Core Entrustable Professional Activities for Entry Into Residency: Curricular Gap or Unrealistic Expectations?
PERCEPTIONS OF RESIDENTS REGARDING CENTRAL LINE PLACEMENT BEFORE AND AFTER SIMULATION TRAINING [Meeting Abstract]
The ACGME Self-Study-An Opportunity, Not a Burden
A SECOND LOOK AT POST-INTERVIEW COMMUNICATION [Meeting Abstract]
Reconsideration of viral protein immunoblotting for differentiation of human herpes simplex viruses
Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are ubiquitous human pathogens that infect their hosts for life and reactivate to cause disease at or near the initial site of infection. As the incidence of genital HSV-1 infections increase, there is an increased demand for valid viral typing diagnostics. In this report, we reconsidered and developed a triple-phase immune-typing procedure that compares differences in electrophoretic mobilities of viral ICP4, ICP27, and VP22 proteins between HSV-1 and HSV-2 strains. We isolated and immunotyped 5 primary HSV-1 strains derived from orofacial, ocular, and genital areas along with 2 primary HSV-2 strains from the genital area. Advantages of this methodology include its general technical simplicity, sensitivity, and ability to definitively type HSV. It is anticipated that this methodology will be useful in distinguishing viruses obtained in clinical cultures.
Herpes simplex virus 2 modulates apoptosis and stimulates NF-kappaB nuclear translocation during infection in human epithelial HEp-2 cells
Virus-mediated apoptosis is well documented in various systems, including herpes simplex virus 1 (HSV-1). HSV-2 is closely related to HSV-1 but its apoptotic potential during infection has not been extensively scrutinized. We report that (i) HEp-2 cells infected with HSV-2(G) triggered apoptosis, assessed by apoptotic cellular morphologies, oligosomal DNA laddering, chromatin condensation, and death factor processing when a translational inhibitor (CHX) was added at 3 hpi. Thus, HSV-2 induced apoptosis but was unable to prevent the process from killing cells. (ii) Results from a time course of CHX addition experiment indicated that infected cell protein produced between 3 and 5 hpi, termed the apoptosis prevention window, are required for blocking virus-induced apoptosis. This corresponds to the same prevention time frame as reported for HSV-1. (iii) Importantly, CHX addition prior to 3 hpi led to less apoptosis than that at 3 hpi. This suggests that proteins produced immediately upon infection are needed for efficient apoptosis induction by HSV-2. This finding is different from that observed previously with HSV-1. (iv) Infected cell factors produced during the HSV-2(G) prevention window inhibited apoptosis induced by external TNFalpha plus cycloheximide treatment. (v) NF-kappaB translocated to nuclei and its presence in nuclei correlated with apoptosis prevention during HSV-2(G) infection. (vi) Finally, clinical HSV-2 isolates induced and prevented apoptosis in HEp-2 cells in a manner similar to that of laboratory strains. Thus, while laboratory and clinical HSV-2 strains are capable of modulating apoptosis in human HEp-2 cells, the mechanism of HSV-2 induction of apoptosis differs from that of HSV-1.
Herpes simplex virus: propagation, quantification, and storage
Herpes simplex virus (HSV) is the prototype of a family of large, enveloped, double-stranded DNA viruses, the Herpesviridae, which cause significant morbidity and mortality in humans. Productive replication of HSV in cells in culture results in definitive changes in cellular physiology and metabolism, ultimately leading to lysis. These definitive aspects of viral-host interactions enable diagnosis of HSV infections. In this unit, a series of methods are described for the propagation, quantification, and storage of HSV. Infectious center assays are used to measure the titers of HSV stocks. In addition, immunological methods are described for documenting the accumulation of viral polypeptides in infected whole cell extracts, as well as in situ using indirect immunofluorescence. These techniques should be beneficial to basic research virologists utilizing standard laboratory HSV strains, as well as clinical microbiologists interested in characterizing HSV isolated from patients.
Nuclear localizations of the herpes simplex virus type 1 tegument proteins VP13/14, vhs, and VP16 precede VP22-dependent microtubule reorganization and VP22 nuclear import
Herpes simplex virus type 1 (HSV-1) induces microtubule reorganization beginning at approximately 9 h postinfection (hpi), and this correlates with the nuclear localization of the tegument protein VP22. Thus, the active retention of this major virion component by cytoskeletal structures may function to regulate its subcellular localization (A. Kotsakis, L. E. Pomeranz, A. Blouin, and J. A. Blaho, J. Virol. 75:8697-8711, 2001). The goal of this study was to determine whether the subcellular localization patterns of other HSV-1 tegument proteins are similar to that observed with VP22. To address this, we performed a series of indirect immunofluorescence analyses using synchronously infected cells. We observed that tegument proteins VP13/14, vhs, and VP16 localized to the nucleus as early as 5 hpi and were concentrated in nuclei by 9 hpi, which differed from that seen with VP22. Microtubule reorganization was delayed during infection with HSV-1(RF177), a recombinant virus that does not produce full-length VP22. These infected cells did not begin to lose microtubule-organizing centers until 13 hpi. Repair of the unique long 49 (UL49) locus in HSV-1(RF177) yielded HSV-1(RF177R). Microtubule reorganization in HSV-1(RF177R)-infected cells occurred with the same kinetics as HSV-1(F). Acetylated tubulin remained unchanged during infection with either HSV-1(F) or HSV-1(RF177). Thus, while alpha-tubulin reorganized during infection, acetylated tubulin was stable, and the absence of full-length VP22 did not affect this stability. Our findings indicate that the nuclear localizations of tegument proteins VP13/14, VP16, and vhs do not appear to require HSV-1-induced microtubule reorganization. We conclude that full-length VP22 is needed for optimal microtubule reorganization during infection. This implies that VP22 mainly functions to reorganize microtubules later, rather than earlier, in infection. That acetylated tubulin does not undergo restructuring during VP22-dependent, virus-induced microtubule reorganization suggests that it plays a role in stabilizing the infected cells. Our results emphasize that VP22 likely plays a key role in cellular cytopathology during HSV-1 infection.