Материал: Bovine Viral Diarrhea Virus Diagnosis, Management, and Control

ity of BVDV to grow in a variety of cell lines but also the potential for it to contaminate cell lines inadvertently. Interestingly, although the virus is capable of infecting many cell lines, cell tropisms do exist, and the viral glycoprotein E2 has been shown to play a role in this cell tropism (Liang et al., 2003). As previously discussed, researchers and diagnosticians must control for possible cell line contamination with BVDV or the results of investigative studies will be compromised.

CONCLUSIONS

The reports of naturally occurring disease indicate that pigs and a variety of domestic and wild ruminants can be infected with BVDV. The virus has been shown to produce congenital infection in pigs, sheep, and goats. In some cases, congenital infection results in persistently infected animals that can be a significant source of viral transmission. Wild ruminants are known to be susceptible to acute infections with BVDV, and limited evidence suggests that cervids may also undergo congenital infections leading to persistent infection (Grondahl et al., 2003). This suggests a new potential for this virus to be shed in large amounts from wild ruminants that could then infect cattle. Current evidence suggests that wild ruminants may serve as a transient source of virus while undergoing acute infections and may possibly be a more prolonged source of virus from persistently infected animals. This would be of greatest concern where these animals are in contact for prolonged periods of time, as opposed to transient fence line contact.

The role of species from which the virus has never been isolated but in which seroconversion has been observed is not clear. The significance of in vitro growth of BVDV in certain cell lines without infection in whole animals is also not known. It is unlikely, however, that these species would be viremic for significant periods of time and thus are unlikely to shed BVDV in the environment. Based on the previous discussion, it seems that non-bovine domestic ruminants and swine remain the greater concern for disrupting management attempts to control the diseases caused by BVDV in cattle.

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INTRODUCTION

Bovine viral diarrhea virus (BVDV) is a ubiquitous pathogen of ruminants that is often associated with severe economic losses. Understanding these viruses, particularly at the cellular and molecular levels, is important to develop new vaccination and treatment strategies. The events that transpire following infection of the host animal are only now beginning to become clear. Upon entry into a susceptible host cell, the virus replicates utilizing viral as well as cellular proteins and machinery. Our understanding of BVDV replication comes from studies of BVDV directly or is extrapolated from studies of classical swine fever virus (CSFV, known previously as hog cholera virus) and other members of the Flaviviridae, particularly hepatitis C virus (HCV). Most proteins encoded by BVDV, CSFV, and HCV, as well as RNA structural motifs, are considered functionally equivalent (Rice, 1996; Branza-Nichita et al., 2001). The two exceptions are the Npro and Erns proteins that are unique to pestiviruses. This chapter describes functions provided by the host cell that are absolutely required for virus replication, including protein translation, protein modification, and transport and release of progeny virus. Results from studies of other pestiviruses and flaviviruses are discussed in context of BVDV and will be considered common to all viruses unless stated otherwise.

RECEPTOR AND VIRUS ATTACHMENT

Arguably, the single most important event in the life cycle of a virus, the “make or break point,” is the attachment of the infectious virus particle to the surface of a susceptible cell. If this does not occur, downstream events such as uptake, release of genome, replication of viral RNA(s) and translation of viral proteins cannot take place. Binding of the

virus to the cell surface has been shown in a number of systems to involve the recognition of specific cellular molecules that are embedded in, or intimately associated with, the cell’s plasma membrane. These specific cellular molecules are usually proteins, although many examples of viruses binding to carbohydrate moieties are known (Jackson et al., 1996; Hilgard and Stockert, 2000; Martínez and Melero, 2000; Dechecchi et al., 2001; Terry-Allison et al., 2001). Virus receptors are normal components of the plasma membrane and serve diverse functions such as internalization of ligands, cell signaling, and cell- to-cell interactions (Mendelsohn et al., 1989; Wykes et al., 1993; Colston and Racaniello, 1994; Roivainen et al., 1994; Agnello et al., 1999). The types and nature of these receptor molecules affects both the host and tissue tropism of the virus. If the receptor molecule of an organism is sufficiently different from that of the normal host, infection will not occur. Additionally, the receptor molecule(s) may be expressed in only a subset of cell types in the host, only at specific points in the cells cycle, or on specific surfaces of polarized cells (Compans, 1995). Any of these factors would limit the infection to specific cells or tissues. This in turn gives rise to the characteristic disease symptoms, lesions, and pathology of the viral infection.

CELLULAR FACTORS

Several groups have studied the interaction of BVDV and the cell surface. Initial studies involved the production of monoclonal antibodies (Mab) directed against cell surface epitopes. Teyssedou et al. (1987) reported the production of a Mab that was reactive against a protein on the surface of MadinDarby bovine kidney (MDBK) cells. When attached, this Mab completely prevented infection by bovine enterovirus-3 (BEV-3) strain 240A and par-

tially inhibited infection by BEV-2 and BVDV. The mechanism behind this incomplete inhibition was unclear and probably did not represent an antibody reactive against the true receptor molecule. This was followed shortly by a report by Moennig et al. (1988) of a bovine-specific Mab that prevents infection of bovine cells with a number of cytopathic strains of BVDV (cp BVDV) while not preventing infection with CSFV, bovine herpesvirus-1, parain- fluenza-3, and pseudorabies virus. Based on these findings, the authors suggested a single receptor molecule for BVDV. Further analysis of the protection afforded by this Mab, using the more sensitive immunoperoxidase method (Collett et al., 1989), revealed small numbers of infected foci in Mabtreated MDBK cultures, indicating the presence of multiple cell surface receptors for BVDV.

Another type of Mab, anti-idiotypic antibodies, were produced to study specific virus-receptor interactions (Xue and Minocha, 1993; Xue and Minocha, 1996, Xue et al., 1997). Anti-idiotypic Mabs were raised against neutralizing Mabs that bound the viral protein anti-gp53 (E2). These anti-idiotypic Mabs thus mimicked the viral epitopes presumed to be associated with binding of the virus to the cellular receptor. The anti-idiotypic Mabs bound to the surface of MDBK cells in a manner similar to that of the virus and inhibited infection by BVDV. A 50 kDa protein was identified in immunoprecipitation experiments as the target of these Mabs (Xue and Minocha, 1993; Minocha et al., 1997). Xue et al. (1997) determined that this receptor molecule was protein in nature because protease treatment of cells resulted in concurrent loss of virus binding to the cell surface. Glycosidase treatment of cells prior to infection was used to demonstrate that glycosylation of the receptor protein was not necessary for binding of virus. The receptor molecule was found on a number of different cell types, both susceptible and nonsusceptible to infection with BVDV (Xue and Minocha, 1993; Xue and Minocha, 1996), indicating a protein of conserved function. The receptor protein was recognized by BVDV on the surface of porcine cells, based on the resulting productive infection. However, the replication of the virus was slower, with progeny virus levels eventually reaching those produced by bovine cells. The difference was probably not a result of cell surface binding and uptake but rather in functions related to replication.

Three additional Mabs (Moennig et al., 1988) that bound to the surface of cells susceptible to infection with BVDV were characterized by Schelp et al. (1995). These Mabs bound to the surface of cultured

cells as well as leukocytes freshly isolated from the blood of cattle and blocked infection with BVDV to varying degrees. When used in immunoprecipitation experiments, all precipitated cellular proteins of 60 and 93 kDa. In follow-up experiments (Schelp et al., 2000), one of these Mabs, BVD/CA 26, recognized a protein consisting of 28 and 56 kDa subunits that, under nonreducing conditions, formed multimers of approximately 200 kDa. The 56 kDa subunit was shown to bind to F-actin, giving some insight into its possible biological function. It is unknown why there was a discrepancy in protein sizes between the two reports. It is also unknown if the 56 kDa protein described here and the 50 kDa protein described by Xue and Minocha (1993) are the same or related proteins.

A different approach examining BVDV binding and uptake was taken by Flores and Donis (1995). They generated an MDBK cell line that was resistant to infection by BVDV. This mutant cell line, CRIB, was developed by infection of a susceptible monolayer of cells with a cytopathic strain of BVDV (Singer strain) and culturing of the survivors. The resulting culture contained no virus (infectious or defective) as determined by cocultivation, animal inoculation, immunofluoresence, western and northern blots, and reverse-transcription polymerase chain reaction (RT-PCR). Infection of CRIB cells with 24 additional strains of BVDV did not result in a productive infection as determined by expression of viral proteins. Transfection of viral RNA or virions in the presence of polyethylene glycol (PEG) did result in a productive infection as measured by immunofluoresence, indicating CRIB cells were defective at virus entry and not at a postuptake function. However, CRIB cells were not resistant to infection by 10 other bovine viruses, indicating that the block of BVDV replication was specific and was not a general antiviral activity.

In additional studies, Flores et al. (1996) reported that resistance of CRIB cells to infection with BVDV was blocked at virus entry and suggested that a cell membrane function that was important in virus uptake following viral attachment was mutated or missing. Using PEG-mediated virus uptake in these cells, the authors concluded that while CRIB cells bound saturating levels of virus, entry was blocked by a defect in endocytosis. This was further demonstrated by blockage of PEG-mediated uptake of virus using inhibitors of endocytosis and endosomal acidification.

Characterization of the cell surface molecule bound by the E2 envelope provided evidence that the low density lipoprotein receptor (LDLR) was the

E2 receptor molecule as well as the mediator of endocytosis of the attached virus particle protein (Agnello et al., 1999). The LDLR is a cell surface endocytic receptor that mediates the uptake of extracellular ligands into the cell (May et al., 2003). Three lines of evidence support the LDLR as a BVDV receptor: complete inhibition of endocytosis by anti-LDLR antibodies, inhibition of endocytosis by phenylarsine oxide (an inhibitor of endocytosis), and inhibition of uptake by chemical methods that prevent lipoprotein/LDLR interactions. In addition, CRIB cells (Flores and Donis, 1995; Flores et al., 1996) lack LDLR based on failure to bind antiLDLR antibodies. The loss of infection by inhibition of endocytosis with anti-LDLR antibody indicates that LDLR-mediated endocytosis may be the main mechanism of virus entry (Agnello et al., 1999). However, the observation that there was a low background of infection associated with high levels of infecting virus indicated the presence of other, lowaffinity receptor molecules.

The possibility of the CD46 molecule as a cell surface receptor for BVDV has been proposed (Rümenapf et al., 2000; Maurer et al., 2002). The CD46 protein is a known receptor for measles virus and herpesvirus 6 (Greenstone et al., 2002). Antibodies against the CD46 molecule, a complement regulatory protein, inhibited infection with BVDV. Expression of the bovine CD46 protein in porcine cells increased plaquing efficiency of cytopathic BVDV fortyto hundredfold. Expression of CD46 in nonpermissive cells did not confer susceptibility.

VIRAL FACTORS

The outer envelope of the BVDV virion contains the structural proteins Erns, E1, and E2. These proteins are highly glycosylated and possess the major antigenic determinants of the virus. For a more complete discussion concerning the biology and structure of these proteins, refer to Chapter 3. Recent work has demonstrated that two of these proteins, Erns and E2, play important roles in the attachment of the virus particle to the cell surface. Hulst and Moormann (1997), working with purified CSFV Erns and E2 synthesized in insect cells, showed that Erns added to susceptible cells prior to infection (100 µg/ml) could irreversibly bind to cells and prevent infection. Purified E2 at 10 µg/ml also provided 100% inhibition of infection but this inhibition was reversible and required additional supplementation of E2 to maintain inhibition. Following removal of E2, infection still occurred, presumably by virus particles bound to the surface of the cells. Treatment

with 100 µg/ml Erns released these particles. The difference between concentration of proteins required to reach complete inhibition and differences in binding properties indicated that these proteins bound to different molecules on the cell surface. These differences also indicate that the E2 binding site is of lower prevalence.

Experiments have demonstrated that dengue virus (Hilgard and Stockert, 2000) and BVDV (Iqbal et al., 2000) Erns molecules first bind to cell surface heparin sulfate proteoglycans and sulfated heparin-like glycosaminoglycans, respectively. This type of initial virus/cell interaction has been reported for a number of viruses that utilize two or more cell surface receptors (Mettenleiter et al., 1990; Okazaki et al., 1991; Jackson et al., 1996; Chen et al., 1997; Jusa et al., 1997; Krusat and Streckert, 1997; Asagoe et al., 1997). Erns binds to both permissive and nonpermissive cells because of the near ubiquitous presence of glycosaminoglycans (Iqbal et al., 2000). Soluble Erns added to medium blocked replication of BVDV. As supporting evidence, Erns binding was not observed when cells were treated with heparinase I or III, when soluble glycosaminoglycans were present, or to Chinese hamster ovary (CHO) cells that did not produce glycosaminoglycans or heparin sulfate. These lines or evidence led to the hypothosis that binding of Erns to cell surface glycosaminoglycans is the initial event in viral attachment. Subsequently, Iqbal and McCauley (2002) showed that the conserved KKLENKSK motif near the C- terminus of the Erns protein mediated binding of the glycosaminoglycan molecule.

The mechanism of release of genomic RNA into the cytoplasm of the cell is unclear but probably involves acidification of endocytic vesicles (Flores and Donis, 1995; Flores et al., 1996). Uptake of virus appears to occur by endocytosis and not by a membrane fusion mechanism. Treatment of BVDVinfected MDBK cells with phenylarsine oxide, an inhibitor of endocytosis and inhibitors of endosome acidification (chloroquine and ammonium chloride) prevented uptake of virus in the presence of PEG (Flores and Donis, 1995). Dengue virus, a member of the Flaviviridae, has been shown to traffic the major clathrin-dependent endocytic pathway during infection (Hilgard and Stockert, 2000). Work with West Nile virus demonstrated that acidification (<pH 6.6) caused rapid loss of viral infectivity (Gollins and Porterfield, 1986). Ammonium chloride also inhibited uncoating of virus and infection, demonstrating the dependence on acid pH in the infection process.