Genomic location and nucleotide sequence of a porcine adenovirus penton base gene

Summary The putative penton base gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and sequenced. The genomic location of the PAV3 penton base was deduced by probing a Southern blot with a polymerase chain reaction generated product containing the human adenovirus type 2 (HAV2) penton base gene. Sequencing revealed an open reading frame (ORF) of 1527 nucleotides coding for a polypeptide of 509 amino acids. However, cDNA analysis indicated an acceptor splice site one nucleotide upstream of the second ATG in the ORF. This produced an ORF of 1452 nucleotides coding for a polypeptide of 484 amino acids with a calculated molecular weight of 54.5 kDa. Comparison with the HAV2 penton base amino acid sequence revealed the putative PAV3 penton base homologue to be 87 amino acids shorter with an overall amino acid homology of approximately 65%. Comparison with the penton base proteins of other HAV types revealed a region between amino acid positions 283 and 379 with no similarity.GenBank Accession No. U24432.     

Construction and characterization of human adenovirus serotype 3 packaged by serotype 7 hexon

Human adenovirus serotype 3 (Ad3) and serotype 7 (Ad7) are important pathogens causing respiratory tract diseases such as acute respiratory disease in pediatric and adult patients, but the immunodominant targets of Ad3- and Ad7-specific neutralizing antibodies (NAbs) remain unclear. A chimeric Ad vector, Ad3/H7, was constructed by replacing the Ad3 hexon gene (H3) with the hexon gene (H7) of Ad7. The chimeric viruses were successfully rescued in HEp-2 cells, and the Ad7 hexon was able to encapsidate the Ad3 genome, and functioned as efficiently as the Ad3 hexon. Furthermore, we tested the host neutralization responses against the viruses using BALB/C mice. Up to 97% of the NAbs produced by mice that were infected with these viruses were specific for the hexon protein in vitro. Preimmunization of mice with one of Ad7 and Ad3/H7 significantly prevented subsequent intranasal infection of the other type in vivo. In contrast, preimmunization of mice with one of Ad3 and Ad3/H7 did not remarkably prevent subsequent infection of the other type. We next evaluated the functional significance of hexon and other structural proteins specific NAbs to suppress the immunogenicity of Ad3/H3 and Ad3/H7 vectors expressing EGFP in mice preimmunized with wild type Ad. Preimmunization of mice with Ad7 evidently suppressed EGFP-specific humoral immune responses elicited by Ad3/H7, and did not exert suppressive effects on Ad3/H3. But contrary to the in vitro neutralization results, EGFP-specific humoral immune responses elicited by Ad3/H7 was remarkably inhibited in Ad3-preimmunization mice. The whole genome of the Ad7 strain was sequenced and aligned with Ad3. The major differences between Ad3 and Ad7 were only observed in the fiber and hexon among all structural proteins, and the variation between the hexons only located in four hypervariable regions (HVRs), HVR-1, -2, -5, and -7. These results thus suggest that Ad3- and Ad7-specific NAbs are directed primarily against the hexon proteins both in vitro and in vivo. But high titer Ad3 fiber-specific NAbs may also play an important role in blunting Ad3 immunogenicity in vivo. These studies contribute to a more profound understanding of Ad immunogenicity and have relevance for the design of novel Ad vaccine.     

Identification, cloning and sequence analysisof the equine adenovirus 1 hexon gene

Summary.  Based on sequence homology with human adenovirus 2 (HAdV2), the hexon gene of equine adenovirus 1 (EAdV1) was identified. HindIII restriction fragments containing the hexon and other viral genes were cloned into the plasmids pUC19 and pBlueScript SK(-) and sequenced. The nucleotide sequence of the hexon gene was completely determined and partial sequence data were obtained for seven other EAdV1 genes. Amino acid (aa) sequence comparison with published adenovirus (AdV) proteins identified the genes for the IIIa, penton, pVII, pVI, 23K proteinase, DNA binding and 100K proteins. The eight EAdV1 genes appeared to be in the same relative order as homologous genes of other AdV. The EAdV1 hexon protein was encoded the hexon-associated pVI upstream and the 23K proteinase gene downstream and comprised 2 742 nucleotides which translated into 913 aa. Similar to other members of the genus Mastadenovirus the EAdV1 hexon yielded two highly conserved genome segments at the N- and C-termini which flanked intermediate variable and hypervariable regions. The majority of the residue differences between EAdV1 and other AdV hexons occurred in two loops that are known for other AdV to protrude from the surface of the nucleocapsid. Amino acid comparisons with other AdV hexons revealed highest homology with HAdV12 hexon with 72% identical and 83% functionally similar residues, followed by bovine AdV3 hexon with 71% identities and 82% functional residue conservation. Phylogenetic analysis suggested that EAdV1 and other AdV do not have an immediate common ancestor. Accepted December 13, 1996 Received October 9, 1996     

Isolation of porcine adenovirus as a candidate of 5th serotype

A strain of cytopathic virus, named strain TG/K79, was isolated from the brain of a newborn piglet, pure Hampshire breed, which died shortly after birth. The physicochemical property of virus was considered to be that of the family Adenoviridae. A significant difference between our isolate and 4 reference porcine adenoviruses was demonstrated by cross-seroneutralization test Differences between TG/K79 and other porcine adenoviruses were also seen in electrophoretic patterns of viral DNA in agarose gel after digested by restiction endonucleases. Two SPF pigs, 2-month-old, experimentally infected via intranasal showed a fever and a hemorrhagic enteritis. A serological survey indicates that at least swine in the farm where the virus was isolated have been highly contaminated.     

Genomic cloning and restriction site mapping of a porcine adenovirus isolate: demonstration of genomic stability in porcine adenovirus

Summary Restriction endonuclease maps were constructed for the genome of a porcine adenovirus (PAV), NADC-1, which was isolated in 1972 from an adult swine. Genomic DNA libraries of NADC-1 Bam HI, Eco RI/Bam HI, and Sph I fragments were cloned into pUC-18. Using the cloned NADC-1 Bam HI and Eco RI/Bam HI fragments as probes, Southern blot hybridizations were performed to human adenovirus 2 (Ad-2) restriction fragments to determine the left-to-right orientation of the Bam HI and Eco RI/Bam HI fragments. Genomic NADC-1 DNA was cleaved with ten restriction endonucleases (RE). Using cloned NADC-1 genomic fragments as probes in Southern blot hybridizations, an RE site map was constructed. Nucleotide sequencing of four clones confirmed several RE sites. The size of the NADC-1 genome was determined to be approximately 32 kbp. The size of Hind III, Xba I, Sma I, Eco RI, Bam HI, Bgl II, Pst I, and Sph I RE fragments from NADC-1 was compared to those from the reference strain of PAV serotype 4 (F 618), and to two recent isolates, NADC-2 and NADC-3. For all restriction enzymes examined, the sizes of the NADC-1 fragments were identical to PAV-4, NADC-2, and NADC-3 fragments, indicating that the NADC-1 isolate is very closely related, if not identical, to PAV-4 and two recent isolates. Southern blot hybridizations also indicated that NADC-1, NADC-2, NADC-3, and PAV-4 are very similar and revealed regions of sequence similarity between NADC-1 and human Ad-2 and human Ad-5.     

Sequence analysis of adenovirus DNA I. Nucleotide sequence at the carboxy-terminal end of the gene for adenovirus type 2 hexon

The nucleotide sequence of a 210 base-pair long fragment of adenovirus type 2 DNA has been determined. The fragment, which is located between map positions 59.0 and 60.2, specifies the codons for 45 amino acids at the carboxy-terminal end of the adenovirus hexon polypeptide. The hexon gene is terminated by a single UAA stop codon which starts 136 nucleotides to the left of the cleavage site for endonuclease BamHI at position 59.0     

Genetic heterogeneity of the hexon gene of adenovirus type 3 over a 9-year period in Korea

Hexon sequences were analyzed in 29 epidemiologically unrelated adenovirus type 3 (Ad3) isolates from the 7 genome types to understand the molecular basis of the genome-type diversity of Ad3 associated with childhood pneumonia in Korea during the period 1991-1999. Nine nucleotide substitutions were observed among the 29 Ad3 strains. Five of the 9 involved amino acid changes in loops 1 (Gly to Val at codon 205 and Thr to Ile at 211) and loop 2 (His to Asn at 417, Thr to Ala at 429, and Ala to Asp at 439). The predicted hydropathic characteristics of this region have been affected by these amino acid changes. The region surrounding codons from 417 to 439 of Ad3a16 and Ad3a18 manifested greater hydrophobicity than the region of other genome types (Ad3a, Ad3a13, Ad3a14, Ad3a15, and Ad3a17). In particular, three amino acid changes in loop 2 were associated with two new genome types, namely, Ad3a16 and Ad3a18, which were recognized during later epidemics in 1998-1999. Phylogenetic relatedness revealed that these two genome types clustered into distinct lineages in the phylogenetic tree. This result suggests that the genetic heterogeneity of Ad3 hexon could play a potential role in the appearance of new genome types and that it could affect the antigenic characteristics of Ad3.     

Pathogenicity and complete genome sequence of a fowl adenovirus serotype 8 isolate

In this study we determined and analyzed the complete nucleotide sequence of the genome of a fowl adenovirus serotype 8 (FAdV-8) isolate and examined its pathogenicity in chickens. The full genome of FAdV-8 was 44,055 nucleotides in length with a similar organization to that of FAdV-1 and FAdV-9 genomes. No regions homologous to early regions E1, E3 and E4 of mastadenoviruses were recognized. Along with FAdV-9, FAdV-8 has only one fiber gene and with regard to sequence composition and genome organization, FAdV-8 is closer to FAdV-9 than to FAdV-1. Moreover, our findings suggest that FAdV-1 of species Fowl adenovirus A as the current type species despite its historical priority is not representative of the genus Aviadenovirus, and that FAdV-8 or FAdV-9 in species Fowl adenovirus E and Fowl adenovirus D, respectively, would be more suitable for that designation. Additionally, pathogenicity of FAdV-8 was studied in specific pathogen free chickens following oral and intramuscular inoculations. Despite lack of clinical signs and pathological changes virus was found in tissues and cloacal swabs of all birds with the highest viral copy numbers present in the cecal tonsils. The highest virus titers in the feces for orally and intramuscularly inoculated chickens were recorded at days 10 and 3 post-infection, respectively.     

Genetic variability of hexon loops 1 and 2 between seven genome types of adenovirus serotype 7

Summary.  In order to understand the evolutionary relationships between different genome types of adenovirus serotype 7, the nucleotide sequences of the hexon loops 1 and 2 from seven genome types have been determined. Their amino acid sequences comprising 473 to 476 residues were consequently analysed. The pairwise comparison of the sequences from seven genome types revealed the existences of two genome type clusters (GTCs). GTC1 includes Ad7p and Ad7p1, and GTC2 contains Ad7b, Ad7c, Ad7d, Ad7g and Ad7h. The amino acid similarity was 98.3–99.8% within a cluster and 93.0–94.5% between two clusters. However, the average amino acid similarity among 16 different human adenovirus serotypes was only 65% with two exceptions, 92.7% between Ad4 and Ad16, and 86.8% between Ad3 and Ad7. Two variable regions were found in the loops 1 and 2. A deletion of nine nucleotides was detected in the variable region 1 of all members of GTC2. According to the alignment of nucleotide sequences, two short direct repeats were found at the deletion junctions, indicating that GTC2 may be derived from GTC1 by illegitimate recombination. In variable region 2, the substitution from 443Leu (Ad7b) to Gln (Ad7d) dramatically affected the hydropathic characters of this region. The region surrounding 443Leu (Ad7b) manifested hydrophobicity whereas the region surrounding Gln (Ad7d) manifested hydrophilicity. Received June 22, 1998 Accepted April 19, 1999     

Development of adenovirus serotype 35 as a gene transfer vector

While 51 human adenoviral serotypes have been identified to date, the vast majority of adenoviral vectors designed for gene transfer have been generated in the adenovirus serotype 5 (Ad5) backbone. Viral infections caused by Ad5 are endemic in most human populations and the majority of humans carry preexisting humoral and/or cellular immunity to Ad5 which may severely limit the use of Ad5-based vectors for gene therapy applications. To circumvent this preexisting Ad5 immunity, we have identified Ad35 as an alternative adenoviral serotype to which the majority of humans do not have neutralizing antibodies. Importantly, Ad35 can be grown to high titers with a low particle-to-PFU ratio. As a prerequisite for the development of Ad35 for use as a gene transfer vector, a genome organization map was constructed using the available Ad35 sequence information, and E1a-deficient Ad35 vectors encoding marker genes were generated. Ad35 biodistribution in mice was assessed following intravenous administration and compared with that of Ad5. Extremely low levels of Ad35 were detected in all organs evaluated, including liver, lung, spleen, and bone marrow, while Ad5 displayed high transduction of these organs. Due to the lack of Ad35 liver tropism, minimal hepatotoxicity was observed in mice treated with Ad35. Furthermore, the half-life of Ad35 in mouse blood was found to be two to three times longer than that of Ad5. These data suggest that either mice do not express the Ad35 cell surface receptor or that Ad35 does not efficiently transduce mouse cells in vivo following systemic delivery. Therefore, to begin to elucidate the Ad35 cell entry mechanisms, in vitro competition studies were performed. These data demonstrated that Ad35 cell entry is CAR independent, and may involve protein(s) expressed on most human cells.     

Antigenic determinants of the hexon of bovine adenovirus type 3

Radioimmunoprecipitation of cattle adenovirus type 3 revealed in its hexon a determinant of narrow specificity (species-specific or subgenus-specific), intersubgenus-specific (common for cattle adenovirus subgroups 1 and 2), genus-specific (common for mammalian adenoviruses) and intergeneric (common for mammalian and avian adenoviruses) antigenic determinants.     

Complete sequence and organization of the human adenovirus serotype 46 genome

Out of 51 human adenoviral serotypes recognized to date, 32 of them belong to species D. Members of species D adenoviruses are commonly isolated from immune suppressed patients (organ transplant) and patients suffering from AIDS. The role of species D adenoviruses in pathogenesis is currently unclear. To derive new insights into the genetic content and evolution of species D adenoviruses and as a first step towards development of human adenovirus serotype 46 (Ad46) as vector, the complete nucleotide sequence of the virus was determined. The size of the genome is 35,178 bp in length with a G+C content of 56.9%. All the early and late region genes are present in the expected locations of the genome. The deduced amino acid sequences of all late region genes, with the exception of fiber, exhibited high degree of homology with the corresponding proteins of other adenoviruses. The deduced amino acid sequences of early regions E1, E3 and E4 showed a high degree of homology with the corresponding proteins of adenoviruses belonging to species D and less homology with the corresponding proteins of adenoviruses of other species. The homologues of Ad5 E3 region genes encoding 12.5K, gp19K, 10.4K, 14.5K and 14.7K are conserved in the genome of Ad46. However, the E3 region of Ad46 lacks genes encoding 6.7K and adenovirus death protein (ADP) but contains two additional open reading frames with a coding capacity of 433 and 281 amino acids. The fiber protein of Ad46 is 200 amino acids smaller than the fiber protein of Ad5 and contains only 10 pseudo-repeats in the shaft region. To facilitate the manipulation of the genome, the complete genome of Ad46 was cloned into a single bacterial plasmid. Following transfection into E1 complementing cell lines, the virus was recovered demonstrating the feasibility of viral genome manipulation for generation of recombinant viruses.     

Nucleotide sequence analysis of the L1 loop variable region of hexon gene of fowl adenovirus 4 isolates from India

Three fowl adenovirus 4 (FAV4) isolates from chicken and one from quail, all from Tamil Nadu, India were analyzed. The L1 loop variable region of hexon gene of these isolates was amplified by PCR and sequenced. The nucleotide sequences (442 bp) and deduced amino acid sequences of the four isolates were compared with those of other isolates of FAV4. The nucleotide sequences of the four isolates had a 98% homology with other Indian isolates and a 96% homology with Belgian and Russian isolates. The amino acid sequences of the four Indian isolates had a more than 98% homology with other Indian isolates and a more than 92% homology with Belgian and Russian isolates. Hence, the variable of L1 loop region of hexon gene was found to be highly homologous in all the FAV4 isolates tested both at nucleotide and amino acid level.     

7型腺病毒疫苗株序列测定及其六邻体特性分析

目的 完成7型腺病毒疫苗株(Ad7v)的全序列测定并阐明其六邻体特征。方法 克隆Ad7v基因组17．5～68．0mu片段，应用Sanger双脱氧法进行DNA序列测定，将编码六邻体蛋白的核苷酸序列输入GenBank数据库中，获得录入号为AF515814。用CLUSTAL.V软件比较Ad7v与其他亚组及同一亚组腺病毒的六邻体蛋白的同源性，分析其抗原性的差异，同时用RasMol2．71软件预测Ad7v六邻体三维结构。结果 AdTv17．5～68．0mu由17596个碱基组成。这段基因r链主要编码晚期基因L1、L2和L3，L链编码E2的一部分。六邻体编码多肽位于L3区，由934个氨基酸残基组成，与其他9个已知的六邻体蛋白序列进行多序列同源性比较，发现Ad7／疫苗株与其他亚组腺病毒的同源性为86．8％(Ad4)～78．5％(Ad5)。在同一亚组中，Ad7疫苗株与Ad3同源性最高，高达95．1％。可变序列主要集中在7个高变区(HVRs)，根据Ad2六邻体三维结构模型预测Ad7六邻体三维结构，发现可变区大部分位于六邻体顶端的I1和I2环中。结论 完成了AdTv的全序列分析，其六邻体序列与Ad5、Ad2等其他亚组病毒有很大差异，这一结果为构建新型腺病毒载体打下了基础。     

Radioimmunological analysis of simian adenovirus SA7 hexon

Direct and competitive radioimmunoassay (RIA) identified several antigenic determinants in hexone of adenovirus (Ad) of lower primates SA7: a species-specific, one common for Ad SA7 and SV38, as well as 2 genus-specific determinants: one common for the studied human Ad types 1 and 6, simian Ad SA7 and SV38, cattle AD of type 3, and a new determinant common for human Ad type 6 and simian Ad SA7 and SV38. It is proposed that the above genus specific determinants be designated alpha 1 and alpha 2, respectively. Indirect evidence of the occurrence in nature of rabbit Ad was obtained as sera from some nonimmunized animals precipitate purified labeled SA7 hexone, i.e. contain antibodies to the genus-specific determinant of the hexone.