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 and neighbouring countries is serotype 8. This serotype has not previously been identified in Europe.
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 at 3:00 am Saturday morning (26th 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, during Saturday (26thAugust).
The Institute for Animal Health’s Pirbright Laboratory, in Surrey, is the European Community Reference Laboratory (CRL) for bluetongue and other diseases. It was the scientists of Pirbright Laboratory that tested thousands of samples during the outbreak of foot-and-mouth disease in the UK in 2001.
Rapid success
Professor Philip Mellor, Head of the European Community Reference Laboratory (CRL) for bluetongue said “The isolation of a bluetongue virus (BTV) serotype new to the region, by Professor Peter Mertens’ Arbovirus Research Group at Pirbright, operating under the umbrella of the CRL and 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 (BTV). This was much faster than the older methods used to identify previous bluetongue 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 BTV 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 European Community Reference Laboratory for bluetongue based 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 type 8; and has made it possible to carry out a phylogenetic analysis which shows that the virus is a western virus, most closely resembling those from 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 CRL for bluetongue at the Institute for Animal Health’s Pirbright Laboratory. Within 24 hours of the samples arriving at Pirbright the initial identification of BTV-specific antibodies 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 dsRNA viral genome. Each of the genes is on a separate piece of the genome – a dsRNA genome segment. The first test, for genome segment 7 (which codes for a conserved protein - VP7 that is serologically similar for all BTV strains) was designed to detect any of the 24 serotypes of BTV. This confirmed the Dutch results (on segment 8 – also conserved), demonstrating that BTV 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 means that the virus had been spread to Dutch sheep by midges in the Netherlands; BTV is transmitted between its hosts only by these biting insects. BTV is an arbovirus, a group of viruses 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 strain in the Netherlands and neighbouring countries belongs to a ‘western’ lineage, that is to say it could 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, originating from the middle or Far East.
The BTV outer capsid is composed of two distinct viral proteins, VP2 and VP5. Antibodies against these proteins, particularly VP2, are able to neutralise the virus and represent an important part of a host animal’s protective immune response. 24 distinct forms of VP2 have previously been identified, and the specificity of their interactions with neutralising antibodies determines BTV serotype. Sequence comparisons of genome segment 2 (which codes for VP2), from multiple isolates of BTV, including the 24 reference strains of different serotypes, have recently been completed by the Arbovirus Research Group. These studies have demonstrated that the sequence of this genome segment can be used reliably to identify the virus type. Based on these sequence analyses, Pirbright researchers had already developed PCR genetic tests that are 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. However, initial and repeated assays for detection of genome segment 2 by these methods were negative, even though the tests for genome segment 7 were positive (all 24 types of BTV give a positive result with the PCR for genome segment 7). This indicated that the BTV strain from the Netherlands was not one of the types previously detected in southern Europe (not types 1, 2, 4, 9 or 16).
Attention was also focussed on genome segment 5, one of the more highly conserved regions of the genome. Sequencing studies of this gene showed that the Northern European BTV isolate was significantly different from any of the recent European BTVs, or the live BTV vaccine strains that have been widely used in Europe. This indicated that it was not simply derived from one of the previous European outbreaks but was likely to be a novel introduction to the region.
On this basis the Pirbright scientists used sequence data which they have recently generated for genome segment 2 of over 300 isolates of BTV, including the 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, to design serotype-specific reagents (primers) for type-specific PCR tests.
The virus in samples of sheep blood from the Netherlands, was used to infect cultured midge cells grown in the laboratory, providing an initial isolate of the new virus strain within 7 days of its arrival at IAH, for further analysis. Genetic material (RNA) from the newly grown virus was used in the novel PCR tests for each of the 24 types of BTV. The only positive results were obtained with three separate PCR assays designed to be specific for BTV-8, identifying it as this serotype.
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The DNA produced in the three positive PCRs was analysed and the sequence of the deduced VP2 protein was compared with that of the BTV reference strains, representing all 24 types. 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
www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/outbreaks.htm
Summary of the known distribution of different BTV types
www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/btv-serotype-distribution.htm
Clinical signs of bluetongue in sheep, from the Institute for Animal Health
Mild disease signs in sheep
www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/disease%20symptomsBT-Mild.ppt
Moderate disease signs in sheep
www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/disease%20symptomsBT-Moderate.ppt
Severe disease signs in sheep
www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/disease%20symptomsBT-Severe.ppt
