In the present study,a total of 24 MAbs were produced against bluetongue virus (BTV) by polyethyleneglycol (PEG) mediated fusion method using sensitized lymphocytes and myeloma cells. All these clones were characteriz...In the present study,a total of 24 MAbs were produced against bluetongue virus (BTV) by polyethyleneglycol (PEG) mediated fusion method using sensitized lymphocytes and myeloma cells. All these clones were characterized for their reactivity to whole virus and recombinant BTV-VP7 protein,titres,isotypes and their reactivity with 24 BTV-serotype specific sera in cELISA. Out of 24 clones,a majority of them (n = 18) belong to various IgG subclasses and the remaining (n = 6) to the IgM class. A panel of eight clones reactive to both whole BTV and purified rVP7 protein were identified based on their reactivity in iELISA. For competitive ELISA,the clone designated as 4A10 showed better inhibition to hyperimmune serum of BTV serotype 23. However,this clone showed a variable percent of inhibition ranging from16.6% with BTV 12 serotype to 78.9% with BTV16 serotype using 24 serotype specific sera of BTV originating from guinea pig at their lowest dilutions. From the available panel of clones,only 4A10 was found to have a possible diagnostic application.展开更多
Twenty five serotypes of Bluetongue virus (BTV) have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue (BT). We have therefore developed a...Twenty five serotypes of Bluetongue virus (BTV) have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue (BT). We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 (VP7) and the non-structural protein (NS1) gene from different serotypes of BTV by DNAstar showed that the 5' end of the NS1 gene is the most conserved region. The primer pairs (P1 and P2) were designed based on the highly conserved region of NS1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus (EHDV) serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes.展开更多
Biting midges of the genus Culicoides(order Diptera,family Ceratopogonidae)are potential biological vectors for the transmission of certain arboviruses among humans,livestock,and wild animals.This study collected a to...Biting midges of the genus Culicoides(order Diptera,family Ceratopogonidae)are potential biological vectors for the transmission of certain arboviruses among humans,livestock,and wild animals.This study collected a total of 405 Culicoides individuals from seven animal farms located in five counties in the border areas of Yunnan Province,China,and examined the Culicoides species composition and the major arboviruses carried by the Culicoides species.The collected Culicoides were classified into seven species with variable abundances:Culicoides arakawae(5.43%,22/405),Culicoides homotomus(1.23%,5/405),Culicoides obsoletus(19.75%,80/405),Culicoides orientalis(17.28%,70/405),Culicoides oxystoma(29.38%,119/405),Culicoides peregrinus(5.68%,23/405),and Culicoides nipponensis(21.23%,86/405).Among the seven species,C.oxystoma and C.nipponensis were distributed in all the five counties with abundances of 13.33–44.87%and 10.00–46.83%,respectively,suggesting that these were the dominant species of Culicoides widespread on animal farms in the border areas.PCR was used to detect major arboviruses in the collected Culicoides specimens,including bluetongue virus(BTV),Japanese encephalitis virus,Dengue virus,Zika virus,African swine fever virus,and African horse sickness virus.Among the tested viruses,only BTV serotype 1 was tested positive in C.oxystoma specimens collected from a buffalo farm.Culicoides oxystoma was the dominant species on animal farms in the sampled areas,but it has not previously been documented as positive for BTV in China.The current results thus suggest that C.oxystoma could be an important vector for BTV transmission in these border areas,which,however,needs to be confirmed by further comprehensive experiments.Overall,the present study provides the first profile of Culicoides species on animal farms in the China,Vietnam,and Myanmar border areas,establishes the prevalence of arboviruses carried by these Culicoides species,and suggests the vector potential of C.oxystoma species for the transmission of BTV.展开更多
文摘In the present study,a total of 24 MAbs were produced against bluetongue virus (BTV) by polyethyleneglycol (PEG) mediated fusion method using sensitized lymphocytes and myeloma cells. All these clones were characterized for their reactivity to whole virus and recombinant BTV-VP7 protein,titres,isotypes and their reactivity with 24 BTV-serotype specific sera in cELISA. Out of 24 clones,a majority of them (n = 18) belong to various IgG subclasses and the remaining (n = 6) to the IgM class. A panel of eight clones reactive to both whole BTV and purified rVP7 protein were identified based on their reactivity in iELISA. For competitive ELISA,the clone designated as 4A10 showed better inhibition to hyperimmune serum of BTV serotype 23. However,this clone showed a variable percent of inhibition ranging from16.6% with BTV 12 serotype to 78.9% with BTV16 serotype using 24 serotype specific sera of BTV originating from guinea pig at their lowest dilutions. From the available panel of clones,only 4A10 was found to have a possible diagnostic application.
基金Hi-Tech Research and Development Program of China (2006AA10Z446)
文摘Twenty five serotypes of Bluetongue virus (BTV) have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue (BT). We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 (VP7) and the non-structural protein (NS1) gene from different serotypes of BTV by DNAstar showed that the 5' end of the NS1 gene is the most conserved region. The primer pairs (P1 and P2) were designed based on the highly conserved region of NS1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus (EHDV) serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes.
基金supported by the National Key Research and Development Program of China(2017YFD0501805)the Natural Science Foundation of Shanghai,China(19ZR1469000)+1 种基金the Shanghai Science and Technology Development Funds(17391901600)the Project of International Science and Technology Cooperation,China(2014DFE30140)。
文摘Biting midges of the genus Culicoides(order Diptera,family Ceratopogonidae)are potential biological vectors for the transmission of certain arboviruses among humans,livestock,and wild animals.This study collected a total of 405 Culicoides individuals from seven animal farms located in five counties in the border areas of Yunnan Province,China,and examined the Culicoides species composition and the major arboviruses carried by the Culicoides species.The collected Culicoides were classified into seven species with variable abundances:Culicoides arakawae(5.43%,22/405),Culicoides homotomus(1.23%,5/405),Culicoides obsoletus(19.75%,80/405),Culicoides orientalis(17.28%,70/405),Culicoides oxystoma(29.38%,119/405),Culicoides peregrinus(5.68%,23/405),and Culicoides nipponensis(21.23%,86/405).Among the seven species,C.oxystoma and C.nipponensis were distributed in all the five counties with abundances of 13.33–44.87%and 10.00–46.83%,respectively,suggesting that these were the dominant species of Culicoides widespread on animal farms in the border areas.PCR was used to detect major arboviruses in the collected Culicoides specimens,including bluetongue virus(BTV),Japanese encephalitis virus,Dengue virus,Zika virus,African swine fever virus,and African horse sickness virus.Among the tested viruses,only BTV serotype 1 was tested positive in C.oxystoma specimens collected from a buffalo farm.Culicoides oxystoma was the dominant species on animal farms in the sampled areas,but it has not previously been documented as positive for BTV in China.The current results thus suggest that C.oxystoma could be an important vector for BTV transmission in these border areas,which,however,needs to be confirmed by further comprehensive experiments.Overall,the present study provides the first profile of Culicoides species on animal farms in the China,Vietnam,and Myanmar border areas,establishes the prevalence of arboviruses carried by these Culicoides species,and suggests the vector potential of C.oxystoma species for the transmission of BTV.
