EPIDEMIOLOGY, GENOMIC CHARACTERISATION AND EVOLUTIONARY ANALYSIS OF ITALIAN H1 SWINE INFLUENZA VIRUSES

Abstract Study n.1 - Swine influenza viruses: virological surveillance in Italy during 1998-2011 Three predominant subtypes of swine influenza viruses (SIVs) circulating in pigs throughout the world are H1N1, H3N2 and H1N2. In Europe over the last years, the epidemiology of SIVs has considerable changed. In particular in Italy, swine monitoring programs have been applied since the nineties and are based on genome detection, virus isolation and sequencing of all respiratory forms. This study reported the swine influenza surveillance programme preformed during the period 1998 – 2011. Investigated samples originated from the entire country but the major part of them was from North Italy where more than 75% of the swine industry is located. In this period 580 SIVs were isolated divided in 243 H1N1, 214 H3N2, 111 H1N2, 12 2009 A/ H1N1 pandemic (H1N1pdm) and 1 H3N1. These results revealed continuous circulation of H1N1, H3N2 and H1N2 viruses and isolation of the H1N1 pdm viruses in pigs starting in 2009. The most frequent subtype was the avian-like H1N1 and the second the H3N2. The H1N2 subtype was rarely isolated in the first part of the investigated period but since 2005, its frequency increased becoming the second subtype in the last three years. In the last two years 12 H1N1pdm strains were isolated in 7 different farms also when these viruses did not circulate in the human population. Moreover it is worth noting the isolation of one H3N1 strain, which resulted from a reassortment between swine H1N1 and H3N2 influenza viruses. Phylogenetic analysis evidenced homogeneity among the recent Italian H1N1 SIVs, distinguishing these from the earlier strains. Also for the H1N2 subtype the presence of a homogeneous group, differing from the earlier strains is reported. Interestingly the neuraminidase (NA) gene of the recent Italian strains showed different characteristics respect to the European H1N2 strains, deriving from the recent human H3N2 viruses. Finally, Italian H3N2 viruses were gradually evolved from A/Port Chalmer/1/73 like human virus, which is considered the most probable ancestor of the swine PCh-73 lineage. These results evidenced reassortment events between swine and human influenza viruses and highlight the need for a stringent surveillance in the pig population with particular attention to the H1N2 subtype and the H1N1 pdm viruses. Study n.2 - First pandemic H1N1 outbreak from a pig farm in Italy The first outbreak of the 2009 A/H1N1 pandemic virus in a swine breeder farm in Italy in November 2009 was reported. Clinical signs observed in sows included fever, depression, anorexia and agalactia, while in piglets diarrhoea and weight loss. The morbidity in sows was approximately 30% and the accumulated mortality rate was similar with those usually reported in piggeries (<10%). Virus was isolated from piglets (A/Sw/It/290271/09) and the sequencing of the whole genome was then performed. Comparison with all pandemic H1N1 sequences available in GenBank shows in A/Sw/It/290271/09 three unique amino-acid (aa) changes in PB2 (S405T), PB1 (K386R) and PA (K256Q), not yet associated to any well characterized phenotype markers of Influenza viruses. All eight aa at positions representing the so-called species-specific swine-human signatures, found in both swine and in the pandemic H1N1v, are also present. The M2 protein displays the C55F and the PA protein the S409N substitutions, both corresponding to enhanced transmission phenotype markers. Phylogenetic analysis showed that the virus was genetically related to the pandemic H1N1 virus. In addition, serological samples were collected from 40 sows, of which 20 resulted positive to the pandemic H1N1 virus by HI test proving a virus circulation in the farm. Study n.3 - Novel H1N2 swine influenza reassortant strain in pigs derived from the pandemic H1N1/2009 virus Swine influenza monitoring programs have been in place in Italy since the 1990’s and from 2009 testing for the 2009 A/ H1N1 pandemic virus (H1N1pdm) virus was also performed on all the swine samples positive for type A influenza. This paper reports the isolation and genomic characterization of a novel H1N2 swine influenza reassortant strain from pigs in Italy that was derived from the H1N1 pdm virus. In May 2010, mild respiratory symptoms were observed in around 10% of the pigs raised on a fattening farm in Italy. Lung homogenate taken from one pig showing respiratory distress was tested for Influenza type A and H1N1pdm by two real time RT-PCR assays. Virus isolation was achieved by inoculation of lung homogenate into specific pathogen free chicken embryonated eggs (SPF CEE) and applied onto Caco-2 cells and then the complete genome sequencing and phylogenetic analysis was performed from the CEE isolate. The lung homogenate proved to be positive for both influenza type A (gene M) and H1N1pdm real time RT-PCRs. Virus isolation (A/Sw/It/116114/2010) was obtained from both SPF CEE and Caco-2 cells. Phylogenetic analysis showed that all of the genes of A/Sw/It/116114/2010, with the exception of neuraminidase (NA), belonged to the H1N1pdm cluster. The NA was closely related to two H1N2 double reassortant SIVs, previously isolated in Sweden and Italy. NA sequences for these three strains were clustering with H3N2 SIVs. The emergence of a novel reassortant H1N2 strain derived from H1N1pdm in swine in Italy raises further concerns about whether these viruses will become established in pigs. The new reassortant not only represents a pandemic (zoonotic) threat but also has unknown livestock implications for the European swine industry. Study n.4. Genomic characterization and evolutionary analysis of H1N2 swine influenza viruses in Italy Three subtypes (H1N1, H1N2, and H3N2) are currently diffused worldwide in pigs. The H1N2 subtype was detected for the first time in Italian pigs in 1998. To investigate the genetic characteristics and the molecular evolution of this subtype in Italy, we conducted a phylogenetic analysis of the HA and NA genes of 53 strains isolated from 1998 to 2012. Moreover the whole genome sequences of 26/53 strains were performed. Phylogenetic analysis of HA and NA genes showed differences between the older (1998-2003) and the more recent strains (2003-2010). The older isolates were closely related to the established European H1N2 lineage, whereas the more recent isolates possessed a different NA deriving from recent human H3N2 viruses. Furthermore, eight Italian H1N2 strains isolated recently in 2008-2012 (A/Sw/It/196875/08, A/Sw/It/310411/09, A/Sw/It/195639/10, A/Sw/It/195399/11, A/Sw/It/274551/11, A/Sw/It/329017/11 A/Sw/It/26654/12 and A/Sw/It/107798/12) showing different features were detected. They were characterised by an HA gene, closely related to the EU H1N1 SIVs. The last two strains exhibited different patterns: A/sw/It/ 58769/10 was an uncommon strain with an HA that is closely related to H1N1 and an NA similar to H3N2 SIVs; A/Sw/116114/10 was a H1N2 reassortant strain derived from 2009 H1N1 pdm viruses. Amino acid analysis revealed interesting features: a deletion of two amino acids (146-147) in the HA gene of the recent isolates and two strains isolated in 1998; the presence of the uncommon aa change (N66S), in the PB1-F2 protein in strains isolated from 2009-2010, which is said to have contributed to the increased virulence. The evolutionary analysis of the HA and NA genes revealed five different reassortment events between human and swine influenza viruses occurred at different times between 1978 and 2000. These results demonstrate the importance of pigs as mixing vessels for animal and human influenza and show the presence and establishment of reassortant strains involving human viruses in pigs in Italy. These findings also highlighted different genomic characteristics of the NA gene the recent Italian strains compared to circulating European viruses.