Typical Adenovirus Research Articles

Structure and N-acetylglucosamine binding of the distal domain of mouse adenovirus 2 fibre.

Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenoviruses, it is a member of the genus Mastadenovirus, family Adenoviridae. The MAdV-2 genome has a single fibre gene that expresses a 787 residue-long protein. Through analogy to other adenovirus fibre proteins, it is expected that the carboxy-terminal virus-distal head domain of the fibre is responsible for binding to the host cell, although the natural receptor is unknown. The putative head domain has little sequence identity to adenovirus fibres of known structure. In this report, we present high-resolution crystal structures of the carboxy-terminal part of the MAdV-2 fibre. The structures reveal a domain with the typical adenovirus fibre head topology and a domain containing two triple β-spiral repeats of the shaft domain. Through glycan microarray profiling, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and site-directed mutagenesis, we show that the fibre specifically binds to the monosaccharide N-acetylglucosamine (GlcNAc). The crystal structure of the complex reveals that GlcNAc binds between the AB and CD loops at the top of each of the three monomers of the MAdV-2 fibre head. However, infection competition assays show that soluble GlcNAc monosaccharide and natural GlcNAc-containing polymers do not inhibit infection by MAdV-2. Furthermore, site-directed mutation of the GlcNAc-binding residues does not prevent the inhibition of infection by soluble fibre protein. On the other hand, we show that the MAdV-2 fibre protein binds GlcNAc-containing mucin glycans, which suggests that the MAdV-2 fibre protein may play a role in viral mucin penetration in the mouse gut.

Construction of recombinant adenoviras expressing RSV F gene

Objective To construct a recombinant adenovirus contained respiratory syncytial virus F gene fragment. Methods The F gene fragment was amplified by RT-PCR. Then the recombinant shuttle plasmid pShuttle-CMV/F was constructed. The iinearized pShuttle-CMV/F was transformed into BJ5183-AD-1 electroporation competent cells which contained plasmid pAdEasy-1. The obtained homologous recom-binant plasmid was named pAdEasy/F, and transfected into 293 cells. Then the recombinant adenovirus rAd/F was obtained, and it was tested by electron microscope, RT-PCR, Western blot and immuno-fluores-cence assay(IFA). The vires titer and genetic stability were also studied initially. Results The recombi-nant adenovirus rAd/F was constructed, which was visualized as typical adenovirus morphology under elec-tron microscope. The transcription and expression of RSV F gene fragment integrated in the rAd/F were con-finned by RT-PCR, Western blot and IFA. Conclusion The recombinant adenovirus rAd/F contained RSV F gene fragment is successfully constructed and lay an important foundation for the further vaccine study. Key words: Respiratory syncytial virus; Fusion protein; Adenovirus vector

Enteritis hemorrágica por adenovirus en pavos

Hemorrhagic enteritis (HE) in 4 to 11 weeks old turkeys is described by the first time in Colombia, based on gross, morphologic and electron microscopic studies. Sick animales presented enteritis and splenomegaly as the main findings. Mononuclear cells of the lamina propria of the small intestine presented intranuclear inclusion bodies. In the spleen these bodies were seen in most of the cells of white and red pulp. The inclusion bodies were slightly eosinophilic and the chromatin was displaced to the nuclear membrane. A basophilic round flattened body, displaced to a nuclear pole, suggested a nucleolus. Ultrastructural studies of the inclusion development from the initial appearance of a cristaloid material to the final formation of large amounts of capsids and virions are described. Negative staing of spleen macerates presented typical adenovirus particles but these particles were not detected in the intestinal content by the same technique.

Isolation of the virus of egg drop syndrome 1976 (EDS-76) in a Japanese outbreak.

We observed outbreaks of an egg drop syndrome similar to EDS-76 over the period from October, 1979 to March, 1980. A hemagglutinating virus (H-162) was isolated from the faces of a hen in a broiler breeding flock and shown to be serologically identical to the EDS-76 virus by the double immunodiffusion and hemagglutination-inhibition (HI) tests. The appearance of HI antibody to this virus was associated with the syndrome. The replication of the H-162 virus was inhibited with 5-iodo-2'-deoxyuridine and its infectivity was resistant to treatment by ethyl ether or chloroform. The virus was stable at pH 3.7 and readily filtered through membrane filters with 100 nm or greater pore sizes, but not through 50-nm filters. The virus multiplied with cytopathic effect in cultures of chick embryo liver and kidney cells and fibroblasts and chiken kidney cells. Eosinophilic inclusion bodies were observed in the nuclei of infected cells. Electron microscopy revealed typical adenovirus particles often aggregated in paracrystalline array forming a cubic lattice in the nuclei of thin-sectioned cells. The virus multiplied to high titers in the allantoic cavity of duck embryos, whereas no viral growth was shown in chicken embryos. The virus agglutinated chicken erythrocytes and the reaction was specifically inhibited with antiserum against the virus.

A sixth species of ovine adenovirus isolated from lambs in New Zealand

Two adenoviruses (WV419/75 and WV757/75), isolated from lambs in New Zealand were compared using neutralisation tests with the five recognised ovine adenovirus species, NINE bovine and four porcine adenoviruses. WV419/75 did not cross-react with any of the viruses tested and represents a new ovine adenovirus species (OAV-6). WV757/75 cross-reacted with bovine adenovirus type 7 (BAV-7) with a homologous to heterologous titre ratio of 16 in one direction only, and also showed a substantial one-way cross reaction in haemagglutination-inhibition tests (WV757 antiserum inhibiting haemagglutination by BAV-7). There was therefore insufficient distinction from BAV-7 virus to allow designation as a separate species. Fluorescent antibody studies with WV419 and WV757 demonstrated virus inclusions in the nuclei of infected cells. These were stained by antiserum to OAV-4 indicating presence of the mammalian group antigen. Thin section electron microscope studies showed typical adenovirus particles and associated inclusions in cell nuclei. The similarity of the two viruses to the bovine subgroup 2 adenoviruses in several of their properties is discussed.

In vitro association of empty adenovirus capsids with double-stranded DNA.

Several lines of evidence suggest that empty adenovirus capsids are preassembled intermediates in the pathway of virion assembly. We have observed that purified empty capsids of subgroup B adenoviruses have a remarkable affinity for DNA in vitro. The products of capsid-DNA association are sufficiently stable, once formed in low-salt solution, to permit purification and characterization in CsCl density gradients. Neither virions nor the DNA-containing incomplete particles of subgroup B adenoviruses can give rise to such in vitro reaction products. The average molecular weight of the empty adenovirus capsids is about 123 X 10(6), consistent with the absence of viral core peptides and a small deficiency of exterior shell polypeptides. Electron microscopy of negatively stained capsids and the capsids bound to DNA reveals a typical adenovirus size and architecture. The particles appear with a surface discontinuity that is presumed to expose the DNA binding site(s). The DNA molecules associated with the empty capsids are susceptible to the actions of DNase and restriction endonucleases. The dependence of rate of capsid-DNA association on DNA length suggests randomly distributed binding sites on the DNA molecules. Although the DNA molecules can successively acquire additional empty capsids, the empty particles themselves are restricted to interactionwith only one DNA molecule. Electron microscopy of the capsid-DNA complexes spread in cytochrome c films shows that the particles are bo-nd along the contour of extended duplex DNA. The amount of DNA within each bound particle appears to be less than 300 base pairs, as estimated by the length of the DNA molecules visible outside of the bound particle. The empty capsid-DNA association product described in this report provides an interesting substrate for further investigation of the DNA packaging process in a defined in vitro system, with extracts or purified components from infected cells.

Ultrastructural Characterisation of isolate 127 of egg drop syndrome 1976 virus as an adenovirus

Virus isolate 127 causing egg drop syndrome 1976 was purified and examined by electron microscopy. In CsCl equilibrium density gradients three bands could be visualised. Two bands at an apparent density of 1.32 g/ml, which could not be separated by fractionation, harboured high amounts of HA-activity and infectivity. Electron microscopic examination of these bands revealed particles of typical adenovirus morphology. The third band at a density of 1.30 g/ml consisted mainly of disrupted particles. This band showed a clear peak of HA-activity but no evidence of radioactivity and negligible amounts of infectivity.

Biological and physical properties of a virus (strain 127) associated with the egg drop syndrome 1976

The biological and physical properties of strain 127 virus, a haemagglutinating virus associated with the egg drop syndrome 1976, are described. Haematoxylin and eosin and immunofluorescent studies demonstrated that virus multiplication took place in the nucleus of cells with production of typical adenovirus inclusions. Thin section electron microscopy showed typical adenovirus particles and associated inclusions accumulating in nuclei. Strain 127 infectivity was stable in monovalent but not divalent cations, and stable to ether treatment and extremes of pH. IDU inhibition indicated presence of DNA. Growth of 127 as evidenced by HA production was better in duck kidney, fibroblast and liver cells, than in fowl cell cultures, while growth in turkey cells was limited to kidney and liver cultures. There was no evidence of growth in a range of mammalian cells. Strain 127 agglutinated erythrocytes from avian but not mammalian species and the haemagglutinin was stable to heating and freezing. There was no crossing in neutralisation or haemagglutination-inhibition tests between 127 and 11 fowl adenoviruses, and no cross-immunofluorescence between 127 and FAV-1. It is concluded that 127 is an avian adenovirus, possibly originating from ducks.

Characteristics of Noncultivable Adenoviruses Associated with Diarrhea in Infants: A New Subgroup of Human Adenoviruses

Virus particles morphologically resembling adenovirus were found in fecal specimens from infants and were examined for cultivability with standard cell culture techniques and for characteristics of human adenoviruses. Specimens from 13 of 15 infants could not be cultivated in cell cultures. The two adenoviruses that were cultivated, types 1 and 31, reacted in the expected manner in all tests. Counterimmunoelectrophoresis with group-specific anti-hexon serum confirmed that the observed particles in the 15 specimens were human adenoviruses. The buoyant density in sucrose of five of the noncultivable adenoviruses in original stool suspensions averaged 1.335 g/cm(3) and that of the two cultivable ones averaged 1.332 g/cm(3); both groups had typical adenovirus morphology by electron microscopy. Treatment of the specimens and of a variety of tissue culture cells with proteolytic and other enzymes did not improve cultivability. Examination of partially purified virus by immunoelectron microscopy did not reveal evidence of immunoglobulin A, G, or M coating on the particles, an indication that coproantibody inhibition was not the cause of noncultivability. Fluorescent-antibody studies with an antihexon conjugate and counterimmunoelectrophoresis studies of serially passaged noncultivable viruses indicated that the viruses are infecting cells but are not undergoing effective replication. Antisera to three of the noncultivable viruses demonstrated homologous reactions in counterimmunoelectrophoresis with the respective immunizing antigens but showed only low levels of hemagglutination-inhibiting and neutralizing activity to a few of the known human adenoviruses. We concluded that the noncultivable viruses in these infant diarrhea cases were indeed human adenoviruses, were not defective particles, were not bound to coproantibody, were infectious but incapable of effective relication in conventional cell cultures, were serologically related to types 11, 17, 32, and 33, and should be considered a new, distinct subgroup.

799 SEVERE ADENOVIRUS 7 PNEUMONIA IN A COMPROMISED HOST: IMMUNOLOGICAL INTERVENTION & SUCCESSFUL OUTCOME

Adenovirus pneumonia in immunologically compromised hosts is usually fatal. A 6 y.o. girl with partial combined immunodeficiency (CID) presented with failure to thrive,high fever,cyanosis & extensive bilateral pneumonia unresponsive to antibiotics. She was neutropenic,lymphopenic & hypogammaglobulinemic(IgG=110mg%) She developed abnormal LFT's & thrombocytopenia.Open lung biopsy was performed.Adenovirus 7 was isolated in 2 days.Histopathology revealed a severe interstitial necrotizing pneumonia with typical Adenovirus inclusions.Two units of fresh frozen plasma(FFP) failed to alter her course.She was given gammaglobulin(GG)(1.8cc per kg)IM.Within 48 hrs. she improved dramatically.By virus neutralization tests the GG contained a high titer (1:240) of Adenovirus 7 antibody(AB). No AB could be detected in either unit of FFP. Recovery coincided with a substantial rise in serum AB titer to Adenovirus 7. Her clinical condition has remained stable over 9 mos. She has received GG(0.7cc/kg) every 3 wks. Evidence of CID persists with poor lymphocyte responses to PHA, Con A, PWM,and in MLC, & no response to Adenovirus 7.Total T-cell E-rosettes have risen from 44% to 75%. That the administration of specific AB resulted in a successful outcome is strengthened by comparison with an almost identical situation in Arabian foals with CID. That the mechanism of recovery was due to AB neutralization or AB-dependent cell-mediated cytotoxicity (ADCC) remains to be determined.

Transformation with specific fragments of adenovirus DNAs I. Isolation of specific fragments with transforming activity of adenovirus 2 and 5 DNA

DNA of human adenoviruses 2 and 5 was cleaved by the restriction endonucleases HsuI, BamHI, HpaI, and SmaI. The resulting fragments were separated and tested for their ability to transform primary baby rat kidney (BRK) cells, using the calcium technique. Fragments with transforming activity were obtained with endo's R·EcoRI (fragments A), BamHI (fragments B of Ad2 and A of Ad5 DNA), and HsuI (fragments G). The transforming fragments all represented the left terminal fragments of the respective restriction endonuclease cleavage products. The smallest fragment found to contain transforming activity was the HsuI G fragment (molecular weight 1.7 · 10 6 for both Ad2 and Ad5 DNA). Transforming activity of both adeno DNAs was abolished by digestion with endo's R·HpaI and SmaI. This indicated that these enzymes cleave into an area essential for transformation. A number of cell lines transformed by restriction endonuclease fragments were established and some of their properties were studied. All adeno DNA fragment-transformed lines were found to grow to only a very low level in 0.33% agarose medium (cloning efficiency < 1%), irrespective of the size of the fragment used to transform the cells. All transformed cell lines were found to react with antiserum against adenovirus subgroup-C specific T antigen, as detected by immunofluorescence. The T antigen pattern in the HsuI G-transformed cells, however, was atypical and differed from the usual pattern, in that the fluorencence was largely localized in the cytoplasm. Selection of HsuI G-transformed cells in 0.33% agarose medium resulted in cell populations containing the typical adenovirus T antigen pattern.

Biological and biophysical characteristics of mouse adenovirus, strain FL.

Mouse adenovirus, strain FL (MAV), is best propagated in primary rather than secondary cultures of mouse kidney cells. Virus release from the cells into the medium is fairly efficient; the average yield per cell is about 1000 TCID50. Morphological and other biophysical characteristics are those of a typical adenovirus: the icosahedral shell with a diameter of 74 nm, fiber projections of 29 nm length, its localization inside the infected kidney cells, a buoyant density in CsC1 of 1.34 g/ml, inhibition of multiplication by inhibitors of DNA synthesis. The thermostability is higher than that of human adenoviruses, whereas, in contrast to these, MAV is inactivated to a great extent by trypsin. Complete or incomplete hemagglutinin or toxin-like activity were not detected. The virus shows no cross-neutralization with human adenoviruses and a one-side cross-reaction with another murine adenovirus strain K-87. Soluble complement-fixing antigen of MAX exhibits a sedimentation rate of 12S identical to the hexon component of human adenoviruses; both antigens show a partial antigenic relationship in tests with appropriate antisera.

Purification and properties of chick embryo lethal orphan virus (an avian adenovirus)

Chick embryo lethal orphan (CELO) virus, an oncogenic avian adenovirus, and one of its antigens (the hexon) were purified from infected allantoic fluid and some of their properties were studied. Particles of CELO virus contained 80.7% protein and 17.3% DNA and had a buoyant density of 1.35 g cm −3 in CsCl. CELO virus DNA was a linear double-stranded molecule with a molecular weight of 30 × 10 6 daltons (greater than that of human adenovirus DNA) and a G + C content of 54%. Particles of CELO virus had typical adenovirus morphology and the hexons appeared to be similar to those of adenovirus type 2. CELO virus pentons, however, differed remarkably from those of other adenoviruses. Pentons isolated from the soluble antigen fraction of infected allantoic fluid, or dissociated by dialysis from particles of CELO virus possessed a base which carried two knobbed fibres, one long (425 Å) and the other short (85 Å). Besides the hexon and penton antigens, CELO virus particles possessed another antigen which was probably one of the internal proteins. The amino acid composition of CELO virus hexon was similar to that of adenovirus type 2 hexon but these two hexons were completely unrelated immunologically. SDS-polyacrylamide gel electrophoresis showed that CELO virus particles contained fewer internal proteins than particles of adenovirus type 2. CELO virus particles, disrupted in various ways, did not show hexons arranged in groups of nine characteristic of adenovirus types 2 and 5.

Examination of adult primary human kidney cell cultures for adventitious viral agents.

SummaryTwelve primary adult human kidney cell cultures were examined by several techniques for the presence of adventitious viral agents. Although no definite cytopathic effects were observed in 11 of the cell cultures, 7 of these exhibited suspicious fluorescent foci after staining by the indirect fluorescent-antibody technique 4 to 7 weeks after initiation of the cultures. Electron microscopy (EM) studies, utilizing negative staining and thin sections, were done on several of the cell cultures, but no virus particles were observed. Passage attempts in primary and continuous cell cultures did not reveal the presence of transmissible viral agents in any of the 11 cell cultures. One cell culture, obtained from a 58-year-old male who died shortly after a myocardial infarction, showed cytopathic changes 28 days after initiation. EM examination of the cell pellet showed typical adenovirus particles which were identified as a type 2 adenovirus. In contrast to observations with subhuman primate tissues, latent ...

In vitro transformation by adenovirus-simiam virus 40 hybrid viruses. IV. Properties of clones isolated from cell lines transformed by adenovirus 2-simiam virus 40 and adenovirus 12-simiam virus 40 transcapsidant hybird viruses.

Clones were isolated from hamster cells transformed by the adenovirus 2-SV40 and adenovirus 12-SV40 transcapsidant hybrid viruses. The clones were characterized with respect to their cytomorphology, virus and antigen content, and the histomorphology of tumors induced by transplantation of the clonal sublines to hamsters. Three different cellular and colonial morphologies were observed. Clones with an SV40 morphology gave rise to tumors predominantly with an SV40 histology, whereas clones with an adenovirus morphology produced typical adenovirus tumors upon transplantation of the transformed cells. Clones which had features of both SV40 and adenovirus transformed cells gave rise to "intermediate" and adenovirus tumors. The results indicate that multiple events occur during transformation and tumorigenesis by the transcapsidant virus populations and provide an explanation for the multiplicity of findings which have been reported with these virus populations.

The Fine Structure of Replicating Simian Adenoviruses in LLC-MK2

The simian adenoviruses SV20, SV30 and SA7 have been found to be oncogenic in the Syrian hamster. The growth characteristics and replicative cycle of these viruses in tissue culture therefore appeared appropriate to investigate. Cesium chloride purified simian adenovirus with an infectivity titer of 100 TCID50, was inoculated into monolayers of LLC-MK2 cells. Cells were fixed in osmium tetroxide and embedded for ultrastructural studies at 1, 3, 6, 9, 18, 24, 48, 72, 120 and 192 hours post-infection.At the first hour post-infection, virus particles were adsorbed to the plasmalemma and found within the peripheral cytoplasm of many LLC-MK2 cells (Fig. 1). Although the first detection of infectious virus occurred at 14 hours and infectivity titers did not reach a maximum until 30 hours, intranuclear virus particles were observed by 3 hours in typical adenovirus crystalline array (Fig. 2) by means of electron microscopy. These typical honeycomb arrayed virus particles at 3 hours provided evidence of significant replication in approximately 5 percent of tissue culture cells examined. Simultaneously, a classical nuclear inclusion manifested by peripheral condensation of nuclear chromatin was evident by light microscopy. As early at 6 to 9 hours, unusual intranuclear concentric membranes formed “tubes” which contained linear arranged virus particles (Fig. 3). In transverse or tangential sections, these “tubes” appeared cochlear-like in shape. In longitudinal section, these intranuclear tubular structures contained individual virus particles at various stages of maturation in a linear arranged order. This arrangement resembled “peas in a pod”.

Adenovirus endocarditis in mice.

Viral endocarditis developed in 24 percent of 50 newborn mice 6 to 8 days after intraperitoneal inoculation with murine adenovirus. Typical adenovirus intranuclear inclusions were seen in heart-valve lesions, and high titers of virus were recovered from heart tissue. Furthermore, adenovirions were directly visualized by electron microscopy within endothelial cells and fibroblasts of the heart valves.

The Pathogenesis and Pathology of Infection in Calves with a Strain of Bovine Adenovirus Type 3

SUMMARY Eight colostrum-deprived calves were inoculated intranasally on the day of birth with 10 ml. of a strain (IVBR 1) of bovine adenovirus Type 3, and with a further 10 ml. of the same virus given intratracheally 24 hr. later. The inoculum had a mean infectivity titre of 104·0 TCID50 per ml. Three calves were killed at 4 days, 3 at 7 days and the remaining 2 calves at 3 months. A further 2 calves were inoculated in the same way with trypsin-treated WBR 1 virus and both were killed at 7 days. This inoculum had an infectivity titre comparable with that of the untreated virus. All calves showed a mild clinical reaction comprising some conjunctival and nasal discharge and a degree of pyrexia. Two calves exhibited respiratory embarrassment. In 6 calves, the onset of pyrexia was accompanied by a diarrhoea lasting 2 to 4 days. Swabs were made daily from conjunctiva, nose and rectum; virus was recovered from the faeces up to 10 days after infection and from the eye and nose up to 11 days. Virus was recovered from the nose of 1 calf after 3 weeks. No viraemia was demonstrated in any of the calves. At necropsy, gross lesions were confined principally to the lungs, which showed areas of collapse, consolidation and emphysema, and were especially marked in those calves inoculated with untreated virus and killed at 4 or 7 days. Lesions were discrete in the lungs of calves killed at 3 months and in those inoculated with trypsin-treated virus. Histological changes in the lungs of calves killed on the fourth and seventh days consisted of a proliferative bronchiolitis and necrosis with bronchiolar occlusion which resulted in alveolar collapse. Typical adenovirus intranuclear inclusions were observed in a variety of tissues including lung, tonsils, tracheal mucosa and in retropharyngeal, mediastinal and bronchial lymph nodes. Residual lesions consisting of scar tissue and some degree of lympho-reticular hyperplasia were seen in the lungs of calves killed 3 months after infection. Virus was recovered from the lung lobes of the calves inoculated with untreated virus and killed at 4 or 7 days, but not from those killed at 3 months or those infected with trypsin-treated virus. Precipitating antibody to the group reactive antigen of adenoviruses was demonstrated by immunodiffusion in the sera of calves 3 to 4 weeks following infection. Such antibody persisted until the animals were killed at 3 months. Two control calves inoculated with virus-free fluid and maintained in pens adjacent to the infected calves remained healthy, free of virus and showed no evidence of disease when killed at 4 and 7 days respectively. The role of adenoviruses in bovine respiratory disease and their possible implication in pneumo-entcritis of calves is discussed.