Venezuela attorney general Tarek Saab says two people close to opposition candidate Maria Corina Machado have been arrested and seven others, including Machado's right-hand Magalli Meda, have warrants out for their detention.
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The arrests are set to complicate an already-confused scenario ahead of the July presidential contest. Machado won the opposition's October nominating contest by a landslide, but is barred from registering her candidacy.
Washington called the move an attack on civil society and said it was inconsistent with an election guarantees deal inked last year that had led the US to relax some sanctions.
"We condemn the arbitrary arrests and warrants issued today for members of the democratic opposition in Venezuela," Brian Nichols, from the US State Department, said on social media.
"We call for the immediate release of these individuals and all those unjustly detained."
Washington has already warned it will allow oil sanction relaxations to expire in April if the socialist government of President Nicolas Maduro does not allow free and fair elections.
Henry Alviarez, the national coordinator of Machado's Vente Venezuela party, and Dignora Hernandez, the party's political secretary, were detained for alleged involvement in planned conspiracies and street violence, Saab said on state television.
"The public ministry asked for nine arrests, two have been carried out," Saab said.
Alviarez had previously had an arrest warrant issued for him in December, but it was withdrawn soon after a US-Venezuela prisoner swap in which some opposition figures, as well as detained Americans, were freed.
Four other Machado campaigners have been arrested over the last three months on conspiracy accusations, among other alleged crimes.
Machado and Vente Venezuela condemned the arrests on X.
"Venezuelans, I ask you for strength and courage in these difficult times. Today, more than ever, we need to be united and firm to continue moving towards our goals," Machado said.
With no sign her ban will be lifted before the July 28 contest, opposition parties have been pressuring Machado to name a substitute who can register by a March 25 deadline. Meda had been one of the people floated as a possible alternate.
Machado has been polling far ahead of Maduro. Various opinion polls have forecast Machado, 56, would pick up at least 50 per cent of votes, while Maduro, a 61-year-old former bus driver, was projected to receive around 20 per cent.
But Venezuela's top court upheld her ban in January, imposed over her support of US sanctions and accusations of corruption that Machado says are an attempt to prevent a democratic vote.
In response, the United States has started re-imposing sanctions and warned about the April expiry of oil sanctions relaxations.
A Washington official said this week the US is still holding out for progress but Caracas had been sending "negative signals."
Australian Associated Press