- Email: [email protected]
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vaccinated cats from FIV-infected cats has caused a dilemma in the use of this vaccine. This problem can be resolved by developing sensitive molecular diagnostics or a vaccine that does not conflict with current FIV diagnostics. Identifying the protective vaccine epitopes should assist in designing a vaccine that is devoid of diagnostic epitopes. Moreover, FIV research on vaccines will provide new insights to HIV vaccine development for humans. The recent discovery of fatal pathogenic FIV strains (10% acute mortality) demonstrates the pathogenic evolution of FIV similar to HIV-1 immunopathogenesis. Hence, FIV infection is not only important for feline medicine but serves as an important small animal model for testing novel antiretroviral drugs, immunotherapy, and vaccine approaches for HIV/AIDS.
See also: Equine Infectious Anemia Virus; Human Immunodeficiency Viruses: Antiretroviral agents; Human Immunodeficiency Viruses: Molecular Biology; Human Immunodeficiency Viruses: Origin; Human Immunodeficiency Viruses: Pathogenesis; Simian Immunodeficiency Virus: Animal Models of Disease; Simian Immunodeficiency Virus: General Features; Simian Immunodeficiency Virus: Natural Infection; Visna-Maedi Viruses.
Further Reading Bachmann MH, Mathiason-Dubard C, Learn GH, et al. (1997) Genetic diversity of feline immunodeficiency virus: Dual infection, recombination, and distinct evolutionary rates among envelope sequence clades. Journal of Virology 71: 4241–4253. Burkhard MJ and Dean GA (2003) Transmission and immunopathogenesis of FIV in cats as a model for HIV. Current HIV Research 1: 15–29. Evermann JE, Howard TH, Dubovi EJ, et al. (2000) Controversies and clarifications regarding bovine lentivirus infections. Journal of the American Veterinary Medical Association 217: 1318–1324. Podell M, March PA, Buck WR, and Mathes LE (2000) The feline model of neuroAIDS: Understanding the progression towards AIDS dementia. Journal of Psychopharmacology 14: 205–213. Snider TG, Hoyt PG, Jenny BF, et al. (1997) Natural and experimental bovine immunodeficiency virus infection in cattle. The Veterinary Clinics of North America. Food Animal Practice 13: 151–176. St-Louis M-C, Cojocariu M, and Archambault D (2004) The molecular biology of bovine immunodeficiency virus: A comparison with other lentiviruses. Animal Health Research Reviews 5: 125–143. Troyer JL, Pecon-Slattery J, Roelke ME, et al. (2005) Seroprevalence and genomic divergence of circulating strains of feline immunodeficiency virus among Felidae and Hyaenidae species. Journal of Virology 79: 8282–8294. Uhl EW, Heaton-Jones TG, Pu R, and Yamamoto JK (2002) FIV vaccine development and its importance to veterinary and human medicine: A review. Veterinary. Immunology and Immunopathology 90: 113–132. Wilcox GE, Chadwick BJ, and Kertayadnya G (1995) Recent advances in the understanding of Jembrana disease. Veterinary Microbiology 46: 249–255.
Bovine Ephemeral Fever Virus P J Walker, CSIRO Livestock Industries, Geelong, VIC, Australia ã 2008 Elsevier Ltd. All rights reserved.
Glossary Epicardium The outer layer of heart tissue. Hypocalcemia A low level of calcium in the circulating blood. Leucopenia A decreased total number of white blood cells in the circulating blood. Pericardial fluid Fluid within a double-walled sac that contains the heart and the roots of the great blood vessels. Sternal recumbency Reclined in a position of comfort on the chest bone. Synovial membranes Connective tissue membranes lining the cavities of the freely movable joints. Thoracic fluid Fluid in the chest cavity.
Introduction Bovine ephemeral fever virus (BEFV) is an arthropodborne rhabdovirus that causes a disabling and sometimes lethal disease of cattle (Bos taurus, Bos indicus, and Bos javanicus) and water buffaloes (Bubalus bubalis). Unapparent infections can also occur in cape buffalo, hartebeest, waterbuck, wildebeest, deer, and possibly goats. Bovine ephemeral fever (BEF) was first recorded in East Africa and Egypt during the late nineteenth century. However, BEF (which is also variously called three-day sickness, bovine enzootic fever, bovine influenza, and stiffsiekte) is thought to have been endemic since antiquity in much of tropical and subtropical Africa, and Asia. As the name suggests, BEF is often characterized by the rapid onset of and recovery from clinical signs that can include a bi- or multiphasic fever, ocular and nasal discharge,
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muscle stiffness, anorexia, rumenal stasis, lameness, and sternal recumbency. Although mortality rates rarely exceed 1–2%, a particularly severe outbreak in Taiwan in 1996 has been reported to have resulted in the death or culling of 11.3% of a population of 110 247 dairy cattle on 516 farms. Severe infections commonly occur in larger, more valuable animals. Morbidity rates may approach 100% with significant economic impacts including loss of milk production, temporary infertility in bulls, abortion, loss of condition in beef herds, and disablement of draft animals at the time of harvest. The economic consequences of BEF can be significant. An outbreak of BEF in Israel in 1999 has been estimated to have cost, on average $280 per lactating cow and $112 per nonlactating cow. In Australia, sweeping epidemics in the 1970s have been estimated to have caused industrywide losses exceeding $200 million in today’s values. Due to limitations on the importation of livestock and semen from animals with evidence of BEFV infection, the disease can also have significant impact on international trade.
Taxonomic Classification BEFV is classified in the order Mononegavirales, family Rhabdoviridae as the type species of the genus Ephemerovirus. Ephemeroviruses have morphological and genetic characteristics common to all rhabdoviruses, including an enveloped, bullet-shaped virion containing a nonsegmented, negative-sense, single-stranded (ss) RNA genome. However, unlike viruses classified in other established rhabdovirus taxa, ephemeroviruses share the unusual characteristic of two type 1 transmembrane glycoproteins (G and GNS) that are related in amino acid sequence and appear to have arisen by gene duplication. Other recognized species in the genus include Berrimah virus (BRMV) and Adelaide River virus (ARV). These viruses have each been isolated from cattle in northern Australia and also appear to be transmitted by biting insects. Tentative species in the genus include Kimberley virus (KIMV), Malakal virus (MALV), and Puchong virus (PUCV), each of which is closely related serologically to BEFV. KIMV has been isolated from Culex annulirostris mosquitoes and Culicoides brevitarsis midges in Australia, and from healthy cattle. MALV was isolated in Sudan and PUCV in Malaysia, each from Mansonia uniformis mosquitoes. Potential vertebrate hosts for these viruses are yet to be identified. Although BEFV is the only ephemerovirus known to be associated with disease, Kotonkan virus (KOTV), which was originally isolated from Culicoides spp. in Nigeria, does cause an ephemeral fever-like illness in cattle. Recent phylogenetic studies using the N gene and segments of the L gene suggest that KOTV, as well as Obodhiang virus (OBOV) which was isolated from
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Mansonia uniformis mosquitoes in Sudan, should also be classified as ephemeroviruses. It has also been suggested that all arthropod-borne rhabdoviruses, including the ephemeroviruses, vesiculoviruses, and a large number of other unclassified rhabdoviruses share a common ancestor and should form a larger taxonomic group for which the name ‘dimarhabdovirus’ (dipteran-mammalian rhabdovirus) has been proposed (Figure 1).
Geographic Distribution BEFV is known to occur in most tropical and subtropical regions of Africa, Asia, and Australia. The disease occurs throughout much of Africa and in Asian countries south of a line that includes Israel, Iraq, Iran, Syria, India, Pakistan, Bangladesh, southern and central China, and east to Taiwan, and southern Japan. It occurs throughout Southeast Asia, parts of New Guinea, and in most of northern and eastern Australia. It does not occur in New Zealand or the islands of the Pacific, or in the Americas, or Europe, where it is considered an important exotic pathogen. There is reported serological evidence of infection in southern Russia but the disease has not been described. The distribution of BEFV is determined by the geographic range of available insect vectors and is limited by international trade restrictions on live animals and semen showing evidence of infection.
Epizootiology Bovine ephemeral fever is a seasonal disease. It occurs principally in the summer and early autumn, and with the onset of the monsoon season in Asia. Outbreaks are usually associated with periods of high rainfall which precipitate the emergence of insect vectors in large numbers. BEFV can also spread in epizootics that follow the pattern of prevailing winds with a general southward movement in the Southern Hemisphere and northward movement above the Equator. Wind-borne movement of insect vectors is the likely mechanism of spread. Vectors include biting midges (Culicoides spp.) and mosquitoes, in which the virus replicates. BEFV has been isolated from Culicoides brevitarsis, Culicoides coarctatus, Anopheles bancroftii, a mixed pool of mosquito species in Australia, and from a mixed pool of biting midges in Africa. The virus has also been recovered from several other species of biting midge and mosquito following experimental infection. The abundance and distribution of insects from which BEFV has been isolated suggests that several major vectors may be involved in transmission. There is no evidence of direct transmission of BEFV between cattle, even when encouraged by smearing nasal or ocular discharges on mucosal surfaces.
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Figure 1 Phylogenetic tree of partial N gene sequences of 20 rhabdoviruses infecting mammals and/or insects. The tree was generated from a Clustal X alignment of the sequences by the neighbor-joining method and presented graphically using Treeview software. The viruses include representatives of three rhabdovirus genera: Ephemerovirus (BEFV, ARV, Obodhiang virus, and Kotonkau virus); Vesiculovirus (VSV Indiana, Chandipura, Isfahan, and Piry viruses); and Lyssavirus (rabies, Mokola, and Lagos bat viruses). Unclassified rhabdoviruses include Sandjimba, Rochambeau, Flanders, Kern Canyon, Mount Elgon bat, Oita, Tupaia and Kolongo, and sigma viruses. Confidence in branch nodes was determined by bootstrap analysis on 1000 replicates (indicated as numbers).
Pathology and Pathogenesis Bovine ephemeral fever is principally an inflammatory disease. The incubation period is normally 2–4 days. Viremia usually persists for 1–3 days and peaks approximately 24 h before the onset of fever. The initial sites of infection are not known but the virus has been isolated from neutrophils and reticuloendothelial cells of the lungs, spleen, and lymph nodes. There is also evidence of infection in synovial membranes, epicardium and aorta, and in cells derived from synovial, pericardial, thoracic, and abdominal fluids. There is not widespread tissue damage. The primary lesion is a vasculitis affecting the endothelium of small vessels of synovial membranes, tendon sheaths, muscles, facia, and skin. The onset of fever and other clinical signs is accompanied by marked leucopenia, relative neutrophilia, elevated plasma fibrinogen,
and elevated levels of cytokines including interferon a, interleukin 1, and tissue necrosis factor. There is also a significant hypocalcemia that is thought to be responsible for sternal recumbency. The major clinical signs can be treated very effectively with anti-inflammatory drugs.
Virion Structure and Morphogenesis BEFV virions are enveloped, bullet-shaped particles (approximately 70 180 nm) containing a precisely coiled, helical nucleocapsid with 35 cross-striations at an interval of 4.8 nm. Virions have a prominent axial channel intruding from the base and typically are more cone-shaped than commonly observed for viruses in other genera of animal rhabdovirus (e.g., lyssaviruses and vesiculoviruses). The envelope contains a single 81 kDa class
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1 transmembrane glycoprotein (G) that forms visible projections on the virion surface. The G protein mediates cell attachment and entry, is the target for virus-neutralizing antibodies, and induces protective antibodies in cattle. Nucleocapsids contain a negative-sense ssRNA genome, a 52 kDa nucleoprotein (N) which is tightly bound to the genome, a large 250 kDa replicase protein (L), and a highly charged 43 kDa replicase cofactor (P). Virions also contain a 29 kDa matrix protein (M) which is a major structural component and appears to lie between nucleocapsids and the inner surface of the lipid envelope (Figure 2). Viral replication is cytoplasmic and morphogenesis occurs primarily at the plasma membrane in association with accumulations of a filamentous, granular, intracytoplasmic matrix. However, late in infection, there is a proliferation of plasma membrane, cells become highly vacuolated, and virions are observed both at the plasma membrane and within cytoplasmic vacuoles. Following budding as cone-shaped extrusions, virions accumulate in extracellular spaces. The general characteristics of viral morphogenesis appear to be similar in infected mammalian cell cultures and mouse neurons.
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BEFV Genome Organization and the Encoded Proteins The 14 900 nt BEFV genome is the largest known for any rhabdovirus and one of the largest and most complex genomes for any nonsegmented, negative-sense RNA virus. The genome comprises 12 genes, flanked by terminal, partially complementary leader (l ) and trailer (t) sequences, and arranged in the order 30 -l-N-P/C-M-GGNS-a1/a2/a3-b-g-L-t-50 . By analogy with other rhabdoviruses, the 30 leader (50 nt) and 50 trailer (70 nt) sequences are likely to have important roles in the initiation and control of replication and transcription, and in nucleoprotein assembly and packaging. The structural protein genes (N, P, M, G, and L) are arranged in the same order as for all other known rhabdoviruses and encode proteins with similar functional characteristics (Figure 3). The N Protein The BEFV N gene encodes the 431 amino acid nucleoprotein (N). The N protein is a highly hydrophilic, RNA-binding protein containing 14.4% basic residues
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(b) Figure 2 (a) Negative-contrast electron micrograph of a purified BEFV virion. (b) Schematic representation of the BEFV virion. Structural proteins N, P, M, G, and L, and the negative-sense ssRNA genome are indicated. The axial channel is also depicted. The size and relative quantities of the proteins do not accurately reflect the content in virions. Scale ¼ 50 nm (a).
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Figure 3 Illustration of the genome organization and transcription strategy of BEFV and ARV. Solid arrows indicate the major transcriptional products. Minor transcripts are indicated by dotted arrows. The length of the ARV L protein is estimated from the alignment of available sequences with those of BEFV.
(lysine, arginine, and histidine) distributed relatively evenly throughout the molecule. Seven of these residues are highly conserved in rhabdoviruses and are involved in RNA-binding and stabilizing the interaction. The N protein also contains 14.6% acidic residues (glutamate and aspartate) that are less evenly distributed with significant clustering in a short domain near the C terminus. A similar acidic domain in the rabies N protein is a phosphorylation site involved in binding P protein to nucleocapsids. The BEFV N protein is also phosphorylated when packaged in virions and contains a similar phosphorylation site in this acidic domain. The P Protein The P gene encodes the 278 amino acid, highly hydrophilic P protein. It corresponds to the polymerase-associated phosphoproteins of rabies virus (RV) and vesicular stomatitis virus (VSV) which are components of nucleocapsids and act as essential cofactors to the L protein during transcription and replication. The BEFV P protein has not been observed to be phosphorylated when extracted from virions but is phosphorylated when expressed from a recombinant baculovirus in insect cells. The BEFV P gene also contains an alternative open reading frame (ORF) encoding a 48 amino acid, highly basic 5.8 kDa polypeptide. This protein has not been detected in BEFV-infected cells and it is not known if it is expressed. However, alternative ORFs in the
P gene occur in vesiculoviruses and are a common feature of many viruses in the Mononegavirales. As in VSV, the BEFV C protein has two potential initiation codons, suggesting it could be expressed in two different forms. The M Protein The M gene encodes the 691 amino acid, basic, hydrophilic protein that corresponds to the matrix protein (M) of rabies and VSV. The M protein is a major component of rhabdovirus virions and has been shown to have important functions in regulation of viral replication and transcription, inhibition of host cell protein synthesis and induction of apoptosis, and in budding of nucleocapsids at cytoplasmic membranes. The BEFV M protein has been shown to be phosphorylated when extracted from virions but not when expressed from a recombinant baculovirus in insect cells. This is consistent with observations that phosphorylation of the VSV M protein occurs at a late stage in viral assembly. The BEFV M protein also contains a ‘late domain’ sequence motif (PPSY) which, in VSV and several other RNA viruses, is essential for efficient budding from infected cells. The G Protein The BEFV G gene encodes the 623-amino-acid virion transmembrane glycoprotein (G). The G protein is a
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class 1 membrane protein. It shares significant amino acid sequence identity with other animal rhabdovirus G proteins and contains a core of conserved cysteine residues suggesting preservation of fundamentally similar secondary structure. The G protein contains five potential N glycosylation sites, three of which appear to align with similar sites in VSV and/or rabies virus. The G protein is responsible for cell docking and entry, and is the target of virus-neutralizing antibodies for which the major binding sites have been defined (see below). The L Protein The L gene encodes the 2144-amino-acid RNA-dependent RNA polymerase (L protein). The L protein is a structural component of nucleocapsids that, in cooperation with the N and P proteins, forms the ribonucleoprotein (RNP) complex that is responsible for replication and transcription of the viral genome. The BEFV L protein shares a high level of sequence similarity with other rhabdovirus L proteins and contains all of the conserved sequence motifs associated with the major functional domains; these include the polymerase catalytic site and other regions involved in replication, transcription, initiation, elongation, and termination, 30 polyadenylation, 50 capping, and cap methylation.
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The Small Nonstructural Proteins Downstream of the GNS gene is a complex region of the genome-encoding proteins that appear to be unique to BEFV and other ephemeroviruses. None of these proteins has yet been detected in virions or BEFV-infected cells. The a-gene coding region contains three long ORFs (a1, a2, and a3). The a1 ORF encodes an 88 amino acid, 10.6 kDa protein. It features a central transmembrane domain comprising 16 hydrophobic amino acids bounded by arginine residues, and a highly basic C-terminal domain in which 12 of 18 amino acids are lysine or arginine residues. This structure suggests a1 may function as a viroporin, a class of proteins that causes cytopathic effects by increasing membrane permeability. The BEFV a1 protein has been shown to be cytotoxic when expressed in insect cells from a recombinant baculovirus vector. The a2 ORF encodes a 116 amino acid, 13.7 kDa polypeptide. It overlaps the a3 ORF which encodes a 51 amino acid, 5.7 kDa polypeptide that contains an unusual triple repeat of isomers of the sequence KLMEE at intervals of four residues. The b-gene encodes a 107 amino acid, 12.3 kDa polypeptide. The g-gene encodes a 114 amino acid, 13.5 kDa polypeptide. The a2, a3, b, and g products share no sequence homology with known viral proteins (other than ARV, see below) and their functions are yet to be determined.
The GNS Protein
BEFV Transcription
The BEFV nonstructural protein genes are located in a 3442 nt region of the genome between the G gene and L gene. Immediately downstream of the G gene is a second gene encoding a class 1 transmembrane glycoprotein (GNS). The 90 kDa GNS protein is abundant in infected cells but has not been detected in virions. It is related in structure and sequence to the BEFV virion G protein and other rhabdovirus G proteins and the evidence suggests that it has arisen by gene duplication. The GNS protein contains eight potential N-glycosylation sites and, as the size by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) is approximately 21 kDa in excess of the calculated molecular weight of the unmodified polypeptide, it appears to be highly glyscosylated. Sequence alignments indicate that it shares 10 of 12 cysteine residues that appear to form the core of secondary structure common to all animal rhabdoviruses. However, the GNS protein does not share antigenic sites with the BEFV G protein and antibody to GNS does not neutralize the infectivity of BEFV produced in either mammalian or insect cells. It has been shown to accumulate at the cell surface in association with amorphous structures but not with budding or mature virions. The function of the GNS is currently unknown.
The RNP complex, comprising the RNA genome and the N, L, and P proteins, is the active replication and transcription unit of the virus. As for other rhabdoviruses, BEFV transcription from the () RNA genome generates 50 methylated, capped, and polyadenylated mRNAs by a progressive mechanism that initiates and terminates at short, conserved sequences flanking each gene. For each of the structural protein genes (N, P, N, G, and L), and the nonstructural glycoprotein gene (GNS), transcription initiates at the sequence UUGUCC and terminates at the polyadenylation signal GUAC [U]7. Transcription of the a-, b-, and g-coding regions is more complex. The a-coding region is translated as an a1-a2-a3 polycistronic mRNA that initiates at UUGUCC but terminates at the variant polyadenylation signal GUUC [U]7. This variant signal appears to cause incomplete termination and partial readthrough of a longer tri-cistronic a-b-g mRNA. The b gene is also immediately followed by a variant polyadenylation signal (GUAC [U]6). However, the truncated (U6) palindrome does not allow transcription termination, and reinitiation does not occur at a UUGUCC sequence located immediately in advance of the g gene. As a result, the b- and g-coding regions are transcribed as a bi-cistronic mRNA that initiates at UUGUCC upstream of the b ORF and terminates at the functional GUAC [U]7
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polyadenylation signal downstream of the g ORF. This polyadenylation signal overlaps the L gene initiation sequence by 21 nt, requiring an upstream repositioning of the polymerase to commence L gene transcription. A similar arrangement for L gene transcription has been observed for several other nonsegmented () RNA viruses.
ARV Genome Organization and Transcription The genome organization and transcription strategy of ARV are similar to those of BEFV. However, there are subtle differences that reveal aspects of genome evolution and the control of gene expression in ephemeroviruses. The ARV genome includes the five structural protein genes that are common to all rhabdoviruses (N, P, M, G, and L). Each of the proteins encoded in these genes is similar in size and shares a high level of sequence identity with the corresponding BEFV proteins. Like BEFV, the ARV P gene also contains an alternative ORF that encodes a basic protein of similar size (7.4 kDa) to the BEFV C protein. The ARV 30 leader sequence (49 nt) is similar in size to the BEFV leader RNA and shares a high level of sequence identity (21/22 nt) in the U-rich terminal domain. The ARV 50 trailer sequence (47 nt) is shorter than the BEFV trailer RNA (70 nt), primarily due to the absence of a 26 nt direct repeat of the BEFV leader sequence that occurs in the BEFV trailer RNA. The function (if any) of this direct repeat is not known. The A-rich 50 terminal region of the ARV trailer RNA shares only moderate sequence identity (15/21 nt) with the BEFV trailer, and is partially complementary (18/21 nt) to the U-rich ARV 30 leader. This complementarity reflects the specificity of interaction of the polymerase with both the () RNA genome and (þ) RNA antigenome during replication. Like BEFV, the ARV genome encodes a second, class 1 transmembrane glycoprotein (GNS) immediately downstream of the G gene. ARV GNS is also nonstructural and shares significant amino acid sequence identity with the BEFV GNS protein, as well as the G proteins of BEFV and other animal rhabdoviruses. ARV GNS has eight potential N-glycosylation sites, four of which appear to align with sites in BEFV GNS. There is a high level of preservation of cysteine and proline residues with BEFV GNS, suggesting a similar folded secondary structure. The ARV and BEFV GNS glycoproteins also appear to have preserved a core of cysteine residues conserved in rhabdovirus G proteins and is crucial for maintaining a fundamentally similar secondary structure. A proline-rich motif that, in VSV, forms a crucial ‘P’ helix in the membrane fusion domain, is also present in the BEFV and ARV G proteins. The ‘P’ helix motif is also present in BEFV GNS but it is absent from the ARV GNS protein. However, unlike the BEFV G protein, BEFV GNS does not induce cell fusion
when expressed in insect cells from a recombinant baculovirus vector. The biological significance of these observations will not be clear until further studies are conducted to better define the functions of ephemerovirus GNS proteins. Between the GNS and L genes, ARV also contains a complex region encoding several proteins of uncertain function. The genes in this region are arranged in the order –GNS-a1/a2-b-L-t-50 . The a1 ORF encodes a membrane-spanning, nonstructural protein with a highly basic C-terminal domain which is similar to the BEFV a1 protein and may well also function as a viroporin. The ARV a2 and b ORFs encode proteins similar in size to the corresponding BEFV proteins but, although the overall sequence similarity is relatively high, there is no significant sequence identity. A 17 kDa protein reported in purified ARV virions is similar in size to that predicted for the b protein. The a3 and g ORFs are not present in the ARV genome. The ARV transcription strategy is somewhat different from that of BEFV. Only the N-gene, L-gene, and b-gene are transcribed solely as monocistronic mRNAs. For the N-gene and L-gene, transcription initiates and terminates at standard UUGUCC and GUAC [U]7 signals flanking each gene. Transcription of the b-gene initiates at the variant UUGUCU sequence and terminates at GUAC [U]7. The P-gene and the M-gene are transcribed both as high abundance monocistronic mRNAs and a lower abundance (approximately 10%) bicistronic P/M mRNA. Each initiates at UUGUCC and the M-gene terminates at GUAC [U]7. However, the P-gene terminates at the leaky variant signal GCAC [U]7. The G-, GNS-, and a-genes are transcribed primarily as a long polycistronic mRNA that initiates at UUGUCG upstream of the G-gene and terminates at GUAC [U]7 following the a-gene. Corrupted termination/polyadenylation signals following the G-gene (GUAC [U]4C [U]2) and the GNS-gene (GUGC [U]2C [U]4) appear to allow a very low level of termination and transcription initiation at UUGUCC signals immediately preceding both the GNS- and a-genes. As for the BEFV gL junction, there is an overlap (22 nt) of the b-L gene junction in ARV, highlighting the importance of polymerase repositioning in the control of L gene expression. There is also a high level of nucleotide sequence identity between the a-b and b-g gene junctions in BEFV, and between the ARV b-L gene junction and the BEFV gL gene junction. This suggests that the BEFV g-gene may have evolved as a consequence of b-gene duplication. It appears, therefore, that gene duplication may have an important role in ephemerovirus evolution.
Antigenic Variation As defined either by cross-protection experiments in cattle, or by cross-neutralization tests in mice, or in cell
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cultures, BEFV exists as a single serotype globally. The relative antigenic stability of BEFV is most likely due to the occurrence of viremia and vector-borne transmission several days prior to the appearance of significant levels of virus-neutralizing antibodies. The BEFV virion G protein is the target of neutralizing antibody and four major neutralization sites have been identified and mapped to the amino acid sequence. Antigenic site G1 is a linear site that maps as two minimal B-cell epitopes at each end of the sequence spanning amino acids Y487 to K503 in the ‘stem’ domain of the G protein. This domain appears to be a unique feature of ephemeroviruses. Antigenic site G2 is conformational. It is located adjacent to two cysteine residues (C172 and C182) that appear to form a disulfide bridge linking a tight glycosylated loop structure in the folded G protein. Site G3 is the major conformational site comprising two partially overlapping elements (G3a and G3b). The site encompasses three different domains of the cysteine-rich ‘head’ structure of the folded G protein spanning Q49 to D57, K215 to E229, and Q265. Similarly complex antigenic sites map to corresponding regions of other animal rhabdoviruses, again supporting the view that essential elements of G protein secondary structure are preserved. Site G4 is a linear site. It has not yet been mapped to the G-protein sequence but it is known to be conserved in BRMV and KIMV which are also neutralized by site G4 monoclonal antibodies. (Amino acid residues are numbered here to include the N-terminal
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signal peptide that is cleaved during maturation of the G protein; Figure 4). Limited natural antigenic variation has been reported between BEFV isolates. Variations in sites G3a and G3b have been identified among 70 Australian BEFV isolates collected from diverse locations between 1956 and 1992. There appears to be a temporal basis for the shift in site G3a which is present in most strains isolated after 1973. Comparison of prototype Australian and Chinese BEFV isolates has also indicated variations in site G3a. In Taiwan, variations have been reported in sites G1 and G3. The pattern of amino acid substitutions indicates that the isolates cluster into those which included the 1984 Taiwanese vaccine strain (Tn73) and those which were isolated after 1986. It is possible that incomplete protection provided by available BEF vaccines is contributing to antigenic instability in the G protein.
Immune Response and Vaccination Natural BEFV infection induces a strong neutralizing antibody response and apparently durable immunity. Following experimental infection, neutralizing IgG antibody appears 4–5 days after the onset of clinical signs and peaks within 1–4 weeks. Although there are some reports that cattle with high levels of neutralizing antibody can be susceptible to experimental challenge, other evidence
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Figure 4 Schematic illustration of the structure of the BEFV G protein showing the locations of the major neutralization sites (G1, G2, and G3) and the predicted fusion domain including the highly conserved poly-proline helix (PPYYPP). Amino acids known to be located in the major antigenic sites are indicated as shaded circles. Disulfide bridges are assigned according to previous predictions from sequence alignments with other rhabdoviruses and from the known crystallographic structure of the low-pH form of the VSV G protein. Amino acids are numbered from the first residue of the translated protein, prior to removal of the N-terminal signal peptide.
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suggests a good correlation between protection and neutralizing antibody. Colostral antibody has also been shown to protect calves against experimental challenge. High levels of cytokines circulate during the acute phase of infection but little is known of the role of innate or adaptive cell-mediated immunity in recovery from infection or protection against natural or experimental challenge. Several forms of live-attenuated, inactivated, subunit, and recombinant BEFV vaccines have been reported and vaccines of varying format are produced for commercial use. Live-attenuated vaccines have been produced in mice and in cell cultures. In general, live vaccines are relatively effective in inducing protection but require at least two doses in adjuvant to generate durable immunity. Inactivated vaccines have been produced by treatment of BEFV with formalin or b-propiolactone, but have generally poor efficacy. Consecutive vaccinations with live-attenuated and killed preparations have also been used with some success. A purified G-protein subunit vaccine delivered in Quil A adjuvant has been shown to provide reliable protection following a two-dose treatment at an interval of 21 days. Recombinant BEFV vaccines employing the BEFV G protein delivered in vaccinia and capripox viral vectors have also been trialed. See also: Animal Rhabdoviruses; Chandipura Virus; Fish Rhabdoviruses; Rabies Virus; Vesicular Stomatitis Virus.
Further Reading Inaba Y, Kurogi H, Takahashi A, et al. (1974) Vaccination of cattle against bovine ephemeral fever with live attenuated virus followed by killed virus. Archiv fur die Gesamte Virusforschung 44: 121–132. Kirkland PD (2002) Akabane and bovine ephemeral fever virus infections. Veterinary Clinics of North America: Food Animal Practice 18: 501–514.
Kongsuwan K, Cybinski DH, Cooper J, and Walker PJ (1998) Location of neutralizing epitopes on the G protein of bovine ephemeral fever rhabdovirus. Journal of General Virology 79: 2573–2578. Kuzmin IV, Hughes GJ, and Rupprecht CE (2006) Phylogenetic relationships of seven previously unclassified viruses within the family Rhabdoviridae using partial nucleoprotein gene sequences. Journal of General Virology 87: 2323–2331. McWilliam SM, Kongsuwan K, Cowley KA, Byrne KA, and Walker PJ (1997) Genome organization and transcription strategy in the complex GNS-L intergenic region of bovine ephemeral fever rhabdovirus. Journal of General Virology 78: 1309–1317. Nandi S and Negi BS (1999) Bovine ephemeral fever: A review. Comparative Immunology, Microbiology, and Infectious Diseases 22: 81–91. St. George TD (1990) Bovine ephemeral fever virus. In: Dinter Z and Morein B (eds.) Virus Infections of Vertebrates, Vol. 3: Virus Infections of Ruminants, pp. 405–415. Amsterdam: Elsevier. Theodoridis A, Giesecke WH, and Du Toit IJ (1973) Effects of ephemeral fever on milk production and reproduction of dairy cattle. The Onderstepoort Journal of Veterinary Research 40: 83–92. Tomori O, Fagbami A, and Kemp G (1974) Kotonkan virus: Experimental infection of Fulani calves. Bulletin of Epizootic Diseases of Africa 22: 195–200. Tordo N, Benmansour A, Calisher C, et al. (2005) Rhabdoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, and Ball LA (eds.) Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 623–644. San Diego, CA: Elsevier Academic Press. Uren MF, St. George TD, and Zakrzewski H (1989) The effect of antiinflammatory agents on the clinical expression of bovine ephemeral fever. Veterinary Microbiology 19: 99–111. Venter GJ, Hamblin C, and Paweska JT (2003) Determination of the oral susceptibility of South African livestock-associated biting midges, Culicoides species, to bovine ephemeral fever virus. Medical and Veterinary Entomology 17: 133–137. Walker PJ (2005) Bovine ephemeral fever in Australia and the World. In: Fu Z and Kaprowski H (eds.) The World of Rhabdoviruses. Current Topics in Microbiology and Immunology, vol. 292, pp. 57–80. Berlin: Springer. Walker PJ, Byrne KA, Riding GA, et al. (1992) The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes. Virology 191: 49–61. Wang YH, McWilliam SM, Cowley JA, and Walker PJ (1994) Complex genome organization in the GNS-L intergenic region of Adelaide River rhabdovirus. Virology 203: 63–72.
Bovine Herpesviruses M J Studdert, The University of Melbourne, Parkville, VIC, Australia ã 2008 Elsevier Ltd. All rights reserved.
History Although some of the clinical diseases caused by herpesviruses in members of the family Bovidae have been recognized for centuries, it was not until the first and probably most important alphaherpesvirus now called bovine herpesvirus 1 (BHV1) was isolated in the late 1950s from the genital disease coital exanthema (also called infectious pustular vulvovaginitis (IPV) in the female) and from the respiratory disease infectious bovine
rhinotracheitis (IBR) that any of these diseases was confirmed to be caused by a herpesvirus. Historically, IPV and its male counterpart infectious pustular balanoposthitis (collectively the male and female diseases are termed coital exanthema or blaschenausschlag) were commonly described diseases in central Europe throughout the nineteenth century. It was common for a single bull in a village to serve all the female cattle in that village and, where distances were small, also in nearby villages, and blaschenausschlag was a frequently observed sequel to mating. The isolation
Bovine and Feline Immunodeficiency Viruses
vaccinated cats from FIV-infected cats has caused a dilemma in the use of this vaccine. This problem can be resolved by developing sensitive molecular diagnostics or a vaccine that does not conflict with current FIV diagnostics. Identifying the protective vaccine epitopes should assist in designing a vaccine that is devoid of diagnostic epitopes. Moreover, FIV research on vaccines will provide new insights to HIV vaccine development for humans. The recent discovery of fatal pathogenic FIV strains (10% acute mortality) demonstrates the pathogenic evolution of FIV similar to HIV-1 immunopathogenesis. Hence, FIV infection is not only important for feline medicine but serves as an important small animal model for testing novel antiretroviral drugs, immunotherapy, and vaccine approaches for HIV/AIDS.
See also: Equine Infectious Anemia Virus; Human Immunodeficiency Viruses: Antiretroviral agents; Human Immunodeficiency Viruses: Molecular Biology; Human Immunodeficiency Viruses: Origin; Human Immunodeficiency Viruses: Pathogenesis; Simian Immunodeficiency Virus: Animal Models of Disease; Simian Immunodeficiency Virus: General Features; Simian Immunodeficiency Virus: Natural Infection; Visna-Maedi Viruses.
Further Reading Bachmann MH, Mathiason-Dubard C, Learn GH, et al. (1997) Genetic diversity of feline immunodeficiency virus: Dual infection, recombination, and distinct evolutionary rates among envelope sequence clades. Journal of Virology 71: 4241–4253. Burkhard MJ and Dean GA (2003) Transmission and immunopathogenesis of FIV in cats as a model for HIV. Current HIV Research 1: 15–29. Evermann JE, Howard TH, Dubovi EJ, et al. (2000) Controversies and clarifications regarding bovine lentivirus infections. Journal of the American Veterinary Medical Association 217: 1318–1324. Podell M, March PA, Buck WR, and Mathes LE (2000) The feline model of neuroAIDS: Understanding the progression towards AIDS dementia. Journal of Psychopharmacology 14: 205–213. Snider TG, Hoyt PG, Jenny BF, et al. (1997) Natural and experimental bovine immunodeficiency virus infection in cattle. The Veterinary Clinics of North America. Food Animal Practice 13: 151–176. St-Louis M-C, Cojocariu M, and Archambault D (2004) The molecular biology of bovine immunodeficiency virus: A comparison with other lentiviruses. Animal Health Research Reviews 5: 125–143. Troyer JL, Pecon-Slattery J, Roelke ME, et al. (2005) Seroprevalence and genomic divergence of circulating strains of feline immunodeficiency virus among Felidae and Hyaenidae species. Journal of Virology 79: 8282–8294. Uhl EW, Heaton-Jones TG, Pu R, and Yamamoto JK (2002) FIV vaccine development and its importance to veterinary and human medicine: A review. Veterinary. Immunology and Immunopathology 90: 113–132. Wilcox GE, Chadwick BJ, and Kertayadnya G (1995) Recent advances in the understanding of Jembrana disease. Veterinary Microbiology 46: 249–255.
Bovine Ephemeral Fever Virus P J Walker, CSIRO Livestock Industries, Geelong, VIC, Australia ã 2008 Elsevier Ltd. All rights reserved.
Glossary Epicardium The outer layer of heart tissue. Hypocalcemia A low level of calcium in the circulating blood. Leucopenia A decreased total number of white blood cells in the circulating blood. Pericardial fluid Fluid within a double-walled sac that contains the heart and the roots of the great blood vessels. Sternal recumbency Reclined in a position of comfort on the chest bone. Synovial membranes Connective tissue membranes lining the cavities of the freely movable joints. Thoracic fluid Fluid in the chest cavity.
Introduction Bovine ephemeral fever virus (BEFV) is an arthropodborne rhabdovirus that causes a disabling and sometimes lethal disease of cattle (Bos taurus, Bos indicus, and Bos javanicus) and water buffaloes (Bubalus bubalis). Unapparent infections can also occur in cape buffalo, hartebeest, waterbuck, wildebeest, deer, and possibly goats. Bovine ephemeral fever (BEF) was first recorded in East Africa and Egypt during the late nineteenth century. However, BEF (which is also variously called three-day sickness, bovine enzootic fever, bovine influenza, and stiffsiekte) is thought to have been endemic since antiquity in much of tropical and subtropical Africa, and Asia. As the name suggests, BEF is often characterized by the rapid onset of and recovery from clinical signs that can include a bi- or multiphasic fever, ocular and nasal discharge,
Bovine Ephemeral Fever Virus
muscle stiffness, anorexia, rumenal stasis, lameness, and sternal recumbency. Although mortality rates rarely exceed 1–2%, a particularly severe outbreak in Taiwan in 1996 has been reported to have resulted in the death or culling of 11.3% of a population of 110 247 dairy cattle on 516 farms. Severe infections commonly occur in larger, more valuable animals. Morbidity rates may approach 100% with significant economic impacts including loss of milk production, temporary infertility in bulls, abortion, loss of condition in beef herds, and disablement of draft animals at the time of harvest. The economic consequences of BEF can be significant. An outbreak of BEF in Israel in 1999 has been estimated to have cost, on average $280 per lactating cow and $112 per nonlactating cow. In Australia, sweeping epidemics in the 1970s have been estimated to have caused industrywide losses exceeding $200 million in today’s values. Due to limitations on the importation of livestock and semen from animals with evidence of BEFV infection, the disease can also have significant impact on international trade.
Taxonomic Classification BEFV is classified in the order Mononegavirales, family Rhabdoviridae as the type species of the genus Ephemerovirus. Ephemeroviruses have morphological and genetic characteristics common to all rhabdoviruses, including an enveloped, bullet-shaped virion containing a nonsegmented, negative-sense, single-stranded (ss) RNA genome. However, unlike viruses classified in other established rhabdovirus taxa, ephemeroviruses share the unusual characteristic of two type 1 transmembrane glycoproteins (G and GNS) that are related in amino acid sequence and appear to have arisen by gene duplication. Other recognized species in the genus include Berrimah virus (BRMV) and Adelaide River virus (ARV). These viruses have each been isolated from cattle in northern Australia and also appear to be transmitted by biting insects. Tentative species in the genus include Kimberley virus (KIMV), Malakal virus (MALV), and Puchong virus (PUCV), each of which is closely related serologically to BEFV. KIMV has been isolated from Culex annulirostris mosquitoes and Culicoides brevitarsis midges in Australia, and from healthy cattle. MALV was isolated in Sudan and PUCV in Malaysia, each from Mansonia uniformis mosquitoes. Potential vertebrate hosts for these viruses are yet to be identified. Although BEFV is the only ephemerovirus known to be associated with disease, Kotonkan virus (KOTV), which was originally isolated from Culicoides spp. in Nigeria, does cause an ephemeral fever-like illness in cattle. Recent phylogenetic studies using the N gene and segments of the L gene suggest that KOTV, as well as Obodhiang virus (OBOV) which was isolated from
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Mansonia uniformis mosquitoes in Sudan, should also be classified as ephemeroviruses. It has also been suggested that all arthropod-borne rhabdoviruses, including the ephemeroviruses, vesiculoviruses, and a large number of other unclassified rhabdoviruses share a common ancestor and should form a larger taxonomic group for which the name ‘dimarhabdovirus’ (dipteran-mammalian rhabdovirus) has been proposed (Figure 1).
Geographic Distribution BEFV is known to occur in most tropical and subtropical regions of Africa, Asia, and Australia. The disease occurs throughout much of Africa and in Asian countries south of a line that includes Israel, Iraq, Iran, Syria, India, Pakistan, Bangladesh, southern and central China, and east to Taiwan, and southern Japan. It occurs throughout Southeast Asia, parts of New Guinea, and in most of northern and eastern Australia. It does not occur in New Zealand or the islands of the Pacific, or in the Americas, or Europe, where it is considered an important exotic pathogen. There is reported serological evidence of infection in southern Russia but the disease has not been described. The distribution of BEFV is determined by the geographic range of available insect vectors and is limited by international trade restrictions on live animals and semen showing evidence of infection.
Epizootiology Bovine ephemeral fever is a seasonal disease. It occurs principally in the summer and early autumn, and with the onset of the monsoon season in Asia. Outbreaks are usually associated with periods of high rainfall which precipitate the emergence of insect vectors in large numbers. BEFV can also spread in epizootics that follow the pattern of prevailing winds with a general southward movement in the Southern Hemisphere and northward movement above the Equator. Wind-borne movement of insect vectors is the likely mechanism of spread. Vectors include biting midges (Culicoides spp.) and mosquitoes, in which the virus replicates. BEFV has been isolated from Culicoides brevitarsis, Culicoides coarctatus, Anopheles bancroftii, a mixed pool of mosquito species in Australia, and from a mixed pool of biting midges in Africa. The virus has also been recovered from several other species of biting midge and mosquito following experimental infection. The abundance and distribution of insects from which BEFV has been isolated suggests that several major vectors may be involved in transmission. There is no evidence of direct transmission of BEFV between cattle, even when encouraged by smearing nasal or ocular discharges on mucosal surfaces.
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Sandjimba
Kolongo
Sigma
Lyssavirus Tupaia
Vesiculovirus
Rabies Piry Lagos bat 996
Isfahan
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Chandipura 721 904
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Mokola
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VSV Indiana
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800 Flanders
Oita
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Mount Elgon bat Kern canyon
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Figure 1 Phylogenetic tree of partial N gene sequences of 20 rhabdoviruses infecting mammals and/or insects. The tree was generated from a Clustal X alignment of the sequences by the neighbor-joining method and presented graphically using Treeview software. The viruses include representatives of three rhabdovirus genera: Ephemerovirus (BEFV, ARV, Obodhiang virus, and Kotonkau virus); Vesiculovirus (VSV Indiana, Chandipura, Isfahan, and Piry viruses); and Lyssavirus (rabies, Mokola, and Lagos bat viruses). Unclassified rhabdoviruses include Sandjimba, Rochambeau, Flanders, Kern Canyon, Mount Elgon bat, Oita, Tupaia and Kolongo, and sigma viruses. Confidence in branch nodes was determined by bootstrap analysis on 1000 replicates (indicated as numbers).
Pathology and Pathogenesis Bovine ephemeral fever is principally an inflammatory disease. The incubation period is normally 2–4 days. Viremia usually persists for 1–3 days and peaks approximately 24 h before the onset of fever. The initial sites of infection are not known but the virus has been isolated from neutrophils and reticuloendothelial cells of the lungs, spleen, and lymph nodes. There is also evidence of infection in synovial membranes, epicardium and aorta, and in cells derived from synovial, pericardial, thoracic, and abdominal fluids. There is not widespread tissue damage. The primary lesion is a vasculitis affecting the endothelium of small vessels of synovial membranes, tendon sheaths, muscles, facia, and skin. The onset of fever and other clinical signs is accompanied by marked leucopenia, relative neutrophilia, elevated plasma fibrinogen,
and elevated levels of cytokines including interferon a, interleukin 1, and tissue necrosis factor. There is also a significant hypocalcemia that is thought to be responsible for sternal recumbency. The major clinical signs can be treated very effectively with anti-inflammatory drugs.
Virion Structure and Morphogenesis BEFV virions are enveloped, bullet-shaped particles (approximately 70 180 nm) containing a precisely coiled, helical nucleocapsid with 35 cross-striations at an interval of 4.8 nm. Virions have a prominent axial channel intruding from the base and typically are more cone-shaped than commonly observed for viruses in other genera of animal rhabdovirus (e.g., lyssaviruses and vesiculoviruses). The envelope contains a single 81 kDa class
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1 transmembrane glycoprotein (G) that forms visible projections on the virion surface. The G protein mediates cell attachment and entry, is the target for virus-neutralizing antibodies, and induces protective antibodies in cattle. Nucleocapsids contain a negative-sense ssRNA genome, a 52 kDa nucleoprotein (N) which is tightly bound to the genome, a large 250 kDa replicase protein (L), and a highly charged 43 kDa replicase cofactor (P). Virions also contain a 29 kDa matrix protein (M) which is a major structural component and appears to lie between nucleocapsids and the inner surface of the lipid envelope (Figure 2). Viral replication is cytoplasmic and morphogenesis occurs primarily at the plasma membrane in association with accumulations of a filamentous, granular, intracytoplasmic matrix. However, late in infection, there is a proliferation of plasma membrane, cells become highly vacuolated, and virions are observed both at the plasma membrane and within cytoplasmic vacuoles. Following budding as cone-shaped extrusions, virions accumulate in extracellular spaces. The general characteristics of viral morphogenesis appear to be similar in infected mammalian cell cultures and mouse neurons.
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BEFV Genome Organization and the Encoded Proteins The 14 900 nt BEFV genome is the largest known for any rhabdovirus and one of the largest and most complex genomes for any nonsegmented, negative-sense RNA virus. The genome comprises 12 genes, flanked by terminal, partially complementary leader (l ) and trailer (t) sequences, and arranged in the order 30 -l-N-P/C-M-GGNS-a1/a2/a3-b-g-L-t-50 . By analogy with other rhabdoviruses, the 30 leader (50 nt) and 50 trailer (70 nt) sequences are likely to have important roles in the initiation and control of replication and transcription, and in nucleoprotein assembly and packaging. The structural protein genes (N, P, M, G, and L) are arranged in the same order as for all other known rhabdoviruses and encode proteins with similar functional characteristics (Figure 3). The N Protein The BEFV N gene encodes the 431 amino acid nucleoprotein (N). The N protein is a highly hydrophilic, RNA-binding protein containing 14.4% basic residues
(a) M
P
L
RNA
G
N
Axial channel
(b) Figure 2 (a) Negative-contrast electron micrograph of a purified BEFV virion. (b) Schematic representation of the BEFV virion. Structural proteins N, P, M, G, and L, and the negative-sense ssRNA genome are indicated. The axial channel is also depicted. The size and relative quantities of the proteins do not accurately reflect the content in virions. Scale ¼ 50 nm (a).
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0
1
2
3
4
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9
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α3
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70 nt trailer RNA
mRNAs 3⬘ ARV
5⬘ N
ORFs
P
M
G
GNS
α1α2 β
L
C
49 nt leader RNA
47 nt trailer RNA
Figure 3 Illustration of the genome organization and transcription strategy of BEFV and ARV. Solid arrows indicate the major transcriptional products. Minor transcripts are indicated by dotted arrows. The length of the ARV L protein is estimated from the alignment of available sequences with those of BEFV.
(lysine, arginine, and histidine) distributed relatively evenly throughout the molecule. Seven of these residues are highly conserved in rhabdoviruses and are involved in RNA-binding and stabilizing the interaction. The N protein also contains 14.6% acidic residues (glutamate and aspartate) that are less evenly distributed with significant clustering in a short domain near the C terminus. A similar acidic domain in the rabies N protein is a phosphorylation site involved in binding P protein to nucleocapsids. The BEFV N protein is also phosphorylated when packaged in virions and contains a similar phosphorylation site in this acidic domain. The P Protein The P gene encodes the 278 amino acid, highly hydrophilic P protein. It corresponds to the polymerase-associated phosphoproteins of rabies virus (RV) and vesicular stomatitis virus (VSV) which are components of nucleocapsids and act as essential cofactors to the L protein during transcription and replication. The BEFV P protein has not been observed to be phosphorylated when extracted from virions but is phosphorylated when expressed from a recombinant baculovirus in insect cells. The BEFV P gene also contains an alternative open reading frame (ORF) encoding a 48 amino acid, highly basic 5.8 kDa polypeptide. This protein has not been detected in BEFV-infected cells and it is not known if it is expressed. However, alternative ORFs in the
P gene occur in vesiculoviruses and are a common feature of many viruses in the Mononegavirales. As in VSV, the BEFV C protein has two potential initiation codons, suggesting it could be expressed in two different forms. The M Protein The M gene encodes the 691 amino acid, basic, hydrophilic protein that corresponds to the matrix protein (M) of rabies and VSV. The M protein is a major component of rhabdovirus virions and has been shown to have important functions in regulation of viral replication and transcription, inhibition of host cell protein synthesis and induction of apoptosis, and in budding of nucleocapsids at cytoplasmic membranes. The BEFV M protein has been shown to be phosphorylated when extracted from virions but not when expressed from a recombinant baculovirus in insect cells. This is consistent with observations that phosphorylation of the VSV M protein occurs at a late stage in viral assembly. The BEFV M protein also contains a ‘late domain’ sequence motif (PPSY) which, in VSV and several other RNA viruses, is essential for efficient budding from infected cells. The G Protein The BEFV G gene encodes the 623-amino-acid virion transmembrane glycoprotein (G). The G protein is a
Bovine Ephemeral Fever Virus
class 1 membrane protein. It shares significant amino acid sequence identity with other animal rhabdovirus G proteins and contains a core of conserved cysteine residues suggesting preservation of fundamentally similar secondary structure. The G protein contains five potential N glycosylation sites, three of which appear to align with similar sites in VSV and/or rabies virus. The G protein is responsible for cell docking and entry, and is the target of virus-neutralizing antibodies for which the major binding sites have been defined (see below). The L Protein The L gene encodes the 2144-amino-acid RNA-dependent RNA polymerase (L protein). The L protein is a structural component of nucleocapsids that, in cooperation with the N and P proteins, forms the ribonucleoprotein (RNP) complex that is responsible for replication and transcription of the viral genome. The BEFV L protein shares a high level of sequence similarity with other rhabdovirus L proteins and contains all of the conserved sequence motifs associated with the major functional domains; these include the polymerase catalytic site and other regions involved in replication, transcription, initiation, elongation, and termination, 30 polyadenylation, 50 capping, and cap methylation.
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The Small Nonstructural Proteins Downstream of the GNS gene is a complex region of the genome-encoding proteins that appear to be unique to BEFV and other ephemeroviruses. None of these proteins has yet been detected in virions or BEFV-infected cells. The a-gene coding region contains three long ORFs (a1, a2, and a3). The a1 ORF encodes an 88 amino acid, 10.6 kDa protein. It features a central transmembrane domain comprising 16 hydrophobic amino acids bounded by arginine residues, and a highly basic C-terminal domain in which 12 of 18 amino acids are lysine or arginine residues. This structure suggests a1 may function as a viroporin, a class of proteins that causes cytopathic effects by increasing membrane permeability. The BEFV a1 protein has been shown to be cytotoxic when expressed in insect cells from a recombinant baculovirus vector. The a2 ORF encodes a 116 amino acid, 13.7 kDa polypeptide. It overlaps the a3 ORF which encodes a 51 amino acid, 5.7 kDa polypeptide that contains an unusual triple repeat of isomers of the sequence KLMEE at intervals of four residues. The b-gene encodes a 107 amino acid, 12.3 kDa polypeptide. The g-gene encodes a 114 amino acid, 13.5 kDa polypeptide. The a2, a3, b, and g products share no sequence homology with known viral proteins (other than ARV, see below) and their functions are yet to be determined.
The GNS Protein
BEFV Transcription
The BEFV nonstructural protein genes are located in a 3442 nt region of the genome between the G gene and L gene. Immediately downstream of the G gene is a second gene encoding a class 1 transmembrane glycoprotein (GNS). The 90 kDa GNS protein is abundant in infected cells but has not been detected in virions. It is related in structure and sequence to the BEFV virion G protein and other rhabdovirus G proteins and the evidence suggests that it has arisen by gene duplication. The GNS protein contains eight potential N-glycosylation sites and, as the size by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) is approximately 21 kDa in excess of the calculated molecular weight of the unmodified polypeptide, it appears to be highly glyscosylated. Sequence alignments indicate that it shares 10 of 12 cysteine residues that appear to form the core of secondary structure common to all animal rhabdoviruses. However, the GNS protein does not share antigenic sites with the BEFV G protein and antibody to GNS does not neutralize the infectivity of BEFV produced in either mammalian or insect cells. It has been shown to accumulate at the cell surface in association with amorphous structures but not with budding or mature virions. The function of the GNS is currently unknown.
The RNP complex, comprising the RNA genome and the N, L, and P proteins, is the active replication and transcription unit of the virus. As for other rhabdoviruses, BEFV transcription from the () RNA genome generates 50 methylated, capped, and polyadenylated mRNAs by a progressive mechanism that initiates and terminates at short, conserved sequences flanking each gene. For each of the structural protein genes (N, P, N, G, and L), and the nonstructural glycoprotein gene (GNS), transcription initiates at the sequence UUGUCC and terminates at the polyadenylation signal GUAC [U]7. Transcription of the a-, b-, and g-coding regions is more complex. The a-coding region is translated as an a1-a2-a3 polycistronic mRNA that initiates at UUGUCC but terminates at the variant polyadenylation signal GUUC [U]7. This variant signal appears to cause incomplete termination and partial readthrough of a longer tri-cistronic a-b-g mRNA. The b gene is also immediately followed by a variant polyadenylation signal (GUAC [U]6). However, the truncated (U6) palindrome does not allow transcription termination, and reinitiation does not occur at a UUGUCC sequence located immediately in advance of the g gene. As a result, the b- and g-coding regions are transcribed as a bi-cistronic mRNA that initiates at UUGUCC upstream of the b ORF and terminates at the functional GUAC [U]7
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polyadenylation signal downstream of the g ORF. This polyadenylation signal overlaps the L gene initiation sequence by 21 nt, requiring an upstream repositioning of the polymerase to commence L gene transcription. A similar arrangement for L gene transcription has been observed for several other nonsegmented () RNA viruses.
ARV Genome Organization and Transcription The genome organization and transcription strategy of ARV are similar to those of BEFV. However, there are subtle differences that reveal aspects of genome evolution and the control of gene expression in ephemeroviruses. The ARV genome includes the five structural protein genes that are common to all rhabdoviruses (N, P, M, G, and L). Each of the proteins encoded in these genes is similar in size and shares a high level of sequence identity with the corresponding BEFV proteins. Like BEFV, the ARV P gene also contains an alternative ORF that encodes a basic protein of similar size (7.4 kDa) to the BEFV C protein. The ARV 30 leader sequence (49 nt) is similar in size to the BEFV leader RNA and shares a high level of sequence identity (21/22 nt) in the U-rich terminal domain. The ARV 50 trailer sequence (47 nt) is shorter than the BEFV trailer RNA (70 nt), primarily due to the absence of a 26 nt direct repeat of the BEFV leader sequence that occurs in the BEFV trailer RNA. The function (if any) of this direct repeat is not known. The A-rich 50 terminal region of the ARV trailer RNA shares only moderate sequence identity (15/21 nt) with the BEFV trailer, and is partially complementary (18/21 nt) to the U-rich ARV 30 leader. This complementarity reflects the specificity of interaction of the polymerase with both the () RNA genome and (þ) RNA antigenome during replication. Like BEFV, the ARV genome encodes a second, class 1 transmembrane glycoprotein (GNS) immediately downstream of the G gene. ARV GNS is also nonstructural and shares significant amino acid sequence identity with the BEFV GNS protein, as well as the G proteins of BEFV and other animal rhabdoviruses. ARV GNS has eight potential N-glycosylation sites, four of which appear to align with sites in BEFV GNS. There is a high level of preservation of cysteine and proline residues with BEFV GNS, suggesting a similar folded secondary structure. The ARV and BEFV GNS glycoproteins also appear to have preserved a core of cysteine residues conserved in rhabdovirus G proteins and is crucial for maintaining a fundamentally similar secondary structure. A proline-rich motif that, in VSV, forms a crucial ‘P’ helix in the membrane fusion domain, is also present in the BEFV and ARV G proteins. The ‘P’ helix motif is also present in BEFV GNS but it is absent from the ARV GNS protein. However, unlike the BEFV G protein, BEFV GNS does not induce cell fusion
when expressed in insect cells from a recombinant baculovirus vector. The biological significance of these observations will not be clear until further studies are conducted to better define the functions of ephemerovirus GNS proteins. Between the GNS and L genes, ARV also contains a complex region encoding several proteins of uncertain function. The genes in this region are arranged in the order –GNS-a1/a2-b-L-t-50 . The a1 ORF encodes a membrane-spanning, nonstructural protein with a highly basic C-terminal domain which is similar to the BEFV a1 protein and may well also function as a viroporin. The ARV a2 and b ORFs encode proteins similar in size to the corresponding BEFV proteins but, although the overall sequence similarity is relatively high, there is no significant sequence identity. A 17 kDa protein reported in purified ARV virions is similar in size to that predicted for the b protein. The a3 and g ORFs are not present in the ARV genome. The ARV transcription strategy is somewhat different from that of BEFV. Only the N-gene, L-gene, and b-gene are transcribed solely as monocistronic mRNAs. For the N-gene and L-gene, transcription initiates and terminates at standard UUGUCC and GUAC [U]7 signals flanking each gene. Transcription of the b-gene initiates at the variant UUGUCU sequence and terminates at GUAC [U]7. The P-gene and the M-gene are transcribed both as high abundance monocistronic mRNAs and a lower abundance (approximately 10%) bicistronic P/M mRNA. Each initiates at UUGUCC and the M-gene terminates at GUAC [U]7. However, the P-gene terminates at the leaky variant signal GCAC [U]7. The G-, GNS-, and a-genes are transcribed primarily as a long polycistronic mRNA that initiates at UUGUCG upstream of the G-gene and terminates at GUAC [U]7 following the a-gene. Corrupted termination/polyadenylation signals following the G-gene (GUAC [U]4C [U]2) and the GNS-gene (GUGC [U]2C [U]4) appear to allow a very low level of termination and transcription initiation at UUGUCC signals immediately preceding both the GNS- and a-genes. As for the BEFV gL junction, there is an overlap (22 nt) of the b-L gene junction in ARV, highlighting the importance of polymerase repositioning in the control of L gene expression. There is also a high level of nucleotide sequence identity between the a-b and b-g gene junctions in BEFV, and between the ARV b-L gene junction and the BEFV gL gene junction. This suggests that the BEFV g-gene may have evolved as a consequence of b-gene duplication. It appears, therefore, that gene duplication may have an important role in ephemerovirus evolution.
Antigenic Variation As defined either by cross-protection experiments in cattle, or by cross-neutralization tests in mice, or in cell
Bovine Ephemeral Fever Virus
cultures, BEFV exists as a single serotype globally. The relative antigenic stability of BEFV is most likely due to the occurrence of viremia and vector-borne transmission several days prior to the appearance of significant levels of virus-neutralizing antibodies. The BEFV virion G protein is the target of neutralizing antibody and four major neutralization sites have been identified and mapped to the amino acid sequence. Antigenic site G1 is a linear site that maps as two minimal B-cell epitopes at each end of the sequence spanning amino acids Y487 to K503 in the ‘stem’ domain of the G protein. This domain appears to be a unique feature of ephemeroviruses. Antigenic site G2 is conformational. It is located adjacent to two cysteine residues (C172 and C182) that appear to form a disulfide bridge linking a tight glycosylated loop structure in the folded G protein. Site G3 is the major conformational site comprising two partially overlapping elements (G3a and G3b). The site encompasses three different domains of the cysteine-rich ‘head’ structure of the folded G protein spanning Q49 to D57, K215 to E229, and Q265. Similarly complex antigenic sites map to corresponding regions of other animal rhabdoviruses, again supporting the view that essential elements of G protein secondary structure are preserved. Site G4 is a linear site. It has not yet been mapped to the G-protein sequence but it is known to be conserved in BRMV and KIMV which are also neutralized by site G4 monoclonal antibodies. (Amino acid residues are numbered here to include the N-terminal
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signal peptide that is cleaved during maturation of the G protein; Figure 4). Limited natural antigenic variation has been reported between BEFV isolates. Variations in sites G3a and G3b have been identified among 70 Australian BEFV isolates collected from diverse locations between 1956 and 1992. There appears to be a temporal basis for the shift in site G3a which is present in most strains isolated after 1973. Comparison of prototype Australian and Chinese BEFV isolates has also indicated variations in site G3a. In Taiwan, variations have been reported in sites G1 and G3. The pattern of amino acid substitutions indicates that the isolates cluster into those which included the 1984 Taiwanese vaccine strain (Tn73) and those which were isolated after 1986. It is possible that incomplete protection provided by available BEF vaccines is contributing to antigenic instability in the G protein.
Immune Response and Vaccination Natural BEFV infection induces a strong neutralizing antibody response and apparently durable immunity. Following experimental infection, neutralizing IgG antibody appears 4–5 days after the onset of clinical signs and peaks within 1–4 weeks. Although there are some reports that cattle with high levels of neutralizing antibody can be susceptible to experimental challenge, other evidence
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CIII
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Y
P
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Figure 4 Schematic illustration of the structure of the BEFV G protein showing the locations of the major neutralization sites (G1, G2, and G3) and the predicted fusion domain including the highly conserved poly-proline helix (PPYYPP). Amino acids known to be located in the major antigenic sites are indicated as shaded circles. Disulfide bridges are assigned according to previous predictions from sequence alignments with other rhabdoviruses and from the known crystallographic structure of the low-pH form of the VSV G protein. Amino acids are numbered from the first residue of the translated protein, prior to removal of the N-terminal signal peptide.
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Bovine Ephemeral Fever Virus
suggests a good correlation between protection and neutralizing antibody. Colostral antibody has also been shown to protect calves against experimental challenge. High levels of cytokines circulate during the acute phase of infection but little is known of the role of innate or adaptive cell-mediated immunity in recovery from infection or protection against natural or experimental challenge. Several forms of live-attenuated, inactivated, subunit, and recombinant BEFV vaccines have been reported and vaccines of varying format are produced for commercial use. Live-attenuated vaccines have been produced in mice and in cell cultures. In general, live vaccines are relatively effective in inducing protection but require at least two doses in adjuvant to generate durable immunity. Inactivated vaccines have been produced by treatment of BEFV with formalin or b-propiolactone, but have generally poor efficacy. Consecutive vaccinations with live-attenuated and killed preparations have also been used with some success. A purified G-protein subunit vaccine delivered in Quil A adjuvant has been shown to provide reliable protection following a two-dose treatment at an interval of 21 days. Recombinant BEFV vaccines employing the BEFV G protein delivered in vaccinia and capripox viral vectors have also been trialed. See also: Animal Rhabdoviruses; Chandipura Virus; Fish Rhabdoviruses; Rabies Virus; Vesicular Stomatitis Virus.
Further Reading Inaba Y, Kurogi H, Takahashi A, et al. (1974) Vaccination of cattle against bovine ephemeral fever with live attenuated virus followed by killed virus. Archiv fur die Gesamte Virusforschung 44: 121–132. Kirkland PD (2002) Akabane and bovine ephemeral fever virus infections. Veterinary Clinics of North America: Food Animal Practice 18: 501–514.
Kongsuwan K, Cybinski DH, Cooper J, and Walker PJ (1998) Location of neutralizing epitopes on the G protein of bovine ephemeral fever rhabdovirus. Journal of General Virology 79: 2573–2578. Kuzmin IV, Hughes GJ, and Rupprecht CE (2006) Phylogenetic relationships of seven previously unclassified viruses within the family Rhabdoviridae using partial nucleoprotein gene sequences. Journal of General Virology 87: 2323–2331. McWilliam SM, Kongsuwan K, Cowley KA, Byrne KA, and Walker PJ (1997) Genome organization and transcription strategy in the complex GNS-L intergenic region of bovine ephemeral fever rhabdovirus. Journal of General Virology 78: 1309–1317. Nandi S and Negi BS (1999) Bovine ephemeral fever: A review. Comparative Immunology, Microbiology, and Infectious Diseases 22: 81–91. St. George TD (1990) Bovine ephemeral fever virus. In: Dinter Z and Morein B (eds.) Virus Infections of Vertebrates, Vol. 3: Virus Infections of Ruminants, pp. 405–415. Amsterdam: Elsevier. Theodoridis A, Giesecke WH, and Du Toit IJ (1973) Effects of ephemeral fever on milk production and reproduction of dairy cattle. The Onderstepoort Journal of Veterinary Research 40: 83–92. Tomori O, Fagbami A, and Kemp G (1974) Kotonkan virus: Experimental infection of Fulani calves. Bulletin of Epizootic Diseases of Africa 22: 195–200. Tordo N, Benmansour A, Calisher C, et al. (2005) Rhabdoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, and Ball LA (eds.) Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 623–644. San Diego, CA: Elsevier Academic Press. Uren MF, St. George TD, and Zakrzewski H (1989) The effect of antiinflammatory agents on the clinical expression of bovine ephemeral fever. Veterinary Microbiology 19: 99–111. Venter GJ, Hamblin C, and Paweska JT (2003) Determination of the oral susceptibility of South African livestock-associated biting midges, Culicoides species, to bovine ephemeral fever virus. Medical and Veterinary Entomology 17: 133–137. Walker PJ (2005) Bovine ephemeral fever in Australia and the World. In: Fu Z and Kaprowski H (eds.) The World of Rhabdoviruses. Current Topics in Microbiology and Immunology, vol. 292, pp. 57–80. Berlin: Springer. Walker PJ, Byrne KA, Riding GA, et al. (1992) The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes. Virology 191: 49–61. Wang YH, McWilliam SM, Cowley JA, and Walker PJ (1994) Complex genome organization in the GNS-L intergenic region of Adelaide River rhabdovirus. Virology 203: 63–72.
Bovine Herpesviruses M J Studdert, The University of Melbourne, Parkville, VIC, Australia ã 2008 Elsevier Ltd. All rights reserved.
History Although some of the clinical diseases caused by herpesviruses in members of the family Bovidae have been recognized for centuries, it was not until the first and probably most important alphaherpesvirus now called bovine herpesvirus 1 (BHV1) was isolated in the late 1950s from the genital disease coital exanthema (also called infectious pustular vulvovaginitis (IPV) in the female) and from the respiratory disease infectious bovine
rhinotracheitis (IBR) that any of these diseases was confirmed to be caused by a herpesvirus. Historically, IPV and its male counterpart infectious pustular balanoposthitis (collectively the male and female diseases are termed coital exanthema or blaschenausschlag) were commonly described diseases in central Europe throughout the nineteenth century. It was common for a single bull in a village to serve all the female cattle in that village and, where distances were small, also in nearby villages, and blaschenausschlag was a frequently observed sequel to mating. The isolation