Complete genome analysis of a novel intertypic recombinant human adenovirus causing epidemic keratoconjunctivitis in Japan

For 4 months from September 2008, 102 conjunctival swab specimens were collected for surveillance purposes from patients across Japan suspected of having epidemic keratoconjunctivitis (EKC). Human adenovirus (HAdV) DNA was detected in 61 samples by PCR, though the HAdV type for 6 of the PCR-positive samples could not be determined by phylogenetic analysis using a partial hexon gene sequence. Moreover, for 2 months from January 2009, HAdV strains with identical sequences were isolated from five conjunctival swab samples obtained from EKC patients in five different regions of Japan. For the analyses of the 11 samples mentioned above, we determined the nucleotide sequences of the entire penton base, hexon, and fiber genes and early 3 (E3) region, which are variable regions among HAdV types, and compared them to those of other HAdV species D strains. The nucleotide sequences of loops 1 and 2 in the hexons of all 11 samples showed high degrees of identity with those of the HAdV type 15 (HAdV-15) and HAdV-29 prototype strains. However, the fiber gene and E3 region sequences showed high degrees of identity with those of HAdV-9, and the penton base gene sequence showed a high degree of identity with the penton base gene sequences of HAdV-9 and -26. Moreover, the complete genome sequence of the 2307-S strain, which was isolated by viral culture from 1 of the 11 samples, was determined. The 2307-S strain was a recombinant HAdV between HAdV-9, -15, -26, -29, and/or another HAdV type; however, the recombination sites in the genome were not obvious. We propose that this virus is a novel intertypic recombinant, HAdV-15/29/H9, and may be an etiological agent of EKC.     

Characterisation of two enteroviruses isolated from Australian brushtail possums (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>) in New Zealand

Summary. Two enteroviruses, designated W1 and W6, were isolated from intestinal contents of Australian brushtail possums (Trichosurus vulpecula) in New Zealand. The genomic sequences of W1 and W6 were 7390 and 7391 nucleotides (nt), respectively. Genetically, possum isolates W1 and W6 were related to bovine enterovirus serotype 2 (BEV-2) strains, especially to the strain PS87/Belfast, based on the capsid protein sequence. However, W1 and W6 formed a clade that was distinct from PS87Belfast based on nucleotide sequences of the 3′ and 5′-non-translated region and in the amino acid sequences of 2A, 3C and 3D. Possum isolates W1 and W6 grew more readily in possum kidney cells than in Madin-Darby bovine kidney (MDBK) cells, suggesting that co-evolution of W1 and W6 with possums has made them more adapted to possum cells.     

Seasonal Effects on the Haematology and Blood Chemistry of Wild Brushtail Possums, Trichosurus vulpecula (Marsupialia: Phalangeridae) in New Zealand

Seasonal adjustments to red cell haematology that support altered metabolic requirements in Australian marsupials are either not expressed, or are minimal in brushtail possums introduced into New Zealand. Raised leucocyte differentials in winter imply seasonal challenges to the immune system. A small increase in plasma glucose, fall in plasma protein, and generally high condition index are consistent with the relatively high quality of habitat enjoyed by the marsupial immigrant.     

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.     

Full genome analysis of a novel adenovirus from the South Polar skua (Catharacta maccormicki) in Antarctica

Adenoviruses have been identified in humans and a wide range of vertebrate animals, but not previously from the polar region. Here, we report the entire 26,340-bp genome of a novel adenovirus, detected by PCR, in tissues of six of nine South Polar skuas (Catharacta maccormicki), collected in Lake King Sejong, King George Island, Antarctica, from 2007 to 2009. The DNA polymerase, penton base, hexon and fiber genes of the South Polar skua adenovirus (SPSAdV) exhibited 68.3%, 75.4%, 74.9% and 48.0% nucleotide sequence similarity with their counterparts in turkey hemorrhagic enteritis virus. Phylogenetic analysis based on the entire genome revealed that SPSAdV belonged to the genus Siadenovirus, family Adenoviridae. This is the first evidence of a novel adenovirus, SPSAdV, from a large polar seabird (family Stercorariidae) in Antarctica.     

Computational analysis and identification of an emergent human adenovirus pathogen implicated in a respiratory fatality

Adenoviral infections are typically acute, self-limiting, and not associated with death. However, we present the genomic and bioinformatics analysis of a novel recombinant human adenovirus (HAdV-D56) isolated in France that caused a rare neonatal fatality, and keratoconjunctivitis in three health care workers who cared for the neonate. Whole genome alignments revealed the expected diversity in the penton base, hexon, E3, and fiber coding regions, and provided evidence for extensive recombination. Bootscan analysis confirmed recombination between HAdV-D9, HAdV-D26, HAdV-D15, and HAdV-D29 in the penton base and hexon proteins, centered around hypervariable loops within the putative proteins. Protein structure analysis of the fiber coding region revealed similarity with HAdV-D8, HAdV-D9, and HAdV-D53, possibly accounting for the ocular tropism of the virus. Based on these data, this virus appears to be a new HAdV-D type (HAdV-D56), underscoring the importance of recombination events in human adenovirus evolution and the emergence of new adenovirus pathogens.     

Cloning of the MHC class II DRB cDNA from the brushtail possum (Trichosurus vulpecula)

The cell-surface glycoproteins encoded by the major histocompatibility complex (MHC) bind to processed foreign antigens and present them to T lymphocytes. Two classes of MHC molecules and their corresponding gene sequences have been extensively studied in eutherian mammals and birds, but data on the marsupial MHC are limited. Marsupials split from eutherian mammals about 125 million years ago and represent a distinct branch in mammalian evolution. Here the cDNA cloning of MHC class II genes of the brushtail possums (Trichosurus vulpecula) is reported. The sequences obtained were found to be relatively conserved when compared to the red-necked wallaby (Macropus rufogriseus) and an South American marsupial, Monodelphis domestica. The T. vulpecula sequence shared an average overall sequence identity of 75.4% at the deduced amino acid level with M. rufogriseus and M. domestica, respectively.     

Sequencing and phylogenetic analysis of the hexon, fiber, and penton regions of adenoviruses isolated from AIDS patients

Sequencing and phylogenetic analysis of the hexon, fiber, and penton regions of adenoviruses isolated between 1986 and 1997 from AIDS patients has been performed. Sequencing the L2 part of the hexon gene of 51 adenoviruses isolated between 1986 and 1997 from AIDS patients revealed only one type each from species A and C and two types from species B with all the remaining isolates from species D. Further sequencing and phylogenetic analysis of the fiber knob region of these species D adenoviruses revealed that 28/46 were intermediate strains with conflicting hexon and fiber sequences. When the penton regions of these intermediate strains were sequenced, it became clear that some had originated from a third adenovirus type presumably by intergene recombination events. Evidence from sequencing the L1 hexon and fiber shaft regions showed no evidence of intragene recombination but penton sequences showed that recombination between the hypervariable region (HVR) and RGD regions was common. Six isolates appear to be from three new adenovirus types. Five AIDS patients showed sequential infection with different adenovirus variants and six such variants were isolated from a single patient in 2 years.     

Characterization of the avian adenovirus penton base.

A portion of the fowl adenovirus 10 (FAV-10) penton base gene was located within a Sau3A genomic DNA fragment by the combination of a plasmid expression library and colony immunoassays with rabbit anti-FAV-10-sera. The coding portion of the sequence contained in the expression vector was mapped to a 11.6-kb Bg/II fragment and more precisely mapped to the right-hand end of the 11.6-kb fragment at map units 37.7 to 41.3. Nucleotide sequence analysis of the region revealed an open reading frame of 1575 bp with translation of the sequence producing a polypeptide of 525 amino acids in length. The polypeptide predicted from the open reading frame would have a molecular weight of 57.4 kDa. Analysis of the amino acid sequence revealed an overall homology of 41.8% with the human adenovirus 2 (HAV-2) penton base. However, a region near the center of the polypeptide (amino acids 219 to 311) showed a significantly greater level of homology to the HAV-2 penton base (71%). Time course experiments using mRNA confirmed that this gene is expressed at late times postinfection. Upon probing genomic DNA from other FAV serotypes the penton base coding region hybridized to all six different serotypes tested, indicating a relatively conserved DNA sequence among a variety of FAVs.     

Immunoreactive domains and integrin-binding motifs in adenovirus penton base capsomer

A panel of nine independent mouse monoclonal antibodies (MAbs) against penton base capsomers of subgenus C adenovirus serotypes 2 (Ad2) and 5 (Ad5) were isolated and characterized. Two of them (1D2 and 5A5), raised against Ad5 virion as the immunogen, bound to sodium dodecyl sulfate (SDS)-resistant and subgenus C-specific epitopes that were not present in subgenus B Ad3 penton base. The 1D2 and 5A5 epitopes were mapped to two distinct regions that did not belong to the main variable region carrying the integrin-binding RGD motif at position 340. For the other seven MAbs, raised against recombinant Ad2 penton base protein (9S-pentamers), the epitopes were sensitive to SDS-denaturation, but reacted with native Ad2, Ad5, and Ad3 penton base. The epitopes recognized by the nine MAbs and by polyclonal antipenton base antibodies defined three major immunoreactive regions. One (I) mapped to the N-terminal domain (residues 116-165); the other two regions were almost symmetrically disposed on both sides of the integrin-binding RGD motif at position 340, within residues 248-270 (II), and within residues 368-427 (III) in the C-terminal domain. Region II overlapped the fiber-binding site in penton base (residues 254-260). None of the MAbs showed any detectable virus neutralization effect, but they all slightly augmented the efficiency of Ad-mediated gene transfer. Although none of their epitopes included the RGD-340 tripeptide, substitutions of the arginine residue in the RGD motif abolished the reactivity of six individual and distant epitopes, suggesting a major conformational role for the RGD-containing domain.     

Histology of the pouch epithelium and the mammary glands during chemically induced oestrus in the brushtail possum (Trichosurus vulpecula)

Changes in the epithelium of the maternal pouch and the mammary gland of brushtail possums (Trichosurus vulpecula) were examined after animals were treated to induce ovulation with follicle-stimulating hormone (FSH), luteinizing hormone (LH), pregnant mares' serum gonadotrophin (PMSG) and oestradiol. The mammary glands were similar in appearance to those described in eutherian mammals and in previous studies on other marsupials. Exposure of possums to these compounds, particularly PMSG, appeared to result in changes in the mammary glands that could be associated with milk/secretion production. In contrast, the pouch epithelium had a similar histological appearance to that of epithelium from other parts of the body regardless of whether the animal was exposed to stimulants. These preliminary observations are discussed in the context of the purported role of the pouch epithelium and the mammary gland in production of secretions at oestrus and provision of immunological protection to the neonatal marsupial.     

Assembly of adenovirus penton base and fiber

Human adenovirus type 2 (H2) fiber isolated from the cellular pool of H2 wild-type (WT) soluble components was found to be capable of assemble in vitro with penton base obtained from the fiber-defective temperature-sensitive (ts) mutant H2 ts-125. This assembly occurred over a relatively broad range of pHs (5.5–9.0) and of ionic strengths (0.05–1.0). The extent of penton formation was estimated by two-dimensional immuno-electrophoresis. The legitimacy of the assembly in vitro was demonstrated by morphological, biochemical, and biological evidence. The assembly of the penton base with the fiber appeared to be a reversible reaction, with a dissociation constant K_D = 2 × 10^?7M, in terms of fiber molarity. This value implied a high affinity of the penton base for the fiber. Comparative digestions by carboxypeptidase of uncombined fiber and of penton base-combined fiber suggested that the recognition site for assembly involved the last 20 amino acids of the C-terminal sequence of the fiber polypeptide chain. The same analysis performed on [¹⁴C]glucosamine-labeled fiber and penton seemed to indicate that the carbohydrate units carried by the fiber were positioned too far from the C-end to be directly involved in the recognition and assembly process of the penton base with the fiber. Chimeric penton produced in vitro by incubating fiber and penton base from different adenovirus serotypes, as well as in vivo by interserotypic ts⁺ recombinants suggested a group specificity of the recognition site on the fiber and a likely highly conserved C-terminal amino acid sequence. Assembly of penton base and fiber in vitro resulted in a significant change, in the antigenicity of the penton base-combined fiber, which was thought to reflect some three-dimensional remolding.     

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     

Removal of the shell coat affects maintenance of epithelia in blastocysts of the Brushtail Possum in vitro

The aim of the present study was to investigate the role of the marsupial shell coat in embryonic development because it may be a potential target for immunocontraceptive control of the brushtail possum. Conceptuses from 52 female possums were collected between 1995 and 1997 in New Zealand and Australia. Development was examined in representative stages from cleavage to the early trilaminar blastocyst. The effect of coat removal by microdissection was examined by comparing development in vivo (n = 29), with development in vitro, both with (n = 10) and without (n = 13) shell coat removal. Conceptuses were monitored and photographed in culture, fixed and examined by transmission electron microscopy. The ultrastructure of uni-, bi- and trilaminar blastocyst stages in vivo and in vitro and of the early stages of hypoblast and embryonic endoderm formation are described for the first time in the possum. Shell coat removal had little impact on most cleavage stages, as the intact mucoid coat appeared to provide structural support to the embryo. Common features of unilaminar coat-free specimens after culture were rounding up and detachment of cells from the blastocyst epithelium and the loss of cell surface projections. The most remarkable features of the shell-free trilaminar blastocysts in comparison with the in vivo and in vitro controls were the hydration of many cells, and the large-scale disruption and modification of numerous epithelia, particularly of the younger, or newly forming populations. The shell appears to be functionally important after blastocyst formation, particularly after breakdown of the mucoid coat. After shell removal, conceptus response in vitro suggested that the shell played a role in maintaining structural integrity. The shell was found to be necessary for normal embryonic development.     

Characterization of the early region 3 and fiber genes of Ad7

The nucleotide sequence and the predicted amino acid sequences for open reading frames (ORFs) encoded in the Bam-Hl D fragment of Ad7 (Gomen) DNA show an organization and conservation of potential polypeptides between Ad3 and Ad7. Five ORFs encoded within early region 3 (E3) and shared with the corresponding region of Ad3 can be identified; four of these potential coding regions also share homology to ORFs found in E3 of Ad2 and Ad5. The fiber gene of late region 5 (L5) is also apparent within this region; S1 mapping experiments show that the 5' and 3' boundaries of the main exon in fiber mRNA lie at each end of the proposed fiber ORF. The predicted amino acid sequence for Ad7 fiber shares 60% amino acid homology to Ad3 fiber, but only 20% to Ad2 fiber. Surprisingly, there are three regions of partial amino acid homology near the N- and C-termini of the predicted fiber gene sequences from Ad2, Ad3, Ad5, and Ad7; these conserved regions may be important for interaction with penton base, for proper folding of the shaft of the molecule, or for recognition of the cellular receptor to which adenovirus attaches during infection.     

Genes for fowl adenovirus CELO penton base and core polypeptides

Summary A 3.5-kilobase DNA fragment of the fowl adenovirus type 1 (CELO), located between map units 31.1 and 39.4 has been determined. The sequence contains the probable CELO equivalents of the IIIa protein, penton base, pVII and pV core protein genes of human adenovirus (HAV). The CELO penton base and major core protein (analog HAV pVII) were found to consist of 514 (56.8 kDa) and 72 amino acids (8.4 kDa), respectively.     

Immunobiology of mycobacterial infections in marsupials

Mycobacterial infections of marsupials are important for two reasons. Firstly, the Australian brushtail possum (Trichosurus vulpecula) serves as the major wildlife reservoir for Mycobacterium bovis in New Zealand and secondly, M. avium is a significant cause of disease in endangered marsupial species held in captivity. Marsupials are highly susceptible to specific mycobacterial infections which may be linked to deficiencies in their cellular immunity. Histopathological inspection of affected tissues indicates that, unlike most eutherians, marsupials are unable to wall off infection sites, resulting in formation of satellite lesions and generalised disease. This review examines possible reasons for the high susceptibility of marsupials to mycobacterial infections and investigates the prospects for developing vaccines to control these diseases.