Experimental Infection of Syrian Hamsters With Aerosolized Nipah Virus.

Background: Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that can cause severe respiratory illness and encephalitis in humans. Transmission occurs through consumption of NiV-contaminated foods, and contact with NiV-infected animals or human body fluids. However, it is unclear whether aerosols derived from aforesaid sources or others also contribute to transmission, and current knowledge on NiV-induced pathogenicity after small-particle aerosol exposure is still limited. Methods: Infectivity, pathogenicity, and real-time dissemination of aerosolized NiV in Syrian hamsters was evaluated using NiV-Malaysia (NiV-M) and/or its recombinant expressing firefly luciferase (rNiV-FlucNP). Results: Both viruses had an equivalent pathogenicity in hamsters, which developed respiratory and neurological symptoms of disease, similar to using intranasal route, with no direct correlations to the dose. We showed that virus replication was predominantly initiated in the lower respiratory tract and, although delayed, also intensely in the oronasal cavity and possibly the brain, with gradual increase of signal in these regions until at least day 5-6 postinfection. Conclusion: Hamsters infected with small-particle aerosolized NiV undergo similar clinical manifestations of the disease as previously described using liquid inoculum, and exhibit histopathological lesions consistent with NiV patient reports. NiV droplets could therefore play a role in transmission by close contact.

Optimized P2A for reporter gene insertion into Nipah virus results in efficient ribosomal skipping and wild-type lethality.

Incorporation of reporter genes within virus genomes is an indispensable tool for interrogation of virus biology and pathogenesis. In previous work, we incorporated a fluorophore into a viral ORF by attaching it to the viral gene via a P2A ribosomal skipping sequence. This recombinant Nipah virus, however, was attenuated in vitro relative to WT virus. In this work, we determined that inefficient ribosomal skipping was a major contributing factor to this attenuation. Inserting a GSG linker before the P2A sequence resulted in essentially complete skipping, significantly improved growth in vitro, and WT lethality in vivo. To the best of our knowledge, this represents the first time a recombinant virus of Mononegavirales with integration of a reporter into a viral ORF has been compared with the WT virus in vivo. Incorporating the GSG linker for improved skipping efficiency whenever functionally important is a critical consideration for recombinant virus design.

Rift Valley Fever Virus MP-12 Vaccine Is Fully Attenuated by a Combination of Partial Attenuations in the S, M, and L Segments.

UNLABELLED: Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and characterized by a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), which has a tripartite negative-stranded RNA genome (consisting of the S, M, and L segments). Further spread of RVF into countries where the disease is not endemic may affect the economy and public health, and vaccination is an effective approach to prevent the spread of RVFV. A live-attenuated MP-12 vaccine is one of the best-characterized RVF vaccines for safety and efficacy and is currently conditionally licensed for use for veterinary purposes in the United States. Meanwhile, as of 2015, no other RVF vaccine has been conditionally or fully licensed for use in the United States. The MP-12 strain is derived from wild-type pathogenic strain ZH548, and its genome encodes 23 mutations in the three genome segments. However, the mechanism of MP-12 attenuation remains unknown. We characterized the attenuation of wild-type pathogenic strain ZH501 carrying a mutation(s) of the MP-12 S, M, or L segment in a mouse model. Our results indicated that MP-12 is attenuated by the mutations in the S, M, and L segments, while the mutations in the M and L segments confer stronger attenuation than those in the S segment. We identified a combination of 3 amino acid changes, Y259H (Gn), R1182G (Gc), and R1029K (L), that was sufficient to attenuate ZH501. However, strain MP-12 with reversion mutations at those 3 sites was still highly attenuated. Our results indicate that MP-12 attenuation is supported by a combination of multiple partial attenuation mutations and a single reversion mutation is less likely to cause a reversion to virulence of the MP-12 vaccine. IMPORTANCE: Rift Valley fever (RVF) is a mosquito-transmitted viral disease that is endemic to Africa and that has the potential to spread into other countries. Vaccination is considered an effective way to prevent the disease, and the only available veterinary RVF vaccine in the United States is a live-attenuated MP-12 vaccine, which is conditionally licensed. Strain MP-12 is different from its parental pathogenic RVFV strain, strain ZH548, because of the presence of 23 mutations. This study determined the role of individual mutations in the attenuation of the MP-12 strain. We found that full attenuation of MP-12 occurs by a combination of multiple mutations. Our findings indicate that a single reversion mutation will less likely cause a major reversion to virulence of the MP-12 vaccine.

Efficient reverse genetics reveals genetic determinants of budding and fusogenic differences between Nipah and Hendra viruses and enables real-time monitoring of viral spread in small animal models of henipavirus infection.

UNLABELLED: Nipah virus (NiV) and Hendra virus (HeV) are closely related henipaviruses of the Paramyxovirinae. Spillover from their fruit bat reservoirs can cause severe disease in humans and livestock. Despite their high sequence similarity, NiV and HeV exhibit apparent differences in receptor and tissue tropism, envelope-mediated fusogenicity, replicative fitness, and other pathophysiologic manifestations. To investigate the molecular basis for these differences, we first established a highly efficient reverse genetics system that increased rescue titers by ≥3 log units, which offset the difficulty of generating multiple recombinants under constraining biosafety level 4 (BSL-4) conditions. We then replaced, singly and in combination, the matrix (M), fusion (F), and attachment glycoprotein (G) genes in mCherry-expressing recombinant NiV (rNiV) with their HeV counterparts. These chimeric but isogenic rNiVs replicated well in primary human endothelial and neuronal cells, indicating efficient heterotypic complementation. The determinants of budding efficiency, fusogenicity, and replicative fitness were dissociable: HeV-M budded more efficiently than NiV-M, accounting for the higher replicative titers of HeV-M-bearing chimeras at early times, while the enhanced fusogenicity of NiV-G-bearing chimeras did not correlate with increased replicative fitness. Furthermore, to facilitate spatiotemporal studies on henipavirus pathogenesis, we generated a firefly luciferase-expressing NiV and monitored virus replication and spread in infected interferon alpha/beta receptor knockout mice via bioluminescence imaging. While intraperitoneal inoculation resulted in neuroinvasion following systemic spread and replication in the respiratory tract, intranasal inoculation resulted in confined spread to regions corresponding to olfactory bulbs and salivary glands before subsequent neuroinvasion. This optimized henipavirus reverse genetics system will facilitate future investigations into the growing numbers of novel henipavirus-like viruses. IMPORTANCE: Nipah virus (NiV) and Hendra virus (HeV) are recently emergent zoonotic and highly lethal pathogens with pandemic potential. Although differences have been observed between NiV and HeV replication and pathogenesis, the molecular basis for these differences has not been examined. In this study, we established a highly efficient system to reverse engineer changes into replication-competent NiV and HeV, which facilitated the generation of reporter-expressing viruses and recombinant NiV-HeV chimeras with substitutions in the genes responsible for viral exit (the M gene, critical for assembly and budding) and viral entry (the G [attachment] and F [fusion] genes). These chimeras revealed differences in the budding and fusogenic properties of the M and G proteins, respectively, which help explain previously observed differences between NiV and HeV. Finally, to facilitate future in vivo studies, we monitored the replication and spread of a bioluminescent reporter-expressing NiV in susceptible mice; this is the first time such in vivo imaging has been performed under BSL-4 conditions.

Cytokine response in mouse bone marrow derived macrophages after infection with pathogenic and non-pathogenic Rift Valley fever virus.

Rift Valley fever virus (RVFV) is the most pathogenic member of the genus Phlebovirus within the family Bunyaviridae, and can cause severe disease in humans and livestock. Until recently, limited information has been published on the cellular host response elicited by RVFV, particularly in macrophages and dendritic cells, which play critical roles in stimulating adaptive and innate immune responses to viral infection. In an effort to define the initial response of host immunomodulatory cells to infection, primary mouse bone marrow derived macrophages (BMDM) were infected with the pathogenic RVFV strain ZH501, or attenuated strains MP-12 or MP-12 based Clone13 type (rMP12-C13 type), and cytokine secretion profiles examined. The secretion of T helper (Th)1-associated antiviral cytokines, chemokines and various interleukins increased rapidly after infection with the attenuated rMP12-C13 type RVFV, which lacks a functional NSs virulence gene. In comparison, infection with live-attenuated MP-12 encoding a functional NSs gene appeared to cause a delayed immune response, while pathogenic ZH501 ablates the immune response almost entirely. These data demonstrate that NSs can inhibit components of the BMDM antiviral response and supports previous work indicating that NSs can specifically regulate the type I interferon response in macrophages. Furthermore, our data demonstrate that genetic differences between ZH501 and MP-12 reduce the ability of MP-12 to inhibit antiviral signalling and subsequently reduce virulence in BMDM, demonstrating that viral components other than NSs play a critical role in regulating the host response to RVFV infection.

Cardiovascular Critical Care: A Perceived Deficiency Among U.S. Trainees.

OBJECTIVE: Acute and chronic cardiovascular comorbidities are common among critically ill individuals. It is unclear if current critical care fellowship trainees feel adequately prepared to manage these conditions. DESIGN: Prospective, cross-sectional survey. PATIENTS OR SUBJECTS: Trainees enrolled in U.S. critical care training programs. SETTING: Accredited pulmonary/critical care, surgery/critical care, anesthesiology/critical care, and stand-alone critical care training programs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A 19-item survey assessing trainee confidence in the management of cardiac critical illness and the performance of cardiac-specific critical care interventions was constructed using Accreditation Council for Graduate Medical Education recommendations as a reference. After validation, the survey was electronically sent to all training programs for dissemination to their trainees. Confidence scores were measured on a Likert scale from 1 to 5. A total of 134 completed surveys were analyzed. Overall, respondents reported lower confidence in managing cardiovascular compared with noncardiovascular diseases in the ICU (4.0 vs 4.6 out of 5). Likewise, they reported lower perceived competence in performing cardiovascular procedures specific to the ICU (2.9 vs 4.5 out of 5). The majority (88%) of those surveyed felt that they would benefit from increased didactic and clinical experience in the management of cardiovascular critical illness. CONCLUSIONS: Current critical care fellows may be unprepared to deal with the increasing prevalence of cardiovascular illness in the ICU. This potential educational gap warrants timely attention to ensure that future graduates have the requisite skills necessary to manage these critically ill patients and presents a unique opportunity to develop multidisciplinary partnerships for enhancing training.

Bilateral entry and release of Middle East respiratory syndrome coronavirus induces profound apoptosis of human bronchial epithelial cells.

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) infects human bronchial epithelial Calu-3 cells. Unlike severe acute respiratory syndrome (SARS)-CoV, which exclusively infects and releases through the apical route, this virus can do so through either side of polarized Calu-3 cells. Infection results in profound apoptosis within 24 h irrespective of its production of titers that are lower than those of SARS-CoV. Together, our results provide new insights into the dissemination and pathogenesis of MERS-CoV and may indicate that the virus differs markedly from SARS-CoV.

The dominant-negative inhibition of double-stranded RNA-dependent protein kinase PKR increases the efficacy of Rift Valley fever virus MP-12 vaccine.

Rift Valley fever virus (RVFV), belonging to the genus Phlebovirus, family Bunyaviridae, is endemic to sub-Saharan Africa and causes a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. MP-12 is the only RVFV strain excluded from the select-agent rule and handled at a biosafety level 2 (BSL2) laboratory. MP-12 encodes a functional major virulence factor, the NSs protein, which contributes to its residual virulence in pregnant ewes. We found that 100% of mice subcutaneously vaccinated with recombinant MP-12 (rMP12)-murine PKRN167 (mPKRN167), which encodes a dominant-negative form of mouse double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in place of NSs, were protected from wild-type (wt) RVFV challenge, while 72% of mice vaccinated with MP-12 were protected after challenge. rMP12-mPKRN167 induced alpha interferon (IFN-α) in sera, accumulated RVFV antigens in dendritic cells at the local draining lymph nodes, and developed high levels of neutralizing antibodies, while parental MP-12 induced neither IFN-α nor viral-antigen accumulation at the draining lymph node yet induced a high level of neutralizing antibodies. The present study suggests that the expression of a dominant-negative PKR increases the immunogenicity and efficacy of live-attenuated RVFV vaccine, which will lead to rational design of safe and highly immunogenic RVFV vaccines for livestock and humans.

Predictors of permanent pacemaker requirement after cardiac surgery for infective endocarditis.

BACKGROUND: Infectious endocarditis is often complicated by conduction abnormalities at the time of presentation. Cardiac surgery is the treatment of choice for many infectious endocarditis patients, but carries an additional risk of persistent postoperative conduction abnormality. We sought to define the incidence and clinical predictors of significant postoperative conduction abnormalities necessitating permanent pacemaker implantation after cardiac surgery for infectious endocarditis. METHODS: All consecutive patients with infectious endocarditis who were surgically treated at Cleveland Clinic from 2007 to 2013 were identified using the Cleveland Clinic Infective Endocarditis Registry and the Cardiovascular Information Registry. Patients with a pre-existing cardiac implantable electronic device were excluded. The primary outcome was the need for permanent pacemaker placement postoperatively for atrioventricular block. Regression analysis was performed to identify risk factors for permanent pacemaker requirement. RESULTS: Among 444 infectious endocarditis patients who underwent cardiac surgery for infectious endocarditis, 57 (13%) required postoperative permanent pacemaker for atrioventricular block. Multivariable analysis identified that prolongation in preoperative PR and QRS intervals, Staphylococcus aureus as the infectious endocarditis organism, the presence of intracardiac abscess, tricuspid valve involvement, and prior valvular surgery independently predicted postoperative permanent pacemaker placement. The developed model exhibited excellent predictive ability (c-statistic 0.88) and calibration. CONCLUSION: Infectious endocarditis cardiac surgery patients often require a postoperative permanent pacemaker. Preoperative conduction abnormality, S. aureus infection, abscess, tricuspid valve involvement, and prior valvular surgery are strong predictors of postoperative permanent pacemaker placement.

Severe acute respiratory syndrome (SARS) coronavirus-induced lung epithelial cytokines exacerbate SARS pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells.

Severe acute respiratory syndrome (SARS), which is caused by a novel coronavirus (CoV), is a highly communicable disease with the lungs as the major pathological target. Although SARS likely stems from overexuberant host inflammatory responses, the exact mechanism leading to the detrimental outcome in patients remains unknown. Pulmonary macrophages (Mphi), airway epithelium, and dendritic cells (DC) are key cellular elements of the host innate defenses against respiratory infections. While pulmonary Mphi are situated at the luminal epithelial surface, DC reside abundantly underneath the epithelium. Such strategic locations of these cells within the airways make it relevant to investigate their likely impact on SARS pathogenesis subsequent to their interaction with infected lung epithelial cells. To study this, we established highly polarized human lung epithelial Calu-3 cells by using the Transwell culture system. Here we report that supernatants harvested from the apical and basolateral domains of infected Calu-3 cells are potent in modulating the intrinsic functions of Mphi and DC, respectively. They prompted the production of cytokines by both Mphi and DC and selectively induced CD40 and CD86 expression only on DC. However, they compromised the abilities of the DC and Mphi in priming naïve T cells and phagocytosis, respectively. We also identified interleukin-6 (IL-6) and IL-8 as key SARS-CoV-induced epithelial cytokines capable of inhibiting the T-cell-priming ability of DC. Taken together, our results provide insights into the molecular and cellular bases of the host antiviral innate immunity within the lungs that eventually lead to an exacerbated inflammatory cascades and severe tissue damage in SARS patients.

Differential virological and immunological outcome of severe acute respiratory syndrome coronavirus infection in susceptible and resistant transgenic mice expressing human angiotensin-converting enzyme 2.

We previously reported that transgenic (Tg) mice expressing human angiotensin-converting enzyme 2 (hACE2), the receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), were highly susceptible to SARS-CoV infection, which resulted in the development of disease of various severity and even death in some lineages. In this study, we further characterized and compared the pathogeneses of SARS-CoV infection in two of the most stable Tg lineages, AC70 and AC22, representing those susceptible and resistant to the lethal SARS-CoV infection, respectively. The kinetics of virus replication and the inflammatory responses within the lungs and brains, as well as the clinical and pathological outcomes, were assessed in each lineage. In addition, we generated information on lymphocyte subsets and mitogen-mediated proliferation of splenocytes. We found that while both lineages were permissive to SARS-CoV infection, causing elevated secretion of many inflammatory mediators within the lungs and brains, viral infection appeared to be more intense in AC70 than in AC22 mice, especially in the brain. Moreover, such infection was accompanied by a more profound immune suppression in the former, as evidenced by the extensive loss of T cells, compromised responses to concanavalin A stimulation, and absence of inflammatory infiltrates within the brain. We also found that CD8(+) T cells were partially effective in attenuating the pathogenesis of SARS-CoV infection in lethality-resistant AC22 mice. Collectively, our data revealed a more intense viral infection and immunosuppression in AC70 mice than in AC22 mice, thereby providing us with an immunopathogenic basis for the fatal outcome of SARS-CoV infection in the AC70 mice.

Niemann-Pick C1 Heterogeneity of Bat Cells Controls Filovirus Tropism.

Fruit bats are suspected to be natural hosts of filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV). Interestingly, however, previous studies suggest that these viruses have different tropisms depending on the bat species. Here, we show a molecular basis underlying the host-range restriction of filoviruses. We find that bat-derived cell lines FBKT1 and ZFBK13-76E show preferential susceptibility to EBOV and MARV, respectively, whereas the other bat cell lines tested are similarly infected with both viruses. In FBKT1 and ZFBK13-76E, unique amino acid (aa) sequences are found in the Niemann-Pick C1 (NPC1) protein, one of the cellular receptors interacting with the filovirus glycoprotein (GP). These aa residues, as well as a few aa differences between EBOV and MARV GPs, are crucial for the differential susceptibility to filoviruses. Taken together, our findings indicate that the heterogeneity of bat NPC1 orthologs is an important factor controlling filovirus species-specific host tropism.

Time-resolved EPR investigation of potential model systems for acrylate polymer main chain radicals based on esters of Kemp's tri-acid.

Methyl esters of Kemp's tri-acid and cyclohexanetricarboxylic acid are structurally similar to acrylate polymers, having the same functionalities and stereoregularities as poly(methylmethacrylate) and poly(methylacrylate), respectively. The photochemistry and free radicals from these model systems have been studied using time-resolved electron paramagnetic resonance spectroscopy with laser flash photolysis at 248 nm. Chemically induced electron spin polarization from the triplet mechanism (net emission) is observed. Well-resolved spectra are obtained at all temperatures for the model system radicals, which are determined to be in the slow motion condition, that is, there is no interconversion of chair conformations. The temperature dependence of the spectra is minimal; some hyperfine lines shift as the temperature increases, but without much broadening. Density functional theory calculations are presented and discussed in support of the experimental data.

Treatment of Diffuse Right Coronary Aneurysmal Disease With Standard Drug-Eluting Stent Scaffolding of Polytetrafluoroethylene-Covered Stents.

Demonstration of a novel technique to safely exclude long aneurysmal segments that require multiple covered stents in a single segment. In addition, these images and video series demonstrate the usefulness of balloon-assisted GuideLiner tracking to pass bulky equipment to the distal segments of heavily diseased vessels.

Randomized Evaluation of Heart Failure With Preserved Ejection Fraction Patients With Acute Heart Failure and Dopamine: The ROPA-DOP Trial.

OBJECTIVES: This study sought to compare a continuous infusion diuretic strategy versus an intermittent bolus diuretic strategy, with the addition of low-dose dopamine (3 µg/kg/min) in the treatment of hospitalized patients with heart failure with preserved ejection fraction (HFpEF). BACKGROUND: HFpEF patients are susceptible to development of worsening renal function (WRF) when hospitalized with acute heart failure; however, inpatient treatment strategies to achieve safe and effective diuresis in HFpEF patients have not been studied to date. METHODS: In a prospective, randomized, clinical trial, 90 HFpEF patients hospitalized with acute heart failure were randomized within 24 h of admission to 1 of 4 treatments: 1) intravenous bolus furosemide administered every 12 h; 2) continuous infusion furosemide; 3) intermittent bolus furosemide with low-dose dopamine; and 4) continuous infusion furosemide with low-dose dopamine. The primary endpoint was percent change in creatinine from baseline to 72 h. Linear and logistic regression analyses with tests for interactions between diuretic and dopamine strategies were performed. RESULTS: Compared to intermittent bolus strategy, the continuous infusion strategy was associated with higher percent increase in creatinine (continuous infusion: 16.01%; 95% confidence interval [CI]: 8.58% to 23.45% vs. intermittent bolus: 4.62%; 95% CI: -1.15% to 10.39%; p = 0.02). Low-dose dopamine had no significant effect on percent change in creatinine (low-dose dopamine: 12.79%; 95% CI: 5.66% to 19.92%, vs. no-dopamine: 8.03%; 95% CI: 1.44% to 14.62%; p = 0.33). Continuous infusion was also associated with greater risk of WRF than intermittent bolus (odds ratio [OR]: 4.32; 95% CI: 1.26 to 14.74; p = 0.02); no differences in WRF risk were seen with low-dose dopamine. No significant interaction was seen between diuretic strategy and low-dose dopamine (p > 0.10). CONCLUSIONS: In HFpEF patients hospitalized with acute heart failure, low-dose dopamine had no significant impact on renal function, and a continuous infusion diuretic strategy was associated with renal impairment. (Diuretics and Dopamine in Heart Failure With Preserved Ejection Fraction [ROPA-DOP]; NCT01901809).

N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN.

Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)-any amino acid (X)-serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: "Gc-large" and "Gc-small", and N1077 was responsible for "Gc-large" band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN.

Attenuation of pathogenic Rift Valley fever virus strain through the chimeric S-segment encoding sandfly fever phlebovirus NSs or a dominant-negative PKR.

Rift Valley fever is a mosquito-borne zoonotic disease affecting ruminants and humans. Rift Valley fever virus (RVFV: family Bunyaviridae, genus Phlebovirus) causes abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or retinitis in humans. The live-attenuated MP-12 vaccine is conditionally licensed for veterinary use in the US. However, this vaccine lacks a marker for the differentiation of vaccinated from infected animals (DIVA). NSs gene is dispensable for RVFV replication, and thus, rMP-12 strains lacking NSs gene is applicable to monitor vaccinated animals. However, the immunogenicity of MP-12 lacking NSs was not as high as parental MP-12. Thus, chimeric MP-12 strains encoding NSs from either Toscana virus (TOSV), sandfly fever Sicilian virus (SFSV) or Punta Toro virus Adames strain (PTA) were characterized previously. Although chimeric MP-12 strains are highly immunogenic, the attenuation through the S-segment remains unknown. Using pathogenic ZH501 strain, we aimed to demonstrate the attenuation of ZH501 strain through chimeric S-segment encoding either the NSs of TOSV, SFSV, PTA, or Punta Toro virus Balliet strain (PTB). In addition, we characterized rZH501 encoding a human dominant-negative PKR (PKRΔE7), which also enhances the immunogenicity of MP-12. Study done on mice revealed that attenuation of rZH501 occurred through the S-segment encoding either PKRΔE7 or SFSV NSs. However, rZH501 encoding either TOSV, PTA, or PTB NSs in the S-segment uniformly caused lethal encephalitis. Our results indicated that the S-segments encoding PKRΔE7 or SFSV NSs are attenuated and thus applicable toward next generation MP-12 vaccine candidates that encode a DIVA marker.

Outcomes and worsening renal function in patients hospitalized with heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) has been described as a disease of elderly subjects with female predominance and hypertension. Our clinical experience suggests patients with HFpEF from an urban population are far more heterogenous, with greater co-morbidities and significant inhospital morbidity. There are limited data on the hospitalization course and outcomes in acute decompensated HFpEF. Hospitalizations for acute heart failure at our institution from July 2011 to June 2012 were identified by International Classification of Diseases, Ninth Revision, codes and physician review for left ventricular ejection fraction ≥50% and were reviewed for patient characteristics and clinical outcomes. Worsening renal function (WRF) was defined as creatinine increase of ≥0.3 mg/dl by 72 hours after admission. Hospital readmission and mortality data were captured from electronic medical records and the Social Security Death Index. Of 434 heart failure admissions, 206 patients (47%) with HFpEF were identified. WRF developed in 40%, the highest reported in HFpEF to date, and was associated with higher blood pressure and lower volume of diuresis. Compared to previous reports, hospitalized patients with HFpEF were younger (mean age 63.2 ± 13.6 years), predominantly black (74%), and had more frequent and severe co-morbidities: hypertension (89%), diabetes (56%), and chronic kidney disease (55%). There were no significant differences in 1- and 12-month outcomes by gender, race, or WRF. In conclusion, we found hospitalized patients with HFpEF from an urban population develop a high rate of WRF are younger than previous cohorts, often black, and have greater co-morbidities than previously described.

Endothelial cell permeability and adherens junction disruption induced by junín virus infection.

Junín virus (JUNV) is endemic to the fertile Pampas of Argentina, maintained in nature by the rodent host Calomys musculinus, and the causative agent of Argentine hemorrhagic fever (AHF), which is characterized by vascular dysfunction and fluid distribution abnormalities. Clinical as well as experimental studies implicate involvement of the endothelium in the pathogenesis of AHF, although little is known of its role. JUNV has been shown to result in productive infection of endothelial cells (ECs) in vitro with no visible cytopathic effects. In this study, we show that direct JUNV infection of primary human ECs results in increased vascular permeability as measured by electric cell substrate impedance sensing and transwell permeability assays. We also show that EC adherens junctions are disrupted during virus infection, which may provide insight into the role of the endothelium in the pathogenesis of AHF and possibly, other viral hemorrhagic fevers.

Favipiravir (T-705) inhibits Junín virus infection and reduces mortality in a guinea pig model of Argentine hemorrhagic fever.

BACKGROUND: Junín virus (JUNV), the etiologic agent of Argentine hemorrhagic fever (AHF), is classified by the NIAID and CDC as a Category A priority pathogen. Presently, antiviral therapy for AHF is limited to immune plasma, which is readily available only in the endemic regions of Argentina. T-705 (favipiravir) is a broadly active small molecule RNA-dependent RNA polymerase inhibitor presently in clinical evaluation for the treatment of influenza. We have previously reported on the in vitro activity of favipiravir against several strains of JUNV and other pathogenic New World arenaviruses. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the efficacy of favipiravir in vivo, guinea pigs were challenged with the pathogenic Romero strain of JUNV, and then treated twice daily for two weeks with oral or intraperitoneal (i.p.) favipiravir (300 mg/kg/day) starting 1-2 days post-infection. Although only 20% of animals treated orally with favipiravir survived the lethal challenge dose, those that succumbed survived considerably longer than guinea pigs treated with placebo. Consistent with pharmacokinetic analysis that showed greater plasma levels of favipiravir in animals dosed by i.p. injection, i.p. treatment resulted in a substantially higher level of protection (78% survival). Survival in guinea pigs treated with ribavirin was in the range of 33-40%. Favipiravir treatment resulted in undetectable levels of serum and tissue viral titers and prevented the prominent thrombocytopenia and leucopenia observed in placebo-treated animals during the acute phase of infection. CONCLUSIONS/SIGNIFICANCE: The remarkable protection afforded by i.p. favipiravir intervention beginning 2 days after challenge is the highest ever reported for a small molecule antiviral in the difficult to treat guinea pig JUNV challenge model. These findings support the continued development of favipiravir as a promising antiviral against JUNV and other related arenaviruses.