Complete genome analysis and identification of highly pathogenic H5N1 influenza A virus from tiger in China

摘要

Wild birds serve as natural reservoir for all subtypes of influenza virus but in thesehosts the viruses can cause no disease signs and are in evolutionary stasis. After transfer tonew avian or mammalian hosts, the viruses evolve rapidly and cause mild respiratory andoccasionally severe diseasE. Rapid rate of evolution thought to be the result of adaptivechanges by virus to grow in abnormal host species. Highly pathogenic avian influenza(HPAI) H5N1 has been reported in several Asian countries including Cambodia, China,Indonesia, Japan, Korea, Laos, Pakistan, Thailand and Vietnam. China is considered ashypothetical influenza epicenter and outbreaks of disease in China in early months of 2004 were widely dispersed. In May 2005, H5N1 virus outbreak occurred in migratory waterfowls at Qinghai lake, western China which caused death of thousands of migratory birds.It has been reported that H5N1 virus has caused fatal infection in dogs, domestic cats,leopards, and tigers; species previously considered to be resistant to disease from influenzaA virus infection. The transmission of avian influenza A virus to mammalian species is ofgreat concern because this may allow the virus to adapt to mammalian hosts and acquirepandemic potential. In July 2005, zoo tigers in Shanghai of China showed typical signs of respiratoryinfection. This finding was alarming as influenza virus infection causing high mortality inzoo tigers was already reported. (A/Tig/SH/O1/2005 (H5N1) virus was isolated from tissuesamples of dead tiger and H5N1 influenza A virus infection was confirmed. Completegenome sequence analysis was performed to understand evolution and origin of the isolatE.Sequence analysis revealed that tiger influenza virus was highly identical to H5N1 virusisolated from migratory duck at Poyang lake, Jiangxi in May 2005. Genotyping resultsshowed that tiger virus isolate belonged to genotype K,G,D,5J,F,1J,F,1E. Phylogeneticanalysis revealed that (A/Tig/SH/O1/2005 (H5N1) virus was closely related to other H5N1 isolates, mainly from southern China. HA gene harbored mutation of R to G at position 323in cleavage site, which might be a host adaptive signature and an important factor inevolution. Genetic analysis and chronological infozznation about NA gene showed deletionof 20 amino acids in stalk region same as found in other HSN1 isolates from hosts likehumans, cats, dogs, leopards, tigers and variety of domestic and wild birds. Unique pattern of mutation which was observed mostly in isolates after year 2004, on both sides of stallsregion deletion is suggested as a step toward adaptation of virus and it seems as virus isselecting sites for nice and balanced cutting of sequence for further shortening of NA stalkregion and for increased virulencE. PB2 gene contained E at position 627 and evidencesindicate that PB2 gene having no substitution of K at 627 position can also cross speciesbarrier and replicate in mammalian hosts like humans and tigers. NS gene showed deletionof 5 amino acids at position 80-84. Sequence ESEV at C terminal of NS which ischaracteristic of HPAI viruses is conserved, furthermore (A/Tig/SH/O1/2005 (H5N1) tiger influenza virus also maintained amino acid A at position 149 of NS gene which canantagonize the induction of host interferon response and is indicative of high virulence ofInfluenza A viruses. Genetic analysis of all gene segments revealed that (A/Tig/SH/O1/2005(H5N1) virus isolated from zoo tiger was highly pathogenic. Amino acid substitutions atHA cleavage site and on both sides of NA stalk region deletion were conserved in most ofthe recent isolates. This finding demands continuous surveillance of future H5N1 isolatesand justifies continuous analysis of emerging influenza A viruses. In order to determine the pathogenesis of the virus infection in mammals, eight micewere inoculated intraperitoneally with A/Tiger/SH/01/2005(H5N1) and two mice wereinjected with sterile PBS (Phosphate buffer saline) as control in a BSL-3 facility, andobserved daily for any clinical signs. At 7 days post challenge, all mice were euthanizedand macroscopic examination was performed to find any lesions in visceral organs. Thenbrain and lung tissues were selected for microscopic examination and virus antigendetection by immunohistochemistry assay with antibody to H5N1 influenza virus. Theresults showed that after five days of infection all mice inoculated with the virus, showedthe clinical sign of nervous system with paralysis more prominent in hind legs, resulting inability to move, weak and emaciated due to lack of feed and water intakE. Gross lesionexamination showed that brain tissue was enlarged when compared to control. Lungs werecongested and more fragile in texture than control group. Histopathological examinationrevealed that bronchiolar and alveolar walls were thickened while brain tissue showeddegenerative changes. Influenza virus antigen expression was detected in both brain andlung tissues. The main lesions in the pulmonary tissue involved alveoli and bronchioles.Results of immunohistochemistry of brain tissue showed positive signal for antigenlocalization in neurons, glial cells and nerve cells of cortex layer. (A/Tig/SH/O1/2005(H5N1) virus had multiple basic amino acids at the HA cleavage site which allowed for theenhanced HA cleavabiiity in many different cell types throughout the visceral organs and itis possible that after getting entry into body influenza virus could spread to extra respiratorytissues by blood. This was first ever report of highly pathogenic influenza A virus infection to bigmammals in China, furthermore the pathogenesis of influenza infection was studied in micetissues and antigen expression was identified in extrapulmonary tissues. This study mayhelp to understand pathogenesis and evolutionary pathways of highly pathogenic H5N1influenza isolates in futurE.

目录

英文文摘

论文说明：List of abbreviations

声明

Acknowledgements

PartA:Chapter 1. A review of evolution and adaptation of influenza A virus to mammalian hosts

1 General introduction and history of influenza viruses

2 Structure and Life cycle of influenza virus

3 Topological and chronological information about highly pathogenic avian influenza viruses(HPAI)

4 Transmission from avian to mammalian hosts

5 Global threat of pandemic and importance of its preparedness

6 Significance of whole genome sequence information

7 Growth of Influenza Virus Sequences in GenBank

References

PartA:Chapter 2: A review of importance of histopathology and immunohistochemistry in detection of pathogenic antigen

1 Principal factors affecting the outcome of immunohistochemistry for detecting cellular antigen

2 Antigens and Antibodies

2.1 Antibody structure

3 Monoclonal and polyclonal Abs

3.1 Polyclonal Abs

3.2 Monoclonal Abs

4 Detection Systems

4.1 Direct methods

4.2 Indirect methods

5 Ag Retrieval

5.1 Heat-induced epitope retrieval

6 IHC on mouse tissues

7 Color of the Ag-Ab reaction

References

PartB:Chapter 3 .Complete genome analysis of highly pathogenic H5N1 influenza A virus from tiger in China

1 INTRODUCTION

1.1 H5N1 Influenza A Viruses

1.2 General features of the Influenza virus genome

1.3 Reasons of reassortment and immunity escape

1.4 Expansion of Host range from avian to mammals

1.5 Surveillance of Domestic Poultry

1.6 Transmission of Influenza Virus to tigers

1.7 Phylogenetic Systematics

2 Materials and Methods

2.1 Virus isolation and propagation

2.2 Virus propagation in MDCK cells

2.3 Virus titration

2.4 RNA extraction and Reverse Transcription

2.5 Polymerase chain reaction(PCR)

2.6 Agarose Gel Electrophoresis and DNA purification

2.7 Genome sequencing

2.8 Phylogenetic Analysis

2.9 Genotyping

2.10 List of abbreviations used in analysis

3 Results and Discussion

3.1 Isolation and amplification of complete genome of A/Tig/SH/01/2005(H5N1)

3.2 Genotype of A/Tig/SH/01/2005(H5N1)

3.3 Phylogenetic Analysis

3.4 Genetic Characterization

References

PartB:Chapter 4.Pathogenic identification of A/Tiger/SH/01/2005(H5N1)in mice

1 Introduction

2 Materials and Methods

2.1 Challenge of virus to mice

2.2 Macroscopic Examination

2.3 Microscopic Examination

3 Results and discussion

3.1 Clinical signs and Macroscopic Examination

3.2 Microscopic examination

References

Conclusion

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