Resurgence of bovine ephemeral fever in mainland Japan in 2015 after a 23-year absence.

In September and October 2015, suspected cases of bovine ephemeral fever (BEF) were reported in the mainland region of Kagoshima Prefecture and on Tanegashima Island. The genome of the BEF virus (BEFV) was detected in the diseased cows and the cows that had recovered. The serum obtained from the affected cows contained high titers of BEFV-neutralizing antibody. In total, 18 affected cows were demonstrated to be infected with BEFV during the outbreak. Our findings showed evidence that BEF occurred in mainland Japan after a 23-year absence. Phylogenetic analysis based on the surface glycoprotein (G) gene revealed that BEFVs detected in the affected cows were genetically distinct from previous Japanese BEFVs, but were close to BEFVs circulating in Taiwan and mainland China in recent years. Amino acid substitution in the neutralizing epitope domains of the G protein was limited between the detected viruses and the vaccine strain (YHL isolate), and high titers of the neutralizing antibody against the YHL isolate were induced in the infected cattle during the disease occurrences. Therefore, current BEF vaccines probably elicit protective immunity against the BEFVs detected in 2015, although their effectiveness should be assessed. Since the BEFV vaccination rates are estimated to be low, a BEF outbreak should be considered a possibility in mainland Japan.

High-resolution melting (HRM) for genotyping bovine ephemeral fever virus (BEFV).

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.

Occurrence of bovine ephemeral fever in Okinawa Prefecture, Japan, in 2012 and development of a reverse-transcription polymerase chain reaction assay to detect bovine ephemeral fever virus gene.

In September 2012, several cows and a calf showed decreased activity, anorexia and fever on Ishigaki Island, Okinawa Prefecture, Japan, and the cases were diagnosed as bovine ephemeral fever (BEF). We isolated BEF virus (BEFV) from one of the affected cows and then determined the complete genome sequence of the G gene, which encodes a class I transmembrane glycoprotein of BEFV. The BEFV isolate in this case, ON-3/E/12, was sorted into the same cluster as other BEFV isolates in Japan, Taiwan and China obtained in 1996-2004 and was most closely related to a 2002 Chinese isolate, JT02L, according to the phylogenetic analysis of the complete G gene. Since inactivated vaccines for BEF available in Japan are considered effective against the ON-3/E/12 isolate as well as other isolates in East Asia from 1996-2004, annual vaccination should be conducted to prevent BEF in Okinawa. Additionally, in this study, we developed an RT-PCR assay to detect the BEFV gene in Japan and neighboring countries. Our assay was able to amplify target sequences in all of the tested BEFV isolates, including 18 isolates in Japan and another isolate in Australia. The assay was found to be useful also for testing RNA samples extracted from bovine peripheral blood mononuclear cells, and the detection limit of the assay was 10 copies per tube. We believe that our assay would be an important tool for the screening of BEFV infection and the diagnosis of BEF.

A reverse-transcription PCR method for detecting all known ephemeroviruses in clinical samples.

Bovine ephemeral fever virus (BEFV) is an economically important vector-borne pathogen of cattle in tropical and sub-tropical regions of Australia, Asia, Africa and the Middle East. Although clinical cases of bovine ephemeral fever are usually attributed to BEFV, definitive diagnosis is rarely performed and at least two other related viruses, kotonkon virus (KOTV; an ephemerovirus) and Fukuoka virus (FUKAV; an unassigned rhabdovirus), can cause similar clinical signs. As vaccines have been developed against BEFV but not against KOTV or FUKAV, a test capable of detecting and differentiating these pathogens would be useful. In the present study, an RT-PCR method using degenerate primers designed to a region of block III of the polymerase (L) gene was developed and optimised for primer annealing temperature and MgCl2 concentration. The RT-PCR detected all known ephemeroviruses and several other closely related insect-transmitted rhabdoviruses, including FUKAV. Viruses could be identified by subsequent sequencing and phylogenetic analysis of the amplicons. BEFV could be detected using tissue culture isolates or cattle blood to a sensitivity of 500 RNA copies per reaction. This test will be useful for establishing the identity of the causative agent of bovine ephemeral fever from field samples and cultured isolates.

A reverse-transcription, loop-mediated isothermal amplification assay for detection of bovine ephemeral fever virus in the blood of infected cattle.

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.

Serological detection of bovine ephemeral fever virus using an indirect ELISA based on antigenic site G1 expressed in Pichia pastoris.

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 GS115 was used as the coating antigen. The optimal concentration of coated antigen was 0.3 microg/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.

A real-time RT-quantative(q)PCR for the detection of bovine ephemeral fever virus.

A quantitative reverse-transcriptase real-time PCR assay, using TaqMan chemistry, for detecting bovine ephemeral virus (BEFV) is described. Available G gene sequences of viral RNA were aligned, and primers and probes were designed to recognize the virus. To quantitate the viruses, cDNA containing the real-time amplicon was prepared with a forward primer carrying the T7 promoter sequences. Run-off transcription from the T7 promoter amplicon template was used to prepare cRNA. Ten-fold dilutions of the run-off viral transcript were used as templates for the reaction in which they served as standards to quantitate unknown viral samples. By using this system it was shown that as few as 10-100 copies of a viral genome could be detected.

Bovine ephemeral fever in Australia and the world.

Bovine ephemeral fever (BEF) is a disabling viral disease of cattle and water buffaloes. It can cause significant economic impact through reduced milk production in dairy herds, loss of condition in beef cattle and loss of draught animals at the time of harvest. Available evidence indicates clinical signs of BEF, which include bi-phasic fever, anorexia, muscle stiffness, ocular and nasal discharge, ruminal stasis and recumbency, are due primarily to a vascular inflammatory response. In Australia, between 1936 and 1976, BEF occurred in sweeping epizootics that commenced in the tropical far north and spread over vast cattle grazing areas of the continent. In the late 1970s, following several epizootics in rapid succession, the disease became enzootic in most of northern and eastern Australia. In Africa, the Middle East and Asia, BEF occurs as also epizootics which originate in enzootic tropical areas and sweep north or south to sub-tropical and temperate zones. The causative virus is transmitted by haematophagous insects that appear to be borne on the wind, allowing rapid spread of the disease. Bovine ephemeral fever virus (BEFV) has been classified as the type species of the genus Ephemerovirus in the Rhabdoviridae. It has a complex genome organization which includes two glycoprotein genes that appear to have arisen by gene duplication. The virion surface glycoprotein (G protein) contains four major antigenic sites that are targets for neutralizing antibody. An analysis of a large number of BEFV isolates collected in Australia between 1956 and 1992 has indicated remarkable stability in most neutralization sites. However, epitope shifts have occurred in the major conformational site G3 and these have been traced to specific mutations in the amino acid sequence. BEFV isolates from mainland China and Taiwan are closely related to Australian isolates, but some variations have been detected. Natural BEFV infection induces a strong neutralizing antibody response and infection usually induces durable immunity. Several forms of live-attenuated, inactivated and recombinant vaccines have been reported but with variable efficacy and durability of protection. The BEFV G protein is a highly effective vaccine antigen, either as a purified subunit or expressed from recombinant viral vectors.

A blocking ELISA for the detection of specific antibodies to bovine ephemeral fever virus.

A blocking ELISA (B/ELISA) for detecting antibodies to bovine ephemeral fever virus (BEFV) in cattle is described. In this test, the binding capacity of a monoclonal antibody specific for an epitope on antigenic site G1 of the BEF virus glycoprotein is blocked in the presence of positive serum. The sensitivity of the B/ELISA was compared with the virus neutralisation (VN) test using a total of 380 sera from cattle. Of these, 118 were from an area known to be free of bovine ephemeral fever, 181 from naturally and experimentally BEFV-infected cattle, 33 sequential serum samples from a sentinel steer from which Berrimah virus (BERV) had been isolated, 9 from a sentinel cow from which Kimberley virus (KIMV) was isolated and a panel of 39 sera supplied as a blind trial. The B/ELISA results overall compared favourably with those of the VN tests. The monospecificity of the test was demonstrated using hyperimmune mouse ascitic fluid to other BEF serogroup viruses, namely KIM and BER viruses and the results showed no significant cross-reaction. The greater simplicity and sensitivity of the test when compared with the VN test makes it the preferred test for the diagnosis and monitoring of clinical bovine ephemeral fever.

Bovine ephemeral fever.

Ephemeral fever remains a viral disease of considerable importance to many countries including Australia. The virus has been only partly characterised and still awaits final classification. Although BEF virus was first thought to contain 6 structural proteins there is increasing evidence to suggest that it contains the 5 proteins characteristic of the Rhabdoviridae. Although BEF is thought to be arthropod borne, the vector has yet to be identified but it is clear from the distribution of BEF that more than one vector is capable of transmitting the disease. Despite rigorous investigation of the clinical signs and the pathology of ephemeral fever, little progress has been made on the pathogenesis of the disease. This has been partly due to the difficulty of propagating BEF virus in vitro and the inability to define the site of replication. However, there is mounting evidence to suggest that BEF is immunopathologic in nature and that the clinical expression of the disease is influenced by the release of one or more mediators of inflammation. The disease is characterised by a number of haematological and biochemical changes and early and prolonged treatment with phenylbutazone is capable of reversing a number of these changes. The intravenous administration of calcium can now be considered a justifiable addition to the treatment regimen together with prolonged phenylbutazone therapy. The vaccines currently available are prepared from either live attenuated or killed virus and may be less than reliable. There appears to be a need for a reliable, inexpensive, cold-chain independent alternative vaccine.

Preliminary studies of the complement fixation test to confirm the diagnosis of bovine ephemeral fever.

A strain of bovine ephemeral fever (BEF) virus isolated in China in 1976 was adapted to growth in tissue cultures. A baby hamster kidney complement fixing (CF) antigen, stable at -20 degrees C for at least 120 days, was prepared from the BEF virus grown in tissue culture and used to test bovine sera for antibodies to that virus. CF antibodies were detected in all of 31 cattle after convalescence from experimental infection with BEF virus, in 208 (98%) of 213 cattle observed to have shown clinical ephemeral fever in an epidemic, in 96 cattle in these herds which did not show clinical signs of ephemeral fever and 16 cattle from herds in northern China outside the epidemic area. The CF antibodies to BEF virus were found to persist in 34 (89%) of 38 cattle which were bled 6 years after natural exposure to ephemeral fever. The CF antigen is economical to prepare and is suitable to differentiate ephemeral fever from other viral infections with which it could possibly be confused on clinical appearance.

Studies on the pathogenesis of bovine ephemeral fever.

A study of the pathogenesis of bovine ephemeral fever confirmed that the major clinical signs were fever lasting no more than 2 days, with increased respiratory rate, dyspnoea and some degree of lameness. Haematological observations revealed a neutrophilia with a left shift and a lymphopaenia at the time of peak clinical reaction. The net result was a slight leucopaenia on the day after this reaction. The most prominent pathological changes involved the lungs and synovial joints. Pulmonary emphysema and alveolar collapse with bronchiolitis, degenerative changes in synovial membranes and increased synovial fluid were observed. Specific fluorescence indicating the presence of BEF viral antigen could be detected at the time of peak clinical response in individual cells in the lungs, spleen and lymph nodes as well as neutrophils. Before and after the peak fever some fluorescence was seen in cells which appeared to be reticular cells in the lymph nodes. Viral isolation in mice could be made from blood, lungs, spleen and lymph nodes over a period of no more than 3 days. It is postulated that viral growth takes place mainly in the reticuloendothelial cells in the lungs, spleen and lymph nodes and not in vascular endothelium or lymphoid cells.