Foot-and-mouth disease virus causes transplacental infection and death in foetal lambs.

Transcription

1 Appendix 6 Foot-and-mouth disease virus causes transplacental infection and death in foetal lambs Eoin Ryan 1,2 *, Jacquelyn Horsington 1, Harriet Brooks 2, Joe Brownlie 2, Zhidong Zhang 1. 1 Pirbright Laboratory, Institute for Animal Health, Ash Road, Woking, Surrey, GU2 NF, UK. 2 Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, AL9 7TA, UK. *eoin-d.ryan@bbsrc.ac.uk Abstract: Introduction: There are field reports of foot-and-mouth disease virus (FMDV) causing severe abortion in sheep and yet there are no published studies of experimental infection of pregnant sheep. This would appear to be the first reported investigation of experimental FMDV pathogenesis in foetal lambs. Materials and Methods: Using FMDV type O/UKG/21/3, we infected pregnant sheep at two stages of pregnancy, 5 days and 75 days, and sequentially killed the dams postinoculation. Foetal lambs, the dams and foetal fluids were examined for virus using viral isolation and real time reverse-transcription polymerase chain reaction. Foetal tissues were also examined for the major cytokines (IFN, IFN, IFN, TNF, IL-1). In situ hybridisation (ISH) was used to visualise FMDV RNA in foetal tissues. Results: Infectious virus and viral RNA were isolated from amniotic fluid and foetuses from 2 onwards. At 7, generalised systemic infections had been established in most foetuses. At 18 in the 75-day foetuses, 5 of 7 foetuses were infected; of these, had died in utero. There was an acute anti-viral cytokine response (IFN, IFN) at 2 and, with the pro-inflammatory cytokines (IFN, TNF and IL-1) increasing from 7. Using ISH, viral RNA was visualised in tongue epithelium, myocardium and skeletal muscle. Discussion: The results show FMDV caused transplacental infection in foetal lambs, and was associated with the deaths of some foetuses. There was an excellent degree of correlation between the increase in cytokines and foetal deaths, and increased cytokine levels were particularly noticeable in the myocardium. The presence of infectious virus in foetal fluids at 18 demonstrates the potential for FMDV-induced abortion to cause further disease transmission, an epidemiological factor previously overlooked. Introduction: Foot-and-mouth disease (FMD) in sheep is milder than in cattle or pigs, with viraemia present for up to three days before the appearance of vesicular lesions, as reviewed by Alexandersen et al (23). During this time the sheep may be pyrexic and distressed with lameness spreading through the flock. Agalactia may occur in ewes. Vesicular lesions occur in the interdigital cleft, along the coronary bands and on the bulb of the heels. Oral lesions are less common but can occur on the dental pad, tongue and gums (Barnett and Cox, 1999, Hughes et al., 22, Pay T. W, 1988). Death may occur in young lambs (Ryan, 2). There are field reports of FMD causing severe outbreaks of abortion in sheep (Geering, 1967, Nazlioglu, 1972) yet there are no published studies of experimental infection of pregnant farm animals. Oppermann (1921), cited by Littlejohn (197), reported that in the 192 epizootic in Germany the average abortion rate was 3-1% but was sometimes up to 37%, and as high as 1% in isolated cases. Abortion occurred in ewes at various stages of gestation and usually began 1 days to six weeks after the start of the outbreak. Parakin (1961), cited by Littlejohn (197), described an outbreak in Russia in which 23 of a flock of 75 ewes aborted, and mentioned that abortion may occur as early as the first to second month of gestation. During the 21 outbreak in the UK, large numbers of abortions were reported in sheep in Dumfries within 2 hours of the onset of lameness in the flock (Reid, 22). Andersen and Campbell (1976) reported inoculation of pregnant mice with an attenuated FMDV type O strain, and concluded that the placenta serves as 38

2 an active site of infection for FMDV in pregnant mice, but the foetus is relatively resistant to infection. The absence of previous experimental data regarding the possible transmission of FMDV transplacentally, its role in causing abortion and the potential of the foetus and associated fluids to act as a reservoir of infection has hindered understanding of the disease. The epidemiological significance of a uterine reservoir of virus has not previously been examined. For these reasons, we carried out a series of experiments to address these issues. In the present study, we demonstrated that FMDV caused transplacental infection and foetal death. Materials and methods: 21 ewes, 7 pregnant 5 days and 1 pregnant 75 days, were used. Three ewes, one pregnant 5 days and two pregnant 75 days, were killed as negative controls and negative control samples of serum were taken from all remaining ewes prior to inoculation. The ewes were inoculated in the coronary band with FMDV O UKG3/21, as described previously (Alexandersen and Donaldson, 22), each animal receiving 5.9 log1 TCID 5 ml -1. Serum samples were collected daily, temperatures were measured and clinical signs noted. Two ewes were killed from each pregnancy group at two, four and seven days post inoculation (). Of the remaining six sheep at day 75 of pregnancy, three were killed at 17 and three at 18 (see table 1). Serum samples were tested for the presence of antibodies to FMDV by an enzyme-linked immunosorbent assay (ELISA) (Ferris, 1987; Ferris et al., 199). At post-mortem examination, tissue samples were taken and put in RNAlater (Ambion) for RNA extraction, 5% (v/v) glycerol in M25 phosphate buffered saline and 1% formalin for histological examination. Amniotic fluid samples were taken and stored at -8 o C. Tissue samples in RNAlater were stored at -2 o C. Due to their smaller size and less developed stage, 8-11 different tissue samples were taken from each foetus at day 5 of gestation, compared to 1 samples per foetus at day 75 of gestation. Automated RNA extraction from samples was performed using a MagNA Pure LC robotic workstation (Roche) and the accompanying nucleic acid isolation kits (Roche), according to the manufacturer s instructions, as described previously (Quan et al., 2). Extracted RNA was stored at -8 o C until used. The level of viral RNA in samples was quantified by real-time RT-PCR as described previously (Alexandersen et al., 21, Reid et al., 21, Zhang and Alexandersen, 23), using a Taqman probe and primers specific for FMDV type O strain UKG 3/21 as described previously (Quan et al., 2). Similarly, levels of IFN, IFN, IFN, TNF and IL-1 were quantified by real-time RT-PCR using specific Taqman probes and primers, as described previously (Zhang et al., 26). Cytokine levels were normalised to the housekeeping gene GAPDH. PCR assays were performed on a Stratagene MX machine. Statistical analysis was carried out using Minitab version 1. The infectivity of tissue samples was determined by inoculation of monolayers of primary bovine thyroid (BTY) cells, essentially as previously described (Snowdon, 1966). Serotyping ELISA was used to confirm the presence of FMDV in the supernatant of the positive tubes (Ferris and Dawson, 1988). In situ hybridisation (ISH) was carried out using a digoxenin-labelled RNA probe to the 3D region of the FMDV genome. ISH was performed on formalin-fixed, paraffin-embedded sections using an mrnalocator ISH kit (Ambion). An RNA ISH probe for SVDV was used as a negative control on all sections, and ISH was also performed on tissues from control foetuses as a further negative control. Results: No vesicular lesions observed on foetuses: None of the 11 foetuses at day 5 of gestation at inoculation had grossly visible pathological changes. In the group of day 75 of gestation at the start of the experiment, of the 19 foetuses, 1 had gross abnormalities, which included multiple cutaneous petechial haemorrhages, reddened peritoneal fluid, subcutaneous oedema, ascites and epicardial petechiae (table 1). Two of these foetuses had no detectable viral RNA and were negative for virus isolation. Three of the foetuses at 17 and one at 18 were not freshly dead at post-mortem examination and were showing signs of autolysis. However, no vesicular lesions were visible on any foetuses. No 381

3 significant pathological lesions were visible on histological examination of foetal and placental tissues. Table 1: The gestational age and gross pathological lesions in foetuses. Foetu s ID Gestati onal age* dp i Gross abnormalities at post-mortem examination Foet us ID Gestation al age* * The gestational age of foetuses at the day the ewes were inoculated. : days post inoculation at the day the ewes were killed. Gross abnormalities at post-mortem examination FA53 5 None FA None FA5 5 2 None FB None FA None FA11 75 Dead before FB None FB11 75 Cutaneous petechial haemorrhages FA56 5 None FA12 75 None FB56 5 None FB12 75 None FA57 5 None FA Cutaneous petechiation FB57 5 None FB Cutaneous petechiation FA None FA Cutaneous petechiation, reddened peritoneal fluid FB None FB None FA None FA Dead, autolyzed, reddened subcutaneous oedema FB None FA None FA5 75 None FA Dead, autolyzed, swollen abdomen FB5 75 None FB Dead, autolyzed, swollen abdomen FA6 75 None FA Cutaneous petechiation, ascites, epicardial haemorrhages FA None FA Cutaneous petechiation FB None FA Dead, autolyzed Viral RNA and infectious virus in foetal tissues during acute infection: Viral RNA levels in tissues from foetuses which were at 5 days gestation at maternal inoculation are shown in table 2. Low levels of viral RNA were detected in a few tissues at 2 and, but at 7 viral RNA was detected in every tissue sample examined from all four foetuses although there was no detectable viraemia in ewes at this time. Among the tissues examined from these foetuses, heart and tongue samples contained the highest level of viral RNA. 382

4 Table 2 Viral RNA in foetal tissues, 5 days gestation Viral RNA load 2 7 foetus FA53* FA5 FA55 FB55 FA56 FB56 FA57 FB57 FA58 FB58 FA59 FB59 ID IV N N N P N N N N P P P P intestine ND heart kidney leg liver lung mouth ND soft ND ND ND ND ND ND ND palate spleen ND ND ND ND tongue ND ND ND ND ND ND * uninfected foetus was killed as control and tissues collected for analysis. viral RNA loads in tissues were quantified by real-time RT PCR and expressed as log 1 copy number per g tissue ND: no samples taken. IV: infectious virus in tissue samples. P: positive, N: negative : days post inoculation at the day the ewes were killed Viral RNA levels in tissues from foetuses which were at 75 days gestation at maternal inoculation are shown in table 3. No viral RNA was detected at 2, and only low levels at. At 7, although there was no detectable viraemia in ewes at this time, viral RNA was detected in every tissue sample from three of the foetuses. Among the tissues examined, muscle and tongue samples contained the highest level of viral RNA. At 17, three of the four foetuses contained viral RNA. All of them were dead and autolyzed at post-mortem examination. At 18, two of three foetuses were positive for viral RNA. One foetus, dead and autolyzed at post-mortem examination, had viral RNA in all tissue samples at a higher level than those in the second foetus, which had survived to this timepoint and which had viral RNA in 8 of 1 tissue samples examined. As shown in tables 2 and 3, infectious virus was isolated from foetuses at both stages of pregnancy. In the 5 day gestation group (n=11), of eight foetuses which were positive for viral RNA, five tested positive for infectious virus and three negative. In the 75 day gestation group, among foetus tissues examined (n=12), virus was only isolated from tissues collected at 7 from the three foetuses (n=3) in which viral RNA had been detected. It was not possible to perform virus isolation on samples from 17 and 18 due to inadvertent sample deterioration during processing. Viral RNA and infectious virus in amniotic fluid samples: Data on viral RNA load and infectious virus in amniotic fluid samples is summarised in table. Viral RNA was detected in 5 of 11 amniotic fluid samples taken from the group at day 5 of gestation at the start of the experiment and in 7 of 19 amniotic fluid samples taken from the group at day 75 of gestation at the start of the experiment. As shown in table, amniotic fluid associated with foetuses which were free of viral RNA (FA5, FB57) could contain detectable level of viral RNA, while the other amniotic fluid samples did not contain detectable viral RNA, although the foetuses associated with them were positive for viral RNA (FA58, FB59) (table 2). Infectious virus was isolated from amniotic fluid samples collected from both groups. The supernatant from all positive virus isolation tubes was confirmed as FMDV type O by serotyping ELISA. 383

5 Table Quantification of viral RNA in amniotic fluid* Foetus ID Viral load IV Foetus ID Viral load IV FA53 # N FA17 N FA5.96 P FB17 N FA55 N FA11 N FB55 P FB11 N FA56 N FA12 N FB56 P FB12 N FA P FA9 N FB P FB9 P FA58 N FA P FB P FB P FA P FA7.69 P FB59 P FA8 N FA5 # N FA1.25 N FB5 # N FB1.28 N FA6 # N FA P FA16 N FA15 N FB16 N FA P *Amniotic fluid samples were collected from both the group at day 5 of gestation at the start of the experiment and the group 75 days pregnant at the start of the experiment. The viral RNA levels were quantified by real-time RT PCR and expressed as log 1 copy number per ml sample. # uninfected foetuses as controls IV: infectious virus in tissue samples. P: positive, N: negative Cytokine responses in foetuses at day 75 of gestation: Levels of IFN and IFN increased in the cervical lymph nodes (LN) of infected foetuses at 2 and by a factor of 3- times control levels. By 7, levels were increased in all tissues by factors ranging from 3. (spleen) to 2 (kidney) times control levels, while the levels in skin increased by 319 and 252 times for IFN and IFN respectively. At 17/18, levels in all tissues increased by factors ranging from 73 (skin) to 18,552 (heart). When ANOVA was carried out, the difference in IFN response between the infected and uninfected foetuses was found to be significant (p<.5) in the coronary band, intestine, heart, liver, lung, soft palate, spleen and tongue. For IFN, a significant (p<.5) difference was found in all tissues except skin and tongue. Scatterplots of log 1 fold increases in levels of IFN are shown in fig. 1 and IFN in fig. 2. Levels of IFN did not increase at 2 and, but there was an increase at 7 in some tissues, with cervical LN and tonsil having 11 times control levels and skin showing an increase of 51 times control levels. At 17/18, levels in all tissues increased by factors ranging from 235 (mandibular LN) to 1,13 (heart) (see fig. 3). A significant (p<.5) difference was found in the IFN response between infected and uninfected foetuses in the cervical LN, heart, liver, muscle and spleen. Levels of TNF increased in the cervical LN by a factor of 1 at 2, but not in other tissues. At, levels in the cervical LN were increased by 2 times, while levels in some other tissues increased by factors of 2 (tonsil) to 8 (intestine), with no increase in coronary band or muscle. At 7, there was an increase of 95 times control levels in skin, with increases in other tissues ranging from 3 times (spleen) to 1 times (cervical LN) control levels. By 17/18, levels in all tissues increased by factors ranging from 112 (mandibular LN) to 2,933 (heart) (see fig. ). The differences in the TNF responses in the cervical LN, heart, lung and spleen were found to be significant (p<.5). Levels of IL-1 did not increase in any tissues, and showed a slight decrease in some tissues, at 2, and 7. At 17/18, levels were increased in all tissues by factors ranging from 25 (coronary band) to 3717 (liver) times control levels (see fig. 5). All tissues except kidney, liver, mandibular LN and tonsil showed a significantly (p<.5) different IL- 1 response in the infected foetuses. 38

6 Viral RNA visualistion in tissues using in situ hybridisation: Positive ISH signal was detected at the highest levels in the skeletal muscle of the tongue and leg, in the cardiac muscle and in the orbital muscles. Positive signal was only occasionally seen in the epithelium of the tongue. Viral RNA was visualised in the cytoplasm of the infected cells. Figure 6 shows positive ISH signal in these tissues. Discussion: FMDV infection in foetuses: Although all ewes developed clinical FMD and viraemia following inoculation, not all foetuses became infected. In some cases, one twin became infected while a co-twin did not. The reason for this phenomenon remains unclear. The results do not demonstrate a minimum maternal viraemia level necessary for transplacental infection, as the ewe with the lowest amount of viral RNA in its serum still transmitted FMDV to its foetus. The gross abnormalities in the foetuses included multiple cutaneous petechial haemorrhages, reddened peritoneal fluid, subcutaneous oedema, ascites and epicardial haemorrhages. These are non-specific findings, and viral replication has not been shown to be directly responsible for them. The finding that two uninfected foetuses exhibited these changes supports this. One foetus killed at 7 (foetus FB13) was positive by real time RT-PCR and virus isolation, yet no gross abnormalities were recognised. No placental abnormalities were recognised. In the infected foetuses at 2 and, a low level of viral RNA was detected in epithelial tissues such as the coronary band, tongue, skin, soft palate and mouth. These are tissues that are the sites of initial virus replication in adult infections (Alexandersen et al., 23). The virus replicated at these sites and spread throughout the body of the foetus. Transplacental infection occurred at 2-, the time of peak maternal viraemia. By 7, the infection had become systemic, with viral RNA detected at high levels throughout the foetus. Amniotic fluid samples associated with infected foetuses tested positive by virus isolation and real-time RT-PCR. The volume of amniotic fluid associated with a sheep foetus is variable and is influenced by the stage of gestation and whether the foetus is a singleton, twin or triplet (Wintour et al., 1986). We estimated the average volume per foetus in this study at day 5 to be 2-3 ml and at 75 days to be 5-7 ml. Thus if an infected foetus and associated fluids were expelled from the ewe after FMD-related abortion, a large amount of FMDV would be released into the surrounding environment. This could have profound effects on the spread of the disease. A pregnant sheep could be infected with FMDV but not show severe clinical signs and thus escape detection. We have shown that up to 18 days later, such a sheep, now fully recovered from disease and showing no clinical signs, may be carrying a foetus and associated foetal fluids containing large amounts of virus. Cytokine response to FMDV infection: The increase in levels of mrna of the anti-viral cytokines IFN and IFN and the pro-inflammatory cytokines IFN, TNF and IL-1 during foetal FMDV infection provides an insight into the pathogenesis of the disease. The initial type 1 interferon (α and β) response occurred at 2 and, when the virus was crossing the placenta and establishing an infection. FMDV has been shown to inhibit type 1 interferon expression by L proteinase blocking both translation and transcription in cell culture (Chinsangaram et al., 1999, de Los Santos et al., 26). This has not been shown in vivo, but a reduction in type 1 interferon production may occur to some degree. Other work has shown an up-regulation of IFN mrna during FMD in cattle (Brown et al., 2). The data presented here agrees with that, showing an acute innate immune anti-viral response to FMDV. This initial type 1 interferon response was followed with a pro-inflammatory response from 7. Although it has been proposed that the ovine foetal immune system has a Th2 bias due to placental progesterone (Entrican, 22), the strong Th1 response shown here suggests this may not be correct. IFN has been shown to be an inhibitor of persistent FMDV infection (Zhang et al., 22), but it also stimulates TNF release. The responses to TNF and IL-1 in foetal lambs have been studied as a model for premature babies (Ikegami et al., 23, Mulrooney et al., 2). IL-1 was associated with chorioamnionitis, 385

7 bronchopulmonary dysplasia and premature parturition, while TNF was a potent inducer of a generalised inflammatory response. The strong correlation between increases in these cytokines and foetal death, particularly in the heart tissue, suggest that cytokine-mediated viral-induced inflammation was a factor in the in utero deaths. Viral RNA visualistion in tissues using in situ hybridisation: The localisation of viral RNA chiefly in the skeletal and cardiac muscle shows that FMDV in foetuses has a different tissue tropism than in adults, and also explains the lack of epithelial vesicular lesions. We have previously reported viral RNA in the myocardium of infected young lambs (Ryan, 2), but not in the myocytes of the tongue, leg and orbital muscles. This study is the first we are aware of to demonstrate the ability of FMDV to cause infection and death in foetal lambs. It establishes that amniotic fluid may contain FMDV up to 18 - a possible mechanism by which infected but recovered pregnant sheep may cause fresh outbreaks of FMD by release of aborted foetuses and associated fluids. Conclusions: FMDV causes transplacental infection and death in foetal lambs. Foetal pathology and death was associated with a pro-inflammatory cytokine response and virus localisation in skeletal and cardiac muscle. Infected foetuses and associated fluids can be infectious up to 18. Recommendations: Further research into infection at different stages of gestation. Research on transplacental transmission in vaccinated sheep. Research on epidemiological risk posed by uterine reservoir of infection. References: Alexandersen, S., and A. I. Donaldson. 22. Further studies to quantify the dose of natural aerosols of foot-and-mouth disease virus for pigs. Epidemiol Infect 128: Alexandersen, S., M. B. Oleksiewicz, and A. I. Donaldson. 21. The early pathogenesis of foot-and-mouth disease in pigs infected by contact: a quantitative time-course study using TaqMan RT-PCR. J Gen Virol 82: Alexandersen, S., Z. Zhang, A. I. Donaldson, and A. J. M. Garland. 23. The pathogenesis and diagnosis of foot-and-mouth disease. J Comp Pathol 129:1-36. Andersen, A. A., and C. H. Campbell Experimental placental transfer of foot-andmouth disease virus in mice. Am J Vet Res 37: Barnett, P. V., and S. J. Cox The role of small ruminants in the epidemiology and transmission of foot-and-mouth disease. Vet J 158:6-13. Brown, C. C., J. Chinsangaram, and M. J. Grubman. 2. Type I interferon production in cattle infected with 2 strains of foot-and-mouth disease virus, as determined by in situ hybridization. Can J Vet Res 6:13-3. Chinsangaram, J., M. E. Piccone, and M. J. Grubman Ability of foot-and-mouth disease virus to form plaques in cell culture is associated with suppression of alpha/beta interferon. J Virol 73: de Los Santos, T., S. de Avila Botton, R. Weiblen, and M. J. Grubman. 26. The leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response. J Virol 8: Entrican, G. 22. Immune regulation during pregnancy and host-pathogen interactions in infectious abortion. J Comp Pathol 126:

8 Ferris, N. P., and M. Dawson Routine application of enzyme-linked immunosorbent assay in comparison with complement fixation for the diagnosis of foot-and-mouth and swine vesicular diseases. Vet Microbiol 16:21-9. Geering W, G Foot and mouth disease in sheep. Aust Vet J. Hughes, G. J., V. Mioulet, R. P. Kitching, M. E. Woolhouse, S. Alexandersen, and A. I. Donaldson. 22. Foot-and-mouth disease virus infection of sheep: implications for diagnosis and control. Vet Rec 15:72-7. Ikegami, M., T. J. Moss, S. G. Kallapur, N. Mulrooney, B. W. Kramer, I. Nitsos, C. J. Bachurski, J. P. Newnham, and A. H. Jobe. 23. Minimal lung and systemic responses to TNF-alpha in preterm sheep. American journal of physiology. Lung cellular and molecular physiology 285:L Littlejohn, A FMD in sheep - part 1. State Veterinary Journal 25:3-12. Mulrooney, N., A. H. Jobe, and M. Ikegami. 2. Lung inflammatory responses to intratracheal interleukin-1alpha in ventilated preterm lambs. Pediatr Res 55: Nazlioglu, M Fmd in Sheep and Goats. Bulletin Office International des Epizooties 77: Pay T. W, F FMD in sheep and goats: a review. FMD Bulletin 26:2-13. Quan, M., C. M. Murphy, Z. Zhang, and S. Alexandersen. 2. Determinants of early foot-and-mouth disease virus dynamics in pigs. J Comp Pathol 131: Reid, H. W. 22. FMD in a parturient sheep flock. Vet Rec 15:791. Reid, S. M., N. P. Ferris, G. H. Hutchings, Z. Zhang, G. J. Belsham, and S. Alexandersen. 21. Diagnosis of foot-and-mouth disease by real-time fluorogenic PCR assay. Vet Rec 19: Ryan, E., Durand, S., Brownlie, J., Alexandersen, S. 2. Presented at the Research Group of the Standing Technical Committe of the European Commission for the Control of Foot-and-Mouth Disease, Chania, Crete, Greece. Snowdon, W. A Growth of foot-and mouth disease virus in monolayer cultures of calf thyroid cells. Nature 21: Wintour, E. M., B. M. Laurence, and B. E. Lingwood Anatomy, physiology and pathology of the amniotic and allantoic compartments in the sheep and cow. Aust Vet J 63: Zhang, Z., J. B. Bashiruddin, C. Doel, J. Horsington, S. Durand, and S. Alexandersen. 26. Cytokine and Toll-like receptor mrnas in the nasalassociated lymphoid tissues of cattle during foot-and-mouth disease virus infection. J Comp Pathol 13: Zhang, Z. D., and S. Alexandersen. 23. Detection of carrier cattle and sheep persistently infected with foot-and-mouth disease virus by a rapid real-time RT- PCR assay. J Virol Methods 111:95-1. Zhang, Z. D., G. Hutching, P. Kitching, and S. Alexandersen. 22. The effects of gamma interferon on replication of foot-and- mouth disease virus in persistently infected bovine cells. Arch Virol 17:

9 Fold increase in log1 IFNalpha mrna levels vs. control CB Cervical LN Heart Intestine Kidney Liver Lung Mandibular LN muscle skin Soft palate Spleen Tongue Fig. 1: Scatterplot of log1 IFNalpha vs Gestational stage = 75 days 2 Tonsil Panel variable: tissue type status infected not infected Fold increase in log1 IFNbeta mrna levels vs. control CB Cervical LN Heart Intestine Kidney Liver Lung Mandibular LN muscle skin Soft palate Spleen Tongue 1 2 Panel variable: Tissue Fig. 2: Scatterplot of log1 IFNbeta vs Gestational stage = 75 days Tonsil status infected not infected Fold increase in log1 IFNgamma mrna levels vs. control CB Cervical LN Heart Intestine Kidney Liver Lung Mandibular LN muscle skin Soft palate Spleen Tongue 1 2 Panel variable: Tissue Fig. 3: Scatterplot of log1 IFNgamma vs Gestational stage = 75 days Tonsil status infected not infected Figures 1-3: Scatterplots showing fold increases compared to controls in IFN, IFN and IFN levels in infected and uninfected foetuses on a log scale. 388

10 Fold increase in log1 TNFalpha mrna levels vs. control CB Cervical LN Heart Intestine Kidney Liver Lung Mandibular LN muscle skin Soft palate Spleen Tongue 1 2 Panel variable: Tissue Fig. : Scatterplot of log1 TNFalpha vs Gestational stage = 75 days Tonsil status infected not infected Fold increase in log1 IL-1alpha mrna levels vs. control CB Cervical LN Heart Intestine Kidney Liver Lung Mandibular LN muscle skin Soft palate Spleen Tongue 1 2 Panel variable: Tissue Fig. 5: Scatterplot of log1 IL-1alpha vs Gestational stage = 75 days Tonsil status infected not infected Figures &5: Scatterplots showing fold increases compared to controls in TNF and IL-1 levels in infected and uninfected foetuses on a log scale. 389

11 6A 6B 6C 6D 6E 6F 6G 6H Figure 6: ISH on sections from FMDV-infected foetuses. A: tongue from foetus FA1, x magnification, and B: tongue from FA1, x, showing abundant positive signal in myocytes. C: tongue from FB9, x, showing viral RNA in basal layer of epithelium and in muscle. D: tongue from FB9, 63x, showing cytoplasmic staining in multinucleated myocytes. E: heart from FB9, x, with diffuse positive signal in myocardium. F: heart from FA58, x, with focal area of viral RNA in myocardium. G: leg section from FA58, x, positive signal in skeletal muscle. H: orbital muscle from FB58, x, positive cytoplasmic staining. 39

12 Table 3: Viral RNA in foetal tissues, 75 days gestation * three uninfected foetuses were killed as controls and tissues collected for analysis. viral RNA loads in tissues were quantified by real-time RT PCR and expressed as log 1 copy number per g tissue. Viral RNA load foetus ID control* FA16 FB16 FA17 FB17 FA11 FB11 FA12 FB12 FA9 FB9 FA13 FB13 FA7 FA8 FA1 FB1 FA1 FA15 FA18 IV N N N N N N N N N N P P P ND ND ND ND ND ND ND coronary band cervical LN ND intestine heart kidney liver lung mandibular ND ND ND ND LN muscle skin spleen ND ND tongue soft palate tonsil ND ND ND ND ND: no samples taken. : days post inoculation at the day the ewes were killed IV: infectious virus in tissue samples. P: positive, N: negative 391