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Human cells in early passage cultures of benign oral tumors, normal oral tissues, normal lung tissues and, especially, in long-term established oral carcinoma cultures were highly susceptible to infection by human adenovirus types 5, 21, and 31. In contrast, replication of each adenovirus type was markedly limited in inoculated cells of newly-established oral squamous cell carcinoma cultures.
Human adenoviruses 1 to 28 were shown to produce a cytopathic effect in Vero cell cultures. Viruses of subgroups III and IV (Ad 1, 2, 5, 6, 12, and 18) were readily passaged in Vero cell cultures and were produced in high amounts. This was also found for Ad 11, 16, and 21, while Ad 3, 4, and 7 showed a lower degree of multiplication and Ad 14 could not be passed serially. For Ad 8, 26, 27, 20, 25, and 28, a multiplication in Vero cells could not be proved, while the remaining serotypes of subgroup II showed a moderate degree of multiplication. The sensitivity of Vero cells to small amounts of virus was lower than that of HeLa cells. No adaption of adenoviruses to Vero cells after 5 Vero passages was observed. Attempts to enhance virus multiplication by coinfection with SV40 failed.
Adenovirus 12 (Ad12) (Huie) (highly oncogenic group A) readily induces tumors in newborn rodents. Since Ad12 is isolated from human fecal samples, we investigated whether it plays a role in the etiology of human gastrointestinal cancer. If Ad12 is a causal agent of human cancer, then human tumors should contain Ad12 transforming genes, as indicated by studies of cells transformed in vitro and in vivo by oncogenic viruses. Ad12 DNA and the Ad12 transforming restriction fragment (EcoRI-C fragment, left 16% of the viral genome) were labeled in vitro to 10(7) to 4 X 10(8) cpm/mug by the nick translation reaction of DNA polymerase of Escherichia coli. The fidelity and sensitivity of these probes were established by (i) analysis of DNA from Ad12-transformed cells and from hamsters with tumors induced by Ad12, (ii) reconstruction experiments with added Ad12 DNA and EcoRI restriction fragments, and (iii) comparison of annealing characteristics with Ad12 probes labeled in vivo. With Ad12 [3H]DNA as probe, no viral DNA sequences were detected in 18 normal gastrointestinal tissues and 34 gastrointestinal tumors, including cancers of the colon, rectum, small intestine, and stomach, under conditions that would detect 0.1 copy of the Ad12 genome per tumor cell. Similar analyses of Ad12-transformed hamster cells and Ad12 primary hamster tumors indicated 6-18 copies per cell of over 90% of the viral genome. With the Ad12 EcoRI-C transforming fragment as probe, no hybridization was detected with 32 human gastrointestinal tumors and five normal tissues; this result excludes 1-2% of the Ad12 genome per tumor cell. Our date are strong evidence that Ad12 is not a major cause of human gastrointestinal cancer. The Ad12 transforming EcoRI-C fragment hybridized (50-68% efficiency) with other Ad12 isolates and with Ad18 and 31 (members of oncogenic group A), but not at all with 28 other human Ad serotypes (manuscript in preparation). Thus other group A members probably are also not involved in human gastrointestinal cancer. No viral DNA sequences were detected in 12 normal lungs and 22 lung tumors, suggesting that respiratory cancer does not involve an Ad12 etiology.
Temperature-sensitive mutants of type 5 adenovirus belonging to eight complementation groups were complemented in mixed infection by type 12 adenovirus, whereas mutants of 7 other groups were not enhanced. In some crosses, phenotypic mixing took place. No evidence of recombination between type 5 ts mutants and type 12 was found.
Group C human adenoviruses (Ads) of serotypes 1, 2, 5, and 6 infect most children and commonly cause latent infections of lymphoid tissues. Ads transform cells into a malignant-like phenotype; the oncogenic genetic information is in the left 8% of the viral genome, in the HindIII-G DNA fragment. We have investigated the molecular basis for group C Ad latent infections in human tonsils as well as whether these viruses are linked to human cancer. Tonsil or cancer DNAs and RNAs were assayed for Ad sequences by liquid-phase saturation-hybridization with in vitro-labeled Ad5 HindIII-G fragment. About 25% of the 52 tonsils analyzed contained DNA or RNA sequences specific to HindIII-G, indicating that Ad transforming sequences are expressed as RNA in tonsils. Southern blotting analysis of four tonsil DNAs revealed multiple copies of the complete Ad genome in a free state and provided evidence for an unusual form of the Ad genome, possibly Ad DNA integrated into cellular DNA. In assays of human cancers, no Ad sequences were detected in DNAs from 26 squamous cell carcinomas (Cas), 3 adenocarcinomas, 4 oat cell Cas, 5 stomach Cas, 5 small intestine Cas, 15 colon Cas, 6 rectum Cas, 5 Hodgkin and 6 non-Hodgkin lymphomas, and 2 breast Cas. Reconstruction experiments indicated that the HindIII-G probe could detect 1 copy per cell of 0.2-0.3% of the viral genome. No HindIII-G-specific sequences were detected in RNAs from 21 squamous cell Cas, 3 oat cell Cas, 2 stomach Cas, or 18 colon Cas. In six other experiments using the complete Ad2 genome as probe, no Ad sequences were found in DNAs from 6 lung Cas, 12 normal lung tissues, 33 gastrointestinal Cas, 19 normal gastrointestinal tissues, 6 Hodgkin lymphomas, 3 breast Cas, or 4 kidney Cas, at a sensitivity of about 1 copy per tumor cell of 5-10% of the Ad2 genome. All Ad-induced cancer cells should contain at least 1 copy of 1-6% of the viral genome, the minimal size of the transforming region, and probably should contain multiple copies of more of the genome. Therefore, our data are definitive evidence against group C Ads being the cause of the cancers tested, which represent about 50% of the cancer incidence in the United States. Of additional interest, we did not detect Ad2 sequences in RNAs from 7 human placentas, 12 normal lungs, or 19 normal gastrointestinal tissues (nor in 44 cancer or 23 tonsil RNAs). Thus, we did not confirm a recent report of the presence of Ad2 RNA in RNAs from human placentas; the possibility that a small population of cells in placenta expresses group C "related" sequences is not ruled out.
The 31 human adenoviruses (Ad) from five groups (A-E) whose DNAs are <20% homologous by molecular hybridization. Ad5 (group C) DNA contains a 55,000-dalton protein probably covalently bound to each 5' terminus. This covalently bound protein may be analogous to polypeptides found in other viral and nonviral systems that are covalently bound to genomic DNAs or RNAs and that are thought to function in DNA or RNA replication. Because of the importance of proteins linked to nucleic acids, we have investigated whether DNAs from all five groups of human adenoviruses have terminal proteins, as well as the peptide relationships among the different terminal proteins. We show here that DNAs from Ad12, 7, 2, 19, and 4, representing Ad groups A-E, respectively, all contain covalently bound proteins of about 55,000 daltons. To investigate the peptide relatedness among the terminal proteins, we prepared microgram quantities of covalently bound protein from Ads in groups A-E and compared their chymotryptic and tryptic (125)I-labeled peptide maps. We find that the covalently bound protein maps of the five Ad groups are highly related and possibly identical. On the other hand, the tryptic and chymotryptic peptide maps of the major virion protein II and the core proteins V and VII of groups B, C, and E Ads show considerable heterology. Assuming that the covalently bound protein is virally coded, the conserved primary sequence of these proteins suggests a major functional role for the protein in Ad replication. Because the genetic origin of the Ad covalently bound proteins is not established, our data are also consistent with the possibility that the protein is coded by a cellular gene.
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.
The 31 human adenovirus (Ad) serotypes form five groups based upon DNA genome homologies: group A (Ad12, 18, 31), group B (Ad3, 7, 11, 14, 16, 21), group C (Ad1, 2, 5, 6), group D (Ad8, 9, 10, 13, 15, 17, 19, 20, 22-30), and group E (Ad4) (M. Green, J. Mackey, W. Wold, and P. Rigden, Virology, in press). Group A Ads are highly oncogenic in newborn hamsters, group B Ads are weakly oncogenic, and other Ads are nononcogenic. However, most or all Ads transform cultured cells. We have studied the homology of Ad5, Ad7, and Ad12 transforming restriction endonuclease DNA fragments with DNAs of 29 Ad types. Ad5 HindIII-G (map position 0-7.3), Ad7 XhoI-C (map position 0-10.8), and Ad12 (strain Huie) EcoRI-C (map position 0-16) and SalI-C (map position 0-10.6) fragments were purified, labeled in vitro (nick translation), and annealed with DNAs of Ad1 to Ad16, Ad18 to Ad24, and Ad26 to Ad31. Hybrids were assayed by using hydroxylapatite. Ad5 HindIII-G hybridized 98 to 100% with DNAs of group C Ads, but only 1 to 15% with DNAs of other types. Ad7 XhoI-C fragment hybridized 85 to 99% with DNAs of group B Ads, but only 6 to 21% with DNAs of other types. Ad12 (Huie) EcoRI-C hybridized 53 to 68% with DNAs of five other Ad12 strains, 53% with Ad18 DNA, 56% with Ad31 DNA, but only 3 to 13% with DNAs of other types. In vitro-labeled Ad12 (Huie) SalI-C hybridized 35 to 71% with DNAs of 6 other Ad12 strains, 44% with Ad18 DNA, 52% with Ad31 DNA, but only 2 to 7% with DNAs Ad7, Ad2, Ad26, or Ad4. When assayed using S-1 nuclease, SalI-C annealed 17 to 44% with DNAs of group A Ads. The melting temperatures of the hybrids of Ad5 HindIII-G with all group C Ad DNAs were 84 degrees C in 0.12 M sodium phosphate (pH 6.8). The melting temperature of the Ad12 (Huie) EcoRI-C hybrid with Ad12 (Huie) DNA was 83 degrees C, but was only 71 to 77 degrees C with DNAs of other group A Ads. Thus, group C and group B Ads both have very homologous transforming regions that are not represented in DNAs of non-group C Ads or non-group B Ads, respectively. Similarily, group A Ads have unique but less homologous transforming regions. These different transforming nucleotide sequences may be reflected in the different oncogenic properties of group A, B, and C Ads.
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Serum specimens from 42 normal dogs and 42 with untreated malignant tumours were assayed for the presence of antibodies to human adenovirus types 5, 21 and 31 and to infectious canine hepatitis (ICH) virus. Radioimmunoassays using human adenovirus antigens showed that 71 per cent (30/42) of all dogs with tumours, but only 19 per cent (8/42) of all normal dogs, were positive for human adenovirus antibody. Canine sera reactive with antigens of one human adenovirus type in radioimmunoassays were also reactive with antigens of the other two types. Dogs bearing malignant lymphoma or squamous cell carcinoma tumours had higher levels of antibody against adenovirus type 5 antigens. Human adenovirus type 5 was neutralised by sera from four tumour-bearing and two normal dogs, while sera from two normal and five tumour-bearing dogs were positive in immunodiffusion tests with human adenovirus antigens. Levels of ICH antibody in sera of normal adult dogs and adult dogs with tumours were not markedly different when measured by radioimmunoassays. Likewise, sera from these two groups of dogs had similar ranges of ICH neutralising antibody titres. In contrast, levels of ICH antibody detected by the serological assays in sera from non-pet, non-vaccinated pups were either markedly low or absent. Possible explanations for the observed increased levels of human adenovirus antibody in sera of tumour-bearing canine pets are discussed.
Human embryonic kidney cells have been transformed by exposing cells to sheared fragments of adenovirus type 5 DNA. The transformed cells (designated 293 cells) exhibited many of the characteristics of transformation including the elaboration of a virus-specific tumour antigen. Analysis of the polypeptides synthesized in the 293 cells by labelling with 35S-methionine and SDS PAGE showed a variable pattern of synthesis, different in a number of respects from that seen in otheruman cells. On labelling the surface of cells by lactoperoxidase catalysed radio-iodination, the absence of a labelled polypeptide analogous to the 250 K (LETS) glycoprotein was noted. Hybridization of labelled cellular RNA with restriction fragments of adenovirus type 5 DNA indicated transcription of a portion of the adenovirus genome at the conventional left hand end.
C57 black mouse cells infected with human adenovirus type 5 (Ad5) produced large amounts of early virus proteins, small amounts of late virus proteins and less than 0.2 infectious units (i.u.)/cell of infectious virus. Many cells died but the cultures recovered. Virus DNA and cellular DNA were synthesized. Some Ad5 DNA sedimented with cell DNA in alkaline sucrose, but virus DNA was rapidly lost from the culture after recovery and none of 28 unselected cloned survivors contained detectable amounts of virus DNA or antigens. Ad5 ts36 was temperature-sensitive for virus DNA replication in mouse cells, but ts125 was detective at 32.5 degrees C as well as at 39.9 degrees C. No difference was detected in the percentage of virus DNA that sedimented in alkali with cell DNA, in mouse cells infected by Ad5 ts+, ts36 or ts125 at 32.5 or 39.9 degrees C. All parts of the virus genome were equally represented in virus DNA that sedimented with cell DNA, in mouse cells infected by Ad5 ts+ or ts36 at either temperature.
We investigated whether human adenovirus type 4 (Ad4) might cause cancer in humans. Cell culture and animal model studies indicated that Ad4-induced human tumors should contain Ad4 DNA sequences. Thus Ad4 DNA was labeled in vitro (sp act, approximately 10(7) 3H counts/min/microgram) and hybridized in liquid phase with DNA extracted from human tumors. No viral sequences were detected in 31 squamous cell carcinomas of the lung, 5 adenocarcinomas of the lung, 4 oat cell carcinomas of the lung, 4 carcinomas of the stomach, 10 carcinomas of the colon, 3 carcinomas of the kidney, 3 carcinomas of the breast, 2 carcinomas of the ovary, and 6 Hodgkin's lymphomas. Reconstruction experiments with added Ad4 DNA indicated that the probe detected about 1 copy of 5% of the Ad4 genome per tumor cell. Therefore, these data were strong evidence (but did not prove) that none of these particular tumors were induced by Ad4.
Human adenoviruses of species B (HAdV-B) can cause upper respiratory tract infections and conjunctivitis, but also severe lower respiratory tract infections (LRTI). Although HAdV-associated LRTI are non-notifiable in Germany, typing data of our Adenovirus Reference Laboratory indicated an HAdV-B3 epidemic in 2023, with 67 samples initially typed as HAdV-B3 compared to < 10/year in the previous years. Circulation of a novel, highly virulent HAdV-B3 strain was suspected and complete viral genomic sequencing performed, revealing a recombinant phylogeny of the penton gene (P), which originated from HAdV-B7, whereas hexon (H) and fiber (F) genes originated from HAdV-B3. Therefore, this virus was acknowledged by the Adenovirus Working Group as the novel recombinant genotype 114 (P7H3F3). Interestingly, BLAST search of the HAdV-B114 prototype sequence showed 99.91% identity to the old HAdV-B genome type 3a. Additionally, multiple complete adenovirus genomic sequences labeled as HAdV-B3 during the last two decades had > 99.8% identity, suggesting long-term circulation of HAdV-B114 although the recombinant phylogeny of its penton region had not been recognized. This detailed analysis of an HAdV epidemic associated with ocular and respiratory infections, including severe LRTI, led to the discovery of a novel genotype HAdV-B114, which is rather a neglected, re-emergent than an emerging virus.
A mutant of human adenovirus type 12, temperature-sensitive for virus replication an for the enhancement of thymidine incorporation in resting human embryonic kidney cells, is described. The mutant was also temperature-sensitive for transformation of rat cells. The results indicate that a viral gene required for the virus growth cycle is also required for transformation.
15 days after experimental infection with human adenovirus 5, C57 Bl/6 murine spleen cells were found to have altered in vitro properties. Proliferative response to phytohemagglutinine was slightly increased while the allogeneic response was decreased and the capability to stimulate allogeneic cells was significantly diminished. The degree of expression of surface antigens responsible for allogeneic stimulation is influenced by many regulatory factors, still poorly defined; alteration of some factors of antigenicity may result, directly or indirectly, from certain viral infections.
Physico-chemical characteristics of human adenovirus type 6 DNA were studied. The sedimentation constant of adenovirus type 6 DNA was shown to be 32.7S. The intrinsic viscosity of DNA was on the average 93.8 dl/g. The average size of the molecular mass determined by the results of sedimentation and viscosimetry was 23.5 X 10(6) daltons which agreed favourably with the value calculated from the data of electron microscopic determination of the length of DNA molecule. The buoyant density of DNA in cesium chloride density gradient was 1.7126 g/cm3. The melting temperatures were 90.5 and 75 degrees C for 1 X SSC and 0.1 X SSC, respectively. Adenovirus type 6 DNA contained 52.6% GC pairs on the average.