O Lopez

A Amy-Klein

C Daussy

Ch Chardonnet

F Narbonneau

M Lours

G Santarelli

86-km optical link with a resolution of 2×10 -18 for RF frequency transfer

RF frequency transfer over an urban 86 km fibre has been demonstrated with a resolution of 2×10 -18 at one day measuring time using an optical compensator. This result is obtained with a reference carrier frequency of 1 GHz, and a rapid scrambling of the polarisation state of the input light in order to reduce the sensitivity to the polarisation mode dispersion in the fibre. The limitation due to the fibre chromatic dispersion associated with the laser frequency fluctuations is highlighted and analyzed. A preliminary test of an extended compensated link over 186 km using optical amplifiers gives a resolution below 10 -17 at 1 day.

Introduction

Besides the current developments of atomic clocks, high resolution frequency distribution and comparison have become a new challenge in the field of frequency metrology. Atomic clocks have improved to a fractional frequency accuracy in the range 10 -15 -10 -16 (see for instance [1][2][3][4]) and fractional accuracies in the 10 -17 range or better are under investigation by several groups. The comparison between collocated clocks has been greatly simplified by the recent development of optical synthesizer based on femtosecond laser frequency comb (see for instance [5]). On the other hand the comparison of distant RF or optical clocks is currently using satellite-based techniques and is limited at a level around 10 -15 over one day of measurement. In the context of fundamental physics tests the development of long-distance high resolution frequency transfer is an important issue. From the comparison of atomic or molecular clocks the temporal variation of fundamental constants can be measured providing a test of the equivalence principle [6][7]. The development of such measurements for a large number of clocks with different atoms and molecules will allow discriminating the variations of the different constants and will improve the reliability of measurements in this controversial field. Astrophysics could also benefit from such frequency transfer development especially for large-base interferometers [8][9][START_REF] Logan | Proc. of IEEE Frequency Control Symposium[END_REF][START_REF] Calhoun | [END_REF][12].

Recent developments gave greater place to optical fibres for high resolution frequency transfer [13]. Progress has been reported in the dissemination of either a RF/microwave [START_REF] Logan | Proc. of IEEE Frequency Control Symposium[END_REF][START_REF] Calhoun | [END_REF][12][14][15] or an optical reference frequency [16][17][18]. Optical transfer is promising and very attractive in the perspective of optical clocks comparison; alternatively a RF system can be easier to implement depending on the local laboratories facilities and the aimed applications.

We have already demonstrated the distribution of a reference signal in the RF domain at 100 MHz through an optical link of 43 km between two laboratories, LPL and LNE-SYRTE [15,19]. Since mechanical perturbation and temperature variation along the fibre slightly perturb the propagation delay on the link, a noise compensation set-up was implemented and described in [14][15]. Two types of noise compensator have been developed, both using a round trip phase correction technique. We will design as electronic compensator the one based on the phase conjugation method; it acts on the phase of the reference signal injected in the link [START_REF] Logan | Proc. of IEEE Frequency Control Symposium[END_REF]. The so-called optical compensator is based on an optomechanical actuator, which acts directly on the optical length of the fibre [8,[START_REF] Calhoun | [END_REF]. With this compensated link, two frequency standards from each laboratory, one in the IR domain (CO 2 /OsO 4 at 30 THz) and the other in the microwave domain (CSO at 12 GHz) have been compared with a resolution of 3x10 -14 at 1s [15]. This paper is describing the improvement of the transfer resolution on the cascaded 86 km link using an upgraded optical compensator. Two major developments have been implemented. To gain on the signal-to-noise ratio, the reference carrier frequency has been increased to 1 GHz. To reduce the sensitivity to polarisation mode dispersion (PMD) in the fibre, the polarisation state of the input light has been scrambled. The further limitation due to the fibre chromatic dispersion is highlighted and analyzed. A preliminary attempt to realize a compensated link over 186 km is also presented.

RF reference dissemination and noise optical compensation.

The schematic of the optical link and its compensation system is shown in Figure 1. The link connecting LPL and LNE-SYRTE is composed of two parallel 43-km dark optical fibres, which have been obtained by splicing a few sections of SMF 28 single mode fibres from the standard telecommunication network. The 86 km link is obtained by cascading the two twin fibres between LPL and LNE-SYRTE. This way the two ends are collocated at LPL, one designated as Local, and the other as Remote.

The input RF signal at 1 GHz is modulating the intensity of a DFB laser diode which is feeding the optical link. The wavelength is about 1.55 µm and the maximum output optical power is 20 mW. After propagation along the link, the modulation is detected at the other end of the fibre with a fast photodiode, and used to synthesize a coherent RF signal at 900 MHz. This signal exhibits the phase fluctuations of the forward signal at the Remote end; it modulates the intensity of a second laser diode, which is feeding the link in the backward direction. Optical circulators are used at each fibre end to discriminate the forward and backward optical signals. The backward signal is detected at Local end with a second photodiode and exhibits phase fluctuations produced by one round trip. The phase comparison of this backward signal and the input signal is realized at 900 MHz and gives a measurement of the round trip phase fluctuations.

The optical polarizations of both laser diodes are scrambled directly at each fibre end, as discussed below. Two different frequencies, 1 GHz and 900 MHz, are used for the forward and backward RF signals in order to eliminate the parasitic signals due to stray optical reflections on connectors and splicing along the link, which are adding noise to the error signal. The stimulated Brillouin Back Scattering (SBS) is also strongly attenuated by the 100 MHz shift between forward and backward signals, much larger than the SBS gain profile (< 10 MHz).

The error signal is processed by a simple analog loop filter and applied to two delay lines, designed as fast and slow ones, at the Local input of the link in order to cancel out the variations of the propagation delay. The fast delay line is achieved with a 15 m length optical fibre that is wrapped around a cylindrical piezoelectrical transducer (PZT). Fast and small variation corrections are applied on the PZT voltage to stretch the fibre with a correction range of about 15 ps. The slow delay line is made of a 4 km optical fibre wrapped around a copper wheel ; the slow corrections are applied by heating the whole spool with a sensitivity of 150 ps/°C and a total dynamic range of 6 ns. The loop bandwidth is limited by the roundtrip propagation delay (0.88 ms) to a few hundreds Hz. The relative stability of the compensated link is measured by analyzing the phase variation between the 1 GHz signals at both Local and Remote end. The phase signal is filtered with a low-pass filter (3 Hz bandwidth) then digitized and the overlapping Allan deviation of the measured phase samples is deduced.

Results and limitations.

Figure 2 displays a sample of the phase fluctuation of the compensated link. The propagation delay fluctuation is around 1 ps over a few days. The fractional frequency stability of the link is displayed on Figure 3 (squares): it is 5x10 -15 at 1 second integration time and 2x10 -18 at one day integration time. For comparison, without any corrections, the stability is 3x10 -14 at 1 second with a floor around 10 -15 after 100s (triangles in Figure 3) limited by the diurnal temperature variations. The three orders of magnitude reduction of the long-term frequency fluctuations demonstrates the effectiveness of the compensation system. The stability at one day is 5 times better than previous implementation of the link at 100 MHz on 43 km (circles in figure 3) [15]. Moreover the average frequency offset between reference signal and transferred signal over the link is zero within the error bars. The better performance of the present system is directly related with the increased of the RF carrier frequency from 100 MHz to 1 GHz (900 MHz for the backward signal). The signal-to-noise ratio of the error signal driving the compensator is increased about a factor 9: fibre phase fluctuations are 9 times higher while detection noise is approximately the same.

The performance of this round trip compensation technique is limited by the parasitic noise on the error signal. Figure 4 shows the phase noise spectral density at 1 GHz of the compensator in closed loop with the 86 km link (upper trace) and with an equivalent optical attenuator instead of the link (lower trace). This last trace displays the noise of the compensation system, which is at the level of -120 dB at 1 Hz. It corresponds to an Allan deviation of 10 -15 at 1 s integration time, which is the aimed stability for the compensated 86km link. However the optical link degrades the noise by nearly 15 dB, leading to a noise of -105 dB at 1 Hz. The bump around 300 Hz is due to the bandwidth of the control loop.

This parasitic excess phase noise originates from the fact that the round trip phase fluctuations do not exactly correspond to twice the forward phase fluctuations due to an asymmetry of the phase noise in forward and backward propagation directions along the link. Due to the chromatic and polarization mode dispersions of the fibre the propagation delay is sensitive to the characteristics of the two optical signals injected in each direction at each end of the link. The two laser diodes have uncorrelated optical frequency fluctuations and polarizations state. We show in the next paragraph how the asymmetry due to the polarization mode dispersion (PMD) can be strongly reduced by scrambling the light polarization state at each end. Thus, in our set-up, the short-term frequency stability is limited by the chromatic dispersion and laser frequency fluctuations. The long-term frequency stability is restricted by the RF electronic components temperature sensitivity in the compensator.

Polarization effects.

The propagation characteristics along the fibre depend on the polarization state through the polarization mode dispersion, PMD (see for instance [START_REF] Joindot | Les Télécommunications par fibres optiques[END_REF]). The PMD is induced by the birefringence of the optical fibre due to the asymmetry of the core and is varying in time because of the mechanical stress induced by vibrations or by temperature variations. Moreover the directions of both fast and slow axes of the link move randomly in time.

Due to PMD, the propagation delay along the link fluctuates. Since two independent laser diodes with different wavelengths and polarisation states are used at each fibre end, these propagation delay fluctuations are uncorrelated for the forward and backward signals along the link. This lack of symmetry of the propagation delay fluctuations degrades the effectiveness of the noise compensator, which can only correct the symmetrical phase fluctuations, since the error signal is the roundtrip phase fluctuations. The main effect is the degradation of the long term stability.

The polarisation related effects are enhanced by the optical compensator itself. Both delay lines induce a parasitic change of the polarization state of the signal injected in the 86km link and thus introduce an unwanted variation in the propagation delay along the link. This variation is caused by the stretching of the correction fibres, and can be much faster than the typical slow variation of PMD.

As mentioned above, polarization effects can be significantly reduced by scrambling the polarization directly at each laser diode output, at a frequency higher than the cut off frequency of the stabilization loop. The polarization scrambling is obtained with a commercial three axes piezoelectric polarization scrambler. Each axis is excited with a low voltage sinusoidal signal tuned to a piezoelectric resonance. Among the possible resonance we chose the non-harmonically related ones (around 60, 100, and 130 kHz respectively). This fast polarisation modulation explores all the polarization states much faster than the propagation delay along the link [START_REF] Noe | [END_REF]. In this case the DGD is averaged and the PMD effects are minimized.

In order to evaluate the limitations due to the PMD or the delay line induced polarisation effects, the following measurements have been performed. The phase fluctuations between the local and remote ends have been first recorded without any scramblers and the corresponding Allan deviation is plot as triangles on Figure 5. In that case, the fast delay line is not used. In fact, we may notice that the delay lines are set directly at the input end of the link. They affect the polarisation from the input of the fibre, and their effect on the propagation delay fluctuations is then enhanced by the PMD. With the PZT fibre stretcher this effect is high enough to perturb the correction loop and prevent its use. Thus for time scale below 100s there is no correction and the fractional frequency stability is that of the free link. For time scale above 100s the stability is degraded by the polarisations effects about one order of magnitude. The bump between 10 3 and 10 4 s is due to the slow thermal compensator, which is also changing the polarisation. Since this Allan deviation is not typical from PMD effects, a second experiment has been performed with only one scrambler at the Local end (that is on the forward way) and no scrambler at the Remote end on the backward way. In that case, both delay lines are used and the polarisations effects due to the delay lines and PMD are minimized on the forward propagation delay. On the backward propagation way however, PMD effects are still effective but they are not enhanced by the delay line induced polarisation effects since the delay lines are at the output end of the backward way. With this scheme we aim to test the PMD of the 86-km fibre link alone. The phase fluctuations are displayed on Figure 2 together with the phase fluctuations of the compensated link with the two scramblers. Let us remind that the compensation system does not distinguish the forward and backward fluctuations and the delay lines are correcting on each way half the total round trip fluctuations. Since the PMD affects solely the backward phase fluctuations, only half of the PMD phase fluctuations can be corrected, and the phase data in closed loop still exhibits half the PMD phase fluctuations on the backward way. It amounts around 2 ps, and thus the effects of PMD on one way is about 4 ps. The Allan deviation plot of the phase data of Figure 2 is displayed as circles on Figure 5 and compared to the Allan deviation plot with two scramblers (squares). The PMD does not affect the short-term stability but diurnal temperature variations of the PMD degrade the long-term frequency stability to a few 10 -17 .

Finally, when scrambling the polarization, the stability (squares in Figures 3 or 5) reaches a value compatible with the laser dispersion noise level (see section 5 below), which demonstrates that the polarization effects are strongly cancelled out.

Chromatic dispersion effects.

The second source of parasitic noise limiting the compensation performance arises from the chromatic dispersion of the fibre associated with the frequency fluctuations of the laser diodes. An additional phase noise, that we will call dispersion noise, is induced along the fibre, and is not correlated in the forward and backward signals. This asymmetrical additional phase noise can not be corrected by our compensation system.

The laser current modulation induces both an amplitude and a frequency modulation (chirp), and the spectrum of both laser diodes is constituted by a carrier at the optical frequency and sidebands separated by the reference RF frequency 2π Ω (= 0.9 or 1 GHz) [22].

The detected signal at the RF frequency results from the beat notes between adjacent lines, which propagate at different velocities due to the dispersion. At first order, the propagation time of two adjacent lines differs from the quantity 0 0

d t D L λ ω ⎛ ⎞ Ω Δ = - ⎜ ⎟ ⎝ ⎠
where D=17 ps/km.nm is the dispersion of the fibre, L the fibre length (in km), 0 λ the laser wavelength (in nm) and 0 2 ω π the laser frequency. In our case at 1 GHz, 12.3

d t p s Δ = -
for the 90-km fibre (including the link and the correction fibre spool). The phase detected at each end of the fibre at 1GHz (respectively 900 MHz) is the contribution of two terms: δω are the uncorrelated frequency fluctuations of each laser diode. The dispersion noise degrades the system phase noise level of around 15 dB (see Figure 4), leading to a noise of -105 dB at 1 Hz.

At the remote end, the remaining phase noise is ( ) Δ . The Allan deviation floor (star plot) due to this effect is displayed in Figure 3 and is very close to the Allan deviation of the compensated link. The small difference partly arises from the additional thermal fluctuations induced by the compensator on the laser diodes. This demonstrates that the laser frequency noise associated with the dispersion of the fibre is presently the main limitation for the resolution of the compensation link. It is worth noting that this contribution to the noise scales linearly with the modulation frequency through d t Δ

and, thus, cannot be reduced by increasing this value. This limitation could be overcome with a stabilisation of the laser diodes frequencies. Extensive efforts had already been done to reduce the laser frequency fluctuations with a better control of the diode temperature and bias current. An active stabilisation is more efficient although not straightforward to implement, especially at long-term. Laser frequency fluctuation of the order of 1 kHz at 1 s would lead to a negligible noise contribution (at the level of 10 -17 ). Finally a more simple solution would consist in adding a negative dispersion fibre of adequate length. This would compensate for the link chromatic dispersion, even if losses will be inserted.

Longer link: the problem of the attenuation.

The above results demonstrate that an RF optical link can be used for metrological purposes to disseminate a RF reference signal between laboratories in the same area. We present a preliminary evaluation of this technique for longer distances, which has been tested by adding fibre spools to the existing 86-km link.

For a longer link, the performance of the noise compensator is still limited by the dispersion noise, which increases proportionally to the fibre length. Moreover the signal-tonoise ratio is further reduced by the attenuation of the fibre spool, which yields a reduction of 40dB on the RF signal for each 100 km of additional fibre. This attenuation can not be compensated by increasing the laser diodes power, since the power in each sideband is already near the threshold of SBS. To overcome this limitation we use Erbium Doped Fibre Amplifiers (EDFA). A preliminary test was done with a 186-km link composed of the 86-km link and 100 km of fibre spools. Two standard telecom unidirectional amplifiers were connected with two optical circulators in order to obtain a full bidirectional configuration. This composite bidirectional EDFA was inserted at half distance of the link and the amplification level was adjusted to compensate the additional optical losses. For such an amplification level (around 20 dB), the EDFA's excess phase noise is equivalent to a few 10 - 15 frequency stability at 1s, which is negligible compared to the chromatic dispersion noise.

Figure 6 shows preliminary results of the relative frequency stability of this 186 km link for two different set-ups, one using the optical compensator and the other an electronic compensator. For this alternative compensator described in [14], the correction is applied directly on the RF source signal. The two systems give about the same results. At short-term, the Allan deviation for the optical compensator is 2×10 -14 at 1 s integration time, whereas the limit due to the laser dispersion is 1×10 -14 at 1s. This small difference could be attributed to any optical or electronic components which have not been carefully optimised for these measurements. The long term frequency stability is better than 10 -17 at one day integration time.

This demonstrates the first realization of a RF frequency transfer below 10 -17 level on distances above 100 km. The resolution is very close to the limit due to the dispersion noise, which demonstrates that EDFA's can be used to compensate for the attenuation. For longer distances the stability is degraded because the fibre phase fluctuations and the loop bandwidth are respectively increasing and reducing with the fibre length. For a 1000 km link around 10 EDFA's should be used to recover the signal, the control bandwidth is reduced to around 10 Hz, and the fibre noise could not be corrected by more than a factor of 10 at 1 s. However the present set-up demonstrates an elementary component which could act as a repeater for frequency dissemination over a multi-section optical link. By cascading a few such systems the stability of around 10 -17 at one day could be maintained over distances up to 1000 km.

Conclusion.

We have demonstrated a 86-km compensated link from an urban network with a frequency stability of 5×10 -15 at one second and 2×10 -18 at one day integration time. These results were obtained after the limiting effects were analyzed and two different AM frequencies used for each propagation direction and light polarization state scrambled. The dispersion of the fibre, associated with the laser frequency fluctuations, gives the present limit of our compensation set-up. This limitation can be overcome by the laser frequency stabilisation or by the cancelling of the chromatic dispersion with a negative dispersion fibre. In that case, an Allan deviation of 10 -15 at 1 s is achievable, limited by the electronic noise of the compensator. For a longer link, the signal attenuation becomes critical. For 186-km, a stability below 10 -17 at one day integration time has been measured using EDFA's and the short-term is limited by the fibre chromatic dispersion. This demonstrates a quite simple repeater for multi-section frequency dissemination over very long distances.

The compensator could be further improved by using a microwave frequency signal around 10 GHz instead of the present 1 GHz signal. The signal-to-noise ratio of the error signal is indeed proportional to the reference frequency. Thus the distance between repeaters for multi-section frequency transfer could be chosen with an additional tolerance of 50 km. The degradation of the error signal due to the EDFA's and the electronic noise is also much less critical. However the set-up and compensator should be adapted to microwave modulation techniques and the stability improvement is not straightforward.

The present results give a new opportunity to compare distant frequency standards. Our compensation system constitutes a robust repeater with 100 % duty cycle and a resolution below 10 -17 at 1 day; it could be used for multi-section frequency transfer up to 1000 km distances. Further developments should render optical links a real alternative to satellite link. One obtains an asymmetrical spectrum [22] :

( ) 0 0 0 1 j t j t j t n n n E E e L L e L e ω ∞ Ω -Ω + - = ⎛ ⎞ = + + ⎜ ⎟ ⎝ ⎠ ∑ with ( ) ( ) ( ) ( ) ( ) ( ) ( )
0 2 2 0 0 0 1 0 0 1 1 1 1 2 2 1 1 1 1 2 2 n a a a a a n n n a n a n a a n a n n n a n a a L M J L M J J M J M M L MJ JM J M M - ∞ = ∞ + + - = ∞ - + = ⎧ = ⎪ ⎪ ⎪ = + + + - ⎨ ⎪ ⎪ = - + + + - ⎪ ⎩ ∑ ∑ ∑ .
After propagation along the fibre, the Fourier components are dephased by the quantities 0 ϕ (for the fundamental) and n ϕ + and n ϕ -(for the n + Ω and n -Ω components respectively).

At low order (in term of chromatic dispersion), we have :

+ - + + - - - =-Δ -ΩΔ -Ω ⎧ ⎪ - = Δ -ΩΔ +Ω ⎪ ⎪ - = -Δ -ΩΔ -Ω ⎨ ⎪ - = Δ -ΩΔ +Ω ⎪ ⎪ ⎩ .
The intensity is detected and filtered at 2π Ω . One obtains:

( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( 1) ( 1) 0 0 0 ( 1) ( 1) 0 0 0 2 c o s2 1 c o s 2 s i n2 1 s i n n n n n d d n n n n n d d n S L L L L n t t t t L L L L n t t t t ω ω ∞ + + + - + - = ∞ + + + - + - = = + + Ω Δ Ω-Δ -Ω + - + Ω Δ Ω-Δ -Ω ∑ ∑
The term in quadrature is resulting from the frequency modulation of the laser, which induces a non-symmetrical spectrum. Note also that the amplitude is depending from the differential time delay d t Δ . The amplitude is thus modulated with the fibre length.

Figure captions

t

  is the propagation time of the laser carrier along the fibre (see detailed calculation in Annex). The fluctuation of 0 t in the first term gives the fibre noise that we want to correct. The second term gives the so-called dispersion noise resulting from the conversion of the laser frequency noise by the fibre chromatic dispersion. The resulting dispersion noise after one round trip is ( )

Fourier

  and m are respectively the amplitude and frequency modulation indexes. In our case, m i =0.7 and m=15, due to the chirp coefficient of the laser diode estimated to η =375 MHz/mA[START_REF] Joindot | Les Télécommunications par fibres optiques[END_REF]22].The field can be developed in Fourier series at the modulation frequency 2π Ω the Fourier serie of the phase, and n M the coefficients of the

Figure 1 :

 1 Figure 1: Block diagram of the 86 km fibre link and the optical compensation system. Both local and remote functions are located in the same place. DFB LD : distributed feedback laser diode.

Figure 2 :

 2 Figure 2: Propagation delay fluctuation of the compensated link (a) with 2 scramblers and (b) with only one scrambler (see section 4); it is obtained from the differential phase fluctuation of the 1 GHz signals at local and remote ends.

Figure 3 :

 3 Figure 3: Fractional frequency stability of the free 86-km link (triangles), the compensated link at 100 MHz (circles) and 1 GHz (squares) and dispersion noise due to the laser diodes frequency fluctuation (stars).

Figure 4 :

 4 Figure 4: Phase noise spectral density of the optical compensation system at 1GHz in closed loop a) with the 86-km optical fibre b) with optical attenuators equivalent to the fibre attenuation.

Figure 5 :

 5 Figure 5: Polarisation effects: fractional frequency stability of the 86-km compensated link without scramblers (and no fast delay line) (triangles), with only one scrambler at input end (circles), and with two scramblers (squares).

Figure 6 :

 6 Figure 6: Preliminary fractional frequency stability of the 186-km link. Triangles: electronic compensator; squares: optical compensator.

Figure 1 Figure 2

 12 Figure 1

  Figure 4

  Figure 5

  Figure 6

Acknowledgments

This work was supported by Ministère de la Recherche and European Space Agency/ESOC.

Appendix :

The laser field is given by ( )


Matthew R Buckwalter

Thi Phan

Nga

Meriadeg Ar Gouilh

Laurence Fiette

Jean-Francois Bureau

Melissa E Laird

Julian Buchrieser

Simona Ozden

Justine Cheval

Marc Eloit

Jean-Claude Manuguerra

Antoine Gessain

Paul T Brey

Arnaud Fontanet

Matthew L Albert
email: albertm@pasteur.fr.

Identification of a Novel Neuropathogenic Theiler's Murine Encephalomyelitis Virus ᰔ

Theiler's murine encephalitis viruses (TMEV) are divided into two subgroups based on their neurovirulence. Persistent strains resemble Theiler's original viruses (referred to as the TO subgroup), which largely induce a subclinical polioencephalomyelitis during the acute phase of the disease and can persist in the spinal cord of susceptible animals, inducing a chronic demyelinating disease. In contrast, members of the neurovirulent subgroup cause an acute encephalitis characterized by the rapid onset of paralysis and death within days following intracranial inoculation. We report herein the characterization of a novel neurovirulent strain of TMEV, identified using pyrosequencing technology and referred to as NIHE. Complete coverage of the NIHE viral genome was obtained, and it shares <90% nucleotide sequence identity to known TMEV strains irrespective of subgroup, with the greatest sequence variability being observed in genes encoding the leader and capsid proteins. The histopathological analysis of infected brain and spinal cord demonstrate inflammatory lesions and neuronal necrosis during acute infection with no evidence of viral persistence or chronic disease. Intriguingly, genetic analysis indicates the putative expression of the L* protein, considered a hallmark of strains within the persistent subgroup. Thus, the identification and characterization of a novel neurovirulent TMEV strain sharing features previously associated with both subgroups will lead to a deeper understanding of the evolution of TMEV strains and new insights into the determinants of neurovirulence.

Theilioviruses have positive-sense, single-stranded RNA genomes and are members of the Picornaviridae family and the Cardiovirus genus. Several serotypes have been identified, causing disease in rodents, such as Theiler's murine encephalomyelitis virus (TMEV) [START_REF] Theiler | Spontaneous encephalomyelitis of mice, a new virus disease[END_REF][START_REF] Theiler | Encephalomyelitis of mice. I. Characteristics and pathogenesis of the virus[END_REF] and rat Theiler-like virus (RTV) [START_REF] Ohsawa | Genetic analysis of a Theiler-like virus isolated from rats[END_REF], and humans, such as Vilyuisk human encephalomyelititis virus (VHEV) [START_REF] Casals | Immunological characterization of Vilyuisk human encephalomyelitis virus[END_REF] and Saffold viruses [START_REF] Chiu | Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections[END_REF][START_REF] Jones | Discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin[END_REF][START_REF] Zoll | Saffold virus, a human Theiler's-like cardiovirus, is ubiquitous and causes infection early in life[END_REF]. TMEV is an enteric pathogen that follows the fecal-oral route of infection; however, when inoculated intracranialy (IC), it can cause severe pathology in susceptible mouse strains. As a natural mouse pathogen, TMEV provides a valuable experimental model for understanding host-pathogen interactions. The TMEV serotype is divided into two subgroups based on the pathogenesis of the disease. Members of the TO subgroup, so named because they resemble Theiler's original strains [START_REF] Theiler | Spontaneous encephalomyelitis of mice, a new virus disease[END_REF], cause a biphasic infection. The early phase is an acute poliomyelitis characterized by viral replication in the neurons of the brain [START_REF] Aubert | Early infection of the central nervous system by the GDVII and DA strains of Theiler's virus[END_REF][START_REF] Lipton | Persistent Theiler's murine encephalomyelitis virus infection in mice depends on plaque size[END_REF]. During the late phase of infection, the virus progresses from the gray to the white matter of the spinal cord, where, depending on the genetics of the mouse strain (4), it can persist for the life of the animal within various cell types [START_REF] Aubert | Identification of Theiler's virus infected cells in the central nervous system of the mouse during demyelinating disease[END_REF][START_REF] Lipton | The predominant virus antigen burden is present in macrophages in Theiler's murine encephalomyelitis virus-induced demyelinating disease[END_REF], causing in some instances chronic demyelination [START_REF] Lipton | Theiler's virus infection in mice: an unusual biphasic disease process leading to demyelination[END_REF]. In contrast, the second subgroup, consisting of the GDVII and FA strains, are neurovirulent, and their pathology is characterized by an acute and rapidly fatal encephalitis [START_REF] Theiler | Encephalomyelitis of mice. I. Characteristics and pathogenesis of the virus[END_REF]. These viruses replicate mainly in the neurons of the brain, and for the few animals that survive primary infection, there does not appear to be viral persistence in the central nervous system (CNS) [START_REF] Lipton | Persistent Theiler's murine encephalomyelitis virus infection in mice depends on plaque size[END_REF]. Both neurovirulent strains were isolated in 1937 by Theiler and Gard; however, full-length sequences and infectious clones have been generated only for the GDVII strain. For the FA strain, only partial genomic sequence covering the 5Ј-untranslated region (5ЈUTR) through the end of the P1 region are available (GenBank accession no. U32924 and M80883). Within these 3,262 bp, there is 99.7% identity to the GDVII strain, which has led to speculation that GDVII and FA actually are separate isolates of the same strain due to the time and place of isolation [START_REF] Michiels | Analysis of the leader and capsid coding regions of persistent and neurovirulent strains of Theiler's virus[END_REF].

Here, we report the phenotype and genetic analysis of the complete genome of a novel neurovirulent strain of TMEV that we have designated NIHE based on its isolation at the National Institute of Hygiene and Epidemiology in Hanoi, Vietnam. We demonstrate that the kinetics of disease and the phenotype of infected animals mimic those published for GDVII and FA strains. Moreover, histopathological analyses demonstrate that tissue and cellular tropism in the CNS are consistent with those observed following the inoculation of neurovirulent strains, including the inability to persist past the acute phase in the small percentage of inoculated animals that do survive. Phylogenetic analysis reveals NIHE to be less divergent from the TO strains than GDVII. Interestingly, we present evidence for the presence of an alternate open reading frame (ORF) which is predicted to encode the full-length L* protein, facilitated by a canonical AUG start sequence and previously shown to be present only in the TO subgroup strains [START_REF] Kong | Alternative translation initiation site in the DA strain of Theiler's murine encephalomyelitis virus[END_REF]. These data illustrate the need for continued investigation to establish the genetic and host immune determinants of neurovirulence and the evolution of viral persistence in this family of viruses.

MATERIALS AND METHODS

Ethics statement. All animal experiments described in the current study were conducted at the Institut Pasteur according to the European Union guidelines for the handling of laboratory animals The protocol was approved by the Institut Pasteur animal care and use committee. Inoculations were performed under anesthesia, and all efforts were made to minimize animal suffering.

Mice and infections. Outbred Swiss mice were bred and maintained in the animal facility of the National Institute of Hygiene and Epidemiology in Hanoi, Vietnam. This colony originated from animals imported from France. The initial phenotype was observed in 2008 following the low-number serial passage of brain homogenate from apparently healthy donors. Samples were transferred to the Institut Pasteur in Paris for further characterization. Female 5-to 7-week-old SJL and C57BL/6 mice were purchased from Charles River Laboratories and housed in our SPF animal facility at the Institut Pasteur in Paris. The handling of mice and experimental procedures were performed in accordance with institutional guidelines for animal care. Inoculations were performed in mice via the intracranial injection of 30 to 35 l of mouse brain homogenates. Specifically, brains from infected animals were prepared as 10% homogenates by mechanical disruption in sterile serum-free Dulbecco's modified essential medium (DMEM). Homogenates were clarified by centrifugation (10,000 ϫ g for 20 min). Control animals received 30 to 35 l of clarified supernatants from naïve animals at the indicated dilutions. All dilutions were prepared in sterile phosphatebuffered saline (PBS) or DMEM.

RNA isolation and preparation for 454 sequencing. Clarified supernatants were generated from the brains of animals demonstrating a disease phenotype. Aliquots of supernatant were combined with the AVL buffer supplied in the QIAamp viral RNA Minikit (Qiagen) and processed according to the manufacturer's specifications. Aliquots were combined, and 454 sequencing was performed by Eurofins MWG Operon (Ebersberg, Germany).

BLAST analysis and bioinformatics. FASTA files containing individual sequencing reads were obtained from the sequencing reactions. The nonredundant protein (nr) and nucleotide (nt) sequence databases were downloaded from NCBI and updated August 2010 prior to query. All sequences were queried against the nr/nt databases using BLASTϩ [START_REF] Camacho | BLASTϩ: architecture and applications[END_REF]. Metagenomic analysis was performed using MEGAN software (version 3.9), which resulted in the grouping of sequences according to phylogeny [START_REF] Huson | MEGAN analysis of metagenomic data[END_REF]. The de novo assembly of sequences greater than 80 bp was performed using the local alignment algorithm within CodonCode Aligner software (version 3.5), and the resulting consensus sequence was checked for inserted/deleted sequences that can be an artifact of 454 sequencing homopolymeric sequences. Twenty-three sites were manually con-firmed and corrected throughout the sequence. The corrected consensus sequence was used in all subsequent comparative analyses.

Histology and immunohistochemistry. Infected or control animals were anesthetized and perfused with PBS followed by 4% paraformaldehyde in PBS. Brains and spinal cords were dissected and fixed in 4% paraformaldehyde overnight at 4°C. Tissues were embedded in paraffin by following established procedures, and 5-m serial sections from the brain and spinal cord were stained with hematoxylin-eosin (HE). Cross-reactivity with TMEV antigens was tested by immunohistochemistry on paraffin sections incubated with a primary rabbit polyclonal antibody raised against Theiler's virus capsid proteins (kindly provided by J. F. Bureau and M. Brahic). Slides were counterstained with hematoxylin.

Phylogenetic analysis and alignment. Nucleotide and associated protein sequences for the following viruses were obtained from GenBank (data concerning three domains were collected: protein sequences coding for the 3D polymerase [3Dpol], P1 [protein 1], and VP1 [viral protein 1]): GDVII (M20562 and NC_001366), FA (U32924 and M80883 combined), DA (M20301), Yale (EU723238), BeAn8386 (M16020), TOB15 (EU718732), VIE415HTR (EU718733), TO4 (U33045 and M80885 combined), WW (M80889 and U33046 combined), Mengo virus (DQ294633), encephalomyocarditis virus (NC_001479), porcine encephalomyocarditis (DQ517424), Saffold virus (FM207487, NC_009448, and EF165067), cardiovirus D (EU681176), human TMEV-like cardiovirus (NC_010810 and EU376394), RTV NGS910 (AB090161), RTV-1 (EU542581 and EU815052), and Vilyuisk (EU723237, M80888, and M94868 combined). Multiple-sequence alignment was performed using the Muscle algorithm implemented within SeaView (version 4.2.4). The 3Dpol data set included 20 taxa and 1,404 characters, the P1 data set had 23 taxa for 2,617 nucleotides, and the VP1 matrix involved 23 taxa for 868 characters.

Phylogenetic reconstruction. The phylogenetic reconstructions of three protein-coding sequences (3Dpol, VP1, and V1) from three sets of nucleotides were inferred separately by a maximum-likelihood analysis on matrixes composed of 20, 20, and 23 taxa, respectively. These phylogenetic analyses were implemented by the BEAST software package (version 1.6.1). As this method required a predetermined evolution model estimated on the data set, we specified as one of the models selected by Jmodeltest [START_REF] Posada | jModelTest: phylogenetic model averaging[END_REF] on each matrix (3Dpol, VP1, and P1). Using the results of Jmodeltest, the three-codon partition model of evolution was used (under the GTR substitution model with a gamma site heterogeneity distribution [GTRϩG]), so that each codon position (first, second, and third) could follow its own evolution rate. The Yule speciation process (specified as tree prior) and constant-population-size coalescent models show similar estimates for each data set. The Monte Carlo Markov chain was set to 5,000,000 states for 3Dpol and VP1 data sets and to 10,000,000 for the P1 matrix to reach adequate prior effective sampling size (ESS) values greater than 300 and 2,000, respectively. Trees were annotated with TreeAnnotator (version 1.4.8) and displayed and edited using FigTree (version 1.3.1).

Genetic analysis. Similarity plots were generated using SimPlot (version 3.5.1) with the following parameters: 200-bp window, 20-bp step, and Kimura 2-parameter distance model [START_REF] Lole | Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination[END_REF].

Nucleotide sequence accession number. The nucleotide sequence reported in this paper was submitted to GenBank and has been assigned the accession no. HQ652539.

RESULTS

Identification of a novel neurovirulent strain of TMEV.

While performing animal experiments involving the serial passage of clarified lysate from mouse brain homogenates, we observed the appearance of a distinct disease phenotype. Following inoculation, several animals displayed a flaccid tail and an abnormal gait, which progressed to unilateral or bilateral limb paralysis 9 to 10 days postinoculation. Following the next passage, 100% of inoculated animals succumbed to infection; continued serial passage has demonstrated consistent lethality. To determine the kinetics of death, groups of C57BL/6 mice (n ϭ 10) were inoculated IC with 10ϫ dilutions (10 Ϫ1 to 10 Ϫ6 ) of infected mouse brain homogenate (IMBH), and survival was monitored (Fig. 1A). Animals receiving a 10 Ϫ1 dilution had a mean survival time of 7 days, whereas animals receiving 10 Ϫ2 , 10 Ϫ3 , and 10 Ϫ4 dilutions had mean survival times of 9, 10, and 11.5 days, respectively. The 50% lethal dose (LD 50 ) was determined to be a 10 Ϫ5 dilution of IMBH. Control animals received a 10 Ϫ1 dilution of 10% brain homogenate derived from naïve animals with no apparent phenotype (Ͼ45 days postinoculation). To quantitatively describe the disease progression in mice, a clinical scoring method was established (Fig. 1B), and disease progression was monitored daily (Fig. 1C). To accurately describe disease progression, only symptomatic animals were included; the number of symptomatic animals per group is indicated in the figure legend.

To determine the identity of the infectious agent, brains from symptomatic mice were pooled and homogenized, and viral RNA was enriched for sequencing. Mass sequencing was performed using 454 GS-FLX technology (Roche). Approximately 700,000 sequencing reads were obtained with an average read length of 330 nucleotides. BLAST analyses were performed, and mouse genomic sequences were excluded. The remaining sequences were blasted against available viral genomes in the NCBI nr/nt databases. Of the identified sequences, 6.9% (ϳ5,100/73,000) showed similarity to members of the Cardiovirus genus, with the greatest sequence identity to TMEV isolates. Sequences were assembled de novo into contigs using a local alignment algorithm (CodonCode Aligner, version 3.5.5). Complete and redundant genomic coverage of a virus (designated NIHE) was obtained with an average of 177 reads per 100-bp segment (Fig. 2). Based on these analyses, a consensus sequence was generated and translated to reveal a full-length polyprotein. The nucleotide and amino acid sequences of NIHE were used to compare percent identities to those of full-length neurovirulent (GDVII) and persistent (BeAn, DA, Yale, TOB15, and Vie415HTR) TMEV strains available in GenBank. Strains with partial sequences were also considered, namely, neurovirulent (FA and ASK-1) and persistent (TO4 and WW) TMEV strains (Tables 1 and2).

Disease pathogenesis is consistent with NIHE being a neuropathogenic strain. To further characterize the pathogenesis of NIHE-mediated disease, histopathological analysis was performed on brains and spinal cords isolated from SJL and C57BL/6 mice 8 days postinoculation with 10 Ϫ1 to 10 Ϫ7 dilutions of IMBH. In the brain, acute inflammatory lesions were observed in several regions, including the cerebral cortex, thalamus, brain stem, and cerebellum, although most of the neuronal destruction and viral antigen was observed in the hippocampus (Fig. 3). In the gray matter, perivascular cuffing was observed and was comprised mainly of mononuclear cells. Many neurons showed characteristics of necrosis and were surrounded by mononuclear cells showing evidence of neuronophagia. Examples of the histopathological findings are shown for the hippocampus (Fig. 3A to C). Infected regions of the spinal cord showed similar, albeit more severe, inflammatory lesions. Perivascular cuffs and neuronal necrosis were observed primarily in the gray matter, but perivascular infiltrates very occasionally were observed in the white matter as well (Fig. 3D). To determine if NIHE is cross-reactive to anti-FIG. 1. Survival and kinetics of disease progression for animals inoculated with NIHE. (A) Five-week-old C57BL/6 mice were inoculated IC with 10ϫ dilutions (10 Ϫ1 to 10 Ϫ6 ) of infected mouse brain homogenates (n ϭ 10 per group); control mice (white boxes) received 10 Ϫ1 brain homogenate derived from noninfected animals. Animals were monitored daily, and Kaplan-Meier curves illustrate the kinetics of survival. (B) Clinical scoring system used to evaluate disease progression in mice following IC inoculation with NIHE. (C) Groups of animals were monitored daily for the presence of disease. Clinical scores for symptomatic animals within each group were averaged. The number of infected animals per group is noted in the figure key. FIG. 2. Complete and redundant coverage of the NIHE genome was obtained by pyrosequencing. Individual sequencing reads (average length of 330 bp) were assembled de novo, and the number of sequencing reads was determined for each 100-bp segment along the length of the genome. An organizational map of the TMEV genome is aligned at the top of the figure. Gene regions are drawn to scale, and P1 (red), P2 (blue), and P3 (green) are highlighted. bodies known to recognize other TMEV strains (31), immunohistochemistry was performed and representative sections are shown, demonstrating significant viral infection in the brain throughout the hippocampus and thalamus (Fig. 3E andF) as well as gray matter of the spinal cord (data not shown). Numerous foci of infected cells, particularly neurons, are evident in both tissues. Severe neuronal necrosis and TMEV staining were observed, particularly in the CA1 and CA2 pyramidal cell layers of the hippocampus, yet the CA3 and dentate gyrus areas were almost completely spared (Fig. 3A andB).

A shift in the tissue tropism from the gray matter to the white matter of the spinal cord is one of the defining characteristics of the persistent TMEV subgroup. The capacity of NIHE to persist in susceptible SJL animals was examined by the histological analysis of the brain and spinal cord 45 days postinoculation, the time point generally considered the chronic phase of disease [START_REF] Mcallister | Genetic mapping of the ability of Theiler's virus to persist and demyelinate[END_REF]. Animals were inoculated IC with increasing dilutions of infected mouse brain homogenate. All animals that received a dilution of Ͼ10 Ϫ4 succumbed during the acute phase of disease, 18% of the animals that received a 10 Ϫ5 dilution died, and all animals that received a Յ10 Ϫ6 dilution remained asymptomatic (data not shown). Histopathological analysis was performed on brain and spinal cord, and no evidence of inflammatory lesions was present in the tissues, and viral antigens were not detected following anti-TMEV staining (data not shown).

Genetic analysis of the NIHE strain in the context of known cardioviruses. To more fully understand the basis for the neurovirulence of NIHE, as well as to determine its relationship to other TMEV group members, we performed a detailed genetic analysis. All strains with full-length sequence data were aligned and compared to the sequence of NIHE (Fig. 4A). The greatest percent identity is seen in the 5ЈUTR. Interestingly, the persistent strain DA maintains the highest identity to NIHE through the sequence coding for the L protein (98% identity of amino acid sequence), but it diverges significantly for other genes (Fig. 4A, green line). Regions that demonstrated the lowest percentage of nucleotide identity between NIHE and other TMEV strains were clustered in the L protein and P1 region, particularly the sequences encoding the capsid proteins VP2 and VP1 (Fig. 4A to C). A more focused comparison of the P1 region allows the inclusion of partially sequenced strains and demonstrates additional dissimilarity, specifically within the VP2 and VP1 regions, with no apparent clustering with either subgroup (Fig. 4B). Interestingly, when the same analysis was performed using the GDVII strain as the reference sequence, a separation within the TO subgroup was observed (Fig. 4C). Specifically, the Yale and TOB15 strains (blue and black lines, respectively) maintain a relatively constant degree of sequence identity throughout this region (ϳ90%). This is in contrast to the cluster of TO4/WW, BeAn, DA, and Vie415HTR strains, which differ from GDVII considerably (70 to 80%). NIHE (red line) appears at an intermediate position between these two groups.

To place genetic differences within NIHE in context with other known cardioviruses, a phylogenetic analysis of three proteins or regions was performed: (i) the well-conserved 3Dpol (RNA-dependent RNA polymerase); (ii) the P1 region, which encodes the four capsid proteins; and (iii) the highly variable VP1 gene (Fig. 5). The phylogenetic analysis of the 3Dpol supports the separation of the human viruses, Thera viruses (rat), and TMEV species. NIHE clusters within the TMEV clade; however, it segregates with Vilyuisk and the mildly neurovirulent Vie415HTR strain, while GD-VII groups with the TO strains. These groupings are well supported based on the high posterior probability for the nodes (Fig. 5A). Differences in the topography of the P1 region and that of 3Dpol are seen. NIHE segregates with ASK-1 and is more closely related to GDVII and FA than to the TO strains (Fig. 5B). The phylogenetic relationships established for the P1 region are echoed in the VP1 analysis, with NIHE segregating with a group comprised mostly of neurovirulent strains (Fig. 5C).

Analysis of the L and capsid proteins. To identify possible genetic determinants of neurovirulence and persistence in the NIHE genome, we aligned and compared the amino acid se-FIG. 4. Sequence similarity for the complete genome and P1 regions of TMEV strains compared to those of NIHE and GDVII. (A) Sequence identity for all five TMEV strains with complete genome sequence information was obtained by comparing 20-bp segments along their entire genome to the sequence of NIHE. (B) Sequence identity of full-length and partially sequenced TMEV strains using NIHE as a reference. (C) Sequence identity of all TMEV stains (including NIHE) compared to the GDVII strain as the reference sequence. All sliding-window graphs were generated using Simplot 3.2 software as described in Materials and Methods. The x axis for all graphs is the nucleotide position, and the y axis is the percent similarity. A genetic map of TMEV is shown at the top of each graph, illustrating the organization and relative size of the individual genomic regions. quence for all coding regions of NIHE to the other complete and partial sequences of TMEV strains. The mean percent identity (compared to NIHE) of the amino acid sequence for the indicated viral protein is shown (Fig. 6). Although significant dissimilarity is seen in the VP2 and VP1 proteins, as predicted by the similarity plots (Fig. 4), the lowest percent similarity is seen in the L (88.8%) and L* (87.3%) proteins. A comparative analysis of the amino acid sequence for the carboxy terminus of the L protein, including several distinct domains, is shown in Fig. 6B.

Using modeling software (Swiss-PDB viewer [START_REF] Guex | SWISS-MODEL and the Swiss-Pdb-Viewer: an environment for comparative protein modeling[END_REF] and RasWin [START_REF] Sayle | RASMOL: biomolecular graphics for all[END_REF]), we compared the predicted structure of the NIHE VP1-VP3 protomer complex to the structures of BeAn and DA strains (Fig. 7). The calculated structure of NIHE VP1, VP2, and VP3 proteins (blue, green, and red, respectively) was overlaid on the BeAn structure (Fig. 7A) [START_REF] Luo | Threedimensional structure of Theiler murine encephalomyelitis virus (BeAn strain)[END_REF]. Differences in the amino acid sequence between these two strains are indicated by white-space-filled molecules (40 amino acid changes), and sequences that are unique to NIHE (compared to all published strains) are shown in yellow (14 amino acid changes). The spatial positioning of CD loop I and CD loop II are shown (Fig. 7A, upper right), with the loops delineated by the lightyellow and purple-filled molecules, respectively. Puff A (yellow) and puff B (light-blue) regions of VP2, as well as a protrusion from VP3 known as the knob, are highlighted (Fig. 7A, lower right). To further understand the relationship between NIHE and the other TMEV strains, specifically within these critical loop structures, we compared the amino acid sequences of selected regions (Fig. 7B).

Genetic evidence for L*. The region that was observed to have the greatest intrastrain variation coded for the L* protein (Fig. 6A). The generation of L* is the result of an additional open reading frame initiated 13 nucleotides downstream of the AUG start site that is responsible for initiating the viral polyprotein [START_REF] Kong | Alternative translation initiation site in the DA strain of Theiler's murine encephalomyelitis virus[END_REF]. This protein is not efficiently produced by the GDVII strain (and, presumably, FA) due to a polymorphism (ACG) in the sequence that initiates translation. Figure 8A depicts the sequence of this region, including the polyprotein FIG. 6. Percent identity for individual proteins of TMEV strains compared to NIHE proteins. Amino acid sequences for all available TMEV strains were compared to the NIHE sequence for each gene product. (A) The percent identity was determined, and whisker plots were generated showing the mean, minimum, and maximum values. Numbers above each plot identify how many amino acids are within each protein. Proteins are presented according to genome structure, and P1 (red), P2 (blue), and P3 (green) regions are colored according to the description in previous figure legends. (B) Amino acid alignments for eight TMEV strains are compared to the sequence of NIHE. The amino acid sequence for the Ser/Thr-rich region, Theilio domain, and L/VP4 cleavage sites are shown for the NIHE L protein along the top row. Dashes indicate identity to the NIHE sequence, and the single-letter codes for nonidentical amino acids are shown. Amino acid positions that have been implicated in a particular phenotype or function are annotated with a superscript number indicating the respective citation. start site and both alternative initiation sites located immediately downstream. The putative sequences coding for the L* protein of available strains were aligned, and a midrooted tree was generated (Fig. 8B). An intrastrain alignment was performed, and the difference between putative coding sequences of NIHE and other strains are shown (Fig. 8C).

DISCUSSION

A novel highly neurovirulent TMEV strain. Our study reports the identification of a novel neurovirulent TMEV strain, the first since Theiler's characterization of the GDVII and FA strains in 1938. In fact, based on the time and location at which FIG. 7. Analysis of the NIHE capsid structure formed by VP1, VP2, and VP3 proteins. Capsid proteins (VP1, VP2, and VP3) were aligned and overlaid onto the three-dimensional structure of the BeAn8386 strain using DeepView/Swiss-pdbViewer (version 4.0.1). (A) Quaternary structure with the individual proteins VP1 (blue), VP2 (green), and VP3 (red) indicated. White space-filled molecules indicate amino acid positions that differ between the sequence of BeAn8386 and NIHE. Amino acid positions that are unique to NIHE (compared to all TMEV strains) are highlighted in yellow. Highly variable regions, loops, puffs, and a canyon or "pit" are annotated. Also shown, to the right, are a close-up of VP1 CD loops I and II and the cluster A region (upper), as well as VP2 puff A and B and the VP3 knob (lower). (B) Amino acid alignments for the indicated region of all TMEV strains compared to the NIHE sequence. Dashes indicate identity to the NIHE sequence, and the single-letter codes for nonidentical amino acids are shown. Amino acid positions and annotations are the same as those described in the legend to Fig. 6. these two strains were discovered, as well as the current understanding of quasispecies diversity, it has been proposed that FA is a derivative of GDVII, thus making NIHE only the second fully annotated neurovirulent TMEV. A strain isolated in Japan, referred to as ASK-1 [START_REF] Casals | Immunological characterization of Vilyuisk human encephalomyelitis virus[END_REF], has been proposed to be a member of the neurovirulent subgroup [START_REF] Bandyopadhyay | A three-nucleotide insertion in the H stem-loop of the 5Ј untranslated region of Theiler's virus attenuates neurovirulence[END_REF][START_REF] Lipton | Differential usage of carbohydrate co-receptors influences cellular tropism of Theiler's murine encephalomyelitis virus infection of the central nervous system[END_REF]; however, no characterization of this strain has been published, and only a partial sequence has been submitted to GenBank [START_REF] Luo | The structure of a highly virulent Theiler's murine encephalomyelitis virus (GDVII) and implications for determinants of viral persistence[END_REF]. FIG. 8. Alternative AUG initiation sequence present for the L* gene within the NIHE genome. Nucleotide sequences were aligned for TMEV strains and compared to the sequence of NIHE. (A) The canonical start sequence (AUG) is outlined in blue, and both downstream initiating codon sequences are outlined in red. Sequences not identical to that of NIHE are highlighted in red. (B) Nucleotide sequences potentially coding for L* were aligned, and a midrooted tree was constructed using the maximum-likelihood method. There were a total of 156 positions in the final data set. Numbers at the nodes represent posterior probabilities, and genetic distance is indicated by the bar at the bottom of the panel. GDVII and FA strains were included in the analysis, although they do not efficiently produce L* (indicated by an asterisk). (C) Nucleotide sequences were aligned for all TMEV strains with available sequence information and compared to the sequence of NIHE. Only amino acid positions with discrepancies between strains are shown. The NIHE sequence is presented along the top row. Dashes indicate identity to the NIHE sequence, and the single-letter codes for nonidentical amino acids are shown.

Several characteristics of the NIHE strain led us to classify it as a member of the neurovirulent subgroup: (i) the rapid onset of disease following IC inoculation (Fig. 1); (ii) marked neuronal destruction in the brain and spinal cord, far greater than that observed for persistent strains (Fig. 3); and (iii) a lack of evidence (pathology or viral antigen) that NIHE persists in the CNS of mice surviving low-dose inoculation (data not shown). We do, however, realize the inherent paradox in studying highly virulent TMEV strains, in that it is difficult to distinguish between the inability of the virus to persist and the failure of a productive infection due to low viral inoculation.

The use of next-generation sequencing (NGS) techniques allowed for the complete, redundant sequencing as well as the de novo assembly of the entire NIHE genome (Fig. 2), suggesting the identification of a novel TMEV strain. The degree of identity shared by NIHE and known TMEV strains is Ͻ90% at the nucleotide level and Ͻ95% in its primary amino acid sequence (Tables 1 and2). This is in contrast to the Ͼ99% identity for quasispecies variation seen for the TO4, WW, and DA strains [START_REF] Michiels | Analysis of the leader and capsid coding regions of persistent and neurovirulent strains of Theiler's virus[END_REF] or the laboratory derivatives of S2 BeAn [START_REF] Kim | A spontaneous low-pathogenic variant of Theiler's virus contains an amino acid substitution within the predominant VP1(233-250) T-cell epitope[END_REF] or GDVII substrains [START_REF] Law | The complete nucleotide sequence of the GDVII strain of Theiler's murine encephalomyelitis virus (TMEV)[END_REF][START_REF] Pevear | Insights into Theiler's virus neurovirulence based on a genomic comparison of the neurovirulent GDVII and less virulent BeAn strains[END_REF].

The discovery of NIHE provides new information about the determinants of neurovirulence versus persistence and offers a novel basis of comparison on which to examine the relevance of amino acid differences previously identified between the GDVII and TO strains. Intriguingly, our analysis suggests that although NIHE has a decidedly neurovirulent phenotype, it shares high sequence identity with the TO strains in many important regions, including L, L*, and capsid proteins.

Features of NIHE L, L*, and capsid proteins segregate with TO stains. The analysis of the sequence comparison between NIHE and the other TMEV sequences demonstrates clusters of differences in the amino acid sequence [START_REF] Fu | Neurovirulence determinants of genetically engineered Theiler viruses[END_REF][START_REF] Jarousse | A single amino acid change determines persistence of a chimeric Theiler's virus[END_REF][START_REF] Lin | Molecular characterization of a nondemyelinating variant of Daniel's strain of Theiler's virus isolated from a persistently infected glioma cell line[END_REF][START_REF] Mccright | Theiler's viruses with mutations in loop I of VP1 lead to altered tropism and pathogenesis[END_REF][START_REF] Sato | A neutralization site of DA strain of Theiler's murine encephalomyelitis virus important for disease phenotype[END_REF][START_REF] Wada | Replacement of loop II of VP1 of the DA strain with loop II of the GDVII strain of Theiler's murine encephalomyelitis virus alters neurovirulence, viral persistence, and demyelination[END_REF][START_REF] Wada | Importance of amino acid 101 within capsid protein VP1 for modulation of Theiler's virus-induced disease[END_REF][START_REF] Zurbriggen | Direct evidence of a role for amino acid 101 of VP-1 in central nervous system disease in Theiler's murine encephalomyelitis virus infection[END_REF]. Although we present clear evidence that NIHE is neurovirulent, we were surprised to observe the number of instances in which the NIHE sequence segregated with the persistent strains. Here, we restrict our discussion to instances in which NIHE segregates with TO strains within regions important for modulating the host antiviral response, viral persistence, or interaction with the coreceptor sialic acid.

The L protein is produced by all members of the Cardiovirus genus and has been implicated in in vivo persistence because it modulates the host antiviral response [START_REF] Delhaye | The leader protein of Theiler's virus interferes with nucleocytoplasmic trafficking of cellular proteins[END_REF][START_REF] Stavrou | Different strains of Theiler's murine encephalomyelitis virus antagonize different sites in the type I interferon pathway[END_REF][START_REF] Van Pesch | The leader protein of Theiler's virus inhibits immediate-early alpha/beta interferon production[END_REF]. Although analysis revealed that the L protein is the second most divergent region across TMEV strains (Fig. 6), there were no unique amino acids predicted by the NIHE sequence. The majority of the sequence variation between NIHE and the other strains was clustered within the Ser/Thr-rich domain (62.5%) and the Theilo domain (79.5%). Interestingly, two positions in the Ser/Thr-rich region (L-55 and L-57) and one in the L-VP4 cleavage site (L-70) have been identified previously as distinguishing between the GDVII and TO strains [START_REF] Michiels | Analysis of the leader and capsid coding regions of persistent and neurovirulent strains of Theiler's virus[END_REF][START_REF] Takano-Maruyama | Theiler's murine encephalomyelitis virus leader protein amino acid residue 57 regulates subgroup-specific virus growth on BHK-21 cells[END_REF]. In all three instances, the NIHE sequence segregates with the TO strains.

A slightly different result emerged when analyzing the L* reading frame. Several nucleotide changes are unique to the NIHE strain, resulting in distinct amino acid sequences at positions L*-123 (Ser), L*-147 (Arg), and L*-153 (Phe) com-pared to sequences of any of the known TMEV strains (Fig. 8C). Again, the strains encoding L* sequences most closely related to NIHE are those produced by the Yale, BeAn, and TOB15 strains. Perhaps the most notable example of this divergence is the observation NIHE, like the TO strains, retains the genetic capacity to efficiently express L* by encoding an AUG sequence at the two initiation positions (Fig. 8). It is to note that some L* transcripts can be produced via the ACG initiation sites, albeit less efficiently [START_REF] Van Eyll | Non-AUG-initiated internal translation of the L* protein of Theiler's virus and importance of this protein for viral persistence[END_REF]. What evolutionary advantage this protein provides to a neurovirulent strain is unclear, particularly given that it is not required to infect neurons during the acute phase of disease. Advantages, however, can be imagined if moderate levels of L* are required by the virus during the natural route of transmission or for the crossing of the blood-brain barrier.

Many intraspecies sequence differences mapped to exposed areas of the viral capsid (Fig. 7). Cluster A, a highly variable region within VP1, initially was believed to segregate between GDVII and persistent strains; however, sequence information for several viruses was not then available. The inclusion of recently published strains and our contribution of the NIHE genome further clarifies these observations. Although GDVII and FA segregate with ASK-1 and NIHE at two positions (VP1-139 and VP1-232), NIHE segregates with a subset of TO strains at positions VP1-136, VP1-142, and VP1-180. In addition to these differences, NIHE also encodes two unique amino acids within this domain (VP1-138 and VP1-140), as shown in Fig. 7.

Perhaps the most functionally relevant example of NIHE sequences similar to those of persistent TO strains is observed in VP2 puff B (within the EF loop). Positions VP2-161, VP2-162, VP2-163, and VP2-174 were demonstrated to be the binding site for sialic acid in the persistent strains [START_REF] Zhou | Sialylation of the host receptor may modulate entry of demyelinating persistent Theiler's virus[END_REF]. Although a cellular receptor for TMEV strains currently is unknown, this region has been considered relevant to persistence due to the observation that Ser at position VP2-162 within the GDVII/FA strains prevents VP1-161 (Glu) from forming a hydrogen bond with sialic acid. Alignment with NIHE and ASK-1 indicate that these nonpersistent strains are identical to the TO strains at these positions. VP2-171, VP2-172, and VP2-173 also influence binding to sialic acid by establishing a structural framework for Gly at VP2-174 to bond with sialic acid [START_REF] Luo | The structure of a highly virulent Theiler's murine encephalomyelitis virus (GDVII) and implications for determinants of viral persistence[END_REF][START_REF] Zhou | Role of sialyloligosaccharide binding in Theiler's virus persistence[END_REF][START_REF] Zhou | Sialylation of the host receptor may modulate entry of demyelinating persistent Theiler's virus[END_REF]. NIHE is unique in this area, encoding Ser-Gln-Thr, whereas Arg-Gln-Ala is encoded in the GDVII/FA strains and Ser-Arg-Thr is in the majority of the TO strains (Fig. 7B). Interestingly, NIHE is identical to ASK-1, but the consequence on the orientation of Gly at VP2-174 remains unknown. Within this domain, there also exist three unique amino acids within NIHE (VP2-156, VP2-158, and VP2-177). None of these residues has been implicated previously in TMEV pathogenicity, and they may reflect either genetic drift in a nonessential position or compensatory mutations that promote viral entry or replication.

Evolutionary comparisons within TMEV. To ascertain information about the relationship of NIHE to other TMEV strains and gain insight into its evolution, we performed the phylogenetic analysis of highly conserved 3Dpol as well as the divergent regions P1 and VP1. The analysis of the 3Dpol gene clearly positions NIHE within the TMEV cluster and demonstrates that NIHE is more closely related to the human Vily-uisk virus and VIE415HTR than the other murine neurovirulent strains (Fig. 5A). The position of NIHE within the topology of the P1 and VP1 phylogenies is well supported and further supports our conclusion of neurovirulence. Furthermore, the addition of the NIHE sequence clarifies the groupings by defining a neurovirulent clade with GDVII, FA, and ASK-1, particularly within the VP1 analysis (Fig. 5C). Evidence of parallel evolutionary paths are predicted for NIHE, ASK-1, and GDVII/FA strains, all of which have obtained neurovirulence, albeit via potentially distinct mutations (Fig. 5B andC). Although it would be difficult to decipher an evolutionary history with such few numbers of sequenced strains, it has been hypothesized that neurovirulent strains have evolved from persistent strains [START_REF] Michiels | Analysis of the leader and capsid coding regions of persistent and neurovirulent strains of Theiler's virus[END_REF].

In summary, our study reports the discovery and characterization of a novel, highly neurovirulent TMEV, the first report of a new strain in this subgroup in more than 70 years. Critically, we characterized the pathogenesis of disease and obtained complete sequencing information without subjecting NIHE to conditions that promote genomic changes (e.g., in vitro expansion, which is to introduce artifactual compensatory mutations). Perhaps the most significant advance of this work is that the discovery of the NIHE strain helps to illuminate the relevance of sequences putatively implicated in the evolution of neurovirulence versus persistence, data that previously had been based solely on comparison with the GDVII strain. NIHE also provides an opportunity to evaluate new positions as potential determinants for neurovirulence, as NIHE contains unique amino acids at several positions, particularly within the capsid proteins. Further investigation will be required to assess to what extent these positions contribute to the extreme phenotype of neurovirulence observed in the NIHE strain.

FIG. 3 .

 3 FIG. 3. Histopathological features and viral tropism within the brain and spinal cord of C57BL/6 mice 8 days after the intracranial inoculation of NIHE virus. (A to D) HE staining. Severe acute necrotizing polioencephalitis and poliomyelitis characterized by perivascular cuffs (arrows), neuronal necrosis (blue stars), satellitosis and neuronophagia (green triangles), and gliosis (black arrowheads). (A) Hippocampus (original magnification, ϫ100). (B) Hippocampus (Ammon's horn) (original magnification, ϫ200). (C) Hippocampus (original magnification, ϫ400). (D) Spinal cord (ventral horns) (original magnification, ϫ200). GM, gray matter of the spinal cord; WM, white matter of the spinal cord. (E and F) Immunostaining against Theiler's virus (strain DA). (E) Many viral neuronal bodies and processes are labeled in the Ammon's horn of the hippocampus and in thalamic nuclei. Original magnification, ϫ20. (F) In the Ammon's horn, many virus-positive neurons are seen and are necrotic in CA1 and CA2; in CA3 only a few neurons are infected and necrotic. Original magnification, ϫ200. CA, Ammon's horn; DG, dentate gyrus; CC, cerebral cortex; Cca, corpus callosum; Lv, lateral ventricle; St, striatum; Th, thalamus; CA1, CA2, and CA3, fields of the Ammon's horn; 3rdV, third ventricle.

FIG. 5 .

 5 FIG. 5. Phylogenetic relationships between NIHE and select cardioviruses based on the nucleotide sequences of 3D pol , P1, and VP1 regions. Sequences were aligned and phylogenetic analysis was performed as outlined in Materials and Methods. Phylogenetic relationships are shown for sequences of the 3D pol (A), P1 (B), and VP1 (C) regions of the genome. Numbers at the nodes represent posterior probabilities, and the genetic distance is indicated by the bar at the bottom of each panel. The position of the NIHE strain is marked in red.

TABLE 1 .

 1 Comparison of nucleotide sequence identities for TMEV strains a Nucleotide sequences were aligned, and a percent identity was determined for each sequence pair. b Partial sequence was obtained by combining sequences U32924 and M80883 (accession numbers), which yielded a 3,265-nt fragment. c Partial sequence was obtained by combining sequences U33045 and M80885 (accession numbers), which yielded a 3,260-nt fragment. d Only a partial sequence (2,649 nt) is available for this strain.

						% Identity to:
	Strain	NIHE	GDVII	FA b	DA	BeAn8386	TOB15	VIE415HTR	Yale	TO4/WW c
	GDVII	88
	FA	87	Ͼ99
	DA	88	88	83
	BeAn8386	88	90	89	89
	TOB15	88	91	91	88	93
	VIE415HTR	85	84	82	86	85	84
	Yale	89	91	90	90	91	93	85
	TO4/WW	85	83	83	Ͼ99	85	83	86	83
	ASK-1 d	87	87	88	81	85	87	81	87	87

a

TABLE 2 .

 2 Comparison of amino acid sequence identities for TMEV strains a

						% Identity to:
	Strain	NIHE	GDVII	FA b	DA	BeAn8386	TOB15	VIE415HTR	Yale	TO4/WW c
	GDVII	95
	FA	94	Ͼ99
	DA	95	94	91
	BeAn8386	95	95	94	95
	TOB15	95	95	94	94	96
	VIE415HTR	93	93	92	94	95	92
	Yale	95	95	94	95	95	96	93
	TO4/WW	93	91	91	Ͼ99	93	92	92	92
	ASK-1 d	96	95	95	95	95	94	94	94	93

a Amino acid sequences were aligned, and the percent identity was determined for each sequence pair. For partial sequences, amino acid numbers refer to the corresponding number of the NIHE strain.

b Only a partial sequence is available; amino acids 1 to 922 were used for comparison. c Only a partial sequence is available; amino acids 1 to 920 were used for comparison. d Only a partial sequence is available; amino acids 62 to 934 were used for comparison.

BUCKWALTER ET AL.J. VIROL.

J. VIROL.

ACKNOWLEDGMENTS

This work was supported by grants from the Agence National de la Recherche (ANR) and an EURYI Award from the European Science Foundation (ESF).

We are grateful to Michel Brahic (Stanford University, Stanford, CA) for the anti-TMEV antibody and to Thomas Michels (Universite Ćatholique de Louvain, Brussels, Belgium) for the anti-L* antibody. We also thank Patric Ave ´(Histotechnologie et Pathologie, Institut Pasteur, Paris, France) for his expert technical assistance in the preparation of the histology slides and Herbert Virgin IV (Washington University School of Medicine, St. Louis, MO) for advice on deep sequencing and data mining. We thank Olivier Schwartz and Felix Rey for their critical reviews of the manuscript.


I. INTRODUCTION

With an increased resolution, the new space-borne SAR images should be an important source of information for glacier activity monitoring, by providing regular measurements such as surface topography, velocity fields or rocks and crevasses cartography. After successful SAR experiments on Arctic and polar region glaciers [START_REF] Fischer | Observation of recent surges of Vatnajkull, Iceland, by means of ERS SAR interferometry[END_REF], the study of the alpine temperate glaciers proved to be more complex. Some experiments carried out with existing space-borne SAR interferometric (InSAR) data have shown that only specific data such as ERS 1/2 tandem couples provide information on well-oriented glaciers at certain time of the year [START_REF] Trouvé | Combining optical and SAR data to monitor temperate glaciers[END_REF]. But the characteristics of the future space-borne SAR missions such as TerraSAR-X (14-day repeat cycle, left/right looking, various incidence angles, up to 2-meter resolution, dual polarisation...) reinforce the potential of interferometric measurements over alpine glaciers.

The existing standard interferometric processors (the Differential Interferometric Automated Process Applied to Survey Of Nature -DIAPASON or the Repeated Orbit Interferometry Package -ROI-PAC) provide a complete D-InSAR processing chain, which was already tested and successfully applied in the context of SAR interferometry to measure accurate displacements (volcanology or seismology). Although these processors are built from standard well-established InSAR algorithms as co-registration of interferograms, phase scaling and unwrapping, this procedure is often error-prone when applied to temperate glacier monitoring because of the complex glacier topography or the phase noise and discontinuities at glacier boundaries.

With the resolution of the data which have been available up to now (about 20 m with ERS images), the amplitude is often difficult to use to extract precise information on specific features. The next generation of SAR satellites should provide meter resolution images and fully polarimetric data, which will be very useful to detect different features and backscattering mechanisms and to improve the extraction of the geometric deterministic component of the interferometric phase by adaptive techniques [START_REF] Vasile | General adaptive neighborhood technique for improving SAR interferometric coherence and phase estimation[END_REF]. The two main limitations of the glacier InSAR measurements are the temporal decorrelation and the need to separate the topographical fringes from the displacement fringes. Among the different techniques which can be used, the safest one consists in using a Digital Terrain Model (DTM) of the target area. The DTM accuracy requirement increases with the baseline between the two SAR acquisitions, which makes interferograms more and more sensitive to the topography, especially in the high relief areas of Alpine glaciers.

In this context, reliable estimates of the phase gradient within the interferogram become more and more necessary with the increase of the SAR resolution: smaller ground resolution cells will reveal the local topographic variations (micro-relief) and larger baselines (smaller altitude of ambiguity) will transform them into local fringe pattern difficult to detect without using adaptive techniques. A previous approach based on the estimation of local frequencies proved to be rather efficient with low resolution data. If correctly estimated, local frequencies can be used in the InSAR processing at different stages such as interferogram filtering or phase unwrapping [START_REF] Trouvé | Improving phase unwrapping techniques by the use of local frequency estimates[END_REF]. In this paper, we propose to improve this approach and to adapt it to the new context of high resolution interferograms. An original 2-step method for estimating local frequencies is presented. Results using two different data sets are presented over the Mer-de-Glace and Leschaux glaciers which are located in the Mont-Blanc region: a real ERS tandem interferogram containing topographic and displacement fringes and a simulated high resolution (HR) interferogram obtained from a HR DTM computed from aerial photographs.

II. HIGH FREQUENCY FRINGE PATTERN ANALYSIS A. Proposed model

In each pixel of the interferogram, the 2D phase signal φ is considered to have two deterministic components, namely the low-resolution phase φ LR which corresponds to low-resolution items (fields, mountains...) and the highresolution phase φ HR which appears in HR data (small local topography variations as ice-cracks or larger rocks), and one random component corresponding to the phase noise φ n . Accordingly, the analytical phase signal can be written as:

e iφ = e iφLR e iφHR e iφn (1)
The first step of the proposed algorithm consists in estimating the low-resolution phase and removing the associated fringe pattern. After reducing the initial resolution of the interferogram, a modified version of the MUSIC algorithm [START_REF] Trouvé | Fringe detection in noisy complex interferograms[END_REF] is applied for estimating the 2D local frequency (f LR x , f LR y ) of a fringe pattern within a squared fixed-size centered neighborhood. This lowresolution frequency is used in each pixel (m, n) of the full resolution interferogram to approximatively flatten the global fringe pattern in surrounding pixels (k, l):

φ fl (k, l) = φ(k, l) -2π((k -m)f LR x + (l -n)f LR y ). (2)
The resulting phase signal e φ fl exhibits the local differences between the 2D sine-wave model and the real HR fringe pattern.

B. Spatial support

In the second step, the local high-resolution frequencies are estimated. The proposed procedure consists in gathering only the pixels which belong to the same HR spatial feature and respect the "local stationarity" hypothesis [START_REF] Touzi | A review of speckle filtering in the context of estimation theory[END_REF]. Under the assumption that small homogeneous regions within the two intensity images correspond to ground areas with an homogeneous cover which should respect the stationarity hypothesis, the multi-resolution technique employed in this paper is the Amplitude-Driven-Adaptive-Neighborhood (ADAN) [START_REF] Vasile | General adaptive neighborhood technique for improving SAR interferometric coherence and phase estimation[END_REF].

C. Auto-correlation functions

Using as spatial support the obtained adaptive neighborhood, the two 1D autocorrelation functions of the LRflattened phase signal given by Eq. 2 are computed. Using 1D notation and a phase model φ(z) = zf z + φ n , the autocorrelation coefficients are defined by: γ(p) = e  (φ(z+p)+φn(z+p)) • e - (φ(z)+φn(z))

(3)

Under the assumption that the phase noise is an independent and identically distributed (iid) random process, we obtain:

γ(p) = K e  2π pfz (4)
where K = e  φn(0) 2 is a real coefficient which does not affect the autocorrelation phase anymore. Accordingly, our algorithm estimates the frequency as:

f z = arg p N (p) + N (p + 1) 2 γ(p + 1).γ(p) (5)
where N (p) is the number of pairs of pixels belonging to to the adaptive neighborhood that can be used to estimate γ(p).

The proposed algorithm applied in each direction allows to obtain the expected values (f HR x , f HR y ) measuring the high resolution component of the fringe pattern local frequency.

III. RESULTS AND DISCUSSION The proposed method has been tested on the Chamonix Mont-Blanc test site which is located in the Alps, near the borders between France, Italy and Switzerland (45 • 50 N, 6 • 51 E). It includes the Aiguille-Verte (4122 m ASL), the Chamonix valley (1000 m ASL) and two well-known instrumented glaciers: Mer-de-Glace and Leschaux.

A. Real ERS 1/2 TANDEM InSAR data

In order to test the feasibility of the proposed method on real data, an ERS 1/2 tandem pair has been selected (Tab. III-A) and processed by the ROI-PAC software. Starting from the RAW data, SLC images are synthesized and corregistered and a 1 × 5 complex multilooking with orbital fringe correction is applied to obtain the amplitude and phase images illustrated in Figs. 1-a,-b. The resulting images have approximately 20-meter ground resolution squared pixels. The fringe pattern which can be observed in the Fig. 1-a has a relatively high frequency due to the small altitude of ambiguity e a . Moreover, along the two glaciers the topographic fringes are superposed on the glacier displacement phase component.

The results of the 2D local frequency estimation performed at low resolution (LR), at high resolution (HR) after LR-flattening, and the final combined estimates are illustrated in Fig. 2 by the orientation of the corresponding phase gradient. The LR result is as expected smooth and robust but it does not follow the local variations of the fringe pattern. This information is obtained in the HR result and the sum of these two estimates provides a robust and precise estimate of the local fringe pattern.

To illustrate the interest and the accuracy of this result, the original noisy interferogram was filtered with the Amplitude-Driven-Adaptive-Neighborhood (ADAN) filter [START_REF] Vasile | General adaptive neighborhood technique for improving SAR interferometric coherence and phase estimation[END_REF], using the deterministic fringe pattern compensation provided by the HR and LR local frequencies estimation (Fig. 1-c). The filtered interferogram preserves well the stationarity in the foldover polluted areas. Moreover, the glacier is not connected with the surrounding terrain and still remains a disconnected phase pattern (useful for eventual D-InSAR phase unwrapping).

B. Simulated TERRASAR-X InSAR data

In order to illustrate the proposed method on HR data over Alpine glaciers, a 2-meter resolution DTM of the Mer-de-Glace [START_REF] Trouvé | Combining optical and SAR data to monitor temperate glaciers[END_REF] has been used to simulate the SAR intensity of TerraSAR-X images using the method proposed in [START_REF] Julea | Simulation of SAR images and radar coding of georeferenced information for temperate glacier monitoring[END_REF]. The parameters used in the simulation are presented in Tab. III-B. Moreover, the altitude information provided by the DTM was radar coded and converted into topographic fringes modulo a tunable altitude of ambiguity. A controlled noise level was also introduced on the resulting high-resolution phase and amplitude images.

Figs. 3-a,-b present respectively the real ERS amplitude (∼ 20-meter resolution) and the simulated TerraSAR-X amplitude (∼ 2-meter resolution). As expected, small relief variations become visible in the HR simulated image and the presence of crevasses is noticed. The simulated topographic interferometric phase is presented in Fig. 3-c.

Most of this information is missed by the LR frequency estimation illustrated in Fig. 4-a. It can be observed in the HR estimates (Fig. 4-b) and in the final result (Fig. 4-c). IV. CONCLUSIONS AND PERSPECTIVES The increased performances of future high resolution SAR systems (TerraSAR-X, COSMO-SKYMED or Radarsat-2) together with the developments of appropriate processing algorithms reinforce the potential of SAR interferometry for temperate glacier periodic monitoring. A new method has been proposed to estimate the local frequencies within a HR interferogram. This method allows to measure the local topographic variations which are useful to benefit from the new High Resolution SAR images. In the context of temperate glacier activity monitoring, it can be applied to improve the processing of two kinds of interferograms: small altitude of ambiguity interferograms to derive very accurate DTM and to compute differences, to obtain for instance volume balances. In this case, HR frequency estimation combined with amplitude driven neighborhood techniques provides very good results since most of the sources of phase nonstationarity are revealed by the SAR amplitude which is mostly influenced by the local slope. It will also be useful for high altitude of ambiguity differential interferograms to obtain HR velocity field measurements which require efficient phase filtering and unwrapping methods. In this case, one difficulty that has to be addressed in future works is the presence of phase non-stationarity which are independent from the amplitude information.

ACKNOWLEDGMENT

High

  Resolution SAR Interferometry: influence of local topography in the context of glacier monitoring G. Vasile * ¶ , I. Petillot * , A. Julea * ¶ , E. Trouvé * , Ph. Bolon * , L. Bombrun † , M. Gay † , T. Landes ‡ , P. Grussenmeyer ‡ , and J.-M. Nicolas § * Université de Savoie, ESIA/LISTIC -BP 806 -F-74016 Annecy Cedex, FRANCE Tel: +33 450 096 548 -Email: {g.vasile|ivan.petillot|andreea.julea|trouve|bolon}@univ-savoie.fr † Institut National Polytechnique de Grenoble/LIS -BP 46 -38402 Saint-Martin-d'Hères, FRANCE Tel: +33 476 826 256 -Email: {lionel.bombrun|michel.gay}@lis.inpg.fr ‡ INSA Strasbourg -Equipe Photogrammétrie et Géomatique -24, Bd de la Victoire, 67084 Strasbourg Cedex, FRANCE Tel: +33 388 144 733 -Email: {tania.landes|pierre.grussenmeyer}@insa-strasbourg.fr § Ecole Nationale Supérieure des Télécommunications, LTCI -46, Rue Barrault -75013 Paris, FRANCE Tel: +33 145 818 129 -Email: nicolas@tsi.enst.fr ¶ Universitatea Politehnica Bucuresti, LAPI -Bucharest, ROMANIA -Tel: +4021 402 4683 Institutul de Stiinte Spatiale -Bucuresti, ROMANIA -Tel/fax +4021 457 44 71Abstract-SAR interferometric data offers the opportunity to measure temperate glacier surface topography and displacement between the two acquisitions. Recently, reliable estimates of the phase gradient given by interferogram local frequencies become mandatory with the increase of the SAR resolution. In this paper, an original 2-step method for estimating local frequencies is proposed. The 2D phase signal is considered to have two deterministic components corresponding to low-resolution fringes and high-resolution patterns due for instance to the micro-relief. The first step of the proposed algorithm consists in the low-resolution phase flattening. In the second step the local high-resolution frequencies are estimated from the phase auto-correlation functions computed on adaptive neighborhoods using only the pixels which belong to the same HR spatial feature and respect the "local stationarity" hypothesis. Results with both real ERS 1/2 tandem and simulated TerraSAR-X interferograms are presented to illustrate the potential of the proposed method.

Fig. 1 .

 1 Fig. 1. TANDEM ERS 1/2 interferogram: a) SAR amplitude, b) original phase, c) ADAN filtered phase.

Fig. 2 .

 2 Fig. 2. Orientation map of the local fringe pattern with ERS: a) lowresolution with the local fringe orientation Look-Up-Table in the left upper corner, b) high-resolution, c) sum between the low-resolution and high-resolution components.

Fig. 3 -Fig. 3 .Fig. 4 .

 334 Fig. 3. The Mer-de-glace glacier: a) ERS 5-look amplitude, b) simulated 1-look TerraSAR-X amplitude, c) simulated 1-look TerraSAR-X phase, d) TerraSAR-X simulated ADAN filtered phase.

TABLE I ERS

 I SAR RAW DATA ACQUIRED OVER THE TEST AREA.

	Date	Satel.	Orbit	Frame Pass	B ⊥	ea
	95.12.31 96.01.01	ERS-1 23328 ERS-2 03655	2673	Des.	208 m 45 m

TABLE II PROCESSING

 II PARAMETERS IN TERRASAR-X IMAGE SIMULATION.

	Parameter	Value
	satellite altitude (km)	514
	step on azimuth direction (m)	2
	step on ground range (m)	2
	step on slant range (m)	1.2
	viewing angle at close range (degrees)	30
	altitude of ambiguity (m)	10
	speckle variance in amplitude image	1
	uniform phase noise distribution	±π/4

This work was supported by the French national project ACI-MEGATOR. The authors wish to thank to the European Space Agency for providing ERS SAR data through the Category 1 proposal No.3525.


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Veterinary Research is now a full Open Access journal

Michel Brémont * , Élodie Coulamy

As previously announced in mid-2010, Veterinary Research became an Open Access (OA) journal, published with BioMed Central, on 1 st January 2011.

OA is defined as free, immediate, permanent and fulltext online access for any user to peer reviewed research. Authors retain the copyright for their articles and grant anyone the right to copy, distribute or display the work provided that it is correctly cited. Some of the expected advantages of OA are increased visibility, wider audience and greater impact, accelerated publication, increased citations, and enhanced availability of online search tools.

Veterinary Research will continue to be supported by INRA and there is no change in the journal's title, scope of interests, editorial management, or peer-review process. The OA business model is relatively new and the cost of OA publication is a much-discussed question. The economic basis of OA is that authors, or rather their home institutions or research funders, are charged a fee to have their work published, instead of the readers being charged to access articles. The number of universities, funding agencies, and other organisations supporting OA and covering OA publication charges is growing rapidly and worldwide. A prominent example is the Harvard-initiated "Compact for Open Access Publishing Equity" [START_REF]Compact for Open Access Publishing Equity[END_REF], and more and more universities are introducing central OA funding mechanisms as the equivalents to library budgets that pay for subscriptions [START_REF][END_REF]. OA publishers like BioMed Central have membership arrangements [START_REF]Brémont and Coulamy: Veterinary Research is now a full Open Access journal[END_REF] with research institutions across the globe.

For Veterinary Research the article processing charge for each accepted manuscript will be 500 euros in 2011. This flat fee, which is supported by INRA and covers additional files (including movies) and data, is a highly competitive charge in the world of OA publishing.

We are convinced that publishing Veterinary Research as an Open Access journal will contribute to maintaining and further developing a high-quality journal in the field of the infectious diseases of animals. We look forward to working with all of you in the near future.

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