Apoptosis induced by bovine ephemeral fever virus

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 20-25 h 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.     

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.     

Antigenic variation of the bovine ephemeral fever virus glycoprotein

Summary Glycoprotein-specific monoclonal antibodies (MAbs) were used to select escape mutants of bovine ephemeral fever (BEF) virus to determine the escape frequency for different epitopes and to construct an epitope map. At least six antigenic sites were detected by this method and escape frequencies between 10^–2 and 10^–8 were recorded. One new non-conformational site was defined by a MAb, 5A5, which neutralized Berrimah and Kimberley viruses as well as three BEF virus strains. Batch to batch variation was detected in the BB7721 strain of BEF virus when tested for MAb neutralization. Eighteen strains of BEF virus, isolated from blood and insects from a variety of locations in Australia over a period of 33 years, were examined using MAbs and at least one epitope could not be detected in strains isolated since 1975. Implications for vaccine development are discussed.     

Vaccination of cattle against bovine ephemeral fever with live attenuated virus followed by killed virus

Summary Serial passage of bovine ephemeral fever virus in hamster lung cells, HmLu-1, deprived rapidly the virus of virulence for cattle with simultaneous loss of the ability to produce neutralizing antibody. The attenuated virus thus obtained, however, could prime cattle to produce neutralizing antibody rapidly to high titers following a subsequent inoculation of formalin-inactivated, AIPO₄ gel-adsorbed vaccine (K). Although one subcutaneous dose of live vaccine (L) prepared from the attenuated virus produces no detectable amounts of neutralizing antibody unless as heavy a dose as 10⁷ TCID₅₀ was used, the priming effect of L vaccine was shown even with 10 TCLD₅₀. Similar priming effect was also shown in mice by intracerebral inoculation of L vaccine. Maximal antibody response of cattle was obtained by one subcutaneous L vaccine dose of lO⁵]TCID₅₀ followed by 3 ml of K vaccine administered intramuscularly at intervals of 2 to 4 weeks. This LK method induced seroconversion in nearly all of initially seronegative cattle, and the neutralizing antibody thus produced persisted much longer than that following two doses of K vaccine given at one-month intervals. The LK method exerted little side effects in cattle. Cows vaccinated during pregnancy delivered healthy calves in term. No adverse effect on milk production was shown. The vaccine virus could not be passaged serially in calves by intravenous inoculation of the blood. Lyophilized L vaccine was stored at 4 C with little loss of infectivity for at least one year. Reconstituted vaccine could be used safely at least for 6 hours when kept at 4 C or even at room temperature.All these findings indicate the efficacy and safety of the LK vaccination method, although the final evaluation rests on large-scale field trials.     

Demonstration of vascular permeability changes in cattle infected with bovine ephemeral fever virus

Five cattle infected with bovine ephemeral fever virus were necropsied on the day after onset of clinical disease, when clinical signs of lameness were most severe. Gross lesions observed included a serofibrinous polyserositis involving the synovial, pericardial, thoracic and abdominal cavities. The associated histological changes consisted primarily of oedema and an influx of neutrophils into affected tissues and fluids. In a further eight infected cattle, increases in permeability of vessels associated with serosal surfaces were demonstrated by labelling with either colloidal carbon or Evans blue. Intravenous injections of carbon provided both macroscopic and histological labelling of affected vessels. Evans blue appeared to be more sensitive than carbon but did not provide a histological marker of vascular permeability and provided labelling of tissues rather than individual vessels. The main sites of increased permeability were synovial, pericardial, thoracic and abdominal serosae.     

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 virus in cell culture and mice

Summary Light, immunofluorescent and electron microscopic observations were carried out sequentially on mice and VERO cell cultures infected with bovine ephemeral fever (BEF) virus. In early harvests from cell culture, 185×73 nm cone-shaped particles with nearly parallel sides predominated; these particles had all other features typical of the Rhabdoviruses (surface projections, envelope, axial channel, precisely coiled helical nucleocapsid with 35 cross-striations at a 4.8 nm interval). Identical particles were the most frequent form in mouse brain. Variation in shape toward more broadly based cone-shapes occurred late in infection and reflected anomalous morphogenesis or particle breakdown. T (truncated) particles were common at all stages of infection. It was concluded that the reported variation in length and shape of particles in various BEF virus isolates is not necessarily releated to strain variation but more likely to varying growth rates and T particle interference associated with varying degrees of adaptation to a host system. BEF viral morphogenesis took place primarily upon plasma membranes in vivo and in cell culture in association with small accumulations of intracytoplasmic matrix. Fusion of viral envelopes was observed, and an early syncytium formation occurred in infected cell cultures. Cytopathology was similarin vivo and in cell culture; a protracted stage of cell rounding was followed by extreme cytoplasmic vacuolation and condensation and then by lysis. Necrotic encephalomyelitis was severe in moribund mice but extraneural sites of viral propagation and damage were not found with any of the techniques employed. Since this strict neurotropism is most likelynot characteristic of naturally acquired BEF virus infection in nature, it was concluded that the mouse was an unsatisfactory host for the further study of pathogenesis.On leave from Center for Disease Control, Atlanta, Georgia, U.S.A.     

Bovine ephemeral fever

Summary Thermal and pH stability of ephemeral fever virus (EFV) were studied. Infectivity of EFV was inactivated within 10 minutes at 56°C, by 18 hours at 37°C, and within 120 hours at 25°C. Infected mouse-brain virus had little titer loss after 30 days' storage at 4°C. Infected culture fluids stored at –35°C lost about 0.04 log units of virus per day. EFV was inactivated within 10 minutes at pH 2.5 and 12.0. Infectivity was lost in 60 minutes at pH 5.1 and in 90 minutes at pH 9.1. EFV was adapted to monkey kidney cell lines (Vero and MS); a plaque assay in Vero cells is described. The virus sized between 100 and 220 m by filtration. Growth studies of EFV in MS cells are described.     

The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes.

A 3789 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located 1.65 kb downstream of the N gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs) which are bounded by putative consensus (AACAGG) and polyadenylation (CATG[A]7) sequences and are separated by an intergenic region of 53 nucleotides. Discrete mRNAs corresponding to each ORF have been identified. The first ORF encodes a polypeptide comprising 623 residues which was identified by peptide sequencing as the virion G protein. The deduced amino acid sequence of the G protein includes putative signal and transmembrane domains and five potential glycosylation sites. The second ORF encodes a polypeptide of 586 amino acids which also has characteristics of a rhabdovirus glycoprotein, including putative signal and transmembrane domains and eight potential glycosylation sites, and appears to correspond to a 90-kDa nonstructural glycoprotein (GNS) identified in BEFV-infected cells (Walker et al. [1991] J. Gen. Virol. 72, 67-74). A database search indicated that both the G and GNS proteins share significant amino acid sequence homology with other rhabdovirus G proteins and with each other. Highest homology scores for each protein were with sigma virus and vesicular stomatitis virus serotypes.     

Antigenic characterization of bovine ephemeral fever rhabdovirus G and GNS glycoproteins expressed from recombinant baculoviruses

Recombinant baculoviruses expressing the BEFV envelope glycoprotein G and non-structural glycoprotein G(NS) were constructed. The G protein expressed in insect cells was located on the cell surface and induced spontaneous cell fusion at mildly acidic pH. The expressed G protein reacted with MAbs to continuous and conformational neutralization sites (G1, G2, G3b and G4), but not to conformational site G3a. The expressed G(NS) protein was also located on the cell surface but did not exhibit fusogenic activity. The G(NS) protein reacted with polyclonal antiserum produced from vaccinia-virus-expressed recombinant G(NS) but did not react with G protein antibodies. A His(6)-tagged, soluble form of the G protein was expressed and purified by Ni(2+)-NTA chromatography. The purified G protein reacted with BEFV-neutralizing MAbs to all continuous and conformational antigenic sites. The highly protective characteristics of the native BEFV G protein suggest that the secreted, baculovirus-expressed product may be a useful vaccine antigen.     

RNA polymerase (L) gene and genome terminal sequences of ephemeroviruses bovine ephemeral fever virus and Adelaide River virus indicate a close relationship to vesiculoviruses

The sequence of the RNA genome of bovine ephemeral fever virus (BEFV) was determined from the start of the L (polymerase) gene to the end of the untranslated 5′ trailer sequence, completing the sequence of the 14 900 nucleotide (nt) genome. The 6470 nt L gene encodes a single long ORF of 2144 amino acids with a deduced molecular weight of 249 766 Da. The 70 nt BEFV 5′ trailer region displays partial terminal complementarity with the 3′ leader sequence and contains a 26 nt direct repeat of the U-rich domain of the 3′ leader region. The 47 nt 5′ trailer region of Adelaide River virus (ARV) displays terminal sequence similarity to the BEFV trailer and partial terminal complementarity with the ARV 3′ leader sequence, but does not contain the direct repeat sequence. The BEFV L protein contains all characteristic sequence motifs of amino acid blocks I–VI, conserved among RNA polymerase proteins of single-stranded (−) RNA viruses, separated by regions of lower homology. Phylogenetic analysis using the complete BEFV L protein sequence indicated a closer relationship to vesicular stomatitis virus than to rabies virus. Sequence comparison of two conserved central domains encompassing blocks II and III and block VI of the BEFV and ARV L proteins indicated they are closely related. An extended phylogenetic analysis using the block III sequence, confirmed the relationship of these ephemeroviruses to vesiculo- and lyssaviruses and to other single-stranded (−) RNA viruses.     

Formalin-inactivated respiratory syncytial virus vaccine induces antibodies to the fusion glycoprotein that are deficient in fusion-inhibiting activity.

The fusion (F) glycoprotein of respiratory syncytial virus (RSV) induces neutralizing antibodies and antibodies that inhibit fusion of infected cells (FI antibody). It was previously shown that infants and children immunized with Formalin-inactivated RSV 20 years ago developed antibodies that bound to the F glycoprotein but were deficient in neutralizing activity. A reexamination of these sera indicated that they were also deficient in FI activity. Thus, Formalin-inactivated RSV vaccine stimulated an unbalanced immune response in which an unusually large proportion of the induced antibodies were directed against nonprotective epitopes rather than against the epitopes that induce functional antibodies, i.e., neutralizing and FI antibodies. This deficiency in stimulation of functional antibodies probably decreased the protective efficacy of the vaccine and could have contributed to potentiation of disease in the vaccines during subsequent RSV infection.     

Sheep-associated malignant catarrhal fever virus: prospects for vaccine development

Sheep-associated malignant catarrhal fever is emerging as a significant problem for several ruminant species worldwide. The inability to propagate the causative agent, ovine herpesvirus 2, in vitro has seriously hindered research efforts in the development of effective programs for control of the disease in clinically susceptible hosts. Recent molecular technologic advances have provided powerful tools for investigating this difficult-to-study virus. Identification of the infectious virus source, establishment of experimental animal models and completion of sequencing the genome for ovine herpesvirus 2 have put us in a position to pursue the development of vaccines for control of the disease. In this review, the authors briefly describe the current understanding of ovine herpesvirus 2 and prospectively discuss vaccine development against the virus.     

Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) for detection of bovine ephemeral fever virus

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/mul of the BEFV plasmid DNA could be detected by the assay. The optimal hybridization conditions at 50 degrees C in 5x 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.     

Neurovirulence of yellow fever 17DD vaccine virus to rhesus monkeys

The yellow fever 17D virus is attenuated and used for human vaccination. Two of its substrains, 17D-204 and 17DD, are used for vaccine production. One of the remarkable properties of this vaccine is limited viral replication in the host but with significant dissemination of the viral mass, yielding a robust and long-lived neutralizing antibody response. The vaccine has excellent records of efficacy and safety and is cheap, used as a single dose, and there are well-established production methodology and quality control procedures which include the monkey neurovirulence test (MNTV). The present study aims at a better understanding of YF 17DD virus attenuation and immunogenicity in the MNVT with special emphasis on viremia, seroconversion, clinical and histological lesions scores, and their intrinsic variability across the tests. Several MNVTs were performed using the secondary seed lot virus 17DD 102/84 totaling 49 rhesus monkeys. Viremia was never higher than the accepted limits established in international requirements, and high levels of neutralizing antibodies were observed in all animals. None of the animals showed visceral lesions. We found that the clinical scores for the same virus varied widely across the tests. There was a direct correlation between the clinical scores in animals with clinical signs of encephalitis and a higher degree of central nervous system (CNS) histological lesions, with an increase of lesions in areas of the CNS such as the substantia nigra, nucleus caudatus, intumescentia cervicalis, and intumescentia ventralis. The histological scores were shown to be less prone to individual variations and had a more homogeneous value distribution among the tests. Since 17DD 102/84 seed virus has been used for human vaccine production and immunization for 16 years with the vaccine being safe and efficacious, it demonstrates that the observed variations across the MNVTs do not influence its effect on humans.     

Yellow fever vaccine

Summary Removal of avian leukosis viruses (ALV), which have contaminated the yellow fever (YF) 17 D vaccine since its development in the middle 1940's, had no effect upon the antigenicity of this vaccine in rhesus monkeys. From results of plaque neutralization tests, the high degree of antigenicity of the 17 D vaccine was confirmed.Pre-existing cross-reacting antibodies to other antigenically related arboviruses did not interfere with the antibody response to YF vaccine. However, administration of YF vaccine did elicit antibodies capable of cross-reacting with West Nile, and less so with Langat, arbovirus antigens.