Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger.

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MICINF4206_proof ■ 24 October 2014 ■ 1/8

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Microbes and Infection xx (2014) 1e8 www.elsevier.com/locate/micinf

Original article

Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger Q4

Shang He a,b,c, Jianzhong Shi d, Xian Qi e, Guoqing Huang a, Hualan Chen d, Chengping Lu a,b,c,* a

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China b Key Lab of Animal Bacteriology, Ministry of Agriculture, Nanjing 210095, China c OIE Reference Laboratory for Swine Streptococcosis, Nanjing 210095, China d Division of Animal Influenza, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150009, China e Institute of the Prevention of Acute Disease, Jiangsu Province Center for Disease Control and Prevention, Nanjing 210009, China Received 31 May 2014; accepted 14 October 2014

Abstract In early 2013, a Bengal tiger (Panthera tigris) in a zoo died of respiratory distress. All specimens from the tiger were positive for HPAI H5N1, which were detected by real-time PCR, including nose swab, throat swab, tracheal swab, heart, liver, spleen, lung, kidney, aquae pericardii and cerebrospinal fluid. One stain of virus, A/Tiger/JS/1/2013, was isolated from the lung sample. Pathogenicity experiments showed that the isolate was able to replicate and cause death in mice. Phylogenetic analysis indicated that HA and NA of A/Tiger/JS/1/2013 clustered with A/ duck/Vietnam/OIE-2202/2012 (H5N1), which belongs to clade 2.3.2.1. Interestingly, the gene segment PB2 shared 98% homology with A/wild duck/Korea/CSM-28/20/2010 (H4N6), which suggested that A/Tiger/JS/1/2013 is a novel reassortant H5N1 subtype virus. Immunohistochemical analysis also confirmed that the tiger was infected by this new reassortant HPAI H5N1 virus. Overall, our results showed that this Bengal tiger was infected by a novel reassortant H5N1, suggesting that the H5N1 virus can successfully cross species barriers from avian to mammal through reassortment. © 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

Keywords: Novel reassortant avian influenza A virus; H5N1; Tiger

1. Introduction The highly pathogenic avian influenza (HPAI) A H5N1 virus was first detected in diseased geese in 1996 [1]. As a consequence of rapid evolution of the virus by genetic drift and genetic shift, a diversity of H5N1 genotypes have been isolated and multiple subtypes have been established. The HPAI A H5N1 virus is highly contagious among birds, and can be deadly, especially in domestic poultry. Recently, HPAI viruses have crossed the species barrier and caused human infections. More than 600 human HPAI A H5N1 cases have

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* Corresponding author. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China. Tel./fax: þ86 2584396517. E-mail address: [email protected] (C. Lu).

been reported to the World Health Organization from 15 countries in Asia, Africa, the Pacific, Europe, and the Near East since November 2003. Approximately 60% of the cases resulted in death (Cumulative number of confirmed human cases for avian influenza A (H5N1) reported to WHO, 2003e2014, http://www.who.int/influenza/human_animal_ interface/H5N1_cumulative_table_archives/en/). HPAI A H5N1 viruses have also been detected in animals other than birds, which cause symptomatic illness, severe disease, and death in most infected animals. The following animals have been reported to be infected: pigs in China, Indonesia, and Vietnam; domestic cats in Germany, Thailand, Iraq, and Australia [2e4]; dogs in Thailand [5]; a wild stone marten in Germany; and tigers and leopards in zoos in Thailand [6,7]]. There is no evidence that the virus has acquired the ability to

http://dx.doi.org/10.1016/j.micinf.2014.10.004 1286-4579/© 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: He S, et al., Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.10.004

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S. He et al. / Microbes and Infection xx (2014) 1e8

be consistently transmitted among mammals. Furthermore, the majority of human infections with HPAI A H5N1 viruses have been associated with direct or indirect contact with infected live poultry or poultry carcasses. In this study, we isolated an influenza virus A from a dead tiger that suffered from respiratory disease in a zoo in Jiangsu province, China in 2013. We studied its genome and pathogenicity in mice, and also detected H5N1 antigens on bronchial epithelial cells of the lungs by immunohistochemistry. Our studies revealed that the virus isolate from the tiger encoded eight gene segments of avian influenza virus origin, were highly pathogenic to mice, and were genetically related to the H5N1 subtype viruses. By contrast, the PB2 segment of the isolate shared high similarity with other influenza viruses, except for the H5N1 subtypes. The discovery of a H5N1 virus from a dead tiger underlines the importance of enforcing H5N1 virus surveillance in mammals, especially carnivorous animals. 2. Materials and methods 2.1. Case reporting In early 2013, a 4-month-old Bengal tiger (Panthera tigris) died unexpectedly in a zoo. The zookeepers reported that there were a total of five Bengal tigers in the zoo, and all of them were reared in one cage. The tigers were fed daily with chicken from the market. The dead tiger had been fed chicken the day before its death, and was found with severe respiratory distress the following morningdthe tiger vomited copious amounts of green-yellow liquid. Its heart stopped beating and respiratory arrest occurred after a few minutes of the emergency aid. The zoo requested that we dissect the body of this tiger to determine the cause of death. Tissue samples, including nose swab, throat swab, tracheal swab, cardiac, liver, spleen, lung, kidney, aquae pericardii, and cerebrospinal fluid, were collected. All samples tested positive for HPAI A H5N1 by real-time fluorescence PCR, while no pathogenic bacteria were detected. Nose swab samples from other four tigers, which were co-housed with the dead tiger, showed no evidence of infection with H5N1 influenza virus. 2.2. Virus isolation The virus was isolated and purified from lung specimens harvested from the dead Bengal tiger by inoculating 10-dayold specific-pathogen-free (SPF) embryonated eggs [8]. The viral subtype was identified by real-time PCR [9]. All experiments involving H5N1 viruses, including animal studies, were performed in a Biosafety Level 3 laboratory at the Harbin Veterinary Research Institute. 2.3. Phylogenetic and molecular analysis To characterize the evolutionary history of the virus isolated from the tiger, whole genome sequencing was conducted. Viral RNA was extracted from allantoic fluid using TRIzol

(Invitrogen, Carlsbad, CA, USA) and was then reversetranscribed. A set of fragment-specific primers (primer sequences are available on request) was used for PCR amplification and sequence analysis. PCR products were purified with a PCR purification kit (Omega, Norcross, GA, USA) and sequenced using the BigDye Terminator V 3.1 Kit with an ABI3500XL DNA sequencer (Life Technologies, Carlsbad, CA, USA). Sequence data were compiled with the SEQMAN program (DNASTAR7.0, Madison, WI, USA). Phylogenetic trees were generated with MEGA 5.2 by the neighborejoining (NJ) method and bootstrap tests (1000 replicates) based on the open reading frame sequences. 2.4. Infection of mice Eight groups of 6-week-old female BALB/c mice (Beijing Experimental Animal Center, Beijing, China) were used for experiments. Seven groups were composed of five mice and one group was composed of three mice. The seven groups of five mice were lightly anesthetized with CO2 and inoculated intranasally with the following concentrations of the virus isolate from the tiger resuspended in 50 ml phosphate buffered saline (PBS): 101.0, 102.0, 103.0, 104.0, 105.0, or 106.0 EID50; the control group received 50 ml PBS alone [8]. The group composed of three mice was also inoculated intranasally with 106.0 EID50. On day 3 post-inoculation (p.i.), the 106.0 EID50 group with three mice were euthanized and organs, including nasal turbinate, lung, kidney, spleen, and brain, were collected and homogenized in 1 ml cold PBS using a Tissue Lyser (QIAGEN, Valencia, CA, USA). Solid debris was pelleted by centrifugation, and either undiluted or 10-fold serially diluted supernatants were inoculated in 10-day-old embryonated eggs. Viral titers in eggs were then calculated using the Reed and Muench method [10]. The remaining groups of mice were monitored daily for weight loss and mortality for 14 days. The viral EID50 for inoculations was calculated using the Reed and Muench method. 2.5. Histopathology and immunohistochemistry of tissues from the tiger To characterize the tissue injury and H5N1 infection status of the dead tiger, tissues were collected and used to make sections for histological and immunohistochemical analyses. The collected tissues were immediately fixed in 10% neutral buffered formalin for histological examination. After 24 h, the fixed tissue samples were processed, paraffin-embedded, and stained with hematoxylin and eosin. Tissues were cut into 3mm sections, mounted on positively charged SuperFrost Plus microscope slides (Menzel, Braunschweig, Germany), dewaxed, and rehydrated. Antigen retrieval was performed by pressure-cooking for 25 min in citrate buffer, pH 6. The sections were incubated with chicken antiserum against type A influenza virus (H5N1; maintained by the Division of Animal Influenza, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) at a 1:2000 dilution. The EnVision AP (DAKO, K1396, Carpinteria, CA, USA)

Please cite this article in press as: He S, et al., Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.10.004

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S. He et al. / Microbes and Infection xx (2014) 1e8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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Table 1 Genetic similarities between the A/Tiger/JiangSu/1/2013 isolate and reference strains available in GenBank. Gene

Region compared (nt)

The virus with the greatest similarity

Similarity (%)

PB2

1e2338

PB1

14e2297

PA

15e2205

NS

1e846

NP

1e1529

HA

1e1705

NA

1e1350

M

1e1017

A/wild duck/Korea/CSM-28/20/2010 (H4N6) A/muscow duck/Vietnam/LBM240/2012 (H3N8) A/muscow duck/Vietnam/LBM/229/2012 (H5N1) A/muscow duck/Vietnam/LBM/230/2012 (H5N1) A/muscow duck/Vietnam/LBM/295/2012 (H5N1) A/muscow duck/Vietnam/LBM/227/2012 (H5N1) A/muscow duck/Vietnam/LBM/228/2012 (H5N1) A/muscow duck/Vietnam/LBM/227/2012 (H5N1) A/muscow duck/Vietnam/LBM/228/2012 (H5N1) A/muscow duck/Vietnam/LBM/229/2012 (H5N1) A/duck/Vietnam/OIE-2202/2012 (H5N1) A/muscow duck/OIE-2215/2012 (H5N1) A/duck/Vietnam/OIE-2202/2012 (H5N1) A/muscow duck/Vietnam/LBM/227/2012 (H5N1) A/grey heron/Hong Kong/1046/2008 (H5N1) A/Shen Zhen/1/2011 (H5N1)

98% 98% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99%

detection system and nuclear fast red (DAKO, K1396) were used as chromogens. Sections were counterstained with Mayer's hematoxylin.

the other viruses from tigers. All gene sequences of the virus isolated from the tiger are available from GenBank under accession numbers KF813110eKF813117.

3. Results

3.3. Molecular characterization of the virus isolate

3.1. Virus isolation

Based on the predicted amino acid sequence, the virus isolate from the tiger encoded 567 amino acids in its HA fragment, and had a multibasic amino acid motif (PQRERRRKKR/GL) at its HA cleavage sites, which is a characteristic of HPAI viruses [12e14]. According to the H5N1 Genetic Changes Inventory (http://www.cdc.gov/flu/ pdf/avianflu/h5n1-inventory.pdf), some mutations of virus proteins were encoded by this isolate. Some mutations (such as Asp94Asn, Ser133Aln, Ser155Asn, Thr156Ala, Gln222Leu, and Gly224Ser) were found in the HA of the virus isolate from the tiger, which could affect fusion and play a role in enhancing the binding of the H5N1 virus to the sialic acid (SA) a-2, 6-Gal receptor [15e18]. This finding implied that the isolate could preferentially bind to SA a-2, 6-Gal linkages of receptors on mammal cells. Glycosylation sites can prevent antibody recognition and virus neutralization. The gain or loss of glycosylation sites near a basic amino acid can also reduce the sensitivity of HA to proteases, thereby increasing its pathogenicity. The HA from the tiger virus isolate had seven glycosylation sites: 26 NNST, 27 NSTE, 39 NVTV, 156 NSSF, 181 NNTN, 209 NPTT, 302 NSSM, 499 NGTY and 558 NGSL. Antivirals are essential for the treatment and prevention of influenza infections. It has been reported that several amino acid mutations in NA, including Glu119Val, Arg293Lys, Asn294Ser, and His274Tyr (numbered according to the NA sequence of the N2 subtype), could confer viral resistance to NA inhibitors (e.g., oseltamivir and zanamivir) [19e22]. The His274Tyr and Asn294Ser mutations were reported to confer resistance to oseltamivir in clinical influenza (H5N1) isolates [23]. The tiger virus NA did have a His274Tyr mutation, the results suggested that the isolated virus should be sensitive to NA inhibitors.

One virus was isolated from the dead Bengal tiger by inoculating ten-day-old SPF embryonated eggs; the virus was named A/Tiger/Jingsu/1/2013 (H5N1). The isolate was determined to be of the H5N1 subtype by real-time PCR analysis. The pathogenicity of the isolate was characterized by inoculating eight 5week-old specific-pathogen-free (SPF) chickens with the virus intravenously. All eight inoculated chickens died within 24 h. Based on the World Organization for Animal Health criteria, this virus was highly pathogenic to chickens [11]. 3.2. Phylogenetic analysis To understand the molecular epidemiology of this the virus isolate from the tiger, we conducted genomic sequencing and a nucleotide BLASTn analysis of the eight virus segments (Table 1), and also carried out a phylogenetic analysis (Fig. 1). In the HA gene tree, the virus isolated from the tiger fell into clade 2.3.2.1. The PB1, PA, NP, and NS segments shared high homology with the A/muscow duck/Vietnam/LBM/227/2012 (H5N1) virus (Table 1). By contrast, the HA and NA genes were more similar to the A/duck/Vietnam/OIE-2202/2012 (H5N1) virus, and the M gene was more similar to the A/grey heron/Hong Kong/ 1046/2008 (H5N1) virus. The PB2 segment shared 98% homology with the A/wild duck/Korea/CSM-28/20/2010 (H4N6) virus, which was derived from a non-HPAI H5N1 virus. Moreover, another 3 isolates from tiger (A/tiger/Shanghai/01/ 2005(H5N1), A/Tiger/Thailand/VSMU-1-SPB/2004(H5N1) and A/tiger/Thailand/2004(H5N1)) had been in the phylogenetic analysis, there was no association between the virus and


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Several amino acid mutations in the polymerases (PB2, PB1, and PA) or NP protein may have important effects on the virulence and adaptation of the H5N1 virus in hosts, including mice. Studies have shown that amino acid substitutions (e.g. Glu627Lys and Asp701Asn of PB2, and Asn319Lys of NP) might increase the virulence of H5N1 viruses in mammals [24e26]. Nevertheless, no amino acid substitutions were found at these residues, except for Lys627Glu of PB2. The NS1 protein plays an important role in the pathogenicity of H5N1 viruses in different hosts. Previous studies have shown that several mutations, such as Pro42Ser, Asp87Glu, Leu98Phe, and Ile101Met, can increase the virulence of H5N1 viruses in mice [27e31]. These mutations were observed in the virus isolated from the tiger. The tiger virus encoded an “avian-like” ESEV motif at the NS1 C-terminal region and a PDZ-domain motif, which might contribute to increased virulence [32,33]. Only one substitution that has been reported to increase virulence in mice, Asn30Asp, occurred in the M1 protein [34]. In summary, all of the amino acid or motif changes and the phenotypic consequences of the gene segments of the virus could contribute to increased virulence or enhance the binding of H5N1 virus to the SA a-2, 6-Gal receptor. 3.4. Mice studies To determine the capacity of H5N1 avian influenza viruses to replicate and be pathogenic in mammals, we infected mice with this virus. We observed pathological symptoms, such as loss of appetite, difficulty breathing, ataxia, shaking, and slow motion in mice after infection. We inoculated eight groups of mice intranasally with 106.0 EID50 virus. We euthanized one group of three mice on day 3 p.i. and collected tissues, including nasal turbinate, lung, spleen, kidney, and brain, for virus titration in eggs. For the other seven groups of mice, we observed these mice for two weeks for changes in body weight or morbidity (Fig. 2). The viral titer in the mice was highest in the lungs, followed by the nasal turbinate and then the other three organs (Fig. 2A). At day 3 p.i., mice inoculated with 10 6.0 EID50 began to die (Fig. 2B). By day 8 p.i., all mice in the experimental groups had died, whereas no mice in the control group died. Mice inoculated with the virus isolate from the tiger exhibited greater than 20% body weight loss (Fig. 2C). These findings indicated that the virus isolated from the tiger could replicate in mice and establish a lethal infection. 3.5. Histopathology and immunohistochemistry of the tiger lung Histological examination showed that necrosis occurred in the central vein area in liver tissue. Additionally, splenic periarterial lymphatic sheath cells were markedly reduced, and

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most cells in this region were T lymphocytes (Fig. 3). Pathological changes in lung bronchioles cells were apparent, whereby epithelial cells “peeled off” and blocked the bronchial lumen. 4. Discussion In this study, the isolate from the tiger was confirmed to be HPAI A virus H5N1 according to sequence analysis. Compared with the previously published sequences of avian influenza virus, the isolated strain A/tiger/Jiangsu/01/ 2013(H5N1) had a high level of homology with the recently identified HPAI H5N1 viruses from Southeast Asia and China, but low levels of homology with isolates from tigers in Shanghai and Vietnam. These findings indicate that the source of the strain is avian rather than from tigers. A retrospective epidemiological survey was adopted to confirm this speculation. The tiger was fed with carcasses of chickens and may have become infected with H5N1 from the infected poultry. Consequently, poultry products that are contaminated with highly pathogenic influenza viruses would be a threat to mammals [35]. After being fed with the chicken carcasses, the tiger died within 24 h. Prior to death, the tiger suffered fever and dyspnea with copious amounts of yellow-green liquid from the nose and mouth. The autopsy established that a large volume of yellow-green liquid was also present in the lungs, and therefore death was likely to have resulted from hypoxia followed by respiratory obstruction from the inflammatory exudate. Histochemistry studies were then conducted. Histologically stained sections showed central vein necrosis in liver tissue, which might have been caused by hypoxia. Indeed, cells in the central vein area of the liver are more likely to become damaged because of a lack of oxygen than those in portal areas. In spleen, lymphocytes in the periarteriolar lymphoid sheaths, which mainly include T lymphocytes and are distinct from those in the lymph nodes, were scare. The scarcity of T lymphocytes might be related to their mobilization and consumption during the progression of some acute viral diseases. Simultaneously, lung bronchial epithelial cells had “peeled off” and blocked the bronchial lumen, which might have caused difficulty in breathing. Thus, we can speculate that after infection with H5N1, T lymphocytes in the lymphoid organs of the tiger were rapidly mobilized by the innate immune response, while the bronchial obstruction in lung resulted in difficulty in breathing because of the lack of oxygen, thereby causing liver necrosis. The difficulty in breathing might have directly contributed to the death of the tiger. Many studies have indicated that pathogenicity is determined not only by the functional integrity of each gene for infection, but also by the interactions between the virus and

Fig. 1. Phylogenetic trees of the HA (a), NA (b), NP (c), M (d), NS (e), PB2 (f), PA (g) and PB1 (h) genes of the virus isolate from the tiger and related reference viruses. The evolutionary relationships among the viruses were estimated by the neighborejoining method with 1000 bootstraps using MEGA version 5.2. The segment PB2 shared higher homology with viruses that were not derived from HPAI H5N1 virus, whereas the other 7 segments shared high homology with the HPAI A H5N1 virus. Please cite this article in press as: He S, et al., Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.10.004

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A

sa Na

l

B

10 8 6 4 2 0 b in tur

100 PBS

Percent survival

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65


Virus titers (Log 10 EID50)

6

ate

ng Lu

lee Sp

n

Kid

y ne

ain Br

80

101 EID50

60

102 EID50

40

104 EID50

103 EID50 105 EID50

20 0

106 EID50

0

2

4

6

8

10

12

14

Days post-challenge

Organs

C

Fig. 2. Virus titers in each organ of mice after challenge with influenza. Influenza virus was detected in nasal turbinate, brains, spleens, kidneys, and lungs of infected mice (A). The virus titer in lung tissue was the highest among the organs that we tested. The survival rate (B) and weight changes (C) of mice inoculated with the H5N1 virus isolated from the tiger are shown. Groups of five mice were intranasally inoculated with 106 EID50 (in a 50 ml volume of PBS) or with PBS as a control and were weighed daily for 14 days.

the host [23,32]. Influenza viruses attach to host cells by the binding of HA to sialosaccharides on the host cell surface. Receptor binding preference is a major factor in determining host species tropism. Abundant SA-a2, 6-Gal and SA-a2, 3-

Gal sialosaccharides are expressed on the surface of tiger epithelial cells in the respiratory and intestinal tracts [36]. The seven genes (PB1, PA, HA, NA, M, NP, and NS ) of this virus showed 99% nucleotide identity with recently identified H5N1

Fig. 3. Histological and immunohistochemical analysis of tissues from the dead tiger. In the liver, the area around the central vein became necrotic (A). In the spleen, the number of lymphocytes around the periaterial lymphatic sheath became reduced (B). In the lung, pathological changes to the bronchiolar cells were obvious and we observed that epithelial cells were shed and blocked the lumen (C). By analyzing lung tissue sections by immunohistochemistry, lung tissues strongly stained positively for HPAI A H5N1 virus antigen in epithelial cells (D). The negative control (E). Please cite this article in press as: He S, et al., Lethal infection by a novel reassortant H5N1 avian influenza A virus in a zoo-housed tiger, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.10.004

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viruses, but one gene (PB2) was derived from a non-H5N1 influenza virus [7]. This indicates that the virus may have derived from a reassortment between the influenza A H5N1 from the Vietnam strain and other subtypes. It remains to be proved whether the virus has enhanced levels of sensitivity and pathogenicity to mammalian cells after this recombination. Many amino acid mutations happened in the fragments of the virus, and the results of the mutations contributed to viral virulence and adaptation in tigers [37]. Taken together, the above findings proved that the tiger died as a result of infection with the influenza A H5N1 virus, but it remains unclear why the other four tigers that were co-housed in the same cage did not become ill or die. Thanawongnuwech et al. previously reported that H5N1 influenza virus transmission occurred between tigers in Thailand in 2004, but this outbreak did not occur for accidental reasons [38]. In the case of this current infection, zoo officials immediately moved the other tigers to other cages when the tiger became symptomatic, which could have prevented the transmission of the virus between animals. In addition, nasal swabs were collected from the four healthy tigers and underwent real-time fluorescent PCR to confirm that the tigers were H5N1-negative. Unfortunately, serum samples were not taken from the four tigers because of difficulties in collection. Therefore, we could not confirm whether these tigers were infected with the virus or not. We speculate that the four tigers did not eat the chicken carcasses contaminated with H5N1, and the early isolation avoided horizontal virus transmission. The evolution of influenza viruses is remarkably dynamic. Influenza viruses evolve rapidly in sequence and undergo frequent reassortments of gene segments [39]. As a consequence of continual genetic reassortment, a diverse collection of H5N1 virus genotypes has been identified since 2001 [12]. HPAI H5N1 viruses have become endemic in poultry in southern China and Southeast Asia since 2003. Genetic analysis revealed that this epidemiology resulted in the establishment of multiple different H5N1 sublineages [40]. Our studies in mice confirmed that the virus isolated from the tiger was highly virulent, which explains why the tiger died within a very short period after feeding on chicken carcasses. This observation was surprising as it demonstrated that the virus had developed enhanced levels of pathogenicity, potentially explained by the genetic reassortment. This observation needs to be verified by further testing. However, this phenomenon indicates that we need to be vigilant against such changes occurring in viruses that are pathogenic to humans. Human H7N9 avian flu infections and deaths occurred in eastern China a month after the death of this tiger, and it is impossible to predict when a novel reassortment will occur between H5N1 and H7N9 influenza viruses or others. References [1] Mukhtar MM, Rasool ST, Song DG, Zhu CL, Hao Q, Zhu Y, et al. Origin of highly pathogenic H5N1 avian influenza virus in China and genetic characterization of donor and recipient viruses. J Gen Virol 2007;88:3094e9.

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