Molecular characterization of bovine ephemeral fever virus in Thailand between 2013 and 2017.

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.

Seroprevalence investigation of bovine ephemeral fever in yaks in Tibetan Plateau of China from 2012 to 2015.

Bovine ephemeral fever (BEF) is caused by the arthropod-borne bovine ephemeral fever virus (BEFV), which is classified in family Rhabdoviridae and genus Ephemerovirus. However, it is still unclear whether yaks from the Tibetan plateau of China are exposed to BEFV. It is the first time that a survey was conducted to investigate the seroprevalence of BEFV infection in yaks (Bos grunniens) on the Tibetan Plateau of China. A total of 1123 serum samples were collected randomly from yaks from 2012 to 2015 and were assayed for BEFV antibodies by enzyme-linked immunosorbent assay (ELISA). The proportions of positive serum samples were assessed among the 1123 samples, as well as factors of geographical origin and years. The results showed that there were 454 serum samples that tested positive for BEFV, and the total positive rate is 40.4 %. The prevalence in 2012, 2013, 2014, and 2015 was 49.3, 36, 44.1, and 34.0 %, respectively, and the difference is statistically significant (P< 0.01). In different regions, the prevalence was ranged from 34.7 to 45.7 % with a significant difference among the different regions of (P < 0.05). Logistic regression analysis showed that yaks in Tibet (Xizang autonomous region) (45.7 %) had 1.6 times (OR = 1.589, 95 % CI = 1.141-2.215, P < 0.01) higher risk of being seropositive compared to yaks in Qinghai province, while no regional difference was found of Sichuan province compared to Qinghai (P > 0.05). The prevalence in 2012 (49.3 %) was more than 1.8 time (OR = 1.880, 95 % CI = 1.350-2.619, P < 0.001) at risk of acquiring the infection compared to the year of 2015. The prevalence of yaks in 2014 (44.1 %) had a 1.5 times (OR = 1.528, 95 % CI = 1.350-2.619, p < 0.001) at risk of being seropositive compared to yaks in 2015, while no year difference was found of 2013 compared to 2015 (P > 0.05). Our study suggests that the yaks from the high plateau are highly infected by BEFV, and geographical origin and years are main risk factors for BEF seroprevalence.

Viral neurotropism, peripheral neuropathy and other morphological abnormalities in bovine ephemeral fever virus-infected downer cattle.

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 virus-infected 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.

Kinetics of selected plasma cytokines during innate-adaptive immune response transition in adult cattle infected with the bovine ephemeral fever virus.

While virus neutralizing antibodies are known to be variably protective against bovine ephemeral fever (BEF) virus (BEFV) infections, the cytokine events that mediate the nascent adaptive immune response have not been defined in cattle. This study determined the plasma kinetics of IL-2, IFN-γ, IL-6, and IL-10 during the period of innate-immune response transition and evaluated the relationship between the virus neutralizing antibody response and viraemia in BEFV-infected cattle. Plasma from four virus-infected and uninfected negative control animals was tested by cytokine-specific immunoenzymatic assays, viraemia monitored by qRT-PCR, and virus neutralizing antibody titres determined using a standard protocol. Unlike the negative controls, plasma IL-6 and IL-10 were increased in all the virus-infected animals starting several days prior to initiation of viraemia. In one animal, plasma IL-2 and IFN-γ were consistently higher than in the other three virus-infected animals and the negative control mean. The animal with the strongest IL-2 and IFN-γ responses had the shortest viraemia while the heifer with the lowest IL-2/IFN-γ indices demonstrated the longest viraemia. Evidently, increase in plasma IL-6 and IL-10 precedes seroconversion during BEFV infections in cattle suggesting the two cytokines may influence immunological events that pave way to B-cell activation and seroconversion. While there is remarkable variability in IL-2 and IFN-γ expression amongst BEFV-infected animals, increased plasma levels of the two cytokines appear to be associated with a shorter viraemia. Ongoing studies will help define the precise role of T cells in anti-BEFV adaptive immune responses.

Risk analysis and seroprevalence of bovine ephemeral fever virus in cattle in the Kingdom of Saudi Arabia.

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that causes disabling clinical signs and major economic losses in cattle and water buffalo. The disease is well documented in Asia, Africa, and the Middle East; however, the seroprevalence of BEFV in different regions and bovine breeds in the Kingdom of Saudi Arabia (KSA) is unknown. The aim of this study was to analyze risk factors which affect the prevalence of antibodies against BEFV in small herds of cattle in four geographical regions of KSA. A total of 1480 serum samples from non-BEFV vaccinated small herds of cattle were collected from the Eastern, Jizan, Qasim, and Riyadh regions (370 samples per region) during the summer of 2010. Serum neutralization test was used to detect antibodies against BEFV. There was a significant effect of region, breed, sex, and age on the seroprevalence of BEFV. Seropositive ratios were 18, 18, 26, and 12 % for the Eastern, Jizan, Qasim, and Riyadh regions, respectively (P = 0.00002); 23.2 % for dairy and 13.7 % for non-dairy breeds (P = 0.00004); 24.4 % for males and 14.6 % for females (P = 0.00004); and 15.4, 29.1, and 11.4 % for animals <1 year, 1-3 years, and >3 years, respectively (P < 0.001). Risk analysis showed a significant effect of different regions of KSA on the seroprevalence of BEFV. Host risk factors (age, sex, and breed) showed also a significant effect on the seroprevalence of BEFV. This indicates active circulation of this virus in small herds of cattle. Insect control strategies and BEFV vaccination programs during the spring are recommended to reduce the spread of BEFV and minimize subsequent economic losses as this is adopted in many enzootic countries.

Kinetics of pro-inflammatory cytokines, interleukin-10, and virus neutralising antibodies during acute ephemeral fever virus infections in Brahman cattle.

While fever and inflammation are hallmark features of bovine ephemeral fever (BEF), the cytokine networks that underlie the acute phase of the disease have not been empirically defined in cattle. This study characterised the plasma kinetics of proinflammatory cytokines (IL-1ß, IL-6, TNF-α) and IL-10 during acute BEF and elucidated on the relationship between the onset of the virus neutralizing antibody response and resolution of viraemia in natural BEF virus (BEFV) infections in cattle. Plasma from three BEFV-infected and three uninfected cattle was tested for the study cytokines by a cELISA, viraemia monitored by qRT-PCR, and virus neutralizing antibody titres determined using a standard protocol. Unlike the negative controls, plasma concentrations of IL-1ß, TNF-α, IL-6, and IL-10 were consistently increased in the three virus-infected animals. Two of the infected heifers were recumbent and pyrexic on the first day of monitoring and increased cytokine production was already in progress by the time viraemia was detected in all the three infected animals. In all the virus-infected heifers, IL-1ß was the most strongly expressed cytokine, IL-6 and IL-10 manifested intermediate plasma concentrations while TNF-α was the least expressed and demonstrated bi-phasic peaks three and five days after the onset of pyrexia. In two of the BEFV-infected heifers, viraemia resolved on the day of seroconversion while in the other infected animal, viral RNA was detectable up to three days after seroconversion. The present data document variable increase in plasma IL-1ß, IL-6, TNF-α, and IL-10 during natural BEFV infections and the fact that upregulation of all but TNF-α precedes seroconversion. In addition to virus neutralising antibodies, it is likely that cytokine-mediated cellular mechanisms may be required for resolution of viraemia in BEF. Considering the anti-inflammatory properties of IL-10, its upregulation may potentially antagonise the fever response in BEFV-infected cattle.

Application of a real-time polymerase chain reaction assay to the diagnosis of bovine ephemeral fever during an outbreak in New South Wales and northern Victoria in 2009-10.

OBJECTIVE: To report the occurrence of an epizootic of bovine ephemeral fever (BEF) in New South Wales (NSW) and northern Victoria in 2009-10 and describe the application of a real-time reverse transcription polymerase chain reaction (qRT-PCR) assay during the outbreak. PROCEDURES: Whole-blood samples from animals exhibiting clinical signs of BEF were requested from district veterinarians in NSW. In addition, samples were submitted from private practitioners in NSW and Victoria. In NSW, samples from animals showing acute clinical signs of BEF were tested using a qRT-PCR assay. Serological testing for BEF diagnosis was undertaken as required. Virus isolation was performed on selected samples in which bovine ephemeral fever virus (BEFV) RNA was detected. Archival serum samples and mosquito homogenates were also tested for BEFV by qRT-PCR. RESULTS: Accessions were received from 121 properties in NSW, with cases of BEF confirmed on 84 properties by qRT-PCR and 20 properties by serology. In northern Victoria, BEF was confirmed on 25 properties based on serological testing. Screening of samples by qRT-PCR enhanced the success of BEFV isolation. BEFV RNA was successfully detected in archival serum samples and a single mosquito homogenate. CONCLUSIONS: The 2009-10 outbreak resulted in the most extensive transmission of BEFV in NSW and Victoria since 1995-96, and follows a smaller outbreak in summer-autumn 2008. The use of qRT-PCR for BEF diagnosis offers veterinarians and cattle owners rapid confirmation of infection (1-2 days) and provides 'real-time' information about the presence of the disease in a district.

Sero-survey on Aino, Akabane, Chuzan, bovine ephemeral fever and Japanese encephalitis virus of cattle and swine in Korea.

Vector-borne arboviruses produce mild to severe symptoms in domestic animals. Bovine ephemeral fever (BEF), Akabane, Aino, and Chuzan virus have been primarily attributed to reproductive disorders or febrile diseases in cattle, and Japanese encephalitis virus (JEV) is mainly associated with reproductive failures in swine. We investigated antibody titers from domestic swine against four bovine arboviruses (BEF, Akabane, Aino, and Chuzan virus) and from cattle against JEV in Korea. While the positive rates for Akabane and BEF were 37.4% and 15.7%, the positive incidence of Chuzan and Aino were relatively low, with positive rates of 3.04% and 0.4%, respectively, based on a virus neutralization assay. Antibody titers against more than one virus were also frequently detected in domestic swine. The incidence of JEV was 51.3% among domestic cattle. In addition, one positive case was detected in the thoracic fluids from 35 aborted calves, based on the hemagglutination inhibition test. Our results indicate that swine are susceptible hosts of bovine arboviruses without showing clinical symptoms in a natural environment. Moreover, we confirmed that JEV could be associated with reproductive failure in pregnant cattle, as were other vector-borne bovine arboviruses assessed in this study.

Sero-survey on Aino, Akabane, Chuzan, bovine ephemeral fever and Japanese encephalitis virus of cattle and swine in Korea

Vector-borne arboviruses produce mild to severe symptoms in domestic animals. Bovine ephemeral fever (BEF), Akabane, Aino, and Chuzan virus have been primarily attributed to reproductive disorders or febrile diseases in cattle, and Japanese encephalitis virus (JEV) is mainly associated with reproductive failures in swine. We investigated antibody titers from domestic swine against four bovine arboviruses (BEF, Akabane, Aino, and Chuzan virus) and from cattle against JEV in Korea. While the positive rates for Akabane and BEF were 37.4% and 15.7%, the positive incidence of Chuzan and Aino were relatively low, with positive rates of 3.04% and 0.4%, respectively, based on a virus neutralization assay. Antibody titers against more than one virus were also frequently detected in domestic swine. The incidence of JEV was 51.3% among domestic cattle. In addition, one positive case was detected in the thoracic fluids from 35 aborted calves, based on the hemagglutination inhibition test. Our results indicate that swine are susceptible hosts of bovine arboviruses without showing clinical symptoms in a natural environment. Moreover, we confirmed that JEV could be associated with reproductive failure in pregnant cattle, as were other vector-borne bovine arboviruses assessed in this study.

Studies on the pathogenesis of bovine ephemeral fever. IV: A comparison with the inflammatory events in milk fever of cattle.

The study of ephemeral fever in cattle has defined a range of haematological and biochemical changes in blood which are characteristic of an inflammatory response. One of the clinical signs of ephemeral fever, a temporary paralysis reversible by treatment with calcium borogluconate, is similar to that in milk fever (parturient paresis), a disease of multiparous dairy cows. Three separate groups of cows were studied. Four multiparous cows were observed and sampled repeatedly during calving, three similar cows and one cow calving for the first time in a dairy herd were sampled daily before and after calving; and, in other dairy herds, seven cows with milk fever were sampled during illness. One of the cows under repeated observation during calving developed milk fever. The results showed that all the inflammatory indicators in blood were present in the multiparous cows at calving and that these were essentially similar to those established in ephemeral fever. The similarities in the four cows sampled repeatedly during the periparturient period were: a rectal temperature rise of 1 to 1.2 degrees C; rise in circulating neutrophils to peaks between 5700 and 11200 l-6; disappearance of eosinophils for 1 day; hypocalcaemia (plasma Ca < 2.0 mM l-1); fall of plasma zinc to low levels immediately after calving (plasma Zn < 500 micrograms l-1); fall of inorganic phosphate (plasma P < 0.9 mM l-1); rises in copper (plasma Cu > 1000 micrograms l-1) and plasma fibrin to > 8.75 g l-1. Plasma glucose peaked at calving between 5.7 and 8.9 mM l-1 then fell to levels ranging between 3.4 and 3.8 mM l-1. Plasma iron rose in one cow to 1220 micrograms l-1, was unchanged in one cow and fell in the other two to 440 and 860 micrograms l-1 respectively. The three multiparous cows which were sampled daily and calved normally showed similar haematological, macro and micromineral changes and fibrin response as did the seven milk fever cases. In the periparturient period, milk fever cows differed from multiparous cows calving normally, in degree but not in kind, of inflammatory response. It is postulated that an inflammatory event occurs in the periparturient period of multiparous cows which partially accounts for the falls in plasma calcium. This can precipitate a paralysis and other hypocalcaemic signs similar to that seen in acute ephemeral fever.

Studies on the pathogenesis of bovine ephemeral fever in experimental cattle. III. Virological and biochemical data.

In an attempt to define the nature of the response of cattle to ephemeral fever infection, a number of indicators of inflammation were monitored during clinical disease. The total Ca, Zn, Fe, Cu, glucose and phosphate in plasma, together with blood ammonia, were assayed relative to changes in the rectal temperature. CaT levels fluctuated markedly and hypocalcaemia occurred in 4 of 8 cattle. Plasma Zn and Fe values fell while plasma Cu levels rose markedly in all cattle. Mean levels of serum NH3 of 20-30 mumol l-1 rose to a peak value of 56 mumoll-1. Plasma glucose levels rose to a peak of 4.6 +/- 0.5 mMl-1 and the plasma phosphate levels fell from 2.4 +/- 0.1 mMl-1 to 1.17 +/- 0.2 mMl-1 during fever. Values of pCO2 fell from a mean of 46.9 +/- 3.6 mmHg to 36.4 +/- 3.1 mmHg and coincided with a rise in pH. Virus was isolated 73 h (+/- 23) after inoculation and persisted until 130 h (+/- 21). The common role of these parameters in generalised inflammation and ephemeral fever is discussed.

An outbreak of bovine ephemeral fever in Nagasaki Prefecture in 1988.

In October, 1988, bovine ephemeral fever (BEF) occurred in Nagasaki Prefecture and throughout Kyushu island, with the exception Miyazaki Prefecture. The first outbreak occurred in Hirado-shi on October 17. The total number of diseased cattle was 24 in 24 farms in Nagasaki Prefecture. The clinical findings were mainly sudden fever, anorexia, and instability in standing. The serum neutralizing antibodies against BEF virus (BEFV) rose in all infected cattle. Twelve strains of the virus were isolated in HmLu-1 cell cultures made directly from the heparinized blood of 17 infected cattle. The buffy coat was mainly collected from the samples and washed three times with phosphate buffered saline. These isolates were all neutralized by an antiserum against BEFV (Yamaguchi strain). With the aid of an electron microscope, a representative of isolates named Hirado-9 with a length of 150 nm was seen in the sample of infected HmLu-1 cell cultures. Both Hirado-9 and Yamaguchi strains reacted with antisera. The outbreak of BEF in 1988 was the first since 1971 in Nagasaki Prefecture. The result proved that BEFV can be easily isolated in HmLu-1 cell culture from the washed blood cells of infected cattle.

Clinical response of cattle to experimental infection with bovine ephemeral fever virus.

As part of a study of the pathology and pathogenesis of bovine ephemeral fever virus infection 44 cattle were infected by the intravenous injection of virulent virus. Thirty-eight animals responded clinically and detailed haematological and serological data were obtained from 10 of them. Inappetence was the only clinical sign observed before the onset of fever. The temperature response was characteristically biphasic, with the second peak occurring 12 to 24 hours after the first. The only consistent haematological response was an increase in the numbers of circulating neutrophils with a concurrent decline in the numbers of mononuclear leucocytes. There were no detectable changes in plasma or blood volume, packed cell volume, red cell count, haemoglobin concentration, serum calcium, magnesium, phosphorus and creatinine concentrations, or aspartate aminotransferase activity. Viraemia was demonstrated on either the first or second day of clinical disease and lasted for at most 48 hours. Low levels of neutralising antibody could be detected within one or two days after the cessation of viraemia. Six antibody-free animals did not respond clinically to injection with virulent virus, and did not develop detectable viraemia or a serum neutralising antibody response.

Studies on the pathogenesis of bovine ephemeral fever in sentinel cattle. II. Haematological and biochemical data.

Twenty-two non-lactating dairy cattle from a sentinel herd previously described (St. George, 1985) were monitored daily during an outbreak of ephemeral fever. Nine developed clinical ephemeral fever between 25 December 1981 and 30 January 1982. There were no subclinical infections with bovine ephemeral fever virus in the group. There were, however, subclinical infections with CSIRO Village, Akabane, Aino, Tinaroo and Kimberley viruses as described by St. George et al. (1984). Six of the nine affected cattle showed a neutrophilia with a concurrent lymphopaenia on the day of pyrexia; however, the differential white cell profile had begun to change up to 24 h prior to leucocytosis. Serum carboxypeptidase values fell by 24 h following the febrile response. Plasma fibrinogen rose rapidly in all six cows. The peak concentration (15.6 +/- 2.70 g l-1) occurred 3 days after pyrexia with the highest individual increase being from 6.05 to 19.6 g l-1. Plasma fibrinogen levels remained elevated for at least 7 days. Serum calcium fell significantly during Day 1 of the disease, the mean decline being 0.22 +/- 0.08 mmol l-1. The greatest individual fall was from 2.33 to 1.92 mmol l-1. None of the affected cattle showed any compensatory change in serum magnesium. There was no change in the normal values of creatinine, urea, gamma-GT, AST and alkaline phosphatase. Bovine ephemeral fever virus was isolated from only four of the six cases, whereas specific antibody was detected in all cattle 3-4 days after recovery.

Serological and biochemical factors in bovine ephemeral fever.

Clinical signs of ephemeral fever, which were observed in individual cattle during two successive epidemics in 1973 and 1976, were related to biochemical, cellular and serological changes in the blood. The rise in peripheral blood neutrophil counts in samples collected from 12 sentinel cattle on a daily basis before, during and after natural disease in the two epidemics to mean peaks of 9.6-12.5 X 10(9) per litre, and fall in counts of lymphocytes to a trough of 5-7 X 10(9) per litre was found to occur on the same day as the fever peak. A fall in serum calcium levels from a normal mean of 2.55 mmol/l to 2.0 mmol/l occurred on the day clinical signs were most pronounced. Serum magnesium levels were affected to only a minor degree. Plasma fibrinogen rose from a normal mean of 5.0 milligrams to a peak of 18 milligrams on the second day of disease and fell towards normal in the week after recovery. Neutralizing antibodies to bovine ephemeral fever virus were detected up to 63 days prior to clinical disease, and the rise of antibody after recovery was secondary in pattern. Serological evidence of a prior infection with an antigenically related virus, Kimberley virus, was found in these animals. In more severe clinical cases of ephemeral fever serum calcium levels were as low as 1.95 mmol/l. Treatment of cattle showing clinical signs of the disease with phenylbutazone and calcium borogluconate was favourable.

The infection of merino sheep with bovine ephemeral fever virus.

Four Merino sheep inoculated intravenously with bovine blood containing ephemeral fever virus showed no clinical signs of ephemeral fever. Two sheep showed a mild haematological response and developed a neutralising antibody which closely paralled that of a steer inoculated at the same time. Leucocytes harvested from these 2 sheep on days 3 and 4 after inoculation with virus reproduced ephemeral fever when inoculated into susceptible steers whilst those harvested on days 1, 2 and 5 did not. Even though this indicates that EFV can multiply in some sheep when they are inoculated intravenously, it cannot be inferred that natural infection occurs.