A Zoonotic Adenoviral Human Pathogen Emerged through Genomic Recombination among Human and Nonhuman Simian Hosts.

Genomics analysis of a historically intriguing and predicted emergent human adenovirus (HAdV) pathogen, which caused pneumonia and death, provides insight into a novel molecular evolution pathway involving "ping-pong" zoonosis and anthroponosis. The genome of this promiscuous pathogen is embedded with evidence of unprecedented multiple, multidirectional, stable, and reciprocal cross-species infections of hosts from three species (human, chimpanzee, and bonobo). This recombinant genome, typed as HAdV-B76, is identical to two recently reported simian AdV (SAdV) genomes isolated from chimpanzees and bonobos. Additionally, the presence of a critical adenoviral replication element found in HAdV genomes, in addition to genes that are highly similar to counterparts in other HAdVs, reinforces its potential as a human pathogen. Reservoirs in nonhuman hosts may explain periods of apparent absence and then reemergence of human adenoviral pathogens, as well as present pathways for the genesis of those thought to be newly emergent. The nature of the HAdV-D76 genome has implications for the use of SAdVs as gene delivery vectors in human gene therapy and vaccines, selected to avoid preexisting and potentially fatal host immune responses to HAdV.IMPORTANCE An emergent adenoviral human pathogen, HAdV-B76, associated with a fatality in 1965, shows a remarkable degree of genome identity with two recently isolated simian adenoviruses that contain cross-species genome recombination events from three hosts: human, chimpanzee, and bonobo. Zoonosis (nonhuman-to-human transmission) and anthroponosis (human to nonhuman transmission) may play significant roles in the emergence of human adenoviral pathogens.

A novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity.

Recombinant adenoviruses are among the most promising tools for vaccine antigen delivery. Recently, the development of new vectors has focused on serotypes to which the human population is less exposed in order to circumvent pre-existing anti vector immunity. This study describes the derivation of a new vaccine vector based on a chimpanzee adenovirus, Y25, together with a comparative assessment of its potential to elicit transgene product specific immune responses in mice. The vector was constructed in a bacterial artificial chromosome to facilitate genetic manipulation of genomic clones. In order to conduct a fair head-to-head immunological comparison of multiple adenoviral vectors, we optimised a method for accurate determination of infectious titre, since this parameter exhibits profound natural variability and can confound immunogenicity studies when doses are based on viral particle estimation. Cellular immunogenicity of recombinant E1 E3-deleted vector ChAdY25 was comparable to that of other species E derived chimpanzee adenovirus vectors including ChAd63, the first simian adenovirus vector to enter clinical trials in humans. Furthermore, the prevalence of virus neutralizing antibodies (titre >1:200) against ChAdY25 in serum samples collected from two human populations in the UK and Gambia was particularly low compared to published data for other chimpanzee adenoviruses. These findings support the continued development of new chimpanzee adenovirus vectors, including ChAdY25, for clinical use.

[Hybrids of human and monkey adenoviruses (adeno-adeno hybrids) that can reproduce in monkey cells: biological and molecular genetic peculiarities]. / Gibridy adenovirusov cheloveka i obez'iany (adeno-adeno gibridy), sposobnye razmnozhat'sia v kletkakh obez'iany: biologicheskie imolekuliarno-geneticheskie osobennosti.

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.

The fingerprints of the host innate immunity on the cells of primary virus-induced tumors.

As shown earlier, the cells transformed in vitro by various oncogenes, during subsequent in vivo growth were gradually replaced by descendant tumor cells, which co-expressed highly increased H(2)O(2)-catabolizing antioxidant activity (H(2)O(2)(CA)), and the ability to release PGE(2) (PGE(S)) in contact with natural killer cells; v-src was the only oncogene, which in vitro induced cells transformation characterised with the expression of [H(2)O(2)(CA)+PGE(S)] phenotype. Expression of [H(2)O(2)(CA)+PGE(S)] phenotype was providing tumor cells with significantly increased resistance to cytotoxic activities of macrophages and NK cells. However, the possible involvement of [H(2)O(2)(CA)+PGE(S)] phenotype in primary carcinogenesis remained obscure. Here, using three models of the primary virus-induced Syrian hamster tumors we demonstrated that Rous Sarcoma Virus-induced tumors arising after short latent period expressed [H(2)O(2)(CA) + PGE(S)] phenotype at appearance. During the latent periods of SV40- and SA7(C8)-induced tumors the cells expressing [H(2)O(2)(CA)+PGE(S)] phenotype gradually replaced the original [H(2)O(2)(CA)+PGE(S)]-phenotype-negative cell populations. The effectiveness of such selection correlated with the duration of in vivo tumor development. Thus it was shown, that selection of tumor cells expressing [H(2)O(2)(CA)+PGE(S)] phenotype is beginning and may be completed during the latent period of primary carcinogenesis. Taken together, data of this and preceding our studies on [H(2)O(2)(CA)+PGE(S)] phenotype demonstrate that in vivo the host innate immunity antitumor reactions are apparently responsible for the selection of rare tumor cell variants capable to defend themselves against CTA of Mph and NK.

Oncoviral DNAs induce transposition of endogenous mobile elements in the genome of Drosophila melanogaster.

Previously, we have shown that particles of Rous sarcoma virus or cloned fragments of RSV cDNA as well as DNA of oncogenic simian adenovirus Sa7, injected into the polar plasm of early Drosophila melanogaster embryos, were able to induce, with high frequency, unstable visible mutations in different groups of genetic loci. The genetic instability of the recovered mutations, i.e., their ability to revert to normal state or to generate new mutant alleles at the affected locus, was manifest in mutant lines through several generations. The molecular analysis undertaken in this study of the yellow-scute loci region which is highly sensitive to the microinjected Sa7 DNA, and of the white locus, that frequently mutates under the influence of RSV cDNA, clearly shows that the induced mutations and reversions are accompanied by insertion/excision of endogenous mobile elements. This conclusion is confirmed by in situ hybridization experiments which demonstrate that the adenovirus DNA is able to change, though with different efficiency, the chromosomal localization of certain Drosophila retrotransposons. These results partially elucidate the molecular mechanism of the genetic instability in D. melanogaster induced by microinjection of oncoviruses into early embryos, implying that is results from mobilization of endogenous transposons which play the role of insertional elements directly causing unstable mutations.

Simian adenovirus type 7 (SA-7) induces tumours of nerve-supporting or paraneural cell origin in newborn hamsters.

Simian adenovirus type 7 (SA-7) was found to induce tumours originating from nerve-supporting or paraneural cells in newborn hamsters, regardless of injection site or tissues. SA-7 induces glioblastomas characterized by definite localization (subependymal regions) and its main cell type, bipolar spongioblast-like cells, in the brain of hamsters inoculated as newborns. When the eyes of newborn hamsters were directly inoculated, SA-7 failed to induce retinoblastoma (0/27), but retro or peri-bulbar SA-7 tumours frequently occurred in tissues closely related to the peripheral nerve apparatus, including the oculomotor nerve or ciliary ganglion. These tumour cells were situated like stromal cells in these nerve tissues. The histological features of the orbital tumours were similar to those of SA-7-induced subcutaneous tumours but not to brain tumours. In contrast with other hamster brain tumours induced by human adenovirus type 12 or human papova JC virus, medulloepithelioma or medulloblastoma, SA-7 induced tumours exhibit distinctive histological and localization characteristics.

[Nucleolar apparatus function and the ribosomal gene activity of mouse embryonic fibroblasts infected by the SA7(C8) virus]. / Sostoianie nukleoliarnogo apparata i aktivnost' ribosomnykh genov émbrional'nykh fibroblastov myshei, zarazhennykh virusom SA7(C8).

Computer morphometry revealed statistically significant (P less than 0.001) decrease in the number and area of nucleoli and ribosomal gene activities (as demonstrated by AgNOR staining) in primary mouse embryonal fibroblast cultures infected by oncogenic adenovirus SA7(C8). Even more drastic increased in these parameters was found when serum-depleted cultures were infected. There were no effects on nucleolar and AgNOR parameters in mock-infected cultures. It is suggested that an increase in the ribosomal gene activity may reflect synthesis of SA7(C8) early gene products in target cells.

Analysis of the interlaboratory and intralaboratory reproducibility of the enhancement of simian adenovirus SA7 transformation of Syrian hamster embryo cells by model carcinogenic and noncarcinogenic compounds.

The intralaboratory and interlaboratory reproducibility of a DNA virus (SA7) transformation enhancement assay was investigated using nine carcinogenic and noncarcinogenic compounds representing a variety of chemical classes. By the use of standardized procedures designed to limit assay variables, replicate assay data were collected in two independent laboratories and analyzed for concurrence. The carcinogens, 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, and N-methyl-N'-nitro-N-nitrosoguanidine yielded reproducible dose-dependent cytotoxicity and positive transformation effects (defined as statistically significant [p less than or equal to 0.05] enhancement of virus transformation at two or more consecutive dose levels) in all experiments in both laboratories. The carcinogens lead chromate, diethylnitrosamine, 4-nitroquinoline-N-oxide, and 2-acetylaminofluorene demonstrated enhancement of SA7 transformation at two or more dose levels in 40-50% of the assays. The noncarcinogenic structural analogs anthracene and pyrene consistently did not produce positive assay responses when tested at dose levels up to the limits of solubility. Good interlaboratory concurrence was demonstrated for these model compounds in the Syrian hamster embryo cell-SA7 assay.

[Changes in the biochemical parameters of lymphocyte functional activity in simian adenovirus SA7(C8)-induced carcinogenesis]. / Izmenenie nekotorykh biokhimicheskikh parametrov funktsional'noi aktivnosti limfotsitov pri kantserogeneze, indutsirovannom adenovirusom SA7(C8) obez'ian.

A functional defect of lymphocytes in hamsters with tumours during carcinogenesis induced by monkey adenovirus SA7(C8) was found and estimated by means of the blast transformation reaction. An increased activity of transport ATPases, the disturbance of cyclic nucleotide levels (cAMP, cGMP) and a decrease in both basal and Con A-activated calcium transportation were observed simultaneously with changes in the activation of immunocompetent cells under Con A influence.

[Enhancement of the transforming activity of simian adenovirus S5 and of the DNA from human adenovirus type I by benzo(a)pyrene and TPA]. / Usilenie transformiruiushchei aktivnosti adenovirusa S5 obez'ian i DNK adenovirusa tipa I cheloveka benz(a)pirenom i TFA.

Benz(a)pyrene in a concentration of 2.5 micrograms/ml produced a 3.3-fold increase in the transforming activity of simian adenovirus S5 in primary cultures of rat kidney cells. Under analogous conditions TPA increased (2.1 times) the transforming activity of DNA of human adenovirus of type I (AdI). PX5 cells (cotransformed by S5 and benz(a)pyrene) induced tumours in 100% of syngeneic and 81% xenogeneic animals. PX4 cells (transformed by S5 only) induced tumours in 60% of syngeneic animals. Cells transformed by DNA AdI and DNA AdI + TPA did not induce tumours in animals.

Chemical enhancement of SA7 virus transformation of hamster embryo cells: evaluation by interlaboratory testing of diverse chemicals.

Twelve chemicals from diverse structural classes were tested under code for their capacity to enhance the transformation of Syrian hamster embryo cells by simian adenovirus SA7 in two independent laboratories. Pretreatment of hamster cells with eight of those chemicals (reserpine, dichlorvos, methapyrilene hydrochloride, benzidine dihydrochloride, diphenylhydantoin, cinnamyl anthranilate, 11-aminoundecanoic acid, and 4,4'-oxydianiline) produced repeatable enhancement of SA7 transformation at two or more consecutive dose levels, which constitutes clear evidence of enhancing activity in this assay. Both toxic and nontoxic doses of each of these chemicals caused enhancement of virus transformation. Two chemicals (2,6-dichloro-p-phenylenediamine and cinnamaldehyde) produced some evidence of enhancing activity (repeatable transformation enhancement at one dose). Dose ranges for cytotoxicity and enhancement of SA7 transformation were similar in both laboratories for all chemicals producing activity. The final two chemicals, chloramphenicol sodium succinate and ethylene thiourea, failed to reproducibly demonstrate either significant cytotoxicity or enhancement of SA7 transformation at concentrations up to 10-20 mM. The test results for these 12 chemicals were combined with the test results for 9 known carcinogens and noncarcinogens in order to evaluate relationships between activity, dose response, and lowest effective enhancing concentration for these compounds, as well as to correlate them with rodent carcinogenesis classifications. The Syrian hamster embryo cell-SA7 system demonstrated reproducible test responses in both intra- and interlaboratory studies and detected 13 out of 15 known rodent carcinogens.

Influence of various parameters on benzo(a)pyrene enhancement of adenovirus SA7 transformation of Syrian hamster embryo cells.

Several of the major variable factors in the Syrian hamster embryo/simian adenovirus SA7 (SHE/SA7) viral enhancement assay were identified and the effects of these parameters on assay sensitivity were assessed. The extent of dose-dependent cytotoxicity and enhancement of SA7 transformation of primary SHE target cells by benzo(a)pyrene was examined through analysis of data obtained from 37 assays performed over a 2-year period. The variables analyzed for contribution to assay sensitivity included the number of SA7-induced transformed SHE cell foci enumerated in ten replicate dishes in the negative control condition (background focus count) (range: 26-139); the age of the SHE cell cultures at the time of exposure to benzo(a)pyrene (range: 72-144 hr postseeding); and the source of the pregnant hamsters used to prepare the primary SHE cells (Wilmington colony vs Lakeview colony, Charles River Laboratories, Inc., Wilmington, MA). The benzo(a)pyrene-induced cytotoxicity and enhancement of SA7 transformation responses were found to be independent of each of these variables, within the range of values tested.

[T- and B-cell functional activity in the immune response in viral carcinogenesis]. / Issledovanie funktsional'noi aktivnosti T- i B-kletok v immunnom otvete pri virusnom kantserogeneze.

The functional activity of the spleen cells, their ability to produce antibody-forming cells on the model of the adoptive transfer, activity of T-helper-cells and B-precursor-cells of antibody producers in the tumour growth induced by monkey SA7 (C8) adenovirus were studied. The inoculation of the tumourigenic virus to the newborn mice CBA, inhibit antibody formation in the system of adoptive transfer already in the latent period with a sharp inhibition at the terminal stages of carcinogenesis. The helper activity is suppressed earlier than the activity of B-precursors of antibody producers. The obtained data indicate a considerable immunodepressive action of monkey adenovirus SA7 (C8).

Aspiration pneumonia with adenovirus infection in a Japanese macaque (Macaca fuscata fuscata).

Pneumonia with aspiration of foreign bodies and adenovirus infection was identified in a wild Japanese macaque which died 1 month after capture. The adenovirus infection was considered to be secondary in importance to the pneumonia.

[Analysis of cell lines obtained from tumors induced in Syrian hamsters by SA7 adenovirus and its DNA]. / Analiz linii kletok, poluchennykh iz opukholei, indutsirovannykh u siriiskikh khomiachkov adenovirusom SA7 i ego DNK.

Four cell lines derived from tumors induced by adenovirus SA7, its DNA (intact and fragmented with restrictase Sal-1), as well as isolated C-Sal-1 fragment (19.5% of genome) containing oncogene were studied. All the cell lines had the phenotypic markers of tumor cells, similar tumorigenicity, and produced T- and S-antigens. The line derived from the tumor induced by intact DNA contained the smallest adenovirus SA7 genome region (12%) which suggests that the information required for the synthesis of T and S antigens of adenovirus SA7 lies within the limits of this genome region.

[Isolation of cell lines transformed by highly oncogenic simian adenovirus SA7 and nononcogenic human adenovirus type 6 and their DNAs]. / Poluchenie linii kletok, transformirovannykh vysokoonkogennym adenovirusom obez'ian SA7 i neonkogennym adenovirusom cheloveka tipa 6 i ikh DNK.

Methods for generation of cell lines transformed by highly oncogenic simian adenovirus SA7, nononcogenic human adenovirus type 6 and their DNAs are described. WAG rat kidney cells were used for transformation. To produce 1 focus of transformation, 1.7 X 10(6) PFU of SA7 virus is required. Intact and fragmented DNA of both viruses may be quite effectively used for transformation. For production of 1 transformation focus 1.2 microgram SA7 DNA and 1.1 microgram type 6 adenovirus DNA is required. In most cases, DNA fragmentation increases the transforming activity which has been shown to be associated with the left genome region of both viruses under study.

[Use of a BCG vaccine from a Soviet substrain for the immunotherapy of tumors caused by SA7 (C8) virus]. / Ispol'zovanie vaktsiny BTsZh iz otechestvennogo subshtamma dlia immunoterapii opukholei, vyzvannykh virusom SA7 (C8).

The oncolytic and immunotherapeutic effect of the national BCG vaccine with respect to undifferentiated sarcoma of hamster induced by the highly oncogenic adenovirus of green monkeys SA7(C8) was studied. Preliminary data on significant positive effect of the vaccine inoculated before tumor formation were obtained. A single inoculation of the vaccine into tumors did not result in tumor regression in any of the cases.

[Biological properties of large-plaque clone 230 of simian adenovirus SA7(C8)]. / Nekotorye biologicheskie svoistva krupnobliashechnogo klona 230 adenovirusa obez'ian SA7(C8).

The clone KB-230 of simian adenovirus SA7(C8) is described differing from the reference SA7(C8) strain and clones KB-2 and MB-1 by the presence of additional recognition sites when treated with different endonucleases. The KB-230 clone differs antigenically in the neutralization test from the MB-1 clone. Some biological and molecular-biological properties of the KB-230 clone were studied. All the simian cell cultures under study were highly sensitive to the cytopathic effect and replication of the KB-230 clone. The reproduction cycle of the KB-230 clone was 12 h, that of KB-2 clone 16 h. The maximum accumulation of virus in the cells was observed by 27-29 h for both clones. The KB-230 clone differed from the KB-2 clone by early development of the CPE (by 9 h of cultivation against 19 h for the latter). The oncogenic activity of the KB-230 clone was less marked than that of the MB-1 clone. The methods of heteroduplex and polypeptide analysis established the difference of the KB-230 clone from the reference SA7(C8) strain and KB-2 and MB-1 clones.

Persistent infection with adenovirus types 5 and 6 in lymphoid cells from humans and woolly monkeys.

Chronic infection with adenovirus types 5 and 6 was established in primary mononuclear leukocytes from human umbilical cord blood and in Epstein-Barr virus (EBV)-transformed B lymphocytes from human umbilical cord blood and from woolly monkey blood. Adenovirus could be recovered from cultures of primary leukocytes and of EBV-transformed lymphocytes for two and three months, respectively, without visible alteration of cell growth. Infection in cultures of EBV-transformed lymphocytes from woolly monkey blood was obliterated by exposure to antibody, but EBV-transformed lymphocytes from human umbilical cord blood contained small amounts of virus for prolonged periods that restored infection in the culture when antibody was removed. Thus, chronic infection of lymphoid cells by some adenoviruses is maintained by at least two mechanisms: cell-to-cell spread of virus in the absence of antibody and intracellular persistence of infectious virus in the presence of antibody.