Successful use of extracorporeal membrane oxygenation for pH1N1-induced refractory hypoxemia in a child with hypoplastic left heart syndrome.

OBJECTIVE: To report the first successful use of extracorporeal membrane oxygenation for acute respiratory distress syndrome secondary to 2009 pH1N1 influenza A infection in a child status post the Fontan operation for hypoplastic left heart syndrome. DESIGN: Individual case report. SETTING: Pediatric intensive care unit. PATIENT: We describe a 7-yr-old boy with a history of the Fontan operation for hypoplastic left heart syndrome admitted with acute respiratory distress syndrome secondary to 2009 pH1N1 influenza A infection. INTERVENTION: Cannulation for venoarterial extracorporeal membrane oxygenation. MEASUREMENTS AND MAIN RESULTS: In this patient with a history of complex congenital heart disease and repair, extracorporeal membrane oxygenation was a successful rescue therapy for refractory pH1N1-induced respiratory failure. CONCLUSION: Extracorporeal membrane oxygenation can be successfully applied for refractory respiratory failure, even in the setting of significant underlying comorbidity. With emerging data to support the role of extracorporeal membrane oxygenation in improving mortality for refractory hypoxemia secondary to pH1N1, it is prudent to strongly consider the use of extracorporeal support in patients with underlying diseases or comorbidities that may have previously precluded them from being candidates for this therapy.

Development of a collaborative program to provide extracorporeal membrane oxygenation for adults with refractory hypoxemia within the framework of a pandemic.

OBJECTIVE: We report the process used to rapidly develop a collaborative adult respiratory extracorporeal membrane oxygenation program as a response to caring for young adult patients with refractory hypoxemia in the setting of the pH1N1 pandemic. DESIGN: Interdisciplinary response of a complex medical system to a public health crisis. PATIENTS, INTERVENTIONS, MEASUREMENTS, AND MAIN RESULTS: After the successful use of extracorporeal membrane oxygenation in young adults with pH1N1-induced acute respiratory distress syndrome refractory to conventional therapies, an adult venovenous extracorporeal membrane oxygenation program was implemented over an 8-wk period. Implementation of this program involved a number of key steps that were crucial in the development process, including administrative and institutional support, multidisciplinary leadership and collaboration, extensive interdisciplinary educational initiatives, and substantial technical modifications. CONCLUSIONS: In the setting of the pH1N1 influenza pandemic, an adult respiratory extracorporeal membrane oxygenation program was successfully developed to complement an established neonatal-pediatric program. This program expansion integrated all of the necessary components involved in the development process from start to finish and confirms that a healthcare system can respond very quickly and successfully to an urgent healthcare need.

Extracorporeal membrane oxygenation for severe refractory respiratory failure secondary to 2009 H1N1 influenza A.

BACKGROUND: Respiratory failure and acute respiratory distress syndrome secondary to H1N1 influenza infection is a source of substantial morbidity and mortality, having caused over 265,000 hospitalizations in the United States in 2009. During the H1N1 pandemic, up to 31% of the H1N1 patients required intensive care unit admission, and many were refractory to maximal conventional therapies. These most critically ill patients may require extracorporeal membrane oxygenation (ECMO) for survival. METHODS: We retrospectively reviewed the medical records of the 7 patients with refractory hypoxemia due to H1N1 influenza who were treated with ECMO in our pediatric intensive care unit. RESULTS: Five of the 7 patients survived to hospital discharge. The cohort's mean age was 21 years, and 4 were female. At admission to the pediatric intensive care unit, 6 had at least one comorbid condition, 6 were mechanically ventilated, and one was in shock. All 7 patients were treated with oral oseltamivir, high-frequency oscillatory ventilation, and inhaled nitric oxide prior to ECMO. Five received intravenous steroids, and 2 were treated with compassionate-use intravenous zanamivir. The mean duration of pre-ECMO ventilation was 8.7 days (range 14 h to 25 d). Mean oxygenation index was 50 (range 26-73) at ECMO cannulation. Six received venovenous ECMO, and one received venoarterial ECMO. The mean duration of ECMO was 432 hours (range 192-890 h). CONCLUSIONS: This series suggests that ECMO is a viable treatment for refractory hypoxemia secondary to H1N1 influenza infection in both pediatric and adult patients.

Real-time RT-PCR differentiation and quantitation of infectious bursal disease virus strains using dual-labeled fluorescent probes.

A real-time RT-PCR assay was developed utilizing dual-labeled fluorescent probes binding to VP4 sequence that are specific to the classical (Cl), variant (V) and very virulent (vv) strains of infectious bursal disease virus (IBDV). The assay was highly sensitive and could detect as little as 3 x 10(2) to 3 x 10(3) copies of viral template. Viral genomic copy number could be accurately assayed over a broad range of 7-8 logs of viral genome. The variant sequence-specific probe was found to be highly specific in detecting isolates classified as variant A, D, E, G and GLS-5, and did not react with classical strains. A total of 130 field and experimental variant strain isolates were tested using this assay. The classical sequence-specific probe also demonstrated high sensitivity and specificity, and positively detected a total of 87 STC isolates, both field and experimental isolates, while differentiating between isolates that were variant and classical strains. The very virulent sequence-specific probe detected positively the Holland vvIBDV isolate and did not react with classical or variant strains. Rapid identification of viral strain is a primary concern to poultry flock health programs to ensure administered vaccines will protect against current strains of virus circulating in the flock. The ability to quantify virus concurrently is also of assistance in identifying the progression of disease outbreaks within the flock.

Outcomes before and after implementation of a pediatric rapid-response extracorporeal membrane oxygenation program.

BACKGROUND: Rapid-response extracorporeal membrane oxygenation (RR-ECMO) has been implemented at select centers to expedite cannulation for patients placed on ECMO during extracorporeal cardiopulmonary resuscitation (ECPR). In 2008, we established such a program and used it for all pediatric venoarterial ECMO initiations. This study was designed to compare outcomes before and after program implementation. METHODS: Between 2003 and 2011, 144 pediatric patients were placed on venoarterial ECMO. Records of patients placed on ECMO before (17 ECPR and 62 non-ECPR) or after (14 ECPR and 51 non-ECPR) RR-ECMO program implementation were retrospectively compared. RESULTS: The peak performance of the ECMO team was assessed by measuring ECMO initiation times for the ECPR patient subgroup (n = 31). There was a shift toward more ECPR initiations achieved in less than 40 minutes (24% pre-RR-ECMO versus 43% RR-ECMO; p = 0.25) and fewer requiring more than 60 minutes (47% pre-RR-ECMO versus 21% RR-ECMO; p = 0.14) after program implementation, although these changes did not reach statistical significance. After multivariable risk adjustment, RR-ECMO was associated with a 52% reduction in neurologic complications for all patients (adjusted odds ratio, 0.48; 95% confidence interval, 0.23 to 0.98; p = 0.04), but the risk of in-hospital death remained unchanged (adjusted odds ratio, 0.99; 95% confidence interval, 0.50 to 1.99; p = 0.99). CONCLUSIONS: Implementation of a pediatric RR-ECMO program for venoarterial ECMO initiation was associated with reduced neurologic complications but not improved survival during the first 3 years of program implementation. These data suggest that development of a coordinated system for rapid ECMO deployment may benefit both ECPR and non-ECPR patients, but further efforts are required to improve survival.

Attenuation of chicken anemia virus by site-directed mutagenesis of VP2.

Chicken anemia virus (CAV) is a significant immunosuppressive pathogen of chickens, but relatively little is known about the effect of specific mutations on its virulence. In order to study the virulence of CAV, an infection model was developed in embryos. Significant growth depression, measured as a reduction in mean body weight, was found for wild-type CAV infection. Infection with wild-type CAV resulted in a significant reduction in thymic and splenic weights and consistently produced severe lesions in the thymus, spleen and bone marrow, as well as haemorrhages. CAVs mutated in the VP2 gene were infectious for embryos, but were highly attenuated with respect to growth depression and CAV-specific pathology. Relative to wild-type infection, viruses Mut C86R, Mut R101G, Mut H103Y, Mut R129G, Mut Q131P, Mut R/K/K150/151/152G/A/A, Mut D/E161/162G/G and Mut E186G were highly attenuated, and viruses Mut L163P and Mut D169G were moderately attenuated. Attenuation of the ability to produce lesions was found consistently for the thymus, spleen and bone marrow, thymic and splenic weights, and for CAV-induced haemorrhage. There was no growth depression associated with infection by the group of highly attenuated mutant viruses and a moderate reduction in mean body weight was only found for virus Mut L163P. These findings show that mutations in the VP2 gene can reduce the virulence of CAV and these mutant viruses may have value as vaccine candidates.

Site-directed mutagenesis of the VP2 gene of Chicken anemia virus affects virus replication, cytopathology and host-cell MHC class I expression.

Chicken anemia virus (CAV) is an immunosuppressive pathogen of chickens. To further examine the role of viral protein 2 (VP2), which possesses dual-specificity protein phosphatase (DSP) activity, in viral cytopathogenicity and its influence on viral growth and virulence, an infectious genomic clone of CAV was subjected to site-directed mutagenesis. Substitution mutations C87R, R101G, K102D and H103Y were introduced into the DSP catalytic motif and R129G, Q131P, R/K/K150/151/152G/A/A, D/E161/162G/G, L163P, D169G and E186G into a region predicted to have a high degree of secondary structure. All mutant constructs were infectious, but their growth curves differed. The growth curve for mutant virus R/K/K150/151/152G/A/A was similar to that for wild-type virus, a second cluster of mutant viruses had an extended latent period and a third cluster of mutant viruses had extended latent and eclipse periods. All mutants had a reduced cytopathogenic effect in infected cells and VP3 was restricted to the cytoplasm. Mutation of the second basic residue (K102D) in the atypical DSP signature motif resulted in a marked reduction in virus replication efficiency, whereas mutation of the first basic residue (R101G) attenuated cytopathogenicity, but did not reduce replication efficiency. Expression of major histocompatibility complex (MHC) class I was markedly downregulated in cells infected with wild-type CAV, but not in those infected with mutants. This study further demonstrates the significance of VP2 in CAV replication and shows that specific mutations introduced into the gene encoding this protein can reduce virus replication, cytopathogenicity and downregulation of MHC I in infected cells.

Mutation of chicken anemia virus VP2 differentially affects serine/threonine and tyrosine protein phosphatase activities.

Novel dual-specificity protein phosphatases (DSPs), which catalyse the removal of phosphate from both phosphotyrosine and phosphoserine/phosphothreonine substrates, have recently been identified in two viruses within the family Circoviridae. Viral protein 2 (VP2) of chicken anemia virus (CAV) and ORF2 of TT virus have been shown to possess DSP activity in vitro. CAV VP2 is unusual in possessing two vicinal cysteines within the protein phosphatase signature motif. The first cysteine residue (C95) within the motif has been identified by mutagenesis as the essential catalytic cysteine. In this study, it was shown that virus mutated at this residue displayed a marked inhibition of growth, with titres reduced 10(4)-fold, and reduced cytopathogenic effect in cell culture, indicating that viral DSP activity may be significant during infection. As with virus mutated at the first cysteine residue, mutation of the second cysteine (C97) within the motif resulted in a marked reduction in viral growth and attenuation of cytopathogenicity in infected cell cultures. However, mutagenesis of this second cysteine only reduced phosphotyrosine phosphatase activity to 70 % of that of wild-type VP2, but increased phosphoserine/phosphothreonine phosphatase activity by as much as 700 %. The differential effect of the C97S mutation on VP2 activity does not appear to have parallels in other DSPs and suggests a unique role for the second cysteine in the function of these viral proteins, particularly in vivo.

Chicken anemia virus VP2 is a novel dual specificity protein phosphatase.

The function of viral protein 2 (VP2) of the immunosuppressive circovirus chicken anemia virus (CAV) has not yet been established. We show that the CAV VP2 amino acid sequence has some similarity to a number of eukaryotic, receptor, protein-tyrosine phosphatase (PTPase) alpha proteins as well as to a cluster of human TT viruses within the Sanban group. To investigate if CAV VP2 functions as a PTPase, purified glutathione S-transferase (GST)-VP2 fusion protein was assayed for PTPase activity using the generalized peptide substrates ENDpYINASL and DADEpYLIPQQG (where pY represents phosphotyrosine), with free phosphate detected using the malachite green colorimetric assay. CAV GST-VP2 was shown to catalyze dephosphorylation of both substrates. CAV GST-VP2 PTPase activity for the ENDpYINASL substrate had a V(max) of 14,925 units/mg.min and a K(m) of 18.88 microm. Optimal activity was observed between pH 6 and 7, and activity was specifically inhibited by 0.01 mm orthovanadate. We also show that the ORF2 sequence of the CAV-related human virus TT-like minivirus (TLMV) possessed PTPase activity and steady state kinetics equivalent to CAV GST-VP2 when expressed as a GST fusion protein. To establish whether these viral proteins were dual specificity protein phosphatases, the CAV GST-VP2 and TLMV GST-ORF2 fusion proteins were also assayed for serine/threonine phosphatase (S/T PPase) activity using the generalized peptide substrate RRApTVA, with free phosphate detected using the malachite green colorimetric assay. Both CAV GST-VP2 and TLMV GST-ORF2 fusion proteins possessed S/T PPase activity, which was specifically inhibited by 50 mm sodium fluoride. CAV GST-VP2 exhibited S/T PPase activity with a V(max) of 28,600 units/mg.min and a K(m) of 76 microm. Mutagenesis of residue Cys(95) to serine in CAV GST-VP2 abrogated both PTPase and S/T PPase activity, identifying it as the catalytic cysteine within the proposed signature motif. These studies thus show that the circoviruses CAV and TLMV encode dual specificity protein phosphatases (DSP) with an unusual signature motif that may play a role in intracellular signaling during viral replication. This is the first DSP gene to be identified in a small viral genome.

Embryonic age influences the capacity for cytokine induction in chicken thymocytes.

Thymocyte responses to functional activation are of relevance to the evaluation of the efficacy of in ovo immunotherapies and vaccines in chickens. In this study we have demonstrated differences in chicken thymocyte responses according to developmental age. RNA samples from stimulated and unstimulated chicken thymocytes were assayed for messenger RNA encoding the cytokines interleukin-1beta (IL-1beta), IL-2, interferon-alpha (IFN-alpha), IFN-beta, IFN-gamma and transforming growth factor-beta4 (TGF-beta4), and also components of the major histocompatibility complex (MHC), beta2-microglobulin (beta2M) and the MHC class I alpha-chain (MHC IA). At embryonic day 14 thymocytes were least responsive to functional activation and differences existed even between thymocyte populations at embryonic day 18 and day 1 post-hatch. The duration of proliferation in response to stimulation was found to increase with increasing embryonic age. Mitogen stimulation of embryonic day 18 and day 1 post-hatch thymocytes induced up-regulation of IFN-gamma, IL-1beta and TGF-beta transcripts, and down-regulation of IFN-alpha, IFN-beta and IL-2 transcripts, with a higher induction of IFN-gamma, IL-1beta and TGF-beta transcripts in more immature T-cell-receptor-negative (TCR-) than TCR+ (TCR1+, TCR2+, or TCR3+) subsets. In contrast, in the mouse and human, both mature and immature thymocytes respond to mitogen stimulation with up-regulation of IL-2. Thymocytes from embryonic day 14 chicks responded to mitogen with a short burst of unsustained proliferation, and transcriptional down-regulation of the cytokines IL-2, IL-1beta, IFN-alpha, IFN-beta and IFN-gamma. These results suggest that embryonic day 14 thymocytes are largely unresponsive to mitogen. Transcripts encoding TGF-beta and type I interferons (IFN-alpha and IFN-beta) were constitutively expressed at high levels in very early thymocytes at embryonic day 14. Thymocytes at embryonic days 14 and 18 and day 1 post-hatch responded to mitogen stimulation with up-regulation of MHC IA transcript. The pattern of beta2M transcription following mitogen stimulation was distinct from that of the globally up-regulated MHC IA transcript, with up-regulation of beta2M transcription observed at embryonic day 18 and day 1 post-hatch but not at embryonic day 14. In thymocyte subsets, up-regulation of beta2M transcription was found to be specific to the CD8+ TCR+ population. The balance of responses in the embryonic thymus suggests that at all stages thymocytes have a reduced capacity for activation in comparison to mature thymocyte populations.