Fatal human respiratory disease associated with the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is characterized by severe lung pathology, including pulmonary edema and ...extensive inflammatory infiltrate. Here, we quantified the cellular immune response to infection in the mouse lung by flow cytometry and demonstrate that mice infected with highly pathogenic (HP) H1N1 and H5N1 influenza viruses exhibit significantly high numbers of macrophages and neutrophils in the lungs compared to mice infected with low pathogenic (LP) viruses. Mice infected with the 1918 pandemic virus and a recent H5N1 human isolate show considerable similarities in overall lung cellularity, lung immune cell sub-population composition, and cellular immune temporal dynamics. Interestingly, while these similarities were observed, the HP H5N1 virus consistently elicited significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages, revealing a potentially critical difference in the pathogenesis of H5N1 infections. Primary mouse and human macrophages and dendritic cells were also susceptible to 1918 and H5N1 influenza virus infection in vitro. These results together indicate that infection with HP influenza viruses such as H5N1 and the 1918 pandemic virus leads to a rapid cell recruitment of macrophages and neutrophils into the lungs, suggesting that these cells play a role in acute lung inflammation associated with HP influenza virus infection.
The proapoptotic PB1-F2 protein of influenza A viruses has been shown to contribute to pathogenesis in the mouse model. Expression of full-length PB1-F2 increases the pathogenesis of the influenza A ...virus, causing weight loss, slower viral clearance, and increased viral titers in the lungs. After comparing viruses from the Hong Kong 1997 H5N1 outbreak, one amino acid change (N66S) was found in the PB1-F2 sequence at position 66 that correlated with pathogenicity. This same amino acid change (N66S) was also found in the PB1-F2 protein of the 1918 pandemic A/Brevig Mission/18 virus. Two isogenic recombinant chimeric viruses were created with an influenza A/WSN/33 virus background containing the PB1 segment from the HK/156/97: WH and WH N66S. In mice infected with WH N66S virus there was increased pathogenicity as measured by weight loss and decreased survival, and a 100-fold increase in virus replication when compared to mice infected with the WH virus. The 1918 pandemic strain A/Brevig Mission/18 was reconstructed with a pathogenicity-reducing mutation in PB1-F2 (S66N). The resultant 1918 S66N virus was attenuated in mice having a 3-log lower 50% lethal dose and caused less morbidity and mortality in mice than the wild-type virus. Viral lung titers were also decreased in 1918 S66N-infected mice compared with wild-type 1918 virus-infected mice. In addition, both viruses with an S at position 66 (WH N66S and wt 1918) induced elevated levels of cytokines in the lungs of infected mice. Together, these data show that a single amino acid substitution in PB1-F2 can result in increased viral pathogenicity and could be one of the factors contributing to the high lethality seen with the 1918 pandemic virus.
Highly pathogenic influenza A viruses, including avian H5N1 viruses and the 1918 pandemic virus, cause severe respiratory disease in humans and animals. Virus infection is followed by intense ...pulmonary congestion due to an extensive influx of macrophages and neutrophils, which can release large quantities of reactive oxygen species potentially contributing to the pathogenesis of lung disease. Here, the role of nitric oxide (NO), a potent signaling molecule in inflammation, was evaluated following highly pathogenic influenza virus challenge in mice. We observed higher levels of NO in mice infected with H5N1 and 1918 viruses as compared to a seasonal H1N1 virus. Mice deficient in inducible NO synthase (NOS2 —/— ) exhibited reduced morbidity, reduced mortality, and diminished cytokine production in lung tissue following H5N1 and 1918-virus challenge, compared with wild-type control mice. Furthermore, systemic treatment of mice with the NOS inhibitor NG-monomethyl-L-arginine delayed weight loss and death among 1918 virus infected mice compared to untreated control animals. This study demonstrates that NO contributes to the pathogenic outcome of H5N1 and 1918 viral infections in the mouse model.
The ongoing threat from Influenza necessitates the development of new vaccine and adjuvant technologies that can maximize vaccine immunogenicity, shorten production cycles, and increase global ...vaccine supply. Currently, the most successful adjuvants for Influenza vaccines are squalene-based oil-in-water emulsions. These adjuvants enhance seroprotective antibody titers to homologous and heterologous strains of virus, and augment a significant dose sparing activity that could improve vaccine manufacturing capacity. As an alternative to an emulsion, we tested a simple lipid-based aqueous formulation containing a synthetic TLR4 ligand (GLA-AF) for its ability to enhance protection against H5N1 infection. GLA-AF was very effective in adjuvanting recombinant H5 hemagglutinin antigen (rH5) in mice and was as potent as the stable emulsion, SE. Both adjuvants induced similar antibody titers using a sub-microgram dose of rH5, and both conferred complete protection against a highly pathogenic H5N1 challenge. However, GLA-AF was the superior adjuvant in ferrets. GLA-AF stimulated a broader antibody response than SE after both the prime and boost immunization with rH5, and ferrets were better protected against homologous and heterologous strains of H5N1 virus. Thus, GLA-AF is a potent emulsion-free adjuvant that warrants consideration for pandemic influenza vaccine development.
Background. Highly pathogenic avian influenza viruses of the H5N1 subtype continue to cross the species barrier to infect humans and cause severe disease. It has been suggested that an exaggerated ...immune response contributes to the pathogenesis of H5N1 virus infection in mammals. In particular, H5N1 virus infections are associated with a high expression of the proinflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α). Methods. We investigated the compounding affects of both cytokines on the outcome of H5N1 virus disease by using triple mutant mice deficient in 3 signaling receptors, TNF-R1, TNF-R2, and IL-1-RI. Results. Triple mutant mice exhibited reduced morbidity and a significant delay in mortality following lethal challenge with a lethal H5N1 virus, whereas no such differences were observed with the less virulent A/PR/8/34 (H1N1) virus. H5N1-infected triple mutant mice displayed diminished cytokine production in lung tissue and a quantifiable decrease of macrophages and neutrophils in the lungs postinfection. Moreover, morphometric analysis of airway sections revealed less extensive inflammation in H5N1-infected triple mutant mice, compared with infected wild-type mice. Conclusions. The combined signaling from the TNF or IL-1 receptors promotes maximal lung inflammation that may contribute to the severity of disease caused by H5N1 virus infection.
The 1918 influenza pandemic was exceptionally severe, resulting in the death of up to 50 million people worldwide. Here, we show which virus genes contributed to the replication and virulence of the ...1918 influenza virus. Recombinant viruses, in which genes of the 1918 virus were replaced with genes from a contemporary human H1N1 influenza virus, A/Texas/36/91 (Tx/91), were generated. The exchange of most 1918 influenza virus genes with seasonal influenza H1N1 virus genes did not alter the virulence of the 1918 virus; however, substitution of the hemagglutinin (HA), neuraminidase (NA), or polymerase subunit PB1 genes significantly affected the ability of this virus to cause severe disease in mice. The 1918 virus virulence observed in mice correlated with the ability of 1918 recombinant viruses to replicate efficiently in human airway cells. In a second series of experiments, eight 1918 1:7 recombinants were generated, in which each Tx/91 virus gene was individually replaced by a corresponding gene from 1918 virus. Replication capacity of the individual 1:7 reassortant viruses was assessed in mouse lungs and human airway cells. Increased virus titers were observed among 1:7 viruses containing individual 1918 HA, NA, and PB1 genes. In addition, the 1918 PB1:Tx/91 (1:7) virus showed a distinctly larger plaque size phenotype than the small plaque phenotype of the 1918 PA:Tx/91 and 1918 PB2:Tx/91 1:7 reassortants. These results highlight the importance of the 1918 HA, NA, and PB1 genes for optimal virus replication and virulence of this pandemic strain.
Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus ...pandemic was the most severe of the modern era. Recent work has recovered the gene sequences of this unusual strain, so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes. However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 haemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. Here we show that of the 32 individuals tested that were born in or before 1915, each showed seroreactivity with the 1918 virus, nearly 90 years after the pandemic. Seven of the eight donor samples tested had circulating B cells that secreted antibodies that bound the 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that showed potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but did not cross-react with HAs of more contemporary human influenza viruses. The antibody genes had an unusually high degree of somatic mutation. The antibodies bound to the 1918 HA protein with high affinity, had exceptional virus-neutralizing potency and protected mice from lethal infection. Isolation of viruses that escaped inhibition suggested that the antibodies recognize classical antigenic sites on the HA surface. Thus, these studies demonstrate that survivors of the 1918 influenza pandemic possess highly functional, virus-neutralizing antibodies to this uniquely virulent virus, and that humans can sustain circulating B memory cells to viruses for many decades after exposure-well into the tenth decade of life.
Laboratory diagnostic testing service delivery and compliance with international standards for laboratory quality are directly influenced by laboratory workforce competency. Many hospital ...laboratories in constrained resource settings such as Cambodia struggle to cope with the training needs of laboratory professionals in an environment of competing healthcare development priorities. Resource-limited countries need an adaptable and effective approach to provide laboratory professionals with job-specific quality oversight training to ensure the accuracy, timeliness, and reliability of diagnostic services.
Here, we describe the results of an in-service training and mentoring program conducted with the Cambodia Ministry of Health at 12 tertiary-level hospital laboratories to drive improvements in laboratory quality management systems toward ISO 15189 accreditation, which demonstrated significant progress between baseline and outcome audits in a concurrent study. This case study describes the program, and evaluates how the four primary activities, including actionable gap assessments and planning, centralized and in situ training curriculum, in-person mentoring, and remote tele-mentoring via video communication technologies, contributed towards quality improvement in the participating laboratories. We evaluated participant responses to Likert scale and free response questions from program and training evaluation surveys, and we used thematic analysis to develop a model of best practices within the program's four primary activities to inform future training approaches. Of these activities, participants agreed most highly that in-person visits and planning based on gap assessments contributed to their learning and ability to improve laboratory operations. Tele-mentoring was rated lowest by participants, who were critical of excessive group dialogue and distraction during web-conferencing; however, feedback suggests both in-person and remote mentoring contribute to continuing education, accountability to action, and peer collaboration and problem solving to improve workforce efforts toward improved quality management systems.
We recommend here a package of in-service training activities for laboratory quality management system improvement initiatives in resource constrained settings that includes needs-based curricula and personalized action plans for participants; interactive and on-site training workshops; and in-person mentoring, complemented with well managed and regular tele-mentoring that focuses on knowledge retention, accountability to goals, and collaborative problem solving. Our model presents an adaptable approach to human resource development for quality improvement in medical laboratories.
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