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1.  Pathophysiologic and Transcriptomic Analyses of Viscerotropic Yellow Fever in a Rhesus Macaque Model 
Infection with yellow fever virus (YFV), an explosively replicating flavivirus, results in viral hemorrhagic disease characterized by cardiovascular shock and multi-organ failure. Unvaccinated populations experience 20 to 50% fatality. Few studies have examined the pathophysiological changes that occur in humans during YFV infection due to the sporadic nature and remote locations of outbreaks. Rhesus macaques are highly susceptible to YFV infection, providing a robust animal model to investigate host-pathogen interactions. In this study, we characterized disease progression as well as alterations in immune system homeostasis, cytokine production and gene expression in rhesus macaques infected with the virulent YFV strain DakH1279 (YFV-DakH1279). Following infection, YFV-DakH1279 replicated to high titers resulting in viscerotropic disease with ∼72% mortality. Data presented in this manuscript demonstrate for the first time that lethal YFV infection results in profound lymphopenia that precedes the hallmark changes in liver enzymes and that although tissue damage was noted in liver, kidneys, and lymphoid tissues, viral antigen was only detected in the liver. These observations suggest that additional tissue damage could be due to indirect effects of viral replication. Indeed, circulating levels of several cytokines peaked shortly before euthanasia. Our study also includes the first description of YFV-DakH1279-induced changes in gene expression within peripheral blood mononuclear cells 3 days post-infection prior to any clinical signs. These data show that infection with wild type YFV-DakH1279 or live-attenuated vaccine strain YFV-17D, resulted in 765 and 46 differentially expressed genes (DEGs), respectively. DEGs detected after YFV-17D infection were mostly associated with innate immunity, whereas YFV-DakH1279 infection resulted in dysregulation of genes associated with the development of immune response, ion metabolism, and apoptosis. Therefore, WT-YFV infection is associated with significant changes in gene expression that are detectable before the onset of clinical symptoms and may influence disease progression and outcome of infection.
Author Summary
Yellow fever virus causes ∼200,000 infections and 30,000 deaths annually in Africa and South America. Although this is an important human pathogen, the basis of yellow fever disease severity remains poorly understood. Rhesus macaques are susceptible to yellow fever and develop similar symptoms as severe as those observed in humans. In this study, we characterized disease progression in this model and observed a profound loss of lymphocytes that preceded the appearance of serum markers of virus-induced liver pathology. This change might provide an early indicator of fatal yellow fever. In addition, we also identified significant changes in gene expression in white blood cells that occur before any measurable disease symptoms and these genetic signatures may provide future targets for antiviral therapeutics and better diagnostics.
PMCID: PMC4238990  PMID: 25412185
2.  Age and Immune Status of Rhesus Macaques Impact Simian Varicella Virus Gene Expression in Sensory Ganglia 
Journal of Virology  2013;87(15):8294-8306.
Simian varicella virus (SVV) infection of rhesus macaques (RMs) recapitulates the hallmarks of varicella-zoster virus (VZV) infection of humans, including the establishment of latency within the sensory ganglia. Various factors, including age and immune fitness, influence the outcome of primary VZV infection, as well as reactivation resulting in herpes zoster (HZ). To increase our understanding of the role of lymphocyte subsets in the establishment of viral latency, we analyzed the latent SVV transcriptome in juvenile RMs depleted of CD4 T, CD8 T, or CD20 B lymphocytes during acute infection. We have previously shown that SVV latency in sensory ganglia of nondepleted juvenile RMs is associated with a limited transcriptional profile. In contrast, CD4 depletion during primary infection resulted in the failure to establish a characteristic latent viral transcription profile in sensory ganglia, where we detected 68 out of 69 SVV-encoded open reading frames (ORFs). CD-depleted RMs displayed a latent transcriptional profile that included additional viral transcripts within the core region of the genome not detected in control RMs. The latent transcriptome of CD20-depleted RMs was comparable to the latent transcription in the sensory ganglia of control RMs. Lastly, we investigated the impact of age on the establishment of SVV latency. SVV gene expression was more active in ganglia from two aged RMs than in ganglia from juvenile RMs, with 25 of 69 SVV transcripts detected. Therefore, immune fitness at the time of infection modulates the establishment and/or maintenance of SVV latency.
PMCID: PMC3719833  PMID: 23698305
3.  Simian Varicella Virus Gene Expression During Acute and Latent Infection of Rhesus Macaques 
Journal of Neurovirology  2011;17(6):600-612.
Varicella zoster virus (VZV) is a neurotropic α-herpesvirus that causes chickenpox during primary infection and establishes latency in sensory ganglia. Reactivation of VZV results in herpes zoster and other neurological complications. Our understanding of the VZV transcriptome during acute and latent infection in immune competent individuals remains incomplete. Infection of rhesus macaques with the homologous simian varicella virus (SVV) recapitulates the hallmarks of VZV infection. We therefore characterized the SVV transcriptome by quantitative real-time reverse transcriptase PCR (RT-qPCR) during acute infection in bronchial alveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMC), and during latency in sensory ganglia obtained from the same rhesus macaques. During acute infection, all known SVV open reading frames (ORFs) were detected and the most abundantly expressed ORFs are involved in virus replication and assembly such as the transcriptional activator ORF 63 and the structural proteins ORF 41 and ORF 49. In contrast, latent SVV gene expression is highly restricted. ORF 61, a viral transactivator and latency-associated transcript, is the most prevalent transcript detected in sensory ganglia. We also detected ORFs A, B, 4, 10, 63, 64, 65, 66 and 68 though significantly less frequently than ORF 61. This comprehensive analysis has revealed genes that potentially play a role in the establishment and/or maintenance of SVV latency.
PMCID: PMC3286235  PMID: 22052378
Herpesvirus; simian varicella virus; gene expression; latency; rhesus macaque
4.  A Flow Cytometry-Based Assay for Quantifying Non-Plaque Forming Strains of Yellow Fever Virus 
PLoS ONE  2012;7(9):e41707.
Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE) on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar) could not be measured by plaque assay (PA), focus-forming assay (FFA), or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6) and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA) to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine). Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses.
PMCID: PMC3446955  PMID: 23028428
5.  Accelerated immune senescence and reduced response to vaccination in ovariectomized female rhesus macaques 
Age  2010;33(3):275-289.
Aging is associated with a general dysregulation in immune function, commonly referred to as “immune senescence”. Several studies have shown that female sex steroids can modulate the immune response. However, the impact of menopause-associated loss of estrogen and progestins on immune senescence remains poorly understood. To help answer this question, we examined the effect of ovariectomy on T-cell homeostasis and function in adult and aged female rhesus macaques. Our data show that in adult female rhesus macaques, ovariectomy increased the frequency of naïve CD4 T cells. In contrast, ovariectomized (ovx) aged female rhesus macaques had increased frequency of terminally differentiated CD4 effector memory T cells and inflammatory cytokine-secreting memory T cells. Moreover, ovariectomy reduced the immune response (T-cell cytokine and IgG production) following vaccination with modified vaccinia ankara in both adult and aged female rhesus macaques compared to ovary-intact age-matched controls. Interestingly, hormone therapy (estradiol alone or in conjunction with progesterone) partially improved the T-cell response to vaccination in aged ovariectomized female rhesus macaques. These data suggest that the loss of ovarian steroids, notably estradiol and progesterone, may contribute to reduced immune function in post-menopausal women and that hormone therapy may improve immune response to vaccination in this growing segment of the population.
PMCID: PMC3168610  PMID: 20814751
Aging; Immune senescence; Ovariectomy; T cells; Vaccine; Estrogen; Progestin
6.  CD4 T Cell Immunity Is Critical for the Control of Simian Varicella Virus Infection in a Nonhuman Primate Model of VZV Infection 
PLoS Pathogens  2011;7(11):e1002367.
Primary infection with varicella zoster virus (VZV) results in varicella (more commonly known as chickenpox) after which VZV establishes latency in sensory ganglia. VZV can reactivate to cause herpes zoster (shingles), a debilitating disease that affects one million individuals in the US alone annually. Current vaccines against varicella (Varivax) and herpes zoster (Zostavax) are not 100% efficacious. Specifically, studies have shown that 1 dose of varivax can lead to breakthrough varicella, albeit rarely, in children and a 2-dose regimen is now recommended. Similarly, although Zostavax results in a 50% reduction in HZ cases, a significant number of recipients remain at risk. To design more efficacious vaccines, we need a better understanding of the immune response to VZV. Clinical observations suggest that T cell immunity plays a more critical role in the protection against VZV primary infection and reactivation. However, no studies to date have directly tested this hypothesis due to the scarcity of animal models that recapitulate the immune response to VZV. We have recently shown that SVV infection of rhesus macaques models the hallmarks of primary VZV infection in children. In this study, we used this model to experimentally determine the role of CD4, CD8 and B cell responses in the resolution of primary SVV infection in unvaccinated animals. Data presented in this manuscript show that while CD20 depletion leads to a significant delay and decrease in the antibody response to SVV, loss of B cells does not alter the severity of varicella or the kinetics/magnitude of the T cell response. Loss of CD8 T cells resulted in slightly higher viral loads and prolonged viremia. In contrast, CD4 depletion led to higher viral loads, prolonged viremia and disseminated varicella. CD4 depleted animals also had delayed and reduced antibody and CD8 T cell responses. These results are similar to clinical observations that children with agammaglobulinemia have uncomplicated varicella whereas children with T cell deficiencies are at increased risk of progressive varicella with significant complications. Moreover, our studies indicate that CD4 T cell responses to SVV play a more critical role than antibody or CD8 T cell responses in the control of primary SVV infection and suggest that one potential mechanism for enhancing the efficacy of VZV vaccines is by eliciting robust CD4 T cell responses.
Author Summary
Varicella zoster virus (VZV) causes chickenpox and establishes a life-long latent infection in humans. VZV can reactivate years later to cause shingles, a debilitating and painful disease. Vaccines against both chickenpox and shingles are available but not 100% efficacious. Two doses of the chickenpox vaccine are required to provide adequate protection and the shingles vaccine reduces the incidence of this disease by 51%. To improve these vaccines, we must identify the components of the immune system that are important for the control of VZV replication. However, the contribution of T versus B cell responses is unknown. Infection of rhesus macaques with simian varicella virus is a robust model of VZV infection. Here, we used this unique animal model to show for the first time that the absence of B cells does not alter disease severity and that the loss of CD8 T cells only results in a mild increase in disease severity. In sharp contrast, the lack of CD4 T cells leads to disseminated varicella. These data highlight the importance of CD4 T cells and suggest that novel vaccines that focus on engendering a more robust CD4 T cell response against VZV might provide better protection from chickenpox and shingles.
PMCID: PMC3213099  PMID: 22102814
7.  Immune senescence in aged nonhuman primates 
Experimental gerontology  2010;45(9):655-661.
Aging is accompanied by a general dysregulation in immune system function, commonly referred to as ‘immune senescence’. This progressive deterioration affects both innate and adaptive immunity, although accumulating evidence indicates that the adaptive arm of the immune system may exhibit more profound changes. Most of our current understanding of immune senescence stems from clinical and rodent studies. More recently, the use of nonhuman primates (NHPs) to investigate immune senescence and test interventions aimed at delaying/reversing age-related changes in immune function has dramatically increased. These studies have been greatly facilitated by several key advances in our understanding of the immune system of old-world monkeys, specifically the rhesus macaques. In this review we describe the hallmarks of immune senescence in this species and compare them to those described in humans. We also discuss the impact of immune senescence on the response to vaccination and the efficacy of immuno-restorative interventions investigated in this model system.
PMCID: PMC2926233  PMID: 20558288
9.  The Global Regulator Ler Is Necessary for Enteropathogenic Escherichia coli Colonization of Caenorhabditis elegans  
Infection and Immunity  2006;74(1):64-72.
Enteropathogenic Escherichia coli (EPEC) is an important cause of infant diarrhea in developing countries and is useful for general investigations of the bacterial infection process. However, the study of the molecular pathogenesis of EPEC has been hampered by the lack of genetically tractable, convenient animal models. We have therefore developed the use of the nematode Caenorhabditis elegans as a small animal model of infection for this diarrheal pathogen. We found that nematodes died faster on nematode growth medium in the presence of EPEC pathogens than in the presence of the laboratory control strain MG1655. Increased numbers of pathogens in the gut, determined by standard plate count assays and fluorescence microscopy using green fluorescent protein-expressing bacteria, correlated with killing. Deletion of the gene encoding the global regulator Ler severely reduced the ability of EPEC to colonize the nematode gut and could be complemented by providing the ler gene on a multicopy plasmid in trans. Neither the type III secretion system nor the type IV bundle-forming pilus was required for colonization. Combined, the similarities and distinct differences between EPEC infection of nematodes and that of humans offer a unique opportunity to study several stages of the infection process, namely, attachment, colonization, and persistence, in a genetically tractable, inexpensive, and convenient in vivo system.
PMCID: PMC1346621  PMID: 16368958
11.  Comparison of agreement between different measures of blood pressure in primary care and daytime ambulatory blood pressure 
BMJ : British Medical Journal  2002;325(7358):254.
To assess alternatives to measuring ambulatory pressure, which best predicts response to treatment and adverse outcome.
Three general practices in England.
Validation study.
Patients with newly diagnosed high or borderline high blood pressure; patients receiving treatment for hypertension but with poor control.
Main outcome measures
Overall agreement with ambulatory pressure; prediction of high ambulatory pressure (>135/85 mm Hg) and treatment thresholds.
Readings made by doctors were much higher than ambulatory systolic pressure (difference 18.9 mm Hg, 95% confidence interval 16.1 to 21.7), as were recent readings made in the clinic outside research settings (19.9 mm Hg,17.6 to 22.1). This applied equally to treated patients with poor control (doctor v ambulatory 21.4 mm Hg, 17.3 to 25.4). Doctors' and recent clinic readings ranked systolic pressure poorly compared with ambulatory pressure and other measurements (doctor r=0.46; clinic 0.47; repeated readings by nurse 0.60; repeated self measurement 0.73; home readings 0.75) and were not specific at predicting high blood pressure (doctor 26%; recent clinic 15%; nurse 72%; patient in surgery 81%; home 60%), with poor likelihood ratios for a positive test (doctor 1.2; clinic 1.1; nurse 2.1, patient in surgery 4.7; home 2.2). Nor were doctor or recent clinic measures specific in predicting treatment thresholds.
The “white coat” effect is important in diagnosing and assessing control of hypertension in primary care and is not a research artefact. If ambulatory or home measurements are not available, repeated measurements by the nurse or patient should result in considerably less unnecessary monitoring, initiation, or changing of treatment. It is time to stop using high blood pressure readings documented by general practitioners to make treatment decisions.
What is already known on this topicProspective studies indicate that ambulatory blood pressure is a much better predictor of adverse outcome and response to treatment than readings made by a doctorPreliminary evidence suggests that measurements by doctors are likely to be higher than those made by nurses, technicians, or patientsNo study has compared all the available measures in a typical primary care setting with ambulatory blood pressure in patients with newly diagnosed and established hypertensionWhat this study addsThe white coat effect associated with measurements by doctors is not an artefact of research studies; it applies equally in primary care and for both initial diagnosis and assessment of controlIf ambulatory measurement is not possible, repeated measurement by a nurse or by the patient will result in much less unnecessary treatment or change in treatment for high blood pressure
PMCID: PMC117640  PMID: 12153923

Results 1-11 (11)