Aspects of the innate and adaptive immune responses to acute infections with BVDV

Veterinary Microbiology 96 (2003) 337–344

Aspects of the innate and adaptive immune responses to acute infections with BVDV L.S. Brackenbury, B.V. Carr, B. Charleston∗ Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN UK

Abstract The immune response can be divided into innate and adaptive components that synergise to effect the clearance of pathogens. Recently, it has been realised that these arms of the immune system do not act independently, the magnitude and quality of the adaptive response is dependent on signals derived from the innate response. Here, we review the innate immune responses to bovine viral diarrhoea virus infections of cattle and relate these changes to immunosuppression and the subsequent development of the adaptive immune response. © 2003 Published by Elsevier B.V. Keywords: Bovine viral diarrhoea virus; Interferon; Immunosuppression; Innate; Adaptive; Immunity

1. Introduction The pestiviruses, bovine viral diarrhoea virus (BVDV), classical swine fever virus and border disease virus of sheep, the flaviviruses and hepatitis C virus comprise a closely related group of small enveloped viruses with a single-stranded, positive sense RNA genome of approximately 12.5 kb. They all have a similar genomic structure and protein composition, the virus particles comprising a single capsid protein surrounded by an envelope containing two or three glycoproteins. BVDV has a worldwide distribution and readily establishes endemic infection in cattle populations. Disease is associated with both acute and persistent infections and, depending on epidemiological circumstances, may manifest as outbreaks affecting large numbers of animals or a continual low incidence of cases within endemically infected herds. Both disease patterns have a major impact on the productivity of affected cattle populations. Two biotypes of the virus, cytopathic and non-cytopathic, are identifiable based on their lytic activity in in vitro cultures (Meyers and Thiel, 1996). The high prevalence of cattle herds ∗

Corresponding author. Tel.: +44-1635-578411; fax: +44-1635-577263. E-mail address: [email protected] (B. Charleston). 0378-1135/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/j.vetmic.2003.09.004

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infected with BVDV in many countries throughout the world is believed to be a consequence of the ability of non-cytopathic BVDV (ncpBVDV) to establish lifelong infections following in utero infection in early pregnancy, thus generating a reservoir of persistently infected animals (Brownlie et al., 1989). The studies conducted within our work programme are aimed at understanding the role of specific immune responses in protection against BVDV and the mechanisms by which the virus modulates host immune responses to favour its own survival. The results of these studies are expected not only to be of strategic value for control of disease caused by BVDV, but also to contribute more generally to understanding the biology of this group of RNA viruses. This paper reviews available information on the innate and adaptive immune responses to acute BVDV infections, illustrated by recent data from our own work.

2. Immunosuppression Although acute infections with ncpBVDV are often asymptomatic or produce only mild clinical symptoms, there is evidence that they result in immunosuppression. This is based on experimental studies in which acute infection of calves with BVDV was found to enhance susceptibility to infection with bovine herpes virus 1 (BHV-1), as well as field observations indicating increased susceptibility after acute infection with BVDV to intercurrent infections (Edwards et al., 1986; Bielefeldt-Ohmann and Babiuk, 1985; Wray and Roeder, 1987). The mechanism of immunosuppression induced by BVDV has not been determined, although there has been considerable speculation, based largely on in vitro observations (Potgieter, 1995). In studies using cattle with an established immune response to attenuated Mycobacterium bovis (BCG) we have demonstrated a transient suppression of this immune response for approximately 14 days after acute infection with ncpBVDV (Charleston et al., 2001a). Similar suppression by infection with ncpBVDV has also been shown for immune responses to Mycobacterium avium paratuberculosis (Theon and Waite, 1990). The interaction between BVDV and M. bovis may have important practical implications. The suppression of immune responses to M. bovis by BVDV may hinder diagnostic tests used to identify infected animals in a herd. The capacity of ncpBVDV to cause immunosuppression could be related to the tropism of the virus for antigen presenting cells (APC) (Sopp et al., 1994). We have performed studies with bovine dendritic cells (DCs) and monocytes infected with ncp and cpBVDV, to investigate whether there are differences in viral growth and cytopathogenicity in these two cell types (Glew et al., 2003). Briefly, ncpBVDV grows to a lower titre in DCs compared to monocytes. Also, ncpBVDV infection of monocytes causes a reduction in the proliferative response of either allotypic or memory CD4+ T cells when mixed with these APC. In contrast, cpBVDV infected DC are not compromised in their capacity to stimulate similar proliferative responses in CD4+ T cells. However, the most striking difference between the two cell types was demonstrated during infection with cpBVDV. Ninety percent of monocytes were killed within 96 h of infection, but DC viability was not affected by cpBVDV infection. We have excluded a role for interferon ␣/␤ in the protection from cytopathic effect, a mechanism proposed for the

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failure of influenza virus to induce a cytopathic effect in human DCs (Cella et al., 1999b). The mechanisms DCs use to survive infection with cpBVDV remain to be elucidated. The capacity to cause immunosuppression has also been related to the ability of ncp virus to block the induction of interferons (IFN)␣␤. ncpBVDV isolates have been shown not to induce IFN␣␤ in vitro (Diderholm and Dinter, 1966; Adler et al., 1997) and to block the induction of IFN␣␤ by double stranded RNA (dsRNA) or infection with other viruses (Rossi and Kiesel, 1980; Schweizer and Peterhans, 2001).

3. Effects of BVDV on interferon production in vivo We have undertaken a series of studies to examine IFN␣␤ responses in vivo. In contrast to the in vitro findings, experimental infection with ncpBVDV was found to induce strong IFN␣␤ responses in both gnotobiotic and conventionally reared calves (Charleston et al., 2002; Fig. 1). Elevated levels of IFN␣␤ were detected in the serum between 1 and 7 days after infection. These results indicate that the immunosuppression caused by BVDV is not associated with low interferon responses. Interestingly, IFN␣␤ was not detectable in the serum of PI animals (data not shown), suggesting that persistent infection may result in down regulation of the response and/or that an adaptive immune response may be required to amplify the IFN␣␤ response. Whereas infection of the early foetus (<120 days) with ncpBVDV results in the establishment of persistent infection, previous studies have shown that cp virus is unable to establish persistent infection (Brownlie et al., 1989). However, it is unclear whether this is due to the absence of cells capable of supporting replication of cp virus at this stage of development or to the ability of the foetus to clear the infection. To determine whether the ability to establish infection in utero is associated with the capacity to induce IFN␣␤, 60-day bovine foetuses were directly infected with ncpBVDV or cpBVDV and the dams and foetuses were slaughtered 3, 5 and 7 days later for post-mortem examination (Charleston et al., 2001b). Both viruses were found to replicate in foetal spleen. IFN␣␤ was found in the amniotic fluid of foetuses infected with cpBVDV, but was undetectable in amniotic fluid of foetuses infected with ncpBVDV. Western blot analysis of foetal spleen samples for Mx protein, a molecule specifically induced by IFN␣␤, revealed strong bands in spleens from foetuses infected with cp virus but only faint bands in spleens from foetuses infected with ncp virus. These results suggest that the failure of ncpBVDV to induce IFN␣␤ may have evolved to enable the virus to establish persistent infection in the early foetus.

4. Adaptive immune responses In view of the evidence that CD4+ T cells play an essential role in immunity to BVDV (Howard et al., 1992), our studies have focussed on examining the kinetics, specificity and functional features of CD4+ T cell responses. Experimental infection of calves with ncpBVDV gives rise to transient viraemia and nasal excretion of virus, with resolution of infection about 12–14 days after infection. In contrast, infection of calves with homologous cpBVDV results in lower titres of virus in nasal secretions and undetectable viraemia

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Fig. 1. Kinetics of IFN␣␤ production in the serum of calves after intranasal challenge infection with 5 × 106 pfu ncpBVDV isolate 11249. Individual samples were analysed in duplicate and the mean value determined. Titres are expressed as the mean of six animals ± S.E.M. (a) Data from gnotobiotic calves; (b) data from conventional calves.

(Lambot et al., 1998). However, virus-specific antibody is first detected shortly after viral clearance of both biotypes. By contrast there is a marked difference in the kinetics of the development of a specific T cell response after ncp and cpBVDV infection. We have found that T cell proliferative responses are not reproducibly detectable until about 6–8 weeks

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Fig. 2. Specific proliferative T cell responses and total antibody responses of calves after primary BVDV infection. Proliferative T cell responses (A)–(C) and total BVDV antibody responses (D)–(F) of calves infected with Pe515ncp (A) and (D), Pe515cp (B) and (E) or NADL (C) and (F). Filled and open circles represent data from separate animals. Experimental details as described previously (Collen and Morrison, 2000).

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after ncpBVDV infection, but were detectable by 3–4 weeks post-infection with cpBVDV (Collen and Morrison, 2000; Fig. 2). The kinetics of the T cell response to ncpBVDV is, therefore, markedly different from that observed with other acute viral infections in cattle. BVDV is clearly a fascinating virus because of its interactions with the immune system and the availability of antigenically homologous pairs of ncp and cp viruses. The dichotomy of the host interaction with the two biotypes is most clearly demonstrated by foetal infection during the first trimester of pregnancy (Brownlie et al., 1989; Charleston et al., 2001b). However, it is now becoming clear that the difference in the capacity of the two biotypes to stimulate the innate immune response is influencing the development of the adaptive immune response (Fig. 3). Examination of serum IFN␣␤ levels after acute intranasal infection with

Fig. 3. Model of the interaction between cpBVDV and ncpBVDV and cells of the innate and adaptive immune system. cpBVDV: infection with cpBVDV at a mucosal or epithelial surface will cause cytokines such as IFN␣ to be expressed from a range of cell types, including DCs. These cytokines will activate effector cells of the innate immune response, eosinophils, macrophages and NK cells. A combination of the direct action of cytokines and effector cell function will limit the replication of virus. The environment created by the cp infection will activate DCs, leading to enhanced antigen capture, maturation and migration. DCs are probably resistant to the lytic effects of cp infection and can traffic to the local lymph node. After migration to the lymph node the antigen laden DCs will present antigen via peptide–MHC complexes to antigen specific lymphocytes. The resulting activated T lymphocytes (e.g. cytotoxic lymphocytes—CTLs, helper T cells) traffic back to the injured tissue to eliminate virus or virus infected cells. Activated B cells will migrate to form germinal centres—GC in the lymph node, where they will mature into plasma cells that produce antibody to neutralise the virus. ncpBVDV: in contrast to cp infection, ncp infection will not stimulate an early cytokine response. Consequently, viral replication is not limited and DCs do not become highly activated. Cell free virus and virus infected DC will drain to the local lymph node. In the lymph node the virus encounters plasmacytoid DCs and large quantities of IFN␣ are produced. The increasing quantities of IFN␣ in the circulation and tissue will enhance the activation of DCs and limit viral replication. However, the ncp virus will have already disseminated throughout the animal; adapted from Palucka and Banchereau (2002).

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either cp or ncpBVDV reveal contrasting findings to in vitro studies. In vivo, ncp infection results in high and prolonged levels of IFN␣␤ production detectable in serum (Charleston et al., 2002), whereas IFN␣␤ is not detectable after cp infection (data not shown). We hypothesise that intranasal cp infections are confined to mucosal and submucosal tissues co-incident with a rapid and potent induction of IFN␣␤. However, despite this constraint on virus dissemination, activation of DCs and processing of viral antigen occurs to stimulate a primary immune response. Indeed, our in vitro studies with cp virus would also suggest that the virus does not kill DCs (Glew et al., 2003), allowing these cells to migrate and initiate an immune response in local lymph nodes. In contrast, infection with ncp virus probably results in a different sequence of events. Ncp virus would not stimulate the innate immune response at the mucosal surface after intranasal challenge. Because innate immunity is not stimulated at the local site, DCs will not become activated and viral growth not restricted. However, when free virus enters the lymph node, interaction with plasmacytoid DCs would result in the induction of IFN␣ (Cella et al., 1999a). These recently recognised plasmacytoid DCs, also called natural interferon producing cells, are a key cell type in the link between the innate and adaptive immune response. This specialised subpopulation of DC precursors are capable of producing very large quantities of IFN␣␤, which influences the development and sensitivity of a number of other DC subpopulations and so shapes the adaptive immune response. Induction of IFN␣ in the lymph node would cause elevation of serum and tissue IFN␣ levels and result in activation and enhanced migration of DCs to stimulate a primary immune response (Howard et al., 1999). Because the elevation of tissue IFN␣ levels will be slower by 24–36 h after ncp infection compared to cp infection, ncp virus will become more readily disseminated and the onset of a protective immune response delayed. The establishment of viraemia for a period of days after acute infection is crucial to the lifecycle of ncpBVDV (Niskanen et al., 2002). It is only by exposure of the early foetus to infectious virus that another persistently infected animal will be born.

5. Conclusion Understanding the early events in the immune response to BVDV will aid the rational design of new vaccines. The quality and onset of the immune response after vaccination will depend in part on how the innate immune response is stimulated. Further investigations of the different responses to cp and ncpBVDV in vivo will highlight the importance of the timing and location of the interaction with cells of the innate immune system to stimulate the development of a rapid, protective immune response.

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