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Novel Virostatic Agents against Bluetongue Virus  [PDF]
Linlin Gu,Volodymyr Musiienko,Zhijun Bai,Aijian Qin,Stewart W. Schneller,Qianjun Li
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0043341
Abstract: Bluetongue virus (BTV), a member in the family Reoviridae, is a re-emerging animal disease infecting cattle and sheep. With its recent outbreaks in Europe, there is a pressing need for efficacious antivirals. We presented here the identification and characterization of a novel virostatic molecule against BTV, an aminothiophenecarboxylic acid derivative named compound 003 (C003). The virostatic efficacy of C003 could be improved via chemical modification, leading to a de novo synthesized compound 052 (C052). The 50% effective concentrations (EC50) of C003 and C052 were determined at 1.76±0.73 μM and 0.27±0.12 μM, respectively. The 50% cytotoxicity concentration (CC50) of C003 was over 100 μM and the CC50 of C052 was at 82.69 μM. Accordingly, the 50% selective index (SI50) of C003 and C052 against BTV was over 57 and 306, respectively. The inhibitory effect of C003/C052 on BTV-induced apoptosis was also confirmed via the inhibition of caspase-3/-7 activation post BTV infection. C003/C052 could inhibit BTV induced CPE even when added as late as 24 h.p.i., indicating that they might act at late stage of viral life-cycle. C003/C052 could reduce over two-logs of both the progeny virus production and the number of genomic viral RNA copies. Interestingly, both the activation of host autophagy and viral protein expression were inhibited post BTV infection when cells were treated with C003 and C052, suggesting that C003/C052 might act as virostatic agents via inhibiting host autophagy activation. Although further investigations might be needed to pin down the exact mechanism of C003/C052, our finding suggested that these compounds might be potent lead compounds with potential novel mechanism of action against BTV.
A sandwich ELISA for the detection of bluetongue virus in cell culture using antiserum against the recombinant VP7 protein  [PDF]
Karam Chand,Sanchay K. Biswas,Baneswar Sing,Ankan De
Veterinaria Italiana , 2009,
Abstract: A polyclonal antibody-based sandwich enzyme-linked immunosorbent assay (s-ELISA) was developed for the detection of bluetongue virus (BTV). The test used antiserum against BTV and antiserum against the bluetongue (BT) core protein. The antiserum against the virus was used as a capture antibody and the antiserum against the protein was used for detection. In this study, antiserum to recombinant viral protein 7 (rVP7) was used as a detection antibody in place of anti-core antiserum. The assay was used to detect the BT serotypes found in India, namely: 1, 2, 9, 15, 18 and 23. The modified sandwich assay was able to detect BTV serotypes in cell culture supernatants. The use of anti-rVP7 antiserum as the detection antibody avoids the tedious and expensive purification of BTV core particles.
Structural models for the design of novel antiviral agents against Greek Goat Encephalitis  [PDF]
Louis Papageorgiou,Styliani Loukatou,Vassiliki Lila Koumandou,Wojciech Makaowski,Vasileios Megalooikonomou,Dimitrios Vlachakis,Sophia Kossida
PeerJ , 2015, DOI: 10.7717/peerj.664
Abstract: The Greek Goat Encephalitis virus (GGE) belongs to the Flaviviridae family of the genus Flavivirus. The GGE virus constitutes an important pathogen of livestock that infects the goat’s central nervous system. The viral enzymes of GGE, helicase and RNA-dependent RNA polymerase (RdRP), are ideal targets for inhibitor design, since those enzymes are crucial for the virus’ survival, proliferation and transmission. In an effort to understand the molecular structure underlying the functions of those viral enzymes, the three dimensional structures of GGE NS3 helicase and NS5 RdRP have been modelled. The models were constructed in silico using conventional homology modelling techniques and the known 3D crystal structures of solved proteins from closely related species as templates. The established structural models of the GGE NS3 helicase and NS5 RdRP have been evaluated for their viability using a repertoire of in silico tools. The goal of this study is to present the 3D conformations of the GGE viral enzymes as reliable structural models that could provide the platform for the design of novel anti-GGE agents.
Characterization of Protection Afforded by a Bivalent Virus-Like Particle Vaccine against Bluetongue Virus Serotypes 1 and 4 in Sheep  [PDF]
Ana Cristina Pérez de Diego, Thimmasandra N. Athmaram, Meredith Stewart, Belén Rodríguez-Sánchez, José Manuel Sánchez-Vizcaíno, Robert Noad, Polly Roy
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0026666
Abstract: Background Bluetongue virus (BTV) is an economically important, arthropod borne, emerging pathogen in Europe, causing disease mainly in sheep and cattle. Routine vaccination for bluetongue would require the ability to distinguish between vaccinated and infected individuals (DIVA). Current vaccines are effective but are not DIVA. Virus-like particles (VLPs) are highly immunogenic structural mimics of virus particles, that only contain a subset of the proteins present in a natural infection. VLPs therefore offer the potential for the development of DIVA compatible bluetongue vaccines. Methodology/Principal Findings Merino sheep were vaccinated with either monovalent BTV-1 VLPs or a bivalent mixture of BTV-1 VLPs and BTV-4 VLPs, and challenged with virulent BTV-1 or BTV-4. Animals were monitored for clinical signs, antibody responses, and viral RNA. 19/20 animals vaccinated with BTV-1 VLPs either alone or in combination with BTV-4 VLPs developed neutralizing antibodies to BTV-1, and group specific antibodies to BTV VP7. The one animal that showed no detectable neutralizing antibodies, or group specific antibodies, had detectable viral RNA following challenge but did not display any clinical signs on challenge with virulent BTV-1. In contrast, all control animals' demonstrated classical clinical signs for bluetongue on challenge with the same virus. Six animals were vaccinated with bivalent vaccine and challenged with virulent BTV-4, two of these animals had detectable viral levels of viral RNA, and one of these showed clinical signs consistent with BTV infection and died. Conclusions There is good evidence that BTV-1 VLPs delivered as monovalent or bivalent immunogen protect from bluetongue disease on challenge with virulent BTV-1. However, it is possible that there is some interference in protective response for BTV-4 in the bivalent BTV-1 and BTV-4 VLP vaccine. This raises the question of whether all combinations of bivalent BTV vaccines are possible, or if immunodominance of particular serotypes could interfere with vaccine efficacy.
Multiserotype Protection Elicited by a Combinatorial Prime-Boost Vaccination Strategy against Bluetongue Virus  [PDF]
Eva Calvo-Pinilla, Nicolás Navasa, Juan Anguita, Javier Ortego
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0034735
Abstract: Bluetongue virus (BTV) belongs to the genus Orbivirus within the family Reoviridae. The development of vector-based vaccines expressing conserved protective antigens results in increased immune activation and could reduce the number of multiserotype vaccinations required, therefore providing a cost-effective product. Recent recombinant DNA technology has allowed the development of novel strategies to develop marker and safe vaccines against BTV. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing VP2, VP7 and NS1 proteins from BTV-4. IFNAR(?/?) mice inoculated with DNA/rMVA-VP2,-VP7-NS1 in an heterologous prime boost vaccination strategy generated significant levels of antibodies specific of VP2, VP7, and NS1, including those with neutralizing activity against BTV-4. In addition, vaccination stimulated specific CD8+ T cell responses against these three BTV proteins. Importantly, the vaccine combination expressing NS1, VP2 and VP7 proteins of BTV-4, elicited sterile protection against a lethal dose of homologous BTV-4 infection. Remarkably, the vaccine induced cross-protection against lethal doses of heterologous BTV-8 and BTV-1 suggesting that the DNA/rMVA-VP2,-VP7,-NS1 marker vaccine is a promising multiserotype vaccine against BTV.
Epidemiological investigation of bluetongue virus antibodies in sheep in Iran  [cached]
Mohammad Khezri,Seyed Mahmoud Azimi
Veterinary World , 2013, DOI: 10.5455/vetworld.2013.122-125
Abstract: Aim: Bluetongue is a non-contagious, infectious viral disease of domestic and wild ruminants; which is believed to have originated in Africa. The epidemiology of Bluetongue virus infection is poorly defined in many parts of the world, including a wide range of Asia and the Middle East. This paper reports the results of a Bluetongue serological survey in sheep from some provinces of Iran during 2007-2008. Material and Methods: A total of 996 sheep sera were collected from 8 provinces in Iran and tested for Bluetongue virus specific using c-ELISA. Results: The results showed that the Bluetongue virus seroprevalence of sheep over the entire study areas was 34.93%, with the highest and lowest prevalence seen in West-Azerbaijan (64.86%) and Qom (12.1%) areas respectively. Conclusion: The results demonstrated a high prevalence of Bluetongue antibodies in Iranian sheep, giving serological evidence of extensive exposure to Bluetongue virus infection in some provinces of the country. [Vet World 2013; 6(3.000): 122-125]
Protection of Spanish Ibex (Capra pyrenaica) against Bluetongue Virus Serotypes 1 and 8 in a Subclinical Experimental Infection  [PDF]
Cristina Lorca-Oró, Joan Pujols, Ignacio García-Bocanegra, Gregorio Mentaberre, José Enrique Granados, David Solanes, Paulino Fandos, Iván Galindo, Mariano Domingo, Santiago Lavín, Jorge Ramón López-Olvera
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036380
Abstract: Many wild ruminants such as Spanish ibex (Capra pyrenaica) are susceptible to Bluetongue virus (BTV) infection, which causes disease mainly in domestic sheep and cattle. Outbreaks involving either BTV serotypes 1 (BTV-1) and 8 (BTV-8) are currently challenging Europe. Inclusion of wildlife vaccination among BTV control measures should be considered in certain species. In the present study, four out of fifteen seronegative Spanish ibexes were immunized with a single dose of inactivated vaccine against BTV-1, four against BTV-8 and seven ibexes were non vaccinated controls. Seven ibexes (four vaccinated and three controls) were inoculated with each BTV serotype. Antibody and IFN-gamma responses were evaluated until 28 days after inoculation (dpi). The vaccinated ibexes showed significant (P<0.05) neutralizing antibody levels after vaccination compared to non vaccinated ibexes. The non vaccinated ibexes remained seronegative until challenge and showed neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of non vaccinated ibexes from 2 to the end of the study (28 dpi) and in target tissue samples obtained at necropsy (8 and 28 dpi). BTV-1 was successfully isolated on cell culture from blood and target tissues of non vaccinated ibexes. Clinical signs were unapparent and no gross lesions were found at necropsy. Our results show for the first time that Spanish ibex is susceptible and asymptomatic to BTV infection and also that a single dose of vaccine prevents viraemia against BTV-1 and BTV-8 replication.
Segment 2 based characterization of a novel Indian Bluetongue virus isolate  [cached]
Minakshi Prasad,Koushlesh Ranjan,Pawan Kumar,Gaya Prasad
Veterinary World , 2013, DOI: 10.5455/vetworld.2013.244-248
Abstract: Aim: The study was conducted to characterize and serotype the novel isolate of bluetongue virus (BTV) isolated from India. Materials and Methods: The BTV isolate was propagated in BHK-21 cell line. Nucleic acid (dsRNA) was extracted using Trizol method and cDNA was prepared using a process called reverse transcription. The cDNA was subjected to group specific PCR using ns1 gene specific primer to confirm the isolate as BTV. The type specific PCR was conducted to confirm the serotype of the virus using vp2 gene specific primers for all the BTV serotype including BTV10. The vp2 gene specific PCR amplicon was sequenced and in-silico restriction enzyme analysis and phylogenetic analysis was conducted. Results: Group specific PCR using ns1 gene specific primers showed a single 274bp amplicon in agarose gel electrophoresis confirmed the sample as BTV. The type specific PCR using BTV10 vp2 gene specific primer showed a single amplicon of 647bp. Remaining BTV serotype specific primers didn't show any amplification. The vp2 gene PCR amplicon was sequenced. The in-silico restriction enzyme analysis of vp2 gene of Indian BTV10 isolate along with other isolates from GenBank database using HindIII, XhoII and ApoI showed a common pattern between Indian and USA isolates. Similarly, phylogenetic analyses using vp2 gene nucleotide as well as deduced amino acid sequence of Indian BTV10 isolate and global isolates showed that Indian and most of the USA isolates placed in a single clad. Conclusion: A novel BTV isolate was isolated and confirmed as BTV serotype 10. Upon molecular analysis Indian BTV10 isolate was found closer to that of USA isolates than other global isolates. [Vet World 2013; 6(5.000): 244-248]
Seroprevalence of Bluetongue Virus in Dairy Herds with Reproductive Problems in Sudan  [PDF]
Amira Mohamed Elhassan,Mohamed Abdalla Fadol,Abdel Rahim Mohamed El Hussein
ISRN Veterinary Science , 2014, DOI: 10.1155/2014/595724
Abstract: The objectives of this cross-sectional study were to determine the seroprevalence of blue tongue virus (BTV) and assess potential risk factors associated with BTV infection in dairy cattle with reproductive problems in Sudan. Serum samples were collected from a total of 784 animals from 37 herds and tested for antibodies against BTV using cELISA. A total of 663 out of 784 (84.57%) sera tested proved positive for BTV antibodies in all farms tested in Khartoum and Gazira States. The prevalence of antibodies was high in both areas being 94.32% in Gazira State and 76.62% in Khartoum State. BTV antibodies prevalence were significantly higher ( ) in older animals than in younger ones. These rates were also significantly higher in the rainy season ( ) and in Gazira State compared to Khartoum State. Sex also showed significant ( ) differences in the seroprevalence, whereby females (74.7%) had higher level than males (9.8%). However, no significant ( ) variations for BTV seroprevalence were observed between breeds. The BTV antibodies prevalence in infertility cases (86.6%) was not significantly different from that found in abortion (74.3%) or neonatal death (66.7%) cases. The high seroprevalence of BTV recorded herein calls for control strategy to be implemented. 1. Introduction Because of the difficulties involved in the diagnosis of the causative agent, bovine fetal loss resulting from abortion and/or infertility can sometimes be challenging to farmers, veterinarian, and diagnostic laboratories [1, 2]. Several bacterial, viral, protozoan, and fungal pathogens had been associated with infertility and abortions in cattle [3]. Viral agents incriminated include bovine herpes-1 (BoHV-1), bovine viral diarrhea virus (BVDV), and bluetongue virus (BTV) [4–6]. Bluetongue virus is the prototype species of the genus Orbivirus, family Reoviridae and occurs almost globally between latitudes 35°S and 50°N. In infected animals, the disease is characterized by various clinical forms, with symptoms ranging from acute to subacute, mild, or inapparent. In addition, this virus is responsible for fetal death, congenital defects, and reproductive failure in cattle [6]. At least 24 immunologically distinct serotypes of the virus are recognized [7–9]. Cattle are readily susceptible to infection with BTV; bovine BTV infections are important because cattle can serve briefly as a source of infection for Culicoides species that biologically transmit BTV to sheep and other ruminants [10]. The sequelae of abortion and the birth of calves with congenital defects like dwarf-like
Status of sheep sera to bluetongue, peste des petits ruminants and sheep pox in a few northern states of India
Veerakyathappa Bhanuprakash,Paramasivaiah Saravanan,Madhusudan Hosamani,Vinayagamurthy Balamurugan
Veterinaria Italiana , 2008,
Abstract: Bluetongue (BT), peste des petits ruminants (PPR) and sheep pox are the most economically important viral diseases of sheep in India. Serum samples obtained from sheep in five northern states of the country were screened for antibody against these agents to explore the extent of spread of these infections. A total of 516 serum samples were screened for the presence of antibodies against BT and PPR viruses. Of these, 155 samples were also tested for antibodies against sheep pox virus. BT antibodies were found in 293 (56.8%) animals, PPR virus antibodies in 215 (41.7%) and sheep pox virus antibodies in 106 (68.3%). Of the serum samples tested, 25.2% were positive for antibodies against all three viruses. These findings clearly demonstrated not only the enzootic nature of disease, but also the co-existence of antibodies to more than one of these viruses which would indicate that concurrent infections were common. Therefore, control measures should focus in combating all three diseases simultaneously by exploring the possibility of a trivalent vaccine or the use of multiple genes expressing vectored vaccine.
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