Quantitative Stability of DNA after Extended Storage of Clinical Specimens as Determined by Real-Time PCR

Jerome, Keith R.; Huang, Meei Li; Wald, Anna; Selke, Stacy; Corey, Lawrence

doi:10.1128/jcm.40.7.2609-2611.2002

Cited by 134 publications

(105 citation statements)

References 7 publications

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“…7C). Taken together, these findings suggest that although HSV-inactivated CTL cannot be stimulated through the TCR, they a TaqMan chemistry used previously published primers and probes against HSV glycoprotein B (gB) gene and the cellular ␤-globin gene (41).…”

Section: Stimulating Hsv-inactivated Ctl Through the Tcr Or With A Phmentioning

confidence: 86%

“…DNA was quantified by spectrophotometry and used as template in TaqMan real-time PCR with primers and fluorogenic probes specific for the HSV glycoprotein B gene or the cellular ␤-globin gene. Reactions were done on an ABI 7700 sequencer (Applied Biosystems, Foster City, CA), and the sequences and concentrations of the primers and probes used were as previously described (41).…”

Section: Quantitative Real-time Pcr Using Taqman Chemistrymentioning

confidence: 99%

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CTL Are Inactivated by Herpes Simplex Virus-Infected Cells Expressing a Viral Protein Kinase

Sloan

Zahariadis

Posavad

et al. 2003

The Journal of Immunology

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Numerous cell-to-cell signals tightly regulate CTL function. Human fibroblasts infected with HSV type 1 or 2 can generate such a signal and inactivate human CTL. Inactivated CTL lose their ability to release cytotoxic granules and synthesize cytokines when triggered through the TCR. Inactivation requires cell-to-cell contact between CTL and HSV-infected cells. However, inactivated CTL are not infected with HSV. The inactivation of CTL is sustainable, as CTL function remains impaired when the CTL are removed from the HSV-infected cells. IL-2 treatment does not alter inactivation, and the inactivated phenotype is not transferable between CTL, distinguishing this phenotype from traditional anergy and T regulatory cell models. CTL inactivated by HSV-infected cells are not apoptotic, and the inactivated state can be overcome by phorbol ester stimulation, suggesting that inactivated CTL are viable and that the signaling block is specific to the TCR. HSV-infected cells require the expression of US3, a viral protein kinase, to transmit the inactivating signal. Elucidation of the molecular nature of this signaling pathway may allow targeted manipulation of CTL function.

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Section: Stimulating Hsv-inactivated Ctl Through the Tcr Or With A Phmentioning

confidence: 86%

Section: Quantitative Real-time Pcr Using Taqman Chemistrymentioning

confidence: 99%

CTL Are Inactivated by Herpes Simplex Virus-Infected Cells Expressing a Viral Protein Kinase

Sloan

Zahariadis

Posavad

et al. 2003

The Journal of Immunology

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“…Oral swabs were collected from mothers, newborns, and all other preschool age children in the home in a standardized manner at each visit. All the swabs were tested by PCR for CMV, EBV, HSV, HHV-6, and HHV-8 using published methods (40)(41)(42)(43)(44)(45). Two infants with persistent CMV shedding beginning in the first week of life were classified as having congenital infections and were excluded from subsequent analyses (11).…”

Section: Methodsmentioning

confidence: 99%

Transient Oral Human Cytomegalovirus Infections Indicate Inefficient Viral Spread from Very Few Initially Infected Cells

Mayer

Krantz

Swan

et al. 2017

J Virol

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Cytomegalovirus (CMV) is acquired by the oral route in children, and primary infection is associated with abundant mucosal replication, as well as the establishment of latency in myeloid cells that results in lifelong infection. The efficiency of primary CMV infection in humans following oral exposure, however, is unknown. We consistently detected self-limited, low-level oral CMV shedding events, which we termed transient CMV infections, in a prospective birth cohort of 30 highly exposed CMV-uninfected infants. We estimated the likelihood of transient oral CMV infections by comparing their observed frequency to that of established primary infections, characterized by persistent high-level shedding, viremia, and seroconversion. We developed mathematical models of viral dynamics upon initial oral CMV infection and validated them using clinical shedding data. Transient infections comprised 76 to 88% of oral CMV shedding events. For this high percentage of transient infections to occur, we identified two mathematical prerequisites: a very small number of initially infected oral cells (1 to 4) and low viral infectivity (Ͻ1.5 new cells infected/cell). These observations indicate that oral CMV infection in infants typically begins with a single virus that spreads inefficiently to neighboring cells. Thus, although the incidence of CMV infection is high during infancy, our data provide a mechanistic framework to explain why multiple CMV exposures are typically required before infection is successfully established. These findings imply that a sufficiently primed immune response could prevent CMV from establishing latent infection in humans and support the achievability of a prophylactic CMV vaccine.IMPORTANCE CMV infects the majority of the world's population and is a major cause of birth defects. Developing a vaccine to prevent CMV infection would be extremely valuable but would be facilitated by a better understanding of how natural human CMV infection is acquired. We studied CMV acquisition in infants and found that infections are usually brief and self-limited and are successfully established relatively rarely. Thus, although most people eventually acquire CMV infection, it usually requires numerous exposures. Our analyses indicate that this is because the virus is surprisingly inefficient, barely replicating well enough to spread to neighboring cells in the mouth. Greater knowledge of why CMV infection usually fails may provide insight into how to prevent it from succeeding. KEYWORDS Epstein-Barr virus, basic reproduction number, cytomegalovirus, founder population, herpes simplex virus, human herpesviruses, mathematical modeling, oral shedding, transient infection

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“…After genital examination, Dacron swabs moistened with sterile water were rubbed on the genital mucosa corresponding to each section of the grid, avoiding touching other sites. Each swab was placed in 1ϫ PCR buffer for HSV PCR (14). Participants were queried about genital symptoms and sexual activity during each visit.…”

Section: Participants and Proceduresmentioning

confidence: 99%

Virologic and Immunologic Evidence of Multifocal Genital Herpes Simplex Virus 2 Infection

Johnston

Zhu

Jing

et al. 2014

J Virol

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Genital herpes simplex virus (HSV) reactivation is thought to be anatomically and temporally localized, coincident with limited ganglionic infection. Short, subclinical shedding episodes are the most common form of HSV-2 reactivation, with host clearance mechanisms leading to rapid containment. The anatomic distribution of shedding episodes has not been characterized. To precisely define patterns of anatomic reactivation, we divided the genital tract into a 22-region grid and obtained daily swabs for 20 days from each region in 28 immunocompetent, HSV-2-seropositive persons. HSV was detected via PCR, and sites of asymptomatic HSV shedding were subjected to a biopsy procedure within 24 h. CD4؉ and CD8 ؉ T cells were quantified by immunofluorescence, and HSV-specific CD4 ؉ T cells were identified by intracellular cytokine cytometry. HSV was detected in 868 (7%) of 11,603 genital swabs at a median of 12 sites per person (range, 0 to 22). Bilateral HSV detection occurred on 83 (67%) days with shedding, and the median quantity of virus detected/day was associated with the number of sites positive (P < 0.001). In biopsy specimens of asymptomatic shedding sites, we found increased numbers of CD8 ؉ T cells compared to control tissue (27 versus 13 cells/mm 2 , P ‫؍‬ 0.03) and identified HSV-specific CD4 ؉ T cells. HSV reactivations emanate from widely separated anatomic regions of the genital tract and are associated with a localized cellular infiltrate that was demonstrated to be HSV specific in 3 cases. These data provide evidence that asymptomatic HSV-2 shedding contributes to chronic inflammation throughout the genital tract. IMPORTANCEThis detailed report of the anatomic patterns of genital HSV-2 shedding demonstrates that HSV-2 reactivation can be detected at multiple bilateral sites in the genital tract, suggesting that HSV establishes latency throughout the sacral ganglia. In addition, genital biopsy specimens from sites of asymptomatic HSV shedding have increased numbers of CD8 ؉ T cells compared to control tissue, and HSV-specific CD4 ؉ T cells are found at sites of asymptomatic shedding. These findings suggest that widespread asymptomatic genital HSV-2 shedding is associated with a targeted host immune response and contributes to chronic inflammation throughout the genital tract. H erpes simplex virus 2 (HSV-2) infects over 500 million people worldwide (1) and is the leading cause of genital ulcer disease (2) and increases the risk of HIV acquisition (3). Herpetic genital ulcers in the immunocompetent person are localized with respect to time and anatomic site (4), consistent with reactivation of virus from the ganglion innervating this region. However, most HSV genital shedding is subclinical, with rapid onset and clearance (5, 6). Such subclinical reactivation may be more widespread in the genital area (7), consistent with frequent leakage of virus into the genital skin and mucosa with localized host containment (8, 9). Mathematical models suggest that observed shedding patterns are the result of ove...

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Quantitative Stability of DNA after Extended Storage of Clinical Specimens as Determined by Real-Time PCR

Cited by 134 publications

References 7 publications

CTL Are Inactivated by Herpes Simplex Virus-Infected Cells Expressing a Viral Protein Kinase

CTL Are Inactivated by Herpes Simplex Virus-Infected Cells Expressing a Viral Protein Kinase

Transient Oral Human Cytomegalovirus Infections Indicate Inefficient Viral Spread from Very Few Initially Infected Cells

Virologic and Immunologic Evidence of Multifocal Genital Herpes Simplex Virus 2 Infection

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