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

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.

Scientific RepoRts | 6:25845 | DOI: 10.1038/srep25845 two more tigers died in the zoo; one 8monthold male tiger died from high fever two days following vomiting and severe respiratory distress, and the other 24monthold female tiger was a sudden death. After the first tiger died, the other tigers were fed daily just with pork and beef, no chicken. But five peacocks died with H5N1 virus infection in the zoo, on June.
All tigers that died had nasal discharge and neurologic signs of infection. Necropsy was performed at once after their death. The lungs and livers were severely congested with hemorrhaging ( Fig. 1A-C, tig1508), the brains had severe congestion; pleural effusion was observed, and serosanguinous exudate was also seen throughout the tracheal and bronchiolar lumen in all deceased tigers. RNA from the lung, liver, pleural effusion, throat and tracheal swab specimens tested positive for the hemagglutinin gene and neuraminidase gene of the H5N1 virus. The homogenates of the RT-PCR positive samples for tigers were centrifuged at lowspeed and either undiluted or 10-fold serially diluted supernatants, and then inoculated into 10-day-old SPF embryonated chicken eggs. The viral titers in the tigers were calculated using the Reed and Muench method and were highest in the lungs (Fig. 1D,E).

Virus isolation and genetic identity analysis.
To analyze the correlation of H5N1 virus infection in tigers and peacocks in the zoo, two peacock isolates (A/ peacock/Yunnan /1 /2015(H5N1) and A/peacock / Yunnan /3 /2015(H5N1)) and three tiger originated virus isolates (tig1404, tig1412 and tig1508 isolated from tigers died on April 8 th , December 15 th , 2014 and August 12 th (the 8monthold male tiger), 2015, respectively.) were chosen for full genome sequencing. Sequence analyses revealed that the three viruses belonged to different clades. Phylogenetic analysis indicated that HA, NA and six internal genes of A/tiger /Yunnan /tig1404 /2014(H5N1) shared more 99% homology (Table 1)

Molecular features associated with AIV virulence, transmissibility, and antiviral resistance.
We analyzed the molecular features of tiger originated viruses associated with H5N1 virus virulence, transmissibility, and antiviral resistance. The three isolates displayed some molecular markers associated with increased virulence and transmission in mammals according to the H5N1 Genetic Changes Inventory (http://www.cdc. gov/ flu/ pdf/ avianflu/ h5n1-inventory.pdf). The isolated hemagglutinin cleavage sites possess a multibasic amino acid cleavage site ( Table 2). Although the three isolates possessed a conserved amino acid motif Q-R-G (Gln Ser Gly equivalent to clades 2.3.4 and 2.3.2) at residues 222-224 (H3 numbering 226-228) of the hemagglutinin protein indicating no substantial changes in avian-like receptor binding preferences 8,9 , the HA of the viruses isolate from the tigers harbored some mutations (such as Asp94Asn in tig1404 and tig1508, Ser133Ala in tig1404,

Virus
Gene Virus with the highest percentage of nucleotide identity tig1412 and tig1508, Thr156Ala in tig1412, Thr188Ile in tig1412 and tig1508, and Lys189Arg in tig1412), which several studies have suggested increased the binding of the H5N1 virus to the sialic acid (SA) α -2, 6-Gal (α 2-6) receptor [10][11][12] . The neuraminidase stalk deletion (49-68 deletion) was detected in the three isolates, which could play a role in enhancing H5N1 virulence in mice 13 . The mutations/substitutions involved in drug resistance and transmission to mammals, were observed in the NA (His254Tyr ) of the three isolates and PB2 (Asp701Asn ) proteins of tig1508 isolate (Table 2) 14,15 . The three isolates possessed Asn30Asp and Thr215Ala mutation in M1 protein, which are associated with increased virulence in mice 16 . The Ser31Asn mutation was exhibited in the M2 protein of the tig1508 virus, which confers resistance to the antiviral drug adamantane and rimantadine 17,18 . The mutations of NS1 protein plays an important role in enhancing the virulence of H5N1 viruses in mice, such as 80-84 deletion, Leu98Phe, Ile101Met and PDZ ligand motif ESEV in 222-225 sites 19,20 , were observed in the three isolates from the tigers and Asp87Glu in the tig1404 and tg1412 isoslates. The N-Glycosylation sites were predicted to examine the sequence context of Asn-Xaa-Ser/Thr sequins by the NetNglyc server 1.0. In addition to seven possible N-glycosylation sites observed for other serotype H5N1 in hemagglutinin proteins 9 , the predicted results of the N-Glycosylation sites indicated that the HA segments of each isolate harbored new N-glycosylation sites: Asn84 and Asn154 in the tig1404 isolate, Asn273 in the tig1412 isolate, and Asn154 in the tig1508 isolate, respectively.
In summary, all of mutations/substitutions of the gene segments of the tiger originated viruses could contribute to the enhancement of virulence or the increase of the H5N1 virus binding to the α 2-6 receptor.

Pathogenicity of the tigers originated H5N1 viruses in mice.
To further characterize the virulence of these novel tigers-originated H5N1 viruses in mammals, we infected mice with these viruses and observed for 10 days for morbidity. The signs of illness, anorexia and dyspnea were observed in the mice inoculated with 10 1 -10 6 ELD 50 and the mice inoculated with 10 6.0 EID 50 began to die at 2 days post infection (dpi); by 8 dpi, all mice in the experimental groups had died (Fig. 4A-C). Among the mice infected with tig1404, tig1412 and tig1508, viruses were positive with RT-PCR and were successfully re-isolated from lung, liver, pleural effusion, throat and tracheal swab, Kidney and Spleen tissues. Viral titers in eggs were calculated and shown in the Fig. 4 (Fig. 4D-F). Virus replication was not detected in any of the direct contact mice in the tig1404 and tig1412 groups. But in the Tig1508 group, virus replication was detected in one direct contact naïve mouse. These results indicated that these

Discussion
In this study, we provide the evidence of fatal H5N1 AIV infection in zoo-housed Tigers in Yunnan Province. In comparisons of previously published nucleotide sequences with those of other avian influenza viruses from public databases, the isolated three tiger-originated-viruses (specially the tig1404 and tig1508 isolate) had a high level of homology with the recently identified HPAI H5N1 viruses, which circulates mainly in chickens and other avians in Yunnan province at the same time 7  Our results suggested that the reassortant different H5N1 virus subclades can successfully cross species barriers from avian to mammal and infect north-east China tigers which might be with the contribution that mutations/substitutions of the gene segments in the tiger originated viruses could enhance virulence or increase the H5N1 virus binding to the α 2-6 receptor.
Evolutionary analysis showed that A/tiger /Yunnan /tig1508 /2015(H5N1) including A/peacock/ Yunnan /1 /2015(H5N1) virus and A/peacock/ Yunnan /3 /2015(H5N1) virus which circulates in peacocks and other poultry is a novel reassortant virus originating from H5N1 and H9N2 subtypes influenza A virus. The HI assay demonstrated antiserum from the RE-6 vaccine strain did not inhibit hemagglutination of clade 2.3.2.1c such as tig1508 and peacock isolates, and this change must be considered when evaluating and selecting prepandemic candidate vaccine viruses for the region.

Materials and Methods
Tissue samples of all deceased tigers, including throat and tracheal swab, lung, liver, spleen, kidney, cardiac with aquae pericardii, and cerebrospinal fluid, were collected to determine the cause of death. Testing for detection of influenza A virus was performed by a reverse transcription PCR method after RNA extraction using a viral RNA kit (Invitrogen, USA) 24 . RNA from the lung, liver, cardiac with aquae pericardii, throat and tracheal swab specimens tested positive for the hemagglutinin gene and neuraminidase gene of the H5N1 virus.
Virus isolation and gene sequencing. To isolate and characterize the H5N1 viruses, isolates from H5N1 virus RNA positive dead tiger's lung samples and peacocks's cloacal swab specimens were injected into 10-day-old specific pathogen free embryonated chicken eggs in a Biosafety Level-3 laboratory. Two peacock isolates (A/ peacock/Yunnan /1 /2015(H5N1) and A/peacock /Yunnan /3 /2015(H5N1)) and three tiger originated virus isolates (tig1404, tig1412 and tig1508 isolated from tigers died on April 8 th , December 15 th , 2014 and August 12 th , 2015, respectively.) were chosen for full genome sequencing. The specific RT-PCRs were performed as described previously 25 . The homogenates of the RT-PCR positive samples for tigers, including the lung, liver, cardiac with aquae pericardii, throat and tracheal swab specimens, were centrifuged at lowspeed (6,000 × g) for 10 min at 4 °C, treated with 100,000 U/ml penicillin and 100 μ g/ml streptomycin, and either undiluted or 10-fold serially diluted supernatants, and then inoculated into 10-day-old SPF embryonated chicken eggs. Viral titers were then calculated using the Reed and Muench method.
Genetic and phylogenetic analysis. The nucleotide sequences were analyzed using DNAman (version 6.0) and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi ). Phylogenetic analyses were performed using the maximum

Pathogenicity of the tigers originated H5N1 viruses in mice.
To further characterize the virulence of these novel H5N1 viruses in mice, groups of five mice under light CO 2 anesthesia were inoculated intranasally with 10 1 −10 6 50% egg lethal dose (ELD 50 ) of tested virus in a volume of 50 μ l. Meanwhile, a group of five mice were inoculated with an equal volume of PBS as negative control. All the mice were monitored for mortality daily for 10 days. For virus infectivity and replication testing, groups of three mice were lightly anesthetized with CO 2 and inoculated intranasally with 10 6 ELD 50 of tested virus in a volume of 50 μ l, every three inoculated mice were euthanized at 2-5 days post-inoculation, and their organs were collected for virus re-isolation and RT-PCR test. To investigate the virus transmission ability, after 24 hours post-inoculation, three naïve mice were placed in direct contact with the inoculated mice. Virus re-isolation was performed in 10-day-old SPF embryonated chicken eggs.

The hemagglutination inhibition (HI) test.
To test the antigenic relationship of three tiger viruses, we examined serologic cross-reactivity between the tiger originated isolates and the diagnostic antigen of the widely used inactivated reassortant vaccine Re-5 and vaccine Re-6 in Yunnan Province (Table 3). A hemagglutination-inhibition test was conducted to test for Re-5 and Re-6 antiserum against tiger's viruses 27 . Forty serum specimens were collected from healthy chickens were vaccinated twice with RE-5 vaccine strain (collection date = 2012). Statistical analysis. The statistical significance of differences between groups was determined using the Student's t-test (Graphpad prism 5). A P value < 0.05 was considered statistically significant. Ethics Statement. The animal experiment was conducted in accordance with the Regulations for the Table 3. Results of HI assays using Re-5 antiserum and Re-6 antiserum for avian influenza A(H5N1) isolated from tigers in Yunnan China, 2014-2015*. * Re-5 (n = 38) and Re-6 (n = 40) antiserum were generated by vaccinating specific-pathogen free chickens with the commercial Re-5 and Re-6 vaccine (Harbin Weike biologic Technology Development Company, Harbin, China). HI titers against the homologous antigen/ virus are shown in boldface. HI, hemagglutination inhibition; NA, not applicable. The titre differences were statistically significant by One Way ANOVA (P < 0.05). † The commercial Re-5 and Re-6 diagnostic antigen (including positive and negative control serum) are from Harbin Weike biologic Technology Development Company, Harbin, China.