The French Tarrasch Variation Pdf Converter

1. French Defense Tarrasch Variation
2. French Tarrasch Variation

The Modern French Tarrasch - download book In the Tarrasch Variation of the French Defence 1 e4 e6 2 d4 d5 3 Nd2 c5, the 4 exd5 line has undergone a genuine re-evaluation in the last Comment 0.

French Defence

a b c d e f g h 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 a b c d e f g h
Moves	1.e4 e6
ECO	C00–C19
Named after	London vs. Paris correspondence match (1834–1836)[1]
Parent	King's Pawn Game

The French Defence is a chessopening characterised by the moves:

1. e4e6

This is most commonly followed by 2.d4 d5, with Black intending ...c5 at a later stage, attacking White's centre and gaining space on the queenside. White has extra space in the centre and on the kingside and often plays for a breakthrough with f4–f5. The French has a reputation for solidity and resilience, although some lines such as the Winawer Variation can lead to sharp complications. Black's position is often somewhat cramped in the early game; in particular, the pawn on e6 can impede the development of the bishop on c8.

• 3Main line: 2.d4 d5
  • 3.13.Nc3
    • 3.1.1Winawer Variation: 3...Bb4
    • 3.1.2Classical Variation: 3...Nf6

Basics[edit]

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Following the opening moves 1.e4 e6, the game usually continues 2.d4 d5 (see below for alternatives). White makes a claim to the centre, while Black immediately challenges the pawn on e4.

White's options include defending the e4-pawn with 3.Nc3 or 3.Nd2, exchanging it with 3.exd5, or advancing the pawn with 3.e5, each of which lead to different types of positions. Note that 3.Bd3 allows 3...dxe4 4.Bxe4 Nf6, after which White must concede to Black either a tempo or the advantage of the two bishops.

General themes[edit]

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The diagram shows for the pawn structure most typical of the French. Black has more space on the queenside, so tends to focus on that side of the board, almost always playing ...c7–c5 at some point to attack White's pawn chain at its base, and may follow up by advancing his a- and b-pawns.

Alternatively or simultaneously, Black will play against White's centre, which is cramping his position. The flank attack ...c7–c5 is usually insufficient to achieve this, so Black will often play ...f7–f6. If White supports the pawn on e5 by playing f2–f4, then Black has two common ideas. Black may strike directly at the f-pawn by playing ...g7–g5. The pawn on g5 may also threaten to advance to g4 to drive away a white knight on f3, augmenting Black's play against the white centre. Another idea is to play ...fxe5, and if White recaptures with fxe5, then Black gains an open f-file for his rook. Then, as White usually has a knight on f3 guarding his pawns on d4 and e5, Black may sacrifice the exchange with ...Rxf3 to destroy the white centre and attack the king. On the other hand, if White plays dxe5, then the a7–g1 diagonal is opened, making it less desirable for White to castle kingside.

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White usually tries to exploit his extra space on the kingside, where he will often play for a mating attack. White tries to do this in the Alekhine–Chatard Attack, for example. Another example is the following line of the Classical French: 1.e4 e6 2.d4 d5 3.Nc3 Nf6 4.Bg5 Be7 5.e5 Nfd7 6.Bxe7 Qxe7 7.f4 0-0 8.Nf3 c5 9.Bd3 (see diagram). White's light-square bishop eyes the weak h7-pawn, which is usually defended by a knight on f6 but here it has been pushed away by e5. If 9...cxd4 (Black does better with 9...f5 or 9...f6), White can play the Greek gift sacrifice 10.Bxh7+ Kxh7 11.Ng5+ Qxg5! 12.fxg5 dxc3 13.Qh5+! where Black has three minor pieces for the queen, which gives him a slight material superiority, but his king is vulnerable and White has good attacking chances.

Apart from a piece attack, White may play for the advance of his kingside pawns (an especially common idea in the endgame), which usually involves f2–f4, g2–g4 and then f4–f5 to use his natural spatial advantage on that side of the board. A white pawn on f5 can be very strong as it may threaten to capture on e6 or advance to f6. Sometimes pushing the h-pawn to h5 or h6 may also be effective. A modern idea is for White to gain space on the queenside by playing a2–a3 and b2–b4. If implemented successfully, this will further restrict Black's pieces.

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One of the drawbacks of the French Defence for Black is his queen's bishop, which is blocked in by his pawn on e6 and can remain passive throughout the game. An often-cited example of the potential weakness of this bishop is S. Tarrasch–R. Teichmann, San Sebastián 1912, in which the diagrammed position was reached after fifteen moves of a Classical French.

Black's position is passive because his light-square bishop is hemmed in by pawns on a6, b5, d5, e6 and f7. White will probably try to exchange Black's knight, which is the only one of his pieces that has any scope. Although it might be possible for Black to hold on for a draw, it is not easy and, barring any mistakes by White, Black will have few chances to create counterplay, which is why, for many years, the classical lines fell out of favour, and 3...Bb4 began to be seen more frequently after World War I, due to the efforts of Nimzowitsch and Botvinnik. In Tarrasch–Teichmann, White won after 41 moves. In order to avoid this fate, Black usually makes it a priority early in the game to find a useful post for the bishop. Black can play ...Bd7–a4 to attack a pawn on c2, which occurs in many lines of the Winawer Variation. If Black's f-pawn has moved to f6, then Black may also consider bringing the bishop to g6 or h5 via d7 and e8. If White's light-square bishop is on the f1–a6 diagonal, Black can try to exchange it by playing ...b6 and ...Ba6, or ...Qb6 followed by ...Bd7–b5.

Main line: 2.d4 d5 [edit]

3.Nc3 [edit]

Played in over 40% of all games after 1.e4 e6 2.d4 d5, 3. Nc3 is the most commonly seen line against the French. Black has three main options, 3...Bb4 (the Winawer Variation), 3...Nf6 (the Classical Variation), and 3...dxe4 (the Rubinstein Variation). An eccentric idea is 3...Nc6!? 4.Nf3 Nf6 with the idea of 5.e5 Ne4; German IM Helmut Reefschlaeger has been fond of this move.

Winawer Variation: 3...Bb4 [edit]

This variation, named after Szymon Winawer and pioneered by Nimzowitsch and Botvinnik, is one of the main systems in the French, due chiefly to the latter's efforts in the 1940s, becoming the most often seen rejoinder to 3.Nc3, though in the 1980s, the Classical Variation with 3...Nf6 began a revival, and has since become more popular.

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3... Bb4pins the knight on c3, forcing White to resolve the central tension. White normally clarifies the central situation for the moment with 4. e5, gaining space and hoping to show that Black's b4-bishop is misplaced. The main line then is: 4... c5 5. a3 Bxc3+ 6. bxc3, resulting in the diagrammed position.

While White has doubled pawns on the queenside, which form the basis for Black's counterplay, they can also help White since they strengthen his centre and give him a semi-open b-file. White has a spatial advantage on the kingside, where Black is even weaker than usual because he has traded off his dark-square bishop. Combined with the bishop pair, this gives White attacking chances, which he must attempt to use as the long-term features of this pawn structure favour Black.

In the diagrammed position, Black most frequently plays 6... Ne7 (The main alternative is 6...Qc7, which can simply transpose to main lines after 7.Qg4 Ne7, but Black also has the option of 7.Qg4 f5 or ...f6. 6...Qa5 has recently become a popular alternative). Now White can exploit the absence of Black's dark-square bishop by playing 7. Qg4, giving Black two choices: he may sacrifice his kingside pawns with 7...Qc7 8.Qxg7 Rg8 9.Qxh7 cxd4 but destroy White's centre in return, the so-called 'Poisoned Pawn Variation'; or he can play 7...0-0 8.Bd3 Nbc6, which avoids giving up material, but leaves the king on the flank where White is trying to attack. Experts on the 7.Qg4 line include Judit Polgár.

If the tactical complications of 7.Qg4 are not to White's taste, 7.Nf3 and 7.a4 are good positional alternatives, and 7.h4 is a more aggressive attempt:

7. Nf3 is a natural developing move, and White usually follows it up by developing the king's bishop to d3 or e2 (occasionally to b5) and castling kingside. This is called the Winawer Advance Variation. This line often continues 7... Bd7 8. Bd3 c4 9. Be2 Ba4 10. 0-0 Qa5 11. Bd2 Nbc6 12. Ng5 h6 13. Nh3 0-0-0. Its assessment is unclear, but most likely Black would be considered 'comfortable' here.

The purpose behind 7. a4 is threefold: it prepares Bc1–a3, taking advantage of the absence of Black's dark-square bishop. It also prevents Black from playing ...Qa5–a4 or ...Bd7–a4 attacking c2, and if Black plays ...b6 (followed by ...Ba6 to trade off the bad bishop), White may play a5 to attack the b6-pawn.

White also has 7. h4, which has the ideas of either pushing this pawn to h6 to cause more dark-square weaknesses in the Black kingside (if Black meets h5 with ...h6, White can play g4-g5), or getting the rook into the game via Rh3–g3.

Black can also gain attacking chances in most lines: against 7.Qg4, Black will attack White's king in the center; whereas against the other lines, Black can often gain an attack with ...0-0-0, normally combined with ...c4 to close the queenside, and then ...f6 to open up the kingside, where White's king often resides. If Black can accomplish this, White is often left without meaningful play, although ...c4 does permit White a4 followed by Ba3 if Black has not stopped this by placing a piece on a4 (for example, by Bd7–a4).

Sidelines[edit]

5th move deviations for White include:

• 5.Qg4
• 5.dxc5
• 5.Nf3
• 5.Bd2

4th move deviations for White include:

• 4.exd5 exd5, transposing to a line of the Exchange Variation, where White may aim to prove that Black's bishop on b4 is misplaced.
• 4.Ne2 (the Alekhine Gambit) 4...dxe4 5.a3 Be7 (5...Bxc3+ is necessary if Black wants to try to hold the pawn) 6.Nxe4 to prevent Black from doubling his pawns.
• 4.Bd3 defending e4.
• 4.a3 Bxc3+ 5.bxc3 dxe4 6.Qg4, another attempt to exploit Black's weakness on g7.
• 4.e5 c5 5.Bd2, again preventing the doubled pawns and making possible 6.Nb5, where the knight may hop into d6 or simply defend d4.
• 4.Bd2 (an old move sometimes played by Rashid Nezhmetdinov, notably against Mikhail Tal)

Deviations for Black include:

• 4...Ne7 although this move usually transposes to the main line.
• 4...b6 followed by ...Ba6, or 4...Qd7 with the idea of meeting 5.Qg4 with 5...f5. However, theory currently prefers White's chances in both lines.
• Another popular way for Black to deviate is 4.e5 c5 5.a3 Ba5, the Armenian Variation, as its theory and practice have been much enriched by players from that country, the most notable of whom is Rafael Vaganian. Black maintains the pin on the knight, which White usually tries to break by playing 6.b4 cxb4 7.Qg4 or 7.Nb5 (usually 7.Nb5 bxa3+ 8.c3 Bc7 9.Bxa3 and White has the upper hand).

Classical Variation: 3...Nf6 [edit]

This is another major system in the French. White can continue with the following options:

4.Bg5[edit]

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White threatens 5.e5, attacking the pinned knight. Black has a number of ways to meet this threat:

• Burn Variation, named after Amos Burn is the most common reply at the top level: 4... dxe4 5. Nxe4 and usually there now follows: 5... Be7 6. Bxf6 Bxf6 7. Nf3 Nd7 or 7... 0-0, resulting in a position resembling those arising from the Rubinstein Variation. However, here Black has the bishop pair, with greater dynamic chances (although White's knight is well placed on e4), so this line is more popular than the Rubinstein and has long been a favourite of Evgeny Bareev. Black can also try 5...Be7 6.Bxf6 gxf6, as played by Alexander Morozevich and Gregory Kaidanov; by following up with ...f5 and ...Bf6, Black obtains active piece play in return for his shattered pawn structure. Another line that resembles the Rubinstein is 5...Nbd7 6.Nf3 Be7 (6...h6 is also tried) 7.Nxf6+ Bxf6.
• 4... Be7 5. e5 Nfd7 used to be the main line and remains important, even though the Burn Variation has overtaken it in popularity. The usual continuation is 6. Bxe7 Qxe7 7. f4 0-0 8. Nf3 c5, when White has a number of options, including 9.Bd3, 9.Qd2 and 9.dxc5. An alternative for White is the gambit6. h4, which was devised by Adolf Albin and played by Chatard, but not taken seriously until the game Alekhine–Fahrni, Mannheim 1914. It is known today as the Albin–Chatard Attack or the Alekhine–Chatard Attack. After 6... Bxg5 7. hxg5 Qxg5 8. Nh3 Qe7 9. Nf4 Nc6 10. Qg4 (the reason for 8.Nh3 rather than 8.Nf3), White has sacrificed a pawn to open the h-file, thereby increasing his attacking chances on the kingside. Black may also decline the gambit in several ways such 6...a6 and 6...f6, but most strong players prefer 6...c5.
• A third choice for Black is to counterattack with the McCutcheon Variation. In this variation, the second player ignores White's threat of e4–e5 and instead plays 4... Bb4. The main line continues: 5. e5 h6 6. Bd2 Bxc3 7. bxc3 Ne4 8. Qg4. At this point Black may play 8...g6, which weakens the kingside dark squares but keeps the option of castling queenside, or 8...Kf8. The McCutcheon Variation is named for John Lindsay McCutcheon of Philadelphia (1857–1905), who brought the variation to public attention when he used it to defeat World Champion Steinitz in a simultaneous exhibition in Manhattan in 1885.^[2][3][4]

4.e5[edit]

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The Steinitz Variation, named after Wilhelm Steinitz, continues with 4. e5 Nfd7. Here 5.Nce2, the Shirov–Anand Variation, prepares to bolster the white pawn centre with c2–c3 and f2–f4; while 5.Nf3 transposes to a position also reached via the Two Knights Variation (2.Nf3 d5 3.Nc3 Nf6 4.e5 Nfd7 5.d4). The main line of the Steinitz is 5. f4 c5 6. Nf3 Nc6 7. Be3. (Instead 7.Nce2 transposes to the Shirov–Anand Variation, while 7.Be2? cxd4 8.Nxd4 Ndxe5! 9.fxe5 Qh4+ wins a pawn for Black.) Here Black may step up the pressure on d4 by playing 7...Qb6 or 7...cxd4 8.Nxd4 Qb6, begin queenside play with 7...a6 8.Qd2 b5, or continue kingside development by playing 7...Be7 or 7...cxd4 8.Nxd4 Bc5. In these lines, White has the option of playing either Qd2 and 0-0-0, or Be2 and 0-0, with the former typically leading to sharper positions due to opposite-side castling when Black castles kingside in both cases.

Rubinstein Variation: 3...dxe4 [edit]

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This variation is named after Akiba Rubinstein and can also arise from a different move order: 3.Nd2 dxe4. White has freer development and more space in the centre, which Black intends to neutralise by playing ...c7–c5 at some point. This solid line has undergone a modest revival, featuring in many GM games as a drawing weapon but theory still gives White a slight edge. After 3... dxe4 4. Nxe4, Black has the following options:

• The most popular line is: 4...Nd7 5.Nf3 Ngf6 6.Nxf6+ Nxf6 when Black is ready for ...c5.
• 4...Bd7 5.Nf3 Bc6 (the Fort Knox Variation) activating the light-square bishop, which is often played by Alexander Rustemov.

Rare sidelines after 3.Nc3[edit]

One rare sideline after 3.Nc3 is 3...c6, which is known as the Paulsen Variation, after Louis Paulsen. It can also be reached via a Caro-Kann Defence move-order (1.e4 c6 2.d4 d5 3.Nc3 e6).

Another rare sideline after 3.Nc3 is 3...Nc6, which was played by Aron Nimzowitsch.

Tarrasch Variation: 3.Nd2 [edit]

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The Tarrasch Variation is named after Siegbert Tarrasch. This move became particularly popular during the 1970s and early 1980s when Anatoly Karpov used it to great effect. Though less aggressive than the alternate 3.Nc3, it is still used by top-level players seeking a small, safe advantage.

Like 3.Nc3, 3.Nd2 protects e4, but is different in several key respects: it does not block White's c-pawn from advancing, which means he can play c3 at some point to support his d4-pawn. Hence, it avoids the Winawer Variation as 3...Bb4 is now readily answered by 4.c3. On the other hand, 3.Nd2 develops the knight to an arguably less active square than 3.Nc3, and in addition, it hems in White's dark-square bishop. Hence, White will typically have to spend an extra tempo moving the knight from d2 at some point before developing said bishop.

• 3... c5 4. exd5 and now Black has two ways to recapture:
  • 4... exd5 this was a staple of many old Karpov–Korchnoi battles, including seven games in their 1974 match, usually leads to Black having an isolated queen's pawn (see isolated pawn). The main line continues 5.Ngf3 Nc6 6.Bb5 Bd6 7.0-0 Nge7 8.dxc5 Bxc5 9.Nb3 Bb6 with a position where, if White can neutralise the activity of Black's pieces in the middlegame, he will have a slight advantage in the ending. Another possibility for White is 5.Bb5+ Bd7 (5...Nc6 is also possible) 6.Qe2+ Be7 7.dxc5 to trade off the bishops and make it more difficult for Black to regain the pawn.
  • 4... Qxd5 is an important alternative for Black; the idea is to trade his c- and d-pawns for White's d- and e-pawns, leaving Black with an extra centre pawn. This constitutes a slight structural advantage, but in return White gains time for development by harassing Black's queen. This interplay of static and dynamic advantages is the reason why this line has become popular in the last decade. Play usually continues 5.Ngf3 cxd4 6.Bc4 Qd6 7.0-0 Nf6 (preventing 8.Ne4) 8.Nb3 Nc6 9.Nbxd4 Nxd4, and here White may stay in the middlegame with 10.Nxd4 or offer the trade of queens with 10.Qxd4, with the former far more commonly played today.
• 3... Nf6 While the objective of 3...c5 was to break open the centre, 3... Nf6 aims to close it. After 4. e5 Nfd7 5. Bd3 c5 6. c3 Nc6 (6...b6 intends ...Ba6 next to get rid of Black's 'bad' light-square bishop, a recurring idea in the French) 7. Ne2 (leaving f3 open for the queen's knight) 7... cxd4 8. cxd4 f6 9. exf6 Nxf6 10. Nf3 Bd6 Black has freed his pieces at the cost of having a backward pawn on e6. White may also choose to preserve his pawn on e5 by playing 4.e5 Nfd7 5.c3 c5 6.f4 Nc6 7.Ndf3, but his development is slowed as a result, and Black will gain dynamic chances if he can open the position to advantage.
• 3... Nc6 is known as the Guimard Variation: after 4.Ngf3 Nf6 5.e5 Nd7 Black will exchange White's cramping e-pawn next move by ...f6. However, Black does not exert any pressure on d4 because he cannot play ...c5, so White should maintain a slight advantage, with 6.Be2 or 6 Nb3.
• 3... Be7 is known as the Morozevich Variation.^[5] A fashionable line among top GMs in recent years, this odd-looking move aims to prove that every White move now has its drawbacks, e.g. after 4.Ngf3 Nf6 5.e5 Nfd7 White cannot play f4, whereas 4.Bd3 c5 5.dxc5 Nf6 and 4.e5 c5 5. Qg4 Kf8!? lead to obscure complications. 3...h6?!, with a similar rationale, has also gained some adventurous followers in recent years, including GM Alexander Morozevich.
• Another rare line is 3... a6, which gained some popularity in the 1970s. Similar to 3...Be7, the idea is to play a waiting move to make White declare his intentions before Black commits to a plan of his own. 3...a6 also controls the b5-square, which is typically useful for Black in most French lines because, for example, White no longer has the option of playing Bb5.

Advance Variation: 3.e5 [edit]

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The main line of the Advance Variation continues 3... c5 4. c3 Nc6 5. Nf3 and then we have a branching point:

• 5... Qb6, the idea is to increase the pressure on d4 and eventually undermine the white centre. The queen also attacks the b2-square, so White's dark-square bishop cannot easily defend the d4-pawn without losing the b2-pawn. White's most common replies are 6.a3 and 6.Be2.
  • 6.a3 is currently the most important line in the Advance: it prepares 7.b4, gaining space on the queenside. Black may prevent this with 6...c4 intending to take en passant if White plays b4, which creates a closed game where Black fights for control of the b3-square. On the other hand, Black may continue developing with 6...Nh6, intending ...Nf5, which might seem strange as White can double the pawn with Bxh6, but this is actually considered good for Black. Black plays ...Bg7 and ...0-0 and Black's king has adequate defence and White will miss his apparently 'bad' dark-square bishop.
  • 6.Be2 is the other alternative, aiming simply to castle. Once again, a common Black response is 6...Nh6 intending 7...cxd4 8.cxd4 Nf5 attacking d4. White usually responds to this threat with 7.Bxh6 or 7.b3 preparing Bb2.
  • 6.Bd3 cxd4 7.cxd4 Bd7 (7...Nxd4?? 8.Nxd4 Qxd4 9.Bb5+) 8.0-0 Nxd4 9.Nxd4 Qxd4 10.Nc3 is the Milner-Barry Gambit. If Black continues 10...Qxe5, White gains an attack with 11.Re1 Qb8 12.Nxd5 or 11...Qd6 12.Nb5.^[6]
• 5... Bd7 was mentioned by Greco as early as 1620, and was revived and popularised by Viktor Korchnoi in the 1970s. Now a main line, the idea behind the move is that since Black usually plays ...Bd7 sooner or later, he plays it right away and waits for White to show his hand. If White plays 6.a3 in response, modern theory says that Black equalises or is better after 6...f6! The lines are complex, but the main point is that a3 is a wasted move if the black queen is not on b6 and so Black uses the extra tempo to attack the white centre immediately.
• 5... Nh6 has recently become a popular alternative; the idea is that 6.Bxh6 gxh6 gains Black a semi-open g-file to attack the White king, or Black can play ...Bg7 to support ...f6 to attack White's pawn on e5. If White doesn't take the knight, it will move to f5 to pressure d4, or (after ...f6) to f7 to pressure e5.

There are alternative strategies to 3... c5 that were tried in the early 20th century such as 3...b6, intending to fianchetto the bad bishop and which can transpose to Owen's Defence or 3...Nc6, played by Carlos Guimard, intending to keep the bad bishop on c8 or d7 which is passive and obtains little counterplay. Also, 4...Qb6 5.Nf3 Bd7 intending 6...Bb5 to trade off the 'bad' queen's bishop is possible.

Exchange Variation: 3.exd5 exd5 [edit]

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Many players who begin with 1.e4 find that the French Defence is the most difficult opening for them to play against due to the closed structure and unique strategies of the system. Thus, many players choose to play the exchange so that the position becomes simple and clearcut. White makes no effort to exploit the advantage of the first move, and has often chosen this line with expectation of an early draw, and indeed draws often occur if neither side breaks the symmetry. An extreme example was Capablanca–Maróczy, Lake Hopatcong 1926, which went: 4.Bd3 Bd6 5.Nf3 Nf6 6.0-0 0-0 7.Bg5 Bg4 8.Re1 Nbd7 9.Nbd2 c6 10.c3 Qc7 11.Qc2 Rfe8 12.Bh4 Bh5 13.Bg3 Bxg3 14.hxg3 Bg6 15.Rxe8+ Rxe8 16.Bxg6 hxg6 17.Re1 Rxe1+ 18.Nxe1 Ne8 19.Nd3 Nd6 20.Qb3 a6 21.Kf1 ½–½.^[7]

Despite the symmetrical pawn structure, White cannot force a draw. An obsession with obtaining one sometimes results in embarrassment for White, as in Tatai–Korchnoi, Beer Sheva 1978, which continued 4.Bd3 c5!? 5.Nf3 Nc6 6.Qe2+ Be7 7.dxc5 Nf6 8.h3 0-0 9.0-0 Bxc5 10.c3 Re8 11.Qc2 Qd6 12.Nbd2 Qg3 13.Bf5 Re2 14.Nd4 Nxd4 0–1.^[8] A less extreme example was Mikhail Gurevich–Short, Manila 1990 where White, a strong Russian grandmaster, played openly for the draw but was ground down by Short in 42 moves.^[9]

To create genuine winning chances, White will often play c2–c4 at some stage to put pressure on Black's d5-pawn. Black can give White an isolated queen's pawn by capturing on c4, but this gives White's pieces greater freedom, which may lead to attacking chances. This occurs in lines such as 3.exd5 exd5 4.c4 (played by GMs Normunds Miezis and Maurice Ashley) and 4.Nf3 Bd6 5.c4, which may transpose to the Petroff. Conversely, if White declines to do this, Black may play ...c7–c5 himself, e.g. 4.Bd3 c5, as in the above-cited Tatai–Korchnoi game.

If c2–c4 is not played, White and Black have two main piece setups. White may put his pieces on Nf3, Bd3, Bg5 (pinning the black knight), Nc3, Qd2 or the queen's knight can go to d2 instead and White can support the centre with c3 and perhaps play Qb3. Conversely, when the queen's knight is on c3, the king's knight may go to e2 when the enemy bishop and knight can be kept out of the key squares e4 and g4 by f3. When the knight is on c3 in the first and last of the above strategies, White may choose either short or long castling. The positions are so symmetrical that the options and strategies are the same for both sides.

Another way to unbalance the position is for White or Black to castle on opposite sides of the board. An example of this is the line 4.Bd3 Nc6 5.c3 Bd6 6.Nf3 Bg4 7.0-0 Nge7 8.Re1 Qd7 9.Nbd2 0-0-0.

Early deviations for White[edit]

After 1.e4 e6, almost 90 percent of all games continue 2.d4 d5, but White can try other ideas. The most important of these is 2.d3 d5 3.Nd2, with a version of the King's Indian Attack. White will likely play Ngf3, g3, Bg2, 0-0, c3 and/or Re1 in some order on the next few moves. Black has several ways to combat this setup: 3...c5 followed by ...Nc6, ...Bd6, ...Nf6 or ...Nge7 and ...0-0 is common, 3...Nf6 4.Ngf3 Nc6 plans ...dxe4 and ...e5 to block in the Bg2, and 3...Nf6 4.Ngf3 b6 makes ...Ba6 possible if White's light-square bishop leaves the a6–f1 diagonal. 2.d3 has been used by many leading players over the years, including GMs Pal Benko, Bobby Fischer and Lev Psakhis.

• 2.f4 is the Labourdonnais Variation, named after Louis-Charles Mahé de La Bourdonnais, the 19th-century French master.^[10] Play can continue 2...d5 3. e5 c5 4. Nf3 Nc6 5.c3 Nge7 6.Na3 Nf5.
• 2.Qe2 is the Chigorin Variation, which discourages 2...d5 because after 3.exd5 the black pawn is pinned, meaning Black would need to recapture with the queen. Black usually replies 2...c5, after which play can resemble the 2.d3 variation or the Closed Variation of the Sicilian Defence.
• 2.Nf3 d5 3.Nc3 is the Two Knights Variation: 3...d4 and 3...Nf6 are good replies for Black.
• 2.c4 (attempting to discourage 2...d5 by Black) is the Steiner Variation. But Black can reply 2...d5 anyway, when after 3.cxd5 exd5 4.exd5 Nf6 the only way for White to hold on to his extra pawn on d5 is to play 5.Bb5+. Black gets good compensation in return for the pawn, however.
• 2.Bb5 has occasionally been tried. Notably, Henry Bird defeated Max Fleissig with the variation during the Vienna 1873 chess tournament.^[11]
• 2.b3 leads to the Réti Gambit after 2...d5 3.Bb2 dxe4, but Black can also decline it with 3...Nf6 4.e5 Nd7 with White going for f4 and Qg4 before putting the knight on f3.

There are also a few rare continuations after 1.e4 e6 2.d4 d5, including 3.Bd3 (the Schlechter Variation), 3.Be3 (the Alapin Gambit), and 3.c4 (the Diemer–Duhm Gambit, which can also be reached via the Queen's Gambit Declined).

Early deviations for Black[edit]

Although 2...d5 is the most consistent move after 1.e4 e6 2.d4, Black occasionally plays other moves. Chief among them is 2...c5, the Franco-Benoni Defence, so-called because it features the ...c7–c5 push characteristic of the Benoni Defence. White may continue 3.d5, when play can transpose into the Benoni, though White has extra options since c2–c4 is not mandated. 3.Nf3, transposing into a normal Sicilian Defence, and 3.c3, transposing into a line of the Alapin Sicilian (usually arrived at after 1.e4 c5 2.c3 e6 3.d4) are also common. Play may also lead back to the French; for example, 1.e4 e6 2.d4 c5 3.c3 d5 4.e5 transposes into the Advance Variation. Another move is 2...b6, which transposes into Owen's Defence or the English Defence. Also possible is 2...f5, the Franco-Hiva Gambit, but this is regarded as dubious.^[12]

French Defense Tarrasch Variation

History[edit]

The French Defence is named after a match played by correspondence between the cities of London and Paris in 1834^[1] (although earlier examples of games with the opening do exist). It was Chamouillet, one of the players of the Paris team, who persuaded the others to adopt this defence.^[13]

As a reply to 1.e4, the French Defence received relatively little attention in the nineteenth century compared to 1...e5. The first world chess championWilhelm Steinitz said 'I have never in my life played the French Defence, which is the dullest of all openings'.^[14] In the early 20th century, Géza Maróczy was perhaps the first world-class player to make it his primary weapon against 1.e4. For a long time, it was the third most popular reply to 1.e4, behind only 1...c5 and 1...e5. However, according to the Mega Database 2007,^[15] in 2006, 1...e6 was second only to the Sicilian in popularity.

Historically important contributors to the theory of the defence include Mikhail Botvinnik, Viktor Korchnoi, Aron Nimzowitsch, Tigran Petrosian, Lev Psakhis, Wolfgang Uhlmann and Rafael Vaganian. More recently, its leading practitioners include Evgeny Bareev, Alexey Dreev, Mikhail Gurevich, Alexander Khalifman, Smbat Lputian, Alexander Morozevich, Teimour Radjabov, Nigel Short, Gata Kamsky, and Yury Shulman.

The Exchange Variation was recommended by Howard Staunton in the 19th century,^[16] but has been in decline ever since. In the early 1990s Garry Kasparov briefly experimented with it before switching to 3.Nc3. Note that Black's game is made much easier as his queen's bishop has been liberated. It has the reputation of giving immediate equality to Black, due to the symmetrical pawn structure.

Like the Exchange, the Advance Variation was frequently played in the early days of the French Defence. Aron Nimzowitsch believed it to be White's best choice and enriched its theory with many ideas. However, the Advance declined in popularity throughout most of the 20th century until it was revived in the 1980s by GM and prominent opening theoretician Evgeny Sveshnikov, who continues to be a leading expert in this line. In recent years, it has become nearly as popular as 3.Nd2; GM Alexander Grischuk has championed it successfully at the highest levels. It is also a popular choice at the club level due to the availability of a simple, straightforward plan involving attacking chances and extra space.

ECO codes[edit]

The Encyclopaedia of Chess Openings includes an alphanumeric classification system for openings that is widely used in chess literature. Codes C00 to C19 are the French Defence, broken up in the following way (all apart from C00 start with the moves 1.e4 e6 2.d4 d5):

• C00 – 1.e4 e6 without 2.d4, or 2.d4 without 2...d5 (early deviations)
• C01 – 2.d4 d5 (includes the Exchange Variation, 3.exd5)
• C02 – 3.e5 (Advance Variation)
• C03 – 3.Nd2 (includes 3...Be7; C03–C09 cover the Tarrasch Variation)
• C04 – 3.Nd2 Nc6 (Guimard Variation)
• C05 – 3.Nd2 Nf6
• C06 – 3.Nd2 Nf6 4.e5 Nfd7 5.Bd3
• C07 – 3.Nd2 c5 (includes 4.exd5 Qxd5)
• C08 – 3.Nd2 c5 4.exd5 exd5
• C09 – 3.Nd2 c5 4.exd5 exd5 5.Ngf3 Nc6
• C10 – 3.Nc3 (includes the Rubinstein Variation, 3...dxe4)
• C11 – 3.Nc3 Nf6 (includes the Steinitz Variation, 4.e5; C11–C14 cover the Classical Variation)
• C12 – 3.Nc3 Nf6 4.Bg5 (includes the McCutcheon Variation, 4...Bb4)
• C13 – 3.Nc3 Nf6 4.Bg5 dxe4 (Burn Variation)
• C14 – 3.Nc3 Nf6 4.Bg5 Be7
• C15 – 3.Nc3 Bb4 (C15–C19 cover the Winawer Variation)
• C16 – 3.Nc3 Bb4 4.e5
• C17 – 3.Nc3 Bb4 4.e5 c5
• C18 – 3.Nc3 Bb4 4.e5 c5 5.a3 (includes the Armenian Variation, 5...Ba5)
• C19 – 3.Nc3 Bb4 4 e5 c5 5.a3 Bxc3+ 6.bxc3 Ne7 7.Nf3 and 7.a4

See also[edit]

References[edit]

1. ^ ^ab'London Chess Club vs. Paris Chess Club, corr. 1834'. Chessgames.com.
2. ^T.D. Harding, French: MacCutcheon [sic] and Advance Lines, Batsford, 1979, pp. 12, 56. ISBN0-7134-2026-X.
3. ^Although many sources refer to John Lindsay McCutcheon and his eponymous variation as 'MacCutcheon', 'McCutcheon' is the correct spelling. Jeremy Gaige, Chess Personalia, McFarland & Company, 1987, pp. 260, 275. ISBN0-7864-2353-6; David Hooper and Kenneth Whyld, The Oxford Companion to Chess (2nd ed. 1992), Oxford University Press, p. 240, p. 478 n. 1205. ISBN0-19-866164-9.
4. ^'Wilhelm Steinitz vs. John Lindsay McCutcheon (1885)'. Chessgames.com.
5. ^'French Defense Tarrasch Variation Morozevich Variation – Chess Opening'. chesstempo.com.
6. ^*Hooper, David; Whyld, Kenneth (1996) [First pub. 1992]. 'Milner-Barry Gambit'. The Oxford Companion to Chess (2nd ed.). Oxford University Press. p. 260. ISBN978-0-19-280049-7.
7. ^'Capablanca vs. Maroczy, Lake Hopatcong 1926'. Chessgames.com.
8. ^'Tatai vs. Korchnoi, Be'er Sheva 1978'. Chessgames.com.
9. ^'Gurevich vs. Short, Manila 1990'. Chessgames.com.
10. ^'C00: French, Labourdonnais variation – 1. e4 e6 2. f4 – Chess Opening explorer'. www.365chess.com.
11. ^'Bird, Henry – Fleissig, Max 1873 , Vienna , Vienna'. chesstempo.com.
12. ^Watson, John: Taming Wild Chess Openings p.67
13. ^Le Palamède edited by St. Amant (1846), p. 20.
14. ^'The Cable Match Between Messrs.Tschigorin and Steinitz'. The International Chess Magazine. 7.1. January 1891. p. 27. Retrieved 16 September 2013.
15. ^'Mega Database 2007'. Archived from the original on 2007-02-08. Retrieved 2007-02-04.
16. ^p369, Howard Staunton, The Chess-Player's Handbook, 1847, H.G.Bohn.

Further reading[edit]

• Berg, Emanuel (2013). Grandmaster Repertoire The French Defence Volume One. Quality Chess. ISBN978-1907982408.
• Berg, Emanuel (2014). Grandmaster Repertoire The French Defence Volume Two. Quality Chess. ISBN978-1907982422.
• Berg, Emanuel (2015). Grandmaster Repertoire The French Defence Volume Three. Quality Chess. ISBN978-1907982859.
• Eingorn, Viacheslav (2008). Chess Explained: The French. Gambit Publications. ISBN978-1-904600-95-4.
• Moskalenko, Viktor (2008). The Flexible French. New In Chess. ISBN978-90-5691-245-1.
• Moskalenko, Viktor (2010). The Wonderful Winawer. New In Chess. ISBN978-90-5691-327-4.
• Moskalenko, Viktor (2015). The Even More Flexible French: Strategic Ideas and Powerful Weapons. New In Chess. ISBN978-9056915742.
• Tzermiadianos, Andreas (2008). How to Beat the French Defence: The Essential Guide to the Tarrasch. Everyman Chess. ISBN9781857445671.
• Vitiugov, Nikita (2010). The French Defence. Chess Stars. ISBN978-954-8782-76-0.
• Watson, John (2012). Play the French 4th ed. Everyman Chess. ISBN978-1857446807.
• Williams, Simon (2011). Attacking Chess The French: A Dynamic Repertoire for Black. Everyman Chess. ISBN978-1857446807.

External links[edit]

The Wikibook Chess Opening Theory has a page on the topic of: French Defence

Associated Data

Supplementary Materials

S1 Fig: Efficient conversion of human recombinant PrP by atypical BSE strains. RT-QuIC reactions were seeded with 10⁻⁴ dilutions of bovine tissue (brainstem), using human recombinant protein. (A) and (D), classical BSE and uninfected bovine tissues. (B) and (E), classical BSE and atypical H-BSE. (C) and (F), classical BSE and atypical L-BSE. Each point represents the mean value of 3 replicate relative fluorescence unit readings.

(PDF)

S2 Fig: Conversion of bovine recombinant PrP by atypical BSE strains. Average data and statistical significance of the individual results presented in S3 Fig are represented here. (A), classical BSE isolates and uninfected bovine samples. (B), classical BSE and atypical H-BSE isolates. (C), classical BSE and atypical L-BSE isolates. Each point represents the mean value of 3 replicate relative fluorescence unit readings, which were averaged over the number of animals in each group. Error bars represent the mean standard deviation (SD). Vertical dashed lines indicate a statistically significant difference of signal between the test groups. *, p<0,05; ****, p<0,0001.

(PDF)

S3 Fig: Conversion of bovine recombinant PrP by atypical BSE strains. RT-QuIC reactions were seeded with 10⁻⁴ dilutions of bovine tissue (brainstem), using bovine recombinant protein. (A) and (D), classical BSE isolates and uninfected bovine samples. (B) and (E), classical BSE and atypical H-BSE isolates. (C) and (F), classical BSE and atypical L-BSE isolates. Each point represents the mean value of 3 replicate relative fluorescence unit readings.

(PDF)

S4 Fig: Western blot analysis of PrP^res content in brain homogenates (A) from non-human primates inoculated with classical or atypical L-BSE and (B) from human sCJD and vCJD patients. Homogenates were subjected to proteinase K digestion and serial dilutions were detected by immunoblotting using Sha31 (primates) or 3F4 (CJD patients) monoclonal antibodies.

(PDF)

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

Abstract

The transmission of classical bovine spongiform encephalopathy (C-BSE) through contaminated meat product consumption is responsible for variant Creutzfeldt-Jakob disease (vCJD) in humans. More recent and atypical forms of BSE (L-BSE and H-BSE) have been identified in cattle since the C-BSE epidemic. Their low incidence and advanced age of onset are compatible with a sporadic origin, as are most cases of Creutzfeldt-Jakob disease (CJD) in humans. Transmissions studies in primates and transgenic mice expressing a human prion protein (PrP) indicated that atypical forms of BSE may be associated with a higher zoonotic potential than classical BSE, and require particular attention for public health. Recently, methods designed to amplify misfolded forms of PrP have emerged as promising tools to detect prion strains and to study their diversity. Here, we validated real-time quaking-induced conversion assay for the discrimination of atypical and classical BSE strains using a large series of bovine samples encompassing all the atypical BSE cases detected by the French Centre of Reference during 10 years of exhaustive active surveillance. We obtained a 100% sensitivity and specificity for atypical BSE detection. In addition, the assay was able to discriminate atypical and classical BSE in non-human primates, and also sporadic CJD and vCJD in humans. The RT-QuIC assay appears as a practical means for a reliable detection of atypical BSE strains in a homologous or heterologous PrP context.

Introduction

Prion diseases are fatal transmissible disorders affecting humans and animals. These neurodegenerative diseases are characterized by brain vacuolization, neuronal loss and accumulation of PrP^Sc, an abnormal isoform of the host-encoded cellular prion protein (PrP^c). The infectious agent is mainly, if not solely, composed of abnormal PrP^Sc and is capable of converting cellular PrP^c into PrP^Sc in an autocatalytical manner []. After proteinase K digestion of PrP^Sc, molecular features of the protease resistant fragment (PrP^res) can be evidenced by Western blot.

Among animals, the classical bovine spongiform encephalopathy (C-BSE) affects cattle. The C-BSE epidemic in the 1980s became a major matter of concern for human health when the variant of Creutzfeldt-Jakob disease (vCJD) appeared as the result of a C-BSE foodborne transmission to humans [–]. Since the C-BSE epidemic, atypical forms of BSE have been reported in cattle []. They present a biochemical signature distinct from C-BSE, with a higher (H-BSE) or lower (L-BSE) apparent molecular weight of unglycosylated PrP^res observed in Western blot []. The annual incidence (1 case per million) and the old age of atypical BSE-affected animals are compatible with a sporadic origin []. When transmitted to primates [–] and transgenic mice expressing a human PrP [, ], L-BSE showed an apparent higher pathogenicity than C-BSE. Pathological and biochemical similarities have been observed between L-BSE in cattle or primates and certain sCJD subtype [, ]. When L-BSE was transmitted to sheep [] and transgenic mice expressing ovine PrP [, ], specific strain properties were retained, yet important PrP^res molecular changes could be observed in some conditions []. Taken together, these studies highlight the importance of a rapid and proper identification of atypical BSE strains in cattle and other species.

Among recent methods developed to amplify prions in vitro, the real-time quaking-induced conversion (RT-QuIC) assay allows the sensitive detection of prion seeding activity in numerous tissues of animal and human origins [–]. The RT-QuIC assay relies upon the conversion of recombinant prion protein (recPrP) by prion-associated seeds into amyloid fibrils in presence of an amyloid-sensitive dye, thioflavine T (ThT). The incorporation of ThT within elongating fibrils is monitored in a multiwell plate in real time.

In two recent studies, this test was used successfully for the discrimination of a few C-BSE and L-BSE samples of cattle [], and for the discrimination of C-, L-, and H-BSE samples of experimentally inoculated cattle []. Here, we took advantage of the extensive collection of classical and atypical BSE isolates identified over a 10 year-period of active surveillance in France to validate the rapid discrimination of classical and atypical BSE samples of cattle using RT-QuIC. Furthermore, we were also able to discriminate sporadic CJD and vCJD in humans as reported previously [, ], and also classical BSE and atypical L-BSE in primates.

Materials and methods

Ethic statement

A written informed consent for autopsy and research use was provided by patient’s relatives, according to the French regulation (L.1232-1 to L.1232-3, Code de la Santé Publique). The brain tissues with the corresponding written informed consent are referred for postmortem diagnosis and research to the French National Neuropathological Network for CJD (funded by the French Government) and to the French National Centre of Reference for prions (funded by the French Institute for Public Health Surveillance). No approval by local ethics committee is required during this procedure.

Sources of tissues

Samples from primates and cattle existed before the study began. BSE brainstem samples were collected during the active surveillance in France and confirmed by discriminatory western blot by the Agence Nationale de Sécurité Sanitaire de l’Alimentation, de l’Environnement et du Travail (ANSES, Lyon, France). In this study, 13 H-BSE and 14 L- BSE isolates, encompassing all the atypical BSE cases collected during the period 2000–2010, were analyzed by RT-QuIC. Fifteen C-BSE cases collected during this period were also included. Among samples collected from fallen stock or after several freeze-and-thaw cycles, autolysis was often observed, affecting 10 out of 13 H-BSE samples, 11 out of 14 L-BSE samples and 6 out of 15 C-BSE samples (Table 1). Brainstems from 8 negative BSE animals collected in 2010 were analyzed as negative controls. Brain tissue was taken from 3 non-CJD patients, 3 iatrogenic CJD (iCJD-hGH) patients resulting from an infection with contaminated growth hormone of human origin, 2 vCJD cases (MM2b) and 4 sCJD cases (MM1, MV1, MV2, and VV2) as defined by their PRNP codon 129 genotype (MM, MV or VV), their PrP^res type (type 1 or 2), and according to the migration pattern of PrP^res on Western blot. Patients were referred to the French National Reference Center for Unconventional Transmissible Agents for CJD, and the diagnosis was confirmed biochemically and neuropathologically. Brain samples from cynomolgus macaques previously inoculated via intracerebral and oral routes with classical and atypical L-BSE isolates [, ] were analyzed blindly by RT-QuIC.

Table 1

Cattle ID	Result of molecular typing by Western blot	Sample shown in Fig 3	Age (year)
01–2437	C-BSE	1	6
01–2579	C-BSE	6
02–2263	C-BSE	2	7
02–2715	C-BSE	8
02–2811	C-BSE	3	6
02–2872	C-BSE	9
02–2992	C-BSE	4	8
04–0881	C-BSE	9
05–0294	C-BSE	9
06–1164	C-BSE	5	12
07–0324	C-BSE	6	12
07–0453	C-BSE	12
09–0170	C-BSE	7	15
09–0335	C-BSE	15
10–0015	C-BSE	6
00–2549	H-BSE	13
01–2604	H-BSE	8
02–0558	H-BSE	8	13
02–2695	H-BSE	11
03–0440	H-BSE	9	16
03–1928	H-BSE	8
03–2095	H-BSE	12
07–0644	H-BSE	10	11
08–0257	H-BSE	18
08–0498	H-BSE	11	8
09–0169	H-BSE	16
09–0497	H-BSE	12	13
10–0161	H-BSE	13
02–2528	L-BSE	8
10–0075	L-BSE	13	15
03–2052	L-BSE	14	13
04–0824	L-BSE	15	19
05–0009	L-BSE	12
06–0931	L-BSE	16	13
07–0012	L-BSE	10
07–1136	L-BSE	17	10
08–0074	L-BSE	11
08–0374	L-BSE	18	15
09–0007	L-BSE	12
09–0397	L-BSE	19	14
09–0481	L-BSE	18
10–0409	L-BSE	20	9

Recombinant prion protein preparation

Human recombinant full length PrP (codon 129M) (recHuPrP; aa 23–231; GenBank accession number no. M13899) and bovine recombinant full length PrP (recBovPrP; aa 25–242; GenBank accession number no. NP_851358) were purified according to the protocol published previously []. Protein concentrations were determined by measuring the absorbance at 280 nm, and aliquots were stored at -80°C until use.

Brain homogenate preparation

The tissues were prepared in PBS (Sigma-Aldrich) containing 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and Complete Protease Inhibitor Cocktail (Roche) to give a final tissue concentration of 10% (w/v). The homogenization was performed in 2ml centrifuge tubes containing ceramic beads using a FastPrep^® 24 instrument (MP Biomedical) for 45 s at speed 6.5. Gross cellular debris were removed from brain homogenates (BH) after a centrifugation at 2000 g for 2 min. Supernatants were collected and stored at -80°C until use.

Normalization of brain homogenates from affected individuals

To assess the analytical sensitivity of our RT-QuIC assay for the detection of sCJD and determine the minimal amount of detectable PrP^Sc, brain homogenates (BH) from CJD patients were normalized using known concentrations of recHuPrP. The samples containing PrP^res were digested with PK 100μg/ml for 1h at 37°C and loaded onto Novex 4–12% Bis-Tris acrylamide gels (Life Technologies), alongside dilutions of recHuPrP (ranging from 10 to 1.25 ng). To compare the detection of the studied prion strains in the different groups of affected individuals (iCJD patients, non-human primates), brain homogenates were analyzed by Western blot (WB) as previously described []. The amount of PrP^res in samples was adjusted with a further dilution to match the amount detected in the sample with the lowest PrP^res signal at the determined dilution.

After electrophoresis, the separated proteins were transferred to nitrocellulose membrane and immunoblotted with anti-PrP mAb 3F4 (Eurogentec) for human samples. It was followed by an incubation with a secondary antibody coupled with horseradish peroxidase (HRP). The HRP activity was revealed using ECL (GE Biosciences), and the blots were exposed to ECL Hyperfilms (GE Biosciences). The films were scanned using a Bio-Rad GS800 densitometer and analyzed using Bio-Rad Quantity One software. The densities of the single Western blot band corresponding to recHuPrP were compared with the combined densities of the 3 bands corresponding to PrP^res. To obtain an estimated amount of PrP^res in 2 μl equivalent to 100 fg of recHuPrP, the corresponding 100% brain tissue dilutions varied from 10⁻⁶ (MM1, MV1, MV2) to 5x10^-7 (MM2b, VV2). For BSE isolates, all the samples were tested by RT-QuIC at the same dilution (10⁻⁴), regardless of the PrP^res level in the sample. A Western blot analysis with TeSeE confirmatory Western blot kit (Bio-Rad) was done to compare relative quantity of PrP^res between groups (H-, C- and L-BSE) and to confirm the presence of PrP^res when no seeding activity was detected by RT-QuIC.

RT-QuIC method

The RT-QuIC assay was prepared as described previously [, ]. Samples were serially diluted 10-fold in PBS containing 1x N2 media supplement (Life Technologies) and 0.1% SDS. The RT-QuIC reaction mixture was prepared in 1X PBS with final concentration of 300 mM NaCl, 1 mM EDTA, 10 μM thioflavin T, 0.1 mg/ml full length recombinant human (23–231) or bovine (25–241) recPrP, and 98 μl of this mixture were distributed in 96-well black bottom optic plates (Nalgene Nunc). Each reaction was seeded in triplicates with 2 μl of tissue dilution and plates were placed in a BMG Fluostar Omega plate reader (BMG Labtech) at 42°C for 70h (280 cycles, each consisting of 1 min shaking at 600 rpm and 1 min at rest, with ThT fluorescence measurement taken every 15-min with a gain setting of 1000).

Data analysis

All RT-QuIC experiments were performed at least three times and produced comparable results. At pertinent time points, the statistical significance of the difference between mean fluorescence or lag phases of analyzed groups was assessed using the non-parametric, unpaired t- test with unequal variance (Welch correction), using GraphPad Prism software v6.0 (San Diego, USA).

Results

Atypical BSE strains seeded more efficiently the conversion of recombinant PrP than classical BSE strain

The seeding efficiency of brain homogenates prepared from cattle affected by atypical and classical BSE was investigated. A total of 15 C-BSE, 14 L-BSE and 13 H-BSE isolates (see details in Table 1), along with 8 negative bovine cases, were analyzed by RT-QuIC. PrP^res Western blot results for each BSE group are illustrated in Fig 1. Although a substantial inter-individual variability was observed within each group, a tendency to a higher PrP^res level was observed in C-BSE samples. A typical run was performed using 2 plates, in which were included the 8 negative samples and a half of each BSE group. In our conditions, negative and classical BSE samples could not be differentiated using recHuPrP (average data, Fig 2A; individual data, panels A & D in S1 Fig). On the contrary, an efficient amplification of atypical BSE samples was obtained, along with a remarkable homogeneity of the lag phase (~10h) for animals of each group (H-BSE and L-BSE) (Fig 2B and 2C; panels B, C, E, F in S1 Fig). Statistical analyses were performed to determine the time at which the increase in fluorescence becomes significant for each BSE group. No difference was observed between uninfected and C-BSE samples (Fig 2A). The difference of fluorescence signal between C-BSE and H-BSE or L-BSE was statistically significant as early as 10h (p<0.01) (Fig 2B) and 12h (p<0.05) (Fig 2C), respectively, and at terminal 70h time point (p<0.0001).

Western blot analysis of PrP^res levels in brainstem homogenates from cattle affected by classical or atypical BSE.

Homogenates were subjected to proteinase K digestion followed by immunoblotting using Sha31 monoclonal antibody. Dilutions of a C-BSE sample (02–2872) are indicated, along with samples from each C-, H- and L-BSE group. The numbers 1 to 20 refer to cattle identity indicated in Table 1.

Conversion of human recombinant PrP by atypical BSE strains.

Average data and statistical significance of the individual results shown in S1 Fig are represented here. (A), classical BSE isolates and uninfected bovine samples. (B), classical BSE and atypical H-BSE isolates. (C), classical BSE and atypical L-BSE isolates. Each point represents the mean value of 3 replicate relative fluorescence unit readings, which were averaged over the number of animals in each group. Error bars represent the mean standard deviation (SD). Vertical dashed lines indicate a statistically significant difference of signal between the test groups. *, p<0,05; **, p<0,01; ****, p<0,0001.

Experiments were repeated using a different recombinant PrP protein. Although homology of sequence between seed and substrate was described as being not mandatory with RT-QuIC method, we used bovine PrP (recBovPrP) to ascertain that the lack of C-BSE amplification we observed was not due to the use of recHuPrP. Similar results were obtained with recBovPrP. While C-BSE and negative samples remained undistinguishable (panel A in S2 Fig, panels A and D in S3 Fig), H-BSE and L-BSE groups were clearly distinct from C-BSE group (panels B and C in S2 Fig; panels B, C, E and F in S3 Fig). The lag phase was in the same range as that obtained with recHuPrP. The difference of fluorescence signal between C-BSE and H-BSE or L-BSE was statistically significant as early as 11h (p<0.05) (panel B in S2 Fig) and 14h (p<0.05) (panel C in S2 Fig), respectively, and at terminal 70h time point (p<0.0001). We next investigated whether such properties of differential amplification between atypical and classical BSE strains were preserved after passage to non-human primates.

Efficient amplification of atypical and classical BSE strains after transmission to non-human primates

Western blot showing the PrP^res content in brain homogenates from macaques with C-BSE and L-BSE are illustrated in S4 Fig (panel A). Both L-BSE and C-BSE strains, which have been transmitted to non-human primates by intracerebral and oral routes [,], seeded efficiently RT-QuIC reactions with human recombinant PrP (Fig 3). An efficient reaction, although with a lower maximum fluorescence level, was also achieved with bovine recombinant PrP (not shown). In both cases, classical BSE samples provided a more efficient seeding material than L-BSE, with a lag phase shorter than 10h. A remarkable homogeneity of the samples within each group was observed, and it was possible to blindly identify 2 groups of samples. No apparent effect of the inoculation route was evidenced. The difference of lag phases (when RFU > 1000) between the 2 groups was statistically significant (p<0.005).

Conversion of human recombinant PrP by C- and L- BSE transmitted to macaques.

RT-QuIC reactions were seeded with 10⁻⁴ and 2.5x10^-5 dilutions of brain tissue from macaques that had been inoculated by oral (per os) or intracranial (IC) routes with C-BSE and L-BSE, respectively. Each point represents the mean value of 3 replicate relative fluorescence unit readings. Horizontal dashed line indicates a statistically significant difference of lag phase between the L-BSE and C-BSE groups. **, p<0.01.

Brain homogenates from sCJD patients seeded more efficiently the conversion of human recPrP than vCJD brain homogenates

We next assessed whether differential seeding activity can be observed in humans using seeds from patients infected with the C-BSE agent and from patients with sporadic CJD. Western blot results showing the PrP^res content in brain homogenates from sCJD and vCJD patients are illustrated in S4 Fig (panel B). Brain homogenates from sCJD patients with different molecular subtypes (MM1, MV1, MV2 and VV2) seeded with the same efficiency RT-QuIC reactions using full-length recHuPrP (Fig 4A), after the normalization of the PrP^res levels. The assay was able to detect the molecular subtype MM1 down to a 10⁻⁹ dilution of 100% brain tissue, corresponding to 100 ag of PrP^res (Fig 4B). In contrast, and despite equivalent amounts of PrP^res (100 fg) used to seed the reaction, vCJD brain homogenates proved less efficient to initiate RT-QuIC reactions, and only a sensitivity down to a 10⁻⁷ brain dilution (equivalent to 10 fg of PrP^res) could usually be achieved in our conditions (Fig 4B). Replacing human recPrP with bovine recPrP did not improve the detection of vCJD (data not shown). Negative results were obtained with normal brain homogenates (NBH), which were diluted 10⁶ fold starting from 100% brain tissue.

Conversion of human recombinant PrP by sCJD seeds.

(A) RT-QuIC was seeded with an estimated amount of 100 fg PrP^res from MM1, MV1, MV2 and VV2 brain homogenates. Non-CJD brain homogenate was diluted at an equivalent dilution (10⁻⁶). (B) Serial dilutions of sCJD MM1, vCJD MM2b and non-CJD seeds were submitted to RT-QuIC. Amounts of PrP^res detected in brain homogenate ranged from the equivalent of 100 fg to 10 ag. Each point represents the mean value of 3 replicate relative fluorescence unit readings.

Brain homogenates from iCJD-hGH patients seeded efficiently the conversion of human recPrP

We also studied the efficiency of our RT-QuIC assay with iatrogenic CJD, an additional infectious form of human prion disease due to a contamination by the peripheral route, after cadaver–derived human growth hormone treatment. Brain homogenates from 3 French iatrogenic cases seeded as efficiently as sCJD MM1 the conversion of human recPrP, as sporadic and iatrogenic seeds were indistinguishable and showed similar lag-phases (<10h) (Fig 5).

Conversion of human recombinant PrP by iCJD seed.

RT-QuIC reactions were seeded with 10⁻⁶ and 8x10^-7 dilutions of brain tissues from one sCJD MM1 and three iCJD-hGH patients (iCJD #1, #2 and #3), respectively. Each point represents the mean value of 3 replicate relative fluorescence unit readings.

Discussion

In this study, we assessed the power of discrimination of BSE strains from cattle using RT-QuIC. Indeed, in two recent studies, this assay was used successfully for the discrimination of a few C-BSE and L-BSE samples from cattle [], and for the discrimination of C-, L-, and H-BSE samples from experimentally inoculated cattle []. Here, we took advantage of the extensive collection of classical and atypical BSE isolates identified over a 10 year-period of active surveillance in France to validate RT-QuIC performances through the analysis of 15 classical and 27 atypical BSE samples from cattle. Classical BSE, from which human vCJD is derived, was particularly inefficient to seed recombinant PrP and could not be differentiated from negative bovine samples. On the contrary, all the atypical BSE samples were readily detected, with the same lag phase, despite very different PrP^res content. Thus, our study confirms previous results obtained with 5 L-BSE and 4 C-BSE natural cases by Orru et al [] and with 3 experimental cases of each BSE subtype by Masujin et al [], using large series of natural cases (15 C-BSE, 14 L-BSE and 13 H-BSE cases). In addition, we further tested the detection of L-BSE and C-BSE after transmission to macaques and compared the seeding properties of vCJD samples to those obtained with sCJD and French iCJD after GH treatment.

Atypical cases of BSE putatively represent sporadic forms of prion disease in cattle, with PrP^sc glycotypes, neuropathology and PrP^sc deposition different from those observed in classical BSE [, ]. In humans, we also observed a differential amplification between seeds from sCJD and vCJD, as previously reported []. Using sCJD brain homogenates, we achieved a sensitivity in a similar range to that reported by others using hamster [] or human recPrP substrates [], without false positive reaction, contrasting with the experience from other laboratories []. Likewise, vCJD homogenates were less efficient to seed reactions than sCJD homogenates. Possible explanations have been discounted, such as case-to-case variations, brain region variation, or inhibitors present in vCJD brain []. In our study, we tested different regions, from 2 different vCJD patients, without any improvement of the detection.

RT-QuIC discrimination may rely on differences in abnormal PrP assemblies sustaining distinct seeding activities that may vary with several factors (sporadic or infectious origin of the disease, affected species, route of infection). We were able to discriminate brain samples from macaques inoculated with C-BSE and with L-BSE. Surprisingly, in macaques, C-BSE showed the most efficient seeding activity while it was relatively inefficient in humans. Differences in PrP amino acid sequence might contribute to this discrepancy. Mature forms of bovine and human PrP share 91.3% of amino acid sequence, and bovine and cynomolgus macaques PrP only 88.7%. Different regions of PrP have been proposed as key domains for fibrillization, such as the S1H1S2 region [, ] or the H2H3 domain []. It was suggested that single amino acid variations in the H2 and H3 domains trigger different oligomerization pathways []. A sequence alignment of bovine and macaque PrP amino acids shows variations at residues 100 and 108 in macaque (and not in human PrP). This central region of PrP contains an amyloidogenic sequence AGAAAAGA that appears critical for PrP^res formation [] and because corresponding synthetic peptides are able to specifically inhibit in vitro formation of PrP^res []. Other variations exist in macaque PrP at residue 143 (H1 domain), residue 155 (6 amino acids before S2 domain), and residue 220 (H3 domain). Hence, it is possible that such amino acid variations in critical regions of the macaque PrP has an impact on the quaternary structure of PrP^sc assemblies and therefore on the related seeding activity. For example, the H187R mutation in the human PrP has a dramatic effect on the protein folding, resulting in a markedly increased propensity to oligomerize [].

However, the unpredictable manner of the biological changes of prion strain properties during interspecies transmission has been extensively described [–]. In more recent studies, when L-BSE isolates were propagated in wild type mice [] and transgenic mice expressing ovine PrP [], strain features closely similar to those of C-BSE agent were observed. In addition, a shift in the biochemical signature of the C-BSE agent was observed in the spinal cord of orally-infected macaques at the preclinical stage []. Altogether, these data highlight the fact that the properties of BSE strains may evolve during interspecies transmission, notably in macaques. Our results suggest that such a strain variation also has an impact on seeding activity.

A main feature of C-BSE-infected brain tissue from various animal models including macaques is the presence of amyloid plaques [–] composed of PrP^Sc fibers which could modulate the seeding activity of these tissues. However, plaques are also observed in the brain of vCJD patients, and vCJD brain homogenates show a poor seeding activity. Another characteristic of C-BSE in macaques is the high proportion of diglycosylated PrP^res, but C-BSE or vCJD PrP^res share the same feature, and yet are not associated with an efficient seeding activity. Likewise, the Western blot type of PrP^res as defined by the molecular mass of the unglycosylated PrP^res after proteinase K digestion cannot account for these different seeding activities, since PrP^res type 2 is distributed in both groups with efficient (sCJD MV2, VV2, C-BSE in macaques) and inefficient (vCJD) seeding properties. It was intriguing that PrP^sc from all the studied natural diseases with a sporadic or presumably sporadic origin (all sCJD subtypes, all atypical BSE isolates) showed a high level of seeding activity, unlike peripherally acquired diseases due to the C-BSE agent (vCJD, C-BSE). To investigate the role of the peripheral route on the selection of PrP^Sc species with seeding activity, we analyzed brain homogenates from iatrogenic CJD cases secondary to growth hormone treatment, which are acquired prion diseases and result from a human-to-human CJD transmission. We found similar and efficient seeding activities for sCJD MM1 and iCJD-hGH cases using RT-QuIC. Moreover, we also found similar and efficient seeding activities in brain homogenates from non-human primates inoculated intracerebrally and via the oral route with C-BSE or L-BSE. Altogether, our data do not support the peripheral route as a main factor influencing the selection of PrP^sc species with low seeding activity. In our hands, it appears that this poorly efficient RT-QuIC profile was limited to the C-BSE agent, which propagated in cattle and humans.

To conclude, we showed that RT-QuIC detects PrP^Sc from a large series of 27 atypical BSE isolates within hours, and provides a promising tool for the diagnosis of these natural diseases and for their discrimination from C-BSE in a homologous and heterologous PrP context.

Supporting information

S1 Fig

Efficient conversion of human recombinant PrP by atypical BSE strains.

RT-QuIC reactions were seeded with 10⁻⁴ dilutions of bovine tissue (brainstem), using human recombinant protein. (A) and (D), classical BSE and uninfected bovine tissues. (B) and (E), classical BSE and atypical H-BSE. (C) and (F), classical BSE and atypical L-BSE. Each point represents the mean value of 3 replicate relative fluorescence unit readings.

(PDF)

S2 Fig

Conversion of bovine recombinant PrP by atypical BSE strains.

Average data and statistical significance of the individual results presented in S3 Fig are represented here. (A), classical BSE isolates and uninfected bovine samples. (B), classical BSE and atypical H-BSE isolates. (C), classical BSE and atypical L-BSE isolates. Each point represents the mean value of 3 replicate relative fluorescence unit readings, which were averaged over the number of animals in each group. Error bars represent the mean standard deviation (SD). Vertical dashed lines indicate a statistically significant difference of signal between the test groups. *, p<0,05; ****, p<0,0001.

(PDF)

S3 Fig

Conversion of bovine recombinant PrP by atypical BSE strains.

RT-QuIC reactions were seeded with 10⁻⁴ dilutions of bovine tissue (brainstem), using bovine recombinant protein. (A) and (D), classical BSE isolates and uninfected bovine samples. (B) and (E), classical BSE and atypical H-BSE isolates. (C) and (F), classical BSE and atypical L-BSE isolates. Each point represents the mean value of 3 replicate relative fluorescence unit readings.

(PDF)

S4 Fig

Western blot analysis of PrP^res content in brain homogenates (A) from non-human primates inoculated with classical or atypical L-BSE and (B) from human sCJD and vCJD patients.

Homogenates were subjected to proteinase K digestion and serial dilutions were detected by immunoblotting using Sha31 (primates) or 3F4 (CJD patients) monoclonal antibodies.

(PDF)

Acknowledgments

The research leading to these results has received funding from the program “Investissements d’avenir” ANR-10-IAIHU-06. This study was funded in part by LFB Biomédicaments and Institut de Veille Sanitaire (InVS).

Funding Statement

This work was funded by “Investissements d’avenir” ANR-10-IAIHU-06; LFB Biomédicaments; Institut de Veille Sanitaire (InVS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

All relevant data are within the paper and its Supporting Information files.

References

French Tarrasch Variation