John C. Hierholzer

DNA restriction analysis of adenovirus prototypes 1 to 41

SummaryDNA restriction patterns of all known human adenovirus prototypes (1 to 41), ordered according to subgenera A to F, are presented for restriction endonucleases BamHI, BgIII, BstEII, HindIII, and SmaI. This catalogue is a prerequisite for typing of adenoviruses by DNA restriction analysis in diagnostic laboratories and for strain identification in reference laboratories. To determine the genetic relationship of human adenoviruses within a subgenus and between subgenera, a pairwise analysis of comigrating fragments was performed. Adenoviruses of subgenus C were closely related. Adenoviruses of subgenus B showed two related clusters of four types each, whereas the numerous serotypes of subgenus D did not show any corresponding clustering. Little comigration was observed between DNA restriction fragments from members of subgenus A or F respectively.

Fatal disseminated adenovirus infection in a renal transplant recipient

A 61 year old woman died of diffuse interstitial adenovirus pneumonia 55 days after receiving a cadaveric renal allograft. The adenovirus was serologically distinct from the 33 known human adenovirus serotypes and appears to represent a new human adenovirus. Pathologic and virological findings indicate that the pneumonia was only one manifestation of a disseminated infection, the source of which may have been a latent adenovirus infection preexisting in the donor kidney. The establishment of the etiologic diagnosis in this case, which was complicated by the presence of oculocutaneous and esophageal herpes simplex virus infection as well as focal pulmonary aspergillosis, required coordinated histopathologic and virological investigation. Our findings demonstrate that severe viral infections in transplant recipients are not caused exclusively by members of the herpesvirus group.

Antigenic relationships among the 47 human adenoviruses determined in reference horse antisera

SummaryReference equine antisera to all 47 serotypes of human adenoviruses presently described have been prepared and evaluated by reciprocal neutralization and hemagglutination-inhibition tests. All tests were carried to endpoint dilutions a minimum of five times in each direction to give accurate values for homologous and heterologous antibody titers. Significant cross-reactions in the horse antisera were compared to similar data obtained from rabbit antisera. Using this analysis, major antigenic relationships exist among types 12–18–31 of subgenus A, types 7–11–14 and 34–35 of subgenus B, types 8–9–10, 10–19–37, 13–38–39, 15–22–42, 20–47, 24–32–33–46, and 29–45 of subgenus D, types 16-4 between subgenera B and E, and types 40–41 of subgenus F. Across all subgenera, types 8, 10, 13, 15, 17, 19, 26, 29, 39, 40, and 43 have antigenic moieties found most frequently in other types, averaging 12 heterologous reactions per type when summing both tests in both directions. Types 20, 30, 32, and 45 exhibit shared determinants slightly less often, with a mean of 8 heterologous reactions per type.

Mapping of base sequence heterologies between genomes from different adenovirus serotypes

Abstract An electron microscopic heteroduplex technique was used to physically map sequence heterologies between genomes from different human adenovirus serotypes. When DNA from serotypes was mixed, denatured, and renatured, characteristic heteroduplex molecules were formed. These molecules, composed of one strand from one serotype and one strand from the other serotype, contained one or more detectable regions of strand separation (or heterology), depending upon denaturing conditions and the combination of type-specific DNA preparations. By increasing denaturing conditions, regions of partial homology could be demonstrated in all interserotypic heteroduplexes. The heterology patterns in heteroduplexes constructed with DNA from nononcogenic serotypes (Ad 1,2, and 5) were relatively simple and nearly similar, whereas both simple and complex patterns were seen in heteroduplexes prepared with DNA from weakly oncogenic serotypes (Ad3,7, and 21). Only complex heterology patterns were found in heteroduplexes prepared with DNA from highly oncogenic serotypes (Ad 12, 18, and 31). As expected, the most extensive heterologies were observed in heteroduplexes prepared with DNA from serotypes belonging to the different serologic groups. Within groups, heteroduplex molecules contained two specific regions in which heterologies were most marked. These regions appeared to map at similar locations among heteroduplexes from all groups. It was found that the integrated SV40 virus DNA in an Ad 7-SV40 hybrid genome was situated in one of these regions.

Molecular epidemiology and restriction site mapping of adenovirus type 3 genome types.

SummaryFrom the United States, the Federal Republic of Germany, and other regions, 168 strains of AV7, isolated between 1961 and 1985, were analyzed by six restriction endonucleases and nine genome types were identified. The enzymes BamHI and HindIII were most discriminative. The genome type D5 (or 7b) predominated with 120 isolates since 1970 in both countries. Strains of D2 (7a) and D4 (7c) were isolated for a limited time, D3 for an extended time period. Several clusters of infections with the same genome type were found. Differences in pathogenicity could not be derived from our data.On the basis of restriction site mapping, most other genome types were similar to D5, one to D2 and one to the prototype (D1). The genomic relation between AV7 and AV3 is discussed and shown by a dendrogram.

Purification and biophysical properties of human coronavirus 229E

Abstract Coronavirus 229E was grown to high titers in diploid fibroblast cells under medium containing twice the normal concentrations of amino acids and vitamins. Growth curves showed maximum virus production at multiplicities of infection of 0.1 and 1; maximum titers of intracellular virus occurred at 22–24 hr and of extracellular virus at 26 hr postadsorption. Tube infectivity titers ranged from 109.0–109.5 TCID50/ml and plaque titers from 1010.2–1010.9 y PFU/ml at the time of peak virus production, when no cytopathology was evident. Virus titer dropped rapidly between 26 and 56 hr, coincident with increasing cytopathology. A single precipitin band was observed in immunodiffusion and immunoelectrophoresis between concentrated virus preparations and antiserum to purified 229E. Neuraminidase and hemagglutinin assays were negative. Virus was purified by two procedures: adsorption to and elution from human “0” erythrocytes and CaHPO4 gel followed by equilibrium sucrose gradient centrifugation, and PEG precipitation followed by equilibrium glycerol/tartrate gradients and rate zonal sucrose or glycerol/tartrate gradients. Final lots of purified virus containing <0.02% of the crude tissue culture proteins had absorption maxima at 256 nm and minima at 241.2 nm and a mean extinction coefficient of E 1cm 1% = 54.3 at 256 nm. The fully corrected sedimentation coefficient for the intact virion was S 20,v 0 = 381 S. PAGE by different techniques revealed seven polypeptides of mean apparent molecular weights between 16,900 and 196,100. Six contained carbohydrate and one contained lipid. Electropherograms of 3H- and 14C-labeled virus were identical to those of stained gels. Two glycoproteins constituting 25% of the virion protein were identified by bromelin digestion as the spike proteins. The density in sucrose and in potassium tartrate was 1.18 g/ml for the virion and 1.15 g/ml for the “despiked” particle.

Extrapulmonary manifestations of adenovirus type 7 pneumonia simulating Reye syndrome and the possible role of an adenovirus toxin.

Three children developed extensive extrapulmonary disease in the course of fatal adenovirus type 7 pneumonia. Several clinical features, including the unexpected onset of coma, suggested the development of Reye syndrome, but biochemical and histopathologic findings were inconsistent with this diagnosis. Virologic and pathologic studies did not reveal evidence of extrapulmonary adenovirus infection, despite clinical involvement of the liver, skeletal muscle, and central nervous system. The detection in premortem sera from all three patients of adenovirus penton antigen, known to be cytotoxic in vitro, suggests a possible mechanism for the production of extrapulmonary pathology in the absence of extrapulmonary virus infection.

Protein composition of coronavirus OC 43

Abstract A human coronavirus, strain OC 43, was propagated in suckling mouse brain and purified 5000-fold with, a 90% yield. Purity of the virus was confirmed by electrophoretic, ultracentrifugal, and electron microscopic procedures. Immunodiffusion and immunoelectrophoresis tests revealed one precipitin line with normal mouse brain, three with purified virus, and four with crude virus when tested against anti-pure virus or anti-crude virus animal serums. The association of a host cell antigen with the virion was confirmed by standard HI and CF tests. Polyacrylamide gel electrophoresis of solubilized purified virus revealed a minimum of six polypeptides with apparent molecular weights of 191,000 (No. 1), 104,000 (No. 2), 60,000 (No. 3), 47,000 (No. 4), 30,000 (No. 5), and 15,000 daltons (No. 6). A seventh band was occasionally found in the 165,000-dalton region of the gels. Four polypeptides contained carbohydrate and one contained lipid. Polypeptide No. 5 comprised 26% of the total viral protein and glycopolypeptide No. 3 comprised 23%. Three other components accounted for most of the remaining protein: polypeptide No. 4 (16%), glycopolypeptide No. 6 (14%), and glycolipopolypeptide No. 1 (13%). Glycopolypeptide No. 2 was 8% of the total protein. Bromelin digestion of the viral projections (spikes) removed glycopolypeptides No. 2 and No. 6. Association of the remaining polypeptides with structural components of the virion is only tentatively postulated. The buoyant density in potassium tartrate of the bromelin-treated virus was 1.15 g/cm3 and of the intact OC 43 virion was 1.18 g/cm3. By analytical ultracentrifugation the corrected sedimentation coefficient (s 0 20w) of the OC 43 virion was determined to be 390 ± 16 S, and the apparent molecular weight (MW a ) was calculated to be 112 ± 5 × 106 daltons.

Analysis of antigenically intermediate strains of subgenus B and D adenoviruses from AIDS patients.

SummaryWe carried out detailed antigenic and restriction enzyme (RE) analyses on the subgenus B and D adenovirus isolates from 48 AIDS patients. These isolates were unusual both in the diversity of serotypes and in the number of intermediate strains identified. All unusual isolates were strain-purified and tested by serum neutralization (SN) and hemagglutination-inhibition (HI) tests with reference horse and rabbit antisera to all the prototype human adenoviruses; conversely, rabbit antisera were prepared to 16 selected strains from this study and tested by SN and HI with all prototype viruses. Among subgenus B strains, 6 DNA variants of Ad 11 isolates were distinguished by endonucleases BamHI, BglII, BstEII, HindIII, and SmaI. The D2 variant of Ad 11 was prominant with 5 isolates, and 7 other isolates differed from D 2 in only 1 or 2 enzymes. HindIII was the most discriminative endonuclease for Ad 11 and related strains. Within subgenus D, there were 16 isolates of intermediate strains, including 4 intermediate types related to new Ad types 43–47. The RE patterns of subgenus D strains showed fragment distributions typical of subgenus D, with various unique patterns. Particular care was taken to analyze multiple strains from the same patient, recovered as much as 8 months apart, for evidence of genetic changes. The possible long-term infections with these viruses may provide the opportunity for mutations and recombination to occur, with the resultant generation of a variety of new strains of adenoviruses.

AN “OUTBREAK” OF JUVENILE DIABETES MELLITUS: CONSIDERATION OF A VIRAL ETIOLOGY

Abstract Huff. J. C., J. C. Hierholzer (Respiratory Virology Section, Center for Disease Control. Atlanta, Ga. 30333) and W. A. Farris. An “outbreak” of juvenile diabetes mellitus: consideration of a viral etiology. Am J Epidemiol 100:277–287. 1974.—Nine of twelve cases of juvenile diabetes mellitus, representing an unusual geographic and temporal cluster, were investigated for evidence that a specific viral infection might be etiologically related to their occurrence. Eight diabetics had experienced recent “viral-like” illnesses, predominantly repiratory, but these illnesses bore no uniform temporal relation to their onsets of diabetes. Diabetics demonstrated no serologic evidence of a recent viral illness common to all. Elevated titers to only one virus, coxsackie B-3, were more prevalent in diabetics than in controls (33% vs. 6%), but geometric mean titers of diabetics to a panel of 26 common viral antigens were similar to those of controls. These data neither support nor negate the hypothesis that infection with a specific virus precipitates juvenile diabetes mellitus.