Phylogenetic Analysis of HA and NA Genes of Swine Influenza Viruses in Serbia in 2016-2018

Abstract

Pigs are very important for the epidemiology of influenza A viruses, being commonly infected with the lineages of most adapted H1N1, H3N2, H1N2 swine subtypes. Epidemiological complexity of swine influenza is increasing by a periodic spillover of human or avian viruses in the pig population when genetic shifts can occur. The objectives of this research were to determine the presence of the influenza A virus in nasal and tracheobronchial swabs and lung tissue samples of ill and dead pigs on commercial farms, to determine circulating subtypes and characterize them through the phylogenetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes. A total of 255 samples collected from 13 farms were analyzed by means of real-time RTPCR. The genome of influenza A virus was detected in 24 samples, which represented a 61.5% prevalence at the farms level (influenza A virus was confirmed in 8 out of 13 farms included in this study). Based on HA and NA gene sequences of 8 viruses, the circulation of H1N1 and H3N2 subtypes of influenza A viruses were determined. In addition, one farm exhibited a time separated circulation of H1N1 and H3N2 virus subtypes. Using Influenza Research Database, our viruses of the H1 subtype were classified into 1C.2.1 and 1A.3.3.2. clade. Based on the nucleotide sequences of HA genes, three viruses of the H1N1 subtype belong to the H1N1pdm09 lineage, and the other four to Eurasian “avian-like” H1avN1 lineage; while based on NA genes sequences, these seven viruses belong to Eurasian “avian-like” H1avN1 lineage. Both HA and NA genes of the virus of the H3N2 subtype belonged to the A/swine/ Gent/1/1984-like H3N2 lineage.

Protein sequence features of H1N1 swine influenza A viruses detected on commercial swine farms in Serbia

“…Lysine at position 251 was described as a virulence factor in H1avN1 viruses (5). The possibly higher virulence of these two viruses could be associated with their having been detected in lung tissue samples of animals that died of swine influenza (27). Substitution D9N, which provides mitochondrial association of PB2 and consequently inhibition of expression of β-interferon and impaired immune response of the host (15) was not present in our isolates.…”

“…Additional research in the form of an inhibition of haemagglutination test should be done to validate this assumption. Previous research on receptor-binding properties of these viruses revealed affinity to the α2-6 human type of receptors (27), indicating a major predisposition for transmission to human hosts.…”

“…The finding of the alternative +1 open reading frame in the PB1 segment revealed that viruses SRB/sw/2017/1 and SRB/sw/2016/5 synthesised PB1-F2 accessory non-structural proteins respectively 90 and 79 AAs long, as did German swIAVs isolated in 2009-2012 (16). Based on the presence and the known functions of the PB1-F2 protein (downregulation of antiviral innate immunity, promotion of pro-inflammatory cytokines, induction of cell death and enhancement of viral polymerase activity) ( 21), we can assume the higher virulence of these two strains and link this to the clinical outcome that they provoked (the deaths of the affected pigs) (27). Concerning the SRB/sw/2017/2, SRB/sw/2016/3 and SRB/sw/2017/7 strains, the PB1 segment of which belonged to the H1N1pdm09 lineage, STOP codons were located at positions 12, 58, and 88, indicating synthesis of a truncated PB1-F2 protein of only 11 AAs.…”

“…Full genome sequences of five swIAV strains were translated into their AA sequences using MEGA 7.0 software (23). The virus strains originated from diseased and dead swine reared on five different commercial farms in Serbia (27) and were characterised by Eurasian "avian-like" swine H1N1 (H1avN1) or reassorted genomes (28) (Table 1). Investigations of the presence of molecular markers related to the lineage characteristics, virulence, crossreactivity with vaccine strains, and resistance to antiviral drugs were performed in the Influenza Research Database (IRD) (50).…”

Influenza surveillance in pigs: balancing act between broad diagnostic coverage and specific virus characterization

“…However, the odds for the initial detection of swIAV was lower in contact wipes compared to dust wipes and oral fluids in the nursery. Referring to tracheobronchial swabs, the burdensome sample procedure for animals and human as well as the low swIAV detection rate [ 53 ] disqualifies this type of sample as a tool for the detection of enzootically infected herds. However, tracheobronchial swabs might be suitable for detecting acutely diseased pigs as all tracheobronchial swabs positive samples in the nursery originated from pigs with clinical signs (data presented in [ 54 ]).…”