Immunological and Other Biological Characteristics of Pentons of Human Adenoviruses

Comparative hemagglutination-enhancement (HE) tests demonstrated diversified patterns of antigenic specificities both in the fiber and vertex capsomer part of pentons of human adenovirus types 3, 11 (subgroup I), 9, 15 (II), 1, 2, 4, 5, 6 (III), and 12. All fibers contained a type-specific antigen. Subgroup II and III fibers, in addition, contained specificities both unique for each subgroup and also common to the two subgroups. Fibers of serotypes 4 and 12 displayed a somewhat deviating behavior. All vertex capsomers tested shared a group-specific part. This was the only antigenic specificity demonstrable for serotype 12. Maximal penton HE titers of all sera were reached in tests with incomplete hemagglutinin of type 11. In addition, maximal HE activity of sera against individual serotypes also was recorded against pentons of other members of the same subgroup. Antigen characteristics of vertex capsomers of type 4 indicated a closer relationship to subgroup I than to subgroup III. The toxin activity of pentons was more sensitive to trypsin treatment than their capacity to function as incomplete hemagglutinin. Homotypic antipenton sera, unabsorbed or absorbed with homotypic fibers to remove antibodies against this component, and, to a varying extent, also heterotypic antipenton sera could neutralize toxin activity. Antifiber sera could neutralize toxin activity of pentons carrying short fibers (10 nm, type 3) but not of those carrying long fibers (28 to 31 nm, type 2). It is concluded that toxin activity is carried by a specific part of vertex capsomers and that cell detachment can be brought about via a direct contact between this component and cell membranes. Fiber-mediated attachment does not seem to be necessary for this biological activity to become expressed.     

Hybrid of Monkey and Human Adenoviruses

Following joint replication of monkey SA7 adenovirus (C8 strain) and human adenovirus type 2 in green monkey kidney tissue culture, a virus possessing the properties of a hybrid was obtained. It was designated Ad2C8. Ad2C8 preparations contained two types of viral particles: human adenovirus type 2, and hybrid particles. The hybrid virions multiplied in green monkey kidney cells in the presence of human adenovirus types 1, 2, and 3, but not 3 and 7, and acquired the capsid of the helper adenovirus. The hybrid can serve as a helper for human adenoviruses. It can apparently induce T antigen of the C8 virus but, in contrast to the latter, does not induce tumors in hamsters.     

Capsid Mosaics of Intermediate Strains of Human Adenoviruses

Antisera against isolated capsid components of intermediate adenovirus strains, types 3-16 (the San Carlos agent) and 15-9 and of "parental" prototype strains were compared in neutralization tests, hemagglutination-inhibition (HI) tests employing soluble and virion-associated hemagglutinin as antigens, and by electron microscopy. Hexons of the intermediate strains were found to be similar to, but not identical to, those of the prototype strains with which a cross-reaction occurred in neutralization tests (types 3 and 15). In contrast, fibers of intermediate strains displayed characteristics relating them to the corresponding components of prototype strains to which a relationship has been found in HI tests. Fibers (and possibly even pentons) of types 9 and 15-9 appeared to be identical, whereas fibers of types 3-16 and 16 displayed antigen specificities of both common and unique nature.     

Detection of Relationships Between Streptococcus faecalis and Lactobacillus casei by Immunological Studies with Two Forms of Malic Enzyme

Antisera prepared against two isologous malic enzymes from Lactobacillus casei strains 64H and M40 were used to survey and categorize the various malic enzymes found within this diverse species. In addition to detecting three major antigenic variants of malic enzyme within this group, both antisera readily reacted with Streptococcus faecalis malic enzyme. The cross-reactions between the L. casei malic enzyme antisera and the S. faecalis malic enzyme indicated that these iso-functional enzymes found in two apparently diverse groups of organisms were immunologically homologous. A scheme proposing a common ancestry for the two species S. faecalis and L. casei based on the results of quantitative immunological studies is presented.     

Quantification of Enterococci and Human Adenoviruses in Environmental Samples by Real-Time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

Immunological Relationships Among Some Species of Extremely Halophilic Bacteria

Rabbits were immunized with four strains of halobacteria, Halobacterium halobium NRL, H. halobium R-1, H. salinarium NRL-9, and H. cutirubrum NRL-10, that had been fixed with formaldehyde. The antisera obtained detected the presence of an antigen common to the Halobacterium genus and, after absorption, detected three distinct antigenic groups within the Halobacterium genus. A fourth group was agglutinated only by unabsorbed sera.     

Occurrence of Human Adenoviruses at Two Recreational Beaches of the Great Lakes

Human adenoviruses (HAdVs) have been related to several waterborne diseases such as acute gastroenteritis, conjunctivitis, and respiratory illness, and it has been shown that an important human exposure pathway is through recreational waters. However, HAdV occurrence at recreational freshwater beaches has not been previously investigated. In this study, a total of 58 water samples were collected from two recreational beaches on Lake Michigan (i.e., Silver Beach and Washington Park Beach) during the summer of 2004. Occurrences of HAdVs in these lake samples were determined using two hexon-based real-time PCR assays (one for monitoring all 51 serotypes of HAdVs and another for specifically detecting F species HAdVs, i.e., serotypes 40 and 41) and compared to an integrated cell culture (ICC) PCR method. The real-time PCR results showed that 8 of 30 Silver Beach samples and 6 of 28 Washington Park Beach samples contained HAdVs, and F species HAdVs were detected in three of these positive samples. The concentrations of HAdVs ranged from (1.7 ± 0.7) × 10¹ to (3.4 ± 0.8) × 10² and from (7 ± 2) × 10⁰ to (3.8 ± 0.3) × 10³ virus particles/liter for Silver Beach and Washington Park Beach, respectively. F species HAdVs were detected at levels ranging from (4.8 ± 0.8) × 10¹ to (4.6 ± 1.5) × 10² virus particles/liter. Approximately 60% of the ICC-PCR analyses agreed with the real-time PCR results. This study revealed the occurrence of HAdVs at Lake Michigan recreational beaches. Given the potential health risks, further assessment regarding sources, virus transport, and survival is needed to improve the safety of the region.     

Immunological Studies of Human Monoamine Oxidases

Antisera have been raised against monoamine oxidase preparations from human placenta and platelets. These antisera have been employed to characterize membrane-bound enzyme from a variety of human sources including liver, heart, and brain. The comparisons were based on a displacement radioimmunoassay system with soluble placental monoamine oxidase, previously labelled specifically with [3H]pargyline, as antigen. All forms of enzyme investigated demonstrated immunological cross reaction; however, the placental enzyme appeared to possess determinants not exhibited by the enzyme from the platelets or other tissues examined.     

Quantification and Stability of Human Adenoviruses and Polyomavirus JCPyV in Wastewater Matrices

Human adenoviruses (HAdV) and human polyomavirus JCPyV have been previously proposed as indicators of fecal viral contamination in the environment. Different wastewater matrices have been analyzed by applying real-time quantitative PCR procedures for the presence, quantity, and stability of a wide diversity of excreted HAdV and JCPyV. High quantities of HAdV and JCPyV were detected in sewage, effluent wastewater, sludge, and biosolid samples. Both viruses showed high stability in urban sewage. These results confirm the suitability of both viruses as indicators of human fecal viral pollution.     

Human Adenoviruses and Coliphages in Urban Runoff-Impacted Coastal Waters of Southern California

A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis.     

Assembly of Adenoviruses

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified incomplete particles of adenoviruses type 2 and 3 revealed that core proteins V and VII and capsid proteins VI, VIII, and X were absent in these particles, but they contained polypeptides not present in complete particles. Two types of incomplete particles were observed in the electron microscope, appearing as deoxyribonucleic acid-less particles with single discontinuities in the capsid structure (about 80%), or more amorphous particles resembling hexon aggregates (about 20%). The amount of incomplete and complete particles increased in parallel during the infectious cycle. Detectable amounts were found at 13 h with a maximum rate of synthesis for both particles at 24 h after infection. (3)H-labeled amino acids were incorporated into incomplete particles without a detectable lag period, but the label appeared in complete particles with a 60- to 80-min lag. Early after the pulse in pulse-chase experiments, the radioactivity was higher for incomplete particles than for complete particles and leveled off before the activity of complete particles reached a maximum. In the adenovirus type 2 system, pulse-chase experiments suggested a precursor-product relationship between incomplete and complete particles. After a short pulse, 19 h postinfection, entrance of (3)H-labeled amino acids into the hexon polypeptide of complete particles was delayed for 80 min, but no delay was observed for the labeling of the hexon polypeptide of incomplete particles. The core polypeptides appear in complete particles without a delay, also suggesting that incomplete particles were precursors to complete particles. Incorporation of (3)H-labeled amino acids into the hexon polypeptide of complete and incomplete particles was drastically decreased by inhibition of protein synthesis with emetine. However, the uptake of label into core proteins of complete particles was only decreased to 50% on inhibition of protein synthesis. The results suggest that incomplete particles are intermediates in virus assembly in vivo and that the assembly of capsid polypeptides into incomplete and complete particles is dependent on continuing protein synthesis.     

Replication and Complementation of Human Adenoviruses and Simian Papovavirus at an Elevated Temperature

The simian papovavirus SV40 replicated as well in simian cells incubated at 41 C as in cells incubated at 37 C, although the latent period was shortened at the elevated temperature. Human adenoviruses differed in their responses to the elevated temperature. Some serotypes, such as 3, 4, 5, 7, 8, 16, and 21, replicated as well, or almost as efficiently, in human cells incubated at 41 C as in cells incubated at 37 C, whereas with other serotypes, such as 1, 2, 6, 12, and 14, maximal yields in cultures incubated at 41 C were much lower than the yields from companion cultures incubated at 37 C. This difference was also detected in simian cells co-infected with SV40 and a human adenovirus; maximal complementation occurred with some serotypes at the elevated temperature but not with other serotypes. The degree of complementation observed in the simian cells at 41 C was directly correlated with the ability of the adenovirus to replicate at 41 C in human cells. Therefore, the capacity of SV40 to serve as a helper virus is not affected by the elevated temperature, showing that the complementation event supplied by the simian virus is heat-stable between 37 and 41 C. Maximal complementation appeared to depend upon a characteristic present in the adenovirus genome.     

Ovine Adenovirus Vectors Overcome Preexisting Humoral Immunity against Human Adenoviruses In Vivo

Recombinant human adenoviruses (hAd) have become widely used as tools to achieve efficient gene transfer. However, successful application of hAd-derived vectors in clinical trials is limited due to immunological and potential safety problems inherent in their human origin. In this study, we describe a recombinant ovine adenovirus (OAV) as an alternative vector for gene transfer in vivo. In contrast to an hAd vector, the OAV vector was not neutralized by human sera. An OAV vector which contained the cDNA of the human alpha1-antitrypsin (hAAT) gene linked to the Rous sarcoma virus promoter was generated and administered systemically to mice. The level and duration of hAAT gene expression was similar to that achieved with an hAd counterpart in both immunocompetent and immunodeficient mice. However, the tissue distribution of the OAV vector differed from that observed for hAd vectors in that the liver was not the dominant target. Significantly, we demonstrated efficient gene transfer with the OAV vector into mice immunized with hAd vectors and vice versa. We also confirm that the immune response to a transgene product can prevent its functional expression following sequential application of a vector. Our results suggest a possible solution to endemic humoral immunity against currently used hAd vectors and should therefore have an impact on the design of improved gene therapy protocols utilizing adenovirus vectors.