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

1743-422X-5-40 1743-422X Research The evolution of human influenza A viruses from 1999 to 2006: A complete genome study Bragstad Karoline kbr@ssi.dk Nielsen P Lars lpn@ssi.dk Fomsgaard Anders afo@ssi.dk

Laboratory of Virus Research and Development, Statens Serum Institut, DK 2300 Copenhagen, Denmark

WHO National Influenza Centre, Statens Serum Institut, DK-2300 Copenhagen, Denmark

Virology Journal 1743-422X 2008 5 1 40 http://www.virologyj.com/content/5/1/40 18325125 10.1186/1743-422X-5-40 09 1 2008 07 3 2008 07 3 2008 2008 Bragstad et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Background

Every year the influenza A virus causes human infection with varying severity depending on the host acquired immunity against the particular virus strain. Three to five million people experience severe illness and 0.25 to 0.5 million people die of influenza yearly worldwide (WHO EB111/10). The influenza virus evades host immunity by accumulation of point mutations (drift) in the major surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA) or by reassortment of segments from different viruses co-infecting the same cell leading to a new stain with a HA (and NA) not seen in the population before (shift). In the worst case, shifts may cause pandemics. There have been three pandemics the last hundred years, the Spanish flu in 1918 (H1N1), the Asian flu in 1957 (H2N2) and the Hong Kong flu in 1968 (H3N2). It is believed that new pandemics emerge through shifts with strains from the avian reservoir, as was the case of the pandemics of 1957 and 1968, or by direct introduction of an avian strain into the human population as suggested for the 1918 pandemic 1. At present only two of the 16 possible HA subtypes (H1 and H3), and two of the nine possible NA subtypes (N1 and N2) are circulating in man. H3N2 and H1N1 influenza A viruses have co-circulated in the human population since the re-emergence of H1N1 in 1977, increasing the possibility for genetic reassortments. The prevalence of the different subtype combinations may vary from season to season. The H3N2 has been the predominant influenza A strain during the last 20 years, with the exception of the 1988–1989 and 2000–2001 seasons where H1N1 infections dominated 2. In the 2000–2001 season a new reassorted human strain, H1N2, emerged in Europe and became established in the autumn 2001 34. The new H1N2 subtype was covered by the 2002–2003 H1 and N2 trivalent vaccine components and because both H1 and N2 viruses had circulated the previous years some degree of herd immunity against the new strain was expected. The H1N2 viruses were not associated with severe influenza illness that season. In 2002, a new lineage A/Fujian/411/02(H3N2)-like emerged in Asia and caused significant outbreaks on every continent 56.

For the northern hemisphere the WHO issues the recommendation for strains to be included in the trivalent vaccine for the next season based on epidemiological data and antigenic and genetic analyses of circulating strains.

Until the recent release of over 1,800 complete influenza A genome sequences from the Influenza Genome Sequencing Project managed by US National Institute of Allergy and Infectious Diseases 78 very few complete genome sequences have been published to the GenBank. Also, there have been limited longitudinal studies of the complete genome of influenza A viruses. The present study characterise the complete genome evolution of H3N2, H1N1 and H1N2 influenza A virus from Denmark spanning seven seasons from 1999 to 2006.

Results

Prevalence of influenza A in Denmark from 1999 to 2006

The relative prevalence of influenza virus varies from season to season. Influenza A H3N2 was the dominating strain in Denmark during the last seven years, with the exception of the 2000–2001 season where the H1N1 viruses dominated, as can be seen in Figure 1.

Figure 1

Relative prevalence of sentinel and routine influenza A viruses in Denmark 1999 to 2006

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.

Only H3N2 viruses were isolated during the 2001–2002 season. In the 2002–2003 season the H3N2 and H1N1 reassorted influenza A virus strain, H1N2, emerged in Denmark, but has not been isolated in Denmark since 2003–2004. Higher prevalence of H1N1 viruses co-circulating with H3N2 viruses was observed the last two seasons, 2004/2005 and 2005/2006.

Genetic evolution of influenza A

H3N2 viruses

Based on phylogenetic analysis of the HA and NA nucleotide sequences from 1999 to 2006 (Figure 2), ten isolates representative for the phylogenetic clustering of sequences from each subtype in each season, as far as possible, were included in the final HA and NA tree (Figure 2) and representatives were chosen for complete genome sequencing. Generally the H3N2 HA and NA genes formed seasonal phylogenetic clusters (Figure 2). However, we observed that strains of different lineages and clusters co-circulated within the same season and that viruses had reassorted with viruses from previous seasons (Figure 2).

Figure 2

Evolutionary relationships of circulating H3N2 influenza A viruses sampled in Denmark from 1999 to 2006

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.

The HA gene of the influenza H3N2 strains from the 1999–2000 season formed a phylogenetic subclade to A/Moscow/10/99(H3N2) and A/Sydney/5/97(H3N2) (represented by A/Memphis/31/98) (Figure 2), located between A/Moscow/10/99 and A/Panama/2007/99 (not shown). The antigenicity of these strains was A/Moscow/10/99(H3N2)-like in a haemagglutination inhibition assay, and will therefore be referred to as Moscow-like throughout this report. The NA and the internal genes were all A/Moscow/10/99(H3N2)-like, with the exception of the matrix (M) gene that clustered as a subclade to the A/New York/55/01-like strains (Figure 3).

Figure 3

Evolutionary relationships of circulating H3N2 and H1N1 influenza A viruses sampled in Denmark from 1999 to 2006

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.

The 2001–2002 season was represented as a monophyletic cluster of A/New York/55/01(H3N2)-like viruses in all genes (Figure 2). The next season, 2002–2003, was characterised by co-circulating lineages. These were of viruses most closely related to A/New York/55/01(H3N2) from the previous season, H1N2 viruses (described in more detail below) and a new H3 lineage, the A/Fujian/411/02(H3N2)-like viruses. The introduction of the A/Fujian/411/02(H3N2)-like viruses caused a "jump" in the evolution of the H3N2 viruses (Figure 2). The HAs in subsequent seasons have evolved from these viruses.

In 2003–2004 the HAs form a subclade to the A/Fujian/411/02(H3N2)-like lineage from 2002–2003. These viruses were reassortants probably acquiring the rest of the genome from the 2001–2002 or 2002–2003 A/New York/55/01(H3N2)-like viruses (Figure 2, 3 and 4) and became the predominant lineage co-circulating with the A/Wellington/1/04(H3N2)-like viruses introduced from the southern hemisphere. One single H1N2 virus isolate was also observed this season. The A/Wellington/1/04(H3N2)-like lineage, the following season (2004/2005), had drifted into a more A/California/7/04(H3N2)-like lineage, causing a revision of the vaccine composition from A/Fujian/411/02(H3N2) to A/California/7/04(H3N2) 9. In 2005–2006 the 2004–2005 A/California/7/04(H3N2)-like lineages continued to circulate together with the slightly different A/Wisconsin/67/05(H3N2)-like viruses (Figure 2). As a result the H3N2 vaccine component for the northern hemisphere 2006–2007 was changed to A/Wisconsin/67/05(H3N2) 9. The A/Fujian/411/02(H3N2), A/Wellington/1/04(H3N2) and A/California/7/04(H3N2)-like viruses all share the same type of NS segments and there are few variations between the Wellington, California and Wisconsin-like strains, especially in the internal genes (Figure 3 and 4). However, the internal genes of the A/Wisconsin/67/05(H3N2)-like viruses, especially the polymerase acidic (PA), nucleoprotein (NP) and M are more closely related to the A/Fujian/411/02(H3N2)-like viruses from 2002–2003 than the A/California/7/04(H3N2) from the previous season (Figure 3 and 4).

Figure 4

Evolutionary relationships of circulating H3N2 and H1N1 influenza A viruses sampled in Denmark from 1999 to 2006

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.

H1N1 viruses

H1N1 viruses dominated the 2000–2001 season in Denmark (Figure 1). Thirteen isolates from this season were available for sequencing, and all were of the H1N1 subtype. These sequences represented two different co-circulating lineages (Figure 4). Lineage I is A/Bayern/7/95(H1N1)-like and lineage II include the H1N1 strains of today and the A/New Caledonia/20/99(H1N1) vaccine reference strain (Figure 4). The phylogenetic trees of NA and the internal genes showed the same topology (Figure 3 and 4). The lineage II strains are characterised by a deletion K130 in HA (K134 in H3 numbering) (Table 1). H1N1 virus was again isolated in 2004–2005 and showed a homogeneous distribution in the lineage II for all genes (Figure 3 and 4). Higher nucleotide variation was observed among the 2005–2006 H1N1 sequences in both HA and NA genes (Figure 4). This could indicate that the H1N1 viruses are in progression, away from the A/New Caledonia/20/99(H1N1)-like viruses.

Table 1

Amino acid changes in H3N2, H1N1 and H1N2 viruses between seasons *

Amino acid

1999–00

2001–02

2002–03

2003–04

2004–05

2005–06

Amno acid

1999–00

2001–02

2002–03

2003–04

2004–05

2005–06

H1N2

Amino acid

H3 no.

2000–01

2004–05

2005–06

H1N2

Amino acid

N2 no.

2000–01

2004–05

2005–06

S(A)

A(S)

L(R)

15 ^Pa

V/I

I(L)

I(T)

M/I

F(L)

L(F)

V(I)

45 ^Pb

H/Y

45 ^c

I(S)

69 ^Cb

L(S)

I(V)

50 ^c

G(R)

G(E)

I(V)

V(I)

I(F)

R/K

H(Y)

V(A)

S/R

75 ^E

Q(H)

F(C)

C(F)

E(K)

H/Q

83 ^E

K(E)

S(P)

A(T)

V(I)

92 ^E

I(T)

100

81 ^Pc

T/P

106

A(V)

N(D)

S(N/D)

120

124

D(E/G)

V/M

112

I(V)

V(I)

143

V(G)

G(V)

130

134

-(K)

S/P

126 ^a

D(N)

150

H(G)

133

136

S(T)

I/V

128 ^b

A(T)

172

K(R)

R(K)

146

149

R(K)

S/R

131 ^a

T(A)

194

V(I)

153 ^Sb

156

G(E)

149

V(I)

144 ^a

N(D)

199 ^b

163 ^Sa

166

K(M)

155

Y/F

145 ^a

S/N

216

V(G)

G(V)

166 ^Ca1

169

V(A)

173

172

K/R

155 ^b

T(H)

221 ^b

K(N)

K(D/E)

168

171

N(K)

188 ^Pd

187

M/L

156 ^b

H(Q)

258

170 ^Ca1

173

E(G)

220

219

K(E)

159 ^b

Y(F)

265

T(I)

I(T)

175

177

222

221

Q(R)

164

L(Q)

267

179

181

V(I)

249 ^Pf

248

G/R

173 ^D

E(K)

307

I(V)

V(I)

183

186

P(S)

254

253

K/R

186 ^b

310

Y(H)

185

188

I(M)

262

261

K/R

188 ^b

D(Y)

329 ^c

N(T)

N(D)

187

190

D(N)

270

269

N/D

189 ^b

S(N)

332 ^c

S(F)

F(S)

190 ^Sb

193

274

273

F/Y

193 ^b

F(S)

370 ^c

S(L)

191

194

L(I)

332 ^Pi

332

199

S(P)

372

S(L)

194

197

T(K)

344 ^Pj

347

D(N)

202

I(V)

385 ^a

N(K)

K(N)

202

205

V(L)

352 ^Pk

355

K/R

222

R(W)

392 ^a

I(T)

215

218

364

367

S(N)

225

D(G)

N(D)

393 ^a

N(K)

237 ^Ca1

240

G/R

389 ^Pm

397

M/V

226 ^D

399 ^a

D(E)

E(D)

239

242

T(S)

396 ^Pm

399

I/M

227 ^D

S(P)

401 ^a

252

255

418

I/M

271

431

253

256

432 ^Pn

432

R(K)

304 ^c

A/P

A(P)

432

Q(E)

267

269

T(I)