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1.
Bluetongue (BT) and/or BT viruses (BTV) have been identified in the Mediterranean basin and the Balkans each year from 1998 to 2002 and in particular BTV serotype 2 in the French Island of Corsica (2000 and 2001). In response to these virus incursions, the French Veterinary Authorities carried out epidemiological studies that included virological, serological and entomological analysis, and two vaccination campaigns performed in the winter of 2000/2001 and the winter and spring of 2001 and 2002. Rapid and reliable serotype differentiation is essential at the start of an outbreak to allow an early selection of vaccine to control the spread of the virus. Thus, molecular tools, that complement conventional methods, have been developed for early detection of infection, determination of the serotype, and differentiation between natural infection and vaccination. Serological results showed that the first vaccination campaign during the winter of 2000/2001 did not provide full protection for all sheep and during the summer of 2001, 335 sheep flocks in Corsica were again infected by BTV 2 (7-fold more that in 2000). Entomological studies have demonstrated that the only proven vector of the disease, Culicoides imicola, was present in the island in 2000 and that it has successfully established itself in Corsica. The safety and immunogenicity of the commercial South African vaccine were studied. Fourteen sheep were vaccinated and then observed for clinical signs. Blood, sera, spleen and lymph nodes were collected and analyzed, and the results confirmed the safety and potency of using this vaccine to protect sheep from clinical disease. As a result, an intensive vaccination campaign was performed during winter and spring 2001/2002. No cases of BT had been observed by the end of summer 2002, indicating that the vaccination campaign has been successful in protecting sheep from infection.  相似文献   

2.
The complete nucleotide sequences of the VP2 segments of bluetongue virus (BTV) isolates recovered from Italy, Greece and Israel, from 1998 to 2003, were determined. Phylogenetic analysis of these sequences, those from related viruses and the South African vaccine strains, were used to determine the probable geographic origin of BTV incursions into Italy. Results indicated that viruses from each of the four serotypes isolated in Italy (2, 4, 9 and 16) possibly had a different origin. Analysis of the bluetongue virus serotype 2 (BTV‐2) isolates gave evidence that this serotype probably moved from Tunisia. BTV‐4 results showed probable incursion from the southwest and not from Greece or Israel. BTV‐9 isolates clearly have an eastern origin (most probably Greece), whereas BTV‐16 isolates are indistinguishable from the BTV‐16 live attenuated vaccine strain. The phylogenetic findings were supported by polyacrylamide gel electrophoresis (PAGE) analysis of the complete amplified genome of each isolate except for BTV‐16 Italian field isolate, which showed a slightly different PAGE profile. A combination of the complete VP2 sequencing and PAGE analysis of complete genomes, allowed not only phylogenetic analysis, but also vaccine detection and assessment of reassortment events.  相似文献   

3.
The complete nucleotide sequences of the VP2 segments of bluetongue virus (BTV) isolates recovered from Italy, Greece and Israel, from 1998 to 2003, were determined. Phylogenetic analysis of these sequences, those from related viruses and the South African vaccine strains, were used to determine the probable geographic origin of BTV incursions into Italy. Results indicated that viruses from each of the four serotypes isolated in Italy (2, 4, 9 and 16) possibly had a different origin. Analysis of the bluetongue virus serotype 2 (BTV-2) isolates gave evidence that this serotype probably moved from Tunisia. BTV-4 results showed probable incursion from the southwest and not from Greece or Israel. BTV-9 isolates clearly have an eastern origin (most probably Greece), whereas BTV-16 isolates are indistinguishable from the BTV-16 live attenuated vaccine strain. The phylogenetic findings were supported by polyacrylamide gel electrophoresis (PAGE) analysis of the complete amplified genome of each isolate except for BTV-16 Italian field isolate, which showed a slightly different PAGE profile. A combination of the complete VP2 sequencing and PAGE analysis of complete genomes, allowed not only phylogenetic analysis, but also vaccine detection and assessment of reassortment events.  相似文献   

4.
The replication of cell culture passaged Australian bluetongue virus (BTV) isolates, serotypes 20 (CSIRO19) and 1 (CSIRO156), and an untyped BTV (CSIRO154) was assessed in eight continuous cell lines (one derived from baby hamster kidney cells, BHK-21; three derived from monkey kidney cells, Vero, LLC-MK2 and CV-1P; a foetal ovine lung and a mouse fibroblast cell line, CSL503 and L929, respectively, a Super-Vero-Porcine stable cell line, SVP; and a mosquito cell line, Aedes albopictus cells) and in 11-day-old embryonated chicken eggs (ECE) at different multiplicities of infection. All three viruses replicated in the cell lines tested, maximum extracellular virus yields being attained from BHK-21 cells at high multiplicities of infection (approximately 10 PFU per cell). Also BHK-21 cells produced much higher yields of virus than the other cell lines tested when low multiplicities of infection were used (approximately 10(-4) PFU per cell). All BTV serotypes multiplied in Singh's Aedes albopictus cells with no cytopathogenic effects over the 4 day period tested. The viruses also replicated in 11-day-old ECE; however, the sensitivity of ECE for growth of the Australian serotypes was not as high as has been reported for BTV isolates in other countries. In all cell culture systems and in ECE, BTV1 and BTV20 replicated more efficiently than did CSIRO154 virus.  相似文献   

5.
Genome segment 10 of bluetongue virus (BTV) serotype 11 UC8 strain was cloned and subsequently hybridized to viral double-stranded RNA extracted from 90 field isolates of BTV serotypes 10, 11, 13, and 17; the prototype strains of BTV 2, 10, 11, 13, and 17; the prototype strain epizootic hemorrhagic disease virus (EHDV) serotype 1; and 4 field isolates of EHDV serotype 2. The 90 field isolates were obtained from different counties in California, Louisiana, and Idaho during the years 1979, 1980, and 1981. The cloned genetic probe hybridized with all the BTV samples tested, showing different degrees of cross-hybridization at the stringency conditions used in this study. This indicated that BTV genome segment 10 has conserved nucleotide sequences among the BTV serotypes 2, 10, 11, 13, and 17. No cross-hybridization signals were detected between the cloned genome segment 10 of BTV 11 UC8 strain and the prototype strain of EHDV serotype 1 and the field isolates of serotype 2. This probe recognized a wide variety of BTV isolates.  相似文献   

6.
After 44 years of epidemiological silence, bluetongue virus (BTV) was reintroduced in Portugal in the autumn of 2004. The first clinical cases of bluetongue disease (BT) were notified in sheep farms located in the South of Portugal, close to the Spanish border. A total of six BTV, five of serotype 4 and one of serotype 2 were isolated from sheep and cattle during the 2004-2006 epizootics. The nucleotide sequence of gene segments L2, S7 and S10 of BTV-4 prototype strain (BTV4/22045/PT04) obtained from the initial outbreak and of BTV-2 (BTV2/26629/PT05) was fully determined and compared with those from other parts of the world. The phylogenetic analysis revealed that BTV4/22045/PT04 is related to other BTV-4 strains that circulate in the Mediterranean basin since 1998, showing the highest identity (99%) with BTV-4 isolates of 2003 from Sardinia and Corsica, whereas BTV2/26629/PT05 is almost indistinguishable from the Onderstepoort BTV-2 live-attenuated vaccine strain and its related field strain isolated in Italy. Since live-attenuated BTV-2 vaccine was never used in Portugal, the isolation of this strain may represent a natural circulation of the vaccine virus used in other countries in Mediterranean Europe.  相似文献   

7.
In October 2000, bluetongue virus was detected on the French island of Corsica. The disease was also reported in Sardinia, Calabria, Sicily and on the Spanish islands of Majorca and Minorca. This paper describes the use of molecular techniques for a rapid identification and serotype determination of serotype 2 of the virus. The nucleotide sequences of segments 2 and 7 of the genome of the Corsican strain were determined and its phylogenetic relationships are described.  相似文献   

8.
动物蓝舌病毒L2基因克隆及序列分析比较   总被引：2，自引：0，他引：2  
对 15株中国动物蓝舌病毒血清 1型 (BTV- 1)野毒株及 1株弱毒疫苗株 L 2基因进行了克隆和测序 ,并进行了系统进化分析。中国 BTV- 1型毒株分为 2个组 :分离年代最早的 Y86 3毒株 (1979年 )为 组 ;1996年以后分离的 14株毒株为 组。 2组之间的同源性是 91.6 %。 组又分为 4个谱系 ,疫苗株 Vac F45与原始株 V6 5 8分别属于 组的第 1和第 3谱系 ,核苷酸同源性 98.6 %。由此表明 ,中国 BTV- 1型毒株在自然进化及鸡胚传代驯化过程中 ,L2基因发生了遗传变异。  相似文献   

9.
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10.
Thirty-two bovine field isolates of bluetongue virus (BTV), 6 field isolates of epizootic hemorrhagic disease virus (EHDV) from deer, 4 BTV prototype serotypes (10, 11, 13, and 17), and 2 EHDV prototype serotypes (1 and 2) were coelectrophoresed, using polyacrylamide gels. Field isolates were obtained from various regions of the United States. Analysis of polyacrylamide gels and scattered plots generated for comparison of migration patterns for different isolates within each serotype of BTV revealed wide variation among the individual segments. The BTV serotypes 10 and 11 had more variation, compared with BTV serotypes 13 and 17, especially for migration of genome segment 5. A definitive correlation was not seen between the double-stranded RNA migration profiles on polyacrylamide gel electrophoresis, geographic origin, herd of origin, or year of collection. One BTV field isolate contained more than 1 electropherotype, with 2 bands at the segment-7 position, and it was further characterized as BTV serotype 11. Segments 2 and 5 of EHDV isolates were more variable in their migration than were the other gene segments. Generally, migration profiles for EHDV double-stranded RNA were more variable, compared with those of BTV isolates. Although a correlation was found between migration profiles and serotype of 2 isolates of EHDV, a study of additional EHDV isolates is required before the diversity of electrophoretic patterns of EHDV can be determined.  相似文献   

11.
The double-stranded RNA genome from 117 field isolates of bluetongue virus (BTV) serotypes 10, 11, 13, and 17 was blotted onto nitrocellulose paper and hybridized with a radioactively labeled cloned copy of DNA genome segment 2 of BTV-17. Viral RNA from BTV prototype strains 2, 10, 11, 13, and 17 were used as controls. The probe hybridized only with the viral RNA from prototype BTV-17 virus and field isolates of BTV-17. There was no cross hybridization with field isolates of BTV serotypes 10, 11, and 13. A complementary DNA probe developed from genes coding for BTV serotype specificity was effectively used in a slot-blot hybridization system for efficiently characterizing the viral serotype.  相似文献   

12.
This paper records the results of a bluetongue virus (BTV) serological survey and reports the first isolation of BTV on the French Island of Reunion. In January 2003, the French Island of Reunion, located off the coast of Madagascar, reported an outbreak of disease in cattle that resembled clinical bluetongue (BT) in sheep. The suspected causal agent was isolated and identified as epizootic haemorrhagic disease of deer virus (EHDV). However, because of the similarity in the clinical signs to those of BT, a retrospective survey against BTV was carried out using sera collected in 2002. Results revealed the presence of antibody in all sera tested indicating that BTV has been resident on the Island since 2002, and probably earlier. Although up to July 2003 no clinical BT had ever been reported in sheep, BTV viral RNA was amplified by RT-PCR from a single sheep blood collected in February that year, which strongly suggested that BTV was currently circulating on the Island. Following a second outbreak of disease in August 2003, this time involving a flock of Merino sheep, infectious BTV was finally isolated, and identified by both traditional and molecular techniques as serotype 3. The nucleotide and amino-acid sequences of the RT-PCR products amplified for BTV segments 7 and 10 from the sheep blood collected in February and August from different areas of the Island, were sufficiently diverse as to suggest that they were of different origins and/or different BTV serotypes.  相似文献   

13.
The 10 double-stranded RNA gene segments of 2 vaccinal strains of bluetongue virus (BTV) serotype 10 that are used in the United States (BTV CA8 California and BT-8 Colorado), and a BTV-10 isolate recently obtained from infected sheep in Washington (state) were characterized by oligonucleotide fingerprint analyses. It was determined that although the 2 BTV-10 vaccinal strains are genotypically distinct, they are closely related both to each other and to the United States prototype BTV-10 virus. The BTV-10 field isolate appears to be a naturally occurring reassortment virus with genome segments derived from both United States prototype BTV-10 and BTV-11 viruses. However, one RNA segment of the isolate was totally unlike the corresponding segments of United States prototype BTV-10, -11, -13 and -17 viruses.  相似文献   

14.
Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), one of OIE listed diseases. Most of the currently available detection methods do not allow discrimination between wild-type CSF viruses and the vaccine strains. This study was designed to develop a multiplex real-time RT-PCR for the quantitative and differential detection of wild-type viruses and C-strain vaccine widely used in China. CSFV specific primers and two differently labeled TaqMan probes for the differentiation of wild-type viruses from C-strain vaccine were designed in the 5'-untranslated region of the viral genome of CSFV. The two TaqMan probes specifically hybridize wild-type viruses of different subgroups and C-strain vaccine, respectively, in the multiplex real-time RT-PCR, with no cross-reaction to a number of non-CSFV porcine viruses. The sensitivity of the assay for detecting wild-type and C-strain-type vaccine viruses was determined to be 41.8 and 81.5copies/microL viral RNA, respectively. Completely correct differentiation of wild-type viruses from C-strain vaccine was achieved when testing reference strains and characterized field isolates of CSFV in China. The multiplex real-time RT-PCR was able to detect the viral RNA in the whole blood samples of experimentally infected pigs as early as 2 days post-infection, 3 to 4 days prior to the onset of clinical signs in co-housed pigs. The agreements between the multiplex real-time RT-PCR and a multiplex RT-nested PCR for detection of wild-type and C-strain-type viruses were 96.9% and 100%, respectively, when detecting 106 different field samples. There is a positive correlation between the titers of C-strain vaccines titrated in rabbits and RNA copies quantitated by the multiplex real-time RT-PCR. The novel assay described here is rapid and sensitive, and is useful for differentiating field strains and C-strain of CSFV in China.  相似文献   

15.
This study reports on an outbreak of disease that occurred in central Algeria during July 2006. Sheep in the affected area presented clinical signs typical of bluetongue (BT) disease. A total of 5245 sheep in the affected region were considered to be susceptible, with 263 cases and thirty-six deaths. Bluetongue virus (BTV) serotype 1 was isolated and identified as the causative agent. Segments 2, 7 and 10 of this virus were sequenced and compared with other isolates from Morocco, Italy, Portugal and France showing that they all belong to a ‘western’ BTV group/topotype and collectively represent a western Mediterranean lineage of BTV-1.  相似文献   

16.
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17.
Serological surveys revealed that some cattle in northern Australia possessed bluetongue virus (BTV) group-reactive (agar gel diffusion precipitin, AGDP, and complement-fixing, CF) antibodies, but not serum neutralizing (SN) antibodies, to BTV20, a new type previously found in Australia. Attempts were made during 1979 to isolate viruses causing these reactions. There was one isolate of a virus (CSIRO 154) and eight isolates of another virus (CSIRO 156) made from the blood of healthy cattle in the Northern Territory. These viruses could not be distinguished from BTV20 by AGDP, CF or fluorescent-abtibody tests and hence were designated members of the bluetongue serogroup. Serotyping was carried out using the plaque-inhibition and plaque-reduction SN tests. CSIRO 156 virus could not be distinguished from BTV1 by any of the SN tests and it was concluded that it was an Australian isolate of the BTV1 serotype. CSIRO 154 virus was found to be related to, but not identical with, BTV6. It is probably not one of the known 20 BTV serotypes and may represent a new BTV serotype. None of the three Australian BTV isolates is known to cause clinical disease in sheep or cattle under natural conditions, and biochemical comparisons with the African BTV serotypes may show differences not revealed by these serological studies.  相似文献   

18.
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19.
Twenty-eight PRRS viruses (PRRSVs) isolated from various pig farms in Korea between 2002 and 2003 were sequenced for open-reading frame (ORF) 5 and/or full-length genome and compared with numerous PRRSVs reported from North America, Europe and Asia. All Korean isolates examined were genetically of the North American genotype. The ORF5 sequence of one isolate was identical to Ingelvac PRRS MLV vaccine virus. ORF5 nucleotide sequence divergence of the remaining 27 Korean PRRSVs from VR-2332, the prototype of the North American PRRSV and parental strain of the MLV vaccine virus, ranged from 1.3% to 12.9%, which corresponded to 2.0% to 14.9% divergence at the amino acid level, raising a concern on the efficacy of the MLV vaccine. Phylogenetic analyses of ORF5 and/or full-length sequences revealed that the Korean PRRSVs formed a clade distinct from PRRSVs reported from other Asian countries (China, Taiwan, Japan, and Thailand). Our study demonstrated that PRRSVs of the North American genotype were introduced to the Korean swine population some time ago and have evolved independently from PRRSV in other Asian countries, suggesting that geographic separation might influence the molecular evolution of PRRSV. This should be taken into consideration when a national PRRS prevention and control policy for international trade is established.  相似文献   

20.
At the end of September 2000, clinical symptoms of Bluetongue appeared in sheep flocks of the Balearic Islands (Spain). The presence of the BTV serotype 2 in tissue and blood samples of affected animals was confirmed by laboratory techniques. A systematic vaccination were carried out in affected areas using a live monovalent serotype 2 vaccine available from Onderstepoort laboratory (South Africa). In order to perform epidemiological studies, a new method to differentiate between the NS1 genes of BTV-2 affecting the Balearic islands and that of the Onderstepoort commercial live virus vaccine (monovalent, serotype 2) has been developed. This procedure is based on the use of an RT-PCR, followed by restriction endonuclease analysis. Epidemiological data of a study carried out in the period January-October 2001 using this procedure are included.  相似文献   

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