Detailed morphological characterisation of Hendra virus infection of different cell types using super-resolution and con
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RESEARCH
Open Access
Detailed morphological characterisation of Hendra virus infection of different cell types using super-resolution and conventional imaging Paul Monaghan1*, Diane Green1, Jackie Pallister1, Reuben Klein1, John White1, Catherine Williams1, Paul McMillan2,3,4,5, Leann Tilley2,3,4,5, Marko Lampe6,7,8, Pippa Hawes9 and Lin-Fa Wang1,10
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. Conclusion: 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. Keywords: Hendra virus, Paramyxovirus, Confocal microscopy, Super-resolution microscopy, M protein, G protein, Cell lines
Background Hendra virus (HeV), along with the closely-related Nipah virus (NiV) and Cedar virus (CedPV), form the Henipavirus genus in the family Paramyxoviridae. Bats are the reservoir host for the henipaviruses and have been the source of a number of spill-over events. HeV outbreaks have so far been restricted to northern Australia [1], but NiV outbreaks * Correspondence: [email protected] 1 CSIRO Australia
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