Triple‐reassortant influenza A virus with H3 of human seasonal origin, NA of swine origin, and internal A(H1N1) pandemic 2009 genes is established in Danish pigs

This report describes a triple‐reassortant influenza A virus with a HA that resembles H3 of human seasonal influenza from 2004 to 2005, N2 from influenza A virus already established in swine, and the internal gene cassette from A(H1N1)pdm09 has spread in Danish pig herds. The virus has been detected in several Danish pig herds during the last 2‐3 years and may possess a challenge for human as well as animal health.


| INTRODUCTION
genes, which have been frequently recovered. 1,3,4 Reassortant viruses with genes from other human seasonal IAV origin and enzootic swIAV are occasionally detected in pigs. Remarkably, these reassortments are typically detected in pigs several years after their human counterpart appeared as seasonal influenza viruses. [5][6][7] In Denmark, a passive surveillance program for swIAV has been conducted since 2011 based on samples submitted for diagnostic purpose from pigs with acute respiratory diseases. The most frequent circulating subtypes are avian-like H1N1 (avH1N1), reassortant IAV with avian-like H1 and internal gene cassette, and with N2 from swineadapted H3N2 (rH1N2) and A(H1N1)pdm09. 8 In addition, a number of different reassorted viruses are found sporadically. 1

| CASE DESCRIPTION
In 2014, samples from a herd located in the central part of Jutland, Denmark, were submitted to the National Veterinary Institute, due to persistent problems with respiratory disease in pigs and reproductive problems in the sow herd. The herd vaccinated sows with the Gripovac3 vaccine (Merial Norden). This vaccine contains three enzootic swine influenza strains: an avian-like H1N1 (A/sw/ Haselunne/IDT2617/2001(H1N1)), a H1N2 strain with a humanlike H1 (A/sw/Bakum/1832/2000 (H1N2)), and a H3N2 strain (A/ sw/Bakum/IDT1769/2003)). IAV was detected in nasal swabs from piglets by an in-house-modified real-time RT-PCR (rtRT-PCR) targeting the matrix segment. 9 Initial capillary electrophoresis sequencing of the HA and NA segment was performed on the amplicons made using modified primers designed by Hoffmann et al. 10 Analysis of the sequences by BLAST search of the EpiFlu database and phylogenetic analysis revealed that closest hit in a BLAST search (95% identity) was to the H3 genes from the human seasonal influenza virus circulating in 2004/05 (designated H3hu05). NA was phylogenetically closely related (~97% identity) to that of rH1N2 viruses routinely found in Danish pigs (designated N2sw). The virus was named: A/swine/ Denmark/1916Denmark/ -1/2014. To our knowledge, this is the first case in Europe of spillover of human H3 surface genes that have been established and found to circulate in swine. Fidelity. Purified PCR products were pooled in equimolar quantity and subjected to next-generation sequencing on the Ion Torrent PGM™ sequencer. The sequences were assembled using CLC genomics workbench 6.5 (Qiagen), and phylogenetic trees were constructed with MrBayes 3.2.6 11 using the CIPRES science gateway 12 from alignments constructed with the MUSCLE algorithm. For all segments, the following parameters were used nset=6, rates=invgamma, ngen=10 000 000 or to convergence of 0.002. The analysis showed that samples from both owners were closely related with 97%-100% nt identity ( Figure 1) but clustering based on owner is also observed, with more than 99% identity when comparing sequences obtained from one owner with exception of sample A/swine/Denmark/13669-4/2014 that had ~97% identity to all other sequences. The analysis also revealed that all the internal genes shared >98% identity to genes of circulating A(H1N1)pdm09 porcine strains ( Figure 2). In Table 1 Table 2). The results revealed that there was no crossreactivity between post-vaccination serum and the H3hu05N2sw isolate ( Table 2). This was expected based on the relatively large difference (81%-82% amino acid identity) in the HA sequence between the H3hu05N3sw viruses and the H3 strain included in the vaccine. 2 Test of samples received in the frame of the national surveillance program in 2015-2016 has revealed that at least seven other production systems located geographically widespread in Denmark were positive for this novel subtype, indicating that this virus has been established in Danish swine (data not shown).

| CONCLUSIONS
We report here the detection of a new triple-reassortant H3N2 influenza virus in swine with H3 gene of seasonal human influenza virus origin, internal genes from A(H1N1)pdm09-like viruses, and NA from contemporary N2 swine viruses. Its genetic makeup is distinct from previously known European swine H3N2 viruses, and the virus was retrospectively detected in a sample from 2013. It is now apparent that the subtype has become established in the Danish pig population.

Human-like H3
Reference strain Avian-like H1 A(H1N1)pdm09 H3 EU swine F I G U R E 2 Phylogenetic tree of the six internal genes of the two isolates that were fully characterized together with reference strains. The coloring is dictated by the HA tree in Figure 1  The reassortment events leading to this virus also remain speculative.
If the H3 gene indeed originated from the human seasonal influenza strains circulating in 2004/2005, this H3 gene have circulated undetected in swine or another host for more than 10 years, and until the emergence of A(H1N1)pdm09, this must have been without the internal genes derived thereof. In our view, a likely scenario is that the H3 gene reassorted with a swine influenza strain with an HXN2-avian-like backbone creating a virus that did not cause severe clinical signs. In 2010 or later, this virus reassorted with A(H1N1)pdm09 creating the H3hu05N2 virus which apparently is capable of inducing severe clinical signs in pigs. Denmark is annually exporting more than 10 million living pigs. As pigs are not routinely tested for IAV in relation to export, it is likely that this virus will spread to other European countries, emphasizing the need of joint European surveillance initiatives such as the former European Union funded ESNIP programs. 1,8 As there was no link between the two independent production systems, the introductions either happened independently from a third source or by transmission between the production systems by other horizontal routes, for example, airborne transmission.
The human-like swine H3N2 virus is distinct from the strains included in all available swine vaccines in Europe and, furthermore, the prevalence of viruses in swine with an H3 gene is very low in pigs in most European countries including Denmark. 8 Thus, the establishment of this new H3N2 virus in pigs could have a significant impact on the swine industry due to lack of population immunity.
Indeed, the respiratory disease in pigs and reproductive failures in sows reported from some of the herds in this study were quite severe despite vaccination of the sows. According to the practitioners we have had contact with, the clinical sigs seen in the herds are comparable to or even more severe than the clinical signs normally encountered during acute outbreak of influenza in Danish swine, so this virus seems to be as virulent or even more virulent than the enzootic circulating strains. Further controlled studies are needed to address this further.
The identification of this new virus with seven of eight genes of human origin including an A(H1N1)pdm09 matrix gene also raises severe concern on the impact on human health. In the United States, swine-adapted H3N2 viruses which also have acquired the A(H1N1) pdm09 matrix gene 13 have been shown to be able to infect humans, albeit with limited human-to-human transmission. The new virus reported here has in addition to the A(H1N1)pdm09 matrix gene also a human-adapted HA gene which may lead to an improved risk of human-to-human transmission if introduced into humans. Studies are ongoing to investigate these further using ferrets as infection models. H3hu# denotes sera from five different sows from the herd infected with A/swine/Denmark/15164-1p1/2014 H3huN2sw. Vacc# is post-vaccination with Gripovac 3 serum provided by the manufacturer of the vaccine IDT. H3ref is the standard sera used by the National Veterinary Institute of Denmark routine diagnostic laboratory.