The emergence of new IAV in humans, pigs and other host species is mostly associated with the reassortment process of two or more IAV strains [22,23,24]. This study intended to clarify the role of pigs in this process. Our data confirmed that pigs can be experimentally co-infected with SIV and AIV and that an exchange of RNA segments may occur.
The vast majority of the isolated strains were Gent/08 (96.49%). The Italy/05 strain comprised 2.19% of the isolates, while the rH1N1 strain was confirmed in 1.32% of the isolates. All of the Italy/05 and rH1N1 strains were isolated from the nasal swabs of different CE pig.
The transmission of the Italy/05 virus in pigs confirmed in our study strongly contrasts with previous studies which showed efficient replication of this virus in pigs which was not transmitted further . In our experiment, the Italy/05 could be transmitted. Moreover, our rH1N1 possessed a majority of avian-origin genes excluding the M or NP genes. It was reported that the M and NP genes have an impact on the transmission capability of IAV [25,26,27]. In the case of the r5 H1N1 isolates, the reassortment process may have occurred independently in two CE piglets or, presumably, in one CE or one FI piglet, which then transmitted the r5 H1N1 to naïve animals.
A study conducted in the 1990s indicated that SIV acts as a helper virus in the process of AIV replication in pigs. In that study, the animals were co-infected with SIV Sw/Hok/2/81 (H1N1) and AIV Dk/Hok/8/80 (H3N8), which showed no ability to replicate in the pig. Apart from SIVs, AIVs and rH3N1 with swine-origin NA, NP and M or NS genes were obtained . The SIV used by the Japanese researchers supported the replication of the AIV, whereas in our study, SIV strongly competed with AIV. Nonetheless, in our study, the infection with Gent/08 and the clinical symptoms such as sneezing and coughing may have facilitated the transmission of Italy/05.
An antigenic shift did not occur in our study and the rH1N1 gained only one RNA segment. This could be connected with competitiveness among the used strains.
All of the rH1N1 were isolated from nasal swabs. This may suggest favourable conditions in the upper respiratory tract for the exchange of IAV RNA segments. A similar conclusion was drawn from the experimental co-infection of ferrets with A/Wyoming/03/03 (H3N2) (low virulence, high transmissivity) and A/Thailand/16/06, (avian-like H5N1) (high virulence, low transmissivity). In that study, a low reassortment (8.9%) efficiency was obtained , which is similar to our findings. This may be caused by phenotypic and genotypic differences of the IAVs used for co-infection.
The ability of IAV to infect different hosts largely depends on the optimal gene constellation . Thus, the capacity for replication and transmission in pigs may increase in the case of reassortants that have compatible genes compared with a wholly AIV. However, without an experimental comparison we cannot assume that our rH1N1 isolates are better adapted to pigs than Italy/05.
In previous studies, 63 possible reassortants of the H5N1 subtypes of human seasonal A/Wyoming/3/2003 (H3N2) and avian-like A/Thailand/16/2004 (H5N1) IAVs were divided into 4 phenotypic groups based on the rescue efficiency. Strains with a similar genomic constellation to our rH1N1 were included in group 1 (r7 H5N1), while strains with good replication efficiency and marginally viable reassortants made up group 4 (r5 H5N1). The authors underlined that all of the strains in group 4 had an NP gene of mammalian-origin, but the addition of an NS gene (r5/8) or NS and M genes (r5/7/8) from the H3N2 virus significantly increased the rescue efficiency of the reassortant .
Another research group generated 254 reassortants of a low pathogenic AIV H5N1 subtype and a HuIV H3N2 subtype . In that study, the strains with M or NP gene of mammalian-origin were included in group 1 (the M gene) and 4 (the NP gene).
Both studies [1, 30] showed that the acquisition of the M gene of mammalian-origin did not significantly affect the functionality of the AIV, while the presence of the NP gene dramatically decreased the activity of the virus. Those results differ from our findings, because 2 of the 3 obtained rH1N1 strains acquired the NP gene. This difference may result from the use of different strains of AIV. It may also be caused by better compatibility of the Gent/08 NP gene with the remaining Italy/05 RNA segments and the congruity of Gent/08 NP with other proteins, especially viral polymerase.
The acquisition of a single gene by the rH1N1 in this study did not increase the frequency of its isolation compared with Italy/05. On the other hand, the results indicate that the rH1N1 viruses have not lost the capability to replicate. Taking into consideration the results of the previous studies , we can assume that acquiring another gene of swine-origin could improve the replication and transmission properties of our rH1N1 and enhance its adaptation in pigs.
The most favourable conditions for the occurrence of reassortment during co-infection in experimental models have not been determined. Therefore, it is not clear what circumstances are needed under natural conditions to promote the emergence of new IAV variants .
In order to exclude certain unfavourable reassortment factors, such as segment mismatch, an American research group  used for co-infection the H3N2 subtype IAV strains with a different genotype. Even then, a theoretically optimal reassortment efficiency was not achieved. They found that parental strains occurred more frequently than expected, suggesting incomplete mixing of RNA segments of parental viruses in co-infected cells, although this may also be related to the small differences in the input of viral particles into single cells . The authors also found that in vivo reassortment occurred less frequently (59%) than in vitro reassortment (88.4%) .
Therefore, the use of two phenotypically and genotypically different strains in our experiment had a significant impact on the low incidence of reassortment, mainly due to RNA segment mismatch and the competitiveness of the Gent/08 and the Italy/05 strains. The low incidence of the reassortment was also affected by the use of an animal model.
In previous in vivo studies , pigs were inoculated with two SIV strains: the classical H1N1 (cSIV) and the triple reassortant H3N2. The majority (85.9%) of the obtained isolates were reassortants. Interestingly, there were no cSIV strains among all the isolates. This may indicate that the cSIV strain replicates less efficiently in pigs compared to the H3N2 SIV strain and the identified reassortants. Despite such a high percentage of reassortants, their transmission to the CE pigs was not confirmed .
The results of that study suggest that there was Italy/05 and Gent/08 reassortment in the CE piglets in our study. This is due to the fact that despite the use of two SIV strains by the American researchers, their reassortants were not transmissible. However, as mentioned above, the confirmed transmission of both IAVs used in our experiment dose not exclude the possibility of reassortant transmission.
Provided there are optimal conditions for reassortment, segment exchange is influenced by the viral strains. The functional compatibility of the synthesised proteins, the match between the sequences initiating RNA segment packing to progeny virions  and/or the efficiency of co-infection impacts in the affinity for various receptors .
The results of this study show that despite the very low efficiency of reassortment, it can occur between genotypically and phenotypically different IAV strains in pigs. This underscores the necessity for enhanced viral surveillance strategies that monitor reassortment events, as well as for experimental studies which will elucidate the the reassortment process.