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Platelet Activation Determines Angiopoietin-1 and VEGF Levels in Malaria: Implications for Their Use as Biomarkers

  • Judith Brouwers ,

    Contributed equally to this work with: Judith Brouwers, Rintis Noviyanti

    Affiliation Department of Clinical Chemistry and Haematology, University Medical Centre, Utrecht, The Netherlands

  • Rintis Noviyanti ,

    Contributed equally to this work with: Judith Brouwers, Rintis Noviyanti

    Affiliation Eijkman Institute for Molecular Biology, Jakarta, Indonesia

  • Rob Fijnheer,

    Affiliation Department of Clinical Chemistry and Haematology, University Medical Centre, Utrecht, The Netherlands

  • Philip G. de Groot,

    Affiliation Department of Clinical Chemistry and Haematology, University Medical Centre, Utrecht, The Netherlands

  • Leily Trianty,

    Affiliation Eijkman Institute for Molecular Biology, Jakarta, Indonesia

  • Siti Mudaliana,

    Affiliation Eijkman Institute for Molecular Biology, Jakarta, Indonesia

  • Mark Roest,

    Affiliation Department of Clinical Chemistry and Haematology, University Medical Centre, Utrecht, The Netherlands

  • Din Syafruddin,

    Affiliation Eijkman Institute for Molecular Biology, Jakarta, Indonesia

  • Andre van der Ven,

    Affiliation Department of Internal Medicine, Radboud University Nijmegen Medical Centre, The Netherlands

  • Quirijn de Mast

    q.demast@aig.umcn.nl

    Affiliations Department of Clinical Chemistry and Haematology, University Medical Centre, Utrecht, The Netherlands, Department of Internal Medicine, Radboud University Nijmegen Medical Centre, The Netherlands

Platelet Activation Determines Angiopoietin-1 and VEGF Levels in Malaria: Implications for Their Use as Biomarkers

  • Judith Brouwers, 
  • Rintis Noviyanti, 
  • Rob Fijnheer, 
  • Philip G. de Groot, 
  • Leily Trianty, 
  • Siti Mudaliana, 
  • Mark Roest, 
  • Din Syafruddin, 
  • Andre van der Ven, 
  • Quirijn de Mast
PLOS
x

Abstract

Introduction

The angiogenic proteins angiopoietin (Ang)-1, Ang-2 and vascular endothelial growth factor (VEGF) are regulators of endothelial inflammation and integrity. Since platelets store large amounts of Ang-1 and VEGF, measurement of circulation levels of these proteins is sensitive to platelet number, in vivo platelet activation and inadvertent platelet activation during blood processing. We studied plasma Ang-1, Ang-2 and VEGF levels in malaria patients, taking the necessary precautions to avoid ex vivo platelet activation, and related plasma levels to platelet count and the soluble platelet activation markers P-selectin and CXCL7.

Methods

Plasma levels of Ang-1, Ang-2, VEGF, P-selectin and CXCL7 were measured in CTAD plasma, minimizing ex vivo platelet activation, in 27 patients with febrile Plasmodium falciparum malaria at presentation and day 2 and 5 of treatment and in 25 healthy controls.

Results

Levels of Ang-1, Ang-2 and VEGF were higher at day 0 in malaria patients compared to healthy controls. Ang-2 levels, which is a marker of endothelial activation, decreased after start of antimalarial treatment. In contrast, Ang-1 and VEGF plasma levels increased and this corresponded with the increase in platelet number. Soluble P-selectin and CXCL7 levels followed the same trend as Ang-1 and VEGF levels. Plasma levels of these four proteins correlated strongly in malaria patients, but only moderately in controls.

Conclusion

In contrast to previous studies, we found elevated plasma levels of Ang-1 and VEGF in patients with malaria resulting from in vivo platelet activation. Ang-1 release from platelets may be important to dampen the disturbing effects of Ang-2 on the endothelium. Evaluation of plasma levels of these angiogenic proteins requires close adherence to a stringent protocol to minimize ex vivo platelet activation.

Introduction

The endothelium plays a central role in the pathophysiology of P. falciparum malaria. Erythrocytes containing mature malaria parasites adhere to the endothelium via a range of endothelial receptors in order to escape removal by the spleen. Endothelial activation is an early feature of malaria, which is likely to favor sequestration of parasitized erythrocytes [1]. Excessive activation may contribute to loss of barrier function of the endothelium and organ dysfunction. Angiogenic proteins are increasingly recognized to be central regulators of endothelial physiology. Vascular endothelial cell growth factor (VEGF) increases the expression of adhesion molecules and coagulation factors and increases vascular permeability [2], [3]. Angiopoietins are other important mediators of angiogenesis. Binding of angiopoietin (Ang)-1 to the Tie2-receptor on endothelial cells maintains endothelial integrity and reduces the effects of inflammation [4], [5]. In contrast, Ang-2 counteracts the protective Ang-1 effects and promotes vascular leakage and inflammation [6], [7]. With endothelial cell activation, cytoadherence and microvascular hypoxia as central features of malaria, it may not come as a surprise that these angiogenic proteins have been studied extensively in malaria. Circulating levels of these proteins have been determined in several studies and, collectively, these studies found reduced Ang-1 levels and elevated Ang-2 levels in patients with malaria compared to healthy controls, while data on VEGF varied across studies [8][10]. Additional studies suggested Ang-1 and Ang-2 to be promising biomarkers to differentiate cerebral from non-cerebral malaria [11], [12].

Both Ang-1 and VEGF are both stored in high quantities in alpha granules from platelets [13] [14] and this is especially relevant when circulating blood concentrations are measured. Ex-vivo platelet activation, which is almost inevitable unless special precautions are taken, may falsely increase blood concentrations. Platelets numbers may also influence plasma concentrations, which is especially relevant for diseases characterized by thrombocytopenia, such as malaria. Indeed, there is increasing evidence that platelets and their released proteins are important regulators of endothelial permeability and this may partly be mediated by these platelet-derived angiogenic proteins [15]. The aim of our study was to determine plasma levels of VEGF, Ang-1, Ang-2 in adult Indonesian patients with Plasmodium falciparum malaria from whom platelet poor plasma was obtained under special conditions to prevent ex vivo platelet activation. A second aim was to correlate plasma levels of these angiogenic proteins with circulating platelet numbers and markers of platelet activation.

Methods

Ethics Statement

This study received ethical clearance for the use of human subject from the Eijkman Institute Research Ethics Committee, Jakarta, Indonesia and all enrolled patients gave written informed consent.

Study Area, Study Population, and Ethics

This study was conducted in the Rumah Sakit Karitas Hospital in Waitabula, West Sumba, East Nusa Tenggara Province, Indonesia, an area of hypo- to meso-endemic P. falciparum and P. vivax malaria transmission. Consecutive patients presenting to hospital with recent or current fever, clinical symptoms of malaria and a positive blood slide for P. falciparum were enrolled in this study following informed consent. All patients were assessed according to a predefined protocol which included a standardized history and physical examination performed by an experienced internist-infectious diseases specialist. All patients were treated with intravenous quinine and an antipyretic (paracetamol). Concurrent administration of antibiotics for severely ill patients was on the discretion of the treating physician. Venous blood for this study was collected at enrollment and on day two and five after enrollment. A group of 25 healthy young adults were recruited among local hospital staff as controls. All controls had no signs or symptoms of any illness and a negative malaria blood slide. Treatment for malaria in the past two months was an exclusion criteria for both malaria patients and controls.

Sample Collection and Laboratory Procedures

Five ml of venous blood was collected in EDTA tubes for a full blood count and malaria slides and in CTAD tubes (Becton-Dickinson Vacutainer Systems; tubes containing citrate and the platelet stabilizing agents theophylline, adenosine, and dipyridamole) for measurement of angiogenic and platelet activation marker proteins. In vitro platelet activation is almost completely absent in whole blood anticoagulated with CTAD [16], [17]. CTAD blood tubes from malaria patients and controls were centrifuged within 30 minutes at 2,000 g for 10 minutes and the top fraction of the plasma was collected, carefully avoiding the buffy coat. The plasma was frozen at −20°C until shipment to a −80°C freezer at the Eijkman Institute in Jakarta. A full blood count was determined by a standard hematology analyzer (Arcus, Diatron, Vienna, Austria). Thick and thin blood smears were stained with Giemsa, and the number of parasites was quantified against 200 white blood cells. Parasite density was calculated using the patient’s white blood cell count. Plasma Ang-1, Ang-2 and VEGF levels were measured by quantitative sandwich enzyme immunoassay technique according to the instructions of the manufacturer (Quantikine, R&D systems, Minneapolis, USA) at the Eijkman Institute. The platelet activation markers P-selectin and CXCL7 (beta-thromboglobulin) were measured in the Department of Clinical Chemistry and Haematology of the University Medical Center Utrecht as described in detail earlier [18].

Statistical Analysis

Data are presented as median followed by interquartile range in parentheses unless otherwise stated. Within the group of malaria patients, the Freidman test with post-tests was used to compare laboratory parameters on the three time points.Mann-Whitney U test was used for comparisons with the controls. Relationships between laboratory parameters were assessed using Spearman correlation coefficient. All analyses were performed with GraphPad version 5.0.

Results and Discussion

A total number of 27 patients with P. falciparum malaria and 25 controls were included. Demographic and clinical characteristics are shown in table 1. Compared to the controls, malaria patients were younger and more often male. Both groups shared the same genetic background. None of the patients and controls had been treated for malaria in the past two months or had used medication in the week before enrollment. Moreover, none had diabetes mellitus or had suffered from a cardiovascular event or tuberculosis in the past. Malaria patients had a significantly lower platelet count and a lower hemoglobin level. According to WHO-criteria, two malaria patients were classified as suffering from cerebral malaria and four as severe malarial anemia.

Figure 1 shows the course of platelet numbers, together with the course of the angiogenic proteins Ang-1, Ang-2 and VEGF and of the platelet activation markers P-selectin and CXCL7. As expected, platelet numbers rose after start of antimalarial treatment on day 0. Ang-2 is stored in Weibel-Palade bodies in endothelial cells and is therefore regarded as a marker of endothelial cell activation. Plasma levels of Ang-2 were elevated at day 0 and decreased upon start of antimalarial treatment to levels comparable to those in controls. In contrast, Ang-1 and VEGF plasma levels were also higher at day 0 compared to levels in controls, but start of antimalarial treatment resulted in a further increase in these proteins. The ratio of Ang-2/Ang-1 in the day 0 sample of patients and controls was similar (1.4 vs. 1.3; p = 0.18). After start of antimalarial treatment, the Ang-2/Ang-1 ratio declined to 0.5 at day 2 and 0.3 at day 5. the six patients with severe malaria had a higher median Ang-2/Ang-1 ratio at day 0 (1.5 vs. 0.8) and day 2 (4.7 vs. 0.5), although these differences did not reach statistical significance.

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Figure 1. Platelet number and plasma levels of angiogenic and platelet activation markers.

Levels of angiopoietin-2, angiopoietin-1, vascular endothelial growth factor (VEGF), soluble P-selectin and CXCL7 were determined in CTAD plasma in 27 patients with febrile P. falciparum malaria at the start of malaria treatment (day 0), day 2 and day 5 and in 25 healthy controls. Data are presented in a scatter dot plot with median and interquartile range. Differences between malaria patients in time were assessed by the Friedman’s test; differences with controls with the Mann-Whitney test. **denotes p<0.01 and *p<0.05.

https://doi.org/10.1371/journal.pone.0064850.g001

The time course of Ang-1 and VEGF resembled the course of the platelet activation markers P-selectin and CXCL7. Correlation analysis, presented in table 2, showed that both Ang-1 and VEGF levels in malaria patients were strongly correlated with P-selectin and especially CXCL7 levels on the three time points. While P-selectin is released by both activated platelets and endothelial cells, CXCL7 is exclusively derived from platelets [19]. There was no significant correlation with either Ang-2, platelets counts or WBC counts. Moreover, in controls, correlations between Ang-1 and VEGF and markers of platelet activation were only weak. These findings suggest that platelet activation is one of the main determinants of plasma levels of Ang-1 and VEGF in malaria patients, but not in healthy controls. The fact that VEGF is made by many different cell types, and its production is upregulated in hypoxic tissues may explain the weaker correlation of VEGF than Ang-1 with other platelet activation markers. We have previously reported that malaria is associated with a normal thrombopoietic activity [20]. Ongoing peripheral clearance of degranulating platelets may explain why plasma levels of these platelet-stored proteins were increased despite the low circulating platelet counts. The rise in circulating platelet counts following start of antimalarial treatment together with ongoing platelet degranulation may also explain the further increase in plasma levels of these proteins, although no significant correlation of platelets numbers with Ang-1 and VEGF plasma levels was found. Age did not correlate with protein levels of any of the measured proteins (data not shown), suggesting that age difference between malaria patients and controls did not influence our findings.

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Table 2. Spearman correlation of angiopoietin-1 and VEGF with other parameters.

https://doi.org/10.1371/journal.pone.0064850.t002

These findings are in contrast to previous studies which showed that patients with malaria had lower Ang-1 levels than healthy controls [8], [9]. Within the group of malaria patients, those with the most severe illness also had lower levels [11], [12]. Data on VEGF in malaria patients varied more across studies with both higher and lower levels reported [10], [21], [22]. Importantly, to our knowledge, in none of the studies, CTAD or citrate was used as anticoagulant. As described above, when measuring platelet-stored proteins like Ang-1 and VEGF, selecting the right anticoagulant and blood collection and processing techniques is of paramount importance. If the goal is to assess physiological plasma levels, EDTA or heparin plasma are less suitable as these anticoagulants are known to cause various degrees of in vitro platelet activation [23], [24], [25]. While sodium citrate as anticoagulant already results in a major reduction in ex vivo platelet activation, CTAD tubes almost completely prevent in vitro platelet activation and several authors have already recommended using CTAD when platelet-stored angiogenesis proteins are being measured [17], [25][28]. When these special measures are not taken, the circulating platelet number may have a strong impact on measured concentrations of platelet-stored proteins. Patients with malaria frequently have thrombocytopenia and platelet numbers have been shown to inversely correlate with severity of illness. Thus, we speculate that the lower circulating platelet numbers together with variable degrees of ex vivo platelet activation was responsible for the lower Ang-1 and VEGF concentrations in previous studies. Serum concentrations of platelet-stored angiogenic proteins may also have prognostic value. Generation of serum results in complete in vitro platelet activation and serum concentrations will largely reflect the total platelet content of angiogenic proteins. Possible drawbacks of using serum for this objective is that clotting processes may not release all platelet-stored angiogenic growth factors into the serum [29] and that serum concentrations may have no additional prognostic value over platelet count, as serum concentrations will heavily depend on platelet counts.

We only used a single centrifugation step to generate platelet poor plasma in our study, while a double centrifugation step is often advised. We assumed that only under special circumstances single centrifugation does not result in near complete removal of platelets from the top layer of the plasma. We tested this assumption by centrifuging venous blood in CTAD tubes from five volunteers using the same procedures as in our study and we found that a singly centrifugation step resulted in no detectable platelets in the plasma in four volunteers and a negligible platelet number (2×109/L) in the remaining volunteer.

Several groups of researchers have investigated the utility of angiopoietins and VEGF as biomarkers to identify patients with severe malaria. Due to its limited sample size, our study did not allow to explore the role of these angiogenic proteins in malaria. In general, we strongly recommend that a similar protocol for measurement of platelet-stored angiogenic proteins is used in future biomarker studies, including use of CTAD plasma and consideration of circulating platelet numbers. These precautions do not apply to Ang-2, which is derived from endothelial cells and of which plasma levels are not affected by platelet activation.

What are the implications of these findings for the pathophysiology of malaria? Increasing evidence supports a central role for platelets in protecting the vasculature during inflammation [15], [30], [31]. The exact mechanisms are still unclear, but soluble factors released from platelets may play a role [15]. The angiopoietin-Tie-2 system has evolved as a central regulator of the activation status and permeability of the vascular lining. An imbalance in the pro-permeability and pro-inflammatory effects of Ang-2 and VEGF and the anti-permeability effects of Ang-1, together with the disrupting effects of pro-inflammatory cytokines on vascular integrity, may contribute to transient plasma leakage. Our current data suggest that there is indeed a distortion of this balance. Ang-1 release from platelets may especially be important in these circumstances to dampen the disturbing effects of Ang-2 on the endothelium. Since both platelet and endothelial activation are common in many infectious diseases, the changes in angiogenic proteins observed in our study may not be specific to malaria.

In conclusion, we show that platelet activation is an important determinant of circulating Ang-1 and VEGF levels in malaria. Optimal biomarkers for malaria severity would rely on inexpensive point-of-care devices using whole blood. The fact that assays measuring these platelet-stored angiogenic proteins require special precautions to avoid inadvertent platelet activation during blood processing and are prone to artifacts limits widespread use of these proteins as possible biomarkers.

Acknowledgments

The authors thank S.A.E. Sebastian for measuring the platelet activation markers and all patients and staff at the Karitas Hospital in Weetabula, Sumba, Indonesia for their support and participation.

Author Contributions

Conceived and designed the experiments: JB RN RF PdG DS AV QM. Performed the experiments: JB RN LT SM MR. Analyzed the data: JB RN QM. Wrote the paper: RN QM.

References

  1. 1. de Mast Q, Groot E, Lenting PJ, de Groot PG, McCall M, et al. (2007) Thrombocytopenia and release of activated von Willebrand Factor during early Plasmodium falciparum malaria. J Infect Dis 196: 622–628.
  2. 2. Kim I, Moon SO, Kim SH, Kim HJ, Koh YS, et al. (2001) Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells. J Biol Chem 276: 7614–7620.
  3. 3. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, et al. (1983) Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219: 983–985.
  4. 4. Thurston G, Rudge JS, Ioffe E, Zhou H, Ross L, et al. (2000) Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med 6: 460–463.
  5. 5. Thurston G, Rudge JS, Ioffe E, Papadopoulos N, Daly C, et al. (2005) The anti-inflammatory actions of angiopoietin-1. EXS 94: 233–245.
  6. 6. Fiedler U, Reiss Y, Scharpfenecker M, Grunow V, Koidl S, et al. (2006) Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation. Nat Med 12: 235–239.
  7. 7. Parikh SM, Mammoto T, Schultz A, Yuan HT, Christiani D, et al. (2006) Excess circulating angiopoietin-2 may contribute to pulmonary vascular leak in sepsis in humans. PLoS Med 3: e46.
  8. 8. Jain V, Lucchi NW, Wilson NO, Blackstock AJ, Nagpal AC, et al. (2011) Plasma levels of angiopoietin-1 and -2 predict cerebral malaria outcome in Central India. Malar J 10: 383.
  9. 9. Lovegrove FE, Tangpukdee N, Opoka RO, Lafferty EI, Rajwans N, et al. (2009) Serum angiopoietin-1 and -2 levels discriminate cerebral malaria from uncomplicated malaria and predict clinical outcome in African children. PLoS One 4: e4912.
  10. 10. Yeo TW, Lampah DA, Gitawati R, Tjitra E, Kenangalem E, et al. (2008) Angiopoietin-2 is associated with decreased endothelial nitric oxide and poor clinical outcome in severe falciparum malaria. Proc Natl Acad Sci U S A 105: 17097–17102.
  11. 11. Conroy AL, Lafferty EI, Lovegrove FE, Krudsood S, Tangpukdee N, et al. (2009) Whole blood angiopoietin-1 and -2 levels discriminate cerebral and severe (non-cerebral) malaria from uncomplicated malaria. Malar J 8: 295.
  12. 12. Conroy AL, Phiri H, Hawkes M, Glover S, Mallewa M, et al. (2010) Endothelium-based biomarkers are associated with cerebral malaria in Malawian children: a retrospective case-control study. PLoS One 5: e15291.
  13. 13. Li JJ, Huang YQ, Basch R, Karpatkin S (2001) Thrombin induces the release of angiopoietin-1 from platelets. Thromb Haemost 85: 204–206.
  14. 14. Mohle R, Green D, Moore MA, Nachman RL, Rafii S (1997) Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets. Proc Natl Acad Sci U S A 94: 663–668.
  15. 15. Nachman RL, Rafii S (2008) Platelets, petechiae, and preservation of the vascular wall. N Engl J Med 359: 1261–1270.
  16. 16. Kuhne T, Hornstein A, Semple J, Chang W, Blanchette V, et al. (1995) Flow cytometric evaluation of platelet activation in blood collected into EDTA vs. Diatube-H, a sodium citrate solution supplemented with theophylline, adenosine, and dipyridamole. Am J Hematol 50: 40–45.
  17. 17. Zimmermann R, Koenig J, Zingsem J, Weisbach V, Strasser E, et al. (2005) Effect of specimen anticoagulation on the measurement of circulating platelet-derived growth factors. Clin Chem 51: 2365–2368.
  18. 18. van Bladel ER, Roest M, de Groot PG, Schutgens RE (2011) Up-regulation of platelet activation in hemophilia A. Haematologica. 96: 888–895.
  19. 19. Fijnheer R, Frijns CJ, Korteweg J, Rommes H, Peters JH, et al. (1997) The origin of P-selectin as a circulating plasma protein. Thromb Haemost 77: 1081–1085.
  20. 20. de Mast Q, de Groot PG, van Heerde WL, Roestenberg M, van Velzen JF, et al. (2010) Thrombocytopenia in early malaria is associated with GP1b shedding in absence of systemic platelet activation and consumptive coagulopathy. Br J Haematol 151: 495–503.
  21. 21. Casals-Pascual C, Idro R, Gicheru N, Gwer S, Kitsao B, et al. (2008) High levels of erythropoietin are associated with protection against neurological sequelae in African children with cerebral malaria. Proc Natl Acad Sci U S A 105: 2634–2639.
  22. 22. Jain V, Armah HB, Tongren JE, Ned RM, Wilson NO, et al. (2008) Plasma IP-10, apoptotic and angiogenic factors associated with fatal cerebral malaria in India. Malar J 7: 83.
  23. 23. Golanski J, Pietrucha T, Baj Z, Greger J, Watala C (1996) Molecular insights into the anticoagulant-induced spontaneous activation of platelets in whole blood-various anticoagulants are not equal. Thromb Res 83: 199–216.
  24. 24. Lei H, Gui L, Xiao R (2009) The effect of anticoagulants on the quality and biological efficacy of platelet-rich plasma. Clin Biochem 42: 1452–1460.
  25. 25. Zimmermann R, Ringwald J, Eckstein R (2009) EDTA plasma is unsuitable for in vivo determinations of platelet-derived angiogenic cytokines. J Immunol Methods 347: 91–92.
  26. 26. Ranieri G, Coviello M, Chiriatti A, Stea B, Montemurro S, et al. (2004) Vascular endothelial growth factor assessment in different blood fractions of gastrointestinal cancer patients and healthy controls. Oncol Rep 11: 435–439.
  27. 27. Starlinger P, Alidzanovic L, Schauer D, Brugger P, Sommerfeldt S, et al. (2011) Platelet-stored angiogenesis factors: clinical monitoring is prone to artifacts. Dis Markers 31: 55–65.
  28. 28. Banks RE, Forbes MA, Kinsey SE, Stanley A, Ingham E, et al. (1998) Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology. Br J Cancer 77: 956–964.
  29. 29. Zimmermann R, Arnold D, Strasser E, Ringwald J, Schlegel A, et al. (2003) Sample preparation technique and white cell content influence the detectable levels of growth factors in platelet concentrates. Vox Sang 85: 283–289.
  30. 30. Ho-Tin-Noe B, Demers M, Wagner DD (2011) How platelets safeguard vascular integrity. J Thromb Haemost 9 Suppl 156–65.
  31. 31. Iannacone M, Sitia G, Isogawa M, Whitmire JK, Marchese P, et al. (2008) Platelets prevent IFN-alpha/beta-induced lethal hemorrhage promoting CTL-dependent clearance of lymphocytic choriomeningitis virus. Proc Natl Acad Sci U S A 105: 629–634.