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SID 5
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Research Project Final Report
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SID 5 (Rev. 3/06)
Project identification
SE2612
Development of RT-PCR and phylogenetic sequence
analyses for bluetongue virus serotype and strain
identification
Contractor
organisation(s)
The Institute for Animal Health,
Pirbright Laboratory,Ash Road,
Pirbright, Woking
Surrey
GU24 0NF
54. Total Defra project costs
(agreed fixed price)
5. Project:
Page 1 of 20
£
start date ................
01 July 2003
end date .................
01 October 2006
6. It is Defra’s intention to publish this form.
Please confirm your agreement to do so. ................................................................................... YES
NO
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Executive Summary
7.
The executive summary must not exceed 2 sides in total of A4 and should be understandable to the
intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together
with any other significant events and options for new work.
British breeds of sheep, horses and other livestock species are among the most susceptible
(world wide) to the diseases caused by the orbiviruses, including particularly bluetongue (BT) and
African horse sickness (AHS). Any outbreak of these diseases in the UK would be likely to cause
high levels of disease and mortality, during mid to late summer when the adults of Culicoides
vectors are most abundant. Increasing international trade in animals and animal products, including
trade, together with changes in the European climate, continue to elevate the threat posed by these
diseases to the UK. This is most clearly illustrated by the recent outbreaks of bluetongue in Europe.
Since 1998 five BTV types have been isolated from outbreaks in Mediterranean Europe and North
Africa, representing the largest epizootic of the disease ever recorded with the loss of over 1.8
million animals. During 2006, a further outbreak, caused by a sixth European type since 1998,
spread across the Netherlands, Belgium, Germany, Luxemburg and North West France. This
distribution is much further north in the region than ever before and is on the same latitudes as
southern England, further emphasising risks to the UK.
The work of this project underpins UK policy through an exploration of the significance of genetic
variation in the bluetongue virus. This has helped to develop improved diagnostic assays for the
virus, as well as methods to distinguish different types and strains of the virus. In order to achieve
the project aims it was initially necessary to develop novel and more effective techniques to
sequence specific orbivirus genes. The development of these methods was entirely successful and
they have been used to generate a molecular epidemiology database for different well-documented
strains of BTV (see www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/BTV-isolates.htm ). This
database can be used to identify which of the previously characterised strains, novel isolate of the
virus are closely related to. This generates a more accurate picture of the distribution, movement,
origins and persistence of individual BTV strains in the field than is possible by conventional
methods, and has established the identities of the different lineages of BTV that have invaded
Europe over the last 8 years. These methods and resources represent a set of new tools for
identification of bluetongue viruses, more rapidly and more effectively than ever before. They are
already helping us to identify and combat the disease, which may arrive in the UK in the near future,
SID 5 (Rev. 3/06)
Page 2 of 20
possibly during the summer of 2007.
In most of Mediterranean Europe and north Africa, outbreaks of BT were initially thought to result
exclusively from virus transmission by the biting midge Culicoides imicola. Although this vector is
still of central importance in southern Europe, BTV has also been transmitted in more northerly
regions (Bulgaria, Serbia, Macedonia Croatia, Holland, Germany, Belgium, Luxemburg and northeast France), beyond the range of C. imicola, indicating involvement of other vector species. The
predominant Culicoides species in these regions belong to C. pulicaris and C, obsoletus groups, both
of which are spread across much of northern Europe, including the UK. However, several of the
BTV serotypes that currently affect southern Europe have not spread to the north, or significantly
beyond the range of C. imicola, suggesting that there may be genetic variation between different
strains of the virus that can influence their transmissibility and spread by different vector species.
Selected strains of the bluetongue virus from different geographical regions were assessed for their
ability to cause a disseminated infection in laboratory reared adults of C.sonorensis (the North
American vector - from the colony at IAH Pirbright). Clear differences were detected in the
efficiency of infection, which do suggest that transmissibility is under at least the partial genetic
control of the virus. The project examined variation in a part of the virus genome (genome segment
10) that codes for a protein (NS3) that is thought to be involved in the efficiency of virus spread
within the insect. Three distinct clusters of Seg-10 sequences were detected that may relate to
environmental factors (e.g. transmission by different insect vector populations). Confirmation of
this hypothesis and attempts to identify markers for transmissibility will require further work.
The segmented nature of the BTV segmented genome, allows different virus strains to exchange
genome segments when they co-infect the same cell, by a process known as reassortment. This
process generates novel progeny virus strains containing genome segments derived from one or
other parental strain. The resulting progeny viruses will have biological characteristics (based on
their genome composition) which are therefore derived from, or may even be distinct from either
parent. Environmental selection of the novel progeny viruses may lead to emergence of strains that
have enhanced transmission characteristics, altered virulence or modified serological properties,
which may be more suited to the local ecosystem. Reassortment is therefore an important factor in
the variability of the virus and its adaptation in new ecosystems. The project has generated sequence
data for multiple genome segments from some of the ‘well-documented BTV isolates’ in the IAH
reference collection, demonstrating that reassortment has occurred in Europe involving on at least
two occasions genome segment 5 (NS1 gene). The biological significance of genome segment
reassortment in the field (within Europe) will require further study.
Live BTV vaccines were originally developed in South Africa and have been used to combat BTV
outbreaks in some European countries. However, questions have been raised concerning the safety
of these vaccines, and their effectiveness as part of a BTV eradication campaign in Europe, rather
than simply for protection of individual vaccinated animals in South Africa (their original purpose).
By developing methods (based on genome segment 2) to distinguish vaccine and field strains the
project has made it possible to assess the frequency of vaccine survival and transmission in the field.
By comparison of multiple genome segments to trace the origins of individual genome segments, it
is also possible to assess the ability of these vaccine strains to reassort (exchange genome segments)
with wild type viruses in the field. These studies have provided data relevant to use live vaccines in
Europe.
Further work to maintain and extend these molecular epidemiology studies for BTV and the other
economically important orbiviruses (e.g. African horse sickness virus (AHSV) and Epizootic
Haemorrhagic disease virus (EHDV)) are essential, to provide similar upgrades and maintenance of
diagnostic capabilities. This is particularly important now because the European climate changes
that have increased the spread and threat posed by BTV in Europe as a whole, have also increased
the risk posed by these other orbiviruses, which are transmitted by the same vector insects.
SID 5 (Rev. 3/06)
Page 3 of 20
Project Report to Defra
8.
As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with
details of the outputs of the research project for internal purposes; to meet the terms of the contract; and
to allow Defra to publish details of the outputs to meet Environmental Information Regulation or
Freedom of Information obligations. This short report to Defra does not preclude contractors from also
seeking to publish a full, formal scientific report/paper in an appropriate scientific or other
journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms.
The report to Defra should include:
 the scientific objectives as set out in the contract;
 the extent to which the objectives set out in the contract have been met;
 details of methods used and the results obtained, including statistical analysis (if appropriate);
 a discussion of the results and their reliability;
 the main implications of the findings;
 possible future work; and
 any action resulting from the research (e.g. IP, Knowledge Transfer).
Project Title: Development of RT-PCR and phylogenetic sequence analyses for
bluetongue virus serotype and strain identification
Introduction
British breeds of sheep, horses and other livestock species are among the most susceptible
(world wide) to the diseases caused by the orbiviruses, including particularly bluetongue (BT) and
African horse sickness (AHS). Any outbreak of these diseases in the UK would be likely to cause
high levels of disease and mortality, during mid to late summer (when the adults of Culicoides
vectors are most abundant), in the serologically-naive animal-populations of the UK. Increasing
international trade in animals and animal products, including trade with areas of the world where
Orbivirus diseases are endemic or cause periodic epizootics, together with changes in the European
climate, continue to elevate the threat posed by these diseases to the UK. This is most clearly
illustrated by the recent incidence of bluetongue in Europe. Since 1998 five BTV serotypes (1, 2, 4,
9 and 16) have been isolated from outbreaks in Mediterranean Europe and North Africa, which
collectively represent the largest epizootic of the disease ever recorded with the loss of over 1.8
million animals. During 2006, a further outbreak, caused by BTV- 8 (the sixth European serotype
since 1998) spread across the Netherlands, Belgium, Germany, Luxemburg and North West France.
This distribution is further north in the region than ever before and is on the same latitudes as
southern England, further emphasising risks to the UK.
The primary strategy for control of Bluetongue and African horse sickness in the UK is to
prevent their initial entry to the country. Although it may be impossible to prevent the entry of
wind bourn infected insect from neighbouring countries in Europe, it may be possible to exclude
infected animals or animal products if they can be rapidly and reliably identified. At the start of this
project BTV and related orbiviruses were detected and identified primarily by serological methods.
Competition ELISA is still used to rapidly identify (within 8 hours) serogroup/species specificity of
antibodies to the virus (BTV, AHSV, EHDV, etc) which are present in serum samples after
approximately 4-6 days pi. However, serum neutralisation tests (SNT) usually depend on virus
isolation to reliably determine orbivirus serotype, and can therefore be much slower (up to two
weeks). It is also impossible to distinguish individual virus lineages or strains within a single
serotype by SNT.
Orbivirus serotype is determined by the two outer capsid proteins, which are encoded by two of
the orbivirus viral genome segments (segments 2 and 6 of BTV, encoding VP2 and VP5
respectively). Since different virus strains can and do exchange genome segments by a process of
reassortment, serological typing methods cannot provide reliable information concerning variations
in the remaining eight segments of the viral genome.
SID 5 (Rev. 3/06)
Page 4 of 20
The distribution and circulation of BTV in the field is almost entirely dependent on transmission
between susceptible mammalian hosts, by adult females of certain Culicoides vector species (biting
midges). The vector competence of individual insects within a geographic region is therefore a
major factor concerning the incidence and/or potential for BT outbreaks. Once the virus has been
ingested by an adult female Culicoides its dissemination and transmission within and by the insect
reflect a complex interplay between the insect and the viral proteins and RNAs, involving infection
of insect gut cells, virus replication, virus release from these cell, and dissemination via the insect
haemoceol to reach and replicate in the salivary glands. Genetic factors, including variation in
either the virus or insect could therefore influence the efficiency of these processes and therefore
the overall ability of a specific insect population or individual, to transmit a specific virus strain or
lineage.
Selective insect breeding experiments, have shown that the characteristics of the transmission
barriers in the insect are inheritable and hence competence is at least partially determined by the
genetics of the individual insect and insect population concerned (Tabachnick, 1996), although the
underlying mechanisms for this influence remain poorly defined. It has also been suggested that
individual strains of BTV may evolve to become locally adapted to specific vector populations or
species of Culicoides, forming a virus-vector ‘episystem’ (Wilson et al 2000). According with this
theory, the distribution of specific bluetongue viruses may be limited to geographic areas where
populations or species of insects that are vector competent for those strains are circulating. For
example, the existence of two epidemiological systems (episystems) has been described in the New
World, characterized by the presence of two different vector species for BTV: C. sonorensis in
North America and C. insignis in South America.
The influence of changes in the viral genome through genetic shift (re-assortment), or the effect of
more subtle changes brought about through genetic drift (accumulation of point mutations over
time), have not been investigated as possible mechanisms influencing the infectivity of specific
virus strains for particular Culicoides vector species.
Part of this study was designed to make an assessment of the levels and significance of genetic
variations between BTV strains, particularly in genome segment 10, which has previously been
implicated in the release (and dissemination) of BTV from infected insect cells. The project will
also make an initial assessment of variations between BTV strains from different geographic
regions in terms of their ability to successfully replicate and be transmitted by Culicoides species
from different epysistems. The results of these studies may have relevance to the introduction of
“exotic” strains of BTV into new regions or continents (e.g. Europe), where their spread and ability
to cause disease would be dependent on indigenous species of Culicoides.
Overall the work of this project underpins UK policy through maintenance of existing diagnostic
capabilities for BTV and development of improved nucleic acid based techniques to identify
individual BTV serotypes and strains more rapidly and accurately than before.
Scientific Objectives as set out in the contract
1. Generate a nucleotide sequence database for genome segment 2 of different BTV serotypes
2. Analyse virus movement and epidemiology of BTV particularly within Europe.
3. Develop RT-PCR assays for serotyping BTV isolates
4. Analyse genome segment 10 of BTV strains from different regions of Europe (different
vectors).
5. Analyse frequency and significance of BTV genome segment reassortment within Europe.
The extent to which the objectives of the project have been met
Objective 1: Generate a nucleotide sequence database for genome segment 2 of different BTV
serotypes
SID 5 (Rev. 3/06)
Page 5 of 20
A database has been established containing full length sequence data for genome segment 2 and 6 from
over 200 isolates of BTV from around the world. Data for other genome segments is also being added.
Although these data which are still being analysed and published, they have formed the basis for long term
molecular epidemiology studies of these economically important viruses, and have made it possible to
determine the origins of the BTV strains that have invaded Europe.
Objective 2: Analyse virus movement and epidemiology of BTV particularly within Europe.
The ability to distinguish individual strains within each BTV serotype, by sequence analyses and
comparison of the resulting data to those previously generated for isolates from well documented origins
(molecular epidemiology studies), provides a way of distinguishing individual lineages of the bluetongue
virus in a manner that was previously impossible by conventional serological diagnostic methods. These
studies have precisely identified genome segment 2 from the different strains of BTV that have invaded
Europe (since 1998), even where they represent distinct lineages or topotypes within a single BTV serotype
(e.g. the different European strains of BTV serotypes 1 and 4). These studies have also made it possible to
distinguish European field strains from the live attenuated BTV vaccines that were used in parts of southern
Europe. Precise identification of different virus isolates makes it possible to track further movements within
Europe as well as their most likely original origins outside Europe.
Objective 3: Develop RT-PCR assays for serotyping BTV isolates
Based on the sequence data generated by the project, primers have been designed to selectively
amplify genome segment 2 from the 24 BTV serotypes. These will only amplify sequences from the
homologous serotype, allowing the virus type to be identified more rapidly (within 24 hours) than by
conventional serological assay (usually more than one week). The primers that were designed for some of the
serotypes were based on sequence data for only a very small number of available isolates, and must therefore
be reviewed when more isolates of these types become available and more sequences are generated.
Comparisons of geographically distinct isolates have demonstrated major and consistent variations between
eastern viruses (the Far East, India, Australia) and western isolates (Africa, North and South America). These
have made it possible to design primers for identification of eastern and western strains. Sequence data
generated for genome segments 1, 5, 7 and 8 were also used to design serogroup specific RT-PCR based
assays for BTV.
Objective 4: Analyse genome segment 10 of BTV strains from different regions of Europe (different
vectors).
Genome segment 10 was analysed from over 80 different isolates of BTV, including isolates
from different areas of Europe where transmission is thought to be mediated by different insect
species. Some of these data have already been published, and further publications are in
preparations. Initial evaluation of infection and transmission of different BTV strains by insects
from the laboratory colony of C. sonorensis, indicate the existence of significant strain variations,
which must therefore be under the genetic control of the virus itself, which may relate to variations
in genome segment 10.
Objective 5: Analyse frequency and significance of BTV genome segment reassortment within
Europe.
By comparing sequence data for other genome segments, particularly genome segment 5 (Seg-5
encoding the NS1) it has been possible to search for genome segment reassortment events that have
occurred within Europe. Indeed these data confirm that reassortment has occurred involving the
European BTV-16 field strain and the BTV-2 vaccine used in Italy. These data also indicate that
other reassortment events involving Seg-5 have occurred between BTV serotypes 9 and 16 in the
eastern Mediterranean region.
The methods used and results obtained
Objective 1: Generate a nucleotide sequence database for genome segment 2 of different BTV
serotypes
SID 5 (Rev. 3/06)
Page 6 of 20
During the early part of this study, novel methods were successfully developed and tested for the
synthesis of cDNA copies of dsRNA viral genomes and their subsequent sequence analysis (Maan
et al submitted). These techniques involve the attachment (ligation) of a self-priming molecule
(described as an ‘anchor-primer’) to both (3’) ends of the dsRNA genome segments. The ligated
RNAs are then purified by gel electrophoresis (to remove and unattached primer molecules),
allowing them to be copied reliably into full length cDNAs with no mis-priming. The cDNAs from
individual genome segments could be separated easily by electrophoresis, and sequenced using a
novel method involving ‘phased primers’ which contain part of the anchor-primer sequence and the
conserved sequences found at the 5’ end of the viral RNAs.
These methods allow direct generation of sequence data for specific virus genome segments
(without a lengthy cloning step) and a very significant ‘speeding up’ of the sequencing step for
virus strain identification. These methods have also made it possible to generate sequence data for
uncharacterised dsRNA genome segments much more rapidly, particularly where the absence of
existing sequence data makes the design of specific primers impossible. Sequencing of the near
terminal region makes it possible to design ‘walking-sequencing’ primers for the remainder of the
cDNA segment. The reliable synthesis of full length cDNAs from specific genome segments is also
of real value for further expression, or biochemical studies, of individual dsRNA virus proteins.
This novel sequencing strategy has now been used successfully to complete the analysis of both
genome segment 2 and 6 (encoding outer capsid proteins VP2 and VP5) as well as some other segments of
representative European isolates of BTV types 1, 2, 4, 8 9 and 16 (the six types that have caused recent
disease outbreaks in Europe). These data have been submitted to the international sequence databases and
form the basis of papers that are either published or in preparation.
Milestone 01/03 for Objective 1 - year three: Carry out nucleotide sequence analyses of
genome segment 2 of at least one isolate of all 24 BTV serotypes.
Genome segments 2 (and 6) have been successfully sequenced (full length) for the reference strains
of each of the 24 BTV serotypes (Maan et al 2007 – see publications). These studies show that Seg2 of the different BTV serotypes can be divided into 24 distinct groups based on nucleotide sequence, and
shows greater and more consistent levels of variation between different BTV serotypes than Seg-6
(www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/btv-seg-2.htm). This separation between serotypes correlates
with data concerning the direct interaction of VP2 with the neutralising antibodies and confirms its dominant
role in determination of virus serotype. These data also confirmed that sequence analyses of Seg-2 can be
used to distinguish the reference strains of the 24 virus types.
Objective 2: Analyse virus movement and epidemiology of BTV particularly within Europe.
An important initial aspect of the project was the establishment of a ‘reference collection’ of well
documented BTV isolates from different locations around the world. This was designed as a resource for
wider molecular epidemiology studies, to support the identification of virus strains both at IAH Pirbright and
in other laboratories around the world. Information concerning isolates stored in the collection is therefore
accessible via the internet, at: www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/BTV-isolates.htm . The
collection contains a large number of geographically and temporally referenced isolates, including the
‘standard’ strains of the 24 BTV types. Isolates have been (and continue to be) added to this collection
(particularly from the outbreaks in Europe) as a primary source of materials for sequencing studies of the
BTV genome. However, it was not always possible to determine (with certainty) exactly when, where and
which species some of the earlier isolates were derived from. These isolates are included in the collection,
with the limited data that are available.
Sequencing studies were carried out for Seg-2 of multiple isolates within each BTV serotype (where
available). Subsequent sequence comparisons demonstrated that virus isolates could not only be separated
into 24 serotype-specific ‘clusters’, but also that within each cluster/serotype they can be consistently
separated into geographically distinct subgroups: viruses from the east (e.g. from India, the Far East and
Australia) and viruses from the west (Africa, North and South America). European strains include
SID 5 (Rev. 3/06)
Page 7 of 20
representatives strains belonging to both of these groups, reflecting the position of Europe as a ‘cross-roads’
between the east and west, and the use of live vaccines in the region, some of which were originally derived
from eastern and some from western virus strains.
The sequence of Seg-2 was almost always more closely related to other isolates of the same serotype (69100% sequence identity) than to isolates of distinct serotypes (41-71%). However, some of the serotypes are
also more closely related to each other (forming into 9 distinct nucleotypes (67-71% identity)), than to
viruses in distinct Seg-2 nucleotypes (41- 61% identity). These data suggests that BTV had already evolved
into different serotypes before it became geographically dispersed, but individual strains within each
serotype continued to accumulated further point mutations in their new geographic locations, leading to
distinct eastern and western strains within each type (with a maximum of ~ 10% sequence variation within
each geographic group). An analysis of these data confirmed that Seg-2 sequences can give a clear
indication of the lineage and therefore original geographic origin of each virus strain. The data also
demonstrate that provided variation into different Seg-2 topotypes is taken into account, it is possible to
reliably identify individual BTV serotypes by sequence analysis of genome segment 2 alone. Our analyses
indicated that it is possible to design primers that are specific for each of the BTV serotypes (although they
may need to be multiplex to include both eastern and western sub groups: see objective 3) .
The sequence data generated for BTV Seg-2 have been submitted to the international sequence database and
form the basis of paper that have either been submitted or are in preparation (see publications). The number
of isolates analysed for each type has largely been determined by their availability, for example very few
isolates were generated from the initial and short lived incursion of an ‘eastern’ BTV-1 into Greece
(GRE2001/07), although more recently additional isolates of a western BTV-1 have been obtained from
Algeria (ALG2006/06) and Morocco (MOR2006/07), which may represent the source of a BTV-1 outbreak in
Sardinia during late 2006 (see www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/BTV1-segment2-tree.htm ). It
has not yet been possible to obtain isolates from Italy, limiting the scope of these phylogenetic studies.
Blood samples and virus isolates were obtained very rapidly from colleagues involved in the outbreaks of
BTV that were initially detected in the Netherlands during August 2006. Sequence analyses of genome
segment 5 initially indicated that this strain was from the west (Africa) and was new to Europe. Subsequent
analysis of Seg-2 (www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/BTV-8-Seg-2-tree.htm) demonstrated that
this strain of BTV-8 was originally from Sub-Saharan Africa but was distinct from all strains of BTV-8 that
had previously been analysed (including the BTv-8 vaccine).
The sequences and epidemiological data obtained show that most of the European strains represents a closely
related group, resulting from either a single introduction into Europe, or possibly repeated introductions from
a single source outside Europe. The only exceptions to this are BTV-1, which has arrived in the
Mediterranean region from two sources (eastern and western strains) and BTV-4, which appear to have been
present in Europe (in Cyprus) for a long period (since 1969). Overall it is clear that a total of six serotypes
of BTV have been introduced into Europe on at least 10 different occasions since 1998, involving
introductions in every year except 2002. The majority of strains (with the exception of the eastern BTV-1)
have established themselves and have spread in the region, demonstrating a fundamental change in the
epidemiology and threat posed by BTV and other orbiviruses (such as African horse sickness virus) in
Europe. These continuing and repeated introductions of novel strains and serotypes of BTV into Europe
have major implications for the future of the disease and development of control strategies in the region.
Phylogenetic comparisons have also confirmed that in the majority of cases there are significant differences
in the sequence of seg-2 between the live vaccine and European field strains (BTV-1, 2, 4, 8 and 9) that are
sufficient to distinguish them reliably. Analyses of the majority of European BTV field strains have
demonstrated that they are distinct from the vaccines and were therefore derived from another source.
However, there is also clear evidence that the BTV-2 vaccine has been transmitted in the field (in Italy)
(Ferrari et al 2005). The BTV-9 vaccine was also isolated from an animal that died two weeks after
vaccination (in Sicily) and that the 2004 outbreak of BT in Sardinia was caused by the BTV-16
vaccine strain. Comparisons between other European BTV-16 field strains and the vaccine show
that although these viruses are distinguishable, they have a very close relationship suggesting a
recent common ancestry. It may be significant that the vaccine strain of BTV -16 has been used for
several years as part of a multivalent vaccination campaign in Israel and the early European field
strains of BTV-16 arrived from the east (from Turkey) .
SID 5 (Rev. 3/06)
Page 8 of 20
Milestone 02/03 for objective 2 – year 3: Analyse nucleotide sequences genome segment 2 of
different non-European isolates for comparison to the European BTV strains (same serotypes)
Genome segment 2 (and segment 6) from over 200 isolates of BTV has now been analysed and this
number continues to climb as additional European and non-European strains become available
(including new isolates from India and Central America/southern USA).
In each case the
sequences generated are full length, to ensure both the quality of comparisons for molecular
epidemiology studies, and the reliability of conclusions drawn concerning the identity, movement
and origins of individual BTV strains.
Objective 3: Develop RT-PCR assays for serotyping BTV isolates
Evaluate published RT-PCR methods and primers for detection and amplification of nonAmerican, non-Australian BTV strains of different serotypes:
At the outset of this project RT-PCR primers had previously been published for genome segment 2
from a limited number of BTV strains and only from certain serotypes. These included North
American strains of BTV-2, 10, 11, 13 and 17 (Wilson & Chase, 1993), and Australian isolates of
BTV-1, 3, 9, 15, 16, 20, 21 and 23 (McColl & Gould, 1991), although the primers had not been
fully evaluated. As an initial stage of the project, these published primers were tested for their
ability to identify and distinguish reference strains of the 24 BTV serotypes. They were also tested
with isolates of the homologous serotype from different geographical areas, to evaluate their ‘type’
specificity.
The primers for North American strains of BTV-11 13 and 17, and the Australian primers for BTV21 and 23, worked well. They each showed serotype specific amplification with their respective
reference strain and did not show significant cross-reaction with reference strains of other BTV
serotypes. However, the primers previously designed for BTV 2 and 10 (N. America), 15 and 16
(Australia), generated multiple and incorrectly sized PCR products with reference strains of the
homologous serotype, while the primers for BTV-1, 3, 9 and 20 showed no amplification with their
respective reference strains. These data demonstrated a need for the development of (more reliable)
RT-PCR based assays for most of the BTV serotypes, and that continued monitoring and
development of such assays would be required in the future, to ensure that they remain both
sensitive and specific to current outbreak strains.
Full length sequence analyses of Seg-2 from all 24 BTV reference strains, and multiple
geographically and temporally distinct isolates of each serotype (where available) have provided a
database that was used to design serotype-specific primers for use in RT-PCR assays. The primers
were initially validated in silico (by comparison to the database) to ensure not only that they would
amplify all previously characterised isolates of the homologous type, but also that they would not
amplify sequences from previously characterised strains of heterologous types. This rational
design of primers has previously been impossible in the absence of a sequence database for Seg-2 of
most of the relevant virus strains.
The primers that were designed for five of the European serotypes (types 1, 2, 4, 9 and 16) were
tested with multiple strains of the homologous virus type from the reference collection. In some
cases it was necessary to design separate primers for eastern and western groups within each
serotype, to ensure reliable amplification. In addition the primers initially designed for the Eastern
European strain of BTV-4, only worked inefficiently with the BTV-4 strain from Morocco and the
western Mediterranean region, necessitating a redesign step. These primers were also evaluated
with strains from distinct but closely related serotypes (belonging to the same Seg-2 ‘nucleotype’)
to ensure they would not cross react with other types.
Where possible primers were also designed to distinguish eastern and western strains of some
serotypes. In cases where the vaccine and field strains belong to different groups, such primers
SID 5 (Rev. 3/06)
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could be used to distinguish them in diagnostic samples. Some initial primers for the first five of
the European vaccine and field strains of BTV are available on the dsRNA virus website at:
www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/btv-S2-primers.htm . However, only relatively
small numbers of isolates > 10, were available for many of the BTV serotypes and in some cases
there are from only a single geographic location. Although it has been possible to design primers
that will identify all of available strains of the other serotypes, it is not yet possible to evaluate their
ability to detect isolates of the homologous type but from other geographical regions. The
continued development of such primers for new isolates will be important to ensure they remain as
effective as possible for use in diagnostic assays.
During the recent outbreaks of BTV-8 in northern Europe and BTV-1 in North Africa, the strains
involved were rapidly identified, using serotype specific primers, with further confirmation by
sequencing and phylogenetic comparisons (www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/BTV1segment2-tree.htm & www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/BTV-8-Seg-2-tree.htm )
Milestone 03/02 for objective 3 - Year 3: Develop and evaluate primers for the specific RT-PCR
amplification and sequencing of the different European BTV serotypes
Primers have been developed and evaluated for all six of the European BTV serotypes (1, 2, 4, 8, 9
and 16) These were evaluated in silico for their specificity against the known sequences of Seg-2
all 24 BTV serotypes, and in assays using distinct strains of the homologous serotype (different
topotypes) and closely related but heterologous BTV-serotypes (same nucleotype). Two
manuscripts are is in preparation describing the design and evaluation of serotype specific primers
for all 24 BTV types.
Objective 4: Analyse genome segment 10 of BTV strains from different regions of Europe
(different vectors).
A large collection of European and non-European BTV isolates from around the world has now
been assembled at IAH Pirbright (see above). Sequence analyses of genome segment 10 from an
initial group European and non-European isolates was carried out during the project. Phylogenetic
analyses show that these sequences are more variable than the majority of the BTV genome
segments (except for Seg-2 and Seg-6, encoding outer capsid proteins VP2 and VP5) and can be
divided into three major clusters that do not correlate with virus serotype. These data indicate that
reassortment has occurred at some point in the past, allowing the different serotypes (which contain
Seg-2 with a distinct ancestry) to appear in each Seg-10 cluster, although we found no direct
evidence of reassortment involving Seg-10 during the recent BTV outbreaks in Greece (see below).
The BTV Seg-10 sequences did not fall into simple east and west phylogenetic groups, in the same
manner as the majority of the other BTV genome segments. This suggests that variation in this
segment may respond to or be influenced by other biological or environmental factors, and it has
been suggested that variations in Seg-10 may reflect different vector species used by the virus.
These sequence data have been submitted to the international sequence databases for allocation of
accession numbers and some of the data are now published (Nikolakaki et al, 2005 – see
publications).
Our analyses included BTV 9 strains from the Balkan region, beyond the most northerly
distribution of Culicoides imicola, as well as from further south in Greece and Turkey, where it
appears likely that C. imicola is the major vector species. The data for segment 2 and 6 show that
these isolates represent a single lineage and there are also relatively few differences in the sequence
of genome segment 10. However, it has not yet been possible to obtain additional isolates of BTV9 from Italy, where the virus would have had greater opportunities to reassort (exchange genome
segments) with other field strains and vaccine strains. We have only been able to obtain further
isolates of European BTV-8 from north of the C. imicola distribution limits in Southern Europe,
SID 5 (Rev. 3/06)
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because the distribution of other serotypes in Europe is usually south of this line) making analyses
of Seg-10 difficult from different European vector regions.
Milestone 04/03, for objective 4 – year 3: Analyse the ability of representative BTV isolates from
different vector regions to cause a fully disseminated infection in laboratory reared Culicoides
The vector competence of laboratory reared C. sonorensis (a North American vector for BTV)
for an Australian and a European strain of BTV-1 was assayed. Based on earlier works (Jennings
and Mellor, 1987 and Fu et al., 1999), BTV transmission in the field will occur if virus titres in the
insect exceed 2.5 log10TCID50/midge. The virus strains used in these experiments were: BTV-1
Greece BHK2[E1/BHK2]KC1 (IAH reference collection code GRE2001/07), and BTV-1 Australia
BHK-3/V4 BHK1/V1[BHK1]KC1 (IAH ref. collection code AUS1979/01 ) . Approximately 250 C.
sonorensis female were orally infected with a sheep blood mixture containing 1:2 BTV-1 Greece
virus, or BTV-1 Australia virus strain. After incubation @ ~ 25-28°C, 120 insects were
homogenised and titrated in BHK-21 cells.
BTV-1 Greece: 9.17% (n=11) of the midges were infected at 7 days post infection. The titre
recorded ranged between 0.76 and ≥2.5 log10 TCID50/ml. 36% (4 out of 11) of the positive midges,
representing 3.33% of the midges tested, had a virus titre above 2.5 log10 TCID50/ml and would
therefore be capable of transmitting the virus.
BTV-1 Australia: 5.88% (n=7) of the midges were infected at 7 days pi, with a virus titre that
ranged between 0.38 and ≥2.5 log10 TCID50/ml. Only 14.3% of the infected insects (1 adult out of 7)
and only 0.8% of the midges titrated, showed a virus titre above 2.5 log10TCID50/ml.
These initial results confirm that BTV strains from different part of the world can replicate and
be transmitting by a Culicoides species from a different geographic origin to the virus. This is
perhaps unsurprising in view of the recent emergence of BTV in central and northern Europe. The
two virus strains used also showed different infection rates and transmissibility rates, in C.
sonorensis. The BTV-1 from Greece generated a significantly higher number of infected midges,
with higher (x 4) vector competence (virus titre above 2.5 logs).
These initial studies indicate that there are differences in the way these virus strains interact
with adults of the same vector species, which must therefore be under at least partial genetic control
of the virus itself. Further investigation of these factors, for example by means of reassortment
studies (to identify the viral gene(s) involved), as well as vector competence and sequencing studies
of other different strains of BTVs from different part of the world, could be used to test the
hypotheses that variations in BTV Seg-10 can influence insect vector competence (in insects from
the colony of Culicoides vectors at IAH Pirbright).
Objective 5: Analyse frequency and significance of BTV genome segment reassortment within
Europe.
In addition to our analyses of genome segments 2 and 6 (encoding the outer capsid proteins of the
BTV particle), several of the other genome segments have been analysed from smaller numbers of
virus strains, particularly different strains of the European serotypes (1, 2, 4, 8, 9 and 16).
Phylogenetic comparisons of BTV genome segments 1, 5, and 8 have also demonstrated a clear
division into eastern and western groups, and that the European outbreak strains include viruses
derived from both groups. This indicates that there are routes of transmission for viruses from both
regions (from the east through Turkey, and from the west from Africa) that allow these viruses to
enter Europe. The repeated arrival of strains by both routes, with new introduction every year since
1998, and at least two new introductions in 2006 alone, indicates that these doorways are still open
and emphasises the nature of the continuing risk of further introductions of the virus into Europe .
SID 5 (Rev. 3/06)
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The European strains include BTV-1, 9 and 16 from the east, and BTV-1, 2, 4 and 8 from the west.
There are also vaccine strains of BTV-2, 4, 9 (west) and BTV-16 (East) that have been used in
Southern Europe. This variety of co circulating strains provides opportunities for different virus
strains to reassort (exchange genome segments) generating still further novel progeny viruses.
Comparisons of sequences for genome segments, 2, 6 and 10 of BTV strains from Greece have
failed to show any clear evidence of reassortment during the outbreaks within Europe. It is possible
that this reflects the decision by the Greek authorities not to use the live attenuated vaccines, as well
as the sporadic and relatively short lived nature of the Greek outbreaks. Indeed Greece can
currently be considered free of the disease.
In Italy, four of the five European serotypes have been co-circulating for several years and the
authorities have made widespread use of the live attenuated vaccine strains. It is therefore much
more likely that animals in Italy will have been exposed to more than one strain of BTV
simultaneously, providing much greater opportunities for genome segment reassortment in the field.
Indeed sequencing of genome segments 2, 5, 6, 7 and 10 of different but defined European BTV
isolates (including the vaccine strains), have provided evidence for genome reassortment involving
the NS1 gene (seg-5) on at least two occasions. It has not been possible to fully evaluate the
frequency of reassortment in Italy because not all strains were available. However, a published
analysis of the NS1 gene from a 2002 field strain of BTV-16 from Italy shows that it has an
identical sequence to that of the ‘western’ BTV-2 vaccine strain that was used in Southern Europe.
This sequence is distinct from that of the original field or vaccine strains of BTV 16, which both
have an eastern origin. This indicates that the BTV-16 strain from Italy 2002, was generated by
reassortment between the BTV-2 vaccine and the earlier BTV-16 field strain (originally from
Turkey).
Our analyses of genome segment 5 from other European BTV isolates have also
demonstrated that the Bulgarian BTV-9 strain (see BUL1999/01) and the Turkish strain of BTV 16
(TUR2000/10) also have identical NS1 genes. Although in this case it is not possible to
immediately identify the reassortment parents and decide which is the progeny strain, it is evident
that another reassortment event involving Seg-5 has taken place.
Discussion
The work of this project underpins UK policy through an exploration of the epidemiological and biological
significance of genetic (nucleotide sequence) variation in the bluetongue virus genome. This has in turn
helped to maintain existing diagnostic capabilities for BTV and supported the development of additional and
improved RT-PCR based assays for detection and identification of BTV (targeting the more highly
conserved genome segments, e.g. segments 1, 5, 7 or 9 encoding virus species specific antigens).
RT-PCR assays have previously been used detect BTV in infected blood samples, indicating that
nucleic acid based methods do not require virus isolation prior to strain identification (26). The
project has also developed methods to detect genome segment 2 of different BTV serotypes
(serotype-specific molecular assays).
In order to achieve the project aims it was initially necessary to develop novel and more effective
techniques to amplify (by RT-PCR) and sequence cDNA copies of specific orbivirus genes. The
development of these methods was entirely successful and they have been used to generate an
sequence data for different well documented and reference strains of BTV, as well as several other
dsRNA viruses.
The BTV outer capsid is composed entirely of VP2 and VP5. VP2 is encoded by BTV genome
segment 2 (Seg-2) and is the most variable of the virus proteins. It interacts with neutralising
antibodies and is the major antigenic determinant of virus serotype. This project has generated
nucleotide sequence data genome segment 2 (which encodes VP2) and genome segment 6 (which
encodes VP5) from a wide range of well documented BTV isolates, both from Europe and around
the world (details of isolates analysed from the reference collection are given at:
www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/BTV-isolates.htm). These data form a database
for ‘molecular epidemiology studies’ that can identify which of the previously characterised strains
SID 5 (Rev. 3/06)
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novel isolates are most closely related to. This generates a more accurate picture of the distribution,
movement, origins and persistence of individual BTV strains in the field than is possible by
conventional serological methods. These studies have established the identities of the different
lineages of BTV that have invaded Europe over the last 8 years. These methods and resources
provide new tools for identification of bluetongue viruses, more rapidly and more effectively than
ever before to help combat the disease. They therefore form an important basis for future diagnostic
work and research projects.
Sequence data were initially generated for Seg-2 of the reference strains of each of the 24 BTV
serotypes, as well as multiple isolates of each serotype from around the world (particularly from the
European outbreaks). Sequences for multiple isolates of individual serotypes (particularly those
that have affected Europe) were subsequently added to the database. Detailed comparisons of
conserved and variable regions of genome segment 2, within and between each serotype, were used
to design primers for use in RT-PCR based typing assays. These can be now be used to make an
initial serotype identification (within a matter of hours) that can be confirmed by subsequent
sequence analysis of the cDNA amplicon and phylogenetic comparisons to the Seg-2 database.
In most of Mediterranean Europe and north Africa, outbreaks of BT were initially thought to result
exclusively from virus transmission by Culicoides imicola. Although this vector is still of central
importance in southern Europe, BTV has also been transmitted in more northerly regions (Bulgaria,
Serbia, Macedonia Croatia, Holland, Germany, Belgium, Luxemburg and north-east France),
beyond the range of C.imicola, indicating involvement of novel vectors. The predominant
Culicoides species in these regions belong to C. pulicaris and C, obsoletus groups, both of which
are spread across much of northern Europe, including the UK. The ability to be transmitted by
other vectors has major implications for the potential spread of the virus and the threat posed to
Europe. However, several of the BTV serotypes that currently affect southern Europe have not
spread to the north, or significantly beyond the range of C. imicola, suggesting that there may be
genetic variation between different strains of the virus that can influence their transmissibility by
different vector species.
Genome segment 10 of BTV and the related orbiviruses, encodes a small non-structural membrane
protein, NS3, that mediates release of virus particles from insect cells. NS3 might therefore
influence the efficiency of virus dissemination within the insect, and therefore potentially both the
efficiency of infection of the salivary gland, and the utilisation of the insect as a vector species. The
project was designed to assesses the levels of variations in genome segment 10 (encoding NS3), as
an initial step towards studies of its potential to influence the virus’s ability to cause a fully a
disseminated infection in the insect. Genome segment 10 of isolates from different geographical
regions was therefore sequenced and compared. Selected strains of the virus were also assessed for
their ability to cause a disseminated infection in laboratory reared adults of C.sonorensis (the North
American vector - from the colony at IAH Pirbright).
NS1 (encoded by BTV genome segment 5) forms ‘tubule’ structures within BTV infected cells.
There is recent evidence to suggest that this protein may also play some role in the cell exit
mechanism in BTV and has been implicated as a determinant of virulence for AHSV in a mouse
model system.
Reassortment events involving NS1 could also have implications for the
transmission of these viruses and their adaptation to new vectors / ecosystems.
It is clear that
reassortment can happen in the field, between field strains and between field and vaccine strains,
however an evaluation of the significance of these events and their effects (if any on the biological
properties of the virus, would require further study.
The segmented nature of the BTV segmented genome, allows different virus strains to exchange
genome segments when they co-infect the same cell, by a process known as reassortment. This
process generates progeny novel virus strains containing genome segments derived from one or
other parental strain. The resulting progeny viruses will have biological characteristics (based on
their genome composition) which are therefore derived from, or may even be distinct from either parent.
SID 5 (Rev. 3/06)
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Environmental selection of the novel progeny viruses may lead to emergence of strains that have enhanced
transmission characteristics, altered virulence or modified serological properties, which may be more suited
to the local ecosystem. Reassortment is therefore an important factor in the variability of the virus
and its adaptation in new ecosystems. The project has generated sequence data for multiple genome
segments from some of the ‘well-documented BTV isolates’ in the IAH reference collection,
demonstrating that reassortment has occurred in Europe involving on at least two occasions genome
segment 6 (NS1 gene). The biological significance of genome segment reassortment in the field
(within Europe) will require further study.
Live BTV vaccines were originally developed in South Africa and have been used to combat BTV
outbreaks in some European countries. However, questions have been raised concerning the safety
of these vaccines, and their effectiveness as part of a BTV eradication campaign in Europe, rather
than simply for protection of individual vaccinated animals in South Africa (their original purpose).
By developing methods (based on genome segment 2) to distinguish vaccine and field strains the
project has made it possible to assess the frequency of vaccine survival and transmission in the
field. By comparison of multiple genome segments to trace the origins of individual genome
segments, it is also possible to assess the ability of these vaccine strains to reassort (exchange
genome segments) with wild type viruses in the field. These studies have provided data relevant to
use live vaccines in Europe.
Main implications of the work
The work of this project underpins UK policy through an exploration of the epidemiological and biological
significance of genetic (nucleotide sequence) variation in the bluetongue virus genome.
The project has also developed novel cDNA synthesis and sequencing techniques that are more
rapid and more reliable for the characterisation dsRNA virus genomes, than existing methods.
Based on these techniques and the establishment of a well documented reference collection for BTV
isolates from around the world, it has been possible to generate a sequence database for molecular
epidemiology studies of BTV. The results of these studies provide conclusive confirmation
concerning BTV serotype for novel isolates by RT-PCR (as described above). However they can
also distinguish different lineages of Seg-2, making it possible to identify different virus strains
within each serotype and track both the movements and geographic origins of specific viruses in a
manner that was previously impossible using conventional serological assays.
These studies have helped to maintain diagnostic capabilities for BTV at IAH and have supported
the development of RT-PCR based assays for detection and identification of BTV in diagnostic
samples (targeting the more highly conserved genome segments, e.g. segments 1, 5, 7 or 9 encoding
virus species specific antigens). Such assays have previously been used to detect BTV in infected
blood, indicating that nucleic acid based methods would not require virus isolation prior to strain
identification (26). A paper describing the group specific assays for identification of BTV by
conventional RT-PCR targeting segment 7 is now in press (Anthony et al 2007) and the segment 1
real time RT-PCR has been submitted (Shaw et al submitted)
The project has also developed RT-PCR based methods to detect and distinguish genome segment 2
of specific BTV serotypes, providing a very much more rapid method for BTV serotype
identification (< 24 hours) than by conventional serum neutralisation assays (~ 2 weeks). Initial
evaluations suggest that these assays are sensitive and more reliable than the conventional
serological assays for BTV type. These methods have been used during recent outbreaks of BTV
in northern Europe and North Africa to identify BTV-8 and BTV-1 respectively. They now form an
essential component of diagnostic test capability for these viruses as IAH Pirbright.
The diagnostic assay systems (RT-PCR) that have been developed as part of this project for BTV
serogroup and serotype identification, represent the current state of the art. Although an initial
evaluation has been made, of the sensitivity and specificity of these assays, particularly for
SID 5 (Rev. 3/06)
Page 14 of 20
detection and discrimination of European isolates and serotypes (which appear to be very good),
this is not yet a fully comprehensive validation as recommended by OIE.
References cited
Ferrari G, De Liberato C, Scavia G, Lorenzetti R, Zini M, Farina F, Magliano A, Cardeti G, Scholl
F, Guidoni M, Scicluna MT, Amaddeo D, Scaramozzino P, Autorino GL. (2005) Active circulation
of bluetongue vaccine virus serotype-2 among unvaccinated cattle in central Italy. Prev Vet Med.
68:103-13.
McColl KA, Gould AR. (1991) Detection and characterisation of bluetongue virus using the
polymerase chain reaction. Virus Res. 21:19-34.
Tabachnick WJ. (1996) Culicoides variipennis and bluetongue-virus epidemiology in the United
States. Annu Rev Entomol. 41: 23-43.
Wilson WC, Chase CC. (1993) Nested and multiplex polymerase chain reactions for the
identification of bluetongue virus infection in the biting midge, Culicoides variipennis. J Virol
Methods. 45:39-47.
Wilson WC, Ma HC, Venter EH, van Djik AA, Seal BS, Mecham JO. (2000) Phylogenetic
relationships of bluetongue viruses based on gene S7. Virus Res. 67:141-51.
Possible future work
The primers and assays that have been developed for identification of BTV serogroup, serotype, and
strains within each serotype, will need to be updated as new sequences become available for other
virus isolates from around the world, or as new virus strains emerge. This continued development
will be essential to maintain the effectiveness of molecular assays and the molecular epidemiology
database to track the distribution and movements of the virus. The current database includes very
few viruses from North or South America, or from Australia and the far east. Negotiations are
currently underway to obtain more of these strains, and this may become easier if it is possible to
establish collaborative international links.
Recently thirty virus isolates (some of them untyped)
have been received from north and South America. Seg-2 of these isolates will need to be
sequenced and added to the database, and the viruses will be added to the reference collection
In order to make a further evaluation of genetic variation and reassortment frequencies of BTV
strains within Europe, it will be necessary to sequence whole genomes from multiple distinct virus
isolates from the region. The sequencing methods developed during this project make that possible.
By identifying the viral genes involved in determination of transmissibility (in future reassortment
and vector competence studies), it may be possible to identify genetic markers for transmission of
specific BTV strains by different vector populations (in specific geographic areas). This might
make it possible to assess the significance and threat posed by the introduction of a novel virus
strain, or shifts in distribution of vector species.
The studies included in this project have been very effective and could be extended to include other
orbiviruses, particularly AHSV and EHDV, which are both transmitted by the same vector species
as BTV. AHSV has caused previous outbreaks of disease in southern Europe and must therefore be
considered as a real threat to the region, particularly in view of recent changes in the distribution
and epidemiology of BTV. EHDV has also caused outbreaks of disease in cattle in Israel and
North Africa during 20906 and may represent a further threat to Europe. Initial studies suggest that
the same approach described here for BTV would be equally effective with these other orbiviruses.
Actions resulting from the work
SID 5 (Rev. 3/06)
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The sequencing technologies have been shared with colleagues at HRI (Wellesbourne), IVEM
(Oxford) and in the United States for the sequence analyses of mushroom X virus and cypoviruses.
These have already provided several additional refereed publications. They will also form an
important basis for future projects.
The realtime RT-PCR and conventional RT-PCR assays for BTV genome segments have been
transferred from the Arbovirus Research Group to the BTV Reference Laboratory at IAH Pirbright.
These assays now form part of the front-line diagnostic capability for these viruses, in the current
fight against importation of the disease from northern Europe. In the event of an outbreak of BT in
the UK (which appears likely during 2007), these assay systems will be vital in our programme to
detect and eradicate the virus.
The identification of recent BTV and EHDV outbreak strains has formed the basis of research
reports to Defra, the EU and to relevant government agencies and laboratories of the countries
involved.
References to published material
9.
This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
SID 5 (Rev. 3/06)
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Refereed Papers
Sushila Maan, Shujing Rao Narender Singh Maan, Simon Anthony, Houssam Attoui, Alan Richard
Samuel and Peter Paul Clement Mertens (2007), Rapid cDNA synthesis and sequencing
techniques for the genetic study of bluetongue and other dsRNA viruses. (Journal of Virological
Methods submitted).
Emmanuel Breard, Corinne Sailleau, Kyriaki Nomikou, Chris Hamblin, Peter Mertens, Philip S Mellor,
Medhi El Harrak, Stephan Zientara (2007) Molecular epidemiology of bluetongue virus serotype 4
isolated in the Mediterranean Basin between 1979 and 2004 Virus Research (in Press).
Maan S., Maan N.S, Samuel A.R., Rao S, Attoui, H., & Mertens P.P.C (2007) Analysis and Phylogenetic
Comparisons of Full-Length VP2 Genes of the Twenty-Four Bluetongue Virus Serotypes. Journal
of General Virology (in Press).
S. Anthony, H. Jones, K.E Darpel, H. Elliott, S. Maan, A. Samuel, P. S. Mellor, P. P. C. Mertens. A
duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes.
Journal of Virological Methods (in Press).
Nikolakaki S. V., Nomikou, K, Koumbati, M, Mangana O, Papanastassopoulou, M., Mertens P. P, C. and
Papadopoulos, O. (2005) Molecular analysis of the NS3/NS3a gene of bluetongue virus isolates
from the 1979 and 1998-2001 epizootics in Greece and their segregation into two distinct groups
Archives of Virology 114 :6-14
Bethan V. Purse,
Philip S. Mellor, David J. Rogers,
Alan R. Samuel, Peter P. C. Mertens
&
Matthew Baylis (2005) Climate Change And The Recent Emergence Of Bluetongue In Europe
Nature Reviews Microbiology 3, 171-181.
H. Huismans, P.P.C. Mertens, P. Roy, C. Patta, G. Gerbier, M. Vitale, G.L. Autorino & M. Papin (2005)
Group 4: Vaccines and vaccination. Veterinaria Italia, 40, 721.
S. Maan, N.S. Maan, K.P. Singh, A.R. Samuel & P.P.C. Mertens (2005) The development of RT-PCR
based assays and sequencing for typing European strains of bluetongue virus and differential
diagnosis of field and vaccine strains. Veterinaria Italia, 40, 552-561.
S. Anthony, S. Maan, A. Samuel, P.S. Mellor & P.P.C. Mertens. (2005) Differential diagnosis of
bluetongue virus using an RT-PCR for genome segment 7. Veterinaria Italia, 40, 546-551.
Maan, A.R. Samuel, N.S. Maan, H. Attoui, S. Rao & P.P.C. Mertens (2005) Molecular epidemiology of
bluetongue viruses from disease outbreaks in the Mediterranean Basin. Veterinaria Italia, 40, 489496.
S. Maan, N.S. Maan, A.R. Samuel, R. O’Hara, A.J. Meyer, S. Rao & P.P.C. Mertens (2005) Completion
of the sequence analysis and comparisons of genome segment 2 (encoding outer capsid protein
VP2) from representative isolates of the 24 bluetongue virus serotypes. Veterinaria Italia, 40,
484-488.
K.P. Singh, S. Maan, A.R. Samuel, S. Rao, A. Meyer, & P.P.C. Mertens (2005) Phylogenetic analysis of
bluetongue virus genome segment 6 (encoding VP5) from different serotypes Veterinaria Italia,
40, 479-483.
H.-H. Takamatsu, P.S. Mellor & P.P.C. Mertens (2005) A potential overwintering mechanism for
bluetongue virus – recent findings Bluetongue virus and disease Veterinaria Italia, 40, 456-461.
Mertens, P.P.C., Diprose, J., Maan, S., Singh, K.P., Attoui, H. & Samuel A.R. (2005) Bluetongue virus
replication, molecular and structural biology. Veterinaria Italia, 40 (3), 426-437.
SID 5 (Rev. 3/06)
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Lager, I.A., Duffy, S., Miquet, J., Vagnozzi, A., Gorchs, C., Draghi, M., Cetrá, B., Soni, C., Hamblin, C.,
Maan, S., Samuel, A.R., Mertens, P.P.C., Ronderos, M. & Ramirez, V.(2005) Incidence and
isolation of bluetongue virus infection in cattle of the Ituzaingó and Santo Tomé Departments,
Corrientes Province, Argentina. Veterinaria Italia, 40 (3), 141-144.
Maan, S., Samuel, A. and Attoui, H. (2005). Orbivirus, Reoviridae. In: Virus Taxonomy, VIIIth Report of
the ICTV (C.M. Fauquet, M.A. Mayo, J. Maniloff, U. Desselberger, and L.A. Ball, eds), 466-483.
Elsevier/Academic Press, London.
Mertens, P. P. C (2004) dsRNA viruses. Virus Research 101, 3-13
Mertens PP, Diprose J. (2004) The bluetongue virus core: a nano-scale transcription machine. Virus Res.
101, 29-43.
H. Takamatsu, P. S. Mellor, P. P. C. Mertens, P. A. Kirkham, J. N. Burroughs & R. M. E. Parkhouse
(2003) A possible overwintering mechanism for bluetongue virus in the absence of the insect
vector. Journal of General Virology 84, 227-35.
Mertens P. P. C. and Mellor P. S. (2003) Bluetongue State Veterinary Journal 13, 18-25
Crafford, J. E., Guthrie, A. J., van Vuuren, M., Mertens, P. P. C, Burroughs, J. N., Howell, P. G. and
Hamblin C. (2003) A group-specific, indirect sandwich ELISA for the detection of equine
encephalosis virus antigen. Journal of Virological Methods 112, 129-135
Green TB, Shapiro A, White S, Rao S, Mertens PP, Carner G, Becnel JJ. (2006) Molecular and biological
characterization of a Cypovirus from the mosquito Culex restuans. J. Invertebr. Pathol. 91 :27-34.
Terry B. Green, Susan White, Shujing Rao, Peter P. C. Mertens, Peter H. Adler, James J. Becnel, (2006)
Biological and Molecular Studies of a Cypovirus from the Blackfly Simulium ubiquitum (Diptera:
Simuliidae) ( Journal of Invertebrate Pathology in press)
Graham RI, Rao S, Possee RD, Sait SM, Mertens PP, Hails RS. (2006) Detection and characterisation of
three novel species of reovirus (Reoviridae), isolated from geographically separate populations of
the winter moth Operophtera brumata (Lepidoptera: Geometridae) on Orkney. J Invertebr Pathol.
91:79-87.
Alexandra Shapiro; Susan White; Shujing Rao; Peter P.C. Mertens; Gerry Carner; James J Becnel (2005)
Molecular and Biological Characterization of a Cypovirus from the mosquito Culex restuans.
Journal of Invertebrate Pathology 91: 27-34.
Alexandra Shapiro, Terry Green, Shujing Rao, Susan White, Gerry Carner, Peter P. C. Mertens, James
Becnel (2005) Morphological and molecular characterization of a cypovirus (Reoviridae) from the
mosquito Uranotaenia sapphirina (Diptera: Culicidae) Journal of Virology 79, 9430-9438.
Promed reports
Peter Mertens: Analysis and typing of EHDV isolates from Morocco, Algeria and Israel 2006, Date: Thu
14 Dec 2006
Peter Mertens: Bluetongue - Europe (21): Bulgaria, BTV-8 suspected Date: Fri 24 Nov 2006
Peter Mertens BLUETONGUE - EUROPE (05) Date: Sat, 16 Sep 2006
Oral Presentations
Strategy for Bluetongue virus Detection and diagnosis in Europe (2006): Talk given by P. Mertens in
Brussels to CVOs SCOFCA at the European Commission on 22/09/2006
SID 5 (Rev. 3/06)
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Molecular epidemiology of BT: 40 minute talk presented by P. Mertens at the Bluetongue Diagnostics
and Epidemiology Workshop (28-29th November 2006: 73, rue Archimède, Brussels) Audience
consisted of the European research groups working on BTV and the Commission.
Natalie Ross-Smith, Jennifer Simpson, Pippa Hawes, Paul Monaghan, and Peter P. C. Mertens (2006) the
role of ns2 in formation of viral inclusion bodies during bluetongue virus infection of mammalian
cells. Talk and Abstract: presented at the 9th dsRNA virus Symposium Cape Town 21-26
October 2006.
Langner, KFA, Darpel KE, Denison, E, leibold, W., Mellor, P.S., Mertens, P.P.C., Nimtz, M., GreiserWilke, I., (2006) Collection and analysis of Culicoides spp. salivary proteins. Talk and Abstract:
presented at the 9th dsRNA virus Symposium Cape Town 21-26 October 2006.
Chiam, R., Rao, S., Mertens, P.P.C., Mellor, P., Blacklaws. B., Davis-Poynter, N. and, Castillo-Olivares,
J., (2006) Equine immune responses to recombinant vaccinia virus- expressed African
horsesickness virus antigens. Talk and Abstract: presented at the 9th dsRNA virus Symposium
Cape Town 21-26 October 2006.
Karin E. Darpel, Paul Mongahan, Kathrin Langner, Simon Anthony, Andrew Shaw, Haru H. Takamatsu,
Philip S. Mellor and Peter P.C. Mertens (2006) Prolonged infection of mammalian hosts by
bluetongue virus: the influence of the insect vector (Culicoides spp). (2006) Talk and Abstract:
presented at the 9th dsRNA virus Symposium Cape Town 21-26 October 2006.
Peter P. C. Mertens, Andrew Shaw and Carrie A Batten (2006) Phylogenetic analysis of Segment 5 (NS1
gene) from representatives of European BTV isolates Talk and Abstract: presented at the 9th
dsRNA virus Symposium Cape Town 21-26 October 2006.
Real-time RT-PCR for bluetongue virus diagnosis and research. (2006) Shaw, A.E., Monaghan, P.,
Anthony, S., Darpel, K., Batten, C, Elliot, H., Jones, H. and Mertens, P.P.C. Talk and Abstract:
presented at the 9th dsRNA virus Symposium Cape Town 21-26 October 2006.
Carrie A. Batten, Eugene van Rooij, Kris De Clerq, Stephan Zientara, Bernd Hoffman, Martin Beer,
Emmanuel Breard, Corinne Sailleau Piet van Rijn, Sushila Maan, Andrew Shaw, Alan Samuel,
Simon Anthony, Karin Darpel, Eva Veronesi, Chris Oura, Philip S. Mellor and Peter P. C.
Mertens (2006) Identification of bluetongue virus from northern Europe (2006). Talk and
Abstract: presented at the 9th dsRNA virus Symposium Cape Town 21-26 October 2006.
Vet Research Club by Peter Mertens , 13th October 2006 - Linean Society London “ Bluetongue in
Europe (1998- 2006) an update”
Simon Anthony, Natalie Ross-Smith, Alan Samuel and Peter Mertens Variation of genome segment 9
from epizootic haemorrhagic disease virus (EHDV); a member of the Orbivirus genus. Abstract
for talk presented at the XIII World Virology Congress, San Francisco July 22-30 2005 V-262,
pp236.
K. Nomikou, A.R. Samuel, S. Maan, K.P. Singh , S. Anthony, S. Nikolakaki and P.P.C. Mertens (2005)
Isolation of BTV-1 in Greece: Molecular Diagnosis and Sequence Analyses based on Genome
Segments 2, 6, 3, 7,and 10. Abstract for poster presented at the XIII World Virology Congress, San
Francisco July 22-30 2005 176 -V-360, pp156.
Karin E. Darpel, Paul Monaghan, Jennifer Simpson, Simon J Anthony, Natalie Ross-Smith, Philip S
Mellor, Haru H Takamatsu, and Peter P.C. Mertens (2005) Prolonged (or persistent) bluetongue
virus infection of mammalian cells. Abstract for poster presented at the XIII World Virolog y
Congress, San Francisco July 22-30 2005 176 -V-312, pp156.
Mertens PPC, Carner GR, Scott S, Winton J, Becnel J, Graham R, Hagiwara K, Shapiro M, Lynn D,
Zeddam JL &, Rao S (2005) Full genome sequence comparison validates electropherotyping for
identification of Cypovirus species Abstract for poster presented at the XIII World Virology
Congress, San Francisco July 22-30 2005 176 -V-360, pp157.
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Ross-Smith, N., Simpson, J., Hawes, P., Monaghan, P., and Mertens P.P.C. (2005) Elucidation of the
role of Non-structural protein 2 in Bluetongue virus infection. Abstract for poster presented at the
XIII World Virology Congress, San Francisco July 22-30 2005 176 -V-298, pp157.
A. R. Samuel, S. Maan and P.P.C. Mertens (2005) Molecular epidemiology of bluetongue Viruses from
Disease Outbreaks in the Mediterranean basin. Abstract for poster and talk presented at the XIII
World Virology Congress, San Francisco July 22-30 20-05. 184- V-504 pp175.
Ross-Smith, N., Simpson, J., Hawes, P., Monaghan, P., and Mertens P.P.C. (2005) Elucidation of the
role of Non-structural protein 2 in Bluetongue virus infection. Abstract for poster presented at the
XIII World Virology Congress, San Francisco July 22-30 2005 176 -V-298, pp157.
A. R. Samuel, S. Maan and P.P.C. Mertens (2005) Molecular epidemiology of bluetongue Viruses from
Disease Outbreaks in the Mediterranean basin. Abstract for poster and talk presented at the XIII
World Virology Congress, San Francisco July 22-30 20-05. 184- V-504 pp175.
Peter P. C Mertens Houssam Attoui, Jonathan Grimes, Dennis H. Bamford and David I. Stuart (2005)
Reovirales or Diplornavirales? A new order for dsRNA viruses based on structure, or genome?
Abstract for poster and talk presented at the XIII World Virology Congress, San Francisco July
22-30 20-05. 81-V-504 pp175.
O’Hara, R.S., Rao, S., and Mertens, P.P.C. (2003) Nucleotide sequence analysis of genome segment 10
from bluetongue viruses isolated in the current European outbreak Abstracts dsRNA Virus
Symposium, Tuscany September 2003.S. Rao, G. Carner, S. W. Scott, M. Shapiro, D. Lynn, J.
Winton, D. Stoltz, K. Hagiwara, T. Omura, S. Maan, A. Samuel, H. Attoui, J, Rodriguez, A.
Meyer, G. Sutton, P.P.C. Mertens (2003) Genome-wide Approach to Sequence Determination and
Identification for Viruses of the Genera Cypovirus, Aquareovirus, Orthoreovirus, and Orbivirus in
the Family Reoviridae Abstract (P7.18) 8th International Symposium on Double-Stranded RNA
viruses Tuscany Sept 13-18 2003 (pp146).
Singh, K.P., Maan, S., Rao, S., Samuel, A.R., Meyer, A and Mertens, P.P.C., (2003) Full length sequence
analysis of genome segment 6(encoding VP5) from different serotypes of bluetongue virus
Reoviridae Abstract (W7.9) 8th International Symposium on Double-Stranded RNA viruses
Tuscany Sept 13-18 2003.
Anthony, S., Maan S., Samuel A., Mellor P. S , Mertens P. P. C. The differential diagnosis of bluetongue
virus: a PCR approach. Abstract (P7.20) 8th International Symposium on Double-Stranded RNA
viruses Tuscany Sept 13-18 2003 (pp148).
Maan S., Maan N., O’Hara R., Samuel A.R. & Mertens P.P.C. (2003) Phylogenetic analysis of genome
segment 2 from representative isolates of the 24 BTV serotypes. Abstract (P7.22) 8 th International
Symposium on Double-Stranded RNA viruses Tuscany Sept 13-18 2003 (pp150).
Samuel A. Maan S., Maan N., & Mertens P. (2003) RT-PCR based assays for typing European strains of
Bluetongue virus and differential diagnosis of field and vaccine strains. Abstract (P7.23) 8th
International Symposium on Double-Stranded RNA viruses Tuscany Sept 13-18 2003 (pp151).
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