Max Arens

Penetrating keratoplasty in rabbits induces latent HSV-1 reactivation when corticosteroids are used

The increased incidence of corneal graft failure in patients with herpes simplex virus (HSV) keratitis may be due in part to reactivation of latent HSV following surgical corneal trauma and postoperative corticosteroid therapy. To determine the onset, frequency, and nature of HSV recurrences following penetrating keratoplasty (PKP), 21 HSV type 1 (HSV-1) latently infected rabbits underwent unilateral autograft PKP. Opposite unoperated eyes served as HSV-1 latently infected controls. Corneal autografts were performed so that immunologic graft rejection would not be confused with recurrent HSV-1 stromal disease. After PKP, 11 of the 21 eyes were treated with dexamethasone. Ocular cultures and slit-lamp examinations were performed daily for the first postoperative 8 days and every other day thereafter for 82 days. Nine (82%) of the 11 dexamethasone-treated PKP eyes, 2 (20%) of the PKP eyes not treated with dexamethasone, and 3 (17%) of the 18 unoperated eyes had positive HSV-1 ocular cultures. Geographic ulcers appeared only in the PKP eyes treated with dexamethasone; 9 (82%) of the 11 PKP eyes treated with dexamethasone developed geographic ulcers. Between the 24th and 90th postoperative days, stromal keratitis appeared in 5 (56%) of the 9 PKP eyes treated with dexamethasone and in 2 (25%) of the 8 PKP eyes not treated with dexamethasone. Autograft PKP with postoperative corticosteroids significantly increased HSV-1 ocular shedding, epithelial ulceration, and stromal keratitis. This experimental model provides a useful tool to further investigate the development and treatment of HSV-1 epithelial and stromal recurrences after PKP.

Characterization of DNA polymerase associated with nuclear membrane fractions from uninfected and adenovirus 2-infected KB cells

We have isolated a nuclear membrane fraction from KB cells infected with human adenovirus 2 that synthesizes exclusively small viral DNA chains (approx. 9 S) in vitro (Yamashita, T., Arens, M. and Green, M. (1975) J. Biol. Chem. 250, 3273-3279). The DNA polymerase activity present in the adenovirus 2 DNA-nuclear membrane complex was purified through chromatography on phosphocellulose and DEAE-cellulose, glycerol gradient centrifuation and DNA-cellulose chromatography. A single peak of enzymatic activity sedimented in glycerol gradients at about 6.7 S which corresponds to a molecular weight of 125000. The enzyme preparation in the step of glycerol gradient centrifugation utilized activated calf thymus, KB cell and adenovirus 2 DNA as template-primer in the presence of Mg2+; Km values for these DNAs were 5.5, 4.0, and 0.8 mug/ml, respectively. The partially purified enzyme preparation was characterized by several criteria which were compared to the properties of the three major mammalian DNA polymerases, alpha, beta, and psi. On the basis of template-primer preference, effect of salt, inhibition by N-ethylmaleimide and Km for dTTP, the DNA polymerase activity from the membrane complex can be distinguished from the alpha and beta DNA polymerases. The elution profile from DNA cellulose revealed a minor peak (I) and a major peak (II) of DNA polymerase activity utilizing poly(A) -(dT)10 as template-primer in the presence of Mn2+ - Peak II from DNA cellulose, which contained about 90% of the total DNA polymerase activity eluted from the column, was 2-3 times as active with poly(A) - (dT)10 as template-primer in the presence of Mn2+ than with activated calf thymus DNA in the presence of Mg2+. On the other hand, peak I had a low ratio of poly(A) - (dT)10 to activated calf thymus DNA activity. DNA polymerase was also purified from the nuclear membrane fraction of uninfected KB cells by the same procedures as those used in enzyme purification from the adenovirus 2 DNA-nuclear membrane complex. A minor peak and a major peak of DNA polymerase activity utilizing poly(A) - (dT)10 as template primer in the presence of Mn2+ were again observed that eluted from DNA cellulose at the same KCl concentrations as peak I and II from adenovirus 2-infected cells. The enzymes of the nuclear membrane fraction of uninfected KB cells could not be differentiated from the enzymes of the adenovirus 2 DNA-nuclear membrane complex through any of the purification steps nor by their template specificities. These results indicate that the predominant enzyme in the adenovirus 2 DNA-nuclear membrane complex and in the KB cell nuclear membrane complex belongs to the class of DNA polymerase psi.

Strategy for efficient detection of respiratory viruses in pediatric clinical specimens

Direct immunofluorescence (IF) with a polyclonal respiratory syncytial virus (RSV)-specific antibody preparation was used for antigen detection during the 1982-1983 RSV season (155 specimens) and gave an overall sensitivity of 94% with 87% specificity compared with viral culture. Indirect IF was used in the 1983-1984 season (265 specimens) and exhibited sensitivity of 96% with specificity of 79%. During these two seasons, 42 of 224 (18.8%) specimens that were IF-negative for RSV grew viruses other than RSV. In the winter of 1984-1985, we screened 297 specimens for RSV by IF and 80 (27%) were positive. Forty-four (20%) of the IF-negative specimens were culture-positive for RSV(2) or other viruses(44). We conclude that, in the interest of cost reduction and expeditious detection of respiratory viruses, once a properly equipped laboratory has become thoroughly familiar with IF techniques, pediatric respiratory specimens can be screened for RSV by IF and only the IF-negative specimens need be inoculated into cell cultures for isolation of virus during the winter respiratory season.

Value of Rapid Diagnosis of Respiratory Syncytial Virus Infection on Management of Small Infants

Respiratory syncytial virus (RSV) is a common cause of infection in infancy and early childhood. A presumptive diagnosis of RSV infection can frequently be made on clinical grounds. Confirmation can be made by viral culture, which may take 3 to 7 days. Immunofluorescent assay (IFA) is a specific and sensitive test that can provide laboratory confirmation of RSV infection the same day. Rapid diagnosis of RSV infection may have implications regarding prevention of nosocomial spread of RSV, early initiation of anti-viral therapy, use of antibiotics, and duration of hospital stay. Data are presented regarding the use of RSV-IFA and its effect on patient management.

CHARACTERIZATION OF AN INFLUENZA VIRUS-RESISTANT MDBK CELL VARIANT

I. ABSTRACT We have established an influenza virus-resistant cell line which was obtained from cultures of MDBK cells that were initially persistently infected with the WSN strain of influenza A and subsequently lost the ability to produce virus. These variant cells are now almost completely refractory to influenza virus infection but retain the ability to support the growth of several other viruses including VSV, Sindbis virus, adenovirus and herpes simplex virus. All evidence indicates that this resistant cell variant was in fact derived from the completely permissive MDBK line. In view of these properties, we have termed these cells MDBK/IV to denote both their origin and their resistance to influenza virus. We have taken several approaches to investigate the biochemical block that these cells have established to specifically prevent the replication of influenza virus. To this end, we are investigating (i) influenza virus binding and biochemical properties of the plasma membrane, (ii) the production of influenza virus-specific positive and negative strand RNA, (iii) the ability of the cells to accurately and efficiently translate influenza virus specific mRNA, and (iv) the possibility that virus-specific information is integrated into the cell genome. Our experiments showed that MDBK/IV cells bound 80% less influenza virus than did the parental line and that this difference could not fully account for the lack of virus production. We have also detected the synthesis of virus-specific polyadenylated positive strand RNA in these cells and are currently characterizing this RNA and investigating its ability to be translated in vivo and in vitro.

Studies with Enzyme Complexes that Synthesize Adenovirus 2 DNA In Vitro

The genomes of the small (SV40 and polyoma) and medium-sized (adenovirus) mammalian DNA viruses lend themselves readily to biochemical and structural analysis. Thus, these viruses have been widely used as tools in model systems in much the samc manner that the small phages have been used for the understanding of chromosomal replication in bacteria. To this end, nuclear and subnuclear replication systems have been sought in order to simplify and further investigate the process of eukaryotic DNA replication. We have selected subnuclear complexes from adenovirus 2 (Ad2) infected KB cells on the basis of their ability to synthesize Ad2 DNA in vitro in a DNA polymerase reaction mixture. We have characterized the in vitro products and some of the proteins and enzymes associated with these complexes.

Adenovirus DNA: Transcription During Productive Infection, Integration in Transformed Cells, and Replication in Vitro

The human adenoviruses provide excellent model systems for studying the molecular biology of the mammalian cell, i.e., the mechanism of DNA replication and RNA transcription and translation, and for analyzing the integration and function of viral genes in transformed cells. Adenoviruses are icosahedral particles that are 80 nm in diameter, weigh 175 million daltons, and contain 9–10 polypeptides and 12–13% DNA (Green, 1970). Adenovirus genomes are linear, duplex DNA molecules of molecular weight 20–25 x 106 (Green, 1970). The interaction of the adenovirus genome with the cell can result in either (i) productive infection (usually human cells) in which thousands of virus particles are replicated and the cell is killed, or (ii) cell transformation (usually rodent cells) in which no virus is formed, but a portion of the viral genome is integrated into cellular DNA, and growth properties and macromolecular synthesis are controlled in an unknown way by information from viral genes.