Fatal influenza A (H5N1) virus Infection in zoo-housed Tigers in Yunnan Province, China

Abstract

From 2014 to 2015, three cases of highly pathogenic avian influenza infection occurred in zoo-housed north-east China tigers (Panthera tigris ssp.altaica) and four tigers died of respiratory distress in succession in Yunnan Province, China. We isolated and characterized three highly pathogenic avian influenza A(H5N1) viruses from these tigers. Phylogenetic analysis indicated that A/tiger /Yunnan /tig1404 /2014(H5N1) belongs to the provisional subclade 2.3.4.4e which were novel reassortant influenza A (H5N1) viruses with six internal genes from avian influenza A (H5N2) viruses. The HA gene of the isolated A/tiger /Yunnan /tig1412 /2014(H5N1) virus belongs to the subclade 2.3.2.1b. The isolated A/tiger /Yunnan /tig1508/2015 (H5N1) virus was a novel reassortant influenza A (H5N1) virus with three internal genes (PB2, PB1 and M) from H9N2 virus and belongs to the subclade 2.3.2.1c.

Figure 1. Autopsy change and The H5N1 viral titers in different organs of the tigers.

Autopsy change in different organs of tig1508 are shown in (A) (liver), (B) (lung) and (C) (heart); the viral titers in different organs of tigers are shown in (D) (tig1404), (E) (tig1412) and (F) (tig1508).

Figure 2. Phylogenetic tree of influenza A (H5N1) hemagglutinin (HA) gene and neuraminidase(NA) gene sequences from the tiger isolates in Yunnan and related reference viruses retrieved from the GenBank database.

The phylogenetic tree was generated in MEGA version 6 (www.megasoftware. net), using maximum likelihood (ML) analysis with 1,000 bootstrap replicates. The HA tree was rooted to prototype strain A/goose/Guangdong/1/96 [Gs/GD]. Scale bar indicates nucleotide substitutions per site. The viruses reported in this paper were highlighted using black triangle and bold italic. Note*:some 2.3.4.4 sub-clades such as 2.3.4.4e have just been proposed but not yet been confirmed by the WHO/OIE/FAO H5N1 nomenclature working group.

Figure 3. Phylogenetic analyses of six internal genes of the tiger isolates in Yunnan and related reference viruses.

The viruses reported in this paper were highlighted using black using black triangle and bold italic. PB2, basic polymerase 2; PB1, basic polymerase 1; PA, acidic polymerase; NP, nucleoprotein; M, matrix protein and NS, nonstructural protein.

Figure 4. Virulence of tigers-originated- H5N1 viruses in mice.

The survival rate of infected mice are shown in (A) (tig1404), (B) (tig1412) and (C) (tig1508); the tigers-originated- H5N1 viruses were detected in lung, liver, pleural effusion, throat and tracheal swab, Kidney and Spleen of infected mice and the viral titers in each organ of mice after challenge with influenza are shown in (D) (tig1404), (E) (tig1412) and (F) (tig1508).