#Influenza #Hemagglutinins #H2, #H5, #H6, and #H11 are not Targets of Pulmonary #Surfactant Protein D: N-glycan subtypes in host-pathogen interactions (J Virol., abstract)

[Source: Journal of Virology, full page: (LINK). Abstract, edited.]

Influenza Hemagglutinins H2, H5, H6, and H11 are not Targets of Pulmonary Surfactant Protein D: N-glycan subtypes in host-pathogen interactions

Lisa Parsons, Yanming An, Li Qi, Mitchell White, Roosmarijn van der Woude, Kevan Hartshorn, Jeffery K. Taubenberger, Robert P. de Vries, John F. Cipollo

DOI: 10.1128/JVI.01951-19



Seasonal influenza carrying key hemagglutinin (HA) head region glycosylation sites can be removed from the lung by pulmonary surfactant protein D (SP-D). Little is known about HA head glycosylation of low pathogenicity A type influenza virus (LPAIV) subtypes. These can pose a pandemic threat through reassortmant and emergence in human populations. Since the presence of head region high mannose glycosites dictates SP-D activity, the ability to predict these glycosite glycan subtypes may be of value. Here we investigate the activities of two recombinant human SP-D forms against representative LPAIV including H2N1, H5N1, H6N1, H11N9, an avian H3N8 and a human seasonal H3N2 subtype. Using mass spectrometry, we determined the glycan subclasses and heterogeneities at each head glycosylation site. Sequence alignment and molecular structure analysis of the HAs were performed for LPIAV strains in comparison to seasonal H3N2 and avian H3N8. Intramolecular contacts were determined between protein backbone and glycosite glycan based on available three-dimensional structure data. We found that glycosite “N165” (H3 numbering) is occupied by high mannose glycans in H3 HA but by complex glycans in all LVIAV HAs. SP-D was not active on LPAIV but was on H3 HAs. Since SP-D affinity for influenza HA depends on the presence of high mannose glycan on the head region our data demonstrate that SP-D may not protect against virus containing these HA subtypes. Our results also demonstrate that glycan subtype can be predicted at some glycosites based on sequence comparisons and three dimensional structural analysis.



Low pathogenicity A type influenza virus (LPAIV) subtypes can reassort with circulating human strains and pandemic viruses can emerge in human populations as was seen in the 1957 pandemic, where an H2 virus reassorted with the circulating H1N1 to create a novel H2N2 genotype. Lung surfactant protein D (SP-D), a key factor in first line innate immunity defence, removes IAV through interaction with hemagglutinin (HA) head region high mannose glycan(s). While it is known that both H1 and H3 HAs, have a key high mannose glycosite(s) in the head region, little is known about such glycosylation of LPAIV strains H2N1, H5N1, H6N1, or H11N9, which may pose future health risks. Here, we demonstrate that the hemagglutinins of LPAIV strains do not have the required high mannose glycans, do not interact with SP-D, and that sequence analysis can predict glycan subtype thus predicting presence or absence of this virulence marker.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Avian Influenza; Influenza A; Reassortant strains; H1N1; H2N2; H2N1; H3N2; H3N8; H5N1; H6N1; H11N9; Viral pathogenesis.


Expression of 9-O- and 7,9-O-Acetyl Modified #Sialic Acid in Cells and Their Effects on #Influenza Viruses (MBio, abstract)

[Source: MBio, full page: (LINK). Abstract, edited.]

Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses

Karen N. Barnard, Brian R. Wasik, Justin R. LaClair, David W. Buchholz, Wendy S. Weichert, Brynn K. Alford-Lawrence, Hector C. Aguilar, Colin R. Parrish

Xiang-Jin Meng, Editor

DOI: 10.1128/mBio.02490-19



Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in a variety of chemically modified forms, including those with acetyl modifications at the C-7, C-8, and C-9 positions. Here, we analyzed the distribution and amounts of these acetyl modifications in different human and canine cells. Since Sia or their variant forms are receptors for influenza A, B, C, and D viruses, we examined the effects of these modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl modified Sia are widely but variably expressed across cell lines from both humans and canines. Although they were expressed on the cell surfaces of canine MDCK cell lines, they were located primarily within the Golgi compartment of human HEK-293 and A549 cells. The O-acetyl modified Sia were expressed at low levels of 1 to 2% of total Sia in these cell lines. We knocked out and overexpressed the sialate O-acetyltransferase gene (CasD1) and knocked out the sialate O-acetylesterase gene (SIAE) using CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O- and 9-O-acetyl Sia expression, confirming previous reports. However, overexpression of CasD1 and knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no change in 7,9-O-acetyl levels, indicating that there are complex regulations of these modifications. These modifications were essential for influenza C and D infection but had no obvious effect on influenza A and B infection.



Sialic acids are key glycans that are involved in many different normal cellular functions, as well as being receptors for many pathogens. However, Sia come in diverse chemically modified forms. Here, we examined and manipulated the expression of 7,9-O- and 9-O-acetyl modified Sia on cells commonly used in influenza virus and other research by engineering the enzymes that produce or remove the acetyl groups.

Keywords: Influenza A; Influenza B; Influenza C; Influenza D; Viral pathogenesis.


Fluorescent #CCHF virus illuminates tissue #tropism patterns and identifies early mononuclear phagocytic cell targets in IFNAR-/- mice (PLOS Pathog., abstract)

[Source: PLOS Pathogens, full page: (LINK). Abstract, edited.]


Fluorescent Crimean-Congo hemorrhagic fever virus illuminates tissue tropism patterns and identifies early mononuclear phagocytic cell targets in IFNAR-/- mice

Stephen R. Welch, Jana M. Ritter, Anita K. McElroy, Jessica R. Harmon, JoAnn D. Coleman-McCray, Florine E. M. Scholte, Gary P. Kobinger, Éric Bergeron, Sherif R. Zaki, Stuart T. Nichol, Jessica R. Spengler , Christina F. Spiropoulou


Published: December 2, 2019 / DOI: https://doi.org/10.1371/journal.ppat.1008183 / This is an uncorrected proof.



Crimean-Congo hemorrhagic fever virus (CCHFV, order Bunyavirales, family Nairoviridae, genus Orthonairovirus) is the tick-borne etiological agent of Crimean-Congo hemorrhagic fever (CCHF) in humans. Animals are generally susceptible to CCHFV infection but refractory to disease. Small animal models are limited to interferon-deficient mice, that develop acute fatal disease following infection. Here, using a ZsGreen1- (ZsG) expressing reporter virus (CCHFV/ZsG), we examine tissue tropism and dissemination of virus in interferon-α/β receptor knock-out (Ifnar-/-) mice. We demonstrate that CCHFV/ZsG retains in vivo pathogenicity comparable to wild-type virus. Interestingly, despite high levels of viral RNA in all organs assessed, 2 distribution patterns of infection were observed by both fluorescence and immunohistochemistry (IHC), corresponding to the permissiveness of organ tissues. To further investigate viral dissemination and to temporally define cellular targets of CCHFV in vivo, mice were serially euthanized at different stages of disease. Flow cytometry was used to characterize CCHFV-associated alterations in hematopoietic cell populations and to classify infected cells in the blood, lymph node, spleen, and liver. ZsG signal indicated that mononuclear phagocytic cells in the lymphatic tissues were early targets of infection; in late-stage infection, overall, the highest levels of signal were detected in the liver, and ZsG was found in both antigen-presenting and lymphocyte cell populations.


Author summary

Human infection by tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV) can result in severe disease with up to 30% case fatality rates. While CCHFV is known to be hepatotropic, the presence and implications of virus in other tissues are less clear. Furthermore, to date, early cellular targets of infection in a CCHFV disease model have not been investigated in detail. Here, using a recombinant reporter CCHFV expressing the fluorescent protein ZsGreen1 (ZsG; CCHFV/ZsG) in interferon-α/β receptor knock-out (Ifnar-/-) mice, which develop acute fatal disease following infection, we investigate both cellular and tissue targets of infection. Importantly, we find that CCHFV/ZsG infection demonstrated comparable pathogenicity to wild-type virus in Ifnar-/- mice. We used in situ visualization of fluorescent signal in tissues to assess viral dissemination throughout the course of infection, and found robust viral signal in reproductive tissues, previously unrecognized as sites of CCHFV infection. We also used flow cytometry to detect intracellular fluorescent signal, and identified initial target cells of CCHFV infection as macrophage and monocyte populations in lymphatic tissues. These findings support a central role of immune cells in early virus dissemination, and a need for further investigations into reproductive tract involvement in human CCHFV infection.


Citation: Welch SR, Ritter JM, McElroy AK, Harmon JR, Coleman-McCray JD, Scholte FEM, et al. (2019) Fluorescent Crimean-Congo hemorrhagic fever virus illuminates tissue tropism patterns and identifies early mononuclear phagocytic cell targets in IFNAR-/- mice. PLoS Pathog 15(12): e1008183. https://doi.org/10.1371/journal.ppat.1008183

Editor: Veronika von Messling, Federal Ministry of Education and Research, GERMANY

Received: June 4, 2019; Accepted: November 1, 2019; Published: December 2, 2019

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was partially supported by an appointment to the Research Participation Program at the Centers for Disease Control and Prevention administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and CDC (S.R.W.), by a CDC foundation project funded by NIAID grant R01AI109008 (E.B.), and by CDC Emerging Infectious Disease Research Core Funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Keywords: CCHF; Viral pathogenesis.


#Structures of #MERS-CoV #spike glycoprotein in complex with sialoside attachment receptors (Nat Struct Mol Biol., abstract)

[Source: Nature Structural and Molecular Biology, full page: (LINK). Abstract, edited.]

Article / Published: 02 December 2019

Structures of MERS-CoV spike glycoprotein in complex with sialoside attachment receptors

Young-Jun Park, Alexandra C. Walls, Zhaoqian Wang, Maximillian M. Sauer, Wentao Li, M. Alejandra Tortorici, Berend-Jan Bosch, Frank DiMaio & David Veesler

Nature Structural & Molecular Biology (2019)



The Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and often lethal respiratory illness in humans, and no vaccines or specific treatments are available. Infections are initiated via binding of the MERS-CoV spike (S) glycoprotein to sialosides and dipeptidyl-peptidase 4 (the attachment and entry receptors, respectively). To understand MERS-CoV engagement of sialylated receptors, we determined the cryo-EM structures of S in complex with 5-N-acetyl neuraminic acid, 5-N-glycolyl neuraminic acid, sialyl-LewisX, α2,3-sialyl-N-acetyl-lactosamine and α2,6-sialyl-N-acetyl-lactosamine at 2.7–3.0 Å resolution. We show that recognition occurs via a conserved groove that is essential for MERS-CoV S-mediated attachment to sialosides and entry into human airway epithelial cells. Our data illuminate MERS-CoV S sialoside specificity and suggest that selectivity for α2,3-linked over α2,6-linked receptors results from enhanced interactions with the former class of oligosaccharides. This study provides a structural framework explaining MERS-CoV attachment to sialoside receptors and identifies a site of potential vulnerability to inhibitors of viral entry.

Data availability

The cryo-EM maps (sharpened and unsharpened) and atomic models have been deposited to the EMDB and wwPDB under accession numbers EMD-20542 and PDB 6Q04 (MERS-CoV S + Neu5Ac), EMD-20829 (MERS-CoV S + Neu5Gc), EMD-20543 and PDB 6Q05 (MERS-CoV S + sLeX), EMD-20544 and PDB 6Q06 (MERS-CoV S + 2,3-SLN) and EMD-20545 and PDB 6Q07 (MERS-CoV S + 2,6-SLN). Source Data are available online for Extended Data Fig. 5.

Keywords: MERS-CoV; Viral pathogenesis.


Viral determinants in #H5N1 #influenza A virus enable productive #infection of #HeLa cells (J Virol., abstract)

[Source: Journal of Virology, full page: (LINK). Abstract, edited.]

Viral determinants in H5N1 influenza A virus enable productive infection of HeLa cells.

Ariel Rodriguez-Frandsen, Laura Martin-Sancho, Anshu P. Gounder, Max W. Chang, Wen-Chun Liu, Paul D. De Jesus, Jessica von Recum-Knepper, Miriam S. Dutra, Nicholas J. Huffmaster, Monica Chavarria, Ignacio Mena, Laura Riva, Courtney B. Nguyen, Saunil Dobariya, Kristina M. Herbert, Christopher Benner, Randy A. Albrecht, Adolfo García-Sastre, Sumit K. Chanda

DOI: 10.1128/JVI.01410-19



Influenza A virus (IAV) is a human respiratory pathogen that causes yearly global epidemics, and sporadic pandemics due to human adaptation of pathogenic strains. Efficient replication of IAV in different species is, in part, dictated by its ability to exploit the genetic environment of the host cell. To investigate IAV tropism in human cells, we evaluated the replication of IAV strains in a diverse subset of epithelial cell lines. HeLa cells were refractory to growth of human H1N1 and H3N2, and low pathogenic avian influenza (LPAIs) viruses. Interestingly, a human isolate of the highly pathogenic avian influenza (HPAI) virus H5N1 successfully propagated in HeLa cells to levels comparable to a human lung cell line. Heterokaryon cells generated by fusion of HeLa and permissive cells supported H1N1 growth, suggesting the absence of a host factor(s) required for replication of H1N1, but not H5N1, in HeLa cells. The absence of this factor(s) was mapped to reduced nuclear import, replication, and translation, and deficient viral budding. Using reassortant H1N1:H5N1 viruses, we found that the combined introduction of nucleoprotein (NP) and hemagglutinin (HA) from H5N1 was necessary and sufficient to enable H1N1 growth. Overall, this study suggests the absence of one or more cellular factors in HeLa cells that results in abortive replication of H1N1, H3N2, and LPAI viruses, but can be circumvented upon introduction of H5N1 NP and HA. Further understanding of the molecular basis of this restriction will provide important insights into virus-host interactions that underlie IAV pathogenesis and tropism.



Many zoonotic avian influenza A viruses have successfully crossed the species barrier and caused mild to life-threatening disease in humans. While human-to-human transmission is limited, there is a risk for these zoonotic viruses to acquire adaptive mutations to efficiently propagate and cause devastating human pandemics. Therefore, it is important to identify viral determinants that provide these viruses with a replicative advantage in human cells. Here, we tested growth of influenza A virus in a subset of human cell lines and found that abortive replication of H1N1 viruses in HeLa cells can be circumvented upon introduction of H5N1 HA and NP proteins. Overall, this work leverages the genetic diversity of multiple human cell lines to highlight viral determinants that could contribute to H5N1 pathogenesis and tropism.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Influenza A; H1N1; H3N2; H5N1; Avian Influenza; Viral pathogenesis.


Characterization of #Usutu virus NS5 protein. #Polymerase activity, protein-protein interaction and cellular localization (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Characterization of Usutu virus NS5 protein. Polymerase activity, protein-protein interaction and cellular localization.

L. Albentosa-González, P. Clemente-Casares, R. Sabariegos, A. Mas

DOI: 10.1128/AAC.01573-19



Usutu virus (USUV) has become increasingly relevant in recent years with large outbreaks that sporadically have affected humans, being reported in wildlife. Similarly to the rest of flaviviruses, USUV contains a positive single-stranded RNA genome which is replicated by the activity of the non-structural protein 5 (NS5). USUV NS5 shows high sequence identity with the remaining viruses in this genus. This permitted us to identify the predicted methyl-transferase domain and the RNA-dependent RNA polymerase domain (RdRpD). Owing to their high degree of conservation, viral polymerases are considered priority targets for the development of antiviral compounds. In the present study, we have cloned and expressed the entire NS5 and the RdRpD in a heterologous system and have used purified preparations for protein characterizations. We have determined the optimal reaction conditions by investigating how variations in different physicochemical parameters, such as buffer concentration, temperature, and pH, affect RNA polymerization activity. We also found that USUV polymerase, but not the full-length NS5, exhibits cooperative activity in the synthesis of RNA, and that the RdRp activity is not inhibited by Sofosbuvir. To further examine the characteristics of USUV polymerase in a more biological context, we have expressed NS5 and the RdRpD in eukaryotic cells and analyzed its subcellular location. NS5 is predominantly found in the cytoplasm, a significant proportion is directed to the nucleus and this translocation involves nuclear location signals (NLS) located, at least, between the MTase and RdRpD domains.

Copyright © 2019 Albentosa-González et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Keywords: Arbovirus; Flavivirus; Usutu virus; Viral pathogenesis.


#Key #aminoacid residues of #neuraminidase involved in #influenza A virus #entry (Pathog Dis., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Pathog Dis. 2019 Nov 8. pii: ftz063. doi: 10.1093/femspd/ftz063. [Epub ahead of print]

Key amino acid residues of neuraminidase involved in influenza A virus entry.

Chen F1, Liu T1, Xu J1, Huang Y1, Liu S1, Yang J1.

Author information: 1 Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China.



Generally, influenza virus neuraminidase (NA) plays a critical role in the release stage of influenza virus. Recently, it has been found that NA may promote influenza virus to access the target cells. However, the mechanism remain unclear. Here, we reported that peramivir indeed possessed anti-influenza A virus (IAV) activity in the stage of viral entry. Importantly, we verified the critical residues of influenza NA involved in the viral entry. As a result, peramivir as an efficient NA inhibitor could suppress the initiation of IAV infection. Furthermore, mutational analysis showed NA might be associated with viral entry via amino acids residues R118, E119, D151, R152, W178, I222, E227, E276, R292 and R371. Our results demonstrated neuraminidase must contain the key amino acid residues can involve in IAV entry.

© FEMS 2019.

KEYWORDS: Influenza A virus; neuraminidase; neuraminidase active site; peramivir; viral entry

PMID: 31702775 DOI: 10.1093/femspd/ftz063

Keywords: Influenza A; Peramivir; Antivirals; Viral pathogenesis.