Material and Methods

Virus propagation

For virus isolationlung tissue samples were triturated with tissue tearor machine (Biospec products Inc. USA) with phosphate-buffered saline containing antibiotics, solid debris was pelleted by centrifugation at 4000g for 5 min. The supernatant fluid was further passed through 0.2 micrometer pore-size syringe filter (Millipore). The filtered homogenate was inoculated into the allantoic cavity of 10-day-old embryonated chicken eggs and incubated for 24–48h at 37oC. Embryonated eggs were chilled at 4oC and the allantoic fluids were harvested. All experiments using infectious virus were conducted in a biosafety level 3 (BSL-3) facility.

Reverse Transcription PCR

RNA was extracted from 500μl infectious allantoic fluid using TRIzol LS reagent (Invitrogen) and eluted in 20μl of water. For cDNA synthesis 13.5μl of RNA was added to reverse transcription reaction mixture (6.5μl),containing 1μl influenza A universal reverse transcription primer (5’-AGCAAAAGCAGG-3’)0.5μM, 4μl of 5X MLV (reverse transcription) buffer and 1μl of 10mM dNTP. Mixture was incubated at 65oC for 10 minutes and after adding 0.5μl of MLV (200u/μl) RT reaction was performed at 37oC for one hour. PCR was performed to amplify gene fragments 3 to 8 asdescribed previously[1],while to amplify full length of gene fragments 1 and 2, two sets of primers were used. (Primer sequences are available upon request). PCR amplification was performed with initial step of denaturation at 94°C for 5 min and then 30 cycles of denaturation at 94°C for 40 sec,annealing at 54°C to 58°C for 45 sec, extension at 72°C for 7 min, followed by final extension at 72°C for 10 min. DNA corresponding in size to the gene segment of interest was excised and purified with the Agarose Gel DNA purification kit (TaKaRa. Japan).

Genome sequencing

The purified PCR product was ligated with pMD18-T vector (TaKaRa. Japan) which is used to clone gene fragments of PCR products containing poly A overhang at 3' to T cloning site of the vector. Ligation product was then transformed to DH5α Escherichia coli cells and incubated at 37oC with ampicillin resistance. Cultures were grown overnight and after picking single colonies at random, plasmids were extracted using the Qiaprep spin miniprep kit (Qiagen). Clones with inserted gene, were identified and confirmed, by enzyme digestion. Genesequences were determined on an automated sequencer in Invitrogen Company in Shanghai in China. The nucleotide sequence of the clones was determined twice.

Phylogenetic analysis and Genotyping

More than thirty strains from variety of avian and mammalian hosts were included in phylogenetic analysis depending on their close similarity among different genes with tiger isolate. Strains isolated from canine and feline species from different geographical locations were also included in analysis to better understand the origin and evolution of virus. Sequence editing, multiple sequence alignment and homology analysis were performed with ClustalW and MEGA software[2]. Neighbor-joining (NJ), Maximum Parsimony (MP) and Minimum Evolution (ME) trees were generated by using MEGA. Estimates of the phylogenies were calculated by performing 500 bootstrap replicates. Nucleotide distance matrices were estimated by Kimura two-parameter algorithm based on the number of total nucleotide substitutions and evolutionary trees for all genes were constructed. We used flu genome website, a specialized bioinformatics tool to define genotype of influenza A viruses. In this method of genotyping each gene segment is analyzed separately and a letter representing lineage for every gene is assigned. Genotyping is defined by sequential combination of lineages for eight gene segments. For HA, NA and NS genes, numbering is given with letter which represents subtype or allele.

Abbreviations used for; Animals: BGs, Bar Headed Goose; Ck, Chicken; Cr, Crow; Dg, Dog; Dk, Duck; Fl, Feline; Gs, Goose; GF, Guinea Fowl; Leo, Leopard; MD, Migratory Duck; Tig, Tiger; TD, Tufted Duck. Places: Aus, Austria; Ay, Anyang; Dag, Dagestan; Den, Denmark; Fj, Fujian; Gd, Guangdong; Ger, Germany; Gx, Guangxi; Gy, Guiyang; HK, Hong Kong; Hb, Hubei; Hn, Hunan; Ind, Indonesia; Jl, Jilin; Jx, Jiangxi; Kor, Korea; Nig, Nigeria; Nvs, Novosibrisk; Osk, Osaka; Qh, Qinghai; Sh, Shanghai; St, Shantou; Thai, Thailand; Tul, Tula

References

1. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR (2001) Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146: 2275-2289

2. Tamura K DJ, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. J Mol Evol 10: 1093

Figure legend

Figure S1. Phylogenetic trees are based on the nucleotide sequences of influenza A virus NA(a) and PA(b) genes. The phylogenetic trees are generated by using the Neighbor-Joining alogrithm. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The evolutionary distances were computed using the Kimura 2-parameter method. Analyses was based on nucleotides 126-1078(953) of NA and 683-2172(1490) of PA gene in the final dataset. Phylogenetic analyses were conducted in MEGA. The tiger virus of this study is indicated by (▲). Abbreviations are listed in materials and methods section.

Table S1. Available lineages for all 8 gene segments of H5N1 avian influenza viruses from flugenome database.

PB2
Lineages / PB1 Lineages / PA Lineages / HA Lineages / NP Lineages / NA Lineages / M Lineages / NS Lineages
G / G / E / 5E / F / 1E / F / *2A
K / E / D / 5G / 1F / 1E
I / 5H / 1G / 1F
5J / 1H
1J

Table S1. Lineages are assigned to each gene segment on the basis of evolutionary rates. The genotype of influenza A viral strain is sequential combination of the lineages for each of the gene segments in a genome. Genotype of A/Tig/SH/01/2005 (H5N1) was interrogated from flugenome database. Letters which are bold and underlined represent the lineage of each gene segment of A/Tig/SH/01/2005 (H5N1). Sequential aggregation of eight assigned gene segment lineages shows that genotype of A/Tig/SH/01/2005 (H5N1) virus is K,G,D,5J,F,1J,F,1E., where 5J at number four position stands for subtype H5 of HA segment and 1J at position six stands for subtype 1 of NA segment.

* Genotype K,G,D,5J,F,1J,F,2A., represented by Gs/Gd/96

Fig S1

S1(a)NA

S1(b)PA