The evolution of human influenza A viruses from 1999 to 2006: a complete genome study

Abstract

Background: Knowledge about the complete genome constellation of seasonal influenza A viruses from different countries is valuable for monitoring and understanding of the evolution and migration of strains. Few complete genome sequences of influenza A viruses from Europe are publicly available at the present time and there have been few longitudinal genome studies of human influenza A viruses. We have studied the evolution of circulating human H3N2, H1N1 and H1N2 influenza A viruses from 1999 to 2006, we analysed 234 Danish human influenza A viruses and characterised 24 complete genomes.

Results: H3N2 was the prevalent strain in Denmark during the study period, but H1N1 dominated the 2000-2001 season. H1N2 viruses were first observed in Denmark in 2002-2003. After years of little genetic change in the H1N1 viruses the 2005-2006 season presented H1N1 of greater variability than before. This indicates that H1N1 viruses are evolving and that H1N1 soon is likely to be the prevalent strain again. Generally, the influenza A haemagglutinin (HA) of H3N2 viruses formed seasonal phylogenetic clusters. Different lineages co-circulating within the same season were also observed. The evolution has been stochastic, influenced by small "jumps" in genetic distance rather than constant drift, especially with the introduction of the Fujian-like viruses in 2002-2003. Also evolutionary stasis-periods were observed which might indicate well fit viruses. The evolution of H3N2 viruses have also been influenced by gene reassortments between lineages from different seasons. None of the influenza genes were influenced by strong positive selection pressure. The antigenic site B in H3N2 HA was the preferred site for genetic change during the study period probably because the site A has been masked by glycosylations. Substitutions at CTL-epitopes in the genes coding for the neuraminidase (NA), polymerase acidic protein (PA), matrix protein 1 (M1), non-structural protein 1 (NS1) and especially the nucleoprotein (NP) were observed. The N-linked glycosylation pattern varied during the study period and the H3N2 isolates from 2004 to 2006 were highly glycosylated with ten predicted sequons in HA, the highest amount of glycosylations observed in this study period.

Conclusion: The present study is the first to our knowledge to characterise the evolution of complete genomes of influenza A H3N2, H1N1 and H1N2 isolates from Europe over a time period of seven years from 1999 to 2006. More precise knowledge about the circulating strains may have implications for predicting the following season strains and thereby better matching the vaccine composition.

Figure 1

Relative prevalence of sentinel and routine influenza A viruses in Denmark 1999 to 2006. The actual numbers of influenza A positive samples for the respective seasons are as follows; 1999–2000 49, 2000–2001 28, 2001–2002 80, 2002–2003 61, 2003–2004 83, 2004–2005 91 and 2005–2006 54.

Figure 2

Evolutionary relationships of circulating H3N2 influenza A viruses sampled in Denmark from 1999 to 2006. The nucleotide coding region trees were generated with maximum parsimony, heuristic random branch swapping search (neighbor joining and maximum likelihood analysis revealed the same tree topology). Bootstrap values of 1000 resamplings in per cent (>70%) are indicated at key nodes. H3N2 HA and NA trees are rooted to A/Beijing/353/89 and A/Beijing/32/92. Reference sequences referred to in the text are shown in bold. The A/Fujian/411/02(H3N2) reference sequence is represented by A/Wyoming/03/03.

Figure 3

Evolutionary relationships of circulating H3N2 and H1N1 influenza A viruses sampled in Denmark from 1999 to 2006. The nucleotide coding region trees were generated with maximum parsimony, heuristic random branch swapping search (neighbor joining and maximum likelihood analysis revealed the same tree topology). Bootstrap values of 1000 resamplings in per cent (>70%) are indicated at key nodes. The trees for H3N2 and H1N1 PB2, PB1, PA and NP genes are mid-point rooted for means of clarity. Reference sequences referred to in the text are shown in bold. The A/Fujian/411/02(H3N2) reference sequence is represented by A/Wyoming/03/03.

Figure 4

Evolutionary relationships of circulating H3N2 and H1N1 influenza A viruses sampled in Denmark from 1999 to 2006. The nucleotide coding region trees were generated with maximum parsimony, heuristic random branch swapping search (neighbor joining and maximum likelihood analysis revealed the same tree topology). Bootstrap values of 1000 resamplings in per cent (>70%) are indicated at key nodes. The trees for H3N2 and H1N1 M and NS and H1N1 HA and NA genes are mid-point rooted for means of clarity. Reference sequences referred to in the text are shown in bold. The A/Fujian/411/02(H3N2) reference sequence is represented by A/Wyoming/03/03.

Figure 5

Fraction of predicted N-linked glycosylation sequons in HA and NA of H3N2 and H1N1 viruses sampled in Denmark seasons 1999 to 2006. Sites with predicted potential threshold values above 0.5 are shown. Sites not shown for H3 (n = 204): 122, N2 (n = 166): 200, 329, 402, H1 (n = 27): 10, position 539 is positively predicted; however this site is located at the cytosolic region of HA and is therefore not glycosylated, N1 (= 30): 455.

Figure 6

(A) Seasonal amino acid distances of H3N2 HA and NA proteins since 1999 and (B) amino acid distances of H3N2 HA and NA from one season to the next. The same trends were observed for nucleotide distances. In 2000–2001 H1N1 viruses only were observed and therefore not included. Distance means were computed as the arithmetic mean of all pair wise distances between two seasons in the inter-season comparisons by the MEGA v.3.1 software [68]