Bluetongue virus in the Netherlands identified as serotype 8 by Institute for Animal Health
Scientists at the Institute for Animal Health's Pirbright Laboratory have shown that the bluetongue virus causing disease in the Netherlands is serotype 8. This serotype has not previously been identified in Europe.
The virus had been sent to IAH Pirbright by the European Commission, as IAH Pirbright is the European Community Reference Laboratory for bluetongue.
After confirming that the disease in the Netherlands was bluetongue, the IAH Pirbright scientists worked day and night over the past week to identify the serotype of the virus. Having eliminated that possibility that the virus was one of the five serotypes previously detected in southern Europe (types 1, 2, 4, 9, and 16), they developed tests for the other 19 types.
The identification of the serotype was done by PCR tests late on Friday night (25th August). The test involves the gene for protein VP2 of the virus. VP2 unequivocally specifies the serotype of the virus. The PCR result was confirmed by sequencing most of genome segment 2 of the virus, on Saturday (26thAugust).
Rapid success
Professor Philip Mellor, Head of the European Community Reference Laboratory (CRL) for bluetongue said “The isolation of a bluetongue virus serotype new to the region, by Professor Peter Mertens’ group at Pirbright, within only eight days of receipt of the first infected sheep material from the Netherlands, is an astonishing feat.” This was achieved not only by round-the-clock working but also by the application of modern diagnostic tests that had been developed at Pirbright. These tests focussed on detecting and sequencing the genes of the bluetongue virus (BTV). This was much faster than the older methods used to identify previous BT outbreaks in southern Europe, which took three to four weeks.
Typing of the virus is important in tracking where the BTV-8 virus came from. The results 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.
The outer surface of the bluetongue virus particle, showing VP2 (red) and VP5 (yellow). (combined cryo-electron microscope reconstruction and atomic model).
Picture: Institute for Animal Health and University of Oxford
Chronology of events
Summary
The Bluetongue European Community Reference Laboratory at IAH Pirbright has, within the space of only eight days since receipt of the first samples from the Netherlands: confirmed the presence of BTV; demonstrated that it was not one of the five serotypes previously detected in Europe; successfully isolated and propagated the virus; has sequenced several genome segments, including that encoding VP2 which determines serotype, identifying the virus as serotype 8; and has done phylogenetic analysis which shows that the virus is a western virus, most closely resembling those of sub-Saharan Africa.
The first suspected cases of bluetongue were observed in a flock of sheep in the Netherlands. The Dutch Central Institute for Animal Disease Control, CIDC-Lelystad, Wageningen UR, sent samples from the affected sheep to the Institute for Animal Health’s Pirbright Laboratory, which is the European Commission’s Reference Laboratory for bluetongue. Within 24 hours of the samples arriving at Pirbright the initial identification of BTV-specific antibodies (by the BTV Reference Centre) was positive, confirming the observations by colleagues in the Netherlands.
At the same time a test for BTV genes was performed (a polymerase chain reaction-based test, PCR). BTV has 10 genes, which collectively form the genome. Each of the genes is on a separate piece of the genome – a genome segment. The first test, for genome segment 7, was designed to detect any of the 24 serotypes of BTV. This confirmed the Dutch results (on segment 8), demonstrating that BT virus was indeed present in the sheep. Since these were Dutch sheep i.e. not imported, this showed that they must have acquired the infection whilst in the Netherlands. This meant that the virus had been spread to Dutch sheep by midges in the Netherlands; BTV is only spread by biting insects. Bluetongue virus is a member of a group of viruses called arboviruses, so called because they are spread (borne) by arthropods, of which midges and mosquitoes are two examples. (arthropod borne).
The Arbovirus Research Group at IAH then showed, by further PCRs, that the BTV in the Netherlands and neighbouring countries was a ‘western’ one, that is to say it would have come originally from Africa or America. This ruled out the European field strains of BTV-1, 9 and 16 which are all eastern viruses e.g. from south-eastern Europe.
One of the proteins exposed at the surface of the virus is virus protein 2 (VP2). It is antibodies against this protein that determine the serotype of a BTV. The genome segment that produces VP2 is genome segment 2. The Pirbright researchers had previously developed PCR genetic tests specific for the five serotypes of BTV that had been detected in Europe in recent years (types 1, 2, 4, 9 and 16) www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/btv-S2-primers.htm. These tests were set up within 24 hours of arrival of the first sheep samples from the Netherlands. The results for genome segment 2 were negative, even though the tests for genome segment 7 (all 24 types of BTV give a positive result with the PCR for genome segment 7). This indicated that the BTV from the Netherlands was not of a type previously detected in southern Europe (not types 1, 2, 4, 9 or 16). The tests were repeated, with the same result.
Attention was also focussed on genome segment 5. Sequencing of part of this gene showed that the BTV was very different from any of the recent European BTVs or the live BTV vaccine strains widely used in Europe. This indicated that the northern European BTV was not simply derived from one of the previous European BTVs but was most likely to be a novel introduction to the region.
On this basis the Pirbright scientists went back to the sequence data which they had recently generated for genome segment 2 of over 300 isolates of BTV, including reference strains of all 24 serotypes. (Reference strains are older isolates that are used as a bench-mark against which all subsequent BTVs are compared). The scientists identified regions of genome segment 2 which were unique for each of the remaining 19 serotypes, in order to use serotype-specific reagents to provide serotype-specific PCR tests.
In order to produce more BTV for the tests, midge cells grown in the laboratory were infected by BTV present in samples of sheep blood from the Netherlands. Genetic material (RNA) from the newly grown virus was used in PCR tests specific for each of the 24 types of BTV. The only positive results were obtained with PCRs specific for type 8 virus, demonstrating the virus was BTV-8.
RNA from the infected KC cells was extracted and tested with the type specific primers for all 24 serotypes, generating cDNA products only with three sets of primers, all of which are derived from BTV-8 sequences (demonstrating that the virus is BTV-8).
The DNA produced in the three positive PCRs was sequenced and the sequence of the VP2 protein was deduced from the genetic sequence. The VP2 sequence was compared with that of the BTV reference strains, representing all 24 types of BTV. This confirmed that the BTV from the Netherlands was indeed BTV-8.
Comparison of the VP2 gene sequences shows that the BTV-8 Dutch isolate is very similar to BTV-8 viruses isolated in sub-Saharan Africa. http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/btv-8.htm
By what path the BTV-8 got to northern Europe remains a mystery. Further epidemiological studies, including genetic analysis, will be required to answer this question.
More information on bluetongue virus, from the Institute for Animal Health
This can be found at http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/, followed by scrolling down the page to Reoviridae, then a little further down to bluetongue virus.
More information on recent bluetongue outbreaks, from the Institute for Animal Health
Summary of bluetongue outbreaks during the last four years
http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/outbreaks.htm
Clinical signs of bluetongue in sheep, from the Institute for Animal Health
Mild disease signs in sheep
http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/disease symptomsBT-Mild.ppt
Moderate disease signs in sheep
http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/disease symptomsBT-Moderate.ppt
Severe disease signs in sheep
http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/disease symptomsBT-Severe.ppt
For more information contact Dave Cavanagh, Press Officer, Institute for Animal Health.
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