
PubMed1976

Intracellular forms of adenovirus DNA. V. Viral DNA sequences in hamster cells abortively infected and transformed with human adenovirus type 12.

The persistence of viral DNA in BHK-21 cells abortively infected with human adenovirus type 12 has been investigated using reassociation kinetics. No indication of an increase in the amount of viral DNA per cell has been found. On the contrary, the amount of intracellular viral DNA sequences decreases rapidly after infection. Thus, free adenovirus type 12 DNA does not replicate in BHK-21 cells. The influence of the multiplicity of infection on the amount of persisting adenovirus type 12 DNA has also been explored. The viral DNA sequences persisting in four lines of hamster cells transformed in vitro by adenovirus type 12 at various multiplicities of infection have been quantitated and mapped by reassociation kinetics experiments using restriction endonuclease fragments of 3H-labeled adenovirus type 12 DNA. All the EcoRI restriction nuclease fragments of the adenovirus type 12 genome are represented in each of the four cell lines. Individual fragments of the viral genome are represented in multiple copies in non-equimolar amounts.

Adenoviruses, Human↗
PubMed2005

[Effect of expression of coxsackie and adenovirus receptor on antitumor activity of genetically modified adenovirus].

BACKGROUND & OBJECTIVE: The effect of adenovirus, a kind of oncolytic virus and a kind of carrier in gene therapy, depends on the expression of Coxsackie and adenovirus receptor (CAR) on cell surface to a large degree. This study was to evaluate relationship of CAR expression to infectivity and efficacy of adenovirus. METHODS: Pathologic specimens from 29 patients in a clinical trial of genetically modified adenovirus (H101) were collected. Expression of CAR in cancer tissues was detected by immuohistochemistry. Expression of CAR on cancer cell membrane was detected by flow cytometry (FCM). Inhibitory effect of H101 on cells was assessed by MTT assay. RESULTS: Positive rate of CAR was significantly higher in patients achieved complete remission (CR) or partial remission (PR) than in patients had stable disease (SD) or progressive disease (PD) [70.0% (7/10) vs. 31.6% (6/19), P=0.048]. CAR expression on different cells was different, and the amount of CAR was positively related to virus infectivity, presented as the inhibitory rate of H101-infected cells (r=0.986). CONCLUSIONS: The expression of CAR closely relates to antitumor activity and efficacy of adenovirus. Also, the efficacy of adenovirus is higher on cancers with increased CAR expression than on those with decreased CAR expression.

Adenoviridae↗
PubMed2005

Susceptibility of B lymphocytes to adenovirus type 5 infection is dependent upon both coxsackie-adenovirus receptor and alphavbeta5 integrin expression.

Human lymphocytes are resistant to genetic modification, particularly from recombinant adenoviruses, thus hampering the analysis of gene function using adenoviral vectors. This study engineered an Epstein-Barr virus-transformed B-lymphoblastoid cell line permissive to adenovirus infection and elucidated key roles for both the coxsackie-adenovirus receptor and alphavbeta5 integrin in mediating entry of adenoviruses into these cells. The work identified a strategy for engineering B cells to become susceptible to adenovirus infection and showed that such a strategy could be useful for the introduction of genes to alter lymphoblastoid-cell gene expression.

Adenovirus Infections, Human↗
PubMed1998

Oral tolerization to adenoviral proteins permits repeated adenovirus-mediated gene therapy in rats with pre-existing immunity to adenoviruses.

Exposure to wild-type adenoviruses is common in humans and results in immune response against adenoviruses. The pre-existing antibodies and a strong secondary humoral and cellular immune response would interfere with gene transfer using recombinant adenoviral vectors. To test whether the secondary immune response can be abrogated by oral tolerization to adenoviral antigens, we immunized bilirubin-UDP-glucuronosyltransferase (BUGT)-deficient jaundiced Gunn rats with a recombinant adenovirus (5 x 10(9) pfu/rat) expressing the human UDP-glucouronosyltransferase (BUGT1) gene (Ad-hBUGT). Transgene expression was shown by reduction of mean serum bilirubin levels from 7.0 mg/dL to 2.3 mg/dL in 14 days, which then increased gradually to pretreatment levels in 6 weeks. All recipients developed antibodies (1:2[10]) and cytotoxic lymphocytes against the adenovirus. For oral tolerization, we administered to the immunized rats protein extracts of a recombinant adenovirus type 5 (1-1.5 mg/day) via duodenostomy tubes 10 to 40 days after the initial virus injection; control rats received bovine serum albumin. In rats fed adenoviral proteins and the BSA-fed controls, the antibody titers decreased to 1:2(7) and 1:2(9), respectively, in 70 days. Lymphocytes from the tolerized rats expressed TGF-beta1 upon exposure to antigen-presenting cells primed with adenoviral antigens, whereas IFN-gamma expression was undetectable. In contrast, lymphocytes from the BSA-treated control rats expressed IFN-gamma but not transforming growth factor beta1 (TGF-beta1). Seventy days after the first injection in the orally tolerized rats, but not in the controls, a second Ad-hBUGT injection caused human BUGT1 expression again, reducing serum bilirubin levels to those observed after the first injection. In the tolerized rats, serum antibody titers and anti-adenoviral cytotoxic lymphocyte activities continued to decline despite the second injection, whereas the antibody levels were boosted in the non-tolerized group. This results show that by preventing the secondary booster response, oral tolerization permits repeated adenovirus-directed gene transfer despite the presence of a residual antibody titer from a previous adenoviral exposure.

Adenoviridae↗
PubMed1981

Construction of an adenovirus-SV40 recombinant producing SV40 T antigen from an adenovirus late promoter.

An adenovirus-SV40 recombinant has been constructed in which the SV40 early region, joined to a copy of the adenovirus major late promoter, was substituted for a small portion of the adenovirus late region and all of early region 3. The inserted sequence functions as a separate transcriptional unit, using the ectopic adenovirus promoter, SV40 splice sites and SV40 polyadenylation site. The RNAs produced lack both the second and third members of the tripartite leader normally found on adenovirus late RNA, yet are translationally active, directing the synthesis of substantial amounts of SV40 T antigen. The recombinant may serve as a model vehicle for the abundant expression of other eucaryotic genes inserted into the adenovirus genome.

Adenoviridae↗
PubMed2003

Detection of replication-competent adenoviruses spiked into recombinant adenovirus vector products by infectivity PCR.

The presence of replication-competent adenovirus (RCA) in clinical lots of adenovirus vectors raises a variety of safety concerns. To detect RCA in adenovirus vector products, the cell culture/cytopathic effect (CPE) method has generally been preferred. However, it is difficult to evaluate the amount of RCA clearly and quantitatively by this method. In addition, the cell culture/CPE method requires large-scale cell culturing and a substantial amount of time. For the purpose of establishing a method to detect RCA more sensitively and rapidly, we developed the infectivity PCR, a hybrid method that combines the infectivity assay and quantitative PCR. This method allows RCA to be quantified by real-time quantitative PCR using primers and a probe designed for E1 DNA. By infectivity PCR, 1 pfu of RCA spiked into 10(9) particles of adenovirus vectors could be detected. In contrast, CPE was observed in the cells infected with 10(4) pfu of RCA spiked into 10(9) particles of adenovirus vectors. The glass-beads method was suitable for extracting DNA rapidly from the RCA-infected cells. These results showed that infectivity PCR combined with the glass-beads-based DNA extraction method was useful for the detection of RCA in adenovirus vector products.

Adenoviridae↗
PubMed2001

A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus.

Fully deleted adenovirus vectors (FD-AdVs) would appear to be promising tools for gene therapy. Since these vectors are deleted of all adenoviral genes, they require a helper adenovirus for their propagation. The contamination of the vector preparation by the helper limits the utility of currently existing FD-AdVs in gene therapy applications. We have developed an alternative system for the propagation of FD-AdVs, in which the adenoviral genes essential for replication and packaging of the vector are delivered into producer cells by a baculovirus-adenovirus hybrid. A hybrid baculovirus Bac-B4 was constructed to carry a Cre recombinase-excisable copy of the packaging-deficient adenovirus genome. Although the total size of the DNA insert in Bac-B4 was 38 kb, the genetic structure of this recombinant baculovirus was stable. Bac-B4 gave high yields in Sf9 insect cells, with titers of 5 x 10(8)p.f.u./ml before concentration. Transfection of 293-Cre cells with lacZ-expressing FD-AdV plasmid DNA followed by infection by Bac-B4 at a MOI of 2000 p.f.u./ml resulted in rescue of the helper-free vector. Subsequent passaging of the obtained FD-AdV using Bac-B4 as a helper resulted in approximately 100-fold increases of the vector titer at each passage. This resulting vector was completely free of helper virus and was able to transduce cultured 293 cells. However, scaling-up of FD-AdV production was prevented by the eventual emergence of replication-competent adenovirus (RCA). Experiments are underway to optimize this system for the large-scale production of helper virus-free FD-AdVs and to minimize the possibility of generation of replication-competent adenovirus (RCA) during vector production. This baculovirus-based system will be a very useful alternative to current methods for the production of FD-AdVs.

Adenoviridae↗