Keywords: religious freedom, evangelical Christianity, US Foreign policy, Egypt, Coptic Church

When President Clinton signed into law the International Religious Freedom Act in 1998, he provided the members of the religious right with a unique opportunity to promote their vision of the world through the publication of annual reports assessing religious freedom worldwide. This paper presents a critical analysis of the Commission"s reports (1998-2013) on sectarian strife in Egypt and discusses the ideological implications of the discourse adopted by the commissioners. In a second step, this discourse will be confronted to the arguments put forward by members of minority groups who oppose any legislation establishing the US as the religious police of the world.

DEFENDING FREEDOM OF RELIGION IN CONFLICT-TORN

COUNTRIES: A BRIEF ANALYSIS OF AMERICAN STRATEGIES IN CONTEMPORARY EGYPT (1998-2013)

DOMINIQUE CADINOT

In assessing the role and image of the American missionary movement during the civil war which in 1860 opposed, in the Levant, Christians and Muslims, author Adele L. Younis came to the conclusion that missionaries were "wedged between fighting factions" and that "consequently, both sides regarded them as impartial" (Younis 1995, 55). Be that as it may, the issue of religious freedom was, at the time, at least in Washington, nobody"s concern. However, since then, major changes have occurred in the relationship between foreign policy and religious affairs. The ratification by President Bill Clinton in 1998 of the International Religious Freedom Act (IRFA) constitutes a pivotal moment in the history of US diplomacy because the new law officially inscribed the promotion of religious freedom in the foreign affairs agenda. It should be recalled that following the 1994 Republican Revolution, both houses of Congress were in the hands of conservative factions who maintained close ties with evangelical and fundamentalist churches (such as the powerful Southern Baptist Convention). Hence emanating from the Christian right 1 which made its mark in the prerogatives of the US Congress, this new piece of legislation allowed the creation of a federal commission (The United State Commission on International Religious Freedom -USCIRF) whose main duties has been to provide lawmakers with an annual report on the conditions of religious freedom in the world in order to impose various economic and other sanctions to offenders 2 . If the constitutionality of initiatives seeking to involve the federal government in the religious affairs of foreign nations is a legitimate question, this analysis will discuss US engagement with religion in foreign policy development particularly in context of conflict-torn countries. More precisely, it will aim to identify, through a critical reading of the USCIRF reports published between 1998 and 2013 3 , the ideological implications of the religious right"s (and their fundamentalist allies) involvement in favor of imperiled minorities abroad. In a second part, the article will confront this discourse to the social reality as it is perceived and analyzed by local opponents to U.S. initiatives. Indeed, in several countries, many citizens have vigorously opposed any interference from the United States. Because Middle-Eastern Christians have been a key target of American missionaries since the early 19th century 4 , we shall look in particular at the reactions of intellectuals and political leaders in Egypt. How can the reluctance of some Egyptian dignitaries to the work of Americans for universal religious freedom be explained, especially when Coptic or Shi"a communities have repeatedly suffered violence at the hands of fellow citizens affiliated to another religious group? Ultimately, our intention is to bring to light the effects of "America"s unavoidable responsibility" 5 on the balance of power between the various communities which form the Egyptian social body.

Incriminations versus ideological discourse: A critical reading of USCIRF"s reports

Although present on the political scene since Ronald Reagan"s first term, evangelical activists filled in 1998 the vacuum left in the corridors of power by the liberals and sought the enactment of a federal policy designed to promote religious freedom abroad. This commitment which main expression was the participation in the drafting of USCIRF"s annual reports, offered the Christian movement the opportunity to promote in official documents its own vision of the world. Each year, countries under observation are classified in two lists: the Watch List, which includes, as its name indicates, countries which ought to be closely monitored, and the Countries of Particular Concern list for those that commit systematic violations of religious freedom. If Iran, Pakistan or China have invariably appeared in every report, Egypt first appeared in the Watch List in 2002, before being reported in the second in 2011, at a time when new popular uprisings eventually toppled President Hosni Mubarak.

In the first place, we note that the commission"s reports are expanded and gradually enriched with illustrations and photographs, but also contain many paragraphs which are simply copied-pasted from one year to the other 6 . The chapter devoted to the state of religious freedom in Egypt grew in length from a single page in 2000 to more than fifteen pages in 2013. Hence, the latest publications contain a selection of the year"s most important political events followed by a complete account of the persecution or discrimination exercised against religious minorities. The communities surveyed by the commission are: the Baha'is (a syncretic religion of Iranian origin), the Shi"a, the Coranists (a current of Islam), the Jewish community and the Jehovah's Witnesses. However, most pages are devoted to Middle-Eastern Christians and to the Coptic Orthodox community precisely. In the 2006 report, for example, three pages deal with the situation of Copts against a half page for each other minority. The observers present a chronological inventory, summarizing in detail the attacks, murders or cases of discrimination against Egyptians of the Christian faith. Are reported for example the violent incidents that took place in the town of Esna in Upper Egypt, the arbitrary arrests, or the failures of the judicial proceedings against those responsible for confessional strife (USCIRF, 2006, 223). This prioritization of the Christian case was, it must be emphasized, one of the reasons which had hampered in 1997 the passage of a first bill called the Wolf-Specter bill. At the time, the evangelical discourse was not yet associated with that of the secular humanists. In June 1997, at a preliminary hearing before the Senate Committee on Foreign Relations, Nina Shea 7 explained that never in history had the Christians suffered as much, and that the secularist-liberal approach for the promotion of human rights had shown its limits: "Christians are the most persecuted religious group in the world today […] why don't we know about it? Well, Mr. Chairman, I think that our own discriminatory attitudes and secular myopia have prevented us from recognizing the problem" (Shea 1998, 1). After a single reading of these thirteen reports, one notices that if the focus of investigation has sometimes widened to include other minorities in a more balanced manner (see the reports of 2010 and 2011), the issue of religious freedom has always and foremost meant, for the members of the commission, the safeguarding of Christianity. The fact that each commission has always included in its midst two or three representatives of the evangelical movement (such as Richard Land of the Southern Baptist Convention), often shouldered by a conservative Catholic (like Charles J. Chaput, the very orthodox archbishop of Philadelphia) 8 , is obviously not without link to the permanent support granted to the Christians of Egypt.

If we now look more closely at the proposed review of the social context in Egypt, we notice that very often the angle adopted is that of news in brief. In fact, the contributors often relate cases of violence which, they explain, occurred following simple rumors or allegations. Thus in the 2008 report, for example, one can read: "In February 2007, Muslim groups reportedly set fire to several Christian-owned shops in southern Egypt due to rumors of a relationship between a Muslim woman and a Coptic Christian man" (USCIRF, 2008, 224). Besides cases of personal vengeance, family vendettas or sexual assaults, very few explanations are provided as to other points of contention, as to more influential political or socio-economic conditions that may have triggered such acts. The role played by faith leaders in the lives of the communities is for instance never acknowledged. There is no mention of Pope Shenouda, patriarch of the Coptic Orthodox Church and privileged spokesman to the Egyptian ruler from 1971 to 2012. Similarly, the Muslim Brotherhood"s schemes to sway public opinion and rise to power after 2011 are never brought into connection with sectarian violence. The same factual paragraph is almost identically reproduced from one year to the other and inserted at the end of the report. In sum, clashes between Muslims and Christians are depicted as if they resulted from an outbreak of irrational hatred, as if they were totally independent from any parochial or sectarian influence. However, in 2000, it is indicated that religious strife had long been aggravated by the Egyptian president who implemented de jure discrimination. The commissioners emphasize in particular the difficulties encountered to obtain government permission and the necessary funds for the construction or restoration of places of worship (USCIRF, 2000, 20). Another contextual explanation, but provided much later in 2005, is the state of emergency imposed by force for more than thirty years and used as a pretext for discriminatory legislation (USCIRF, 2005, 108). The accusations formulated by the members of the commission thus only focus on the regulatory function of the state and usually leave aside the mediating function of civil society. Hence, the portrait made of Egyptian civil society excludes the political dimension and endorses the model of a society structured around religious communities such as it was promoted by the national authorities until 2011. As a result, in the ideological model shaped by the American commission, the religious freedom of the individual overshadows collective initiatives. The free and devout individual is the only vector of social harmony. This focus on the individual is, according to Sebastien Fath, a basic element of the American evangelical rhetoric: "Contrary to mainline Protestantism, [this rhetoric] favors the individual more than the institution, more than the infrastructure" (Fath 2004, 66). But above all, it encourages, according to Mokhtar Ben Barka, "an indifference to issues pertaining to social justice" (Ben Barka 1998, 20). Indeed, in the 2008 report, no mention is made of the national strike organized by the workers of the textile mill Ghazl El-Mahalla, the largest to engulf Egypt since the 1950"s. The implicit effect of American action is therefore to incite the Egyptian believers"s empowerment so that they may eventually transcend "the hierarchies of power, knowledge or fortune" (Keppel 2004, 61). In all the commission"s reports, a following chapter is devoted to the issue of religious proselytizing. As it has been extensively documented, proselytism is of paramount importance for evangelicals. Being "born again" means being an example, and spreading one"s faith. Mokhtar Ben Barka concurs, explaining that: "The zeal of the converter does not stop at all with those who are near him. The borders of the world are the limits. The good news must reach the whole of mankind" (Ben Barka 2008, 94). Although, Section 107 of IRFA has granted any US citizen equal access to American missions abroad for religious activities, the commission"s reports do not, paradoxically, provide any explicit account of soul-saving activities. Thus avoiding any clear reference, the commission simply alludes to the difficulties encountered by American "foreign workers" who under various guises have tried to transform Muslims and Catholics alike into born-again disciples of Christ 9 . The taking of hostages in Egypt or in other countries of the Arab World of itinerant pastors, such as Reverend Michel Louis founder of a Pentecostal church in Boston and abducted while on a missionary trip in 2012 10 , proves the not easily quantifiable presence of these mercenaries of faith. Following the paragraph devoted to religious proselytizing, all reports deplore the discrimination or persecution suffered by those Egyptian Muslims who have converted to Christianity. No example of violence against Egyptian Christians converted to Islam is mentioned. The proposed analysis of the motives for conversion remains equally superficial. Only cases of marital conversion are mentioned. The reader learns that in matters of divorce, the Christian orthodox Church is rather strict since it only validates the procedure in cases of proven adultery. This explains why many Copts convert to Islam in the first place, and once divorce is obtained, reconvert to Christianity. If the commissioners remain discrete on the rigidity of clerical authorities, they criticize the administration"s refusal to provide re-converts with new identity documents reflecting their adoption of the Christian faith. Since the beginning of 2000, hundreds of Christian re-converts have indeed initiated legal actions to have their religion recognized in official documents 11 . But, besides this utilitarian practice, no analysis is offered on the motivations other than matrimonial which have led these people to recant their original faith. Yet, though the process of conversion remains a subject of heated debate, many scholars have laid bare the intricacy of motives. As demonstrated, for example, by the work of Raymond F. Paloutzian, J. T. Richardson and L. R. Rambo, conversion may constitute an effective resource to renegotiate identity and status: "Conversion results in profound, life transforming changes in mid-level functions such as goals, feelings, attitudes, and behaviors, and in the more self-defining personality functions such as identity and life meaning " [START_REF] Paloutzian | Religious conversion and personality change[END_REF]Rambo 1999, 1070). More specifically, in a study devoted to the phenomenon of conversion to Islam in Egypt, Laure Guirguis observed: " [Christians converts] see in Islam a way [...] to get rid of the self-hatred instilled in the individual Copt by the multiplication of signs of inferiority in the collective space" (Guirguis, 2012, 76). The complexity of identity issues related to conversion is therefore not taken into account in the depiction of the Egyptian confessional landscape, where faith often determines social status. Although the main architecture and the broad content of the reports remain unchanged, some variations in the field of investigation may however be observed. While in 2003, the overwhelming majority of commissioners shared rather conservative moral values (whether they were Catholic, Protestant or Jewish) 12 , the report focused exclusively on freedom of religion. The following year, the appointment by Democrat Nancy Pelosi, Speaker of the House of Representatives, of two new commissioners -Patti Chang, a feminist activist from California and Khaled Abu El Fadl, a jurist and scholar of Islamic law-coincides with a widening of the analytical focus. Thus the 2003 version announces: "Serious problems of discrimination against a number of religious groups remain widespread in Egypt" (USCIRF 2003, 15). In contrast, the 2004 report first confirms that: "Serious problems of discrimination and other human rights violations against members of religious minorities remain widespread in Egypt", but continues with the following comment: "Egypt has a poor overall human rights record that includes repressive practices that seriously violate freedom of thought, conscience, and religion" (USCIRF 2004, 72). This discrete yet meaningful variation reveals the disagreements which intermittently opposed the newly-appointed members of the commission. It also reflects the political beliefs or moral values of the various administrations which have held sway between 1998 and 2013. However, in spite of these moments of tension, in the editions of 2008, 2009, 2010 and 2013 the last paragraph bears a title which clearly privileges freedom of religion over any other freedoms: "Promoting Freedom of Religion and Belief and related human rights ". It becomes obvious, as Dominique Decherf, diplomat and French author also noted, that the American legislation on freedom of religion is quite distinct from the international human rights law from which it avowedly draws its legitimacy 13 (Decherf 2002, 7). As we have seen, the act isolates a single article and elevates religious freedom over other equally valuable rights. Yet Article 18 of the Universal Declaration of Human Rights stipulates, in this order, that; "Every person has the right to freedom of thought, conscience and religion" (United Nations 1948, 5). In fact, IRFA imbalances the architecture of these texts, in granting a privileged status to the freedom of worship, when freedom of conscience is designed by the secular community as the faculty left to each to adopt -or not-the religious, but also philosophical or political doctrines that he deems good, and to act accordingly. The American fundamentalist discourse, even if it has sometimes been moderated, therefore permeates the content of each report ; the chapters or the topics covered being predominantly the reflection of an ideology aimed at giving a spiritual basis to the social body. The message as it is articulated in the reports encourages individuals to define their identity not by membership to a collective and national culture, but by the sole identification to a spiritual order. The question of religious identification being particularly salient in the contemporary history of Egypt, it is now appropriate to look at the reactions of indigenous opponents and to examine the arguments set forth to oppose this prioritization of rights.

An ideological model against social reality: the reasons for rejection

The year which preceded the vote of the American law, President Hosni Mubarak was renewed in his functions after obtaining 97% of the votes. Scoring a similar landslide, he was re-elected in 1999 and 2005. If the verdict of the ballot box was many times called into question in the street, the authoritarian regime maintained the state of emergency and resisted until the constraint resignation of its leader on February 11, 2011. Over the past two decades, the regime pressured upon the social fabric to assert its authority. As a result, political parties, along with religious congregations, competed to obtain governmental favors. Such is the observation of American sociologist Mohammed Bamyeh: "The government was regarded, by its elites and constituents alike, as simply a source of situational favors" (Bamyeh 2012, 56). It was in such particular context that on March 22, 2001 an American delegation led by Elliott Abrams, President of USCIRF, traveled to Cairo in order to conduct their investigations. The reception organized at the United States embassy, it was reported, was only attended by two representatives from the Egyptian civil society, one of the guests later denying his presence at the embassy (Langohr 2001, 1). In truth, the visit of the commissioners raised the indignation of Coptic as well as Muslim dignitaries. Relaying their dismay, the Egyptian press lambasted American officials for justifying at home what they condemn abroad. Al-Ahram, Egypt"s most distributed daily, called attention to certain practices such as the use of "secret evidence" authorized by the Anti-Terrorism and Effective Death Penalty Act of 1996 which allowed the holding in detention of the accused without any court order or review (Abdel-Latif 2001, 1). In addition, several Egyptian associations for the defense of human rights emphasized the double talk of the US commission who does not decry the curtailment of the Palestinians" civil rights in the Occupied Territories (Langohr 2001, 2). But beyond these somewhat predictable criticisms, what are the effects of the American action on the civil and sectarian strife tearing contemporary Egypt? In the first place, the effects are most readily observable on inter-religious tensions. Although victims of repeated violence and discrimination, some Copts have explained that their community is an integral part of the Egyptian nation and that it should not be regarded as a minority since its existence precedes the Muslims" arrival in the 7th century. Consequently, by focusing on the Copts, the commission inevitably isolates these populations who then become scapegoats and guilty of treason. The accusation is even more widespread that Coptic communalism has been cultivated by members of the Christian elite who argue that this ancient connection to Pharaonic history excludes de facto any affiliation with the Arab world 14 . Such is the opinion expressed by the bishop of Al-Qusiya in 2008: "If you say to a Copt that he is Arab, you offend him. We are not Arabs, we are Egyptians. I am very pleased to be Egyptian and I will never accept to be called an Arab, because ethnically I"m not" (quoted in Guirguis 2012, 70). In addition, the position taken by Pope Shenouda III and the synod of bishops in favor of the re-election of H. Mubarak in 2005 similarly poisoned relationships between Christians and Muslims, especially among the leaders of the grassroots coalition Kifâya founded in 2004 15 . Outraged by the Patriarch"s stance, the writer and founding member of the movement Alaa al Aswany writes: "Pope Shenouda represents a spiritual authority and not a political authority. He uses his religious attributes to impose his political point of view to the Copts. By doing this, he curtails their right to change opinion" (Al Aswany 2011, 157). In consequence, it appears that the singling out of the Copts has had the other effect of strengthening Coptic communalist sentiments and has therefore encouraged sectarianism in the Egyptian political life. The American approach resembles for many Egyptians, the strategy adopted in their time by colonizing powers (ottoman or European) which consisted in pitting Christians against Muslims with a view to divide and conquer. Accordingly, it is possible to imagine that the persecution inflicted to Christians in 2005, for example, were not simply or solely motivated by religious antagonisms, but also perhaps by strategic disputes as to the best course of action in ousting the autocrat. The attackers, mentioned in the report, may have been animated by a resentment of a believer as much as that of a citizen. In the end, it becomes clear that the ideology promoted by the Commission encourages the formation of an Egyptian society composed mainly of religious communities, excluding or downgrading any other form of social tie and especially any other forms of mobilization. In this context, how can the creation of political parties including both Christians and Muslims be achieved? A goal which the Neo-Wafd party, heir of the oldest secular and liberal party, has pursued for years and whose favorite motto claims: "Religion is for God and the homeland is for all" (quoted in Mazourki 2013, 50). Along the same line, philosopher Murad Wahba, very critical of the fundamentalist movements, argued in the newspaper Al-Ahram : "Being a believer is not the same thing as being a citizen" (Wahba 2013, 1), thereby showing the existence, in the Egyptian civil society, of a Muslim secularist movement opposed to any exploitation of religion and to any violation of the national identity. However, the American mission does not only weigh on inter-confessional relations. The ignorance of the Christian secular tradition has mobilized, here too, many intellectuals or political leaders. In condemning the singling out of the Copts, historian Vivian Ibrahim recalls that the community in question is far from forming a monolithic group: "There is an inherent assumption in most analysis and imagery that Copts act in a unified and consistent manner" (Ibrahim 2012, 1). It must be borne in mind that the divide between lay and religious Copts worsened dramatically in 1971 after the enthronement of Shenouda III who consolidated his power within the church by including political matters within his patriarchal functions. Determined to reign without sharing, Shenouda marginalized the secular Copts by, for example, taking hold of the management of many cultural or charitable associations originally created by the laity (Guirguis 2012, 137). The secularist component however, refuses that the church be the only one to define Coptic family law, particularly in matters of divorce. Because they were evicted from the community scene and opposed to the political positions of the patriarch in favor of the regime, the Coptic laity in the early days of the 2011 revolution took to the streets to defend the rule of law. The American mission in Egypt as it has been carried out by the commission has therefore also contributed to heightened tensions within the Christian community itself. In fact, for secular Egyptians, marxist or liberal, Christians or Muslims, the hope for unity which culminated with the revolution has been hampered by schemes and policies aimed at dividing the constituents of civil society. In summary, the fundamental reason why the activities of the American commission in Egypt have met and continue to meet with an opposition is the fact that the act of 1998 privileges a vertical rather than a horizontal stratification of society. The American commitment in favor of endangered minorities leads one to believe that faith should pervade holistically every aspect of life over other principles such as class or culture.

Conclusion

The vision of America as a nation gifted with a special mission is one of the constants of American foreign policy. During the 1990"s, the new Christian right intended to spread their culture and values by winning the war of ideas; the "new American century", from the name of the manifesto conjured up by neoconservatives, was hailed as the most enviable model to the peoples of the planet. The enactment of IRFA therefore enabled evangelical Christians and their allies to associate their spiritual agenda to worldly concerns, a new strategy by a religious movement which has always considered America as the agent of the divine will. That said, it has since been shown that evangelicals in reality, between 1998 and 2008, only exercised a limited influence on foreign policy, because there has always been within the diplomatic apparatus a current of opposition to any religious legitimization of federal action; an attitude which Thomas Farr, one of the most vocal defenders of IRFA, calls "the secularist culture of American foreign service" (Farr 2009, 960). In fact, T. Farr was forced to conclude in 2009: "The outcome of religious freedom has remained unconnected to the broader imperative of US foreign policy. In most cases, no action was taken with respect to designated countries" (Farr 2009, 957). It has indeed been observed that, for example, in the wake of the September 11 th , 2001 attacks, American diplomacy has preferred to silence its accusations to losing valuable alliances in its conduct of the war on terror 16 . Still, it remains that the intervention of external actors on the ground can sometimes be counterproductive especially when civil war is fought along sectarian lines. In seeking the salvation of suffering and persecuted Middle-Eastern Christians, the commission has contributed indirectly to their precariousness. Moreover, the study of certain strategies exploited to promote national values abroad demonstrates that, in the process, American society transplants some of the social issues that it has recurently struggled with. The model developed by members of the commission recommend that social issues be looked at through the narrow prism of religion when faith is only one element at work within a multifaceted context on the ground. The traditional blindness of Americans (especially if they are evangelicals) to class issues thus reduces the debate on social injustice at the single paradigm of communalism and forecloses any questioning of structured inequalities.

  Latif, Omayma. 2001. US Commission faces closed doors. Al-Ahram Weekly On-Line, March 22-28 2001. Available from http://weekly.ahram.org.eg/2001/526/eg1.htm. Internet. Retrieved 15 December 2014. Al Aswany, Alaa. 2011. Chroniques de la révolution égyptienne. Paris : Actes Sud.

	Works Cited
	Abdel-

Notes 1 The Christian Right, as M. Ben Barka reminds us, drew support from fundamentalist Protestants and politically conservative Catholics, Jews and Mormons. 2008, 7. 2 For a much fuller analysis, see Allen Hertzke 2004. 3 All IRF reports can be found on the website of the State Department"s Office For International Religious Freedom: www.uscirf.gov. 4 The first American Christian missionaries settled in the Levant in 1820. See Heather Jane Sharkley 2008. 5 The expression was used by President Clinton in a speech on foreign policy: "Speech at the United States Institute of Peace. See Rubinstein, Shayevich and Zlotnikov 2000. 6 All reports are available at : www.uscirf.gov. 7 Nina Shea is the director of the Center for Religious Freedom/Freedom House and has served on the commission since


L Nicolas

M Furstoss

M A Galland
email: galland@selene.mecaflu.ec-lyon.fr.

Analogy Electromagnetism -Acoustics: Validation and Application to Local Impedance Active Control for Sound Absorption

Keywords: Acoustics, Microwave propagation, Finite elements, Modeling

An analogy between electromagnetism and acoustics is presented in 2D. The propagation of sound in presence of absorbing material is modeled using an open boundary microwave package. Validation is performed through analytical and experimental results. Application to impedance active control for sound absorption is finally described.

I. INTRODUCTION

The original form of active noise control consisted in sound pressure reduction by the superposition of an acoustic signal 180 degrees out of phase with the unwanted sound [START_REF] Lueg | Process of silencing sound oscillations[END_REF]. The additional sound is emitted by a secondary source. This method provides an attractive low frequency alternative to the inefficient passive treatments of using absorbent materials. In recent years, due to advances in digital signal processing hardware, active control technology has become an industrial reality.

By locating the active "noise reducer" behind a resistant cloth, the acoustic dissipation is increased by friction in the low frequency range [START_REF] Olson | Electronic sound absorber[END_REF]. This method appears to be efficient in two main cases. The first one concerns the reduction of the low frequency noise radiated by inlet or exhaust ducts. In this case, it is almost impossible to apply active control methods based on interference principle because no perfect correlation exists between primary and secondary waves. For such a case, active absorption achieved along the duct walls by local control set-ups is a very promising alternative. The second case concerns the active sound absorption for noise reduction in enclosed sound fields. A general formulation has been proposed for this problem by considering two different approaches [START_REF] Nelson | Active control of sound[END_REF]: local and global control of sound. In the first of these strategies, the aim remains to achieve pressure releases at a set of discrete locations. No attention is paid to the overall acoustic field in the volume. Applications of this strategy concern for example the noise reduction at the passenger heads in car or propeller aircraft cabins. The second approach, on the contrary, attempts to minimize the overall acoustic level ( the total acoustic energy inside the cavity). Nevertheless, experimental difficulties (especially concerning the energy estimation) limit its application to realistic environments. A good alternative consists in changing the boundary conditions and thus the wall impedances to reduce the total acoustic energy.

Various set-ups have been developed by many research teams to achieve active control of the impedance [START_REF] Furstoss | Surface impedance control for sound absorption: direct and hybrid passive/active strategies[END_REF], which is defined on a surface by the ratio between pressure and normal velocity. Two different strategies have been proposed: the first one is named direct impedance control because it involves sensors to estimate both pressure and velocity close to the secondary source. However, its implementation is limited by major drawbacks such as bandwidth limitations and difficulties for extending absorbing surfaces. The second strategy combines active control and porous material, in order to improve its absorbent behavior. In a previous study [START_REF] Furstoss | Surface impedance control for sound absorption: direct and hybrid passive/active strategies[END_REF] we have highlighted that this association allows the active control to be reduced to a simple pressure release if the porous layer characteristics are well suited. These hybrid passive/active systems then enable feedback control techniques to be implemented and extended absorbing coatings to be achieved.

In order to optimize such absorbers and their location, attention is now paid to precisely modelize their behavior. Only numerical computation can simulate the acoustic propagation in unbounded domains in the presence of a porous material. Boundary elements methods have been extensively used to determine pressure fields in radiation problems. But up to now, absorption is generally taken into account by introducing impedance boundary conditions. This cannot exactly modelize propagation phenomena occurring in porous media. On the other hand, modeling sound absorbing material has been developed with a good accuracy only over the past ten years. The adaptation of a 2D package previously developed for open boundary microwaves [START_REF] Nicolas | Modélisation 2D par Éléments Finis de phénomènes micro-ondes en milieu ouvert[END_REF] has allowed us to associate porous material and free field radiation.

The objective of this paper is to show how an analogy between acoustics and electromagnetism has made possible the use of this package to modelize our acoustic problem. In a first section, we present the analogy used between acoustics and electromagnetism. The second section deals with the validation of the analogy by first studying the simple case of a plane wave impinging on a porous layer in a wave guide. A numerical simulation of the set-up used to measure the surface impedance of a porous layer in free field is also presented to validate the analogy for unbounded domain. The last section deals with the development of large surfaces of active absorbent and show the interest of using numerical simulations to optimize this set-up.

II. 2D ANALOGY ACOUSTICS -ELECTROMAGNETISM

The acoustic behavior of a material layer has to be taken into account with the use of propagation models in porous media. Many classical materials are assumed to have a motionless frame. Thus, they are replaced on a macroscopic scale by an equivalent fluid having a complex dynamic density ρ e and a complex dynamic bulk modulus K. Solving Helmholtz equation enables their acoustic behavior to be described by a complex wave number k and a complex characteristic impedance Zc. The frequency dependence of these parameters is given by modeling. Usually the model proposed by Allard [START_REF] Allard | Propagation of sound in porous media[END_REF] is used because it is well suited for low frequencies. In this model ρ e and K are functions of five physical characteristics of the material. For very classical porous absorbers such as fiberglass, this number may be reduced to one single, the resistivity.

In the harmonic case, the acoustic pressure verifies the Helmholtz's equation [START_REF] Lueg | Process of silencing sound oscillations[END_REF], which can be compared to the scalar wave equation for the magnetic field H z in TM polarization:

( ) ( ) Δ Δ p K p e z + H + 2 H 2 z ω ρ ω ω ω με = ≡ = 0 0
This leads to the analogy presented in Table I, where the acoustic pressure p is equivalent to the magnetic field H z . The acoustic velocity v is then in quadrature with the electric field E. On the contrary of the usual analogy (p≡E z [START_REF] Ingard | Fundamentals of waves and oscillations[END_REF]), this allows the boundary conditions to be equivalent for a perfectly reflecting boundary in acoustics (n.v=0) and for a perfect electric conductor (nxE=0). Equivalence can also naturally be found between the acoustic intensity vector and the Poynting vector, between the acoustic and the electromagnetic energies, and between the surface impedances.

There is no reason for this analogy to be not valid in 3D. However, the different nature of the fields -scalar in acoustics and vector in electromagnetism-makes the use of an electromagnetic package more complicated in this case.

III. VALIDATION OF THE ANALOGY.

We have developed previously a general 2D Finite Element (F.E.) package for the modeling of open boundary microwave problems [START_REF] Nicolas | Modélisation 2D par Éléments Finis de phénomènes micro-ondes en milieu ouvert[END_REF]. The formulation is given by [START_REF] Olson | Electronic sound absorber[END_REF]. It handles conducting, permeable, dielectric and absorbing materials. The open boundary is taken into account either by coupling with the boundary element method or by using absorbing boundary condition (Engquist-Majda for a rectangular outer boundary or Bayliss-Turkel for a circular boundary). Second order triangles are used for the F.E. discretization. Far field is computed by boundary element method or by harmonic series expansion [9]. Applications of such a formulation are the design of antennas or the modeling of electromagnetic scattering.

∇ ∇ + ⎡ ⎣ ⎢ ⎤ ⎦ ⎥ ∫ ∫∫ ∫∫ W j H j W H j W H n z z z . . 1 1 ωε ωμ ωε ∂ ∂ d - d = W. J d Ω Γ Ω Γ Ω Ω (2)
The same formulation is used for acoustic problems. The analogy is validated using two methods: by comparison with analytical results in the case of a standing wave tube, and by comparison with experimental measurements of the surface normal acoustic impedance of porous materials.

A. The Standing Wave Tube

The standing wave tube has been used for a long time to measure the reflection coefficient of materials. It is a closed boundary problem. The absorbing material is set at one extremity of the tube, and a plane acoustic wave propagates parallel to the axis of the tube (fig. 1). An analytical solution for this standing wave problem is given in [START_REF] Rayleigh | Theory of Sound[END_REF]. An excellent

μ permeability K ≡ 1/μ (1)
agreement is found between analytical and numerical results, either for the pressure (fig. 2) or for the velocity.

B. Numerical Simulation of an Impedance Measurement Experimental Set-up

A plane piston, modelized as a constant speed acoustic source, is located above a fiberglass panel supported by a rigid floor. Fig. 3 shows the computed acoustic pressure when the frequency of the source is 200 Hz and 800 Hz. Obviously, the plane wave hypothesis is more satisfied when the wavelength decreases. The computed surface impedances obtained at the middle of the panel surface are compared in fig. 4. A very good agreement is found from 200 Hz to 1000 Hz with the results provided by the model under plane wave assumption. Moreover, the same impedance value has been computed on the whole surface. Some differences only appear for points located near edges. This is probably due to the proximity of the computation domain boundaries.

V. APPLICATION TO THE LOCAL IMPEDANCE ACTIVE CONTROL

A. Numerical Modeling of the Surface Impedance control of a Porous Material by Pressure Minimization

A primary source is located above a porous material supported by a rigid floor (fig. 5). A secondary source is located inside the cavity, allowing to cancel the acoustic pressure at the point O. The primary source has a constant velocity V 0 and the secondary source has a constant velocity λV 0 . The global acoustic pressure in any point of the space is then obtained by superposition of the pressures due to both sources (p 1 : primary, p 2 : secondary): p(x,y) = p 1 (x,y) + λp 2 (x,y)

The coefficient λ is found by applying the zero pressure condition at the point O. To modelize such a device, three computations are thus necessary: the pressure field separately produced by each source is first computed. This allows to calculate λ. A third computation cancels the pressure at the point O corresponding to the control microphone position.

A local absorption coefficient may be calculated from the knowledge of the normal impedance on the surface of the absorbent.. Fig. 6 shows this coefficient along the porous surface. A good agreement between the numerical results and the experimental measurements may be found above the secondary source. The absorption is perfect in this area. On the other hand, it seems to be underestimated by the F.E. outside this area. This is certainly due to the 3D nature of the device, and to the anisotropy of the absorbent, which is important for this configuration.

B. Increase of the Active Surface

Extended absorbing surfaces may be built by assembling elementary active cells behind a large porous material sample. Fig. 7 shows the example of a 3×3 cells array. Each of them includes a loudspeaker which is controlled in order to cancel the pressure at a microphone located at the rear face of the porous material. The 2D FE modeling is performed by considering a single cross section. As previously, the pressure field separately produced by each source is first computed. A linear combination allows then to cancel the pressure at the points corresponding to the control microphone positions. From fig. 8, the influence of active absorption on pressure fields appears clearly: stationary waves produced by the rigid floor are canceled with active control, and the primary source radiates almost as in free field. Experimental results are confirmed by the numerical results (fig. 9). The minimum and maximum values of absorption are especially well predicted by the numerical modeling. Slight differences are due to bad experimental conditions: for example, the absorbent may close incompletely the cells because of the presence of wires.

CONCLUSION

We have shown in this paper how an analogy between two different disciplines of the physic can be made, validated, and used to adapt a numerical formulation. In our case, electromagnetics has been used to design local impedance active sound control systems. Furthermore, we could imagine that, if active control is used for sound absorption, equivalent system could also be used for an electromagnetic compatibility purpose, by reducing electromagnetic radiations with appropriated antennas and sources. Such approach is already used in low frequency (boat demagnetization) and it requires certainly to be studied for higher frequencies.

Fig. 4 .

 4 Fig. 4. Normal surface impedance of a porous material.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Geometry of the standing wave tube and acoustic pressure at 200 Hz and 400 Hz.

Fig. 3 .Fig. 5 .

 35 Fig. 3. Acoustic pressure in presence of a porous material. Left: f=200 Hz, right: f=800 Hz

Fig. 6 .Fig. 7 .Fig. 8 .Fig. 9 .

 6789 Fig. 6. Absorption coefficients along the absorbent -f = 400 Hz.


Laurent Dacheux
email: laurent.dacheux@pasteur.fr

Florence Larrous

Rachel Lavenir

Anthony Lepelletier

Abdellah Faouzi

Cécile Troupin

Jalal Nourlil

Philippe Buchy

Herve Bourhy

Dual combined real-time reverse transcriptionpolymerase chain reaction assay for the diagnosis of lyssavirus infection

Keywords: Rabies, lyssavirus, molecular diagnosis, real-time reverse-transcription polymerase chain reaction (RT-qPCR), human, animal, skin, saliva, intra-vitam, post-mortem, zoonosis, TaqMan, SYBR Green

¶ These authors contributed equally to this

Introduction

Rabies is an almost invariably fatal form of acuteprogressive encephalomyelitis that kills an estimated 59,000 humans each year, mostly in low-income Asian and African countries [START_REF] Hampson | Estimating the global burden of endemic canine rabies[END_REF]. This zoonosis is transmittedto humans by rabid animals biting, scratching or lickingmucous membranes or damaged skin. Moreover, some cases of human-to-human transmission have been described following the transplantation of organs or tissues from donors with undiagnosed rabies [START_REF] Basavaraju | Encephalitis caused by pathogens transmitted through organ transplants, United States, 2002-2013[END_REF][START_REF] Maier | Management and outcomes after multiple corneal and solid organ transplantations from a donor infected with rabies virus[END_REF], and exceptional laboratory cases of human rabies followingaerosol contamination [START_REF] Winkler | Airborne rabies transmission in a laboratory worker[END_REF][START_REF]Follow-up on rabies -New York[END_REF].

The principal etiological agent, responsible for almost all human rabies cases, is Rabies virus (RABV), the prototype species of the genus Lyssavirus in the family Rhabdoviridae [START_REF] Dacheux | Human rabies encephalitis prevention and treatment: progress since Pasteur's discovery[END_REF][START_REF] Who | Expert Consultation on Rabies -Second report[END_REF].Thirteen other species of this genus have been identified:Lagos bat virus (LBV), Mokola virus (MOKV), Duvenhage virus (DUVV), European bat lyssavirus 1(EBLV-1) and 2 (EBLV-2), Australian bat lyssavirus (ABLV), West Caucasian bat virus (WCBV), Irkut virus (IRKV), Aravan virus (ARAV), Khujand virus (KHUV), Shimoni bat virus (SHIBV), Ikoma lyssavirus (IKOV) and Bokeloh bat lyssavirus (BBLV) [START_REF] Ictv ; King | Virus taxonomy: classification and nomenclature of viruses: Ninth Report of the International Committee on Taxonomy of Viruses[END_REF]. Another new potential lyssavirus species, Lleida bat lyssavirus, (LLBV) has also recently been identified in bats in Spain [START_REF] Ceballos | Novel lyssavirus in bat, Spain[END_REF].

Most of these viruseswereisolated from bats, suggesting that lyssaviruses may have originated in Chiroptera [START_REF] Badrane | Host switching in Lyssavirus history from the Chiroptera to the Carnivora orders[END_REF].

RABV is the lyssavirus with the widest distribution worldwide, and with the broadest spectrum of vectors or animal reservoir hosts from the ordersCarnivora and Chiroptera.The domestic dog isamajorreservoir host of RABV, implicated in almost all cases of human rabies [START_REF] Who | Expert Consultation on Rabies -Second report[END_REF][START_REF] Knobel | Re-evaluating the burden of rabies in Africa and Asia[END_REF]. However, several other animal species can act as reservoirs for RABV transmission, depending on the geographic area considered. RABV displays also broad genetic diversity, mainly depending of animal hosts and geographic origin.

In animals, rabies is diagnosed post-mortem, on a brain specimen, with the fluorescent antibody test (FAT), the designated reference technique of the World Health Organization (WHO) and the Organization for Animal Health (OIE) [START_REF] Who | Expert Consultation on Rabies -Second report[END_REF][START_REF]Manual of Diagnostic Tests and Vaccines for Terrestrial Animals[END_REF]. This method is also the gold standard for the post-mortem diagnosis of human rabies. Many molecular methods have been developed for the intra-vitamdiagnosis of human rabies. These methods include reversetranscription (RT)-PCR [START_REF] Wacharapluesadee | Ante-and post-mortem diagnosis of rabies using nucleic acid-amplification tests[END_REF][START_REF] Fooks | Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century[END_REF][START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF][START_REF] Crepin | Intravitam diagnosis of human rabies by PCR using saliva and cerebrospinal fluid[END_REF] and real-time RT-PCR (RT-qPCR) [START_REF] Fooks | Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century[END_REF][START_REF] Wakeley | Development of a real-time, TaqMan reverse transcription-PCR assay for detection and differentiation of lyssavirus genotypes 1, 5, and 6[END_REF][START_REF] Hoffmann | Improved safety for molecular diagnosis of classical rabies viruses by use of a TaqMan real-time reverse transcription-PCR "double check" strategy[END_REF][START_REF] Coertse | Improved PCR methods for detection of African rabies and rabies-related lyssaviruses[END_REF][START_REF] Fischer | Molecular double-check strategy for the identification and characterization of European Lyssaviruses[END_REF], but none of the techniques developed to date can deal with the full diversity of the Lyssavirus genus and/or have been validated for use with all animal and human specimens collected in the field.

We describe here the development of a one-step pan-lyssavirus detection technique, based on a dual combinedreal-time reverse-transcriptionPCR assay(combo RT-qPCR). This assay includes a pan-RABV RT-qPCR probe-based technique able to detect all representatives of the broad genetic diversity of RABV, using two degenerate TaqMan ® probes. A SYBR ® Green RT-qPCR assay carried out in parallel can detect all the other lyssaviruses tested, in addition tosome RABV isolates. This combo RT-qPCR dedicated to pan-lyssavirus detection assay was found to have highsensitivity and specificity and was able to detect a large panel of animal lyssavirus samples.Italso provided accuratepost-mortem or intra-vitam diagnoses of human rabies. The pan-RABV RT-qPCR was then evaluated in two national reference laboratories in enzootic countries, where its utility and efficacy for rabies diagnosis and surveillance were demonstrated.

Materials and Methods

Ethics Statement

Human samples were obtained from routine diagnostic activities at the National Reference

Primer design

A region of the polymerase (L) gene (nucleotide positions 7170 to 7419 according to the Pasteur virus genome; GenBank accession number M13215) conserved among lyssavirus genomes was selected as the target of the combo RT-qPCR technique, based on the results of previous studies [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF][START_REF] Bourhy | Phylogenetic relationships among rhabdoviruses inferred using the L polymerase gene[END_REF]. This target sequence was amplified from a selected panel of lyssavirus isolates by a previously described RT-hnPCR method [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF], and the PCR products were Sanger sequenced and analyzed with Sequencher 5.0 (Gene Codes Corporation) software. The probes and primers of the combo RT-qPCR assay weredesigned on the basis of a ClustalX 2.1 multiple nucleotide alignment [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF]. All primers were designed with OligoAnalyzer 3.1 software, available from http://eu.idtdna.com/calc/analyzer, and checked in silico for the formation of hairpins, self-dimers and hetero-dimers and sequency analogy, by blast analysis (BlastN on the NCBI database).

Virusesand biological samples from animals and humans

The lyssavirus isolates analyzed were used as viral suspensions or as naturally (original brain samples from rabid animals) or experimentally (brains of newborn suckling mice inoculated with the original infected samples) infected animal tissues. All were obtained from the NRC-R or from the WHO Collaborating Center for Reference and Research on Rabies archive, both locatedat Institut Pasteur, Paris, France, and from the Institut Pasteur du Cambodge at Phnom Penh, Cambodia.Viral suspensions were generated with baby hamster kidney cells (BSR cells) [START_REF] Sato | Plaque formation of herpes virus hominis type 2 and rubella virus in variants isolated from the colonies of BHK21/WI-2 cells formed in soft agar[END_REF], and titrated on the same cells with five-fold serial dilutions, the results beingexpressed as fluorescent focus units per mL (FFU/mL).Animal brain samples that had tested negative for rabies virus with the FAT were also obtained from the NRC-R.

Human samples were obtained from routine diagnostic activities at the NCR-RofInstitut Pasteur, Paris, at the Institut Pasteur du Cambodge (Phnom Penh, Cambodia), at the Institut Pasteur du Maroc (Casablanca, Morocco) or from the NCR-R repository.

RNA isolation

Total RNA was extracted from titrated viral suspensionswith TRI Reagent LS (Molecular Research Center, Cincinnati, Ohio, USA) after the serialdilution of supernatants of infected cell cultures (BSR cells) [START_REF] Sato | Plaque formation of herpes virus hominis type 2 and rubella virus in variants isolated from the colonies of BHK21/WI-2 cells formed in soft agar[END_REF]in a dilution solution prepared from brain specimens from rabiesnegative dogs. These specimens were homogenized in culture medium (DMEM) and clarified by centrifugation at 3000 rpm for 5 minutes. For each virus stock previously titrated,serial 10fold dilutions were prepared in order to obtain from 1x10 6 to 1 fluorescent focus-forming units (FFU) in a final volume of 0.2 mLfor extraction. Extractions were performed in accordance with the kit manufacturer's protocol. We extracted total RNA from negative and positive (naturally or experimentally infected) animals in TRI Reagent (Molecular Research Center, Cincinnati, Ohio, USA), in accordance with the manufacturer's instructions.

For human samples and depending of their availability, at least onetype of sample(among skin biopsy, saliva andcerebrospinal fluid or CSF) was used per patient for totalRNAextraction, as previously described [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF]. Briefly, skin biopsy samples were dissociated with sterile scissors and incubated in 180 μL of ALT tissue lysis buffer and 20 μL of proteinase K (Qiagen, Courtaboeuf, France) at 37°C for 3 h, with gentle shaking. The resulting suspension was then mixed with 0.8mL of TRI Reagent LS (Molecular Research Center, Cincinnati, Ohio, USA) and RNA was extracted according to the manufacturer's instructions. RNA was extracted from saliva samples,collected using saliva swabsor directly after spiting into a tube (a volume of 0.2 mL was used in this latter case) or from CSF samples (0.2 mL),with TRI Reagent LS (Molecular Research Center, Cincinnati, Ohio, USA), using 2µL glycogen (5 mg/mL; Life Technologies, Saint Aubin, France) as a coprecipitant. RNA was finally dissolved in 50μL of nuclease-free water and stored at -70°Cuntiluse.

Preparation of cloned-target plasmids

Viral RNA was extracted from selected RABV isolates (n=9) and from one or two representative LBV, MOKV, DUVV, EBLV-1, EBLV-2 and ABLV lyssaviruses, reversetranscribed and amplified with the PVO5m/PVO9 primers, as previously described [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF]. The PCR products were then inserted into the TopoTA vector according to the manufacturer's recommendations (Life Technologies, Saint Aubin, France).For the other species and isolates of lyssavirus (WCBV, ARAV, KHUV, IRKV, SHIBV, IKOV, Ozernoe and BBLV), which were not available at the time of this study,the nucleotide region corresponding to the PVO5m/PVO9 fragment was synthesized (Eurofins Genomics, Ebersberg, Germany) based on the reference sequences in GenBank. This region was then inserted into the TopoTA vector, as described above.A large-scale plasmid preparation procedure was carried out and the plasmids obtained were used as a DNA matrix for determining the efficiency and the limit of quantification of the combo RT-qPCR assay.

Pan-RABV RT-qPCR assay

This one-step, probe-basedreal-time RT-PCR assay (pan-RABV RT-qPCR) was performed with the Superscript III Platinum One-Step RT-qPCR kit (Life Technologies, Saint Aubin, France),as recommended by the manufacturer, with only minor modifications. Real-time PCR, which was optimized fora final reaction volume of 20 μL, was performed with10 μL 2x Reaction Mix, 1.5 μL nuclease-free water, 1 μL of each primer Taq3long and Taq17revlong (10 μM), 0.4 μL SuperScript ® III RT/Platinum ® Taq Mix, 0.3 μLof each probe RABV4 and RABV5 (10 μM), 0.25 μL MgSO 4 (50 mM), 0.2 μL RNasin ® recombinant ribonuclease inhibitor (Promega, Charbonnieres, France), 0.05 μL ROX TM reference dye and 5 μL RNA template (previously diluted 1:10 in nuclease-free water).Amplification was carried out according to the following program:1 cycle of heating at 45°C for 15 min and 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 61°C for 1 min, during which fluorescence values were recorded.All reactions were carried out as technical duplicates in Thermo Scientific 96well plates (Life Technologies, Saint Aubin, France), with an Applied Biosystems 7500 Real-Time PCR System (Life Technologies, Saint Aubin, France). For each RT-PCR, a quantification cycle number (C q ) was determined as the PCR cycle number at which the fluorescence of the reaction exceeded a value considered to be significantly higher than background by the software associated with the Applied Biosystems 7500 Real-Time PCR System (Life Technologies, Saint Aubin, France).The efficiency (E), slope and correlationcoefficient (R 2 ) were also determined withthis software. All reactions were carried out as technical duplicates. A cutoff ≥ 38 was defined for negative results.

Pan-lyssavirus RT-qPCR assay

This assay was performed withthe SuperScript III Platinum SYBR ® Green One-Step qRT, as recommended by the manufacturer (Life Technologies, Saint Aubin, France),with the sameminor modificationsas indicated for the pan-RABV assay. In particular, this real-time PCR was optimized for a final volume of 20 μL,using the same mixture composition and the sameamount of diluted sample. The primers used were Taq5long and Taq16revlong and the probes were replaced with nuclease-free water.Amplification was performed on asimilar thermocycler,as follows: 15 minutes at 45°C, 3 minutesat 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 55°C, during which fluorescence values were recorded. After the 40 cycles of amplification, a melting analysis was carried out to check the product amplified by determining itsspecific melting temperature (increase 0.01°C/s,55-95°C). As previously indicated, the efficiency (E), slope and correlationcoefficient (R 2 ) were also determined withthe software associated with Applied Biosystems 7500 Real-Time PCR System (Life Technologies, Saint Aubin, France). All reactions were carried out as technical duplicates. For this assay, a positive reaction was not based on the Cq value but exclusively on the melting temperature (Tm) value and the shape of the melting curve, both compared to positive and negative controls.

Universal internal control system

The quality of RNA extraction was checkedwith theheterologous internal universal control system based on in vitro transcribed eGFP-RNA described by [START_REF] Hoffmann | A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses[END_REF] [START_REF] Hoffmann | A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses[END_REF].

Working dilutions containing 2x10 5 copies/μL in nuclease-free water were prepared and stored as aliquotsat -70°C. We directly spiked each sample with a total of 10 6 copies (5 μL) during the extraction step, after adding the TRI Reagent. We then detected eGFP RNA by RT-qPCR, according to a slightly modified version of the method used in the original study [START_REF] Hoffmann | A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses[END_REF]. In particular, the conditions of amplification were identical to those used with the pan-RABV RT-qPCR assay. The mixture composition and thermal conditions were identical to those for the pan-RABV RT-qPCR assay, and we used the EGFP1-F and EGFP2-R primers and the EGFP1-FAM probe. A part of the animal and human samples which were retrospectively analysed in this study (i.e. already extracted before the implementation of this internal control) were not spiked with this internal control.

Other controls used in the combo RT-qPCR assay

For each assay,we performed two negative controls, with nuclease-free water and a calibrated total RNA suspension (0.2 μg/μL)obtained by extraction from a pool ofnegative dog brainspecimens.Two positive controls for the pan-RABV RT-qPCR were also used, consisting oftotal RNA extracted from suckling newborn mousebrainsinfected with the CVS strain and diluted 1:10 in the calibrated RNA suspension (0.2 μg/μL).Serial log 10 dilutions were tested, and two successive serial dilution points were selected in the linear range of the amplification curve determined vi. Similar positive controls were carried outfor the pan-lyssa RT-qPCR assaywith theEBLV-1 lyssavirus (isolate 8918FRA).

RThemi-nested PCR (RT-hnPCR)

Reverse transcription was performed, as previously described, in a final volume of 30 μL [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF].

We then subjected 2 µL of complementary DNA (cDNA) to amplification by hnPCR, with 10 pmol of primers PVO5m/PVO9 in the first round and primers PVO5m/PVO8 in the second round. The amplification reactions contained 2 U AmpliTaq DNA Polymerase (Life Technologies, Saint Aubin, France), 10 nmol of each nucleotide triphosphate, and 62.5 nmol magnesium, as previously indicated [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF].

International interlaboratory trial evaluation

The combo RT-qPCR assay was evaluated by the NRC-R in 2014, in an interlaboratory trial organized by the European Union reference laboratory for rabies, which is located in Nancy, France [START_REF]Inter-laboratory test for rabies diagnosis[END_REF]. The FAT and RT-hnPCR were also evaluated in parallel in this trial. The test panel consisted of nine anonymous samples of freeze-dried homogenized brains, either unifected or infected with various lyssavirus species. Details of this trial have been provided elsewhere [START_REF]Inter-laboratory test for rabies diagnosis[END_REF].

Results

Probe and primer design

Due to the limited number of nucleotide sequences corresponding to the targeted region of the polymerasegene of lyssavirusavailable frompublic databases at the time of this study, a large panel of RABV isolates was selected and sequenced, so as to obtain at least one prototype sequence for each of the principal phyloclades previously defined (Table S1) [START_REF] Kissi | Genetic polymorphism in the rabies virus nucleoprotein gene[END_REF][START_REF] Bourhy | The origin and phylogeography of dog rabies virus[END_REF][START_REF] Pant | Recent emergence and spread of an Arctic-related phylogenetic lineage of rabies virus in Nepal[END_REF]. We also selected and sequenced a panel of isolates from other lyssavirus species. The probes and primers for the combo RT-qPCR assay were designed from two different sequence datasets.

The first dataset contained102RABV partial polymerase gene sequences, including 93newly acquired sequences, and was used to design the pan-RABV RT-qPCR assay, which was based on the Taq3long and Taq17revlong primers and the TaqMan ® probes RABV4 and RABV5 (Fig1) (Table 1). These primers amplified a 143-nucleotide sequence. To complete the evaluation of the spectrum of detection of these primers and probes of the pan-RABV RT-qPCR assay, we analyzed in silico another dataset of 91 partial polymerase gene sequences of RABV isolates originated from the New World region (the Americas)and which have been recently published and available in GenBank (Fig S1). The genetic diversity observed in this dataset was high, leading to the presence of cumulative mistaches for some isolates.

For the pan-lyssa RT-qPCR assay, we used another dataset contained 45 sequences representing all the other lyssavirus species and including 22 new sequences. On the basis of multiple nucleotide alignment data, we defined a single set of primers, Taq5long and Taq16revlong (Fig 2 ), amplifing a119-nucleotide region.Due to the high diversity of sequences in all the different lyssavirus species, no consensual probe was designed and we used these primers with an intercalating dye (SYBR ® Green). Asterisks indicted partial polymerase sequences obtained in this study. A description of the RABV isolates included in this multiple alignment is provided in Table S1. S1. [START_REF] Crepin | Intravitam diagnosis of human rabies by PCR using saliva and cerebrospinal fluid[END_REF]

Determination of the assay acceptance parameters

We first evaluated theintrinsic parameters of the comboRT-qPCR assay withcloned-target plasmids representative of the 14 recognized lyssavirus species(Table2) [START_REF] Ictv ; King | Virus taxonomy: classification and nomenclature of viruses: Ninth Report of the International Committee on Taxonomy of Viruses[END_REF]. We analyzed nine RABV isolates belonging to different phylogenetic clades and/or lineageswiththe pan-RABV RT-qPCR assay. The mean efficiency (E) and correlation coefficient (R 2 ) values obtained were 97% (±9%) and 0.98 (±0.012), respectively. The limit of quantification ranged from 10 to 10 4 target copiesper assay, with the lowest values obtained for isolates 8693GAB and 03003IND and the highest for isolates 08338GAM and 9105USA. Seventeen other lyssavirus isolates were target-cloned and tested with the pan-lyssa RT-qPCR assay. The mean efficiency and R 2 values obtained were 106% (±25%) and 0.964 (±0.033), respectively. The lowest limit of quantification value was obtained with one isolate of EBLV-1 (02007DAN)

and WCBV, at one target copy per assay, and the highest was obtained with one isolate of IRKV, at10 5 target copies (efficiency was also lowest for this isolate, at 47%).

18/40 Total number includes samples tested in Cambodia, France and Morocco on all the patients, negative and positive for rabies. b One more skin biopsy sample was detected with pan-RABV RT-qPCR (n=43) compared to RT-hnPCR (n=42). c 8 saliva samples from a same patient were not detected but one saliva sample from another patient was detected, both with pan-RABV RT-qPCR compared to RT-hnPCR, with a total of n=20 and n=27 saliva samples detected for pan-RABV RT-qPCR and RT-hnPCR, respectively. d Including all patients, with rabies-confirmed and negativepatients.

For skin specimens, the combo RT-qPCR identified 43 samples as positive, versus42 for the reference technique, RT-hnPCR, giving a sensitivity of 102%. In total,41 samples gave positive results in thepan-RABV RT-qPCR, with a mean Cq value of 31.53 (SD±4.38). Two biopsies from patient 28tested negative in thepan-RABV assay but positive in the pan-lyssa assay (Table S4). This patient wasinfected with a rabies virus belonging to the Africa 2 phylogenetic clade [START_REF] Contou | Severe ketoalkalosis as initial presentation of imported human rabies in france[END_REF]. The skin biopsy specimens ofpatients2and 31 tested positive with thepan-RABV RT-qPCR, with Cq values of 35.5 and 33.9, respectively,but negative byRT-hnPCR (Table S4). For patient 48, a skin biopsyspecimen tested negative with the pan-RABV RT-qPCR but gave a weak positive signal with the reference technique.

For saliva, 20 samples tested positive with thepan-RABV RT-qPCR, with a mean Cq value of 33.93 (SD±3.00), versus 27 withthe RT-hnPCR technique. All these samples were stored frozen (70°C) before testing. The samples that tested positive by RT-hnPCR included eight saliva samples collected from the same patient (patient 28)from Mali andtesting negative in thecombo RT-qPCR assay but not with the reference technique, resulting in a sensitivity of 74% with saliva (Table 5) (Table S4). For patient 16, one saliva sample tested positive with thepan-RABV RT-qPCR, with a Cq value of 37.42, but negative with the RT-hnPCR (Table S4).

For CSF, all results were concordant between the two assays and the meanCq value was25.3 (SD±0.14) (Table 5)(Table S4). In addition, one aspirate of bronchial secretions tested negative with the pan-RABV RT-qPCR but positive byRT-hnPCR (Table S4) [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF]. assay.The third isolate almost certainly gave negative results due to the small amount of material extracted, given the weak reaction obtained in the RT-hnPCR.All the lyssavirus species tested (34 isolates belonging to 7 different species) with the pan-lyssa RT-qPCR assay were successfully detected, even those associated with a high limit of detection and/or quantification such as LBV and MOKV lyssavirus. This combo RT-qPCR provides a test with a large spectrum of detection which is not covered by all fluorescent conjugated antibodies commercially available. All the assays gave negative results for all of the negative control materials, indicating a high specificity.

To complete the evaluation of the performance of our pan-RABV RT-qPCR assay, we analyzed in silico the targeted nucleotide regionof a dataset of RABV isolates originated from New World area, circulating mainly in bats, skunks or raccoons, and which were recently available in GenBank. Indeed, the primers and probes were initially designed based on a limited number of such isolates.A high genetic diversity was found in isolates originated from the Americas, as observed in silico with the presence of cumulative mistmaches in the primers and/or probes hybridization regions, which could interfere with their detection (Figure S1).

Although we demonstrated in our study that the pan-RABVRT-qPCR assay allowed the positive detection of different prototype isolatesfrom this region (such as 9704ARG and 9105USA), further evaluation of this assay has to be performed on a larger and more representative dataset of isolates from New World area.

We evaluated the utility of this method for the diagnosis of human rabies, using one of the largest collections of positive and negative biological samples ever assembled. Our test detected all65cases of human rabies tested.Strong concordance (98.1%) was observed between the results of RT-qPCR assaysand the RT-hnPCR considered as the reference technique.In particular, both the overall sensitivity and specificity of the combo RT-qPCR assay were high, with 92.7% and 100%, respectively. Compared to the RT-hnPCR, this combo RT-qPCR assayperformed in our conditions presented the advantage to limit crosscontamination (one-step technique), to be time-saving (two to three times faster) for a similare cost.

We confirmed the interest of skin biopsy and saliva samples for the intra-vitam diagnosis of rabies in humans [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF][START_REF] Crepin | Intravitam diagnosis of human rabies by PCR using saliva and cerebrospinal fluid[END_REF]. They allow the diagnosis in 100% of the rabid patients, with 91.5% of skin biopsies and 54% of saliva samples scored positive, in accordance with previous results [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF]. This technique requires only small amounts of RNA, with only 3 µL required for the assay.

This could be a key advantage in cases in which material is precious or in short supply, such as CSF collected from children or saliva swabs from bats.

We demonstrated the applicability of this test in enzootic areas, in two reference laboratories locatedin Morocco and Cambodia. Finally this technique was included in aninternational interlaboratory trial in 2014, in which it provided 100% concordant results [START_REF]Inter-laboratory test for rabies diagnosis[END_REF]. This demonstrates that this test, also not directly applicable in rural settings as most of other molecular methods, can be easily implemented in enzootic countries in laboratories equipped with real-time PCR thermocyclers. In these labs, it could then be considered as a valuable alternative in labs not able to perform FAT or as an interesting confirmatory/back up technique following FAT when needed. This technique could also been a useful complement of rapid and in field diagnostic methods recenlty developed such as rapid immunodiagnostic test (RIDT) (Léchenne et al., in revision in PlosNTD). Compared to other methods of diagnosis for rabies infection, our combo RT-qPCR assay presented the common advantages and limitations of other RT-qPCR methods used for the diagnosis of rabies, and which have been reviewed elsewhere [START_REF] Wacharapluesadee | Ante-and post-mortem diagnosis of rabies using nucleic acid-amplification tests[END_REF][START_REF] Fooks | Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century[END_REF][START_REF] Dacheux | More accurate insight into the incidence of human rabies in developing countries through validated laboratory techniques[END_REF][START_REF] Duong | Laboratory diagnostics in dog-mediated rabies: an overview of performance and a proposed strategy for various settings[END_REF].

This study further supports the WHO recommendations asking for the collection of one skin biospsy and/or 3 saliva samples in patients suspected of rabies [START_REF] Who | Expert Consultation on Rabies -Second report[END_REF][START_REF] Dacheux | More accurate insight into the incidence of human rabies in developing countries through validated laboratory techniques[END_REF].

  Center for Rabies (NCR-R) of Institut Pasteur, Paris, at the Institut Pasteur du Cambodge (Phnom Penh, Cambodia), at the Institut Pasteur du Maroc (Casablanca, Morocco) or from the NCR-R repository. The NCR-R repository has been registered for research purposes and declared, in accordance with French regulations (article L.1243-3 in relation to the French Public Health Code), to both the French Ministry for Research and a French Ethics Committee, both of which approved and registered the biobank (declaration number DC-2009-1067; collection No. 12).

Figure 1 .

 1 Figure 1. Multiple alignment of the 102 partial polymerase sequences of RABV species, with

Figure 2 :

 2 Figure 2: Multiple alignment of 45 partial polymerase sequences from lyssaviruses other than

Table 1 : Oligonucleotide sequences of primers and probes used in the combo RT-qPCR (combination of pan-RABV and pan- 350 lyssa RT-qPCR assays) and in the internal control eGFP-based RT-qPCR assay.

 1 /40

	351
	352
	353
	Application	Reference	Name	Type	Length	Sequence (5'-3')	Sense Position
				Taq3long	Primer	22	ATGAGAAGTGGAAYAAYCATCA	S	7273-7294 a
				Taq17revlong	Primer	25	GATCTGTCTGAATAATAGAYCCARG	AS	7390-7414 a
	Pan-RABV RT-qPCR assay (TaqMan ® -based)	This study	RABV4	Probe (FAM/TAMRA)	29	AACACY TGATCBAGKACAGARAAYACATC	AS	7314-7342 a
				RABV5	Probe (FAM/TAMRA)	32	AGRGTGTTTTCY AGR ACWCAYGAGTTTTTYCA	S	7353-7384 a
	Pan-lyssa RT-qPCR assay (SYBR ® Green-based)	This study	Taq5long Taq16revlong	Primer Primer	23 25	TATGAGAAATGGAACAAYCAYCA GATTTTTGAAAGAACTCATGKGTYC	S AS	a 7272-7294 7366-7390 a
				EGFP1F	Primer	20	GAC CAC TAC CAG CAG AAC AC	S	637-656 b
	eGFP internal control assay	Hoffmann et al., 2006	EGFP2R EGFP	Primer Probe (VIC/TAMRA)	19 22	GAA CTC CAG CAG GAC CAT G AGC ACC CAG TCC GCC CTG AGC A	AS S	b 768-750 703-724 b
	354
	355	a According to the Pasteur virus (PV) RABV genome sequence (GenBank accession number M13215).
	356	b According to the cloning vector pEGFP-1 sequence (GenBank accession number U55761).
	357

Table 2 : Intrinsic parameters of the combo RT-qPCR assay using target-cloned plasmids. 371

 2

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Table 3 : Intrinsic parameters of the combo RT-qPCR assay using titrated viral suspensions.

 3

	390
	391
	Species	Isolate	Location	Host	Assay	Slop e	R 2	Efficiency (%)	Limit of detection (FFU/mL) a	Corresponding Cq max	Limit of quantification (FFU/mL) a
	RABV	CVS b	-	Lab strain Pan-RABV -2.46 0.900	155	50	36.5	50
		8743THA	Thailand	Human	Pan-RABV -3.46 0.995	95	50	36	50
		9147FRA	France	Red fox	Pan-RABV -3.31 0.980	100	50	36	50
		9704ARG	Argentina	Bat	Pan-RABV -3.64 0.981	88	500	34	500
	LBV	8619NGA	Nigeria	Bat	Pan-lyssa -3.33 0.998	100	1500	NA c	15000
	MOKV	86100CA M	Cameroon	Shrew	Pan-lyssa -3.42 0.986	96	5000	NA	5000
	DUVV	87020SA South Africa	Bat	Pan-lyssa -3.04 0.984	113	500	NA	5000
		86132SA South Africa	Human	Pan-lyssa -3.42 0.990	96	38	NA	3850
	EBLV-1 8918FRA	France	Bat	Pan-lyssa -3.67 0.998	87	390	NA	390
	EBLV-2 02053SWI Switzerland	Bat	Pan-lyssa -3.04 0.994	113	500	NA	500
	ABLV	9810AUS	Australia	Bat	Pan-lyssa -3.59 0.994	90	500	NA	500
	392
	393
	Species	Isolate	Location	Host	Assay	Slop e	R 2	Efficiency (%)	Limit of detection (FFU/mL) a	Corresponding Cq max	Limit of quantification (FFU/mL) a
	RABV	CVS b	-	Lab strain Pan-RABV -2.46 0.900	155	50	36.5	50
		8743THA	Thailand	Human	Pan-RABV -3.46 0.995	95	50	36	50
		9147FRA	France	Red fox	Pan-RABV -3.31 0.980	100	50	36	50
		9704ARG	Argentina	Bat	Pan-RABV -3.64 0.981	88	500	34	500
	LBV	8619NGA	Nigeria	Bat	Pan-lyssa -3.33 0.998	100	1500	NA	15000
	MOKV	86100CA M	Cameroon	Shrew	Pan-lyssa -3.42 0.986	96	5000	NA	5000
	DUVV	87020SA South Africa	Bat	Pan-lyssa -3.04 0.984	113	500	NA	5000

Table 4 : Sensitivity and spectrum of detection of the combo RT-qPCR assay using a dataset of positive lyssavirus samples.

 4

	a ND : Not done

Table 5 :

 5 Evaluation of the intrinsic parameters of the combo RT-qPCR assay for the diagnosis of human rabies, using the RT-hnPCR as the referent method.

			Combo RT-qPCR result
	Type of sample	Total number a	No. pos. samples/Total no.of samples(%) From all patients From pos. patients	Mean Cq value ± SD	Sensitivity Specificity	Positive predicted value	Negative predicted value
	Sample Skin biopsy	67	43/67(64.2)	43/47 (91.5)	31.53 ± 4.38	102 b	100	100	96.3
	Saliva	120	20/120 (16.7)	20/37 (54)	33. 93 ± 3.00	74 c	100	100	93.4
	CSF	12	3/12 (25)	3/7 (42.9)	25.3 ± 0.14	100	100	100	100
	Brain biopsy	12	10/12 (83.3)	10/10 (100)	21.4 ± 5.56	100	100	100	100
	Total	211	76/211 (36)	76/101 (75.2)	30.70 ± 5.7	92.7	100	100	95.6
	Patient d	65	41/65 (63.1)	41/41 (100)	41/65 (61.5)	100	100	100	100
	a

Acknowledgments

We would like to thank Ousmane Faye and Martin Faye for providing us with the Senegalese samples.

Author summary

Rabies is an infectious disease of humans and other mammals caused by lyssaviruses. It has one of the highest mortality rates of any infectious disease. Rabies has been known since We then evaluated theintrinsic parameters of this comboRT-qPCR assay withtitrated viral suspensionsrepresentative of the main lyssavirus species(Table 3).Four RABV isolates (CVS, 8743THA, 9147FRA and 9704ARG) were testedinthe pan-RABV assay. The mean efficiency and R 2 values were 109% (±23%) and 0.964 (±0.032), respectively, with similarvalues for the limits of detection and quantification, ranging from 50 to 500 FFU/mL of the initial sample before extraction. At least one representative virus from each of themain other lyssavirus species was analyzed with the pan-lyssa assay (Table 3). The mean efficiency and correlation coefficient values were as good as those for the TaqMan ® based-assay, at99% (±8%) and 0.992 (±0.004), respectively. The limit of detection ranged from 38 FFU/mL for one isolate of DUVV (86132SA)to 5,000 FFU/mL for MOKV (isolate 86100CAM) whereas the limit of quantification ranged from 390 FFU/mL for EBLV-1 (isolate 8918FRA) to 15,000 FFU/mL for LBV (isolate 8619NGA).

Determination of the threshold of positivity for pan-RABV RT-qPCR.

The cut-off value of the threshold of positivity for the pan-RABV RT-qPCR assay was based on the results obtained from a panel of all negative samples confirmed negative samples (human and animal).

We first analyzed a panel of 40 primary brain samples from 10 different animal species (Table S3). All had previously tested negative with the reference technique, FAT. All these samples tested in the pan-RABV RT-qPCR assay, provided a mean Cq value of 39.79 associated to a standard deviation (SD) of 0.31, and a minimum Cq value of 38.41.

In parallel, a total of 97 human negative samples (including brain and skin biopsies, saliva and CSF samples) were tested with the pan-RABV RT-qPCR assay (Table S4).These samples were previously confirmed negative by conventional RT-PCR techniques performed during routine diagnosis by the national reference centre for rabies in France and in Cambodia. The mean Cq value obtained was39.95 (SD±0.23), with a minimum Cq value of 38.66.

Taking together all these samples (n=137), we obtained a mean Cq value of 39.92 (SD±0.29).

Based on these results we determined the cut-off value for the threshold of detection at 38, corresponding to the mean Cq from which were substracted nearly 6 SD.

Determination of the sensitivity, specificity and spectrum of detection of the combo RT-qPCR assay for the post-mortem diagnosis of animal rabies

Both the pan-RABV and pan-lyssa RT-qPCR assays were evaluated witha large panel of positive animal samples, each previously been confirmed withthe reference technique,FAT. We selected121 RABV isolates consideredrepresentative of the main phyloclades, subclades and lineagescirculating worldwide (Table 4)(Table S2) for testing. Eighty corresponded tooriginal brain tissue samples collected from 12 different animal species, whereas the others were amplified by inoculatingsuckling newborn mice. All the different genetic lineages of RABV tested gave positive results in thepan-RABV RT-qPCR assay, except for three isolates from Senegal belonging to Africa 2 clade(Table 4) (Table S2). The mean Cq value was 19.52 (SD±5.42), with maximum and minimum Cq values of 36.37 and 9.88, respectively.The sensitivity and spectrum of detection of the pan-lyssa RT-qPCR assay were evaluated with 34 isolates from sevendifferent lyssavirus species: LBV (n=2), MOKV (n=3), DUVV (n=3), EBLV-1 (n=18), EBLV-2 (n=6), ABLV (n=1)and BBLV (n=1) (Table 4) (Table S2). Five of the samples were primary brain tissuesfrom bats and a catinfected with the EBLV-1 lyssavirus species, and one was from a bat infected with BBLV. Positive detection was obtained for all the isolates tested, with a unique and well-defined dissociation curve, with a melting temperature Tm, centered on77.75°C (SD±1.06). We also tested 49 RABV isolates with the pan-lyssa RT-qPCR:41testedpositive, with Cq values below 21.Two of the three samples from Senegalthat tested negative in TaqMan ® assays yielded positive results in thisSYBR ® Green assay. Only two isolates not belonging to the RABV species were detected with the pan-RABV RT-qPCR assay (1 from EBLV-1 and the other from EBLV-2) (Table 4) (Table S2). Finally, all isolates (n=155) except one (RABV isolate 14011SEN) gave positive results in the combo RT-qPCR compared to the FAT, leading to a high sensitivity(99.3%) (Table 4) (Table S2).

The overall specificity of the combo RT-qPCR assay was complete (100%) when compared to FAT. Indeed, specificity was determined on a panel of 40 primary brain samples from 10 different animal species (Table S3), which have been previously tested negative with the reference technique, FAT. All these samples tested negative in the pan-RABV RT-qPCR assay, with a Cq value higher than the cut-off value of 38. The same samples were tested with the pan-lyssa RT-qPCR assay and all yielded negative results with non-specific dissociation curves.

Determination of thesensitivity and specificity of the combo RT-qPCR assay for the diagnosis of human rabies

We assessed the utility of thecombo RT-qPCR assay for human rabies diagnosis, using a largecollection of positive and negative biological samples collected from 65 different patients (Table S4). Samples were obtained for post-mortem or intra-vitam diagnosis in France, Cambodia and Morocco. All samples were tested with the pan-RABV assay and the results obtained were compared with RT-hnPCR, used as the reference technique [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF]. Some samples were also tested with the pan-lyssa RT-qPCR assay (Table S4). In total, 12 samples for post-mortem (all brain biopsies) and 199 samples for intra-vitam diagnosis were tested with the pan-RABV RT-qPCR assay, including skin biopsy specimens (n=67), saliva (n=120) and CSF (n=12)(Table 5) (Table S4).

For post-mortem diagnosis, 12 brain specimens were tested and results were positive with both theRT-hnPCR and thepan-RABV RT-qPCR techniques, with a mean Cq value of 21.4 (SD±5.56) (Table 5) (Table S4).

Compared to the RT-hnPCR, the specificity of the pan-RABV RT-qPCR assay was 100% for all type of negative samples tested, as well as for the pan-lyssa RT-qPCR assay, with nonspecific dissociation curves(Table 5) (Table S4).

The sensitivity and specificity of the combo RT-qPCR weretherefore 100% for brain and skin specimens and well as forCSF.A lower sensitivity was obtained for saliva (74%). The specificity was also 100%. No discordance was noticed at the patient level. Overall, 41 of the 65 patients tested gave positive results for both assays.

Implementation of the pan-RABV RT-qPCR in local settings, in two reference laboratories in enzootic areas

Two national reference laboratories for rabieslocatedin Pasteur Institutes in enzootic areas, Morocco and Cambodia, tested thepan-RABV RT-qPCR assay in their environments. The results for the pan-RABV assaywere compared with those for RT-hnPCR, used locally as a reference, for skin biopsies (n=5) and brain biopsies (n=4) in Morocco, and saliva (n=5), skin biopsies (n=35) and brain biopsies (n=4) in Cambodia (Table S5). The overall sensitivity and specificity were 97.7 and 100%, respectively. Concordance was excellent, with a Kappa value of 0.9 for the two techniques for human diagnosis, with only one saliva sample from Cambodia testing negative in thepan-RABVRT-qPCR assay butweakly positive byRT-hnPCR (Tables S4 andS5).

Evaluation of the combo RT-qPCR assay in aninternational interlaboratory trial

Finally, we evaluated the comboRT-qPCR assayin an interlaboratory trial testing FAT, RT-qPCR and RT-hnPCR on nine anonymous samples; the results obtained were concordant with those expected(Table S6) [START_REF]Inter-laboratory test for rabies diagnosis[END_REF].

Discussion

The clinical diagnosis of rabies remains challenging, and is often unreliable, particularlyin humans, and confirmation by laboratory methods is therefore required [START_REF] Who | Expert Consultation on Rabies -Second report[END_REF][START_REF] Dacheux | More accurate insight into the incidence of human rabies in developing countries through validated laboratory techniques[END_REF]. Laboratory testing is based on the post-mortem analysis of brain samples, the gold standard test being the FAT, which can be used together with virus isolation in therabies tissue culture infectious test (RTCIT) [START_REF] Who | Expert Consultation on Rabies -Second report[END_REF][START_REF]Manual of Diagnostic Tests and Vaccines for Terrestrial Animals[END_REF][START_REF] Bourhy | Comparative field evaluation of the fluorescent-antibody test, virus isolation from tissue culture, and enzyme immunodiagnosis for rapid laboratory diagnosis of rabies[END_REF][START_REF] Dean | The fluorescent antibody test[END_REF]. However, these techniques remain impossible or of limited value for theintra-vitam diagnosis of rabies. Alternative new techniques have been proposed over the last two decades, particularly since the advent of molecular techniques, such as reverse transcription-polymerase chain reaction (RT-PCR) [START_REF] Wacharapluesadee | Ante-and post-mortem diagnosis of rabies using nucleic acid-amplification tests[END_REF][START_REF] Fooks | Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century[END_REF][START_REF] Dacheux | More accurate insight into the incidence of human rabies in developing countries through validated laboratory techniques[END_REF]. Various protocols have been developed for detecting lyssavirus RNA in different tissue samples by conventional PCR or real-time quantitative PCR (qPCR). Several RT-qPCR techniques for the detection of RABV have been described, based onTaqMan ® assays targeting a conserved region of nucleoprotein genes [START_REF] Wacharapluesadee | Ante-and post-mortem diagnosis of rabies using nucleic acid-amplification tests[END_REF][START_REF] Fooks | Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century[END_REF][START_REF] Hoffmann | Improved safety for molecular diagnosis of classical rabies viruses by use of a TaqMan real-time reverse transcription-PCR "double check" strategy[END_REF]. Other assays are dedicated to lyssavirus species, and are designed to discriminate between RABV and EBLV-1 and 2 [START_REF] Fooks | Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century[END_REF][START_REF] Wakeley | Development of a real-time, TaqMan reverse transcription-PCR assay for detection and differentiation of lyssavirus genotypes 1, 5, and 6[END_REF] or to detect RABV, LBV, MOKV and DUVV [START_REF] Coertse | Improved PCR methods for detection of African rabies and rabies-related lyssaviruses[END_REF], for example. A SYBR ® Green-basedassay was recently developed for the detection of all lyssavirus species, but it was not tested on fieldsamples or on a broad range of lyssavirus species [START_REF] Hayman | A universal real-time assay for the detection of Lyssaviruses[END_REF].Another molecular diagnosis protocol based on a combination of TaqMan ® -based and SYBR ® Green-based assayswas recently used on animal samples infected withlyssaviruses known to be circulatingin Europe [START_REF] Fischer | Molecular double-check strategy for the identification and characterization of European Lyssaviruses[END_REF].

We developed a dual combined RT-qPCR method for pan-lyssavirus detection insamples, for intra-vitam diagnosis in humansandpost-mortem diagnosis in animals. This assay is based on two complementary techniques. The first is probe-based (TaqMan ® ) RT-qPCR for detection of the RABV species (pan-RABV RT-qPCR), whereas the second uses an intercalating dye (SYBR ® Green) for the detection of other lyssavirus species (pan-lyssa RT-qPCR). The primers of both systems and the probes for the pan-RABV assay were based on a region of the polymerase gene known to be conserved; they were degenerate, to facilitate annealing with a broad spectrum of lyssavirus isolates (Table 1, Figures 1 and2) [START_REF] Dacheux | A reliable diagnosis of human rabies based on analysis of skin biopsy specimens[END_REF][START_REF] Bourhy | Phylogenetic relationships among rhabdoviruses inferred using the L polymerase gene[END_REF].

Weevaluated the intrinsic parameters of our combo RT-qPCR assaywithcloned-target plasmids and titrated viral suspensions. We were able to detect all the lyssavirus species tested with viral suspensions (n=11) or plasmids (n=26). The only lyssavirus isolate not tested with this assay was Lleida bat lyssavirus (LLEBV) recently identified in a bat in Spain, for which the sequence of the full genome, including the polymerase gene, was not available [START_REF] Ceballos | Novel lyssavirus in bat, Spain[END_REF].

For both assays, the limits of detection were compatible with rabies diagnosis, with values ranged from 38 to 5.10 3 FFU/mL and from1 to 10 5 target copies/mL, for viral suspensions and cloned-target plasmids, respectively.

The performance parameters for this combined assay were evaluated with a large panel of fieldanimal samples representative of the overall genetic variability encountered inRABV species and extending to all lyssavirus species charactarized to date. We tested121 animal samples with our combo RT-qPCR assay.Only three of the isolates tested(RABV from the Africa 2 clade, originating from Senegal) testednegative in pan-RABV RT-qPCR assay. A combination of at least one mismatch withthe forward primer and in each of the two probes was observed for these samples, and more generally for RABV isolates belonging to the Africa 2 clade, potentially accounting for the lower sensitivity for thisphylogenetic clade (Figure 1). We tested 50 RABV isolates in our pan-lyssa RT-qPCR system, and 40 gave positive results. This system also detectedhiglyinfected RABV samples (Cq<21) and a large proportion of RABV isolates from theAfrica 2 clade. Indeed, two of the three isolates from the Africa 2 clade giving negativepan-RABV RT-qPCR results were detected with this

Supporting Information Legends

Table S1 : Description of lyssavirus isolates used for the design of primers and probes of the combo RT-qPCR assay (combination of pan-RABV RT-qPCR and pan-lyssa RT-qPCR assays).

Table S2 : Description of samples used for the post-mortem diagnosis of animal rabies and results of the analytical sensitivity of the combo RT-qPCR assay.

Table S3 : Results of the analytical specificity of the combo RT-qPCR assay for the post-mortem diagnosis of animal rabies.

Table S4 : Diagnostic sensitivity and specificity of the combo RT-qPCR compared to the RT-hnPCR for the diagnosis of human rabies.

Table S5 : Results of the implementation of the pan-RABV RT-qPCR in local settings of two national reference laboratories for rabies localized in Morocco and in Cambodia.

Table S6 : Evaluation of the combo RT-qPCR assay in an international interlaboratory trial

Table S7 : Description of RABV isolates originated from New World region and used for the multiple alignment in Table S7, corresponding to the in silico evaluation of primers and probes of the pan-RABV RT-qPCR assay.