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

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

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

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

Phylogenetic analyses of SFTSV M genomic segments identified in patients and ticks.

<p>Phylogenetic analyses of SFTSV M genomic segments identified in patients and ticks.</p

Timelines of key events for three SFTSV infections in southeastern China.

<p>Timelines of key events for three SFTSV infections in southeastern China.</p

The Clinical features of patients involved in a family cluster occurred in 2014, Southeastern China.

<p>*Data are peak or nadir measurement during hospitalization</p><p>The Clinical features of patients involved in a family cluster occurred in 2014, Southeastern China.</p

Laboratory findings of 26 H5N1 cases^* on initial testing, at hospital admission, and during hospitalization, China.

*<p>Indicates denominators for testing of fewer cases than full group.</p>†<p>Abbreviations and normal range: WBC, white blood cell, 4.0–10.0×10⁹ cells per L, leukopenia (abnormal) was defined as leukocyte count less than 4×10⁹ per L; LYM, lymphocyte count, 0.8–4.0×10⁹ cells per L, lymphopenia (abnormal) was defined as lymphocyte count less than 0.8×10⁹ per L; PLT, platelet count, 100–300×10⁹ platelets per L, thrombocytopenia (abnormal) was defined as platelet count less than 100×10⁹ per L.</p>‡<p>Abbreviations and normal range: ALT, alanine aminotrasferase, 0.0–45.0 U/L; AST, aspartate aminotransferase, 0–45 U/L; Albumin, 35.0–55.0g/L; Creatinine, 36.0–144.0 µmol/L; CK, creatine kinase, 25–190 U/L, abnormal was defined as >130 IU/L for males and >110 IU/L for females; CK-MB, creatine phosphokinase isoenzymes, 0–25 U/L; LDH, Lactic dehydrogenase, 110–250 U/L; Plasma glucose concentration, 3.33–5.55 mmol/L for <15 years, 3.89–5.83 mmol/L for adults (16–59 years), 4.44–6.38 mmol/L for age >60 years, hyperglycemia (abnormal) was defined as plasma glucose concentration above the upper limit.</p>#<p>Abbreviations and normal range: PT, prothrombin time, 11–13 second, abnormal was defined as 3 seconds longer than the upper range of normal; APTT, activated partial thromboplastin time, 26–36 second, abnormal was defined as 3 seconds longer than the upper range of normal; FIB, fibrinogen, 2.0–4.0 g/L, abnormal was defined as <2.0 g/L.</p>§<p>Normal ranges: total protein (below 120 mg/L); red blood cells (0 to 1 per average high powered field [HPF×400)); white blood cells (1–4 per HPF×400)</p>¶¶<p>NA demotes not applicable.</p

Comparison of demographic and clinical features of 17 fatal and 9 nonfatal H5N1 cases, China.

*<p>Medians were compared between fatal and survival cases with the Wilcoxon rank sum test. For categorical variables, percentages of cases in each category were compared with Fisher's exact test.</p>†<p>NA demotes not applicable.</p>‡<p>Two fatal H5N1 cases had underlying medical conditions, including a 24-year-old pregnant woman <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002985#pone.0002985-Shu1" target="_blank">[28]</a> and a 16-year-old male with a 10-year history of minimal change glomerulopathy. Two surviving H5N1 cases had underlying medical conditions, including a 26-year-old pregnant woman and a 44-year-old female with a ten-year history of chronic bronchitis [unpublished data, China CDC].</p>#<p>A higher proportion of cases survived that received any antiviral treatment compared to those that did not receive antivirals (67% [8/12 patients] vs 7% [1/14 patients], p = 0.003), and with a positive linear association: the Gamma coefficient equals 0.664 (p = 0.005) which indicate a positive correlation between antiviral therapy and disease outcome.</p>$<p>High-dose corticosteroid use was defined as ≥250 mg hydrocortisone or equivalent intravenous (IV) administration daily. For children <13 years old, high-dose corticosteroid use was defined as ≥5 mg hydrocortisone or equivalent IV/kg/day.</p>¶<p>[]: Indicates denominators for testing of fewer cases than full group.</p

Initial chest radiographic findings and progression during hospitalization of 26 H5N1 cases, China.

*<p>NA demotes not applicable</p