Table Of ContentMethods in
Molecular Biology 1442
Ralph A. Tripp
Patricia A. Jorquera
Editors
Human
Respiratory
Syncytial Virus
Methods and Protocols
M E T H O D S I N M O L E C U L A R B I O L O G Y
S eries Editor
J ohn M . W alker
S chool of Life and Medical Sciences
U niversity of Hertfordshire
H atfield, Hertfordshire, AL10 9AB, U K
F or further volumes:
h ttp://www.springer.com/series/7651
H uman Respiratory
Syncytial Virus
M ethods and Protocols
E dited by
R alph A . T ripp and Patricia A. Jorquera
College of Veterinary Medicine, University of Georgia, Athens, GA, USA
E ditors
R alph A . T ripp P atricia A . Jorquera
C ollege of Veterinary Medicine C ollege of Veterinary Medicine
U niversity of Georgia U niversity of Georgia
A thens , G A , U SA A thens , GA , USA
I SSN 1064-3745 ISSN 1940-6029 (electronic)
M ethods in Molecular Biology
I SBN 978-1-4939-3685-4 ISBN 978-1-4939-3687-8 (eBook)
D OI 10.1007/978-1-4939-3687-8
L ibrary of Congress Control Number: 2016933106
© Springer Science+Business Media New York 2 016
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P refa ce
R espiratory syncytial virus (RSV) is the prototypic member of the P neumovirus genus of the
P aramyxoviridae family [1]. RSV has a negative-sense single-stranded RNA genome com-
prised of 15,222 nucleotides that encodes for 10 major species of mRNA and 11 proteins.
The genome is transcribed sequentially from NS1 to L, with reduced expression levels along
its length. Three proteins are found on the envelope surface, the heavily glycosylated G,
fusion F, and small hydrophobic SH protein. The G and F proteins are the major antigens
involved in virus neutralization. However, the G protein is highly variable, which allows for
the classifi cation of RSV strains into different clades based on its nucleotide sequence [2].
R SV is the leading cause of lower respiratory tract infections such as bronchiolitis and
bronchopneumonia, as well as hospitalizations in infants, elderly, and immunocompro-
mised individuals [3]. No effective licensed therapies are readily available, but existing and
emerging point-of-care diagnostics and investigational RSV-specifi c antiviral inhibitors
offer promising progress [4]. Prophylactic antibodies have been successfully developed
against RSV [5]. However, their use is restricted to a small group of infants considered at
high risk for developing severe RSV disease. No RSV vaccine has been licensed to date, but
the availability of new tools to study the virus-host interaction, pathogenesis of RSV dis-
ease, and the ability to construct a wide variety of vaccines using different vaccine platforms
suggests that an RSV vaccine should be feasible [5].
T his book is intended to summarize the current techniques that have made this prog-
ress possible, ranging from protocols for virus growth, isolation, quantifi cation, and gen-
eration of recombinant RSV virus to procedures for the effi cient characterization of the
host immune response to RSV infection. These techniques are used in numerous laborato-
ries around the world and are, thus, the building blocks that support the majority of RSV
virus research.
Athens, GA, USA Ralph A. Tripp
Patricia A. Jorquera
R eferences
1 . T eng MN, Collins PL (1998) Identifi cation of the respiratory syncytial virus proteins required for for-
mation and passage of helper-dependent infectious particles. J Virol 72(7):5707–5716.
2 . A nderson LJ et al (1985) Antigenic characterization of respiratory syncytial virus strains with monoclonal
antibodies. J Infect Dis 151(4):626–633.
3 . L eung TF et al (2014) Epidemiology and risk factors for severe respiratory syncytial virus infections
requiring pediatric intensive care admission in Hong Kong children. Infection 42(2):343–350.
4 . S imoes EA et al (2015) Challenges and opportunities in developing respiratory syncytial virus therapeu-
tics. J Infect Dis 211(Suppl 1):S1–S20.
5 . B roadbent L et al (2015) Respiratory syncytial virus, an ongoing medical dilemma: an expert com-
mentary on respiratory syncytial virus prophylactic and therapeutic pharmaceuticals currently in clinical
trials. Infl uenza Other Respir Viruses 9(4):169–178.
v
C ontents
P reface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
C ontributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
1 H uman Respiratory Syncytial Virus: An Introduction . . . . . . . . . . . . . . . . . . . 1
P atricia A . Jorquera, L ydia A nderson, and R alph A . Tripp
2 R SV Growth and Quantification by Microtitration and qRT-P CR Assays. . . . . 13
H ayat C aidi, J ennifer L . H arcourt, and L ia M . H aynes
3 Q uantification of RSV Infectious Particles by Plaque Assay
and Immunostaining Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3
P atricia A . J orquera and R alph A . T ripp
4 D etection of RSV Antibodies in Human Plasma by Enzyme Immunoassays . . . 4 1
S amadhan J . J adhao and L arry J . A nderson
5 S ecretory Expression and Purification of Respiratory Syncytial Virus G
and F Proteins in Human Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
S amadhan J . J adhao and L arry J . A nderson
6 D evelopment of Human Monoclonal Antibodies Against Respiratory
Syncytial Virus Using a High Efficiency Human Hybridoma Technique . . . . . 6 3
G abriela A lvarado and J ames E . C rowe Jr.
7 R espiratory Syncytial Virus (RSV): Neutralizing Antibody,
a Correlate of Immune Protection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
P edro A . P iedra, A nne M . H ause, and L etisha A ideyan
8 Host Factors Modulating RSV Infection: Use of Small Interfering RNAs
to Probe Functional Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
L eon C aly, H ong-mei L i, and D avid J an
9 I n Vitro Modeling of RSV Infection and Cytopathogenesis
in Well-Differentiated Human Primary Airway Epithelial Cells
(WD-PAECs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 19
L indsay B roadbent, R emi V illenave, H ong G uo-Parke, I sobel D ouglas,
M ichael D. S hields , and U ltan F . P ower
1 0 R everse Genetics of Respiratory Syncytial Virus . . . . . . . . . . . . . . . . . . . . . . . . 1 41
C hristopher C . S tobart , A nne L . H otard , J ia M eng , and M artin L . M oore
1 1 U se of Minigenome Systems to Study RSV Transcription . . . . . . . . . . . . . . . . 1 55
M ichael N . T eng and K im C . T ran
1 2 S creening for Host Factors Directly Interacting
with RSV Protein: Microfluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
S arit K ipper , D orit A vrahami, M onika B ajorek, and D oron G erber
1 3 A Proteomic-Based Workflow Using Purified Respiratory Syncytial
Virus Particles to Identify Cellular Factors as Drug Targets . . . . . . . . . . . . . . . 1 75
T ra N guyen H uong, B oon H uan T an, and R ichard J . S ugrue
vii
viii Contents
1 4 M icroRNA Profiling from RSV-Infected Biofluids, Whole Blood,
and Tissue Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 95
Lydia A nderson, P atricia A . J orquera, and R alph A . T ripp
1 5 M ouse and Cotton Rat Models of Human Respiratory
Syncytial Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
P enny A . R udd, W eiqiang C hen, and S uresh M ahalingam
1 6 I n Vivo Assessment of Airway Function in the Mouse Model . . . . . . . . . . . . . 2 19
A zzeddine D akhama and E rwin W . G elfand
1 7 E valuation of the Adaptive Immune Response
to Respiratory Syncytial Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
C ory J . K nudson, K ayla A . W eiss , M egan E . S toley , and S teven M . V arga
I ndex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 45
C ontributors
L ETISHA A IDEYAN • D epartments of Molecular Virology and Microbiology , B aylor College
of Medicine, H ouston, T X, U SA
G ABRIELA A LVARADO • D epartments of Pathology, Microbiology and Immunology,
The Vanderbilt Vaccine Center , V anderbilt University Medical Center, Vanderbilt
University, N ashville, T N, U SA
L ARRY J . A NDERSON • D epartment of Pediatrics, School of Medicine, E mory University,
A tlanta , G A, U SA
L YDIA A NDERSON • D epartment of Infectious Diseases, College of Veterinary Medicine,
Animal Health Research Center , U niversity of Georgia, A thens, G A, U SA
D ORIT A VRAHAMI • M ina and Everard Goodman Faculty of Life Sciences, Nanotechnology
Institute, B ar Ilan University, R amat Gan, I srael
M ONIKA B AJOREK • F aculty of Medicine, Section of Virology, I mperial College London,
L ondon, U K
L INDSAY B ROADBENT • D entistry and Biomedical Sciences, School of Medicine, Centre
for Infection and Immunity , Q ueen’s University Belfast, B elfast, U K
H AYAT C AIDI • G astroenteritis and Respiratory Viruses Laboratory Branch, Division
of Viral Diseases, National Center for Immunization and Respiratory Diseases , C enters
for Disease Control and Prevention (CDC) , A tlanta , G A, U SA
L EON C ALY • B iochemistry and Molecular Biology , M onash University, C layton, V IC ,
A ustralia
W EIQIANG C HEN • E merging Viruses and Infl ammation Research Group, Institute
for Glycomics, G riffi th University , N athan, Q LD , A ustralia
J AMES E . C ROWE J R • D epartments of Pathology, Microbiology and Immunology, Pediatrics,
The Vanderbilt Vaccine Center , V anderbilt University Medical Center, Vanderbilt
University, N ashville, T N, U SA
A ZZEDDINE D AKHAMA • A ssociate Professor at National Jewish Health and University of
Colorado Anschutz Medical Campus, C O, U SA
I SOBEL D OUGLAS • T he Royal Belfast Hospital for Sick Children, B elfast Health and Social
Services Trust, B elfast, U K
E RWIN W . G ELFAND • D epartment of Pediatrics, N ational Jewish Health, D enver, C O, U SA
D ORON G ERBER • M ina and Everard Goodman Faculty of Life Sciences, Nanotechnology
Institute, B ar Ilan University, R amat Gan, I srael
H ONG G UO-PARKE • D entistry and Biomedical Sciences, School of Medicine, Centre
for Infection and Immunity , Q ueen’s University Belfast, B elfast, U K
J ENNIFER L . H ARCOURT • G astroenteritis and Respiratory Viruses Laboratory Branch,
Division of Viral Diseases, National Center for Immunization and Respiratory Diseases ,
C enters for Disease Control and Prevention (CDC) , A tlanta , G A, U SA
A NNE M . H AUSE • D epartments of Molecular Virology and Microbiology , B aylor College
of Medicine, H ouston, T X, U SA; T ranslational Biology and Molecular Medicine,
B aylor College of Medicine, H ouston, T X, U SA
ix
