Molecular evolutionary analysis of the influenza A(H1N1)pdm, May-September, 2009: temporal and spatial spreading profile of the viruses in Japan

Abstract

Background: In March 2009, pandemic influenza A(H1N1) (A(H1N1)pdm) emerged in Mexico and the United States. In Japan, since the first outbreak of A(H1N1)pdm in Osaka and Hyogo Prefectures occurred in the middle of May 2009, the virus had spread over 16 of 47 prefectures as of June 4, 2009.

Methods/principal findings: We analyzed all-segment concatenated genome sequences of 75 isolates of A(H1N1)pdm viruses in Japan, and compared them with 163 full-genome sequences in the world. Two analyzing methods, distance-based and Bayesian coalescent MCMC inferences were adopted to elucidate an evolutionary relationship of the viruses in the world and Japan. Regardless of the method, the viruses in the world were classified into four distinct clusters with a few exceptions. Cluster 1 was originated earlier than cluster 2, while cluster 2 was more widely spread around the world. The other two clusters (clusters 1.2 and 1.3) were suggested to be distinct reassortants with different types of segment assortments. The viruses in Japan seemed to be a multiple origin, which were derived from approximately 28 transported cases. Twelve cases were associated with monophyletic groups consisting of Japanese viruses, which were referred to as micro-clade. While most of the micro-clades belonged to the cluster 2, the clade of the first cases of infection in Japan originated from cluster 1.2. Micro-clades of Osaka/Kobe and the Fukuoka cases, both of which were school-wide outbreaks, were eradicated. Time of most recent common ancestor (tMRCA) for each micro-clade demonstrated that some distinct viruses were transmitted in Japan between late May and early June, 2009, and appeared to spread nation-wide throughout summer.

Conclusions: Our results suggest that many viruses were transmitted from abroad in late May 2009 irrespective of preventive actions against the pandemic influenza, and that the influenza A(H1N1)pdm had become a pandemic stage in June 2009 in Japan.

Figure 1. Geographical distribution of virus isolates of influenza A(H1N1)pdm in Japan.

Figure 2. Bayesian coalescence phylogeny of influenza A(H1N1)pdm full-genome sequences from the world.

The branch length of the phylogeny is in units of time. The scale bar indicating 30 days of the branch length is drawn at the bottom of the tree. The clusters described in the previous report are annotated by brackets on the right of the tree. The sequences from Japan are colored with red. The sequences from Central-East Asia except for Japan, South-East Asia, Europe, New York, and Central-North America except for New York are colored with orange, yellow, purple, blue and green, respectively. Shadows on the terminal lineages show Japanese micro-clades. Date with arrows pointing out a node of the tree shows tMRCA of the monophyletic group. MC represents micro-clade.

Figure 3. Distance-based neighbor joining phylogeny of influenza A(H1N1)pdm in early phase of the pandemic with particular swine influenza strains.

The upper panel shows the phylogeny using NA-MP that was derived from Eurasian avian-like swine H1N1 virus. The lower panel shows the phylogeny using PB2-PB1-PA-HA-NP-NS that was derived from the triple reassortant swine H1N2/3. Bold name shows Narita/1 which was the first confirmed case in Japan. Both trees were re-rooted with swine influenza strains. The scale bar in the units of the number of base substitutions per site is drawn at the bottom of each tree.

Figure 4. Schematic representation of the estimated profile of A(H1N1)pdm epidemic in Japan.

The vertical and the horizontal axes show geographic localities and the times of viruses collection, respectively. The geographic localities are aligned in order of their latitude and the longitude as north-east to south-west. Seventy-five isolates of A(H1N1)pdm viruses from Japan are plotted by small symbols. The most recent common ancestor of twelve micro-clades inferred from the analysis are plotted by large symbol. Shape of the symbols indicate the cluster of each virus belonged; circle shows cluster 1, lozenge shows cluster 2, six-pointed star shows cluster 1.2 and five-pointed star shows cluster 1.3. The number below the large symbol shows tMRCA date. The number in the large symbol shows the micro-clade. The member of each micro-clade and their MRCA are linked with curves using different colors. The viruses which cannot classify any micro-clades are represented by gray symbols. A virus with a single asterisk is Narita/1 that was the first confirmed case in Japan. A virus with a double asterisk is A/Japan/PR1070 that had no epidemiological information.