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Emerging Viruses 2016
12 TH September 2016
Lady Brodie Room, St Hilda’s College, Cowley Place
Oxford, United Kingdom
Email: [email protected]
Web: http://lpmhealthcare.com/emerging -viruses-2016
Twitter: @LPMHealthcare | @InfluenzaOxford | Hashtag: #EmergVir16
TABLE OF CONTENTS
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12:
General Information
Information for Presenters
Insurance and Liability
Disclaimer
Map
Podium Agenda
Podium Abstracts
Poster Abstracts
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GENERAL INFORMATION
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Event Dates:
12 September 2016
Event Website:
http://lpmhealthcare.com/emerging-viruses-2016
Venue:
Lady Brodie Room, St Hilda’s College, Cowley Place, Oxford, United Kingdom, Cowley Place,
Oxford, OX4 1DY, England, UK
Tel: +44 (0) 1865 276884, Website: http://www.st-hildas.ox.ac.uk
Registration Desk:
Located outside the meeting hall
Name Badges:
The College requests all delegates to wear name badges while on the premises to avoid any
confusion.
Refreshments/Lunch:
Outside the meeting hall
Mobile Phones:
As a courtesy to speakers and participants, please switch off your mobile phone during oral
presentations.
Speaker Presentations:
We will not be distributing speaker presentations. Therefore, if you interested in
presentation slides of any speakers, please get in touch with them directly.
Internet access:
Please use edurom if you can. Otherwise, WiFi Code and instructions for internet access via
your laptop/mobile device can be obtained at the time of registration.
Health and Safety:
Please do not leave your belongings unattended or in passageways and familiarise yourself
with emergency exits.
Smoking:
In addition to any local venue regulations, UK no-smoking regulations apply on the College
premises.
INFORMATION FOR PRESENTERS
SPEAKERS:
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Presentation standard will be data projection from a central PC. Your presentation is best brought in a
memory stick.
Macintosh will not be available. Therefore, if you are a Macintosh user please bring your own.
As a courtesy to other speakers and attendees please finish your talk absolutely within your allocated
time slot. (Guide: For a 20 minute talk, prepare 12-14 slides maximum ; for a 30 minute talk, prepare
20-22 slides maximum; allow 3-4 minutes for questions). Please check the agenda below for your
presentation schedule.
POSTER DISPLAY:
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Please leave your poster at the registration desk when you register.
There is no specific poster session. Instead the posters will be displayed in the
registration/refreshment area for full duration of the meeting.
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INSURANCE AND LIABILITY:
Participants are responsible for taking appropriate insurance cover (including health insurance) in connection with their
attendance of this event. The event organisers and hosts are not responsible for personal accidents, any travel costs, or
the loss of private property, and will not be liable for any claims. Event participants shall be responsible for compensating
any loss, should they cause any damage to the host’s property or the venue.
DISCLAIMER:
The information specified in oral and poster presentations, written abstracts, biographies and exhibitions come from
diverse sources and it is not in the capacity of event organisers to validate it, and is provided on an ‘as-is’ basis.
Responsibility for the literary and scientific content of the abstracts and the presentations, both oral and poster, remains
with the authors and the presenters. Therefore, the event organisers accept no responsibility for literary or scientific
correctness of this information, and shall accept no liability of any kind, should any of the information be incorrect. The
event organisers and hosts make no representation or warranty of gain of business or profits as a result of use of services
or information provided in connection with the even and shall not be liable for any direct or indirect damages, loss of
business, employment, profits or anticipated savings resulting from the use of the services or information provided in
connection with the event, in any country or court of law. Furthermore, the materials contained in the event handbook
are provided on the understanding that speakers or presenters have the right to their presentation in this manner.
Therefore, event organisers and hosts shall not be liable for infringement of third party rights by an event presenter,
participant, sponsor, supporter or exhibitor.
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This handbook is for use by the Emerging Viruses 2016 Oxford (12 September 2016) participants only.
© 2016 Copyright Information: Textual and graphical contents of this handbook are copyright of presenters, sponsors,
instructors and/or LibPubMedia Ltd, unless explicitly stated otherwise. No part of this handbook may be reproduced, distributed
or transmitted in any form or by any means, electronic or mechanical, including but not limited to, photocopy, recording, or any
other information storage or retrieval system, without the prior written permission of the legal copyright owners.
To train station
Bus stops to
London & airports
Bus stop from
London & airports
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PODIUM AGENDA (subject to change)
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Monday 12 September 2016 | Lady Brodie Room, Hall Building, St Hilda’s College
0930:
1020:
Registration, networking and welcome coffee
Welcome and Housekeeping
Session 1: Chair Dr Simon Scott
1030: Dr Janet Daly, University of Nottingham, UK
New twists on serological assays for emerging Bunya- and Flavi- viruses
1100: Dr Jason Long, Imperial College London, UK
Species difference in ANP32A underlies influenza A virus polymerase host restriction
1130: Dr Chinedu Ugwu, University of Cambridge, UK
Understanding the varying roles of Antigen presenting cells (APC) in emerging virus zoonoses
1150: Dr Alain Kohl, University of Glasgow, UK
Zika virus: properties and interactions with host cells
1220: Lunch, networking, posters
Session 2: Chair Dr Edward Wright
1300: Professor Jonathan Heeney, University of Cambridge, UK
Reservoirs and the evolution of Zoonotic Pathogens
1330: Professor Ian Goodfellow, University of Cambridge, UK
Provision of lab support during and after the 2013-16 Ebola epidemic in Sierra Leone
1400: Miss Pramila Rijal, University of Oxford, UK
Isolation and characterization of human monoclonal antibodies to Ebola Virus glycoprotein with an aim for therapeutic use
1420: Professor Sunetra Gupta, University of Oxford, UK
The role of epitopes of limited diversity in the antigenic evolution of influenza
1450: Dr Keith Grehan, University of Kent, UK
Viral Pseudotypes and In-silica analysis tools for studies of pathogen host switching
1510: Dr Ashley C Banyard, Animal and Plant Health Agency, UK
The challenge of emerging lyssaviruses
1540: Refreshments, networking, posters
Session 3: Chair Dr Nigel Temperton
1610: Professor Maria Zambon (Keynote), Public Health England, UK
Emerging viruses: The UK response
1640: Dr Lorraine McElhinney, Animal and Plant Health Agency, UK
Increased detection of hantaviruses in the UK
1710: Dr Mark Page, The National Institute for Biological Standards and Control (NIBSC), UK
Responding to the need for reference standards in an outbreak situation
1740: Miss Emma Bentley, University of Westminster, UK
Exploiting pseudotypes for emerging virus research
1800: Close
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PODIUM ABSTRACTS
New twists on serological assays for emerging Bunya- and Flavi-viruses
Janet M Daly1, Ben Maddison2, Barnabas King3, Rachael Tarlinton1, Kevin Gough1
1
School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, LE12 5RD, UK
ADAS Biotechnology Group, School of Veterinary Medicine and Science, University of Nottingham, Sutton, UK
3
School of Life Sciences, University of Nottingham, Nottingham, NG7 2UH, UK
2
Serological assays are an important component of the response to emerging viruses for diagnosis, particularly
of viruses that can only be transiently detected in body fluids, and surveillance to monitor the spread of virus.
The emergence of Schmallenberg virus (SBV) in Europe in 2011 sparked a demand for serological assays that
was rapidly met by a commercially-available ELISA. How the latest techniques and modern technologies can be
applied to address the need for diagnostic tests for veterinary use that are more affordable and user-friendly
(allowing them to be used pen-side), while retaining sensitivity and specificity, will be presented. Many of
these same criteria are equally applicable to the development of diagnostic tests for human disease in lowand middle-income countries – the WHO assured criteria are that a test should be affordable, sensitive,
specific, user-friendly, robust and rapid, equipment-free and deliverable (to remote areas). The translation of
what we have learned from development of tests for SBV to other emergent viruses such as Zika virus will be
also be discussed.
Species difference in ANP32A underlies Influenza A virus polymerase host restriction
Jason Long, Giotis Stathis, Oliver Moncorgé, Rebecca Cocking, Ruth Elderfield, Mike Skinner and Wendy
Barclay
Imperial College London, UK
Influenza A viruses originate in wild birds, and have crossed over to human hosts in pandemic events after
which their ability to accumulate drift mutations allows them to continue to circulate causing seasonal
epidemics. Despite a large avian reservoir, pandemics are infrequent because avian viruses require adaptation
at several different steps to enable an efficient replication cycle in mammals. We have discovered the
underlying mechanism for the host range adaptation by the virus polymerase. Avian influenza polymerases do
not function efficiently in human cells. Twenty five years ago the notorious mutation of the PB2 subunit at
position 627 was first described to adapt the influenza polymerase to mammalian cells. However, until now,
the mechanism by which this adaptation overcomes the host range barrier has remained elusive. We screened
hamster:chicken radiation hybrid cells using a minireplicon polymerase assay for their ability to support avian
influenza polymerase activity. Using microarray analysis, genes present in positive and negative hybrids were
compared, narrowing the list of possible cellular factors responsible to a specific length of a chicken
chromosome. By cloning a selection of these genes we identified a highly conserved protein present in avian
and mammalian species but with a key sequence difference in the form of a repeat/insertion in the bird
genomes that result in an additional stretch amino acids in the avian homologue. Overexpressing the chicken
homologue rescued avian influenza polymerase activity in human cells, and also increased avian influenza virus
replication in infected cells. When the avian sequence was inserted into the human homolog, the resulting
chimeric protein could now facilitate avian influenza polymerase activity. The importance of this cellular factor
in supporting virus replication was further confirmed by knockdown in human and avian cells. It appears that
avian influenza viruses have evolved to utilize this factor to support their replication cycle but are unable to
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utilize the mammalian homologue unless they undergo PB2 adaptation. This then explains the host range
barrier and why PB2 mutation is the most potent way to overcome it.
Understanding the varying roles of Antigen presenting cells (APC) in
emerging virus zoonoses
Chinedu A Ugwu1, Barbara Blacklaws1, Andrew Bosworth2 Miles W Carrol2, Jonathan L Heeney1
1
2
Department of Veterinary Medicine, University of Cambridge, CB3 OES, Cambridge, UK
Public Health England, Porton Down, Salisbury SP4 0JG, UK
Antigen presenting cells (APCs) are a diverse population of specialised cells of the immune system that survey
the host environment for the presence of harmful self and foreign antigen in order to initiate appropriate
immune responses. They include subtypes of dendritic cells (DCs), monocyte/macrophages, and B cells
amongst others. Dendritic cells and macrophages are two of the professional APCs in the body. While DC are
known for their ability to induce adaptive T cell responses, macrophages are recognised for their phagocytic
activity and ability to mediate wound repair. Because of their role as sentinels they often inadvertently serve
as targets for most emerging virus zoonoses, including agents of viral hemorrhagic fever (VHF). Ebola and Lassa
fever viruses are two important RNA viruses implicated in viral viral hemorrhagic fever syndrome with recent
outbreaks in West Africa. Both viruses infect and replicate in APC and the outcome of their interaction
determines the type of immune response elicited. These interactions also contribute to the
immunopathologies associated with the disease. While Ebola produces a cytokine storm, infection with Lassa
virus results in immunosuppression. We are using a minigenome virus like particle (trVLP) model in an in vitro
system to study how two different viruses use different mechanisms to produce the same clinical outcome of
viral hemorrhagic fever and to understand the role of APC in shaping this outcome. This data will provide a
better understanding of the important early events that influence the outcome of viral haemorrhagic fever
infections, while opening new opportunities for early therapeutic interventions and improved clinical
management.
Zika virus: properties and interactions with host cells
Alain Kohl
MRC-University of Glasgow Centre for Virus Research, 464 Bearsden Road, Glasgow G61 1QH, Scotland, UK
Zika virus is an emerging arbovirus linked to neurological disorders and congenital Zika syndrome. It is
transmitted by mosquitoes though in some cases sexual transmission has been described. Research on the
biology and genetics of this virus had been neglected for many decades as it was not associated with
significant human disease. There is limited information on viral sequences, virus-host interactions and other
aspects relating to the biology of this virus. Here I will present data on the viral sequence properties of a Zika
virus isolate from Brazil and virus interactions with host immune responses.
Reservoirs and the evolution of Zoonotic Pathogens
Jonathan L Heeney
The Lab of Viral Zoonotics, Department of Veterinary Medicine, University of Cambridge, Cambridge, UK
What viral and host dynamic changes occur that allow zoonotic spill-overs and facilitate viral adaptation to a
new host species? Here I will use examples of a spectrum of viral infections (eg; Rabies, Influenza, HIV-1, HIV-2
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and Ebola) to address the question of the evolution of zoonotic viral infections in the context of those known
to adapt and to become persistent human infections (in populations and individuals). I will end with examples
from the most recent Ebola epidemic in West Africa and will use evidence of the scale of the West Ebola
outbreak that gave an unprecedented number of survivors, enabling a recognisable persistent human
reservoir. The West African human Ebola reservoir gave rise to post-outbreak flare-ups, infections that were
especially evident at the tail end of the epidemic. I’ll end with considerations and possible adaptive
requirements that enable non-integrating RNA viruses to establish persistent infections within the human
population.
Provision of lab support during and after the 2013-16 Ebola epidemic in
Sierra Leone
Ian Goodfellow
University of Cambridge, UK
Abstract currently unavailable
Isolation and characterization of human monoclonal antibodies to Ebola
Virus glycoprotein with an aim for therapeutic use
Pramila Rijal1, Sean Elias2, Victoria O’Dowd3, Daniel Lightwood3, Matthew Edmans2, Julie Xiao1, Simon C
Mendelsohn2, Samara R Machado1, Catherine Cherry2, Kuan Huang4, Xiao-Ning Xu5, Terry Bakker3, Tess
Lambe2, Gillian Burgess3, Simon J Draper2, Alain R Townsend1
1
Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
Jenner Institute, University of Oxford, Oxford, UK
3
UCB, Slough, UK
4
Division of Pediatric Infectious Diseases, Chang Gung Children’s Hospital, Taiwan
5
Imperial College, Chelsea & Westminster Hospital, London, UK
2
The recent Ebola outbreak in 2014 in West Africa resulted in more than 28,000 reported cases with >11,000
deaths. Ebola virus belongs to the Filoviridae which are enveloped and contain a non-segmented, singlestranded negative sense RNA genome. Glycoprotein (GP) is the only surface glycoprotein and the main target
of host immunity. We have isolated and characterized 84+ monoclonal antibodies from 11 human volunteers
vaccinated with a priming dose of ChAd3-EBOVZ (a chimp adenovirus vector encoding Zaire GP; developed by
GSK/NIH) and booster MVA-EBOZ BN/Filo (a modified vaccinia encoding GP from Zaire and Sudan Ebola and
Marburg virus). About half of the antibodies neutralize an Ebola surrogate virus we have developed, composed
of an inactivated influenza core coated in a recent EBOV-Zaire strain GP that can be handled in BSL 2
containment. Neutralization IC50 titers range from 0.1 to 40 μg/ml. Most of the neutralizing mAbs have high
avidity. VH gene usage is diverse within the collection of mAbs, and within individuals. These mAbs are close to
germline sequence and even the mAbs generated from memory cells carry few somatic mutations, as
expected for a response to a novel antigen. Almost one tenth of the antibodies recognize the mucin-like
domain (MLD) of the glycoprotein and are non-neutralizing. Some mAbs have been defined, based on binding
cross-inhibition, to recognize non-overlapping epitopes in the glycan cap, and GP1-GP2 interface or base of the
GP. This epitope mapping is in accordance with the results of binding of some mAbs to GP epitopes on a yeast
display library. There are also many antibodies cross-reactive to Sudan and Bundibugyo GP. One example is
mAb 66-3-9C binding to a conserved peptide GEWAFWET/N in GP1. We have formulated cocktails of
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neutralizing and cross-reactive antibodies targeting independent sites which should prevent the selection of
escape mutants. We are ready to test them for protection in the guinea pig animal model.
The role of epitopes of limited diversity in the antigenic evolution of
influenza
Sunetra Gupta
Professor of Theoretical Epidemiology, Department of Zoology, University of Oxford, Oxford, UK
Targets of natural immunity on the influenza virus are currently seen to be polarised between (i) highly
immunogenic (and protective) epitopes of high variability, and (ii) invariant epitopes of low immunogenicity.
Together, these form the backbone of the theory of ‘antigenic drift’ whereby the virus population slowly and
incrementally acquires changes in highly variable epitope regions requiring vaccines directed against these
sites to be continuously updated, with the only other alternative being seen as the artificial boosting of
immunity to invariant epitopes of low natural efficacy. By contrast, we propose that the antigenic evolution of
influenza is best explained by postulating the existence of highly immunogenic epitopes of limited variability
(Recker et al., PNAS 2007) – a theory that has come to be known as ‘antigenic thrift’. Under these
circumstances, most influenza strains are in competition with each other because they share epitopes in
regions of limited variability. Thus, although new strains may be generated constantly through mutation, most
of these cannot expand in the host population due to pre-existing immune responses against their less variable
epitopes. An important corollary of this model is that universal vaccines may be constructed by identifying
such protective epitopes of low variability: these vaccines would also have the potential to protect against
newly emerging strains of influenza.
Viral Pseudotypes and In-silica analysis tools for studies of pathogen host
switching
Keith Grehan1, 2, Edward Wright2, Nigel Temperton1
1
2
University of Kent, Chatham, Kent, UK
University of Westminster, London, UK
Novel viral diseases are frequently the result of host switching or “spillover”, in which a virus established in
one species infects a new host species. With their short generation times and relatively high mutation rates
RNA viruses are exceptionally accomplished at rapidly adapting to the changing environment of their hosts.
It is this constant adaptation that has led to numerous incidents of host switching by viruses and of
particular importance is the zoonotic viruses that switch from non-human hosts to human hosts often with
severe consequences. Members of the Filoviridae, such as Ebola virus (likely spread from Bats to humans
and other primates) the Coronaviridae such as Middle East respiratory syndrome coronavirus MERS-CoV
(found in camels and humans) are examples of zoonotic viruses that have made a switch from a non-human
animal hosts to humans, often causing a major public health treat in the process. In order to understand the
processes that drive these switching events it is essential that we develop an understanding of the
evolutionary pressures that shape the viral genomes and allow certain viruses to make the shift from one
host to another. The development of in-silica tools to study selective pressure and adaptation has opened
up the possibility for virologists to study viral evolution in greater detail than ever before, and the increasing
use of tools such as viral pseudotypes allows the results of such analysis to be rapidly and safely tested in a
“wet lab” environment. In this study we investigate the potential for in-silica analysis of selective pressure,
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as measured via the ratio of non- synonymous (dN) to synonymous (dS) mutations, to inform wet lab
experiments studying mutagenesis of both the MERS-CoV and Ebola virus pathogens.
The challenge of emerging lyssaviruses
Ashley C Banyard1, Denise A Marston1, Anthony R Fooks1,2
1
2
Wildlife Zoonoses and Vector Borne Diseases, Animal and Plant Health Agency, Addlestone, Surrey, UK
Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool, UK
The lyssaviruses constitute a group of viruses that cause 100% fatality following the establishment of
symptomatic infection. The archetypal lyssavirus is rabies virus, historically one of the most feared pathogens
on the planet, and one that globally causes over 65,000 deaths every year. Alongside rabies virus, the genus
comprises a number of genetically related viruses that cause fatal encephalitis clinically indistinguishable from
rabies. Whilst the human burden of these non-rabies lyssaviruses remains unclear, fatalities have been
reported. Furthermore, novel lyssaviruses continue to be isolated from different species globally although
most commonly, novel isolates are described in bats. Tools that can be used to either pre-immunise individuals
(vaccines) against rabies or treat potential exposures to virus (vaccines and rabies immunoglobulin) in
prodromal periods as post-exposure treatments have been available for decades. Despite these tools, the
continued annual mortality rate remains, predominantly through a lack of availability of these preparations in
areas where the virus is endemic. Importantly, whilst currently available pre- and post-exposure tools are able
to protect against all detected strains of rabies, their ability to protect individuals from the other lyssaviruses
varies significantly. The lyssavirus glycoprotein is the sole target for virus neutralising antibodies induced by
vaccination and targeted by rabies immunoglobulin yet divergent lyssaviruses are not neutralised by these
preparations. Here we describe the current situation regarding the continued discovery of novel lyssaviruses
and the challenges to the development of tools to counteract potential infection with antigenically divergent
members of this important group of viruses.
Emerging viruses: The UK response
Maria Zambon
Public Health England, UK
Each new virus epidemic brings misery to affected human populations, in unique ways. In the last 15 years, we
have experienced the emergence and spread of Severe Acute Respiratory Syndrome (SARS), H5N1 and H7N9
influenza A viruses, pandemic influenza A(H1N1)pdm09, Middle Eastern Respiratory Syndrome (MERS) and
Ebola virus disease, and most recently in 2015–16, Zika virus. The wider societal impact that such infectious
disease events can cause has been amply demonstrated with Ebola virus in West Africa. Each of the viruses
mentioned above occupies a different ecological niche, with diverse impact on the human population as a
result of transmission characteristics, host immune response and disease pathogenesis. Serious
complications and deaths from Zika virus infection have not been common: most infections are
asymptomatic or very mild, although there is an association with neurological complications such as
Guillain–Barré syndrome. The key issue, however, is the impact of infection on pregnancy. Proportionate
public health control measures for populations are required in each instance of a possible emerging virus.
For most emerging viruses, classical control measures of contact tracing and quarantine will eventually
break chains of transmission between humans following zoonotic infection, when human-to-human
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transmission occurs and infectiousness is related to symptomatic illness, but this is not always the case.
Recent examples of national responses to a range of possible emerging infections in 2015/16 including
parechovirus, hepatitis E virus and Zika will be discussed.
Increased Detection of Hantaviruses in the United Kingdom
Lorraine M McElhinney1,2, Denise A Marston1, Ellen Murphy2, Robert Smith3, Tim Brooks2,4, Charlotte
Featherstone5, Anthony R Fooks1,2
1
Wildlife Zoonoses and Vector Borne Diseases, Animal & Plant Health Agency (Weybridge), UK
Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool University, UK
3
Public Health Wales (Cardiff), UK
4
Public Health England (Porton), UK
5
Animal & Plant Health Agency (Thirsk), UK
2
Hantavirus species are the etiological agents of haemorrhagic fever with renal syndrome (HFRS) and are
responsible for thousands of human cases per year in Europe. The majority of HFRS cases in Europe are due to
the bank vole associated hantavirus, Puumala Virus. The hantavirus associated with brown rats (Seoul Virus,
SEOV) is responsible for increasing numbers of urban hantavirus infections worldwide. However, the
prevalence of rodent and human cases associated with SEOV in Europe have long been considered to be low
and speculated to be driven by the sporadic introduction of infected brown rats (Rattus norvegicus) via ports.
SEOV was previously believed to cause mild to moderate HFRS but has recently been linked to a number of
severe HFRS cases with acute renal failure in the UK. SEOV has been identified in both wild and pet rats in the
UK, causing disease in individuals exposed to the rat excreta. In July 2015, an outbreak of SEOV associated
HFRS in South Wales was traced to a number of domestic and commercial facilities breeding rats for reptile
food. The outbreak data will be presented. Our findings suggest that SEOV, whilst localized, may be more
prevalent in European brown rats than previously believed and may contribute to a greater number of the
reported HFRS cases in Europe.
Responding to the need for reference standards in an outbreak situation
Page M1, Mattiuzzo G1, Myhill S1, Ashall J1, Hassall, M1, Wilkinson D1, Wright E2, Temperton N3, Efstathiou S1,
Minor P1
1
National Institute for Biological Standards and Control, South Mimms, Herts, UK
Viral Pseudotype Unit, Faculty of Science and Technology, University of Westminster, London, UK
3
University of Kent, UK
2
During human epidemic disease outbreaks, methods to evaluate the disease are required to be in place in the
shortest time frame possible. These include diagnostic assays, such as nucleic acid amplification technologies,
and serology assays to monitor both candidate vaccine efficacy and disease epidemiology. Such assays are
rarely in place when an outbreak occurs or they are not fully calibrated or validated, leading to uncertainties in
data reliability. Central to validating assay results is the availability of suitable control or reference materials,
such as WHO endorsed International Standards. To address the problem of providing reference materials
contemporaneously to the epidemic, we have developed a platform technology based around lentiviral
vectors and have applied this to the recent Ebola outbreak. Lentiviral vectors were used to produce synthetic
NAT reagents by packaging genomic RNA into virus like particles. A particular advantage is that the reagents
are safe, non-infectious and non-replicative and therefore overcomes biosafety issues associated with high
hazard group viruses requiring appropriate containment facilities. The reagents can also be used as a reference
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materials to control both the extraction process andthe amplification reaction. Having the ability to freeze-dry
these reference materials increases both the lifetime of the standard and its utility in resource limited
laboratories where sample storage may be inadequate. For the antibody reagents lentiviral vectors were
utilised to produce pseudotyped viruses. These were used principally for assessing antibody neutralising
activity in plasma/serum of infected and convalescent individuals or vaccinees. This guided the selection of
candidate antibody standards. Therefore, the lentiviral platform offers a flexible solution when establishing
assay standards and can be readily applied to RNA viruses such as those on the WHO priority pathogens list.
Exploiting Pseudotypes for Emerging Virus Research
Emma M Bentley1, Giada Mattiuzzo2, Ruqiyo Ali1, Daniel Horton3, Davide Corti4,5, Ashley C Banyard6, Anthony R
Fooks6,7, Mark Page2 and Edward Wright1
1
Faculty of Science and Technology, Viral Pseudotype Unit, University of Westminster, London, UK
National Institute for Biological Standards and Control (NIBSC), Potters Bar, UK
3
School of Veterinary Medicine, University of Surrey, Guildford, Surrey, UK
4
Humabs BioMed SA, 6500 Bellinzona, Switzerland
5
Institute for Research in Biomedicine, Università della Svizzera Italiana, 6500 Bellinzona, Switzerland
6
Wildlife Zoonoses and Vector Borne Diseases, Animal and Plant Health Agency, Addlestone, Surrey, UK
7
Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool, UK
2
Emerging viruses pose a continuing threat to human and animal health. In the last 30 years there has been a
steady increase in the number of outbreaks, which is highlighted by a recent increase in research advances in
the development of efficacious vaccines, antivirals and improved diagnostics. It is also reflected in heightened
media coverage surrounding outbreaks. Studies that require the handling of highly pathogenic viruses are
generally restricted by the need for these to be undertaken in high containment, BSL 3/4 facilities. This
bottleneck can be alleviated by the use of replication-defective pseudotyped virus (PV), which can be handled
in low BSL 1/2 containment laboratories. In response to the recent outbreak of Ebola virus disease in West
Africa, we sought to develop a panel of filovirus PVs. Highest titres were achieved using lentiviral cores and
transfecting HEK 293T cells. Evaluation of target cells also identified HEK 293T cells as most permissive to
infection, yet CHO-K1 cells offered the clearest neutralisation data. This filovirus PV neutralisation assay
offered a highly accessible, low-containment method for serological assessments, such as that needed for
vaccines and antiviral drugs. Further to this we have undertaken work demonstrating the inherent flexibility of
the PV platform. While lyssaviruses had previously pseudotyped efficiently, the Arctic-like rabies virus (AL
RABV) lineage failed to generate infectious PV. Construction of chimeric AL RABV glycoproteins, splicing the
ecto- and transmembrane domains with the cytoplasmic domain of vesicular stomatitis virus, led to a
significant increase in PV titres (11.3 – 83.3 fold; p <0.0005). This did not alter the serological profile of the
glycoprotein and allowed neutralisation assays to be undertaken. Consequently, the efficacy of current
vaccines and antivirals against this RABV lineage was tested.
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POSTER ABSTRACTS
Establishing a WHO International Reference Reagent for Ebola virus
antigen
Sophie H Myhill, Giada Mattiuzzo, Mark Page, James Ashall, Dianna Wilkinson, Gillian Cooper, Kaetan Ladhani
and Stacey Efstathiou
Department of Virology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK
NIBSC is developing an antigen Reference Reagent for Ebola virus (EBOV) to monitor the performance of
diagnostic assays, as part of the World Health Organization’s (WHO) response to the 2013-2016 EBOV
outbreak in Western Africa and to provision for any further outbreaks. While the preferred Ebola diagnostic
assays used during the outbreak have been PCR-based methods, these could take up to 5 days from collection
of the sample, transport to special facilities and analysis. At the clinic, to give a fast diagnosis to patients
suspected of having Ebola virus infection, point of care (POC) tests would allow a more rapid, although less
sensitive and less specific, alternative. To date, three POC antigen capture tests have approved for emergency
deployment by the WHO, all based on the detection of EBOV viral protein 40 (VP40) as the target antigen.
Candidate materials were either produced in-house at NIBSC or donated from commercial organisations; these
underwent preliminary testing by an anti-EBOV VP40 ELISA developed in-house or using the approved POC
kits. Based on a consensus obtained from the results, nine preparations were formulated in human serum and
freeze-dried. A collaborative study was organised involving 8 laboratories from 5 different countries
worldwide, which tested the blind-coded samples in 3 independent assays. Based on data gained from the
collaborative study, a panel of 3 preparations have been recommended for endorsement as reference
reagents for Ebola virus antigen: a purified VP40 protein, a second preparation of VP40 protein at a
challenging concentration to control for assay sensitivity and a negative control. A report summarising the
collaborative study results and discussion will be submitted to the WHO Expert Committee on Biological
Standardization in October 2016 who will consider the establishment of this panel as reference materials for
Ebola virus antigen.
Establishment of reference material for serology of Zika virus infection
James Ashall, Giada Mattiuzzo, Mark Hassall, Sophie Myhill, Mark Page, Stacey Efstathiou
Department of Virology, National Institute of Biological Standards and Controls, Potters Bar, Hertfordshire, UK
Accurate diagnosis of Zika virus (ZIKV) infection is essential not only to minimise further transmission but also
to improve the understanding of viral epidemiology. ZIKV is usually associated with mild symptoms however
during the ongoing outbreak in South America, the rise of Zika-associated cases of microcephaly and
neurological disorders has prompted the WHO to declare an epidemic of international concern. Serological
tests are used for testing patients for zika infection due to short viraemia in plasma; however they generally
lack specificity and struggle to differentiate between the closely related Dengue virus. This highlights the needs
for specific reagents to assure assay performance. NIBSC has been establishing serological reference reagents
to ZIKV, according to our remit from the WHO. Possible candidates will include human infected/convalescent
© EMERGING VIRUSES 2016 | 12TH SEPTEMBER 2016 | OXFORD | UK
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plasma, human polyclonal antibodies from transgenic bovines and monoclonal antibodies. The candidate
material will be evaluated at NIBSC using an in-house developed anti-ZIKV ELISA, and a plaque reduction
neutralisation assay. We have also created monoclonal antibodies to ZIKV by hybridoma system, these may be
humanised by fusing the constant region of IgG or IgM. To test for cross-reactivity these candidates will be
tested on commercial kits and in-house PRNT for anti-dengue antibodies and other arboviruses. The candidate
materials provided as a blinded sample panel will be evaluated in a multicentre international collaborative
study to determine the most suitable material for use as a run control and primary reference reagent. The
results of this study will be taken to the WHO Expert Committee on Biological Standardization with the goal of
establishing the first serological international standard to ZIKV.
© EMERGING VIRUSES 2016 | 12TH SEPTEMBER 2016 | OXFORD | UK