Genome analysis of a novel avian atadenovirus reveals a possible horizontal gene transfer.

We report the discovery and characterization of a novel adenovirus, Zoothera dauma adenovirus (ZdAdV), from a wild bird species, Zoothera dauma (Scaly thrush). This new atadenovirus was discovered by metagenomic sequencing without virus cultivation. Analyses of the full genome sequence revealed that this new virus is a distinct member of the genus Atadenovirus and represents a novel species. ZdAdV has a genome of 34,760 bp with 28 predicted genes and 39% GC content. ZdAdV is the first atadenovirus to contain ORF19, a gene previously found only in aviadenoviruses. Phylogenetic analysis of ORF19 suggests that it was acquired by ZdAdV through horizontal gene transfer from an aviadenovirus. By analyzing all orthologous genes of aviadenovirus, mastadenovirus, atadenovirus, and siadenovirus, we also found potential horizontal gene transfer for the E4 gene in Pigeon aviadenovirus B. Our study widens our knowledge concerning the genetic diversity and evolutionary history of atadenoviruses and their potential for cross-species transmission.

Complete Genome Sequencing of an Embryonated Chicken Egg-Adapted Duck atadenovirus A.

Egg drop syndrome (EDS) is prevalent in industrial poultry globally. This disease is caused by Duck atadenovirus A or EDS virus (EDSV), a member of the genus Atadenovirus under the family Adenoviridae. The disease is attributed to significant economic losses in the poultry industry worldwide due to a drop in egg production, reduction in egg quality, and failure to reach maximum egg production. Oil-adjuvant inactivated vaccines, which are widely used in the poultry industry, provide good protection for immunized chickens against EDS. This study aimed to genetically and phylogenetically analyze the full-length genome of an embryonated chicken egg-adapted EDSV strain 127. After extraction of viral DNA from the allantoic fluid, overlapping fragments of the viral genome sequence were generated by polymerase chain reaction (PCR) using 25 pairs of primers. Purified PCR products were subjected to complete genome sequencing by the next-generation sequencing (NGS) approach. The nucleotide homology observed between genomes of the studied strain and that of the original strain 127 (NC_001813) of laying chickens was 99.9%. Its genome was 33,213 bp in length, with a G + C content of 43.01%. A comparison of the genome sequence of the egg-adapted virus with strain 127 revealed only three non-synonymous single-nucleotide polymorphisms (SNPs) between these viral genome sequences. Two mutations of S320G and I62K out of these SNPs were found within the coding regions of fiber and hypothetical proteins which may play a role in the adaptation of EDSV in the embryonated chicken eggs. The full genome sequencing of EDSV using NGS techniques provides insights into the discovery of genetic variants. Moreover, the genome sequence information of the EDSV provides valuable data for vaccine development in near future.

Molecular characterization of a bovine adenovirus type 7 (Bovine Atadenovirus F) strain isolated from a systemically infected calf in Germany.

Bovine adenovirus 7 (BAdV-7) is an unclassified member of the genus Atadenovirus with a worldwide distribution and has been reported to induce clinical disease of varying severity in infected cattle, ranging from asymptomatic infections to severe enteric or respiratory disease. In this study, we used next-generation sequencing to obtain the first complete genome sequence of a European strain of BadV-7, from pooled spleen and liver tissue obtained from a deceased newborn Limousin calf. Histopathological analysis and electron microscopy showing systemic lesions in multiple organs with intranuclear amphophilic inclusions observed in endothelial cells in multiple peripheral tissues. Virus isolation was readily achieved from tissue homogenate using bovine esophagus cells (KOP-R), a strategy which should facilitate future in vitro or in vivo BAdV-7 studies. Phylogenetic analysis of available genome sequences of BAdV-7 showed that the newly identified strain groups most closely with a recent BAdV-7 strain, SD18-74, from the USA, confirming that this newly identified strain is a member of the Atadenovirus genus. The fiber gene was found to be highly conserved within BAdV-7 strains but was highly divergent in comparison to Ovine adenovirus 7 (OAdV-7) (39.56% aa sequence identity). Furthermore, we report a variable region of multiple tandem repeats between the coding regions of E4.1 and RH5 genes. In summary, the presented pathological and molecular characterization of this case suggests that further research into the worldwide molecular epidemiology and disease burden of BAdV-7 is warranted.

First Report on Detection and Molecular Characterization of Adenoviruses in the Small Indian Mongoose (Urva auropunctata).

Using a broad-range nested PCR assay targeting the DNA-dependent DNA polymerase (pol) gene, we detected adenoviruses in 17 (20.48%) out of 83 fecal samples from small Indian mongooses (Urva auropunctata) on the Caribbean island of St. Kitts. All 17 PCR amplicons were sequenced for the partial pol gene (~300 bp, hereafter referred to as Mon sequences). Fourteen of the 17 Mon sequences shared maximum homology (98.3-99.6% and 97-98.9% nucleotide (nt) and deduced amino acid (aa) sequence identities, respectively) with that of bovine adenovirus-6 (species Bovine atadenovirus E). Mongoose-associated adenovirus Mon-39 was most closely related (absolute nt and deduced aa identities) to an atadenovirus from a tropical screech owl. Mon-66 shared maximum nt and deduced aa identities of 69% and 71.4% with those of atadenoviruses from a spur-thighed tortoise and a brown anole lizard, respectively. Phylogenetically, Mon-39 and Mon-66 clustered within clades that were predominated by atadenoviruses from reptiles, indicating a reptilian origin of these viruses. Only a single mongoose-associated adenovirus, Mon-34, was related to the genus Mastadenovirus. However, phylogenetically, Mon-34 formed an isolated branch, distinct from other mastadenoviruses. Since the fecal samples were collected from apparently healthy mongooses, we could not determine whether the mongoose-associated adenoviruses infected the host. On the other hand, the phylogenetic clustering patterns of the mongoose-associated atadenoviruses pointed more towards a dietary origin of these viruses. Although the present study was based on partial pol sequences (~90 aa), sequence identities and phylogenetic analysis suggested that Mon-34, Mon-39, and Mon-66 might represent novel adenoviruses. To our knowledge, this is the first report on the detection and molecular characterization of adenoviruses from the mongoose.

Identification and genetic characterization of an isolate of bovine adenovirus 7 from the United States, a putative member of a new species in the genus Atadenovirus.

The bovine adenovirus 7 (BAdV-7) isolate SD18-74 was recovered from lung tissue of calves in South Dakota. The 30,043-nucleotide (nt) genome has the typical organization of Atadenovirus genus members. The sequence shares over 99% nt sequence identity with two Japanese BAdV-7 sequences, followed by 74.9% nt sequence identity with the ovine adenovirus 7 strain OAV287, a member of the species Ovine atadenovirus D. SD18-74 was amplified in both bovine and ovine primary nasal turbinate cells, demonstrating greater fitness in bovine cells. The genomic and biological characteristics of BAdV-7 SD18-74 support the inclusion of the members of the BAdV-7 group in a new species in the genus Atadenovirus.

Molecular Characterisation of a Novel and Highly Divergent Passerine Adenovirus 1.

Wild birds harbour a large number of adenoviruses that remain uncharacterised with respect to their genomic organisation, diversity, and evolution within complex ecosystems. Here, we present the first complete genome sequence of an atadenovirus from a passerine bird that is tentatively named Passerine adenovirus 1 (PaAdV-1). The PaAdV-1 genome is 39,664 bp in length, which was the longest atadenovirus to be sequenced, to the best of our knowledge, and contained 42 putative genes. Its genome organisation was characteristic of the members of genus Atadenovirus; however, the novel PaAdV-1 genome was highly divergent and showed the highest sequence similarity with psittacine adenovirus-3 (55.58%). Importantly, PaAdV-1 complete genome was deemed to contain 17 predicted novel genes that were not present in any other adenoviruses sequenced to date, with several of these predicted novel genes encoding proteins that harbour transmembrane helices. Subsequent analysis of the novel PaAdV-1 genome positioned phylogenetically to a distinct sub-clade with all others sequenced atadenoviruses and did not show any obvious close evolutionary relationship. This study concluded that the PaAdV-1 complete genome described here is not closely related to any other adenovirus isolated from avian or other natural host species and that it should be considered a separate species.

Real-time fluorescence loop-mediated isothermal amplification assay for direct detection of egg drop syndrome virus.

BACKGROUND: Egg drop syndrome (EDS), caused by the adenovirus "egg drop syndrome virus" (EDSV) causes severe economic losses through reduced egg production in breeder and layer flocks. The diagnosis of EDSV has been done by molecular tools since its complete genome sequence was identified. In order to enhance the capabilities of the real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay, we aimed to apply the method for direct detection of the EDSV without viral DNA extraction. In order to detect the presence of the EDSV DNA, three pairs of primers were designed, from the conserved region of fiber gene of the EDSV. RESULTS: For our assay, test and control samples were directly used in the reaction mixture in 10-fold serial dilution. The target DNA was amplified at 65 °C, which yield positive results in a relatively short period of 40-45 min. The method reported in this study is highly sensitive as compared to polymerase chain reaction (PCR) and showed no sign of cross-reactivity or false positive results. The RealAmp accomplished specific identification of EDSV among a variety of poultry disease viruses. CONCLUSIONS: The direct RealAmp can be used to detect the presence of EDSV. As our result showed, the RealAmp method could be suitable for the direct detection of other DNA viruses.

Structure of a Reptilian Adenovirus Reveals a Phage Tailspike Fold Stabilizing a Vertebrate Virus Capsid.

Although non-human adenoviruses (AdVs) might offer solutions to problems posed by human AdVs as therapeutic vectors, little is known about their basic biology. In particular, there are no structural studies on the complete virion of any AdV with a non-mammalian host. We combine mass spectrometry, cryo-electron microscopy, and protein crystallography to characterize the composition and structure of a snake AdV (SnAdV-1, Atadenovirus genus). SnAdV-1 particles contain the genus-specific proteins LH3, p32k, and LH2, a previously unrecognized structural component. Remarkably, the cementing protein LH3 has a trimeric ß helix fold typical of bacteriophage host attachment proteins. The organization of minor coat proteins differs from that in human AdVs, correlating with higher thermostability in SnAdV-1. These findings add a new piece to the intriguing puzzle of virus evolution, hint at the use of cell entry pathways different from those in human AdVs, and will help development of new, thermostable SnAdV-1-based vectors.

Whole-genome sequences of Odocoileus hemionus deer adenovirus isolates from deer, moose and elk are highly conserved and support a new species in the genus Atadenovirus.

We present the first complete genome sequence of Odocoileus hemionus deer adenovirus 1 (OdAdV-1). This virus can cause sporadic haemorrhagic disease in cervids, although epizootics with high mortality have occurred in California. OdAdV-1 has been placed in the genus Atadenovirus, based on partial hexon, pVIII and fibre genes. Ten field isolates recovered from naturally infected mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginiana) and moose (Alces alces) from Wyoming, black-tailed deer (Odocoileus hemionus columbianus) from California, and Rocky Mountain elk (Cervus elaphus nelsoni) from Colorado and Washington state were sequenced. The genome lengths ranged from 30 620 to 30 699 bp, contained the predicted proteins and gene organization typical of members of genus Atadenovirus, and had a high percentage of A/T nucleotides (66.7 %). Phylogenic analysis found that the closest ancestry was with ruminant atadenoviruses, while a divergence of the hexon, polymerase and penton base proteins of more than 15 % supports classification as a new species. Genetic global comparison between the 10 isolates found an overall 99 % identity, but greater divergence was found between those recovered from moose and elk as compared to deer, and a single variable region contained most of these differences. Our findings demonstrate that OdAdV-1 is highly conserved between 10 isolates recovered from multiple related cervid species, but genotypic differences, largely localized to a variable region, define two strains. We propose that the virus type name be changed to cervid adenovirus 1, with the species name Cervid atadenovirus A. Sequence data were used to develop molecular assays for improved detection and genotyping.

Hyperplastic stomatitis and esophagitis in a tortoise (Testudo graeca) associated with an adenovirus infection.

A 2-year-old female, spur-thighed tortoise (Testudo graeca) was presented with poor body condition (1/5) and weakness. Fecal analysis revealed large numbers of oxyurid-like eggs, and radiographs were compatible with gastrointestinal obstruction. Despite supportive medical treatment, the animal died. At gross examination, an intestinal obstruction was confirmed. Histopathology revealed severe hyperplastic esophagitis and stomatitis with marked epithelial cytomegaly and enormous basophilic intranuclear inclusion bodies. Electron microscopy examination revealed a large number of 60-80 nm, nonenveloped, icosahedral virions arranged in crystalline arrays within nuclear inclusions of esophageal epithelial cells, morphologically compatible with adenovirus-like particles. PCR for virus identification was performed with DNA extracted from formalin-fixed, paraffin-embedded tissues. A nested, consensus pan-adenovirus PCR and sequencing analysis showed a novel adenovirus. According to phylogenetic calculations, it clustered to genus Atadenovirus in contrast with all other chelonian adenoviruses described to date. The present report details the pathologic findings associated with an adenovirus infection restricted to the upper digestive tract.

Molecular characterization of a lizard adenovirus reveals the first atadenovirus with two fiber genes and the first adenovirus with either one short or three long fibers per penton.

UNLABELLED: Although adenoviruses (AdVs) have been found in a wide variety of reptiles, including numerous squamate species, turtles, and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (Heloderma horridum). Here we report the full genomic and proteomic characterization of the latter, designated lizard adenovirus 2 (LAdV-2). The double-stranded DNA (dsDNA) genome of LAdV-2 is 32,965 bp long, with an average G+C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome were largely concordant with those in other atadenoviruses, except for four novel open reading frames (ORFs) at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits shared with SnAdV-1 further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein compositions of the virions. The two fiber genes produce two fiber proteins of different sizes that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers nor a mixed number of fibers per vertex had previously been reported for adenoviruses or any other virus. IMPORTANCE: Here we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins-one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton base. This observation raises new intriguing questions on virus structure. How can the triple fiber attach to a pentameric vertex? What determines the number and location of each vertex type in the icosahedral particle? Since fibers are responsible for primary attachment to the host, this novel architecture also suggests a novel mode of cell entry for LAdV-2. Adenoviruses have a recognized potential in nanobiomedicine, but only a few of the more than 200 types found so far in nature have been characterized in detail. Exploring the taxonomic wealth of adenoviruses should improve our chances to successfully use them as therapeutic tools.

Development of a new real-time polymerase chain reaction assay to detect Duck adenovirus A DNA and application to samples from Swiss poultry flocks.

Between 2008 and 2012, commercial Swiss layer and layer breeder flocks experiencing problems in laying performance were sampled and tested for infection with Duck adenovirus A (DAdV-A; previously known as Egg drop syndrome 1976 virus). Organ samples from birds sent for necropsy as well as blood samples from living animals originating from the same flocks were analyzed. To detect virus-specific DNA, a newly developed quantitative real-time polymerase chain reaction method was applied, and the presence of antibodies against DAdV-A was tested using a commercially available enzyme-linked immunosorbent assay. In 5 out of 7 investigated flocks, viral DNA was detected in tissues. In addition, antibodies against DAdV-A were detected in all of the flocks.

Crystallization of the C-terminal domain of the fibre protein from snake adenovirus 1, an atadenovirus.

Adenovirus fibre proteins play an important role in determining viral tropism. The C-terminal domain of the fibre protein from snake adenovirus type 1, a member of the Atadenovirus genus, has been expressed, purified and crystallized. Crystals were obtained belonging to space groups P2(1)2(1)2(1) (two different forms), I2(1)3 and F23. The best of these diffracted synchrotron radiation to a resolution of 1.4 Å. As the protein lacks methionines or cysteines, site-directed mutagenesis was performed to change two leucine residues to methionines. Crystals of selenomethionine-derivatized crystals of the I2(1)3 form were also obtained and a multi-wavelength anomalous dispersion data set was collected.

Epidemiology of egg drop syndrome virus in ducks from South Korea.

Egg drop syndrome virus (EDSV) is an important pathogen of poultry that decreases egg production in chickens and causes respiratory disease in goslings. In 2011, we obtained serum samples from 139 domestic Pekin ducks, 416 one-day-old Pekin ducklings, and 75 wild ducks (67 mallards and 8 pintails) to survey their exposure to EDSV. A total of 123 of 139 sera (88.5%) from Pekin ducks, 396 of the ducklings (95.2%), and 16 of 67 mallards (23.9%) were positive. Field cases of EDSV in wild and domestic ducks were investigated. Six cases from domestic Pekin ducks were identified by PCR detection and were used for virus isolation and molecular analysis. Phylogenetic analyses of the partial hexon and full fiber genes showed that the D11-JW-012 and D11-JW-017 strains among 6 isolates belonged to different clusters compared with other known strains including the 127 strain. We assessed cell growth efficiency by hemagglutination (HA) titers and cytopathic effects in duck embryo liver cells and chicken embryo liver (CEL) cells to investigate host adaptation. The D11-JW-017 strain propagated more in chicken embryo liver than the D11-JW-012 strain and the field isolate from chickens. Our results demonstrate the high prevalence of EDSV in wild and domestic ducks in South Korea and provide information on EDSV from ducks that showed variable adaptability in chickens.

Respiratory disease due to current egg drop syndrome virus in Pekin ducks.

Severe acute respiratory symptoms with coughing, dyspnea, and gasping were reported in two flocks of 9-day-old Pekin ducklings from different provinces. Gross lesions, white exudate and mucous membrane congestion in the trachea as well as blue to purple color changes and sclerosis in lungs were observed. Histological lesions revealed that the trachea and bronchial epithelium were hyperplastic and infiltrated by neutrophil granulocytes. Egg drop syndrome virus (EDSV) was differentially diagnosed using polymerase chain reaction, and the strains were isolated from tracheas and lungs by inoculation of 10-day-old embryonated duck eggs. The virus isolates were designated strain D11-JW-012 and D11-JW-017. The clinical and pathological signs were reproduced by intra-tracheal inoculation of the isolates in 3-day-old ducklings. Although the two isolates produced similar clinical signs, pathological lesions and ciliostasis, the D11-JW-017 strain resulted in more severe clinical signs with progressive symptoms compared to those of D11-JW-012 strain-infected ducklings. We suggest that different EDSV strains with mild or severe to moderate pathogenicity coexist and have potential risks in poultry. Hereby, we report an EDSV infection in ducklings.

Detection of known and novel adenoviruses in cattle wastes via broad-spectrum primers.

The critical assessment of bovine adenoviruses (BAdV) as indicators of environmental fecal contamination requires improved knowledge of their prevalence, shedding dynamics, and genetic diversity. We examined DNA extracted from bovine and other animal waste samples collected in Wisconsin for atadenoviruses and mastadenoviruses using novel, broad-spectrum PCR primer sets. BAdV were detected in 13% of cattle fecal samples, 90% of cattle urine samples, and 100% of cattle manure samples; 44 percent of BAdV-positive samples contained both Atadenovirus and Mastadenovirus DNA. Additionally, BAdV were detected in soil, runoff water from a cattle feedlot, and residential well water. Overall, we detected 8 of 11 prototype BAdV, plus bovine, rabbit, and porcine mastadenoviruses that diverged significantly from previously reported genotypes. The prevalence of BAdV shedding by cattle supports targeting AdV broadly as indicators of the presence of fecal contamination in aqueous environments. Conversely, several factors complicate the use of AdV for fecal source attribution. Animal AdV infecting a given livestock host were not monophyletic, recombination among livestock mastadenoviruses was detected, and the genetic diversity of animal AdV is still underreported. These caveats highlight the need for continuing genetic surveillance for animal AdV and for supporting data when BAdV detection is invoked for fecal source attribution in environmental samples. To our knowledge, this is the first study to report natural BAdV excretion in urine, BAdV detection in groundwater, and recombination in AdV of livestock origin.

A unique novel reptilian paramyxovirus, four atadenovirus types and a reovirus identified in a concurrent infection of a corn snake (Pantherophis guttatus) collection in Germany.

In 2009, 26 clinical samples (organs and oral/cloacal swabs) from a total of 24 corn snakes (Pantherophis guttatus) from a single owner were sent to our laboratory to be tested for the presence of viruses. Paramyxoviruses (PMV), adenoviruses (AdV) and reoviruses were detected by RT-PCR, PCR and virus isolation methods. Three snakes were infected with all three viruses at the same time, while two other snakes had a double infection (PMV and reo, AdV and reo) and nine other snakes had a single infection with any of the three viruses. No viruses were detected in 10 animals. All isolated reoviruses were identical to one another and to the reptilian orthoreovirus isolate 55-02 in the partial RNA dependent RNA polymerase (RDRP) gene sequence. AdV partial polymerase sequences represented four different types, one of which was first described here: most similar to SnAdV-1, while the other three were identical to known types: SnAV-1, -2 and -3. However, the detected single PMV differed distinctly from described reptile PMV and was a new type. According to partial L gene, HN gene and U gene sequences it may be the first described representative of a third squamatid PMV cluster: "group C" within the proposed reptilian PMV genus "Ferlavirus". Nucleotide identity values for the L gene of the new PMV compared to group A viruses range between 76.5 and 80.3%, and between 80.5 and 81.2% compared to group B viruses. For the HN gene, these values were similar: 78.2-80% (A) and 79.9-80.5% (B) and somewhat lower for the U gene: 72.7-75.4% (A) and 69.7-70% (B). No reports on the prevalence of concurrent viral infection in captive snake populations have been published so far. The possibility of concurrent infection with several different viruses and subsequent consequences for animal health should be kept in mind when testing reptile samples for viruses.

Efficient replication and generation of recombinant bovine adenovirus-3 in nonbovine cotton rat lung cells expressing I-SceI endonuclease.

BACKGROUND: The rigorous evaluation of recombinant bovine adenovirus (BAdV)-3 as a gene delivery vector requires quick and efficient method of isolating recombinants. This requires both a suitable cell line and an efficient method of rescuing recombinant BAdV-3. To facilitate rapid isolation of recombinant BAdV-3, we have developed an efficient system for generating recombinants using newly identified nonbovine cell line permissive for replication of BAdV-3. METHODS: Nonbovine cotton rat lung (CRL) cells in comparison to Madin-Darby bovine kidney cells and VIDO R2 cells were analyzed for the production of progeny virus and DNA transfection efficiency. In addition, lentiviral expression system was used to generate stable nonbovine CRL cell line expressing endonuclease I-SceI as examined by western blotting. Transfection of this cell line with circular or linear plasmid containing full-length BAdV-3 genome was used to generate recombinant BAdV-3. RESULTS: We demonstrate that nonbovine CRL cells are permissive for replication of BAdV-3 and can be efficiently transfected with plasmid DNA. Second, we constructed CRL cell line (VIDO DT1) expressing an intron-encoding endonuclease I-SceI. Finally, we demonstrate that transfection of VIDO DT1 cells with a circular plasmid containing recombinant BAdV-3 genome flanked by I-SceI recognition sites can efficiently rescue recombinant virus. CONCLUSIONS: The use of circular molecular clones together with I-SceI endonuclease expressing, BAdV-3 permissive CRL cell line not only increased the viral genome transfection efficiency, but also reduced the viral rescue time and amount of DNA required for rescuing recombinant BAdV-3s.

Induction of both cellular and humoral immunity following a rational prime-boost immunization regimen that incorporates recombinant ovine atadenovirus and fowlpox virus.

Recombinant fowlpox viruses (rFPV) and ovine atadenoviruses (rOAdV) are being developed as safe, nonpathogenic, prophylactic and therapeutic vaccine vectors. There is scope, however, to improve the limited immune responses elicited by each of these vaccine vectors. Using previously determined and optimized routes of administration and viral doses, we characterized the primary adaptive immune responses elicited by recombinant variants of each virus. We demonstrate the contrasting nature of the response elicited by each recombinant virus. Whereas rFPV generates predominately cell-mediated immunity to our nominal target antigen, ovalbumin (OVA), rOAdV drives strong humoral responses. By defining the time taken to achieve maximal cytotoxic T cell responses and by studying the different patterns and kinetics of major histocompatibility complex class I-restricted OVA antigen expression postimmunization, we proposed a heterologous prime-boost regimen of immunization with rOAdV followed by rFPV. The subsequent experimental results showed that this approach produced robust cell-mediated and humoral immune responses against OVA that, importantly, were accompanied by weak anti-viral vector antibody responses. These results, therefore, represent a novel and potentially clinically applicable way to achieve broadly based and effective immunity to the antigens encoded by vectored vaccines.

Ovine atadenovirus, a novel and highly immunogenic vector in prime-boost studies of a candidate HIV-1 vaccine.

Ovine adenovirus type 7 (OAdV) is the prototype member of the genus Atadenovirus. No immunity to the virus has so far been detected in human sera. We describe the construction and evaluation of a candidate HIV-1 vaccine based on OAdV and its utilisation alone and in combination with plasmid-, human adenovirus type 5 (HAdV5; a Mastadenovirus)-, and modified vaccinia Ankara (MVA)-vectored vaccines. All vectors expressed HIVA, an immunogen consisting of HIV-1 clade A consensus Gag-derived protein coupled to a T cell polyepitope. OAdV.HIVA was genetically stable, grew well and expressed high levels of protein from the Rous sarcoma virus promoter. OAdV.HIVA was highly immunogenic in mice and efficiently primed and boosted HIV-1-specific T cell responses together with heterologous HIVA-expressing vectors. There were significant differences between OAdV and HAdV5 vectors in priming of naïve CD8(+) T cell responses to HIVA and in the persistence of MHC class I-restricted epitope presentation in the local draining lymph nodes. OAdV.HIVA primed T cells more rapidly but was less persistent than AdV5.HIVA and thus induced a qualitatively distinct T cell response. Nevertheless, both vectors primed a response in mice that reduced viral titres in a surrogate challenge model by three to four orders of magnitude. Thus, OAdV is a novel, underexplored vaccine vector with potential for further development for HIV-1 and other vaccines. The data are discussed in the context of the latest HIV-1 vaccine developments.