Origin and Characteristics of Internal Genes Affect Infectivity of the Novel Avian-Origin Influenza A (H7N9) Virus

<div><p>Background</p><p>Human infection with a novel avian-origin influenza A (H7N9) virus occurred continuously in China during the first half of 2013, with high infectivity and pathogenicity to humans. In this study, we investigated the origin of internal genes of the novel H7N9 virus and analyzed the relationship between internal genes and infectivity of the virus.</p> <p>Methodology and Principal findings</p><p>We tested the environmental specimens using real-time RT-PCR assays and isolated five H9N2 viruses from specimens that were positive for both H7 and H9. Results of recombination and phylogeny analysis, performed based on the entire sequences of 221 influenza viruses, showed that one of the Zhejiang avian H9N2 isolates, A/environment/Zhejiang/16/2013, shared the highest identities on the internal genes with the novel H7N9 virus A/Anhui/1/2013, ranging from 98.98% to 100%. Zhejiang avian H9N2 isolates were all reassortant viruses, by acquiring NS gene from A/chicken/Dawang/1/2011-like viruses and other five internal genes from A/brambling/Beijing/16/2012-like viruses. Compared to A/Anhui/1/2013 (H7N9), the homology on the NS gene was 99.16% with A/chicken/Dawang/1/2011, whereas only 94.27-97.61% with A/bramnling/Beijing/16/2012-like viruses. Analysis on the relationship between internal genes and the infectivity of novel H7N9 viruses were performed by comparing amino acid sequences with the HPAI H5N1 viruses, the H9N2 and the earlier H7N9 avian influenza viruses. There were nine amino acids on the internal genes found to be possibly associated with the infectivity of the novel H7N9 viruses. </p> <p>Conclusions</p><p>These findings indicate that the internal genes, sharing the highest similarities with A/environment/Zhejiang/16/2013-like (H9N2) viruses, may affect the infectivity of the novel H7N9 viruses.</p> </div

Comparative process on the amino acids associated with high infectivity of the novel H7N9 virus.

<p>The green and blue circles represent the novel H7N9 viruses and the earlier H7N9 AIVs respectively. The black circle contains the HPAI H5N1 viruses and the H9N2 AIVs. The infectivity of each group is marked behind the viruses. The yellow regions represent the amino acids associated with high infectivity of the novel H7N9 virus. </p

Evolutionary process of the novel H7N9 virus isolated in China in 2013.

<div><p>Reassortment events on the six internal genes of the novel H7N9 viruses were analyzed based on the entire sequences of 221 influenza viruses using the RDP, GENECONV and MaxChi suites within the RDP3 software. In order to enhance the credibility of the results, the highest P value was set as 0.01. </p> <p>Note: A/brambling/Beijing/16/2012 (H9N2) -like strains including A/chicken/Shangdong/01/2009, A/chicken/Jiangsu/Q3/2010, A/chicken/Zhejiang/611/2011, A/chicken/Shanghai/C1/2012, A/chicken/Zhejiang/329/2011 and A/chicken/China/AH-10-01/2010. A/environment/Zhejiang/16/2013 (H9N2) -like strains including A/environment/Zhejiang/9/2013, A/environment/Zhejiang/13/2013, A/environment/Zhejiang/14/2013, A/environment/Zhejiang/16/2013. A/chicken/Dawang/1/2011 (H9N2)-like strains including A/chicken/Shuanggou/1/2011 (H9N2) and A/pigeon/Xuzhou/1/2011. The eight genes of different strains were shown in different color.</p></div

Phylogenetic trees for PB2 and NS genes of the novel H7N9 viruses.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081136#pone-0081136-g002" target="_blank">Figure 2A and 2B</a> was constructed based on the amino acid sequences of PB2 and NS genes respectively. These trees were all estimated using the Neighbor-joining (NJ) method of MEGA software (Version 5.0) with the bootstrap analysis of 1000 replications. The novel H7N9 isolates are marked with a triangle and the H9N2 virus strains isolated from environment specimens in Zhejiang province in 2013 are marked with a circle.</p

Time-scaled phylogeographic history of epidemic Japanese encephalitis virus (JEV) genotype I (GI).

Boxes with gradient shading indicate clades GIa and GIb; posterior probabilities for their ancestral nodes are shown. Tip colors represent different sampling locations, according to the map at upper left. The base layer of the modified maps are sourced from Natural Earth, and download in GeoJSON format from website (https://geojson-maps.ash.ms/).</p

Root-to-tip regression analyses of phylogenetic temporal signals.

Root-to-tip regression analyses of phylogenetic temporal signals.</p

Amino acid markers and corresponding nucleotide changes identified for different clades.of JEV GI.

Amino acid markers and corresponding nucleotide changes identified for different clades.of JEV GI.</p

Sample information for Japanese encephalitis virus complete genome sequences identified in the study.

Sample information for Japanese encephalitis virus complete genome sequences identified in the study.</p

Evidence for adaptive selection on the full open reading frame (ORF) of the JEV genome according to different assessment models.

Evidence for adaptive selection on the full open reading frame (ORF) of the JEV genome according to different assessment models.</p

Fig 2 -

Bayesian skyline plots showing the demographic history of JEV GI including (A) GI, (B) GIa, (C) GIb, (D) GIb clade 1, (E) GIb clade 2, and (F) sub-clades NS2a-151V and NS4-169K. Each y-axis represents virus effective population size (Ne), which is a measure of genetic diversity based on the number of genomes effectively contributing to new infections. Each x-axis represents time in calendar years. Blue lines are median estimates; purple lines are 95% highest posterior density (HPD) intervals.</p