Jennifer L. Tipper

Higher Level of Replication Efficiency of 2009 (H1N1) Pandemic Influenza Virus than Those of Seasonal and Avian Strains: Kinetics from Epithelial Cell Culture and Computational Modeling

ABSTRACT The pathogenicity and transmission of influenza A viruses are likely determined in part by replication efficiency in human cells, which is the net effect of complex virus-host interactions. H5N1 avian, H1N1 seasonal, and H1N1 2009 pandemic influenza virus strains were compared by infecting human differentiated bronchial epithelial cells in air-liquid interface cultures at relatively low virus particle/cell ratios. Differential equation and computational models were used to characterize the in vitro kinetic behaviors of the three strains. The models were calibrated by fitting experimental data in order to estimate difficult-to-measure parameters. Both models found marked differences in the relative values of p, the virion production rate per cell, and R 0, an index of the spread of infection through the monolayer, with the values for the strains in the following rank order (from greatest to least): pandemic strain, followed by seasonal strain, followed by avian strain, as expected. In the differential equation model, which treats virus and cell populations as well mixed, R 0 and p varied proportionately for all 3 strains, consistent with a primary role for productivity. In the spatially explicit computational model, R 0 and p also varied proportionately except that R 0 derived for the pandemic strain was reduced, consistent with constrained viral spread imposed by multiple host defenses, including mucus and paracrine antiviral effects. This synergistic experimental-computational strategy provides relevant parameters for identifying and phenotyping potential pandemic strains.

Integrative "omic" analysis of experimental bacteremia identifies a metabolic signature that distinguishes human sepsis from systemic inflammatory response syndromes.

RATIONALE Sepsis is a leading cause of morbidity and mortality. Currently, early diagnosis and the progression of the disease are difficult to make. The integration of metabolomic and transcriptomic data in a primate model of sepsis may provide a novel molecular signature of clinical sepsis. OBJECTIVES To develop a biomarker panel to characterize sepsis in primates and ascertain its relevance to early diagnosis and progression of human sepsis. METHODS Intravenous inoculation of Macaca fascicularis with Escherichia coli produced mild to severe sepsis, lung injury, and death. Plasma samples were obtained before and after 1, 3, and 5 days of E. coli challenge and at the time of killing. At necropsy, blood, lung, kidney, and spleen samples were collected. An integrative analysis of the metabolomic and transcriptomic datasets was performed to identify a panel of sepsis biomarkers. MEASUREMENTS AND MAIN RESULTS The extent of E. coli invasion, respiratory distress, lethargy, and mortality was dependent on the bacterial dose. Metabolomic and transcriptomic changes characterized severe infections and death, and indicated impaired mitochondrial, peroxisomal, and liver functions. Analysis of the pulmonary transcriptome and plasma metabolome suggested impaired fatty acid catabolism regulated by peroxisome-proliferator activated receptor signaling. A representative four-metabolite model effectively diagnosed sepsis in primates (area under the curve, 0.966) and in two human sepsis cohorts (area under the curve, 0.78 and 0.82). CONCLUSIONS A model of sepsis based on reciprocal metabolomic and transcriptomic data was developed in primates and validated in two human patient cohorts. It is anticipated that the identified parameters will facilitate early diagnosis and management of sepsis.

Respiratory Syncytial Virus Impairs Macrophage IFN-α/β– and IFN-γ–Stimulated Transcription by Distinct Mechanisms

Macrophages are the primary lung phagocyte and are instrumental in maintenance of a sterile, noninflamed microenvironment. IFNs are produced in response to bacterial and viral infection, and activate the macrophage to efficiently counteract and remove pathogenic invaders. Respiratory syncytial virus (RSV) inhibits IFN-mediated signaling mechanisms in epithelial cells; however, the effects on IFN signaling in the macrophage are currently unknown. We investigated the effect of RSV infection on IFN-mediated signaling in macrophages. RSV infection inhibited IFN-beta- and IFN-gamma-activated transcriptional mechanisms in primary alveolar macrophages and macrophage cell lines, including the transactivation of important Nod-like receptor family genes, Nod1 and class II transactivator. RSV inhibited IFN-beta- and IFN-gamma-mediated transcriptional activation by two distinct mechanisms. RSV impaired IFN-beta-mediated signal transducer and activator of transcription (STAT)-1 phosphorylation through a mechanism that involves inhibition of tyrosine kinase 2 phosphorylation. In contrast, RSV-impaired transcriptional activation after IFN-gamma stimulation resulted from a reduction in the nuclear STAT1 interaction with the transcriptional coactivator, CBP, and was correlated with increased phosphorylation of STAT1beta, a dominant-negative STAT1 splice variant, in response to IFN-gamma. In support of this concept, overexpression of STAT1beta was sufficient to repress the IFN-gamma-mediated expression of class II transactivator. These results demonstrate that RSV inhibits IFN-mediated transcriptional activation in macrophages, and suggests that paramyxoviruses modulate an important regulatory mechanism that is critical in linking innate and adaptive immune mechanisms after infection.

Neurovirulence of H5N1 infection in ferrets is mediated by multifocal replication in distinct permissive neuronal cell regions.

Highly pathogenic avian influenza A (HPAI), subtype H5N1, remains an emergent threat to the human population. While respiratory disease is a hallmark of influenza infection, H5N1 has a high incidence of neurological sequelae in many animal species and sporadically in humans. We elucidate the temporal/spatial infection of H5N1 in the brain of ferrets following a low dose, intranasal infection of two HPAI strains of varying neurovirulence and lethality. A/Vietnam/1203/2004 (VN1203) induced mortality in 100% of infected ferrets while A/Hong Kong/483/1997 (HK483) induced lethality in only 20% of ferrets, with death occurring significantly later following infection. Neurological signs were prominent in VN1203 infection, but not HK483, with seizures observed three days post challenge and torticollis or paresis at later time points. VN1203 and HK483 replication kinetics were similar in primary differentiated ferret nasal turbinate cells, and similar viral titers were measured in the nasal turbinates of infected ferrets. Pulmonary viral titers were not different between strains and pathological findings in the lungs were similar in severity. VN1203 replicated to high titers in the olfactory bulb, cerebral cortex, and brain stem; whereas HK483 was not recovered in these tissues. VN1203 was identified adjacent to and within the olfactory nerve tract, and multifocal infection was observed throughout the frontal cortex and cerebrum. VN1203 was also detected throughout the cerebellum, specifically in Purkinje cells and regions that coordinate voluntary movements. These findings suggest the increased lethality of VN1203 in ferrets is due to increased replication in brain regions important in higher order function and explains the neurological signs observed during H5N1 neurovirulence.

Bik Mediates Caspase-Dependent Cleavage of Viral Proteins to Promote Influenza A Virus Infection

Influenza virus induces apoptosis in infected cells to promote viral replication by manipulating the host cell death signaling pathway. Although some Bcl-2 family proteins play a role in the replication of influenza A virus (IAV), the role of cell death pathways in the viral replication cycle is unclear. We investigated whether deficiency of the proapoptotic Bcl-2 family protein, Bik, plays a role in IAV replication. IAV replication was attenuated in mouse airway epithelial cells (MAECs) from bik(-/-) compared with bik(+/+) mice, as indicated by reduced viral titers. Bik(-/-) MAECs showed more stable transepithelial resistance after infection than did bik(+/+) MAECs, were less sensitive to infection-induced cell death, and released fewer copies of viral RNA. Similar results were obtained when Bik expression was suppressed in human airway epithelial cells (HAECs). Bik(+/+) mice lost weight drastically and died within 8 days of infection, whereas 75% of bik(-/-) mice survived infection for 14 days and were 10-fold less likely to die from infection compared with bik(+/+) mice. IAV infection activated caspase 3 in bik(+/+) but not in bik(-/-) MAECs. Cleavage of viral nucleoprotein and M2 proteins were inhibited in bik(-/-) MAECs and when caspase activation was inhibited in HAECs. Furthermore, Bik deficiency impaired cytoplasmic export of viral ribonucleoprotein. These studies suggest a link between Bik-mediated caspase activation and cleavage of viral proteins. Thus, inhibition of proapoptotic host factors such as Bik and downstream mediators of cell death may represent a novel approach to influenza treatment.

Influenza-mediated reduction of lung epithelial ion channel activity leads to dysregulated pulmonary fluid homeostasis

Severe influenza (IAV) infection can develop into bronchopneumonia and edema, leading to acquired respiratory distress syndrome (ARDS) and pathophysiology. Underlying causes for pulmonary edema and aberrant fluid regulation largely remain unknown, particularly regarding the role of viral-mediated mechanisms. Herein, we show that distinct IAV strains reduced the functions of the epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane regulator (CFTR) in murine respiratory and alveolar epithelia in vivo, as assessed by measurements of nasal potential differences and single-cell electrophysiology. Reduced ion channel activity was distinctly limited to virally infected cells in vivo and not bystander uninfected lung epithelium. Multiple lines of evidence indicated ENaC and CFTR dysfunction during the acute infection period; however, only CFTR dysfunction persisted beyond the infection period. ENaC, CFTR, and Na,K-ATPase activities and protein levels were also reduced in virally infected human airway epithelial cells. Reduced ENaC and CFTR led to changes in airway surface liquid morphology of human tracheobronchial cultures and airways of IAV-infected mice. Pharmacologic correction of CFTR function ameliorated IAV-induced physiologic changes. These changes are consistent with mucous stasis and pulmonary edema; furthermore, they indicate that repurposing therapeutic interventions correcting CFTR dysfunction may be efficacious for treatment of IAV lung pathophysiology.