Can one negative yelp review (defamation about our Business) from disgruntled former employee count as defamation case?What are the chance of winning?

Yes, absolutely. In Virginia, all that is required to maintain a cause of action for defamation is (a) publication (b) of an actionable statement (c) with the requisite intent. (See https://www.virginiadefamationlawyer.com/2013/02/defamation-of-character-libel-and-slander-law-in-virginia.html). If a former employer poses as a customer and posts a false review containing highly damaging statements about your business, there is a good chance that all three of those elements have been satisfied.