Bluetongue - Netherlands, Belgium, Germany (08): BTV-8 origin

International Society for Infectious Diseases
<http://www.isid.org>

[1]
Date: Fri, 15 Sep 2006

There has been speculation in ProMED-mail postings and elsewhere as
to the origin of the bluetongue virus type 8 [BTV-8] which has
emerged in Netherlands, Belgium, Germany and France. One post
[20060828.2448] stated: "The European Commission was informed by the
Community Reference Laboratory for Bluetongue in Pirbright (UK) over
the weekend that the virus responsible for the recent bluetongue
outbreak in the Netherlands has been identified, through genetic
testing, as being BTV-8. This strain of virus has never been reported
in Europe before, and the disease recently detected in the
Netherlands, Belgium, and Germany is now considered to be of exotic
origin. 1st results suggest that the BTV-8 is of a sub-Saharan
lineage, although the precise origin is not yet known, nor is the way
in which the virus was introduced into Europe."

Is it accurate to state that "this strain of virus has never been
reported in Europe before" when Bulgaria resorted to vaccination in
1999/2000, using a pentavalent (serotypes 3, 8, 9, 10 and 11) [live
attenuated vaccine. - Mod.AS] imported from South Africa? If I am not
mistaken the vector of bluetongue (BTV type 9 not 8 presumably) in
Bulgaria and the Balkans was not _Culicoides imicola_ but was almost
certainly _C. obsoletus_ which is well represented in Netherlands,
Germany, Belgium, France and other European countries.

It is certainly tempting to relate the 2 events yet your posting
"BLUETONGUE - NETHERLANDS, BELGIUM, GERMANY (06): BTV-8" includes the
following didactic statement [made by the European Commission
Reference Laboratory for Bluetongue, Pirbright. - Mod.AS]: "The
results [of virus typing] show that the Dutch isolate is NOT
descended from vaccine forms of the bluetongue virus that have been
used in many parts of southern Europe including Bulgaria, Italy,
Corsica, Spain, and also in South Africa, in recent years. The gene
sequence data points to an origin in sub-Saharan Africa."

Elsewhere [archived 20060831.2478] Peter Mertens is quoted as
indicating that "The genetic fingerprint of the virus found this week
is closest to that of a virus isolated in Nigeria in 1982, which
means it almost certainly came from Africa."

Are we really confident enough to discount a relationship between the
use of BTV-8 vaccine in Bulgaria and the emergence of BTV-8 in the
Netherlands? Given the serious implications of this decision I am
reluctant to dismiss the possibility so easily. I was not aware that
the pentavalent vaccine with a BTV-8 (component) had been used
anywhere else in Europe than in Bulgaria but if it was (as indicated
above) then I would be even more reluctant to stop exploring the connection.

--
Dr Peter Roeder BVetMed, MSc, PhD, MRCVS
Animal Health Officer (Virology)
GREP Secretary
Animal Health Service
Animal Production and Health Division
Food and Agriculture Organization of the United Nations
Vialle delle Terme di Caracalla
00100 Rome, Italy


Dr Roeder's comments have been forwarded by us to the Community
Reference Laboratory for Bluetongue in Pirbright (UK). Their response
is included in the following item.

******
[2]
Date: Fri, 15 Sep 2006


Regarding Dr Roeder's suggestion that bluetongue virus serotype 8
(BTV-8) in the Netherlands (strain NET2006/01) might be associated in
some way with the use of BTV-8 as a component of a multi strain
vaccine in Bulgaria in 1999/2000:

Although there is evidence that BTV-2 and BTV-16 vaccines have been
involved in outbreaks of disease in Southern Europe, it appears to be
very unlikely that the BTV-8 vaccine was a source for the recent
northern European outbreak.

The type-8 vaccine was used in Bulgaria in 1999, quite a long time
ago, while all of the viruses isolated from Bulgaria were
subsequently typed as BTV-9, suggesting that BTV-8 did not establish
a focus of infection in the region. Furthermore, the Greeks typed
their viruses as BTV 1, 4, 9 or 16 -- confirmed by analyses at the
Community Reference Laboratory (CRL) here at Pirbright -- with no
indication of type 8. Additionally, the Italian viruses were typed
as BTV 2, 4, 9 and 16 -- again with no indication of type 8. These
data suggest that BTV-8 was not circulating undetected in southern
Europe for the 6 years following the Bulgarian vaccination.

Our initial sequencing studies of the highly conserved NS1 gene of
the Netherlands virus (NET2006/01), showed that it is distinct from
the previous European isolates of BTV-1, 2, 4, 9 and 16 (strains from
outbreaks in the Mediterranean region). These studies showed that it
is also distinct from the vaccine strains of these serotypes that
were used in Southern Europe, indicating that the outbreak strain
represented a novel introduction to Europe.

We then tried to amplify genome segment 2 (which is the most variable
of the BT genes -- encoding the main serotype specific antigen) from
NET2006/01, using primers designed from each of the 24 BTV
serotypes. Only 3 sets of primers worked, all of which were designed
from BTV-8 sequences, clearly indicating that the new virus was also
serotype 8. To confirm this we sequenced the amplified cDNAs and have
compared them to Segment 2 sequences from the 3 other BTV-8 strains
that we have in our reference collection. The resulting phylogenetic
tree is available at
<http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/BTV-8-Seg-2-tree.htm>.

You will see that there was a close relationship with BTV-8 from West
Africa south of the Sahara (Nigeria-1982) but a less close
relationship with the Type-8 reference strain from South Africa.
Other studies at IAH Pirbright have separately demonstrated a very
close relationship between the reference and vaccine strains of
BTV-8, that approaches identity. For these reasons we believe that
there is virtually no chance that the South African vaccines were the
cause of this outbreak in northern Europe. It is more likely to have
been derived from another African strain ( e.g. Nigeria 1982) than
directly from either the vaccine or reference strains of BTV-8.

We are currently working on a full genome analysis of NET2006/01,
which will allow us to compare each of its genome segments to those
of other BTV isolates.

In relation to the involvement of cattle in the northern European
outbreak, it is quite correct that this appears to be a new
development, the Americans for instance have said that only 0.01
percent of infected cattle exhibit any clinical signs. However, one
of the earlier BT papers indicates something different should BTV get
into an entirely new location. quote: ".....on the other hand, among
highly susceptible bovine populations, the first introduction of the
disease will produce both clinical symptoms and occasional
mortality." [It is] also worth noting that the South Africans graded
BTV serotypes 1, 3, 6, 8 and 9 as being "very pathogenic". So, is
this really a new phenomenon or just a recent manifestation of an old
observation, reflecting the movement of the virus to a new area,
containing a susceptible and naive host population?

With regard to the vectors, _C. imicola_ is absent from Bulgaria,
the Balkans, northern and NW Greece and European Turkey, while _C.
obsoletus/pulicaris_ are hugely abundant. We therefore originally
suggested that species within these complexes were the vectors in
these areas (see Mellor and Wittmann 2002). This followed on from our
work in 1976 in Cyprus when we isolated BTV-4 from _C. obsoletus_,
and in 1986 when we isolated AHSV [African horse sickness virus] from
mixed pools of obsoletus and pulicaris (both viruses use the same
vectors) -- all published.

Additionally, and subsequent to these suggestions that
obsoletus/pulicaris were competent vectors, Italian colleagues from
IZS Terramo (Savini et al in 2003) recorded multiple isolations of
BTV from _C. obsoletus_ in mainland Italy. Working with colleagues
at IZS Palermo (Caracappa et al 2003) we also recorded multiple
isolations of BTV from _C. pulicaris_ in Sicily. Furthermore Simon
Carpenter here at IAH has shown that BTV-9 replicates to
transmissible levels in certain populations of obsoletus and
pulicaris in the UK -- all published.

--
Prof. Peter Mertens & Prof. Philip Mellor
Arbovirus Research Group
Department of Epidemiology,
Institute for Animal Health,
Ash Road Pirbright Woking Surrey,
GU24 0NF, UK

dsRNA Viruses web site
<http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins>

The data provided by IAH are impressive; for a final, round-up
picture, it will be helpful to obtain the results of the contemplated
full genome analysis of the Dutch BTV-8 strain NET2006/01 and
subsequent comparisons of each of its genome segments to those of
other BTV-8 isolates, including the vaccine strain. It will also be
useful to include among the compared isolates, if possible, the BTV-8
strain which was suspected to be involved in clinical BT cases in
South African cattle during 1996. This event has been described in:
G.H. Gerdes (2004). A South African overview of the virus, vectors,
surveillance and unique features of bluetongue. Vet. Ital., 40 (3),
39-42. < http://www.te.izs.it/vet_italiana/index.html>.

Dr Roeder's question about the possibility that the pentavalent
live-attenuated vaccine including BTV-8 was used in European
countries additional to Bulgaria, remains to be answered.