Maternal and Fetal Response to Fetal Persistent Infection with Bovine Viral Diarrhea Virus

Authors

  • Thomas R. Hansen,

    1. Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
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  • Natalia P. Smirnova,

    1. Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
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  • Hana Van Campen,

    1. Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
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  • Megan L. Shoemaker,

    1. Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
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  • Andrey A. Ptitsyn,

    1. Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
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  • Helle Bielefeldt-Ohmann

    1. School of Veterinary Science, University of Queensland, Gatton Campus, Qld, Australia
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  • Presented as invited paper at the 29th Annual American Society of Reproductive Immunology Meeting, Orlando, FL, June 9, 2009. Dr. Hansen and Dr. Smirnova are co-first authors on this paper.

Thomas R. Hansen, Traubert Professor and Director, Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Campus Delivery 1683, Fort Collins, CO 80523, USA.
E-mail: thomas.hansen@colostate.edu

Abstract

Citation Hansen TR, Smirnova NP, Van Campen H, Shoemaker ML, Ptitsyn AA, Bielefeldt-Ohmann H. Maternal and fetal response to fetal persistent infection with bovine viral diarrhea virus. Am J Reprod Immunol 2010

Problem  Infection of naïve pregnant cows with non-cytopathic (ncp) bovine viral diarrhea virus (BVDV) results in transplacental infection of the fetus. Infection of the pregnant cow with ncp BVDV late in gestation (after day 150) results in transient infection (TI), as both the dam and fetus can mount an immune response to the virus. In contrast, if the fetus is infected with ncp BVDV early in gestation (before day 150), the fetal immune system is undeveloped and unable to recognize the virus as foreign. This results in induction of immune tolerance to the infecting BVDV strain and persistent infection (PI).

Methods  Infection of naïve pregnant heifers with ncp BVDV2 on day 75 was hypothesized to induce differential gene expression in white blood cells of the dams and their fetuses, adversely affecting development and antiviral immune responses in PI fetuses.

Results  Gene expression differed in maternal blood cells in the presence of PI versus uninfected fetuses. PI adversely affected fetal development and antiviral responses, despite protective immune responses in the dam.

Conclusion  Fetal PI with BVDV alters maternal immune function, compromises fetal growth and immune responses, and results in expression of maternal blood biomarkers that can be used to identify cows carrying PI fetuses.

Introduction

Bovine viral diarrhea virus (BVDV) was described in cattle from Canada and also in dairy cattle in New York in 1946.1 Worldwide it infects >80% of the cattle population.1,2 The United States alone suffers losses of more than $400 million per year, designating BVDV as the most costly bovine viral disease in the country.3–5 Control of the virus is an ongoing problem because vaccination programs are not universally implemented and it has been impossible, until recently, to distinguish seroconverted vaccinated pregnant cattle from those cattle that seroconverted in response to infection but also carried a PI fetus. A better understanding of the mechanisms underlying persistent BVDV infection in addition to further development of maternal markers for pregnant cattle with PI fetuses may greatly contribute to eradicating the virus.

The clinical manifestations of acute infections caused by cytopathic (cp) and ncp BVDV are similar for both BVDV1 and 2 strains and range from sub-clinical or unapparent infections to embryonic death, abortions, congenital defects and stillborn calves. Acutely infected animals usually recover and eliminate the virus within 10–14 days post-infection (p.i.). After birth, some PI calves fail to thrive, have reduced fertility and show immunosuppression, resulting in death because of superimposed infections during the first year of life6 or the development of mucosal disease.7 Surviving PI calves continually shed the virus and infect other cattle, thus ensuring the survival of the virus in the herd or the population.2 Detection and removal of pregnant cows carrying PI fetuses, as a potential reservoir of the BVDV infection, would greatly benefit the successful implementation of control programs.

The proposed mechanism of BVDV PI is an evasion of the adaptive immune response because of the presence of the virus during selection of T lymphocytes, which see the viral antigen as part of the host antigen repertoire.8,9 This lack of recognition of the virus by the adaptive immune system results in a failure to clear the infection and a persistent viremia. A potential evasion of the innate immune system in persistent BVDV infection might occur through failure of activation of the antiviral type I interferon (IFN) response.10–16 The innate immune system is triggered by the recognition of pathogen-associated molecular patterns (PAMPs) such as viral RNA.17 Activation of the type I IFN pathway in response to viral infection stimulates an autocrine and paracrine signal transduction cascade resulting in an antiviral state in the host cells and the triggering of an adaptive immune response.18 Type I IFN production is activated by pattern recognition receptors such as Toll-like receptors and RNA helicases, which signal the presence of PAMPs and initiate downstream signaling leading to the induction of cytokines.19 This review describes induction of a massive type I IFN response in response to acute ncp BVDV infection that also is present at more discrete but chronic levels in PI fetuses and steers. After clearing the virus, cows that have transmitted BVDV transplacentally to their fetuses have suppressed expression of chemokine receptor 4 (CXCR4) and associated signal transduction genes. These maternal and fetal responses to BVDV infection are presented as compromised maternal and fetal immune responses, and in context of fetal intra-uterine growth restriction.

Development of an Animal Model to Examine Maternal and Fetal Responses to Fetal PI with BVDV

The only way to accurately generate cows carrying PI fetuses is to experimentally infect them with ncp BVDV prior to development of the fetal immune system (Fig. 1). Eighteen weaned Hereford heifers (approximately 7 months of age) were obtained from a source that did not vaccinate against BVDV. Pregnancy was confirmed by ultrasound examination 35–40 days after insemination. The ncp BVDV2 strain 96B222220 was used for viral challenge of pregnant heifers as previously described in.21 While there were no fundamental differences in the pathology caused by BVDV1 and 2 in adult cattle or infected fetuses,22 previous experiments have shown slightly better rate of transplacental infection of fetuses with BVDV2. Each heifer (n = 6 per treatment group) was inoculated intranasally with 2 mL of the viral stock aliquot at 4.4 log10 TCID50/mL or with media.

Figure 1.

 Experimental Design. Pregnant heifers were inoculated with ncpBVDV on day 75 of gestation to generate persistently infected (PI), on day 175 to generate transiently infected (TI), or were not infected to generate control fetuses. Blood samples were collected on days indicated for serology and detection of BVDV, as well as for total blood cell mRNA isolation. Black stars indicate days when viremia was detected. Microarray screens were completed on blood cell mRNA on days 160 and 190. Blood cell gene expression in heifers carrying PI compared to control fetuses was completed on day 160. Maternal blood cell gene expression also was compared for all groups on day 190. Adapted from,34 with permission.

Three days after viral challenge, there was no detectable BVDV viral RNA in blood of heifers inoculated with ncp BVDV2 on day 75 or day 175 of pregnancy. Viral RNA was amplified from blood of heifers on day 7 p.i. in both PI and TI groups (days 82 and 182 of pregnancy), indicating that viremia occurred by day 7 p.i. (Fig. 1). BVDV RNA was detected in spleen of TI and PI fetuses on day 190 of gestation with significantly higher amounts of viral RNA in PI versus TI fetuses (data not shown).

BVDV antigen was not found in the maternal blood samples at any time point tested or in fetal blood and ear notch extracts of TI and control fetuses. High BVDV antigen levels in the blood and ear notch extracts of PI fetuses confirmed their infection status (data not shown). These data are consistent with the hypothesis that fetuses infected with ncp BVDV early in gestation and before the development of the immune system do not recognize the foreign origin of the infecting virus and are unable to mount strong innate and adaptive immune responses to the virus, thus permitting the persistence of BVDV.23

Low viral neutralizing antibody activity in heifers with PI fetuses was detected on day 90 of pregnancy (day 15 p.i.), gradually increased over time and reached very high titers by day 190 of pregnancy (day 115 p.i.; data not shown). Serum neutralizing antibodies were not detected in heifers with TI fetuses on day 15 p.i. (day 190 of pregnancy), or in blood of any of the fetuses, which probably reflects limited time for seroconversion in TI dams and fetuses and lack of antibody production in PI fetuses. Control heifers remained seronegative for BVDV throughout the experiment.

Differential Blood Cell Gene Expression in Pregnant Heifers with Control, TI or PI Fetuses

Microarray Analysis of Blood Cells from Heifers on Day 190 of Gestation Revealed Differential Gene Expression

Microarray analysis of white blood cells (after the removal of red blood cells) of pregnant heifers on day 190 of gestation revealed 96 differentially expressed genes (fold change >1.5, P < 0.05) in a 3-way analysis (control versus TI versus PI, Fig. 1). Among the highly up-regulated genes in the TI group were IFN stimulated genes (ISGs) such as IFN-stimulated gene 15 kD (ISG15), double-stranded RNA (dsRNA)-dependent protein kinase R (PKR), oligoadenylate synthetase 1 (OAS-1), myxovirus resistance factor 2 (MX2), ISG44, ISG28, as well as several genes from the JAK/STAT pathway. Cytosolic sensor for dsRNA–retinol-induced gene I (RIG-I), considered to be one of the key elements for the detection and elimination of different viruses, including paramyxoviruses, influenza and Japanese encephalitis virus,24,25 was also up-regulated in TI heifers. Because ISG15 is considered a prominent marker for the cascade of events that occur during the innate immune response,26 expression of ISG15 mRNA in whole blood (including all blood cell populations) of pregnant heifers during the course of ncp BVDV2 infection was studied using qRT-PCR (Fig. 2) A dramatic up-regulation of ISG15 mRNA was observed in blood of heifers during the acute (by day 3) stage of infection, when compared with the low baseline ISG15 mRNA expression level in blood from control heifers throughout the experiment. This strong up-regulation of ISG15 expression in maternal blood from both TI and PI groups was transient with a return to comparatively physiologic levels by day 7 p.i. However, ISG15 mRNA expression was still significantly higher in blood of heifers of both PI and TI groups on day 15 post-viral challenge (days 90 and 190 of pregnancy, respectively), when compared with the ISG15 level in blood of control heifers. Differences in the magnitude of ISG15 up-regulation between the TI and PI groups might reflect individual variation of immune responses to the viral infection as well as responses mediated based on the different stages of pregnancy. Even though heifers of the PI group were carrying viremic PI fetuses and subject to continuous exposure to the virus transplacentally, maternal ISG15 expression returned to the baseline expression level by day 45 p.i. (day 120 of pregnancy) and did not differ from the ISG15 expression level in blood from the control heifers throughout the rest of the experiment.

Figure 2.

 Relative expression of ISG15 mRNA in blood cells of pregnant heifers by using qRT-PCR. Profile for all time points of infection (a), and days 90, 182 and 190 (b). Please note the difference in the scale in (a) and (b). *P < 0.05, **P < 0.01, ***P < 0.001. The stars represent blood samples with significantly elevated antiviral activity. Adapted from,21 with permission.

Antiviral Type I IFN Activity was Detected in Blood of TI Heifers and Fetuses

Because ISGs were induced following acute infection with BVDV, an antiviral assay was used to determine if type I IFN also was up-regulated. Serum from heifers inoculated with ncp BVDV2 in early pregnancy (day 75) showed type I IFN antiviral activity by day 7 p.i when tested in a vesicular stomatitis virus assay. Serum from heifers of the TI group presented substantial type I IFN antiviral activity by day 3 p.i. (day 178 of pregnancy). Likewise, the fetal serum at day 15 post-maternal BVDV inoculation (day 190 of gestation; TI fetuses) contained high type I IFN bioactivity in contrast to serum from the six control fetuses (data now shown).21

Many viruses have evolved ways to either evade or interfere with specific members of the type I IFN cascade. Several in vitro studies have indicated that BVDV may have the ability to interrupt the type I IFN response by either actively interfering with or failing to induce the IFN regulatory factors (IRF) 3 and 7.10,11,27 Evidence also exists that BVDV may be able to inhibit the effects of protein kinase R (PKR) by preventing the molecule’s activation by dsRNA.13 Viral interference with the type I IFN pathway, in conjunction with the adaptive immunotolerance of PI, would allow the virus to persist unchallenged by the host immune response. However, in contrast to in vitro studies that have shown a lack of type I IFN activity in ncp BVDV infection, our group21and others28,29 demonstrate production of type I IFN in response to ncp BVDV infection in vivo. This type I IFN response has been identified in acutely infected animals but has not yet been elucidated in PI animals. In fact, several groups have implicated viral interference with type I IFN signaling as a contributing factor to the ability of the virus to establish PI.15,30 We tested whether PI animals exhibited differential gene expression and a type I IFN response.

Microarray Analysis of Blood Cells from Heifers on Day 160 of Gestation

The “classic” approach to microarray analysis, based on inference of potential biomarkers (differentially expressed genes) either by a fold change or by stringent statistical tests (such as t-test or SAM,31 have certain drawbacks. In particular, a stringent selection of reproducible candidate markers leaves little data for the pathway analysis, which can be rendered statistically underpowered. A modification of analysis pipeline to release more data from inference to pathway analysis stage has been advocated.32 In this strategy, more relaxed J5 metric33 was employed here to maximize the overall efficiency of analysis. When applied to Affymetrix microarray screen of d 160 blood cell (red blood cells removed) mRNA from pregnant heifers carrying PI (n = 5) and control non-infected (n = 4) fetuses, this analysis revealed differential expression of approximately 1000 genes from numerous pathways.34 Several members of the type I IFN pathway and members of the chemokine family and chemokine receptors were differentially expressed in blood of PI heifers. Chemokine C-X-C motif receptor 4 (CXCR4) was one of the most significantly down-regulated genes in PI mothers. This was confirmed in whole blood mRNA using qRT-PCR for the duration of the in vivo experiment (Fig. 3). Because CXCR4 was the most consistently down-regulated blood cell gene in heifers carrying PI fetuses, associated signal transducers and targeted maternal blood cells were examined further. Significant down-regulation of CXCR4 mRNA expression was detected with qRT-PCR in blood cells of heifers 7 days after BVDV inoculation of the PI group. CXCR4 expression remained suppressed for 90 days and then returned to baseline expression level by day 175 of gestation (Fig. 3).

Figure 3.

 Relative expression of CXCR4 mRNA in blood of pregnant heifers carrying control, TI or PI fetuses. qRT-PCR revealed that down-regulation of CXCR4 occurs by the time of viremia in infected heifers (day 7 p.i.), and expression remains down-regulated for about 3 months after inoculation. Fold change (control vs PI) is between 2.1 and 2.6, respectively, on days 82–160. **P < 0.01, ***P < 0.001. Adapted from,34 with permission.

The CXCR4 receptor is a G-protein-coupled 7-transmembrane receptor that is expressed on immune cells, platelets and the cells of the central nervous system. It has a unique specific endogenous ligand called stromal derived factor 1 (SDF-1), CXCL12 in new nomenclature. Disruption of CXCL12/CXCR4 interaction impacts multiple biologic processes, such as hematopoiesis, cardiogenesis, vasculogenesis, neuronal development and immune cell trafficking.35,36 Defects in CXCR4 can also lead to embryonic lethality because of the serious developmental defects and defects in B cell lymphopoeisis, bone marrow colonization and cardiac septum formation. CXCR4 expression is suppressed in response to several viral infections, including late stage HIV and human herpesvirus six infection (reviewed in37). Several signal transduction and transcription factors downstream from CXCR4 were examined to determine if down-regulation of CXCR4 is functionally uncoupled with downstream signal transduction pathways in blood cells from mothers carrying PI fetuses (Fig. 4).

Figure 4.

 Down-regulation of CXCR4 and associated signal transduction in blood of heifers (red blood cells removed) with PI fetuses. Microarray analysis on day 160 revealed down-regulation of CXCR4 (fold change 0.57). Several downstream members of the pathway are also down-regulated: guanine nucleotide binding protein (G-protein); leukocyte common antigen precursor, tyrosine phosphatase (CD45) (required for T-cell activation through the antigen receptor); proto-oncogene tyrosine-protein kinase LCK (essential for the selection and maturation of developing T-cell in the thymus and in mature T-cell function, which plays a key role in TCR-linked signaling pathways); E3 ubiquitin-protein ligase CBL (negative regulator of signaling pathways); dual specificity protein kinases MEK1/2 (play a critical role in mitogen growth factor signal transduction), and mitogen-activated protein kinase 1 ERK2 (required for initiation of translation) are down-regulated in blood of mothers with PI fetuses. Adapted from,34 with permission.

Because CXCR4 down-regulation was sustained for approximately 90 days of gestation and was functionally coupled to down-regulation of so many key branch points in signal transduction, further investigation of the signal transduction pathway was undertaken. Down-regulation of several TCR pathway members in blood of heifers carrying PI fetuses also was determined using microarray screen and qRT-PCR: CD3ζ chain, ZAP-70 and MEK1/2 were significantly down-regulated, while there was a trend for TCRγ down-regulation.34 The CD8 was also down-regulated in blood of heifers carrying PI fetuses. The decreased expression of CD8 might be caused by down-regulation of the TCR pathway and reflective of impaired cellular defense responses in pregnant dams. Exactly, why CXCR4 and TCR are down-regulated so long after clearance of the infection is unknown. Rapid down-regulation of the CXCR4 receptor after infection might reflect direct action of the virus on transcription of the CXCR4 gene. However, CXCR4 remains down-regulated even after seroconversion following BVDV infection. Thus, a factor(s) might be released by the PI fetus, which then enters maternal blood to cause down-regulation of the CXCR4 receptor. Disruption of CXCL12/CXCR4 signaling could have deleterious consequences on both development of the fetus and post-natal responses to infections or inflammatory responses.

Suppressed Growth and Differential Gene Expression in PI Fetuses

Persistent BVDV Infection Adversely Affected Fetal Growth and Development

PI fetuses had significantly lower weight at C-section and were smaller when compared by heart girth and crown rump length with fetuses from TI and control groups. The ponderal index, commonly used as an indicator of fetal growth,38 was significantly lower in PI fetuses compared to fetuses of TI and control groups, indicating significant intrauterine growth retardation of the PI fetuses. Gross morphological analysis of the fetal bones revealed thickening of the femoral cortical bone in three of 6 PI fetuses, leading to a significantly smaller internal diameter and volume of intramedullar space. Clinical cases of osteopetrosis in calves born with persistent BVDV infection were described previously.39 Osteopetrosis significantly reduces the medullar space of the bone and can be a restricting factor for the developing bone marrow in PI animals. However, in the present cases, there was no histologic evidence of osteopetrosis and the change is interpreted as a delay in development rather than a frank pathological process.

Gross and histopathologic changes were only seen in fetuses infected at gestational age of 75 days and retrieved at gestational age of 190 days (PI fetuses). While viral antigen could be detected in most tissues from PI fetuses, the primary target organs for histopathologic changes were brain, liver and spleen. In the brain, viral antigen was detected in neurons, oligodendrocyte precursors and infiltrating macrophages. Histological changes included leukomalacia and macrophage infiltration of meninges and neuropil. Other histological changes or deviations from normal fetal development noted in the PI fetuses were increased extramedullary hematopoiesis in the liver, mild decrease in the frequency of megakaryocytes in liver and spleen, mild epi- and myocarditis, and precocious development of the peripheral lymphoid tissues, most notably the lymph nodes,22 the latter corroborating earlier studies.40

We have described a significant intrauterine growth restriction and malformation of the long bones in PI fetuses collected at day 190 of gestation (see21). Malformations of the central nervous system have also been described, including a demyelination of the nerves, ataxia, tremors and abnormal stance.7,41,42 PI animals often appear immunosuppressed as adults, with an increased susceptibility to respiratory infections; however, the evidence for this defect has been circumstantial, and the mechanisms have yet to be elucidated.8,9

Type I IFN Pathway Genes Were Up-Regulated in Blood of PI Steers and in TI and PI Fetuses.

Two classes of receptors serve to detect viral RNA and initiate the antiviral response; Toll-like receptors are located on the cellular membranes of immune cells, whereas the RNA helicases RIG-I and melanoma differentiation protein 5 (MDA5) are located in the cytosol.25,43 RIG-I and MDA5 are ubiquitously expressed, and their binding to double-stranded RNA (dsRNA) results in activation of the receptor and the initiation of a signaling cascade. A third RNA helicase, LGP2, is induced by type I IFN and seems to serve as a negative feedback by binding to both RIG-I and IFN-β promoter stimulator, thus inhibiting their action.44 Members of the type I IFN pathway serve a variety of roles that contribute to the immune defense including growth-suppressive and anti-apoptotic effects, which promote cell survival during viral infection. ISGs produced in response to the recognition of viral infection include ISG15, PKR, OAS-1 and MX2. ISG15 has been a molecule of particular interest because of its intense up-regulation in maternal blood during BVDV infection.21 ISG15 is an ubiquitin-like protein with the ability to conjugate to a variety of intracellular proteins and thus influence their activity through post-translational modification and enhancing the antiviral response, including regulation of IRF-3 and PKR.45,46

Shoemaker et al.47 examined blood cell RNA collected from naturally infected PI steers and age-matched control uninfected steers. Microarray analysis of blood cell mRNA (red blood cell removed) revealed 294 genes that were differentially regulated in PI vs. control steers (P < 0.05, >1.5 fold). Type I IFN pathway genes including ISG15, MX2, OAS-1 and PKR, cytosolic dsRNA sensor genes such as RIG-I and MDA5, recently implicated in type I IFN gene regulation upon cytoplasmic dsRNA stimulation,25,43 and RNA helicase LGP2, a negative regulator of RIG-I function, were significantly up-regulated in blood from PI steers. qRT-PCR on whole blood confirmed that RIG-I and MDA-5, which activate production of type I IFN, also were highly up-regulated in blood of TI fetuses 15 days p.i., and, to a lesser degree, in the blood of PI fetuses 115 days p.i. (Fig. 5). LGP2 was also significantly up-regulated in blood of TI and PI fetuses and steers. All four ISGs tested (ISG15, MX2, PKR and OAS-1) were highly up-regulated in PI steer blood and in blood of TI fetuses. Expression of MX2 and PKR was significantly up-regulated in blood of PI fetuses; however, while ISG15 and OAS-1 expression showed the same trend, the difference between PI and control fetal blood was not significant because of high variation between animals. Analysis of the gene expression in fetal spleen with qRT-PCR revealed up-regulation of all tested ISGs in TI fetuses. This finding is consistent with increased antiviral activity in blood from TI fetuses. Our interpretation is that infection with ncp BVDV induces a robust type I IFN antiviral response in acutely infected animals, including developing fetuses.

Figure 5.

 Cellular responses to infection with viral RNA. Infection with BVDV causes up-regulation of IFN and ISGs in fetal TI and PI as well as in steer PI blood cells. Adapted from,47 with permission.

Up-regulation of ISG15 in Day 190 Fetal Spleen Following TI and PI

Western blot analysis of proteins from fetal spleen revealed high amounts of both conjugated and free ISG15 protein in TI fetuses (Fig. 6a), while low ISG15 was detected in PI or control fetuses. Western blot for ubiquitin-like activating enzyme, E1 like (UBE1L), known to act as ISG15 conjugating enzyme, revealed UBE1L expression in the spleen of TI fetuses (Fig. 6a), while no significant amount of UBE1L was detected in control or PI fetuses. Quantitative analysis of the Western blot confirmed significant and very profound up-regulation of ISG15 protein, both free and conjugated, in spleen of TI fetuses (Fig. 6b).

Figure 6.

 ISG15 and conjugation to targeted proteins is up-regulated in spleens from TI when compared to PI and control fetuses. (a) Western blot analysis of ISG15 and UBE1L protein in fetal spleen. (b) Quantitative analysis of free and conjugated ISG15 and UBE1L in spleen of infected fetuses. Recombinant bovine ISG15 (rboISG15) served as a positive control. Image Quant 5.2 software was used to quantify the intensity of protein bands. (c) Immuno-histochemical localization of ISG15 in fetal spleen. Controls included Japanese encephalitis virus antibody and omission of primary antibody, neither of which showed staining. Adapted from,47 with permission.

Immunohistochemical localization of ISG15 in control spleens was discretely limited to reticular and macrophage-like cells. A much more diffuse pattern of ISG15 antigen was detected in the PI and TI spleens, with strong signals localized to endothelial cells of the sinusoids and blood vessels, reticular cells and macrophage-like cells. The intensity of ISG15 staining in the TI spleen was notably stronger than in the PI.

IFNs regulate the cell cycle by inhibiting G1 phase and by lengthening G1, G2 and S phases.48 Cell cycle can also be mediated through IFNs by down-regulating cyclins and cyclin-dependant kinases.49 The antiproliferative action of IFN also is medicated by PKR, which inactivates eiF-2α and up-regulates cyclin-dependant kinase inhibitor p21/WAF1 resulting in inhibition of protein synthesis.50–53 Antiproliferative responses induced by IFN impede synthesis of viral proteins and delay growth of host cells. However, chronic expression of growth inhibitory genes may impair growth and development of a PI animal. For example, we have demonstrated that intrauterine growth restriction occurs in PI fetuses21 and this might be associated with chronic exposure to type I IFN.

PI animals typically have normal growth and do not always demonstrate obvious outward signs of physical debilitation. However, a slight inhibition of growth during fetal development or post-natal life can have drastic economic consequences. Leukocyte profiles and antigen-presenting capabilities of PI animals have been reported to be normal.54,55 Extensive study of the innate response has not been thoroughly examined with respect to a contribution to a weak immune response. It is possible that this continued stimulation of the IFN pathway may decrease the ability of the innate antiviral system to respond to other viruses, thus increasing their susceptibility to infections later in life.

Conclusions

BVDV causes viral diarrhea in herds of cattle and leads to significant economical loss in the USA and other countries.56–58 The BVDV genome is prone to high mutation rates because of the absence of proofreading activity of the viral replicase and genetic recombination between viruses of different genotypes.59 Genomic mutations create antigenic diversity of the viral strains, which promotes survival of the virus by allowing re-infection of host populations with a heterogeneous strain,60 although genetically invariant BVDVs can be found within the same herd for several years. BVDV represents a useful model for studying the complex virus–host interactions in intrauterine infections of the fetus. The vast majority of studies on PI calves are based on clinical or field observations, or following short-term-induced experimental infection in vivo,55,61–63 with only a few studies focused on transplacental infection of fetuses by viral challenge of their mothers at different stages of pregnancy.64–67

One unexpected finding from the microarray comparison of blood cell gene expression between heifers carrying PI vs. healthy fetuses was the very significant down-regulation of the CXCL12/CXCR4 signaling pathway by the time of established viremia, which lasted for approximately 90 days following infection with BVDV. While CXCR4 has been reported to decline in response to other viral infections,37 there are no reports of a possible role of the CXCL12/CXCR4 pathway in BVDV pathogenesis.

We conclude that PI fetuses, whose dams were infected with ncp BVDV at day 75 of gestation, are able to mount an IFN response to the infecting virus, but because of insufficient development of the immune system and the consequent lack of BVDV antigen recognition they are not able to clear the virus. Persistence of the virus causes prolonged stimulation of type I IFN pathway genes: up-regulation of ISGs occurred to varying degrees even 115 days p.i. (in PI fetuses) and after birth (PI steers). Presence of chronic stimulation of the IFN pathway as reflected by ISG expression in both PI steer and fetal blood, as well as a microarray gene expression profile may prove detrimental for the PI host. Microarray analysis of PI steers compared to uninfected controls revealed a gene pattern that was reflective of an active IFN response; in addition to up-regulation of ISGs (e.g. MX2, ISG15), downstream effects of the cascade were evident. In adult animals and, presumably, immunocompetent fetuses the type I IFN response is transient and returns to basal level after the clearance of the virus. However, in PI fetuses with undeveloped adaptive immunity, the persisting virus is not recognized as foreign, which leads to lifelong persistent infection, and, as we have shown, to chronic up-regulation of type I IFN. Because type I IFN can act as a growth-suppressive cytokine,68 a long-term up-regulation of ISGs may contribute to the intrauterine growth restriction, seen in animals with persistent BVDV infection. The function of ISGs induced following BVDV infection will require future study, particularly in terms of implications for fetal growth and development of the immune response.

The maternal blood cell response to fetal persistent infection with BVDV continues to be a focus of our experiments. In addition, experiments are planned that will help clarify how the PI fetus regulates maternal immune and platelet cell function and when and why fetal growth is impaired as a result of the inability to resolve viral infection. Better management of BVDV infections would have dramatic benefits for the cattle industry, especially in reference to eliminating the PI reservoir. Congenital BVDV infection of bovine fetuses is a reproducible model by which one can study fetal infections leading to PI, immunologic consequences and fetal pathology. A better understanding of BVDV pathogenesis and the mechanisms underlying the establishment and maintenance of persistent BVDV infection will greatly contribute to establishing methods of control of the virus.

Acknowledgments

Research was funded by USDA-NIFA-AFRI grants 2004-35204-17005 and 2008-35204-04652. Authors thank Ms. Kathleen Austin, Dr. Alberto van Olphen, Dr. Don Montgomery and Dr. Hyungchul Han (University of Wyoming) for help with collection of tissues and preliminary experiments contributing to this review.

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