title: Detailed morphological characterisation of Hendra virus infection of different cell types using super-resolution and conventional imaging.
abstract: BACKGROUND
  Hendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells.
  
  
  METHODS
  A range of primary cells and immortalised cell lines were infected with HeV, fixed at various time points post-infection, labelled for HeV proteins and imaged by confocal, super-resolution and transmission electron microscopy.
  
  
  RESULTS
  Significant differences were noted in viral protein distribution depending on the infected cell type. At 8 hpi HeV G protein was detected in the endoplasmic reticulum and M protein was seen predominantly in the nucleus in all cells tested. At 18 hpi, HeV-infected Vero cells showed M and G proteins throughout the cell and in transmission electron microscope (TEM) sections, in pleomorphic virus-like structures. In HeV infected MDBK, A549 and HeLa cells, HeV M protein was seen predominantly in the nucleus with G protein at the membrane. In HeV-infected primary bovine and porcine aortic endothelial cells and two bat-derived cell lines, HeV M protein was not seen at such high levels in the nucleus at any time point tested (8,12, 18, 24, 48 hpi) but was observed predominantly at the cell surface in a punctate pattern co-localised with G protein. These HeV M and G positive structures were confirmed as round HeV virions by TEM and super-resolution (SR) microscopy. SR imaging demonstrated for the first time sub-virion imaging of paramyxovirus proteins and the respective localisation of HeV G, M and N proteins within virions.
  
  
  CONCLUSIONS
  These findings provide novel insights into the structure of HeV and show that for HeV imaging studies the choice of tissue culture cells may affect the experimental results. The results also indicate that HeV should be considered a predominantly round virus with a mean diameter of approximately 280 nm by TEM and 310 nm by SR imaging.
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. paul.monaghan@csiro.au.
authors.forename: Paul
authors.initials: P
authors.lastname: Monaghan
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. diane.green@csiro.au.
authors.forename: Diane
authors.initials: D
authors.lastname: Green
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. jackie.pallister@csiro.au.
authors.forename: Jackie
authors.initials: J
authors.lastname: Pallister
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. reuben.klein@csiro.au.
authors.forename: Reuben
authors.initials: R
authors.lastname: Klein
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. john.white@csiro.au.
authors.forename: John
authors.initials: J
authors.lastname: White
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. catherine.williams@csiro.au.
authors.forename: Catherine
authors.initials: C
authors.lastname: Williams
authors.affiliation: Department of Biochemistry and Molecular Biology, Melbourne, Australia. mpj@unimelb.edu.au.|ARC Centre of Excellence for Coherent X-ray Science, Melbourne, Australia. mpj@unimelb.edu.au.|Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, VIC, 3010, Australia. mpj@unimelb.edu.au.|Current Address: Biological Optical Microscopy Platform, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, VIC, 3010, Australia. mpj@unimelb.edu.au.
authors.forename: Paul
authors.initials: P
authors.lastname: McMillan
authors.affiliation: Department of Biochemistry and Molecular Biology, Melbourne, Australia. ltilley@unimelb.edu.au.|ARC Centre of Excellence for Coherent X-ray Science, Melbourne, Australia. ltilley@unimelb.edu.au.|Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, VIC, 3010, Australia. ltilley@unimelb.edu.au.|Current Address: Biological Optical Microscopy Platform, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, VIC, 3010, Australia. ltilley@unimelb.edu.au.
authors.forename: Leann
authors.initials: L
authors.lastname: Tilley
authors.affiliation: Leica Microsystems, CMS GmbH, Ernst-Leitz Strasse 17-37, Wetzlar, Germany. lampe@embl.de.|Current Address: European Molecular Biology Laboratory, Meyerhofstr 1, D-69117, Heidelberg, Germany. lampe@embl.de.|Current Address: Translational Lung Research Center (TLRC), Department Translational Pulmonology, University of Heidelberg, Im Neuenheimer Feld 350, D-69120, Heidelberg, Germany. lampe@embl.de.
authors.forename: Marko
authors.initials: M
authors.lastname: Lampe
authors.affiliation: Pirbright Institute, Pirbright, Woking, Surrey, GU240NF, UK. pippa.hawes@pirbright.ac.uk.
authors.forename: Pippa
authors.initials: P
authors.lastname: Hawes
authors.affiliation: CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, VIC, 3220, Australia. linfa.wang@csiro.au.|Duke-NUS Graduate Medical School, Singapore, Singapore. linfa.wang@csiro.au.
authors.forename: Lin-Fa
authors.initials: LF
authors.lastname: Wang
mesh_terms: D000818:Animals; D002460:Cell Line; D045404:Hendra Virus / Q000502:physiology* / Q000648:ultrastructure*; D006801:Humans; D008853:Microscopy; D061848:Optical Imaging; D019065:Virus Assembly