virus in the west of Iran

Aim: The objective of this study was conducted to determine the seroprevalence and S7 gene characterization of bluetongue virus (BTV) of sheep in the West of Iran, during 2007-2008. Materials and Methods: A total 372 sheep blood samples were collected from known seropositive regions in the West of Iran. Anti-BTV antibodies were detected in the serum samples by group specific, C-ELISA. Extractions of the dsRNA from whole blood samples were carried out. The One-step RT-PCR kit was used for the detection of S7 BTV gene in the blood samples. PCR products of the first amplification (RT-PCR) were used; template in the nested PCR. Products were separated by 1.2% Agarose gel electrophoresis. Nested PCR products of S7 segment from positive samples and the reference strain; BTV1 (RSA vvvv/01) were prepared for sequencing. All sequences were subjected to multiple sequence alignments and phylogenetic analysis. Results: The results showed widespread presence of the anti-BTV antibodies in the province's sheep population, where 46.77% of the tested sera were positive on C-ELISA. Bluetongue viruses were diagnosed in some animals by RT-PCR and nested PCR, by targeting S7 segment. This genome segment was sequenced and analyzed in four samples as a conserved gene in BTV serogroup. This group was very similar to the West BTV strains from USA, Africa and Europe. This clustered was categorized with BTV4 from Turkey. Conclusion: Increases in epidemic disease may constitute a serious problem for Iran's rural economy in future, and the situation is likely to worsen in the next few years as the proportion of unvaccinated livestock increases.


Introduction
occurs as far as 50 North and most recently, northern Europe [3,4].Bluetongue (BT) is a non-contagious, arthropod However, the distribution of specific insect borne viral hemorrhagic disease of ruminants, particularly vectors and different BTV serotypes differs remarkof sheep and occasionally cattle and some species of ably throughout the world, so specific vectors exist deer [1].It occurs mostly during periods of high with specific constellations of BTV serotypes and temperature and rainfall, and usually disappears with topotypes in relatively distinct global ecosystems [5]. the first frost or severe cold weather [2].Hemato-There are 24 distinct BTV serotypes currently recogphagous Culicoides insects are biological vectors that nized, and the recently described Toggenburg transmit BTV from infected to susceptible ruminants, Orbivirus (TOV) proposed to be a 25th serotype [6].thus the global distribution of BTV coincides with the Due to its economic impact, BT is an OIE listed distribution of competent Culicoides insect vectors disease.Economic losses associated with BTV and appropriate climatic conditions.Specifically, infection are through reduction in productivity and BTV exists in an extensive band that includes tropical, death; and indirectly through trade losses due to subtropical, and temperate regions of the world 0 0 animal movement restrictions, and restrictions on the between latitudes of approximately 40 North and 35 export of cattle semen [7].In sheep, the clinical signs South.Exceptions include regions of Asia and western may include fever, excessive salivation, depression, North America, where BTV infection of ruminants dyspnea and panting.Initially, animals have a clear without EDTA were stored at 4°C and serum nasal discharge; later, the discharge becomes separation was achieved by centrifuging, and then mucopurulent and dries to a crust around the nostrils.stored at -20°C until required.The samples with EDTA The muzzle, lips and ears are hyperemic, and the lips were stored at 4°C. and tongue may be very swollen.The tongue is C-ELISA: Anti-BTV antibodies were detected in the occasionally cyanotic and protrudes from the mouth.serum samples by group specific, C-ELISA, using ID-The coronary bands on the hooves are often hyperemic Vet kit (Monpellier-France).The test is based on the and the hooves painful; lameness is common and competition between test sera and an anti-VP7 MAb animals may slough their hooves if they are driven.for a VP7 antigen previously bound to the solid phase Pregnant ewes may abort their fetuses, or give birth to of an ELISA plate."dummy" lambs [2].In sheep, the severity of disease varies with the breed of sheep, virus strain, and Extraction of viral RNA: Extractions of the dsRNA environmental stresses.The morbidity rate can be as from whole blood samples were carried out using the high as 100% in this species.The mortality rate is viral RNA Mini kit (QIAamp®viral RNA Mini Kit, usually 0-30%, but can be up to 70% in highly USA) according to manufacturer's instruction.The susceptible sheep [2].
to the prepared master mix.In the RT-PCR, extracted The purpose of this study was to use a valid RT-RNA initially reverse-transcribed at 45ºC for 30 min., PCR method to detect any BTV isolates in blood followed by a step at 95ºC for 15min.Forty amplifisamples.Then we evaluate the genotypic variation of cation cycles were performed at 95ºC for 1 min, 45ºC this gene among PCR positive samples and compared for 1min and 72ºC for 2min.The PCR cycles were them with BTV strains that were isolated in other parts terminated by final extension step at 72ºC for 10 min. of the world.
Nested PCR: PCR products of the first amplification

Materials and Methods
(RT-PCR) were used; template in the nested PCR.The A total 372 sheep blood samples were collected mixture of the master mix contained, 5µl of 10x PCR from known seropositive regions in the West of Iran, buffer, 1µl dNTPs (10mM), 1µl MgCl (50mM), 1µl 2 where 135 were from Kurdistan and 237 originated (20pmol) of each IntS7F&R primers, 0.5µl Taq from Ilam.Bleeding was from the Jugular vein and polymerase (2.5U), 35µl RNase free water and 5µl of vacutainer tubes with and without EDTA were used for template that was added to the reaction at the end.The blood and sera collection, respectively.The samples thermal cycler (Master cycler personal, Eppendorf) was set to amplify the nested fragment as follows: first (MWG, Germany).Both strands of each sample were step was 95ºC for 1min, then 30 cycles were sequenced by forward and reverse primers.performed at 95ºC for 1 min, 59ºC for 1 min and 72ºC Computer analysis of the sequences: All sequences for 1min.The reaction stopped by extension at 72ºC were subjected to multiple sequence alignments and for 10 min.phylogenetic analysis using Cluster W program [15].Analysis of PCR products: Products were separated The sequence identity matrix was calculated using by 1.2% Agarose gel electrophoresis, and stained for Bioedit program (Bio Edit Sequence Alignment Editor 20min in Ethidium bromide (1µg/ml).The gels were Copywrite®1997-2007 Tom Hall).The resulting analyzed using Gel Documentation System (Bio Docdendrogram was viewed and edited by Tree View It Imaging system, UK). (1.6.6)software.
PCR product sequencing: Nested PCR products of Results S7 segment from positive samples and the reference Mention the ELISA test under materials and strain; BTV1 (RSA vvvv/01) were prepared for methods, and briefly explain its principle and the sequencing.The BTV1 strain was received from the nature of the antigen used to coat the plates.One Institution for Animal Health, Pirbright, UK and used hundred and seventy four serum samples were positive as positive control.The amplified products were for BTV-specific antibodies by cELISA test.The total purified from the Agarose gel (High pure PCR product BTV prevalence in western Iran was 46.77% (Table purification Kit, Roche, USA), and then sent for 1), where prevalence in Kurdistan and Ilam were sequencing at MWG DNA Sequencing Services 51.85% and 43.88% respectively.BTV strains that  used for nucleotide sequence comparison were listed Iran, the specific virus serotypes and vector insects in Table 2.The alignment of sequences showed that that occur within the region remain uncharacterized, each samples more than 84% overall nucleotide as they are in adjacent countries such as Kazakhstan similarity was determined.However, between these [3].The vast majority of infections with bluetongue samples 71-78%, homogeneity of S7 gene can be seen.are clinically in apparent.In a percentage of infected The result of sequence identity evaluation between sheep and occasionally other ruminants, more sever detected this group and BTV strains from GeneBank disease can occur [2].Iran's strategic location in the are shown in Table 2.This Group consisted of four South-East of Europe makes it an important potential samples beside of BTV1 (RSA vvvv/01).The source of BTV strains and serotypes that might members of this cluster showed 84-99% similarity infiltrate adjacent areas [16], thus more intensive with each other.The western Iranian BTV sequences epidemiological investigations are clearly warranted co-clustered with those of American (BTV?-502172within the region [4].BTV genome segment 7 was USA, BTV?-600558-USA), African (BTV1-S.chosen as a target gene for an RT-PCR assay because it Africa, BTV1-RSAvvvv/01) and some European codes for the major BTV species specific and (BTV4-Corsica, BTV4-Greece, BTV8-Netherlands, immuno-dominant antigen VP7, and it is therefore BTV1-Portugal) strains.They had 71-77% identity considered likely to show variations that mimic the with BTV9/16-Turkey but 82-87% with BTV4antigenic variation of different orbivirus strains and Turkey.The similarity of these sequences (KO215, I9, species as detected in serogroup-specific serological I84, I90) with BTV1 (RSA vvvv/01) was determined assays.96-99%.The identity of the BTV1 (RSA vvvv/01) S7 Seg-7 is therefore also considered unlikely to sequence and the newly generated ones with that of the show cross-reactions between Orbivirus species that out group, Epizootic hemorrhagic disease virus might be detected in more conserved DNA genes.(EHDV), were 56-57% and 53-55% respectively Since Seg-7 is highly conserved, differences between (Figure -1).
BTV isolates (even from different geographic origins)

Discussion
are in most cases likely to be relatively small, often representing changes in the third base position (and BTV exists throughout many parts of the world thus maintaining the conservation at the amino acid including, the America, Africa, southern Asia and level).Oligonucleotide primers targeting the near northern Australia.While, the virus is occasionally terminal regions of the segment 7 genome fragments present in some areas in the southern part of Europe, were designed for detecting many BTV strains for recent developments indicate that it may be extending which sequences were available in GenBank, with the its range northwards into areas of Europe that have exception of the highly divergent serotypes 7, 15 and never affected before was attributed mainly to climate 19 sequences.It were previously reported that Chinese change and was linked to the northern expansion of the and Australian strains of BTV-15 could show up to major Old World vector Culicoides imicola (Kieffer), 30% genetic divergence in Seg-7, from isolates of which is an Afro-Asiatic species of biting midge [16].
other BTV serotypes [22].Molecular techniques have; This study reports, for the first time, the prevalence of compared to a valuable and exclusive application BT antibodies (46.77%) in sheep in the West of Iran.
comparing to the other diagnostic methods.For The various proportions of seropositive animals came example, it provides the opportunity to trace the origin from Northern districts.This may be attributed to the of BTV in outbreak situations and study their genetic presence of many insects in these areas.Climatic variation [23].Previous studies have showed that East factors play an important role in the occurrence of and West BTV topotype had specific characters in the BTV infection in animals and influence the size of majority of genome segments studied, especially the vector populations and periods of their seasonal ones [24].The ability to differentiate isolates from activity [17].
different geographical origins based on genomic Higher seropositivity rates 76.44%, 54.1%, and sequences has been demonstrated for other 48.4%, detected in the East-Azerbaijan in the North of Orbiviruses like Epizootic haemorrhagic disease virus Iran [18], Saudi Arabia [19], and Pakistan [20], (EHDV) [25].Wilson et al., (2000) compared the respectively.However, lower incidence rates 8.3%, genetic diversity of S7 segment among isolates from 29.5%, and 34.7%, reported from India [21], and the the USA, Caribbean Basin and Central America (west West-Azerbaijan in the North-West of Iran [4], Southgroup of BTV) and found several distinct clads [26].Eastern Turkey [13], respectively.Although BTV In this study, the source of BTV2, BTV3 and infection of sheep is clearly widespread in northwest BTV17 was presented.BTV2-OnaB showed 99.8% dary disease and there are several reasons that could identity with BTV2-S.Africa, but only 79.4% with explain the observed similarities between the viruses BTV11-US.BTV3 strains which circulated in Caribbean from western Iran and Turkey, such as the long border Basin and Central America had nearly complete through which vertebrate and invertebrate hosts are identical S7 gene with BTV3S.Africa.These data transported between the two countries.Moreover, the confirmed that the viruses in the related regions can be ecosystems in the two countries are similar and can originated from South Africa.But BTV17 in Caribbean support the same vectors.In the previous study BTV4-Basin and Central America probably came from US. Turkey was grouped with other European (Greece, Because they are phylogentically near to BTV17-US.Spain, Italy, Bulgaria and Corsica) and African strains During the 2004-2006 BT outbreaks in Portugal, (Morocco), suggesting that the BTV4 strain which molecular investigations were performed to trace the invaded Europe and the Eastern Mediterranean region origin of the outbreak strains.BTV genome segments since 1999 belonged to the western BTV lineage S7, L2 and S10 for molecular analysis of BTV2 and [29,30].Co-clustering reference strain BTV1 (RSA/ BTV4 isolated, where it was shown that the homology vvvv01) with this group, which originated from west at nucleotide level between the two isolates was less (South Africa), supported the categorization of these than 75%, but 99.9% and 99.3% with the South viruses with west origin BTV strains.However, some African BTV2 and Cortisone/Italian BTV4 other investigator made evidence that invasion of respectively.It was concluded that the two Portuguese BTV1 lineage to Europe could be form bout east and viruses had distant origins [27].Mann et al., (2008) west sources [30][31][32].By assessment of the genetic compared segment 7 of 41 BTV strains from all diversity of BTV gene, lots of valuable information around of world.They found S7 gene indicated about epidemiology of this virus can be collected.In significant level of variation.They found the viruses our study we concluded that probably there are west can be segregated into six clads, three in western and BTV strains in the West of Iran. the others in eastern topotype.The authors suggested Conclusions that the source of the north European BTV8 Increases in epidemic disease may constitute a (Netherlands 2006/04) came from west.Because serious problem for Iran's rural economy in future, and genome segment 7, like the other genes, showed very the situation is likely to worsen in the next few years as close relationship with this category.For example BTV8 the proportion of unvaccinated livestock increases.had 97% sequence identity with BTV1-Honduras and Hence, there is a need to act now to strengthen veterinary BTV1-S.Africa from western topotype [28].
services in rural areas.In this study the high percentage of homology between the nucleotide sequence of S7 gene and Author's contribution published BTV strains in GenBank, confirmed the M Khezri prepared samples.SM Azimi implemented identity of detected agents as BTV.We have attempted the study design and analyzed data.M Khezri drafted to investigate the genetic variation of detected BTV in and revised the manuscript.All the authors read and the West of Iran by sequencing of seg-7.This result approved final Manuscript.was consistent with previous studies that reported the variability of S7 gene in BTV up to 30% [22,26,28].Acknowledgments According to the epidemiology of BTV in the world, We are very grateful to the staff of the Veterinary the situation of Middle East is unique.Because it is Division of Agricultural, and Natural Resources Research between east and west hemisphere, and may be Center of Kurdistan, particularly Dr. Baharake invaded by BTV strains that circulated in these two Mohammadian, Babak Rokhzad, Homan Khanbabaie.macro-environments.In addition, this area can play an This study was supported with a grant of Razi Vaccine important role for in transferring BTV strains between and Serum Research Institute.these two ecosystems.Therefore, it can be anticipated that both East and West BTV strains are found in this References part of the world, possibly because of the extensive 1.
animal transportation in this part of the country, and Studies of the Antigenic relationships between reassortment ability of BTV may be explained it.

Table - 1
. Seroprevalence of bluetongue antibodies in sheep in the West of Iran

Table - 2
. The result of sequence identity analysis between samples in the West of Iran and other BTV strains from Gene bank