Bovine respiratory syncytial virus (BRSV) is a negative-strand RNA virus classified in the family Paramyxoviridae, order Mononegavirales (Collins et al.,2001) (Table 1). Paramyxoviridae are subdivided into two subfamilies. The Paramyxovirinae includes Sendai virus, measles virus, mumps virus, Newcastle disease virus, simian virus 5 and human parainfluenza viruses while the second subfamily Pneumovirinae is represented by the genera Pneumovirus and Metapneumovirus. Pneumoviruses differ from other paramyxoviruses in several aspects: (1) eight or ten encoded mRNAs are typical for pneumoviruses compared with six or seven for paramyxoviruses; (2) proteins not present in paramyxoviruses are: NS1, NS2, M2-1 and M2-2; (3) pneumoviruses lack the V, D and C proteins present in some paramyxoviruses; (4) the unusual mucin-like G attachment protein of pneumoviruses is structurally distinct from the hemagglutinin-neuraminidase (HN) or the hemagglutinin (H) attachment proteins of other paramyxoviruses .
The genus Metapneumovirus consists of human metapneumovirus and avian pneumovirus (APV). These viruses lack the NS1 and NS2 genes and have a different gene order with respect to the F and M2 genes (Collins et al.,2001).
Table 1: Family Paramyxoviridae
Genus: Meta- pneumovirus Human meta- pneumovirus Avian pneumovirus
Subfamily: Pneumovirinae Genus: Pneumovirus Bovine respiratory syncytial virus Human respiratory syncytial virus (RSV) Caprine RSV Ovine RSV Pneumonia virus of Mice (PVM)
Henipa viruses: Hendravirus Nipahvirus Tupaia- Paramyxovirus
Genus: Morbillivirus Measles virus Canine distemper virus Phocine distemper virus Rinderpest virus Peste des petites- ruminants virus
Genus: Rubulavirus Mumps virus Human parain- Fluenza virus types 2 and 4 Simian virus 5 (canine parain- fluenza virus type 2) Newcastle disease virus
Family: Paramyxoviridae Subfamily: Paramyxovirinae Genus: Respirovirus Human parain- fluenza virus types 1 and 3 Bovine para- influenza virus type 3 Sendai virus
2.1.1 Epidemiology and clinical factors
HRSV and BRSV are closely related and show common epidemiological, clinical and pathological characteristics. They are the most common and important cause of lower respiratory tract illness in calves and young infants (Van der Poel et al., 1994). BRSV was first diagnosed in 1967 by Paccoud and Jacquier in Switzerland. The virus was first detected in Japan, Belgium and Switzerland, and it was isolated later in England and USA (Murphy et al., 1999). The presence of BRSV in cattle herds has been recognized worldwide (Bryson et al.,1978). Severe respiratory symptoms in calves are almost exclusively observed during autumn and winter seasons (Van der Poel et al., 1994; Murphy et al., 1999).
Calves that are born early in the year are most likely to suffer from the early syndrome in their first summer before they are weaned. The clinical signs associated with the early syndrome typically include coughing, nasal and ocular discharge, and an elevated temperature. The severity of the early syndrome is usually quite mild and the calves will often completely recover. However, the early syndrome can predispose the calves to secondary bacterial infection leading to a more severe and often fatal pneumonia. The early signs are often mild and transient and would include such features as a high-pitched dry cough, clear nasal and ocular discharge, mild anorexia, and mild depression (Backer et al., 1997). The later signs of this disease are often more evident and include increased temperature, severe anorexia, and significant weight loss. Often the eyes and surrounding tissues will swell.
2.1.2 Pathogenesis, pathology and immune response
Initially, an animal inhales the aerosolized virus. The virus is able to infect both ciliated and nonciliated cells of the upper and lower respiratory tract. Cytopathological changes in these cells result in a necrotizing bronchiolitis. Numerous cytokines and chemokines are released by infected cells and serve as chemoattractants for inflammatory cells, particularly neutrophils. BRSV is often associated with secondary bacterial infections suggesting either an immuno-suppressive activity of the virus or a role in producing mechanical damage allowing the entry of bacterial pathogens or a combination of both.
In experimentally infected calves the virus causes complete loss of the ciliated epithelium 8-10 days after infection so that pulmonary clearance is compromised facilitating secondary infections. At necropsy, subpleural and interstitial emphysema may be seen in all lobes of the lungs. A characteristic finding is the presence of syncytia in the lungs which are usually larger than those associated with parainfluenza virus type 3 infection (Murphy et al.,1999).
Both cell-mediated and antibody-mediated immune responses contribute to the efficient protection of animals. A hallmark of RSV infection is that the immune response is short-lived and reinfections are common. Neutralizing antibodies are induced by the F and G proteins as evaluated in calves vaccinated with recombinant vacinia virus encoding these proteins. High titers of neutralizing antibodies are induced by the F protein, whereas the G protein only induces a low level of complement-dependent neutralizing antibodies (Taylor et al., 1997; Thomas et al., 1998). Maternal antibodies are commonly present in calves but do not provide complete protection against infection (Kimman et al., 1998). Although infection can occur in the presence of neutralizing antibodies, they provide some protection as high antibody titers decrease the severity of the disease in both children and calves (Kimman and Westenbrink, 1990). Attempts to develop a vaccine against HRSV were not successful so far. In vaccination studies, increased lung pathology was observed after application of formalin-inactivated preparations associated with an Arthus-type reaction, antibody mediated enhancement of macrophage infection, complement activation, and hypersensitivity reactions. This immunopathology may be a consequence of a predominant Th2 response of helper cells with the preferential release of inflammatory cytokines (Murphy et al., 1999). For more than 18 years BRSV vaccines have been developed and used in some European countries (Valarcher et al., 2000). Modified-live virus (MVL) and inactivated single fraction and combination vaccines are currently available; nevertheless, their efficacy and potential disease enchancing properties remain controversial (Backer et al., 1997;
Bowland and Shewen, 2000). It has been shown that MLV and inactivated BRSV vaccines stimulate distinctively different antibody responses in cattle. Differences in T lymphocyte responses have also been reported, and it has been suggested that natural infection and MLV vaccines stimulate T helper 1 (TH-1)-type responses, whereas inactivated BRSV stimulates T helper 2 (Th-2)-type responses (Gerschwin et al., 1998; West et al., 1999, 2000)
2.1.3 Morphology and genome structure
BRSV virions include irregular spherical particles from 150 to 300 nm in diameter (Fig. 1). Virion preparations can include a high content of filamentous forms 60 to 100 nm in diameter and up to 10 µm in length (Berthiaume et al., 1974). The virion contains an helical nucleocapsid enveloped by a lipid bilayer derived from the host-cell plasma membrane. BRSV lacks the hemagglutination and neuraminidase activities present in several members of the Paramyxovirinae. However, the absence of an hemagglutinin is not a general hallmark of pneumoviruses because PVM is able to agglutinate erythrocytes.
Figure 1. Schematic structure of BRSV
The BRSV genome is a single-stranded, negative-sense RNA of 15 200 nucleotides, and encode 11 proteins (Fig. 2) (Buchholz et al., 1999). BRSV transcription and replication are similar to those of other Mononegavirales members.
The viral genomic RNA is tightly encapsidated by the N protein and serves as a template for replication and transcription. The N, P, and L proteins, together with the RNA genome are the virus-specific components required for RNA replication (Grosfeld et al., 1995; Yu et al., 1995). It has been shown that these components
L
P N G
F
RNA M SH
M2
lipid envelope
protein (Collins et al., 1996; Fearns and Collins, 1999 ). RNA replication involves the synthesis of a full-length, positive-sense, encapsidated RNA, called the antigenome, which serves as the template for the synthesis of negative-strand genomes.
Transcription initiates at the 3’ genomic promoter and copies the genes by a linear, sequential, start-stop mode that yields subgenomic mRNAs (Kuo et al., 1996;
Zamora and Samal, 1992). Each gene begins with a conserved 10 nt start (GS) motif that directs initiation of transcription, and ends with a 12-13 nt end (GE) motif that directs polyadenylation and release of the mRNA (Kuo et al.,1997). For the members of the Mononegavirales, the gene order appears to be a important factor dictating the relative molar amounts of viral mRNAs synthesized during virus infection (Collins et al., 1983). The efficiency of gene transcription decreases with increasing distance from the promoter (Barik, 1992). The first nine BRSV genes are separated by intergenic regions of variable length and sequence, the role of which in transcription is not clear (Hardy and Wertz, 1999). The last two genes, M2 and L overlap, by 67 nt and are expressed as separate mRNAs. The M2 mRNA contains two open reading frames (ORFs) that slightly overlap (Collins et al.,1990) (Figure 2); the upstream ORF1 encodes the M2-1 protein and the downstream ORF2 the M2-2 protein which appears to be involved in regulating the balance between transcription and RNA replication (Bermingham and Collins, 1999).
Figure 2. Schematic structure of the BRSV genome
2.1.4 BRSV proteins
The BRSV genome encodes eleven proteins:
N is the major nucleocapsid protein. It binds tightly to genomic and antigenomic RNA.
NS1
3‘ 5‘
NS2 N P M SH G F M2-1/2 L
L is the major polymerase subunit.
P is the most heavily phosphorylated RSV protein. It serves as a polymerase cofactor.
M2-1 is encoded by the 5’ proximal ORF of the M2-mRNA and serves as a transcription elongation factor.
M2-2 is encoded by the second internal ORF of the M2-mRNA and is a putative negative regulatory factor for replication and transcription (Bermingham and Collins, 1999).
There are three viral transmembrane glycoproteins:
SH (small hydrophobic protein) is a small protein of unknown function (Olmsted and Collins, 1989; Samal and Zamora, 1991). SH is dispensable for virus replication in cell culture (Bukreyev et al., 1997). However, recombinant RSV lacking SH are attenuated in vivo (Bukreyev et al., 1997; Whitehead et al., 1999).
G is the attachment glycoprotein, a heavily glycosylated type II membrane protein that does not share any sequence or structural homologies with attachment proteins of other paramyxoviruses (Satake et al., 1985; Wertz et al., 1985). It lacks hemagglutinatin and neuraminidase activities. It shows a high degree of species- and strain-specific antigen variation that has led to the definition of serological subgroups (Lerch et al., 1990; Mallipedi and Samal, 1993; Furze et al., 1994). The receptor-binding activity of the G protein was demonstrated using G-specific antibodies that inhibited virus attachment (Levine et al., 1987).
F glycoprotein mediates membrane fusion and syncytium formation. It is the major antigen inducing neutralizing antibodies.
M is the non-glycosylated matrix protein and has a central role in organizing the virus envelope (Teng and Collins, 1998). Recent studies suggest that the M protein may play a role in early stages of infection by inhibiting host cell transcription (Ghildyal et al., 2003).
NS1 and NS2 are small proteins detected in only trace amounts in purified virions and therefore have been considered to be nonstructural. NS1 is a putative negative regulatory factor for replication and transcription. Recombinant RSV lacking NS1 or NS2 are infectious and replicate in cell cultures (Collins et al., 2001) but show marked reduction in replication efficacy in vivo (Whitehead et al., 1999; Teng et al., 2000). These genes have been shown to function as interferon antagonists
2.1.5 RSV fusion protein
The RSV F protein is synthesized as a non-active precursor F0 which is cleaved in the trans-Golgi network by the cellular protease furin. The cleavage occurs at two conserved furin consensus cleavage sites and results in the generation of three products: a large subunit F1 with a highly conserved hydrophobic N terminus, the fusion peptide; the small subunit F2, and a glycosylated peptide of 27 amino acids (pep27) (Gonzales-Reyes et al., 2001; Zimmer et al., 2001, 2002). Pep 27 was found to be dispensable for viral replication in vitro. It is subject to additional posttranslational modifications and is secreted by the infected cells as a biologically active tachykinin (Zimmer et al., 2003). The two subunits F1 and F2 are arranged in a disulfide-linked complex that represents the biologically active form of the F protein.
The F protein is a type I membrane protein that contains a hydrophobic transmembrane domain near the C-terminus of the F1 subunit. The carboxyterminal domain is composed of 24-amino acids and is located in the cytoplasm. The classical role of the fusion (F) protein is to direct viral penetration by a fusion event between the viral envelope and the host cell plasma membrane whereby the nucleocapsid is delivered into the cytoplasm. In addition, when the F protein is expressed on the cell surface of infected cells, it mediates fusion with neighboring cells resulting in giant, multinucleated syncytia.
The pneumovirus and paramyxovirus F proteins do not show significant sequence similarity (Collins et al., 1984). However, in both genera the fusion proteins have similar structural features like a similar length, and the location of hydrophobic domains, heptad repeats, and cysteine residues. A common feature of fusion proteins is that upon proteolytic activation an hydrophobic fusion peptide is exposed that triggers the fusion reaction (Dutch et al., 2000). The RSV F protein can mediate entry and syncytium formation in the absence of other RSV proteins. This feature discriminates it from most other paramyxovirus fusion proteins which require the assistance of the homologous attachment protein to show fusion activity. Although the RSV F protein can mediate syncytium formation by itself, coexpression of the G and SH proteins has been shown to increase fusion efficiency (Heminway et al., 1994).
2.1.6 Heparin binding of RSV
Heparan sulfate and chondroitin sulfate B belong to a group of glycosaminoglycans (GAGs). These two types GAGs are major binding sites for respiratory syncytial virus on the cell-surface. Glycosaminoglycans are unbranched polymers of repeating disaccharide units. Some of them are found as a proteoglycans covalently linked to membrane proteins on the cell surface of most mammalian cells. One of the prominent physico-chemical properties of GAGs is the presence of a large and varying number of negative charges. Heparin is frequently used as a convenient analog of heparin sulfate for experimental purposes (Lindahl et al., 1994). The heparin-binding properties of RSV were reported for the first time by Krusat and Streckert in 1997, who showed that heparin is a potent antagonist of RSV infection. A cellular protein receptor for RSV has not been identified, however there is evidence that cell surface glycosaminoglycans (GAGs) are important for efficient infection, at least in vitro. It was shown that RSV infection can be inhibited by preincubation of the virus with soluble GAGs such as heparin. In addition, RSV infection was significantly reduced following treatment of the cells with enzymes that remove GAGs from the cell surface. It was also shown that mutant cell lines with defects in GAGs synthesis are not efficiently infected by RSV (Krusat and Streckert, 1997; Bourgeois et al., 1998 Hallak et al., 2000; Karger et al., 2001; Techaarpornkul et al., 2002). Binding of RSV to GAGs is primary mediated by the G protein (Krusat and Streckert, 1997). Feldman et al. (1999) suggested that region with clusters of positively charged amino acids that share similarity with heparin-binding domains from other viral and mammalian proteins represents the major GAG-binding domain.
However, subsequent studies using recombinant viruses showed that this site can be deleted without affecting infectivity or susceptibility to neutralization by soluble GAGs (Teng and Collins, 2002). Analysis of the G protein C-terminus revealed that the postulated heparin-binding domain expands to residue 187-231 (Shields et al., 2003) confirming the work of Teng and Collins (2002) that the HBD is not confined to the previously reported residues 184-198 (Feldman et al., 1999). Another report suggested that heparin-like structures associated with the G protein are involved in RSV infection (Bourgeois et al., 1998), but these data have not been confirmed.
It was shown that heparan sulfate and dermatan sulfate are the two cell surface GAGs which are important for efficient RSV infection (Hallak et.al., 2000a).
other GAGs that do not contain IdUA did not inhibit RSV infection. Furthermore, sulfation at specific GAG positions and the length of the saccharide chains have also been found to be important for RSV infection (Hallak et al., 2000b).
The observation that the biologically derived cold-passaged mutant cp-52 which lacks the G and the SH proteins is still dependent on heparin suggests that the F protein also binds to GAGs on the cell surface to initiate infection (Feldman et al., 2000). A similar observation was made when the G gene was deleted from HRSV and BRSV (Karger et al., 2001; Techaarpornkul et al., 2002). When the GAG dependence of a recombinant virus with F protein as its only glycoprotein was compared to an isogenic virus containing the complete genome, it turned out that attachment and infection by both viruses required cell surface heparan sulfate. All these data suggest that both G and F protein bind to HS. A region in the F protein responsible for heparin binding have not been identified so far.