Recombinant Tula hantavirus shows reduced fitness but is able to survive in the presence of a parental virus: analysis of consecutive passages in a cell culture

Plyusnina, Angelina; Plyusnin, Alexander

First, we designed RT-PCR primers able to discriminate between non-recombinant (V-type) and recombinant (REC-type) types of TULV S RNA. The resullts presented in Fig. 1 show that the primer pairs designed to generate the 118 bp-long products from either V-type or REC-type S RNA amplified, indeed, homologous sequences only, whether these were taken along (lines 1 and 6) or mixed with the heterologous sequences (lines 3 and 7). Using the two specific RT-PCR conditions, the presence of V-type and REC-type S RNA was monitored on ten sequential passages of the mixture of TULV02 and RecTULV5 variants (Fig. 2 ). S RNA of V-type was seen on all passages ( Fig. 2A , lines 1-10). In contrast, S RNA of REC-type, was detected up to the fifth passage (Fig. 2B , lines 1-5), and then disappeared (Fig. 2B , lines 6-10). An alternative approach to check the presence of the two different types of S RNA using specific primer pairs at the stage of nested PCR gave exactly the same result. The V-type S RNA was detected during all ten passages while the REC-type totally disappeared after the 5 th passage (data not shown). These data confirmed our earlier observation [11] that the transfection-mediated HRec yields functionally competent and stable virus, recTULV. The purified and pre-passaged recombinant virus, however, presented no real match to the original cell adapted variant, TUL02, it terms of fitness. Taking into account that the in situ formed recombinant S RNA disappeared from the mixture after four passages [11] , one should conclude that the lower competitiveness of the recombinant virus seen earlier did not result from its "immature" status. When, under similar experimental settings, TUL02 has been passaging in the presence of another isolate, TULV/Lodz, none of the two viruses was able to establish a dominance during ten consecutive passages (Plyusnin et al., unpublished data).