Characterization of Influenza A (H7N9) Viruses Isolated from Human Cases Imported into Taiwan

<div><p>A novel avian influenza A (H7N9) virus causes severe human infections and was first identified in March 2013 in China. The H7N9 virus has exhibited two epidemiological peaks of infection, occurring in week 15 of 2013 and week 5 of 2014. Taiwan, which is geographically adjacent to China, faces a large risk of being affected by this virus. Through extensive surveillance, launched in April 2013, four laboratory-confirmed H7N9 cases imported from China have been identified in Taiwan. The H7N9 virus isolated from imported case 1 in May 2013 (during the first wave) was found to be closest genetically to a virus from wild birds and differed from the prototype virus, A/Anhui/1/2013, in the MP gene. The other three imported cases were detected in December 2013 and April 2014 (during the second wave). The viruses isolated from cases 2 and 4 were similar in the compositions of their 6 internal genes and distinct from A/Anhui/1/2013 in the PB2 and MP genes, whereas the virus isolated from case 3 exhibited a novel reassortment that has not been identified previously and was different from A/Anhui/1/2013 in the PB2, PA and MP genes. The four imported H7N9 viruses share similar antigenicity with A/Anhui/1/2013, and their HA and NA genes grouped together in their respective phylogenies. In contrast with the HA and NA genes, which exhibited a smaller degree of diversity, the internal genes were heterogeneous and provided potential distinctions between transmission sources in terms of both geography and hosts. It is important to strengthen surveillance of influenza and to share viral genetic data in real-time for reducing the threat of rapid and continuing evolution of H7N9 viruses.</p></div

Genotyping of the four imported H7N9 viruses of Taiwan.

<p>Genotyping of the four imported H7N9 viruses of Taiwan.</p

Phylogenetic relationships for (A) PB2, (B) PB1, (C) PA, (D) HA, (E) NP, (F) NA, (G) MP and (H) NS of the four imported H7N9 viruses identified in this study.

<p>The phylogenetic trees were constructed using the Maximum likelihood method with 1000 bootstrap replications. Branch values of more than 70 are indicated. Genome sequences of H7N9 viruses isolated from humans, poultry and the environment during the two epidemic waves in China were obtained from the NCBI and GISAID databases and were used as references. The classification of specific evolutionary clades, including the clades 1, 2 and 3, is indicated. The four imported viruses of Taiwan, which were isolated during the first or second waves of the H7N9 epidemic, were indicated by purple and blue colors, respectively. Viruses indicated by the green colors were the H9N2 lineages with genetic constellation related to the H7N9 viruses. Viruses indicated by the red colors were H7N9 strains isolated from Hong Kong and Guangdong in China during the second wave of the H7N9 epidemic.</p

Geographical locations in mainland China of the four laboratory-confirmed cases of influenza A (H7N9) before arrival in Taiwan.

<p>The locations of the cases are represented by dots, with a different color for each case. The location of Taihu Lake is marked with a symbol, as indicated.</p

Information about the four confirmed H7N9 cases.

<p>The time points (dates) of previous chicken exposure, arrival in Taiwan, illness onset hospitalization and discharge for each case are indicated by symbols, as shown in the key. Provinces and cities where each imported case carried out daily activities before arrival in Taiwan are indicated in gray shadow boxes.</p

Phylogenetic and Evolutionary History of Influenza B Viruses, which Caused a Large Epidemic in 2011–2012, Taiwan

<div><p>The annual recurrence of the influenza epidemic is considered to be primarily associated with immune escape due to changes to the virus. In 2011–2012, the influenza B epidemic in Taiwan was unusually large, and influenza B was predominant for a long time. To investigate the genetic dynamics of influenza B viruses during the 2011–2012 epidemic, we analyzed the sequences of 4,386 influenza B viruses collected in Taiwan from 2004 to 2012. The data provided detailed insight into the flux patterns of multiple genotypes. We found that a re-emergent TW08-I virus, which was the major genotype and had co-circulated with the two others, TW08-II and TW08-III, from 2007 to 2009 in Taiwan, successively overtook TW08-II in March and then underwent a lineage switch in July 2011. This lineage switch was followed by the large epidemic in Taiwan. The whole-genome compositions and phylogenetic relationships of the representative viruses of various genotypes were compared to determine the viral evolutionary histories. We demonstrated that the large influenza B epidemic of 2011–2012 was caused by Yamagata lineage TW08-I viruses that were derived from TW04-II viruses in 2004–2005 through genetic drifts without detectable reassortments. The TW08-I viruses isolated in both 2011–2012 and 2007–2009 were antigenically similar, indicating that an influenza B virus have persisted for 5 years in antigenic stasis before causing a large epidemic. The results suggest that in addition to the emergence of new variants with mutations or reassortments, other factors, including the interference of multi-types or lineages of influenza viruses and the accumulation of susceptible hosts, can also affect the scale and time of an influenza B epidemic.</p> </div

Phylogentic analysis of HA genes and distribution of various genotypes.

<p>(A) Phylogenetic topology of the HA genes from multiple genotypes of influenza B viruses from 2007 to 2012 in Taiwan. The phylogenetic trees were constructed using the neighbor-joining method with 1000 bootstrap replications. Branch values of more than 70 are indicated. Based on the phylogenetic analyses and the amino acid signatures of HA genes, the circulating Yam88 viruses were classified into three genotypes, TW08-I, TW08-II, and TW08-III, which are represented by the viruses B/Taiwan/3798/2007, B/Taiwan/30/2008 and B/Taiwan/2563/2008, respectively. The major amino acid signatures of each genotype are as the following: 63K, 123A and 266M for TW08-I; 165I, 180Y, 244D and 266M for TW08-II; and 244S and 266M for TW08-III. (B) The monthly distribution of various genotypes. The monthly numbers of each genotype are shown as a line chart. The inserts indicate the ratios of the three genotypes in 2008, 2010 and 2011.</p

Data from the laboratory-based surveillance network in Taiwan from 2004 to 2012.

<p>(A) The monthly distributions of the influenza isolates, including A(H1N1), A(H3N2), A(H1N1)pdm09 and influenza B viruses, are shown as bars. Positive rates of confirmed cases are also shown in the line chart. (B) The dynamic changes in influenza B activity. The monthly positive rates of influenza B represent the percentage of confirmed influenza B cases among all reported and tested cases in a specific month. The predominance of influenza B was calculated by dividing the number of all influenza viruses by the number of confirmed influenza B viruses. (C) The lineage distribution of influenza B viruses. The Victoria (Vic87) and Yamagata (Yam88) lineages isolated from 2004 to 2012 were classified based on their HA genes. The numbers of influenza B viruses analyzed each month are shown as bars, and the monthly ratio of each lineage is shown as a line chart.</p

Antigenic analysis of the Yamagata lineage influenza B viruses by hemagglutination inhibition assay.

*<p>Passage history of virus was indicated by the number; e.g. viruses of second generation propagated in MDCK cells from clinical specimens were indicated as C2.</p

Diagram representing the evolutionary histories of Yamagata-lineage influenza B viruses in Taiwan.

<p>The genome compositions of various genotypes are based on the phylogenetic topographies in Fig. 3, and the genotype definitions and representative viruses are described in the legends of Fig. 2 and 3. The gene segments of the viruses are presented in the order of PB1, PB2, PA, HA, NP, NA, M, and NS (from top to bottom). The circulation time period of each genotype is also illustrated. Blue arrows represent possible evolutionary pathways of genotype development. Green arrows represent reassortment between BE98 and existing genotypes. For example, the TW04-II was generated through reassortment between CA00 virus, providing PB2, PA, NP and NS segments and BE98 virus, providing PB1, HA, NA and MP segments. The red arrow indicates the acquisition of segments from YA98 through reassortment. CA00: B/Canada/16188/2000-like viruses; BE98: B/Beijing/76/98-like viruses; YA98: B/Yamanashi/166/98-like viruses.</p