Bovine Ephemeral Fever Virus Infection Research Articles

Stretching the wings further- susceptibility of Culex pipiens Linnaeus to bovine ephemeral fever virus infection under experimental conditions

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.

Role of cervids in the epidemiology of bovine ephemeral fever virus infection in the Republic of Korea: A cross-sectional retrospective study.

Bovine ephemeral fever (BEF) is a viral disease in cattle and buffaloes, with subclinical involvement in various ruminant species. This study aimed to investigate bovine ephemeral fever virus (BEFV) transmission in deer in the Republic of Korea (ROK) and the potential risk factors associated with seropositivity. We conducted a retrospective cross-sectional serological survey of neutralising antibodies against BEFV in cervid sera collected from the ROK. The seroprevalence of BEFV was estimated to be 10.8% (95% confidence interval [CI] = 8.5-14.1), demonstrating that exposure to this virus is prevalent among farmed and free-ranging cervids in the ROK. The results revealed that age class and geographic location affected seroprevalence. Older age and the presence of neighbouring ruminant farms were significant risk factors (odds ratio [OR] = 2.394, 95% CI = 1.195-4.796) and (OR = 1.533, 95% CI = 1.026-2.288), respectively. We also observed that the individual likelihood of positivity in the southern provinces was significantly higher than that in the northern provinces (OR = 1.744, 95% CI = 1.001-3.037). There were also significant differences in the seroprevalence of cervids between the western and eastern provinces (OR = 2.021, 95% CI = 1.047-3.900). Factors that were not significantly associated with BEFV antibody prevalence included herd size and species (p > 0.05). These results suggest that cervid species may serve as important reservoirs for the transmission of BEFV, highlighting the need for closer monitoring of BEFV infections in cervids in the ROK.

Isolation and genetic characterization of bovine ephemeral fever virus from epidemic-2020 in Turkey.

Bovine ephemeral fever virus (BEFV) infection occurs seasonally in many tropical and subtropical regions of Africa, Asia (including the Middle East), and Australia while it is exotic in Europe. In this study, the epidemiology of BEFV infection in Turkey that bridges southeastern Europe and Asia, geographically, was investigated according to the comparison of the nucleotide sequences of the virus caused the last epidemic in 2020 with those of the strains previously detected in Turkey as well as BEFV strains from other countries. In the phylogenetic analysis, based on an alignment of full-length G gene sequences, BEFVs from epidemic-2020 were located in Middle Eastern lineage and appear to represent most closely related BEFVs from India-2018 and 2019. The findings will contribute to a better understanding of BEFV epidemiology in Turkey.

Epidemiological Assessment of Risk Factors Associated with Bovine Ephemeral Fever Virus Exposure among Sheep and Goats in South Korea

Background: Bovine ephemeral fever virus (BEFV) is an arthropod-borne virus classified as a type species of the genus Ephemerovirus, family Rhabdoviridae. BEFV is the causative agent of bovine ephemeral fever (BEF), a non-contagious disease of acute febrile clinical signs in cattle and water buffalo. Some species might act as reservoir hosts, and antibodies to BEFV have also been found in asymptomatic sheep, goats, pigs, and many wild animals. This study aimed to conduct a retrospective cross-sectional serological screening in South Korea to address BEFV seroprevalence and identify risk factors for becoming seropositive for the virus in sheep and goats. Materials, Methods & Results: The apparent prevalence rates were considered to be the animal-level prevalence, defined as the proportion of serum neutralization test (SNT)-positive animals out of the total number of animals tested in the study area, and flock prevalence was defined as the proportion of SNT-positive flocks out of the total number of tested flocks in the area. A flock was classified as positive if at least one animal was SNT-positive. At the national level in 2011, 28 of 177 flocks (15.8%; 95% CI, 11.2−21.9%) and 71 of 498 heads (14.3%, 95% CI: 11.5-17.6%) that were analyzed showed serum neutralizing antibodies against BEFV. Our results revealed that age class, vector control, and geographic location affected seroprevalence to differing extents. In the univariate analysis, older age was a significant risk factor (OR, 2.327; 95% CI, 1.147-4.721; P = 0.017 in adults). The management risk factor attributes showed that preventive measures, such as routine application of insecticides in farms, decreased the odds of seropositivity for BEFV (OR, 0.514; 95% CI, 0.267-0.991; P = 0.044). Vector control was a significant protective factor, while animal species, flock size, and flock structure were not significantly associated. Differences in seroprevalence between variations in the presence of ruminant farms, lakes, or rice paddies within a 1-km radius or type of land use were not statistically significant (P > 0.05). We observed a significant difference in the individual likelihood of being positive in the southern provinces with respect to that in the northern provinces (OR, 2.166; 95% CI, 1.228-3.824; P = 0.007). Differences in seroprevalence between variations in the eastern and western regions were not statistically significant (P > 0.05). The retrospective study results showed that the virus was widely distributed in sheep and goats in South Korea, with seropositive rates ranging from 7.8% to 19.7% between 2003 and 2008.Discussion: This is the first report of circulating antibodies against BEFV in sheep and goats in South Korea. The serological prevalence of BEFV infection in sheep and goats was significantly different between different age cohorts, vector control, and geographical locations: it was higher in the older group and the southern and western regions of South Korea. Determination of seropositivity rates often leads to an understanding of virus circulation dynamics and is useful in the formulation of disease control measures. Our results demonstrated that vector control was a significant protective factor; therefore, the summer control of vectors could be better implemented in provinces with elevated seropositivity rates. The results of this seroprevalence study may serve as a basis for future epidemiological studies on BEFV infection in South Korea.

Cell apoptosis regulated by interaction between viral gene alpha 3 and host heterogeneous nuclear ribonucleoprotein K facilitates bovine ephemeral fever virus replication

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus and causes bovine ephemeral fever of cattle and water buffalo in worldwide. Previous studies have demonstrated that infection with BEFV leads to induction of host cellular apoptosis. However, the role of apoptosis in viral replication and the interaction between viral genes and host genes involved in the process of BEFV-induced apoptosis remains unclear. Herein we investigated the interaction between viral non-structural protein α3 and cellular heterogeneous nuclear ribonucleoprotein K (hnRNP K) in the BEFV-induced apoptosis and its role in virus replication. Overexpression of α3 gene activated caspase 3 and consequently cleaved PARP, ultimately lead to apoptosis. Moreover, virus titer of BHK-21 cells infected with BEFV and then treated respectively by the pan-caspase inhibitor (Z-VAD-FMK) and apoptosis inducer (CCCP) was determined, the results showed that apoptosis promoted viral replication. In addition, knockdown of hnRNP K gene promoted BEFV replication, whereas overexpression of hnRNP K gene had the opposite effects. More importantly, overexpression of hnRNP K inhibited virus-induced apoptosis. Subsequently, it was found that hnRNP K suppressed BEFV replication via degrading viral α3 gene and further inhibited apoptosis induced by α3 gene. Finally, the expression of hnRNP K protein was significantly down-regulated upon BEFV infection, and degradation of hnRNP K protein in BHK-21 cells infected with BEFV was mediated by viral activation of caspase 3. Taken together, these results suggest that apoptosis takes a pivotal role in BEFV replication, and interaction between viral α3 gene and host hnRNP K gene in BEFV-induced apoptosis facilitates BEFV replication.

Bovine ephemeral fever virus triggers autophagy enhancing virus replication via upregulation of the Src/JNK/AP1 and PI3K/Akt/NF-\u03baB pathways and suppression of the PI3K/Akt/mTOR pathway

Autophagy plays an important role in cellular response to pathogens. However, the impact of the autophagy machinery on bovine ephemeral fever virus (BEFV) infection is not yet determined. A recent study in our laboratory demonstrated that BEFV triggers simultaneously the PI3K/Akt/NF-κB and Src/JNK-AP1 pathways in the stage of virus binding to enhance virus entry. In this work, we report that BEFV induces autophagy via upregulation of the PI3K/Akt/NF-κB and Src/JNK/AP1 pathways in the early to middle stages of infection and suppresses the PI3K/Akt/mTOR pathway at the late stage of infection. To activate NF-κB, BEFV promotes degradation of IκBα and activates Akt to stimulate NF-κB translocation into the nucleus. Immunoprecipitation assays revealed that BEFV disrupts Beclin 1 and Bcl-2 interaction by JNK-mediated Bcl-2 phosphorylation, thereby activating autophagy. Overexpression of Bcl-2 reversed the BEFV-induced increase in the LC3 II levels. Suppression of autophagy either by knockdown of autophagy-related genes with shRNAs or treatment with a pharmacological inhibitor 3-MA reduced BEFV replication, suggesting that BEFV-induced autophagy benefits virus replication. Our results revealed that the BEFV M protein is one of the viral proteins involved in inducing autophagy via suppression of the PI3K/Akt/mTORC1 pathway. Furthermore, degradation of p62 was observed by immunoblotting, suggesting that BEFV infection triggers a complete autophagic response. Disruption of autophagosome-lysosome fusion by depleting LAMP2 resulted in reduction of virus yield, suggesting that formation of autolysosome benefits virus production.

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.

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.

Bovine Ephemeral Fever Virus Uses a Clathrin-Mediated and Dynamin 2-Dependent Endocytosis Pathway That Requires Rab5 and Rab7 as Well as Microtubules

The specific cell pathways involved in bovine ephemeral fever virus (BEFV) cell entry have not been determined. In this work, colocalization of the M protein of BEFV with clathrin or dynamin 2 was observed under a fluorescence microscope. To better understand BEFV entry, we carried out internalization studies with a fluorescently labeled BEFV by using a lipophilic dye, 3,30-dilinoleyloxacarbocyanine perchlorate (DiO), further suggesting that BEFV uses a clathrin-mediated endocytosis pathway. Our results suggest that clathrin-mediated and dynamin 2-dependent endocytosis is an important avenue of BEFV entry. Suppression of Rab5 or Rab7a through the use of a Rab5 dominant negative mutant and Rab7a short hairpin RNA (shRNA) demonstrated that BEFV requires both early and late endosomes for endocytosis and subsequent infection in MDBK and Vero cells. Treatment of BEFV-infected cells with nocodazole significantly decreased the M protein synthesis and viral yield, indicating that microtubules play an important role in BEFV productive infection, likely by mediating trafficking of BEFV-containing endosomes. Furthermore, BEFV infection was strongly blocked by different inhibitors of endosomal acidification, suggesting that virus enters host cells by clathrin-mediated and dynamin 2-dependent endocytosis in a pH-dependent manner.

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.

Apoptosis induction in BEFV-infected Vero and MDBK cells through Src-dependent JNK activation regulates caspase-3 and mitochondria pathways

Our previous report demonstrated that bovine ephemeral fever virus (BEFV)-infected cultured cells could induce caspase-dependent apoptosis. This study aims to further elucidate how BEFV activates the caspase cascade in bovine cells. BEFV replicated and induced apoptosis in Vero and Madin-Darby bovine kidney (MDBK) cells, and a kinetic study showed a higher efficiency of replication and a greater apoptosis induction ability of BEFV in Vero cells. Src and c-Jun N-terminal kinase (JNK) inhibitor, but not extracellular signal-regulated kinase (ERK) or p38 inhibitor, alleviated BEFV-mediated cytopathic effect and apoptosis. In BEFV-infected Vero and MDBK cells, BEFV directly induced Src tyrosine-418 phosphorylation and JNK phosphorylation and kinase activity, which was inhibited specifically by SU6656 and SP600125, respectively. The caspase cascade and its downstream effectors, Poly (ADP-ribose) polymerase (PARP) and DFF45, were also activated simultaneously upon BEFV infection. In addition, cytochrome c, but not Smac/DIABLO, was released gradually from mitochondria after BEFV infection. SU6656 suppressed Src, JNK, and caspase-3 and -9 activation, as well as PARP and DFF45 cleavage; SP600125 reduced JNK and caspase-3 and -9 activation, as well as PARP and DFF45 cleavage. Taken together, these results strongly support the hypothesis that a Src-dependent JNK signaling pathway plays a key role in BEFV-induced apoptosis. The molecular mechanism identified in our study may provide useful information for the treatment of BEFV.

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.

Bovine ephemeral fever virus-induced apoptosis requires virus gene expression and activation of Fas and mitochondrial signaling pathway

Although induction of apoptosis by bovine ephemeral fever virus (BEFV) in several cell lines has been previously demonstrated by our laboratory, less information is available on the process of BEFV-induced apoptosis in terms of cellular pathways and specific proteins involved. In order to determine the step in viral life cycle at which apoptosis of infected cells is triggered, chemical and physical agents were used to block viral infection. Treatment of BHK-21 infected cells with ammonium chloride (NH4Cl) or cells infected with UV-inactivated BEFV was seen to abrogate virus apoptosis induction, suggesting that virus uncoating and gene expression are required for the induction of apoptosis. Using soluble death receptors Fc:Fas chimera to block Fas signaling, BEFV-induced apoptosis was inhibited in cells. BEFV infection of BHK-21 cells results in the Fas-dependent activation of caspase 8 and cleavage of Bid. This initiated the dissipation of the membrane potential and the release of cytochrome c but not AIF or Smac/DIABLO from mitochondrial into cytoplasm leading to activation of caspase 9. Combined activation of the death receptor and mitochondrial pathways results in activation of the downstream effecter caspase 3 leading to cleavage of PARP. Fas-mediated BEFV-induced apoptosis could be suppressed by the overexpression of Bcl-2 or by treatment with caspase inhibitors and soluble death receptors Fc:Fas chimera. Taken together, this study provided first evidence demonstrating that BEFV-induced apoptosis requires viral gene expression and occurs through the activation of Fas and mitochondrion-mediated caspase-dependent pathways.

Suppression of bovine ephemeral fever virus by RNA interference

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.

Epidemiology of bovine ephemeral fever virus infection in Taiwan

Sick animals with excessive nasal discharges and protruding tongue as a result of dyspnea were observed in the August of 1996. Eight strains of BEF virus were isolated from heparinized blood samples of the affected cattle. Most of the affected cattle were difficult to be treated and had a poor prognosis. A total of 516 farms in the 9 districts of Taiwan were affected in 1996. Among a population of 110,247 dairy cattle, 14,993 (13.6%) cattle were found to be clinically ill. During the epidemic, 1,685 (11.3%) affected cattle were culled or dead after the onset of the disease. Furthermore, a strain of Ibaraki virus was isolated from the blood sample of a sick cattle that showed pyrexia, labored respiration and solitary behavior in the affected farm. The cattle with Ibaraki virus infection had typical symptoms of BEF at the early stage of the disease, but neither stomatitis nor pharyngoesophageal paralysis was observed at the onset of the disease. The outbreak was presumably brought about by the low level or non-immune status of a large cattle population due to the negligence of BEF vaccination. Therefore, the disease easily recurred in Taiwan after a typhoon episode in the August of 1996, which resulted in the proliferation of biting midges in the field. No difference in the antigenicty was found between the new and the previous isolates of BEF virus. As analyzed by cross neutralization test, the isolated BEF viruses showed no relationship to the Kimberley and Berrimah viruses that were isolated from the blood of cattle and related to BEF virus in Australia. We have tried to advise farmers that they must vaccinate their cattle annually to prevent BEF outbreak in the future.

Vaccinia virus-expressed bovine ephemeral fever virus G but not G(NS) glycoprotein induces neutralizing antibodies and protects against experimental infection.

Two related glycoproteins (G and G(NS)) encoded in the bovine ephemeral fever virus (BEFV) genome were expressed from recombinant vaccinia viruses (rVV). Both proteins were detected in lysates of rVV-infected cells by labelling with D-[6-3H]glucosamine or by immuno-blotting. The recombinant G protein (mol. mass 79 kDa) appeared slightly smaller than the native G protein but reacted with monoclonal antibodies directed against all defined neutralizing antigenic sites (G1, G2, G3a, G3b and G4). The recombinant G(NS) protein (mol. mass 90kDa) was identical in size to the native G(NS) protein and failed to react by immuno-fluorescence with anti-G protein monoclonal or poly-clonal antibodies. Antisera raised in rabbits against rVV-G or rVV-G(NS) both reacted strongly by immuno-fluorescence and immuno-electron microscopy with BEFV-infected cells. The G protein was localized intracellularly in the endoplasmic reticulum/Golgi complex and at the cell surface associated with budding and mature virus particles. The G(NS) protein also localized intracellularly in the endoplasmic reticulum/Golgi complex; however, at the cell surface it was associated with amorphous structures and not with budding or mature virions. Rabbits vaccinated with rVV-G developed high levels of antibodies which neutralized BEFV grown in either mammalian or insect cells. Cattle vaccinated with rVV-G also produced neutralizing antibodies and were protected against experimental BEFV infection. In contrast, rVV-G(NS) vaccinated rabbits and cattle failed to produce neutralizing antibodies and, after challenge, BEFV was isolated from two-thirds of the vaccinated cattle.

Epidemiologic analysis of spatial clustering of bovine ephemeral fever outbreaks. II. Principal component analysis.

The principal component analysis (PCA) was applied to analyze a correlation matrix of three variables on epidemic data of bovine ephemeral fever (BEF) outbreaks. These original data were summarized from the official outbreak report of Fukuoka Prefecture. The first and the second principal components of the PCA were interpreted as the infectious potency due to BEF virus and the prevention against BEF virus infection, respectively. The BEF outbreak areas were able to be classified epidemically into 4 groups by using the two principal components. The valuable epidemiological insights can be reasonably obtained from an application of the PCA. The results provided an important information for a further BEF vaccination campaign in the western part of Japan.

The pathogenesis of bovine ephemeral fever virus infection of adult mice

Infection of adult mice with small doses of neuro-virulent bovine ephemeral fever virus caused a rapidly fatal encephalitis, the course of which was largely unaffected by treatment with either cyclophosphamide or anti-lymphocyte serum. Athymic mice died earlier than their thymic littermates. Mice could be protected by treatment with defective virus before infection or by passive administration of antibody if given less than 48 h after infection. Taken together these results emphasize the important protective role of antibody present at the time of infection. Viral isolation and immuno-fluorescent studies showed that the virus is confined to the central nervous system. Regular infection of suckling and adult mice by extra-cerebral routes was not possible.

The Role of Neutrophils in Bovine Ephemeral Fever Virus Infection of Cattle

Bovine ephemeral fever is a rhabdoviral disease characterized by a pronounced neutrophilia and an influx of neutrophils into serosae and serosal fluids. Studies were undertaken on the effect of bovine ephemeral fever virus infection of cattle with an induced neutropenia. An equine antiserum to bovine neutrophils was produced using neutrophils derived from a bovine udder. This antiserum was used to deplete circulating neutrophils in three calves that were infected with virulent bovine ephemeral fever virus. The calves became viremic without developing clinical signs and without developing detectable antibodies to bovine ephemeral fever virus. Three months later, after restoration of blood neutrophil levels to normal values, two of these calves were again infected with virulent bovine ephemeral fever virus. Both calves developed viremia, clinical disease, and serum-neutralizing antibodies to bovine ephemeral fever virus.