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The purpose of this study was to investigate the antibody response to the bovine ephemeral fever virus (BEFV) vaccine in Japanese Black calves. Twenty‐eight Japanese Black calves, which were raised on an ordinal farm, were divided into two groups. Fifteen calves received the inactivated BEFV vaccine at 12 and 16 weeks of age (vaccination group), and 13 calves did not receive the vaccine (non‐vaccination group). Blood samples were obtained at 0, 4, 8, 12, 16, 20, 24, 28, and 32 weeks of age. As the results, in the vaccination group, the antibody titers at 16, 20, 24, 28, and 32 weeks of age were significantly higher than those at 0, 4, 8, and 12 weeks of age (p < 0.01). Additionally, antibody titer in the vaccination group increased after 16 weeks of age and showed a significantly higher level than that in the non‐vaccination group throughout the remaining experimental period (p < 0.01). These results might be helpful in establishing a vaccination program against BEFV in calves. [ABSTRACT FROM AUTHOR]

Background: Bovine ephemeral fever (BEF) is an arthropod-borne viral disease caused by the BEF virus (BEFV). This single-stranded RNA virus that affects cattle and water buffalo is endemic in tropical and subtropical regions including Iran. While BEF is a major disease of cattle in Iran, information regarding its agent, molecular characterization, and circulating viruses are highly limited. The current study aimed to, firstly, determine the genetic and antigenic characteristics of BEFV strains in Khuzestan province in Southwest of Iran in 2018 and 2020 and, secondly, to compare them with strains obtained from other areas. Results: By phylogenetic analysis based on the Glycoprotein gene, BEFV strains were divided into four clusters of Middle East, East Asia, South Africa, and Australia; in which the 2018 and 2020 Iranian BEFV strains were grouped in the Middle East cluster with the Turkish, Indian, and Israeli strains. Depending on the chronology and geographical area, the outbreaks of Turkey (2020), Iran (2018 and 2020), and India (2018 and 2019) are proposed to be related. These BEFVs had the highest identity matrix and the lowest evolutionary distance among the studied strains. Multiple sequence alignment of G1, G2, and G3 antigenic sites showed that these neutralizing epitopes are highly conserved among the strains of the Middle East cluster; however, the strains previously identified in Iran differed in three amino acids placed in G1 and G2 epitopes. Conclusion: The findings revealed that BEFVs circulating in the Middle East are closely related phylogenetically and geographically. They also have similar antigenic structures; therefore, developing a vaccine based on these strains can be effective for controlling BEF in the Middle East. [ABSTRACT FROM AUTHOR]

Aims: Vaccines for bovine ephemeral fever virus (BEFV) are available but are difficult to produce, expensive or suffer from genetic instability. Therefore, we designed constructs encoding C‐terminally truncated forms (transmembrane anchoring region deleted) of glycoproteins G and GNS such that they were secreted from the cell into the media to achieve high‐level antigen expression, correct glycosylation pattern and enable further simple purification with the V5 epitope tag. Methods and Results: In this study, synthetic biology was employed to create membrane‐bound and secreted forms of G and GNS glycoprotein. Mammalian cell culture was employed as an antigen expression platform, and the secreted forms of G and GNS protein were easily purified from media using a highly effective, single‐step method. The V5 epitope tag was genetically fused to the C‐termini of the proteins, enabling detection of the antigen through immunoblotting and immunomicroscopy. Our data demonstrated that the C‐terminally truncated form of the G glycoprotein was efficiently secreted from cells into the cell media. Moreover the immunogenicity was confirmed in mice test. Conclusions: The immuno‐dot blots showed that the truncated G glycoprotein was present in the total cell extract, and was clearly secreted into the media, consistent with the western blotting data and live‐cell images. Our strategy presented the expression of secreted, epitope‐tagged, forms of the BEFV glycoproteins such that appropriately glycosylated forms of BEFV G protein was secreted from the BHK‐21 cells. This indicates that high‐level expression of secreted G glycoprotein is a feasible strategy for large‐scale production of vaccines and improving vaccine efficacy. Significance and Impact of the Study: The antigen expression strategy designed in this study can produce high‐quality recombinant protein and reduce the amount of antigen used in the vaccine. [ABSTRACT FROM AUTHOR]

Bovine Ephemeral fever virus (BEFV) is endemic in South Africa and has a negative economic impact on the meat and dairy industries. Bovine ephemeral fever or three‐day stiff‐sickness is controlled through annual vaccination with a live attenuated virus manufactured by Onderstepoort Biological Products (South Africa). We announce the genome sequences of two South African Bovine Ephemeral Virus strains; the live attenuated vaccine strain (14 876 nucleotides) and a field strain (14 883 nucleotides). A mutation in the alpha 3 open reading frame rendered the gene non‐functional in both genomes. Phylogenetic analysis based on the glycoprotein gene showed that the two strains clustered with the South African lineage. [ABSTRACT FROM AUTHOR]

Bovine ephemeral fever (BEF), caused by the bovine ephemeral fever virus (BEFV), is associated with an acute febrile infection in cattle and widespread in tropical and subtropical areas, leading to great economic losses to cattle and milk industry. However, no efficacious BEF vaccine is currently available in China. Herein, we generated a recombinant rabies virus (RABV) expressing BEFV glycoprotein (LBNSE‐BG), utilizing a reverse genetics system based on the recombinant rabies virus strain LBNSE. It was found that mice immunized with LBNSE‐BG produced robust neutralizing antibodies against both BEFV and RABV, and developed complete protection from lethal RABV challenge. Further studies showed that LBNSE‐BG activated more dendritic cells (DCs), B cells and T cells in immunized mice than the parent virus LBNSE. Collectively, these findings demonstrate that the recombinant LBNSE‐BG described here has the potential to be developed as a cost‐effective and efficacious bivalent vaccine for cattle use in endemic areas of BEF and rabies. [ABSTRACT FROM AUTHOR]

Bovine ephemeral fever virus (BEFV) is an evolving arbovirus reported across tropical, subtropical and temperate climatic zones globally. This study reveals prominent BEFV outbreaks in India, emerging annually during monsoons in subtropical areas accompanied by a congenial abundance of the vector population. PCR‐based detection of viral genomic RNA in the blood samples collected during outbreaks of 2018–2019 for the first time confirmed the presence of BEFV in India. Phylogenetic analysis based on the glycoprotein gene of BEFV showed the current isolates to have high sequence homology with Middle Eastern lineage with nearly 97%, identity to Turkey (BEFV Ad12/TUR) and Israel (Israel 2006) isolates. [ABSTRACT FROM AUTHOR]

This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. Experiments performed in both intact cells and digitonin-permeabilized cells revealed that M protein targets the nucleolus and requires carrier, cytosolic factors or energy input. By employing sequence and mutagenesis analyses, we have determined both nuclear localization signal (NLS) 6KKGKSK11 and nuclear export signal (NES) 98LIITSYL TI106 of M protein that are important for the nucleocytoplasmic shuttling of M protein. Furthermore, we found that both lamin A/C and chromosome maintenance region 1 (CRM-1) proteins could be coimmunoprecipitated and colocalized with the BEFV M protein. Knockdown of lamin A/C by shRNA and inhibition of CRM-1 by leptomycin B significantly reduced virus yield. Collectively, this study provides novel insights into nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1 and by a transcription- and carrier- and energy-dependent pathway. • The M protein of BEFV shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. • We have determined both nuclear localization signal (NLS) and nuclear export signal (NES) of M protein for the nucleocytoplasmic shuttling of M protein. • Nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1. • Knockdown of lamin A/C and inhibition of CRM-1 significantly reduced virus yield. [ABSTRACT FROM AUTHOR]

• We reported on the expression of secretory G protein of bovine ephemeral fever virus (BEFV) by mammalian stable cells. • The secretory GΔTM protein reacted with a natural BEF convalescent bovine serum. • It induced neutralising antibody in the guinea pig immunisation model. • This is an attractive antigen production platform for development of BEFV vaccine and diagnostic assays. Bovine ephemeral fever virus (BEFV) causes an acute febrile disease in cattle and water buffalo. The disease has an impact on dairy and beef production in tropical and subtropical countries. Vaccination is used for disease prevention and control. In this study, we developed a recombinant lentivirus to produce mammalian stable cells expressing histidine-tagged BEFV G protein with a deleted transmembrane domain (GΔTM) as a secretory protein. In addition, guinea pigs were immunised with the purified GΔTM protein and booster immunised at a 3-week interval. The mammalian stable cells were able to continuously produce GΔTM protein for a minimum of 25 passages. All of the mammalian stable cells expressing GΔTM protein could react specifically with a BEFV convalescent bovine serum. Serum samples from the immunised guinea pigs could react strongly and specifically with the purified GΔTM protein. Moreover, post-immunised guinea pig sera contained antibodies that could neutralise BEFV. These results indicate that the G protein without a transmembrane domain can be used as a subunit vaccine for the prevention and control of BEFV. The availability of the mammalian stable cells, which constitutively express GΔTM protein, could facilitate the potential use of the secretory protein for BEFV diagnosis and vaccine development. [ABSTRACT FROM AUTHOR]

Epitope-G1 of bovine ephemeral fever virus (BEFV) G glycoprotein has been genetically and antigenically conserved among various isolates of BEFV and only reacts with anti-BEFV neutralising antibodies. Therefore, it is a candidate antigen for development of the enzyme linked immunosorbent assay (ELISA) for serological identification bovine ephemeral fever (BEF)-infected animals. The aim of this study was to produce a monoclonal antibody (MAb) against recombinant G1 antigen expressed into Escherichia coli. For this purpose, somatic cell hybrids between SP2/0 myeloma cells and spleen cells derived from Balb/c mice immunized with maltose-binding protein (MBP)-G1 fusion protein were established. After three rounds of cloning, the stability of antibody secretion in the positive clones was confirmed by ELISA and the reactivity of the MAbs against recombinant G1 was verified by Western blot analysis. The specific MAbs produced against recombinant G1 antigen in this study could be used for establishing BEFV diagnostic experiments in the future. [ABSTRACT FROM AUTHOR]

Highlights • This is the first report on genetics of bovine ephemeral fever virus (BEFV) in Southeast Asia and the connection of BEFV circulation in Asia. • Thai BEFVs were divided into two clusters in the second and third sub-clades of East Asia lineage. • BEFVs that caused high motility rate in cattle are in the third sub-clade. • P' ORF of Thai BEFV contains a premature stop codon resulting in a shorter ORF of 64 instead of 147 nucleotides. • The complete genome of Thai (LRI0045) and Chinese (J02L) BEFVs share several common characteristics. Abstract Bovine ephemeral fever (BEF) is an arthropod-borne disease caused by bovine ephemeral fever virus (BEFV), a negative sense, single-stranded RNA virus. BEFV is endemic in tropical and sub-tropical regions including Thailand, a country in mainland Southeast Asia. However, there are few studies on BEFV and no available information regarding molecular characteristics of BEFV in Thailand. Therefore, the aims of this study were to genetically characterize Thai BEFVs and reveal their evolutions by phylogenetic analysis of G gene ectodomain sequences. From 2013 to 2017, blood samples were collected from bovine that matched with BEF case definition from three regions of Thailand. Thai BEFV G genes and a whole genome of an isolate, East Asia/TH/LRI0045/2016, were sequenced and characterized. Additionally, their phylogenies were constructed. This is the first report on genetics of BEFV in Southeast Asia. G ectodomain encoding region of Thai BEFV found during 2013–2017 are closely related to the second and third sub-clades of East Asia lineage. In addition, we observed mutation in the putative P' ORF of all Thai BEFVs which generated a premature stop codon. Thai G gene sequences are closely related to those of mainland Chinese and Taiwanese isolates. The whole genomic sequences of Thai BEFV and East Asia/China/JT02 L/2002 possess common characteristics, suggesting shared evolutionary relationship between East and Southeast Asian strains. Further studies on relationship between animal translocation, circulation of BEFV in Greater Mekong subregion and acquisition of more G gene sequences may improve understanding of BEFV epidemiology in mainland Southeast Asia. [ABSTRACT FROM AUTHOR]

The envelope glycoprotein (protein G) of bovine ephemeral fever virus (BEFV) has been identified as a plausible vaccine candidate against the BEF disease. In the present study, G1 epitope of the G glycoprotein gene was cloned in an eukaryotic expression vector, pcDNA3.1(+), under the control of the human cytomegalovirus (CMV) promoter. The pcDNA3.1-G1 construct was transfected into human embryonic kidney 293 (HEK 293) cell line and the expression efficiency was verified by immunofluorescence staining of transfected cells and Western blot analysis. The results indicated that G1 protein was expressed by the recombinant pcDNA3.1-G1 construct in the transfected cells. The recombinant plasmid constructed in this study can be used as a DNA vaccine to evaluate its potential for immunogenicity and protection against BEF virus in animal models. [ABSTRACT FROM AUTHOR]

Bovine ephemeral fever (BEF) is a common febrile disease in cattle in tropical countries causing loss of milk production and reproductive performance. Recently, severe outbreaks have occurred in many countries in East Asia, the Middle East, and Southeast Asia, especially in the rainy season, while limited vaccines and diagnostic tools are available. Thus, the aim of this study was to produce a secreted, soluble form of BEFV G glycoprotein in insect cells for further applications. BEFV G gene expressing transmembrane-deleted G glycoprotein (GΔTM) was constructed and cloned into the baculovirus vector. The recombinant GΔTM protein was expressed in High5 insect cells and then purified by affinity chromatography. The purified GΔTM reacted specifically with a convalescent bovine serum. Four 8-week-old Wistar rats were injected subcutaneously with the purified GΔTM protein. Postimmunized rat serum samples also strongly reacted with the G protein by western blot and immunoperoxidase mono player assay (IPMA). These results indicated the potential use of the transmembrane-deleted G protein to develop protein based-diagnostic tools for BEFV control programs. [ABSTRACT FROM AUTHOR]

• Cx. pipiens mosquitoes have the potential to act as a vector of BEFV. • BEFV may has a temporary nature and short viremia in its vectors. • BEFV may be vertically transmitted from Cx. pipiens female to her female offspring. • Vertical transmission may compensate for short viremia and will ensure persistence. Bovine ephemeral fever (BEF) is a significant viral disease of cattle in the tropical, subtropical, and temperate climatic zones. This disease is also known as three-day sickness due to the spontaneous recovery of the cattle within a short period (usually 3–5 days). Despite its short duration, the disease may have a considerable impact. It can cause heavy economic losses, primarily due to decreased milk production, lowered fertility in bulls, and even fatality in severe cases. The virus is suspected to be transmitted by haematophagous insects (mainly mosquitoes and Culicoides biting midges); however, the identity of a competent vector for BEFV remains a mystery. Here, we investigated whether BEFV may replicate for a short duration in Culex pipiens Linnaeus, 1758, the most prevalent mosquito species in Israel and a potential vector of this virus to Israeli cattle. We applied nested- qPCR to test BEFV abundance in Cx. pipiens every 24 h for 14 consecutive days post-infection. Additionally, we collected eggs laid by BEFV-infected females and investigated BEFV abundance in the different developmental stages of F1 mosquitos. Our results suggest that Cx. pipiens mosquitoes have the potential to act as a vector of BEFV and also indicate that BEFV may be vertically transmitted from Cx. pipiens female parent to her female offspring. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G1 protein) , is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G1 protein with high-affinity and inhibit BEFV replication. Methods: The purified BEFV G1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G1 was assayed by ELISA. Then the roles of specific G1-binding peptides in the context of BEFV infection were analyzed. Results: The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained and the T18 and T25 clone had a higher affinity to G1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Conclusion: Two antiviral peptide ligands binding to bovine ephemeral fever virus G1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents. [ABSTRACT FROM AUTHOR]

In recent years there have been several major outbreaks of bovine ephemeral disease in the Middle East, including Israel. Such occurrences raise the need for quick identification of the viruses responsible for the outbreaks, in order to rapidly identify the entry of viruses that do not belong to the Middle-East BEFV lineage. This challenge was met by the development of a high-resolution melt (HRM) assay. The assay is based on the viral G gene sequence and generation of an algorithm that calculates and evaluates the GC content of various fragments. The algorithm was designed to scan 50- to 200-base-long segments in a sliding-window manner, compare and rank them using an Order of Technique of Preference by Similarity to Ideal Solution (TOPSIS) the technique for order preference by similarity to ideal solution technique, according to the differences in GC content of homologous fragments. Two fragments were selected, based on a match to the analysis criteria, in terms of size and GC content. These fragments were successfully used in the analysis to differentiate between different virus lineages, thus facilitating assignment of the viruses’ geographical origins. Moreover, the assay could be used for differentiating infected from vaccinated animales (DIVA). The new algorithm may therefore be useful for development of improved genotyping studies for other viruses and possibly other microorganisms. [ABSTRACT FROM AUTHOR]

Background The potential tissue replication sites and specific cell types that support in vivo virus survival beyond the acute phase of bovine ephemeral fever virus ( BEFV) infection have not been fully defined in cattle. To clarify the knowledge gap, tissue specimens were tested after collection from an adult steer necropsied 1 week after acute BEF. Case report Significant necropsy findings included fibrinoproliferative synovitis in the stifle joints and fibrin clot-laden fluid in serous body cavities. Moderate numbers of infiltrating neutrophils were demonstrated in sections of the prefemoral lymph nodes and haemal node, and lymphoid hyperplasia in the spleen, haemal node and prefemoral lymph nodes. Viral RNA was detected by qRT-PCR in fresh spleen, haemal node, prefemoral lymph node, synovial fluid and in several spleen-derived cell cultures. BEFV was isolated from autogenously derived splenic primary cell cultures 6 days after cessation of viraemia, and characteristic bullet-shaped virions were confirmed by electron microscopy of an ultrathin haemal node section. In sections of the spleen, haemal node and other tissues, immunohistochemistry demonstrated BEFV antigens that were intracellularly associated with probable histiocytic cells. Conclusion BEFV has preferential tropism for bovine lymphoid tissues and the spleen and haemal node may be potential sites for post-viraemic virus replication. [ABSTRACT FROM AUTHOR]

Objective This study assessed the neurotropism of bovine ephemeral fever (BEF) virus (BEFV) and described histomorphological abnormalities of the brain, spinal cord and peripheral nerves that may causally contribute to paresis or paralysis in BEF. Methods Four paralysed and six asymptomatic but virusinfected cattle were monitored, and blood and serum samples screened by qRT-PCR, virus isolation and neutralisation tests. Fresh brain, spinal cord, peripheral nerve and other tissues were qRT-PCR-tested for viral RNA, while formalin-fixed specimens were processed routinely and immunohistochemically evaluated for histomorphological abnormalities and viral antigen distribution, respectively. Results The neurotropism of BEFV was immunohistochemically confirmed in the brain and peripheral nerves and peripheral neuropathy was demonstrated in three paralysed but not the six aneurological but virus-infected animals. Wallerian degeneration (WD) was present in the ventral funicular white matter of the lumbar spinal cord of a paralysed steer and in cervical and thoracic spinal cord segments of three paralysed animals. Although no spinal cord lesions were seen in the steer euthanased within 7 days of illness, peripheral neuropathy was present and more severe in nerves of the brachial plexuses than in the gluteal or fibular nerves. The only steer with WD in the lumbar spinal cord also showed intrahistiocytic cell viral antigen that was spatially distributed within areas of moderate brain stem encephalitis. Conclusion The data confirmed neurotropism of BEFV in cattle and documented histomorphological abnormalities in peripheral nerves and brain which, together with spinal cord lesions, may contribute to chronic paralysis in BEFV-infected downer cattle. [ABSTRACT FROM AUTHOR]

Inactivated vaccine is considered safe and used for prevention of bovine ephemeral fever in several endemic countries. To differentiate between BEFV-infected and vaccinated animals, we developed an ELISA capable of detecting infection-related antibodies against BEFV. Recombinant proteins, including N, P, M, L, G NS , α2, β and γ, were expressed in E. coli and screened by Western blotting and ELISA. The results showed G NS , α2 and β specifically reacted with sera from BEFV infected cattle but not sera from vaccinated cattle. A DIVA ELISA based on a C-terminal truncated form of G NS was developed, with 100% sensitivity and 98.0% specificity at a sample to positive-control optical density ratio (S/P) threshold of 0.18. Specificity analysis showed that the assay has no cross-reactivity with antisera of other common bovine viruses. Anti-G NS antibody appears at 3–4 days post infection (dpi) and persists up to 240–300 dpi in the experimentally infected cattle. Sero-epidemiological survey using sera collected from vaccinated cattle in an endemic area in Jiangsu Province revealed sero-positive rate of 2.36% (6/254), indicating that the DIVA ELISA could be used as a reliable diagnostic tool for differentiating BEFV infected from vaccinated animals. • The G NS , α2 and β proteins specifically reacted with sera from BEFV infected cattle but not sera from vaccinated cattle. • The C-terminal and N-terminal truncated recombinant G NS proteins had similar reactivity with sera from BEFV infected cattle. • A DIVA ELISA was developed based on the C-terminal truncated recombinant G NS protein with 100% sensitivity and 98.0%. [ABSTRACT FROM AUTHOR]

Bovine ephemeral fever virus (BEFV) is a typical species of the genus Ephemerovirus in the family Rhabdoviridae. Today, prevailing BEFV can be divided into three phylogeographic lineages, East Asia, Mideast, and Australia. In this study, we provide evidence that the whole East Asia lineage originates from a homologous recombination (HR) between the Mideast and Australia lineages that probably occurred in the 1940s. To our knowledge, HR has not been proposed before as the genetic mechanism of BEFV. According to the HR event and Bayesian estimation, the three BEFV lineages might originate from Africa, and may have spread to Asia and Australia through the Mideast. In addition, the population of the virus may have augmented significantly in the 2000s, suggesting that the risk for outbreaks of BEFV may be high at present. [ABSTRACT FROM AUTHOR]

This study investigated the geographical distribution and molecular analysis of Culicoides species in the Southern and Southeastern Turkey during the 2012 outbreak of bovine ephemeral fever (BEF). The midge specimens caught by Onderstepoort-type light traps from livestock farms were tested for molecular evidence of existence of viral genome. Blood specimens were collected from clinically BEF-suspected acute febrile cattle. Total nucleic acid samples obtained from field specimens were checked against the BEF virus G gene and Culicoides internal transcribed spacer 1 (ITS-1) gene. A total of 20,845 Culicoides specimens (20,569 ♀♀, 276 ♂♂) comprising 11 species ( Culicoides badooshensis, Culicoides circumscriptus, Culicoides gejgelensis, Culicoides imicola, Culicoides kibunensis, Culicoides longipennis, Culicoides newsteadi, Culicoides nubeculosus, Culicoides odiatus, Culicoides punctatus, Culicoides schultzei, Culicoides spp.) were collected. C. schultzei (18,032) was found as the dominant species and followed by C. imicola (1,857), C. nubeculosus complex (545), and C. circumscriptus (259), respectively. C. kibunensis was identified as new species for this region. PCR positivity of BEF was found 37.14 % (13/35) in blood samples whereas no viral genome was obtained from Culicoides specimens. Culicoides spp. ITS-1 gene sequences were analyzed phylogenetically with GenBank ITS-1 sequences. Molecular homology of Culicoides ITS-1 gene was ranged between 62.74 and 71.39 %. The results described first molecular detection and phylogenetic analysis of Culicoides ITS-1 gene with reference to the 2012 BEF outbreak in Turkey. [ABSTRACT FROM AUTHOR]

Bovine ephemeral fever is an arthropod-borne bovine viral disease caused by infection with bovine ephemeral fever virus which belongs to genus Ephemerovirus within the family Rhabdoviridae . In this study, serological data and virological information about the disease and the virus, spanning from 2001 to 2013, were employed to analyze the relationships of bovine ephemeral fever epizootics to population immunity and virus variation. National and regional surveillance data indicated that 2 of the 3 major epizootics and 87% regional outbreaks were associated with lower neutralizing antibody titers and immunity coverage, reflecting the importance of population immunity for the control of bovine ephemeral fever. Phylogenetic analysis and sequence comparison demonstrated that Taiwanese bovine ephemeral fever viruses were >96.0% and >97.6% similar to the East Asian isolates in nucleotide and amino acid sequences, respectively. These analyses supported that the Taiwanese viruses shared the same gene pool with the strains of the other East Asian countries, mainly Japan. [ABSTRACT FROM AUTHOR]

Highlights: [•] The first single primer pair PCR able to detect all ephemerovirus genus members. [•] This PCR, in conjunction with sequencing, can determine the virus species. [•] This PCR can detect BEFV in both tissue culture isolates and clinical cattle blood. [•] The sensitivity was 500 RNA copies per reaction for BEFV. [•] For KOTV the sensitivity was between 500 and 5000 RNA copies per reaction. [Copyright &y& Elsevier]

Background: The glycoprotein (G) gene sequences of bovine ephemeral fever virus (BEFV) strains derived from mainland China have not been compared with those of the isolates from other countries or areas. Therefore, the G genes of four BEFV isolates obtained from mainland China were amplified and sequenced. A phylogenetic tree was constructed in order to compare and analyze the genetic relationships of the BEFV isolates derived from mainland China and different countries and areas. Results: The complete BEFV G gene was successfully amplified and sequenced from four isolates that originated from mainland China. A total of fifty-one BEFV strains were analyzed based on the G gene sequence and were found to be highly conserved. A phylogenetic tree showed that the isolates were grouped into three distinct lineages depending on their source of origin. The antigenic sites of G1, G2 and G3 are conserved among the isolates, except for several substitutions in a few strains. Conclusions: The phylogenetic relationships of the BEFV isolates that originated from mainland China, Taiwan, Japan, Turkey, Israel and Australia were closely related to their source of origin, while the antigenic sites G1, G2 and G3 are conserved among the BEFV isolates used in this work. [ABSTRACT FROM AUTHOR]

Abstract: A novel reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) assay for the detection of bovine ephemeral fever virus (BEFV) was developed and evaluated in this study. The RT-LAMP assay exhibited higher sensitivity when compared with conventional reverse-transcription polymerase chain reaction (RT-PCR) and virus isolation methods. The specificity of the assay was determined by digestion of the RT-LAMP products with restriction enzyme and detection of BEFV serogroup rabies virus (RV). Using RT-LAMP, RT-PCR and virus isolation methods, 36 blood samples were tested and the results indicated that RT-LAMP could detect early infection with BEFV. The RT-LAMP method is useful for the diagnosis of BEFV infection in blood samples. [ABSTRACT FROM AUTHOR]

Non-segmented, negative-sense RNA viruses (NNSVs) depend on Akt (protein kinase B) for efficient replication. Infection with bovine ephemeral fever virus (BEFV) increases Akt phosphorylation. This study examined the effect of inhibition of phosphatidylinositol 3-kinase (PI3K)-Akt signalling on BEFV replication, since PI3K is the major upstream regulator of Akt. Treatment of BEFV-infected cells with two specific PI3K inhibitors (wortmannin and LY294002) enhanced replication of BEFV when compared to the effects of Akt inhibitors Ill and IV. BEFV antagonised the effects of PI3K inhibitors on Akt dephosphorylation. Inhibition of roTOR by rapamycin also enhanced replication of BEFV. The results provide evidences that inhibition of PI3K and mTOR has positive effects on replication of BEFV. [ABSTRACT FROM AUTHOR]

An indirect ELISA for the serological detection of bovine ephemeral fever virus (BEFV) infection in cattle is described in which a glycosylated protein of approximately 25 kDa (including the G1 antigenic site of the virus glycoprotein) expressed in Pichia pastoris GSI 15 was used as the coating antigen. The optimal concentration of coated antigen was 0.3 µg/well at a serum dilution of 1:40. The optimal positive threshold value of the assay was 1.88, as derived from receiver operating characteristic curve analysis. The test had 100% sensitivity and 96.7% specificity when compared with a micro-neutralisation test using 336 positive and 180 negative sera to BEFV, respectively. The inter-assay and intra-assay coefficients of variation for 15 sera were both <5.8% and there was no evidence of cross-reactivity between the tested coating antigen and antibodies to related rabies virus. The ELISA is an inexpensive and rapid serological detection method that would be suitable for screening for BEFV infection on a large scale. [ABSTRACT FROM AUTHOR]

Abstract: The G gene encoding the neutralization antigen of bovine ephemeral fever virus (BEFV) was characterized in order to define the virus''s molecular epidemiology in Japan and the genetic relationships among the Japanese, Taiwanese and Australian isolates. The nucleotide and amino acid sequences of the gene were highly conserved among the Japanese strains, regardless of the year of isolation, and were closely related to the Taiwanese strains. By phylogenetic analysis, the Japanese and Taiwanese strains were classified clearly into three chronological clusters: 1966, 1984–1989 and 1996–2004, indicating that the epidemics of bovine ephemeral fever may occur almost simultaneously in both countries by the same genotype. On the other hand, the Australian strains were distantly related to these East Asian strains and placed in the independent fourth cluster of the phylogenetic tree. It is suggested that three amino acid substitutions at residues 224, 271 and 499 in the neutralizing epitopes, of which two generate new glycosylation sequences, are responsible for antigenic variations of bovine ephemeral fever virus. The cross-neutralization test using the bovine ephemeral fever virus isolated in Japan demonstrated that the vaccine developed based on the oldest Japanese strain, YHL, appears to still be effective for controlling bovine ephemeral fever in Japan. [Copyright &y& Elsevier]

Abstract: RNA interference (RNAi) was used to suppress bovine ephemeral fever virus (BEFV). Plasmids expressing continuously shRNAs were used against G gene of BEFV to induce RNA interference in cultured cells. A GFP reporter assay was established to determine the efficiency and specificity of siRNA and the potential of BEFV to hamper RNAi. Two of five small interfering RNAs (siRNAs) were shown to suppress BEFV. Suppression of the G gene of BEFV corresponded with reduction of viral plaques and progeny titer. The results suggest that RNAi has the potential for use in suppression of BEFV infection with possible therapeutic implications. [Copyright &y& Elsevier]

Abstract: A rapid, sensitive, and specific assay, RAPID-BAP assay, was developed to detect and quantify the G protein-encoding gene of bovine ephemeral fever virus (BEFV). This new technique uses a nested PCR and magnetic bead-based DNA probing assay. The optimal conditions for the assay were examined. By applying a nested PCR, a minimum of 1 copy/μl of the BEFV plasmid DNA could be detected by the assay. The optimal hybridization conditions at 50°C in 5× SSC and 0.5% SDS with a 20-min incubation allowed clear discrimination between negative and positive controls. The assay was also highly specific as all negative controls failed to show any positive detection. The diagnostic sensitivity of the RAPID-BAP assay, real-time RT-PCR, and conventional RT-PCR in the detection of 34 clinical blood samples suspected to have BEFV infections were 72.73, 36.36, and 18.18%, respectively. The results indicated that the RAPID-BAP assay developed in this study was more sensitive than the conventional RT-PCR and real-time RT-PCR assays for the detection of BEFV. The novel RAPID-BAP assay is an excellent diagnostic tool with high sensitivity, specificity, and fast turnaround time. [Copyright &y& Elsevier]

Abstract: The potential significance of bovine ephemeral fever virus (BEFV)-induced apoptosis and involved viral molecules was fully unknown. In the present study, evidence is provided demonstrating that bovine ephemeral fever virus induces apoptosis in several cell lines. Five types of assays for apoptosis were used in examining BEFV-infected cells. (1) Assay for DNA fragmentation, (2) nuclear staining with acridine orange, (3) ELISA detection of cytoplasmic histone-associated DNA fragment, (4) terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labelling (TUNEL) assay of BEFV-infected cells, (5) observation of blebbing of the plasma membrane and the formation of apoptotic bodies of apoptic cells by scanning electron microscope. The level of lactate dehydrogenase (LDH) in BEFV-infected cells was increased significantly after 20–25h post-infection. Caspases-2, -3, -4, -6, -8, -9, and -10 were activated in BEFV-infected BHK-21 cells. To determine further whether BEFV-induced apoptosis was caspase-dependent, the effect of the tripeptide pan-ICE (caspase) inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyketone on the inhibition of apoptosis in BEFV-infected BHK-21 cells, was investigated. Apoptosis could be blocked by the caspase inhibitor (Z-VAD-fmk), indicating that BEFV induces caspase-dependent apoptosis in cultured cells. [Copyright &y& Elsevier]

Abstract. A total of 10 607 Culicoides midges (Diptera: Ceratopogonidae) were fed on either sheep or horse blood containing not less than 6.5 log10 TCID50 /ml of bovine ephemeral fever virus (BEFV). Insects were collected during two consecutive summers from two distinct climatic areas. Two seed viruses, originating from either South Africa or Australia, were used separately in the feeding trials. Blood-engorged females were incubated at 23.5°C for 10 days and then individually assayed in microplate BHK-21 cell cultures. Of the 4110 Culicoides that survived, 43% were C. (Avaritia ) imicola Kieffer and 27% were C. (A. ) bolitinos Meiswinkel. The remainder represented 18 other livestock-associated Culicoides species. Although BEFV was detected in 18.9% of midges assayed immediately after feeding, no virus could be detected after incubation. The absence of evidence of either virus maintenance or measurable replication suggests that most of the abundant livestock-associated Culicoides species found in South Africa are refractory to oral infection with BEFV. Future studies should be carried out using species of mosquitoes that are associated with cattle in the BEF endemic areas. [ABSTRACT FROM AUTHOR]

Background: Bovine ephemeral fever virus (BEFV) causes fever and muscle stiffness in cattle, resulting in negative economic impact for cattle and dairy farms. During the manufacturing process of inactivated vaccine for virus control, it is important to determine the virus titer, but traditional methods such as plaque assay and TCID50 assay require days of waiting time. We sought to develop a quick dot blot assay for BEFV titering. Results: Three different kinds of BEFV antigens were prepared to raise primary antibodies for BEFV detection in dot blot assays: 1) purified BEFV particles, 2) Escherichia coli (E. coli)-expressed BEFV G1 region, and 3) E. coli-expressed BEFV N protein. Results showed that antibodies raised against purified BEFV particles can detect BEFV particles, but it also showed a high background level from the proteins of BHK-21 cells. Antibodies raised against E.coli-expressed BEFV G1 region could not detect BEFV particles in dot blot assays. Finally, antibodies raised against E.coli-expressed BEFV N protein detected BEFV particles with a high signal-to-noise ratio in dot blot assays. Conclusions: E.coli-expressed N protein is a suitable antigen for the production of antiserum that can detect BEFV particles with a high signal-to-noise ratio. A dot blot assay kit using this antiserum can be developed as a quick and economical way for BEFV titering. [ABSTRACT FROM AUTHOR]

Abstract: The gene encoding antigenic site G1 of bovine ephemeral fever virus (BEFV) was highly expressed in the host cell Escherichia coli. An indirect G1-ELISA with the recombinant protein as the coating antigen was established to detect antibodies against BEFV. The result revealed that the optimal concentration of the coated antigen was 0.5μg/well and the dilution of serum was 1:20. It was optimal that sera with P/N value⩾2.2 were considered positive, P/N value⩽2.0 negative, and between 2.0 and 2.2 ambiguous. The G1-ELISA method gave a sensitivity of 97.6% and a specificity of 98.6% by testing 590 field serum samples. These results suggest that the G1-ELISA may be a good alternative tool for seroepidemiological surveys. [Copyright &y& Elsevier]