Seroepidemiology of bluetongue disease in small ruminants of northeast of Iran

Objective:To estimate the prevalence and distribution of bluetongue vims antibody in sheep and goats in 25 townships of Khorasan Razavi.Bluetongue is an infectious,non-contagious,arthropod born viral disease of ruminants and has been reported from most of the tropical and subtropical regions of the world.Methods:A total number of 1034 serum samples from sheep and goats were collected and transmitted to Serological Laboratory of Veterinary Council of Khorasan Razavi.Serums were screened for the presence of group-specific bluetongue virus antibody using competitive Enzyme Linked Immuno Sorbent Assay(c-ELISA).Kesults:The seropositivitv of sheep and goats for bluetongue was found to be 89.2%.The highest prevalence rate was seen in Taybad.Khalil-abad and Torbat-jam(100%)and the least prevalence rate was seen in Jovein(55%).Conclusions:The results showed that the majority of animals in the north-east of Iran are infected with bluetongue vims.High correlation between abortion history and seroposivity emphasize the economical importance of bluetongue virus in the sheep herds of the region.

Detection of Bluetongue Virus Group-specific Antigen Using Monoclonal Antibody Based Sandwich ELISA

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA.The MAb,designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study.The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein.The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits.The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India,1,2,15,17,18 and 23.The assay could detect BTV antigen as early as day 8 in blood.It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood,washed RBCs,buffy coat and plasma.A total of 102 field samples from animals,suspected of being infected with BTV,were tested and 29.42% were positive.The blood samples were also amplified in cell culture which improved the sensitivity of the assay.Results confirmed that the sELISA is rapid and specific.

Production and Characterization of Monoclonal Antibodies to Bluetongue Virus

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.

A Pair of Novel Primers for Universal Detection of the NS1 Gene from Various Bluetongue Virus Serotypes

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.

ISG20 inhibits bluetongue virus replication

ISG20 is an interferon-inducible exonuclease that inhibits virus replication.Although ISG20 is thought to degrade viral RNA,the antiviral mechanism and specificity of ISG20 remain unclear.In this study,the antiviral role of ovine ISG20(o ISG20)in bluetongue virus(BTV)infection was investigated.It was found that BTV infection upregulated the transcription of ovine ISG20(o ISG20)in a time-and BTV multiplicity of infection(MOI)-dependent manner.Overexpression of o ISG20 suppressed the production of BTV genome,proteins,and virus titer,whereas the knockdown of o ISG20 increased viral replication.o ISG20 was found to co-localize with BTV proteins VP4,VP5,VP6,and NS2,but only directly interacted with VP4.Exonuclease defective o ISG20 significantly decreased the inhibitory effect on BTV replication.In addition,the interaction of mutant o ISG20 and VP4 was weakened,suggesting that binding to VP4 was associated with the inhibition of BTV replication.The present data characterized the anti-BTV effect of o ISG20,and provides a novel clue for further exploring the inhibition mechanism of double-stranded RNA virus by ISG20.

蓝舌病病毒一步RT-PCR检测方法的建立

为建立蓝舌病病毒（BTV）群特异性核酸检测方法,本研究针对BTV S10基因保守区域设计并合成1对特异性引物,建立了一步RT-PCR检测方法,并分别进行了特异性试验、敏感性试验以及临床样品的检测.实验结果表明：该方法对BTV1～24型均有特异性反应,而对茨城病毒（IBAV）、中山病毒（CV）和赤羽病病毒（AKAV）无交叉反应,具有良好的特异性;最低检出量为102 TCID50/mL.此外,该方法能有效的从羊的抗凝血样品中检测出BTV核酸.本研究所建立的一步RT-PCR检测方法可以用于BTV的快速检测及流行病学调查.

Importance of Viral Disease in Dairy Cow Fertility

Many viral diseases are endemic in cattle populations worldwide. The ability of many viruses to cross the placenta and cause abortions and fetal malformations is well understood. There is also significant evidence that viral infections have additional actions in dairy cows, which are reflected in reduced conception rates. These effects are, however, highly dependent on the time at which an individual animal first contracts the disease and are less easy to quantify. This paper reviews the evidence relating to five viruses that can affect fertility, together with their potential mechanisms of action. Acute infection with non-cytopathic bovine viral diarrhea virus (BVDV) in mid-gestation increases abortion rates or causes the birth of persistently infected calves. BVDV infections closer to the time of breeding can have direct effects on the ovaries and uterine endometrium, which cause estrous cycle irregularities and early embryo mortality. Fertility may also be reduced by BVDV-induced immunosuppression, which increases the susceptibility to bacterial infections. Bovine herpesvirus (BHV)-1 is most common in pre-pubertal heifers, and can slow their growth, delay breeding, and increase the age at first calving. Previously infected animals subsequently show reduced fertility. Although this may be associated with lung damage, ovarian lesions have also been reported. Both BHV-1 and BHV-4 remain latent in the host following initial infection and may be reactivated later by stress, for example associated with calving and early lactation. While BHV-4 infection alone may not reduce fertility, it appears to act as a co-factor with established bacterial pathogens such as Escherichia coli and Trueperella pyogenes to promote the development of endometritis and delay uterine repair mechanisms after calving. Both Schmallenberg virus (SBV) and bluetongue virus (BTV) are transmitted by insect vectors and lead to increased abortion rates and congenital malformations.BTV-8 also impairs the development of hatched blastocysts;furthermore, infection around the time of breeding with either virus appears to reduce conception rates. Although the reductions in conception rates are often difficult to quantify, they are nevertheless sufficient to cause economic losses, which help to justify the benefits of vaccination and eradication schemes.

Detection of arboviruses in Culicoides(Diptera:Ceratopogonidae)collected from animal farms in the border areas of Yunnan Province,China

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.