Overreliance on the hexon gene, leading to misclassification of human adenoviruses

The genome of human adenovirus (HAdV) D30 was sequenced in depth. Sequence assembly and analysis revealed two distinct viral sequences with identical hexon genes, which were the same as the one previously reported for HAdV-D30. However, one of the two viruses was found to be a recombinant of HAdV-D29. Exclusive reliance on serum neutralization can lead to mischaracterization of adenoviruses and miss coinfections. Whole-genome sequencing remains the gold standard for proper classification of HAdVs.     

Phylogenetic analysis of the main neutralization and hemagglutination determinants of all human adenovirus prototypes as a basis for molecular classification and taxonomy

Human adenoviruses (HAdV) are responsible for a wide spectrum of diseases. The neutralization epsilon determinant (loops 1 and 2) and the hemagglutination gamma determinant are relevant for the taxonomy of HAdV. Precise type identification of HAdV prototypes is crucial for detection of infection chains and epidemiology. epsilon and gamma determinant sequences of all 51 HAdV were generated to propose molecular classification criteria. Phylogenetic analysis of epsilon determinant sequences demonstrated sufficient genetic divergence for molecular classification, with the exception of HAdV-15 and HAdV-29, which also cannot be differentiated by classical cross-neutralization. Precise sequence divergence criteria for typing (<2.5% from loop 2 prototype sequence and <2.4% from loop 1 sequence) were deduced from phylogenetic analysis. These criteria may also facilitate identification of new HAdV prototypes. Fiber knob (gamma determinant) phylogeny indicated a two-step model of species evolution and multiple intraspecies recombination events in the origin of HAdV prototypes. HAdV-29 was identified as a recombination variant of HAdV-15 (epsilon determinant) and a speculative, not-yet-isolated HAdV prototype (gamma determinant). Subanalysis of molecular evolution in hypervariable regions 1 to 6 of the epsilon determinant indicated different selective pressures in subclusters of species HAdV-D. Additionally, gamma determinant phylogenetic analysis demonstrated that HAdV-8 did not cluster with -19 and -37 in spite of their having the same tissue tropism. The phylogeny of HAdV-E4 suggested origination by interspecies recombination between HAdV-B (hexon) and HAdV-C (fiber), as in simian adenovirus 25, indicating additional zoonotic transfer. In conclusion, molecular classification by systematic sequence analysis of immunogenic determinants yields new insights into HAdV phylogeny and evolution.     

Genetic analysis of a novel human adenovirus with a serologically unique hexon and a recombinant fiber gene

In February of 1996 a human adenovirus (formerly known as Ad-Cor-96-487) was isolated from the stool of an AIDS patient who presented with severe chronic diarrhea. To characterize this apparently novel pathogen of potential public health significance, the complete genome of this adenovirus was sequenced to elucidate its origin. Bioinformatic and phylogenetic analyses of this genome demonstrate that this virus, heretofore referred to as HAdV-D58, contains a novel hexon gene as well as a recombinant fiber gene. In addition, serological analysis demonstrated that HAdV-D58 has a different neutralization profile than all previously characterized HAdVs. Bootscan analysis of the HAdV-D58 fiber gene strongly suggests one recombination event.     

Computational and serologic analysis of novel and known viruses in species human adenovirus D in which serology and genomics do not correlate

In November of 2007 a human adenovirus (HAdV) was isolated from a bronchoalveolar lavage (BAL) sample recovered from a biopsy of an AIDS patient who presented with fever, cough, tachycardia, and expiratory wheezes. To better understand the isolated virus, the genome was sequenced and analyzed using bioinformatic and phylogenomic analysis. The results suggest that this novel virus, which is provisionally named HAdV-D59, may have been created from multiple recombination events. Specifically, the penton, hexon, and fiber genes have high nucleotide identity to HAdV-D19C, HAdV-D25, and HAdV-D56, respectively. Serological results demonstrated that HAdV-D59 has a neutralization profile that is similar yet not identical to that of HAdV-D25. Furthermore, we observed a two-fold difference between the ability of HAdV-D15 and HAdV-D25 to be neutralized by reciprocal antiserum indicating that the two hexon proteins may be more similar in epitopic conformation than previously assumed. In contrast, hexon loops 1 and 2 of HAdV-D15 and HAdV-D25 share 79.13 and 92.56 percent nucleotide identity, respectively. These data suggest that serology and genomics do not always correlate.     

Phylogenetic analysis and structural predictions of human adenovirus penton proteins as a basis for tissue-specific adenovirus vector design

The penton base is a major capsid protein of human adenoviruses (HAdV) which forms the vertices of the capsid and interacts with hexon and fiber protein. Two hypervariable loops of the penton are exposed on the capsid surface. Sequences of these and 300 adjacent amino acid residues of all 51 HAdV and closely related simian adenoviruses were studied. Adjacent sequences and predicted overall secondary structure were conserved. Phylogenetic analysis revealed clustering corresponding to the HAdV species and recombination events in the origin of HAdV prototypes. All HAdV except serotypes 40 and 41 of species F exhibited an integrin binding RGD motif in the second loop. The lengths of the loops (HVR1 and RGD loops) varied significantly between HAdV species with the longest RGD loop observed in species C and the longest HVR1 in species B. Long loops may permit the insertion of motifs that modify tissue tropism. Genetic analysis of HAdV prime strain p17'H30, a neutralization variant of HAdV-D17, indicated the significance of nonhexon neutralization epitopes for HAdV immune escape. Fourteen highly conserved motifs of the penton base were analyzed by site-directed mutagenesis of HAdV-D8 and tested for sustained induction of early cytopathic effects. Thus, three new motifs essential for penton base function were identified additionally to the RGD site, which interacts with a secondary cellular receptor responsible for internalization. Therefore, our penton primary structure data and secondary structure modeling in combination with the recently published fiber knob sequences may permit the rational design of tissue-specific adenoviral vectors.     

Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells

The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC), caused by viruses within human adenovirus species D (HAdV-D), is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD) profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling.     

Construction and Characterization of a Novel Recombinant Attenuated and Replication-Deficient Candidate Human Adenovirus Type 3 Vaccine: ‘‘Adenovirus Vaccine Within an Adenovirus Vector'

Human adenoviruses(HAd Vs) are highly contagious and result in large number of acute respiratory disease(ARD) cases with severe morbidity and mortality. Human adenovirus type 3(HAd V-3) is the most common type that causes ARD outbreaks in Asia,Europe, and the Americas. However, there is currently no vaccine approved for its general use. The hexon protein contains the main neutralizing epitopes, provoking strong and lasting immunogenicity. In this study, a novel recombinant and attenuated adenovirus vaccine candidate against HAd V-3 was constructed based on a commercially-available replication-defective HAd V-5 gene therapy and vaccine vector. The entire HAd V-3 hexon gene was integrated into the E1 region of the vector by homologous recombination using a bacterial system. The resultant recombinants expressing the HAd V-3 hexon protein were rescued in AD293 cells, identified and characterized by RT-PCR, Western blots, indirect immunofluorescence, and electron microscopy. This potential vaccine candidate had a similar replicative efficacy as the wild-type HAd V-3 strain. However, and importantly, the vaccine strain had been rendered replication-defective and was incapable of replication in A549 cells after more than twentygeneration passages in AD293 cells. This represents a significant safety feature. The mice immunized both intranasally and intramuscularly by this vaccine candidate raised significant neutralizing antibodies against HAd V-3. Therefore, this recombinant,attenuated, and safe adenovirus vaccine is a promising HAd V-3 vaccine candidate. The strategy of using a clinically approved and replication-defective HAd V-5 vector provides a novel approach to develop universal adenovirus vaccine candidates against all the other types of adenoviruses causing ARDs and perhaps other adenovirus-associated diseases.     

Computational Analysis of Human Adenovirus Type 22 Provides Evidence for Recombination among Species D Human Adenoviruses in the Penton Base Gene

Recombination in human adenoviruses (HAdV) may confer virulence upon an otherwise nonvirulent strain. The genome sequence of species D HAdV type 22 (HAdV-D22) revealed evidence for recombination with HAdV-D19 and HAdV-D37 within the capsid penton base gene. Bootscan analysis demonstrated that recombination sites within the penton base gene frame the coding sequences for the two external hypervariable loops in the protein. A similar pattern of recombination was evident within other HAdV-D types but not other HAdV species. Further study of recombination among HAdVs is needed to better predict possible recombination events among wild-type viruses and adenoviral gene therapy vectors.Adenoviruses were first isolated in 1953 (18, 31) and currently cause an array of human diseases. These include respiratory, gastrointestinal, and ocular surface infections, opportunistic infections in immune-deficient individuals, and possibly, obesity (9, 13, 14, 20, 40). Adenoviruses have also recently been used as gene therapy vectors (19, 35). Thus, while adenoviruses continue to cause significant morbidity and mortality in the human population, their existence also provides a potential benefit for the treatment of patients with an even broader range of ailments.Since the characterization of the first human adenovirus (HAdV), 53 types have been identified and subsequently classified into seven species (A to G) on the basis of serology, restriction endonuclease digestion patterns, and to a lesser degree, genotyping. Recently, high-throughput sequencing technology has made whole-genome sequencing both rapid and affordable (27). However, the genomic sequences of 23 out of 53 HAdV types remain to be determined, with most of those within species D.Species D HAdV type 22 (HAdV-D22) was originally isolated from a child in 1956 (3). While it is not clear what role HAdV-D22 plays in human disease, one report revealed a possible tropism for the eye (32). Recently, recombination with HAdV-D22 has been identified as the source of a novel HAdV, HAdV-D53, causing an outbreak of keratoconjunctivitis in Germany (37). HAdV-D22 recombination was also identified in a possible variant of HAdV-D53 that was isolated from a patient in Japan (2). Therefore, HAdV-D22 has shown the propensity to recombine with other viruses, with clinically important consequences. The emergence of new pathogenic HAdV genotypes, along with continued interest in HAdVs as vectors for human gene therapy, make adenovirus recombination a critically important issue.HAdV-D22 was acquired from the American Type Culture Collection (Manassas, VA). The complete genome of the prototype strain AV-2711 (ATCC VR-1100) was sequenced on an Applied Biosystems (Foster City, CA) 3730 XL DNA sequencer in the Laboratory for Genomics and Bioinformatics at the University of Oklahoma Health Sciences Center using a previously described protocol (29). The sequence was validated by sequencing on an ABI SOLiD DNA sequencer. Sequences from both methodologies were 100% identical and provided 7,727-fold coverage for the genome.The mVISTA Limited Area Global Alignment of Nucleotides (LAGAN) tool (http://genome.lbl.gov/vista/index.shtml) was used to align and compare the whole HAdV genomes (6) of HAdV-D22 and each of the other nine completely sequenced HAdV-Ds. Analysis revealed sequence diversity in the penton base, hexon, E3, and fiber open reading frames (Fig. ​(Fig.1).1). Surprisingly, comparison of HAdV-D22 to HAdV-D19 strain C (30) and HAdV-D37 (29) revealed considerable sequence conservation in the penton base gene (Fig. ​(Fig.11 and 2A and B).Open in a separate windowFIG. 1.Global pairwise sequence alignment of HAdV-D22 with nine other HAdV-D types. The percent sequence conservation is reflected in the height of each data point along the y axis. A conserved sequence in the penton base open reading frame is designated by an asterisk. GenBank accession numbers are as follows: HAdV-D8, AB448768; HAdV-D9, AJ854486; HAdV-D19, EF121005; HAdV-D26, EF153474; HAdV-D37, DQ900900; HAdV-D46, AY875648; HAdV-D48, EF153473; HAdV-D49, DQ393829; and HAdV-D53, FJ169625.Open in a separate windowFIG. 2.Multisequence alignment of HAdV-D penton base genes. Nucleotide alignment of the bp 397 to 594 (A) and bp 694 to 1089 (B) regions of the penton base gene.Based on the sequence conservation seen in the penton base, Simplot 3.5.1 (http://sray.med.som.jhmi.edu/SCRoftware/simplot/) and Recombination Detection Program (RDP) version 3.34 (http://darwin.uvigo.es/rdp/rdp.html) were used to identify possible recombination sites (21, 24). Bootscan analysis identified two possible recombination loci in the HAdV-D22 penton base, the first in HAdV-D37, encompassing nucleotides 400 to 600, and the second in HAdV-D19 at nucleotides 750 to 1350 (Fig. ​(Fig.3A).3A). In silico amino acid analysis showed that these two probable recombination areas code for the two variable loops in the penton base protein (Fig. ​(Fig.3A),3A), located on the exterior of the viral capsid (16, 44).Open in a separate windowFIG. 3.Bootscan analysis of HAdV-D penton base genes. Comparison of HAdV-D22 (A) with completely sequenced HAdV types. Similar comparisons of the same region were performed with HAdV-D9 (B), HAdV-D49 (C), HAdV-A12 (D), HAdV-B3 (E), HAdV-C2 (F), or HAdV-F41 (G) as the reference type. HAdV-D53 was left out of the analysis due to the 100% identity of its penton base to that of HAdV-D37. The axes of all panels are as labeled in panel A. GenBank accession numbers are as follows: HAdV-A12, NC_001460; HAdV-B3, DQ086466; HAdV-C2, AC_000007; and HAdV-F41, DQ315364.We extended our investigation to determine if these recombination sites were common to penton base genes of other HAdV genomes. We found evidence for recombination between HAdV-D9 and HAdV-D26 in both the nucleotide 400 to 600 and 750 to 1350 regions (Fig. ​(Fig.3B),3B), between HAdV-D49 and HAdV-D48 in the nucleotide 400 to 600 region, and between HAdV-D49 and HAdV-D8 in the nucleotide 750 to 1350 region (Fig. ​(Fig.3C).3C). A similar pattern was observed in one or both of these nucleotide regions for HAdV-D8, -17, -19, -26, -37, and -48 (data not shown). Remarkably, this pattern of recombination in the penton base gene was unique to HAdV species D (Fig. 3D, E, F, and G).Bootstrap-confirmed neighbor-joining phylogenetic trees of HAdV-D penton base genes were constructed with Molecular Evolutionary Genetics Analysis (MEGA) 4.0.2 (http://www.megasoftware.net/index.html) to examine viral evolution in HAdV-D (34). Analysis of the entire penton gene revealed a close relationship of HAdV-D22 and HAdV-D19 strain C (Fig. ​(Fig.4A)4A) (30). Additional phylogenetic trees were constructed to encompass the two proposed recombination sites within this gene. Phylogenetic analysis of the nucleotide 400 to 600 region revealed a close identity among HAdV-D22, HAdV-D37, and HAdV-D53 (Fig. ​(Fig.4B).4B). Analysis of the nucleotide 750 to 1350 region of the penton base gene revealed a close identity of HAdV-D22 and HAdV-D19 (Fig. ​(Fig.4C4C).Open in a separate windowFIG. 4.Phylogenetic analysis of HAdV-D penton base genes. Bootstrap neighbor-joining tree designed using MEGA 4.0.2. The Gonnet protein weight matrix in ClustalX alignment was used, along with complete deletion options. Percent bootstrap confidence levels (1,000 replicates) are shown on the relevant branches. (A) Analysis of sequenced HAdV-D penton base genes. (B) Analysis of the nucleotide 400 to 600 region of sequenced HAdV-D penton base genes. (C) Analysis of the nucleotide 750 to 1350 region of sequenced HAdV-D penton base genes.In summary, comparison of the HAdV-D22 genome to other sequenced HAdV-D genomes identified substantial sequence divergence in the penton, hexon, E3, and fiber open reading frames. Differences between HAdV-D genomes in these areas have previously been described by us in reports on HAdV-D19 strain C and HAdV-D37 genomes (29, 30). However, the sequence conservation among HAdV-D22, HAdV-D19, and HAdV-D37 in the penton base gene was unexpected and suggests recombination among these viruses. Bootscan analysis identified recombination events at two regions within the penton base gene, encompassing nucleotides 400 to 600 and 750 to 1350. Further analysis of other HAdV-D penton genes suggests that these areas represent hot spots for recombination.The propensity for recombination among different HAdV-Ds in the penton base gene can be understood in the context of penton base function. The adenovirus penton base acts as the ligand for a secondary attachment event that is critical to host cell internalization (38) and thus is critical to infection. The penton base protein contains two hypervariable loops located on the surface of the viral capsid (16, 44). One loop contains the host cell integrin binding Arg-Gly-Asp (RGD) motif mediating attachment to host cell integrins (8, 38). The RGD motif is conserved in almost every HAdV penton base protein, with the exception of HAdV-F40 and HAdV-F41, which do not use host cell integrins for internalization (1, 11). The second exterior loop, known as the variable loop (HVL1), has no known function. Both of these regions are highly variable among different HAdV types. We identified recombination around both of these loops for multiple viruses within HAdV-D (Fig. ​(Fig.3A3A).In recent epidemiological studies, patients were identified with coincident clinical infections with two or more adenovirus types (17, 36). Simultaneous infection by more than a single HAdV type is possible because of conserved host receptor affinity, common tissue tropisms, and an absence of immunity across serotypes. Our data, along with evidence from previous studies (22, 23, 37, 43), identified recombination events among viruses with similar tissue tropisms, providing evidence that the restriction of tissue tropism might determine in part the observed recombination within adenoviruses of the same species. Recently, a novel HAdV, HAdV-D53, was isolated from an outbreak of keratoconjunctivitis. Subsequent analysis revealed recombination among HAdV-D22, HAdV-D37, HAdV-D8, and a previously unknown adenoviral sequence, suggesting the potential for the emergence of new pathogens, with important ramifications for human disease.Previous work provides evidence for recombination in HAdVs (10, 12, 22, 25, 26, 37, 39, 41-43), but the mechanisms of recombination have yet to be identified. Recombination may result from selective pressure from the host immune system relative to surface capsid proteins, a nucleotide motif that directs cellular recombination machinery to the local sites on the viral DNA, or a combination of both. Two eukaryotic recombinases, RAD51 and Dmc1, both homologues of the bacterial recombinase RecA, act in host cell DNA recombination (5, 33). RAD51 mediates recombination during mitosis, while Dmc1 acts during meiosis. RAD51 is of potential interest because it colocalizes with promyelocytic leukemia nuclear bodies in the nucleus (4). Adenoviral proteins E1A and E4 Orf3 have been shown to interact with promyelocytic leukemia nuclear bodies, which play an important role in adenoviral replication (7, 15). An interaction between RAD51 and adenoviral DNA has not been studied. A proposed motif consisting of CCNCCNTNNCCNC was recently identified as being associated with loci of recombination and genome instability in humans (28). Although not present in the viruses we studied, sequencing of other viruses within HAdV-D may yet reveal a consensus site for recombination. The elucidation of recombination mechanisms for HAdVs should allow a better understanding of adenoviral evolution.In conclusion, our analysis of the penton base gene of HAdV-D identified a potential paradigm for adenovirus recombination and the emergence of pathogenic strains. An in depth understanding of adenovirus recombination and evolution is critical to ensure the safety of adenoviral gene therapy.     

Sequence analysis of hexon gene from adenovirus KR95 inducing hydropericardium syndrome in chickens

The nucleotide sequence of a part of the HindIII-D fragment (3300 b.p.) of adenovirus KR95 DNA has been determined. Analysis of the nucleotide sequence disclosed a continuous ORF for hexon gene (2814 b.p.) coding the 937 residue protein, part of ORF for the C-terminal region of pVI polypeptide, including 114 residues and the beginning of ORF coding 25 N-terminal residues for viral endoproteinase. Comparison of predicted KR95 hexon sequence and 8 mammalian and avian adenovirus hexon sequences revealed the highest homology between KR95 strain and avian adenoviruses FAV10 and FAV1 (91.1 and 80.1%, respectively). The results were used for creating a test system on the basis of the polymerase chain reaction. The system was used in analysis of fowl samples obtained from 12 poultry farms in Russia. The sequences of hexon gene amplified fragments in the isolated strains and similar fragments of other mammalian and avian adenoviruses have been compared.     

Immunological Basis of the Adenovirus 8-9 Cross-Reaction

The dedecon and hexon components of adenovirus types 8 and 9 have been extensively purified for use in establishing the basis of the cross-reaction between these types. Dodecons, the complete hemagglutinins, were purified 304- to 362-fold by fluorocarbon extraction, calcium phosphate batch chromatography, and ion-exchange column chromatography. Hexons, the group complement-fixation (CF) antigens, were purified 230- to 240-fold by erythrocyte adsorption, ion-exchange chromatography, and exclusion chromatography. Component antisera prepared in rabbits were tested in reciprocal fashion with crude virus and dodecon and hexon components. By hemagglutination-inhibition (HI), the dodecons of types 8 and 9 demonstrated the same predominantly one-sided relationship characteristic of the crude antigens. Some neutralizing activity was associated with both dodecons and hexons of each type. However, combining anti-dodecon and anti-hexon sera or producing antisera against the combined dodecon-hexon components resulted in neutralizing titers which were identical to titers obtained with antisera against the crude virus harvests. Dedecons of each type appear to share at least one antigenic determinant with hexons of the same type, and this determinant may reside on the vertex capsomere. Hexons possess group- and type-specific determinants, as shown by CF, neutralization, and immunodiffusion tests, and may exhibit some minor relationship between types 8 and 9. The results with the purified components are consistent with the predominantly one-sided antigenic relationship between types 8 and 9 in the conventional HI tests and the largely type-specific relationship by neutralization tests.     

Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.

To expand the utility of recombinant adenovirus vectors for gene therapy applications, methods to alter native viral tropism to achieve cell-specific transduction would be beneficial. To this end, we are pursuing genetic methods to alter the cell recognition domain of the adenovirus fiber. To incorporate these modified fibers into mature virions, we have developed a method based on homologous DNA recombination between two plasmids. A fiber-deleted, propagation-defective rescue plasmid has been designed for recombination with a shuttle plasmid encoding a variant fiber gene. Recombination between the two plasmids results in the derivation of recombinant viruses containing the variant fiber gene. To establish the utility of this method, we constructed a recombinant adenovirus containing a fiber gene with a silent mutation. In addition, we generated an adenovirus vector containing chimeric fibers composed of the tail and shaft domains of adenovirus serotype 5 and the knob domain of serotype 3. This modification was shown to alter the receptor recognition profile of the virus containing the fiber chimera. Thus, this two-plasmid system allows for the generation of adenovirus vectors containing variant fibers. This method provides a rapid and facile means of generating fiber-modified recombinant adenoviruses. In addition, it should be possible to use this system in the development of adenovirus vectors with modified tropism to allow cell-specific targeting.     

Stability of the structure and antigenic determinants of adenovirus type 1 native hexon to proteases

Hexon capsomers of human adenovirus type 1 (h1) labeled by iodine 125 were digested in a native state (trimers) by trypsin, chymotrypsin or papain, and the resulting hydrolysates were analyzed by SDS-PAGE. In each case, a discrete and temporally stable pattern of relatively large fragments was revealed. The degree of hexon polypeptide hydrolysis was maximal for papain, intermediate for chymotrypsin and minimal for trypsin, the largest fragments in the digest being 32, 40 and 80 kD, respectively. At room temperature, all the electrophoretically discernible hexon proteolytical fragments were held together in structures resembling intact hexon trimers and could be regarded as "hexon cores", of which papain hexon cores were the most stable during SDS-PAGE. Radioimmunoprecipitation analysis revealed a complete absence of native hexon antigenicity in thermodenaturated fragments of hexon protease digests, while native trypsin, chymotrypsin and papain hexon cores could be precipitated by hexon-specific antibodies. The immunoprecipitated material contained all of the hexon fragments found in appropriate hexon cores and retained the structure of the original cores. Trypsin, chymotrypsin and papain hexon cores were shown to possess at least part of native Ad h1 hexon antigenic determinants of each of the following specificities: species-specific (epsilon), cross-reactive with hexon of human adenoviruses (h3 and h6), simian adenovirus (sim 16), bovine adenoviruses (bos 3 and bos 7) and avian adenovirus (Aviadenovirus gal 1 or CELO). Thus, the full spectrum of known hexon antigenic determinants (species-specific to intergenus-crossreactive) is at least portly stable against protease attack of native hexon capsomers.     

Isolation and Characterization of Adenoviruses Persistently Shed from the Gastrointestinal Tract of Non-Human Primates

Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported) together with 22 complete adenovirus genomes available from GenBank revealed that (a) the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b) there was evidence for intraspecies recombination between adenoviruses, and (c) the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors.     

Novel adenoviruses in wild primates: a high level of genetic diversity and evidence of zoonotic transmissions

Adenoviruses (AdVs) broadly infect vertebrate hosts, including a variety of nonhuman primates (NHPs). In the present study, we identified AdVs in NHPs living in their natural habitats, and through the combination of phylogenetic analyses and information on the habitats and epidemiological settings, we detected possible horizontal transmission events between NHPs and humans. Wild NHPs were analyzed with a pan-primate AdV-specific PCR using a degenerate nested primer set that targets the highly conserved adenovirus DNA polymerase gene. A plethora of novel AdV sequences were identified, representing at least 45 distinct AdVs. From the AdV-positive individuals, 29 nearly complete hexon genes were amplified and, based on phylogenetic analysis, tentatively allocated to all known human AdV species (Human adenovirus A to Human adenovirus G [HAdV-A to -G]) as well as to the only simian AdV species (Simian adenovirus A [SAdV-A]). Interestingly, five of the AdVs detected in great apes grouped into the HAdV-A, HAdV-D, HAdV-F, or SAdV-A clade. Furthermore, we report the first detection of AdVs in New World monkeys, clustering at the base of the primate AdV evolutionary tree. Most notably, six chimpanzee AdVs of species HAdV-A to HAdV-F revealed a remarkably close relationship to human AdVs, possibly indicating recent interspecies transmission events.     

Intertypic modular exchanges of genomic segments by homologous recombination at universally conserved segments in human adenovirus species D

Human adenovirus species D (HAdV-D), which is composed of clinically and epidemiologically important pathogens worldwide, contains more taxonomic “types” than any other species of the genus Mastadenovirus, although the mechanisms accounting for the high level of diversity remain to be disclosed. Recent studies of known and new types of HAdV-D have indicated that intertypic recombination between distant types contributes to the increasing diversity of the species. However, such findings raise the question as to how homologous recombination events occur between diversified types since homologous recombination is suppressed as nucleotide sequences diverge. In order to address this question, we investigated the distribution of the recombination boundaries in comparison with the landscape of intergenomic sequence conservation assessed according to the synonymous substitution rate (d_S). The results revealed that specific genomic segments are conserved between even the most distantly related genomes; we call these segments “universally conserved segments” (UCSs). These findings suggest that UCSs facilitate homologous recombination, resulting in intergenomic segmental exchanges of UCS-flanking genomic regions as recombination modules. With the aid of such a mechanism, the haploid genomes of HAdV-Ds may have been reshuffled, resulting in chimeric genomes out of diversified repertoires in the HAdV-D population analogous to the MHC region reshuffled via crossing over in vertebrates. In addition, some HAdVs with chimeric genomes may have had the opportunity to avoid host immune responses thereby causing epidemics.     

MKK6重组腺病毒的构建与制备

构建腺病毒穿梭载体pAd RSV ,并将p3 8MAPK (mitogen activatedproteinkinase)的上游激酶MKK6(mitogen activatedproteinkinasekinase 6)及其持续激活和无活性的突变体基因亚克隆到该穿梭载体 .通过与腺病毒DNA(pJM17)在能够表达E1的HEK 2 93细胞同源重组生成了能够表达MKK6信号分子的重组腺病毒 .PCR结果表明 ,这些重组腺病毒DNA的插入片段大小是正确的 .而且 ,通过感染COS 7细胞 ,用免疫激酶活性测定证实这些重组的腺病毒能够表达具有功能的蛋白质 .     

Genome Sequence of Human Adenovirus Type 55, a Re-Emergent Acute Respiratory Disease Pathogen in China

Human adenovirus type 55 (HAdV-B55) is an acute respiratory disease (ARD) pathogen first completely characterized in China (2006). This is a unique Trojan horse microbe with the virus neutralization attribute of a renal pathogen and the cell tropism and clinical attributes of a respiratory pathogen, bypassing herd immunity. It appeared to be an uncommon pathogen, with earlier putative, sporadic occurrences in Spain (1969) and Turkey (2004); these isolates were incompletely characterized using only two epitopes. Reported here is the genome of a second recent isolate (China, 2011), indicating that it may occur more frequently. The availability of this HAdV-B55 genome provides a foundation for studying adenovirus molecular evolution, the dynamics of epidemics, and patterns of pathogen emergence and re-emergence. These data facilitate studies to predict genome recombination between adenoviruses, as well as sequence divergence rates and hotspots, all of which are important for vaccine development and because HAdVs are used for epitope and/or gene delivery vectors.     

Nanoporous crystals of chicken embryo lethal orphan (CELO) adenovirus major coat protein, hexon

CELO (chicken embryo lethal orphan) virus is an avian adenovirus that is being developed as a gene transfer vector. Its trimeric major coat protein (942 residues, 106,709 Da) has 42% sequence identity to human adenovirus type 2 (AdH2) hexon and 45% to AdH5 hexon. For structural studies, the growth of CELO virus has been optimized, and its hexon purified and crystallized. The hexon crystals, the first non-human example, diffract to 3.9 A resolution. Molecular replacement using the AdH5 model was used to identify the location of the CELO hexon within the unit cell. There is one hexon monomer in the asymmetric unit of the trigonal space group P321 (a=b=157.8 A, c=114.2 A, gamma=120 degrees) and the solvent content is 67.8%. The hexons pack in a hexagonal honeycomb so that large approximately 100 A diameter channels run through the entire crystal. This remarkable property of the crystals lends itself to their exploitation as a nanomaterial. Structural studies on CELO will elucidate the differences between avian and human adenoviruses and contribute to a better understanding of adenoviruses with non-human hosts.     

A rapid generation of adenovirus vector with a genetic modification in hexon protein

The generation of hexon-modified adenovirus vector has proven difficult. In this paper, we developed a novel method for rapid generation of hexon-modified adenoviral vector via one step ligation in vitro followed by quick white/blue color screening. The new system has the following features. First, eGFP expression driven by the CMV promoter in E1 region functions as a reporter to evaluate the tropism of hexon-modified adenovirus in vitro. Second, it has two unique restriction enzyme sites with sticky ends located in the hexon HVR5 region. Third, a lacZ expression cassette under the control of plac promoter is placed between the two restriction enzyme sites, which allows recombinants to be selected using blue/white screening. To prove the principle of the method, genetically modified adenoviruses were successfully produced by insertion of NGR, RGD or Tat PTD peptide into hexon HVR5. Furthermore, the transduction efficiency of the Tat PTD modified virus was shown to be a significant enhancement in A172 and CHO-K1 cells. In conclusion, the novel system makes the production of truly retargeted vectors more promising, which would be of substantial benefit for cancer gene therapy.