A. Sally Davis

MultiDrug-Resistant 2009 Pandemic Influenza A(H1N1) Viruses Maintain Fitness and Transmissibility in Ferrets

BACKGROUND The 2009 influenza A(H1N1) pandemic called attention to the limited influenza treatment options available, especially in individuals at high risk of severe disease. Neuraminidase inhibitor-resistant seasonal H1N1 viruses have demonstrated the ability to transmit well despite early data indicating that resistance reduces viral fitness. 2009 H1N1 pandemic viruses have sporadically appeared containing resistance to neuraminidase inhibitors and the adamantanes, but the ability of these viruses to replicate, transmit, and cause disease in mammalian hosts has not been fully characterized. METHODS Two pretreatment wild-type viruses and 2 posttreatment multidrug-resistant viruses containing the neuraminidase H275Y mutation collected from immunocompromised patients infected with pandemic influenza H1N1 were tested for viral fitness, pathogenicity, and transmissibility in ferrets. RESULTS The pretreatment wild-type viruses and posttreatment resistant viruses containing the H275Y mutation all demonstrated significant pathogenicity and equivalent viral fitness and transmissibility. CONCLUSIONS The admantane-resistant 2009 pandemic influenza A(H1N1) virus can develop the H275Y change in the neuraminidase gene conferring resistance to both oseltamivir and peramivir without any loss in fitness, transmissibility, or pathogenicity. This suggests that the dissemination of widespread multidrug resistance similar to neuraminidase inhibitor resistance in seasonal H1N1 is a significant threat.

Global Rinderpest Eradication: Lessons Learned and Why Humans Should Celebrate Too

Mota and Qaranyo, why are they notplowed?. I came from there to here withoutseeing an ox.-Line from Ethiopian poem[1, 2], 1890sAfter more than a decade of effortby the Global Rinderpest EradicationProgramme (GREP) of the UN Foodand Agricultural Organization (FAO), theOrganisation mondiale de la sante´(Office international des e´pizooties[OIE]), and the International AtomicEnergy Agency (IAEA), among others[3–5], the veterinary disease rinderpest(Figure 1) was declared eradicated on25 May 2011 [6]. Since rinderpest virusdoes not cause human disease, why isthis achievement important to humans?Since at least the Roman era, rinder-pest (German for ‘‘cattle plague’’) hasbeen indirectly responsible for countlesshuman deaths resulting from agriculturallosses that led to famine and disease. TheEthiopian poem cited above refers to anAfrican rinderpest panzootic that causedrapid loss of virtually all of the cattle,buffaloes, elands, and wild swine, as wellas many sheep, goats, and wildlife species,such as antelopes, gazelles, giraffes,hartebeest, and wildebeest (the ‘‘GreatEthiopian Famine’’ of 1887–1892 [2, 7–11]). Rinderpest virus (RPV) not onlyinfects cattle but also infects

Characterizing and Diminishing Autofluorescence in Formalin-fixed Paraffin-embedded Human Respiratory Tissue.

Tissue autofluorescence frequently hampers visualization of immunofluorescent markers in formalin-fixed paraffin-embedded respiratory tissues. We assessed nine treatments reported to have efficacy in reducing autofluorescence in other tissue types. The three most efficacious were Eriochrome black T, Sudan black B and sodium borohydride, as measured using white light laser confocal Λ2 (multi-lambda) analysis. We also assessed the impact of steam antigen retrieval and serum application on human tracheal tissue autofluorescence. Functionally fitting this Λ2 data to 2-dimensional Gaussian surfaces revealed that steam antigen retrieval and serum application contribute minimally to autofluorescence and that the three treatments are disparately efficacious. Together, these studies provide a set of guidelines for diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. Additionally, these characterization techniques are transferable to similar questions in other tissue types, as demonstrated on frozen human liver tissue and paraffin-embedded mouse lung tissue fixed in different fixatives.

The use of nonhuman primates in research on seasonal, pandemic and avian influenza, 1893-2014.

Attempts to reproduce the features of human influenza in laboratory animals date from the early 1890s, when Richard Pfeiffer inoculated apes with bacteria recovered from influenza patients and produced a mild respiratory illness. Numerous studies employing nonhuman primates (NHPs) were performed during the 1918 pandemic and the following decade. Most used bacterial preparations to infect animals, but some sought a filterable agent for the disease. Since the viral etiology of influenza was established in the early 1930s, studies in NHPs have been supplemented by a much larger number of experiments in mice, ferrets and human volunteers. However, the emergence of a novel swine-origin H1N1 influenza virus in 1976 and the highly pathogenic H5N1 avian influenza virus in 1997 stimulated an increase in NHP research, because these agents are difficult to study in naturally infected patients and cannot be administered to human volunteers. In this paper, we review the published literature on the use of NHPs in influenza research from 1893 through the end of 2014. The first section summarizes observational studies of naturally occurring influenza-like syndromes in wild and captive primates, including serologic investigations. The second provides a chronological account of experimental infections of NHPs, beginning with Pfeiffers study and covering all published research on seasonal and pandemic influenza viruses, including vaccine and antiviral drug testing. The third section reviews experimental infections of NHPs with avian influenza viruses that have caused disease in humans since 1997. The paper concludes with suggestions for further studies to more clearly define and optimize the role of NHPs as experimental animals for influenza research.

Validation of Normal Human Bronchial Epithelial Cells as a Model for Influenza A Infections in Human Distal Trachea

Primary normal human bronchial/tracheal epithelial (NHBE) cells, derived from the distal-most aspect of the trachea at the bifurcation, have been used for a number of studies in respiratory disease research. Differences between the source tissue and the differentiated primary cells may impact infection studies based on this model. Therefore, we examined how well-differentiated NHBE cells compared with their source tissue, the human distal trachea, as well as the ramifications of these differences on influenza A viral pathogenesis research using this model. We employed a histological analysis including morphological measurements, electron microscopy, multi-label immunofluorescence confocal microscopy, lectin histochemistry, and microarray expression analysis to compare differentiated NHBEs to human distal tracheal epithelium. Pseudostratified epithelial height, cell type variety and distribution varied significantly. Electron microscopy confirmed differences in cellular attachment and paracellular junctions. Influenza receptor lectin histochemistry revealed that α2,3 sialic acids were rarely present on the apical aspect of the differentiated NHBE cells, but were present in low numbers in the distal trachea. We bound fluorochrome bioconjugated virus to respiratory tissue and NHBE cells and infected NHBE cells with human influenza A viruses. Both indicated that the pattern of infection progression in these cells correlated with autopsy studies of fatal cases from the 2009 pandemic.

In vivo evaluation of pathogenicity and transmissibility of influenza A(H1N1)pdm09 hemagglutinin receptor binding domain 222 intrahost variants isolated from a single immunocompromised patient

The influenza A(H1N1)pdm09 virus has circulated worldwide and continued to cause complicated infections and deaths. Reports have identified an increased prevalence of the hemagglutinin receptor binding domain D222G mutation in viruses isolated from individuals who have suffered such severe infections, but this association is still unclear. Virus isolated from a nasopharyngeal wash of a severely ill immunocompromised patient at the time of diagnosis contained the D222, but isolates collected later in his course from a bronchoalveolar lavage contained primarily the G222 mutation and was mixed with a minor population of D222. These clinical isolates were compared to a G222 plaque purified virus in the ferret model. The G222 predominant clinical isolate was the most pathogenic in ferrets and developed the most diversity at the 222 amino acid position during infection, suggesting that increased diversity and not a specific polymorphism at HA 222 may be important in predicting pathogenic potential.

Contemporary Avian Influenza A Virus Subtype H1, H6, H7, H10, and H15 Hemagglutinin Genes Encode a Mammalian Virulence Factor Similar to the 1918 Pandemic Virus H1 Hemagglutinin

ABSTRACT Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backbone, differing only by hemagglutinin subtype expressed. Viruses expressing the avian H1, H6, H7, H10, and H15 subtypes were pathogenic in mice and cytopathic in normal human bronchial epithelial cells, in contrast to H2-, H3-, H5-, H9-, H11-, H13-, H14-, and H16-expressing viruses. Mouse pathogenicity was associated with pulmonary macrophage and neutrophil recruitment. These data suggest that avian influenza virus hemagglutinins H1, H6, H7, H10, and H15 contain inherent mammalian virulence factors and likely share a key virulence property of the 1918 virus. Consequently, zoonotic infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals. IMPORTANCE Influenza viruses from birds can cause outbreaks in humans and may contribute to the development of pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its main surface protein, an H1 subtype hemagglutinin, was identified as a key mammalian virulence factor. In a previous study, a 1918 virus expressing an avian H1 gene was as virulent in mice as the reconstructed 1918 virus. Here, a set of avian influenza viruses was constructed, differing only by hemagglutinin subtype. Viruses with the avian H1, H6, H7, H10, and H15 subtypes caused severe disease in mice and damaged human lung cells. Consequently, infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals, and therefore surveillance for human infections with these subtypes may be important in controlling future outbreaks. Influenza viruses from birds can cause outbreaks in humans and may contribute to the development of pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its main surface protein, an H1 subtype hemagglutinin, was identified as a key mammalian virulence factor. In a previous study, a 1918 virus expressing an avian H1 gene was as virulent in mice as the reconstructed 1918 virus. Here, a set of avian influenza viruses was constructed, differing only by hemagglutinin subtype. Viruses with the avian H1, H6, H7, H10, and H15 subtypes caused severe disease in mice and damaged human lung cells. Consequently, infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals, and therefore surveillance for human infections with these subtypes may be important in controlling future outbreaks.

Pneumocystis Encodes a Functional Endo-β-1,3-glucanase That is Expressed Exclusively in Cysts

β-1,3-glucan is a major cell wall component of Pneumocystis cysts. We have characterized endo-β-1,3-glucanase (Eng) from 3 species of Pneumocystis. The gene eng is a single-copy gene that encodes a protein containing 786 amino acids in P. carinii and P. murina, and 788 amino acids in P. jirovecii, including a signal peptide for the former 2 but not the latter. Recombinant Eng expressed in Escherichia coli was able to solubilize the major surface glycoprotein of Pneumocystis, thus potentially facilitating switching of the expressed major surface glycoprotein (Msg) variant. Confocal immunofluorescence analysis of P. murina-infected mouse lung sections localized Eng exclusively to the cyst form of Pneumocystis. No Eng was detected after mice were treated with caspofungin, a β-1,3-glucan synthase inhibitor that is known to reduce the number of cysts. Thus, Eng is a cyst-specific protein that may play a role in Msg variant expression in Pneumocystis.

1918 Influenza receptor binding domain variants bind and replicate in primary human airway cells regardless of receptor specificity.

The 1918 influenza pandemic caused ~50 million deaths. Many questions remain regarding the origin, pathogenicity, and mechanisms of human adaptation of this virus. Avian-adapted influenza A viruses preferentially bind α2,3-linked sialic acids (Sia) while human-adapted viruses preferentially bind α2,6-linked Sia. A change in Sia preference from α2,3 to α2,6 is thought to be a requirement for human adaptation of avian influenza viruses. Autopsy data from 1918 cases, however, suggest that factors other than Sia preference played a role in viral binding and entry to human airway cells. Here, we evaluated binding and entry of five 1918 influenza receptor binding domain variants in a primary human airway cell model along with control avian and human influenza viruses. We observed that all five variants bound and entered cells efficiently and that Sia preference did not predict entry of influenza A virus to primary human airway cells evaluated in this model.