Successful virus purification depends on obtaining high titers of virus in host plants and having methods to efficiently extract biologically active virus from infected tissue. The latter is most critical since virus particles must retain their infectivity while facing the harsh oxidative environment during plant tissue homogenization. Ideally, purification methods should be optimized to obtain a high titer while maintaining biological activity for each virus species under investigation. Purified BYDV (later to be recognized as BYDV-MAV) from Coast black oats was first reported by Rochow and Brakke [36] , using 0.1 M pH 7 phosphate buffer with no additives included in the homogenization buffer. Pierpoint [37] identified a number of substances (ascorbate, ethyl xanthate diethyldithiocarbamate, cysteine, 2mercaptobenzothiazole) that prevented oxidation of polyphenolic compounds in homogenized, infected plant tissue (which can be observed as browning of the plant homogenate). As a result, reducing agents became commonplace additives to virus extraction buffers and, in many cases, with positive outcomes. In contrast to our current study, the addition of sodium sulfite and EDTA were necessary for successful purification of the potyvirus, peanut mottle virus [38] . When purifying BYDV-PAV, Hammond (1983) incorporated sodium sulfite in the extraction buffer to prevent browning, and its addition did not affect infectivity nor did reducing agents affect other luteovirids such as the potato leafroll virus (PLRV) [14] or soybean dwarf virus (B. Tian, personal communication). However these studies did not examine the virus transmissibility in the absence of reducing agents so it is unknown whether sodium sulfite imparts any negative (or positive) effects on the transmissibility of other purified luteovirids. Those reviewing plant virus purification protocols [24] cautiously warn against adoption of a one-size-fits-all protocol for purifying plant viruses. Together with the previous works, our data show such caution should be duly noted by those purifying luteovirids. The vectorvirus specificity that defines the luteovirids undoubtedly reflects the complex chemical nature of protein interactions mediated by the CP and RTP. Care should be taken in understanding how the chemistry of the purification protocol can impart biochemical changes in the virion that will alter virus-host and virus-vector protein interactions. The action of sulfite on disulfides (e.g. cystines) can be represented by the equilibrium equation: R.S.S.R + SO 3 22 « RS 2 + RS.SO 3 2 . Above pH 9, the equilibrium constant does not favor the total cleavage of the disulfide bond, and large excesses of sulfite are required to drive the reaction to completion. However, at lower pH values (e. g. pH 7 of the homogenization buffer used in these virus preparations) the thiol predominates over the thiolate ion resulting in more complex reaction kinetics and more favorable equilibriums [39] . In general, the equilibrium constant can be shifted to the right by any process that removes the thiol and in the presence of divalent metal ions, known to stabilize several icosahedral viruses [40] , and all thiol and disulfide groups can be readily converted to S-sulfonates by a process that reduces the metal ion [41] . The reaction of sulfite with protein disulfides is further complicated by an acute sensitivity to the ionic atmosphere in the neighborhood of the disulfide bond. For example, anionic disulfides have been shown to react much more slowly than neutral or cationic disulfides [42] . For cystines (disulfides) having two positively charged flanking amino acids, the rate constant for reaction of the negative disulfide is 132,000 s 21 M 21 as compared to one having two neutral neighbors, 367 s 21 M 21 , a 10 6 -fold range in rate constants [43] . Furthermore, the rate of reaction can be greatly affected by steric factors, and the disulfide bonds of many proteins show great variability in their susceptibility to cleavage by sulfite in the absence of denaturants such as guanidine hydrochloride or urea [44, 45, 46, 47] . Thus, it can be expected that in the absence of denaturants, the thiols and disulfides most susceptible to modification by sulfite would be those that are most exposed and that have positively charged amino acids as their nearest neighbors.